Eosinophils: important players in humoral immunity.

Eosinophils perform numerous tasks. They are involved in inflammatory reactions associated with innate immune defence against parasitic infections and are also involved in pathological processes in response to allergens. Recently, however, it has become clear that eosinophils also play crucial non-inflammatory roles in the generation and maintenance of adaptive immune responses. Eosinophils, being a major source of the plasma cell survival factor APRIL (activation and proliferation-induced ligand), are essential not only for the long-term survival of plasma cells in the bone marrow, but also for the maintenance of these cells in the lamina propria which underlies the gut epithelium. At steady state under non-inflammatory conditions eosinophils are resident cells of the gastrointestinal tract, although only few are present in the major organized lymphoid tissue of the gut - the Peyer's patches (PP). Surprisingly, however, lack of eosinophils abolishes efficient class-switching of B cells to immunoglobulin (Ig)A in the germinal centres of PP. Thus, eosinophils are required to generate and to maintain mucosal IgA plasma cells, and as a consequence their absence leads to a marked reduction of IgA both in serum and in the gut-associated lymphoid tissues (GALT). Eosinophils thus have an essential part in long-term humoral immune protection, as they are crucial for the longevity of antibody-producing plasma cells in the bone marrow and, in addition, for gut immune homeostasis.

The long-term survival of plasma cells.

Plasma cells sustain antibody production and hence are an essential part of immune protection. In the mucosa-associated lymphoid tissues plasma cells secrete IgA antibodies which protect the organism from invasion by pathogenic bacteria while in the bone marrow they produce the antibodies which guarantee long-term humoral immune protection. The various lymphoid organs provide specific microenvironments which support plasma cell survival. In particular, in the bone marrow, highly specialized survival niches are established by the underlying stromal reticular cells which permit plasma cells to survive for years. In some situations, however, the antibody may be detrimental to the organism. In those auto immune diseases, where plasma cells play a pathological role by producing the auto antibodies, new strategies are needed to interfere with the lifespan of plasma cells and thus to diminish their numbers. The recent finding that eosinophils are essential for the long-term survival of plasma cells in the bone marrow provides a new therapeutic target to modulate the plasma cell survival niche.

Genetics of the idotype of BALB/c myeloma S117: multiple chromosomal loci for Vh genes encoding specificity for group A streptococcal carbohydrate.

A small proportion of the antibodies to Group A streptococcal carbohydrate (A-CHO) elicited in BALB/c mice by immunization with Group A streptococci, has idiotypic determinants in common with the BALB/c myeloma protein S117 which has specificity for N-acetyl-glucosamine, the major antigenic determinant of A-CHO. The expression of these idiotypic determinants is under the control of a gene which is linked to the Ig-1a+ allotype locus in strain BALB/c and in other strains carrying the same Ig-1 haplotype. This gene (S117+) segregates in breeding experiments as if it were an allele to the gene A5A+ which controls the expression of the A5A idiotype in association with antibodies to A-CHO in strain A/J and which is linked to the Ig-1e allotype locus. Another possible allele, linked to the Ig-1c allotype locus, controls the expression of both S117 and A5A cross-reactive determinants (S117cr, A5Acr). The distribution of these idiotypic determinants in various lines that carry recombinant Ig-1 haplotypes suggests that the A5A and S117 loci are nonallelic and map at different positions in the Ig-1 region. The data suggest complex pseudollelic relationships between different Ig-1 haplotypes that allow the expression of the same genes in allelic and in nonallelic fashion.

Anti-CD2 antibodies induce T cell unresponsiveness in vivo.

The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo.

Idiotypic analysis of lymphocytes in vitro. II. Genetic control of T-helper cell responsiveness to anti-idiotypic antibody.

When the IgG1 fraction of anti-idiotypic antibodies raised in guinea pigs is injected into mice, sensitization of idiotypic T and B lymphocytes occurs (1-3). In the present study we analyze the genetic requirements for T-helper cell sensitization by anti-idiotypic antibody. This was done by measuring, in a suitable panel of mouse strains, helper cell responsiveness to two anti-idiotypic reagents which recognize distinct, strain-specific idiotypes, namely the A5A and the S117 marker. Whenever helper cell sensitization by anti-idiotypic antibody was successful, helper function could be specifically inhibited by the same and only the same anti-idiotype. This indicates that helper cells induced by anti-idiotypic antibody express idiotypic determinants on their receptors for antigen. Helper cell sensitization by anti-idiotypic antibody was found in all strains expressing the corresponding or a cross-reactive idiotype at the immunoglobulin level. Idiotype-negative strains were always unresponsive to anti-idiotypic stimulation. In addition, responsiveness did not depend on the H-2 haplotype. Since the A5A and the S117 idiotype are markers for V genes in the heavy-chain linkage group, the present results support the view that the same genes in the Ig-1 complex code for variable portions of immunoglobulins and T-helper cell receptors.

