Cornelis L. Verweij

Is there a future for TNF promoter polymorphisms

The in vitro study of TNF promoter polymorphism (SNP) function was stimulated by the numerous case–control (association) studies of the polymorphisms in relation to human disease and the appearance of several studies claiming to show a functional role for these SNPs provided a further impetus to researchers interested in the role of TNF in their disease of interest. In this review we consider case–control studies, concentrating on the autoimmune and inflammatory diseases rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, and asthma, and on infectious diseases including malaria, hepatitis B and C infection, leprosy and sepsis/septic shock. We also review the available evidence on the functional role of the various TNF promoter polymorphisms. In general, case–control studies have produced mixed results, with little consensus in most cases on whether any TNF polymorphisms are actually associated with disease, although results have been more consistent in the case of infectious diseases, particularly malaria. Functional studies have also produced mixed results but recent work suggests that the much studied −308G/A polymorphism is not functional, while the function of other TNF polymorphisms remains controversial. Studies of the TNF region are increasingly using extended haplotypes that can better capture the variation of the MHC region.

TNF-308A and HLA–DR3 alleles contribute independently to susceptibility to systemic lupus erythematosus

OBJECTIVEnTo evaluate the respective contributions of tumor necrosis factor (TNF) promoter polymorphisms and HLA-DR alleles to susceptibility to systemic lupus erythematosus (SLE).nnnMETHODSnTNF-238G/A and 308G/A promoter polymorphisms and HLA-DRB1 alleles were determined in 99 consecutive Caucasian SLE patients and 177 Caucasian controls. Standard and Mantel-Haenszel odds ratios were calculated to assess the magnitude of the susceptibility factors. The presence or absence of the SLE classification criteria was determined and correlated with the TNF promoter and HLA-DRB1 genotypes.nnnRESULTSnThe frequency of the TNF-308A/A and 308G/A genotypes was significantly higher in SLE patients (odds ratio 5.0). Conversely, TNF-238G/A and 238A/A genotypes were equally prevalent in SLE patients and controls. The HLA-DR3 specificity (DRBI*0301 allele) was significantly more prevalent in the SLE population (odds ratio 4.4). Stratification to correct for interdependence of the 2 loci confirmed the association of both TNF-308A and HLA-DR3 with SLE (Mantel-Haenszel odds ratio 3.2 and 2.4, respectively). No correlation was found between TNF promoter and HLA-DRB1 genotypes and any SLE classification criterion or disease manifestation.nnnCONCLUSIONnTNF-308A and HLA-DR3 alleles are independent susceptibility factors for SLE.

Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis.

OBJECTIVEnTo understand the regulation of anti-citrulline-containing peptide antibody (anti-CCP) production in rheumatoid arthritis (RA), production of anti-CCP by B cells derived from peripheral blood (PB), bone marrow (BM), and synovial fluid (SF) was examined.nnnMETHODSnPurified PB and SF B cells were isolated by negative selection and then cultured in the absence or presence of L-CD40 ligand cells and interleukin-10 or anti-CD3-activated T cells. Total IgM and IgM-anti-CCP were detected after 14 days of culture by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to analyze the frequency of cells that spontaneously produced IgM-anti-CCP in BM and SF B cells.nnnRESULTSnIgM-anti-CCP autoantibodies were induced in PB B cells from healthy controls and RA patients following coculture with activated T cells or application of the CD40 activation system, whereas no production could be detected when PB B cells were cultured in the absence of a stimulus. SF and BM B cells from anti-CCP-seropositive RA patients, but not anti-CCP-seronegative patients, actively produced IgM-anti-CCP without stimulation. The frequency of spontaneous production of IgM-anti-CCP among the IgM-secreting cells ranged from 2.2% to 25%.nnnCONCLUSIONnThese results indicate the presence of B cell precursors for anti-CCP autoantibodies that are able to produce antibodies upon stimulation in the PB B cell repertoire of healthy controls and patients with RA. In contrast, B cells that actively secreted anti-CCP were specifically present in the BM and SF compartment of anti-CCP-seropositive RA patients. The local presence of anti-CCP-secreting cells in the inflamed joints provides evidence for an antigen-driven maturation of CCP-specific B cells at the site of inflammation in RA.

