The effect of Mycoplasma arthritidis infection on the kinetics of colloidal carbon clearance in mice

Abstract The activity of phagocytes from A/J mice was estimated by the carbon clearance test following injection of Mycoplasma arthritidis . Phagocytic activity was significantly depressed 12 h post-infection ( P =0.001) and returned to normal values at 24 h. For animals examined 2 and 7 days post-infection, the overall phagocytic activity increased significantly ( P <10⁻⁴). Phagocytic activity gradually decreased and returned to that of the control group by the end of the fourth week. The relative weights of liver and spleen were significantly increased from the 2nd day post infection ( P =0.0028 and P =0.0014 respectively) and remained increased until the end of the experiment. The early depressive effect on phagocytic activity may be related to superantigen activity with the production of mediators such as macrophage deactivating factor. The later expansion of the macrophage population might bring about the stimulation of autoreactive clones of T and B cells and be responsible for the chronic arthritis that developed in the mycoplasma treated mice.     

Crucial cytokine interactions in nitric oxide production induced by Mycoplasma arthritidis superantigen

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57Bl/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies.     

The effect of Mycoplasma arthritidis infection on the phagocytic activity of macrophages in rats and mice

The phagocytic activity of mononuclear phagocytes of A/J mice and Wistar rats was estimated by the carbon clearance test following injection of Mycoplasma arthritidis. In mice, the overall phagocytic activity was significantly increased at the end of the first week (P less than 0.0001), but the increase was marginal by the third and fourth weeks after injection. A significant increase in the relative weight of liver and spleen was observed even when phagocytic activity had returned to levels similar to those of controls (P less than 0.001). In rats, the overall phagocytic activity was significantly increased until the fourth week (P less than 0.00001). There was not, however, an increase in the relative weight of liver and spleen as observed for the mice. The results are discussed in the context of factors contributing to the pathogenic mechanisms responsible for differences in the patterns of arthritis due to mycoplasma observed in mice and rats.     

Effect of Mycoplasma arthritidis on mouse and rat lymphoid cells in experimental Mycoplasma infection]

Early stages of mycoplasma infection of mice and rats were accompanied by suppression of the populations of rosette- and plaque-forming cells. Later the character and dynamics of the immune response to M. arthritidis differed in mice and rats. In mice mycoplasma infection was accompanied by stimulation of rosette-forming cells with some suppression of the plaque-forming cells from the 7th to the 36th day of infection. In rats by the 7th day the number of plaque- and rosette-forming cells decreased in comparison with control, and the immune response was restored by the 15h day; at later periods the immune response of the infected rats exceeded the normal level considerably. The cellular and humoral immune reactions proved to depend on the mycoplasma dose.     

The vir gene of bacteriophage MAV1 confers resistance to phage infection on Mycoplasma arthritidis

Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn4001T and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all vir-containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.     

Crystal structure of Mycoplasma arthritidis mitogen complexed with HLA-DR1 reveals a novel superantigen fold and a dimerized superantigen-MHC complex

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular TCR Vbeta elements. Here we report the crystal structure of MAM complexed with a major histocompatibility complex (MHC) antigen, HLA-DR1, loaded with haemagglutinin peptide 306-318 (HA). The structure reveals that MAM has a novel fold composed of two alpha-helical domains. This fold is entirely different from that of the pyrogenic superantigens, consisting of a beta-grasped motif and a beta barrel. In the complex, the N-terminal domain of MAM binds orthogonally to the MHC alpha1 domain and the bound HA peptide, and to a lesser extent to the MHC beta1 domain. Two MAM molecules form an asymmetric dimer and cross-link two MHC antigens to form a plausible, dimerized MAM-MHC complex. These data provide the first crystallographic evidence that superantigens can dimerize MHC molecules. Based on our structure, a model of the TCR2MAM2MHC2 complex is proposed.     

