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The Journal of Immunology, 1999, 162: 2299-2307.
Copyright © 1999 by The American Association of Immunologists

Bacterial Superantigens Induce Down-Modulation of CC Chemokine Responsiveness in Human Monocytes Via an Alternative Chemokine Ligand-Independent Mechanism1

Rahbar Rahimpour*,{ddagger}, Gordon Mitchell{dagger},{ddagger}, Masud H. Khandaker*,{ddagger}, Chen Kong{ddagger}, Bhagirath Singh*, Luoling Xu{ddagger}, Atsuo Ochi§, Ross D. Feldman{dagger}, J. Geoffrey Pickering{dagger}, Bruce M. Gill{ddagger} and David J. Kelvin2,*,{ddagger}

Departments of * Microbiology and Immunology and {dagger} Physiology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada; and {ddagger} Laboratory of Molecular Immunology and Inflammation and § The John P. Robarts Research Institute, London, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylococcal superantigens (SAgs) are very potent T cell mitogens, but they can also activate monocytes by binding directly to MHC class II molecules in a manner independent of TCR coengagement. Induction of proinflammatory cytokines and chemokine expression in monocytes by superantigens has recently been reported. Here we report that superantigen stimulation of human peripheral blood monocytes results in a rapid, dose-dependent, and specific down-regulation of chemokine (macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), monocyte chemotactic protein-1 and MIP-1ß) binding sites (e.g., CCR1, CCR2, and CCR5), which correlates with a concomitant hyporesponsiveness of human monocytes to these CC chemokine ligands. This down-regulation occurs 15–30 min following superantigen stimulation and is specific to chemokine receptors, in that binding and responsiveness of monocytes to the chemoattractant formyl-tripeptide FMLP are not affected. We further demonstrate that SAg-induced down-modulation of chemokine binding and monocyte hyporesponsiveness to the chemokines MIP-1{alpha}, monocyte chemotactic protein-1, and MIP-1ß is mediated through cellular protein tyrosine kinases, and the down-modulation can be mimicked by an MHC class II-specific mAb. Additionally, our observations indicate that SAg-induced loss of chemokine binding and monocyte responsiveness is probably mediated by secreted serine proteinases. Bacterial SAg-induced down-modulation of chemokine responsiveness represents a previously unrecognized strategy by some bacteria to subvert immune responses by affecting the intricate balance between chemokine and chemokine receptor expression and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukocyte extravasation from the circulation into infected and inflamed tissue is an essential facet of a functional immune system. The interaction between chemoattractant cytokines (chemokines) and their respective target cell receptors plays a pivotal role in this process. Chemokines are a superfamily of small molecular mass (8–12 kDa) proteins that have been implicated in bacterial and viral pathogenesis 1 . These molecules have been grouped into two main subfamilies of CXC and CC, based on the spacing between the first two of the four conserved cysteines. The recent discoveries of lymphotactin 2 and neurotactin (fractalkine) 2, 3 have led to further expansion of the chemokine family into four subfamilies of CXC, CC, C, and CX3C. Further categorization within the major subfamilies of CXC and CC is based on the functional and structural characteristics of their respective members. Members of the CC chemokine subfamily, which include the monocyte chemotactic proteins (MCP-13 to MCP-5), macrophage inflammatory proteins (MIP-1{alpha}, MIP-1ß, and MIP-1{gamma}), and RANTES, are known to attract and activate monocytes and lymphocytes 1, 4 , while members of the CXC subfamily primarily attract neutrophils. Chemokine expression is induced by bacterial endotoxin and inflammatory cytokines (IL-1, TNF-{alpha}, and IL-6) 5, 6, 7 and can be inhibited by anti-inflammatory cytokines or hormones such as IL-10 and glucocorticoids 8 . Recent discoveries have shown that members of the CC subfamily of chemokines have an inhibitory role in HIV infection 9, 10, 11 .

Chemokine function is mediated through specific cell surface receptors. These receptors are members of the seven transmembrane domain rhodopsin-like family of receptors that are coupled to GTP binding proteins in their inactive state 12, 13 . Ligand binding results in the dissociation of G proteins leading to activation of a variety of downstream signal transduction pathways that entail intracellular calcium mobilization, mitogen-activated protein kinase activation, and STAT protein activation 14, 15, 16 . The end result of these signals is cytoskeletal changes that initiate the migration of leukocytes toward the chemokine gradient. The migration of leukocytes into infected/damaged tissue is an essential part of the defense and repair process. Selected pathogenic micro-organisms have developed means by which they are able to intervene with this process.

