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(Journal of Leukocyte Biology. 2001;70:80-86.)
© 2001 by Society for Leukocyte Biology

Induction of interleukin-8 in human neutrophils after MHC class II cross-linking with superantigens

Li Lei, Julia Altstaedt, Maren von der Ohe, Thomas Proft, Ulrike Gross and Lothar Rink

Institute of Immunology and Transfusion Medicine, University of Lübeck School of Medicine, D-23538 Lübeck, Germany

Correspondence: Dr. Lothar Rink, Institute of Immunology and Transfusion Medicine, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: Rink{at}immu.mu-luebeck.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils have been shown to express major histocompatibility complex class II (MHC II) after stimulation. However, reports concerning the functional effect of MCH II expression are still lacking. In our hands, granulocyte-monocyte colony-stimulating factor (GM-CSF) alone and in combination with interferon (IFN)-{gamma}, but not IFN-{gamma} or interleukin (IL)-3, induced a significant level of expression of human leukocyte antigen DR on neutrophils. The addition of staphylococcal enterotoxin E to neutrophils resulted in a significant increase in IL-8 production only after prestimulation with GM-CSF alone or in combination with IFN-{gamma} but had no effect on neutrophils preincubated with IFN-{gamma} alone or IL-3. Staphylococcal enterotoxin A, another bivalent superantigen, also stimulated production of IL-8 by preincubated polymorphonuclear neutrophils, whereas staphylococcal enterotoxin A mutants that are not able to cross-link MHC II molecules failed to induce IL-8 production. Taken together, our results clearly demonstrate that after induction of MHC II, neutrophils are able to respond to MHC II-specific stimulation. These findings support the ideas that the induced MHC II complex is completely functional and that neutrophils may be able to present antigens.

Key Words: major histocompatibility complex (MHC) • neutrophils • superantigens


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Major histocompatibility complex class (MHC) II plays a central role in the activation of antigen-specific immune responses. In contrast to MHC I, which is expressed by nearly all cell types except erythrocytes, MHC II molecules are detectable only on a limited number of cell populations, including antigen-presenting cells, such as monocytes, but not granulocytes [1 ]. Despite recent evidence of the production and release of cytokines by mature polymorphonuclear neutrophils (PMNs) [1 2 3 ] the role of this cell subpopulation in the immune response still remains elusive [1 , 4 ].

In 1987, Matsumoto et al. reported that interferon (IFN)-{gamma} induces the expression of MHC II on PMNs [5 ], but these data could not be reproduced by Buckle and co-workers [6 ]. However, in 1993, Gosselin et al. demonstrated the induction of MHC II on neutrophils by granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-{gamma}, and interleukin (IL)-3, with GM-CSF being the most effective stimulus [7 ]. The MHC II isotype primarily induced in their experiments was human leukocyte antigen (HLA)-DR [7 ], which is most frequently associated with antigen presentation [8 ]. This observation was recently confirmed by Fanger et al. [9 ], who showed that HLA-DR-expressing PMNs are able to activate T lymphocytes when challenged with superantigens (SAgs) but not tetanus toxoid.

Bacterial SAgs have proven to be excellent tools for investigating specific functions of the immune system. These highly mitogenic soluble proteins simultaneously bind to MHC II and the T-cell receptor (TCR) Vß region, causing profound stimulation of T cells [10 ]. Some SAgs, such as toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin B (SEB), exclusively bind to the MHC II {alpha}-chain [10 ], whereas streptococcal pyrogenic exotoxin C binds only to the MHC II ß-chain [11 ]. Staphylococcal enterotoxins A (SEA) and E (SEE) combine the two binding modes, which enables them to cross-link MHC II molecules on the cell surface [10 , 12 ]. It has recently been shown that this mechanism can induce T-cell-independent cytokine gene expression in murine and human monocytes [13 14 15 ].

