Mast cells (MCs) and eosinophils are thought to play important roles in evoking allergic inflammation. Cell-type–specific gene expression was screened among 12 000 genes in human MCs and eosinophils with the use of high-density oligonucleotide probe arrays. In comparison with other leukocytes, MCs expressed 140 cell-type–specific transcripts, whereas eosinophils expressed only 34. Among the transcripts for expected MC-specific proteins such as tryptase, major basic protein (MBP), which had been thought to be eosinophil specific, was ranked fourth in terms of amounts of increased MC-specific messenger RNA. Mature eosinophils were almost lacking this transcript. MCs obtained from 4 different sources (ie, lung, skin, adult peripheral blood progenitor–derived and cord blood progenitor–derived MCs, and eosinophils) were found to have high protein levels of MBP in their granules with the use of flow cytometric and confocal laser scanning microscopic analyses. The present finding that MCs can produce abundant MBP is crucial because many reports regarding allergic pathogenesis have been based on earlier findings that MBP was almost unique to eosinophils and not produced by MCs.

Introduction

Mast cells (MCs)1 and eosinophils2 are thought to play important roles in evoking allergic inflammation. Human MCs contribute to allergic inflammation by releasing a variety of mediators and cytokines.3-6 On the other hand, human eosinophils are considered to play a role in the bronchoconstriction of asthma patients through the release of leukotriene C47 and also to damage the bronchial epithelial cells through the release of granule proteins such as major basic protein (MBP).8 These 2 cell types also contribute to allergic inflammation by activating each other. Eosinophil MBP can induce MC degranulation,9 whereas human MCs can activate eosinophils through IgE-dependent production of cytokines such as interleukin-5 (IL-5).10 

A draft reading of all human genome sequences has been completed.11,12 It is expected that in the near future, we will resolve previously unanswered questions such as the probability of development of various diseases by screening for single nucleotide polymorphisms over the whole genome sequence. Comprehension of the genome has also accelerated understanding of the transcriptome,13 all of the transcripts present in a cell, and the proteome,14 which controls all of the regulatory elements in a cell. Until recently, it required much time and labor to measure the expression levels of genes even for 100 transcripts. By using recently developed techniques, however, such as cDNA microarrays,15 oligonucleotide expression probe arrays,16 and serial analysis of gene expression,17 such systemic analysis of transcriptomes has become practical.

Among the newly developed techniques, high-density oligonucleotide expression probe array (Genechip; Affymetrix, Santa Clara, CA) is designed to measure the absolute levels of more than 10 000 transcripts regardless of the cell type by using the same set of inner standards on a 1.2-cm2 glass chip. The competition with another cell type required for cDNA microarray assay is not required with the Genechip.16,18-20 Thus, we can compare the expression levels of more than 10 000 transcripts even in different cell types by using the high-density oligonucleotide probe array. In the present study, we used the Genechip to measure and compare cultured human MCs and purified eosinophils for their expression levels of more than 10 000 transcripts. We found that MBP, which has previously been reported to be present only in eosinophils and basophils,21 was abundantly expressed in MCs. We also confirmed that MBP is expressed at both the transcript and protein levels in various types of MCs.

Materials and methods

Subjects

All human subjects in this study provided written informed consent, which was approved by the Ethical Review Board at their hospitals. Nonphagocytic mononuclear cells were separated from adult peripheral blood (PB) or umbilical cord blood (CB) samples by density-gradient centrifugation using Lymphocyte Separation Medium (Organon Teknika, Durham, NC) after depletion of phagocytes with silica (Immuno Biological Laboratories, Fujioka, Japan). The interface containing mononuclear cells was collected. Lineage-negative (Lin) cells were negatively selected from the peripheral mononuclear cells using a magnetic separation column (MACS II; Miltenyi Biotec, Bergisch Gladbach, Germany) and a mixture of magnetic microbead-conjugated antibodies against CD4, CD8, CD11b, CD14, and CD19 (Miltenyi Biotec), according to the manufacturers' instructions. CD34+ cells were positively selected from CB-derived mononuclear cells using a CD34+ cell isolation kit (Miltenyi Biotec).

Cytokines and antibodies

Recombinant IL-3 was purchased from Intergen (Purchase, NY), and rIL-6 was kindly provided by Kirin Brewery (Maebashi, Japan). Bulk vials of recombinant human stem cell factor (SCF) were purchased from PeproTech EC (London, England). Recombinant IL-4 was purchased from R&D Systems (Minneapolis, MN). Antihuman tryptase monoclonal antibody (MoAb) was purchased from Chemicon (Temecula, CA). Two different anti-MBP MoAbs were purchased from Nichirei (BMK-13; Tokyo, Japan)22 and from Chemicon (AHE-2).23 

Cell culture

The cells were suspended in Iscoves modified Dulbecco minimal essential medium (IMDM; Gibco BRL, Grand Island, NY) supplemented with 1% insulin-transferrin-selenium (Gibco BRL), 50 μM 2-ME (Gibco BRL), 1% penicillin+streptomycin (Gibco BRL), and 0.1% bovine serum albumin (BSA; Sigma, St Louis, MO) (complete IMDM). The Lin106 PB cells were suspended in 0.3 mL of complete IMDM. The cells were mixed well with 2.7 mL serum-free Iscoves methylcellulose medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 200 ng/mL SCF, 50 ng/mL IL-6, and 1 ng/mL IL-3, as described previously.20 The cell suspension was inoculated at 0.3 mL per well to 24-well plates (Iwaki Glass, Tokyo, Japan) at 37°C in 5% CO2. Every 2 weeks, 0.3 mL fresh methylcellulose medium containing 100 ng/mL SCF and 50 ng/mL IL-6 was layered over the methylcellulose cultures. After 6 weeks, the whole cells were retrieved by dissolving the methylcellulose medium with phosphate-buffered saline (PBS). The cells were then suspended and cultured in complete IMDM supplemented with 100 g/mL SCF, 50 ng/mL IL-6, and 2% fetal calf serum (FCS; Cansera, Rexdale, ON, Canada) in 25-cm2flasks (Iwaki Glass). CB-derived CD34+ cells were cultured in the complete IMDM supplemented with 100 ng/mL SCF, 50 ng/mL IL-6, and 2% FCS (Cansera) in 25- or 75-cm2 flasks (Iwaki Glass), as described elsewhere.24 

Purification of eosinophils, neutrophils, and mononuclear cells

Eosinophils, neutrophils, and mononuclear cells were separated from venous blood of normal volunteers. Eosinophils were isolated by using Percoll (1.090 g/mL; Pharmacia, Uppsala, Sweden) density centrifugation. The eosinophils were further purified by negative selection with anti-CD16–bound micromagnetic beads, as described previously.25 After this negative selection, the mean eosinophil purity was consistently greater than 99%. Neutrophils were isolated by 2-step density centrifugation. In brief, eosinophils were eliminated by Percoll density centrifugation as a first step. The buoyant fraction was collected and overlaid on Ficoll-Paque (1.077 g/mL; Pharmacia) to eliminate mononuclear cells. The mean neutrophil purity was consistently greater than 99%, and the viability was consistently greater than 95%. Mononuclear cells usually contained lymphocytes with 20% monocytes and 2% basophils.

