Sylke Kaltenhäuser

Association of PTPN22 1858 single-nucleotide polymorphism with rheumatoid arthritis in a German cohort: higher frequency of the risk allele in male compared to female patients

The functional single-nucleotide polymorphism (SNP) of the gene PTPN22 is a susceptibility locus for rheumatoid arthritis (RA). The study presented here describes the association of the PTPN22 1858T allele with RA in a German patient cohort; 390 patients with RA and 349 controls were enrolled in the study. For 123 patients, clinical and radiographic documentation over 6 years was available from the onset of disease. Genotyping of the PTPN22 1858 SNP was performed using an restriction fragment length polymorphism PCR-based genotyping assay. The odds ratio to develop RA was 2.57 for carriers of the PTPN22 1858T allele (95% confidence interval (CI) 1.85–3.58, p < 0.001), and 5.58 for homozygotes (95% CI 1.85–16.79). The PTPN22 1858T allele was significantly associated not only with rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) positive RA, but also with RF and anti-CCP negative disease. The frequency of the PTPN22 1858T allele was increased disproportionately in male patients (53.8% compared to 33.0% in female patients, p < 0.001), and the resulting odds ratio for male carriers was increased to 4.47 (95% CI 2.5–8.0, p < 0.001). Moreover, within the male patient population, the rare allele was significantly associated with the HLA-DRB1 shared epitope (p = 0.01). No significant differences in disease activity or Larsen scores were detected. The results provide further evidence that the PTPN22 1858T allele is associated with RA irrespective of autoantibody production. The increased frequency of the risk allele in male patients and its association with the shared epitope indicate that the genetic contribution to disease pathogenesis might be more prominent in men.

Interaction between Transmembrane TNF and TNFR1/2 Mediates the Activation of Monocytes by Contact with T Cells

Monocytes and monocytic cells produce proinflammatory cytokines upon direct cell contact with activated T cells. In the autoimmune disease rheumatoid arthritis, the pivotal role of TNF-α implies that the interaction between transmembrane TNF-α (mTNF) and the TNF receptors (TNFR1 and TNFR2) might participate in the T cell contact-dependent activation of monocytes. Accordingly, treatment of rheumatoid arthritis by administration of a TNF-α-blocking Ab was found to significantly decrease TNF-α production by monocytes. Several lines of evidence indicated that signaling through TNFR1/2 and through mTNF (reverse signaling) is involved in TNF-α production by monocytes after T cell contact: 1) blocking mTNF on activated T cells leads to a significant reduction in TNF-α production; 2) down-regulation of TNFR1/2 on monocytes by transfection with small interfering RNA results in diminished TNF-α production; 3) blocking or down-regulating TNFR2 on activated T cells inhibits TNF-α production, indicating that mTNF on the monocyte surface mediates signaling; 4) ligation of mTNF on monocytes by surface TNFR2 transfected into resting T cells induces TNF-α production due to reverse signaling by mTNF; and 5) ligation of mTNF on monocytes by a soluble TNFR2:Ig receptor construct induces TNF-α production due to reverse signaling. In conclusion, we identified mTNF and TNFR1/2 as interaction partners contributing to TNF-α production in monocytes. Both pathways initiated by mTNF-TNFR interaction are likely to be inhibited by treatment with anti-TNF-α Abs.

Clonally expanded CD4+CD28null T cells in rheumatoid arthritis use distinct combinations of T cell receptor BV and BJ elements