We have recently established a protocol for extraction of unstimulated neutrophils with high purity from buffy coats [3 ], making it possible to study this subpopulation under defined conditions. This report describes the use of SEE, SEA, and MHC II-binding mutants of SEA to study the interaction between SAgs and MHC II-positive human neutrophils to elucidate the functionality of the expressed MHC II molecules.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell isolation
Neutrophils were isolated from buffy coats of healthy donors as described previously [3 ]. Briefly, the cells were separated by applying them to two density gradients of Percoll (Pharmacia, Freiburg, Germany) after sedimentation through hydroxyethyl starch (Plasmasteril; Fresenius AG, Bad Homburg, Germany). Remaining erythrocytes were removed by hypotonic lysis. The cell concentration was adjusted to 3 x 106/mL in RPMI 1640 medium (BioWhittaker, Verviers, Belgium) containing 10% low-endotoxin fetal calf serum (PAA Laboratories, Coelbe, Germany) and supplemented with 2 mM L-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (all from Biochrom KG, Berlin, Germany).

Cell preparation purity
The purity of the isolated cell population was determined by May-Gruenwald–Giemsa staining (Merck, Darmstadt, Germany) of cytospin preparations and by flow cytometry analysis (Coulter XL; Coulter Electronics, Krefeld, Germany). The mean purity ± SD achieved by our isolation technique was 97% ± 3.7 neutrophils; the contaminating cells were found to be mainly eosinophils (~1%).

Flow cytometry analysis
Preparations were made directly after isolation and after each stimulation interval to determine the purity and degree of activation of the neutrophils and the level of expression of HLA-DR. The cells were pelleted, resuspended in phosphate-buffered saline containing 1% bovine serum albumin (Fluka Chemie AG, Buchs, Switzerland), and incubated with fluorescein isothiocyanate-coupled CD66b (formerly CD67; Immunotech, Hamburg, Germany) and phycoerythrin-conjugated CD62L (alternative names, LECAM-1 and L-selectin; PharMingen, Hamburg, Germany) or HLA-DR–phycoerythrin (Immunotech) for 15 min. As negative controls, we used fluorescein isothiocyanate-conjugated mouse immunoglobulin G1 and phycoerythrin-conjugated mouse immunoglobulin G1 (Immunotech). The cells were washed again with phosphate-buffered saline containing 1% bovine serum albumin, and fixation was done automatically in a Multi-Q-Prep apparatus (Coulter Electronics).

Generation of SEA and mutants
Wild-type SEA (SEAwt) and SEA mutants SEA-H187A/H225A, SEA-F47S, and SEA-F47S/H225A were produced as previously described [12 , 13 ]. All plasmids were a generous gift from J. D. Fraser, Auckland, New Zealand. The recombinant SAgs were further purified with an endotoxin-removing gel (Pierce, Rockford, IL) to avoid potential contamination with lipopolysaccharide.

Stimulation of the cells
Stimulation was done in 5-mL polypropylene tubes (Greiner GmbH, Frickenhausen, Germany) for 24, 48, and 72 h. Directly after isolation, GM-CSF (10 U/mL), IFN-{gamma} (10 U/mL), or IL-3 (100 U/mL) (all from Pharma Biotechnologie, Hannover, Germany) alone or GM-CSF and IFN-{gamma} (10 U/mL each) were added to the cultures. Controls remained unstimulated. At the same time and after 24 or 48 h of incubation, the neutrophils were additionally stimulated with SEE (250 ng/mL; Toxin Technology Inc., Madison, WI). Controls with the different stimuli but without SEE were incubated under the same conditions. As a positive control, neutrophils were also stimulated with zymosan (0.16 g/mL; Serva).

After 48 h of prestimulation with GM-CSF, neutrophils or PMN preparations with contaminating peripheral blood mononuclear cells (PBMCs) due to a lesser degree of purification (3x106 cells/mL) were stimulated with SEAwt, SEA-H187A/H225A, SEA-F47S, or SEA-F47S/H225A (all at 250 ng/mL). No additional stimulus was used for the negative control, and zymosan was used as a positive control (at 0.16 g/mL after 48 h of GM-CSF prestimulation). Supernatants were harvested after 24, 48, and 72 h.

