The rheumatoid arthritis susceptibility polymorphism PTPN22 C1858T is not associated with leflunomide response or toxicity.

WHAT IS KNOWN AND OBJECTIVE: A common polymorphism (C1858T) in the gene that encodes the protein tyrosine phosphatase non-receptor type 22 (PTPN22) is associated with altered T-cell responses and increased susceptibility to rheumatoid arthritis (RA) and other autoimmune diseases. Teriflunomide, the active metabolite of leflunomide, reduces T-cell responses through inhibition of tyrosine kinase p56LCK. We examined a potential association between PTPN22 genotype and response or toxicity to leflunomide in Caucasian RA patients taking leflunomide in combination with other disease-modifying antirheumatic drugs (DMARDs). METHODS: Patients enrolled in the Royal Adelaide Hospital RA inception cohort and taking leflunomide were eligible for inclusion. Participants were followed for 12 months after leflunomide initiation or until either another DMARD was added or leflunomide was ceased. Clinical response according to change in 28-joint Disease Activity Score (DAS28) and cessation due to toxicity were assessed. RESULTS AND DISCUSSION: A total of 94 participants were included in the study, 75 of whom carried the CC genotype, 18 the CT, whereas one individual carried the TT genotype. Over the first 12 months of leflunomide treatment, there was no statistically significant relationship between carrying the T allele and change in DAS28 (-0·84 vs. -1·15, P = 0·446) nor with cessation of leflunomide treatment due to side effects (P = 0·433). These results indicate that PTPN22 C1858T genotype has no effect on response or toxicity outcomes in leflunomide-treated RA patients. WHAT IS NEW AND CONCLUSION: This is the first study to evaluate the biologically plausible hypothesis that PTPN22 genotype might be a predictor of response/toxicity to leflunomide therapy. Despite this, PTPN22 genotype was not associated with leflunomide response or toxicity in patients with RA.

Characterization of thoracic duct cells that transfer polyarthritis.

Polyarthritis may result from the haematogenous distribution of arthritogenic effector lymphocytes that emerge in the efferent lymph and pass through the thoracic duct (TD) to the circulation. We therefore examined whether TD cells collected from rats in the late prodrome of adjuvant-induced arthritis (AA) could transfer polyarthritis adoptively and whether these cells included a subpopulation of arthritogenic cells that could be identified phenotypically. Unfractionated TD cells collected from donor rats 9 days after adjuvant inoculation were injected intravenously into normal syngeneic recipients in numbers equivalent to the overnight harvest from a single donor. TD cell subpopulations, equivalent in number to proportions in the same inoculum, were prepared by negative selection. Unfractionated TD cells transferred polyarthritis without in vitro stimulation or conditioning of recipient animals. Abrogation of arthritogenicity by depletion of alpha/beta TCR(+) cells showed that the polyarthritis was transferred by T cells. Negatively selected CD4(+) but not CD8(+) TD cells transferred AA. An arthritogenic subpopulation of CD4(+) T cells, enriched by either negative or positive selection, expressed the activation markers CD25 (IL-2 receptor alpha), CD71 (transferrin receptor), CD134 (OX40 antigen) and MHC class II. Cells expressing these markers were more numerous in TD lymph from arthritic rats than in lymph from normal rats and they included the majority of large CD4(+) T cells. Thus, arthritogenic effector T cells bearing activation markers are released into the central efferent lymph in the late prodrome of AA. Recruitment of these arthritogenic cells to synovium probably determines the polyarticular pattern of AA.

Recruitment of mononuclear leucocytes to osteoarthritic human synovial xenografts in the ears of SCID mice.

A system has been established to assess the recruitment of 99mTc-hexamethylpropylene amine oxamine (99mTc-HMPAO)-labelled PBMC and [125I]iododeoxyuridine-labelled Con A stimulated lymphoblasts to allogeneic human synovial xenografts in the ears of SCID mice. Successful engraftment of osteoarthritic synovium was achieved in approximately 90% of cases and a connection between the human microvasculature of the xenograft and the circulation of the mouse was shown. Cells were delivered to the xenograft by a system of regional vascular perfusion, thus avoiding the major murine vascular beds. The accumulation of 99mTc-HMPAO-labelled PBMC in mouse ears was monitored in real time. Direct injection of xenograft-bearing ears with recombinant human TNF-alpha, 7 h prior to perfusion, increased the accumulation of both PBMC and lymphoblasts in cytokine-injected ears compared to contralateral control-injected ears. Autoradiography revealed the presence of [125I]iododeoxyuridine-labelled lymphoblasts associated with human microvasculature within the xenograft. However, the increased accumulation of lymphoblasts in cytokine-injected ears occurred in the tissues surrounding the xenograft, where lymphoblasts were associated more often with murine than human vessels. Although the system described offers advantages over similar models, the propensity for mouse endothelium to interact with human leucocytes is likely to be a generic disadvantage for models of human leucocyte recruitment to xenografts in immunodeficient mice.

