Effects of the lpr/lpr mutation on T and B cell populations in the lamina propria of the small intestine, a mucosal effector site.

MRL mice, which develop a lymphoproliferative disease characterized by increased numbers of alpha/beta T cell receptor+ (TCR+) B220/6B2+CD4-CD8- T cells [lymphoproliferation (lpr) T cells], were studied for the effect of the lpr/lpr mutation on the mucosal immune system in the gastrointestinal (GI) tract. We analyzed the effect of the lpr gene mutation on T and B cell populations in the Peyer's patches (PP) and the lamina propria lymphocytes (LPLs), as examples of major IgA inductive and effector tissues in the GI tract respectively. Normal mouse PP contain B cells committed to IgA (surface IgA+) but only low numbers of B cells producing IgA. However, enhanced spontaneous IgA and IgG synthesis occurs in the PP of MRL mice. Further, we have now shown that PP of MRL mice are populated by lpr T cells. Interestingly, lpr T cells were not present in significant numbers in LPLs of MRL mice, even in older animals. Of interest was the finding that the ratio of CD4+ to CD8+ T cells in the lamina propria was lower in MRL when compared with control mice, and the CD8+ T cell subset actually predominates in LPLs of autoimmune mice. In addition, the number of gamma/delta TCR+ T cells in LPL of MRL lpr/lpr mice was significantly increased, especially in MRL lpr/lpr mice at 6 and 12 weeks of age. When the isotype distribution of B cells in LPLs was analyzed, no changes were noted in MRL lpr/lpr mice in comparison with MRL +/+ or normal control mice, and the pattern was IgA much greater than IgM greater than IgG. These results show that although increased numbers of CD8+ T cells and gamma/delta TCR+ cells occur in the LPLs of MRL mice, a normal distribution of plasma cell isotypes (IgA much greater than IgM greater than IgG) is found in this mucosal compartment. Further, Ipr T cells do not develop in the lamina propria compartment of the GI tract.     

The relative distribution of IgM and IgA cells in intestinal mucosa and lymphoid tissues of the young unweaned pig and their significance in ontogenesis of secretory immunity

A comparative study of cells synthesizing immunoglobulins IgA and IgM in intestinal mucosa and various lymphoid tissues of unweaned piglets has been made by immunofluorescence.

The lamina propria of the small intestine contained as many cells synthesizing IgM as those producing IgA. In all other lymphoid organs examined, including Peyer's patches, the number of IgM cells was significantly higher.

The largest population of intestinal immunocytes occurred in the lamina propria of the duodenum where the counts were ten times greater than in the jejunum or ileum.

The relevance of these findings to the earliest stages of development of secretory immunity in the young pig are considered.

     

IgVH gene analysis suggests that peritoneal B cells do not contribute to the gut immune system in man

The contribution of peritoneal B cells to the intestinal lamina propria plasma cell population is well documented in mice, but unknown in humans. We have analyzed immunoglobulin (Ig) genes of human peritoneal B cells, because such genes show distinctive characteristics in mucosal B cells, particularly highly mutated variable regions. Here, we report the characteristics of variable region genes used by IgM, IgA and IgG in peritoneal cells. We focused on the properties of IgV(H)4-34 to allow comparisons of like-with-like between different isotypes and cells from different immune compartments. We observed that the IgM genes were mostly unmutated, and that the mutated subset had less mutations than would be expected in a mucosal B cell population. Likewise, the IgV(H)4-34 genes used by IgA and IgG from peritoneal B cells had significantly lower numbers of mutations than observed in the mucosal counterparts. Other trends observed, while not reaching statistical significance, followed the trend of peripheral B cells. The peritoneal B cell population had more IgA1 than IgA2 sequences, and there was no dominance of J(H)4 in the IgA from peritoneum or spleen, in contrast to the mucosal sequences. Overall, this study suggested that human peritoneal B cell are either peripheral or mixed in origin; they are unlikely to represent an inductive compartment for the mucosal B cell system.     

