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Biomedical subjects

B R Champion

Publications and source records attributed to B R Champion.

At least 37 records · Page 2Linked to original sources

Immunoregulation in the common marmoset, Calithrix jaccus: functional properties of T and B lymphocytes and their response to human interleukins 2 and 4.

Non-human primates have been used to study immune function to a much lesser extent than readily available strains of inbred rodents. Nevertheless, in situations where it might be desirable, but impossible, to study human immune responses in vivo, lower primates could provide an acceptable alternative. In order to extent the knowledge of T- and B-lymphocyte function in lower primates, the common marmoset Callithrix jaccus was used as an experimental model. The functional similarities between this species and humans at the level of T-B co-operation in the antibody response were examined, and xenoreactive T-lymphocyte clones were obtained from marmoset spleen cells using Epstein-Barr virus (EBV)-transformed human B cells as stimulators. These clones could act as helper cells when co-cultured with human B lymphocytes, inducing the secretion of both IgM and IgG. Lymphokine production by mitogen-stimulated marmoset T-cell clones was also examined. Interleukins (IL) 2 and 4 activities were detected in clone supernatants using bioassays and interferon-gamma (IFN-gamma) was detected using a solid-phase ELISA system. However, SDS-PAGE analysis of biosynthetically labelled marmoset and human T-cell clone supernatant proteins revealed major differences between the soluble T-cell products of the two species. The proliferative responses of marmoset T and B cells to recombinant human IL-2 and IL-4 were also examined. Stimulation of [3H]thymidine uptake was detected in both T cell- and anti-IgM-stimulated B-cell cultures with both of the lymphokines. These results suggests that the key components of the antibody response are functionally conserved between lower primates and man and that the common marmoset may be useful as an in vivo model of immune function, particularly with regard to the role of interleukins such as IL-2 and IL-4.

Animals↗

Functional and phenotypic properties of T-cell clones which regulate IgE synthesis.

We have generated a panel of T-lymphocyte clones from a patient suffering from the hyper IgE syndrome, and have attempted to correlate the ability of each to help IgE responses in vitro with the profile of lymphokines secreted after mitogenic stimulation. Clones which showed positive IgE helper activity released larger amounts of interleukin-4 (IL-4) than the non-helpers, which tended to release more interleukin-2 (IL-2). Surprisingly, all clones released moderate amounts of gamma interferon (IFN), which has been shown to inhibit the action of IL-4 on B cells. The clones were analysed by indirect immunofluorescence using monoclonal antibodies to CDw29 and CD45R (4B4 and 2H4 respectively). Those T cells which could provide strong helper activity for all isotypes, expressed high levels of CDw29 and low CD45R. These data suggest that these CD4-positive T cells expressing surface antigen of the 'memory' subset i.e. CDw29, are involved in IgE isotype regulation by virtue of their ability to secrete IL-4 upon antigenic stimulation.

Antigens, Differentiation↗

Functional and phenotypic analysis of human T-cell clones which stimulate IgE production in vitro.

Peripheral blood mononuclear cells (PBMC) from a patient suffering from the hyper IgE syndrome were used to generate phytohaemagglutinin (PHA)-expanded T-cell clones (all CD4+, CD8-, CD23-). A selection of the clones was tested for their ability to help IgE secretion by culturing with normal B cells in the presence of solid-phase antibody to CD3. Supernatants were harvested on Day 7 and assayed by ELISA for IgE, IgG and IgM. Lymphokine secretion by the clones was assessed by culturing clones for 24 hr with solid-phase antibody to CD3 followed by assay of the supernatants for IL-2, IL-4 and interferon-gamma (IFN-gamma) production. In addition, clones were analysed by flow cytometry for CDw29 and CD45R expression. Initial experiments with seven clones indicated that those clones that could help IgE secretion also stimulated optimal IgG and IgM responses. All clones appeared to secrete IL-2, IL-4 and IFN-gamma, although the amounts of each varied. These results confirm recent findings that human T-cell clones do not fall into Tinf (Th1) and Th (Th2) type subsets as described in the mouse. There was no clear correlation between the lymphokines secreted by the clones and their capacity to help IgE production. However, the helper function of the clones for all isotypes, including IgE, appeared to be related to the level of expression of the surface antigen CDw29.