Displacement of Linker for Activation of T Cells from the Plasma Membrane Due to Redox Balance Alterations Results in Hyporesponsiveness of Synovial Fluid T Lymphocytes in Rheumatoid Arthritis

The T lymphocytes that reside in the synovium of the inflamed joints in patients with rheumatoid arthritis display severe hyporesponsiveness upon antigenic stimulation, which is probably due to their constant subjection to high levels of oxidative stress. Here we report that the synovial fluid T lymphocytes exert severely impaired phosphorylation of the adaptor protein linker for activation of T cells (LAT), a crucial component of the TCR-mediated signaling pathways. In healthy T lymphocytes, LAT is a membrane-bound protein and becomes phosphorylated by ζ-associated protein of 70 kDa (ZAP-70) upon TCR engagement. The molecular basis underlying the deficient phosphorylation of LAT and consequently the hyporesponsiveness of the synovial fluid T lymphocytes lies in the membrane displacement of LAT. We demonstrate that the subcellular localization of LAT is sensitive to changes in the intracellular levels of the antioxidant glutathione. The membrane anchorage of LAT, and consequently the phosphorylation of LAT and the cellular activation of the synovial fluid T lymphocytes upon TCR engagement, is restored in synovial fluid T lymphocytes after supplementation of the intracellular glutathione levels with N-acetyl-l-cysteine. These data suggest a role for the membrane displacement of LAT in the hyporesponsiveness of the synovial fluid T lymphocytes as a consequence of oxidative stress.

Allele-specific quantification of tumor necrosis factor alpha (TNF) transcription and the role of promoter polymorphisms in rheumatoid arthritis patients and healthy individuals.

Interindividual variation in the expression of tumor necrosis factor α (TNF) suggests the existence of functionally distinct TNF alleles that could play a role in susceptibility to TNF associated diseases such as rheumatoid arthritis (RA). To determine whether differential expression of TNF alleles exists, the relative contribution of TNF alleles in total TNF RNA production in peripheral blood mononuclear cells (PBMC) of healthy individuals and synovial tissue of RA patients was analyzed. By using a Taiu2009I restriction fragment length polymorphism (RFLP) located at position +489 in the first intron of the gene, the relative contribution of each allele in precursor transcript production in heterozygous individuals could be measured. By means of this method we studied whether differences exist between TNF alleles in TNF pre-mRNA production. The relative contribution of TNF alleles to the non-spliced RNA pool was measured in PBMC of healthy individuals which were stimulated with LPS, PMA and anti-CD3 and anti-CD28 monoclonal antibodies for different time periods. Moreover, synovial biopsy material of RA patients was analyzed. The results of this study do not reveal a difference in the contribution of distinct TNF alleles in TNF pre-mRNA production upon in vitro and physiological stimulation conditions in healthy individuals and RA patients. Since some of the individuals whose PBMC were tested were also heterozygous for either −308, −1031, −863, −857 TNF promoter/enhancer single nucleotide polymorphisms (SNPs), the data argue against functional relevance of these TNF promoter/enhancer SNPs in the regulation of transcription. In conclusion, the data do not provide evidence for the existence of transcriptionally distinct TNF alleles to explain interindividual variation in TNF expression.

Responsiveness to anti-tumour necrosis factor α therapy is related to pre-treatment tissue inflammation levels in rheumatoid arthritis patients

Objective: The response of rheumatoid arthritis (RA) patients to treatment with neutralising antibodies to tumour necrosis factor α (TNFα) is highly variable. The underlying mechanism for therapy responsiveness is currently unknown. We therefore evaluated the relationship between baseline molecular profiles of synovial tissues from RA patients and the clinical response to treatment with infliximab. Methods: Synovial biopsies were obtained by arthroscopy from 18 RA patients with active disease (28 joint count Disease Activity Score (DAS28)⩾3.2) before initiation of treatment with infliximab. All patients were on stable methotrexate treatment. Clinical response at 16 weeks was defined as a reduction in DAS28 of ⩾1.2, non-response as reduction in DAS28 <1.2. Large-scale gene expression profiling using microarrays was performed on synovial tissue samples. To identify biological processes in synovial biopsies that could discriminate between responders and non-responders, we performed pathway analysis on the expression profiles. Results: A total of 12 patients responded to therapy, while 6 patients failed to fulfil the response criteria. We identified several biological processes, related to inflammation, which were up-regulated in patients who responded to therapy, compared to those who did not show clinical improvement. Conclusion: These results indicate that patients with a high level of tissue inflammation are more likely to benefit from anti-TNFα treatment.

A subtype of multiple sclerosis defined by an activated immune defense program.