SLy2 controls the antibody response to pneumococcal vaccine through an IL‐5Rα‐dependent mechanism in B‐1 cells

The adaptor protein SLy2 (Src homology domain 3 lymphocyte protein 2) is located on human chromosome 21 and was reported to be among a group of genes amplified in Down's syndrome (DS) patients. DS patients characteristically show an impaired immunity to pneumococcal infections. However, molecular mechanisms linking gene amplifications with specific DS phenotypes remain elusive. To investigate the effect of SLy2 gene amplification on the mammalian immune system, we studied SLy2 overexpressing transgenic‐SLy2 (TG) mice. We found that baseline immunoglobulin M (IgM) levels as well as IgM responses following Pneumovax immunizations were reduced in TG mice. Moreover, B‐1 cells, the major natural IgM‐producing population in mice, were reduced in the peritoneal cavity of TG mice, while other immune cell compartments were unaltered. Mechanistically, SLy2 overexpression attenuated the expression of the IL‐5 receptor α chain on B‐1 cells, resulting in decreased B‐1 cell numbers and decreased differentiation into Ab‐secreting cells. Since B‐1 cells essentially contribute to immunity against Streptococcus pneumoniae, the present study provides a novel molecular link between SLy2 expression and pneumococcal‐specific IgM responses in vivo. These studies suggest that the adaptor protein SLy2 is a potential future target for immunomodulatory strategies for pneumococcal infections.     

Gluten induces coeliac-like disease in sensitised mice involving IgA, CD71 and transglutaminase 2 interactions that are prevented by probiotics

Coeliac disease (CD) is a malabsorptive enteropathy resulting from intolerance to gluten. Environmental factors and the microbiota are suggested to have critical roles in the onset of CD. The CD71 IgA receptor on epithelial cells is responsible for abnormal retrotranscytosis of IgA-gluten peptide complexes from the intestinal lumen into the lamina propria, inducing intestinal inflammation. However, understanding the role of gluten in the CD physiopathology has been hindered by the absence of relevant animal models. Here, we generated a mouse model for CD to study the factors controlling its pathogenesis as well as to investigate the influence of oral delivery of probiotics on disease development. Gluten sensitivity was established by feeding three generations of BALB/c mice a gluten-free diet (G-) followed by gluten challenge (G+) for 30 days. The G+ mice developed villous atrophy, crypt hyperplasia and infiltration of T cells and macrophages in the small intestine. Inflammation was associated with an overexpression of CD71 on the apical side of enterocytes and an increase of plasma cells producing IgA, which colocalised with the CD71. Moreover, IgA colocalised with the transglutaminase 2 (TG2), the production of which was increased in the lamina propria of G+ mice. These mice displayed increased production of cyclooxygenase-2 (COX-2), pro-inflammatory cytokines and IL-15, as well as anti-gliadin and anti-TG2 autoantibodies. The commensal flora-isolated presumptive probiotic Saccharomyces boulardii KK1 strain hydrolysed the 28-kDa α-gliadin fraction, and its oral delivery in G+ mice improved enteropathy development in association with decrease of epithelial cell CD71 expression and local cytokine production. In conclusion, the G+ BALB/c mouse represents a new mouse model for human CD based on histopathological features and expression of common biomarkers. The selected probiotic treatment reversing disease development will allow the study of the role of probiotics as a new therapeutic approach of CD.     

Antibody-secreting cell responses in the mouse liver.

