INFECrION AND IMMUNITY, JUlY 1992, p. 2969-2975 0019-9567/92/072969-07$02.00/0

Vol. 60, No. 7

Effects of Staphylococcal Enterotoxin B Rodent Mast Cells

on

JACK KOMISAR,* JACINTO RIVERA, ADAYMEE VEGA, AND JEENAN TSENG Department of Experimental Pathology, Division of Pathology, Walter Reed Army Institute of Research,

14th and Dahlia Streets N. W., Washington, D. C. 20307-5100 Received 21 February 1992/Accepted 2 May 1992

Staphylococcal enterotoxin B (SEB) was tested in rodent mast cell cultures for the release of serotonin. Both rat RBL-2H3 mast cells and murine peritoneal cells released serotonin after SEB stimulation in culture. Release of serotonin in RBL-2H3 cells depended on the concentration of SEB; an appreciable release was seen at 50 ,ug/ml. The release of serotonin was not due to cell death. Serotonin release could be enhanced by bradykinin but not by vasoactive intestinal peptide, substance P, lipopolysaccharide from Salmonella typhimurium, the calcium ionophore A23187, acetylcholine, adenosine, 5-hydroxyeicosatetraenoic acid, indomethacin, or phorbol myristate acetate. SEB bound directly to the membrane of RBL-2H3 mast cells, and the SEB-binding site, the presumptive receptor, appeared to be a protein. The SEB receptor could not be capped under membrane-capping conditions, and serotonin release could not be enhanced by attempts to cross-link the receptor. These results suggest that mast cells may be an important cell type involved in SEB toxicosis and that release of serotonin may be enhanced by activation of the kinin-kallikrein system. The mechanism whereby staphylococcal enterotoxins toxic shock and other syndromes is unknown. Marrack and coworkers (32) suggested that the pathological reaction (weight loss and immunosuppression) in mice to staphylococcal enterotoxin B (SEB) is caused by T cells that are able to respond to SEB by mitosis. However, th6 toxicity of SEB and the related toxin staphylococcal enterotoxin C1 in monkeys is partially dissociated from the mitogellic effects of these molecules (45, 46). Thus, cells other t1ii T cells may also be involved in SEB toxicosis. For examplde there is some indirect evidence that mast cells may also contribute to some of the toxic reactions to SEB. Scheuber and coworkers (41) intradermally injected SEB into monkeys and found an immediate-type skin reaction that was presumably caused by mast cells because it was inhibitable by histamine inhibitors. We sought to study the interaction of mast cells with SEB. The RBL-2H3 rat leukemic cell line (3) and peritoneal mast cells from mice were used to determine whether SEB has a direct effect on mast cells. Most of the work was done with RBL-2H3 cells because of their homogeneity and the ease of obtaining large numbers of cells. In addition, there was no necessity to rule out immunoglobulin E-mediated phenomena (as there would be if fresh mast cells were used exclusively). We have found that rodent mast cells do in fact respond to SEB by releasing serotonin and that this response is enhanced by bradykinin. The SEB-binding site or receptor on mast cells appears to be a protein. cause

MATERIALS AND METHODS RBL-2H3 cells (3) were the kind gift of Reuben Materials. Siraganian, National Cancer Institute, Bethesda, Md. SEB was produced by the method of Schantz and coworkers (40) and was obtained in lyophilized form from the U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Md. Acetylcholine chloride, adenosine, biotin-N-hydroxysuccinimide, calcium ionophore A23187, concanavalin A, 5-hydroxyeicosatetraenoic acid (HETE), indomethacin, *

Corresponding author.

