Expressing Neutrophils: Proliferation in the Presence of Superantigen, But Not Tetanus Toxoid

The primary function of polymorphonuclear neutrophils (PMN) in the immune response appears to be acute phagocytic clearance of foreign pathogens and release of inflammatory mediators. Consistent with their assumed lack of major histocompatibility complex (MHC) class II expression, PMN have not been considered to play a role in antigen presentation and T-cell activation. However, recent reports have shown that human PMN can express MHC class II molecules both in vitro and in vivo after stimulation with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). Thus, under appropriate conditions, PMN could play a significant role in immune regulation, including T-cell activation. In this report, we demonstrate that human class II-expressing PMN can serve as accessory cells in superantigen (SAg)-mediated T-cell activation. This accessory activity for SAg presentation was present only after induction of MHC class II expression, and was especially pronounced following culture of PMN with GM-CSF plus IFN-gamma, which acted synergistically to induce MHC class II molecules on PMN. Moreover, the level of MHC class II expression and the magnitude of SAg-induced T-cell responses were found to be highly correlated and distinctly donor dependent, with PMN from some donors repeatedly showing fivefold higher responses than PMN from other donors. On the other hand, culture of PMN with GM-CSF plus IFN-gamma under conditions that resulted in optimal MHC class II expression did not enable them to function as antigen-presenting cells for either intact tetanus toxoid (TT) or for a TT peptide. These results delineate a new pathway for T-cell activation by SAg that may play an important role in the severity of SAg-induced inflammatory responses. They also identify a donor-specific polymorphism for induction of PMN MHC class II expression which may be of significance for therapies involving GM-CSF and IFN-gamma.

OLYMORPHONUCLEAR neutrophils (PMN) play an important role in the effector arm of host immune derecently shown that human PMN can be induced to express fense through the clearance of immune complexes, the MHC class II molecules both in vitro and in vivo.2][3] PMN can synthesize a number of with either granulocyte-macrophage colony-stimulating faccytokines, such as interferon-a (IFN-a), interleukin-1 (ILtor (GM-CSF), IFN-g, or IL-3 results in low-level expres-1), IL-6, IL-8, and tumor necrosis factor-a, as well as constision of HLA-DR. 11,12In addition, significant HLA-DR extuitively express various surface proteins, including Fc repression has been observed on PMN from patients treated ceptors, CR1, CR3, and major histocompatibility complex with either GM-CSF or IFN-g. 13,14Given that PMN are the (MHC) class I molecules. 2-9However, PMN do not normally predominant cell type in many inflammatory reactions, and express MHC class II and, therefore, are believed not to play that HLA-DR expression, and subsequent Ag presentation, a direct role in CD4 / T-cell activation.
is key in initiating or suppressing most cellular immune Presentation of antigen (Ag) to CD4 / T cells is most responses, we examined the ability of HLA-DR-expressing frequently associated with professional antigen-presenting PMN to present two major types of Ag, each with significells (APC) that constituitively express MHC class II such cantly different requirements for initiation of T-cell activaas B cells, macrophages, and dendritic cells.However, sevtion.Tetanus toxoid (TT), a soluble protein Ag which requires processing before presentation, was not presented by In contrast, T-cell activation was observed in the presence bert C. Englert Cell Analysis Laboratory, which was established of superantigen (SAg) and GM-CSF/IFN-g-treated PMN.
with a grant from the Fannie E. Rippel Foundation, and is supported SAg normally does not require processing, and under some in part by the core grant of the Norris Cotton Cancer Center (CAcircumstances appears not to require second signal molecules 23108), or at Albany Medical Center in the Cellular Immunology be present on the APC. 15boratory.

These studies suggest that PMN may play an active and
Address reprint requests to Edmund J. Gosselin Corp (Seattle, WA).IFN-g was a gift from Genentech, Inc (San from the HISTOPAQUE 1077 layer were then incubated for 30 minutes at 4ЊC with the mouse MoAbs OKT8, AML223, FMC63, Francisco, CA).One unit of GM-CSF is equivalent to 0.025 ng of protein.One unit of IFN-g is equivalent to 0.25 ng of protein.
