Diverse regulatory pathways for IgA synthesis in the gut.

Immunoglobulin (Ig) A is the most abundant antibody isotype in mucosal secretions. In this study we summarize recent advances in our understanding of the compartments and mechanisms of intestinal IgA synthesis. We discuss the pathways leading to the generation of IgA(+) B cells in follicular and extra-follicular structures, by T-cell-dependent and T-cell-independent mechanisms.

Intestinal IgA synthesis: a primitive form of adaptive immunity that regulates microbial communities in the gut.

Our intestine is colonized by an impressive community of commensals that has profound effects on the immune functions. The relationship between gut microbiota and the immune system is one of reciprocity: Commensals have important contributions in nutrient processing and education of the immune system, and, conversely, the immune system, particularly gut-associated lymphoid tissues (GALT), plays a key role in shaping the repertoire of gut microbiota. In this chapter we attempt to discuss the mechanisms that underlie this reciprocity and emphasize the key role of mucosal IgA in maintenance of an appropriate segmental distribution of microbiota, which is necessary for immune homeostasis.

In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.

One of the front lines of the immune defence is the gut mucosa, where immunoglobulin- (IgA) is continuously produced to react with commensal bacteria and dietary antigens. It is generally accepted that, after antigenic stimulation in the Peyer's patches, IgA+ lymphoblasts (B220+IgA+) migrate through the lymph and blood circulation, and eventually home to the lamina propria of the intestine. Mice that lack activation-induced cytidine deaminase (AID) are defective in class switch recombination (CSR) and somatic hypermutation. CSR changes the immunoglobulin heavy chain constant region (CH) gene being expressed from Cmu to other CH genes, resulting in a switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA. AID-/- mice also secrete large amounts of immunoglobulin-mu (IgM) into faeces, and accumulate B220-IgM+ plasma cells as well as B220+IgM+ cells in the gut. Here we show that lamina propria B220+IgA+ cells have just completed CSR, as they still express both AID and transcripts from circular DNA that has been 'looped-out' during CSR. Lamina propria IgM+ B cells seem to be pre-committed to switching to IgA+ in vitro as well as in vivo. Culturing lamina propria IgM+ B cells together with lamina propria stromal cells enhances preferential switching and differentiation of B cells to IgA+ plasma cells. We conclude that IgA+ cells in the gut lamina propria are generated in situ from B220+IgM+ lymphocytes.

A hallmark of active class switch recombination: transcripts directed by I promoters on looped-out circular DNAs.

To specify when and where Ig class switch recombination (CSR) takes place, a good molecular marker closely associated with active CSR is required. CSR is accompanied by deletion of circular DNA from the Ig heavy chain locus. The circular DNA contains a DNA segment between Smu and a target S region including its I promoter, which is driven by specific cytokine stimulation before CSR. We found that the specific I promoter is still active in looped-out circular DNA and directs production of I-Cmu transcripts termed "circle transcripts." Reverse transcription-PCR demonstrated transient induction of specific circle transcripts upon CSR in a murine lymphoma cell line, CH12F3-2A, as well as spleen B cells. Production of the circle transcripts appeared to depend on expression of activation-induced cytidine deaminase (AID), an essential factor for CSR. A comparison of kinetics between circle transcripts and circular DNA showed more rapid disappearance of circle transcripts. Thus, circle transcripts may serve as a hallmark for active CSR in vitro and in vivo.

Migration and differentiation of autoreactive B-1 cells induced by activated gamma/delta T cells in antierythrocyte immunoglobulin transgenic mice.

