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The Journal of Immunology, 2007, 178, 8097-8106
Copyright © 2007 by The American Association of Immunologists, Inc.
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Monocyte Chemoattractant Protein-1 Production by Intestinal Myofibroblasts in Response to Staphylococcal Enterotoxin A: Relevance to Staphylococcal Enterotoxigenic Disease1

Irina V. Pinchuk*, Ellen J. Beswick, Jamal I. Saada*, Giovanni Suarez, John Winston*, Randy C. Mifflin*, John F. Di Mari*, Don W. Powell*,{dagger} and Victor E. Reyes2,§

* Department of Internal Medicine, {dagger} Departments of Neuroscience and Cell Biology, {ddagger} Departments of Microbiology and Immunology, and § Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Staphylococcal enterotoxins A and B (SEA and SEB)3 are major enterotoxins associated with food-poisoning outbreaks in the US (~80 and 10%, respectively). This form of food poisoning is severe: 10% of the affected individuals require a visit or an admission to the hospital (1, 2, 3, 4, 5, 6). Staphylococcal enterotoxin food poisoning leads to inflammatory changes throughout the gastrointestinal (GI) tract with severe lesions in the jejunum and ileum (7, 8, 9, 10).

Staphylococcal enterotoxins act through two distinct mechanisms: as potent GI toxins and as superantigens (11, 12). Unlike conventional Ags, staphylococcal superantigens are characterized by their ability to bypass normal intracellular Ag processing and bind directly to their major receptor, MHC class II molecules, outside of the peptide-binding groove. The formed MHC class II-staphylococcal enterotoxin complex induces an excessive release of proinflammatory mediators (MCP-1, IL-8, IL-6, TNF-{alpha}, RANTES) by MHC class II+ APCs leading to the recruitment of T cells to the site of inflammation (13, 14, 15, 16). The MHC class II-staphylococcal enterotoxin complex interacts with large populations of T cells expressing certain Vbeta elements as part of their TCR and thus initiates polyclonal T cell activation (up to 20–50% of the entire T cell repertoire rather than the 0.01% fraction stimulated by conventional Ags). The vast numbers of T cells that become activated contribute with a high level of proinflammatory cytokine and chemokine secretion. All these events lead to fever, hypotension, and diarrhea and may end in toxic shock (16, 17, 18).

Although an understanding of the mode of action of staphylococcal superantigen has progressed significantly, little is known about the mechanisms responsible for the initiation of pathogenesis associated with staphylococcal enterotoxin diarrheal disease and how professional immune cells are recruited to the sites of intestinal mucosal inflammation. Considering that staphylococcal enterotoxin-MHC class II molecule interaction is required for the initiation of the staphylococcal enterotoxin-associated immunopathogenesis, MHC class II+ cells are candidates for a pivotal role in the initiation of staphylococcal enterotoxin-associated pathogenesis. In the normal human GI mucosa, the expression of MHC class II molecules is attributed to conventional APCs, such as CD68+ macrophages and dendritic cells that are present in the mucosal lamina propria and submucosal areas (19, 20, 21). Another cell population that could bind staphylococcal enterotoxins shortly after exposure are intestinal epithelial cells, but their class II MHC expression is not prominent, except during inflammation, and has been distinctly observed only in the normal duodenum (22, 23, 24, 25).

We recently reported that subepithelial intestinal myofibroblasts (IMFs) from normal mucosa express MHC class II molecules and these cells represent about ~12–15% of the lamina propria cell population (26, 27). IMFs are fibroblast-like mesenchymal (stromal) cells that express CD90/Thy-1 (fibroblast/myofibroblast marker in human tissue), {alpha}-smooth muscle actin, vimentin, and myosin H chain, but do not express desmin (smooth muscle marker) and epithelial cytokeratins (28, 29, 30). These cells are located directly subjacent to the epithelial basement membrane. IMFs form an interface between the epithelium and lamina propria immune cells and have been implicated in the regulation of mucosal inflammation (31, 32). Thus, given the unique subepithelial location of MHC class II+ IMFs with respect to mucosal lymphocytes, as well as the fact that staphylococcal enterotoxins can cross the intestinal epithelial barrier in immunologically active form (33, 34) and increase epithelial permeability (35, 36, 37), it is important to study the role of IMFs in the initiation of the staphylococcal enterotoxin-associated immunopathogenesis.

In the studies presented herein, we evaluated the role of MHC class II+ IMFs in the inflammatory response to the staphylococcal superantigens, SEA and SEB. We demonstrated that staphylococcal enterotoxins engage MHC class II molecules on primary isolates of IMFs. SEA cross-linking of MHC class II molecules on IMFs resulted in a distinct inflammatory mediator pattern that includes MCP-1, IL-8, and IL-6. MCP-1 is the earliest chemokine produced by SEA-stimulated IMFs. Therefore, MCP-1 may play an important role in the initiation of inflammatory injury associated with SEA-associated diarrheal disease. Intestinal subepithelial myofibroblasts may be a key contributor to the cytokine/chemokine-mediated pathogenesis of staphylococcal enterotoxin-associated inflammation in the intestinal mucosa.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

Purified SEA and SEB were purchased from Toxin Technology and reconstituted in sterile endotoxin- and LPS-free 5 mM phosphate buffer (pH 7.2). Staphylococcal enterotoxin biotinylation, where indicated, was performed using the FluoReported MiniBiotin-XX Protein Labeling kit from Molecular Probes according to the manufacturer’s instructions.