phorbol myristate acetate, Salmonella typhimurium phenolextracted lipopolysaccharide, substance P, vasoactive intestinal peptide (VIP), Tyrode's solution, silver stain for electrophoresis, swine skin gelatin, and tunicamycin were purchased from Sigma Chemical Company, St. Louis, Mo. Bradykinin was purchased from Peninsula Laboratories, Belmont, Calif. An acrylamide-bisacrylamide (37.5:1 ratio) mixture, Coomassie blue, and sodium dodecyl sulfate (SDS) were purchased from Bio-Rad, Richmond, Calif. RPMI 1640 medium was purchased from GIBCO, Life Technologies, Grand Island, N.Y. Fetal bovine serum, averaging 0.7 endotoxin units per ml of endotoxin according to the manufacturer, was purchased from Hyclone Laboratories, Logan, Utah. BALB/c mice were purchased from Charles River Laboratories, Wilmington, Mass. Recombinant N-glycanase was purchased from Genzyme, Boston, Mass. Twenty-fourwell culture plates, no. 3424, were purchased from Costar, Cambridge, Mass. Tritiated serotonin {5-[1,2-3H(N)]hydroxytryptamine binoxalate} was purchased from NEN Research Products, Boston, Mass. Scinti-Verse, the scintillation fluid, was purchased from Fisher Scientific, Fair Lawn, N.J. Fluorescein-avidin was from Vector Laboratories, Burlingame, Calif., and R-phycoerythrin-avidin was from Molecular Probes, Eugene, Oreg. Capillary microslides were purchased from Vitro Dynamics, Rockaway, N.J. MRC OX-6 (33) ascites fluid was purchased from Sera-Lab, Crawley Down, Sussex, United Kingdom. This antibody reacts with major histocompatibility complex (MHC) class II antigens of several rat strains, including the Wistar rat (34), from which the RBL parent cell line was derived (25). Serotonin release assay. The serotonin release assay was conducted according to the method of Liu and coworkers (28), with slight modifications. Briefly, RBL-2H3 cells were cultured in RPMI 1640 medium (10% fetal bovine serum). Two days before the assay, the cells were seeded at 105 cells per well in a 24-well plate. At 16 to 18 h before the assay, 2 ,uCi of tritiated serotonin was added to each well. On the day of the assay, the cells were washed two times with Tyrode's solution containing 0.5% gelatin from swine skin. Then, 1 ml of the same solution was added to each well, and SEB was 2969

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KOMISAR ET AL.

added to make appropriate concentrations. The plates were incubated for 1 h at 37°C in an atmosphere of 7% CO2. Then, the supernatants were removed and centrifuged at 400 x g. A 6/10 volume of 1 ml of each supernatant was added to 7 ml of scintillation fluid and counted in a scintillation counter (Rackbeta; LKB, Bromma, Sweden). Except when noted for some experiments, six wells of cells were employed for each datum in this study. Total release was determined by lysing the cells with 3% Triton X-100 in distilled water. For assays of serotonin release by murine mast cells, BALB/c mice were sacrificed by cervical dislocation and then injected intraperitoneally with 10 ml of Tyrode's solution containing 0.5% gelatin and 50 U of heparin per ml. The peritoneal cells were then drained with an 18-gauge needle. Between 10 and 15% of these cells are mast cells, i.e., they stained metachromatically with toluidine blue by the procedure of Humason (22). The cells were washed twice with Tyrode's solution (0.5% gelatin), and then 2 x 106 cells per tube (12 by 75 mm) in a volume of 1 ml of Tyrode's solution were incubated for 1 h at 37°C with [3H]serotonin. The cells were then washed with the Tyrode's solution and incubated with SEB in the Tyrode's solution for 1 h in a 37°C water bath. Then, the cells were centrifuged at 400 x g for 7 min, and the supernatants were counted in a scintillation counter as described above. Substances reported to enhance serotonin or histamine release in vitro were used at the following concentrations: A23187, 5 ,ug/ml; acetylcholine, 10' M; adenosine, 10-4 M; bradykinin, 10-4 M; concanavalin A, 10 ,ug/ml; HETE, 1.1 ,uM; indomethacin, 10-6 M; lipopolysaccharide, 10 to 100 ,ug/ml; phorbol myristate acetate, 10 ng/ml; substance P, 10-4 M; and VIP, 10 ,uM. Percent release was calculated by the following formula: Percent release = [(stimulated release - unstimulated release)/(total release - unstimulated release)] x 100, in which total release is the radioactivity released by lysis of the cells with 3% Triton X-100. Biotinylation of SEB. Biotinylation was performed by a modification of the procedure of Hsu and coworkers (20). Fifty microliters of 0.1 M biotin N-hydroxysuccinimide in N,N-dimethylformamide was added to 2 mg of SEB dissolved in 0.2 ml of 0.1 M NaHCO3 (pH 8.3). The mixture was allowed to react for 3 h at room temperature and then dialyzed against four changes of phosphate-buffered saline over the course of 72 h. Biotinylated SEB was passed through a 0.22-,m-pore-size filter before use. Immunofluorescence. Cells were washed in Hanks' balanced salt solution (HBSS) containing 0.1% bovine serum albumin (BSA) and 0.05% sodium azide and incubated on ice for 30 min with various dilutions of biotinylated SEB. The cells were then washed two times in the HBSS solution and then incubated in an ice bath with 50 ,ul of 30 ,ug of fluorescein-avidin per ml. The cells were placed in a capillary microslide and observed under a Zeiss ACM (Carl Zeiss, Thornwood, N.Y.) or Leitz Orthoplan (Ernst Leitz, Wetzlar, Germany) fluorescence microscope equipped with the necessary optics or analyzed in a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San