Antigens, Differentiation↗

Selective autoimmune response to the chicken-specific structures of thyroglobulin in Obese strain chickens.

The characteristics of thyroglobulin (Tg) autoantibodies in Obese strain (OS) chickens with thyroiditis have been defined and compared with those of polyclonal antibodies to chicken Tg produced by immunizing normal chickens and a rabbit, and with mouse monoclonal antibodies (MoAb) to chicken Tg. Chicken Tg autoantibodies (aAb), when tested against Tg from 24 species all showed specificity for chicken Tg which ranged from absolute to limited although in most instances cross-reactions with Tgs of other species were either absent or at a low level. Antibodies to chicken Tg produced by immunization showed a similar limited range of cross-reactions. Four of five chTg-MoAbs were specific for chicken Tg and the fifth was almost so. In competitive experiments, the polyclonal rabbit antibody could fully inhibit binding of all chicken Tg-aAb to chicken Tg but not vice versa. It was inferred that polyclonal rabbit Tg antiserum includes antibodies to all the epitopes seen by chicken Tg-aAb and many more besides. In similar experiments with four chicken Tg-MoAbs, the binding of one chicken Tg-aAb was unaffected, and three other patterns of inhibitions were defined. The binding to chicken Tg of a fifth chicken Tg-MoAb was enhanced rather than inhibited by chicken Tg-aAb. Some but not all chicken Tg-aAb preparations could differentiate between Tgs containing different amounts of thyroxine. We conclude that the autoantibodies to Tg in OS chickens are directed in the main against determinants unique to the species. Not all the species-specific determinants are involved in the autoimmune response but the number of epitopes involved is at least four. In these respects the immune response to Tg in OS chickens resembles that in autoimmune thyroid disease in humans. The conformation of chicken Tg may be affected by combination with antibody or by the content of thyroid hormone.

Animals↗

Critical role of iodination for T cell recognition of thyroglobulin in experimental murine thyroid autoimmunity.

We have used two clonotypically distinct thyroglobulin (Tg)-specific, I-Ak restricted monoclonal T cell populations to investigate the role of thyroid peroxidase-catalyzed iodination in Tg recognition by autoreactive T cells. The results showed that these T cells could recognize Tg only it it was sufficiently iodinated. Unlike normal mouse Tg, noniodinated mouse Tg was unable to induce significant thyroid lesions but could trigger the production of Tg autoantibodies. In these experiments, the importance of T cell recognition of iodination-related epitopes was emphasized by the inability of serum antibodies to distinguish Tg on the basis of iodine content, whether they were induced with normal or noniodinated Tg. Therefore, thyroid peroxidase-dependent modification of Tg would appear to be central to its recognition by autoreactive T cells and hence its capacity to induce autoimmune thyroid lesions.

Amitrole↗

High efficiency antigen presentation by thyroglobulin-primed murine splenic B cells.

B cells primed in vivo with mouse or rat thyroglobulin present these antigens at very low concentrations to CH9, an Ly 1+2- T cell hybridoma specific for mouse and rat thyroglobulin. Presentation measured by interleukin 2 release from CH9 is sensitive to treatment with a monoclonal antibody eliminating splenic B cells but is unaffected by anti-Thy-1.2 or 33D1 (which destroy T cells and dendritic cells, respectively). Presentation is specific for the priming antigen and is blocked by preincubation of the B cells with sheep anti-mouse F(ab')2. We suggest that in this system, primed B cells present thyroglobulin and that this may represent a means by which an initial triggering event priming both B and T cells could allow maintenance of autoreactive responses in vivo in the presence of low concentrations of circulating antigen.