NaturalMycoplasma arthritidis infection in mice

B6C3F₁ mice from a hybrid production colony frequently were serologically positive by enzyme-linked immunosorbent assay (ELISA) and consistently negative by culture forMycoplasma pulmonis. Subsequently, 162 mice were obtained and intensively studied using an expanded group of cultural procedures, ELISA, and histopathology. Lesions attributable to mycoplasma infection were not found, butMycoplasma arthritidis was isolated from 20 mice. TheM. pulmonis ELISA was positive (IgM, IgG, or both) in 113 mice. Selected sera were tested simultaneously in both theM. pulmonis ELISA and in an ELISA usingM. arthritidis antigen, and were found to be positive in both the IgM and IgG classes in both ELISAs. Thus, cross-reacting antibody was produced in mice naturally infected withM. arthritidis, confirming previous observations based on experimental infections. To our knowledge, this is the first report of naturalM. arthritidis infection in laboratory mice.     

Immunological responses of the rat to Mycoplasma arthritidis

Arthritis was produced in rats by the intravenous injection of Mycoplasma arthritidis. Metabolic inhibiting antibody and indirect hemagglutinating antibody could not be detected in the sera of arthritic or convalescent animals. Nonmurine species of mycoplasma were capable of inducing metabolic inhibiting antibody in the rat. A hypothesis based upon the possible occurrence of heterogenetic antigens common to M. arthritidis and rat tissue was brought forward to explain these findings. Complement-fixing antibody to M. arthritidis was detected 3 to 4 days after injection and subsequently rose to high levels, depending upon the severity of arthritis and number of organisms injected. Animals that had recovered from intravenous or subcutaneous inoculation with M. arthritidis were resistant to subsequent infections by the organism. Immunity could be passively transferred by the intravenous injection of convalescent serum. Adsorption of the convalescent serum with antigen greatly reduced the complement fixation titer but did not significantly alter the protective properties of the serum. The presence of complement-fixing antibody could not be related to the development of immunity. An avirulent strain of M. arthritidis and a strain previously classified as M. hominis type 2 were capable of inducing resistance to subsequent injection by virulent M. arthritidis.     

Characterization of the Lysogenic Bacteriophage MAV1 from Mycoplasma arthritidis

The lysogenic bacteriophage MAV1, which is associated with the arthritogenicity of Mycoplasma arthritidis, was characterized. Several strains of M. arthritidis were examined for their ability to support growth of MAV1. A PFU assay was developed, and the sensitivity of phage to various chemical treatments was assayed. The most notable result was the resistance of MAV1 to proteinase K. The MAV1 genome is a double-stranded, linear DNA molecule of about 16 kb. The site of MAV1 DNA integration in the host chromosome was investigated. The ends of MAV1 DNA were cloned from three independent lysogens shown to have MAV1 DNA inserted at different sites in the host. The nucleotide sequences of the ends of the MAV1 genome and of the MAV1 DNA-chromosomal DNA junctions from each of three lysogens were determined. Sequences flanking the integrated prophage and the ends of native MAV1 DNA were determined, allowing the identification of the phage DNA (attP) and bacterial DNA (attB) recombination sites. Analysis of the left MAV1 DNA-chromosomal DNA junction sites showed a single-base heterogeneity located within MAV1 DNA sequences immediately adjacent to the attB sequence. A model for MAV1 integration-excision is proposed.     

Hematologic changes in chronic arthritis of mice induced by Mycoplasma arthritidis.

The arthritis caused by iv injection of M. arthritidis in mice was found to be associated with neutrophilia and lymphopenia without a change in the total WBC concentration. A mild anemia developed which was characterized by hypoferremia and plentiful RE iron but with an increased plasma total iron-binding capacity. This anemia therefore differs from the anemia of chronic disorders and indeed from any anemia which occurs in man.     

Persistence of Mycoplasma arthritidis in the body of mice of different strains

Mice belonging to 3 strains were shown to differ greatly in their sensitivity to M. arthritidis. This organism persisted for a year in (C57BL/6 X A/Sn)F1 mice, for 6 weeks in BALB/c mice and for 1-3 weeks in C57BL/6 mice. The sensitivity of mice to M. arthritidis infection and the persistence of the infective agent in the organs of the animals were found to depend on the state of their cell-mediated immunity.     

