Accessory Function of Human Mononuclear Phagocytes Lymphocyte Responses to the Superantigen Staphylococcal Enterotoxin 6’



of Medicine,

Case Western Reserve University and University Cleveland, Ohio 441064389



26, 1991; accepted January


of Cleveland,

27, 1992

The role of the cytokines IL- I (Y, IL- 10, and IL-6 and the cell adhesion molecules ICAM- 1, LFA- 1 (a and @, and Mac- 1 as accessory molecules for stimulation of T cells by the superantigen staphylococcal enterotoxin B (SEB) was examined. Both blood monocytes and alveolar macrophages were used as accessory cells because these cells differ in patterns of cytokine expression and thus potentially in accessory cell function for superantigens. The blastogenic response of highly purified T cells to SEB was reconstituted with either monocytes or alveolar macrophages. IL-1 secretion was increased comparably in monocytes and alveolar macrophages by SEB, but IL-6 was not stimulated by SEB. IL-la plus IL-l@ reconstituted the response of T cells to SEB but required the addition of accessory cells. The cell adhesion molecules ICAM-I and LFA-I but not Mac-l also functioned as accessory molecules for SEB-induced cluster formation and lymphocyte blastogenesis. Thus, not only must this superantigen bind to Class II MHC on accessory cells as is well known, but also SEB requires at least certain cytokines (IL-1 (Yand IL-l@ produced by accessorycells and cell adhesion molecules (ICAM- 1 and LFA-1 ) for activation of T lymphocytes. 0 1992 Academic

Press, Inc.

INTRODUCTION Mitogens, antigens, and the more recently described superantigens differ in their ability to activate lymphocytes. Mitogens such as phytohemagglutinin are polyclonal activators of T lymphocytes. Antigens interact with only specific T cell receptors so that a low percentage of T cells are capable of reacting. Activation of T cells by superantigens is exemplified by staphylococcal enterotoxin(s) (SE)3 (reviewed in (1, 2)). SE comprise a group of structurally related, serologically distinct proteins (SEA, SEB, SEC, SED, and SEE) produced by certain strains of Staphylococcus aureus (3, 4). In vivo, SE-induced immune activation is thought to play an important role in the patho’ This work was supported by Grants AC-08330, HL-01829 and HL-4357 1, and by Clinical Research Center Grant MOl-RROOO-80-29 from the National Institutes of Health, Public Health Service. ’ To whom correspondence should be addressed at Dept. of Medicine, University Hospitals of Cleveland, 2074 Abington Rd., Cleveland, OH 44106-4389. 3 Abbreviations used: ICAM-1, intercellular adhesion molecule- 1; LFA- 1, lymphocyte function-associatedI; PMB, polymyxin B; SE, staphylococcal enterotoxin(s); SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B. 466 0008-8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form resewed




genesis of S. aureus-induced food poisoning and possibly toxic shock syndrome (5, 6). SE interact with the T cell receptor by means of a specific set of VP elements (79). The term superantigen was suggested to refer to this type of receptor-ligand interaction because the repertoire of responder T cells expressing VP is so much greater than that of T cells responsive to antigens (7). The interest in superantigens stems not only from the clinical implications for elucidating the pathogenesis of certain diseases but also from the recognition that these molecules could serve as tools for better understanding the immune system in general. Mitogens, antigens, and superantigens also differ in their accessory cell requirements for lymphocyte activation. Mononuclear phagocytes such as blood monocytes and tissue macrophages as well as other cell types (B cells, dendritic cells) function as accessory cells for T cell responses to various stimuli. For T cell responses to mitogens, neither processing of the stimulus by accessory cells nor self-recognition through products of the MHC genes is required (10). The cytokines IL-1 and IL-6, however, act as amplifying factors for stimulation of T cells by mitogens ( 11, 12). Accessory cell requirements for stimulation of T cell responses to antigens may differ depending on the activating signal. In general, however, early steps include binding and processing of the antigen by the antigen presenting accessory cell (1315). Lymphocytes are activated by antigens upon interaction with processed protein epitopes displayed on the surface of the antigen presenting cell. This antigen-specific recognition is restricted by determinants encoded in the MHC which are presented by the accessory cell along with processed antigen to the lymphocyte ( 16- 19). T cell activation in response to antigens is further mediated by cytokines such as IL-1 and IL-6. Binding of T lymphocytes to accessory cells occurs through expression on T cells of cell adhesion molecules such as lymphocyte function-associated (LFA)- 1 and CD2 antigens (20-22). The intercellular adhesion molecule (ICAM)- I is a ligand for LFA1 (23, 24) and LFA-3 binds to CD2 (25). The interaction of these receptor-ligand complexes is regulated by lymphocyte activation through various mechanisms and leads to greatly increased sensitivity of T cells to stimuli (26, 27). Such interactions have been studied most thoroughly for antigenic and mitogenic stimuli (reviewed (28, 29)). Accessory cell requirements for stimulation by superantigens is less well understood. Unlike antigens, superantigen such as SE do not require processing by accessory cells for stimulation of T cells (30, 3 1). Nevertheless, SE must bind to accessory cells (3 1) and do so with high affinity to MHC Class II molecules in a genetically unrestricted manner (30, 32, 33). Whether cell adhesion antigens or cytokines are required as accessory molecules for T cell activation by superantigens has not been fully evaluated. Kotb et al. found that the superantigens streptococcal M protein and SEB stimulated purified human T lymphocytes independently of accessory cells when the combination of phorbol myristate acetate, IL- 1, and IL-6 was added (34) but the relationship between cytokines released by accessory cells in coculture with lymphocytes and accessory cell function for lymphocyte responses to SEB was not studied. The ability of another enterotoxin SEA plus cytokines to stimulate T cell proliferation in the absence of accessory cells is controversial (3 1, 35). A dependence on LFA- 1 for the formation of homotypic aggregation of B cells in response to the superantigen toxic shock syndrome toxin-l was described by Mourad et al. (36). These data raised the possibility that




