Class II MHC-expressing myofibroblasts play a role in the immunopathogenesis associated with staphylococcal enterotoxins.

Food poisoning due to staphylococcal enterotoxins (SEs) affects hundreds of thousands of people each year. Little is known about how SEs initiate immune responses and cause pathogenesis. Here, we demonstrate that cultured human intestinal myofibroblasts (IMFs) bind SEs in an MHC class II-dependent fashion. IMFs respond to SE exposure with increased secretion of IL-6, IL-8, and TNF-alpha. A significant proliferative T cell response was observed when MHC class II-expressing IMFs were pulsed with SEA and cocultured with human CD4(+) T cells. In conclusion, our findings support the hypothesis that IMFs may play an important role in pathology associated with staphlococcocal enterotoxigenic disease.

Intestinal myofibroblasts and immune tolerance.

Stromal cells, such as myofibroblasts and fibroblasts, represent a significant fraction of MHC class II-positive cells in the normal human colonic lamina propria, suggesting they may play an important role in CD4(+) T cell regulation in a tolerogenic environment. The aim of this study was to examine whether human colonic myofibroblasts (CMFs) phenotypically and functionally resemble conventional antigen-presenting cells (APCs). Our results support the hypothesis that intestinal myofibroblasts are a novel, nonprofessional APC phenotype important in modulating mucosal T cell responses. Given their strategic location, we propose that intestinal myofibroblasts play a critical role in mediating tolerance to luminal antigens.

Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis.

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.

Helicobacter pylori modulates lymphoepithelial cell interactions leading to epithelial cell damage through Fas/Fas ligand interactions.

Helicobacter pylori causes a common chronic infection of humans that leads to epithelial cell damage. Studies have shown that apoptosis of the gastric epithelium is increased during infection and this response is associated with an expansion of gastric T-helper type 1 (Th1) cells. We report that gastric T cells contribute to apoptosis of the epithelium by a Fas/Fas ligand (FasL) interaction. Fas receptor expression was detected on freshly isolated gastric epithelial cells by flow cytometry and immunohistochemistry, and this level of expression was increased during infection with H. pylori. The expression of Fas receptor on three gastric epithelial cell lines was increased by H. pylori, either alone or in combination with gamma interferon or tumor necrosis factor alpha. The role of Fas in apoptosis of gastric epithelial cell lines was evidenced by DNA fragmentation after cross-linking of Fas with specific antibodies. FasL expression was detected by immunohistochemistry on mononuclear cells in gastric biopsy specimens of infected but not uninfected subjects. Gastric T-cell lines were also shown to express FasL, as evidenced by reverse transcription-PCR and killing of target cells expressing Fas receptor. Moreover, these T-cell lines were capable of killing cultured gastric epithelial target cells and antibodies that block the interaction between Fas receptor and FasL inhibited this cytotoxic activity. These observations demonstrate that local Th1 cells may contribute to the pathogenesis of gastric disease during H. pylori infection by increasing the expression of Fas on gastric epithelial cells and inducing apoptosis through Fas/FasL interactions.

Expression of IL-10 receptors on epithelial cells from the murine small and large intestine.

The appearance of chronic intestinal inflammation in IL-10 knockout mice suggests IL-10 may inhibit adverse responses to luminal antigen. Moreover, this inflammation is associated with an increase in class II MHC molecule expression on intestinal epithelial cells. Thus, the role of IL-10 regulation in epithelial cell function was investigated. Using RT-PCR, it was shown that intestinal epithelial cells express mRNA for both subunits of the IL-10 receptor-signaling complex. In addition, biotinylated IL-10 was shown to bind to both cultured and freshly isolated intestinal epithelial cells prepared from the small or large intestine. This binding appeared specific as it was blocked by neutralizing antibodies to IL-10 but not the isotype control. Moreover, an excess of native IL-10 also inhibited the binding of radiolabeled IL-10. To evaluate whether IL-10 mediated any functions through this receptor, epithelial cells were cultured with IL-10 alone or with IFN-gamma plus IL-10. IL-10 alone had no detectable effects on epithelial cell growth or their expression of class II MHC molecules but it did antagonize the effect of IFN-gamma on the viability of cultured cells. In addition, IL-10 blocked the IFN-gamma-induced expression of class II MHC molecules on cultured epithelial cells. These results suggest that IL-10 binds to a specific receptor on intestinal epithelial cells and may regulate the contribution of epithelial cells to the inflammatory and immune response in the digestive tract.

