The Mycoplasma arthritidis T-cell mitogen, MAM: a model superantigen

The superantigens are receiving a great deal of attention as a new group of potent immunomodulatory molecules. They are produced by diverse microbial agents including staphylococci, streptococci and mycoplasmas and are also encoded by murine tumor viruses (the Mls antigens). Superantigens activate T cells by a unique pathway which can lead to modification of the T-cell repertoire and induction of autoimmunity. Here, Barry Cole and Curtis Atkin review their observations on the Mycoplasma arthritidis superantigen, MAM, and discuss how MAM might contribute to the acute and chronic inflammatory disease mediated by this organism.     

Modulation of cytokine profiles by the Mycoplasma superantigen Mycoplasma arthritidis mitogen parallels susceptibility to arthritis induced by M. arthritidis

Mycoplasma arthritidis mitogen (MAM) is a potent superantigen secreted by M. arthritidis, an agent of murine arthritis. Here we compare the abilities of MAM to induce a panel of cytokines in vitro and in vivo in BALB/c and C3H/HeJ mouse strains that differ in susceptibility to mycoplasmal arthritis. Splenocytes from both mouse strains produced high levels of all cytokines by 24 h following in vitro exposure to MAM. No differences in cytokine profiles were seen irrespective of the MAM dose. However, there were striking differences in cytokine profiles present in supernatants of splenocytes that had been collected from mice after intravenous (i.v. ) injection of MAM and subsequently rechallenged with MAM in vitro. Splenocytes collected 24 and 72 h after i.v. injection of MAM and challenged in vitro with MAM showed the most marked divergence in the secreted cytokines. Type 1 cytokines were markedly elevated in C3H/HeJ cell supernatants, whereas they were depressed or remained low in BALB/c cell supernatants. In contrast, the levels of type 2 cytokines were all greatly increased in BALB/c cell cultures but were decreased or remained low in C3H/HeJ supernatants. Interleukin-12 mRNA and protein was also markedly elevated in C3H/HeJ mice, as were the levels of immunoglobulin G2a. The data indicate a major skewing in cytokine profiles to a type 1 inflammatory response in C3H/HeJ mice but to a protective type 2 response in BALB/c mice. These cytokine changes appear to be associated with the severe arthritis in C3H/HeJ mice following injection of M. arthritidis in comparison to the mild disease seen in injected BALB/c mice.     

Characterization of the metabolism inhibition antigen of Mycoplasma arthritidis.

The Mycoplasma arthritidis antigen(s) responsible for eliciting metabolism-inhibiting antibodies in rabbits has been partially characterized. Metabolism-inhibiting activity was absorbed from rabbit antisera by intact M. arthritidis cells and membranes but much less so by the soluble cytoplasmic fraction, indicating that the antigen is located on the outer membrane surface. It was stable to periodate and lipid extraction but labile to heat and proteolytic enzymes, indicating that it is protein in nature. Finally, it is most likely a tightly bound integral rather than a peripheral membrane protein, since it was not extracted by low-ionic-strength solutions or by the nonionic detergents Triton X-100, Nonidet P-40, and Tween 20. It was solubilized by both the anionic agent sodium deoxycholate and the zwitterionic detergent Zwittergent. Two two monoclonal antibodies with metabolism-inhibiting activity were produced. One recognized a 45,000-dalton surface protein; however, the other recognized an antigen which is probably of cytoplasmic origin, indicating that more than one cell component may be involved in the metabolism-inhibiting antibody response.     

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.     

T cell lines responding to Mycoplasma arthritidis and chondrocytes in the Mycoplasma arthritidis infection of rats

CD 4+ T cell lines responding specifically to Mycoplasma (M.) arthritidis were established from spleen and lymph nodes of a Lewis rat infected with M. arthritidis. The T cell response to M. arthritidis was MHC class II-restricted. M. arthritidis-reactive T cell lines also responded to syngeneic chondrocytes suggesting that M. arthritidis and chondrocytes may share a common antigenic structure. The T cell lines reacting with chondrocytes may play an important role in the chronic stage of the M. arthritidis-induced arthritis of rats by maintaining immune reactions initiated by M. arthritidis antigens and originally established to eliminate the mycoplasmas. These autoimmune reactions could perpetuate after disappearance of the mycoplasmas and possibly last for the entire lifetime.     

Induction of interleukin-6 in murine bone marrow-derived macrophages stimulated by the Mycoplasma arthritidis mitogen MAS.