Jose, Calif.). For studying capping and patching of SEB receptors,

RBL-2H3 cells were incubated with biotinylated SEB in an ice bath. Fluorescein-conjugated rabbit anti-SEB was added, and the cells were incubated in an incubator at 37°C for 5 min. The cells were then washed two times at 4°C with HBSS containing 0.3% sodium azide and 2% fetal bovine serum. Subsequently, the cells were observed by the mi-

INFECT. IMMUN.

croslide technique under a Zeiss ACM or Leitz Orthoplan fluorescence microscope. RBL-2H3 cells cultured in RPMI 1640 medium (10% fetal bovine serum) were removed from flasks by using a cell scraper and divided into two aliquots. Those to be used for trypsin treatment were washed two times in HBSS with 0.1% sodium azide, while cells to be used for N-glycanase treatment were washed two times with HBSS containing azide and 0.1% BSA. Each aliquot was pelleted in a 15-ml conical centrifuge tube, and the medium was aspirated off. A total of 200,000 cells per tube in a volume of about 100 Rl was treated for 1 h at 37°C with 1 U of recombinant N-glycanase in HBSS containing Ca2+ and Mg2+ or for 30 min or 1 h with 0.1% trypsin in the HBSS solution. The cells were then washed and incubated in an ice bath with 50 ,ul of 100 ,ug of biotinylated SEB per ml in HBSS containing azide for 0.5 h. The cells were then stained for 0.5 h in an ice bath with either 50 pul of 20 jig of R-phycoerythrin-avidin or 50 ,ul of 30 ,ug of fluorescein-avidin per ml, washed two times, and analyzed by flow cytometry. Inhibition of N-glycosylation. To inhibit N-linked glycosylation, RBL-2H3 cells were grown overnight in RPMI 1640 medium containing tunicamycin. Tunicamycin was dissolved in 50% ethanol, referred to as the vehicle, at a concentration of 2 mg/ml and then further diluted for use at 5.0 ug/ml in culture medium. An equivalent concentration of ethanol in medium was used for the vehicle control. One million cells in 5 ml of culture medium were seeded to each of three flasks. One flask of cells was untreated, one received tunicamycin in 50% ethanol in a volume of 12.5 p.l, for a final concentration of S ,ug/ml in the flask, and the third, the vehicle control, received 7 ,ul of 95% ethanol. The flasks were incubated overnight at 37°C in a humidified incubator under 7% CO2. The cells were then scraped off the flasks, and 2 x 105 cells were placed in a tube. The cells were stained with 50 ,l (per tube) of 1 mg of biotinylated SEB per ml for 30 min in an ice bath, washed with HBSS containing 0.1% BSA, and then 1 ,ug of R-phycoerythrin-avidin in a volume of 50 pul was added to each tube. The cells were incubated with the phycoerythrin-avidin for 30 min in an ice bath, washed with HBSS, and analyzed in a flow cytometer.

Electrophoresis. SDS-polyacrylamide gel electrophoresis (PAGE) was performed with 15% acrylamide made from a 37.5:1 mixture of acrylamide-bisacrylamide in 0.375 M Tris, pH 8.8, with 1% SDS. The electrode buffer contained 3 g of Tris base per liter, 14.4 g of glycine per liter, and 1.66 g of SDS per liter. Two 11.5-cm gels were electrophoresed simultaneously for 18 h at a 10-mA constant current. One was stained with Coomassie blue, and the other was stained with silver stain. Statistical analysis. Mast cells treated with SEB were compared with those not treated with SEB, and mast cells treated with SEB plus an enhancer of serotonin release were compared with cells treated with SEB alone, using Student's t test for groups with separate variances. P values obtained from the experiments were calculated for a two-tailed test. RESULTS Purity of staphylococcal enterotoxin preparation. To verify that the SEB used in this study was pure and undegraded, we examined it on two SDS-PAGE gels. One was stained with Coomassie blue, while the other was stained with silver stain. The results were the same. The gel stained with Coomassie blue is shown in Fig. 1. There was one major band of about 30 kDa, with a barely visible band of 17 kDa.