Murine mAbs against human HLA-DR (clone L243) were purchased from BD Biosciences. Fluorochrome-conjugated forms of IgG1{kappa}, IgG2a, IgG2b isotype controls, ZO-1 (clone 1), streptavidin, anti-MCP-1-neutralizing mAbs (clone 5D3-F7), and human recombinant MCP-1 were purchased from BD Pharmingen. Functional grade murine IgG1{kappa}, IgG2a, and IgG2b isotypes were purchased from eBioscience. Anti-HLA-DR rat mAbs (clone YD1/63.4.10) were purchased from Abcam. Alexa Fluor (AF) 488-labeled donkey anti-mouse IgG (H+L), AF594-labeled rabbit anti-rat IgG (H+L), and AF633-conjugated streptavidin were purchased from Molecular Probes. The hybridomas secreting anti-human MHC class II mAbs (clones IVA12 and L243) were obtained from the American Type Culture Collection and were used to produce the corresponding Abs. Recombinant human IFN-{gamma} and anti-CCR2 mAbs (clone 48607) were obtained from R&D Systems.

Primary myofibroblast cultures

Normal colonic 18Co myofibroblast primary isolates were obtained from the American Tissue Culture Collection (ATCC no. CRL 1459, passages 11–15). 18Co was used in the study as a representative IMF. Additional primary cultures of subepithelial small IMFs were generated according to the method described by Mahida et al. (38, 39) from full-thickness fresh human tissue samples. Full-thickness fresh human tissue samples were obtained from discarded surgical resection material of small intestine in compliance with protocols approved by the University of Texas Medical Branch Institutional Review Board. Areas of uninvolved intestinal tissue from patients undergoing colectomy for colon cancer were studied. The purity of isolated cells was analyzed by immunohistochemistry and flow cytometry analysis as described below. Studies were performed with primary IMFs at passages 3–9. Cells were cultured at 37°C in 5% CO2 atmosphere in complete MEM, which contain MEM base supplemented with nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin/streptomycin (Cellgro), and 10% heat-inactivated FBS (Sigma-Aldrich).

Isolation of PBMC

PBMC were prepared from the blood of healthy donors by density gradient centrifugation over Ficoll-Paque Plus (Amersham Biosciences) according to the manufacturer’s instructions.

Superantigen-binding assay

IMF monolayers were treated for 1 wk with IFN-{gamma} (100 U/ml) to maximize MHC class II expression. IFN-{gamma} was then removed and cells were rested for 24 h in MEM medium. Biotinylated SEA or SEB (2.5 µg/ml) were added in the presence or absence of the anti-MHC class II Abs (5 µg/ml of each mAbs), which include anti-HLA-DR, DP, DQ (clone IVA12), and anti-HLA-DR (clone L243) mAbs. Isotype controls were included in the experimental design. Cells were incubated for 1 h at 37°C, then washed twice with PBS. The cells were detached with detaching solution (Sigma-Aldrich) and resuspended at 106 cells/ml in PBS. Cells were stained with PE-conjugated streptavidin for 30 min, as described below in the flow cytometry analysis.

Coculture of IMFs with colonic epithelial cell line

A coculture system of intestinal epithelial cell line, CaCo-2, and IMFs has been established according to the modified method of Willemsen et al. (40). IMFs were grown on glass coverslip in 24-well plates at 30–50% confluency, then treated for 1 wk with 100 U/ml IFN-{gamma} to enhance MHC class II expression. IFN-{gamma} containing MEM medium was then removed and the cells were rested for 24 h in MEM medium. Then, these MHC class II-expressing IMFs were used in coculture experiments.

The colonic epithelial cell line CaCo-2 was obtained from ATCC and was grown separately from IMFs at 100% confluency on the top of 24-well plate filter inserts with a pore size of 0.4 µm (BD Biosciences). Transepithelial resistance (TER; {Omega} · cm2) across coculture or monolayer of cells was measured using an epithelial voltohometer (Millicell-ERS; Millipore). Epithelial cells were used when TER was ~700–1000 {Omega} · cm2. The epithelial cells grown in the transwell insert were then transferred on the top of a coverslip covered with IMFs. Biotinylated staphylococcal enterotoxins (2.5 µg/ml) were added to the apical surface of epithelial cells in transwell. The epithelial barrier crossing by staphylococcal enterotoxins and its binding to HLA-DR on IMF was analyzed 18 h later by using immunostaining followed by confocal microscopy.

Confocal microscopy

IMFs and/or epithelial cell lines (in the presence/absence of biotinylated staphylococcal enterotoxins) were grown according to the coculture protocol described above. Then, cells were fixed and permeabilized in 100% cold methanol for 20 min at 4°C.

IMFs were stained with streptavidin conjugated to AF633 and blocked with normal mouse and rabbit serum (1/10 in PBS) for 30 min at room temperature. Next, IMFs were stained with rat anti-human HLA-DR (1/400) mAbs or rat IgG2a isotype control (same dilution) at room temperature for the same time period. Samples were then probed with AF592-conjugated rabbit anti-rat IgG Abs (1/400) for 30 min.

Epithelial cells grown in transwell inserts were stained for AF488-conjugated anti-ZO-1 mAbs (1/300) or mouse normal IgG1 isotype (same dilution) at room temperature for the same amount of time. Conjugation of anti-ZO-1 mAbs with AF488 fluorochrome was performed using a Zenon Mouse IgG Labeling kit (Molecular Probes) according to the manufacturer’s instructions.

Confocal microscopy was performed with a Zeiss LSM510 META laser scanning confocal microscope (Carl Zeiss). AF488 staining was observed using an excitation wavelength of 488 nm and an emission wavelength of 505–530 nm (argon/ion laser). The AF594 staining were visualized with an excitation wavelength of 543 nm and an emission at 585–615 nm (green helium/neon laser). The AF633 staining was detected with an excitation wavelength of 633 nm and an emission wavelength longer then 650 nm (red helium/neon laser). Each staining step was followed by three washes with PBS.

