In Situ Expression of Interleukin-10 in Noninflamed Human Gut and in Inflammatory Bowel Disease

A dysregulated secretion of contra-inflammatory cytokines such as interleukin-10 (IL-10) could play a role in the pathogenesis of inflammatory bowel disease (IBD). We have investigated the expression of IL-10 in gut tissues from patients with Crohn’s disease (CD), ulcerative colitis (UC) and controls by mRNA in situ hybridization and immunohistochemistry. Intestinal epithelial cells were found to express IL-10 mRNA and IL-10 protein in all of the tissues investigated without any major differences in the expression patterns. However, compared with noninflamed gut, significantly increased numbers of mononuclear cells (MNCs) producing IL-10 were present in inflamed gut, both in CD and UC. This cytokine was expressed most prominently by inflammatory infiltrates enriched in macrophages, although T cells seem to contribute to its production as well. Elevated IL-10 expression in IBD was mainly detected in the submucosa, whereas IL-10 production by lamina propria cells remained comparably low. In contrast, the expression of IL-1β mRNA was preferentially increased in the lamina propria. Our data argue against a general deficiency in IL-10 production in IBD. The results suggest rather that the local production of IL-10 by mucosal MNCs in IBD is insufficient to down-regulate pro-inflammatory cytokines such as IL-1β in the lamina propria compartment.     

IL-10-dependent partial refractoriness to Toll-like receptor stimulation modulates gut mucosal dendritic cell function

The default response of the intestinal immune system to most antigens is the induction of immunological tolerance, which is difficult to reconcile with the constant exposure to ligands for TLR and other pattern recognition receptors. We showed previously that dendritic cells (DC) from the lamina propria of normal mouse intestine may be inherently tolerogenic and here we have explored how this might relate to the expression and function of Toll-like receptors (TLR). Lamina propria (LP) DC showed higher levels of TLR 2, 3, 4 and 9 protein expression than spleen and MLN DC, with most TLR-expressing DC in the gut being CD11c(lo), class II MHC(lo), CD103(-), CD11b(-) and F4/80(-). TLR expression by lamina propria DC was low in the upper small intestine and higher in distal small intestine and colon. Freshly isolated lamina propria DC expressed some CD40, CD80, CD86 and functional CCR7. These were up-regulated on CD11c(lo), but not on CD11c(hi) LP DC by stimulation via TLR. However, there was little induction of IL-12 by either subset in response to TLR ligation. This was associated with constitutive IL-10 production and was reversed by blocking IL-10 function. Thus, IL-10 may maintain LP DC in a partially unresponsive state to TLR ligation, allowing them to have a critical role in immune homeostasis in the gut.     

Role of SIRPα in regulation of mucosal immunity in the intestine

Mononuclear phagocytes such as dendritic cells (DCs) and macrophages in the lamina propria (LP) are thought to be important for both induction of inflammatory responses and maintenance of immunologic tolerance in the mammalian intestine. The molecular mechanisms by which these cells regulate intestinal immunity have remained poorly understood, however. Signal regulatory protein α (SIRPα) is a transmembrane protein that is specifically expressed in DCs, macrophages and neutrophils. Here, we show that SIRPα is abundant in CD11c(+) CD11b(+) LP cells of the mouse intestine. Whereas SIRPα did not appear to be important for the steady-state homeostasis of mucosal immunity in the intestine, the flagellin-stimulated production of IL-17 or interferon (IFN)-γ by LP cells of SIRPα mutant (MT) mice that lack the cytoplasmic region of the protein was markedly decreased compared with that observed with wild-type cells. Moreover, the flagellin-induced production of IL-6 by LP cells from SIRPα MT mice was also greatly reduced. SIRPα MT mice were also resistant to the development of colitis induced by IL-10 deficiency. Our data thus suggest that SIRPα expressed on CD11c(+) LP cells is important for the production of IL-17 or IFN-γ in the LP as well as for the development of colitis induced by IL-10 deficiency.     