Regulation of the mucosal immune response.

Infectious diseases continue to exact an extensive toll on populations living closest to the equatorial regions of the globe. A substantial proportion of these infections gain access to the host via the mucosal tissues. Thus, the development of new vaccines that enhance mucosal immunity is considered to be of paramount importance in order to prevent or limit the impact of these infections. Mucosal immune responses must discriminate between commensal flora within the lumen and potential pathogens. These responses are highly adapted to induce protection without excessive amounts of inflammation. The balances that regulate mucosal immune and inflammatory responses have to be understood if effective mucosal immunity is to be induced through local immunization. This review will summarize some of the unique properties of mucosal immune responses and focus on recent advances that have significantly influenced our understanding of the regulation of immune and inflammatory responses following infection.

The role of the invariant chain in mucosal immunity.

The invariant chain (Ii) due to its intimate association with major histocompatibility complex (MHC) alpha and beta chains is a determining element in the development of immune responses. Ii plays a major role in the assembly, the intracellular transport and peptide selection by class II MHC. A segment of Ii designated as CLIP (class II-associated Ii peptide) binds into the antigen binding site of class II MHC molecules until class II MHC reach intracellular compartments that contain peptides from internalized antigens. This association limits the self endogenous peptides that can bind to class II MHC molecules. The removal of CLIP from class II MHC catalyzes the binding of antigenic peptides and their subsequent cell surface expression. An isoform of Ii, known as chondroitin sulfate-modified Ii (IiCS), that is surface-expressed enhances T cell activation while acting as a coreceptor for CD44. The expression of class II MHC molecules by mucosal epithelial cells has generated interest in the role that these cells may have in mucosal immunity. Since in classical antigen-presenting cells (APC) the biology of class II MHC is regulated by Ii, it is necessary to bring into perspective the known functions of Ii in conventional APC to understand the role that Ii may play in mucosal epithelial cells as potential regulators of local immune responses.

The effect of class II major histocompatibility complex expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells: a mechanism for T helper cell type 1-mediated damage.

Helicobacter pylori infection is associated with gastric epithelial damage, including apoptosis, ulceration, and cancer. Although bacterial factors and the host response are believed to contribute to gastric disease, no receptor has been identified that explains how the bacteria attach and signal the host cell to undergo apoptosis. Using H. pylori as "bait" to capture receptor proteins in solubilized membranes of gastric epithelial cells, class II major histocompatibility complex (MHC) molecules were identified as a possible receptor. Signaling through class II MHC molecules leading to the induction of apoptosis was confirmed using cross-linking IgM antibodies to surface class II MHC molecules. Moreover, binding of H. pylori and the induction of apoptosis were inhibited by antibodies recognizing class II MHC. Since type 1 T helper cells are present during infection and produce interferon (IFN)-gamma, which increases class II MHC expression, gastric epithelial cell lines were exposed to H. pylori in the presence or absence of IFN-gamma. IFN-gamma increased the attachment of the bacteria as well as the induction of apoptosis in gastric epithelial cells. In contrast to MHC II-negative cell lines, H. pylori induced apoptosis in cells expressing class II MHC molecules constitutively or after gene transfection. These data describe a novel receptor for H. pylori and provide a mechanism by which bacteria and the host response interact in the pathogenesis of gastric epithelial cell damage.

Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype.