Interaction between MHC II on PMNs and TCRs via SAgs was demonstrated by using the mouse DOIS 19 T-cell hybridoma, which expresses human Vß 6.5 (kindly provided by B. Fleischer, Hamburg, Germany [18a]) as a T-cell model and as an IL-2-producing responder cell line. PMNs were prestimulated with GM-CSF for 48 h and then incubated for a further 24 h with Vß 6.5 cells (3x106/mL) and the stimuli SEAwt, SEA-F47S, and phytohemagglutinin (50 µL/mL; Wellcome, Darford, UK). Controls were incubated without further stimulation. PMN activation and T-cell activation were determined by measuring human IL-8 and murine IL-2 (mIL-2) production, respectively, by enzyme-linked immunosorbent assay (ELISA).

ELISA
Harvested supernatants were stored at -80°C until use and were thawed only once for cytokine detection. To determine the levels of IL-8 and mIL-2 in the supernatants, we used ELISA kits obtained from Bender MedSystems (Vienna, Austria). All ELISAs were quantified using an Anthos ELISA reader (Labotec, Salzburg, Austria).

Statistical analysis
To exclude prestimulated neutrophils, only preparations with a CD62L expression level of >=97% were included in the study.

The Kolmogorow-Smirnov goodness-of-fit test was used to evaluate the normal distribution of the measured cytokine amounts. In the case of normal distributions of spot checks, the statistical significance of differences was analyzed by Student’s t-test using the program Sigma Plot from Jandel Scientific. Otherwise, the two-tailed Wilcoxon matched-pairs signed-rank test and the Pearson test were carried out using the program SPSS for Windows (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of MHC II on neutrophils
Flow cytometry was used to determine the expression of HLA-DR on purified neutrophils at different time points before and after stimulation with various cytokines. We recently established a protocol for the preparation of PMNs from buffy coats [3 ] that yields cells of very high purity with only minimal contamination by MHC II-positive cells such as monocytes [16 ]. The purity of the isolated neutrophils was >=98.5% as determined by flow cytometry using the pan-granulocyte marker CD66b [17 ] and by May-Gruenwald–Giemsa staining of cytospin preparations.

There was no detectable HLA-DR expression from unstimulated or IFN-{gamma}-stimulated neutrophils, and stimulation with IL-3 resulted in only marginal expression of HLA-DR, even after 72 h. On the other hand, stimulation with GM-CSF induced HLA-DR expression in about 10% of the cells after 72 h. Interestingly, the most efficient HLA-DR expression (~4% and ~17% positive cells after 48 and 72 h, respectively) was observed after induction with a combination of GM-CSF and IFN-{gamma} (Fig. 1 ).



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  		Figure 1. Time kinetics of HLA-DR expression on neutrophils. HLA-DR on neutrophils (106/mL) was determined by flow cytometry directly after drawing of the blood (0 h) and after 24, 48 and 72 h of stimulation with GM-CSF, IFN-{gamma}, GM-CSF plus IFN-{gamma}, or IL-3; controls remained unstimulated. Values are means ± SE (n = 5; *, P < 0.05).

 
In accordance with the findings of other investigators [5 , 7 , 9 ] the expression of HLA-DR in response to GM-CSF alone or GM-CSF plus IFN-{gamma} differed between PMNs obtained from different donors (Table 1 ).


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  		Table 1. Correlation of HLA-DR Expression and IL-8 Production

 
Production of IL-8
We have recently shown that IL-8 is the only proinflammatory cytokine produced by pure preparations of neutrophils [3 ]. Therefore, all subsequent cytokine measurements were restricted to IL-8. After stimulation with GM-CSF or GM-CSF plus IFN-{gamma} for 72 h, the neutrophils released significantly larger amounts of IL-8 (~1.11 ng/mL) than unstimulated controls (which exhibited no detectable IL-8 production), whereas stimulation with IL-3 or IFN-{gamma} alone did not have any significant effects on their IL-8 production (Fig. 2 ). In contrast to HLA-DR expression, no significant difference in IL-8 release was observed between cells stimulated with GM-CSF plus IFN-{gamma} and those stimulated with GM-CSF alone.