Type 1 T-helper cell predominance and interleukin-12 expression in the gut of patients with Crohn's disease.

Crohn's disease (CD) is a chronic bowel inflammatory disorder in which the pathogenic role of immune alterations has been suggested, but the immunologic mechanisms responsible for the inflammatory reaction are still poorly understood. We investigated the profile of cytokine secretion by T-cell clones generated from gut tissue specimens of four patients with active CD, five patients with ulcerative colitis, and four patients with noninflammatory gut disorders (NIGDs). The great majority of CD4+ T-cell clones generated from the gut of patients with CD produced high levels of interferon-gamma (IFN-gamma) but low or undetectable amounts of interleukin-4 (IL-4), whereas substantial proportions of CD4+ T-cell clones derived from the gut of patients with either ulcerative colitis or NIGDs produced IL-4 in addition to IFN-gamma. The immunohistochemical analysis revealed high numbers of activated CD4+ T cells showing IFN-gamma but not IL-4 reactivity, as well as substantial proportions of IL-12-containing macrophages, in the intestinal lamina propria and muscularis propria of patients with CD, whereas these cells were very rare or undetectable in patients with NIGDs. Culturing T cells from gut biopsy specimens of a patient with CD in the presence of a neutralizing anti-IL-12 antibody down-regulated the development of IFN-gamma-producing CD4+ T cells. These findings suggest that a critical event in the initiation of bowel inflammatory lesions in CD may involve up-regulation of IL-12 production, resulting in conditions that maximally promote type 1 T-helper immune responses.     

Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation

Dietary proteins harbour bioactive peptides that can be released by a fermentation process. Fish proteins are a valuable and little-exploited source of potentially active biopeptides. The aim of this research was to evaluate the effects of a commercially available fermented fish protein concentrate (Seacure) (FPC) derived from a fermentation process, on the mucosal immune response in a murine model. BALB/c mice received the FPC or the non-fermented powder at different concentrations (0.20, 0.25 or 0.30mg/ml) for 2, 5 or 7 consecutive days. At the end of each feeding period, histological studies of the gut were carried out and the phagocytic activity of peritoneal macrophages, the number of IgA+ cells in the small intestine lamina propria and bronchial tissue and the number of IL-4+, IL-6+, IL-10+, IFNgamma+ and TNFalpha+ cells in the small intestine lamina propria were determined. Different accumulative doses of FPC did not induce any inflammatory immune response and the normal morphology of the small intestine was not affected. Phagocytic activity of peritoneal macrophages was enhanced following FPC administration at 0.3mg/ml for 7 consecutive days. The number of IgA+ cells increased in the small intestine lamina propria but not in the bronchial tissue. IL-4, IL-6 and IL-10 were all significantly increased in the lamina propria of the small intestine of animals that received FPC. At the same time, some pro-inflammatory cytokines such as IFNgamma and TNFalpha also increased, but the intestinal homoeostasis was maintained and no tissue damage was observed. We conclude that FPC is an immunomodulating food with a demonstrated capacity to enhance non-specific host defense mechanisms.     

Macrophages expressing the scavenger receptor CD163: a link between immune alterations of the gut and synovial inflammation in spondyloarthropathy