EFFECTS OF SEB ON RODENT MAST CELLS

VOL. 60, 1992

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Stimulus FIG. 2. Dose response of serotonin release by RBL-2H3 cells. Each bar represents the means and standard errors of the means of six wells. FIG. 1. SDS-PAGE gel of SEB used in this study. SEB was prepared according to the method of Schantz and coworkers (40). The gel was 15% acrylamide and was run as described in Materials and Methods and stained with Coomassie blue. SEB is in the right lane, and shows up as a single band at about 28 kDa, with a faint smaller band probably representing proteolytic degradation.

The 30-kDa band is intact SEB, while the smaller, 17-kDa band is presumably a proteolytic fragment. Most SEB preparations show some nicking, presumably as a result of enzymatic action or chemical hydrolysis either during fermentation or subsequent purification (47). The pattern shown in Fig. 1 suggests that our SEB preparation was relatively pure. Release of serotonin by RBL-2H3 mast cells. The RBL-2H3 mast cell line releases histamine and serotonin in response to antigen if the cells are coated with immunoglobulin E antibody to the antigen (3). The cells respond to several chemicals in the absence of immunoglobulin E, including A23187 and ionomycin (9), thapsigargin (a tumor promoter that releases cytoplasmic Ca + stores) (56), human neutrophilderived histamine-releasing activity (55), and cobra venom cardiotoxin (29). To test whether SEB could cause the release of serotonin, [3H]serotonin-labeled RBL-2H3 cells were incubated with various concentrations of SEB, and release of serotonin was measured in a beta counter. Several experiments were conducted. A representative result is shown in Fig. 2. SEB consistently caused RBL-2H3 cells to release serotonin. The release of serotonin increased with increasing SEB concentration. This release of serotonin was not due to cell death, since there was no significant difference in cell viability in cultures with and without SEB and viability was always greater than 96% by trypan blue exclusion (data not shown). Serotonin release is not enhanced by SEB cross-linking. Many responses of cells depend on cross-linking of surface receptors. We considered the possibility that the response of RBL-2H3 cells was dependent on aggregated SEB that would cross-link the receptors or binding sites. If so, this phenomenon might be mimicked and the sensitivity of the assay might consequently be enhanced by using a small concentration of biotinylated SEB and cross-linking it in the assay system with avidin. Figure 3 shows another serotonin release assay with RBL-2H3 cells. A total of 10 ,ug of SEB per ml was below the threshold of the assay without crosslinking, and there was no enhancement of serotonin release

when avidin was used to cross-link the biotinylated SEB on the cell membrane. In addition, in a separate experiment, the reaction of RBL-2H3 cells to SEB was not enhanced by cross-linking SEB with anti-SEB serum (data not shown). Serotonin release by murine peritoneal cells. Since RBL2H3 cells are leukemic cells, it was desirable to test the effect of SEB on freshly isolated murine peritoneal mast cells. Figure 4 shows that unfractionated murine peritoneal cells also secrete serotonin in response to SEB. Enhancement of serotonin release by bradykinin. A wide variety of substances have been reported to induce histamine release or to enhance the response of mast cells to substances that induce histamine release in vitro (1, 6, 17, 21, 27, 29, 30, 31, 35, 48, 50, 52; for a review, see reference 49). Neuropeptides have also been suggested to trigger mast cells to release histamine in vivo (10). SEB combined with possible enhancers, including bradykinin, lipopolysaccharide, substance P, and VIP was thus tested with RBL-2H3 cells. Table 1 shows that bradykinin was an enhancer of SEB-induced serotonin release from RBL-2H3 cells. VIP, substance P, A23187, acetylcholine, adenosine, HETE, indomethacin, lipopolysaccharide, concanavalin A, and phor-

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Stimulus FIG. 3. Stimulation of serotonin release by 50 ,ug of SEB per ml and failure of cross-linking by biotin to enhance serotonin release by biotinylated SEB-stimulated RBL-2H3 cells. Each bar represents the means and standard errors of the means of three wells.

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KOMISAR ET AL.

