Autoantibodies to HLA B27 in the sera of HLA B27 patients with ankylosing spondylitis and Reiter's syndrome. Molecular mimicry with Klebsiella pneumoniae as potential mechanism of autoimmune disease.

Ankylosing spondylitis (AS) and Reiter's syndrome (RS) both show a strong correlation with the HLA B27 haplotype. We studied whether sharing of homologous amino acid sequences in the HLA B27 antigen with an invading microbe might occur, and if so, what is the biological significance of such homology. In a computer search of the Dayhoff data bank, we found a homology of six consecutive amino acids between HLA B27.1 antigen residues 72-77 and Klebsiella pneumoniae nitrogenase residues 188-193. These shared sequences are hydrophilic, suggesting locations on molecules exposed to the cell surface. Immunochemical analysis showed that 18 of 34 sera from patients with RS (53%) and 7 of 24 sera from patients with AS (29%) contained antibodies that bound to a synthesized peptide sequence representing residues 69-84 of HLA B27.1. In contrast, only 1 of 22 sera from healthy, B27+ controls (5%) contained antibodies to this peptide (p less than 0.01). Sera from three HLA B27- patients with RS did not possess antibodies to the HLA B27 peptide. Additionally, greater than 40% of HLA B27 patients with AS or RS had antibodies to Klebsiella residues 184-193, while none of the normal nonarthritic HLA B27 haplotype subjects did. Our results suggest that an autoimmune response(s) directed against HLA B27.1 may be a pathogenic mechanism in a subset of patients with AS and RS. Further, this response may initially be induced against Klebsiella pneumoniae, a microorganism that shares sequence homology with HLA B27.

Ia determinants on stimulated human T lymphocytes. Occurrence on mitogen- and antigen-activated T cells.

Human T-cell blasts were generated by stimulation with mitogens and antigens. A proportion of these blasts expressed Ia antigens detectable by immunofluorescence with both allo- and hetero-antiserums. The maximal expression of Ia antigens was delayed and usually occurred after the peak of blastogenesis. Among the three mitogens used, pokeweed mitogen (PWM) was most effective in giving a high percentage and intense Ia staining of T-cell blasts. Phytohemagglutinin and concanavalin A blasts gave weaker and lower percentages of Ia staining. Activation by alloantigens and soluble antigens such as tetanus toxoid and purified protein derivative resulted in Ia expression on T cells comparable to PWM stimulation. Depletion of Ia+ cells from freshly isolated T cells with anti-Ia and complement decreased subsequent Ia expression, suggesting that a proportion of Ia+ blasts were derived from Ia-bearing peripheral blood T cells. When the specificities of the Ia antigens on T-cell blasts were examined with alloantiserums, it was evident that the T blasts expressed similar HLA-DR determinants to those on B cells from the same donor; occasional minor differences between stimulated T cells and autologous B-cell lines or fresh B cells were encountered.

Two types of Ia-positive T cells. Synthesis and exchange of Ia antigens.

Two distinct types of Ia-positive T cells have been described. One type represents a blastoid T cell responding from stimulation by mitogens, antigens, and in allogeneic and autologous mixed lymphocyte culture reactions. This is a large cell that is strongly positive for Ia antigens as measured by a variety of different antisera. The other general type is a smaller cell with a lower expression of Ia antigens that is found at low levels in normal peripheral blood and is markedly elevated in various pathological states. It also rises rapidly after inoculation with tetanus toxoid and PPD in sensitized individuals. This cell does not incorporate thymidine and is enriched in the Tgamma fraction; it can be markedly concentrated from normal lymphocytes, and current evidence indicates that it is a T cell. The marked elevation of this cell in the blood of patients with rheumatoid arthritis is of special interest. Considerable evidence indicates that, at least in certain instances, the Ia antigens are synthetized by the cells that carry them. Incorporation of labeled amino acid experiments and the in vitro translation results presented above indicate this. However, the ready exchange of Ia antigens between cells in the experiments described indicates that uptake from other cells may be a significant source.