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  		Figure 2. IL-8 background production of neutrophils. Neutrophils (3 x 106/mL) were stimulated with GM-CSF, IFN-{gamma}, GM-CSF plus IFN-{gamma}, or IL-3 for 24, 48, or 72 h; controls remained unstimulated. IL-8 production was measured by ELISA. Values are means ± SE (n = 5).

 
Certain SAgs, such as SEA and SEE, can trigger MHC II-expressing monocytes in a T-cell-independent mode by cross-linking MHC II molecules [10 ]. To investigate whether MHC II cross-linking also generates a cell transducing signal in neutrophils, SEE was added to the PMN cultures after various periods of preincubation with GM-CSF, IFN-{gamma}, a combination of GM-CSF and IFN-{gamma}, or IL-3. The neutrophils were exposed to SEE for 24 h. The production of IL-8 after stimulation with the different combinations of stimuli was measured by ELISA. Addition of SEE to neutrophils preincubated with GM-CSF alone for 48 h resulted in a further significant increase in IL-8 production to 2.06 ng/mL, compared with 1.11 ng/mL of IL-8 after the length of incubation without SEE (Fig. 3 ). Preincubation with GM-CSF and IFN-{gamma} also resulted in increased production of IL-8 by PMNs compared with that of controls, but the extent was less and not significant (Fig. 3) , whereas this combination of stimuli induced the highest levels of HLA-DR (Fig. 1) . Addition of SEE to IFN-{gamma}- or IL-3-prestimulated cultures did not result in significantly increased IL-8 release (Fig. 3) .



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  		Figure 3. IL-8 production of neutrophils after stimulation with SEE. Neutrophils (3x106/mL) were stimulated with SEE for 24 h after prestimulation with GM-CSF, IFN-{gamma}, GM-CSF plus IFN-{gamma}, or IL-3 for 48 h; controls remained untreated before addition of the SEE. Secretion of IL-8 was measured by ELISA. Values are means ± SE (n=5). *, P < 0.05.

 
The time kinetics of IL-8 production after exposure of the neutrophils to SEE (Fig. 4 ) was comparable to the kinetics of HLA-DR induction (Fig. 1) , with a slightly higher cytokine release already evident after 48 h of incubation but a significantly higher level of IL-8 production exhibited after 72 h. The release of IL-8 after stimulation with SEE correlated with the HLA-DR expression after incubation with GM-CSF (r =0.8251, P<0.0001) and GM-CSF plus IFN-{gamma} (r=0.6014, P< 0.01), whereas controls and neutrophils preincubated with IFN-{gamma} alone or IL-3 did not show any correlation. Comparison of levels of HLA-DR expression and IL-8 release after stimulation of the individual donors for 72 h also correlated significantly (Table 1 ; r=0.9, P<0.022), suggesting that SAg stimulation strongly depends on the expression of HLA-DR on PMNs.



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  		Figure 4. Time kinetics of IL-8 production by neutrophils after stimulation with SEE. Neutrophils (3x106/mL) were stimulated with GM-CSF, IFN-{gamma}, GM-CSF plus IFN-{gamma}, or IL-3 for 24, 48, or 72 h, and SEE was added immediately or after prestimulation of the neutrophils for 24 or 48 h. Incubation time with SEE was 24 h; controls received only SEE. IL-8 release was determined by ELISA. Values are means ± SE (n=5). *, P < 0.05.

 
Zymosan, a strong stimulator of neutrophils [3 ], was used as a positive control. In contrast to stimulation with SEE, IL-8 release after addition of zymosan was significantly increased in all experimental subsets. Table 1 shows IL-8 levels after 24 h of stimulation with zymosan, when IL-8 release reached its maximum.

There was no correlation between IL-8 production after zymosan stimulation and HLA-DR expression (Table 1) , indicating that differences in HLA-DR expression and reaction to SEE stimulation are not due to a decreased vitality of neutrophils in the individual samples.