Given the heterogeneous nature of multiple sclerosis (MS), we applied DNA microarray technology to determine whether variability is reflected in peripheral blood (PB) cells. In this study, we studied whole-blood gene expression profiles of 29 patients with relapsing-remitting MS (RRMS) and 25 age- and sex-matched healthy controls. We used microarrays with a complexity of 43K cDNAs. The data were analyzed using sophisticated pathway-level analysis in order to provide insight into the deregulated peripheral immune response programs in MS. We found a remarkable elevated expression of a spectrum of genes known to be involved in immune defense in the PB of MS patients compared to healthy individuals. Cluster analysis revealed that the increased expression of these genes was characteristic for approximately half of the patients. In addition, the gene signature in this group of patients was comparable with a virus response program. We conclude that the transcriptional signature of the PB cells reflects the heterogeneity of MS and defines a sub-population of RRMS patients, who exhibit an activated immune defense program that resembles a virus response program, which is supportive for a link between viruses and MS.

Neuropsychiatric systemic lupus erythematosus is associated with imbalance in interleukin 10 promoter haplotypes

OBJECTIVE To investigate the association of interleukin 10 (IL10) promoter polymorphisms and neuropsychiatric manifestations of systemic lupus erythematosus (SLE). METHODS IL10 haplotypes of 11 healthy volunteers were cloned to confirm that in the Dutch population, only the three common haplotypes (-1082/-819/-592) GCC, ACC and ATA exist. The IL10 promoter polymorphisms of 92 SLE patients and 162 healthy controls were determined. The medical records of the SLE patients were screened for the presence of neuropsychiatric involvement. RESULTS All cloned haplotypes were either GCC, ACC or ATA. Forty two SLE patients had suffered from neuropsychiatric manifestations (NP-SLE). In NP-SLE patients, the frequency of the ATA haplotype is 30% versus 18% in the controls and 17% in the non-NP-SLE group (odds ratios 1.9, p=0.02, and 2.1, p=0.04, respectively), whereas the GCC haplotype frequency is lower in the NP-SLE group compared with controls and non-NP-SLE patients (40% versus 55% and 61%, odds ratios 0.6, p=0.02 and 0.4 p=0.006). The odds ratio for the presence of NP-SLE is inversely proportional to the number of GCC haplotypes per genotype when the NP-SLE group is compared with non-NP-SLE patients. CONCLUSIONS The IL10 locus is associated with neuropsychiatric manifestations in SLE. This suggests that IL10 is implicated in the immunopathogenesis of neuropsychiatric manifestations in SLE.

Treatment with monoclonal anti-tumor necrosis factor α antibody results in an accumulation of Th1 CD4+ T cells in the peripheral blood of patients with rheumatoid arthritis

OBJECTIVEnIn rheumatoid arthritis (RA), treatment with tumor necrosis factor alpha (TNFalpha) binding agents has proven to be highly effective. Downregulation of the proinflammatory cytokine cascade and a reduced migration of leukocytes into the joints have been proposed as modes of action of TNFalpha blockade. We investigated whether alterations in the number of circulating pro- and antiinflammatory T cell subsets contribute to the therapeutic effect of monoclonal antibodies (mAb) against TNFalpha in RA patients.nnnMETHODSnPhenotypic analysis of peripheral blood T cell subsets was performed on blood from RA patients before and after treatment with an anti-TNFalpha mAb.nnnRESULTSnAn accumulation of primed CD45RA- T cells of both the CD4+ and the CD8+ T cell population was seen shortly after treatment. Most notably, within the CD4+,CD45RA- T cell subset, the number of interferon-gamma-producing T cells was significantly increased after anti-TNFalpha mAb treatment, resulting in a significant rise in the Th1:Th2 ratio. In addition, an increase in the number of CD4+ T cells expressing the homing receptor CD49d in high density was observed after treatment, which correlated positively with the increase in the Th1:Th2 ratio. Conclusion. We show that the Th1:Th2 ratio in the peripheral blood is raised by anti-TNFalpha mAb treatment.

Association of the TNF +489 polymorphism with susceptibility and radiographic damage in rheumatoid arthritis

Multiple genetic factors contribute to susceptibility to rheumatoid arthritis (RA). The extent of variability in disease presentation in RA may be related to genetic heterogeneity. In this study we investigated the association of the TNF gene polymorphism at position +489 with susceptibility to and severity of RA. Analysis of the frequency of the +489 A and G alleles in a group of 293 consecutive RA patients and 138 healthy controls revealed a significant decrease of the A allele. The +489 GA patients had a 3.9 times decreased chance of having erosive disease than +489 GG patients. These results were confirmed in a prospective study using a cohort of 112 patients who were followed for 12 years. The progression rate of the erosion score over 12 years expressed as Sharp score for X-rays of hands and feet was 3.4 per year for the GA-genotyped patients and 12.1 for the GG-genotyped patients. These associations were independent of rheumatoid factor and HLA-shared epitope positivity. In conclusion, these data suggest that the intron TNF +489 polymorphism is associated with susceptibility to and disease severity of RA independently of HLA-shared epitope-positive alleles.