Intracytoplasmic staining for MBP

Intracytoplasmic staining of eosinophils and CB- and PB-derived MCs was done by the method previously reported.26 Briefly, the cells were fixed with 4% paraformaldehyde, washed, and incubated with PBS-saponin containing 0.1% BSA for 1 hour at 37°C. These cells were then incubated with 50 mg/mL human IgG (ICN Biomedicals, Aurora, OH). Cells were then incubated with 3 μg/mL mouse anti-MBP MoAb (Nichirei) for 1 hour at 4°C, washed, and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti–mouse IgG (Becton Dickinson, San Jose, CA) for 30 minutes at 4°C. After staining, all cells were washed and resuspended in PBS containing 0.1% BSA. Control cells were stained with an irrelevant mouse IgG1 (Coulter Immunology, Hialeah, FL). Cell analysis was performed using FACScan and CellQuest software (BD Immunocytometry Systems, San Jose, CA). The distribution of histograms was evaluated as the coefficient of variation.

Confocal laser scanning microscopy

Cells treated with anti-MBP MoAbs (BMK-13) were stained with FITC-conjugated anti–mouse IgG. Confocal imaging was performed with a Fluoview FV300 confocal laser scanning unit (Olympus, Tokyo, Japan) mounted on the inverted-type fluorescence microscope (IX 70; Olympus) with a 60× oil-immersion lens. The images were collected as 1-mm-thick optical sections. In some experiments, differential interference images were also obtained. In one experiment, lung tissue was obtained at surgery (lobectomy) from a patient suffering from multiple lung cysts after informed consent was acquired. The lung tissue was prepared following the method described by Jaffe et al.5 In brief, tissue fragments were incubated in collagenase type II (2 μg/mL) at 37°C for 3 hours. Single-cell suspensions were obtained from the fragments. MCs were semipurified using the MACS system and anti–c-kit MoAb (Pharmingen). MCs were further purified to greater than 99.9% by culturing them in the serum-free IMDM supplemented with 100 ng/mL SCF for 4 weeks.

Immunohistochemical analysis for MBP and tryptase

Skin biopsy samples were obtained from patients with severe atopic dermatitis after informed consent as described above. Sections of 4 μm thick fixed with acetone at 4°C for 5 minutes were incubated with 10% normal goat serum, FITC-labeled anti-MBP MoAb (Nichirei), biotinylated antitryptase MoAb (Chemicon), and rhodamine-conjugated anti–mouse IgG. Negative controls were performed using isotype-matched unrelated antibodies. The results were examined under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany, and Nikon, Tokyo, Japan).

Degranulation assay

MCs were sensitized with 1 μg/mL human myeloma IgE (generously provided by Dr Kimishige Ishizaka, La Jolla, CA) at 37°C for 48 hours in the presence of IL-4. After washing, cells were suspended in Tyrode solution (pH 7.4) containing 124 mM NaCl, 4 mM KCl, 0.64 mM NaH2PO4, 1 mM CaCl2, 0.6 mM MgCl2, 10 mM HEPES, and 0.03% human serum albumin. The cells were preincubated for 10 minutes and then challenged with either 1.5 μg/mL rabbit anti–human IgE (Dako, Glostrup, Denmark) or control Tyrode solution at 37°C for 30 minutes.

Genechip expression analysis

Gene expression was screened for with the Genechip Human Genome U95A probe array (Affymetrix, Santa Clara, CA), which contains the oligonucleotide probe set for approximately 12 000 genes, according to the manufacturer's protocol (Expression Analysis Technical Manual) and previous reports.16,18-20 Total RNA (3-10 μg) was extracted from approximately 107 cells. Double-stranded cDNA was synthesized by using a SuperScript Choice system (Life Technologies, Rockville, MD) and a T7-(dT)24 primer (Amersham Pharmacia Biotech, Buckinghamshire, England). The cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates with the use of a BioArray HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). The biotinylated cRNA was hybridized with a U95A probe array for 16 hours at 45°C. After washing, the hybridized biotinylated cRNA was stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR) and then scanned with the HP Gene Array Scanner. The fluorescence intensity of each probe was quantified using a computer program, Genechip Analysis Suite 3.3 (Affymetrix). The expression level of a single mRNA was determined as the average fluorescence intensity among the intensities obtained by 16 to 20 paired (perfectly matched and single nucleotide–mismatched) primers consisting of 25-base oligonucleotides. If the intensities of mismatched primers are very high, gene expression is judged to be absent even if a high average fluorescence is obtained with the Genechip Analysis Suite 3.3 program.

Reverse transcriptase–polymerase chain reaction for the MBP transcript

Total RNA was isolated from the Isogen (Nippon Gene, Osaka, Japan) solution in which harvested MCs, peripheral eosinophils, or CB-derived immature eosinophils were dissolved. The immature eosinophils were obtained by culturing CB CD34+ cells in the presence of IL-5 plus IL-3, as described previously.27,28 The RNA was DNase-treated, converted to cDNA by reverse transcription (RT) using a kit from Invitrogen (San Diego, CA), and subjected to polymerase chain reaction (PCR) amplification. The primer sequences for MBP were as follows: 5′-GTG CTA AGA CGC TGC CTG AG-3′ for the 5′ primer and 5′-TTT CAG TGG GTT GAC GGC-3′ for the 3′ primer, spanning a fragment of 440 bp. The primer sequences for β-actin were as follows: 5′-TGA CGG GGT CAC CCA CAC TGT GCC-3′ for the 5′ primer and 5′-TAG AAG CAT TTG CGG TGGACG ATG-3′ for the 3′ primer, spanning a fragment of 661 bp (Continental Laboratory Product, San Diego, CA). PCR amplification was performed using a thermal cycler (Geneamp PCR System 9700; PE Biosystems) with an initial denaturation cycle for 1 minute at 94°C; 40 amplification cycles for 1 minute at 94°C, 1 minute at 55°C, and 2 minutes at 72°C; and a final extension phase consisting of one cycle of 10 minutes at 72°C. PCR products were visualized on 0.8% agarose gel (BRL Life Technologies) containing 0.05 μg/mL ethidium bromide (Sigma).

Statistical analysis

Statistical significance between paired groups was determined by the paired Student t test and considered significant forP < .05. Values are expressed as the mean ± SEM.

Results

Detection of MC-specific transcripts by Genechip

As shown in Table 1, we selected MC-specific transcripts by comparing the expression of 12 000 genes among PB-derived MCs, eosinophils, neutrophils, and mononuclear cells containing lymphocytes, monocytes, and basophils with the use of Genechip. Compensation between samples was done with housekeeping genes by referring to the inner standards (legend to Table 1). The MC-specific transcripts were selected by subtracting the compensated values for eosinophils, neutrophils, and mononuclear cells from those for PB-derived MCs. After elimination of redundant transcripts, 140 transcripts were found to be highly MC specific (the subtracted values were greater than 5% of the housekeeping genes).

Table 1.