To elucidate the origins of biliary IgA antibodies, we investigated the isotype and specificity of antibody-secreting cells (ASC) in the liver in comparison with the spleen and intestinal lamina propria of mice immunized by peroral or parenteral routes. The profile of specific IgM, IgG1, IgG2a, and IgA ASC in the liver resembled that of the spleen rather than the lamina propria, regardless of the route of immunization. Peroral immunization increased the proportion of specific IgA ASC in all three organs. However, liver mononuclear cells (MNC) contained a higher proportion of total IgA-secreting cells than spleen cells. After immunization, the number and proportion of B220+ B cells were increased in the liver but not in the spleen. Although the predominant isotype of Ig and specific antibody in bile in response to immunization by either route was IgA, IgM and IgG were clearly detectable. However, specific activities of biliary antibodies relative to total Ig isotype were generally higher than in serum. The predominance of IgA-secreting cells in the liver and the large amount of IgA secreted in the bile resemble the situation at other secretory sites of the mucosal immune system. However, specific antibody-secreting cells appear to accumulate in the liver promptly after immunization, regardless of isotype, and contribute locally produced antibodies to the bile.     

Generation of precursor,immature, and mature murine B1‐cell lines from c‐myc/bcl‐xL‐overexpressing pre‐BI cells

Deregulated expression of c‐myc and bcl‐xL is long known to generate transformed B cells in humans and mice. We overexpressed these genes to induce in vitro and in vivo differentiation of fetal liver‐derived mouse pre‐BI cells to B1‐lineage pre‐BII‐like, immature and mature B‐cell lines, and to Ig‐secreting cells. In vitro, doxycycline‐controlled c‐myc/bcl‐xL‐overexpressing CD19⁺CD93⁺c‐kikt⁺IgM^? pre‐BI cells differentiate to and survive as CD19⁺CD93⁺c‐kit^?IgM⁺ immature B1 cells. Timed CpG stimulation of these oncogene‐overexpressing pre‐B or immature B1 cells generates either CD19⁺CD93^lowc‐kit^?IgM^?SLC^? pre‐BII‐like or IgM⁺MHCII⁺CD73⁺CD80⁺CD40⁺ mature B1‐cell lines and IgM‐secreting B1 cells in vitro and fixes their state of differentiation. All cell lines are clonable, but a majority of immature and mature B1‐cell clones eventually reach a nonproliferating, surviving G₀‐state. Transplanted in vivo, c‐myc/bcl‐xL‐overexpressing pre‐B cells expand to mature B1 cells, and to IgM‐ and IgA‐secreting plasmablasts and plasma cells. Within 2 months, plasmablasts have expanded most prominently in BM and spleen, indicating that the host selectively expanded development of these transformed plasma cells. The sIgM⁺ B1‐cell lines and clones offer the possibility to study their roles in the development of B1‐Ab repertoires, of B1‐cell‐mediated autoimmune diseases and of B1‐cell malignancies.     

Rotavirus-specific intestinal immune response in mice assessed by enzyme-linked immunospot assay and intestinal fragment culture.

Primate rotavirus strain RRV and bovine strain WC3 or reassortants made between these animal viruses and human rotaviruses have been administered to infants as candidate vaccines. We compared RRV and WC3 in a murine model of oral infection. We determined the relative capacities of these viruses to induce a virus-specific humoral immune response by intestinal lymphocytes as tested by enzyme-linked immunospot assay, intestinal fragment culture, and enzyme-linked immunosorbent assay of intestinal contents. We found that inoculation of mice with RRV induced higher frequencies of virus-specific immunoglobulin A (IgA)-secreting cells in the lamina propria, greater quantities of virus-specific IgA in intestinal fragment cultures, and greater quantities of virus-specific IgA in intestinal secretions than did inoculation with WC3 or inactivated RRV (iRRV). The induction of an IgA response in serum was predictive of an IgA response among intestinal lymphocytes after inoculation with RRV but not WC3. In addition, large quantities of IgG, IgA, and IgM not specific for rotavirus were produced in fragment cultures from mice inoculated with RRV but not in cultures from mice inoculated with WC3 or iRRV. Possible mechanisms of RRV-induced polyclonal stimulation of intestinal B cells are discussed.     