Peripheral blood Ia-positive T cells. Increases in certain diseases and after immunization.

The Ia antigens, usually expressed primarily on B lymphocytes, are found on a small percentage of normal peripheral blood T cells (average 2.6% by fluorescence and 10.8% by rosette assay). Elevated levels up to 40% by both assays were observed in a high proportion of patients with rheumatoid arthritis. Increases also were found in patients with systemic lupus erythematosus and various types of infections. The increases were evident with a specific heteroantiserum, a hybridoma reagent, and DR specific alloantisera. Normal levels were present in multiple sclerosis and an assortment of metabolic and other disorders. A rise in similarly positive T cells occurred in normal individuals after immunization with tetanus toxoid or PPD. The cells primarily involved in all of these instances were small lymphocytes, which stained relatively weakly with the fluorescent reagents and were readily distinguishable from T-cell blasts. They were found to be enriched in isolated T gamma fractions but were also found in other T cells. The accumulated evidence indicated that these cells represent an expansion of one or more subsets of T cells found in normal individuals, and that their level in the peripheral blood may serve as an index of immunological stimulation.

Glucocorticoids administered in vivo inhibit human suppressor T lymphocyte function and diminish B lymphocyte responsiveness in in vitro immunoglobulin synthesis.

The effects of corticosteroid given in vivo on human lymphocyte subpopulation function were investigated using an in vitro system of pokeweek mitogen-stimulated immunoglobulin production. Peripheral blood lymphocytes were obtained from normal volunteers before and 4 h after the intravenous administration of methylprednisolone. Unfractioned peripheral blood lymphocytes showed a consistent decrease (mean congruent with 50%) in immunoglobulin and total protein synthesis after steroid administration. Utilizing separated thymus-derived (T) and bone marrow-derived (B) lymphocyte fractions, the pathophysiology of this alteration in immunoglobulin production was elucidated. B lymphocytes obtained after steroid treatment showed a markedly diminished immunoglobulin response (20% of normal) to normal T lymphocytes and to normal T cells that had been irradiated to remove suppressor T lymphocyte function. All major classes of immunoglobulin (IgG, IgM, and IgA) were affected. T lymphocytes procured after steroid administration were capable of providing normal amounts of T cell help for B cells in immunoglobulin production. However, suppressor T lymphocyte activity, observed with normal T lymphocytes at high T to B cell ratios, was absent from the post-steroid T lymphocytes. This loss of suppressor T lymphocyte function was not due to the presence of excess help as irradiated pre- and poststeroid T cells provided equal amounts of helper activity. On recombining the poststeroid treatment B cells, which are hyporesponsive in immunoglobulin synthesis, with the posttreatment T lymphocytes, which lack suppressor activity, diminished amounts of immunoglobulin were produced which correlate well with the effects observed with unseparated cells. Thus, corticosteroids have differential effects on the lymphocyte populations involved in immunoglobulin biosynthesis. B cell responsiveness is diminished, suppressor T lymphocyte activity is removed, and helper T lymphocyte function is unaffected.

Human lymphocyte subpopulations. Effect of corticosteroids.

Normal subjects given 60 mg of prednisone orally at 8:00 a.m. developed a transient lymphopenia at 2:00 p.m. To define the populations of lymphocytes affected the number and type of lymphocytes in the peripheral blood were assayed. "Late" and "early" spontaneous sheep red blood cell rosettes were used as markers for thymus-derived (T) lymphocytes and one of its subpopulations, respectively. Receptors for aggregated gammaglobulin and complement identified bursal-equivalent or bone marrow-derived (B) lymphocytes and one of its subpopulations, respectively. 6 h after administration of 60 mg of prednisone, the blood samples showed a decrease in proportion of T cells from 69.2 +/- 2.1% to 55.9 +/- 2.8% (average +/- SE) and an increase in B-cell proportion from 21.3 +/- 2.0% to 44.8 +/- 4.1%. The changes of "early" rosettes and complement receptor lymphocytes also paralleled these. In all cases the absolute numbers of T cells and of B cells were decreased by prednisone. The density gradient distribution of the lymphocytes did not change after prednisone. These data indicate that both T and B lymphocytes are affected by the prednisone but that the T cell lymphopenia was more pronounced. The lymphopenia might reflect either sequestration in the marrow and/or transient arrest of recirculation.