A common strategy for bacteria, viruses, and other micro-organisms that induce tissue damage, and hence an immune response, is to evade immune surveillance and function by producing a variety of molecules that subvert immune responses 17 . Bacteria such as Bordatella pertussis produce exotoxins that block the signal transduction of G protein-coupled chemotactic receptors 18, 19 , while endotoxins from Gram-negative bacteria down-regulate CXC and CC chemokine receptor expression thereby impairing immunomodulatory functions of human neutrophils and monocytes 20, 21, 22 . Given the importance of chemokine-mediated activation in regulating neutrophil and monocyte migration and function, we hypothesized that Gram-positive bacteria, which lack the endotoxin LPS, have evolved mechanisms for blocking chemokine receptor-mediated functions of monocytes and phagocytes as a possible strategy for immunologic evasion. We tested this hypothesis by examining the effect of superantigenic staphylococcal enterotoxin stimulation of monocytes on cellular chemokine receptor expression and function.

Staphylococcal superantigenic enterotoxins (staphylococcal enterotoxins A, B, C, D, E, and H) are a family of small molecular mass (22–30 kDa) proteins that are produced by the Gram-positive bacteria Staphylococcus aureus 23, 24, 25 and have been implicated as virulence factors during enteric infections. Contrary to the classical Ags, superantigens (SAgs) do not undergo intracellular Ag processing but directly engage both MHC class II and the TCR outside their respective peptide-binding grooves 26, 27 .

Here we investigated whether SAg stimulation of human monocytes could modulate the cell surface expression of chemokine receptors and hence alter monocyte responses to chemokines. Our findings indicate that bacteria-derived SAgs have the ability to activate protein tyrosine kinase-dependent pathways upon engagement of MHC class II, which induces a rapid, agonist-independent mechanism of down-modulating chemokine receptors and responsiveness of monocytes to chemokine ligands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Staphylococcal enterotoxins (SEA and SEB) were purchased from Sigma (St. Louis, MO). Anti-HLA DR (DK22) Ab and the isotype match control Ab (IgG1) were purchased from Dako (Carpinteria, CA). Anti-SEB mAb was purchased from Toxin Technology (Gainesville, FL). Recombinant chemokines were purchased from PeproTech (Rocky Hills, NJ). 125I-labeled chemokines were obtained from Mandel Scientific (Guelph, Canada). Proteinase inhibitors were purchased from Sigma, except for Pefabloc which was obtained from Boehringer Mannheim (Indianapolis, IN). Culture medium consisted of RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, and 1.5% penicillin/streptomycin/fungizone mixture (BioWhittaker, Rockville, MD). PBS and HBSS were also purchased from BioWhittaker.

Isolation of leukocytes

Human leukocytes were isolated from freshly drawn heparinized blood from randomly selected healthy donors. PBMC populations were obtained as described previously 20 . Total cells were overlaid on an equal volume of Histopaque (Sigma) and centrifuged at 1600 rpm for 30 min at room temperature. Buffy interface cells (PBMC) were collected and washed in isotonic PBS. The PBMC population was further enriched by 46% (v/v) Percoll gradient (Pharmacia, Piscataway, NJ) separation. Cells were washed in PBS and resuspended in RPMI 1640 (10% FBS; BioWhittaker), and viable cells were enumerated by vital staining (trypan blue exclusion). The resultant cell population was assessed for purity of isolated cells by morphology and FACS analysis and was typically comprised of >98% monocytes (Mac-1+, HLA-DR+, CD14+, CD4+, CD16+, CD32+).

Binding assay

Cells (2 x 106/sample) were resuspended in 200 µl of binding medium (RPMI 1640/1% BSA) and incubated for 1 h at room temperature with 50,000 cpm (144 pg) 125I-labeled chemokine ligand (Mandel Scientific) with or without a 100-fold excess of competitive unlabeled (cold) ligand to measure nonspecific binding. Cells were then spun (12,000 rpm, 1 min) through an 800-µl cushion of 10% (w/v) sucrose in PBS. The pellet was dried and then counted using an LKB gamma counter (Fisher, Pittsburgh, PA). Specific binding was then calculated by subtracting the nonspecific counts from the total counts.