Intestinal resistance to cholera toxin in mouse. Antitoxic antibodies and desensitization of adenylate cyclase

Mice were immunized perorally with cholera toxin (CT), cholera B-subunit (CB), or buffer as control. The response of anti-CT antibodies of the IgG, IgA and IgM class in bile, IgA being predominating, were similar in both immunized groups. The same number of anti-CT containing plasma cells (ACC) were determined in the intestinal lamina propria of CT - as well as of CB-immunized mice 20 days after the last immunization, while ACC at day 4 in the CB group were 50% higher than in the CT group. In contrast to the vigorous antibody response to CT in both groups of immunized mice, only animals immunized with CT displayed resistance to CT-induced intestinal hypersecretion and to CT stimulation of adenylate cyclase. The CB-treated group responded to CT with fluid accumulation and enzyme activation similar to controls. The results suggest that intestinal resistance to CT in mouse is due to desensitization of adenylate cyclase rather than to CT-neutralizing antibodies.     

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.     

肠道黏膜免疫

肠道免疫系统由大量弥散分布在肠黏膜上皮内和固有层的免疫细胞和免疫分子、以及诸如peyer’spatches(PP)等肠相关性淋巴组织等组成。小肠PP结为肠黏膜免疫主要诱导部位,肠黏膜上皮和固有层为主要效应部位。B细胞、T细胞在PP结诱导后,分化、成熟,并移行到黏膜效应部位,发挥免疫效应功能。     

Role of local IgA antitoxin-producing cells for intestinal protection against cholera toxin challenge

We examined in mice, perorally immunized with cholera toxin (CT) or cholera B subunit (CTB), the association between protection against intestinal toxin challenge and frequency and function of gut mucosal IgA antitoxin-forming cells. The in vitro production of IgA antitoxin by isolated cells and the toxin-neutralizing ability of culture supernatants were determined. Repeated oral immunizations with CT gave rise to high numbers of IgA antitoxin 'spot-forming' cells (SFC) in the lamina propria as well as to protection against challenge with CT in ligated intestinal loops. In contrast, mice immunized with purified CTB, gave poor IgA antitoxin SFC responses in the lamina propria and little or no protection. When a small amount of CT was used to adjuvant the response to CTB, many IgA antitoxin SFC were found; however, protection in intestinal loops remained poor. This discrepancy was explained by the predominant localization of antitoxin SFC in the proximal small intestine following oral CTB/CT-adjuvant immunization, whereas relatively few SFC were found further down in the intestine where the loop-protection test was performed. Thus, when lamina propria plasma cells were isolated from challenged loops and cultured in vitro, they released only low titers of IgA antitoxin and CT-neutralizing antibodies in culture supernatants; this was in contrast to cells from optimally immunized mice which gave supernatants with high IgA antitoxin and toxin-neutralizing antibody titers. Increasing the dose of CT, added as adjuvant to the CTB, resulted in better protection and higher numbers of IgA antitoxin SFC in more distal parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and phenotype of murine rotavirus-specific B cells induced by intranasal immunization with 2/6 virus-like particles

Virus-like particles containing the rotavirus (RV) internal proteins VP2 and VP6 (2/6-VLP) have been shown to induce serum and fecal antibodies as well as protection in mice after intranasal administration with a mutant of E. coli toxin, LT-R192G. To better understand the origin of fecal IgA induced by this protocol, we studied the RV-specific B cell response in systemic and mucosal lymphoid tissues using a flow cytometry assay that allows quantification and phenotypic characterization of RV-specific B lymphocytes. We also assessed the RV-specific antibody-secreting cells in the spleen and intestinal lamina propria (ILP). A remarkably high frequency of RV-specific B cells was found in the respiratory lymphoid tissues and spleen, of which only a minority expressed the alpha4beta7 integrin (intestinal homing receptor). In contrast, but in accordance with alpha4beta7 expression at the induction site, a very low response was observed in intestinal lymphoid tissues (mesenteric lymph nodes and ILP), which did not increase after a second immunization. Thus, intranasal immunization with a nonreplicating antigen does not induce an important number of RV-specific B cells with an intestinal homing profile.     