Flow cytometry

IMFs were detached from the culture flasks by treatment with Detaching Solution (Sigma-Aldrich) at 37°C for 15 min, then washed two times with cold PBS. IMFs were stained for various surface markers. Single-color immunostaining was performed according to a standard surface FACS staining BD Pharmingen protocol as described previously (27). Flow cytometry analysis was performed using FACScan and FACSCanto cytometers (BD Biosciences). Single-color FACS analysis was performed by gating on the typical forward and side light scatter characteristics of the cells being analyzed and histograms were plotted for each Ab to determine the mean fluorescence intensity (MFI). Nonspecific staining was determined for each Ab by using isotype controls or fluorochrome-conjugated streptavidin (when required). Samples were considered negative if the percentage of positive cells was 2% or less. At least 10,000 events were scanned for each experimental condition. Flow cytometry data were analyzed with WinMDI 2.8 software (The Scripps Research Institute, La Jolla, CA).

Real-time RT-PCR

Total cellular RNA was isolated using RNeasy RNA isolation kit (Qiagen) according to the manufacturer’s instructions. The sample concentration was measured by spectrophotometry at 260 nm and RNA quality checked on a 1% agarose gel. Reverse transcription (RT) real-time PCR was performed according to the Applied Biosystems two-step RT real-time PCR protocol. All reagents were purchased from Applied Biosystems. The RT reaction mixture included random 2.5 µM hexamers, 500 µM dNTPs, 0.4 U/µl of the RNase inhibitors, 5.5 mM MgCl2, MultiScribe Reverse Transcriptase (3.125 U/µl), and its buffer and 1 µg of cellular RNA. RT mix final volume of 50 µl was adjusted using RNase and DNase free H2O (Sigma-Aldrich). The RT step was performed using a thermocycler GeneAmp PCR system 9700 (Applied Biosystems) according to the following protocol: 10 min at 25°C, 60 min at 37°C, 5 min at 95°C. The cDNA samples obtained were stored at –20°C (if necessary) and used for the PCR step. The PCR mix was prepared using the TaqMan Universal PCR Master Mix (Applied Biosystems). The appropriate assays-on-demand gene expression assay mix (Applied Biosystems) for human 18S and costimulatory signals (a x20 mix of unlabeled PCR primers and TaqMan MGB probe, FAM dye labeled) and 2 µl of cDNA was added to the PCR mix. The reactions were conducted in a 20-µl final volume using GeneAmp 5700 Sequence Detection System (Applied Biosystems) according to the following protocol: 2 min at 50°C, 10 min at 95°C (1 cycle), and 15 s 95°C and 1 min at 60°C (40 cycles). The negative controls were included in the RT real time two-step reaction. The endpoint used in real-time PCR quantification, cycle threshold (CT), is defined as the PCR cycle number that crosses the signal threshold. CT values range from 0 to 40, with the latter number assumed to represent no product formation. Quantification of cytokine gene expression was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold difference relative to the human housekeeping gene, 18S rRNA. To calculate the fold-change (increase or decrease), the CT value of 18S rRNA was subtracted from CT value of the target cytokine gene to yield the {Delta}CT. Change in the expression of the normalized target gene as a result of an experimental conditions is expressed as 2–{Delta}{Delta}CT where {Delta}{Delta}CT = {Delta}CT experimental samples – {Delta}CT biological control.

Cytokine expression and production

Cytokine production was measured in the supernatants of IMFs incubated in the presence or absence of staphylococcal enterotoxins by using appropriate cytokine ELISA kits (BD Pharmingen and BD Biosciences) and Bioplex Cytokine Preconfigured Human Th1/Th2 Panel Array kit (Bio-Rad) according to the manufacturer’s instructions. The Bio-Plex Array Reader and 96-well plate microplate platform were used as a reading system using xMAP detection technology (Bio-Rad).

Chemotaxis assay

ChemoTx system (NeuroProbe) was used as a chemotaxis assay using a modified Boyden chemotaxis chamber technique as suggested by the manufacturer’s instruction. First, cell culture supernatants from staphylococcal enterotoxin-treated or untreated IMFs were loaded into the microtiter plate. Serial dilutions of recombinant human MCP-1 were used as a standard controls. Anti-MCP-1 Abs (R&D Systems) was added when necessary (to test the specificity of the reaction). All the conditions were tested in triplicate. A filter with pores of 8 µm in size was then applied to cover the wells containing the cell culture supernatants (or standard controls). PBMC were washed three times with PBS and resuspended in plain culture medium. Aliquots of cell suspension (5 x 105 cells/50 µl) were mounted onto the filter. Migration of leukocytes in the microplate lower compartment was detected by using MTS solution from CellTiter 96 AQueous MTS reagent (Promega) according to the manufacturer’s instructions. Briefly, after a 2-h incubation at 37°C, the filter was carefully removed, and the wells of the microtiter plate were stained with MTS tetrazoliun compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salts and an electron coupling reagent phenazine methosulphate solutions by incubation at 37°C for an additional 4 h. The plates were then read at 490 nm using an ELISA reader (Spectra MAX 340PC; Molecular Devices). Standard curves of cell number vs OD490 were realized. Results were expressed as a chemotactic index, which represents the mean number of cells that migrated through the membrane in the presence of a chemokine gradient in experimental conditions divided by the mean number of randomly migrating cells in the absence of chemotaxis inducing agent (e.g., culture supernatant from untreated IMFs).

Statistical analysis

Unless otherwise indicated, the results were expressed as the mean ± SE of data obtained from at least three independent experiments done with triplicate sets in each experiment. Differences between means were evaluated by ANOVA using Student’s t test for multiple comparisons. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
SEA and SEB superantigens bind to MHC class II molecules expressed by IMFs

MHC class II molecules represent the major binding sites for staphylococcal superantigens (41, 42). Therefore, all intestinal cells expressing MHC class II are potentially important in the initiation of staphylococcal enterotoxin-associated GI pathogenesis. We recently reported that colonic IMFs express MHC class II molecules in vitro and in situ. Staphylococcal enterotoxigenic enteritis is mostly associated with inflammation throughout the small intestine. Therefore, in these studies, we first analyzed the expression of MHC class II molecules by small intestinal IMF primary isolates.