BACKGROUND & AIMS: Studies have shown that gastric T cells are increased during Helicobacter pylori infection. The purpose of this study was to characterize the human gastric T-cell responses in the presence or absence of H. pylori. METHODS: T-cell surface antigens were examined by immunohistochemistry or after isolation for evaluation of surface antigens and cytoplasmic cytokines using flow cytometry. RESULTS: CD4+ and CD8+ T cells were increased in situ during infection with H. pylori. Freshly isolated gastric T cells expressed cytoplasmic interferon gamma (IFN-gamma) and interleukin (IL)-2 after a brief stimulation. Simultaneous four-color flow cytometry demonstrated that both CD8+ and CD4+ T cells expressed IFN-gamma. Because stimulation through CD30 favors the induction of IL-5 and Th2 cells, gastric and colonic T cells were examined for CD30 expression. Consistent with the notion that Th2 cells are found in the intestine, CD30 was evident throughout the lamina propria of the colon but was virtually absent in the stomach. Furthermore, freshly isolated gastric T cells produced little IL-4 and virtually no IL-5 or tumor necrosis factor beta. CONCLUSIONS: These observations show that gastric T cells resemble the Th1 type, which may explain their failure to induce immunity to H. pylori and their ability to contribute to the pathogenesis of gastric disease.

How does Helicobacter pylori cause mucosal damage? The inflammatory response.

The role for Helicobacter pylori in the pathogenesis of disease provides the conundrum that only a subset of subjects infected with H. pylori will ever develop peptic ulcer or gastric cancer. Thus, variation in strain as well as environmental or host factors converge in the gastroduodenal milieu and control the final outcome of infection. The host immune and inflammatory response is emerging as an important element in the pathogenesis of these gastric diseases. The ideal host response provides protection to clear an infection without causing excessive amounts of inflammation that could compromise the integrity and function of host cells. This review will cover four main questions: (1) What are the mucosal immune/inflammatory responses that confer protection without damaging the host? (2) How do the gastric immune responses during infection with H. pylori differ from this ideal scenario? (3) Do these responses contribute to autoimmune-mediated damage to gastric tissue? (4) Can immunomodulation through vaccination enhance protective, nondestructive responses that prevent or treat infection or, at least, attenuate inflammation?

Differential stimulation of interleukin-12 (IL-12) and IL-10 by live and killed Helicobacter pylori in vitro and association of IL-12 production with gamma interferon-producing T cells in the human gastric mucosa.

The objective of these experiments was to examine the ability of Helicobacter pylori to stimulate interleukin-10 (IL-10) or IL-12 and select for either Th1 or Th2 cells. Gastric biopsy specimens were collected from patients who were categorized with respect to the presence of H. pylori and gastric disease as well as their age, gender, medications, and other factors. As Th1 and Th2 cells are selected by IL-12 and IL-10, respectively, biopsy specimens were screened for mRNA and protein for these cytokines. Although mRNA for IL-12 and IL-10 was detected in biopsy specimens obtained from both infected and uninfected patients, IL-12 protein predominated. Levels of IL-10 and IL-12 in gastric tissue did not change in response to infection. Moreover, gamma interferon (IFN-gamma)-producing T cells were found in both the infected and the uninfected gastric mucosa. Stimulation of peripheral blood leukocytes from either infected or uninfected donors with various concentrations of live or killed H. pylori induced immunoreactive IL-12 and IL-10. After stimulation with live H. pylori, IL-12 levels increased more than 30-fold, whereas IL-10 levels increased only 2- to 5-fold, compared to cells stimulated with medium alone. Interestingly, killed H. pylori induced significantly more IL-10 (P < 0.05) than live H. pylori, while recombinant urease only induced IL-10. These results demonstrate that live H. pylori selectively stimulates the induction of IL-12 and Th1 cells that produce IFN-gamma, whereas preparations used in oral vaccines induce more IL-10 and may favor Th2 cell responses.

Expression of B7-1 and B7-2 costimulatory molecules by human gastric epithelial cells: potential role in CD4+ T cell activation during Helicobacter pylori infection.