Detection of Mycoplasma arthritidis and Mycoplasma fermentans antibodies in rheumatoid arthritis patients by an immunoenzyme method

The optimum parameters of the immunoenzyme assay system for the identification of antibodies to M. arthritidis and M. fermentans in the sera of patients with rheumatoid arthritis have been established. The investigation has shown that the products obtained by the ultrasonic disintegration of the biomass of M. arthritidis and M. fermentans can be used as soluble antigens for adsorption on the polystyrene surface of plates. The use of the immunonenzyme assay, specially modified, has made it possible to establish that antibodies to M. arthritidis can be detected in 6.5% of cases, antibodies to M. fermentans, in 41.9% of cases and the association of antibodies to M. arthritidis and M. fermentans, in 41.9% of cases. At the same time antibodies to M. arthritidis have been found to belong mainly to IgM and antibodies to M. fermentans, to IgG or to IgG and IgM simultaneously.     

Mycoplasma arthritidis superantigen (MAM)-induced macrophage nitric oxide release is MHC class II restricted,interferongamma dependent,and toll-like receptor 4 independent

Mycoplasma arthritidis causes arthritis in rodents that resembles human rheumatoid arthritis. It produces a superantigen (MAM) that stimulates production of cytokines by making a bridge between lymphocyte T-cell receptor with the appropriate Vbeta chain, and H-2 1-Ealpha MHC class II molecules. Here we studied MAM-induced nitric oxide (NO) production in mouse peritoneal macrophages and found that it was: (1) time and concentration dependent, (2) possibly derived from inducible NOS synthase since it was reduced significantly by amino guanidine pretreatment, (3) restricted to H-2(K) (C3H/HePas and C3H/HeJ) and H-2(d) strains (BALB/c), (4) independent of TLR4 signaling since the coisogenic strains C3H/HePas and C3H/HeJ (TLR4 deficient) produced similar levels of NO following MAM stimulation, (5) potentiated by lipopolysaccharide, and (6) dependent on the presence of nonadherent peritoneal cells. Neutralization of interferon-gamma (IFNgamma in the peritoneal cell cultures with monoclonal antibodies abolished MAM-induced NO production. Addition of rIFNgamma to the adherent cells substituted the nonadherent cells for MAM-induced NO production. A macrophage cell line, J774A.1 (H-2(d)), also produced NO upon MAM stimulation but only when BALB/c spleen lymphocytes were added. Thus, in murine macrophages, MAM induces NO production that is dependent on signaling through MHC class II molecules and IFNgamma but independent of TLR4 expression.     

Identification of an isoschizomer of the HhaI DNA methyltransferase in Mycoplasma arthritidis

The genome of Mycoplasma arthritidis strain 158 has modified cytosine residues at AGCT sequences that render the DNA resistant to digestion with the AluI restriction endonuclease. The DNA methyltransferase responsible for the base modification has previously been designated MarI. From the complete genome sequence of M. arthritidis , we identify Marth_orf138 as a candidate marI gene. Marth_orf138 was cloned in Escherichia coli and its TGA codons converted to TGG. DNA isolated from E. coli cells expressing the modified Marth_orf138 gene was degraded by the AluI nuclease, indicating that Marth_orf138 does not code for MarI. However, the DNA from E. coli was found to have acquired resistance to the restriction endonuclease HhaI. Genomic DNA from M. arthritidis was also found to be resistant to HhaI (recognizes GCGC). The M. arthritidis isoschizomer of the HhaI DNA methyltransferase, coded by Marth_orf138, is designated MarII. Transformation of M. arthritidis was not significantly affected by modification of plasmid at HhaI sites, indicating that the mycoplasma lacks a restriction endonuclease that recognizes GCGC sites.     

Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis

Previous attempts to introduce transposon Tn4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn4001C and Tn4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn4001. Both Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.