Recruitment of lymphoblasts derived from peripheral and intestinal lymph to synovium and other tissues in normal rats and rats with adjuvant arthritis.

Recruitment of [125I]iododeoxyuridine-labeled syngeneic lymphoblasts from thoracic duct (TD) lymph into periarticular tissues has been examined after intravenous administration to normal rats and to rats with adjuvant-induced arthritis. Uptake of label was observed in the inflamed paws of arthritic rats and cells were located in synovium and periarticular bone marrow by autoradiography. Uptake was greater with lymphoblasts from donors in the late prodromal phase of adjuvant-induced arthritis (arthritic donors) than from normal donors. With arthritic donors, recruitment of lymphoblasts from TD lymph was greater than from mesenteric duct lymph, suggesting that most of the joint-seeking lymphoblasts in arthritic rats arose in peripheral lymphoid tissues. Lymphoblasts from arthritic donors were also detected in the synovium of paws from normal rats. Recovery of lymphoblasts was monitored in other tissues; this revealed, in arthritic recipients, competition among extra-articular sites of inflammation (adjuvant injection site, draining lymph nodes, and lymph nodes draining affected joints), the lungs, and the inflamed synovium for recruitment of lymphoblasts from arthritic donors. In contrast, while some lymphoblasts from normal donors were recruited to inflamed joints, the small intestine was the main site of recruitment. The results reflect the known propensity of T lymphoblasts generated in peripheral lymphoid tissues to enter inflamed tissues. However, some mesenteric duct lymphoblasts also entered inflamed synovium. The observed pattern of recruitment of lymphoblasts to synovium is pertinent to the pathogenesis of arthritis, the potential roles of arthritogenic and "bystander" lymphocytes and the known links between the joints and inflammation in the intestine.

Distribution of class II major histocompatibility antigens in enterocytes of the rat jejunum and their association with organelles of the endocytic pathway.

The subcellular distribution of class II major histocompatibility molecules has been investigated in enterocytes in the rat jejunum. Studies have been carried out at both light microscope and ultrastructural levels using an indirect immunoperoxidase technique and monoclonal antisera against rat class II molecules. The results show that the class II molecules have also been demonstrated on the baso-lateral cell membranes of enterocytes, but are absent from the brush borders. The distribution of class II molecules has also been studied during enterocyte maturation. The results suggest that enterocytes might be capable of intracellular processing of antigens and suggest that the encounter between class II molecules and processed antigens within the endocytic pathways of cells may be a general phenomenon in both 'professional' and 'non-professional' antigen-presenting cells.

Subcellular distribution of class II major histocompatibility antigens in enterocytes of the human and rat small intestine.

Class II antigens encoded within the major histocompatibility complex (MHC) have been examined at the ultrastructural level in enterocytes from both man and rat. A protocol has been developed for fixation which, in conjunction with a pre-embedding indirect immunoperoxidase technique preserves antigenicity of the class II molecules and allows detection of intracellular antigen. Details of the technique are provided and discussed in relation to the general paucity of information available on ultrastructural localization of class II MHC molecules. Class II MHC antigens have been identified on the basolateral cell membranes of enterocytes in both species and they have also been found in association with intracellular organelles that have the appearance of multivesicular bodies and secondary lysosomes. These observations link class II molecules with the endocytic pathway in enterocytes and suggest a possible role in the handling of gut antigens. The findings may have a more general significance in relation to the site of engagement between processed antigen and MHC molecules in specialized antigen-presenting cells.

Specificity of a mouse monoclonal antibody raised against acute myeloid leukaemia cells for mast cells in human mucosal and connective tissues.

A mouse monoclonal antibody raised against acute myeloid leukaemia cells (YB5.B8 monoclonal antibody; Gadd, S. J. and Ashman, L. K. (1985): Leukaemia Res. 9, 1329-1336) has been found by an indirect immunoperoxidase technique to bind to scattered cells in frozen sections from a number of human tissues. They have been identified as mast cells in fixed sections of skin, tonsil and duodenum by simultaneous staining of glycosaminoglycan with Alcian blue in 0.7 N HCl. The antibody does not distinguish mast cells in mucosal tissues from those in connective tissue, although the level of expression by cells at both sites appears to be heterogeneous. With the exception of low affinity binding to B lymphocytes, no other bone marrow-derived cells were found to bind the antibody. In particular, basophils and eosinophils were not stained, suggesting that they are not related closely to mast cells and that the antigen detected by YB5.B8 monoclonal antibody is not an IgE Fc receptor. Therefore, among all mature haemopoietic lineages, the antibody is specific for mast cells.