Vitamin A metabolism and mucosal immune function are distinct between BALB/c and C57BL/6 mice

The vitamin A metabolite retinoic acid (RA) has been reported to suppress Th1 responses and enhance Th2 responses. Here, we investigated whether differences in vitamin A metabolism could underlie the differences between C57BL/6 and BALB/c mice, which are reportedly seen as Th1 and Th2 responders, respectively. BALB/c mice were shown to have higher intestinal epithelial expression of RALDH1 (where RALDH is retinaldehyde dehydrogenase), and, consequently, higher RALDH activity in MLN‐DCs, leading to an increased ability to induce IgA class switching in B cells. Furthermore, within BALB/c mice, induction of IgA secretion as well as increased accumulation of regulatory T cells (Treg) in the intestinal lamina propria was observed. Additionally, as BALB/c mice are more resistant to dextran sulphate sodium (DSS) induced colitis, mice that lacked vitamin A in their diet had a more severe form of DSS‐induced colitis compared to control mice. Therefore, the level of RA production and consequently the degree of RA‐mediated signaling is crucial for the efficiency of the mucosal immune system.     

Differential activation requirements of isotype-switched B cells

In the present studies, we compared the activation requirements of sIgM⁺/sIgD⁺ B cells with those of isotype-switched sIgM⁻/sIgA⁺ B cells. We found that whereas sIgM⁺ B cells respond to T cell-independent (TI) and T cell-dependent (TD) Ag with no significant bias toward one stimulus, sIgA⁺ B cells were deficient in their ability to respond to antigen receptor cross-linking but responded remarkably well to TD stimuli. Thus, dextran-conjugated anti-IgA antibody (anti-IgA-dextran), anti-kappa-dextran, or various immobilized anti-IgA antibodies (Ab) induced only low-level IgA B cell proliferation and no IgA secretion in the presence of various lymphokines; in marked contrast, sIgA⁺ B cells responded to cognate and noncognate T cell stimulation as well as to stimulation by CD40 ligand-bearing fibroblasts by secreting large amounts of IgA (up to 240 000 ng/ml per 10⁵ cells). This pattern of sIgA⁺ B cell responsiveness was noted with both germinal center peanut agglutinin^hi (PNA^hi) and non-germinal center PNA^lo B cells. In confirmation of these results, whole Peyer's patch or lamina propria cell populations containing less than 15% sIgA⁺ B cells stimulated with a noncognate T cell stimulus or T cell membranes secreted mainly IgA (68%–94% of the total Ig secreted) and relatively little IgM. The strict T cell dependence of IgA B cell activation and differentiation provides important insights into immune responses of mucosal tissues and must be considered in the development of vaccines, particularly those designed to stimulate mucosal tissues containing large numbers of isotype-switched B cells.     

A Sensitive Method for the Detection of Specific Antibody Production in Different Isotypes from Single Lamina Propria Plasma Cells

A sensitive and reproducible method for the detection of specific antibody production (or total immunoglobulin secretion) at the single cell level from isolated lamina propria lymphocytes was developed. The cells were prepared from mouse intestinal mucosa by enzyme extraction with collagenase, and antibody secretion was demonstrated with a solid phase enzyme-linked immunospot (ELISPOT) assay. Oral immunizations with cholera toxin or keyhole limpet haemocyanin to mice gave high numbers of highly antigen-specific spot-forming cells (SFC) among isolated lamina propria lymphocytes. Spots were shown to result from active synthesis of immunoglobulin in vitro. The variation in SFC numbers between individual animals after a given protocol of oral immunizations was found to be 25% and between equal groups analysed on different occasions, 12%. Kinetics of primary as well as secondary immune responses after oral immunizations with cholera toxin were easily monitored. A single dose of cholera toxin gave rise to 230 antitoxin SFC/10(7) isolated lamina propria lymphocytes. Each additional dose stimulated to increasing numbers of specific SFC with roughly 7000 antitoxin SFC/10(7) cells after five immunizations. Monitoring of day-by-day responses after oral booster immunizations demonstrated peak SFC numbers on day 8 after antigen administration. The total number of immunoglobulin-secreting (Ig) cells and the isotype distribution of specific SFC could also be determined. In the peak antitoxin response, 8% of the isolated total Ig-secreting lamina propria cells were active against cholera toxin, and of these 80% were producing IgA. This method has also been successfully used in humans and rabbits to demonstrate specific antibody production by single lamina propria plasma cells.     