Migration assay

Cell migration was determined in a microchemotaxis chamber (Neuroprobe, Cabin John, MD). Cells were resuspended at 1.5 x 106 cells/ml in the migration medium (RPMI 1640 (without L-glutamine, 2-ME, or antibiotics) with 25 mM HEPES, and 1% (w/v) BSA (Sigma)). Chemokines were dissolved in migration medium at 10 nM (MCP-1 and MIP-1{alpha}), 20 nM (MIP-1ß), and 10-7 M FMLP, and 28 µl was placed in the bottom chamber wells. The negative control wells only contained the migration medium. The chemotaxis chamber was then assembled with a 0.5-µm pore size polycarbonate membrane and a silicon gasket separating the upper from the lower wells (Neuroprobe). The upper wells were filled with 50 µl of the resuspended cells. The chamber was then incubated in a humidified 37°C, 5% CO2 incubator for 1.5 h. Nonmigrating cells were removed from the cell suspension side of the membrane using PBS. Membranes were subsequently stained using a Diff-Quick staining kit (International Reagents, Gibbstown, NJ). Migrated cells were enumerated from three randomly selected, high power, oil immersion fields.

Calcium mobilization assay

Stimulated cells were labeled with a 3-µM final concentration of indo-1/AM (Sigma) for 30 min at 37°C. Cells were washed thoroughly and resuspended in HBSS at 5 x 106/ml. Cells were then stimulated with chemokines at the concentrations described in figure legends. The mobilization of calcium was measured using a 355-nm wavelength for excitation and 405- and 485-nm wavelengths for emission using a dual wavelength fluorometer (model RF-M2004, Photon Technology International, Indianapolis, IN). The data are presented as relative fluorescence.

Cytokine quantification

Human PBMCs were either stimulated with SEB (1 µg/ml) or left unstimulated and incubated at 37°C for different times in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, and 1.5% penicillin/streptomycin/fungizone mixture. Supernatants were collected and stored at -20°C. Cytokine levels were quantified using an ELISA kit (Quantikine, R&D Systems, Stillwater, MN) according to the manufacturer’s protocol. Standard curves were generated, and concentrations of cytokines were calculated as picograms per milliliter of culture supernatant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SAg stimulation of human monocytes induced down-modulation of cell-surface chemokine binding sites

To investigate whether the stimulation of human monocytes with bacterial SAgs would alter ligand binding to cell surface chemokine receptors, we first examined whether treatment of purified PBMCs with SEA or SEB would modulate cell surface binding of MIP-1{alpha}, MCP-1, and MIP-1ß. Our observations show that the treatment of PBMCs with either SEA or SEB resulted in an SAg dose-dependent inhibition of cell surface binding for 125I-labeled MIP-1{alpha}, MCP-1, and MIP-1ß to PBMCs (Fig. 1Go). Since MIP-1{alpha} binds to CCR1 and CCR5 10, 28, 29 , MCP-1 binds to CCR2 30 , and MIP-1ß binds to the CCR5 receptor 10, 31 , the data presented in Fig. 1Go indicate that both SEA and SEB induce the modification of chemokine binding sites within CCR2, CCR5, and possibly CCR1. We next examined the kinetics of SAg-induced down-regulation of chemokine binding and found that SAg treatment of PBMCs significantly reduced MIP-1{alpha}, MCP-1, and MIP-1ß binding (Fig. 1GoD) within 15–30 min posttreatment.



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  		FIGURE 1. SAg-induced down-modulation of chemokine binding to human PBMCs. SAg stimulation induced a dose-dependent inhibition of binding of 125I-labeled chemokines MIP-1{alpha} (A), MCP-1 (B), and MIP-1ß (C) to the monocyte cell surface. Freshly isolated human PBMCs were stimulated for 2 h at 37°C with 0.1, 1, or 10 µg/ml of either SEA or SEB. Binding of radioiodinated chemokines was then measured in a binding assay. SEB-induced inhibition of binding occurs rapidly within 15–30 min posttreatment (D). One microgram per milliliter of SEA or SEB was used in D. After stimulation, specific binding of chemokine ligand was measured as stated in Materials and Methods. The data shown are representative of five independent experiments performed in triplicate.

 
To determine the specificity of SAg-induced down-regulation of chemokine receptors, we examined the effect of SEA and SEB on binding of [3H]FMLP tripeptide to PBMCs. Although not a member of the chemokine superfamily, this peptide is a very potent leukocyte chemoattractant and is known to signal through a seven-transmembrane, G protein-coupled receptor 32 . SAg stimulation had no effect on [3H]FMLP binding (Fig. 2Go), indicating that SAg down-modulation is restricted to a subset of chemotactic seven-transmembrane domain receptors.



