Dendritic cells are potent antigen-presenting cells for microbial superantigens.

Dendritic cells are a small subset of human blood mononuclear cells that are potent stimulators of several T cell functions. Here we show they are 10-50-fold more potent than monocytes or B cells in inducing T cell responses to a panel of superantigens. Furthermore, dendritic cells can present femtomolar concentrations of superantigen to T cells even at numbers where other antigen-presenting cells (APCs) are inactive. Although dendritic cells express very high levels of the major histocompatibility complex products that are required to present superantigens, it is only necessary to pulse these APCs for 1 hour with picomolar levels of one superantigen, staphylococcal enterotoxin B, to maximally activate T cells. Our results suggest that very small amounts of superantigen will be immunogenic in vivo if presented on dendritic cells.

Helper T cell-dependent human B cell differentiation mediated by a mycoplasmal superantigen bridge.

Experimentally induced murine graft-vs.-host disease may be characterized by hypergammaglobulinemia, autoantibody formation, and immune complex-mediated organ system damage that mimics SLE. These autoimmune phenomena are mediated by abnormal Th-B cell cooperation, across MHC disparities, in which donor-derived allospecific Th cells recognize and interact with MHC class II antigens on the surface of recipient B cells. Microbial toxins, termed superantigens, which bind to MHC class II molecules and activate selected T cells based on TCR variable gene usage, may induce a similar form of Th-B cell interaction. In the present study, we generated and characterized human Th cell lines reactive with the Mycoplasma arthritidis superantigen (MAM). The essential observation is that resting human B cells bind MAM and present it to superantigen-reactive autologous or allogeneic Th cells, resulting in both Th cell activation and a consequent polyclonal Ig response by the superantigen-bearing B cells.

Characterization of human T cells reactive with the Mycoplasma arthritidis-derived superantigen (MAM): generation of a monoclonal antibody against V beta 17, the T cell receptor gene product expressed by a large fraction of MAM-reactive human T cells.

While all known microbial superantigens are mitogenic for human peripheral blood lymphocytes (PBL), the functional response induced by Mycoplasma arthritidis-derived superantigen (MAM) is unique in that MAM stimulation of PBL consistently results in T cell-dependent B cell activation characterized by polyclonal IgM and IgG production. These immunostimulatory effects of MAM on the humoral arm of the human immune system warranted a more precise characterization of MAM-reactive human T cells. Using an uncloned MAM reactive human T cell line as immunogen, we have generated a monoclonal antibody (mAb) (termed C1) specific for the T cell receptor V beta gene expressed by the major fraction of MAM-reactive human T cells, V beta 17. In addition, a V beta 17- MAM-reactive T cell population exists, assessed by MAM, induced T cell proliferation and cytotoxic T cell activity. mAb C1 will be useful in characterizing the functional properties of V beta 17+ T cells and their potential role in autoimmune disease.

The sequence of the Mycoplasma arthritidis superantigen, MAM: identification of functional domains and comparison with microbial superantigens and plant lectin mitogens.

Mycoplasma arthritidis, an agent of chronic proliferative arthritis of rodents, secretes a potent soluble superantigen, MAM, that is active for both murine and human T and B lymphocytes. We now report the complete nucleotide and amino acid sequence of MAM and show it to be distinct from other proteins and not closely related phylogenetically to other superantigens. Two functional domains on MAM are identified based on the ability of peptides encompassing these regions to inhibit lymphocyte proliferation by the intact MAM molecule. One of these domains shares short sequences or epitopes with other microbial superantigens. The second domain contains the consensus legume lectin motif-beta, which is important for T cell activation by concanavalin (Con) A. MAM and Con A peptides containing this motif are functionally cross reactive, suggesting a novel secondary pathway for T cell activation by MAM.

Influence of genes of the major histocompatibility complex on ulcerative dermal necrosis induced in mice by Mycoplasma arthritidis.

All mouse strains injected s.c. with Mycoplasma arthritidis developed severe abscesses in the subdermal tissues. However, M. arthritidis strain 14124 P10 also induced an ulcerative dermal coagulation necrosis in mouse strains expressing the k and d haplotypes but not in those expressing the b, q, or s haplotypes. The use of inbred and congenic mouse strains established that the ulcerative necrosis was associated with the haplotypes expressed at the H2 major histocompatibility complex (MHC). The gene restriction seen could be partially overcome by using a more virulent mouse-passaged strain of M. arthritidis (158 P10P9). The data suggest that genes of the MHC function by rendering certain mouse strains more susceptible to an as yet unidentified necrotizing moiety. The close histologic resemblance of the dermal necrosis induced by M. arthritidis to certain human diseases such as necrotizing fasciitis, the ulcerative lesions induced by Mycobacterium ulcerans, and the crepitant and gangrenous cellulitides may therefore provide a unique model to study the genetic factors and mechanisms of pathogenesis in these latter human conditions.

