Abstract

The treatment of healthy donors with granulocyte colony-stimulating factor (G-CSF) and dexamethasone results in sufficient numbers of circulating granulocytes to prepare granulocyte concentrates for clinical purposes. Granulocytes obtained in this way demonstrate relatively normal functional behavior combined with a prolonged life span. To study the influence of mobilizing agents on granulocytes, we used oligonucleotide microarrays to identify genes that are differentially expressed in mobilized granulocytes compared with control granulocytes. More than 1000 genes displayed a differential expression pattern, with at least a 3-fold difference. Among these, a large number of genes was induced that encode proteins involved in inflammation and the immune response, such as C-type lectins and leukocyte immunoglobulin-like receptors. Because mobilized granulocytes have a prolonged life span, we focused on genes involved in the regulation of apoptosis. One of the most prominent among these was CAST, the gene encoding calpastatin. Calpastatins are the endogenous inhibitors of calpains, a family of calcium-dependent cysteine proteases recently shown to be involved in neutrophil apoptosis. Transcriptional activity of the CAST gene was induced by G-CSF/dexamethasone treatment both in vivo and in vitro, whereas the protein expression of CAST was stabilized during culture. These studies provide new insight in the genotypic changes as well as in the regulation of the immunologic functions and viability of mobilized granulocytes used for clinical transfusion purposes.

Introduction

Granulocyte concentrates constitute a promising adjuvant tool in the treatment of neutropenic and immunocompromised patients experiencing life-threatening infections in which the exclusive use of modern antimicrobial drugs and additional growth factors is ineffective.1-3  Donor stimulation with a combination of granulocyte colony-stimulating factor (G-CSF) and dexamethasone has become the standard procedure to increase the number of neutrophils in the circulation of the donors and, thus, to collect a sufficient amount of cells for the preparation of granulocyte concentrates.4 

In addition to its important function in granulopoiesis, G-CSF has been reported to modulate several granulocyte functions in vitro. For instance, G-CSF increases the chemotactic ability, enhances cell adhesion to vascular endothelium, promotes phagocytosis, and primes the NADPH-oxidase activity (for a review, see Eyles et al5 ). However, glucocorticosteroids such as dexamethasone have been suggested to suppress some granulocyte functions, including mobility, adhesion, and microbial killing.6-8 

Despite all the aforementioned studies, we and others have found that donor granulocytes, when mobilized for transfusion purposes, show virtually normal functional characteristics in vitro in the presence of some minor phenotypic changes, whereas their life span is consistently prolonged.9,10 

Both G-CSF and dexamethasone are well-established prosurvival factors for neutrophilic granulocytes,11-13  and this effect involves various survival signaling pathways. G-CSF has been shown to increase in neutrophils the mRNA expression of A1/Bfl-1, an antiapoptotic member of the Bcl-2 family of proteins.14  The prosurvival effect of dexamethasone in neutrophils has been connected to the stabilization of Mcl-1, another antiapoptotic Bcl-2 family member, during neutrophil culture.15  Furthermore, G-CSF inhibits the mitochondria-dependent activation of caspase-3 in neutrophils via control of the calpain-dependent degradation of the X-linked inhibitor of apoptosis (XIAP).16,17  In these studies, the antiapoptotic effect of G-CSF was shown to depend on de novo protein synthesis.

Calpains form a family of calcium-dependent cysteine proteases of which calpain-1 (μ-calpain, calpain I), calpain-2 (m-calpain, calpain II), and the natural inhibitor of calpains, calpastatin, are ubiquitously expressed. Several studies have implicated calpain activity in spontaneous neutrophil apoptosis,18,19  and there is a growing number of indications that calpains play an important role in the early phase of programmed cell death.17,20 

Although some of the major functional characteristics relevant for host defense seem well preserved in the donor granulocytes, the extent to which other relevant properties of these cells are altered by the in vivo preactivation is as yet unclear. To understand how G-CSF and dexamethasone may induce the granulocytes to obtain their increased survival capacity, we performed a comparative study with the use of Agilent microarrays (Agilent Technologies Netherlands, Amstelveen, The Netherlands) coupled with real-time reverse transcription–polymerase chain reaction (RT-PCR), flow cytometry, and immunoblotting. In the present study, we demonstrate that mobilization of granulocytes with G-CSF and dexamethasone has a considerable impact on gene expression of these cells, with more than 1000 genes being strongly affected. These changes could be partially mimicked by in vitro culture of neutrophils in the presence of G-CSF and dexamethasone. However, more than 75% of changes in gene expression were unique for in vivo mobilization. The affected genes encoded proteins involved in cellular transcriptional activity and protein synthesis, immune response and inflammation, as well as cell survival and apoptosis. We show that among the genes involved in control of cell apoptosis, treatment with G-CSF and dexamethasone induced an increased expression of calpastatin, the endogenous inhibitor of calpains. This increased expression also could be accomplished by culturing neutrophils with G-CSF/dexamethasone in vitro. Furthermore, calpains are demonstrated to contribute importantly to neutrophil apoptosis, whereas the increase in calpastatin mRNA and protein levels directly corresponds to the prolonged lifespan of neutrophils when treated with G-CSF/dexamethasone in vivo or in vitro.

Methods

Experimental design

Granulocytes from 3 different healthy donors (2 males and 1 female) were studied before and after treatment with G-CSF and dexamethasone. Donors received G-CSF (5 μg/kg subcutaneously) and dexamethasone (8 mg orally). The study was approved by the ethical medical committee at Sanquin Research and Landsteiner Laboratory and was conducted with informed consent given in accordance with the Declaration of Helsinki. Blood samples were taken just before the donor treatment (control sample, fresh) and 16 to 20 hours after G-CSF and dexamethasone administration (in vivo treatment).

Neutrophils from the control sample were isolated and directly prepared for RNA isolation (see the section “RNA isolation, amplification, labeling, and hybridization”) or cultured overnight in the absence (control sample, apoptosis) or presence of G-CSF (Amgen Europe, Breda, The Netherlands) and dexamethasone (Sigma-Aldrich, St Louis, MO; in vitro treatment). Thus, 4 pools of RNA were obtained for comparison (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article).

Granulocyte isolation and culture

Heparinized venous blood was collected from the donors, and the granulocytes were isolated as described.21,22  In short, the granulocytes and mononuclear cells were separated over isotonic Percoll with a specific density of 1.076 g/mL. Erythrocytes in the pellet were lysed in ice-cold medium containing 155 mmol/L NH4Cl, 10 mol/L KHCO3, and 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA), pH 7.4. Granulocytes were washed and resuspended in Hepes-buffered saline solution (HBSS, containing 132 mmol/L NaCl, 6.0 mmol/L KCl, 1.0 mmol/L CaCl2, 1.0 mmol/L MgSO4, 1.2 mmol/L potassium phosphate, 20 mmol/L Hepes, 5.5 mmol/L glucose, and 0.5% (wt/vol) human serum albumin, pH 7.4). The purity of granulocytes isolated with this method was more than 95%. Overnight culture was performed in HBSS with or without addition of 100 ng/mL G-CSF and 1 μmol/L dexamethasone.

RNA isolation, amplification, labeling, and hybridization

Total cellular RNA was extracted from a minimum of 20 × 106 cells with TRIzol reagent (Invitrogen, Breda, The Netherlands) according to the protocol provided by the manufacturer, with the following minor modifications. An additional phenol-chloroform extraction was performed and the isopropanol precipitation at −20°C was facilitated by the addition of 20 μg/mL glycogen (Roche Applied Science, Almere, The Netherlands). Purity and integrity of the RNA samples were confirmed on the Agilent 2100 bioanalyzer (Agilent Technologies Netherlands) using the RNA 6000 Nano LabChip kit. Finally, mRNA was amplified with the MessageAmp II Kit (Applied Biosystems/Ambion, Foster City, CA). Labeling, hybridization, and data extraction were performed at ServiceXS (Leiden, The Netherlands), as has been described elsewhere.23 

Microarray imaging and data analysis

The microarray slides were scanned with the Agilent dual-laser DNA microarray scanner. Default settings of Agilent Feature Extraction preprocessing protocols were used to obtain normalized expression values from the raw scans. Exact protocol and parameter settings are described in the Agilent Feature Extraction Software User Manual 8.5 (http://chem.agilent.com/scripts/LiteraturePDF.asp?iWHID = 37 629). The default Agilent normalization procedure, called Linear & Lowess, was applied. Rosetta Resolver (Rosetta Biosoftware, Seattle, WA) was used for analysis of the data. Genes were defined as differentially transcribed if the average expression level changed at least 3-fold compared with unstimulated cells (0 hours, control sample) in all 3 donors. The microarray data have been deposited in National Center for Biotechnology Information's Gene Expression Omnibus24  and are accessible through GEO Series accession number GSE12841 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12841).

Quantitative RT-PCR validation of gene expression

PCR amplification was performed on a LightCycler instrument (Roche Applied Science), and analyzed with LightCycler Software version 3.5 (Roche Molecular Biochemicals, Mannheim, Germany). The reaction was performed with Lightcycler FastStart DNA MasterPLUS SYBR Green I (Roche Diagnostics, Indianapolis, IN). The annealing temperature used for all primers was 65°C. The reaction mixture consisted of 4 μL of cDNA, 1 μL of relevant primer combination, and 4 μL of SYBR Green I mix in a total volume of 20 μL. All amplified cDNA was compared with the standard within the same run, and in every run the same standard was used, although there was very little variation in the standard between runs.

For amplification, the following LightCycler protocol was used. The chemical cleft of the Taq polymerase was removed by preincubation for 10 minutes at 95°C; the template was amplified for 40 cycles, with annealing of the primers at 65°C. The fluorescence was measured at the end of each cycle at 72°C. At the end of 40 cycles, a melting curve was generated to determine the unique features of the DNA amplified. The specific size of the product was determined on a 1% (wt/vol) agarose gel. Subsequently, the obtained band was purified using the GFX PCR DNA and Gel Band purification kit (Amersham Biosciences, a division of GE Healthcare, Little Chalfont, United Kingdom) according to the manufacturer's instructions to remove excess dNTPs and primers. The product was sequenced by Big-dye Terminator Sequencing and ABI Prism software (Applied Biosystems, Foster City, CA). The sequence was verified with BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) to determine specificity. All products obtained were unique and had no overlap with other isoforms.

Standard curves and relative quantitation

As a source of cDNA for standard curves to which all samples were normalized, neutrophils were isolated from an apheresis buffy coat obtained from the Sanquin Blood Bank North-West Region (Amsterdam, The Netherlands). Serial 10-fold dilutions from the cDNA obtained were made to which each sample was quantified with the method described in Technical Note No. LC 13/2001 (Roche Applied Science), as has been described elsewhere.25 

Immunostaining and FACS analysis

Cell surface expression of various receptors on granulocytes was assayed in total leukocyte samples by flow cytometry (ie, fluorescence-activated cell-sorting [FACS]), with saturating concentrations of commercially available monoclonal antibodies (MoAbs), either directly labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indirectly labeled with allophycocyanin (APC). CD52-FITC, CD55-FITC, CD59-FITC, and CD69-PE were from Sanquin Reagents (Amsterdam, The Netherlands); EMR3-FITC and CD177-unconjugated were from AbD Serotec (Oxford, United Kingdom); and goat F(ab′)2 anti–mouse-IgG-APC was from Southern Biotech (Birmingham, AL).

Samples were analyzed on an LSRII flow cytometer equipped with FACSDiva software (BD, Franklin Lakes, NJ). Cells were gated based on their forward and side scatter, and 10 000 gated events were collected per sample (100% positive staining for CD16 and negative for CD36 [monocytes] or CD56 [NK cells] confirmed purity of the analyzed population).

Annexin V binding

To detect apoptosis, cells were labeled for 10 minutes on ice with FITC-labeled annexin V (Bender Med Systems, Vienna, Austria), diluted 1:500 in HBSS, and supplemented with 2.5 mM CaCl2. Annexin V labeling was followed by a single wash step with the same medium, whereupon the cells were resuspended in HBSS 2.5 mM CaCl2 containing 1 μg/mL propidium iodide (PI; Sigma-Aldrich). After an additional 5 minutes on ice, the samples were analyzed on a FACScan flow cytometer (BD). Surviving cells were defined as the cells in the lower left quadrant that stained negative for both annexin V and PI. A total of 10 000 events were collected for each sample, and data were analyzed with the use of CellQuest Pro software (BD).

Western blot analysis

Total cell lysates were prepared by treating the cells with a lysis buffer (250 mmol/L sucrose, 70 mmol/L KCl, 0.5% Triton X-100 (vol/vol), 0.5% β-octylglucoside (vol/vol), 2 mmol/L NaVO4, 1 mmol/L NaF, 1 mM EDTA, supplemented with a complete protease inhibitor cocktail mix (PIM; Roche Diagnostic, Almere, The Netherlands) and 2 mmol/L diisopropylfluorophosphate (DFP; Fluka Chemica, Steinheim, Switzerland) in phosphate-buffered saline [PBS]) for 30 minutes on ice. Afterward, samples were mixed with 4× Laemli sample buffer (LSB; 50 mmol/L Tris-HCl, pH 6.8, 10% glycerol [vol/vol], 5 mmol/L DTT [DL-dithiothreitol, Sigma] 1% β-mercaptoethanol, 1% sodium dodecylsulfate [SDS; m/v], 10 μg/mL bromophenol blue) and boiled for 15 minutes at 95°C. All samples were stored at −20°C before subjection to SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Samples were run on 10%, 1.5-mm polyacrylamide gels in a protean-3 mini system (Bio-Rad Laboratories, Veenendaal, The Netherlands). The equivalent of 1.5 × 106 cells was loaded in each lane.

After electrophoresis, proteins were transferred to polyvinyl difluoride membranes (PVDF, Bio-Rad), which were subsequently blocked for 60 minutes with blocking buffer (5% nonfat dry milk [m/v, Elk; Campina, Zaltbommel, The Netherlands] in Tris-buffered saline, 0.1% Tween-20 [vol/vol]). The membranes were immune-labeled with specific antibodies in blocking buffer containing 2 mmol/L NaN3 overnight at 4°C. After washing, the membranes were labeled with fluorescently labeled secondary antibodies (either IRDye 800CW or IRDye 700 [Li-COR Bioscience, Lincoln, NE]), and the protein bands were visualized with the Odyssey Infrared Imaging System (Li-COR), and analyzed with the accompanying software (version 2.1).

Statistics

Significantly regulated genes were selected with Rosetta Resolver (Rosetta Biosoftware, Seattle, WA). Genes with a fold change greater than or equal to 3, together with a P value cutoff of .01 (by 1-way analysis of variance [ANOVA] test with the Benjamini-Hochberg false-discovery rate correction), were considered significantly different across the different cell populations. Genes differentially expressed in mobilized granulocytes were categorized by reported or putative functions by the use of the OntoExpress program (http://vortex.cs.wayne.edu/projects.htm#Onto-Express). Graphs were drawn and statistical analysis was performed with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Data were evaluated by paired, one-tailed Student t test where indicated. The results are presented as the mean plus or minus SEM, as indicated.

Results

G-CSF/dexamethasone mobilization of neutrophils induces global changes in gene expression

Treatment of healthy donors with a combination of a single dose of G-CSF and dexamethasone has been described to result in an increase in the number of circulating neutrophils within 2 to 4 hours after administration, which peaks within 12 to 16 hours (Figure 1A) and returns back to normal levels within 48 to 72 hours.26  Granulocytes for transfusion usually are collected during the peak phase of mobilization (ie, after overnight treatment).

Figure 1

Effect of G-CSF and dexamethasone on granulocytes. (A) Comparison of the granulocyte concentration per milliliter of blood and the EMR3 expression level in time, after donor treatment with G-CSF and dexamethasone (representative graph). PMNs indicate polymorphonuclear leukocytes; ○, EMR3 expression; ■, PMNs per milliliter. (B) Changes in granulocyte gene expression after stimulation with G-CSF and dexamethasone. At 18 to 20 hours after stimulation of the donors or culture of granulocytes with G-CSF and dexamethasone in vitro, neutrophil gene expression was determined by the use of Agilent Whole Humane Genome microarrays. Numbers refer to differentially expressed genes in neutrophils isolated 18 to 20 hours after the administration of G-CSF and dexamethasone in vivo and/or those cultured overnight in medium supplemented with G-CSF and dexamethasone.

Figure 1

Effect of G-CSF and dexamethasone on granulocytes. (A) Comparison of the granulocyte concentration per milliliter of blood and the EMR3 expression level in time, after donor treatment with G-CSF and dexamethasone (representative graph). PMNs indicate polymorphonuclear leukocytes; ○, EMR3 expression; ■, PMNs per milliliter. (B) Changes in granulocyte gene expression after stimulation with G-CSF and dexamethasone. At 18 to 20 hours after stimulation of the donors or culture of granulocytes with G-CSF and dexamethasone in vitro, neutrophil gene expression was determined by the use of Agilent Whole Humane Genome microarrays. Numbers refer to differentially expressed genes in neutrophils isolated 18 to 20 hours after the administration of G-CSF and dexamethasone in vivo and/or those cultured overnight in medium supplemented with G-CSF and dexamethasone.

We used a microarray approach to analyze gene expression patterns in mobilized cells in comparison with the cells obtained from the same healthy control donors before G-CSF/dexamethasone administration. The administration of G-CSF/dexamethasone results in both mobilization of immature neutrophils from the bone marrow, as indicated by staining with EMR3, a myeloid-specific member of the epidermal growth factor-7-transmembrane (EGF-TM7) family of adhesion class TM7 receptors, which has been recently described as a marker for mature granulocytes (Figure 1A),27  as well as direct effects on the already circulating cells. To estimate the contribution of the latter, we also performed in vitro G-CSF/dexamethasone incubations and analyses on neutrophils taken before the in vivo treatment (see also the scheme for experimental design in Figure S1). This step also allows the evaluation of changes in gene expression associated with the G-CSF/dexamethasone–induced delay in apoptosis.