Top 50 most-increased mast cell–specific transcripts

Accession
number* 
Name of transcript (protein) MCs
(%AD
Eo
(%AD) 
MNC
(%AD) 
Neu
(%AD) 
Fold
change 
M33494 Tryptase 197.6 0.9 0.8 1.2 205.3 
M25915 Clusterin 134.0 3.1 9.4 1.2 29.5 
M16117 Cathepsin G 76.7 0.7 0.7 < 0.1 167.0 
Z26248 Eosinophil granule MBP 67.9 0.5 < 0.1 135.6  
M63138 Cathepsin D 107.4 9.1 20.5 12.9 7.6  
AF150241 Prostaglandin D2 synthase 59.7 < 0.1 < 0.1 < 0.1 9324.9 
M73720 Carboxypeptidase A 57.5 0.5 0.9 < 0.1 123.6  
X76534 Glycoprotein (transmembrane) NMB 47.4 0.1 0.1 < 0.1 425.5 
AF002672 Breast cancer suppressor candidate 1 45.1 < 0.1 0.1 < 0.1 1145.4 
M68891 GATA-binding protein 2 56.7 4.8 8.5 0.8 12.0  
L76465 15 hydroxy-prostaglandin dehydrogenase 38.4 0.6 0.9 < 0.1 79.4 
D11139 Tissue inhibitor of metalloproteinases 52.3 3.3 10.4 3.3 9.2 
Y00451 5-aminolevulinate synthase 40.4 1.6 2.5 2.2 19.1  
U18009 Membrane protein of cholinergic synaptic vesicles 43.4 2.6 6.7 1.31 12.4 
AB006780 Galectin-3 49.4 3.9 13.6 0.7 8.2 
D16583 L-histidine decarboxylase 34.8 2.1 1.5 < 0.1 28.6 
M89796 FcMR I β chain 29.4 0.2 0.5 < 0.1 125.2 
M26683 Interferon-γ treatment inducible 34.4 4.6 0.5 1.0 16.8 
R92331 Metallothionein 1E 41.1 3.4 5.3 4.4 9.4 
L78833 BRCA1, Rho7, and vatIgenes 35.1 2.2 3.3 2.3 13.4 
X06182 c-kitproto-oncogene 27.0 < 0.1 0.1 < 0.1 483.1 
X56667 Calretinin 28.3 1.5 0.8 1.3 23.9 
AF022813 NAG-2, transmembrane-4 superfamily 29.5 < 0.1 5.5 < 0.1 16.0 
D86358 Siglec6 23.3 < 0.1 0.2 < 0.1 411.6 
D87119 GS3955 putative serine/threonine kinase 38.6 1.5 13.2 0.9 7.4  
M94345 Capping protein (actin filament) 39.2 6.4 9.6 0.9 7.0 
X51956 Phosphopyruvate hydratase 24.3 0.2 1.9 < 0.1 34.0 
AB000584 TGF-beta superfamily 22.7 0.3 0.2 0.3 85.7  
AF001294 Tumor suppressing subtransferable candidate 3 22.9 1.0 0.3 < 0.1 48.9  
J04988 90-kd heat shock protein 50.8 2.9 26.1 0.8 5.1  
AL050224 RNA polymerase I 22.7 < 0.1 1.0 1.2 29.4 
M28225 Monocyte chemoattractant peptide-1 23.2 2.1 0.3 0.6 23.5  
L35594 Dynactin subunit 20.5 0.2 0.1 < 0.1 209.4  
H68340 RNA helicase-related protein 25.2 1.7 3.5 < 0.1 14.4 
M69136 Chymase 22.1 0.4 0.6 1.3 29.4 
M15518 Tissue-type plasminogen activator 19.9 < 0.1 < 0.1 0.1 242.5 
U50136 Leukotriene C4synthase 39.9 17.2 1.8 2.1 5.7  
X64559 Tetranectin (plasminogen-binding protein) 21.9 1.5 1.6 < 0.1 21.2 
R93527 Metallothionein 1H 31.6 3.8 5.6 3.6 7.3 
X75252 Phosphatidylethanolamine binding protein 25.3 0.6 7.7 0.3 8.8  
X67951 Peroxiredoxin 1 35.0 3.4 15.1 < 0.1 5.6 
M93311 Metallothionein 3 30.0 2.7 8.4 2.6 6.6 
AF038844 MKP-1–like protein tyrosine phosphatase 16.4 < 0.1 0.6 < 0.1 80.0 
U03688 Dioxin-inducible cytochrome P450 (CYP1B1) 23.3 2.0 5.6 0.3 8.8  
U78027 Bruton's tyrosine kinase 26.9 6.2 4.0 1.3 7.0 
K01383 Metallothionein 1A 19.0 < 0.1 3.5 0.4 14.7 
M19481 Follistatin 24.4 3.1 1.7 4.4 7.9 
M69177 Monoamine oxidase B 15.0 < 0.1 0.2 0.2 125.0  
X06948 FcεRI α chain 27.6 2.2 11.3 < 0.1 6.1 
AF045229 Regulator of G-protein signaling 10 24.8 0.4 10.7 0.2 6.6  
 P1 cre recombinase protein (inner standard 1)1-153 81.5 131.5 106.2 97.4  
 P1 cre recombinase protein (inner standard 2) 62.4 109.2 79.9 56.3  
 Mean of the 6 AD levels of housekeeping genes 12 071 7759 16 498 9779  
Accession
number* 
Name of transcript (protein) MCs
(%AD
Eo
(%AD) 
MNC
(%AD) 
Neu
(%AD) 
Fold
change 
M33494 Tryptase 197.6 0.9 0.8 1.2 205.3 
M25915 Clusterin 134.0 3.1 9.4 1.2 29.5 
M16117 Cathepsin G 76.7 0.7 0.7 < 0.1 167.0 
Z26248 Eosinophil granule MBP 67.9 0.5 < 0.1 135.6  
M63138 Cathepsin D 107.4 9.1 20.5 12.9 7.6  
AF150241 Prostaglandin D2 synthase 59.7 < 0.1 < 0.1 < 0.1 9324.9 
M73720 Carboxypeptidase A 57.5 0.5 0.9 < 0.1 123.6  
X76534 Glycoprotein (transmembrane) NMB 47.4 0.1 0.1 < 0.1 425.5 
AF002672 Breast cancer suppressor candidate 1 45.1 < 0.1 0.1 < 0.1 1145.4 
M68891 GATA-binding protein 2 56.7 4.8 8.5 0.8 12.0  
L76465 15 hydroxy-prostaglandin dehydrogenase 38.4 0.6 0.9 < 0.1 79.4 
D11139 Tissue inhibitor of metalloproteinases 52.3 3.3 10.4 3.3 9.2 
Y00451 5-aminolevulinate synthase 40.4 1.6 2.5 2.2 19.1  
U18009 Membrane protein of cholinergic synaptic vesicles 43.4 2.6 6.7 1.31 12.4 
AB006780 Galectin-3 49.4 3.9 13.6 0.7 8.2 
D16583 L-histidine decarboxylase 34.8 2.1 1.5 < 0.1 28.6 
M89796 FcMR I β chain 29.4 0.2 0.5 < 0.1 125.2 
M26683 Interferon-γ treatment inducible 34.4 4.6 0.5 1.0 16.8 
R92331 Metallothionein 1E 41.1 3.4 5.3 4.4 9.4 
L78833 BRCA1, Rho7, and vatIgenes 35.1 2.2 3.3 2.3 13.4 
X06182 c-kitproto-oncogene 27.0 < 0.1 0.1 < 0.1 483.1 
X56667 Calretinin 28.3 1.5 0.8 1.3 23.9 
AF022813 NAG-2, transmembrane-4 superfamily 29.5 < 0.1 5.5 < 0.1 16.0 
D86358 Siglec6 23.3 < 0.1 0.2 < 0.1 411.6 
D87119 GS3955 putative serine/threonine kinase 38.6 1.5 13.2 0.9 7.4  
M94345 Capping protein (actin filament) 39.2 6.4 9.6 0.9 7.0 
X51956 Phosphopyruvate hydratase 24.3 0.2 1.9 < 0.1 34.0 
AB000584 TGF-beta superfamily 22.7 0.3 0.2 0.3 85.7  
AF001294 Tumor suppressing subtransferable candidate 3 22.9 1.0 0.3 < 0.1 48.9  
J04988 90-kd heat shock protein 50.8 2.9 26.1 0.8 5.1  
AL050224 RNA polymerase I 22.7 < 0.1 1.0 1.2 29.4 
M28225 Monocyte chemoattractant peptide-1 23.2 2.1 0.3 0.6 23.5  
L35594 Dynactin subunit 20.5 0.2 0.1 < 0.1 209.4  
H68340 RNA helicase-related protein 25.2 1.7 3.5 < 0.1 14.4 
M69136 Chymase 22.1 0.4 0.6 1.3 29.4 
M15518 Tissue-type plasminogen activator 19.9 < 0.1 < 0.1 0.1 242.5 
U50136 Leukotriene C4synthase 39.9 17.2 1.8 2.1 5.7  
X64559 Tetranectin (plasminogen-binding protein) 21.9 1.5 1.6 < 0.1 21.2 
R93527 Metallothionein 1H 31.6 3.8 5.6 3.6 7.3 
X75252 Phosphatidylethanolamine binding protein 25.3 0.6 7.7 0.3 8.8  
X67951 Peroxiredoxin 1 35.0 3.4 15.1 < 0.1 5.6 
M93311 Metallothionein 3 30.0 2.7 8.4 2.6 6.6 
AF038844 MKP-1–like protein tyrosine phosphatase 16.4 < 0.1 0.6 < 0.1 80.0 
U03688 Dioxin-inducible cytochrome P450 (CYP1B1) 23.3 2.0 5.6 0.3 8.8  
U78027 Bruton's tyrosine kinase 26.9 6.2 4.0 1.3 7.0 
K01383 Metallothionein 1A 19.0 < 0.1 3.5 0.4 14.7 
M19481 Follistatin 24.4 3.1 1.7 4.4 7.9 
M69177 Monoamine oxidase B 15.0 < 0.1 0.2 0.2 125.0  
X06948 FcεRI α chain 27.6 2.2 11.3 < 0.1 6.1 
AF045229 Regulator of G-protein signaling 10 24.8 0.4 10.7 0.2 6.6  
 P1 cre recombinase protein (inner standard 1)1-153 81.5 131.5 106.2 97.4  
 P1 cre recombinase protein (inner standard 2) 62.4 109.2 79.9 56.3  
 Mean of the 6 AD levels of housekeeping genes 12 071 7759 16 498 9779  

