A monoclonal antibody specific for rat intestinal lymphocytes

A monoclonal antibody, RGL-1, was produced by fusion of NSI myeloma cells with spleen cells of a mouse immunized with isolated rat intraepithelial lymphocytes (IEL). SDS-PAGE analysis revealed that RGL-1 precipitated two major noncovalently bound chains of about m.w. 100,000 and 125,000, and a minor component of m.w. 200,000. Examination of both tissue sections and isolated cells indicated that RGL-1 stained the majority of the lamina propria lymphocytes and IEL but only very few cells (less than 2%) in the lymphoid organs and small numbers of lymphocytes in other mucosae. In the small intestine, RGL-1 stained lymphocytes with the helper (W3/25) as well as the cytotoxic/suppressor (OX8) phenotype. The antibody reacted with 95% of the granular IEL but with less than 0.1% of the blood large granular lymphocytes. Although mature IgA plasma cells in the lamina propria were RGL-1-, some large IgA-containing cells were weakly positive. In the gut-associated lymphoid tissues (GALT), studies combining immunofluorescence and autoradiography indicated that 56 and 73% of rapidly dividing cells of mesenteric lymph nodes and of thoracic duct lymph (TDL) stained with RGL-1, respectively. In addition, 90 to 100% of the IgA-containing blasts of MLN and 75% of those of TDL were labeled by RGL-1. In contrast, rapidly dividing cells of spleen and of peripheral lymph nodes did not stain with RGL-1. Because RGL-1 can be demonstrated on both intestinal lymphocytes and their immediate precursors in the GALT, its expression may be related to the homing of lymphocytes into the gut mucosa.     

Gut-associated lymphoid tissue, T cell trafficking, and chronic intestinal inflammation

The etiologies of the inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) have not been fully elucidated. However, there is very good evidence implicating T cell and T cell trafficking to the gut and its associated lymphoid tissue as important components in disease pathogenesis. The objective of this review is to provide an overview of the mechanisms involved in naive and effector T cell trafficking to the gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes and intestine in response to commensal enteric antigens under physiological conditions as well as during the induction of chronic gut inflammation. In addition, recent data suggests that the GALT may not be required for enteric antigen-driven intestinal inflammation in certain mouse models of IBD. These new data suggest a possible paradigm shift in our understanding of how and where naive T cells become activated to yield disease-producing effector cells.     

Specific attachment of mesenteric IgA lymphoblasts to specialized endothelium of intestinal mucosa lamina propria capillaries

The bulk of IgA secreted in the gut is mostly contributed by locally dwelling plasma cells derived from B cells originating in the gut-associated lymphoid tissues (GALT). These IgA cells originate in Peyer's patches and recirculate, returning to the gut upon maturity. The precise mechanism of homing to secretory mucosae is to date not fully understood. It has been demonstrated, however, that specialized endothelium of small vascular spaces in peripheral nodes (PN) and endothelia of mucosal vessels are the site of receptor recognition for B and T cells. In their sojourn, IgA blasts have been shown to stop momentarily in mesenteric nodes (MN) before proceeding to their final destination, the lamina propria (LP) of the gut mucosa. They then develop into IgA-secreting plasma cells. In the present work, we show that IgA MN lymphoblasts, when compared to PN lymphoblasts, attach preferentially to LP venule and capillary endothelium, The B-cell maturation in the mesenteric lymph nodes, where IgA is the sole membrane-bound immunoglobulin, allows attachment of most of these cells. Our work suggests that the site of exit of IgA cells from the circulation are these specialized lamina propria venules and capillaries.     