By the use of Agilent Whole Human Genome microarrays, we screened approximately 32 000 gene transcripts. This analysis revealed that approximately 1000 genes were differentially expressed in the cells after the administration in vivo of G-CSF/dexamethasone compared with those isolated before mobilization (Figure 1B and Document S1). Even more genes were differentially expressed after treatment in vitro of neutrophils with G-CSF/dexamethasone (Figure 1B and Document S1).

Approximately 150 genes were similarly regulated in both experimental systems (in vivo vs in vitro, Table 1), but still more than 800 genes demonstrated a unique expression pattern for the in vivo stimulation.

Table 1

Genes similarly regulated by the treatment with G-CSF/dexamemethasone in vivo and in vitro

Sequence ID Primary sequence name Sequence code Sequence description Fold change
 
In vivo In vitro 
1507161 CD163 NM_004244 CD163 molecule 54.27 11.88 
1984958 CD177 CR592446 CD177 molecule 24.38 8.50 
1519170 GADD45A NM_001924 Growth arrest and DNA-damage-inducible, alpha 20.49 10.75 
1515523 SLC1A3 NM_004172 Solute carrier family 1 (glial high affinity glutamate transporter), 16.98 11.45 
1521404 IL18R1 NM_003855 Interleukin 18 receptor 1 16.54 8.19 
1523267 FKBP5 NM_004117 FK506 binding protein 5 13.12 7.36 
1528485 SORT1 NM_002959 Sortilin 1 12.11 20.42 
1534791 SAMSN1 NM_022136 SAM domain, SH3 domain and nuclear localization signals, 1 11.87 8.34 
1506996 ADORA3 NM_020683 Adenosine A3 receptor 10.90 10.49 
1525991 TRPS1 NM_014112 Trichorhinophalangeal syndrome I 9.13 4.45 
1538316 ACN9 NM_020186 ACN9 homolog (Saccharomyces cerevisiae) 9.13 6.87 
1508652 KLF9 NM_001206 Kruppel-like factor 9 8.65 7.46 
1524454 XBP1 NM_005080 X-box binding protein 1 7.48 6.51 
1510813 IRAK3 NM_007199 Interleukin-1 receptor-associated kinase 3 7.28 10.56 
1531325 ORF1-FL49 NM_032412 Putative nuclear protein ORF1-FL49 7.04 4.25 
1516682 CAST NM_001750 Calpastatin 6.97 10.64 
1506968 ST6GALNAC3 NM_152996 ST6 6.56 4.94 
1513738 SPFH1 NM_006459 SPFH domain family, member 1 6.39 6.20 
1991986 TRPM2 NM_001001 188 Transient receptor potential cation channel, subfamily M, member 2 6.38 13.63 
1989099 GRINA NM_000837 Poly (ADP-ribose) polymerase family, member 10 5.60 11.34 
1522658 VNN1 NM_004666 Vanin 1 5.35 4.47 
1533591 SIRT5 NM_012241 Sirtuin 4.60 4.50 
1521022 HIPK2 AK074291 Homeodomain interacting protein kinase 2 4.28 8.24 
1505831 CIAS1 NM_004895 Cold autoinflammatory syndrome 1 4.10 19.21 
1527074 PEX11G NM_080662 Peroxisomal biogenesis factor 11 gamma 4.06 4.21 
1507368 CCND3 NM_001760 Cyclin D3 3.69 5.58 
1519571 CDC42EP3 NM_006449 CDC42 effector protein (Rho GTPase binding) 3 3.58 7.45 
1531872 TMEM140 AK056910 Transmembrane protein 140 3.48 6.02 
1534101 IL1R1 NM_000877 Interleukin 1 receptor, type I 3.35 6.15 
1511860 ZDHHC2 NM_016353 Zinc finger, DHHC-type containing 2 3.24 4.21 
1530609 QSCN6 NM_002826 Quiescin Q6 3.08 4.22 
1518979 CPD NM_001304 Carboxypeptidase D 3.03 4.63 
1506387 MAPK6 NM_002748 Mitogen-activated protein kinase 6 3.03 5.03 
1515049 ZNF278 NM_032051 Zinc finger protein 278 −3.05 −4.11 
1983183 NDUFB6 NM_182739 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6, 17 kDa −3.10 −4.04 
1507683 FANCA NM_000135 Fanconi anemia, complementation group A −3.19 −4.79 
1985202 IGKC BC067092 Immunoglobulin kappa constant −3.21 −5.50 
1522438 GLIPR1 U16307 GLI pathogenesis-related 1 (glioma) −3.25 −12.71 
1525398 RFX5 NM_000449 Selenium binding protein 1 −3.25 −5.02 
1522187 TOPORS NM_005802 Topoisomerase I binding, arginine/serine-rich −3.26 −4.96 
1536170 TST NM_003312 Thiosulfate sulfurtransferase (rhodanese) −3.28 −6.47 
1530334 CST3 NM_000099 Cystatin C −3.29 −5.23 
1534756 PSMB10 NM_002801 Proteasome (prosome, macropain) subunit, beta type, 10 −3.30 −5.60 
1519302 STAT1 NM_139266 Signal transducer and activator of transcription 1, 91 kDa −3.39 −10.76 
1522898 MVP AK097472 Major vault protein −3.51 −4.58 
1524812 CEP63 NM_025180 Centrosomal protein 63kDa −3.54 −4.22 
1538148 RAB37 NM_175738 RAB37, member RAS oncogene family −3.55 −5.17 
1986068 RNF43 CR620892 Ring finger protein 43 −3.60 −7.06 
1984701 CARD9 NM_052813 Caspase recruitment domain family, member 9 −3.61 −27.75 
1523761 CBR1 NM_001757 Carbonyl reductase 1 −3.61 −4.75 
1511505 RABGAP1L AB007940 RAB GTPase activating protein 1-like −3.63 −10.78 
1511424 EMR3 NM_032571 Egf-like module containing, mucin-like, hormone receptor-like 3 −3.71 −8.61 
1513189 TRERF1 NM_033502 Transcriptional regulating factor 1 −3.76 −16.11 
1990225 IGF2BP2 AA451676 Insulin-like growth factor 2 mRNA binding protein 2 −3.79 −6.35 
1513264 CST5 NM_001900 Cystatin D −3.82 −5.63 
1990830 FAM111A BU684362 Family with sequence similarity 111, member A −3.83 −8.95 
1534340 CD46 NM_002389 CD46 molecule, complement regulatory protein −3.83 −6.11 
1517555 CYP27A1 NM_000784 Cytochrome P450, family 27, subfamily A, polypeptide 1 −3.88 −7.43 
1516190 SAMHD1 NM_015474 SAM domain and HD domain 1 −3.89 −8.86 
1528817 MME NM_007289 Membrane metalloendopeptidase (neutral endopeptidase) −3.92 −8.11 
1536250 CCT2 NM_006431 Chaperonin containing TCP1, subunit 2 (beta) −4.04 −5.43 
1515232 POLB NM_002690 Polymerase (DNA directed), beta −4.07 −5.28 
1527068 PSME1 NM_006263 Proteasome (prosome, macropain) activator subunit 1(PA28 alpha) −4.15 −4.84 
1986072 COL27A1 AI382322 Collagen, type XXVII, alpha 1 −4.21 −4.54 
1532664 PDLIM1 NM_020992 PDZ and LIM domain 1 (elfin) −4.25 −5.30 
1524915 OR51E1 NM_152430 Olfactory receptor, family 51, subfamily E, member 1 −4.27 −4.44 
1507227 P2RY14 NM_014879 Purinergic receptor P2Y, G-protein coupled, 14 −4.28 −12.99 
1508056 EGR3 NM_004430 Early growth response 3 −4.41 −4.50 
1515615 PRRG4 NM_024081 Proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) −4.42 −4.38 
1517506 EGR2 NM_000399 Early growth response 2 (Krox-20 homolog, Drosophila) −4.67 −14.32 
1513793 CATSPER2 NM_172097 Cation channel, sperm associated 2 −4.74 −4.19 
1533707 RG9MTD2 NM_152292 RNA (guanine-9-) methyltransferase domain containing 2 −4.80 −7.17 
1507738 FBP1 NM_000507 Fructose-1.6-bisphosphatase 1 −4.82 −7.32 
1535598 NCOA4 NM_005437 Nuclear receptor coactivator 4 −4.87 −21.40 
1507851 CTGLF1 NM_133446 Centaurin, gamma-like family, member 1 −4.88 −5.47 
1506502 S100A4 NM_002961 S100 calcium binding protein A4 −4.91 −16.73 
1522210 TIGD7 NM_033208 Tigger transposable element derived 7 −5.02 −10.17 
1522120 MATN1 AK057725 Matrilin 1, cartilage matrix protein −5.06 −4.48 
1519593 IFIT5 NM_012420 Interferon-induced protein with tetratricopeptide repeats 5 −5.07 −11.79 
1533259 RBP7 NM_052960 Ubiquitination factor E4B (UFD2 homolog, yeast) −5.16 −12.38 
1532326 DDX58 NM_014314 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 −5.17 −14.04 
1510042 NHS NM_198270 Nance-Horan syndrome −5.40 −12.90 
1983408 ZFP36L1 CD619445 Zinc finger protein 36, C3H type-like 1 −5.41 −4.09 
1518324 FAM111A NM_022074 Family with sequence similarity 111, member A −5.59 −5.82 
1990540 CLU NM_203339 Clusterin −5.66 −5.71 
1520999 DGKG NM_001346 Diacylglycerol kinase, gamma 90 kDa −5.71 −5.82 
1983869 ADAM28 NM_014265 ADAM metallopeptidase domain 28 −5.76 −7.39 
1518750 PARP16 NM_017851 Poly (ADP-ribose) polymerase family, member 16 −5.84 −11.81 
1505761 TNFAIP2 NM_006291 Beta-amylase −5.90 -9.81 
1534912 CMTM5 NM_181618 Chemokine-like factor superfamily 5 −5.91 −4.63 
1508430 SLC25A22 NM_024698 Solute carrier family 25 (mitochondrial carrier: glutamate), −5.94 −13.23 
1525314 S100A10 NM_002966 S100 calcium binding protein A10 −5.94 −6.72 
1521056 GP1BB NM_000407 Septin 5 −5.97 −4.76 
1539206 SPECC1 NM_152904 Spectrin domain with coiled-coils 1 −6.24 −4.94 
1527296 HERC5 NM_016323 Hect domain and RLD 5 −6.27 −7.29 
1506913 MBIP NM_016586 MAP3K12 binding inhibitory protein 1 −6.28 −21.40 
1515002 CYSLTR1 NM_006639 Cysteinyl leukotriene receptor 1 −6.45 −9.25 
1512448 ISOC1 NM_016048 Isochorismatase domain containing 1 −6.51 −7.66 
1513897 GIMAP5 NM_018384 GTPase, IMAP family member 5 −6.56 −4.68 
1520165 AF242507 AF242507 Homosapiens m-tumor necrosis factor-alpha induced protein mRNA, −6.58 −5.74 
1534914 MPEG1 AK074166 macrophage expressed gene 1 −6.60 −5.82 
1988054 RAD50 AK128882 RAD50 homolog (S cerevisiae−6.71 −6.23 
1513813 G0S2 NM_015714 G0/G1 switch 2 −6.74 −10.55 
1530332 MX1 NM_002462 Myxovirus (influenza virus) resistance 1, −6.76 −12.23 
1522509 OR2B2 NM_033057 Olfactory receptor, family 2, subfamily B, member 2 −6.88 −4.91 
1991493 PARP14 NM_017554 Poly (ADP-ribose) polymerase family, member 14 −7.03 −4.29 
1527378 CSF1R NM_005211 Colony stimulating factor 1 receptor, −7.08 −8.69 
1514500 CEP57 BC039711 Centrosomal protein 57 kDa −7.08 −7.41 
1522188 PTGS2 NM_000963 Prostaglandin-endoperoxide synthase 2 −7.22 −90.22 
1537251 OR52K3P AF143328 Olfactory receptor, family 52, subfamily K, member 3 pseudogene −7.25 −5.96 
1514502 CCDC37 NM_182628 Coiled-coil domain containing 37 −7.27 −5.51 
1528770 RPGRIP1 NM_020366 Retinitis pigmentosa GTPase regulator interacting protein 1 −7.32 −6.15 
1987032 TREX1 NM_016381 Three prime repair exonuclease 1 −7.45 −5.26 
1510272 RNASE3 NM_002935 Ribonuclease, RNase A family, 3 (eosinophil cationic protein) −7.61 −11.42 
1991208 TUSC1 NM_001004125 Tumor suppressor candidate 1 −7.62 −5.54 
1539129 SDPR NM_004657 Serum deprivation response (phosphatidylserine binding protein) −7.80 −5.25 
1538745 SGK NM_005627 Serum/glucocorticoid regulated kinase −7.90 −5.90 
1524314 IFI44 NM_006417 Interferon-induced protein 44 −7.91 −10.63 
1989096 SSBP3 NM_001009955 Single stranded DNA binding protein 3 −8.76 −4.70 
1520076 MFI2 BC032042 Antigen p97 −8.93 −6.15 
1518480 DDHD1 AK091528 DDHD domain containing 1 −9.02 −8.19 
1512017 RNASE2 NM_002934 Ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin) −9.05 −8.41 
1536355 LPAL2 AK125910 Homo sapiens cDNA FLJ43922 fis −9.08 −13.45 
1533135 CXCL10 NM_001565 Chemokine (C-X-C motif) ligand 11 −9.10 −8.41 
1522417 TRIO AY358688 Triple functional domain (PTPRF interacting) −9.21 −10.68 
1518425 PSME2 NM_002818 Proteasome (prosome, macropain) activator subunit 2 (PA28 beta) −9.29 −12.60 
1512119 KNTC2 NM_006101 Kinetochore associated 2 −9.33 −4.25 
1506804 PLD4 NM_138790 Phospholipase D family, member 4 −9.53 −4.93 
1520503 NKX3–1 NM_006167 NK3 transcription factor related, locus 1 −9.56 −7.38 
1533140 NME5 NM_003551 nucleoside-diphosphate kinase −9.99 −8.02 
1536919 HLA-DMB NM_002118 Major histocompatibility complex, class II, DM beta −10.73 −4.66 
1538875 AF15Q14 NM_020380 AF15q14 protein −10.83 −7.68 
1519782 LAP3 NM_015907 Leucine aminopeptidase 3 −11.23 −24.38 
1529525 LYSMD2 NM_153374 LysM, putative peptidoglycan-binding, domain containing 2 −11.50 −5.09 
1516830 P2RY10 NM_014499 Purinergic receptor P2Y, G-protein coupled, 10 −11.67 −4.14 
1519975 PRSS33 NM_152891 Protease, serine, 33 −11.84 −5.57 
1507811 PMAIP1 NM_021127 Phorbol-12-myristate-13-acetate-induced protein 1 −12.76 −6.44 
1506299 MMP12 ENST000003 26227 Macrophage metalloelastase precursor −12.98 −7.44 
1536513 IFIT3 NM_001549 Interferon-induced protein with tetratricopeptide repeats 3 −13.08 −9.40 
1513158 HRH4 NM_021624 Histamine receptor H4 −13.66 −6.92 
1537338 FGL2 NM_006682 Fibrinogen-like 2 −13.76 −31.09 
1509838 GBP1 NM_002053 Guanylate binding protein 1, interferon-inducible, 67kDa −13.91 −11.20 
1512112 EPSTI1 NM_033255 Epithelial stromal interaction 1 (breast) −14.02 −9.27 
1522895 GOLPH4 NM_014498 Golgi phosphoprotein 4 −14.30 −12.18 
1508419 CD52 NM_001803 CD52 molecule −14.95 −4.38 
1506912 RARRES1 NM_002888 Retinoic acid receptor responder (tazarotene induced) 1 −15.02 −9.44 
1509737 IFIT2 NM_001547 Interferon-induced protein with tetratricopeptide repeats 2 −15.06 −31.11 
1505505 CCL23 NM_005064 Chemokine (C-C motif) ligand 23 −15.06 −4.22 
1518780 AF131788 AF131788 Clone 24875 mRNA sequence −16.78 −12.00 
1522258 ISG15 NM_005101 Interferon, alpha-inducible protein (clone IFI-15K) −18.16 −4.81 
1519399 GBP5 NM_052942 Guanylate binding protein 5 −18.22 −10.65 
1531078 RSAD2 NM_080657 Radical S-adenosyl methionine domain containing 2 −18.54 −8.17 
1515740 SLC45A3 NM_033102 Solute carrier family 45, member 3 −26.27 −28.60 
1517997 IFIT1 NM_001548 Interferon-induced protein with tetratricopeptide repeats 1 −34.47 −35.07 
1533457 FCER1A NM_002001 Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide −61.82 −31.20 
1532327 CLC NM_001828 Charcot-Leyden crystal protein −69.08 −16.00 
Sequence ID Primary sequence name Sequence code Sequence description Fold change
 