MCs indicates mast cells; Eo, eosinophil; MNC, mononuclear cell; Neu, neutrophil; MBP, major basic protein; TGF, transforming growth factor; MKP, mitogen-activated protein kinase phosphatase; AD, average difference.

*

The GenBank accession number (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) of each transcript is shown.

The level of gene expression was obtained as the AD using Genechip software. The percentages of the specific AD level versus the mean AD level of 6 probe sets for housekeeping genes (β-actin and glyceraldehyde-3-phosphate dehydrogenase) were then calculated. The mean percent AD levels obtained from PB-derived MCs (n = 6), eosinophils (Eo, n = 2), mononuclear cells (MNC, n = 4), and neutrophils (Neu, n = 1) are shown.

The fold change was obtained by dividing the MC (mast cell) value by the mean of the 3 other transcript levels (Eo, MNC, and Neu). The transcripts showing at least a 5-fold change are listed in the table.

F1-153

Inner standards such as PI cre recombinase protein were added to cRNA of the target sample just before the hybridization (see “Materials and methods”) to evaluate the difference between samples. The inner standards were found at similar levels (< 2-fold change) after compensation with the housekeeping genes.

It is interesting that the top 50 PB-derived MC-specific transcripts shown in Table 1 include several genes related to remodeling of tissues, such as tissue-type plasminogen activator (the 36th most-increased transcript in Table 1)29 and tissue inhibitor of metalloproteinases (the 12th most-increased transcript in Table 1),30 as reported previously. Clusterin, an anti-inflammatory multifunctional protein, also called apolipoprotein J,31 was ranked as the second most-increased transcript in Table 1. Clusterin present in MCs is considered to play a role in regression of hemangioma, the most frequent tumor of infancy.32 

Detection of eosinophil-specific transcripts by Genechip

By employing the same protocol as for selecting MC-specific transcripts, we judged 34 transcripts to be highly eosinophil specific (we compared eosinophils versus MCs, neutrophils, and mononuclear cells). The top 30 transcripts are shown in Table2. CRTH2, a chemoattractant receptor specifically expressed on type-2 helper T cells and eosinophils,33 was judged to be eosinophil specific. Many increased transcripts in eosinophils overlapped with those in neutrophils and mononuclear cells containing basophils, whereas MCs tended to have unique transcripts compared with neutrophils, mononuclear cells, and eosinophils.

Table 2.