As we observed previously for colonic myofibroblasts, primary small intestinal IMF cultures expressed low levels of MHC class II molecules, as detected by Western blot analysis (data not shown). Treatment of IMFs with 100 U/ml IFN-{gamma}, a cytokine expressed in tonic levels in the normal intestinal mucosa (43), for 5–7 days significantly enhanced surface MHC class II molecule expression, which was detected by flow cytometry (Fig. 1A).


Figure 1
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  		FIGURE 1. Staphylococcal enterotoxins bind to IMFs in a MHC class II-dependent manner. IMFs were cultured for 7 days in the presence of 100 U/ml IFN-{gamma} to maximize MHC class II expression. IFN-{gamma} was removed and cells were rested for 24 h in medium without IFN-{gamma} before experiments. A, Flow cytometry analysis of HLA-DR expression by primary cultured IMFs. Isotype control (filled histogram) was included and superimposed on HLA-DR staining (open histogram). One of five representative experiments with IMF12 is shown. B, Binding of biotinylated SEA and SEB (2.5 µg/ml) to IMFs in presence/absence of anti-HLA DR, DQ, DP Ab mixture (clone IVA12 and L243, 5 µg/ml of each) or isotype control mixture (IgG1{kappa} and IgG2a, 5 µg/ml of each). Staphylococcal enterotoxin binding was visualized by staining with PE-conjugated streptavidin. The results are expressed as a MFI above background staining with PE-conjugated streptavidin in absence of staphylococcal enterotoxins. Results are calculated as the mean value of five experiments ± SEs. *, p < 0.05.

 
We then investigated IMF capacity to bind SEA and SEB through MHC class II molecules. Biotinylated SEA and SEB (2.5 µg/ml) were used to determine whether HLA-DR+ IMFs were able to bind superantigens. We observed that staphylococcal enterotoxins bind to IMFs (18Co and IMF12) via MHC class II, because the addition of anti-MHC class II Abs abolished this interaction (Fig. 1B) whereas isotype control Abs did not have any effect. Similar results were observed for two other IMF primary isolates, IMF9 and IMF11A (data not shown). Interestingly, SEB binding to MHC class II molecules was significantly lower than that observed for SEA. However, the binding of both superantigens was comparable to that observed using the monocyte cell line THP-1, which was used as a positive control (data not shown).

Staphylococcal enterotoxins cross a CaCo-2 monolayer epithelial barrier and bind to subjacent MHC class II+ IMFs in coculture

To understand how these in vitro observations of staphylococcal enterotoxins binding to IMFs are relevant to in vivo events, we determined whether staphylococcal enterotoxins can cross the epithelial barrier and bind MHC class II molecules on IMFs. A coculture system of intestinal epithelial cell line CaCo-2 and IMF12 was established to mimic the layers of cells encountered by staphylococcal enterotoxins as described by Willemsen et al. (40). The biotinylated SEA or SEB (2.5 µg/ml) was added to the apical surface of CaCo-2 cells in the transwell after establishment of a high-resistance epithelial barrier in coculture (TER = 710 ± 105 {Omega} · cm2). Staphylococcal enterotoxin crossing of the epithelial barrier and its binding to HLA-DR on IMF12 was revealed 18 h later. At this time point, no significant perturbation in the epithelial barrier function was observed. CaCo-2 TER was equal to that observed prior to staphylococcal enterotoxin addition. In addition, no significant reorganization in tight junction-associated protein ZO-1 was detected (Fig. 2A). Despite the presence of unaltered epithelial barrier resistance, we observed significant staphylococcal enterotoxin binding to MHC class II molecules on subjacent myofibroblasts (Fig. 2, B–G). The orange-yellow staining in Fig. 2, D and G, resulting from merged images, indicates staphylococcal enterotoxins and HLA-DR colocalization on IMFs in the coculture. The possibility of nonspecific staining in our detection system was ruled out by immunostaining of MHC class II+ IMF 12 with HLA-DR matched isotype Ab and streptavidin labeled with AF633 in the absence of staphylococcal enterotoxin treatment. No significant fluorescence was observed in these controls (Fig. 2, H–J). Thus, SEA and SEB may cross the epithelial barrier and bind MHC class II molecules on IMFs.


Figure 2
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  		FIGURE 2. SEA and SEB cross the CaCo-2 epithelial barrier and bind HLA-DR on subjacent intestinal subepithelial myofibroblasts IMF12. A, ZO-1 expression by CaCo-2 in IMF/CaCo-2 coculture was detected by anti-ZO-1 mAbs conjugated with AF488 (Molecular Probes). B, Binding of biotinylated SEA and (E) SEB to IMFs shown in green. C and F, HLA-DR expression on IMFs is shown in red. Merging images show colocalization (orange-yellow color) of SEA and HLA-DR (D) and SEB and HLA-DR (G) MHC class II+ IMF12 stained with streptavidin labeled with AF633 but not exposed to SEB (H) and HLA-DR-matched isotype Ab (I) were used as a control. J, Transmission image of cells. Confocal microscopy was performed by using a Zeiss LSM510 META laser scanning confocal microscope (Carl Zeiss). CaCo-2/IMF12 coculture was performed by a slightly modified method of Willemsen et al. (40 ). TER was 710 ± 105 Ohm when staphylococcal enterotoxins (2.5 µg/ml) was added to the apical surface of CaCo-2 in the transwell. Staphylococcal enterotoxins binding to HLA-DR on IMF12 (pretreated with IFN-{gamma} as described in Materials and Methods) after crossing the epithelial barrier was revealed 18 h later. The biotinylated staphylococcal enterotoxins was detected by streptavidin labeled with AF633 (Molecular Probes). HLA-DR was detected using rat monoclonal as a primary and AF594-labeled rabbit anti-rat IgG (H+L) as a secondary Ab (Molecular Probes). Calibration bar, 10 µm.