In vivo In vitro 
1507161 CD163 NM_004244 CD163 molecule 54.27 11.88 
1984958 CD177 CR592446 CD177 molecule 24.38 8.50 
1519170 GADD45A NM_001924 Growth arrest and DNA-damage-inducible, alpha 20.49 10.75 
1515523 SLC1A3 NM_004172 Solute carrier family 1 (glial high affinity glutamate transporter), 16.98 11.45 
1521404 IL18R1 NM_003855 Interleukin 18 receptor 1 16.54 8.19 
1523267 FKBP5 NM_004117 FK506 binding protein 5 13.12 7.36 
1528485 SORT1 NM_002959 Sortilin 1 12.11 20.42 
1534791 SAMSN1 NM_022136 SAM domain, SH3 domain and nuclear localization signals, 1 11.87 8.34 
1506996 ADORA3 NM_020683 Adenosine A3 receptor 10.90 10.49 
1525991 TRPS1 NM_014112 Trichorhinophalangeal syndrome I 9.13 4.45 
1538316 ACN9 NM_020186 ACN9 homolog (Saccharomyces cerevisiae) 9.13 6.87 
1508652 KLF9 NM_001206 Kruppel-like factor 9 8.65 7.46 
1524454 XBP1 NM_005080 X-box binding protein 1 7.48 6.51 
1510813 IRAK3 NM_007199 Interleukin-1 receptor-associated kinase 3 7.28 10.56 
1531325 ORF1-FL49 NM_032412 Putative nuclear protein ORF1-FL49 7.04 4.25 
1516682 CAST NM_001750 Calpastatin 6.97 10.64 
1506968 ST6GALNAC3 NM_152996 ST6 6.56 4.94 
1513738 SPFH1 NM_006459 SPFH domain family, member 1 6.39 6.20 
1991986 TRPM2 NM_001001 188 Transient receptor potential cation channel, subfamily M, member 2 6.38 13.63 
1989099 GRINA NM_000837 Poly (ADP-ribose) polymerase family, member 10 5.60 11.34 
1522658 VNN1 NM_004666 Vanin 1 5.35 4.47 
1533591 SIRT5 NM_012241 Sirtuin 4.60 4.50 
1521022 HIPK2 AK074291 Homeodomain interacting protein kinase 2 4.28 8.24 
1505831 CIAS1 NM_004895 Cold autoinflammatory syndrome 1 4.10 19.21 
1527074 PEX11G NM_080662 Peroxisomal biogenesis factor 11 gamma 4.06 4.21 
1507368 CCND3 NM_001760 Cyclin D3 3.69 5.58 
1519571 CDC42EP3 NM_006449 CDC42 effector protein (Rho GTPase binding) 3 3.58 7.45 
1531872 TMEM140 AK056910 Transmembrane protein 140 3.48 6.02 
1534101 IL1R1 NM_000877 Interleukin 1 receptor, type I 3.35 6.15 
1511860 ZDHHC2 NM_016353 Zinc finger, DHHC-type containing 2 3.24 4.21 
1530609 QSCN6 NM_002826 Quiescin Q6 3.08 4.22 
1518979 CPD NM_001304 Carboxypeptidase D 3.03 4.63 
1506387 MAPK6 NM_002748 Mitogen-activated protein kinase 6 3.03 5.03 
1515049 ZNF278 NM_032051 Zinc finger protein 278 −3.05 −4.11 
1983183 NDUFB6 NM_182739 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6, 17 kDa −3.10 −4.04 
1507683 FANCA NM_000135 Fanconi anemia, complementation group A −3.19 −4.79 
1985202 IGKC BC067092 Immunoglobulin kappa constant −3.21 −5.50 
1522438 GLIPR1 U16307 GLI pathogenesis-related 1 (glioma) −3.25 −12.71 
1525398 RFX5 NM_000449 Selenium binding protein 1 −3.25 −5.02 
1522187 TOPORS NM_005802 Topoisomerase I binding, arginine/serine-rich −3.26 −4.96 
1536170 TST NM_003312 Thiosulfate sulfurtransferase (rhodanese) −3.28 −6.47 
1530334 CST3 NM_000099 Cystatin C −3.29 −5.23 
1534756 PSMB10 NM_002801 Proteasome (prosome, macropain) subunit, beta type, 10 −3.30 −5.60 
1519302 STAT1 NM_139266 Signal transducer and activator of transcription 1, 91 kDa −3.39 −10.76 
1522898 MVP AK097472 Major vault protein −3.51 −4.58 
1524812 CEP63 NM_025180 Centrosomal protein 63kDa −3.54 −4.22 
1538148 RAB37 NM_175738 RAB37, member RAS oncogene family −3.55 −5.17 
1986068 RNF43 CR620892 Ring finger protein 43 −3.60 −7.06 
1984701 CARD9 NM_052813 Caspase recruitment domain family, member 9 −3.61 −27.75 
1523761 CBR1 NM_001757 Carbonyl reductase 1 −3.61 −4.75 
1511505 RABGAP1L AB007940 RAB GTPase activating protein 1-like −3.63 −10.78 
1511424 EMR3 NM_032571 Egf-like module containing, mucin-like, hormone receptor-like 3 −3.71 −8.61 
1513189 TRERF1 NM_033502 Transcriptional regulating factor 1 −3.76 −16.11 
1990225 IGF2BP2 AA451676 Insulin-like growth factor 2 mRNA binding protein 2 −3.79 −6.35 
1513264 CST5 NM_001900 Cystatin D −3.82 −5.63 
1990830 FAM111A BU684362 Family with sequence similarity 111, member A −3.83 −8.95 
1534340 CD46 NM_002389 CD46 molecule, complement regulatory protein −3.83 −6.11 
1517555 CYP27A1 NM_000784 Cytochrome P450, family 27, subfamily A, polypeptide 1 −3.88 −7.43 
1516190 SAMHD1 NM_015474 SAM domain and HD domain 1 −3.89 −8.86 
1528817 MME NM_007289 Membrane metalloendopeptidase (neutral endopeptidase) −3.92 −8.11 
1536250 CCT2 NM_006431 Chaperonin containing TCP1, subunit 2 (beta) −4.04 −5.43 
1515232 POLB NM_002690 Polymerase (DNA directed), beta −4.07 −5.28 
1527068 PSME1 NM_006263 Proteasome (prosome, macropain) activator subunit 1(PA28 alpha) −4.15 −4.84 
1986072 COL27A1 AI382322 Collagen, type XXVII, alpha 1 −4.21 −4.54 
1532664 PDLIM1 NM_020992 PDZ and LIM domain 1 (elfin) −4.25 −5.30 
1524915 OR51E1 NM_152430 Olfactory receptor, family 51, subfamily E, member 1 −4.27 −4.44 
1507227 P2RY14 NM_014879 Purinergic receptor P2Y, G-protein coupled, 14 −4.28 −12.99 
1508056 EGR3 NM_004430 Early growth response 3 −4.41 −4.50 
1515615 PRRG4 NM_024081 Proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) −4.42 −4.38 
1517506 EGR2 NM_000399 Early growth response 2 (Krox-20 homolog, Drosophila) −4.67 −14.32 
1513793 CATSPER2 NM_172097 Cation channel, sperm associated 2 −4.74 −4.19 
1533707 RG9MTD2 NM_152292 RNA (guanine-9-) methyltransferase domain containing 2 −4.80 −7.17 
1507738 FBP1 NM_000507 Fructose-1.6-bisphosphatase 1 −4.82 −7.32 
1535598 NCOA4 NM_005437 Nuclear receptor coactivator 4 −4.87 −21.40 
1507851 CTGLF1 NM_133446 Centaurin, gamma-like family, member 1 −4.88 −5.47 
1506502 S100A4 NM_002961 S100 calcium binding protein A4 −4.91 −16.73 
1522210 TIGD7 NM_033208 Tigger transposable element derived 7 −5.02 −10.17 
1522120 MATN1 AK057725 Matrilin 1, cartilage matrix protein −5.06 −4.48 
1519593 IFIT5 NM_012420 Interferon-induced protein with tetratricopeptide repeats 5 −5.07 −11.79 
1533259 RBP7 NM_052960 Ubiquitination factor E4B (UFD2 homolog, yeast) −5.16 −12.38 
1532326 DDX58 NM_014314 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 −5.17 −14.04 
1510042 NHS NM_198270 Nance-Horan syndrome −5.40 −12.90 
1983408 ZFP36L1 CD619445 Zinc finger protein 36, C3H type-like 1 −5.41 −4.09 
1518324 FAM111A NM_022074 Family with sequence similarity 111, member A −5.59 −5.82 
1990540 CLU NM_203339 Clusterin −5.66 −5.71 
1520999 DGKG NM_001346 Diacylglycerol kinase, gamma 90 kDa −5.71 −5.82 
1983869 ADAM28 NM_014265 ADAM metallopeptidase domain 28 −5.76 −7.39 
1518750 PARP16 NM_017851 Poly (ADP-ribose) polymerase family, member 16 −5.84 −11.81 
1505761 TNFAIP2 NM_006291 Beta-amylase −5.90 -9.81 
1534912 CMTM5 NM_181618 Chemokine-like factor superfamily 5 −5.91 −4.63 
1508430 SLC25A22 NM_024698 Solute carrier family 25 (mitochondrial carrier: glutamate), −5.94 −13.23 
1525314 S100A10 NM_002966 S100 calcium binding protein A10 −5.94 −6.72 
1521056 GP1BB NM_000407 Septin 5 −5.97 −4.76 
1539206 SPECC1 NM_152904 Spectrin domain with coiled-coils 1 −6.24 −4.94 
1527296 HERC5 NM_016323 Hect domain and RLD 5 −6.27 −7.29 
1506913 MBIP NM_016586 MAP3K12 binding inhibitory protein 1 −6.28 −21.40 
1515002 CYSLTR1 NM_006639 Cysteinyl leukotriene receptor 1 −6.45 −9.25 
1512448 ISOC1 NM_016048 Isochorismatase domain containing 1 −6.51 −7.66 
1513897 GIMAP5 NM_018384 GTPase, IMAP family member 5 −6.56 −4.68 
1520165 AF242507 AF242507 Homosapiens m-tumor necrosis factor-alpha induced protein mRNA, −6.58 −5.74 
1534914 MPEG1 AK074166 macrophage expressed gene 1 −6.60 −5.82 
1988054 RAD50 AK128882 RAD50 homolog (S cerevisiae−6.71 −6.23 
1513813 G0S2 NM_015714 G0/G1 switch 2 −6.74 −10.55 
1530332 MX1 NM_002462 Myxovirus (influenza virus) resistance 1, −6.76 −12.23 
1522509 OR2B2 NM_033057 Olfactory receptor, family 2, subfamily B, member 2 −6.88 −4.91 
1991493 PARP14 NM_017554 Poly (ADP-ribose) polymerase family, member 14 −7.03 −4.29 
1527378 CSF1R NM_005211 Colony stimulating factor 1 receptor, −7.08 −8.69 
1514500 CEP57 BC039711 Centrosomal protein 57 kDa −7.08 −7.41 
1522188 PTGS2 NM_000963 Prostaglandin-endoperoxide synthase 2 −7.22 −90.22 
1537251 OR52K3P AF143328 Olfactory receptor, family 52, subfamily K, member 3 pseudogene −7.25 −5.96 
1514502 CCDC37 NM_182628 Coiled-coil domain containing 37 −7.27 −5.51 
1528770 RPGRIP1 NM_020366 Retinitis pigmentosa GTPase regulator interacting protein 1 −7.32 −6.15 
1987032 TREX1 NM_016381 Three prime repair exonuclease 1 −7.45 −5.26 
1510272 RNASE3 NM_002935 Ribonuclease, RNase A family, 3 (eosinophil cationic protein) −7.61 −11.42 
1991208 TUSC1 NM_001004125 Tumor suppressor candidate 1 −7.62 −5.54 
1539129 SDPR NM_004657 Serum deprivation response (phosphatidylserine binding protein) −7.80 −5.25 
1538745 SGK NM_005627 Serum/glucocorticoid regulated kinase −7.90 −5.90 
1524314 IFI44 NM_006417 Interferon-induced protein 44 −7.91 −10.63 
1989096 SSBP3 NM_001009955 Single stranded DNA binding protein 3 −8.76 −4.70 
1520076 MFI2 BC032042 Antigen p97 −8.93 −6.15 
1518480 DDHD1 AK091528 DDHD domain containing 1 −9.02 −8.19 
1512017 RNASE2 NM_002934 Ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin) −9.05 −8.41 
1536355 LPAL2 AK125910 Homo sapiens cDNA FLJ43922 fis −9.08 −13.45 
1533135 CXCL10 NM_001565 Chemokine (C-X-C motif) ligand 11 −9.10 −8.41 
1522417 TRIO AY358688 Triple functional domain (PTPRF interacting) −9.21 −10.68 
1518425 PSME2 NM_002818 Proteasome (prosome, macropain) activator subunit 2 (PA28 beta) −9.29 −12.60 
1512119 KNTC2 NM_006101 Kinetochore associated 2 −9.33 −4.25 
1506804 PLD4 NM_138790 Phospholipase D family, member 4 −9.53 −4.93 
1520503 NKX3–1 NM_006167 NK3 transcription factor related, locus 1 −9.56 −7.38 
1533140 NME5 NM_003551 nucleoside-diphosphate kinase −9.99 −8.02 
1536919 HLA-DMB NM_002118 Major histocompatibility complex, class II, DM beta −10.73 −4.66 
1538875 AF15Q14 NM_020380 AF15q14 protein −10.83 −7.68 
1519782 LAP3 NM_015907 Leucine aminopeptidase 3 −11.23 −24.38 
1529525 LYSMD2 NM_153374 LysM, putative peptidoglycan-binding, domain containing 2 −11.50 −5.09 
1516830 P2RY10 NM_014499 Purinergic receptor P2Y, G-protein coupled, 10 −11.67 −4.14 
1519975 PRSS33 NM_152891 Protease, serine, 33 −11.84 −5.57 
1507811 PMAIP1 NM_021127 Phorbol-12-myristate-13-acetate-induced protein 1 −12.76 −6.44 
1506299 MMP12 ENST000003 26227 Macrophage metalloelastase precursor −12.98 −7.44 
1536513 IFIT3 NM_001549 Interferon-induced protein with tetratricopeptide repeats 3 −13.08 −9.40 
1513158 HRH4 NM_021624 Histamine receptor H4 −13.66 −6.92 
1537338 FGL2 NM_006682 Fibrinogen-like 2 −13.76 −31.09 
1509838 GBP1 NM_002053 Guanylate binding protein 1, interferon-inducible, 67kDa −13.91 −11.20 
1512112 EPSTI1 NM_033255 Epithelial stromal interaction 1 (breast) −14.02 −9.27 
1522895 GOLPH4 NM_014498 Golgi phosphoprotein 4 −14.30 −12.18 
1508419 CD52 NM_001803 CD52 molecule −14.95 −4.38 
1506912 RARRES1 NM_002888 Retinoic acid receptor responder (tazarotene induced) 1 −15.02 −9.44 
1509737 IFIT2 NM_001547 Interferon-induced protein with tetratricopeptide repeats 2 −15.06 −31.11 
1505505 CCL23 NM_005064 Chemokine (C-C motif) ligand 23 −15.06 −4.22 
1518780 AF131788 AF131788 Clone 24875 mRNA sequence −16.78 −12.00 
1522258 ISG15 NM_005101 Interferon, alpha-inducible protein (clone IFI-15K) −18.16 −4.81 
1519399 GBP5 NM_052942 Guanylate binding protein 5 −18.22 −10.65 
1531078 RSAD2 NM_080657 Radical S-adenosyl methionine domain containing 2 −18.54 −8.17 
1515740 SLC45A3 NM_033102 Solute carrier family 45, member 3 −26.27 −28.60 
1517997 IFIT1 NM_001548 Interferon-induced protein with tetratricopeptide repeats 1 −34.47 −35.07 
1533457 FCER1A NM_002001 Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide −61.82 −31.20 
1532327 CLC NM_001828 Charcot-Leyden crystal protein −69.08 −16.00 

Confirmation of microarray data by RT-PCR and flow cytometry

We used LightCycler RT-PCR to verify the changes in gene expression as identified by the microarray analysis. We selected 6 representative genes, encoding well-characterized cell surface receptors, from the microarray dataset for subsequent confirmation by RT-PCR and flow cytometry (Figure 2A). For these genes, there was a strong correlation (r = 0.94) between mRNA levels determined by either microarray or RT-PCR analysis. Mobilization of granulocytes resulted in increased expression levels of complement regulatory factors (CD55 and CD59) and a strong increase in the level of CD177 mRNA, as well as the simultaneous down-regulation of CLEC2A (CD69), CD52, and EMR3 mRNA levels.

Figure 2

G-CSF/dexamethasone mobilization changes the phenotype of granulocytes. (A) Light-cycler confirmation of representative genes. Genes (n = 6 identified as differentially expressed by Agilent Whole Humane Genome microarrays (□) were selected from the overall dataset for confirmation by light cycler real-time PCR () after mobilization with G-CSF/dexamethasone. Data represent the mean ± SD of fold changes in gene expression from the 3 donors used in microarray experiments. Microarray data are presented as the mean fold change of the 3 donors. (B) Flow cytometric analysis of different neutrophil surface receptors. Neutrophils isolated from control donors (□) and those treated with G-CSF/dexamethasone (■) were analyzed for the expression of various surface receptors. Cells were stained with directly labeled monoclonal antibodies and measured by flow cytometry. Results represent the data from 3 independent experiments (mean ± SEM). *P < .05 (significant difference).

Figure 2

G-CSF/dexamethasone mobilization changes the phenotype of granulocytes. (A) Light-cycler confirmation of representative genes. Genes (n = 6 identified as differentially expressed by Agilent Whole Humane Genome microarrays (□) were selected from the overall dataset for confirmation by light cycler real-time PCR () after mobilization with G-CSF/dexamethasone. Data represent the mean ± SD of fold changes in gene expression from the 3 donors used in microarray experiments. Microarray data are presented as the mean fold change of the 3 donors. (B) Flow cytometric analysis of different neutrophil surface receptors. Neutrophils isolated from control donors (□) and those treated with G-CSF/dexamethasone (■) were analyzed for the expression of various surface receptors. Cells were stained with directly labeled monoclonal antibodies and measured by flow cytometry. Results represent the data from 3 independent experiments (mean ± SEM). *P < .05 (significant difference).