Stimulation of GM-CSF-pretreated neutrophils by SEA mutants
To further elucidate whether MHC II cross-linking is the cause of increased IL-8 release after stimulation with a bivalent SAg, MHC II-binding mutants of SEA were tested on purified prestimulated PMNs. GM-CSF was used for prestimulation in all further experiments because it proved to be the best inducer of HLA-DR expression on neutrophils.

The double mutant SEA-H187A/H225A lacks the zinc-binding motif, which is required for binding to the MHC II ß-chain via a coordinated zinc. SEA-F47S is defective for binding to the MHC II {alpha}-chain, and in the double mutant SEA-F47S/H225A, both binding sites are knocked out [12 ]. Thus, only SEAwt binds to both the {alpha}- and ß-chains, thereby making the cross-linking of MHC II molecules on the cell surface possible.

Preincubated neutrophils were stimulated with SEAwt and the three SEA mutants for 72 h (Fig. 5 ). Like SEE, SEAwt stimulated neutrophils to produce significantly increased levels of IL-8 compared with those of the GM-CSF-preincubated control. Similar amounts of IL-8 were observed after incubation with zymosan. On the other hand, the three SEA mutants all failed to induce IL-8 production above background levels of GM-CSF-prestimulated cells. This result strongly suggests that cross-linking of MHC II is an important step in the activation of neutrophils because all SEA mutants lack this binding mode. The time kinetics studies (Fig. 6 ) had similar results. Zymosan stimulation resulted in IL-8 production already after 24 h. After 48 h, SEAwt-stimulated samples also showed increased IL-8 levels. The highest level of IL-8 production was found after 72 h of incubation with both SEAwt and zymosan. The SEA mutants did not increase the production of IL-8 above the background.



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  		Figure 5. IL-8 production of neutrophils after stimulation with SEA and SEA mutants. After 48 h of prestimulation with GM-CSF, neutrophils (3x 106/mL) were stimulated with SEA-F47S/H225A, SEA-H187A/H225A, SEA-F47S, SEAwt, or zymosan (Zy) for 24 h. Controls remained unstimulated after preincubation. IL-8 production was measured by ELISA. A significant level of IL-8 was detected after stimulation with zymosan or SEAwt in comparison with the control (*, P<0.05), with SEAwt-stimulated samples showing a significant effect compared to SEA mutants (#, P<0.05). Values are means ± SE (n=5).

 


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  		Figure 6. Time kinetics of IL-8 production after stimulation with SEA and SEA mutants. GM-CSF-prestimulated neutrophils (3x106/mL) were stimulated with zymosan (Zy), SEAwt, SEA-H187A/H225A, SEA-F47S, or SEA-F47S/H225A for 24, 48, and 72 h. Controls remained unstimulated after preincubation. IL-8 production was measured by ELISA. Results from one representative experiment out of five are shown.

 
Although the SEA-H187A/H22A and SEA-F47S mutants are defective in MHC II cross-linking, they are still functional stimulators of T cells when presented by MHC II [12 , 13 , 18 ]. Figure 7 shows the influence of these mutants on a GM-CSF-prestimulated culture of PMNs with a lower degree of purity. This culture contained about 24% PBMCs, including T cells. Incubation with zymosan and SEAwt increased IL-8 levels about threefold above those of the controls. The two mutants, SEA-H187A/H225A and SEA-F47S, still exhibited a twofold increase in IL-8 production compared with the levels of the control and the SEA-F47S/H225A mutant.



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  		Figure 7. IL-8 production of T-cell-contaminated neutrophils after stimulation with SEA and SEA mutants. After preincubation with GM-CSF, neutrophil preparation with 24% contaminating PBMCs were stimulated with SEA-F47S/H225A, SEA-H187A/H225A, SEA-F47S, SEAwt, or zymosan (Zy) for 72 h. Controls remained unstimulated after preincubation. IL-8 production was measured by ELISA. Data from one representative experiment are shown.