Top 30 increased eosinophil-specific transcripts

Accession
number* 
Name of transcript (protein) Eo
(%AD
MCs
(%AD) 
MNC
(%AD) 
Neu
(%AD) 
Fold
change 
X55988 Eosinophil-derived neurotoxin 86.8 0.2 7.3 0.5 32.2 
L01664 Charcot-Leyden crystal protein 78.0 < 0.1 9.2 0.2 24.5 
X81479 Glycoprotein EMR1 hormone receptor 59.1 1.7 5.2 2.8 18.4  
M84526 Complement factor D 45.9 0.9 10.9 4.8 8.3  
J04130 Macrophage inflammatory protein-1 β 36.8 < 0.1 6.0 1.9 14.0 
D90144 Macrophage inflammatory protein-1 α 41.5 0.2 4.7 8.2 9.5  
AJ004832 Neuropathy target esterase 36.0 3.2 8.7 3.0 7.2 
U09937 Urokinase-type plasminogen receptor 43.6 9.3 4.6 8.6 5.8 
X70326 MARCKS-related protein 42.1 2.7 10.0 10.9 5.4  
U81375 Solute carrier family 29 29.4 10.7 0.5 < 0.1 7.9 
AB008535 CRTH2 18.2 0.6 0.4 < 0.1 56.6 
M23892 Arachidonate 15-lipoxygenase 17.9 0.3 0.5 0.3 46.9 
AA131149 S100 calcium-binding protein P 18.5 0.2 0.9 1.5 21.1  
AB018339 Synaptic nuclei expressed gene 1β 24.3 0.8 7.3 0.4 8.5 
AF079167 Lectin-like oxidized LDL receptor 13.5 < 0.1 < 0.1 0.2 128.9 
D13626 G-protein–coupled receptor for UDP-glucose 14.2 1.1 1.0 0.7 15.0 
M34455 Indoleamine 2,3-dioxygenase 13.5 0.8 1.1 0.9 14.1 
X74328 Cannabinoid receptor 2 13.0 0.3 0.9 1.7 13.7  
L77730 A3 adenosine receptor 10.2 0.4 < 0.1 < 0.1 55.5 
S59049 Regulator of G-protein signaling 1 12.0 2.3 1.0 0.1 10.2  
L41816 CAM kinase I 10.2 1.7 1.1 < 0.1 10.7  
U21931 Fructose-1, 6-bisphosphatase 1 15.9 1.0 6.6 1.2 5.4 
U28694 CCR3 16.1 0.3 1.0 8.0 5.2 
M75914 IL-5 receptor α chain 8.8 0.7 0.7 1.1 10.5 
AF010193 SMAD7 10.9 2.2 2.4 0.2 6.7 
AF000545 Purinergic receptor P2Y10 14.1 0.3 3.7 4.4 5.1  
U48250 Protein kinase C binding protein 2 6.2 0.1 0.1 0.4 30.4 
S68134 Cyclic AMP-responsive element modulator, β unit 13.2 5.6 1.6 0.4 5.2  
L19314 Phosphorylase kinase, β unit 7.3 1.5 0.1 < 0.1 12.7 
L08044 Trefoil factor 3 (intestinal) 5.6 < 0.1 < 0.1 < 0.1 132.9 
 P1 cre recombinase protein (inner standard 1)2-153 131.5 81.5 106.2 97.4  
 P1 cre recombinase protein (inner standard 2) 109.2 62.4 79.9 56.3  
 Mean of the 6 ADvalues 7759 12 071 16 498 9779  
Accession
number* 
Name of transcript (protein) Eo
(%AD
MCs
(%AD) 
MNC
(%AD) 
Neu
(%AD) 
Fold
change 
X55988 Eosinophil-derived neurotoxin 86.8 0.2 7.3 0.5 32.2 
L01664 Charcot-Leyden crystal protein 78.0 < 0.1 9.2 0.2 24.5 
X81479 Glycoprotein EMR1 hormone receptor 59.1 1.7 5.2 2.8 18.4  
M84526 Complement factor D 45.9 0.9 10.9 4.8 8.3  
J04130 Macrophage inflammatory protein-1 β 36.8 < 0.1 6.0 1.9 14.0 
D90144 Macrophage inflammatory protein-1 α 41.5 0.2 4.7 8.2 9.5  
AJ004832 Neuropathy target esterase 36.0 3.2 8.7 3.0 7.2 
U09937 Urokinase-type plasminogen receptor 43.6 9.3 4.6 8.6 5.8 
X70326 MARCKS-related protein 42.1 2.7 10.0 10.9 5.4  
U81375 Solute carrier family 29 29.4 10.7 0.5 < 0.1 7.9 
AB008535 CRTH2 18.2 0.6 0.4 < 0.1 56.6 
M23892 Arachidonate 15-lipoxygenase 17.9 0.3 0.5 0.3 46.9 
AA131149 S100 calcium-binding protein P 18.5 0.2 0.9 1.5 21.1  
AB018339 Synaptic nuclei expressed gene 1β 24.3 0.8 7.3 0.4 8.5 
AF079167 Lectin-like oxidized LDL receptor 13.5 < 0.1 < 0.1 0.2 128.9 
D13626 G-protein–coupled receptor for UDP-glucose 14.2 1.1 1.0 0.7 15.0 
M34455 Indoleamine 2,3-dioxygenase 13.5 0.8 1.1 0.9 14.1 
X74328 Cannabinoid receptor 2 13.0 0.3 0.9 1.7 13.7  
L77730 A3 adenosine receptor 10.2 0.4 < 0.1 < 0.1 55.5 
S59049 Regulator of G-protein signaling 1 12.0 2.3 1.0 0.1 10.2  
L41816 CAM kinase I 10.2 1.7 1.1 < 0.1 10.7  
U21931 Fructose-1, 6-bisphosphatase 1 15.9 1.0 6.6 1.2 5.4 
U28694 CCR3 16.1 0.3 1.0 8.0 5.2 
M75914 IL-5 receptor α chain 8.8 0.7 0.7 1.1 10.5 
AF010193 SMAD7 10.9 2.2 2.4 0.2 6.7 
AF000545 Purinergic receptor P2Y10 14.1 0.3 3.7 4.4 5.1  
U48250 Protein kinase C binding protein 2 6.2 0.1 0.1 0.4 30.4 
S68134 Cyclic AMP-responsive element modulator, β unit 13.2 5.6 1.6 0.4 5.2  
L19314 Phosphorylase kinase, β unit 7.3 1.5 0.1 < 0.1 12.7 
L08044 Trefoil factor 3 (intestinal) 5.6 < 0.1 < 0.1 < 0.1 132.9 
 P1 cre recombinase protein (inner standard 1)2-153 131.5 81.5 106.2 97.4  
 P1 cre recombinase protein (inner standard 2) 109.2 62.4 79.9 56.3  
 Mean of the 6 ADvalues 7759 12 071 16 498 9779  

MARCKS indicates myristoylated alanine-rich C kinase substrate; LDL, low-density lipoprotein; UDP, uridine 5′-diphosphate; CAM kinase, calmodulin-dependent protein kinase.

*

The GenBank accession number (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) of each transcript is shown.

The level of gene expression was obtained as the AD using Genechip software. The percentages of the specific AD level versus the mean AD level of 6 probe sets for housekeeping genes (β-actin and glyceraldehyde-3-phosphate dehydrogenase) were then calculated. The mean percent AD levels obtained from cultured eosinophils (Eo, n = 2), PB-derived MCs (n = 6), mononuclear cells (MNC, n = 4), and neutrophils (Neu, n = 1) are shown.

The fold change was obtained by dividing the eosinophil value (Eo) by the mean of the 3 other transcript levels (MCs, MNC, and Neu). The transcripts showing at least a 5-fold change are listed in the table.

F2-153

Inner standards such as P1 cre recombinase protein were added to cRNA of the target sample just before the hybridization (see “Materials and methods”) to evaluate the difference between samples. The inner standards were found at similar levels (< 2-fold change) after compensation with the housekeeping genes.

Abundant expression of MBP transcripts in MCs but not in eosinophils

Of particular interest is that MBP ranked as the fourth most-increased transcript in MCs but not in eosinophils. Indeed, it has been reported by others34 and by us28 that the level of MBP transcripts in CB-derived developing eosinophils decreases after complete maturation. We also used Genechip to measure the transcripts of major granule proteins in eosinophils derived from 6 patients with atopic dermatitis. The percentage expression levels (per housekeeping gene) of Charcot-Leyden crystal protein, eosinophil-derived neurotoxin, eosinophil cationic protein, and MBP in these eosinophils were, respectively, 150.8% ± 3.4%, 110.1% ± 11.0%, 51.4% ± 21.2%, and 1.6% ± 0.7%, which are relatively higher than the values shown in Table 2.