The objective of this study was to investigate CD163+ macrophages in the synovial membrane of patients with spondyloarthropathy (SpA). Immunohistochemistry was performed on synovium of 17 SpA and 18 rheumatoid arthritis (RA) patients, on colonic biopsies of 16 SpA patients and ten healthy controls, and on paired synovial biopsies of eight SpA patients, before and after anti-TNFalpha therapy. Phenotype and cytokine production were analysed by flow cytometry. CD163+ macrophages were increased in the synovial lining and sublining in SpA versus RA, as well as in colonic lamina propria in SpA versus controls. The number of CD163+ macrophages in the synovial sublining correlated with C-reactive protein levels and erythrocyte sedimentation rate. Paralleling the increase of CD163, HLA-DR was increased in the synovial lining and sublining of SpA. In contrast, the co-stimulatory molecules CD80 and CD86 and the dendritic cell markers CD1a and CD83 were scarce in SpA synovium. Flow cytometry indicated that CD163+ macrophages expressed high levels of HLA-DR and could produce in vitro tumour necrosis factor alpha (TNFalpha) but not interleukin-10 (IL-10). Finally, anti-TNFalpha therapy in vivo induced a decrease of CD163+ macrophages in the synovial lining and sublining. In conclusion, macrophages expressing the scavenger receptor CD163 are increased in synovium and in colonic mucosa in SpA, highlighting the relationship between joint and gut in this disease. The correlation with inflammatory parameters, the expression of HLA-DR, the production of TNFalpha but not IL-10, and the reduction by anti-TNFalpha therapy support a role for CD163+ macrophages in the synovial inflammation in SpA.     

Functional analysis of TCR gamma delta+ T cells in tumour-infiltrating lymphocytes (TIL) of human pancreatic cancer.

The effect of HIV infection on intestinal lamina propria macrophage subsets was investigated in 41 patients at various stages of HIV infection (asymptomatic HIV infection, n = 17; AIDS, n = 24). Duodenal biopsies taken from HIV patients at endoscopy were snap frozen and cryostat sections cut for immunohistochemical staining. MoAbs CD68 (EBM11, pan-macrophage marker), RFD1 (antigen-presenting cells) and RFD7 (mature phagocytic macrophages) were used to identify cell subsets using indirect immunoperoxidase or alkaline phosphatase. Double immunofluorescence using MoAbs to HIV proteins (p24, p17 and gp120) and RFD1 were used to identify HIV-infected antigen-presenting cells. Double immunofluorescence was also used to identify macrophages that expressed both RFD1 and RFD7 ('suppressor' macrophages). Intensity of HLA-DR expression in lamina propria cells was investigated using a MoAb to HLA-DR directly conjugated to glucose oxidase. The results show that there was no difference in overall density of macrophages, but there was a significant decrease in dendritic cells (RFD1+) in all clinical stages of HIV. There was no difference in the density of RFD7+ macrophages, nor was there a difference intensity of HLA-DR expression in lamina propria cells. Only four HIV-infected cells were positively identified in the 41 patients. This result suggests that the antigen-presenting arm of mucosal immune defences may be seriously compromised in HIV infection, and represents a further insult to mucosal immunity already impaired as a result of loss of CD4+ T lymphocytes. This may contribute to development of opportunist infection in the gut.     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

Antigen-specific and polyclonal CD4+ lamina propria T-cell lines: phenotypic and functional characterization.

Surface phenotype and function of lamina propria CD4+ T cells have been evaluated. In addition, long-term, antigen-specific and polyclonal lamina propria CD4+ T-cell lines have been generated and characterized. Lamina propria CD4+ T cells represent approximately 30% of lamina propria lymphocytes and are responsive to a variety of T-cell mitogens, including anti-CD3, concanavalin A, phytohaemagglutinin and pokeweed mitogen. In each case, however, lamina propria T cells are less responsive to these mitogens than spleen T cells. Freshly isolated lamina propria T cells produce substantial amounts of interleukin-2 (IL-2), interleukin-4 (IL-4), gamma interferon and to a lesser extent interleukin-5 (IL-5). Antigen-specific lamina propria CD4+ T-cell lines were generated by orally immunizing animals with antigen (KLH) in conjunction with cholera toxin as an oral adjuvant. Polyclonal lamina propria CD4+ T-cell lines were generated from unimmunized animals using anti-CD3 as a polyclonal stimulus. Both antigen-specific and polyclonal CD4+ T-cell lines were Thy-1+, alpha beta TCR+ and CD8-. The antigen-specific CD4+ T-cell line when stimulated by anti-CD3 and PMA produces predominantly IL-2, IL-4 and gamma interferon, with very little IL-5. In contrast, the polyclonal CD4+ T-cell line when similarly stimulated produces predominantly IL-4 and IL-5, with very little IL-2 and no detectable gamma interferon. In summary, lamina propria CD4+ T cells have been evaluated and in vitro conditions have been determined for successful generation of lamina propria CD4+ T-cell lines.     