3G8, IV.3, and 22 (10 mg/mL).The cells were washed three times with PBS supplemented with 2.5% FBS (PBS-FBS) and eluted Antibodies used in these studies included: IVA12, an IgG1 anti-HLA-DR,-DP,-DQ; L243, an IgG2a anti-HLA-DR; OKT3, an through the goat-anti-mouse Ig and goat-anti-human Ig column to obtain a population of CD4 / T lymphocytes that were identified by IgG2a anti-CD3; OKT8, an IgG2a anti-CD8 (all from ATCC, Rockville, MD); FMC63, an IgG2a anti-CD19 (generous gift of Dr Heddy flow cytometric analysis to be greater than 94% CD4 / , with the contaminating cells being mainly CD8 / T lymphocytes.CD4 / T Zola, Child Health Research Institute, Adelaide Medical Centre for Women and Children, North Adelaide, South Australia); P3, an IgG1 cells were either used immediately in assays or cryopreserved for future use.isotype control; AML223, an IgG2b anti-CD14; 22.2, an IgG1 anti-CD64; IV.3, an IgG2b anti-CD32; 3G8, an IgG1 anti-CD16 (all Flow cytometry.For analysis of FcgRI/CD64 and class II upregulation by cytokines, PMN were isolated as described above and provided by Medarex, Inc, Annandale, NJ); anti-B7-1 and anti-B7-2 (Ancell Corp, Bayport, MN), and fluorescein isothiocyanate resuspended in AIM-V containing 2.5% pooled human serum (PHS) and 250 U/mL G-CSF.PMN (5 1 10 5 ) were added to each well of (FITC)-labeled 531, an IgG1 anti-CD66b (AMAC, Inc, Westbrook, ME).FITC-labeled F(ab) 2 goat-anti-mouse Ig and FITC-labeled a 96-well plate (Costar, Cambridge, MA) followed by addition of various combinations of IL-3, GM-CSF, and IFN-g.Concentrations goat-anti-rabbit Ig secondary antibodies used in these studies were obtained from Caltag Laboratories, Inc (San Francisco, CA).Anti-of each cytokine are presented in the figure legends.The final volume for each well was adjusted to 200 mL, and the PMN were incubated human HLA-DR monoclonal antibody (MoAb) used in Western blot analysis was purchased from DAKO Corp (Carpinteria, CA).
for 44 to 45 hours at 37ЊC in 5% CO 2 before staining.The viability of PMN following 2-day incubation in 2.5% PHS and 250 U/mL of Alkaline phosphatase conjugated goat-anti-mouse IgG antibody and fetal bovine serum (FBS) were purchased from HyClone Labora-G-CSF was consistently greater than 95%.Analysis was performed on a standard FACScan flow cytometer (Becton Dickinson, San Jose, tories, Inc (Logan, UT).Rabbit polyclonal anti-SEA IgG and the SAg SEA, SEB, SEC1, SEE, were all purchased from Toxin Technologies CA) with 5,000 cells analyzed per sample unless otherwise indicated.For staining, 20 mL of cells were combined in a 96-well flat bottom (Sarasota, FL), while the TSST-1 was graciously donated by Dr J. Parsonnet (Dartmouth Medical School, Lebanon, NH).The Ag (TT) plate with 20 mL of human IgG (12 mg/mL; Sigma) to block Fc binding of the MoAbs and 20 mL of primary MoAb (60 mg/mL).was purchased from Accurate Chemical Corp (Westbury, NY).The TT peptide TT830-844 (QYIKANSKFIGITEL) was synthesized and Cells were then incubated on a rotator at 4ЊC for 1 hour.After three washes with 200 mL PBS containing 2 mg/mL of bovine serum purified to greater than 95% purity by Peptidogenic Corporation (Livermore, CA), and is a universal T-cell epitope for TT-specific albumin (BSA) (PBS-BSA), 40 mL of FITC-labeled F(ab) 2 goatanti-mouse Ig (20 mg/mL) was added for an additional 1 hour.Sam-T cells. 16ell isolation.Peripheral blood mononuclear cells (PBMC) ples were then washed three times, resuspended in PBS-BSA for immediate analysis, or PBS-BSA supplemented with 1% paraformal-were obtained from healthy donors and were separated into mononuclear cell and granulocyte fractions using HISTOPAQUE 1077 dehyde for analysis 1 to 7 days later, depending on availability of the flow cytometer.Although most samples were analyzed within (Sigma, St Louis, MO) layered over HISTOPAQUE 1119 (Sigma) according to the manufacturer's instructions.To further enrich the 48 hours of fixation, for those samples analyzed at later time points, background fluorescence remained relatively low, and our ability to PMN population used in TT presentation studies, residual monocytes were removed by adherence and subsequent treatment with L-Leu-measure class II expression was unaffected by the added storage time.SEA binding studies were performed by incubating the PMN cine Methyl Ester (LME) (Sigma).Both histochemical and flow cytometric analysis demonstrated that the resulting PMN population with 25 nmol/L SEA at 4ЊC for 1 hour.PMN were washed three times with PBS-BSA, then incubated with human adsorbed rabbit was 95% to 98% pure.In the case of studies involving SAg, the granulocyte fraction was further enriched by immunodepletion.Cells polyclonal anti-SEA (60 mg/mL) for an additional 1 hour.After three washes with PBS-BSA, PMN were stained with 40 mL of FITC-were incubated for 30 minutes at 4ЊC with the mouse MoAbs L243, FMC63, and OKT3 (10 mg/mL) for 