CB-derived MCs showed lower levels of α- and β-chains of high-affinity receptors for IgE (FcεRI)20 and chymase26 transcripts than PB-derived MCs. However, they had a similar level of MBP (49.9% ± 2.8%, n = 3) compared with adult-type cultured cells (67.9% ± 31.6%, n = 3). Three other transcripts of eosinophil proteins (ie, Charcot-Leyden crystal protein, eosinophil-derived neurotoxin, and eosinophil cationic protein) were judged to be absent in MCs (data not shown).

Using RT-PCR, we also detected mRNA expression of MBP in PB- and CB-derived MCs and in immature eosinophils (Figure1).

Fig. 1.

Expression of transcripts for MBP in MCs and eosinophils with RT-PCR.

The transcripts for MBP (440 bp) and those for β-actin (661 bp) are shown in lanes 1 to 4 and lanes 5 to 8, respectively. The transcripts were obtained from PB-derived MCs (lanes 1, 5), CB-derived MCs (lanes 2, 6), PB-derived primary eosinophils (lanes 3, 7), and CB-derived immature cultured eosinophils (lanes 4, 8).26,27Similar results were obtained with 2 other experiments.

Fig. 1.

Expression of transcripts for MBP in MCs and eosinophils with RT-PCR.

The transcripts for MBP (440 bp) and those for β-actin (661 bp) are shown in lanes 1 to 4 and lanes 5 to 8, respectively. The transcripts were obtained from PB-derived MCs (lanes 1, 5), CB-derived MCs (lanes 2, 6), PB-derived primary eosinophils (lanes 3, 7), and CB-derived immature cultured eosinophils (lanes 4, 8).26,27Similar results were obtained with 2 other experiments.

The presence of immunoreactive MBP in MC granules

We asked whether MBP is also detected as a protein in MCs. As shown in Figure 2, MBP was detected in saponin-permeabilized MCs by flow cytometry.

Fig. 2.

Flow cytometric analysis of intracytoplasmic MBP in MCs and eosinophils.

Representative results obtained with permeabilized CB-derived MCs (A) and eosinophils (B) are shown. The histograms indicated as shaded areas with bold lines were obtained using the anti-MBP MoAb (BMK-13), whereas those indicated as open areas with thin lines were obtained using control antibody. The experiments using another anti-MBP MoAb (AHE-2) derived from a different hybridoma gave similar histograms. The mean fluorescence intensity ratios of MBP to control in PB- and CB-derived MCs and eosinophils were, respectively, 14.3 ± 2.1 (n = 10) and 2.4 ± 0.5 (n = 3). The levels of coefficient of variation for MCs and eosinophils were, respectively, 86.2% ± 6.4% and 37.4% ± 5.3% (n = 10 and n = 3), indicating that the intensities of MBP fluorescence in MCs varied widely compared with those in eosinophils.

Fig. 2.

Flow cytometric analysis of intracytoplasmic MBP in MCs and eosinophils.

Representative results obtained with permeabilized CB-derived MCs (A) and eosinophils (B) are shown. The histograms indicated as shaded areas with bold lines were obtained using the anti-MBP MoAb (BMK-13), whereas those indicated as open areas with thin lines were obtained using control antibody. The experiments using another anti-MBP MoAb (AHE-2) derived from a different hybridoma gave similar histograms. The mean fluorescence intensity ratios of MBP to control in PB- and CB-derived MCs and eosinophils were, respectively, 14.3 ± 2.1 (n = 10) and 2.4 ± 0.5 (n = 3). The levels of coefficient of variation for MCs and eosinophils were, respectively, 86.2% ± 6.4% and 37.4% ± 5.3% (n = 10 and n = 3), indicating that the intensities of MBP fluorescence in MCs varied widely compared with those in eosinophils.

Using a confocal laser scanning microscope, we found MBP not only in purified eosinophils (Figure 3D), but also in CB-derived MCs (Figure 3A,B) and in lung-derived purified MCs (Figure 3C) under the condition that more than 99% of the cells were tryptase positive.

Fig. 3.

Immunofluorescent staining of MBP in MCs and eosinophils.

Confocal fluorescence image with anti-MBP MoAb (A) and differential interference contrast image (B) of permeabilized CB-derived cultured MCs (1-μm section). Confocal fluorescence images with anti-MBP MoAb of permeabilized lung-derived purified MCs (C) and permeabilized PB-derived purified eosinophils (D). MBP was also detected even in unpermeabilized CB-derived cultured MCs, but only after anti-IgE challenge. (E) Confocal fluorescence and differential interference contrast images were synthesized in the figure. (F) Double staining for MBP (FITC) and tryptase (rhodamine) of a skin biopsy sample obtained from a patient with severe atopic dermatitis. Green images represent MBP-only positive cells, whereas orange-yellow images show MBP and tryptase double-positive cells. Tryptase-only positive cells (red images) were not found in this study, including samples from 2 patients with atopic dermatitis and 2 normal control subjects. Use of a control antibody confirmed all of the positive staining findings to be specific for anti-MBP and antitryptase antibodies.

Fig. 3.

Immunofluorescent staining of MBP in MCs and eosinophils.

Confocal fluorescence image with anti-MBP MoAb (A) and differential interference contrast image (B) of permeabilized CB-derived cultured MCs (1-μm section). Confocal fluorescence images with anti-MBP MoAb of permeabilized lung-derived purified MCs (C) and permeabilized PB-derived purified eosinophils (D). MBP was also detected even in unpermeabilized CB-derived cultured MCs, but only after anti-IgE challenge. (E) Confocal fluorescence and differential interference contrast images were synthesized in the figure. (F) Double staining for MBP (FITC) and tryptase (rhodamine) of a skin biopsy sample obtained from a patient with severe atopic dermatitis. Green images represent MBP-only positive cells, whereas orange-yellow images show MBP and tryptase double-positive cells. Tryptase-only positive cells (red images) were not found in this study, including samples from 2 patients with atopic dermatitis and 2 normal control subjects. Use of a control antibody confirmed all of the positive staining findings to be specific for anti-MBP and antitryptase antibodies.

We also tested whether MBP in granules of IgE-sensitized MCs is released by anti-IgE stimulation. We could not detect, using flow cytometry, a significant decrease of MBP fluorescence in permeabilized PB- and CB-derived cultured MCs after degranulation. Yet, using confocal laser scanning microscopy, we found MBP on the outside of these unpermeabilized MC membranes after anti-IgE stimulation (Figure3E), suggesting that MBP is released from granules to at least the outside of the membrane when the cells are suspended without aggregation, and that this immunoreactive MBP is quite an adhesive protein. Thirty-three percent to 43% of the histamine content was specifically released from these MCs by anti-IgE stimulation. We never detected MBP in unpermeabilized MCs when anti-IgE stimulation was not performed.

Double staining for tryptase and MBP revealed that most of the cells at the site of allergic inflammation in skin were double positive (Figure3F).