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  		FIGURE 2. Effect of SAg or MHC class II-specific mAb stimulation on FMLP binding to its cell surface receptor. Purified human PBMCs (107 cells) were stimulated with SEA (1 µg/ml), SEB (1 µg/ml), or DK22 (20 ng/ml) or were left unstimulated (Medium) for 2 h at 37°C. Cells were washed thoroughly, and specific cell surface binding of [3H]FMLP was determined as described in Materials and Methods. The mean value and the SEM from four separate experiments are shown.

 
SAg stimulation diminishes chemokine receptor-mediated monocytic functions

Chemokines bind their respective receptors and induce a signaling cascade manifested by transient elevations in the intracellular calcium concentration. Therefore, to assess the effect of SAg stimulation on chemokine-induced monocytic responsiveness, we next examined whether SAg stimulation of purified PBMCs would modulate chemokine-induced Ca2+ mobilization or chemokine-directed chemotaxis in these cells. Using standard chemotaxis assays 33 , we observed that SAg pretreatment of purified PBMCs significantly antagonized directed migration of monocytes to MIP-1{alpha}, MCP-1, and MIP-1ß (Table IGo). SAg stimulation of human PBMCs did not alter their FMLP-directed migration (data not shown). To further investigate SAg-induced modulation of functional chemokine responses, we also examined the effect of SAg stimulation on chemokine-induced Ca2+ mobilization in monocytes. SEA or SEB stimulation of PBMCs for 1 h resulted in the abrogation of MIP-1{alpha}- and MCP-1-induced Ca2+ mobilization, but did not alter Ca2+ mobilization induced by FMLP (Fig. 3Go). Similar data were obtained for MIP-1ß-induced calcium mobilization.


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  		Table I. SEB suppresses chemokine-directed monocyte migration1

 


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  		FIGURE 3. Analysis of chemokine-induced intracellular calcium mobilization in SAg or MHC class II-specific mAb stimulation of human PBMCs. Fresh PBMCs (107/ml) were stimulated with SEA (1 µg/ml), SEB (1 µg/ml), DK22 (20 ng/ml), or medium as the control for 1 h at 37°C. Cells were loaded with indo-1/AM (3 µM) for 30 min at 37°C and then washed and suspended in HBSS. MIP-1{alpha} (10 ng/ml), MCP-1 (10 ng/ml), or FMLP (10-7 M)-induced Ca2+ mobilization in these cells was then measured in a fluorometer. Representative results from four separate identical experiments are shown.

 
SAg effects are mediated through MHC class II and require PTK-dependent signaling

SEA and SEB have been shown to bind HLA-DR, -DP, -DQ, and -DM haplotypes of MHC class II 24, 34 . If the SAg-induced down-regulation of both chemokine receptor binding and chemokine directed responsiveness is the result of PBMC activation by SAg binding directly to MHC class II, then we would hypothesize that activation of monocytes with MHC class II-specific Abs should mimic the effects of SEA and SEB 24, 34 . Pretreatment of PBMCs with the anti-HLA-DR mAb DK22 resulted in a dose-dependent inhibition of MIP-1{alpha}, MCP-1, and MIP-1ß binding (Fig. 4Go, A–C), dose-dependent inhibition of chemokine-induced migration (Fig. 4GoD), and ablation of chemokine-stimulated Ca2+ mobilization (Fig. 3Go). Treatment with an isotype-matched control Ig had no effect on chemokine binding or function. Data obtained on kinetics of the inhibitory action of the anti-HLA-DR Ab (data not shown) correlated with those shown for SAgs.



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  		FIGURE 4. Dose-dependent down-modulation of chemokine binding to human monocytes following stimulation with MHC class II-specific Ab. Cells (107/ml) were stimulated with 2, 20, and 200 ng/ml of MHC class II-specific mAb (mouse IgG2a anti-HLA DR, DK22) for 2 h at 37°C. Specific binding of 125I-labeled MIP-1{alpha} (A), MCP-1 (B), and MIP-1ß (C) was measured in comparison with the effect of an isotype-matched control (IgG2a, 200 ng/ml) Ab as described in Materials and Methods. The data shown are representative of five separate identical experiments.