Animals↗

T-cell hybridomas specific for self and foreign thyroglobulins.

We have used somatic cell fusion techniques to produce and characterize murine T-cell hybridomas with specificity for self and foreign thyroglobulin (Tg). Two interleukin-2 (IL-2)-releasing I-Ak-restricted hybrid clones with specificity for self determinants on syngeneic Tg were derived from Tg-specific T-cell lines. These two autoreactive hybridomas were independently derived and were clonotypically distinct as determined by restriction fragment length polymorphism of the Ti beta-chain gene, but showed a similar pattern of cross-reactivity against rat and human (but not porcine) Tg. A third T-cell hybridoma showed a previously unknown specificity for the immunizing (non-inbred) Tg, but not for syngeneic Tg, indicating responsiveness to an allelic determinant. Although T-cell hybridization techniques have previously had only minimal application in experimental autoimmunity, this represents an approach to the study of Tg-specific T-cell responses at the molecular level.

Animals↗

Recognition of thyroglobulin autoantigenic epitopes by murine T and B cells.

We have used a large panel of thyroglobulins (Tg) prepared from a wide range of mammalian species to study the Tg autoantigenic epitopes recognized by populations of monoclonal and polyclonal murine T and B cells. This approach showed the existence of at least six different epitopes; three recognized by T cells (in association with I-Ak on antigen-presenting cells) and three by B cells (monoclonal antibodies). The majority of serum and monoclonal autoantibodies were found to be highly specific for mouse Tg, with some cross-reactive binding to rat Tg. In contrast, T-cell lines/clones and hybridomas recognized cross-reactive epitopes on Tg that were highly conserved throughout most of the mammalian orders. Moreover, two hybrid clones, which showed similar patterns of cross-reactivity, differed in their responsiveness to tryptic digests of human Tg. Thus, autoreactive T and B cells recognize distinct areas of the Tg molecule.

Animals↗

Helper and suppressor activities of an autoreactive mouse thyroglobulin-specific T-cell clone.

An autoreactive T-cell clone, MTg9B3, specific for mouse thyroglobulin, has been characterized. This clone has properties consistent with it an being autoreactive T helper cell. It is I-A restricted, with a surface phenotype of Thy 1+, L3T4+, Ly2-, and produces a number of non-specific lymphokine activities following specific triggering with antigen. Furthermore, antigen-stimulated clone cells were capable of providing bystander help in SRBC-specific antibody responses. Surprisingly, MTg9B3 cells had a profound suppressive effect on thyroglobulin autoantibody responses in vitro.

Animals↗

Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis: comparison of the effects of recombinant gamma-interferon on human monocytes and murine peritoneal macrophages.

When cultured in 20% heat-inactivated human serum, human monocytes from seven donors were not on average significantly different from non-activated murine peritoneal cells (cultured simultaneously and in an identical manner) in their ability to inhibit BCG and, when calculated relative to growth of bacilli in the same medium without macrophages, to enhance the growth of Mycobacterium tuberculosis. Recombinant gamma-interferon caused marked inhibition of virulent M. tuberculosis by murine (BALB/c) peritoneal macrophages. This effect was seen, whether the cells were cultured in 10% fetal calf serum or in 20% heat-inactivated normal human serum, with or without the addition of iron supplements. However, unlike murine cells, the addition of crude lymphokine or recombinant gamma-interferon to human monocytes caused only weak inhibition of M. tuberculosis, and in some instances, gamma-interferon caused enhancement of growth of the bacilli. Monocytes were only slightly more effective if precultured for 4-8 days before the addition of the activating stimulus. This relative failure to develop anti-mycobacterial mechanisms occurred in spite of the activation of the cells as shown by a massive increase in reduction of nitro-blue tetrazolium inducible by phorbol myristate acetate.

