Allele specific regulation of cytokine genes: monoallelic expression of the IL-1A gene.
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Alleles↗
Biomedical subjects
C L Verweij
Publications and source records attributed to C L Verweij.
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 alpha (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 Tai I 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.
Alleles↗
Differential requirements for induction of total immunoglobulin and physiological rheumatoid factor production by human peripheral blood B cells.
Rheumatoid factors (RFs) are autoantibodies directed against the Fc part of IgG. Considerable evidence exists that there are two classes of RFs, pathological and physiological. Whereas pathological RFs are associated with disease, physiological RFs are considered to be a normal component of the immune response. RF(+) precursor B cells present as part of the B cell repertoire of healthy individuals are held responsible for the production of physiological RFs, which is a transient phenomenon with a clear correlation with an initiating stimulus such as immunization or exposure to an infection. Here we demonstrate a difference in the regulatory control of total Ig and RF production by peripheral blood (PB) B cells of both healthy controls (HC) and patients with rheumatoid arthritis (RA). Highly purified B cells from HC and patients with RA were cocultured with T cells stimulated with immobilized anti-CD3 mAb. Similar to IgM production, IgM-RF production was shown to be dependent on CD40 cross-linking. However, activation of PB B cells in the CD40 system in the presence of IL-2, IL-4, IL-10, combinations of these cytokines or supernatant of anti-CD3-stimulated T cells failed to induce detectable IgM-RF, whereas total IgM production was considerable. From these results we conclude that conditions to activate physiological RF(+) B cells require additional contact besides CD40--CD40L interactions between T and B cells. Since the requirements for RF production were similar using PB B cells from HC and patients with RA it is suggested that the regulatory properties of RF(+) precursors in the PB B cell compartment is equal among these groups. Together, these results indicate that conditions for the induction of total Ig and physiological RFs are different.
Arthritis, Rheumatoid↗
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 zeta-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.
Adaptor Proteins, Signal Transducing↗
TNF-308A and HLA-DR3 alleles contribute independently to susceptibility to systemic lupus erythematosus.
OBJECTIVE: To evaluate the respective contributions of tumor necrosis factor (TNF) promoter polymorphisms and HLA-DR alleles to susceptibility to systemic lupus erythematosus (SLE). METHODS: TNF-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. RESULTS: The 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. CONCLUSION: TNF-308A and HLA-DR3 alleles are independent susceptibility factors for SLE.
Adult↗
Regulation of synovial B cell survival in rheumatoid arthritis by vascular cell adhesion molecule 1 (CD106) expressed on fibroblast-like synoviocytes.
OBJECTIVE: B lymphocytes accumulate in the inflamed joints of patients with rheumatoid arthritis (RA) and are responsible for production of high amounts of (auto)-antibodies. The aim of this study was to determine the capacity of fibroblast-like synoviocytes (FLS) to contribute to the accumulation of synovial fluid (SF) B cells by extending their life span. METHODS: Highly purified SF B cells were cultured with FLS in the presence or absence of blocking antibodies directed against cell adhesion molecules, and cell viability was determined after various time intervals by trypan blue, annexin V, propidium iodide, or Hoechst staining. Phenotypic characterization of peripheral blood and SF B cells and FLS was carried out by flow cytometry. RESULTS: Synovial B cells, which consist predominantly of memory B cells and plasma cells (PC), undergo spontaneous cell death by apoptosis upon removal from their in vivo environment, despite expression of Bcl-2. Coculture with FLS rescued synovial B cells from apoptosis in a cell contact-dependent manner. Blocking studies using monoclonal antibodies demonstrated a role for the molecular interaction of SF B cells with vascular cell adhesion molecule 1 (VCAM-1; CD106) in FLS-induced survival. The ability of FLS to induce SF B cell survival was not related to the rheumatoid origin since FLS from non-RA patients had similar properties. CONCLUSION: These findings indicate a crucial role for FLS in the survival of synovial B cells at the site of inflammation in RA through the interaction with VCAM-1 expressed on FLS. Consequently, memory B cells and PC accumulation arise and persist not only as a result of maturation and recruitment of these cells, but also by active prevention from cell death by the microenvironment.
Antibodies, Blocking↗
Genomic organization of the human interleukin-12 receptor beta2-chain gene.