30 minutes at 4ЊC, washed three labeled F(ab) 2 goat-anti-rabbit Ig (20 mg/mL) for another 1 hour, washed three times with PBS-BSA, then resuspended in PBS-BSA times with phosphate-buffered saline (PBS) supplemented with 2.5% FBS (PBS-FBS), and eluted through goat-anti-mouse Ig and anti-supplemented with 1% paraformaldehyde until flow cytometric analysis.Relative binding was measured by the mean fluorescence inten-human Ig columns.Resulting PMN purity was monitored by FACScan analysis to confirm that no MHC class II / APC were present in sity (MFI) of positive cells over that of the isotype control for each treatment.the PMN population before incubation with cytokines.The PMN population was greater than 99.9% pure after negative selection, Ag presentation assays.Presentation of TT or TT peptide to Ag-specific T-cell lines was performed in a manner similar to that with the contaminating cells being eosinophils.Monocytes were used as control APC and were isolated via adherence.Specifically, PBMC previously described. 17PMN were incubated with or without MHC class II-inducing cytokines for approximately 24 hours, then cyto-were resuspended in AIM-V to a concentration of 5 1 10 6 cells/ mL, and 100 mL added/well to a 96-well plate for 1 hour at 37ЊC.kines were washed away and cells were resuspended in AIM V containing 2.5% autologous serum and 250 U/mL G-CSF.Concen-Nonadherent cells were then removed by three washes with HEPESbuffered RPMI containing 0.1% human albumin (Sigma).TT-spe-trations of each cytokine are indicated in individual figures.However, the concentrations usually ranged between 50 and 100 U/mL, cific and TT peptide-specific T cells were isolated using a modification of methods previously described. 17Briefly, fresh PBMC (30 1 which results in maximal MHC class II induction after 44 hours of incubation time 11 (and data not shown).In addition, pulsing PMN 10 6 ) were stimulated with 5 mg/mL TT or TT peptide in 50 mL AIM-V medium for 3 days, at which point 10 U/mL recombinant for 24 hours with cytokines is sufficient to induce maximal MHC class II expression by 44 hours (data not shown).PMN were then human (rh) IL-2 and 1.25 mL of autologous serum (2.5%) were added for an additional 7 to 11 days at, which time T cells were added to individual wells at concentrations ranging from 1.25 to 5 1 10 5 /well.To eliminate the possibility that proliferation observed harvested, washed, and stored in liquid nitrogen until needed.Before use, Ag-specific T cells were adhered for 1 hour at 37ЊC and subse-might be due to replication of contaminating lymphocytes within the PMN population, PMN were irradiated with 2,000 rad in all quently treated with LME to remove residual monocytes.Normal T lymphocytes used in SAg studies were enriched using CD4 / T-cell experiments presented in this report.However, similar results were also obtained when using nonirradiated PMN (data not shown).In isolation columns (Pierce, Rockford, IL).Briefly, mononuclear cells some instances irradiated monocytes or PBMC were substituted for mL IFN-g for all donors examined.In addition, as observed PMN as control APC.After addition of Ag and T cells (1 1 10 5 / with GM-CSF treatment alone, 11 donors could still be distinwell), plates were incubated for 72 hours, followed by a 24-hour guished based on the relative amounts of MHC class II they [ 3 H]TdR pulse.Cells were then obtained using a Skatron harvester expressed after GM-CSF/IFN-g treatment (Table 1).To con-(Sterling, VA), and counted on a Beckman scintillation counter firm that the donor-dependent expression of HLA-DR was (Wallac Inc, Gaithersburg, MD). in fact specific for HLA-DR, we compared HLA-DR induc-SAg assays were performed similarly, in AIM-V medium contion on PMN to that of CD64 (FcgRI), which is modulated taining G-CSF supplemented with 2.5% PHS.PMN were isolated on PMN by both GM-CSF and IFN-g.When treated with as described above and cultured in 6-well plates (Costar) at a conceneither GM-CSF, IFN-g, or the combination of these cytotration of 1 1 10 6 /mL for 44 to 45 hours in either the presence or absence of 100 U/mL GM-CSF plus 100 U/mL IFN-g.The PMN kines, there appeared to be no correlation between the exwere harvested, washed once, and then 1 1 10 5 PMN were placed pression of HLA-DR and CD64, in that both HLA-DR low in 96-well plates (Costar) with 5 1 10 4 CD4 / T lymphocytes in and HLA-DR high donors showed similar levels of CD64 ex-  PMN lack the necessary second signals to activate T cells, In contrast, a twofold to threefold increase in 3 H-thymidine GM-CSF/IFN-g-treated and untreated PMN from two doincorporation was observed in response to TT when the same nors whose PMN had previously failed to stimulate T-cell TT-specific T cells used above were combined with TT and proliferation, as well as PMN from two additional donors, professional APC (ie, irradiated PBMC or monocytes).
