P.J.S. van Kooten

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis.

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RBhighCD4+ T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RBhighCD4+ cells from CD28−/− mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RBhighCD4+ cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-γ by lamina propria CD4+ cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25+CD4+ cells from CD28−/− mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RBhigh cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.

Increased Numbers of CD4+ and CD8+ T Cells in Lesional Skin of Cats with Allergic Dermatitis

The aim of this study was to characterize T cells in the skin of cats with an allergic dermatitis histologically compatible with atopic dermatitis, since T cells play an important role in the pathogenesis of atopic dermatitis in humans. We observed a significantly greater number of T cells in lesional skin of domestic short-haired cats with allergic dermatitis (n = 10; median age 5.8 years) than in the skin of healthy control animals (n = 10; median age 5.0 years). In the skin of the healthy control animals, one or two CD4+ cells and no CD8+ cells were found. A predominant increase of CD4+ T cells and a CD4+/CD8+ ratio (mean ± SD: 3.9 ± 2.0) was found in the lesional skin of 10 cats with allergic dermatitis. The CD4+/CD8+ cell ratio in the skin of healthy control animals could not be determined because of the absence of CD8+ cells. The CD4+/CD8+ cell ratio in the peripheral blood of 10 cats with allergic dermatitis (mean ± SD: 1.9 ± 0.4) did not differ significantly from that in 10 healthy control animals (2.2 ± 0.4). The CD4+/CD8+ cell ratio and predominance of CD4+ T cells in the lesional skin of cats with allergic dermatitis is comparable to that found in atopic dermatitis in humans. In addition, the observed increase of CD4+ T cells in the nonlesional skin of cats with allergic dermatitis compared to the skin of healthy cats is similar to what is seen in humans. Cytokines produced by T cells and antigen-specific T cells are important mediators in the inflammatory cascade resulting in atopic dermatitis in humans. This study is a first step to investigate their role in feline allergic dermatitis.

Experimental immunization with anti-rheumatic bacterial extract OM-89 induces T cell responses to heat shock protein (hsp)60 and hsp70; modulation of peripheral immunological tolerance as its possible mode of action in the treatment of rheumatoid arthritis (RA)

OM‐89 is a bacterial (Escherichia coli) extract used for oral administration in the treatment of RA. Given the evidence that immunity to bacterial heat shock antigens plays a critical role in the immunomodulation of arthritis and possibly inflammation in general, the purpose of the present studies was to evaluate the presence and immunogenicity of hsp in OM‐89. Furthermore, we studied the effects of OM‐89 in an experimental arthritis, where hsp are known to have a critical significance in disease development. In rats immunization with OM‐89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of both E. coli and mycobacterial origin. Conversely, immunization with hsp antigens was also found to induce T cell reactivity specific for OM‐89. Based on this and the antigen specificity analysis of specific T cell lines, hsp70 (DnaK) turned out to be one of the major immunogenic constituents of OM‐89. Parenteral immunization with OM‐89 was found to reduce resistance to adjuvant arthritis (AA), whereas oral administration was found to protect against AA. Given the arthritis‐inhibitory effect of oral OM‐89 in AA, it is possible that peripheral tolerance is induced at the level of regulatory T cells with specificity for hsp. This may also constitute a mode of action for OM‐89 as an arthritis‐suppressive oral drug.

The CD28/CTLA-4-B7 signaling pathway is involved in both allergic sensitization and tolerance induction to orally administered peanut proteins

Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.

Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein.

Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the IgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive. mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. Immuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F10B4 to a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed stocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.

Balancing the immune system: Th1 and Th2

CD4+ T cells are subdivided into Th1 and Th2 cells. Their relative presence or activation is thought to have a regulatory effect on immune behaviour. Until recently, the relative suppression of Th1 cells by the relative increase of Th2 activities, was thought to be a main mechanism of keeping or restoring the balance in a diseased immune system. Nowadays, however, a specialised subset of regulatory T cells is held to be responsible for the main effects of securing a balanced immune system. It is possible that heat shock proteins (hsps) are relevant antigens driving such regulation. Heat shock proteins are known to be immunodominant antigens of bacteria. They are evolutionarily strongly conserved proteins present in all eukaryotic and prokaryotic cellular organisms and are upregulated by several forms of stress. Despite (the paradigm of) self tolerance, hsp-epitopes homologous to endogenous host hsp sequences have been implicated as T cell epitopes to endow cross reactive, hsp specific T cells with the capacity to regulate inflammation, such as in experimentally induced autoimmune diseases. Such T cells were found to produce regulatory cytokines like IL10, in contrast with T cells induced with other conserved microbial proteins that are not upregulated by stress. Hsps have been implicated in immune regulation not only as upregulated targets of adaptive immunity during inflammatory stress, but recently also as triggering factors for innate immunity through activation via Toll-like receptors (TLRs). Th1 cells or proinflammatory T cells are known to produce cytokines with proinflammatory activities. Therefore they are supposed to be critically involved in inflammatory conditions such as autoimmune arthritis. Th2 or helper T cells are known to produce cytokines that help B cells to become activated and to switch their class of antibody. Some of the cytokines produced by Th2 cells (IL4, IL5, IL10, and IL13) also have immune …

In vitro stimulation of goat peripheral blood lymphocytes: optimization and kinetics of the response to mitogens and to allogeneic lymphocytes.

Abstract Experiments were performed to establish the optimal concentrations for specific and non-specific lymphocyte transformation in vitro, in cultures containing 105−106 caprine lymphocytes. A microculture system was used in conjunction with a semiautomatic sample harvester. The assay was optimized for mitogen concentration (PHA-P, Con A, PWN and LPS) and allogeneic cell number, number of responding cells, incubation time and amount of tracer. The effect of addition of serum and the cytotoxic effect of phytomitogens on cultured cells is discussed.

Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis

In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2‐deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR‐transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen‐specific activation of splenocytes was enhanced by MDP with respect to IFN‐γ production, which would be consistent with the Th1‐mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG‐induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.

Trypanosome mediated suppression of humoral and cell-mediated immunity in goats

Abstract Using antibody formation against tetanus toxoid and reactivity in mixed-lymphocyte cultures as parameters for B and T lymphocyte reactions, experimental infection with Trypanosoma vivax in goats produced suppression of both functions. Suppression during infection was also demonstrated by in vitro mitogen-induced lymphocyte transformation. In some cases during early stages of the infection, marked increase in lymphocyte reactivity was found. The latter finding could not be related to the mitogenic activity of trypanosomes, since extracts from these organisms were not significantly stimulatory in vitro.

A monoclonal antibody recognizing a differentiation marker on rat gonocytes

Abstract Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (μ, kappa), was obtained which specifically reacted with gonocytes in the fetal testis. No significant cross-reactivity with other tissues from the 18-day p.c. embryo was found. MAb 4B6.3E10 was reactive with rat gonocytes from 17-day p.c. until the day of birth. Germ cells at later stages of testis development did not show any labelling. The epitope recognized by 4B6.3E10 is a carbohydrate as periodate treatment leads to a loss of reactivity of the antibody. By SDS-PAGE and Western blotting of proteins extracted from a testicular membrane fraction from 18-day p.c. testes, MAb 4B6.3E10 was found to recognize at least 3 protein moieties with apparent molecular weights in the ranges of 80–100, 120, 160–180 kDa (either under reducing- or non-reducing conditions). The results suggest that MAb 4B6.3E10 recognizes a specific differentiation marker for fetal rat gonocytes.