Animals↗

Cyclosporine affects the function of antigen-presenting cells.

The immunosuppressant drug cyclosporine (CsA) is known to affect T-cell function. We have studied the effect of CsA on the specific proliferative response of T-cell lines to antigen. In addition to blocking IL-2 release by specifically activated T-cell lines, CsA also affected the ability of irradiated spleen cells to present preprocessed antigen to T-cell lines. Irradiated spleen cells pulsed with antigen for 2 hr were able to stimulate a proliferative response in T-cell lines. Following a 2-hr pulse with CsA, antigen presentation by these irradiated spleen cells was reduced significantly, suggesting that CsA not only affects T cells, but also affects the function of antigen-presenting cells.

Animals↗

Antigenic determinants of human thyroglobulin differentiated using antigen fragments.

Human thyroglobulin (Tg) was digested with V8 protease and the fragments separated by high performance liquid chromatography (HPLC). The antigenic relationship of the fragments was investigated using mouse monoclonal antibodies to human Tg. The binding of Hashimoto's disease autoantibodies to the fragments was measured by radioimmunoassay. This demonstrated that a minority of the patients recognize an epitope on Tg which others do not. The epitopes identified by the autoantibodies were substantially destroyed in the smaller fragments tested, but these smaller fragments were more efficient stimulators of Tg-specific T-cell lines than the larger fragments which carry the antibody binding determinants. This suggests that the parts of the Tg molecule which stimulate autoimmune B cells differ from those which stimulate T cells.

Animals↗

Autoreactive T-cell lines specific for mouse thyroglobulin.

Autoreactive T-cell specific for mouse thyroglobulin have been established and characterized. These Lyt 1+ T cells proliferated specifically in response to thyroglobulin presented by syngeneic irradiated spleen cells. The antigen-presenting cell requirements of these autoreactive T cells appeared to be the same as those for foreign antigen (PPD) reactive T cells. All lines tested required antigen-presenting cells compatible at the I-A subregion of the H-2 complex. Both T-cell types responded to antigen presented by peritoneal cells and splenic dendritic cells, but only gave optimal responses when whole spleen cells were used. The cross-reactivity patterns of responses to mouse, rat, pig and human thyroglobulins indicated that at least two different epitopes could be recognized by the autoreactive T cells. Furthermore, these epitopes appeared to be different from those recognized by the majority of serum autoantibodies to mouse thyroglobulin.

Animals↗

I-A restricted activation by T cell lines of anti-tuberculosis activity in murine macrophages.

Tuberculosis and leprosy remain two of the world's most significant diseases. Immunity involves the activation of macrophages by lymphokines but the details are unknown because there has been no objective assay for the relevant effector function using human pathogens. We previously reported the use of tritiated-uracil uptake by surviving mycobacteria as a measure of the anti-mycobacterial effect of human monocytes. We describe here the use of a modification of this assay to measure control of the proliferation of Mycobacterium tuberculosis in murine peritoneal macrophages. A bacteriostatic effect can be induced in macrophages infected with M. tuberculosis, by adding small numbers of Ly 1 +2- T cells from in vitro lines derived from immunized mice. The phenomenon is dependent on compatibility at the I-A locus of the major histocompatibility complex (MHC) and mediated by soluble factors. Such T cells also recognise and activate macrophages infected with other mycobacterial pathogens. Thus, T cells recognising shared mycobacterial antigens are active. The findings have implications for MHC linked susceptibility to mycobacterioses and the hypothesized ability of cross-reactive environmental mycobacteria to abrogate or pre-empt the protective efficacy of subsequent BCG vaccination.

Animals↗

Immunity to homologous collagens and cartilage proteoglycans in rabbits.