Human B cell differentiation induced by microbial superantigens: unselected peripheral blood lymphocytes secrete polyclonal immunoglobulin in response to Mycoplasma arthritidis mitogen.

Microbial superantigens (SA) activate a significant portion of the T cell repertoire based on their dual avidity for MHC class II antigens and T cell receptor (TCR) epitopes common to products of one or several TCR beta chain variable gene families. While SA that induce massive T cell proliferation and cytokine secretion have been implicated in clinical syndromes characterized by shock and generalized immunosuppression, SA activation of a more restricted T cell response may also have significant, perhaps immunostimulatory, effects on the immune system. To investigate this issue, we measured 3H-thymidine incorporation and polyclonal IgM and IgG secretion by normal human peripheral blood mononuclear cells (PBMC) cultured with a panel of microbial SA, including the Staphylococcus aureus-derived SA, SEA, SEB, SEC-1, SEC-2, SEC-3, SEE, TSST-1, and the Mycoplasma arthritidis-derived SA, MAM. The S. aureus-derived SA induce vigorous proliferation by PBMC, while optimal MAM-induced proliferation is significantly lower in magnitude. In all 12 subjects tested, mitogenic concentrations of MAM reproducibly stimulate unselected PBMC to secrete polyclonal IgM and IgG. In contrast, the S. aureus-derived SA induce Ig production only in cultures containing isolated B cell populations and either very low numbers of untreated autologous T cells, larger numbers of X-irradiated autologous T cells, or very low concentrations of the SA. No difference in the activation of helper (CD4) versus suppressor/cytotoxic (CD8) T cells by MAM and the S. aureus-derived SA was noted. Taken together, these data suggest that MAM's capacity to induce B cell differentiation correlates with its induction of a relatively weak proliferative response by unselected human T cells. MAM-like SA, when encountered in vivo, may result in a significant perturbation of the human immune system and potentially contribute to clinical syndromes characterized by immunostimulation and hypergammaglobulinemia.

T-cell receptors and collagen induced arthritis in H-2r mice.

Mouse strains B10, B10.RIII, RIIIS/J and the F1 and backcross progeny arising from them were tested for susceptibility to porcine type II collagen-induced arthritis (PII-CIA). The clinically severe arthritis of rapid onset that is characteristic of PII-immunized B10.RIII mice developed predominantly in hybrid offspring that had inherited at least one copy of wild type T cell receptor (TCR) genes (V beta b genotype) from the B10 or B10.RIII parent. The results indicate that, in the development of PII-CIA, mice expressing the H-2r/r haplotype preferentially utilize TCR V beta genes that are normally encoded within the TCR V beta genomic deletion region of RIIIS mice (V beta c). After aggressive immunization with PII, the use of alternative TCR V beta genes, encoded outside of the RIIIS deletion region, produced a high IgG antibody response that was cross-reactive with mouse type II collagen (MII) and equivalent to that of B10.RIII mice, but only a very mild, late onset arthritis of 56% (27/48) incidence in RIIIS male mice and 28% (10/35) incidence in RIIIS female mice. In comparison, B10.RIII mice routinely developed early onset of PII-CIA of significantly higher incidence (100%; p < 0.005) and four-fold greater severity, even after milder immunization protocols. The data are compatible with the proposal that the clinically weak CIA response of RIIIs mice may be primarily antibody driven while the severe CIA of B10.RIII mice reflects the added inflammatory effects of collagen-reactive effector-T cells in the joint.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic arthritis of rabbits induced by mycoplasmas. IV. Protective effect of immunization and prior infection.

Rabbits exposed to Mycoplasma arthritidis either by active immunization with killed mycoplasmas or by primary infection in the right knee were protected against intra-articular challenge with viable M. arthritidis in the left knee. This protection extended to the challenged knees of preinfected rabbits even while highly active inflammation persisted in the initially injected joints. We propose that the protection is mediated by antibody, probably metabolic-inhibiting antibody, present in the joint at the time of challenge.

Citrate anticoagulation and cell washing for intraoperative autotransfusion in the baboon.

Extracorporeal citrate was used for anticoagulation during autotransfusion of baboons. A cell-washing plasmaphoresis procedure was added in one group of animals in order to remove activated clotting materials. Both groups became hypocoagulable, but the cell-washed group had less evidence of disseminated intravascular coagulation as well as lower plasma hemoglobin levels. Citrate anticoagulation plus cell washing is a potential alternative to heparinization for autotransfusion.

Expression of metabolism-inhibition antibodies against Mycoplasma arthritidis in rats.