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bol myristate acetate did not induce serotonin release and did not enhance SEB-induced serotonin release from RBL2H3 cells (data not shown). Receptor for SEB on RBL-2H3 cells. Since mast cells respond to SEB by releasing serotonin, one would expect that they would have a binding site or receptor for SEB. To detect the receptor, biotinylated SEB was incubated with RBL-2H3 cells and stained with fluorescein-conjugated avidin and examined under a fluorescence microscope. The SEB-stained cells showed stippled staining on their cell membranes. The cells differed from one another in their fluorescence intensity. When RBL-2H3 cells were stained under capping conditions, the stained cells did not show capping; the cells remained stippled. Thus, the RBL-2H3 cells have a binding site or receptor for SEB. To further characterize the SEB binding site, RBL-2H3 cells were incubated with biotinylated SEB and then stained with phycoerythrin-conjugated avidin and were analyzed in a FACScan flow cytometer. Two populations of cells were seen. The majority of cells had low fluorescence, while a minor population had bright fluorescence (Fig. 5A). When the cells were treated with phycoerythrin-avidin alone (Fig. SB), most of the cells were not bright enough to be on scale. Therefore, there was very little nonspecific binding of phycoerythrin-avidin. When the RBL-2H3 cells were treated TABLE 1. Effect of bradykinin on the response of RBL-2H3 cells to SEB Stimulus Stimulus

~~~Tritiated

serotinin released (cpm)a

% Reas Release

8,484 +/- 347 12,192 +/-1,093 14,695 +/-650 8,780 +/-879 16,796 (two wells) 18,866 +/- 495 96,960

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with N-glycanase, there was a slight change in the flow cytometry pattern, compared with untreated cells (Fig. 5C). In contrast, when RBL-2H3 cells were treated with trypsin, the pattern changed significantly. The vast majority of cells became unstainable with SEB (Fig. 5D). This result suggests that the binding site for SEB on mast cells is probably a protein. To determine whether carbohydrates were involved in the SEB binding site, tunicamycin was used to inhibit N-linked glycosylation of the RBL-2H3 cells. The RBL-2H3 cells were then stained with biotinylated SEB and then by phycoerythrin-avidin. The result is shown in Fig. 6. There was a slight reduction in the proportion of brightly staining cells in the treated group compared with in the vehicle control, but

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Vehicle control

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Control SEB (100 ,ug) SEB (200 ,ug) Bradykinin Bradykinin + 100 ,ug of SEB Bradykinin + 200 pg of SEB Triton X-100

a Counts per minute are the means and standard errors of the means of four to six wells, except for bradykinin plus 100 ,ug of SEB, which was tested in two wells. The results were tested by Student's t test for the difference between two means (two tailed), with separate variances. The results with 100 and 200 ,ug of SEB are significantly different from the control result (P < 0.05 and P < 0.0001, respectively). The result for bradykinin alone is not significantly different from that for the control. The result for bradykinin plus 200 pg of SEB is significantly different from that for 200 pg of SEB alone (P < 0.0001).

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Log phycoerythrin fluorescence FIG. 6. Comparison of cells grown in 5 jig of tunicamycin per ml and cells grown with an equivalent amount of the ethanol used to dissolve the tunicamycin (vehicle control).

VOL. 60, 1992

the difference was not statistically significant. Thus, the SEB binding site on mast cells appears to be protein in nature. Because SEB is known to bind to major histocompatibility complex (MHC) class II antigens (11, 26), we tried to inhibit the binding of SEB to RBL-2H3 cells with MRC OX-6, an antibody to a nonpolymorphic determinant of MHC class II (33, 34). There was no inhibition detectable under a fluorescence microscope. Furthermore, the anti-MHC antibody could not be detected on the RBL-2H3 cells by a fluoresceinlabeled anti-rat immunoglobulin antibody. Therefore, the cells do not bear detectable MHC class II antigens, and the binding site of SEB on RBL-2H3 cells is probably not MHC class II. DISCUSSION We have shown in the present study that SEB can stimulate rat and mouse mast cells to release serotonin and that this effect is enhanced by bradykinin. The binding site for SEB appears to be a protein which is not a class II major histocompatibility antigen. This result suggests that besides T cells, mast cells may be among the other cell types involved in SEB toxicosis. Although we did not identify the cells that release serotonin in our peritoneal cell preparations, it is likely that the serotonin-releasing cells were mast cells. In the body, serotonin is