The in vitro incorporation of [3H]-thymidine into spleen cells was used to show that rabbits with experimentally-induced inflammatory arthritis of 1-4 months duration often develop cellular immunity to purified homologous cartilage proteoglycans, type I and III collagens and, less frequently, to type II collagen. Responses to collagens were primarily directed to antigenic determinants exposed on peptides in degraded molecules, whereas responses to proteoglycans were seen with both native and degraded molecules. These in vitro blastogenic responses were shown to be completely dependent on the presence of T lymphocytes in the cultures. This expression of immunity was not demonstrable in rabbits with long duration arthritis (7-12 months) with any of the antigens tested. Some rabbits injected with homologous proteoglycans demonstrated T-cell-dependent cellular, but not humoral, immunity to the injected antigens. In contrast, rabbits injected with heterologous human proteoglycans developed cellular and humoral immunity to the immunizing proteoglycan, but failed to develop cellular immunity to rabbit proteoglycan. Some of these rabbits did, however, produce circulating antibodies which reacted with rabbit proteoglycans. Moreover, antibodies produced by immunizing rabbits with proteoglycans from bovine, chicken and rat cartilages and the hyaluronic acid binding region from rat cartilage proteoglycan often cross-reacted with rabbit proteoglycan, indicating that there are species-common antigenic determinants present in these proteoglycans and in the hyaluronic acid binding region and that these are recognizable in rabbit proteoglycans by rabbit antibodies. This ability to induce selectively cellular or humoral immunity to proteoglycans should be useful for future investigations of the role of such immunity in the pathogenesis of arthritis, although induction of immunity to proteoglycan was not accompanied by any demonstrable synovitis in these rabbits as has been observed in some strains of rats and mice for type II collagen.

Animals↗

Immunity to homologous type III collagen after partial meniscectomy and sham surgery in rabbits.

Cellular immunity to rabbit types I, II, and III collagen was detected after both partial medial meniscectomy and sham operation in rabbits when an in vitro 3H-thymidine incorporation assay was used. The blastogenic responses were commonly directed towards peptides derived from collagens by cyanogen bromide cleavage used to mimic proteinase degradation products. The responses to type III collagen peptides were by far the strongest and were seen in most rabbits that were operated on. We suggest that the responses to types I and II collagen were caused by a cross-reaction with type III collagen peptides. This conclusion was supported by the observation that spleen cells from rabbits directly immunized with homologous type III collagen peptides in Freund's complete adjuvant responded strongly to the immunizing peptides and also cross-reacted with types I and II collagen peptides. Immunity to cartilage proteoglycans was observed primarily in rabbits that had undergone meniscectomy and had severe cartilage degeneration. These results indicated that immunity to collagens will develop merely as a result of joint surgery, whereas immunity to proteoglycans is largely dependent upon an osteoarthritic lesion.

Animals↗

Age-related changes in the antigenicity of human articular cartilage proteoglycans.

Rabbits were immunized with adult or foetal human articular cartilage proteoglycans and cell-mediated and antibody immunity assessed in vitro. Cellular immunity to proteoglycans of different ages was measured by the stimulation of 3H-thymidine incorporation into cultured spleen cells. Antibodies to proteoglycans in the rabbit sera were detected with an enzyme-linked immunosorbent assay. The ability of adult and foetal proteoglycans to preabsorb antibody activity in this assay was compared. The results from both the cellular and antibody studies support the same conclusion, that adult proteoglycans express all foetal antigenic determinants and have additional determinants not seen on foetal proteoglycans. The reactivity of rabbit spleen cells to proteoglycans of different ages indicated that these determinants appear during maturation when the major changes in proteoglycan structure occur. The results did not appear to be due to differences in chondroitin sulphate content of the preparations, since the same observations were made with chondroitinase ABC-digested proteoglycans. The observations might be explained by other differences in glycosylation between the preparations or by postulating the synthesis during maturation of an additional proteoglycan species with a different core-protein. Such changes in the antigenicity of articular cartilage proteoglycans with age could account for the development of autoimmunity to proteoglycans in inflammatory joint diseases and may play a role in their pathogenesis.

Adolescent↗