were analyzed for the presence of B7-1 and B7-2 using flow Therefore, we considered the possibility that HLA-DR excytometry.Little or no expression of either B7-1 or B7-2 pression might still be limiting, and that a further increase was observed on human PMN regardless of whether the in HLA-DR expression might enhance the ability of these above PMN were incubated with GM-CSF and IFN-g (Fig cells to function as APC. 4).Addition of IL-2 to compensate for this apparent lack of Preliminary studies in our laboratory using combinations second signal molecules also failed to facilitate Ag presentaof cytokines had suggested that the level of MHC class II tion by HLA-DR / PMN (data not shown).on human PMN could be further increased by treating PMN SAg-induced T-cell activation by HLA-DR / PMN.As a with a combination of GM-CSF and IFN-g.Therefore, we result of our failure to demonstrate presentation of TT or TT conducted additional studies to confirm this observation.In peptide by HLA-DR / PMN, we examined the ability of fact, GM-CSF and IFN-g were synergistic for upregulation HLA-DR / PMN to stimulate T cells using SAg.Use of SAg of MHC class II expression on human PMN, when compared bypasses the requirement for Ag processing, as well as the with MHC class II induction by either GM-CSF or IFN-g need for second signal molecules in some instances. 15We alone (Fig 1).Analysis using anti-HLA-DR-, DP-, and DQinitially determined, using flow cytometry, whether PMN specific antibodies showed that this synergistic upregulation treated with 100 U/mL GM-CSF/IFN-g could bind the bacteof PMN MHC class II was predominantly of the HLA-DR rial SAg staphylococcal enterotoxin A (SEA).PMN from an isotype (data not shown), as is the case when using GM-HLA-DR low responder (Fig 5A and B) and an HLA-DR high CSF alone. 11Induction of HLA-DR expression by GM-CSF/ responder (Fig 5C and D) were cultured for 44 hours in the IFN-g was dose-dependent, with a maximum induction being observed at 50 U/mL of GM-CSF combined with 50 U/ presence of GM-CSF/IFN-g , and then incubated with SEA.PMN class II induction after 44 hours of culture in the presence of 100 U/mL GM-CSF/IFN-g is presented for three HLA-DR high responders and three HLA-DR low responders.For personal use only.on October 27, 2017.by guest www.bloodjournal.orgFrom of multiple donors, and relatively high levels of HLA-DR expression by PMN, TT peptide-specific T cells also failed to respond, unless monocytes were provided as APC (Fig 3).Therefore, we considered two additional explanations for the lack of TT presentation by MHC class II expressing PMN: (1) that second signal molecules required for T-cell activation are absent on PMN, and (2) that differences might exist between HLA-DR on PMN and that on monocytes.It is now generally recognized that in order for T cells to proliferate in response to Ag they usually require that second signal molecules such as B7-1 and B7-2 be present on APC with which they interact. 19,20In the absence of such accessory molecules, the autocrine growth factor IL-2 is not produced and T cells are thus unable to proliferate.Flow cytometry analysis of PMN confirmed that little or no B7.1 or B7.2 is present on PMN whether or not they are treated with cytokines.However, it appears that the absence of B7-1 and B7-2 by itself is not sufficient to explain the lack of TT presentation, because addition of exogenous IL-2 also did not result in a T-cell proliferative response to TT (data not decreased considerably in the presence of an anti-HLA-DR MoAb (L243), but not control antibodies specific for either CD14 (AML223) or CD64 (22.2) (Fig 7).Thus, despite the relative lack of second signal molecules, and inability of HLA-DR / PMN to present TT or TT peptide, HLA-DR / PMN are capable of initiating HLA-DR-restricted, SAgdependent T-cell proliferation.