LFA-1 and or its ligand ICAMmay serve as contributing accessory molecules for T cell activation in response to SEB. The objective of this study was to examine the properties of accessory cells including secretion of the cytokines IL- 1(Y, and IL- 10, and IL-6 and expression of the surface adhesion molecules ICAMand LFA-1 involved in SE-induced T lymphocyte activation using SEB as the stimulus. The accessory cells used included the composite of those within peripheral blood mononuclear cells (PBMC), blood monocytes, and alveolar macrophages. Alveolar macrophages differ from monocytes in their pattern of cytokine expression (37) and conceivably expression of surface adhesion molecules. More than one type of accessory cell was used to gain more insight regarding requirements of SEB for cytokines and cell adhesion molecules. PBMC responded well to SEB and could be rendered accessory cell dependent by a series of purification steps. Monocytes and alveolar macrophages reconstituted the response of accessory celldependent lymphocytes to SEB, and at limiting numbers alveolar macrophages were significantly superior as compared to monocytes. The cell adhesion molecules ICAM1 and LFA- 1 and the cytokines IL- 101and IL- 1p functioned as accessory molecules for SEB-induced lymphocyte activation. METHODS Preparation of PBMC and rnonocytes. Venipuncture was performed on healthy volunteers aged 18-40. One hundred twenty milliliters of heparinized blood was obtained and diluted 1: 1 in RPM1 1640 culture medium (M.A. Bioproducts, Walkersville, MD) containing 50 U/ml penicillin (Squibb, Princeton, NJ), 5 &ml gentamicin (M.A. Bioproducts), and 2 mM L-glutamine (GIBCO, Grand Island, NY). This culture medium is referred to as supplemented medium. PBMC were separated from whole blood at room temperature on Ficoll-Paque separation medium (Pharmacia Fine Chemicals, Piscataway, NJ) and washed three times in supplemented medium at 4°C. To separate monocytes from lymphocytes, PBMC were plated onto 100 X 60 mm tissue culture grade petri dishes (Becton-Dickinson, Lincoln Park, NJ) precoated with 0.5 ml heat-inactivated pooled human serum. Then, 5-7 ml PBMC at 10’ cells/ml was added to petri dishes in culture medium plus 10% pooled human serum and was incubated at 37°C in 5% C02/humidified air for 1 hr. Nonadherent cells were removed by repeated washing with 10% fetal calf serum in supplemented medium. Adherent cells were covered with cold (4’C) Hanks’ balanced salt solution without calcium or magnesium (GIBCO) and incubated at 4°C for 30 min. Adherent cells were removed from plates by scraping gently with a rubber policeman. The adherent cells were >95% viable by exclusion of trypan blue and were 90-95% positive for peroxidase (38), >80% positive for nonspecific esterase (39) and appeared morphologically to be monocytes by Wright’s stain. These cells subsequently are referred to as monocytes. For accessory cell assays, monocytes were irradiated with 5000 rad of y radiation from a cobalt source. Bronchoalveolar lavage. Bronchoalveolar lavage was performed on healthy, nonsmoking volunteers to obtain alveolar macrophages as described previously (40). Subjects were excluded if they had any acute or chronic pulmonary disease or if they had an upper respiratory tract infection within 6 weeks; subjects who were on medications including chronic use of aspirin or nonsteroidal anti-inflammatory agents also were excluded. Ages ranged from 20 to 46. Informed consent was obtained in writing from




each subject. The nasooropharynx was anesthetized with 4% lidocaine. An Olympus BF type 4B2 flexible fiberoptic bronchoscope (Olympus Corp. of America, New Hyde Park, NY) was passed through the nose and trachea while instilling 2% lidocaine and wedged into a segment of the right middle lobe. Then, 240 ml of 0.9% sterile saline were instilled in 20- to 30-ml aliquots into one segment and the same volume into an adjacent segment and aspirated to retrieve bronchoalveolar cells. Lavage fluid was centrifuged at 500g for 10 min and the pellet of cells suspended in supplemented medium. Bronchoalveolar cells from these healthy subjects were 90-95% nonspecific esterase positive, 6- 10% sheep red blood cell rosetting, and < 1% granulocytes as assessed by Wright’s stain; and these cells are referred to as alveolar macrophages. As with monocytes, for accessory cell assays, alveolar macrophages were irradiated with 5000 rad of y radiation. Preparation of T cells. The plastic nonadherent cells were sedimented and the cells resuspended in 2 ml of supplemented medium. Since monocytes and B cells adhere to nylon wool (41) to begin to render the plastic nonadherent cells accessory cell dependent, cells were incubated at 37°C for 1 hr on 600-mg nylon well columns (Polysciences, Inc., Wanington, PA) which had been preincubated for 1 hr in supplemented medium with 10% fetal calf serum. Nonadherent cells were eluted with 10 ml of the same medium. The nylon wool nonadherent cells were further purified by depletion of HLA-DR-positive cells as described previously (40) using mouse monoclonal antibody OKIal (Ortho Diagnostic Systems, Raritan, NJ) and Low-Tox-H rabbit complement (Pel-Freez, Brown Deer, WI). Nylon wool nonadherent cells (15 X lo6 cells/reaction) were incubated for 30 min at room temperature with OKIal at a 1:20 dilution and then for 30 min with complement at a 1:3 dilution and washed twice. These Ia-negative cells retained approximately 10% of the optimal response of unseparated mononuclear cells to SEB even without added back monocytes. Therefore, to deplete further the contaminating accessory cells, the Ia-negative cells were subjected to a discontinuous density gradient sedimentation step using Percoll (Pharmacia) by a modification of the procedure described by Timonen et al. (42) and as described previously (43). Percoll was adjusted to isotonicity by addition of one part 10X Hanks’ balanced salt solution to nine parts Percoll. Four concentrations of Percoll in medium (40,43.5,47, and 54%) were prepared. To adjust Percoll concentrations to the appropriate density, the refractive indices were measured using a refractometer (Bausch and Lomb) and adjusted to 1.3427, 1.3432, 1.3437, and 1.3448 for the 40, 43.5, 47, and 54% concentrations, respectively. Two milliliters of each Percoll concentration was carefully layered successively from highest to lowest density into a 15-ml conical polystyrene tube. Cells (6-8 X 107) were suspended in 2 ml medium and layered over the gradient and the cells sedimented at 400g for 30 min at 4°C. The cells above 47% density were excluded and the cells at the 47-54% interface and below were collected and washed three times in medium. Viability was >95% as assessed by exclusion of trypan blue dye. Assay of T lymphocyte blastogenesis. T cells were suspended at lo6 cells/ml in supplemented medium with 20% heat-inactivated pooled human serum. T cells ( 105, 100 &well) were cultured in triplicate in flat-bottom 96-well microtiter plates (BectonDickinson) alone or with various numbers of monocytes in 100 ~1 of supplemented medium/well with or without SEB (Sigma, St. Louis, MO). The cells were incubated for 72 hr at 37°C in 5% COz/humidified air. Twenty-four hours before termination of culture, 1 &i of methyl[3H]thymidine, 5 Ci/mmol (Amersham, Arlington Heights,