Protection against Cholera Toxin after Oral Immunization is Thymus-Dependent and Associated with Intestinal Production of Neutralizing IgA Antitoxin

We studied whether gut mucosal IgA antitoxin production as well as the acquired protection against cholera toxin (CT) after oral immunization with CT are both thymus-dependent immune manifestations. In contrast to normal BALB/c mice, nude, athymic mice did not respond to oral immunizations with CT with either IgA antitoxin-producing cells (SFC) in the lamina propria or protection against challenge with CT in ligated intestinal loops. However, when nude mice were first reconstituted by grafting of syngeneic thymus glands, both IgA antitoxin SFC in the lamina propria and protection were stimulated by oral immunizations with CT and the response were of similar magnitude to those of normal mice after immunizations. During in vitro culture, isolated lamina propria lymphocytes from immunized but not from control mice concomitantly and proportionally produced IgA antitoxin and CT-neutralizing activity. We conclude that intestinal antitoxin formation and protection against toxin challenge after oral immunization with CT are both critically thymus-dependent and therefore likely to be under T-cell control.     

CCR6 promotes steady‐state mononuclear phagocyte association with the intestinal epithelium,imprinting and immune surveillance

The intestinal lamina propria (LP) contains antigen‐presenting cells with features of dendritic cells and macrophages, collectively referred to as mononuclear phagocytes (MNPs). Association of MNPs with the epithelium is thought to play an important role in multiple facets of intestinal immunity including imprinting MNPs with the ability to induce IgA production, inducing the expression of gut homing molecules on T cells, facilitating the capture of luminal antigens and microbes, and subsequent immune responses in the mesenteric lymph node (MLN). However, the factors promoting this process in the steady state are largely unknown, and in vivo models to test and confirm the importance of LP‐MNP association with the epithelium for these outcomes are unexplored. Evaluation of epithelial expression of chemoattractants in mice where MNP–epithelial associations were impaired suggested CCL20 as a candidate promoting epithelial association. Expression of CCR6, the only known receptor for CCL20, was required for MNPs to associate with the epithelium. LP‐MNPs from CCR6^−/− mice did not display defects in acquiring antigen and stimulating T‐cell responses in ex vivo assays or in responses to antigen administered systemically. However, LP‐MNPs from CCR6‐deficient mice were impaired at acquiring luminal and epithelial antigens, inducing IgA production in B cells, inducing immune responses in the MLN, and capturing and trafficking luminal commensal bacteria to the MLN. These findings identify a crucial role for CCR6 in promoting LP‐MNPs to associate with the intestinal epithelium in the steady state to perform multiple functions promoting gut immune homeostasis.     

Transient secretory IgA deficiency in mice after cyclophosphamide treatment

Cyclophosphamide (Cy), an alkylating agent widely used in chemotherapy of leukemia and cancer, causes a well-documented toxicity on hematopoietic and lymphoid cells. Neutropenia is thought to be the main factor involved in infectious complications following antimitotic chemotherapy. Little is known on the effects of these therapies on the mucosal associated lymphoid system which is one of the main barriers against environmental pathogenic agents. The present study examined the effects of a single administration of Cy (200 mg/kg) on murine T and B cell populations of Peyer's patches (PPs), IgA secretion in the proximal part of the small intestine, and plasma cells of the lamina propria. Cy induced in mice a transient decrease in the T and B cell populations of the PPs with a drastic fall of B cell counts and a profound decrease of intestinal IgA secretion due to a reduction of lamina propria plasma cells. This transient secretory IgA deficiency may contribute to the infectious complications following antimitotic chemotherapy.