Discussion

To evaluate the significance of proteins present in MCs and eosinophils, we examined genes selectively transcribed in these cells by using high-density oligonucleotide probe arrays that allow measurement of approximately 12 000 kinds of transcripts at once. We found 34 eosinophil-specific transcripts, whereas PB-derived MCs expressed 140 cell-type–specific transcripts under the same criteria of selection. This difference may be due to the fact that the MC lineage is somewhat different from the myeloid lineage including eosinophils and basophils.35 Four isotypes of metallothioneins,36 antiapoptotic proteins, were ranked as the 19th, 39th, 42nd, and 46th most-increased transcripts among the MC-specific genes (Table 1). The expression of these proteins has been reported to be IL-6 dependent,37 suggesting that these proteins might have been up-regulated by IL-6 in the MC culture. Thus, one may claim that increased transcripts in MCs simply reflect the effect of the culture condition. Indeed, when eosinophils cultured with IL-5 for 6 hours were substituted for the unprimed eosinophils shown in Tables 1 and 2, eosinophil-specific transcripts increased to 57 and MC-specific transcripts decreased to 132. However, MC-specific transcripts were still more than double the eosinophil-specific transcripts (data not shown).

The transcript for MBP is almost absent in purified PB eosinophils, whereas transcripts for 4 other major eosinophil granular proteins were expressed at higher levels. Because immature CB-derived eosinophils express MBP mRNA but lose it after maturation,28,34 PB eosinophils seem to be mature enough to produce more MBP. A similar dissociation between protein and mRNA after maturation is also found for histidine decarboxylase38 and chymase26in human MCs.

MBP was ranked as the fourth most-increased transcript in human MCs when approximately 12 000 genes were examined. The average expression level (average difference in fluorescence intensities between 20 perfectly matched and 20 mismatched probes) of MBP transcripts was 59% (average of 6 experiments) of that of the housekeeping genes. As reported previously,20 the expression levels of more than 10% housekeeping genes were highly reproducible using this method. In addition, using RT-PCR, we detected mRNA of MBP in cultured MCs.

In an earlier immunohistochemical study using a polyclonal antibody against MBP, it was reported that the protein is detected only in eosinophils and, in a lesser amount, in basophils, but not in MCs.21 Later, the same group found that MBP is deposited in tissues without eosinophil infiltration in the IgE-mediated immediate-type reaction.39 However, they explained the results as occurring because eosinophils became invisible after degranulation.39 In another report, they also indicated that MBP is detected in some MC types, although they speculated that MCs incorporated free MBP.40 We found that both immunoreactive protein and mRNA of MBP are abundant in MC granules. MBP was always detected in all MC types in this study, including CB-derived, PB-derived, and lung and skin MCs using the 2 MoAbs derived from different hybridomas. The MBP levels varied between individual MCs, whereas the protein levels in eosinophils were relatively constant, as shown in Figure 1. Some MCs might be judged to be negative for MBP if tested by the less sensitive methods employed in the earlier studies.

MBP has been thought to play a key role in allergic inflammation because this highly basic protein is deposited on the damaged lung epithelium of patients with asthma and in the skin of patients with atopic dermatitis.41-43 We could show that MBP present in MC granules was released at least to the outside of the membrane along with histamine release. There are 2 types of immunoreactive MBP, 14-kd MBP and 24-kd proform MBP,44 and the antibody that can differentiate these specific domains is not commercially available. Proform MBP is not cytotoxic because it contains a highly acidic domain in addition to a highly basic MBP domain. We did not have a suitable antibody applicable to immunoblotting for differentiating the 2 MBP proteins. Thus, it is uncertain whether the immunoreactive protein found in MC granules is MBP or proform MBP. In any case, because MBP is assumed to conjugate with highly sulfated heparin proteoglycan of MCs in the event of degranulation, the biologic significance of MBP present in MCs remains to be determined. Moreover, the biologic significance of MBP present in eosinophils remains controversial because, in a murine model of asthma, MBP was recently reported not to contribute to allergic pathogenesis.45 Yet, the present finding that MCs can produce abundant immunoreactive MBP is crucial because many reports regarding allergic pathogenesis have been based on earlier findings that MBP is almost unique to eosinophils and not produced by MCs.21,39,40 

In conclusion, we found MBP abundantly expressed in human MCs while we were screening for gene expression among the transcriptomes. This result was unexpected and was somewhat different from our aim. Such unexpected findings may result in changing our understanding of cell biology as well as experimental procedures.

We would like to thank Dr Kiyoshi Kawashima, Dr Shigenobu Shoda, and the staff of the Department of Obstetrics, Gyoda Chuo Hospital, for their continuous support by generously providing umbilical cord blood. We thank Dr Harumi Mukai and Dr Haruhiko Ninomiya at the University of Tsukuba and Dr Kentaro Yoshimatsu at Tsukuba Research Laboratory, Eisai Company Limited, for valuable discussion; and Ms Noriko Hashimoto at National Children's Medical Research Center for her skillful technical assistance.

Supported in part by a grant from the Organization for Pharmaceutical Safety and Research and the Ministry of Health, Labour, and Welfare (the Millennium Genome Project, MPJ-5).

T.N. and K.M. contributed equally to this work.

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 U.S.C. section 1734.