IL) was added to each well. The wells were harvested onto glass filter paper discs and 3H content/well was assessed by liquid scintillation spectrophotometry. Bioassay fir IL-Z. The IL-l bioassay used was mitogenesis of mouse thymocytes (44) and was performed as previously described (45). Briefly, thymocytes were prepared from 8- to lZweek-old female C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME). The IL- 1 standard was prepared from mononuclear cells adherent to plastic and stimulated with LPS. Experimental supematants and the standard were diluted serially ( 1:2- 1:256). The dilutions were added to wells containing 1.5 X 1O6thymocytes. Triplicate wells were prepared for each variable. Incorporation of [3H]thymidine was measured after 3 days in culture at 37°C. IL-l activity in U/ml was determined by modified probit analysis (46). Bioassay for IL-6. The IL-6 bioassay was mitogenesis of culture supernatants for the B9 murine plasmacytoma cell line (47) kindly provided by Dr. L. Aarden, University of Amsterdam, The Netherlands. B9 cells were suspended in supplemented medium plus 25 mM Hepes, 4 &ml 2-mercaptoethanol, and 10% fetal calf serum. Cells (5 X 103) in 100~1 volumes were added to each well of 96-well microtiter plates. Supernatants of monocytes or alveolar macrophages were added in 100 yl supplemented medium/well using serial dilutions from 1:5 to 1:625 in triplicate. Human recombinant IL-6 (Genzyme Corp., Boston, MA) was used as a standard. The plates were incubated at 37°C for 72 hr and incorporation of [3H]thymidine was measured. The bioactivity of IL-6 in half-maximal units was determined by probit analysis. Inhibition of lymphocyte blastogenesis by antibodies against cytokines. Monocytes were suspended in supplemented medium at 5 X lo5 cells/ml in microcentrifuge tubes. Neutralizing polyclonal antibodies against recombinant IL- 1a, IL- 10, and IL6 (R & D Systems, Minneapolis, MN) were added to the monocyte suspension alone or in combination at final concentrations of 0.6-50 pg/ml. These concentrations were 6-50X those which neutralized 50% of the maximum mitogenic response of recombinant cytokines IL- 1(Y and IL-l/3 for DlO cells and IL-6 for T 1165 cells (IL-6) as determined by R & D Systems. The mixture of cells plus antibodies was maintained at 4°C for 1 hr before addition to lo5 purified T cells with or without SEB. Cells were incubated at 37°C for 72 hr in microtiter plates and assay of [3H]thymidine incorporation performed. Expression of surface molecules. Monoclonal antibodies against LFA- 1a (TS l/22), LFA-l/3 (TSl/lS), MAC-la (LM2), and ICAM- (RRl) were obtained as a generous gift from Dr. Robert Rothlein (Boehringer Ingelheim Incorporated, Ridgefield, CT). Monocytes (5 X 105) or alveolar macrophages were incubated with or without antibodies to ICAM- 1, LFA- la, LFA- 16, MAC-l (Y, and OKT3 (Ortho-Mune, Raritan, NJ) at final concentrations of 3.4-84 pg/ml for 30 min at 4°C washed, exposed to FITC-conjugated goat anti-mouse IgG (Cappel), and incubated again for 30 min at 4°C and washed. Cells were fixed in freshly prepared 2% paraformaldehyde, washed again, and kept in 100 ~1 Hanks’ balanced salt solution. Fluorescence emission was read on an Ortho Cytofluorograph 11s(Becton-Dickinson, Braintree, MA). Inhibition of blastogenesis and cluster formation by antibodies to surface adhesion molecules. Monoclonal antibodies against cell adhesion molecules were the same as those used for determining expression of surface cell adhesion molecules. Control antibodies against fibroblast growth factor (FGF) were obtained from R & D Systems. Monocytes were preincubated with these antibodies at 4°C using final concentrations of 3.4-84 pg/ml. Monocytes then were added to T cells with or without SEB and






t3H]thymidine incorporation determined. Cluster formation was assessed using an inverted light microscope. Statistical analysis. Statistical significance was determined by the paired t test. Significance was established as P < 0.05. RESULTS Accessory,function qf monocytes and alveolar macrophages for SEB-stimulated lymphocytes. In preliminary experiments, the optimal concentration for responses of PBMC