Expression of arthritis-causing HLA-B27 on Hela cells promotes induction of c-fos in response to in vitro invasion by Salmonella typhimurium.

HLA-B27 confers a very strong genetic predisposition to development of a reactive arthritis after infection by bacteria such as Salmonella typhimurium. This study examines the role of HLA-B27 in the initiation of the earliest host activities after exposure to Salmonella, namely activation of the immediate early genes in the epithelial cells. Our major finding is that in Hela cells, the expression of c-fos was induced by Salmonella invasion only when the cells expressed the transfected HLA-B27 gene, but not the HLA-A1 gene or a truncated HLA-B27 gene lacking the exons encoding the cytoplasmic domain. C-fos is potentially capable of complexing with members of the c-jun family to become the AP-1 transcription complex. Parallel to c-fos expression, we found that only with the HLA-B27 transfectant was there expression of AP-1. AP-1 potentially controls the expression of a large number of genes. On screening a panel of proinflammatory molecules, we found that Salmonella invasion induced expression of monocyte chemoattractant protein-1 in the HLA-B27 cells. Since each of these separate positive findings belong to the same cascade of events after cell activation, together they reinforce the hypothesis that HLA-B27 plays a modulatory role in the early signal transduction events induced by Salmonella invasion. This hypothesis adds another item to the list of allele-specific activities carried out by HLA class I molecules. If similar activation also occurs in the joints, it may play a major role in arthritis.

The nucleotide and deduced amino acid sequence of the cationic 19 kDa outer membrane protein OmpH of Yersinia pseudotuberculosis.

The OmpH proteins of enteric bacteria are recently described, small (16 kDa), cationic outer membrane proteins. Because a Yersinia pseudotuberculosis cell envelope protein of this size has been found to cross-react serologically with the human histocompatibility antigen HLA-B27 (B*2701), the sequence of Y. pseudotuberculosis OmpH was determined by sequencing the gene region which encodes mature OmpH. A protein consisting of 143 amino acid residues was found. It was 96% homologous with the OmpH of Y. enterocolitica and 62% homologous with that of Escherichia coli. Two separate OmpH regions had sequence similarity with B*2701; they were identical in both Yersinia species.

Human lymphocytes subpopulations: rabbit red blood cell rosettes.

When human lymphocytes were incubated with rabbit red blood cells (RRBC) in vitro at 4 degrees C overnight, they adhered to form rosettes. 44 +/- 4.5% of thymocytes and o.7 +/- o.3% of peripheral blood lymphocytes (PBL) formed these rosettes. The rosette-forming cells (RFC) increased to 10--40% when the PBL were stimulated by mitogens. The increases were detected 4 h after culture and reached a maximum at 24 h, thus preceding significant increases in DNA syntheses. In another set of experiments, PBL were stimulated by a range of concentrations of each of 5 mitogens. The concentrations whick led to high DNA synthesis rates also generated high percentages of the rosettes. Lastly, PBL were activated by mitogens and the effect of inhibitors of DNA synthesis, protein synthesis and ATPase activity investigated. Only the latter could inhibit the generation of the RFC. In conclusion, 1y RRBC rosettes in mitogen stimulated human PBL were markers of activated lymphocytes, 2) the percentages of RFC could constitute a reliable index of mitogenic responses, and 3) this index was independent of the usual criteria of protein and DNA synthesis.

Discovery of potent cyclic GMP phosphodiesterase inhibitors. 2-Pyridyl- and 2-imidazolylquinazolines possessing cyclic GMP phosphodiesterase and thromboxane synthesis inhibitory activities.