 
The strong inflammatory response induced by the interaction of IMFs with SEA is MHC class II dependent

Cytokine and chemokine production is a hallmark of staphylococcal enterotoxin-associated immunopathogenesis. The engagement of MHC class II molecules on professional APCs by staphylococcal enterotoxins results in the production of proinflammatory cytokines/chemokines, including IL-1beta, IL-6, IL-8, TNF-{alpha}, MCP-1, and RANTES (13, 16, 44, 45, 46). Therefore, to characterize the panel of cytokine/chemokines produced by IMFs as a result of the engagement of MHC class II molecules on the surface of IMFs by staphylococcal enterotoxin, we used Bioplex cytokine arrays, as described in Materials and Methods. After 48 h, exposure of primary cultured isolates IMF12 and 18Co to SEA, we observed a robust increase in the proinflammatory cytokine IL-6 and the chemokines IL-8 and MCP-1, as well as a moderate increase in the growth factors G-CSF and GM-CSF (Table I). In contrast to SEA, SEB had no significant effect on the production of these cytokines or chemokines. The increase in IL-6, IL-8, and MCP-1 expression by IMFs exposed to SEA was confirmed by using standard ELISAs (Fig. 3).


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  		Table I. SEA ligation of MHC class II molecules on IMFs induces strong proinflammatory cytokine/chemokine responses

 

Figure 3
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  		FIGURE 3. MCP-1, IL-8, and IL-6 inflammatory response of IMFs to SEA is MHC class II dependent. Exposure of IMF12 (pretreated with IFN-{gamma} as described in Materials and Methods) to SEA (2.5 µg/ml) resulted in significant secretion of chemokines MCP-1 and IL-8 and cytokine IL-6 at 48 h as measured by ELISA. These responses were abolished upon blocking of MHC class II with anti-HLA DR, DQ, and DP Ab mixture (clone IVA12 and L243, 5 µg/ml of each). No significant decrease in the proinflammatory mediators was observed when appropriate isotype control Abs were used. Data represent mean ± SEs from the results of triplicates in five experiments. *, p < 0.01.

 
We next explored the molecular basis of the SEA-induced cytokine/chemokine profile (MCP-1, IL-8, and IL-6) on IMFs. Because MHC class II molecules represent the major binding sites for staphylococcal superantigens (41), we examined whether the SEA induction of the proinflammatory mediators on IMFs was MHC class II dependent. The production of SEA-induced MCP-1, IL-8, and IL-6 was inhibited by 80–90% in the presence of anti-MHC class II-blocking Abs (Fig. 3). The observed blocking was specific, because no inhibition of the SEA-induced response was observed in the presence of a control IgG isotype. Similar responses were obtained with three other primary isolates of IMF that were examined (data not shown). Taken together, these data indicate that the inflammatory response of IMFs to SEA is MHC class II dependent.

Cross-linking of MHC class II molecules on IMFs with anti-MHC class II biotinylated Abs mimics the inflammatory cytokine pattern induced by SEA

We observed that both enterotoxins, SEA and SEB, were able to bind MHC class II molecules on IMFs (Fig. 1B). However, only SEA was able to induce a pronounced inflammatory response (e.g., MCP-1, IL-8, and IL-6) by IMFs. It has been demonstrated previously that SEB interacts with the {alpha}-chain of MHC class II molecules, while the SEA simultaneously binds to both {alpha}- and beta-chains, thus enabling the cross-linking of different MHC class II complexes on APCs. This cross-linking capacity has been shown to be important for optimal cytokine induction by APCs (41, 42). Therefore, we next examined whether cross-linking of MHC class II molecules with biotinylated anti-HLA-DR mAbs (clone L243) could induce inflammatory response in IMFs similar to what was observed with SEA. As shown in Fig. 4, the cross-linking of MHC class II molecules by biotinylated anti-HLA-DR mAbs induced robust MCP-1, IL-8, and IL-6 responses on IMF12. Similar results to those shown on Fig. 4 were obtained with the 18Co primary myofibroblast isolate (data not shown). These experiments suggested that not only binding but also efficient MHC class II cross-linking on IMFs is required to induce strong proinflammatory MCP-1, IL-8, and IL-6 responses.


Figure 4
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  		FIGURE 4. Cross-linking of MHC class II on intestinal subepithelial myofibroblasts with anti-MHC class II biotinylated Abs mimics the proinflammatory response induced by SEA. MCP-1, IL-8, and IL-6 production by IMFs was measured in cell culture supernatants by ELISA. IMF12 cells (pretreated with IFN-{gamma} as described in Materials and Methods) were incubated with biotinylated anti-HLA-DR Abs (5 µg/ml) for 48 h in the presence or absence of streptavidin (as a cross-linking agent). Data represent mean ± SEs from the results of duplicates in five experiments. *, p < 0.01.

 
SEA induced a triphasic pattern of cytokine/chemokine expression by IMFs

To better understand the cytokine/chemokine response of IMFs during SEA stimulation, we examined the kinetics of MCP-1, IL-6, and IL-8 expression by IMF12 exposed to SEA. In these assays, we analyzed both level of RNA expression by using real-time RT-PCR and the level of secreted MCP-1, IL-6, and IL-8 proteins in culture supernatants by using a standard ELISA (Fig. 5). SEA induced in IMFs a triphasic pattern of cytokine/chemokine expression.


Figure 5
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  		FIGURE 5. Time course of MCP-1, IL-8, and IL-6 expression/production by MHC class II+ IMFs following SEA exposure. IMF12 were pretreated with IFN-{gamma} (100 U/ml) for 7 days to maximize MHC class II expression, then rested for 24 h before exposure to 2.5 µg/ml SEA. A, Relative quantitative real-time RT-PCR analysis of IL-6, IL-8, and MCP-1 mRNA expression by IMF12. mRNA levels for each inflammatory mediator for each time point were normalized to 18S and compared with the initial level of mRNA in untreated IMF12 (control) at the 5-min time point. The calculation of {Delta} fold increase was performed as described in Materials and Methods. B, Production of IL-6, IL-8, and MCP-1 was measured in the culture supernatants using standard ELISAs. Data represent mean ± SEs from duplicates in three experiments. *, p < 0.05.