Using normal and transgenic (Tg) mice, we have shown that peritoneal B-1 cells are activated by administration of cytokines or lipopolysaccharide and migrate to other lymphoid organs where they differentiate into antibody-secreting cells. However, little is known about the process of B-1 cell migration and differentiation in vivo. We developed a mouse line by crossing the antierythrocyte antibody Tg mice (HL mice) with TCR-gamma/delta Tg mice specific for a self-thymus leukemia (TL) antigen in the recombination activating gene (RAG)2(-/-) background. In the presence of the self-antigen, Tg gamma/delta T cells increased in number and manifested activated phenotypes. Peritoneal B-1 cells in these mice migrated into mesenteric lymph nodes and differentiated into autoantibody-secreting cells, resulting in strong autoimmune hemolytic anemia. Furthermore, transfer of RAG2(-/-) x HL bone marrow or peritoneal cells into the peritoneal cavity of RAG2(-/-) x TCR-gamma/delta Tg mice gave rise to donor-derived B-1 cells in mesenteric lymph nodes, and these cells produced the autoantibody. Thus, this study demonstrates that the migration of B-1 cells and differentiation into the antibody-secreting cells can be induced by noncognate T cell help and implies the possibility that gamma/delta T cells may induce B-1 cell differentiation in vivo.

T-Independent immune response: new aspects of B cell biology.

Recent results emphasize the roles of T-independent antibody response in humoral defenses, for which B1 cells and marginal zone B cells are mostly responsible. We discuss how these cells are activated, migrate, and differentiate into antibody-producing cells in various lymphoid tissues. Based on recent findings in each of these areas of B cell biology, we propose a possible mechanism for peripheral tolerance of autoreactive B cells at target organs.

Generation, expansion, migration and activation of mouse B1 cells.

We have studied the expansion, activation, homing and antibody production of B1 cells in two different mouse models. One is the HL transgenic mouse, which carries Ig genes encoding the anti-red blood cell autoantibody (4C8) and develops autoimmune hemolytic anemia by the activation of autoreactive B1 cells that escape from clonal deletion and expand in the peritoneal cavity (PEC). The other model is represented by alymphoplasia (aly) mice, which carry a point mutation in the gene encoding NF-kappaB-inducing kinase (NIK) and have drastically reduced immunoglobulin serum levels, in spite of their peritoneal cavity containing a large number of B1 cells. We have found that a) expression levels of the B-cell antigen receptor (BCR) influence the size of the B1 -cell compartment and efficiency of allelic exclusion and B2-cell deletion; b) antibody production of B1 cells is closely related with their migration from PEC to other lymphoid organs and is dependent on NIK; and c) infection, lipopolysaccharide stimulation, cytokine administration or T-cell activation by noncanonical antigens induces migration and differentiation of peritoneal B1 cells into antibody-producing cells. We describe a scenario where most of B1 and B2 differences are due to a distinct activation threshold of BCR and antigen repertoire.

Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.

Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.

Increased frequency of surface IgA-positive plasma cells in the intestinal lamina propria and decreased IgA excretion in hyper IgA (HIGA) mice, a murine model of IgA nephropathy with hyperserum IgA.

Because abnormalities of mucosal immunity have been suggested in human IgA nephropathy, we examined the involvement of mucosal immunity in IgA deposition to the kidney in hyper IgA (HIGA) mice, which was established as a mouse model for human IgA nephropathy with hyperserum IgA. The number of surface IgA+B220- lymphocytes in the intestinal lamina propria (LP) of HIGA mice increased 2.7-fold at 30 wk of age as compared with those at 10 wk of age, whereas normal mice did not show such increase. The surface IgA+B220- LP lymphocytes spontaneously secreted IgA in culture. Morphological studies showed that the surface IgA+B220- lymphocytes of murine intestinal LP are identical with plasma cells (PCs). About 20% of IgA+B220- PC in LP expressed both Mac-1 and CD19, suggesting that they may derive from peritoneal B-1 cells. Cell cycle study on intestinal IgA-PCs using bromodeoxyuridine revealed no difference between HIGA mice and normal mice, suggesting that the high frequency of IgA-producing PCs in HIGA mice is not due to enhanced proliferation or prolonged survival of IgA-producing PCs in LP. In addition, IgA secretion into the gut lumen of HIGA mice decreased drastically (to one forth) with aging. These data suggest that the increased number of intestinal IgA-producing PCs and the down-regulation of IgA excretion into the intestinal lumen might synergistically contribute to the hyperserum IgA in HIGA mice and resultant IgA deposition to the kidney.