Involvement of interleukin 18 in Crohn's disease: evidence from in vitro analysis of human gut inflammatory cells and from experimental colitis models

An imbalance of immunoregulatory factors and/or cells contributes to uncontrolled mucosal T cell activation and inflammation in Crohn's disease (CD). Bioactive interleukin (IL)-18 has been shown to be produced by macrophages in CD lesions. We report here that T cells freshly isolated from inflamed tissue of CD patients (and not T cells from control intestinal tissue) were responsive to IL-18. In the presence of IL-18, these T cells produced more interferon (IFN)-gamma and less IL-10. To analyse further the role of IL-18 in this disease, an acute and a chronic model of murine colitis were used. IL-18 mRNA was significantly enhanced in trinitrobenzene sulphonic acid (TNBS) induced colitis, and treatment with IL-18 binding protein (IL-18BPa), which neutralizes IL-18 bioactivity, significantly reduced the severity of colitis. However, IL-18BPa did not affect the course of chronic colitis in CD45RBhighCD4+ T cell reconstituted SCID mice. Production of IFN-gamma in lamina propria mononuclear cell cultures from IL-18BPa-treated SCID mice was decreased, but at the same time fewer lamina propria CD4+ T cells harvested from IL-18BPa-treated mice compared to non-treated mice were in apoptosis. We conclude that IL-18 clearly has a modulatory role in the inflammatory cascade of CD and experimental colitis by affecting IFN-gamma and IL-10 production, and apoptosis. In view of the divergent effects of IL-18 neutralization in the two different murine colitis models, it is unlikely that IL-18 is at the top of this cascade.     

A C-Type Lectin MGL1/CD301a Plays an Anti-Inflammatory Role in Murine Experimental Colitis

Inflammatory bowel disease is caused by abnormal inflammatory and immune responses to harmless substances, such as commensal bacteria, in the large bowel. Such responses appear to be suppressed under healthy conditions, although the mechanism of such suppression is currently unclear. The present study aimed to reveal whether the recognition of bacterial surface carbohydrates by the macrophage galactose-type C-type lectin-1, MGL1/CD301a, induces both the production and secretion of interleukin (IL)-10. Dextran sulfate sodium salt (DSS) was orally administrated to mice that lacked MGL1/CD301a (Mgl1^−/− mice) and their wild-type littermates. Mgl1^−/− mice showed significantly more severe inflammation than wild-type mice after administration of DSS. MGL1-positive cells in the colonic lamina propria corresponded to macrophage-like cells with F4/80-high, CD11b-positive, and CD11c-intermediate expression. These cells in Mgl1^−/− mice produced a lower level of IL-10 mRNA compared with wild-type mice after the administration of DSS for 2 days. Recombinant MGL1 was found to bind both Streptococcus sp. and Lactobacillus sp. among commensal bacteria isolated from mesenteric lymph nodes of DSS-treated mice. Heat-killed Streptococcus sp. induced an increase in IL-10 secretion by MGL1-positive colonic lamina propria macrophages, but not the macrophage population from Mgl1^−/− mice. These results strongly suggest that MGL1/CD301a plays a protective role against colitis by effectively inducing IL-10 production by colonic lamina propria macrophages in response to invading commensal bacteria.     