 
SEB engagement of MHC class II stimulates a signal transduction cascade involving protein kinase C and protein tyrosine kinase (PTK) activation 35 . SAg-induced tyrosine phosphorylation of ZAP-70 and stimulation of p72syk have been reported as early as 1–3 min posttreatment 35 . To investigate whether the SAg-induced down-regulation of chemokine binding and chemokine-directed responsiveness is dependent on PTK signaling, we tested the ability of PTK inhibitors to antagonize SAg down-modulation of both chemokine binding and chemokine-mediated intracellular Ca2+ mobilization. Preincubation of freshly isolated human PBMCs with the PTK inhibitor genistein (30 µg/ml) or herbimycin A (5 µM) for 30 min before SAg stimulation abrogated the SEB-induced down-regulation of MCP-1 binding (Fig. 5Go) as well as MIP-1{alpha} and MIP-1ß binding (data not shown). Similarly, anti-HLA-DR (DK22) Ab-mediated receptor down-modulation was antagonized by the PTK inhibitors (data not shown). PTK inhibitors also blocked the SAg-induced reduction of monocyte responsiveness to chemokine stimulation. Subsequent experiments demonstrated that genistein (Fig. 6Go) and herbimycin A (data not shown) markedly attenuated the SAg impairment of MCP-1-induced Ca2+ mobilization.



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  		FIGURE 5. Effect of protein tyrosine kinase inhibitors on SEB-induced down-regulation of MCP-1 binding. Cells (107/ml) were pretreated with genistein (Gen; 30 µg/ml) or herbimycin A (HmA; 5 µM) for 30 min before stimulation with SEB (1 µg/ml) for an additional 1 h. Specific binding of radioiodinated MCP-1 was then assessed by standard binding assay. The mean and SD from five separate identical experiments are shown. p < 0.001 represents a statistical difference compared with the control nonstimulated group.

 


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  		FIGURE 6. Genistein blocked SEB-induced inhibition of calcium mobilization in response to MCP-1. Freshly isolated PBMCs (107/ml) were left untreated or were pretreated with genistein (30 µg/ml) for 30 min before SEB stimulation (1 µg/ml) for 1 h at 37°C. Cells were washed twice in HBSS, and MCP-1-induced (10 ng/ml) and FMLP-induced (10-7 M) intracellular mobilization of calcium was assessed as described previously. The data shown are representative of three separate identical experiments.

 
Evidence for an agonist-independent mechanism in superantigen-induced down-regulation of receptors

Previous studies of chemokine receptor regulation have indicated that ligand binding-induced sequestration of chemokine receptors involves the phosphorylation of receptor carboxyl-terminal serine/threonine residues, resulting in the internalization of bound receptors 36, 37, 38 . To determine whether SAg actions are mechanistically mediated by agonist-induced receptor sequestration, such that SAg stimulation elicits a substantial release of chemokines that could effectively alter chemokine binding and chemokine-directed responsiveness, we examined the kinetics of chemokine and cytokine secretion from SEB-stimulated PBMCs. Fig. 7Go shows that SEB stimulation of PBMCs resulted in increased secretion of MCP-1, TNF-{alpha}, and IL-1, but only 2–4 h after SEB treatment. These results indicate that down-regulation of chemokine binding or chemokine-directed responsiveness of monocytes is not due to the effect of released chemokines or cytokines (MCP-1, TNF, and IL-1), since the SAg-induced down-regulation of binding and responsiveness occurs before (by 30 min or less) any observable increase in MCP-1, TNF-{alpha}, and IL-1ß expression. In addition, dose-response experiments using exogenously added MCP-1 to determine the effective ligand concentration required to induce receptor sequestration were performed. In these experiments MCP-1 ligand concentrations had to exceed 0.5 ng/ml in culture to begin to significantly diminish iodinated ligand binding (Fig. 8Go). This effective concentration is substantially greater than what was detected by the MCP-1-specific ELISA in cell culture medium after 2 h of SEB stimulation. Thus, these observations strongly suggest that the SAg-induced down-regulation of chemokine receptors employs an alternative pathway, distinct from the recognized ligand/agonist-dependent sequestration pathway.



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  		FIGURE 7. Analysis of MCP-1, TNF-{alpha}, and IL-1ß concentrations in the SEB-stimulated PBMC cell culture supernatants. Fresh PBMCs (107/ml) were stimulated with SEB (1 µg/ml) for the times indicated at 37°C. Cell culture supernatants were saved, and the concentration of each cytokine was measured in triplicate using the Quantikine (R&D Systems) ELISA system. The mean values and the corresponding SDs from three separate experiments are shown.