Human gastric mucosal epithelial cells display class II MHC, the expression of which is increased during Helicobacter pylori infection. These observations suggest that the gastric epithelium may participate as antigen-presenting cells (APC) during local immune responses. The increase in class II MHC expression occurs in parallel with an elevation in gastric CD4+ T cell numbers within and adjacent to the epithelium. Since the expression of either B7-1 (CD80) or B7-2 (CD86) on APC is required for the activation of T cells, it was important to establish human gastric epithelial cells expressed those surface ligands. The expression of B7-1 and B7-2 was detected on human gastric epithelial cell lines and freshly isolated epithelial cells from gastric biopsies with specific antibodies. B7-2 expression was higher than B7-1 at both protein and transcript levels and was increased after crosslinking class II MHC molecules on IFNgamma-treated epithelial cells and in cells pretreated with the combination of IFNgamma and H. pylori. Similarly, B7-2 expression was higher on gastric epithelial cells from H. pylori-infected tissues compared with those from uninfected specimens. To determine the function of these molecules on gastric epithelial cells, antibodies to B7-1 and B7-2 were shown to reduce the ability of the cells to stimulate alloreactive CD4+ T cells. These observations are the first to demonstrate that B7-1 and B7-2 are expressed on mucosal epithelial cells in situ. Thus, the expression of B7-1 and B7-2 by epithelial cells may allow them to act as APC in regulating local responses such as those that occur during infection with H. pylori.

Antigen presentation of mucosal pathogens: the players and the rules.

A vast number of infectious pathogens gain entry into the host through mucosal surfaces, which have a much greater total surface area than the skin. Since the mucosa is continuously exposed to those pathogens, the development of an effective local immune response is of utmost importance. An obligatory step in the development of most immune responses is the presentation of antigens by specialized accessory cells, termed antigen-presenting cells (APC) to T lymphocytes. The recognition of antigens by T cells is largely determined by how the antigens are handled by the APC. Complex antigen-processing events generate a selected set of peptides which ultimately become associated with MHC molecules. The type of MHC molecules that bind the peptides in turn determine what T lymphocyte subset recognizes the peptides. Thus, an understanding of the molecular and cellular processes preceding the T cell recognition event is a prerequisite for understanding how mucosal immune responses develop, as well as for investigating alternative approaches to vaccine development and therapeutic strategies to control autoimmune diseases. This review discusses the cell biology of antigen processing and how various APC populations may participate in mucosal responses.

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha.

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.

Structural analysis of invariant chain subsets as a function of their association with MHC class II chains.

Respective subsets of human invariant chain (Ii), as identified with antibodies to two different epitopes, were characterized as a function of their associations with major histocompatibility complex (MHC) class II alpha,beta chains and intracellular processing. E1 antiserum to Ii(183-193) and VIC-Y1 monoclonal antibody to an N-terminal determinant identified Ii(E1) and Ii(VIC) populations, respectively. Ii proteins comprise several species which have been defined with either genomic or post-translational processes: Ii itself; IpN and IpO, which represent the glycosylated forms on asparagine or threonine/serine, respectively; gamma 2 and gamma 3, which originate from an alternative initiation site for transcription; and p41, which has a 64-amino-acid insert which originated from an additional exon placed after the sixth exon of Ii. Immunoprecipitates of detergent-solubilized protein complexes from [35S]methionine-labeled Raji cells showed that Ii(E1) consisted of Ii, p41, IpN, and immature alpha chain, while Ii(VIC) consisted of Ii, processed Ii with N- and O-linked glycosylation (IpN,IpO), p41, and associated MHC class II alpha,beta chains. In the MHC class II-deficient P3HR-1 cells, Ii(E1) and Ii(VIC) were virtually identical. Ii(E1) was resistant to cathepsin B digestion while Ii(VIC) was sensitive. In pulse-chase radiolabeling experiments with brefeldin A (BFA)-treated cells, Ii(VIC) progressively became resistant to endoglycosidase H (endo H) and had a longer half-life than that in cells not treated with BFA, but Ii(E1) remained susceptible to endo H and its half-life was unaffected by BFA. Since BFA redistributes Golgi enzymes to the endoplasmic reticulum, this observation suggests that Ii(E1) is protected from processing enzymes while Ii(VIC) is not. These studies define association of Ii with MHC class II molecules when certain epitopes on Ii are exposed or not. These differences relate to intracellular transport of Ii and to its release for the binding of antigenic peptides.