 
This demonstrates that contamination of PMN preparations leads to false results. In a T-cell-containing environment, PMNs might be activated by a presentation of SEAwt, via MHC class II {alpha}- or ß-chain to the TCR, the presentation of SEA-H187A/H225A, by the MHC class II {alpha}-chain to the TCR, or the presentation of SEA-F47S by the MHC class II ß-chain to the TCR. This means, that the PMNs are activated by the cross-link of MHC class II and the TCR; the mutants cannot cross-link MHC molecules, because one MHC-binding site is missing in each mutant. Because contaminating cells could as well be IL-8 producing, we further incubated pure PMNs with Vß 6.5-bearing cells that had been obtained from a TCR-negative mouse T-cell line transfected with a mouse TCR containing the human Vß 6.5 element [18a ]. These cells produce mIL-2 on activation via the TCR and serve as a T-cell model with a cell purity of 100%. Vß 6.5 is the appropriate chain of the TCR for the binding of SEA. GM-CSF-preincubated neutrophils and Vß 6.5-bearing cells were incubated with SEAwt or SEA-F47S; controls remained unstimulated (Fig. 8 ). Again, SEAwt stimulated PMNs to produce IL-8. Furthermore, PMNs were stimulated by SEA-F47S to produce IL-8 in this T-cell-containing environment. This demonstrated that if no cross-link is possible, PMNs are still able to be stimulated by interaction of MHC II and TCR via SAgs. On the other hand, the increased amounts of mIL-2 in the supernatants showed that Vß 6.5-bearing cells are activated by SEAwt and SEA-F47S in the presence of PMNs, representing T-cell activation by SAgs via MHC II-bearing neutrophils (Table 2 ).



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  		Figure 8. IL-8 production by PMNs in the presence of Vß 6.5-bearing cells after stimulation with SEA or an SEA mutant. PMNs (3x106/mL) were preincubated for 48 h with GM-CSF. Afterward, human Vß 6.5-bearing mouse T cells (3x106/mL) and either SEAwt or SEA-F47S were added for a further 48 h. IL-8 in the supernatants was measured by ELISA. Values are means ± SE (n=3). *, P < 0.01; #, P < 0.03.

 

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  		Table 2. Stimulation of Murine T-Cells by PMN-Presented SEA

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented in this paper show that highly purified neutrophils can be induced to express HLA-DR by stimulation with GM-CSF alone or in combination with IFN-{gamma} but not with IFN-{gamma} alone or IL-3. These findings are in contrast to the results of Matsumoto et al. [5 ] and Gosselin et al. [7 ], who reported HLA-DR induction in neutrophils after stimulation with IL-3 or IFN-{gamma}. This discrepancy might be explained by the use of different protocols for neutrophil purification. The neutrophil preparations used in this study had purities of >98%, ensuring only minimal contamination with MHC II-bearing cells.

It is interesting that IFN-{gamma} alone did not induce HLA-DR expression, but it obviously enhanced the stimulating effect of GM-CSF on the expression of HLA-DR on neutrophils, confirming the results of Fanger et al. [9 ]. A possible explanation is that, in contrast to GM-CSF, IFN-{gamma} is not able to switch on genes for MHC II expression in neutrophils but has an enhancing influence once the genes are activated.

IL-8 production correlated well with HLA-DR expression, with GM-CSF being the most effective stimulator. IL-3 and IFN-{gamma} did not induce IL-8 expression. The addition of SEE or SEA to GM-CSF-prestimulated, but not to unstimulated, neutrophils significantly increased IL-8 release, suggesting that interaction of SAg and MHC II on neutrophils triggered a signal into the cell. For many years, SAgs have been characterized as potent T-cell stimulators that cross-link MHC II molecules on antigen-presenting cells with TCRs on T cells. However, recently it has been shown that bivalent SAgs, such as SEA and SEE, can stimulate antigen-presenting cells in a T-cell-independent mechanism by binding to both the {alpha}-chain and the ß-chain of MHC II, subsequently cross-linking MHC II molecules on the cell surface [14 ]. In contrast, no further increase in IL-8 production was seen after stimulation with the {alpha}-chain-defective SEA-F47S mutant or the ß-chain-defective SEA-H187A/H225A mutant. Both mutants have been shown to be functional proteins that can cross-link MHC II and TCR, resulting in significant T-cell proliferation [12 , 13 ]. The results therefore suggest that MHC II cross-linking is a prerequisite for neutrophil stimulation by SAgs in a T-cell-depleted environment. Furthermore, it is likely that SAgs bound to MHC II on neutrophils also bind to TCRs when T cells are present.