Innate and specific gut-associated immunity and microbial interference

Certain bacterial species isolated from the gastrointestinal microbial communities release low-molecular-weight peptides into milk products using bacteria-derived proteases that degrade milk casein, and thereby generate peptides, triggering immune responses. The intestinal microbial communities contributes to the processing of food antigens in the gut. The present study was designed to investigate the immunomodulatory effects of microbial interference to determine whether casein degraded by probiotic bacteria-derived enzymes could modulate the cytokine production and peripheral blood mononuclear cells in atopic infants with cow or other synthetic milk allergy. Without hydrolyzation, casein reduced the production of interleukin-4, which indicates that probiotics modify the structure of potentially harmful antigens and thereby alter the mode of their immunogenicity. Intraluminal bacterial antigens have been reported to elicit specific responses in the gut-associated lymphoid tissue (GALT) through the binding capacity of intraluminal bacterial antigens to epithelial cells, which allows antigen entry via enterocytes and aids in evading the tolerance function in Peyer's patches. Such tonic immune responses in the GALT may allow control of the metabolic activity and balance of the gut microbial communities.     

THE DISTRIBUTION AND DIFFERENTIATION OF LYMPH-BORNE IMMUNOBLASTS AFTER INTRAVENOUS INJECTION INTO SYNGENEIC RECIPIENTS

Thoracic duct lymph from inbred, hooded rats was collected 3–5 days after antigenic stimulation of the caudal lymph nodes. During this period the lymph contained 10–15% of large, basophilic lymphoid blast cells (immunoblasts). By incubating the lymph cells at 38.C with radioactive DNA precursors, either ³H-thymidine or ¹²⁵I-deoxyuridine, the immunoblasts became labelled but the small lymphocytes did not. The lymph cells were then washed and injected intravenously into syngeneic recipients which were killed after various intervals up to 24 hr so that the radioactivity of their organs could be assayed by scintillation counting and autoradiography.
The main finding was that in animals killed after 4 or more hours the small gut always contained most of the recoverable activity and autoradiographs showed that this was because the injected cells had infiltrated the lamina propria in large numbers. Earlier, many of the injected cells were retained temporarily in the lungs, liver and spleen but many of them soon left those organs and entered the lamina propria of the small gut.
An electron microscope study of autoradiographs showed that 24 hr after injection the cells which entered the lamina propria of the gut had differentiated into plasma cells so that they displayed abundant, lamellar endoplasmic reticulum.     

Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin

Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine. This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice. Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels. Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma. Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT. Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells. Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site. Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches. Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone. We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time. The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues. We propose that CT accomplishes these effects by altering the regulatory environment within GALT.     

Regulation of immune response at intestinal and peripheral sites by probiotics

The gut associated lymphoid tissue (GALT) should protect intestinal mucosa against pathogens, but also avoid hypersensitivity reactions to food proteins, normal bacterial flora and other environmental macromolecules. The interaction between epithelial cells and microflora is fundamental to establish gut mucosal barrier and GALT development. The normal colonization of intestine by commensal bacteria is thus crucial for a correct development of mucosal immune system. Probiotic bacteria are normal inhabitants of microflora and may confer health benefits to the host. The modification of the intestinal microflora towards a healthier probiotics enriched microflora may generate beneficial mucosal immunomodulatory effects and may represent a new strategy to cure intestinal and allergic diseases. The health benefits may be specific for different probiotic strains. Ongoing research is providing new insights into the probiotic beneficial effects and related mechanisms. This review represents an update of immunomodulatory activity of different probiotics and of the more accredited mechanisms underlying such activities. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Teleost intestinal immunology