to SEB was found to be 1 pg/ml and peak responses were observed after 3 days of stimulation. PBMC responded vigorously to 1 pg/ml SEB: incorporation of [3H]thymidine in the absence of SEB was < 1000 cpm and in the presence of SEB was 36,561 * 5916 cpm, means + SE, n = 14. When monocytes were depleted from mononuclear cells by adherence to plastic, the remaining nonadherent cells retained reactivity to SEB (incorporation of [3H]thymidine 30-80% of the response of PBMC) suggesting that these cells were contaminated with accessory cells such as monocytes, dendritic cells, B cells, or large granular lymphocytes. We therefore rigorously depleted accessory cells from the plastic nonadherent cells by adherence to nylon wool, treatment with OKIal plus complement and density gradient sedimentation to retrieve high density cells as described under Methods. These high density, OKIal-negative cells were >99% T lymphocytes by reactivity with OKT3 and contained no detectable monocytes or large granular lymphocytes as described previously (43) and will be referred to hereafter as lymphocytes. When stimulated with SEB these high density lymphocytes failed to undergo a blastogenic response (< 1000 cpm, Fig. 1). The addition of graded numbers of monocytes to lymphocytes reconstituted the response. Responses were significant when low numbers of monocytes were added back (1:20 ratio of monocytes to lymphocytes, P < 0.025) and peaked at 1:2. As with monocytes, graded numbers of alveolar macro-


c w -





FIG. I. Accessory function of blood monocytes and alveolar macrophages for lymphocyte responses to SEB. Irradiated monocytes or alveolar macrophages were added to 10’ lymphocytes at the ratios shown. Cells were stimulated with SEB (1 rg/ml). Incorporation of [3H]thymidine was measured after 3 days in culture at 37°C. The data shown are the mean cpm f SE, n = 12, for all ratios except 1:20, in which case n = 5. The responses of lymphocytes to SEB were significantly higher when alveolar macrophages were added as compared to monocytes at ratios of 1:20 (P < 0.05) 1:lO (P < 0.04) and 1:5 (P < 0.01).




phages added to lymphocytes reconstituted the response to SEB (Fig. 1). Moreover, the response of lymphocytes was higher in the presence of alveolar macrophages at limitingnumbersofalveolarmacrophages(ratiosofalveolarmacrophagestolymphocytes 1:20- 1:5) as compared to monocytes suggesting differences in expression of accessory molecules for SEB-induced responses between these two groups of mononuclear phagocytes. The response of lymphocytes to SEB, however, was comparable when alveolar macrophages and monocytes were added to lymphocytes at the ratio of 1:2. Not shown on Fig. 1, the response decreased from peak responses by 29% when alveolar macrophages were added to lymphocytes at the ratio of 1: 1, II = 4. The decrease, however, was not significant (P > 0.05). For either added monocytes or alveolar macrophages, the response of lymphocytes to SEB was comparable whether responder lymphocytes were allogeneic or autologous. In a representative of three experiments, at a ratio of 1:5 monocytes/macrophages to lymphocytes, incorporation of [3H]thymidine in SEB-stimulated autologous T cells plus monocytes was 14,9 12 2 2207, allogeneic T cells was 18,598 + 1604; autologous T cells plus alveolar macrophages was 30,830 ? 1252, and allogeneic T cells plus alveolar macrophages was 3 1,7 16 + 44 15. The addition of either monocytes or alveolar macrophages to unstimulated T cells did not induce a blastogenic response (< 1500 cpm) after 3 days in culture ensuring that a mixed leukocyte reaction had not occurred. Thus, the high density T cells represented an accessory cell-dependent group of T lymphocytes fully responsive to SEB when autologous or allogeneic monocytes or alveolar macrophages were added back. These cells therefore were suitable as the responder cell population to examine the role of accessory molecules, such as cytokines and cell adhesion molecules, in the accessory function of monocytes or alveolar macrophages for lymphocyte responses to SEB. Accessory role of cytokines in stimulation of lymphocytes with SEB. To evaluate the role of cytokines in the accessory function of monocytes and alveolar macrophages for SEB, we first assessedthe relative ability of these mononuclear phagocytes to secrete IL-1 and IL-6 in response to SEB. The release of cytokines in response to SEB also was compared to that induced by LPS. SEB and LPS were preincubated with or without polymyxin B (PMB) and then were added to monocytes or alveolar macrophages (Table 1). In three preliminary experiments, the peak of IL- 1 and IL-6 bioactivity was found to be at 24 hr (data not shown). Supernatants of LPS-stimulated monocytes and alveolar macrophages contained IL-1 activity and this activity was completely absent in supernatants of cells in which LPS was preincubated with PMB which is known to bind to and inactivate LPS. Supernatants of either monocytes or alveolar macrophages stimulated for 24 hr with 1 pg/ml SEB, the concentration of SEB used in the accessory assays, had little or no IL- 1 activity (Table 1). In contrast, supernatants of both monocytes and alveolar macrophages stimulated with 10 pg/ml SEB contained IL- 1 activity and the measured levels were comparable between the two cell types. PMB did not inactivate the IL-linducing capacity of SEB demonstrating that the IL-1 measured was not due to contamination of the SEB preparation with LPS. Since the C3H/HeJ mice used in these experiments are specifically resistant to LPS, not unexpectedly the SEB alone also stimulated the thymocytes to proliferate (10 pg/ml SEB, IO-20 U/ml IL-1 activity; 1 rg/ml SEB, no activity) but the activity was always lower than that in supernatants of SEB-stimulated cells.




Stimulation of IL- 1 and IL-6 Bioactivities by LPS and SEB in Monocytes and Alveolar Macrophages Stimulus”


Alveolar macrophages IL-I activity (U/ml)*

None LPS 10 pg/ml LPS + PMB SEB 1 &ml SEB 10 pg/ml SEB 10 &ml + PMB

0 35 + 6 2+2 8+6 92 + 42 121 * 53

0 21 k9 0 0 17 ? 22 107k21 IL-6 activity (U/ml)*

None SEB 10 wg/ml

1412 F 435 1120 + 249

838 f 205 1334 + 505

a LPS and SEB were preincubated for 1 hr at 37°C with or without PMB. Stimuli then were added to cells. Cells were cultured at 0.5 X lo6 cells/ml at 37” for 24 hr. * Supematants then were collected and assayed for IL-l activity using the C3H/HeJ mouse thymocyte assayor for IL-6 activity using a B9 plasmacytoma cell line. IL-6 activity in supematants of cells stimulated with 1 rg/ml SEB was comparable to that in supernatants of unstimulated cells and to cells stimulated with IO pg/ml SEB, n = 3 (data not shown). Data are expressed as means + SE, n = 5.