Cell-free supernatant of cultures from Mycoplasma arthritidis (MAS) functions as an extremely potent T-cell mitogen for human and murine lymphocytes. The T-cell response is dependent on the presence of accessory cells, presenting the intact E2 molecule on the cell surface. Until now, pure MAS protein has not been available. We developed a new multi-step method for MAS purification. The main steps in this protocol are ammonium sulfate precipitation, anion exchange and hydroxyapatite chromatography followed by gel filtration. With this efficient protocol we obtained fractions of extremely potent mitogenic properties, the purification rate was about 5 x 10(5). Although this protease-sensitive mitogenic activity was highly enriched, we failed to detect the protein by sensitive staining methods of SDS-PAGE. In previous studies, we showed that MAS induces the synthesis of interferon gamma in human and murine lymphocyte cultures. Here we demonstrate that MAS induces interleukin-6 (IL-6) in murine bone-marrow derived macrophage cultures. Since IL-6 is also induced by endotoxin, we used C3H/HeJ mice, which are known to be LPS-nonresponders, in all our studies.     

Human T-cell responses to Mycoplasma arthritidis-derived superantigen.

When injected into mice, Mycoplasma arthritidis causes a chronic arthritis that resembles rheumatoid arthritis, histologically. The organism produces a superantigen termed Mycoplasma arthritidis mitogen or MAM, that in humans preferentially expands T cells whose antigen receptors express V beta 17. T cells with this phenotype appear to be increased in rheumatoid synovial effusions. We describe a novel approach to isolating and characterizing human MAM-reactive T-cell lines and determining their T-cell receptor (TCR) V beta usage. Lines were prepared from T cells that clustered with dendritic cells during a 2-day exposure to MAM. Cluster and noncluster fractions of T cells were then expanded by using feeder cells and a polyclonal mitogen. Most of the MAM reactivity was found in dendritic T-cell clusters, as were most of the T cells expressing TCR V beta 17. After expansion, 76% of the cluster-derived T-cell lines were MAM reactive, while no reactivity was seen in cell lines derived from the noncluster fraction. Of the MAM-reactive lines, 49% expressed V beta 17 on some or all of the cells. Cell lines from both cluster and noncluster fractions were analyzed for TCR V beta mRNA expression by PCR amplification. Other V beta genes (5.1, 7, 8, 12, and 20) were found to be expressed by lines that were MAM reactive, although these were not a major component of the cluster-derived T cells. Some non-cluster-derived lines expressed V beta s 17, 12, and 7, but these proved to be nonreactive to MAM. Therefore, dendritic cells can be used to immunoselect and characterize T cells that express superantigen-reactive TCRs.     

Association of Mycoplasma arthritidis mitogen with lethal toxicity but not with arthritis in mice

Mycoplasma arthritidis induces an acute to chronic arthritis in rodents. Arthritis induced in mice histologically resembles human rheumatoid arthritis and can be associated with lethal toxicity following systemic injection. The M. arthritidis mitogen (MAM) superantigen has long been implicated as having a role in pathogenesis, but its significance with respect to toxicity and arthritogenicity in mycoplasma-induced disease is unclear. To study the pathogenic significance of MAM, M. arthritidis mutants that overproduced or failed to produce MAM were developed. MAM overproduction and knockout mutants were more and less mitogenic, respectively, than the wild-type strain. The degree of mitogenic activity correlated with lethal toxicity in DBA/2J mice. In contrast, histopathological studies detected no correlation between MAM production and the severity of arthritis induced in DBA/2J and CBA/J mice.     

Clonal analysis of human T cell activation by the Mycoplasma arthritidis mitogen (MAS)

Mycoplasma arthritidis produces an as yet undefined soluble molecule (MAS) that has a potent mitogenic effect on T cells of several species. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by this mitogen. Reactivity to MAS is clonally expressed among T cell receptor (TcR) alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TcR alpha/beta chain-negative T lymphocyte clones expressing the CD3-associated TcR gamma chain. MAS is able to induce cytotoxicity and/or proliferation in these T cell clones. For triggering of these T cells, regardless of their phenotype of specificity, the presence of autologous, allogeneic or xenogeneic major histocompatibility complex (MHC) class II molecules on accessory cells or target cells is necessary. However, T cells do not immunologically recognize MAS on class II molecules, since a direct action of MAS on the T cells themselves can be demonstrated. Triggering of T cells by MAS can be blocked by monoclonal antibodies against CD2, CD3 and the TcR alpha/beta chain dimer. We discuss as a possible explanation that MAS is a functionally bivalent molecule cross-linking TcR and MHC class II molecules. Thus, the mechanism of T cell activation by MAS has striking similarities to the mechanisms by which Staphylococcal enterotoxins activate T cells. It is intriguing that a similar mitogenic principle has been developed by two evolutionary distinct pathogenic microorganisms.     