Because all transcripts selected for the validation of the microarray data by quantitative RT-PCR represented surface receptors expressed on the plasma membrane, we determined the protein expression levels by flow cytometry (Figure 2B). Both complement regulatory factors CD55 (also known as decay-accelerating factor [DAF]) and CD59 were found to be present on fresh neutrophils. Their expression increased significantly after treatment with G-CSF/dexamethasone in vivo. Granulocyte mobilization also resulted in the increased expression levels of CD177 (also known as the human neutrophil antigen [HNA]-2a or NB1). Increased expression of CD177 on neutrophils has previously been reported in response to G-CSF treatment for hematopoietic stem cell mobilization.28  Control neutrophils clearly expressed the GPI-anchored proteins CD52 and, to a lesser extent, CD69, whereas the surface expression was either diminished (CD52) or had become completely null (CD69) on donor neutrophils mobilized in vivo, confirming the microarray data.

The expression of EMR3 also was found to be lower on the surface of in vivo mobilized cells. This may correspond with the lower maturation state of the granulocytes “purged” from the bone marrow by the combined treatment with G-CSF and dexamethasone, as also indicated by the concomitant neutrophilic left shift (data not shown).

Regulation of different biologic processes by G-CSF/dexamethasone mobilization

Significant numbers of genes with different expression patterns between the control granulocytes and those isolated after G-CSF/dexamethasone administration fell into 6 main functional gene ontology (GO) categories: (1) transcription and regulation of transcription, (2) signal transduction, (3) immune responses, (4) cell survival and apoptosis, (5) cell adhesion and motility, and (6) cell metabolism (Table 2). Similar analysis has been applied to the genes presented in Table 1 to compare these categories of gene patterns (Table S1).

Table 2

G-CSF/dexamethasone mobilization of granulocytes modulates changes in expression profile of genes involved in different biological processes