Idiotypic analysis of lymphocytes in vitro. I. Specificity and heterogeneity of B and T lymphocytes reactive with anti-idiotypic antibody.

Guinea pig anti-idiotypic antibodies (anti-Id) of the IgG1 class, directed to an A/J antibody to Group A streptococcal carbohydrate (A-CHO), or directed to a BALB/c myeloma protein that binds the same antigen, stimulate B-precursor cells as well as T-helper cells when injected into mice of the appropriate strain. The strain-specific induction of both precursor and helper activity was detected by in vitro secondary responses of primed spleen cells to A-CHO or to 2,4,6-trinitrophenyl (TNP) upon challenge with Group A streptococcal vaccine (Strep.A) or with TNP-Strep.A, respectively. B- and T-cell populations primed with anti-Id were uniform with respect to the binding of antigen and of anti-Id. This was in contrast to cells primed with Strep.A, which were heterogenous. Taken together, B and T cells that possess the same antigen-binding specificity share idiotypic determinants, reveal the same idiotypic polymorphism, and may display similar degrees of heterogeneity with respect to the binding of antigen and anti-Id. Since the anti-Id used in this study detect Id determinants associated with the heavy chain of the variable region of mouse antibodies, the data suggest that this region of the immunoglobulin molecule is shared between T- and B-cell antigen receptors.

Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity.

Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs))/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PK(cs) activity.

Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease. The salivary glands of patients with Sjögren's syndrome.

Structures resembling germinal centers are seen in the salivary glands of patients with Sjögren's syndrome, but it is not known whether the microenvironment of these cell clusters is sufficient for the induction of a germinal center response. Therefore, we cloned and sequenced rearranged Ig V genes expressed by B cells isolated from sections of labial salivary gland biopsies from two Sjögren's syndrome patients. Rearranged V genes from B cells within one cell cluster were polyclonal and most had few somatic mutations. Two adjacent clusters from another patient each contained one dominant B cell clone expressing hypermutated V genes. None of the rearranged V genes was found in both clusters, suggesting that cells are unable to migrate out into the surrounding tissue and seed new clusters. The ratios of replacement to silent mutations in the framework and complementarity determining regions suggest antigen selection of high-affinity mutants. These results show that an antigen-driven, germinal center-type B cell response is taking place within the salivary glands of Sjögren's syndrome patients. In view of the recent demonstration of a germinal center response within the rheumatoid synovial membrane and the existence of similar structures in the target tissues of other autoimmune diseases, we propose that germinal center- type responses can be induced in the nonlymphoid target tissues of a variety of autoimmune diseases.

Somatic mutation and memory.

The germinal center plays a crucial role in the development of the memory B cell. The repertoire of the antigen-specific B cell is shaped in this microenvironment. Self-specific B cells escaping normal regulation may develop into high affinity pathogenic Ig-producing cells.

Eosinophils are required to suppress Th2 responses in Peyer's patches during intestinal infection by nematodes.

Infections with enteric nematodes result in systemic type 2 helper T (Th2) responses, expansion of immunoglobulin (Ig)G1 antibodies, and eosinophilia. Eosinophils have a supportive role in mucosal Th2 induction during airway hyperreactivity. Whether eosinophils affect the local T-cell and antibody response in the gut-associated lymphoid tissue during enteric infections is unknown. We infected eosinophil-deficient ΔdblGATA-1 mice with the Th2-inducing small intestinal nematode Heligmosomoides polygyrus and found that parasite fecundity was decreased in the absence of eosinophils. A lack of eosinophils resulted in significantly augmented expression of GATA-3 and IL-4 by CD4+ T cells during acute infection, a finding strictly limited to Peyer's patches (PP). The increase in IL-4-producing cells in ΔdblGATA-1 mice was particularly evident within the CXCR5+PD-1+ T-follicular helper cell population and was associated with a switch of germinal centre B cells to IgG1 production and elevated serum IgG1 levels. In contrast, infected wild-type mice had a modest IgG1 response in the PP, whereas successfully maintaining a population of IgA+ germinal center B cells. Our results suggest a novel role for eosinophils during intestinal infection whereby they restrict IL-4 responses by follicular T helper cells and IgG1 class switching in the PP to ensure maintenance of local IgA production.