Cathepsin B cleavage and release of invariant chain from MHC class II molecules follow a staged pattern.

A staged pattern of cathepsin B cleavage of MHC class II alpha, beta-bound invariant (Ii) chain and release of fragments was defined. Charge-loss mutations in the Ii chain were created in three clusters of cathepsin B putative cleavage sites R78K80K83K86, K137K143, and R151K154. Products of HLA-DR1 alpha, beta and wild type (WT) or mutant Ii genes, co-transfected into COS1 cells, were cleaved by cathepsin B and immunoprecipitated by antibodies either to MHC class II chains or to different Ii epitopes. In WT Ii, cathepsin B digestion generated two forms of p21 Ii fragments: a p21 recognized by anti-C-terminus antibodies and a p21 recognized by an antibody to a determinant near the N-terminus. C-terminal p21 was released from MHC class II alpha, beta chains upon its formation while N-terminal p21 remained associated with MHC class II alpha, beta chains. Mutations at K137K143 inhibited the generation of N-terminal p21 by cathepsin B. Mutation at R78K80K83K86 led to an accumulation of MHC class II-bound N-terminal p21 without the appearance of MHC class II-bound p14, p10, and p6 fragments after cathepsin B digestion. These results indicate that cathepsin B cleaves wild type Ii first about K137K143 to produce a MHC class II-associated N-terminal p21, which is then cleaved about R78K80K83K86 to generate p14, p10 and finally p6 which still associates with MHC class II alpha, beta chains. This pattern of staged cleavage and release of Ii might be related to a concerted mechanism regulating the binding of antigenic peptides to MHC class II molecules.

More efficient peptide binding to MHC class II molecules during cathepsin B digestion of Ii than after Ii release.

The binding of a T cell-presented peptide to MHC class II alpha,beta chains occurs as a concurrent process with the release of the associated invariant chain (Ii) by cathepsin B. Ii was digested by cathepsin B from solubilized, MHC class II alpha,beta,Ii complexes in the presence of N-hydroxysuccinimidyl-4-azidobenzoate-conjugated, 125I-labeled, influenza virus matrix (18-29) peptide. The peptide was crosslinked where it became bound. This HLA-DR1-restricted peptide bound about three times more efficiently to class II alpha,beta chains of DR1-positive B cells when present during cathepsin B digestion of Ii than when added afterward, also at pH 5.0. Binding was competed by similarly DR-restricted peptides. Cathepsin D cleaved Ii but did not enhance peptide binding. However, a trace level of cathepsin D, added to the assay for peptide binding in the presence of cathepsin B, further enhanced peptide binding about three times. These experiments support an hypothesis for the staged release of Ii fragments by cathepsin D and cathepsin B, catalyzing at one point the insertion of a peptide into the antigen binding site formed by class II alpha and beta chains.

The role of the local immune response in the pathogenesis of peptic ulcer formation.

Mucosal immune responses are designed to provide local protection against infection, without inducing excessive amounts of inflammation that would alter epithelial integrity or function. It has become clear that the epithelium not only serves as a barrier to exclude pathogens, but also initiates host responses to infection. Gastric epithelial cells infected with Helicobacter pylori can respond within hours to produce inflammatory mediators that recruit and activate neutrophils. The gastric epithelium can also be recognized by local T-cells, resulting in their activation and ability to induce epithelial damage. During infection with H. pylori, there is a remarkable increase in the level of local IgG antibodies, which may also recognize and damage the epithelium. Thus, activated neutrophils, T-cells and auto-antibodies may contribute to a weakened epithelial barrier that allows luminal acid and other factors to contribute to peptic ulceration. The epithelium appears to play a key role in the initiation of the local inflammatory and immune responses that may contribute to the more serious sequelae associated with H. pylori infection.