Alymphoplasia (aly)-type nuclear factor kappaB-inducing kinase (NIK) causes defects in secondary lymphoid tissue chemokine receptor signaling and homing of peritoneal cells to the gut-associated lymphatic tissue system.

Alymphoplasia (aly) mice, which carry a point mutation in the nuclear factor kappaB-inducing kinase (NIK) gene, are characterized by the systemic absence of lymph nodes and Peyer's patches, disorganized splenic and thymic architectures, and immunodeficiency. Another unique feature of aly/aly mice is that their peritoneal cavity contains more B1 cells than normal and aly/+ mice. Transfer experiments of peritoneal lymphocytes from aly/aly mice into recombination activating gene (RAG)-2(-/-) mice revealed that B and T cells fail to migrate to other lymphoid tissues, particularly to the gut-associated lymphatic tissue system. In vivo homing defects of aly/aly peritoneal cells correlated with reduction of their in vitro chemotactic responses to secondary lymphoid tissue chemokine (SLC) and B lymphocyte chemoattractant (BLC). The migration defect of aly/aly lymphocytes was not due to a lack of expression of chemokines and their receptors, but rather to impaired signal transduction downstream of the receptors for SLC, indicating that NIK is involved in the chemokine signaling pathway known to couple only with G proteins. The results showed that the reduced serum levels of immunoglobulins (Igs) and the absence of class switch to IgA in aly/aly mice are due, at least in part, to a migration defect of lymphocytes to the proper microenvironment where B cells proliferate and differentiate into Ig-producing cells.

Plasmid profile analysis and antibiotic resistance of Salmonella strains from clinical isolates in Cluj-Napoca.

Resistance patterns, plasmid profiles and the genetic resistance determinants were investigated in 38 isolates of Salmonella enterica serotype Typhimurium and 19 isolates of Salmonella enterica serovar Enteritidis derived from children hospitalized in two clinics in Cluj-Napoca, during the period of 1995-1997. Incidence of plasmid and antibiotic resistance was very high in Salmonella typhimurium isolates. All strains were resistant to almost all antibiotics tested but susceptible to the third generation cephalosporines and fluoroquinolones. We identified three resistance patterns and six plasmid profiles. Each plasmid profile was characterized by the presence of two large plasmids of 150-180 Kbp. Approximately 60% of strains harbored three or four small plasmids of 1.3 to 9.5 Kbp. The plasmids of 8.5 Kbp encoded resistance to beta-lactam antibiotics and were non-conjugative. The other small plasmids were cryptic and also non-conjugative. Salmonella enteritidis isolates were susceptible to many antibiotics, except Tetracycline and Trimethoprim-Sulfamethoxazole. We identified three different resistance patterns but nine plasmid profiles. All plasmid profiles were characterized by the presence of a large plasmid (> 100 Kbp). The number and the diversity of small plasmids were higher than in S. typhimurium strains. There was no parallelism between resistance and plasmid profile: for the same resistance pattern a number of two or three plasmid profiles were found. Our conclusions are that Salmonella typhimurium strains were multiresistant to antibiotics and that many genetically different strains of Salmonella typhimurium and Salmonella enteritidis were responsible for gastroenteritis in children from Cluj County. The increasing antibiotic resistance highlights the need for more refined methods in genetic and epidemiological characterization of bacteria involved in gastrointestinal infections.

Plasmid profile analysis and restriction enzyme analysis in characterizing Shigella flexneri isolates from an outbreak.

Shigella flexneri strains which are multiply resistant to antimicrobial agents were isolated from 11 children from an orphanage in Cluj-Napoca during an epidemiological investigation initiated by the Department of Epidemiology. Plasmid profile analysis and restriction endonuclease analysis were used in conjunction with biotyping, serotyping and antimicrobial susceptibility testing for identifying epidemiological related isolates. All strains were serotype 2a and with one exception all of them showed the same resistotype. Plasmid profile analysis differentiated S. flexneri isolate into four patterns, with two common plasmids of 3.5 and 1.9 kb. This study indicates that this outbreak was caused by at least two different strains of S. flexneri which were not differentiated by the classical technique-biotyping, serotyping and antimicrobial susceptibility pattern.