Since the concentration of SEB (1 pg/ml) used in accessory cell assays was less than that which stimulated detectable levels of IL- 1 activity by either monocytes or alveolar macrophages (10 pg/ml), we assessedwhether cocultures of lymphocytes and accessory cells resulted in higher levels of IL- 1 produced in response to SEB (1 pg/ml) than those observed in supernatants of monocytes or alveolar macrophages alone. PBMC were used for this purpose; cells were stimulated with or without SEB for 24 hr and supernatants collected. IL- 1 activity in supernatants of unstimulated cells was 0 U/ml; SEB (1 pg/ml) 7 + 3; SEB (10 pg/ml) 342 + 119, IZ = 5. Thus, there were detectable albeit low levels of IL- 1 activity at SEB (1 pg/ml) in supernatants of PBMC and the levels of IL-l activity in cells stimulated with SEB ( 10 pg/ml) were higher than those for monocytes or alveolar macrophages alone (P < 0.05). Although there appeared to be a slight increase in IL-6 production in supernatants of SEB-stimulated cells over that of unstimulated cells (Table I), the increase was not statistically significant (P > 0.1 for both monocytes and alveolar macrophages) and the activity in supernatants of SEB-stimulated monocytes and alveolar macrophages also did not differ (P > 0.2). Thus, SEB could stimulate both monocytes and alveolar macrophages to produce IL- 1 but not IL-6. The concentration of SEB that stimulated IL- 1 production, however, was higher than the concentration used to stimulate blastogenesis so that the role of IL- 1 as an accessory molecule in this system was unclear. To clarify the role of IL- 1 and IL-6 as molecules relevant to accessory cell function, the effect of neutralizing antibodies against IL- 1q IL- 1p, and IL-6 on accessory cell function of monocytes was examined. Alveolar macrophages were not used to examine the role of these cytokines in accessory cell function because SEB-stimulated IL-l activity was comparable between monocytes and alveolar macrophages. First, we verified that the antibodies used were, in fact, neutralizing of established bioactivities of




these cytokines. The mitogenic activity of supernatants from LPS-stimulated monocytes for mouse thymocytes was not inhibited by anti-IL- 1LYat any concentration tested but anti-IL-1P at 50 pg/ml inhibited the response by 25%. Furthermore, the combination of anti-IL-la (2.5 pg/ml) and IL- l/3 (50 pg/ml) completely neutralized the activity and recombinant IL-la or IL-10 reconstituted the response (Table 2). Thus, the anti-ILla and anti-IL- l/3 used in these studies when used in combination functioned as neutralizing antibodies for the mouse thymocyte assay. The neutralizing capacity of anti-IL-6 was confirmed as abrogation of mitogenesis of B9 cells to recombinant IL6 (data not shown). These cytokine neutralizing antibodies then were used to probe the role of respective cytokines in the accessory function of monocytes for SEB-stimulated lymphocytes. Monocytes were preincubated with various concentrations of antibodies against IL1a, IL- 10, and IL-6, and then the antibodies, cells, and SEB were added to lymphocytes. There was no effect of anti-IL- 1(Y,anti-IL- l,& or anti-IL-6 when added singly to cultures at concentrations of 0.6-50 pg/ml (n = 12). The combination of anti-IL-6 plus either anti-IL- 1cy or anti-IL- 1p also had no effect on accessory function of monocytes (n = 4). When anti-IL-la and anti-IL-l/3 were used in combination, however, accessory function of monocytes for SEB-stimulated lymphocytes was inhibited by 89% (Table 3). Furthermore, either recombinant IL- 1a or IL- l/3 completely restored the response of lymphocytes to SEB. The concentration of antibodies neutralizing accessory function was the same which inhibited the thymocyte assay. Antibodies against IL- 1(Yand ILl/3 together but not alone also inhibited PBMC responses to SEB (I pg/ml) and the response was reconstituted with recombinant IL-l N or IL- I/3 (data not shown). Thus, IL- 1cyor IL- l/3 is required for accessory cell function for SEB such that when either is present accessory cell function is exhibited. In two additional experiments recombinant cytokines were added to SEB-stimulated T cells without added monocytes to determine the accessory cell dependence of SEB. Recombinant IL- 101,IL- 1p, and IL- 1 were added at 6 and 50 pg/ml to SEB-stimulated TABLE 2 Neutralizing Effect of Antibodies against IL- 1cyand IL- 1p on Mouse Thymocyte Responses to a Mitogenic Stimulus Addition to mouse thymocytes” Medium only Mitogenic supernatant Mitogenic supernatant plus anti IL-la anti IL- 1p anti IL-la + anti IL-l@ anti IL-10 t anti IL-lb + rlL-la anti IL- I01 + anti IL- I p + rlL- l/3

Incorporation of [ ‘Hlthymidine (means + SD)b 468 + 166 229,259 + 11,958 208.005 173.787 2.825 254,8 1I 229,662

+ + ir + +

17,466 10,419 368 36,422 6,907

’ Mitogenic supernatant was prepared from monocytes stimulated at IO6 cells/ml with LPS ( 10 pg/ml). Supematant (50 ~1) was added to mouse thymocytes with or without antibodies against IL-lo (2.5 fig/ml), IL-10 (50 fig/ml), or both IL-la and IL-I@ with or without rIL-lol(l2 fig/ml) or rIL-P (12 pg/ml). b Incorporation of [3H]thymidine was measured after 3 days in culture in microtiter plates at 37°C. Data shown are the means + SD of triplicate wells from a representative of four experiments.