Teleosts clearly have a more diffuse gut associated lymphoid system, which is morphological and functional clearly different from the mammalian GALT. All immune cells necessary for a local immune response are abundantly present in the gut mucosa of the species studied and local immune responses can be monitored after intestinal immunization. Fish do not produce IgA, but a special mucosal IgM isotype seems to be secreted and may (partly) be the recently described IgZ/IgT. Fish produce a pIgR in their mucosal tissues but it is smaller (2 ILD) than the 4–5 ILD pIgR of higher vertebrates. Whether teleost pIgR is transcytosed and cleaved off in the same way needs further investigation, especially because a secretory component (SC) is only reported in one species. Teleosts also have high numbers of IEL, most of them are CD3-?⁺/CD8-α⁺ and have cytotoxic and/or regulatory function. Possibly many of these cells are TCRγδ cells and they may be involved in the oral tolerance induction observed in fish. Innate immune cells can be observed in the teleost gut from first feeding onwards, but B cells appear much later in mucosal compartments compared to systemic sites. Conspicuous is the very early presence of putative T cells or their precursors in the fish gut, which together with the rag-1 expression of intestinal lymphoid cells may be an indication for an extra-thymic development of certain T cells. Teleosts can develop enteritis in their antigen transporting second gut segment and epithelial cells, IEL and eosinophils/basophils seem to play a crucial role in this intestinal inflammation model. Teleost intestine can be exploited for oral vaccination strategies and probiotic immune stimulation. A variety of encapsulation methods, to protect vaccines against degradation in the foregut, are reported with promising results but in most cases they appear not to be cost effective yet. Microbiota in fish are clearly different from terrestrial animals. In the past decade a fast increasing number of papers is dedicated to the oral administration of a variety of probiotics that can have a strong health beneficial effect, but much more attention has to be paid to the immune mechanisms behind these effects. The recent development of gnotobiotic fish models may be very helpful to study the immune effects of microbiota and probiotics in teleosts.     

Cutting Edge: 4-1BB Controls Regulatory Activity in Dendritic Cells through Promoting Optimal Expression of Retinal Dehydrogenase

Dendritic cells (DC) in the gut promote immune tolerance by expressing retinal dehydrogenase (RALDH), an enzyme that promotes retinoic acid, which aids differentiation of Foxp3(+) inducible regulatory T cells (iTreg) in the intestinal mucosa. How RALDH expression is regulated is unclear. We found that 4-1BB (CD137), a member of the TNFR family, together with CD103, marked mesenteric lymph node DC with the highest level of RALDH activity, and ligation of 4-1BB maintained RALDH expression in these gut DC. Moreover, 4-1BB signals synergized with those through TLR2 or GM-CSFR to promote RALDH activity in undifferentiated DC. Correspondingly, 4-1BB-deficient mice were impaired in their ability to generate iTreg in the GALT when exposed to oral Ag, and 4-1BB-deficient mesenteric lymph node DC displayed weak RALDH activity and were poor at promoting iTreg development. Thus, our data demonstrate a novel activity of 4-1BB in controlling RALDH expression and the regulatory activity of DC.     

In vivo depletion of CD11c+ cells impairs scrapie agent neuroinvasion from the intestine

Following oral exposure, some transmissible spongiform encephalopathy (TSE) agents accumulate first upon follicular dendritic cells (DCs) in the GALT. Studies in mice have shown that TSE agent accumulation in the GALT, in particular the Peyer's patches, is obligatory for the efficient transmission of disease to the brain. However, the mechanism through which TSE agents are initially conveyed from the gut lumen to the GALT is not known. Studies have implicated migratory hemopoietic DCs in this process, but direct demonstration of their involvement in vivo is lacking. In this study, we have investigated the contribution of CD11c(+) DCs in scrapie agent neuroinvasion through use of CD11c-diptheria toxin receptor-transgenic mice in which CD11c(+) DCs can be specifically and transiently depleted. Using two distinct scrapie agent strains (ME7 and 139A scrapie agents), we show that when CD11c(+) DCs were transiently depleted in the GALT and spleen before oral exposure, early agent accumulation in these tissues was blocked. In addition, CD11c(+) cell depletion reduced susceptibility to oral scrapie challenge indicating that TSE agent neuroinvasion from the GALT was impaired. In conclusion, these data demonstrate that migratory CD11c(+) DCs play a key role in the translocation of the scrapie agent from the gut lumen to the GALT from which neuroinvasion subsequently occurs.     