Moderate cyclic GMP phosphodiesterase (cGMP-PDE, PDE V) inhibitor 2-phenyl-4-anilino-quinazoline (1) was identified utilizing MultiCASE assisted drug design (MCADD) technology. Modification of compound 1 was conducted at the 2-, 4-, and 6-positions of the quinazoline ring for enhancement of cGMP-PDE inhibitory activity. The 6-substituted 2-(imidazol-1-yl)-quinazolines are 1000 times more potent in in vitro PDE V enzyme than the well-known inhibitor zaprinast. The 6-substituted derivatives of 2-(3-pyridyl)quinazoline 84 and 2-(imidazol-1-yl)quinazoline 86 exhibited more than 1000-fold selectivity for PDE V over the other four PDE isozymes. In addition, cGMP-PDE inhibitors 64, 65, and 73 were found to have an additional property of thromboxane synthesis inhibitory activity.

A ribosomal protein of Yersinia pseudotuberculosis having partial epitope identity with HLA-B27.

The phenotype HLA-B27 is common in patients who develop reactive arthritis after having an infection. One hypothesis concerning the pathogenesis of reactive arthritis is that molecular mimicry between HLA-B27 and certain bacterial components might be involved. It is known that an infection with Yersinia is commonly associated with reactive arthritis in B27 positive patients. Therefore, we were interested to investigate whether cross-reactivity between Yersinia and HLA-B27 exists. A gene library of Yersinia pseudotuberculosis was created in the plasmid vector pUC13. One of the resulting clones contained a gene encoding an intracytoplasmic protein that seems to have partial epitope identity with HLA-B27. It reacted in western blot. ELISA and immunoprecipitation with three different HLA-B27 specific monoclonal and polyclonal antibodies of the IgG and IgM class. However DNA-sequencing of the cloned Yersinia gene and the predicted amino acid sequence revealed only a very remote similarity with HLA-B27 in the primary structure. Instead, an extremely high degree of similarity with the ribosomal protein L4 of the S10 operon of Escherichia coli was identified indicating that the protein encoded by the cloned Y. pseudotuberculosis gene is a corresponding ribosomal protein.

An animal model to study serum immunoglobulin response to infection by Yersinia enterocolitica.

Using an Elisa method, we tested whether the serum immunoglobulin response of CBA/H mice, orally infected by a serotype 03 strain of Yersinia enterocolitica was of sufficient magnitude to serve as an experimental model. We found that the dose of bacteria administered was important. At a dose of 10(10)-10(12) bacteria per mouse, easily detectable IgM, IgG and IgA antibody titers could be obtained. At 10(8) bacteria per mouse, although diarrhea occurred and Yersinia was shed into the stool, the antibody titers were minimal.

Absence of lymphotoxic antibodies in patients with Yersinia-induced arthritis, Reiter's syndrome and ankylosing spondylitis.

Serum samples from patients with Reiter's syndrome, ankylosing spondylitis, Yersinia-induced arthritis and Yersinia infections not followed by arthritis were collected and assayed for complement-dependent cytotoxicity, using a standard dye-exclusion microcytotoxicity technique. A total of eighty-nine serum samples derived from these patients were compared to those of forty control subjects, using as target cells six different cultured cell line cells and peripheral blood mononuclear cells isolated from the blood samples of four subjects. Serum samples from only five of the subjects demonstrated cytotoxicity. This did not depend on whether the target cells were HLA-B27 or not. Hence, if antilymphocytic auto-antibodies are present in these patients, they will require a different technique for their detection.

A preliminary analysis of the membrane proteins of arthritis-causing Yersinia.