 
MCP-1 was induced early by SEA in IMF12: mRNA level as well as protein expression increased rapidly until the maximal expression within 13 h. By 24 h poststimulation, the MCP-1 mRNA level was close to the basal level (Fig. 5A). MCP-1 protein secretion was sustained longer; it peaked at 48 h and returned to the basal level at 72 h following stimulation with SEA (Fig. 5B).

IL-8 production by IMF12 stimulated with SEA was observed at 24–48 h poststimulation and was associated with a mid-phase chemokine release. Low levels of IL-8 mRNA were initially detected at 13 h poststimulation and dramatically increased at 24 h with a small increase in expression by 48 h after stimulation (Fig. 5A). IL-8 protein secretion paralleled what was observed at the mRNA level during the same time periods (Fig. 5B). By 72 h, IL-8 mRNA was no longer detectable and the amount of IL-8 protein in culture supernatants was decreased to a level comparable to that of 24 h after stimulation (Fig. 5).

IL-6 production was associated with a late phase of proinflammatory response induced by SEA in human primary IMF12 cultures. Increases in this cytokine mRNA were first detectable at 24 h poststimulation, dramatically increased by 48 h and remained at the same level for the next 24 h (72 h poststimulation) (Fig. 5A). In contrast to MCP-1 and IL-8, a robust increase in IL-6 protein secretion was observed only at 72 h post-SEA stimulation (Fig. 5B). Similar kinetics of MCP-1, IL-8, and IL-6 induction in response to SEA stimulation were observed for 18Co myofibroblasts.

MCP-1 is responsible for IL-8 and IL-6 production induced by SEA stimulation of IMFs

In experiments described in the previous section, we observed that MCP-1 was the earliest chemokine induced by SEA stimulation of MHC class II+ IMFs. This chemokine is known to play a pivotal role in the attraction of monocytes and T cells to the local inflammatory site during the early steps of different inflammatory diseases, including diseases associated with microbial superantigens. Moreover, MCP-1 has been shown to stimulate IL-8 and IL-6 production in professional APCs (44, 45, 46, 47, 48). Therefore, we investigated whether MCP-1 is responsible for the production of mid- and late-phase cytokines IL-8 and IL-6, respectively, induced by SEA stimulation of IMFs.

The biological effects of MCP-1 are mostly mediated by binding to the CC chemokine receptor CCR-2 (49, 50). Therefore, to understand the responsiveness of IMFs to MCP-1, we analyzed the surface expression of CCR-2 by flow cytometry on IMFs. A high constitutive expression of CCR-2 was detected on the IMF 12 primary isolate (Fig. 6A). Because all IMFs used in the previous experiments were pretreated with 100 U/ml IFN-{gamma}, to enhance MHC class II expression, we also examined whether IFN-{gamma} affects CCR-2 expression on IMFs. After 7 days of IFN-{gamma} treatment surface expression of CCR-2 on IMFs remained significant, although a small decrease in expression was observed (Fig. 6A). Similar results were obtained with four other primary IMFs, including the 18Co primary isolate (data not shown).


Figure 6
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  		FIGURE 6. IMFs express CCR-2 and produce IL-8 and IL-6 in response to MCP-1 stimulation. A, Surface CCR-2 expression by IMF12 before and after IFN-{gamma} treatment (as described in Materials and Methods) was analyzed by flow cytometry. Constitutive expression of CCR-2 was detected. CCR-2 surface expression remains significant after 7 days of IFN-{gamma} treatment (100 U/ml). B, IMF12 responds to the MCP-1 treatment (50 ng/ml) by production of IL-6 and IL-8. IMF12 was treated with MCP-1 ({blacksquare}) or not ({square}). Cytokine production in the culture supernatants was measured 48 h later using standard ELISAs. Data represent mean ± SEs of triplicates from three experiments. *, p < 0.05.

 
Because IMFs express the CCR-2 receptor, we examined directly the effect of MCP-1 on IL-8 and IL-6 production by IMFs. Stimulation of IMFs with MCP-1 alone induced significant increases in IL-8 and IL-6 production in culture supernatants that was comparable to what was induced by SEA (Fig. 6B).

To further investigate the role of MCP-1 in the SEA-mediated IMF inflammatory response, we determined whether blocking of the MCP-1 major receptor, CCR-2, would impair the MCP-1 or SEA induced IL-8 and IL-6 expression by using real-time RT-PCR. Stimulation of IMF12 with MCP-1 or SEA for 24 h in presence of anti-CCR-2-blocking Abs inhibited IL-8 and IL-6 production by ~50% (Fig. 7, A and B, respectively). However, the SEA-induced production of both IL-6 and IL-8 were abolished in the presence of anti-MCP-1-neutralizing mAbs in the period 24 h postexposure (Fig. 7). Moreover, at 48–72 h postexposure to SEA, the levels of IL-6 and IL-8 expression remained significantly decreased in the presence of MCP-1-neutralizing mAbs (data not shown). The strong decrease, but not abrogation, of IL-6 and IL-8 expression during the longer exposure of IMFs to SEA in the presence of anti-MCP-1 mAbs, might be due to low stability of the anti-MCP-1 mAbs or presence of an alternative pathway in IMFs to the IL-8 and IL-6 induction by SEA. Similar data were obtained when 18Co myofibroblasts were used in the experiments (data not shown). These data suggest that MCP-1 produced by subepithelial IMFs may play a central role in initiation of intestinal inflammatory injury associated with SEA enterotoxigenic disease.