Adrenergic regulation of lymphocyte proliferative response in cultures with T-cell mitogens

The effects of epinephrine, β-adrenergic agonist terbutaline sulfate, and cAMP phosphodiesterase inhibitor theophylline on proliferative response of peripheral blood lymphocytes from healthy subjects were studied in cultures with phytohemagglutinin and concanavalin A. Both adrenergic agonists inhibited lymphocyte blastogenesis, but the effect of epinephrine was more pronounced. Translated fromByulleten' Eksperimental' noi Biologii i Meditsiny, Vol. 128, No. 8, pp. 207–209, August, 1999     

Chronic proliferative arthritis of mice induced by Mycoplasma arthritidis: demonstration of a cell-mediated immune response to mycoplasma antigens in vitro.

Lymphocytes taken from mice chronically infected with Mycoplasma arthritidis exhibited a significant blastogenic response as measured by (3H)thymidine uptake when exposed in vitro to M. arthritidis antigens. The lymphocytes taken from 9 of 12 control mice of similar age exhibited an inhibition of (3H)thymidine uptake when exposed to M. arthritidis antigens.     

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.     

Factors Influencing the Susceptibility of Mice to Mycoplasma arthritidis

Concentrated broth constituents markedly enhanced the development of abscesses after subcutaneous injection of Mycoplasma arthritidis. Under appropriate conditions, both virulent and avirulent strains of M. arthritidis produced toxic effects in mice, as detected by the development of necrotic abscesses and death of the animals. The treatment of mice at birth with M. arthritidis antigens rendered the animals more susceptible to infection with M. arthritidis later in life, as evidenced by increased arthritis after intravenous injection. The injection of M. arthritidis into pregnant mice resulted in a high incidence of abortions. Comparative studies using different mouse strains showed that DBA mice were the most susceptible and BALB/c and C57BL mice were the most resistant to infection by M. arthritidis.     

Mechanisms of attachment of Mycoplasma arthritidis to host cells in vitro.

Although other investigators have reported that Mycoplasma arthritidis failed to attach to several types of mammalian cells in vitro, we showed that it attached well to rat synovial fibroblasts, lung cells, and skin cells but not to kidney cells, suggesting that receptor sites are unequally expressed or distributed among different rat tissues. M. arthritidis also attached poorly to canine kidney and to baby hamster kidney cells, although it did attach to human fetal lung and fetal amnion cells. Four M. arthritidis strains, two virulent and two avirulent, all attached equally well to rat lung cells. Attachment was inhibited by trypsinization, suggesting that the major adhesins are protein. Fab' fractions of rabbit antisera against four M. arthritidis strains partially inhibited adherence of both homologous and heterologous strains, although not to the same extent, indicating some degree of antigenic heterogeneity among their adhesins. A filter-cloned variant of M. arthritidis 158p10p9, designated LC1, attached poorly compared with the parent strain. Missing from this variant were two proteins migrating within the 81- to 90-kDa range by polyacrylamide gel electrophoresis; in their place was a 24-kDa antigen that may be a truncated version of one of these proteins. A monoclonal antibody that partially inhibited attachment recognized all these peptides by Western immunoblotting. An additional attachment-inhibiting monoclonal antibody recognized a 71-kDa antigen present in both low-adherence and fully adherent populations. The low-adherence variant LC1 induced slightly but significantly less arthritis in Lewis rats than did a fully adherent clone.     

Lymphocyte activation. V. Quantitation of the proliferative responses to mitogens using defined T and B cell populations

Although mitogens are frequently used for polyclonal lymphocyte stimulation in both experimental and clinical studies, biological tests of this kind encounter several technical difficulties. The relative importance of some of these were studied by using `calibration curves' for mitogenic stimulation. Thymocytes from cortisone-treated mice (T cells) and spleen cells from T cell-deprived mice (B spleen cells) were mixed in different known proportions and stimulated by Concanavalin A (Con-A), which is a T cell mitogen, or lipopolysaccharide (LPS), which is a mouse B cell mitogen.