DISCUSSION
2][13][14] However, it has not been determined whether PMN can use this HLA-DR to present Ag.Our data indicate that HLA-DR expressing PMN cannot effectively process and present TT.Neither GM-CSF, nor GM-CSF/ IFN-g-treated PMN stimulated TT-specific T-cell lines to proliferate (Fig 2 and data not shown).This result is apparently not due to an overall inability of the PMN to function, because we found that PMN cultured under identical conditions for 44 to 48 hours exhibited normal oxidative burst activity, as well as the ability to phagocytose IgG-coated red blood cells, confirming prior studies in which PMN cultured for up to 48 hours with IFN-g without the other cytokines functioned normally. 18Another potential explanation for the failure of HLA-DR expressing PMN to present TT could be  molecule with the TCR, ultimately leading to T-cell activation. 31,32Given this, and the number of potential clinical 2) and was inhibited in the presence of an anti-HLA-DR MoAb (Fig 7).The ability of PMN to stimulate SAg-induced applications which use GM-CSF and IFN-g, it will be important to better understand the full consequences of T-cell proliferation suggests that required accessory molecules, such as intracellular adhesion molecule-1 and vascular HLA-DR induction on PMN in vivo in response to these cytokines. 13,14SAg such as Staphylococcus aureus enterotoxins, toxic cell adhesion molecule-1, 21,22 are expressed by PMN in sufficient amounts to stimulate T-cell activation in the absence shock syndrome toxin (TSST-1), and exfoliative toxin are a group of well-characterized proteins implicated in the pathogene-of B7-1 and B7-2 .Furthermore, these results are similar to  35 The exact mechanisms through which these SAg initiate such pathophysiological conditions remain incompletely understood.However, it is thought that such symptoms are primarily related to the binding of SAg to MHC class II molecules on monocytes/macrophages, followed by Tcell activation and cytokine release.In addition, due to the fact that there are multiple Vb specificities, a given SAg can stimulate more than 10% of all T cells.This is in contrast to the possible one per thousand to one per million T cells capable of being stimulated by a particular Ag through conventional Ag presentation.Thus, significantly increasing the number of SAg-presenting cells through induction of HLA-DR on PMN could have serious consequences for patient outcome, particularly where there is risk of exposure to SAg-expressing bacteria.Moreover, the threshold for a significant T-cell response to SAg by different individuals may in some instances be related to their capacity to express class II on PMN.Although doses of SAg were purposely chosen to elicit T-cell responses with PMN from both high-and PMN enter inflammatory sites in very large numbers, expression of HLA-DR on PMN and their subsequent interaction with SAg could significantly exacerbate such diseases, dependent on the level of donor expression.Although these studies suggest presentation of soluble protein Ag, such as TT, may not represent a serious concern, further studies using other Ag are needed.This work represents the first delineation of the functional capabilities of HLA-DR / PMN, and represents the first significant step in understanding what impact these cells may have on immune regulation.Our data document a polymorphism among individuals with respect to induction of MHC class II on PMN and consequently on the capacity of an individual's PMN to support SAg-mediated T-cell activation.These observations may have particular relevance for patients treated with IFN-g and/ or GM-CSF, or for patients with SAg-induced inflammatory responses.
either the absence or presence of SAg.The final concentration of pression (Fig 1).This suggests that the difference in HLA-SAg used in each assay was determined by titrating each of the SAg DR expression between donors resides at a stage downstream tested, and selecting the lowest SAg concentration which elicited from cytokine-receptor signaling.Although IL-3 also inthe greatest T-cell proliferative response.In the case of SEA, SEB, duces low levels of MHC class II expression on human SEC1, and SEE, 1 nmol/L SAg was selected.A concentration of 10 PMN, addition of IL-3 to PMN GM-CSF/IFN-g cultures nmol/L was required to induce a maximal T-cell response when resulted in no significant enhancement of PMN HLA-DR using TSST-1.After 66 hours of incubation with SAg, the wells expression (data not shown).