Fanger et al. [9 ] demonstrated that MHC II molecules on neutrophils are functional because T cells can be activated in the presence of SAgs. This is evidence of an interaction of MHC II and TCR via SAgs. Indeed, we found that SEAwt and our SEA mutants were able to activate MHC II-bearing neutrophils if a TCR was present (Figs. 7 and 8) , indicative of an interaction between MHC II and TCR via SAgs.

Moulding et al. [19 ] demonstrated that neutrophil preparations containing low levels of PBMCs reacted upon stimulation with SEA, SEB, and TSST-1. In T-cell-depleted neutrophil preparations, there were still low-level effects detectable, but after MHC II-expressing cells were depleted, SAgs remained without effect. Moulding et al. concluded that neutrophils depend on the presence of a small number of contaminating PBMCs. However, SEB and TSST-1 bind only to the {alpha}-chain of MHC II and should react only in the presence of T cells. Kum et al. [20 ] reported that differing results for TSST-1 are explained by contamination of the SAg preparation with, for instance, staphylococcal lipase. To show that our SEA and SEA mutant preparations were free from any contamination, we generated a double mutant which does not bind to MHC II molecules at all. Any effect seen would indicate that a contaminating stimulant was present within this SAg preparation. SEA F47S/H225A did not increase IL-8 production in neutrophils; thus, we concluded that our preparations of SEA and SEA mutants were free of contamination. In agreement with our data, Moulding et al. [19 ] found that after MHC II-expressing cells were depleted, no activation followed SAg stimulation. This was evidenced by our results with the SEA mutants showing that cross-linking of MHC II molecules is the mechanism by which SAgs stimulate neutrophils.

The primary role of neutrophils is in the immediate, innate host response at the site of infection, phagocytosis of microorganisms, after migration from the bloodstream. However, the results of this study suggest that the function of this cell subpopulation might be more complex. After stimulation, neutrophils might also play an important role in the presentation of antigens (and SAgs) to T cells. These findings might also lead to further insight into the role of SAgs in disease. Sriskandan et al. [21 ] recently showed that intraperitoneal administration of SPE-A in mice resulted in a dose-dependent increase in neutrophils at the site of inoculation within 6 h.

SAgs are thought to be at least partly responsible for the development of autoimmune diseases, especially rheumatoid arthritis [22 , 23 ], but the pathological mechanism responsible is still unclear [22 ]. T cells, which have thus far been the focus of research on rheumatoid arthritis [24 ], seem to play a major role in the perpetuation of the process rather than in its induction [22 ]. We have shown here that neutrophils can be stimulated by SAgs once MHC II expression on their surface is induced. In the early stage of acute joint inflammation, neutrophils were found to be the predominant cell type in the synovial fluid [24 , 25 ]. Furthermore, GM-CSF, which is a potent stimulus of MHC II expression on neutrophils [4 , 5 , 7 , 9 ], has also been found in the synovial fluid of rheumatoid arthritis patients [26 ], providing the main condition for activation of neutrophils by SAgs.

Taken together with the data of Fanger et al. [9 ], who recently stated that neutrophils seem to be able to present SAgs to T cells and thus stimulate them, our results clearly showing that neutrophils are activated by bivalent SAgs present an interesting new point in the discussion about the pathogenesis of SAg-induced disease. Interesting and challenging experiments have to follow to verify this completely new idea of the functional possibilities of neutrophils in response to an infection.


   ACKNOWLEDGEMENTS
 
We thank Kerstin Borchert for her expertise in flow-cytometric analysis and Una Doherty for critically reading the manuscript.

Received February 24, 1998; revised February 21, 2001; accepted February 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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