IL-10 enhances IL-2-induced proliferation and cytotoxicity by human intestinal lymphocytes

IL-10 modulation of human intestinal T lymphocyte functions was studied for the first time. Lymphocyte proliferation was determined by 3H-thymidine incorporation; cytokine production, by ELISA; expression of surface markers, by immunofluorescence and flow cytometric analysis; and cytotoxicity, by lysis of 51Cr-labelled target cells. IL-10 blocked phytohaemagglutinin (PHA)-induced activation and proliferation of CD8+ T cells from the epithelium and lamina propria. It was a greater inhibitor of IL-2, interferon-gamma, and tumour necrosis factor-alpha production than were IL-4 or transforming growth factor-beta. In contrast, IL-10 enhanced IL-2-stimulated proliferation of both CD4+ and CD8+ T cells by increasing cell division after activation. It also augmented IL-2- but not IL-15-induced cytotoxicity of intestinal lymphocytes against colon cancer by a mechanism independent of natural killer cells. In conclusion, IL-10 blocking of proinflammatory cytokine secretion probably reduces intestinal inflammation. IL-10 augmentation of IL-2-induced cytotoxicity may help to maintain host defence.     

A new assay for anti-DNA antibodies in serum which includes the measurement of anti-Z-DNA.

The role of T cell activation on the immunoregulatory function of lymphocytes in the lamina propria of the normal intestine was investigated. Lymphocytes were isolated from different sites in non-human primates, and their cell surface phenotypes, response to exogenous IL-2, and immunoregulatory function in pokeweed mitogen stimulated cultures were determined. The proportions of Leu-3+ (CD4) and Leu-2+ (CD8) lymphocytes in isolated lamina propria cells were similar to peripheral blood and spleen, but the proportion of Leu-3+ cells was significantly higher in mesenteric lymph node lymphocytes. The proportion of IL-2 receptor positive cells was significantly higher in lamina propria compared with peripheral blood, spleen and mesenteric lymph nodes. Increased IL-2 receptor expression was found on both Leu-3+ and Leu-2+ lamina propria T cells. In addition, although lamina propria T cells had a lower proliferative response to Con A, they had a significantly higher response when cultured with recombinant IL-2, indicating that they have increased expression of functional IL-2 receptors. The helper function of lamina propria T cells for pokeweed mitogen stimulated immunoglobulin synthesis was enhanced by recombinant IL-2. Although Leu-2+ lymphocytes in the lamina propria had increased IL-2 receptor expression, suppressor function of lamina propria T cells was similar to that of spleen cells, and was not enhanced by addition of exogenous IL-2. Thus, T cells in the lamina propria have increased expression of functional IL-2 receptors, and recombinant IL-2 enhances the helper, but not the suppressor function of lamina propria T cells for immunoglobulin synthesis.     

Commensal microbiota induce LPS hyporesponsiveness in colonic macrophages via the production of IL-10

Several subsets of innate immune cells, all with unique properties, reside within the intestinal lamina propria. However, compared with intestinal dendritic cells (DCs), intestinal macrophages are less well characterized. In this study, we examined the properties of macrophages in the colonic lamina propria (LMφ). Colonic DCs (LDC) showed LPS-induced production of IL-12p40. In contrast, LMφ showed constitutive IL-10 production and unresponsiveness to LPS in terms of inflammatory cytokine production. Comparison of the gene expression profiles between LMφ and LDC revealed that LMφ preferentially expressed IL-10-related genes. LMφ obtained from mice lacking IL-10 or Stat3 showed hyperproduction of tumour necrosis factor (TNF)-α and IL-6 in response to LPS. IL-10 production in the large intestine was mainly induced by LMφ and regulatory T cells and was dependent on the presence of commensal microbiota. Accordingly, LMφ from germ-free mice showed less production of IL-10 and increased levels of LPS-induced TNF-α and IL-6 production. Taken together, these results demonstrate that the activity of LMφ to produce pro-inflammatory cytokines is negatively regulated through commensal microbiota-dependent IL-10 production in the large intestine.     