References

References
1
Ishizaka
T
Ishizaka
K
Activation of mast cells for mediator release through IgE receptors.
Progress in Allergy.
Kallos
P
34
1984
188
235
Karger AG
Basel, Switzerland
2
Gleich
GJ
Adolphson
CR
The eosinophilic leukocyte: structure and function.
Adv Immunol.
39
1986
177
253
3
Schwartz
LB
Mast cells: function and contents.
Curr Opin Immunol.
6
1994
91
97
4
Baghestanian
M
Hofbauer
R
Valent
P
et al. 
The c-kit ligand stem cell factor and anti-IgE promote expression of monocyte chemoattractant protein-1 in human lung mast cells.
Blood.
90
1997
4438
4449
5
Jaffe
JS
Glaum
MC
Schulman
ES
et al. 
Human lung mast cell IL-5 gene and protein expression: temporal analysis of upregulation following IgE-mediated activation.
Am J Respir Cell Mol Biol.
13
1995
665
675
6
Toru
H
Pawankar
R
Ra
C
Yata
J
Nakahata
T
Human mast cells produce IL-13 by high-affinity IgE receptor cross-linking: enhanced IL-13 production by IL-4-primed human mast cells.
J Allergy Clin Immunol.
102
1998
491
502
7
Weller
PF
Lee
CW
Foster
DW
Corey
EJ
Austen
KF
Lewis
RA
Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4.
Proc Natl Acad Sci U S A.
80
1983
7626
7630
8
Kita
H
Adolphson
CR
Gleich
GJ
Eosinophils.
Allergy: Principles and Practice.
5th ed.
Middleton
E
Jr
Reed
CE
Ellis
EF
Adkinson
NF
Jr
Yunginger
JW
Busse
WW
1
1998
242
260
Mosby
St Louis, MO
9
Patella
V
de Crescenzo
G
Marone
G
et al. 
Eosinophil granule proteins activate human heart mast cells.
J Immunol.
157
1996
1219
1225
10
Tachimoto
H
Ebisawa
M
Saito
H
et al. 
Activated human mast cells release factors supporting the survival of eosinophils in vitro.
Int Arch Allergy Immunol.
113
1997
293
294
11
Venter
JC
Adams
MD
Myers
EW
et al. 
The sequence of the human genome.
Science.
291
2001
1304
1351
12
International Human Genome Sequencing Consortium
Initial sequencing and analysis of the human genome.
Nature.
409
2001
860
921
13
Velculescu
VE
Madden
SL
Zhang
L
et al. 
Analysis of human transcriptomes.
Nat Genet.
23
1999
387
388
14
Apweiler
R
Biswas
M
Zdobnov
E
et al. 
Proteome analysis database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes.
Nucleic Acids Res.
29
2001
44
48
15
Duggan
DJ
Bittner
M
Chen
Y
Meltzer
P
Trent
JM
Expression profiling using cDNA microarrays.
Nat Genet.
21
1999
10
14
16
Lipshutz
RJ
Fodor
SPA
Gingeras
TR
Lockhart
DJ
High density synthetic oligonucleotide arrays.
Nat Genet.
21
1999
20
24
17
Chen
H
Centola
M
Altschul
SF
Metzger
H
Characterization of gene expression in resting and activated mast cells.
J Exp Med.
188
1998
1657
1668
18
Der
SD
Zhou
A
Williams
BRG
Silverman
RH
Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays.
Proc Natl Acad Sci U S A.
95
1998
15623
15628
19
Alon
U
Barkai
N
Levine
AJ
et al. 
Broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by oligonucleotide arrays.
Proc Natl Acad Sci U S A.
96
1999
6745
6750
20
Iida
M
Matsumoto
K
Saito
H
et al. 
Selective down-regulation of high-affinity IgE receptor (FcεRI) α-chain messenger RNA among transcriptome in cord blood-derived versus adult peripheral blood-derived cultured human mast cells.
Blood.
97
2001
1016
1022
21
Leiferman
KM
Gleich
GJ
Ackerman
SJ
et al. 
Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells.
J Immunol.
136
1986
852
855
22
Moqbel
R
Barkans
J
Bradley
BL
Durham
SR
Kay
AB
Application of monoclonal antibodies against major basic protein (BMK-13) and eosinophil cationic protein (EG1 and EG2) for quantifying eosinophils in bronchial biopsies from atopic asthma.
Clin Exp Allergy.
22
1992
265
273
23
Skubitz
KM
Christiansen
NP
Mendiola
JR
Preparation and characterization of monoclonal antibodies to human neutrophil cathepsin G, lactoferrin, eosinophil peroxidase, and eosinophil major basic protein.
J Leukoc Biol.
46
1989
109
118
24
Tachimoto
H
Ebisawa
M
Saito
H
et al. 
Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-γ.
J Allergy Clin Immunol.
106
2000
141
149
25
Matsumoto
K
Schleimer
RP
Saito
H
Iikura
Y
Bochner
BS
Induction of apoptosis in human eosinophils by anti-Fas antibody treatment in vitro.
Blood.
86
1995
1437
1443
26
Ahn
K
Takai
S
Saito
H
et al. 
Regulation of chymase production in human mast cell progenitors.
J Allergy Clin Immunol.
106
2000
321
328
27
Saito
H
Hatake
K
Ishizaka
T
et al. 
Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins.
Proc Natl Acad Sci U S A.
85
1988
2288
2292
28
Hashida
R
Ogawa
K
Saito
H
et al. 
Gene expression accompanying by differentiation of cord-blood derived CD34+ cells to eosinophils.
Int Arch Allergy Immunol.
125(suppl 1)
2001
1
5
29
Sillaber
C
Baghestanian
M
Valent
P
et al. 
The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis.
J Immunol.
162
1999
1032
1041
30
Zhang
J
Gruber
BL
Marchese
MJ
Zucker
S
Schwartz
LB
Kew
RR
MC tryptase does not alter matrix metalloproteinase expression in human dermal fibroblasts: further evidence that proteolytically-active tryptase is a potent fibrogenic factor.
J Cell Physiol.
181
1999
312
318
31
Calero
M
Rostagno
A
Matsubara
E
Zlokovic
B
Frangione
B
Ghiso
J
Apolipoprotein J (clusterin) and Alzheimer's disease.
Microsc Res Tech.
50
2000
305
315
32
Hasan
Q
Ruger
BM
Tan
ST
Gush
J
Davis
PF
Clusterin/apoJ expression during the development of hemangioma.
Hum Pathol.
31
2000
691
697
33
Nagata
K
Hirai
H
Takano
S
et al. 
CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s).
FEBS Lett.
459
1999
195
199
34
Gruart
V
Truong
MJ
Capron
M
et al. 
Decreased expression of eosinophil peroxidase and major basic protein messenger RNAs during eosinophil maturation.
Blood.
79
1992
2592
2597
35
Kempuraj
D
Saito
H
Nakahata
T
et al. 
Characterization of mast cell-committed progenitors present in human umbilical cord blood.
Blood.
93
1999
3338
3346
36
Vasak
M
Hasler
DW
Metallothioneins: new functional and structural insights.
Curr Opin Chem Biol.
4
2000
177
183
37
Penkowa
M
Giralt
M
Carrasco
J
Hadberg
H
Hidalgo
J
Impaired inflammatory response and increased oxidative stress and neurodegeneration after brain injury in interleukin-6-deficient mice.
Glia.
32
2000
271
285
38
Kuramasu
A
Saito
H
Suzuki
S
Watanabe
T
Ohtsu
H
Mast cell/basophil-specific transcriptional regulation of human L-histidine decarboxylase gene by CpG methylation in the promoter region.
J Biol Chem.
273
1998
31607
31614
39
Zweiman
B
Atkins
PC
von Allmen
C
Gleich
GJ
Release of eosinophil granule proteins during IgE-mediated allergic skin reactions.
J Allergy Clin Immunol.
87
1991
984
992
40
Butterfield
JH
Weiler
D
Peterson
EA
Gleich
GJ
Leiferman
KM
Sequestration of eosinophil major basic protein in human mast cells.
Lab Invest.
62
1990
77
86
41
Filley
WV
Holley
KE
Kephart
GM
Gleich
GJ
Identification by immunofluorescence of eosinophil granule major basic protein in lung tissues of patients with bronchial asthma.
Lancet.
ii
1982
11
16
42
Gleich
GJ
Flavahan
NA
Fujisawa
T
Vanhoutte
PM
The eosinophil as a mediator of damage to respiratory epithelium: a model for bronchial hyperreactivity.
J Allergy Clin Immunol.
81
1988
776
781
43
Leiferman
KM
Ackerman
SJ
Sampson
HA
Haugen
HS
Venencie
PY
Gleich
GJ
Dermal deposition of eosinophil-granule major basic protein in atopic dermatitis: comparison with onchocerciasis.
N Engl J Med.
313
1985
282
285
44
Popken-Harris
P
Checkel
J
Gleich
GJ
et al. 
Regulation and processing of a precursor form of eosinophil granule major basic protein (ProMBP) in differentiating eosinophils.
Blood.
92
1998
623
631
45
Denzler
KL
Farmer
SC
Lee
JJ
et al. 
Eosinophil major basic protein-1 does not contribute to allergen-induced airway pathologies in mouse models of asthma.
J Immunol.
165
2000
5509
5517

Author notes

Hirohisa Saito, Department of Allergy & Immunology, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan; e-mail:hsaito@nch.go.jp.