present, in addition to in mast cells, primarily in enterochromaffin cells (which are not likely to be present in large quantities in peritoneal washes) in the gastrointestinal tract, in platelets (which are confined to the vascular system), and in the central nervous system (4, 44). Our peritoneal cell preparations contained 10 to 20% mast cells as identified by metachromatic staining; T cells, B cells, leucocytes, and macrophages were also present, but they are known to be generally serotonin nonproducers. Nevertheless, we cannot rule out the possibility that other peritoneal cells reacted to SEB and influenced the response of peritoneal mast cells. The mechanism whereby SEB causes toxicosis is unknown. SEB has been called a "superantigen" because of its ability to stimulate murine and human T cells expressing particular VP variable elements of the antigen receptor, regardless of the Va elements (5, 18, 24, 53). Marrack and coworkers (32) have found that weight loss and immunosuppression in SEB-injected mice is mediated through T cells that are capable of responding to SEB by dividing. Presumably, these T cells release various lymphokines, which in large quantities are toxic to a variety of cells. However, there is evidence that toxicity and T-cell mitogenicity are not necessarily associated with one another. For example, reduction of the disulfide loop of staphylococcal enterotoxin A (36) or toxoiding SEB with formaldehyde (45) does not completely destroy mitogenic activity but abolishes emetic activity. Furthermore, Spero and Morlock (46) showed that for SEC1, toxic and mitogenic activities are contained on different fragments of the molecule. Although the role of tumor necrosis factor alpha in enterotoxicosis is not known, it is of interest that Grossman and colleagues (14) abolished mitogenic activity of SEA and SEB by complete reduction and alkylation of the disulfide loops but that class IImediated stimulation of tumor necrosis factor alpha production by monocytes was not affected. Scheuber and coworkers (41) proposed that SEB acts as a nonimmunological mast cell stimulator. They reported that intradermal injection of SEB in monkeys gives an immediate-type skin reaction (42). This skin reaction, which is

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manifested by mast cell degranulation, is inhibited by H2 blockers of histamine, as is emesis. Hi blockers and an inhibitor of cysteinyl leukotrienes inhibit only the skin reaction. In addition, Jett and coworkers (23) found an increase in the levels of some products of the arachidonic acid cascade in monkeys challenged with SEB. All of these results suggest that mediators released by mast cells play an important role in SEB toxicosis. The present work supports the idea that SEB can act on mast cells directly to release mediators and that SEB toxicosis involves various cell types. It is well known that mast cells can release a variety of mediators of inflammation and shock (for a review, see reference 15). In addition, it has been shown recently that both mouse peritoneal mast cells (13) and RBL-2H3 cells (37) produce tumor necrosis factor. This is interesting because SEB-induced shock resembles endotoxic shock (8). On the other hand, there is good evidence that T-cell responses can produce some symptoms of SEB intoxication (32). Therefore, the toxicosis induced by SEB may be a very complex phenomenon, involving several cell types, mediators, and their interactions. The toxicosis might be exacerbated by mediators from one cell type acting on other cell types. For example, interleukin 10, which is produced from activated T cells (and perhaps other cells), can enhance mast cell growth (51). Conversely, histamine has effects on suppressor, helper, and cytotoxic T cells (for a review, see reference 54). We do not know why high concentrations of SEB (a minimum of 50 ,ug/ml) were necessary to trigger serotonin release reproducibly. Other workers have needed supraphysiologic concentrations of stimulators to see effects in vitro. For example, Shanahan and coworkers (43) used 10-4 M substance P (135 ,ug/ml) to stimulate peritoneal mast cells. The explanation for the need for a high concentration of stimulants for triggering mast cells may be that signal transduction with a purified cell population is inefficient. We do not believe that the effect was due to a contaminant in the SEB preparation, because the SEB that we used for the present study is very pure, as demonstrated by SDS-PAGE, and we were able to see a similar stimulatory effect with purified SEB from two other sources. Nevertheless, the sensitivity of mast cells in vivo to SEB may be greater than what we see in vitro because of the synergistic effect of various mediators and perhaps higher local concentrations of SEB associated with some cell types that interact with mast cells. We did observe an enhancement of serotonin release by