TABLE 3 Neutralizing Effect of Antibodies against IL-la and IL-l/3 on Accessory Function of Monocytes for SEB-Induced Lymphocyte Blastogenesis Addition to SEB-stimulated lymphocytes Medium only Monocytes Monocytes plus anti-IL- 1N anti-IL- l/3 anti-IL- 1o( + anti IL- 16 anti-IL-la + anti-IL-10 + rIL-lcu anti-IL-la + anti-IL-10 + rIL-l@

Incorporation of [3H]thymidine (mean cpm f SD) 888 t 149 100,329 f 53 15 91.833 88,602 11,121 112,318 111,947

+ f f + +

4091 3949 801 2359 3225

’ Monocytes were precultured for 1 hr at 4°C with or without antibodies against IL- 101(2.5 pg/ml), ILlp (50 pg/ml), or both and with or without recombinant IL-la (12 pg/ml) or recombinant IL-10 (12 pg/ ml). The monocytes (and antibodies and recombinant cytokines) were added to lymphocytes at a ratio of 1:2 in triplicate. Then, I pg/ml SEB was added to each well and incorporation of [3H]thymidine assayed after 3 days in culture at 37°C. Incorporation of [‘Hlthymidine in unstimulated cells was 589 f 320. The experiment shown is representative of 12 experiments.

T cells either alone or in combination (IL- 1(Yplus IL- 1p, IL- 1(Yplus IL-6, IL- 1p plus IL-6, or IL-la plus IL- 10 plus IL-6). SEB-stimulated T cells did not respond to SEB alone (500 cpm) and when monocytes were added the response was 45,556 t 2400. When any of the recombinant cytokines either alone or in the combinations listed were added to SEB-stimulated T cells without added monocytes, there was no change in the response of the T cells to SEB (maximum change in cpm was ~200, n = 2). These experiments demonstrated that accessory cells are required for T cell responses to SEB and that cytokines alone cannot simulate accessory cells in this activity. Accessory role ojcell adhesion molecules in stimulation of lymphocytes by SEB. ICAM- 1 and LFA- 1 are involved in a variety of cell-cell and cell-matrix interactions. The role of these cell adhesion molecules in accessory cell function for superantigens, however, has not been investigated. Since the increased accessory cell function of alveolar macrophages as compared to monocytes could not be explained on the basis of increased release of IL- 1 or IL-6 in response to SEB, the role of ICAM- 1 and LFA1(Y and LFA- l/3 in accessory function of monocytes and alveolar macrophages was assessed. First, the expression of these cell adhesion molecules by monocytes and alveolar macrophages was compared and then the functional significance evaluated. Table 4 shows the percentage of positive monocytes and alveolar macrophages stained with monoclonal antibodies directed against ICAM- 1, LFA- 1a, LFA- l/3, Mac- 1, and OKT3. The percentage of cells staining with OKT3, which is present on T lymphocytes. was 2-5%, demonstrating a low level of contamination of the monocyte/macrophage populations with T cells. Monocytes expressed high levels of LFA- 1(Y, LFA- 10, and Mac- 1. Monocytes also expressed ICAM- 1 but the percentage of positive cells was lower than for the other markers tested. The percentage of alveolar macrophages expressing LFA- 1cy and LFA-l@ and Mac- 1 was comparable to monocytes. The percentage of alveolar macrophages expressing ICAM- 1, however, was significantly higher than that of monocytes (P < 0.007).




TABLE 4 Percentage of Mononuclear Phagocytes Expressing Adhesion Molecules Monoclonal antibodies” (% positive) ICAM- 1

LFA- 101

LFA- I fl

Mac- lo1


7 f 2b 39 f 9

84 f 4 87 rt 4

96 f 4 82? 11

86 + 4 60 + 20

3&2 2*2

Monocytes Alveolar macrophages

’ Monocytes or alveolar macrophages were incubated with monoclonal antibodies against ICAM-1, LFAla, LFA-I& MAC-la, and OKT3 all at 40 pg/ml for 30 min followed by exposure to FITC-conjugated goat anti-mouse IgG as described under Methods. The fluorescence emission was read on a cytofluorograph II and the percentage of cells specifically expressing each surface molecule calculated (W cells staining with first antibody - % cells staining with second antibody only). b Data shown are means + SE, n = 4.

Next, antibodies directed against these adhesion molecules were preincubated with monocytes or alveolar macrophages and then the antibodies and monocytes added to lymphocytes and incorporation of [3H]thymidine assessed (Fig. 2). Antibody against fibroblast growth factor was used as an isotype-matched irrelevant control antibody. Anti-FGF antibody did not inhibit the accessory function of either monocytes or alveolar macrophages (Fig. 2). In cultures of monocytes, antibodies to both Mac-l and ICAMdecreased lymphocyte responses to SEB, but the inhibition was not significant (P < 0.1). Anti-LFA- 1LYand LFA- 1fl added to monocytes both significantly inhibited the blastogenic responses (P < 0.05). Mac-l did not decrease the accessory




a-FGF a-Mac-l a-ICAM-I a-LFA-Ia a-LFA-lb 0









FIG. 2. Effect of antibodies against adhesion molecules on accessory function of monocytes and alveolar macrophages for lymphocyte responses to SEB. The blastogenic response of SEB-stimulated lymphocytes ( IO5 cells) to which 50% monocytes or alveolar macrophages were added is expressed as 100% response to SEB. Antibodies were added at a concentration of 40 @g/ml to monocytes or alveolar macrophages for 1 hr and then the monocytes and antibodies were added to the lymphocytes. Antibody against FGF was used as a control antibody. Data are expressed as percentage of optima1 response (100%) + SE, n = 4. The response of lymphocytes to SEB was significantly decreased when anti-LFA-la and anti-LFA-l/3 were added to cocultures of monocytes and lymphocytes (P < 0.05) and by anti-LFA-la (P < 0.05) anti-LFA-l/3 (P < O.OOS),and anti-ICAM- 1 (P < 0.05) when added to cocultures of alveolar macrophages and lymphocytes. Anti-FGF and anti-Mac- I did not significantly inhibit responses by lymphocytes to SEB when either monocytes or alveolar macrophages were added back.