Figure 7
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  		FIGURE 7. Blocking of MCP-1 signaling reduces MCP-1 (A) and SEA (B) induced IL-8 and IL-6 production by IMFs. MCP-1 or SEA induced IL-8 and IL-6 expressions were measured by real-time RT-PCR. Blocking the MCP-1 major receptor CCR-2 with 10 µg/ml anti-CCR-2 mAbs (clone 48607) significantly reduced expression of IL-8 and IL-6. Mediators expression was abrogated by 10 µg/ml anti-MCP-1-neutralizing mAbs (clone 5D3-F7). Expression of IL-8 and IL-6 by IMFs was analyzed 24 h poststimulation with MCP-1 or SEA. Results are presented as a fold increase in IL-8 and IL-6 mRNA level treatment, normalized to 18S RNA, and relative to untreated control. The calculation of {Delta} fold increase was performed as described in Materials and Methods. Results represent mean ± SEs of duplicates from four experiments. *, p < 0.05.

 
MCP-1 produced by IMF, in response to SEA stimulation, induces chemotaxis of PBMC

Chemotactic signals are considered to be of critical importance for the recruitment of leukocytes during the initiation of staphylococcal enterotoxin-associated immunopathogenesis (15, 51). We demonstrated above that MHC class II engagement by SEA on IMFs results in high expression of at least two chemokines, MCP-1 and IL-8. We therefore examined the functional activity of chemoattractants produced by IMFs in response to staphylococcal enterotoxin stimulation. To this end, we measured the migration of PBMC in response to cell culture supernatants of IMFs exposed to staphylococcal enterotoxins. Culture supernatants from IMF12 exposed to SEA for 24–48 h significantly increased (p < 0.05) PBMC transmigration in vitro, which was significantly decreased by the addition of anti-MCP-1-neutralizing mAbs during the chemotaxis assay (Table II). No chemotactic activity was observed when SEA (2.5 µg/ml) alone was used instead of supernatants. Moreover, no transmigration of PBMC was observed when supernatants from the culture of SEA-stimulated IMF were used in the presence of anti-MCP-1-neutralizing Abs. These data suggest that MCP-1 produced by IMFs in response to SEA may contribute to the PBMC migration. Thus, MCP-1 might be a key player in the initiation of local intestinal inflammation during staphylococcal enterotoxin enterotoxigenic disease.


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  		Table II. Transmigration of PBMC induced by supernatants from SEA-stimulated IMFs is reduced by neutralization of MCP-1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
During the pathogenesis of staphylococcal enterotoxigenic diarrheal disease, T cells, professional APCs, and neutrophils are recruited to the sites of local intestinal inflammation and the disease is associated with an intensive cytokine response (46, 51). However, the actual sources of the cytokine/chemokines that recruit these professional immune cells to the mucosal inflammatory site remain unclear. Recent studies suggest that stromal cells, in particularly fibroblast and myofibroblasts, represent potential mediators of Ag presentation and T cell attraction to the inflammatory site (52, 53). Fibroblast and myofibroblasts have been shown to produce a number of important mediators during inflammatory responses, including IL-6, IL-8, M-CSF, and GM-CSF (54, 55).

Considering that MHC class II molecules are receptors for staphylococcal enterotoxins, cells that express these molecules are candidates for a pivotal role in the initiation of staphylococcal enterotoxin-associated pathogenesis. We recently reported that subepithelial IMFs constitutively express MHC class II in the normal colon (26, 27). In the studies presented herein, we evaluated whether human subepithelial IMFs can be among the initiators of the local intestinal inflammatory responses to staphylococcal enterotoxins.

We recently demonstrated that class II+ colonic myofibroblasts can function as nonprofessional APCs for allogeneic and Ag-specific T cell activation (27). In contrast to constitutive expression of MHC class II in vivo, the expression of these molecules on primary myofibroblast isolates is drastically reduced during in vitro passages. However, treatment of colonic IMF with low doses of IFN-{gamma} significantly increases MHC class II expression (27). Similar to our previous findings, in this study, we first confirmed that IFN-{gamma} treatment maximizes MHC class II expression on small intestinal IMF primary cultures. We also demonstrated that SEA and SEB bind to MHC class II molecules on primary IMF isolates in vitro. Interestingly, binding efficiency of SEA to class II molecules on IMFs was higher than that of SEB for all IMFs that were examined. This difference might be due to the fact that SEA has the ability to engage either chain of the class II MHC complex and could thus have more binding sites than SEB, which can bind to only the beta-chain of class II MHC complexes. In addition, SEA and SEB binding are differentially sensitive to structural heterogeneity in MHC class II-associated cofactors, as a consequence of the differential regulation of expression of Ag-processing cofactors, including CD74 and HLA-DM, on different APCs (56, 57).

It has been reported that staphylococcal superantigen stimulation of professional immune cells in vitro evokes human epithelial transport and barrier abnormalities (36, 58). Moreover, multiple lines of evidence have accumulated to suggest that staphylococcal enterotoxins can cross the epithelial barrier in an immunologically intact form through transcytosis (33, 34). Thus, the probability that staphylococcal enterotoxin can directly interact with the second layer of nonprofessional immunological defense, e.g., IMFs, is high. To verify that, we determined whether staphylococcal enterotoxins can cross the epithelial barrier and bind MHC class II molecules on IMFs, using an epithelial cell myofibroblast coculture system which mimics the intestinal mucosa (40). In our coculture model, we did not observe significant change in TER of epithelial cells when staphylococcal enterotoxin was added to the coculture for 18 h. These observations are in accord to the experiments of McKay et al. (36, 58) suggesting that the presence of immune cells producing IFN-{gamma} and TNF-{alpha} is required to evoke barrier abnormality. The mucosal MHC class II-expressing APCs are essential to normal health and function and play a vital role in virtually all immune-associated processes, having either beneficial or deleterious effects. Therefore, having up to 15% of the lamina propria cells (e.g., IMFs), in addition to professional APCs, constitutively expressing MHC class II molecules that are capable of binding staphylococcal enterotoxins may inappropriately foster the process of superantigens presentation and leave the mucosa in a state prone to inflammation during the initiation of staphylococcal enterotoxigenic disease. Thus, our findings suggest the possible relevance of the staphylococcal enterotoxin engagement of MHC class II molecules on IMFs to the pathophysiology of staphylococcal enterotoxin-associated diarrheal disease.