The outer membrane proteins (OMP) of fifteen strains of Yersinia were separated from the inner membrane (IMP) and intracytoplasmic (ICP) proteins. The molecular composition of these fractions was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The OMP of all fifteen strains exhibited a uniform pattern markedly distinct from the IMP and ICP. There was, however, heterogeneity in the OMP among the various bacteria, based on the apparent molecular weights of several of the major proteins. The two most prominent major proteins are easily distinguished because their apparent molecular weights varied substantially depending on the temperature at which the membranes were solubilized. Using the apparent molecular weights of these two proteins as a basis for comparison, the Yersinia organisms of serotype 3 presented a unique electrophoretic pattern, different from those of non-serotype 3 Yersinia organisms. Four of the nine serotype 3 organisms analyzed here were isolated from patients who developed arthritis subsequent to infection. However, no obviously unique features could be distinguished. A major focus of research effort is currently being devoted to the examination of the immune response generated against the Yersinia organisms by patients with Yersinia-induced arthritis. Most of the studies, so far, have utilized whole bacteria as the antigens. The data presented in this paper provide the basis of future analysis of such immune responses to individual molecules of Yersinia.

Requirements for HLA-B*2705-binding peptides with special regard to the transporter associated with antigen processing (TAP).

OBJECTIVE: To refine the algorithms governing peptide presentation by HLA-B*2705 by analyzing: (i) the specificity of the human transporter associated with antigen processing (TAP) for HLA-B27 binding peptides; and (ii) the peptide binding affinity to HLA-B*2705. METHODS: TAP-translocation was measured with a labeled reporter peptide containing an N-linked glycosylation acceptor site in Streptolysin O-permeabilized cells for a panel of HLA-B27 binding peptides. Peptide binding affinity was determined by peptide-induced stabilization of empty HLA-B*2705 expressed by the TAP-deficient cell line T2-B27. RESULTS: Human TAP preferentially translocated analogues with residues leucine, isoleucine, methionine and arginine as the carboxy-terminal amino acids, whereas analogues with aspartic acid and serine were translocated poorly. The binding affinity to HLA-B*2705 of the poorly translocated aspartic acid and serine analogues was about 100-fold less compared to the parent HLA-B27 binding peptide. CONCLUSIONS: Human TAP shows considerable specificity for the C-terminus of potential HLA-B27 ligands. Nonamer peptides with aspartic acid and serine at the C-terminus are poorly translocated by the TAP and have low binding affinity for HLA-B*2705, and are therefore unlikely to become presented by HLA-B*2705.

A monoclonal anti-HLA-B27 antibody which is reactive with a linear sequence of the HLA-B27 protein is useful for the study of molecular mimicry.

In the search for cross-reactivity between bacteria and HLA-B27, three groups of investigators have identified several bacterial envelope proteins which are reactive with the monoclonal anti-HLA-B27 antibodies B27.M1 and B27.M2. Since these two antibodies react poorly with HLA-B27-derived synthetic peptides, it is not possible to locate the reactive epitopes on the HLA-B27 using synthetic peptides. Here, we introduce Ye-2, a monoclonal anti-HLA-B27 antibody which, unlike B27.M1 and B27.M2, is reactive with a synthetic peptide derived from residues 63-84 of HLA-B27.1. Analysis with a cross-reactive peptide derived from residues 226-244 of bovine carbonic anhydrase suggests that only a few of the amino acid residues in the HLA-B27-derived peptide are responsible for the reactivity. This antibody should be a useful adjunct in a preliminary assessment of whether a bacterial protein mimics HLA-B27 in primary structure.

The modulation of chlamydial replication by HLA-B27 depends on the cytoplasmic domain of HLA-B27.