 


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  		FIGURE 8. Effect of increasing concentrations of MCP-1 on CCR2 sequestration in monocytes. Fresh human PBMCs (107/ml) were treated with increasing concentrations of MCP-1 for 30 min at 37°C. Cells were then washed thoroughly and used in a standard binding assay as previously described. Mean values for MCP-1-specific binding and the corresponding SEM from three separate experiments are shown. p < 0.01 represents a significant difference compared with the control group, in which no MCP-1 was added.

 
Chemokine receptor down-regulation is mediated by secreted serine proteinases

Based on the evidence that SAg engagement of MHC class II initiates rapid down-modulation of surface chemokine binding, in a time frame that precludes agonist-dependent mechanisms, we hypothesized SAg could be activating a proteolytic pathway that targets monocyte chemokine receptors for degradation. Proteinase-dependent cleavage and shedding of cell surface receptors from leukocytes following their activation have been well documented 39, 40, 41 . Therefore, we examined the involvement of cellular proteinases by treating purified PBMCs with a variety of proteinase inhibitors.

SEB-mediated reduction of chemokine binding was inhibited by pretreating cells with various serine proteinase inhibitors (Pefabloc, chymostatin, N-{alpha}-p-tosyl-L-lysine chloromethyl ketone (TLCK), 3,4-dichloroisocoumarin), which contrasts with the lack of inhibition by the metalloproteinase inhibitor 1,10-phenanthroline (Fig. 9Go). These findings demonstrated that certain serine proteinase inhibitors effectively attenuated the SAg-induced down-modulation of chemokine receptors. However, the serine proteinase inhibitor leupeptin only moderately attenuated the SEB-induced down-modulation of MCP-1 binding (Fig. 9Go), suggesting the participation of a discrete subset of serine proteinases in the SAg-induced down-regulation of receptors.



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  		FIGURE 9. Effect of pretreatment of PBMCs with proteinase inhibitors on SEB-induced down-regulation of MCP-1 binding. Human monocytes (107 cells/ml) were pretreated with each serine proteinase inhibitor (Pefabloc, chymostatin, and leupeptin) or with the metalloproteinase inhibitor 1,10-phenanthroline (1,10-phen) at the indicated concentrations for 30 min before SEB stimulation (1 µg/ml) for 1 h at 37°C. Specific binding of radioiodinated MCP-1 was then assessed in a binding assay. The mean and the corresponding SEM from three separate experiments are shown. p < 0.01 represents statistical differences compared with the SEB-stimulated group.

 
To address the question of whether receptor degradation was facilitated by secreted proteinases, our initial experimental approach was formulated from the data shown above (Figs. 5Go and 6Go) that indicated that inhibition of PTK activity effectively blocked the SAg-induced chemokine receptor down-modulation and monocyte responsiveness to the cognate chemokines. Unstimulated monocyte cultures were pretreated with genistein before replacing the culture medium with conditioned medium obtained from cells incubated for 1 h in the presence or the absence of SEB. The results (Fig. 10Go) show that pretreatment of monocytes with PTK inhibitors before their exposure to conditioned culture medium from SAg-stimulated PBMCs did not inhibit the SAg down-modulation of MCP-1 binding (CCR2). Furthermore, the lack of down-modulation of chemokine binding by conditioned medium from unstimulated (control) monocyte cultures indicates that SAg stimulation is required to generate sufficient release of binding suppression activity in the culture medium. A plausible explanation for these data, which differs from the inhibition of ligand binding loss observed in Fig. 5Go, is that the conditioned culture medium contains sufficient receptor-degrading proteinases released by the primary cell culture upon SEB stimulation. Additionally, conditioned medium obtained from monocytes incubated for 1 h in the presence of SEB and PTK inhibitors and then added to fresh monocytes did not reduce MCP-1 binding (data not shown), further confirming that PTK inhibitors prevent the ability of SEB-conditioned medium to reduce chemokine binding in monocytes.



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  		FIGURE 10. Effect of culture supernatant from SEB-stimulated human monocytes on MCP-1 binding to fresh monocytes. Human PBMCs (107/ml) were treated with SEB (1 µg/ml) for 1 h at 37°C. The cell culture supernatant was collected and added to fresh monocytes (107/ml) that had been pretreated with PTK inhibitor (genistein (Gen), 30 µg/ml; herbimycin A (HmA), 5 µM) and incubated for 1 h at 37°C. Specific binding of radioiodinated MCP-1 was then measured. The mean and SEM from three separate experiments are shown. p < 0.001 represents a statistical difference compared with the control group, which was treated with supernatant (S/N) from unstimulated monocytes. The differences between bound MCP-1 for the two groups (HmA+S/N and Gen+S/N) were not statistically different.