Histopathology and molecular pathology of synovial B-lymphocytes in rheumatoid arthritis.

B-cells of the rheumatoid synovial tissue are a constant part of and, in some histopathological subtypes, the dominant population of the inflammatory infiltrate, located in the region of tissue destruction. The pattern of B-cell distribution and the relationship to the corresponding antigen-presenting cells (follicular dendritic reticulum cells: FDCs) show a great variety. B-cells may exhibit (i) a follicular organization forming secondary follicles; (ii) follicle-like patterns with irregularly formed FDC networks, and (iii) a diffuse pattern of isolated FDCs. Molecular analysis of immunoglobulin VH and VL genes from human synovial B-cell hybridomas and synovial tissue demonstrates somatic mutations due to antigen activation. The FDC formations in the synovial tissue may therefore serve as an environment for B-cell maturation, which is involved in the generation of autoantibodies. An autoantibody is defined as "pathogenic" if it fulfills the Witebsky-Rose-Koch criteria for classical autoimmune diseases: definition of the autoantibody; induction of the disease by transfer of the autoantibody; and isolation of the autoantibody from the disease-specific lesion. B-cells from rheumatoid synovial tissue show specificity for FcIgG, type II collagen, COMP, sDNA, tetanus toxoid, mitochondrial antigens (M2), filaggrin and bacterial HSPs. The contributions of these antigens to the pathogenesis of RA are still hypothetical. A possible contribution could derive from crossreactivity and epitope mimicry: due to crossreaction, an antibody directed originally against a foreign infectious agent could react with epitopes from articular tissues, perpetuating the local inflammatory process. The characteristic distribution pattern, the localisation within the area of tissue destruction, the hypermutated IgVH and IgVL genes, and their exclusive function to recognize conformation-dependent antigens suggest a central role for B-cells in the inflammatory process of rheumatoid arthritis. Therefore, the analysis of synovial B-cell hybridomas and experimental expression of synovial IgVH and IgVL genes will help to characterise the antigens responsible for the pathogenesis of rheumatoid arthritis.

Role of B cells in the pathogenesis of rheumatoid arthritis: potential implications for treatment.

In patients with rheumatoid arthritis, chronic inflammation in affected joints may lead to the development of tertiary lymphoid tissue. A micro-environment is generated in the synovial membrane which supports the activation and differentiation of B cells into plasma cells. Through a process of affinity maturation, plasma cells may be generated locally which secrete antibodies of high affinity. Rheumatoid arthritis is characterised by autoantibodies specific for self immunoglobulin. These rheumatoid factors form large antigen/antibody complexes which may enhance the process of joint destruction. The poor prognosis of rheumatoid factor-positive patients is indicitive of the critical role of immunoglobulin complexes in the continuous stimulation of the immune system and thus of the inflammatory processes. In general, treatment of patients with rheumatoid arthritis aims at suppressing inflammation. The currently most successful reagents are those which interfere with the network of cytokines, such as tumour necrosis factor or interleukin-1 receptor antagonists. Only recently have immunosuppressive therapies targeted directly at the B cell response been developed. These first studies suggest that therapies which directly affect the humoral immune response are of great therapeutic potential in the treatment of patients with rheumatoid arthritis.

[Do B cells play an important role in the pathogenesis of rheumatoid arthritis?]. / B-Lymphozyten -- sind sie wichtig für die Pathogenese der Rheumatoiden Arthritis?

The role of B cells for the pathogenesis of rheumatoid arthritis (RA) has been debated for a long time. Here we show that chronic inflammation in the affected joints leads to the development of ectopic germinal centers. A micro-environment is established which supports B cell activation and differentiation. Plasma cells may develop which secrete autoantibodies of high affinity directly into the synovial tissue. Antigen/antibody complex formation, the activation of the complement cascade and the stimulation of macrophages may contribute to the destruction of joints. Furthermore, B cells are efficient antigen presenting cells. They seem to play a pivotal role in the activation of synovial T cells and the induction of cytokine secretion. The success of B cell depletion therapy by using the monoclonal antibody Rituximab further emphasized the importance of B cells in the pathogenesis of RA.