Clonally expanded, autoreactive CD4+CD28null cells can be found in the peripheral blood of patients with rheumatoid arthritis and have been shown to be associated with severeextra‐articular disease manifestations. We investigated the size of the CD4+CD28null compartment and the TCR β chain repertoire of expanded CD4+ clonotypes in 94 rheumatoid arthritis patients by complementarity‐determining region 3 (CDR3) length analysis (spectratyping) in the BV6 and BV14 TCR families, with primers specific for three arbitrarily chosen β chain joining elements (BJ1S2, BJ2S3 and BJ2S7). The spectratyping results showed a strong correlation of the size of the CD4+CD28null compartment with the detected number of BV14 clonotypes, whereas no association with BV6 oligoclonality was found. Only clones using the BV14–BJ1S2 and BV14–BJ2S3 combinations contributed to this correlation, however, whereas BV14–BJ2S7 clones did not. This preferential correlation implies a role for the TCR β chain in stimulating clonal outgrowth and argues against the previously suggested superantigenic stimulation of in‐vivo‐expanded clones. Instead, since no evidence for shared antigen specificity could be detected, clonal expansion of T cells in rheumatoid arthritis might be influenced by the BJ elements because of changes in the flexibility of the protein backbone of the β‐chain.

Ex Vivo Homeostatic Proliferation of CD4+ T Cells in Rheumatoid Arthritis Is Dysregulated and Driven by Membrane-Anchored TNFα

The systemic CD4+ T cell compartment in patients with rheumatoid arthritis (RA) is characterized by TCR repertoire contraction, shortened telomere lengths, and decreased numbers of recent thymic emigrants, suggesting a disturbed CD4+ T cell homeostasis. In mice, homeostatic proliferation of peripheral CD4+ T cells is regulated by TCR interaction with self peptide-MHC complexes (pMHC) and can be reproduced in vitro. We have established an ex vivo model of homeostatic proliferation, in which self-replication of human CD4+ T cells is induced by cell-cell contact with autologous monocytes. In healthy individuals, blockade of TCR-pMHC class II contact resulted in decreased CD4+ T cell division. In contrast, homeostatic proliferation in RA patients was not inhibited by pMHC blockade, but increased during the initial culture period. The anti-TNF-α Ab cA2 inhibited homeostasis-driven ex vivo proliferation in healthy controls and in RA patients. In addition, treatment of RA patients with infliximab decreased the ex vivo rate of homeostatic proliferation of CD4+ T cells. Our results suggest a disturbed regulation of CD4+ T cell homeostasis leading to the repertoire aberrations reported in RA. Membrane-anchored TNF-α appears to be a cell-cell contact-dependent stimulus of homeostatic proliferation of CD4+ T cells, possibly favoring self-replication of autoreactive CD4+ T cells in patients with RA.

The contact-mediated response of peripheral-blood monocytes to preactivated T cells is suppressed by serum factors in rheumatoid arthritis

Stimulation of monocytes/macrophages after cell contact with preactivated T cells has been suggested to contribute to the excessive TNF-α production in rheumatoid arthritis (RA). In this study, T cell-contact-dependent TNF-α production by peripheral-blood monocytes in vitro was investigated and found to be significantly lower in treated and untreated patients with RA than in healthy controls. This suppression was not due to a general deficiency of monocytes to respond, because responses to lipopolysaccharide were comparable in patients and controls. In agreement with the pivotal role of TNF-α in RA, T cell-dependent induction of TNF-α in synovial macrophages was fivefold to tenfold higher than in peripheral-blood monocytes from either patients or controls. The decreased response of peripheral-blood monocytes from patients with RA was found to be mediated by inhibitory serum factors, because the addition of patient sera to monocytes from healthy controls suppressed TNF-α response in the co-culture assay. Preincubation of monocytes from healthy controls with RA serum was sufficient to suppress the subsequent TNF-α response in T cell co-cultures, indicating that inhibitory factors do indeed bind to monocyte surfaces, which might represent a regulatory counter-action of the immune system to the long-standing and consuming autoimmune process in RA. There are some indications that apolipoprotein A-1 might be part of this regulatory system.

Differences in B cell regulation in DRB1 shared epitope positive and negative rheumatoid arthritis

Aim of the study was the analysis of systemic B cell activity and of the size of the B lymphocyte compartment in patients with rheumatoid arthritis (RA)