 
To further verify that the SAg-mediated reduction of chemokine binding is probably due to the secretion of one or more proteinases that target select chemokine receptors for degradation, we added conditioned medium from monocytes stimulated with SEB to paraformaldehyde-fixed monocytes, since fixed cells are incapable of receptor internalization nor should they release proteins extracellularly by regulated secretion. The results (Fig. 11Go) demonstrated that pretreatment of freshly fixed monocytes with conditioned culture medium from SEB-stimulated monocyte cultures causes a marked decrease in chemokine binding by fixed cells. Notably, if the serine proteinase inhibitor chymostatin or Pefabloc was added to the conditioned medium just before incubating the medium with freshly fixed monocytes, the down-modulation of binding was essentially blocked. Thus, the findings from both the fixed cell data and the previous PTK inhibitor approach (Fig. 10Go) strongly indicate that SAg induces the activation of an alternative, agonist-independent pathway that involves the nascent release of serine proteinase-like activity that rapidly degrades chemokine receptors.



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  		FIGURE 11. Analysis of the effect of cell culture supernatant from SEB-stimulated human monocytes on binding of MCP-1 to paraformaldehyde-fixed monocytes. Freshly isolated monocytes (107 cells/ml) were stimulated with SEB (1 µg/ml) for 1 h at 37°C. The culture supernatant from untreated (control medium) or SEB-stimulated cells was collected and treated with Pefabloc (3 mM) or chymostatin (1 mM) or left untreated (SEB medium) before adding it to fresh monocytes prefixed with 1% (w/v, PBS) paraformaldehyde. Cultures were incubated for an additional 30 min. Specific binding of MCP-1 was then measured. The mean ± SEM from four separate experiments are shown. p < 0.001 represents statistically different MCP-1 binding compared with that of cells treated with SEB-stimulated monocyte supernatant (SEB medium).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Subversion of the immune system is an important strategy developed by different infectious micro-organisms as a mechanism to evade innate and acquired immune responses. Here we demonstrate that Gram-positive bacterial SAgs have the capacity to perturb immune responses by abrogating CC chemokine-mediated monocyte responses. SAgs produced by Gram-positive Staphylococcus aureus (SEA, SEB, SEC1–3, SED, SEE, and SEH) are slightly basic, globular toxins 25, 26 that are stable in the gastric environment and gain access into the bloodstream via transcytosis 23 . On B cells and monocytes, bacterial SAg engages the MHC class II molecule outside the conventional peptide binding groove at multiple contact sites, such that the binding interfaces are dictated by the specific SAg and MHC haplotype involved 42 . SAg engagement of MHC class II induces ZAP70 and p72syk activation 35 . The pathological effects mediated by SAgs are derived from the induced secretion of substantial amounts of proinflammatory cytokines, including IL-1, IL-6, and TNF-{alpha} 43 .

Stimulation of monocytes with either SEA or SEB resulted in an inhibition of CC chemokine-directed migration and Ca2+ mobilization, thus demonstrating that Gram-positive bacteria-derived SAg can suppress functional responsiveness of monocytes to chemokines. The effect of SAg on monocyte function corroborates with the observed SAg-induced inhibition of CC chemokine binding to monocytes. Our data indicate that the mechanism by which these SAgs regulate chemokine responsiveness entails MHC class II-regulated activation of a tyrosine kinase signaling pathway. This is based on our observations that 1) anti-HLA-DR Abs can mimic SAg induced down-modulation of chemokine responsiveness and cell surface binding of CC chemokines; 2) protein tyrosine kinase inhibitors can reverse the suppressive effect of SAgs; and 3) PTK inhibitors are similarly effective at inhibiting the suppressive actions of anti-HLA Abs. The fact that others have demonstrated that SAg stimulation leads to activation of p72syk 35 together with our finding that PTK inhibitors can reverse SAg actions support a key role for PTKs in mediating SAg modulation of monocyte function. Indeed, PTK activation appears to be a critical event for regulation of monocyte responsiveness to chemokines, in that PTK-dependent pathways can effectively regulate interactions of various chemokine receptors and ligands. We have recently found that CXCR1 and CXCR2 down-regulation by Gram-negative bacterial LPS is also dependent on tyrosine kinase signaling 22 . Thus, two different classes of bacterial toxins with very diverse molecular structures down-modulate ligand binding of disparate chemokine subsets through engagement of molecular pathways that mutually involve activation of protein tyrosine kinase signaling. Collectively, these findings indicate that PTK-dependent pathways are critically involved in regulating surface expression of CC chemokine receptors, and thus, PTK signaling appears to pivotally modulate chemokine-mediated responses.