bradykinin, and many known enhancers of histamine release by mast cells have been reported elsewhere (for a review, see reference 49). As reported by Arock and coworkers (1) for rat bone marrow-derived mast cells, bradykinin did not stimulate RBL-2H3 cells by itself. Although SEB comes from a gram-positive organism and hence is unlikely to have much endotoxin contamination, there was a possibility that endotoxin contamination of reagents or serum might have influenced the outcome of the experiments. We have shown that the reaction of RBL-2H3 cells to SEB is not enhanced by S. typhimurium lipopolysaccharide and that lipopolysaccharide by itself does not cause serotonin release. This makes it unlikely that the serotonin release that we observed is caused by lipopolysaccharide contamination of the reagents and culture media. This result is consistent with the observations of Ohno and coworkers (37), who observed that lipopolysaccharide does not induce histamine release from RBL-2H3 cells. If SEB stimulates serotonin secretion by RBL-2H3 cells, then it should bind to them as well. Olenick and coworkers

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(38) found that it does, and we have independently observed the same phenomenon. The only well-defined cell surface receptors for SEB are MHC class II antigens. T-cell receptors do not bind detectably to staphylococcal enterotoxins unless the enterotoxins are complexed with MHC molecules (12, 39). Since there was a report that some murine peritoneal mast cells that are treated with gamma interferon bear MHC class II antigens (2), we tried to inhibit the binding of SEB to RBL-2H3 cells with an antibody to a nonpolymorphic determinant of MHC class II antigens. There was no observable inhibition. Furthermore, the antibody did not stain the cells. Therefore, we believe that the SEB-binding site or receptor on RBL-2H3 cells is not an MHC class II antigen. This finding is consistent with the reports of other workers that enterotoxins also bind to non-MHC receptors

(7, 19). To chemically characterize the SEB-binding site or receptor, we treated the RBL-2H3 cells with trypsin or N-glycanase or grew them in tunicamycin. Trypsin abolished the binding of biotinylated SEB to RBL-2H3 cells, while the effect of N-glycanase was negligible. Tunicamycin had a slight effect on the binding of SEB to the cells, but the difference was not statistically significant. Tunicamycin did not affect the viability of the cells. Therefore, we believe that SEB binds to a protein on RBL-2H3 cells. In a variety of inflammatory reactions, bradykinin is produced by the activation of the kinin system. In the kinin system, bradykinin is produced from high-molecular-weight kininogen by the catalytic action of kallikrein (16). Bradykinin is a powerful vasodilator; it causes increases of vascular permeability and smooth muscle contraction. Although it is not known whether bradykinin is produced during SEB toxicosis, the finding that bradykinin enhances mast cells to release serotonin is very interesting and requires further studies. REFERENCES 1. Arock, M., P. Devillier, G. Luffau, J. J. Guillosson, and M. Renoux. 1989. Histamine-releasing activity of endogenous peptides on mast cells derived from different sites and species. Int. Arch. Allergy Appl. Immunol. 89:229-235. 2. Banovac, K., D. Neylan, J. Leone, L. Ghandur-Mnaymneh, and A. Rabinovitch. 1989. Are the mast cells antigen presenting cells? Immunol. Invest. 18:901-906. 3. Barsumian, E. L., C. Isersky, M. G. Petrino, and R. P. Siraganian. 1981. IgE-induced histamine release from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing cell lines. Eur. J. Immunol. 11:317-323. 4. Broide, D. H. 1991. Inflammatory cells: structure and function, p. 141-153. In D. P. Stites and A. I. Terr (ed.), Basic and clinical immunology. Appleton and Lange, East Norwalk, Conn. 5. Choi, Y., B. Kotzin, L. Herron, J. Callahan, P. Marrack, and J. Kappler. 1989. Interaction of Staphylococcus aureus toxin "superantigens" with human T cells. Proc. Natl. Acad. Sci. USA 86:8941-8945. 6. Delmich, K., D. Eichelberg, and W. Schmutzler. 1985. The effects of adenosine and of some adenosine analogues on the concanavalin A or acetylcholine-induced histamine release from human adenoidal mast cells. Agents Actions 16:141-143. 7. Dohlsten, M., G. Hedlund, S. Segren, P. A. Lando, T. Herrmann, A. P. Kelly, and T. Kalland. 1991. Human major histocompatibility complex class II-negative colon carcinoma cells present staphylococcal superantigens to cytotoxic T lymphocytes: evidence for a novel enterotoxin receptor. Eur. J. Immunol. 21:1229-1233. 8. Elsberry, D. D., D. A. Rhoda, and W. R. Beisel. 1969. Hemodynamics of staphylococcal B enterotoxemia and other types of shock in monkeys. J. Appl. Physiol. 27:164-169. 9. Fewtrell, C., D. Lagunoff, and H. Metzger. 1981. Secretion from

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