Sequence ID Primary sequence name Sequence code Sequence description Fold change 
Transcription and regulation of transcription     
    1514893 CCNA1 NM_003914 Cyclin A1 21.01 
    1983249 SMARCA3 BF513730 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 3 10.43 
    1513827 MLXIP AB020674 MLX interacting protein 8.66 
    1519529 MSL3L1 NM_078629 Male-specific lethal 3-like 1 (Drosophila) 6.75 
    1521872 MEF2A NM_005587 MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A) 6.47 
    1985292 ETS2 BX433326 V-ets erythroblastosis virus E26 oncogene homolog 2 5.59 
    1525013 BATF NM_006399 Basic leucine zipper transcription factor, ATF-like 5.58 
    1537803 ENO1 NM_001428 Enolase 1, (alpha) 5.50 
    1533218 HIPK2 BC041926 CDNA clone IMAGE:5300349 5.07 
    1522326 PHTF1 NM_006608 Putative homeodomain transcription factor 1 4.80 
    1533591 SIRT5 NM_012241 Sirtuin (silent mating type information regulation 2 homolog) 5 (Saccharomyces cerevisiae) 4.60 
    1512793 ZNF167 NM_018651 Zinc finger protein 167 4.30 
    1511846 PLAG1 NM_002655 Pleomorphic adenoma gene 1 4.26 
    1512354 ZNF438 AK095436 hypothetical protein LOC220929 4.20 
    1507417 DACH1 NM_080759 Dachshund homolog 1 (Drosophila) 4.04 
    1526663 ZNF438 NM_182755 Zinc finger protein 438 4.02 
    1537859 ZBTB20 NM_015642 Zinc finger and BTB domain containing 20 3.77 
    1522362 ZNF658 NM_033160 Zinc finger protein 658 3.75 
    1534837 ZNF254 NM_004876 Zinc finger protein 539 3.73 
    1507368 CCND3 NM_001760 Cyclin D3 3.69 
    1509049 VENTX NM_014468 VENT homeobox homolog (Xenopus laevis) 3.61 
    1985753 CRTC3 AK090443 CREB regulated transcription coactivator 3 3.58 
    1529278 XRN2 NM_012255 5′-3′ exoribonuclease 2 3.43 
    1522102 E2F2 NM_004091 E2F transcription factor 2 3.36 
    1517472 CCNC NM_005190 Cyclin C 3.07 
    1535521 KLF6 U51869 Kruppel-like factor 6 3.03 
    1537902 TSC22D1 NM_183422 TSC22 domain family, member 1 −3.01 
    1983175 MCM7 NM_182776 MCM7 minichromosome maintenance deficient −3.02 
    1529350 SSRP1 NM_003146 Structure specific recognition protein 1 −3.03 
    1505187 RELB NM_006509 V-rel reticuloendotheliosis viral oncogene homolog B, nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (avian) −3.04 
    1537144 DEXI AK055529 dexamethasone-induced transcript −3.07 
    1516882 MEF2C NM_002397 MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C) −3.09 
    1527019 CHAF1A NM_005483 Chromatin assembly factor 1, subunit A (p150) −3.25 
    1535620 NCOR2 NM_006312 Nuclear receptor co-repressor 2 −3.34 
    1523857 ZNF395 NM_018660 Zinc finger protein 395 −3.34 
    1512878 ZMYND11 NM_006624 Zinc finger, MYND domain containing 11 −3.35 
    1991240 HMGA1 NM_145904 High mobility group AT-hook 1 −3.37 
    1531990 HEY1 NM_012258 Hairy/enhancer-of-split related with YRPW motif 1 −3.39 
    1533586 ELK1 NM_005229 ELK1, member of ETS oncogene family −3.50 
    1515979 NR4A3 NM_173199 Nuclear receptor subfamily 4, group A, member 3 −3.58 
    1510333 THRA NM_003250 Thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian) −3.68 
    1537025 BTG2 NM_006763 BTG family, member 2 −3.73 
    1513189 TRERF1 NM_033502 Transcriptional regulating factor 1 −3.76 
    1511905 JARID2 NM_004973 Jumonji, AT-rich interactive domain 2 −3.81 
    1526826 ATF3 NM_004024 Activating transcription factor 3 −3.82 
    1523890 RASSF7 NM_003475 Ras association (RalGDS/AF−6) domain family 7 −3.84 
    1533807 MAFF NM_012323 V-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian) −3.89 
    1525862 JUN NM_002228 V-jun sarcoma virus 17 oncogene homolog (avian) −3.94 
    1507971 NRL NM_006177 Neural retina leucine zipper −4.09 
    1533916 LEF1 NM_016269 Lymphoid enhancer-binding factor 1 −4.22 
    1506607 ASF1B NM_018154 ASF1 antisilencing function 1 homolog B (S cerevisiae−4.36 
    1509496 AES NM_198969 Amino-terminal enhancer of split −4.39 
    1533535 TAF4 NM_003185 TAF4 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 135kDa −4.40 
    1508056 EGR3 NM_004430 Early growth response 3 −4.41 
    1533262 BRF1 BC016743 BRF1 homolog, subunit of RNA polymerase III transcription initiation factor IIIB (S cerevisiae−4.48 
    1533571 PHF6 NM_032458 PHD finger protein 6 −4.53 
    1517506 EGR2 NM_000399 Early growth response 2 (Krox−20 homolog, Drosophila) −4.66 
    1989375 SMAD3 NM_005902 SMAD, mothers against DPP homolog 3 (Drosophila) −4.97 
    1507856 MYB NM_005375 V-myb myeloblastosis viral oncogene homolog (avian) −5.06 
    1528623 OLIG2 NM_005806 Oligodendrocyte lineage transcription factor 2 −6.11 
    1524585 GATA1 NM_002049 GATA binding protein 1 (globin transcription factor 1) −6.64 
    1991493 PARP14 NM_017554 Poly (ADP-ribose) polymerase family, member 14 −7.03 
    1510055 PCGF5 BC007377 Polycomb group ring finger 5 −7.57 
    1510369 ASCL2 NM_005170 Achaete-scute complex-like 2 (Drosophila) −7.72 
    1522208 ZNF584 NM_173548 Zinc finger protein 584 −7.93 
    1523937 RPS6KA5 NM_004755 Ribosomal protein S6 kinase, 90 kDa, polypeptide 5 −8.07 
    1529851 ETV7 NM_016135 Ets variant gene 7 (TEL2 oncogene) −8.56 
    1506298 GATA6 NM_005257 GATA binding protein 6 −8.65 
    1989096 SSBP3 NM_001009955 Single stranded DNA binding protein 3 −8.76 
    1520503 NKX31 NM_006167 NK3 transcription factor related, locus 1 (Drosophila) −9.56 
    1522991 ZNF649 NM_023074 Zinc finger protein 649 −11.07 
    1514700 NR4A1 NM_002135 Nuclear receptor subfamily 4, group A, member 1 −92.91 
Signal transduction     
    1513543 GPR84 NM_020370 G protein-coupled receptor 84 25.51 
    1505483 EDG1 NM_001400 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 1 18.05 
    1511321 PTGER2 NM_000956 Prostaglandin E receptor 2 (subtype EP2), 53 kDa 8.26 
    1511570 ARHGAP24 AK130576 Rho GTPase activating protein 24 7.28 
    1523886 PDE4D NM_006203 Phosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce homolog, Drosophila) 6.64 
    1527053 MYO10 BC041694 Myosin X 6.48 
    1512860 MS4A4A NM_024021 Membrane-spanning 4-domains, subfamily A, member 4 6.12 
    1539087 DLC1 NM_182643 Deleted in liver cancer 1 5.96 
    1528643 MS4A6A NM_022349 Membrane-spanning 4-domains, subfamily A, member 6A 5.87 
    1524040 GPR141 NM_181791 G protein-coupled receptor 141 5.28 
    1989101 EDARADD NM_080738 EDAR-associated death domain 4.64 
    1508615 EXT1 NM_000127 Exostoses (multiple) 1 4.30 
    1521881 STK3 NM_006281 Serine/threonine kinase 3 (STE20 homolog, yeast) 3.79 
    1510158 ITPKC NM_025194 Inositol 1.4,5-trisphosphate 3-kinase C 3.63 
    1519571 CDC42EP3 NM_006449 CDC42 effector protein (Rho GTPase binding) 3 3.58 
    1534557 DPYSL3 NM_001387 Dihydropyrimidinase-like 3 3.54 
    1536781 RPS6KA3 NM_004586 Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 3.53 
    1533999 HHIP NM_022475 Hedgehog interacting protein 3.50 
    1512578 GPR97 NM_170776 G protein-coupled receptor 97 3.42 
    1514738 SLC26A6 NM_022911 Inositol hexaphosphate kinase 2 3.28 
    1517434 IL4R NM_000418 Interleukin 4 receptor 3.21 
    1539209 GABBR1 NM_001470 Gamma-aminobutyric acid (GABA) B receptor, 1 3.19 
    1511435 STK16 NM_003691 Serine/threonine kinase 16 3.17 
    1520958 PRKAR2A BC002763 Inositol hexaphosphate kinase 2 3.12 
    1506387 MAPK6 NM_002748 Mitogen-activated protein kinase 6 3.03 
    1523162 ITPK1 NM_014216 Inositol 1.3,4-triphosphate 5/6 kinase −3.05 
    1507956 GNGT2 NM_031498 Guanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 2 −3.09 
    1514326 TAGAP NM_138810 T-cell activation GTPase activating protein −3.09 
    1530414 GPR171 NM_013308 G protein-coupled receptor 171 −3.16 
    1527339 P2RY5 NM_005767 Purinergic receptor P2Y, G-protein coupled, 5 −3.17 
    1517355 ARHGAP25 BC039591 Rho GTPase activating protein 25 −3.23 
    1520830 KIT NM_000222 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog −3.28 
    1517660 KLRK1 NM_007360 Killer cell lectin-like receptor subfamily C, member 4 −3.37 
    1514077 KLRB1 NM_002258 Killer cell lectin-like receptor subfamily B, member 1 −3.43 
    1518328 GRP NM_002091 Gastrin-releasing peptide −3.64 
    1516849 EPHB1 NM_004441 EPH receptor B1 −3.80 
    1514753 PRKAG2 NM_016203 Protein kinase, AMP-activated, gamma 2 non-catalytic subunit −4.09 
    1512127 SOCS1 NM_003745 Suppressor of cytokine signaling 1 −4.13 
    1518864 FLT3LG NM_001459 Fms-related tyrosine kinase 3 ligand −4.26 
    1524915 OR51E1 NM_152430 Olfactory receptor, family 51, subfamily E, member 1 −4.27 
    1507227 P2RY14 NM_014879 Purinergic receptor P2Y, G-protein coupled, 14 −4.28 
    1523084 GUCA1A NM_000409 Guanylate cyclase activator 1A (retina) −4.37 
    1525046 PILRA NM_178273 Paired immunoglobulin-like type 2 receptor alpha −4.48 
    1534223 PPP1R12B NM_032105 Protein phosphatase 1, regulatory (inhibitor) subunit 12B −4.66 
    1518048 GPR34 NM_005300 G-protein–coupled receptor 34 −4.78 
    1534320 ARRB1 NM_004041 Arrestin, beta 1 −5.02 
    1534974 KLRD1 NM_002262 Killer cell lectin-like receptor subfamily D, member 1 −5.16 
    1525314 S100A10 NM_002966 S100 calcium binding protein A10 −5.94 
    1530940 PRKAR2B NM_002736 Protein kinase, cAMP-dependent, regulatory, type II, beta −5.97 
    1522410 IL2RB NM_000878 Interleukin 2 receptor, beta −6.06 
    1518652 CD74 NM_004355 CD74 molecule, major histocompatibility complex, class II invariant chain −6.23 
    1515002 CYSLTR1 NM_006639 Cysteinyl leukotriene receptor 1 −6.45 
    1530332 MX1 NM_002462 Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) −6.76 
    1522509 OR2B2 NM_033057 Olfactory receptor, family 2, subfamily B, member 2 −6.88 
    1984459 PDE4B AB209081 Phosphodiesterase 4B, cAMP-specific −6.89 
    1527378 CSF1R NM_005211 Colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) oncogene homolog −7.08 
    1527858 NRGN NM_006176 Neurogranin (protein kinase C substrate, RC3) −7.49 
    1524392 ANGPT1 NM_001146 Angiopoietin 1 −8.84 
    1516837 MS4A3 NM_006138 Membrane-spanning 4-domains, subfamily A, member 3 −9.24 
    1989999 IL5RA NM_175725 Interleukin 5 receptor, alpha −9.66 
    1524637 GPR162 NM_019858 Triosephosphate isomerase 1 −10.10 
    1519217 P2RY2 NM_176072 Purinergic receptor P2Y, G-protein coupled, 2 −11.66 
    1516830 P2RY10 NM_014499 Purinergic receptor P2Y, G-protein coupled, 10 −11.67 
    1518123 CD83 NM_004233 CD83 molecule −11.67 
    1517216 KLRF1 NM_016523 Killer cell lectin-like receptor subfamily F, member 1 −12.68 
    1512228 EBI2 NM_004951 Epstein-Barr virus–induced gene 2 (lymphocyte-specific G protein-coupled receptor) −13.11 
    1513158 HRH4 NM_021624 Histamine receptor H4 −13.66 
    1537338 FGL2 NM_006682 Fibrinogen-like 2 −13.76 
    1515714 GNG11 NM_004126 Guanine nucleotide binding protein (G protein), gamma 11 −16.57 
    1527111 GPR44 NM_004778 G protein-coupled receptor 44 −23.51 
    1528909 RGS1 NM_002922 Regulator of G-protein signaling 1 −32.65 
Immune responses     
    1507161 CD163 NM_004244 CD163 molecule 54.27 
    1531927 CYP1B1 NM_000104 Cytochrome P450, family 1, subfamily B, polypeptide 1 46.19 
    1984958 CD177 CR592446 CD177 molecule 24.38 
    1521404 IL18R1 NM_003855 Interleukin 18 receptor 1 16.54 
    1983056 MERTK U08023 C-mer proto-oncogene tyrosine kinase 16.00 
    1532739 CLEC5A NM_013252 C-type lectin domain family 5, member A 13.44 
    1511835 CLEC4D NM_080387 C-type lectin domain family 4, member D 12.02 
    1535646 CST7 NM_003650 Cystatin F (leukocystatin) 11.43 
    1506996 ADORA3 NM_020683 Adenosine A3 receptor 10.89 
    1530098 IL18RAP NM_003853 Interleukin 18 receptor accessory protein 10.79 
    1532148 LAIR1 NM_021706 Leukocyte-associated immunoglobulin-like receptor 1 10.45 
    1506562 HP NM_005143 Haptoglobin 10.31 
    1505718 IL1R2 NM_004633 Interleukin 1 receptor, type II 9.95 
    1507483 LILRA5 NM_181879 Leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 5 7.56 
    1524454 XBP1 NM_005080 X-box binding protein 1 7.48 
    1510813 IRAK3 NM_007199 Interleukin−1 receptor-associated kinase 3 7.28 
    1538778 TLR5 NM_003268 Toll-like receptor 5 5.97 
    1521047 CR1 NM_000651 Complement component (3b/4b) receptor 1 (Knops blood group) 5.87 
    1520792 MAPK14 NM_139013 Mitogen-activated protein kinase 14 5.75 
    1535716 CD59 NM_203330 CD59 molecule, complement regulatory protein 5.67 
    1513648 FCER1G CB529629 Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide 5.26 
    1521687 TLR4 BF670973 Toll-like receptor 4 5.15 
    1530114 MAP2K6 NM_002758 Mitogen-activated protein kinase kinase 6 4.87 
    1533789 SERPINB1 NM_030666 Serpin peptidase inhibitor, clade B (ovalbumin), member 1 4.84 
    1522193 FCAR NM_002000 Fc fragment of IgA, receptor for 4.52 
    1527121 PROK2 NM_021935 Prokineticin 2 4.33 
    1505831 CIAS1 NM_004895 Cold autoinflammatory syndrome 1 4.09 
    1524842 IL10 NM_000572 Interleukin 10 3.89 
    1538909 IL1RAP NM_002182 Interleukin 1 receptor accessory protein 3.80 
    1506853 CHIT1 NM_003465 Chitinase 1 (chitotriosidase) 3.48 
    1531091 CD55 NM_000574 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 3.39 
    1534101 IL1R1 NM_000877 Interleukin 1 receptor, type I 3.34 
    1515424 CYBB S67289 Cytochrome b-245, beta polypeptide (chronic granulomatous disease) 3.29 
    1985998 ALOX5 BQ548663 Arachidonate 5-lipoxygenase 3.16 
    1510372 IRAK4 NM_016123 Interleukin-1 receptor-associated kinase 4 3.10 
    1514172 CD48 NM_001778 CD48 molecule 3.09 
    1527340 TLR8 NM_016610 Toll-like receptor 8 3.07 
    1511452 SERPING1 NM_000062 Serpin peptidase inhibitor, clade G (C1 inhibitor), member 1, (angioedema, hereditary) −3.00 
    1530131 IRF2BP2 BC020516 Interferon regulatory factor 2 binding protein 2 −3.03 
    1512742 CLEC7A NM_197954 C-type lectin domain family 7, member A −3.06 
    1528251 NCR3 NM_147130 Natural cytotoxicity triggering receptor 3 −3.13 
    1513772 HLA-DRB3 NM_022555 Major histocompatibility complex, class II, DR beta 1 −3.17 
    1518778 ALOX15 NM_001140 Arachidonate 15-lipoxygenase −3.24 
    1524640 OAS3 NM_006187 2'−5'-oligoadenylate synthetase 3, 100 kDa −3.31 
    1985491 HLA-DRB1 X12544 Major histocompatibility complex, class II, DR beta 1 −3.31 
    1519302 STAT1 NM_139266 Signal transducer and activator of transcription 1, 91 kDa −3.39 
    1509654 CTSW NM_001335 Cathepsin W (lymphopain) −3.45 
    1992017 PLAUR NM_001005377 Plasminogen activator, urokinase receptor −3.50 
    1526588 LY75 NM_002349 Lymphocyte antigen 75 −3.51 
    1536508 LST1 NM_007161 Leukocyte specific transcript 1 −3.64 
    1515890 FAIM3 NM_005449 Interleukin 24 −3.65 
    1511424 EMR3 NM_032571 Egf-like module containing, mucin-like, hormone receptor-like 3 −3.71 
    1534340 CD46 NM_002389 CD46 molecule, complement regulatory protein −3.83 
    1537056 GBP3 NM_018284 Guanylate binding protein 1, interferon-inducible, 67 kDa −3.86 
    1517555 CYP27A1 NM_000784 Cytochrome P450, family 27, subfamily A, polypeptide 1 −3.88 
    1516190 SAMHD1 NM_015474 SAM domain and HD domain 1 −3.89 
    1519488 HLA-DQB2 NM_182549 Major histocompatibility complex, class II, DQ beta 2 −3.94 
    1983904 IL32 NM_001012631 Interleukin 32 −3.99 
    1505541 GZMB NM_004131 Granzyme B −4.04 
    1536691 GZMK NM_002104 Granzyme K (granzyme 3; tryptase II) −4.67 
    1522713 GZMA NM_006144 Granzyme A −4.80 
    1510975 HLA-DRB5 NM_002125 Major histocompatibility complex, class II, DR beta 1 −5.13 
    1527466 HLA-DPA1 NM_033554 Major histocompatibility complex, class II, DP alpha 1 −5.16 
    1516918 B2M AK022379 Beta−2-microglobulin −5.43 
    1538818 HLA-DQB1 NM_002123 Major histocompatibility complex, class II, DQ beta 1 −5.87 
    1521122 SPON2 NM_012445 Spondin 2, extracellular matrix protein −5.95 
    1517271 PF4V1 NM_002620 Platelet factor 4 variant 1 −6.48 
    1508682 HLA-DPB1 NM_002121 Major histocompatibility complex, class II, DP beta 1 −6.67 
    1529151 OASL NM_003733 2′−5′-oligoadenylate synthetase-like −7.10 
    1987518 CYB561 NM_001017916 Cytochrome b-561 −7.39 
    1524314 IFI44 NM_006417 Interferon-induced protein 44 −7.91 
    1533656 DEFA4 NM_001925 Defensin, alpha 4, corticostatin −9.04 
    1526794 HLA-DRA NM_019111 Major histocompatibility complex, class II, DR alpha −10.12 
    1536919 HLA-DMB NM_002118 Major histocompatibility complex, class II, DM beta −10.73 
    1525510 PF4 NM_002619 Platelet factor 4 (chemokine (C-X-C motif) ligand 4) −11.03 
    1514544 DEFA3 NM_005217 Defensin, alpha 1 −11.85 
    1520461 CD69 NM_001781 CD69 molecule −28.65 
    1528045 IL8 NM_000584 Interleukin 8 −35.22 
    1533457 FCER1A NM_002001 Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide −61.82 
Cell survival and apoptosis     
    1519170 GADD45A NM_001924 Growth arrest and DNA-damage-inducible, alpha 20.49 
    1531073 CARD12 NM_021209 Caspase recruitment domain family, member 12 11.37 
    1513896 BIRC1 NM_004536 Baculoviral IAP repeat-containing 1 10.55 
    1525148 ANXA1 NM_000700 Annexin A1 7.39 
    1533289 ANXA3 NM_005139 Annexin A3 7.33 
    1516682 CAST NM_001750 Calpastatin 6.97 
    1532823 PECR NM_018441 Peroxisomal trans−2-enoyl-CoA reductase 5.32 
    1539226 CARD6 NM_032587 Caspase recruitment domain family, member 6 5.15 
    1531614 ELMO2 NM_022086 Engulfment and cell motility 2 4.67 
    1512780 LGALS8 NM_006499 Lectin, galactoside-binding, soluble, 8 (galectin 8) 4.24 
    1505831 CIAS1 NM_004895 Cold autoinflammatory syndrome 1 4.09 
    1521881 STK3 NM_006281 Serine/threonine kinase 3 (STE20 homolog, yeast) 3.79 
    1526256 DDAH2 NM_013974 Dimethylarginine dimethylaminohydrolase 2 3.75 
    1522394 TNFRSF10A NM_003844 Tumor necrosis factor receptor superfamily, member 10a 3.69 
    1988846 AIM1 NM_001624 Absent in melanoma 1 3.52 
    1509305 LTB4R NM_181657 Leukotriene B4 receptor 3.20 
    1984749 HIP1 BC082970 Huntingtin interacting protein 1 3.19 
    1534293 LGALS3 NM_002306 Lectin, galactoside-binding, soluble, 3 (galectin 3) 3.03 
    1535837 SQSTM1 NM_003900 Sequestosome 1 −3.01 
    1537407 NFKBIE NM_004556 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon −3.12 
    1987065 TNFRSF25 NM_148968 Homo sapiens tumor necrosis factor receptor superfamily −3.36 
    1516125 PRDX2 NM_005809 Peroxiredoxin 2 −3.40 
    1531960 GSTP1 NM_000852 Glutathione S-transferase pi −3.42 
    1984701 CARD9 NM_052813 Caspase recruitment domain family, member 9 −3.61 
    1532501 PIM2 NM_006875 Pim−2 oncogene −3.76 
    1528041 IL31RA NM_139017 Interleukin 31 receptor A −3.91 
    1533374 PHLDA2 NM_003311 Pleckstrin homology-like domain, family A, member 2 −3.97 
    1526625 CARD4 NM_006092 Caspase recruitment domain family, member 4 −3.99 
    1520783 DYRK2 NM_006482 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 −4.29 
    1516378 TNFSF12 NM_153012 Tumor necrosis factor (ligand) superfamily, member 12 −4.29 
    1530737 LGALS2 NM_006498 Lectin, galactoside-binding, soluble, 2 (galectin 2) −4.47 
    1510435 LGALS12 NM_033101 Lectin, galactoside-binding, soluble, 12 (galectin 12) −4.47 
    1523074 HSPA9B NM_004134 Heat shock 70-kDa protein 9B (mortalin-2) −5.16 
    1532326 DDX58 NM_014314 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 −5.17 
    1508150 AOC3 NM_003734 Amine oxidase, copper containing 3 (vascular adhesion protein 1) −5.52 
    1538822 IFI6 NM_022873 Interferon, alpha-inducible protein (clone IFI-6–16) −5.56 
    1990540 CLU NM_203339 Clusterin −5.66 
    1517391 BIRC3 NM_001165 Baculoviral IAP repeat-containing 3 −6.55 
    1538745 SGK NM_005627 Serum/glucocorticoid regulated kinase −7.90 
    1523311 BIRC4BP NM_017523 XIAP associated factor-1 −9.09 
    1510278 NGFRAP1 NM_014380 Nerve growth factor receptor (TNFRSF16) associated protein 1 −9.35 
    1533140 NME5 NM_003551 Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) −9.98 
    1533249 HIG2 NM_013332 hypoxia-inducible protein 2 −15.17 
Cell adhesion and motility     
    1536096 MMP8 NM_002424 Matrix metallopeptidase 8 (neutrophil collagenase) 97.77 
    1519153 OLFM4 NM_006418 Olfactomedin 4 23.81 
    1514453 CD44 NM_000610 CD44 molecule (Indian blood group) 10.05 
    1532879 EMILIN2 NM_032048 Elastin microfibril interfacer 2 9.85 
    1521082 CEACAM1 NM_001712 Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) 5.97 
    1509526 MMP9 NM_004994 Matrix metallopeptidase 9 5.64 
    1522658 VNN1 NM_004666 Vanin 1 5.35 
    1984856 ADAM15 NM_207191 ADAM metallopeptidase domain 15 (metargidin) 3.79 
    1507356 F5 NM_000130 Coagulation factor V (proaccelerin, labile factor) 3.78 
    1516175 JAK2 NM_004972 Janus kinase 2 (a protein tyrosine kinase) 3.78 
    1508488 SPP1 NM_000582 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 3.72 
    1523771 ITGAM NM_000632 Integrin, alpha M (complement component 3 receptor 3 subunit) 3.52 
    1508621 PVRL2 NM_002856 Poliovirus receptor-related 2 (herpesvirus entry mediator B) 3.42 
    1531024 SIGLEC9 NM_014441 Sialic acid binding Ig-like lectin 9 3.34 
    1505504 VIM NM_003380 Vimentin 3.34 
    1986801 LAMB3 NM_001017402 Laminin, beta 3 3.11 
    1518582 CEACAM6 BC005008 Carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen) −3.07 
    1506966 SIGLEC8 NM_014442 Sialic acid binding immunoglobulin-like lectin 8 −3.16 
    1534776 CXCL1 NM_00–1511 Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) −3.20 
    1520341 AZU1 NM_001700 Azurocidin 1 (cationic antimicrobial protein 37) −3.23 
    1520433 ITGB7 NM_000889 Integrin, beta 7 −3.59 
    1505769 TUBB1 NM_030773 Tubulin, beta 1 −4.35 
    1528734 ICAM2 NM_000873 Intercellular adhesion molecule 2 −5.24 
    1991393 CXCR4 NM_001008540 Chemokine (C-X-C motif) receptor 4 −5.90 
    1989016 CCL3L3 NM_001001437 Chemokine (C-C motif) ligand 3-like 3 −5.98 
    1510688 CCL3 NM_002983 Chemokine (C-C motif) ligand 3 −6.35 
    1517696 CCL5 NM_002985 Chemokine (C-C motif) ligand 5 −7.71 
    1533135 CXCL10 NM_001565 Chemokine (C-X-C motif) ligand 11 −9.10 
    1505505 CCL23 NM_005064 Chemokine (C-C motif) ligand 23 −15.06 
    1526404 CCL4 NM_002984 Chemokine (C-C motif) ligand 4 −15.45 
    1533611 PPBP NM_002704 Pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) −16.30 
    1515169 ITGA4 NM_000885 Integrin, alpha 4 (antigen CD49D), alpha 4 subunit of VLA-4 receptor −16.74 
    1537942 CCR3 NM_001837 Chemokine (C-C motif) receptor 3 −19.63 
Cell metabolism     
    1538977 HPGD NM_000860 Hydroxyprostaglandin dehydrogenase 15-(NAD) 36.27 
    1989284 PFKFB2 NM_001018053 6-phosphofructo-2-kinase/fructose-2.6-biphosphatase 2 31.40 
    1986330 ATP9A NM_006045 ATPase, Class II, type 9A 25.82 
    1511217 LDHA NM_005566 Lactate dehydrogenase A 10.11 
    1527335 PCSK9 NM_174936 Proprotein convertase subtilisin/kexin type 9 7.95 
    1991955 PLB1 CR600701 Phospholipase B1 7.59 
    1983476 ATP13A3 AJ306929 ATPase type 13A3 7.56 
    1513367 DPH5 NM_0–15958 DPH5 homolog (S cerevisiae6.38 
    1526202 NMNAT2 NM_0–15039 Nicotinamide nucleotide adenylyltransferase 2 6.31 
    1536086 DPYD NM_000110 Dihydropyrimidine dehydrogenase 6.11 
    1990338 ATP11B NM_014616 ATPase, Class VI, type 11B 6.09 
    1515968 ALPL NM_000478 Alkaline phosphatase, liver/bone/kidney 5.59 
    1506037 MGAM NM_004668 Maltase-glucoamylase (alpha-glucosidase) 5.41 
    1532823 PECR NM_018441 Peroxisomal trans-2-enoyl-CoA reductase 5.32 
    1986414 ATP2B4 NM_001001396 ATPase, Ca2+ transporting, plasma membrane 4 4.99 
    1506589 PFKFB3 NM_004566 6-phosphofructo-2-kinase/fructose-2.6-biphosphatase 3 4.75 
    1512399 AGPAT6 NM_178819 1-acylglycerol-3-phosphate O-acyltransferase 6 (lysophosphatidic acid acyltransferase, zeta) 4.74 
    1523383 PGD NM_002631 Phosphogluconate dehydrogenase 4.70 
    1519383 ATP6V1C1 NM_001695 ATPase, H+ transporting, lysosomal 42 kDa, V1 subunit C1 4.11 
    1520655 SMPDL3A NM_006714 Sphingomyelin phosphodiesterase, acid-like 3A 4.09 
    1989848 ACSL4 NM_004458 Acyl-CoA synthetase long-chain family member 4 4.02 
    1522656 LDLR NM_000527 Low-density lipoprotein receptor 3.63 
    1538541 DHRS9 NM_005771 Dehydrogenase/reductase (SDR family) member 9 3.55 
    1514810 ATP1A3 NM_152296 ATPase, Na+/K+ transporting, alpha 3 polypeptide 3.53 
    1524666 FABP5 NM_001444 Fatty acid binding protein 5 (psoriasis-associated) 3.38 
    1538372 ATP1A1 NM_000701 ATPase, Na+/K+ transporting, alpha 1 polypeptide 3.38 
    1524650 GNS NM_002076 Glucosamine (N-acetyl)-6- 3.37 
    1523432 ACSL1 NM_001995 Acyl-CoA synthetase long-chain family member 1 3.35 
    1529572 SGSH NM_000199 N-sulfoglucosamine sulfohydrolase (sulfamidase) 3.29 
    1538734 ACSL3 NM_004457 Acyl-CoA synthetase long-chain family member 3 3.27 
    1985998 ALOX5 BQ548663 Arachidonate 5-lipoxygenase 3.16 
    1527407 GAPDH NM_002046 Glyceraldehyde3-phosphate dehydrogenase 3.16 
    1534240 SULT1B1 D89479 Sulfotransferase family, cytosolic, 1B, member 1 3.10 
    1983183 NDUFB6 NM_182739 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 −3.10 
    1525237 ALDH6A1 NM_005589 Aldehyde dehydrogenase 6 family, member A1 −3.22 
    1518778 ALOX15 NM_001140 Arachidonate 15-lipoxygenase −3.24 
    1523761 CBR1 NM_001757 Carbonyl reductase 1 −3.61 
    1510672 MCCC1 NM_020166 Methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) −4.01 
    1990453 NDUFV2 BI520375 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24 kDa −4.31 
    1521732 ATP12A NM_001676 ATPase, H+/K+ transporting, nongastric, alpha polypeptide −4.74 
    1526184 LDHB NM_002300 Lactate dehydrogenase B −6.08 
    1512448 ISOC1 NM_016048 Isochorismatase domain containing 1 −6.51 
    1989369 METTL7A BC004492 Methyltransferase like 7A −6.99 
    1518627 FA2H NM_024306 Fatty acid 2-hydroxylase −7.75 
    1506804 PLD4 NM_138790 Phospholipase D family, member 4 −9.53 
    1521949 SORD NM_003104 Sorbitol dehydrogenase −10.20 
    1512822 SULF2 NM_018837 Sulfatase 2 −21.22 