OBJECTIVE: The intracellular persistence of viable Chlamydia trachomatis (CT) within the joint is thought to initiate and maintain the inflammatory process in CT-induced arthritis. CT-induced arthritis is associated with HLA-B27. Recently it was shown that HLA-B27, besides being a T-cell restriction element, can directly influence the invasion and/or replication of enterobacteriae and alters salmonella-induced signal transduction. It was the aim of this study to analyze the effect of HLA-B27 on CT-invasion and replication in human host cells. METHODS: Human Hela cells and Hela cells transfected with either HLA-B27 cDNA or controls (HLA-A1 cDNA; HLA-B27 mutant = HLA-B27 without cytoplasmic tail; B27Q10 = HLA-B27 Exon 1-4 linked to Exon 5 of murine Q10) were infected with CT. By direct immunofluorescence chlamydial invasion was determined 4 hours post infection (p.i.), chlamydial replication 2 days and 4 days p.i. The number of infective CT in the different cell lines was determined by titration of the cell lysates on Hep-2 cells with subsequent immunoperoxidase staining. RESULTS: Invasion was not affected by HLA-B27. However, formation of chlamydial inclusion bodies and replication was suppressed by HLA-B27. Genetically engineered mutants of HLA-B27 (HLA-B27 mutant, B27Q10) lacking the cytoplasmic tail of HLA-B27 did not affect replication. CONCLUSION: The reduction of chlamydial replication by HLA-B27 depends on the cytoplasmic domain of HLA-B27, thus providing a new hypothesis for chlamydial persistence in HLA-B27 positive reactive arthritis.

Epitope analysis of an HLA-B27-derived synthetic peptide: a possible approach to analyzing HLA class I antigens.

A monoclonal antibody, F3H7, was generated by immunizing mice with a synthetic peptide corresponding to residues 63-84 of the B*2705 allele of the HLA-B27 antigens. The reactive epitope and the contact residues on the peptide were localized by ELISA using a large panel of overlapping peptides as well as peptides with substituted amino acids. Residues corresponding to R75, D77 and L78 on the HLA-B27 protein appeared to be critical. The clarity of these results indicate that this is a potentially useful approach to the study of HLA class I epitopes.

The genetically-engineered secretory B27/Q10 chimeric molecule inhibits HLA-B27 restricted alloreactive T-lymphocytes.

OBJECTIVES: Intracellularly persisting bacterial infections and high association with HLA-B27 are the hallmarks of reactive arthritis. Soluble HLA-B27 molecules are induced by bacterial infection; however their biological role in arthritis is unknown. It was the aim of this study to generate soluble HLA-B27 molecule and to analyze its effect on cytotoxic HLA-B27 alloreactive CD8+ T-lymphocytes in order to better understand potential functional links between persistent infection and HLA-B27 association. METHODS: Using PCR Exons 1 through 4 of HLA-B*2705 were fused to Exon 5 of the soluble murine MHC class I variant Q10 and stably transfected into Hela-cells. Transfectants were analyzed using specific PCR, RT-PCR and intracellular and extracellular staining with anti-HLA-B27 monoclonal antibody ME1. Secretion of B27Q10 in the supernatant was examined by isoelectric focusing (IEF). The effect of B27Q10 on T-cells was analyzed using either HLA-B27- or HLA-A2-restricted alloreactive T-cells in a standard 51Cr-release assay. RESULTS: PCR and RT-PCR demonstrated the DNA and mRNA of B27Q10 in the transfectants. By intracellular and extracellular staining with ME1 B27Q10-molecule was detected intracellularly but was not expressed in the cell membrane. Using IEF soluble B27Q10-molecules were found in supernatants of transfectants in a concentration of up to 1.342 microg/ml. Soluble B27QJO-molecule inhibited specifically the cytotoxicity of HLA-B27-restricted alloreactive T-cells by about 30%. CONCLUSION: The secretory non-membrane-expressed molecule B27Q10 inhibits HLA-B27 specific T-cells. The inhibition of cytotoxic T-cells by bacteria induced soluble HLA-B27 may thus enable bacterial persistence.

The pathogenesis of HLA-B27 arthritis: role of HLA-B27 in bacterial defense.

The way in which a host accommodates invasive facultative intracellular bacteria must be the key to the development of reactive arthritis. Investigators have analyzed the bacterial events at several levels: invasion into host cells, intracellular survival, translocation from the sites of infection to the joints, residence in the joints, and evasion of host defense. Because HLA-B27 is present in higher incidence in patients with reactive arthritis and is an essential gene in the related ankylosing spondylitis, the role of HLA-B27 in host defense is also assumed to be important in the development of reactive arthritis. This review summarizes the various studies in this field.