We have confirmed earlier observations showing that the amount and specific activity of [³]thymidine have to be adjusted to the culture method used. A linear relationship between the responding cell number and incorporated thymidine was promoted when the extracellular thymidine concentration was constant during the labelling period (16 hr). In our system this could be achieved by adding sufficient amount of thymidine in the form of isotope of relatively low specific activity (50 mCi/mmol). In contrast, when isotope of very high specific activity was used, a low number of responsive cells gave almost `normal' incorporation values.

The validity of the use of mitogens was also tested with the T6 chromosome marker method and cell surface immunoglobulin analysis. It was found that the selective responses of T and B cells to T or B cell specific mitogens were maintained in cultures containing varying numbers of T and B cells. The only indication that B cell activating or `potentiating' factors are released by Con-A-stimulated T lymphocytes was observed in cultures with a high proportion (75%) of `T' cells.

Radio-autographic analysis revealed that under the culture conditions in which the majority of T cells from the spleen were stimulated by Con-A, only 20–30% of B cell population responded to LPS and pokeweed mitogen (PWM).

     

Induction of cytokines in human whole blood cultures by a mitogen derived from Mycoplasma arthritidis and by staphylococcal enterotoxin B.

Mycoplasma arthritidis produces a so-far only partially characterized soluble material (MAS) that has a potent mitogenic effect on T lymphocytes of several species. Similar to staphylococcal enterotoxins and a number of related toxins secreted by other species of bacteria, nanogram quantities of these so-called superantigens are sufficient to induce significant amounts of cytokines in the supernatant of lymphocyte cultures. Induction of interleukin-6 (IL-6) by MAS in murine bone marrow-derived macrophages has recently been described. In our study, we examined the differential effects of MAS and Staphylococcus aureus enterotoxin B (SEB) on human blood cells. When compared to MAS, SEB induced a higher proliferative response and, accordingly, a higher release of IFN-gamma. In contrast, large amounts of the macrophage products IL-1, IL-6 and tumor necrosis factor alpha (TNF-alpha) were observed in supernatants of cell cultures stimulated with MAS, whereas only small amounts were induced by SEB. Staphylococci and mycoplasmas are responsible for a number of diseases with various symptoms in man and animals. Our results suggest that SEB and MAS show different qualities in lymphocyte and macrophage stimulation which may be relevant in the pathogenesis of diseases.     

T-dependence of human B lymphocyte proliferative response to mitogens.

Human peripheral blood and tonsil lymphocytes were fractionated on anti-Ig-coated Sephadex columns or by centrifugation after rosetting with native sheep erythrocytes. Both methods allowed the recovery of B and T-enriched populations the purity of which was checked by fluorescein-labelled anti-Ig serum, E and EAC rosette formation, and heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to PHA, Con A, PWM, and ALS was not found different from that of unfractionated cells, whereas no response of the B cells could be observed to these mitogens providing that no contaminating T cells were present. Addition of T lymphocytes to these unresponsive B cells allowed them to respond to phytomitogens, but not to ALS. X-irradiated T cells could, to some extent, replace the diving T lymphocytes; no T-replacing factor could be found in cell-free supernatants from T cells, whether or not they had been activated by mitrogens. This model of B-T cooperation appears useful for studying the differentiation and maturation of human B lymphocytes.     

Binding of sodium [195Au]aurothiomalate to Mycoplasma arthritidis.

The binding of sodium [195Au]aurothiomalate (ATM) to whole cells of Mycoplasma arthritidis has been measured within the temperature range of 4 to 53 degrees C. The time course (kinetics) and the effect of varying the total concentration of free ATM in the suspension at 37 degrees C were also measured. The extent of binding at 37 degrees C leveled off after 30 min at approximately 65 nCi of [195Au]ATM per mg of membrane protein. The amount of ATM bound per cell appeared to be directly proportional to the concentration of free ATM in the range of 0.25 to 0.60 muCi of [195Au]ATM per ml. An Arrhenius plot showed two distinct regions with slopes of -0.56 degrees K/mg of protein (5 to 22 degrees C) and -2.78 degrees K/mg of protein (22 to 53 degrees C). The break in the Arrhenius plot corresponds to a temperature known to be related to a sudden change in mycoplasma membrane fluidity. Estimations of Scatchard binding constants and number of binding sites per membrane protein molecule resulted in 0.8 to 1.4 sites per molecule in the intact organisms compared to 1.4 to 1.8 sites in membrane fragments. The latter were purified by repeated washings in phosphate-buffered saline after alkaline lysis of the cells. It is suggested that ATM reacts with available protein sulfhydryl groups in the mycoplasmal membrane.