Basal lymphoid aggregates in ulcerative colitis colon: a site for regulatory T cell action

Regulatory T cells seem to play a central role in maintaining immune tolerance in the gut mucosa. Previously we have shown that interleukin (IL)-10 is produced at high levels in the inflamed colonic tissue of ulcerative colitis (UC) patients. The cellular source was CD4+ T cells, suggesting local activation of regulatory T cells. The present study was performed to determine whether the frequency of regulatory T cells is increased in UC colon and whether they are present in the basal lymphoid aggregates, the prominent microanatomical structure in UC colon. Colonic tissue specimens from UC and control patients were analysed for frequencies of lamina propria lymphocytes expressing the regulatory T cell markers forkhead box protein 3 (FoxP3), CD25 and glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) as well as CD28, CD4 and CD3 by using marker specific reagents in immunomorphometry. Two-colour immunohistochemistry was used for detection of CD25/IL-10, FoxP3/IL-10 and CD25/FoxP3 double-positive cells. GITR+ and FoxP3+ cells were present in normal colon mucosa, although at a relatively low frequency, and were located preferentially within the solitary follicles. UC was associated with significantly increased frequencies of CD25+, GITR+ and FoxP3+ lamina propria lymphocytes both within the basal lymphoid aggregates and in the lamina propria outside. Many of the CD25+ cells co-expressed FoxP3 as well as IL-10, suggesting that these are indeed IL-10 secreting regulatory T cells, activated in an attempt to counteract the inflammation. Increased frequency of regulatory T cell subtypes seems insufficient to control the disease activity in UC.     

Effect of long-term continuous consumption of fermented milk containing probiotic bacteria on mucosal immunity and the activity of peritoneal macrophages

The effect of the long-term administration of commercial fermented milk containing probiotic bacteria in the mucosal immune response and peritoneal macrophages was analyzed. BALB/c mice were fed with fermented milk for 98 consecutive days. Small and large intestines were removed for histology; IgA, CD4, CD8 cells and cytokines-producing cells were counted. The influence on the immune cells associated with bronchus and mammary glands as well as on peritoneal macrophages was also analyzed. Continuous oral administration of fermented milk increased IgA+ cells in both parts of the intestine (small and large intestine). IL-10, a regulatory cytokine, increased in the intestinal cells in most samples. TNFalpha, IFNgamma and IL-2 producing cells were also enhanced. Values for CD4 and CD8(+) cell populations in lamina propria of the intestine were increased in relation to the control throughout the assay. No modifications in the histology of intestines were observed. Long-term consumption of fermented milk enhanced intestinal mucosa immunity, mediated by IgA+ cells and by cytokine production. This improvement of gut immunity was maintained and down-regulated by cytokines such as IL-10, preventing gut inflammatory immune response. The effect of this fermented milk on mucosal sites distant to the gut, such as bronchus and mammary glands, showed that in both tissues the increase in IgA+ cells was only observed at the beginning of the continuous consumption and no modifications in the number of cytokine positive cells were found. Similar observations were found when phagocytic activity of peritoneal macrophages was measured. It was demonstrated that the most evident effect of long-term consumption of fermented milk was observed in the intestine. Immunodulatory effects and the maintenance of intestinal homeostasis without secondary effects after long-term administration of fermented milk were also observed.     

Immunobiology of the human penile urethra.

The human penile urethra contains numerous IgA and J chain-positive plasma cells, and the epithelium expresses secretory component, the transport molecule for polymeric IgA, indicating that this region is an active site of secretory IgA-mediated immune defense. At the distal tip, the mucosae of the meatus and fossa navicularis contain intraepithelial dendritic cells but few macrophages, whereas the urethra proper contains many macrophages within the lamina propria and epithelium, but no dendritic cells. T lymphocytes are abundant and ubiquitous in all regions of the urethra. Both CD8+ and CD4+ subpopulations of T lymphocytes are present in the lamina propria and epithelium, although CD8+ cells predominate. The majority of T lymphocytes are positive for CD45RO (memory marker), and many are also positive for the alpha E beta 7 integrin (mucosal-associated antigen). These data indicate that the human urethra is a highly dynamic immunocompetent tissue possessing all the necessary elements for antigen presentation and both humoral and cellular mucosal immune responses. Furthermore, the urethra resembles other mucosal surfaces in terms of lymphocyte subpopulations, segregation of phenotypes and expression of antigenic determinants characteristic of mucosal lymphocytes. It is likely that this region plays a dominant role in protecting the male urogenital tract against ascending infections, and should be targeted in vaccination strategies directed against sexually transmitted diseases.     