Insulin-dependent diabetes and gut dysfunction: the BB rat model.

Accumulating data indicate that intestinal dysfunction and dysregulation of the gut immune system may play a role in the development of type 1 diabetes. This review deals with the occurrence of gut damage and dysfunction in BB rats, an animal model of spontaneous immune type 1 diabetes, placing special emphasis on the effect of diet on the incidence of diabetes in BB rats, the identification of a type 1 diabetes-related protein from wheat, and preliminary observations documenting anomalies in the inductive tissues of the gut immune system (Peyer's patch cells and mesenteric lymph node cells) and pancreatic lymph node cells of diabetes-prone BB rats. In addition to histological evidence of gut damage, the review will also draw attention to altered intestinal disaccharidase activity, changes in intestinal peroxidase activity, glucagon-like peptide 1 anomalies, and perturbation of both intestinal permeability and mucin content in BB rats. In all these cases, the findings in rats fed a diabetes-promoting diet are compared to those collected in animals receiving a protective diabetes-retardant diet.     

Intestinal microflora and diversification of the rabbit antibody repertoire

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1-2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.     

Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.     

Man the barrier! Strategic defences in the intestinal mucosa

Immunologists typically study the immune responses induced in the spleen or peripheral lymph nodes after parenteral immunization with antigen and poorly defined experimental adjuvants. However, most antigens enter the body through mucosal surfaces. It is now clear that the microenvironment in these mucosal barriers has a marked influence on the immune response that ultimately ensues. Nowhere is the microenvironment more influential than in the gut-associated lymphoid tissue (GALT). The GALT must constantly distinguish harmless antigens that are present in food or on commensal bacteria from pathogenic assault by microbes. It is perhaps not surprising, then, that the GALT contains more lymphocytes than all of the secondary lymphoid organs combined.     

Proliferative kinetics of large and small intraepithelial lymphocytes in the small intestine of the mouse.

The proliferative kinetics of the intraepithelial lymphocytes (IL) of the mouse intestine have been evaluated. By inducing mitotic arrest it was found that large IL - constituting about 50% of the IL - showed a mitotic rate of 2.3. Autoradiographic results obtained after two different schedules of 3H-thymidine injections showed that 30% of the large IL were in DNA synthesis, and that the large IL were renewed at a rate comparable to that of blast cells from Peyer's patches, mesenteric lymph nodes and thoracic duct lymph. The small IL were renewed very rapidly compared to small lymphocytes of peripheral lymphoid tissues, although small lymphocytes with lifespans of several weeks were also present in the epithelial sheet. By the use of intestinal perfusion, in vivo, it was estimated that the loss of lymphocytes from intestinal villi into the lumen of the gut was negligible, and it is concluded that the most probable kinetic model for the majority of IL is: B and T lymphoblasts invade the epithelium and undergo mitosis. B lymphoblasts give rise predominantly to plasma cells, and T lymphoblasts give rise to small lymphocytes - probably long-lived - which reenter the circulation.     

T-cell help for the IgA response: the function of T cells from different lymphoid organs in regulating the proportions of plasma cells expressing various isotypes

Differential distribution of IgA-specific primed Lyt 2- T cells (TH) in favor of gut-associated lymphoid tissue (GALT) has been proposed to account for the high proportion of IgA-producing plasma cells at mucosal versus nonmucosal sites. We find, however, that GALT TH primed enterically with sheep red blood cells (SRBC) contain no more help for IgA responses than peripheral lymph node (PN) TH primed subcutaneously. Moreover, GALT TH are only poorly primed by enterically administered soluble protein antigen and therefore provide less help for all isotypes than PN TH primed subcutaneously with the same antigen. On the other hand, supernatants of GALT TH stimulated with concanavalin A (Con A) in vitro do help higher IgA:IgG plaque-forming cell (PFC) ratios in cultures with 2,4, 6-trinitrophenyl-SRBC (TNP-SRBC) than supernatants from PN and spleen, indicating that, when appropriately stimulated, GALT TH are capable of promoting relatively higher IgA responses than TH from other sources. Responses elicited by either SRBC-primed TH or splenic Con A supernatants in the presence of TNP-SRBC contained higher IgA:IgG PFC ratios than those elicited by linked recognition in the presence of haptenated soluble protein carrier.     