Shared antigens between Mycoplasma arthritidis and rat tissues may be responsible for the lack of metabolism-inhibition (MI) and other neutralizing antibodies in rats with M arthritidis-induced arthritis. We were not able to confirm such antigens or to detect cross-reacting antigens between M arthritidis and rat lymphocytes, thymocytes, and muscle tissue. Antisera of rabbit origin to rat lymphocytes, thymocytes, and skeletal muscle reacted by ELISA with M arthritidis only when the mycoplasmal antigens were prepared from organisms grown in medium containing horse serum. Such activity could be completely absorbed by horse serum. These antisera to rat tissues also failed to react by radioimmunoprecipitation with M arthritidis surface antigens. In addition, antibody activity against homologous antigens could not be absorbed by M arthritidis. Similarly, antisera of rabbit origin against M arthritidis failed to react by ELISA specifically with rat lymphocytes, thymocytes, and skeletal muscle or to react by radioimmunoprecipitation with 125I-labeled rat lymphocyte antigens. These rat tissues could not specifically absorb antibodies against M arthritidis from antisera of rabbit origin. These findings suggest that the lack of MI antibodies in rats probably can not be explained by rat tissue antigens that cross-react with M arthritidis MI antigens. Finally, antisera of rat origin against M arthritidis and other rat tissue components failed to block rabbit MI activity against M arthritidis, thus arguing against steric hindrance as a means of preventing recognition of MI antigens.

Engagement of Toll-like receptors by mycoplasmal superantigen: downregulation of TLR2 by MAM/TLR4 interaction.

Mycoplasma arthritidis mitogen (MAM) is a superantigen (SAg) from M. arthritidis, an agent of murine toxic shock syndrome and arthritis. We previously demonstrated that C3H/HeJ and C3H/HeSnJ mice that differ in expression of TLR4 differed in immune reactivity to MAM. We show here that MAM directly interacts with TLR2 and TLR4 by using monoclonal antibodies to TLR2 and TLR4 which inhibit cytokine responses of THP-1 cells to MAM. Also, using macrophages from C3H substrains and TLR2-deficient mice, we confirmed that both TLR2 and TLR4 are used by MAM. Levels of IL-6 in supernatants of MAM-challenged macrophages were higher in mice which expressed only TLR2, lesser with both TLR2 and TLR4, and absent in mice lacking both TLR2 and TLR4. In addition, expression of TLR2 and TLR4 was moderately upregulated in wild-type cells but cells lacking TLR4 showed a fivefold increase in TLR2 expression. Further, blockade of TLR4 on macrophages of C3H/HeN mice with antibody greatly increased expression of TLR2 and release of IL-12p40 in response to MAM. These results indicate that the SAg, MAM, interacts with both TLR2 and TLR4 and that TLR4 signalling might downregulate the MAM/TLR2 inflammatory response.

Genetic control of T cell activation induced by Mycoplasma arthritidis.

At least three genes are now known to influence T-lymphocyte activation induced by the soluble mitogen derived from Mycoplasma arthritidis (MAM). The I-E region of the murine major histocompatibility complex (MHC) codes for the synthesis of the E alpha chain of the I-E molecule, which acts as a receptor for MAM. Mouse, rat and human E alpha molecules have a similar structure, and lymphocytes from all of these species can be activated by MAM. However, lymphocytes from the BN rat, which also express this molecule, fail or respond only weakly to MAM and lectin mitogens due to the influence of a non-MHC gene(s). The RIIIS mouse strain also expresses the E alpha receptor site for MAM, but possesses a recessive non-MHC gene(s) that is associated with an inability of lymphocytes to respond to MAM without influencing their responses to lectin mitogens. There is evidence that in the BN rat and the RIIIS mouse there is a defect in T cell interactions with the mitogen/accessory cells complex. Evidence is also presented that T-lymphocyte activation in vivo may predispose mice to the toxic and necrotizing properties of viable M. arthritidis.

H2 gene control and biological activities of a T-cell mitogen derived from Mycoplasma arthritidis: a review.

Mycoplasma arthritidis generates a soluble, non-dialysable, polyclonal mitogen which is active for murine and human T lymphocytes in the presence of an adherent, radio-resistant, Ia-bearing accessory cell population. Genetic analysis has established that the I-E sub-region of the murine H2 gene complex controls responses to the mitogen and that this control is exercised at the level of the Ia-bearing accessory cell. Lymphocyte proliferation, induction of cytotoxic lymphocytes, and interferon induction are all under Ir gene control and appear to be dependent upon binding of the mitogen to a specific Ia antigen present on a subset of splenic cells. This mycoplasma mitogen provides a new model system to define the mechanisms of Ir gene control of lymphocyte activation.