Host defense against cholera toxin is strongly CD4+ T cell dependent.

This study investigates the role of CD4+ T cells in host defense against cholera enterotoxin-induced diarrhea. Antitoxin immunoglobulin A formation and gut protection against cholera toxin (CT) following oral immunizations with CT were evaluated in normal mice and mice that had been depleted of CD4+ T cells by in vivo treatment with specific anti-CD4 monoclonal antibodies. Flow cytometer analysis demonstrated that anti-CD4 monoclonal antibody effectively eliminated CD4+ T cells in the spleen, mesenteric lymph nodes, and Peyer's patches. In contrast, lamina propria lymphocytes demonstrated only some decrease in CD4+ T-cell numbers following antibody treatment. However, CD4 expression of individual lamina propria lymphocytes was strongly down-regulated. Depletion of CD4+ T cells performed prior to oral immunization with CT completely inhibited the ability to respond to CT. No antitoxin production, as detected at the single-cell level by the ELISPOT technique, was found in the spleen, mesenteric lymph nodes, or Peyer's patches, nor did we observe serum antitoxin responses in these mice. Control mice demonstrated strong antitoxin responses in all locations following oral immunization with CT. Anti-CD4 antibody treatment also effectively inhibited the antitoxin immunoglobulin A response in the lamina propria to CT as well as blocked the ability to develop gut protection against CT challenge of ligated intestinal loops after oral CT immunization. Thus, in vivo CD4+ T-cell depletion rendered these mice unable to develop protective immunity in the gut following oral immunization with CT. Moreover, CD4+ T-cell depletion effectively inhibited the antitoxin immune response in the gut lamina propria, mesenteric lymph nodes, Peyer's patches, and spleen when performed prior to both priming and booster immunizations with CT. This study clearly demonstrates the requirement of functional CD4+ T cells in the gut immune system for the development of host defense against CT-induced disease. Our data also reinforce the concept of a strong association between gut protection against CT and local production of neutralizing immunoglobulin A antitoxin.     

Inhibition of Murine Splenic and Mucosal Lymphocyte Function by Enteric Bacterial Products

Previously we showed that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated human peripheral blood and lamina propria mononuclear cells. The aims of the present study were to determine whether EPEC-inhibitory factors have similar effects on murine lymphoid populations in order to further delineate the mechanisms of alteration of cytokine production. Preexposure to EPEC lysates inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-γ) production by murine spleen cells, but IL-10 production was increased. The inhibition was not due to increased apoptosis and was not blocked by neutralizating antibodies against IL-10 or transforming growth factor β (TGF-β). EPEC lysates also inhibited mitogen-stimulated IL-2 and IFN-γ production by CD11b-depleted spleen cells, IL-2 and IL-4 production by intraepithelial and Peyer’s patch lymphocytes, IL-2 production by the human T-cell line Jurkat, and antigen-stimulated IL-2 production by murine spleen cells. Lysates obtained from Shiga-like toxin-producing E. coli, E. coli RDEC-1, Citrobacter rodentium, and an EPEC espB insertion mutant all inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid cells. In conclusion, lysates of EPEC and related bacteria directly inhibit cytokine production by lymphoid cells from multiple sites by a mechanism that does not increase apoptosis or result from secondary effects of IL-10 or TGF-β.