The gut mucosal viral reservoir in HIV-infected patients is not the major source of rebound plasma viremia following interruption of highly active antiretroviral therapy

Interruption of suppressive highly active antiretroviral therapy (HAART) in HIV-infected patients leads to increased HIV replication and viral rebound in peripheral blood. Effects of therapy interruption on gut-associated lymphoid tissue (GALT) have not been well investigated. We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4(+) T cells in peripheral blood was observed, while gut mucosal CD4(+) T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients. Sequence analysis of rebound virus suggested that GALT was not the major contributor to the postinterruption plasma viremia nor were GALT HIV reservoirs rapidly replaced by HIV rebound variants. Our data suggest an early establishment and persistence of viral reservoirs in GALT with minimal diversity. Early detection of and therapy for HIV infection may be beneficial in controlling viral evolution and limiting establishment of diverse viral reservoirs in the mucosal compartment.     

Immunostimulatory activity of Bacillus spores

Bacillus species, typically Bacillus subtilis, are being used as probiotics and mounting evidence indicates that Bacillus species are important for development of a robust gut-associated lymphoid system (GALT). We used a number of gut isolates of Bacillus incorporating three species, B. subtilis, Bacillus licheniformis and Bacillus flexus to evaluate the nature of interaction between spores and the GALT. In mice orally administered with spores, evidence of cell proliferation was determined in the germinal centers of Peyer's patches. Stimulation of antigen-presenting cells and T lymphocytes was also markedly enhanced. Cytokines were shown to be induced in spleens and mesenteric lymph nodes of mice including the proinflammatory cytokines, tumour necrosis factor-alpha and IL-6. We also demonstrated that vegetative cells of B. subtilis can stimulate expression of the toll-like receptor (TLR) genes for TLR2 and TLR4. However, we were able to show that spores could not stimulate either and must, by default, interact with another TLR and by this mechanism help activate innate immunity.     

Differential homing commitments of antigen-specific T cells after oral or parenteral immunization in humans

Animal experiments show that lymphocytes activated in the intestine circulate through mesenteric lymph nodes, lymphatics, and blood, returning to the gut. Homing into intestinal lamina propria is mediated by lymphocyte surface homing receptors, mainly the alpha4beta7-integrin. We studied in humans whether intestinal T cells entering the blood upon antigenic activation would exhibit homing commitments to the gut. Volunteers were immunized with keyhole limpet hemocyanin (KLH) first orally and then parenterally or only parenterally, and the expression of alpha4beta7 on T cells specific for KLH or tetanus toxoid was studied. Circulating T cells were depleted of alpha4beta7+ cells by immunomagnetic selection. This depletion removed a significant proportion of the KLH-specific cells (mean decrease in proliferative response of 71%) in the orally immunized volunteers. No difference in the KLH-induced proliferation was found between the total and the alpha4beta7-depleted populations in volunteers parenterally immunized with KLH, regardless of whether a preceding mucosal priming had taken place or not. In both immunization groups, the depletion of alpha4beta7+ cells had no influence on the proliferative response to tetanus toxoid. We conclude that, in contrast to T cells activated by parenteral immunization, gut-derived T cells have preferential homing commitments to the gut. This commitment was no longer observed after a subsequent parenteral Ag administration. Besides showing that the site of Ag encounter determines the expression of homing receptors, the present study is the first to provide evidence for a circulation of newly activated Ag-specific intestinal T cells back to the gut in humans.