Sequence ID Primary sequence name Sequence code Sequence description Fold change 
Transcription and regulation of transcription     
    1514893 CCNA1 NM_003914 Cyclin A1 21.01 
    1983249 SMARCA3 BF513730 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 3 10.43 
    1513827 MLXIP AB020674 MLX interacting protein 8.66 
    1519529 MSL3L1 NM_078629 Male-specific lethal 3-like 1 (Drosophila) 6.75 
    1521872 MEF2A NM_005587 MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A) 6.47 
    1985292 ETS2 BX433326 V-ets erythroblastosis virus E26 oncogene homolog 2 5.59 
    1525013 BATF NM_006399 Basic leucine zipper transcription factor, ATF-like 5.58 
    1537803 ENO1 NM_001428 Enolase 1, (alpha) 5.50 
    1533218 HIPK2 BC041926 CDNA clone IMAGE:5300349 5.07 
    1522326 PHTF1 NM_006608 Putative homeodomain transcription factor 1 4.80 
    1533591 SIRT5 NM_012241 Sirtuin (silent mating type information regulation 2 homolog) 5 (Saccharomyces cerevisiae) 4.60 
    1512793 ZNF167 NM_018651 Zinc finger protein 167 4.30 
    1511846 PLAG1 NM_002655 Pleomorphic adenoma gene 1 4.26 
    1512354 ZNF438 AK095436 hypothetical protein LOC220929 4.20 
    1507417 DACH1 NM_080759 Dachshund homolog 1 (Drosophila) 4.04 
    1526663 ZNF438 NM_182755 Zinc finger protein 438 4.02 
    1537859 ZBTB20 NM_015642 Zinc finger and BTB domain containing 20 3.77 
    1522362 ZNF658 NM_033160 Zinc finger protein 658 3.75 
    1534837 ZNF254 NM_004876 Zinc finger protein 539 3.73 
    1507368 CCND3 NM_001760 Cyclin D3 3.69 
    1509049 VENTX NM_014468 VENT homeobox homolog (Xenopus laevis) 3.61 
    1985753 CRTC3 AK090443 CREB regulated transcription coactivator 3 3.58 
    1529278 XRN2 NM_012255 5′-3′ exoribonuclease 2 3.43 
    1522102 E2F2 NM_004091 E2F transcription factor 2 3.36 
    1517472 CCNC NM_005190 Cyclin C 3.07 
    1535521 KLF6 U51869 Kruppel-like factor 6 3.03 
    1537902 TSC22D1 NM_183422 TSC22 domain family, member 1 −3.01 
    1983175 MCM7 NM_182776 MCM7 minichromosome maintenance deficient −3.02 
    1529350 SSRP1 NM_003146 Structure specific recognition protein 1 −3.03 
    1505187 RELB NM_006509 V-rel reticuloendotheliosis viral oncogene homolog B, nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (avian) −3.04 
    1537144 DEXI AK055529 dexamethasone-induced transcript −3.07 
    1516882 MEF2C NM_002397 MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C) −3.09 
    1527019 CHAF1A NM_005483 Chromatin assembly factor 1, subunit A (p150) −3.25 
    1535620 NCOR2 NM_006312 Nuclear receptor co-repressor 2 −3.34 
    1523857 ZNF395 NM_018660 Zinc finger protein 395 −3.34 
    1512878 ZMYND11 NM_006624 Zinc finger, MYND domain containing 11 −3.35 
    1991240 HMGA1 NM_145904 High mobility group AT-hook 1 −3.37 
    1531990 HEY1 NM_012258 Hairy/enhancer-of-split related with YRPW motif 1 −3.39 
    1533586 ELK1 NM_005229 ELK1, member of ETS oncogene family −3.50 
    1515979 NR4A3 NM_173199 Nuclear receptor subfamily 4, group A, member 3 −3.58 
    1510333 THRA NM_003250 Thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian) −3.68 
    1537025 BTG2 NM_006763 BTG family, member 2 −3.73 
    1513189 TRERF1 NM_033502 Transcriptional regulating factor 1 −3.76 
    1511905 JARID2 NM_004973 Jumonji, AT-rich interactive domain 2 −3.81 
    1526826 ATF3 NM_004024 Activating transcription factor 3 −3.82 
    1523890 RASSF7 NM_003475 Ras association (RalGDS/AF−6) domain family 7 −3.84 
    1533807 MAFF NM_012323 V-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian) −3.89 
    1525862 JUN NM_002228 V-jun sarcoma virus 17 oncogene homolog (avian) −3.94 
    1507971 NRL NM_006177 Neural retina leucine zipper −4.09 
    1533916 LEF1 NM_016269 Lymphoid enhancer-binding factor 1 −4.22 
    1506607 ASF1B NM_018154 ASF1 antisilencing function 1 homolog B (S cerevisiae−4.36 
    1509496 AES NM_198969 Amino-terminal enhancer of split −4.39 
    1533535 TAF4 NM_003185 TAF4 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 135kDa −4.40 
    1508056 EGR3 NM_004430 Early growth response 3 −4.41 
    1533262 BRF1 BC016743 BRF1 homolog, subunit of RNA polymerase III transcription initiation factor IIIB (S cerevisiae−4.48 
    1533571 PHF6 NM_032458 PHD finger protein 6 −4.53 
    1517506 EGR2 NM_000399 Early growth response 2 (Krox−20 homolog, Drosophila) −4.66 
    1989375 SMAD3 NM_005902 SMAD, mothers against DPP homolog 3 (Drosophila) −4.97 
    1507856 MYB NM_005375 V-myb myeloblastosis viral oncogene homolog (avian) −5.06 
    1528623 OLIG2 NM_005806 Oligodendrocyte lineage transcription factor 2 −6.11 
    1524585 GATA1 NM_002049 GATA binding protein 1 (globin transcription factor 1) −6.64 
    1991493 PARP14 NM_017554 Poly (ADP-ribose) polymerase family, member 14 −7.03 
    1510055 PCGF5 BC007377 Polycomb group ring finger 5 −7.57 
    1510369 ASCL2 NM_005170 Achaete-scute complex-like 2 (Drosophila) −7.72 
    1522208 ZNF584 NM_173548 Zinc finger protein 584 −7.93 
    1523937 RPS6KA5 NM_004755 Ribosomal protein S6 kinase, 90 kDa, polypeptide 5 −8.07 
    1529851 ETV7 NM_016135 Ets variant gene 7 (TEL2 oncogene) −8.56 
    1506298 GATA6 NM_005257 GATA binding protein 6 −8.65 
    1989096 SSBP3 NM_001009955 Single stranded DNA binding protein 3 −8.76 
    1520503 NKX31 NM_006167 NK3 transcription factor related, locus 1 (Drosophila) −9.56 
    1522991 ZNF649 NM_023074 Zinc finger protein 649 −11.07 
    1514700 NR4A1 NM_002135 Nuclear receptor subfamily 4, group A, member 1 −92.91 
Signal transduction     
    1513543 GPR84 NM_020370 G protein-coupled receptor 84 25.51 
    1505483 EDG1 NM_001400 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 1 18.05 
    1511321 PTGER2 NM_000956 Prostaglandin E receptor 2 (subtype EP2), 53 kDa 8.26 
    1511570 ARHGAP24 AK130576 Rho GTPase activating protein 24 7.28 
    1523886 PDE4D NM_006203 Phosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce homolog, Drosophila) 6.64 
    1527053 MYO10 BC041694 Myosin X 6.48 
    1512860 MS4A4A NM_024021 Membrane-spanning 4-domains, subfamily A, member 4 6.12 
    1539087 DLC1 NM_182643 Deleted in liver cancer 1 5.96 
    1528643 MS4A6A NM_022349 Membrane-spanning 4-domains, subfamily A, member 6A 5.87 
    1524040 GPR141 NM_181791 G protein-coupled receptor 141 5.28 
    1989101 EDARADD NM_080738 EDAR-associated death domain 4.64 
    1508615 EXT1 NM_000127 Exostoses (multiple) 1 4.30 
    1521881 STK3 NM_006281 Serine/threonine kinase 3 (STE20 homolog, yeast) 3.79 
    1510158 ITPKC NM_025194 Inositol 1.4,5-trisphosphate 3-kinase C 3.63 
    1519571 CDC42EP3 NM_006449 CDC42 effector protein (Rho GTPase binding) 3 3.58 
    1534557 DPYSL3 NM_001387 Dihydropyrimidinase-like 3 3.54 
    1536781 RPS6KA3 NM_004586 Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 3.53 
    1533999 HHIP NM_022475 Hedgehog interacting protein 3.50 
    1512578 GPR97 NM_170776 G protein-coupled receptor 97 3.42 
    1514738 SLC26A6 NM_022911 Inositol hexaphosphate kinase 2 3.28 
    1517434 IL4R NM_000418 Interleukin 4 receptor 3.21 
    1539209 GABBR1 NM_001470 Gamma-aminobutyric acid (GABA) B receptor, 1 3.19 
    1511435 STK16 NM_003691 Serine/threonine kinase 16 3.17 
    1520958 PRKAR2A BC002763 Inositol hexaphosphate kinase 2 3.12 
    1506387 MAPK6 NM_002748 Mitogen-activated protein kinase 6 3.03 
    1523162 ITPK1 NM_014216 Inositol 1.3,4-triphosphate 5/6 kinase −3.05 
    1507956 GNGT2 NM_031498 Guanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 2 −3.09 
    1514326 TAGAP NM_138810 T-cell activation GTPase activating protein −3.09 
    1530414 GPR171 NM_013308 G protein-coupled receptor 171 −3.16 
    1527339 P2RY5 NM_005767 Purinergic receptor P2Y, G-protein coupled, 5 −3.17 
    1517355 ARHGAP25 BC039591 Rho GTPase activating protein 25 −3.23 
    1520830 KIT NM_000222 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog −3.28 
    1517660 KLRK1 NM_007360 Killer cell lectin-like receptor subfamily C, member 4 −3.37 
    1514077 KLRB1 NM_002258 Killer cell lectin-like receptor subfamily B, member 1 −3.43 
    1518328 GRP NM_002091 Gastrin-releasing peptide −3.64 
    1516849 EPHB1 NM_004441 EPH receptor B1 −3.80 
    1514753 PRKAG2 NM_016203 Protein kinase, AMP-activated, gamma 2 non-catalytic subunit −4.09 
    1512127 SOCS1 NM_003745 Suppressor of cytokine signaling 1 −4.13 
    1518864 FLT3LG NM_001459 Fms-related tyrosine kinase 3 ligand −4.26 
    1524915 OR51E1 NM_152430 Olfactory receptor, family 51, subfamily E, member 1 −4.27 
    1507227 P2RY14 NM_014879 Purinergic receptor P2Y, G-protein coupled, 14 −4.28 
    1523084 GUCA1A NM_000409 Guanylate cyclase activator 1A (retina) −4.37 
    1525046 PILRA NM_178273 Paired immunoglobulin-like type 2 receptor alpha −4.48 
    1534223 PPP1R12B NM_032105 Protein phosphatase 1, regulatory (inhibitor) subunit 12B −4.66 
    1518048 GPR34 NM_005300 G-protein–coupled receptor 34 −4.78 
    1534320 ARRB1 NM_004041 Arrestin, beta 1 −5.02 
    1534974 KLRD1 NM_002262 Killer cell lectin-like receptor subfamily D, member 1 −5.16 
    1525314 S100A10 NM_002966 S100 calcium binding protein A10 −5.94 
    1530940 PRKAR2B NM_002736 Protein kinase, cAMP-dependent, regulatory, type II, beta −5.97 
    1522410 IL2RB NM_000878 Interleukin 2 receptor, beta −6.06 
    1518652 CD74 NM_004355 CD74 molecule, major histocompatibility complex, class II invariant chain −6.23 
    1515002 CYSLTR1 NM_006639 Cysteinyl leukotriene receptor 1 −6.45 
    1530332 MX1 NM_002462 Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) −6.76 
    1522509 OR2B2 NM_033057 Olfactory receptor, family 2, subfamily B, member 2 −6.88 
    1984459 PDE4B AB209081 Phosphodiesterase 4B, cAMP-specific −6.89 
    1527378 CSF1R NM_005211 Colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) oncogene homolog −7.08 
    1527858 NRGN NM_006176 Neurogranin (protein kinase C substrate, RC3) −7.49 
    1524392 ANGPT1 NM_001146 Angiopoietin 1 −8.84 
    1516837 MS4A3 NM_006138 Membrane-spanning 4-domains, subfamily A, member 3 −9.24 
    1989999 IL5RA NM_175725 Interleukin 5 receptor, alpha −9.66 
    1524637 GPR162 NM_019858 Triosephosphate isomerase 1 −10.10 
    1519217 P2RY2 NM_176072 Purinergic receptor P2Y, G-protein coupled, 2 −11.66 
    1516830 P2RY10 NM_014499 Purinergic receptor P2Y, G-protein coupled, 10 −11.67 
    1518123 CD83 NM_004233 CD83 molecule −11.67 
    1517216 KLRF1 NM_016523 Killer cell lectin-like receptor subfamily F, member 1 −12.68 
    1512228 EBI2 NM_004951 Epstein-Barr virus–induced gene 2 (lymphocyte-specific G protein-coupled receptor) −13.11 
    1513158 HRH4 NM_021624 Histamine receptor H4 −13.66 
    1537338 FGL2 NM_006682 Fibrinogen-like 2 −13.76 
    1515714 GNG11 NM_004126 Guanine nucleotide binding protein (G protein), gamma 11 −16.57 
    1527111 GPR44 NM_004778 G protein-coupled receptor 44 −23.51 
    1528909 RGS1 NM_002922 Regulator of G-protein signaling 1 −32.65 
Immune responses     
    1507161 CD163 NM_004244 CD163 molecule 54.27 
    1531927 CYP1B1 NM_000104 Cytochrome P450, family 1, subfamily B, polypeptide 1 46.19 
    1984958 CD177 CR592446 CD177 molecule 24.38 
    1521404 IL18R1 NM_003855 Interleukin 18 receptor 1 16.54 
    1983056 MERTK U08023 C-mer proto-oncogene tyrosine kinase 16.00 
    1532739 CLEC5A NM_013252 C-type lectin domain family 5, member A 13.44 
    1511835 CLEC4D NM_080387 C-type lectin domain family 4, member D 12.02 
    1535646 CST7 NM_003650 Cystatin F (leukocystatin) 11.43 
    1506996 ADORA3 NM_020683 Adenosine A3 receptor 10.89 
    1530098 IL18RAP NM_003853 Interleukin 18 receptor accessory protein 10.79 
    1532148 LAIR1 NM_021706 Leukocyte-associated immunoglobulin-like receptor 1 10.45 
    1506562 HP NM_005143 Haptoglobin 10.31 
    1505718 IL1R2 NM_004633 Interleukin 1 receptor, type II 9.95 
    1507483 LILRA5 NM_181879 Leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 5 7.56 
    1524454 XBP1 NM_005080 X-box binding protein 1 7.48 
    1510813 IRAK3 NM_007199 Interleukin−1 receptor-associated kinase 3 7.28 
    1538778 TLR5 NM_003268 Toll-like receptor 5 5.97 
    1521047 CR1 NM_000651 Complement component (3b/4b) receptor 1 (Knops blood group) 5.87 
    1520792 MAPK14 NM_139013 Mitogen-activated protein kinase 14 5.75 
    1535716 CD59 NM_203330 CD59 molecule, complement regulatory protein 5.67 
    1513648 FCER1G CB529629 Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide 5.26 
    1521687 TLR4 BF670973 Toll-like receptor 4 5.15 
    1530114 MAP2K6 NM_002758 Mitogen-activated protein kinase kinase 6 4.87 
    1533789 SERPINB1 NM_030666 Serpin peptidase inhibitor, clade B (ovalbumin), member 1 4.84 
    1522193 FCAR NM_002000 Fc fragment of IgA, receptor for 4.52 
    1527121 PROK2 NM_021935 Prokineticin 2 4.33 
    1505831 CIAS1 NM_004895 Cold autoinflammatory syndrome 1 4.09 
    1524842 IL10 NM_000572 Interleukin 10 3.89 
    1538909 IL1RAP NM_002182 Interleukin 1 receptor accessory protein 3.80 
    1506853 CHIT1 NM_003465 Chitinase 1 (chitotriosidase) 3.48 
    1531091 CD55 NM_000574 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 3.39 
    1534101 IL1R1 NM_000877 Interleukin 1 receptor, type I 3.34 
    1515424 CYBB S67289 Cytochrome b-245, beta polypeptide (chronic granulomatous disease) 3.29 
    1985998 ALOX5 BQ548663 Arachidonate 5-lipoxygenase 3.16 
    1510372 IRAK4 NM_016123 Interleukin-1 receptor-associated kinase 4 3.10 
    1514172 CD48 NM_001778 CD48 molecule 3.09 
    1527340 TLR8 NM_016610 Toll-like receptor 8 3.07 
    1511452 SERPING1 NM_000062 Serpin peptidase inhibitor, clade G (C1 inhibitor), member 1, (angioedema, hereditary) −3.00 
    1530131 IRF2BP2 BC020516 Interferon regulatory factor 2 binding protein 2 −3.03 
    1512742 CLEC7A NM_197954 C-type lectin domain family 7, member A −3.06 
    1528251 NCR3 NM_147130 Natural cytotoxicity triggering receptor 3 −3.13 
    1513772 HLA-DRB3 NM_022555 Major histocompatibility complex, class II, DR beta 1 −3.17 
    1518778 ALOX15 NM_001140 Arachidonate 15-lipoxygenase −3.24 
    1524640 OAS3 NM_006187 2'−5'-oligoadenylate synthetase 3, 100 kDa −3.31 
    1985491 HLA-DRB1 X12544 Major histocompatibility complex, class II, DR beta 1 −3.31 
    1519302 STAT1 NM_139266 Signal transducer and activator of transcription 1, 91 kDa −3.39 
    1509654 CTSW NM_001335 Cathepsin W (lymphopain) −3.45 
    1992017 PLAUR NM_001005377 Plasminogen activator, urokinase receptor −3.50 
    1526588 LY75 NM_002349 Lymphocyte antigen 75 −3.51 
    1536508 LST1 NM_007161 Leukocyte specific transcript 1 −3.64 
    1515890 FAIM3 NM_005449 Interleukin 24 −3.65 
    1511424 EMR3 NM_032571 Egf-like module containing, mucin-like, hormone receptor-like 3 −3.71 
    1534340 CD46 NM_002389 CD46 molecule, complement regulatory protein −3.83 
    1537056 GBP3 NM_018284 Guanylate binding protein 1, interferon-inducible, 67 kDa −3.86 
    1517555 CYP27A1 NM_000784 Cytochrome P450, family 27, subfamily A, polypeptide 1 −3.88 
    1516190 SAMHD1 NM_015474 SAM domain and HD domain 1 −3.89 
    1519488 HLA-DQB2 NM_182549 Major histocompatibility complex, class II, DQ beta 2 −3.94 
    1983904 IL32 NM_001012631 Interleukin 32 −3.99 
    1505541 GZMB NM_004131 Granzyme B −4.04 
    1536691 GZMK NM_002104 Granzyme K (granzyme 3; tryptase II) −4.67 
    1522713 GZMA NM_006144 Granzyme A −4.80 
    1510975 HLA-DRB5 NM_002125 Major histocompatibility complex, class II, DR beta 1 −5.13 
    1527466 HLA-DPA1 NM_033554 Major histocompatibility complex, class II, DP alpha 1 −5.16 
    1516918 B2M AK022379 Beta−2-microglobulin −5.43 
    1538818 HLA-DQB1 NM_002123 Major histocompatibility complex, class II, DQ beta 1 −5.87 
    1521122 SPON2 NM_012445 Spondin 2, extracellular matrix protein −5.95 
    1517271 PF4V1 NM_002620 Platelet factor 4 variant 1 −6.48 
    1508682 HLA-DPB1 NM_002121 Major histocompatibility complex, class II, DP beta 1 −6.67 
    1529151 OASL NM_003733 2′−5′-oligoadenylate synthetase-like −7.10 
    1987518 CYB561 NM_001017916 Cytochrome b-561 −7.39 
    1524314 IFI44 NM_006417 Interferon-induced protein 44 −7.91 
    1533656 DEFA4 NM_001925 Defensin, alpha 4, corticostatin −9.04 
    1526794 HLA-DRA NM_019111 Major histocompatibility complex, class II, DR alpha −10.12 
    1536919 HLA-DMB NM_002118 Major histocompatibility complex, class II, DM beta −10.73 
    1525510 PF4 NM_002619 Platelet factor 4 (chemokine (C-X-C motif) ligand 4) −11.03 
    1514544 DEFA3 NM_005217 Defensin, alpha 1 −11.85 
    1520461 CD69 NM_001781 CD69 molecule −28.65 
    1528045 IL8 NM_000584 Interleukin 8 −35.22 
    1533457 FCER1A NM_002001 Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide −61.82 
Cell survival and apoptosis     
    1519170 GADD45A NM_001924 Growth arrest and DNA-damage-inducible, alpha 20.49 
    1531073 CARD12 NM_021209 Caspase recruitment domain family, member 12 11.37 
    1513896 BIRC1 NM_004536 Baculoviral IAP repeat-containing 1 10.55 
    1525148 ANXA1 NM_000700 Annexin A1 7.39 
    1533289 ANXA3 NM_005139 Annexin A3 7.33 
    1516682 CAST NM_001750 Calpastatin 6.97 
    1532823 PECR NM_018441 Peroxisomal trans−2-enoyl-CoA reductase 5.32 
    1539226 CARD6 NM_032587 Caspase recruitment domain family, member 6 5.15 
    1531614 ELMO2 NM_022086 Engulfment and cell motility 2 4.67 
    1512780 LGALS8 NM_006499 Lectin, galactoside-binding, soluble, 8 (galectin 8) 4.24 
    1505831 CIAS1 NM_004895 Cold autoinflammatory syndrome 1 4.09 
    1521881 STK3 NM_006281 Serine/threonine kinase 3 (STE20 homolog, yeast) 3.79 
    1526256 DDAH2 NM_013974 Dimethylarginine dimethylaminohydrolase 2 3.75 
    1522394 TNFRSF10A NM_003844 Tumor necrosis factor receptor superfamily, member 10a 3.69 
    1988846 AIM1 NM_001624 Absent in melanoma 1 3.52 
    1509305 LTB4R NM_181657 Leukotriene B4 receptor 3.20 
    1984749 HIP1 BC082970 Huntingtin interacting protein 1 3.19 
    1534293 LGALS3 NM_002306 Lectin, galactoside-binding, soluble, 3 (galectin 3) 3.03 
    1535837 SQSTM1 NM_003900 Sequestosome 1 −3.01 
    1537407 NFKBIE NM_004556 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon −3.12 
    1987065 TNFRSF25 NM_148968 Homo sapiens tumor necrosis factor receptor superfamily −3.36 
    1516125 PRDX2 NM_005809 Peroxiredoxin 2 −3.40 
    1531960 GSTP1 NM_000852 Glutathione S-transferase pi −3.42 
    1984701 CARD9 NM_052813 Caspase recruitment domain family, member 9 −3.61 
    1532501 PIM2 NM_006875 Pim−2 oncogene −3.76 
    1528041 IL31RA NM_139017 Interleukin 31 receptor A −3.91 
    1533374 PHLDA2 NM_003311 Pleckstrin homology-like domain, family A, member 2 −3.97 
    1526625 CARD4 NM_006092 Caspase recruitment domain family, member 4 −3.99 
    1520783 DYRK2 NM_006482 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 −4.29 
    1516378 TNFSF12 NM_153012 Tumor necrosis factor (ligand) superfamily, member 12 −4.29 
    1530737 LGALS2 NM_006498 Lectin, galactoside-binding, soluble, 2 (galectin 2) −4.47 
    1510435 LGALS12 NM_033101 Lectin, galactoside-binding, soluble, 12 (galectin 12) −4.47 
    1523074 HSPA9B NM_004134 Heat shock 70-kDa protein 9B (mortalin-2) −5.16 
    1532326 DDX58 NM_014314 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 −5.17 
    1508150 AOC3 NM_003734 Amine oxidase, copper containing 3 (vascular adhesion protein 1) −5.52 
    1538822 IFI6 NM_022873 Interferon, alpha-inducible protein (clone IFI-6–16) −5.56 
    1990540 CLU NM_203339 Clusterin −5.66 
    1517391 BIRC3 NM_001165 Baculoviral IAP repeat-containing 3 −6.55 
    1538745 SGK NM_005627 Serum/glucocorticoid regulated kinase −7.90 
    1523311 BIRC4BP NM_017523 XIAP associated factor-1 −9.09 
    1510278 NGFRAP1 NM_014380 Nerve growth factor receptor (TNFRSF16) associated protein 1 −9.35 
    1533140 NME5 NM_003551 Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) −9.98 
    1533249 HIG2 NM_013332 hypoxia-inducible protein 2 −15.17 
Cell adhesion and motility     
    1536096 MMP8 NM_002424 Matrix metallopeptidase 8 (neutrophil collagenase) 97.77 
    1519153 OLFM4 NM_006418 Olfactomedin 4 23.81 
    1514453 CD44 NM_000610 CD44 molecule (Indian blood group) 10.05 
    1532879 EMILIN2 NM_032048 Elastin microfibril interfacer 2 9.85 
    1521082 CEACAM1 NM_001712 Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) 5.97 
    1509526 MMP9 NM_004994 Matrix metallopeptidase 9 5.64 
    1522658 VNN1 NM_004666 Vanin 1 5.35 
    1984856 ADAM15 NM_207191 ADAM metallopeptidase domain 15 (metargidin) 3.79 
    1507356 F5 NM_000130 Coagulation factor V (proaccelerin, labile factor) 3.78 
    1516175 JAK2 NM_004972 Janus kinase 2 (a protein tyrosine kinase) 3.78 
    1508488 SPP1 NM_000582 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 3.72 
    1523771 ITGAM NM_000632 Integrin, alpha M (complement component 3 receptor 3 subunit) 3.52 
    1508621 PVRL2 NM_002856 Poliovirus receptor-related 2 (herpesvirus entry mediator B) 3.42 
    1531024 SIGLEC9 NM_014441 Sialic acid binding Ig-like lectin 9 3.34 
    1505504 VIM NM_003380 Vimentin 3.34 
    1986801 LAMB3 NM_001017402 Laminin, beta 3 3.11 
    1518582 CEACAM6 BC005008 Carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen) −3.07 
    1506966 SIGLEC8 NM_014442 Sialic acid binding immunoglobulin-like lectin 8 −3.16 
    1534776 CXCL1 NM_00–1511 Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) −3.20 
    1520341 AZU1 NM_001700 Azurocidin 1 (cationic antimicrobial protein 37) −3.23 
    1520433 ITGB7 NM_000889 Integrin, beta 7 −3.59 
    1505769 TUBB1 NM_030773 Tubulin, beta 1 −4.35 
    1528734 ICAM2 NM_000873 Intercellular adhesion molecule 2 −5.24 
    1991393 CXCR4 NM_001008540 Chemokine (C-X-C motif) receptor 4 −5.90 
    1989016 CCL3L3 NM_001001437 Chemokine (C-C motif) ligand 3-like 3 −5.98 
    1510688 CCL3 NM_002983 Chemokine (C-C motif) ligand 3 −6.35 
    1517696 CCL5 NM_002985 Chemokine (C-C motif) ligand 5 −7.71 
    1533135 CXCL10 NM_001565 Chemokine (C-X-C motif) ligand 11 −9.10 
    1505505 CCL23 NM_005064 Chemokine (C-C motif) ligand 23 −15.06 
    1526404 CCL4 NM_002984 Chemokine (C-C motif) ligand 4 −15.45 
    1533611 PPBP NM_002704 Pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) −16.30 
    1515169 ITGA4 NM_000885 Integrin, alpha 4 (antigen CD49D), alpha 4 subunit of VLA-4 receptor −16.74 
    1537942 CCR3 NM_001837 Chemokine (C-C motif) receptor 3 −19.63 
Cell metabolism     
    1538977 HPGD NM_000860 Hydroxyprostaglandin dehydrogenase 15-(NAD) 36.27 
    1989284 PFKFB2 NM_001018053 6-phosphofructo-2-kinase/fructose-2.6-biphosphatase 2 31.40 
    1986330 ATP9A NM_006045 ATPase, Class II, type 9A 25.82 
    1511217 LDHA NM_005566 Lactate dehydrogenase A 10.11 
    1527335 PCSK9 NM_174936 Proprotein convertase subtilisin/kexin type 9 7.95 
    1991955 PLB1 CR600701 Phospholipase B1 7.59 
    1983476 ATP13A3 AJ306929 ATPase type 13A3 7.56 
    1513367 DPH5 NM_0–15958 DPH5 homolog (S cerevisiae6.38 
    1526202 NMNAT2 NM_0–15039 Nicotinamide nucleotide adenylyltransferase 2 6.31 
    1536086 DPYD NM_000110 Dihydropyrimidine dehydrogenase 6.11 
    1990338 ATP11B NM_014616 ATPase, Class VI, type 11B 6.09 
    1515968 ALPL NM_000478 Alkaline phosphatase, liver/bone/kidney 5.59 
    1506037 MGAM NM_004668 Maltase-glucoamylase (alpha-glucosidase) 5.41 
    1532823 PECR NM_018441 Peroxisomal trans-2-enoyl-CoA reductase 5.32 
    1986414 ATP2B4 NM_001001396 ATPase, Ca2+ transporting, plasma membrane 4 4.99 
    1506589 PFKFB3 NM_004566 6-phosphofructo-2-kinase/fructose-2.6-biphosphatase 3 4.75 
    1512399 AGPAT6 NM_178819 1-acylglycerol-3-phosphate O-acyltransferase 6 (lysophosphatidic acid acyltransferase, zeta) 4.74 
    1523383 PGD NM_002631 Phosphogluconate dehydrogenase 4.70 
    1519383 ATP6V1C1 NM_001695 ATPase, H+ transporting, lysosomal 42 kDa, V1 subunit C1 4.11 
    1520655 SMPDL3A NM_006714 Sphingomyelin phosphodiesterase, acid-like 3A 4.09 
    1989848 ACSL4 NM_004458 Acyl-CoA synthetase long-chain family member 4 4.02 
    1522656 LDLR NM_000527 Low-density lipoprotein receptor 3.63 
    1538541 DHRS9 NM_005771 Dehydrogenase/reductase (SDR family) member 9 3.55 
    1514810 ATP1A3 NM_152296 ATPase, Na+/K+ transporting, alpha 3 polypeptide 3.53 
    1524666 FABP5 NM_001444 Fatty acid binding protein 5 (psoriasis-associated) 3.38 
    1538372 ATP1A1 NM_000701 ATPase, Na+/K+ transporting, alpha 1 polypeptide 3.38 
    1524650 GNS NM_002076 Glucosamine (N-acetyl)-6- 3.37 
    1523432 ACSL1 NM_001995 Acyl-CoA synthetase long-chain family member 1 3.35 
    1529572 SGSH NM_000199 N-sulfoglucosamine sulfohydrolase (sulfamidase) 3.29 
    1538734 ACSL3 NM_004457 Acyl-CoA synthetase long-chain family member 3 3.27 
    1985998 ALOX5 BQ548663 Arachidonate 5-lipoxygenase 3.16 
    1527407 GAPDH NM_002046 Glyceraldehyde3-phosphate dehydrogenase 3.16 
    1534240 SULT1B1 D89479 Sulfotransferase family, cytosolic, 1B, member 1 3.10 
    1983183 NDUFB6 NM_182739 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 −3.10 
    1525237 ALDH6A1 NM_005589 Aldehyde dehydrogenase 6 family, member A1 −3.22 
    1518778 ALOX15 NM_001140 Arachidonate 15-lipoxygenase −3.24 
    1523761 CBR1 NM_001757 Carbonyl reductase 1 −3.61 
    1510672 MCCC1 NM_020166 Methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) −4.01 
    1990453 NDUFV2 BI520375 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24 kDa −4.31 
    1521732 ATP12A NM_001676 ATPase, H+/K+ transporting, nongastric, alpha polypeptide −4.74 
    1526184 LDHB NM_002300 Lactate dehydrogenase B −6.08 
    1512448 ISOC1 NM_016048 Isochorismatase domain containing 1 −6.51 
    1989369 METTL7A BC004492 Methyltransferase like 7A −6.99 
    1518627 FA2H NM_024306 Fatty acid 2-hydroxylase −7.75 
    1506804 PLD4 NM_138790 Phospholipase D family, member 4 −9.53 
    1521949 SORD NM_003104 Sorbitol dehydrogenase −10.20 
    1512822 SULF2 NM_018837 Sulfatase 2 −21.22 