Direct activation of the J774.1 Murine macrophage cell line by mycoplasma arthritidis.

Viable Mycoplasma arthritidis and supernatants from M. arthriditis cultures produced marked morphological changes in the J774.1 continuous macrophage line similar to those seen during activation of these cells by Mycobacterium bovis BCG cell walls. The mycoplasma-treated macrophages developed pronounced tumoricidal activity against syngenic 3T12-3 target cells and developed an enhanced capacity for the killing of intracellular listeriae, including both virulent and laboratory-maintained strains. Increased acid phosphatase levels and [14C]glucosamine uptake were also seen. We conclude that mycoplasmas can profoundly alter the functions of macrophages, an event which may not only have in vivo significance with regard to disease pathogenesis, but which may pose considerable problems for in vitro work when unsuspected mycoplasma contamination is present.

I-E/I-C region-associated induction of murine gamma interferon by a haplotype-restricted polyclonal T-cell mitogen derived from Mycoplasma arthritidis.

Cell-free supernatants from cultures of Mycoplasma arthritidis induced significant levels of interferon when cocultured with murine splenic cells. On the basis of physicochemical characteristics and antibody neutralization studies, the antiviral substance was identified as gamma interferon. Use of inbred and congenic mouse strains established that splenic cells from mice expressing the H2k and H2d haplotypes produced interferon in response to M. arthritidis culture supernatants, but those from mice with H2b and H2q haplotypes did not. Further studies with recombinant mouse strains established that interferon induction by the mycoplasma supernatant was associated with the haplotype expressed at the I-E/I-C subregion of the murine major histocompatibility complex. The specificity seen for interferon induction was identical with that reported earlier for induction of cytotoxic lymphocytes and for lymphocyte proliferation in response to the mitogen. All of these reactions appear to be dependent upon binding of the mitogen to specific I-E/I-C region-coded products present on splenic cell surfaces. The observations presented introduce the concept that microbial mitogens or their lymphokine products might modify immune responses and defense mechanisms of the naive host in a genetically restricted manner.

Immunosuppressive properties of the Mycoplasma arthritidis T-cell mitogen in vivo: inhibition of proliferative responses to T-cell mitogens.

We have previously shown that Mycoplasma arthritidis produces a soluble T-cell mitogen (MAM) which is active for most mouse strains that express the alpha chain of the I-E molecule (E alpha) encoded within the murine major histocompatibility complex. The lymphocytes from mice injected intravenously with the MAM exhibited a marked decrease in their ability to respond in vitro to MAM, to phytohemagglutinin, or to concanavalin A T-cell mitogens. Suppression could only be induced in MAM-responsive mouse strains and was most marked 1 to 4 days postinjection. Splenic and node cells and, to a lesser extent, thymic cells from MAM-injected mice could inhibit the ability of lymphocytes from normal mice to respond to MAM and lectin mitogens. A minimum of 2.5 x 10(4) viable cells was required for significant transfer of suppression, and no major histocompatibility complex restrictions were seen. Unlike concanavalin A-induced suppressor cells, which consist of a CD4-, CD8+ T-cell subset, suppressor cells induced by MAM were due to a CD4+ CD8- subset. We hypothesize that MAM may play a role in M. arthritidis-mediated disease by both its inflammatory and immunosuppressive properties.

Triggering and exacerbation of autoimmune arthritis by the Mycoplasma arthritidis superantigen MAM.

OBJECTIVE: It has been postulated that superantigens might play a role in the human rheumatic diseases, by activation of self-reactive T cells or by induction of autoantibodies. The Mycoplasma arthritidis superantigen MAM, which is derived from a naturally occurring murine arthitogenic mycoplasma, uses certain V beta chains of the murine T cell receptor (TCR) that have been proposed to be involved in murine collagen-induced arthritis (CIA). The present study was designed to determine whether MAM influences the course of arthritis mediated by immunization with porcine type II collagen (PII). METHODS: MAM or phosphate buffered saline (PBS) was injected locally or systemically into mice convalescing from CIA or mice suboptimally immunized with collagen. RESULTS: In contrast to PBS, MAM caused an exacerbation of arthritis in mice that were recovering from CIA. MAM also triggered arthritis onset in mice that had been suboptimally immunized with PII up to 160 days previously. Injection of MAM during the onset phase of CIA also triggered and enhanced the severity of arthritis in mice given low doses of PII. CONCLUSION: MAM can both trigger and exacerbate murine autoimmune arthritis induced by immunization with type II collagen. Since T cells bearing the same V beta TCRs as are used by MAM have been found to comprise a major portion of the activated cells in the synovial tissue of patients with rheumatoid arthritis, it is possible that superantigens similar to MAM may play a role in this human disease.