function of alveolar macrophages. As with monocytes, anti-LFA-lcw and anti-LFAl@ decreased accessory function of alveolar macrophages (P < 0.05 and 0.005, respectively). The decreased accessory function of alveolar macrophages for lymphocyte responses to SEB in the presence of anti-ICAM- 1 was, however, statistically significant (P < 0.05). Cell adhesion molecules are required for the development of clusters observed when lymphocytes are activated with certain stimuli (48). To further evaluate the role of cell adhesion molecules in accessory function for SEB, the effect of antibodies against ICAM- 1, LFA- 1(Y, and LFA- 1p and, as a control, anti-Mac- 1 on the blastogenic response and on cluster formation of PBMC to SEB was examined. Incorporation of [3H]thymidine in PBMC stimulated with SEB (1 pg/ml) was inhibited by antibodies to cell adhesion molecules: percentage of suppression in the presence of anti-ICAM1 was 52 k 14%; anti-LFA- 1(Ywas 42 + 13%; anti-LFA- 1p was 48 +- 11%; and antiMac-l was 10 5 4%, y1 = 3. Cluster formation was inhibited similarly. Unstimulated cells showed no clusters (Fig. 3). Clusters began to form within 4 hr after stimulation with SEB but were not clearly distinct until 12 hr; enlarged likely through cell proliferation between 24 and 36 hr; and remained for the duration of the 72-hr culture period. SEB-stimulated cells demonstrated that approximately 50% of the cells were in clusters. Few cells were observed to be in clusters in SEB-stimulated cells to which antibodies against ICAM-1, LFA-la, and LFA-l/3 had been added (lo-20% of the cells in clusters). The number of cells in clusters observed when SEB-stimulated cells were cultured in the presence of anti-Mac-l was similar to the number seen with SEB alone. DISCUSSION The role of cytokines and cell adhesion molecules in accessory function for T cell responses to the superantigen SEB was examined using monocytes and alveolar macrophages as accessory cells. We found that both monocytes and alveolar macrophages functioned as accessory cells for lymphocyte responses to SEB and that, at limiting numbers of cells, accessory function of alveolar macrophages was greater than that of monocytes for this stimulus. The cytokines IL-l cx and IL- lp and the cell adhesion antigens ICAM- and LFA-1 (CXand 0) functioned as accessory molecules for SEB. SE, such as SEA and SEB, have long been recognized as potent stimuli of T lymphocytes (49, 50) and have received much investigative attention as regards their mechanism of action. Peavy et al. suggested many years ago that SEB acts more like a mitogen than an antigen stimulating a large percentage of T cells in vitro (5 1). Carlsson et al. demonstrated that unlike antigens, processing of SEA is not required but that the enterotoxin must bind to accessory cells for activation of T cells to occur (31). Fleischer et al. confirmed that processing of enterotoxins is not required and showed further that expression of autologous or allogeneic Class II MHC molecules on either the accessory cell or target cell is required (30). Direct binding of SEA and SEB to Class II MHC was shown by Fraser (33); and binding of SEA to Class II MHC to form a complex that interacts with the T cell receptor was demonstrated by Mollick and colleagues (32). The role of cytokines in SE-induced T cell activation has not been elucidated. Ikejima et al. demonstrated that culture supernatants of strains of Staphylococcus aureus causing toxic shock syndrome are potent inducers of IL-l, whereas those of




FIG. 3. Effect of antibodies against adhesion molecules on cluster formation of SEB-stimulated PBMC. PBMC were incubated without SEB (A) or with SEB (I &ml) (B). Alternatively, antibodies against cell adhesion molecules and Mac-l were added to SEB-stimulated cells: anti-ICAM(C), anti-LFA-lol (D), anti-LFA-IP (E), and anti-Mac-l (F). Photomicrographs were taken after cells were cultured at 37°C for 2 days. The concentration of antibodies used was 40 &ml. Magnification of original is 15X. A camera artifact is carried over in the photographs and should be ignored




FIG. 3-Conrinwd

strains not causing this syndrome do not stimulate production of this cytokine (52). Other studies have demonstrated that purified toxic shock syndrome toxin- 1 stimulated IL-l production by monocytes (53,54). SEA stimulates IL-1 production through par-






ticipation of T cells (55). Not surprisingly, we found that SEB stimulates IL- 1. The relevance of this finding to T cell activation by SEB then was investigated. The concentration of SEB stimulating IL- 1 production by accessory cells was higher than the concentration in which accessory function and T cell activation was first observed.