Genes that were found to be differentially expressed after in vivo treatment with G-CSF-dexamethasone were analyzed with use of the OntoExpress program (http://vortex.cs.wayne.edu/projects.htm#Onto-Express), and categorized into functional profiles. The majority of analyzed genes are categorized into 6 main functional categories: transcription, signal transduction, immune response, cell faith & apoptosis, cell adhesion and mobility, and metabolism. Results are presented as the mean fold-increase or -decrease of 3 separate donors (comparison is with freshly isolated neutrophils t = 0).

Twenty-six genes involved in the regulation of transcription or the process of transcription itself (eg, CCNA1, SMARCA3, and MEF2A) were up-regulated after administration of G-CSF and dexamethasone. However, the expression of more than 40 of such genes was reduced, with NR4A1, NR4A3, and ZNF649 being most strongly affected. This finding suggests that granulocytes obtained from G-CSF/dexamethasone-treated donors express a transcriptional program that diverges from normal, circulating neutrophils. These data are indicative of coordinated changes in transcription and suggest the existence of both positive and negative regulatory mechanisms in the G-CSF/dexamethasone–induced transcriptional response.

Granulocyte mobilization has an effect on genes involved in inflammation

The expression of dozens of genes encoding molecules involved in the process of inflammation and the immune response was shown to be modulated by the in vivo administration of G-CSF/dexamethasone (Table 2). For example, the transcription of the gene CYBB was strongly induced. This gene encodes gp91phox, the enzymatic subunit of the NADPH oxidase enzyme complex that is responsible for superoxide production and contributes to microbial killing by neutrophils (Table 2).

The expression of several receptors responsible for ligand or pathogen sensing was induced as well, as indicated, for example, by the increase in transcripts for several C-type lectins such as CLEC5A, also known as myeloid DAP12-associated protein (MDL-1) and involved in the activation of cells,29  or CLEC4D (CLECSF8), which is also expressed on myeloid cells and has been suggested to take part in the process of endocytosis.30  Expression of another set of immune receptors that is associated with the activation of cells, eg CD48, LAIR1 (leukocyte-associated Ig-like receptor 1) and LILRA5 (LIR9, a member of the immunoglobulin-like receptor family), also was induced after mobilization.

However, we observed a significant reduction in the expression of genes traditionally associated with the adaptive immune response (eg, HLA-DQB2, HLA-DRB5, HLA-DPA1, HLA-DRA, HLA-DMB, and granzymes; Table 2).

In addition, the transcriptome of mobilized cells was characterized by the modulation of a cluster of genes coding for cytokines/chemokines and/or their receptors. Within the chemokine system, we identified CCL3, CCL4, CCL5, CXCL1, CXCL10, as well as the stroma-derived factor-1 receptor CXCR4, and CCR3, as being repressed by G-CSF and dexamethasone treatment at the transcriptional level. Moreover, the transcripts for Annexin-1 and -3 were highly induced by the G-CSF/dexamethasone treatment. Unexpectedly, we also found a strong reduction in IL8 transcripts after mobilization, whereas to our knowledge, no defects in the release of IL-8 from mobilized granulocytes have been reported to date.