were pulsed for 18 to 20 hours with [ 3 H]TdR to measure T-cell proliferation.For antibody blocking assays, each MoAb was incu-Analysis of TT and TT peptide presentation by GM-CSF/ bated with the PMN and T lymphocytes 30 minutes before SAg IFN-g-treated PMN.Having determined that higher levaddition.els of HLA-DR expression by human PMN could be achieved (Fig 1), three additional experiments monitoring RESULTS T-cell activation in response to TT were conducted using GM-CSF/IFN-g-treated PMN from HLA-DR high responding PMN HLA-DR induction by GM-CSF/IFN-g.Initial atdonors.As shown by data from one of these three experitempts directed at demonstrating TT presentation by HLAments, the HLA-DR / PMN again failed to stimulate TT-DR / PMN focused on the use of human PMN treated with specific T cells while at the same time autologous monocytes GM-CSF, because previous studies showed that GM-CSF supported TT-induced T-cell proliferation (Fig 2).To deterwas the optimal cytokine for inducing HLA-DR expression mine if the problem was at the level of Ag processing, further on cultured human PMN. 11Therefore, we performed several experiments were conducted which examined the ability of experiments with cells from three different donors examining HLA-DR / PMN to present preprocessed TT, in the form of the ability of GM-CSF-treated PMN to stimulate TT-spe-TT peptide, to peptide-specific T cells.Again, no significant cific T-cell proliferation in response to TT.However, in each T-cell response was observed (Fig 3), despite relatively high instance GM-CSF-treated PMN failed to generate a TTlevels of HLA-DR expresssion by PMN (Fig 3, inset), and specific T-cell proliferative response significantly above that the ability of these same T cells to respond to TT peptide of untreated PMN, or GM-CSF-treated PMN incubated with presented by adhered monocytes (Fig 3).To determine if TT-specific T cells in the absence of TT (data not shown).

Fig 1 .
Fig 1. Enhanced induction of PMN class II expression by GM-CSF plus IFN-g.PMN from both an HLA-DR low responder and an HLA-DR high responder were cultured in the presence of various concentrations of GM-CSF (ࡗ), IFN-g (), or GM-CSF/IFN-g (q).After 44 hours of culture, the PMN were stained with either MoAb IVA12 (anti-MHC class II) or MoAb 22 (anti-CD64) before flow cytometric analysis.The level of PMN class II induction differed markedly for HLA-DR low responder (A) and the HLA-DR high responder (B) but was synergistically enhanced for both donors when compared to stimulation with either GM-CSF or IFN-g alone.Regulation of PMN CD64 expression was similar for both donors (C and D).Surface expression levels of both class II and CD64 are presented as mean fluorescence intensity and represent the average of duplicate samples Ô SD.

Fig 2 .
Inability of GM-CSF/IFN-g-treated (HLA-DR " ) PMN to stim-* The sampling period is the number of days between the first and ulate TT-specific T cells.Purified monocytes (Mf; 5 Ì 10 4 /well) were last sample tested for each donor.used as control APC.PMN were first incubated for 24 hours in the † The number of PMN demonstrating class II expression levels presence of GM-CSF/IFN-g (50 U/mL), and then added to wells of a above the isotype control are presented as the % positive cells { 96-well plate at concentrations of 0, 1.25, or 2.5 Ì 10 5 cells/well.SEM.TT-specific T cells were than added, and the Ag presentation assay ‡ The MFI represents the level of PMN class II expression above performed as described in Materials and Methods.[ 3 H]TdR uptake isotype control and is the average for all the samples examined { was measured at 72 to 96 hours.Data are expressed as cpm Ô SD of duplicate samples.SEM.

Table 2 . Correlation Between SAg-Induced T-Cell Activation and PMN Class II Expression
-cell proliferation is presented for four separate experiments, each using one HLA-DR high and HLA-DR low responding pair.As described in Materials and Methods, 1 1 10 5 PMN cultured in the presence or absence of 100 U/mL of GM-CSF/IFN-g for 44 hours were incubated with 5 1 10 4 CD4 / T lymphocytes and 10 nmol/L SEA.Values reflect the amount of [ 3 H]TdR incorporated following an 18 to 20-hour pulse and are the mean of triplicate samples { SD.
* The level of class II induction for both the unstimulated and GM-CSF/IFN-g-stimulated PMN was determined by FACScan analysis and is presented as the MFI.†PMN SAg-induced T-cell proliferation is presented as counts per minute (cpm).