This apparent discrepancy may be attributed to the fact that levels of IL-l sufficient for accessory function are at or below the sensitivity of the bioassay used. Another possibility is that more IL- 1 may be produced when accessory cells and T cells are in coculture. In this regard, PBMC produced more IL-l in response to SEB than did monocytes or alveolar macrophages cultured separately. Alternatively, surface IL- 1 relevant to accessory function which would not be detected in the supernatants used for the IL-l assays was conceivably sufficient to provide accessory cell activity. The evidence that IL-l is required for SEB-stimulated activation of T cells is that SEB-induced IL- 1 production and antibodies against IL- 1u and IL- 1p inhibited accessory cell function and this function was reconstituted with recombinant cytokines. Interestingly, antibodies against both IL-la and IL-lp were required for inhibition but either recombinant IL-la or IL-l/3 reconstituted the response indicating that if either cytokine was present accessory function was manifest. Alveolar macrophages did not secrete more IL- 1 activity in response to SEB than monocytes so the increased accessory function for SEB noted at limiting numbers of alveolar macrophages could not be explained on the basis of differences in production of IL- 1. IL-6 was produced constitutively by both monocytes and alveolar macrophages and SEB did not stimulate further secretion of IL-6. Furthermore, antibodies to IL-6 alone or in combination with those against IL- 1(Yand IL- 1p did not decrease accessory cell function. Therefore, IL-6 had no discernable role as an accessory molecule for SEB-induced T cell activation. Although up to 50 pg/ml of anti-IL-6 was used, we cannot exclude, however, that higher concentrations of antibodies would have inhibited accessory cell function. Also apparent from these studies, cytokines alone could not substitute for accessory cells for lymphocyte responses to SEB. Recombinant IL- 1a!, IL- lp, and IL-6 added singly, in combinations of two, or all three did not stimulate accessory cell-dependent T cells to respond to SEB in the absence of added back accessory cells. Kotb et al. demonstrated accessory cell independence of T cells for the superantigen streptococcal M and SEB when the combination of PMA, IL- 1, and IL-6 was used (34). Considering these data with our own suggests to us that PMA may have been the key molecule in the stimulation of T cells noted by Kotb et al. and that PMA but not natural cytokines may substitute for accessory cells in the response of T cells to SEB. Continuous culture with the antibodies against IL-la and IL-lp was deemed necessary since production and secretion of cytokines is a dynamic process such that short exposure of antibodies to either cell type alone would likely have had either little or irrelevant effects. Since the antibodies were present throughout the period of culture, it is not certain whether these antibodies were acting specifically on products of mononuclear phagocytes or of lymphocytes. The lymphocytes were dependent on the accessory function of added monocytes. IL- la and IL-ID are expressed little if at all by T lymphocytes, whereas monocytes express high levels such that neutralization of responses to SEB by antibodies against these cytokines was likely an effect on products of the monocytes in culture. IL-6 is expressed by both monocytes and lymphocytes but SEB did not stimulate IL-6 secretion at least at the concentrations tested nor did anti-IL-6 block accessory cell function. The cell adhesion molecules ICAMand LFA-1 (both the CYand the /3 subunits) also served as accessory molecules for SEB-induced responses as demonstrated by their expression by monocytes and alveolar macrophages and by inhibition of accessory function using antibodies directed against them. LFA-1 is a member of the integrin family of cell adhesion molecules and has a broad distribution and multiple functions




(21). Monoclonal antibodies to either the LFA-la or -/3 chain inhibit a wide variety of adhesion-dependent leukocyte functions including cytolysis of cells bearing foreign antigens and helper T cell functions (21, 29). Monoclonal antibodies against LFA-1 inhibit T cell activation when cell-cell interaction is required between antigen presenting cells and T cells or T and B cells for mitogenic stimuli including PHA, PMA, and Con A and antigenic stimuli including alloantigens, tetanus toxoid, diphtheria toxoid, and PPD (21, 29, 56-58). There also is strong evidence that LFA-1 plays a role in signal transduction (29). In the current study, monoclonal antibodies against both LFA-la and LFA- lp but not Mac-l inhibited the response of T cells to SEB when either monocytes or alveolar macrophages were used as accessory cells. When added directly to PBMC, anti-LFA- 1cyand anti-LFA- 10 inhibited blastogenesis and cluster formation of SEB-stimulated PBMC. Since the monoclonal antibodies against LFA-1 were present continuously in culture, it is not possible to conclude whether the inhibition of blastogenesis or cluster formation occurred by reaction with accessory cells and/or responder cells. Since T cells failed to respond to SEB without added accessory cells, the data suggest, however, that antibodies to these cell adhesion molecules were directed at least in part to antigens expressed on mononuclear phagocytes. ICAMis one of the ligands for LFA-1 (23). The role of ICAMin immune or inflammatory responses is suggested by the finding that IL- 1 and interferon-y increase ICAM- 1 expression on fibroblasts (59). We found that alveolar macrophages expressed significantly higher levels of ICAM- I than did monocytes. Antibodies against ICAM1 inhibited accessory function of alveolar macrophages; inhibition of accessory function of monocytes by anti-1CAM-l was not statistically significant perhaps because of the large standard deviation noted. It is possible that the observed increased accessory function of alveolar macrophages as compared to monocytes could be attributed to the increased basal expression of ICAM- by these tissue macrophages. On the other hand, it is possible that ICAM- 1 on monocytes also may be an accessory molecule for SEB. Antibodies against ICAM- 1 inhibited responses of PBMC to SEB. Expression of ICAM- 1 is modulated by adherence and by cytokines (IL- 1, interferon-y) themselves likely generated during the response to SEB. Thus, the expression of ICAMunder basal conditions may not adequately reflect levels expressed during culture. ICAMalso is a ligand for LFA- 1 and may be expressed by monocytes but this possibility was not investigated (60). These many considerations, notwithstanding, it is clear that the cell adhesion molecules ICAM- 1 and LFA- 1 are required for maximal stimulation of T cells by SEB in the presence of accessory cells. Superantigens such as SE which are potent T cell stimulators have been implicated as pathogenetic in toxic shock syndrome, food poisoning, and also autoimmune diseases. Other bacterial products considered to be superantigens have been implicated in the causation of autoimmune diseases; for example, Klebsiella sp. in ankylosing spondylitis (6 1) and streptococcal M protein in rheumatic fever and rheumatic heart disease (62). Thus, the clinical significance of superantigens is becoming more and more apparent. The clinical importance of superantigens and their intrigue as models to better understand the immune system provide ample reasons to understand further their mechanisms of action. Our study indicates that certain cytokines and cell adhesion antigens known to be important accessory molecules for antigens and mitogens also have an obligatory role in the response of lymphocytes to the superantigen SEB.




ACKNOWLEDGMENTS We are grateful to Dr. Robert Rothlein, Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT) for providing the monoclonal antibodies against ICAM-1, LFA-la, LFA-I& and MAC-la. The B9 murine plasmacytoma cell line was the kind gift of Dr. L. Aarden, University of Amsterdam, The Netherlands. We also thank Cecily Lewis for secretarial assistance and Dr. Jerrold J. Ellner for his critique of this manuscript.

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Accessory function of human mononuclear phagocytes for lymphocyte responses to the superantigen staphylococcal enterotoxin B.

The role of the cytokines IL-1 alpha, IL-1 beta, and IL-6 and the cell adhesion molecules ICAM-1, LFA-1 (alpha and beta), and Mac-1 as accessory molec...
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