Modulation of genes involved in the apoptotic machinery

Because mobilized granulocytes show delayed apoptosis upon in vitro culture,9,10  we carefully examined genes involved in the regulation of cell fate or apoptosis (Table 2). One group of molecules that is known to be regulated at the transcriptional level and is involved in the apoptotic process is the group of galectins. Galectins form a family of lectins that are defined by their ability to recognize β-galactose and the presence of consensus amino-acid sequences.31  Different members of this family have been shown to modulate various steps of the inflammatory response, such as cell-matrix interactions, cell trafficking, cell survival, cell-growth regulation, chemotaxis, and proinflammatory cytokine secretion.32,33  We observed a strong induction of galectin-8 (LGAL8) and moderate induction of galectin-3 (LGALS3), as well as a reduction of galectin-2 and -12 (LGALS2, LGALS12).

Most striking was the strong induction observed for the transcript of GADD45A, a member of the Gadd45 family of genes that have been implicated in stress signaling in response to physiologic or environmental stressors, resulting in either cell-cycle arrest, DNA repair, cell survival, and senescence or apoptosis. Hematopoietic cells from Gadd45a-deficient mice are more susceptible to apoptosis induced by ultraviolet (UV) radiation or genotoxic stress.34,35 

Expression of the CAST gene encoding calpastatin was also strongly induced in mobilized cells. Whereas calpain activity has previously been shown to be involved in neutrophil cell death,17,20  calpastatin is a well-documented and specific endogenous inhibitor of calpains.36  It is encoded by a single gene that produces several isoforms via differential splicing or use of promoters. Calpastatins are known to be associated with an antiapoptotic effect in neutrophils,20  and we have recently observed that inhibition of calpain activity by G-CSF strongly affects neutrophil survival.17  Because mobilized cells have been reported to have a better survival capacity, calpastatin was investigated in greater detail.

Induction of calpastatin correlates with a delay in apoptosis

G-CSF causes rapid neutrophilia but is also known to act as a survival factor on mature, circulating neutrophils both in vitro16,17,37  and in vivo.9  Dexamethasone, although shown to induce apoptosis in various cell types, including lymphocytes, monocytes,38  and eosinophils,15,39  also acts as a prosurvival factor for neutrophils.13,40  This finding is in line with our observation that, upon in vitro culture, granulocytes obtained from donors treated with a combination of the 2 drugs showed a prolonged life span after overnight culture compared with neutrophils isolated from the blood of the same donors before treatment (Figure 3). The same extent of survival was achieved by the in vitro addition of G-CSF and dexamethasone to the culture medium during overnight culture (data not shown).

Figure 3

Prolonged survival of granulocytes isolated from donors mobilized with G-CSF/dexamethasone. Granulocytes were isolated from healthy, control donors (□) and from the same donors treated with G-CSF and dexamethasone (■), and cultured for 20 hours in HBSS. Afterward, the cell viability was assessed by the use of annexin V/PI staining. Results represents data from 6 independent experiments (mean ± SD). *P < .05 (significant difference).

Figure 3

Prolonged survival of granulocytes isolated from donors mobilized with G-CSF/dexamethasone. Granulocytes were isolated from healthy, control donors (□) and from the same donors treated with G-CSF and dexamethasone (■), and cultured for 20 hours in HBSS. Afterward, the cell viability was assessed by the use of annexin V/PI staining. Results represents data from 6 independent experiments (mean ± SD). *P < .05 (significant difference).

The calpastatin transcripts in neutrophils were significantly induced by treatment with G-CSF/dexamethasone, either when applied in vivo or in vitro (Table 1). First, we used RT-PCR to confirm the increase in transcription of the CAST gene that was observed in the microarray analysis. Human calpastatin is expressed as 12 different isoforms of various lengths. The major variation between the isoforms is found in the first 11 exons. The probe set in the microarray only recognizes the main high molecular weight isoforms. We have designed various primer pairs to detect all different isoforms separately in neutrophils and found that all the known transcripts described thus far were expressed (not shown). However, evidence from the literature indicates that all isoforms are able to inhibit calpain activity.41  Therefore, we decided to use a primer set that recognizes all different isoforms in our experiments to confirm the microarray data.

Neutrophils from individuals treated with a combination of G-CSF and dexamethasone, as well as neutrophils cultured with this combination in vitro, both displayed an increase in CAST gene expression (Figure 4A). In freshly purified neutrophils, several isoforms of calpastatin are expressed. In agreement with previously published work, also in other cells, the molecular sizes of these isoforms were found at 110, 108, 90, and 41 kDa.17,42  The most abundant isoforms expressed in neutrophils were the high molecular weight (HMW) forms found as 110- and 108-kDa bands. Neutrophils isolated from granulocyte transfusion donors expressed increased levels of HMW calpastatins compared with the control cells (Figure 4B).

Figure 4

Modulation of calpain/calpastatin pathway by treatment with G-CSF/dexamethasone. (A) Changes in the expression of the CAST gene after treatment with G-CSF and dexamethasone in vivo () or in vitro (■) were measured by the light cycler RT-PCR. Results are presented as the mean ± SEM from 3 independent donors. (B) Different expression of calpastatin after administration of G-CSF and dexamethasone. Samples of neutrophils isolated from control donors (□) and donors stimulated with G-CSF and dexamethasone (■) were subjected to SDS-PAGE and analyzed on Western blots, stained with antibodies against calpastatin. Equal amounts of cells (1.5 × 106) were dissolved in sample buffer, and loaded in each lane. After staining with fluorescently labeled secondary antibody, the blots were scanned with Odyssey and analyzed with Licor Odyssey 2.1 software. Signals were normalized for p38 expression. The intensity of the 2 highest bands recognized by the antibody, which represent 2 major forms of calpastatin in neutrophils, was analyzed. Results represent data from 5 different donors (mean ± SEM). *P < .05 (significant difference). A representative blot comparing calpastatin levels in control cells and mobilized neutrophils is presented. (C) Samples of neutrophils incubated in the presence or absence of G-CSF and dexamethasone were taken at the indicated times and analyzed by Western blot. When indicated, CHX (10 μg/mL) was added to prevent new protein synthesis. Results represent data from 5 independent experiments (mean ± SEM). *P < .05 (significant difference). (D) Samples of neutrophils incubated in the presence or absence of calpain inhibitor 3 (CI3, 20 μmol/L) or caspase inhibitor zVAD (20 μmol/L) were taken at the indicated time points and analyzed by Western Blot. Results represent data from 5 independent experiments (mean ± SEM). *P < .05 (significant difference). (E) Inhibition of Calpains prolongs neutrophil viability. Neutrophils were isolated from healthy donors and cultured overnight alone or with the addition of G-CSF (10 ng/mL), CI3 (20 μmol/L), or zVAD (20 μM). When indicated, CHX (10 μg/mL) was added to prevent new protein synthesis. Cells negative for annexin V staining were considered to be alive. Results represent data from 6 independent experiments (mean ± SEM). *P < .05 (significant difference).

Figure 4

Modulation of calpain/calpastatin pathway by treatment with G-CSF/dexamethasone. (A) Changes in the expression of the CAST gene after treatment with G-CSF and dexamethasone in vivo () or in vitro (■) were measured by the light cycler RT-PCR. Results are presented as the mean ± SEM from 3 independent donors. (B) Different expression of calpastatin after administration of G-CSF and dexamethasone. Samples of neutrophils isolated from control donors (□) and donors stimulated with G-CSF and dexamethasone (■) were subjected to SDS-PAGE and analyzed on Western blots, stained with antibodies against calpastatin. Equal amounts of cells (1.5 × 106) were dissolved in sample buffer, and loaded in each lane. After staining with fluorescently labeled secondary antibody, the blots were scanned with Odyssey and analyzed with Licor Odyssey 2.1 software. Signals were normalized for p38 expression. The intensity of the 2 highest bands recognized by the antibody, which represent 2 major forms of calpastatin in neutrophils, was analyzed. Results represent data from 5 different donors (mean ± SEM). *P < .05 (significant difference). A representative blot comparing calpastatin levels in control cells and mobilized neutrophils is presented. (C) Samples of neutrophils incubated in the presence or absence of G-CSF and dexamethasone were taken at the indicated times and analyzed by Western blot. When indicated, CHX (10 μg/mL) was added to prevent new protein synthesis. Results represent data from 5 independent experiments (mean ± SEM). *P < .05 (significant difference). (D) Samples of neutrophils incubated in the presence or absence of calpain inhibitor 3 (CI3, 20 μmol/L) or caspase inhibitor zVAD (20 μmol/L) were taken at the indicated time points and analyzed by Western Blot. Results represent data from 5 independent experiments (mean ± SEM). *P < .05 (significant difference). (E) Inhibition of Calpains prolongs neutrophil viability. Neutrophils were isolated from healthy donors and cultured overnight alone or with the addition of G-CSF (10 ng/mL), CI3 (20 μmol/L), or zVAD (20 μM). When indicated, CHX (10 μg/mL) was added to prevent new protein synthesis. Cells negative for annexin V staining were considered to be alive. Results represent data from 6 independent experiments (mean ± SEM). *P < .05 (significant difference).

Next, the calpastatin protein levels were monitored during neutrophil spontaneous apoptosis in the presence or absence of G-CSF and dexamethasone. Culturing of the cells resulted in reduced levels of all isoforms, but especially the HMW forms of calpastatin were diminished (Figure 4C and Figure S2), suggesting proteolysis of the protein during apoptosis. The addition of G-CSF and dexamethasone to culture media resulted in an increase in the protein expression after 8 hours of culture and prevented the absolute loss of the protein after 24 hours in culture. Inhibition of new protein synthesis by the addition of cycloheximide (CHX, 10 μg/mL) prevented the increase and led to the total loss of protein expression after prolonged culture, similar to the control cells (Figure 4C and Figure S2A). The reduction in CAST protein level was apparently caused by degradation by either the calpains that have overcome the inhibitory effect of the calpastatin43  or by caspases activated via a different, calpain-independent route,42  because the addition of either the cell-permeable calpain inhibitor III (CI3) or the cell-permeable pan-caspase inhibitor zVAD partially prevented the loss of calpastatin during overnight culture (Figure 4D and Figure S2B). Moreover, the combined addition of CI3 or zVAD to the cells cultured with G-CSF/dexamethasone induced an additive effect, with zVAD having a more pronounced effect (data not shown).

Inhibition of calpains prevents neutrophil apoptosis

To confirm the involvement of calpain activity in neutrophil apoptosis, cells were incubated overnight in the presence of the combination of G-CSF/dexamethasone, the cell-permeable calpain inhibitor III (CI3), or the cell-permeable pan-caspase inhibitor zVAD as a positive control, whereas cell viability was measured by annexin V staining and verified by cellular morphology (data not shown). As reported before,11,15  the addition of G-CSF/dexamethasone to the culture medium decreased the number of apoptotic cells after overnight culture. This prosurvival effect was abolished by the addition of CHX. In addition, CI3 delayed neutrophil apoptosis to a similar extent (Figure 4E). Together, these data suggest a direct link between the calpain activity, endogenous calpastatin levels, and apoptosis.

Discussion

Mobilization of granulocytes by the administration of G-CSF and corticosteroids is a well-established procedure to achieve the number of granulocytes required for transfusion to neutropenic patients with severe, nonresponsive infections.2,4  We studied the effects of G-CSF and dexamethasone administration on the gene expression pattern of granulocytes to be used for transfusion. We found that a high number of genes was differentially regulated after in vivo mobilization of granulocytes. Approximately 20% of those genes changed similarly upon in vitro culture of granulocytes with the combination of G-CSF and dexamethasone. Still, the expression of more than 700 genes was changed significantly only after in vivo administration, which is perhaps not surprising. It seems reasonable to assume that, within the time frame of the experiment (18-20 hours), secondary donor factors released upon administration of the mobilizing agents, from either granulocytes themselves or from other cell types, could have contributed to the observed changes in granulocyte gene expression. In this context, it is important to realize that the G-CSF receptor is not only present on precursor and mature neutrophilic granulocytes, but also on monocytes and platelets, as well as on endothelial cells and on adult neuronal stem cells.44-46  In contrast, the glucocorticoid receptors are ubiquitously expressed and known to induce or modulate gene transcription in various cell types.47 

An additional or, in some cases alternative, explanation may be that the granulocytes recently mobilized from the bone marrow represent a less mature neutrophilic phenotype, as indicated by the lower expression of EMR3, a late marker of granulocytic differentiation,27  and the distinct left shift of the mobilized neutrophils. Another supportive feature of the induced neutrophil egress from the bone marrow was found in the increased transcriptional activity of the genes MMP9 and MMP8, both encoding matrix metalloproteases. The products of these MMP genes are important for creating a localized and highly enriched proteolytic environment in the bone marrow, thus facilitating the release of granulocytes from the stromal microenvironment.48 

Finally, there may be combinatorial effects of G-CSF and dexamethasone and other environmental factors specific for the in vivo or in vitro situation. In any case, it seems reasonable to assume that most, if not all, of the gene regulatory effects observed upon the in vitro treatment with G-CSF and dexamethasone represent direct effects of these factors on granulocytes, but there are multiple other explanations possible for the effects found only in the in vivo or only in the in vitro condition, which may relate to differences in cell composition and/or the combinatorial effects of specific (experimental) conditions.

The basic effector functions of granulocytes collected for transfusion after administration of G-CSF and dexamethasone, including interaction with endothelial cells, migration, respiratory burst or killing capacity, seem to be unaffected.4,10,49,50  However, the life span of those cells is prolonged upon in vitro culture as well as in vivo. In addition, supplementation of the culture medium of previously untreated control cells with G-CSF/dexamethasone also prolongs cell survival. This apparently direct effect depends on gene expression and new protein synthesis because it is abolished by the addition of cycloheximide.

From the groups of genes differentially regulated in mobilized granulocytes, we focused on the regulatory genes of apoptosis. The most impressive change was observed for calpastatin, the endogenous inhibitor of calpains. Inhibition of calpains with pharmacologic inhibitors results in a significant delay of neutrophil apoptosis.19  We have recently found that G-CSF is implicated in the regulation of calpain activity by influencing the intracellular levels of Ca2+ upon in vitro culture of neutrophils,17  an effect depending on new protein synthesis. It has been proposed that, upon mild stimulation, calpastatin binds to calpains, preventing the proteases from degrading their substrates. However, the intramolecular, autolytic activation of calpains is not prevented by calpastatin binding,43,51  and if the levels of intracellular Ca2+ increase even higher, so stimulation becomes stronger, the protease will degrade its inhibitor and thereafter proceed to cleave other substrates. Calpastatin degradation has been shown to occur during spontaneous neutrophil apoptosis by activated calpains or proapoptotic caspases (eg, caspase-3 and -7).20,42,52  Calpastatin expression also has been shown to be preferentially induced in neutrophils isolated from patients with cystic fibrosis simultaneously with decreased levels of pro-calpain-1, coinciding with a delay in neutrophil cell death.20 

Increased levels of mRNA for calpastatin were detected in mobilized neutrophils, isolated from granulocyte donors pretreated for 18 to 20 hours with G-CSF/dexamethasone as well as in neutrophils cultured in vitro in the presence of G-CSF/dexamethasone. A similar increase in calpastatin levels was observed at the protein level when mobilized neutrophils were compared with the control cells.

Immunoblot analysis of neutrophil lysates showed a loss of calpastatin expression upon in vitro culture. The addition of G-CSF and dexamethasone to the culture media increased the level of calpastatin after 8 hours of incubation, which was prevented by cycloheximide. However, after 24 hours in culture, the cells treated with prosurvival factors such as G-CSF and dexamethasone also displayed strongly reduced levels of calpastatin, albeit still significantly greater than cultured control cells. It appears that the stronger and prolonged activation of calpains allows these proteases to overcome the inhibitory effect of cellular calpastatin, which eventually results in its cleavage. Pharmacologic inhibition of calpains partially preserved calpastatin expression during 24 hours in culture, albeit to a lesser extent than with G-CSF and dexamethasone. Most likely, the activated caspases are responsible for this effect because neutrophils incubated in the presence of a pan-caspase inhibitor also sustained their level of calpastatin. Moreover, the combination of G-CSF/dexamethasone with zVAD led to an additive increase in calpastatin levels and survival of neutrophils.

Taken together, our data suggest that the calpain/calpastatin system plays an important role in neutrophil apoptosis. Inhibition of calpain activation by increasing the levels of its endogenous inhibitor may be one of the prosurvival mechanisms by which G-CSF and dexamethasone delay the apoptosis in neutrophils used for transfusion purposes. Finally, we showed that intracellular levels of calpastatin correlate with the viability of neutrophils in vitro and that pharmacologic inhibition of calpains decreases the rate of apoptosis during 24 hours of incubation. A role for calpastatin in apoptosis in neutrophils is supported by data demonstrating that a reduction in its expression levels by antisense nucleotides accelerates apoptosis.53 

In summary, we have shown that mobilization of granulocytes for transfusion by the combination of G-CSF and dexamethasone strongly alters the gene expression pattern in circulating neutrophils compared with untreated donor neutrophils. The characteristics of cells used for transfusion can now be studied based on the changed transcriptional program. Using the changed transcript levels, we can now recognize that mobilized neutrophils display changes in subtle functional aspects that previously remained unidentified. These changes include genes involved in immune reactivity, motility, signal transduction, and gene transcription, as well as cell viability.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

We gratefully acknowledge Prof D. Roos for critical reading and discussion of the manuscript and Dr Perry Moerland for excellent assistance with the bioinformatics and statistics of the microarray analysis.

This work was supported by a grant from the Sanquin Foundation for Cellular Blood Product Development (PPO-C-03-011-2003).

Authorship

Contribution: A.D. designed and performed research, analyzed data, and wrote the paper; B.J.v.R., J.G., and A.T.J.T helped perform parts of the research; O.R.F.M. helped analyze data; and F.B., T.K.v.d.B., and T.W.K. supervised the project and reviewed the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Agata Drewniak, Department of Blood Cell Research, Phagocyte Laboratory, Sanquin Research and Landsteiner Laboratory, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands; e-mail: a.drewniak@sanquin.nl.

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Supplemental data