Expression of proteinases following monocyte activation is well documented 44 . Proteinases have been shown to play a pivotal role in monocyte extravasation and tissue migration 44 . Previous observations of LPS-activated neutrophils and monocytes revealed that SAg-challenged cells evince induction or augmentation of proteolytic activity associated with metalloproteinases, serine/cysteine proteinases, and endo- and amino-peptidases, leading to modification of the cell surface composition and cellular responses 44 . Recently, it has been noted 22, 40 that proteinase activity associated with activated neutrophils is responsible for the cleavage of various cell surface molecules, including CD14, CD16, CD25, CD43, CD44, and TNF-{alpha} receptors. Not only does the proteinase-mediated shedding of surface molecules permit alterations of cell responses, but in some instances the cleaved/shed molecule also manifests functional aspects as well 45, 46 . Increasing evidence that proinflammatory stimuli induce the release of potent proteinase inhibitors 47, 48 underscores a key role for proteinases in dynamically modulating cell responses. Moreover, proteinases have been implicated in the regulation of IL-8R levels on LPS-activated neutrophils 38 .

Our finding that conditioned culture medium from SAg-stimulated monocyte cultures markedly down-regulated CC chemokine binding to paraformaldehyde-fixed monocytes, while unstimulated conditioned medium did not, was pivotal in resolving the pathway employed by bacterial SAg to rapidly down-modulate chemokine binding and concomitantly induce functional desensitization to chemokines. These data suggest that secreted proteinases may be the primary effector molecules responsible for down-modulation of ligand binding and minimize the role of intracellular pathways, such as agonist-driven receptor internalization or secondary release of binding modulators, in the observed loss of ligand binding. Previous reports and our findings support the idea that secreted proteinases are responsible for down-regulation of chemokine receptors and suggest two possibilities of chemokine receptor modulation by SAg stimulation. Proteolytic cleavage of the receptor may inactivate the chemokine binding site, leaving nonfunctional receptors on the cell surface. Alternatively, SAg-stimulated proteinase activity may induce substantial degradation and loss of chemokine receptors from the cell surface. Future studies will focus on the resolution of this question by performing experiments to detect degradation products and monitor the localization of CC chemokine receptors in response to SAg stimulation.

From these findings, we propose a model in which the SAg-induced down-modulation of chemokine receptors most likely mimics a normal physiological process brought about by the cognate interaction between peptide-bound MHC class II molecules and the TCR. Under these conditions down-regulation of CC chemokine receptors may prevent paracrine or autocrine chemokine receptor-mediated activation, since Ag-dependent activation induces the release of chemokines from Ag-bound MHC class II+ cells and activated T cells 1 . Therefore, attenuation of chemokine-mediated responses may prevent or mitigate inappropriate responses upon monocyte activation. Future studies using MHC class II cytoplasmic deletion mutants will address the importance of MHC class II signaling in the regulation of chemokine receptor cell surface expression upon TCR:Ag:MHC class II engagement.


   Acknowledgments
 
We thank Anne Leaist, Joe Andrews, Bill DeYoung, Anu Santhanagopal, Dr. S. J. Dixon, and Nancy Schmidt for their assistance.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada (to R.D.F. and D.J.K.), the Juvenile Diabetes Foundation International (to D.J.K.), and the Heart and Stroke Foundation of Canada (to D.J.K.). D.J.K. is a Medical Research Council Scholar. Back

2 Address correspondence and reprint requests to Dr. David Kelvin, John P. Robarts Research Institute, University of Western Ontario, London, Ontario, Canada N6G 2V4. E-mail address: Back

3 Abbreviations used in this paper: MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; SAg, superantigen; SEA, Staphylococcus enterotoxin A; SEB, Staphylococcus enterotoxin B; PTK, protein tyrosine kinase. Back

Received for publication July 6, 1998. Accepted for publication November 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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