Our data indicate that ligation of MHC class II molecules with SEA results in the significant production of MCP-1, IL-6, and IL-8. Although little is known about immunopathogenesis of SEA-associated diarrheal disease, production of IL-6, IL-8, and TNF-{alpha} has been reported during the inflammatory response associated with airway SEA exposure (13, 46, 51). Increase in these proinflammatory mediators may lead to the epithelial and endothelial damage, in parallel with an increase in neutrophil and professional immune cell influx. Therefore, it is possible that similar events may have a place during immunopathogenesis of SEA diarrheal disease. Importantly, no significant increase in the IL-6, IL-8, and MCP-1 was noted when IMFs were exposed to SEB. This may be due to the fact that SEB interacts only with the {alpha}-chain of MHC class II molecules and is unable to cross-link them. In contrast, SEA simultaneously ligates both {alpha}- and beta-chains of MHC class II resulting in the cross-linking of two different MHC class II complexes on APCs (41). Interestingly, in our experiments the same proinflammatory pattern was induced in IMFs by cross-linking of MHC class II Ag with anti-HLA-DR, -DP mAb, clone IVA12. Therefore, our observations suggest that cross-linking of MHC class II molecules on IMFs may be a requirement for the induction of the proinflammatory response, as has been previously observed for B cells and synovial fibroblasts (16, 41).

Our study clearly showed that multimerization of MHC class II molecules by SEA induced a triphasic pattern of cytokine/chemokine expression by IMFs. The early response (4–13 h) was associated with MCP-1 release and followed by secretion of IL-8 (13–48 h) and a later increase in IL-6 levels (24–72 h). Induction of CXC and CC chemokines, including IL-8 and MCP-1, in human peripheral blood monocytes and fibroblast-like synoviocytes by staphylococcal superantigens has been reported (16, 45). Moreover, the role of IL-8 as a key mediator of neutrophil transmigration to the SEA-induced mucosal inflammatory site has been established in staphylococcal enterotoxin-associated airway diseases (13, 15). However, the role of these chemokines in staphylococcal enterotoxin-associated diarrheal disease is less understood and only partially examined (44, 51, 59, 60, 61). Importantly, our experiments demonstrate that MCP-1 was the earliest chemokine induced by SEA in IMFs. MCP-1 mainly acts through its CCR-2 and specifically attracts monocytes/macrophages and memory T cells and is known to be one of the earliest chemokines to be produced in different acute and chronic inflammatory diseases. MCP-1 has been reported to regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis (62). Although, the role of this proinflammatory mediator in staphylococcal enterotoxin-associated diseases remains elusive, our experiments suggests that MCP-1, produced by subepithelial IMFs in response to SEA stimulation, may be responsible for IL-8 and IL-6 production during the initiation of inflammatory injury associated with SEA-associated diarrheal disease. The production of IL-8 and IL-6 by IMFs upon SEA stimulation was abrogated in presence of MCP-1-neutralizing Abs. However, we observed that CCR-2-blocking Abs only partially blocked the production of these proinflammatory mediators by MCP-1- or SEA-stimulated IMFs, which may suggest promiscuity in MCP-1 receptors (63) or different CCR-2 isoform expression on IMFs, as it has been previously described for monocytes (64). Our experiments also demonstrated that supernatants from SEA-stimulated IMF cultures (24 h) induced significant transmigration of PBMC. This transmigration was decreased to a basal level when we tested supernatants from IMF cultures exposed to SEA in the presence of anti-MCP-1-neutralizing mAbs. These data indicated that MCP-1 produced by IMFs in response to SEA stimulation was biologically active and might be the earliest participant of the chemokine network that contributes to the selective homing and mucosal accumulation of leukocytes in SEA-induced intestinal inflammation.

Taken together, these results indicate that IMFs may be key contributors to the cytokine bolus produced in response to SEA. Moreover, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease. Further investigations of IMF role in early staphylococcal enterotoxin-induced responses, especially in the production of proinflammatory mediators, might provide insights into the mechanisms by which SEA may initiate and/or propagate the inflammatory pathological processes associated with SEA food poisoning. Such insights may lead to the design of new therapeutic strategies for the management of this disease.


   Acknowledgments
 
We thank Dr. Leoncio Vergara of the University of Texas Medical Branch Optical Imaging Laboratory for his assistance and expertise with the confocal microscopy studies. We thank Mark Griffin of the Gulf Coast Digestive Diseases Center Immunology Core for his assistance with flow cytometry analyses.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK55783), the John Sealy Memorial Endowment Fund, the University of Texas Medical Branch, Gastrointestinal Research Interdisciplinary Program, the James W. McLaughlin Endowment Fund, and the Gulf Coast Digestive Diseases Center (DK56338). Back

2 Address correspondence and reprint requests to Dr. Victor E. Reyes, University of Texas Medical Branch, Children’s Hospital, Room 2.300, 301 University Boulevard, Galveston, TX 77555. E-mail address: vreyes{at}utmb.edu Back

3 Abbreviations used in this paper: SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; GI, gastrointestinal; IMF, intestinal myofibroblast; AF, Alexa Fluor; MFI, mean fluorescence intensity; TER, transepithelial resistance; RT, reverse transcription; CT, cycle threshold. Back

Received for publication September 8, 2006. Accepted for publication March 29, 2007.


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