The interleukin-12 receptor (IL-12R) is composed of two subunits, referred to as beta1 and beta2. Both chains are necessary for high-affinity IL-12 binding and signalling, although only the IL-12Rbeta2 chain contains the intracellular tyrosine residues responsible for STAT4 activation. This study presents the intron-exon organization of the human IL-12Rbeta2-chain gene. Polymerase chain reaction (PCR) primers designed across the cDNA (U46198) were used to trace introns, by comparing PCR product sizes obtained using cDNA and genomic DNA as templates. PCR products spanning introns were sequenced to determine the exact splice sites and flanking regions. The coding region of the gene was found to consist of 15 exons and 14 introns. All intron-exon boundaries are consistent with the consensus sequence for splice junctions (5' GT/AG 3'). Comparison of the intron-exon organization with the human GCSFR gene indicated a remarkably well conserved genomic organization between these two class I cytokine receptors. Interestingly, we identified an alternatively spliced mRNA, encoding a putative, truncated protein, lacking all signalling potential.
Alternative Splicing↗
Are differences in interleukin 10 production associated with joint damage?
OBJECTIVE: Constitutive differences between individuals in cytokine production may determine the variation in the course of inflammatory arthritis. METHODS: The association between interleukin 10 (IL-10) production and joint destruction was studied by comparing IL-10 mRNA content in synovial biopsies from seven patients with destructive joint disease and six patients with non-destructive joint disease. The IL-10 mRNA content was 0.4 +/- 0.6 arbitrary units in erosive joints compared with 2.3 +/- 1.2 arbitrary units in non-erosive joints (P: < 0.03, Mann-Whitney U:-test). As this difference suggested that IL-10 production was associated with joint destruction, we tested whether the IL-10 locus determined the extent of joint damage. RESULTS: Innate differences in IL-10 production are locus-dependent. In line with these data, we showed that innate differences in IL-10 protein production were also present as differences in IL-10 mRNA levels. We tested if polymorphisms in the promoter of IL-10 were associated with the extent of joint damage. DISCUSSION: In a cohort study of female rheumatoid arthritis patients followed for 12 yr, the extent of joint destruction differed significantly between patients with different IL-10 genotypes. In patients with the -1082AA genotype who were studied prospectively, the mean increase in radiographic damage score (modified Sharp score of X-rays of hands and feet) during the first 6 yr was 9 +/- 9 per yr vs 19 +/- 16 per yr for patients with the genotype -1082GG (P: < 0.02). In line with these data, cultures of endotoxin-stimulated whole blood from 158 donors showed that the presence of the allele associated with less joint destruction correlated with slightly higher IL-10 production. CONCLUSIONS: Both the immunogenetic and the synovial biopsies suggest that a variation in IL-10 production is associated with joint destruction.
Adult↗
Functional disparity of distinct CD28 response elements toward mitogenic responses.
Activation of T cells through the antigen-specific T-cell receptor in combination with a costimulatory signal results in efficient cytokine gene transcription. The CD28-induced signal represents a major costimulatory signal for T cells. A CD28 response element, named CD28RE, was first identified in the interleukin-2 (IL-2) promoter region. Here we demonstrate that the NF-kappaB sequence in the IL-6 promoter functions as a CD28 response element. Mutations in this sequence rendered the IL-6 promoter unresponsive to CD28 costimulation. Moreover, this element could replace the IL-2 CD28RE in conferring CD28 responsiveness to the IL-2 promoter. In analogy to the known CD28 response elements IL-2 CD28RE, IL-8 CD28RE, and the human immunodeficiency virus-1 (HIV-1) NF-kappaB motif, the IL-6 NF-kappaB motif efficiently bound c-Rel, c-Rel/NFKB1, and the recently identified inducible T-cell factor NF-MATp35. However, the IL-6 NF-kappaB sequence together with the IL-8 CD28RE and HIV-1 NF-kappaB sequence differed from the IL-2 CD28RE in the binding of NF-kappaB/Rel family proteins. Although the IL-2 CD28RE exerted selective binding with c-Rel and c-Rel/NFKB1, the other CD28REs allowed efficient binding of a wide range of NF-kappaB/Rel family proteins. The difference in binding specificity correlated with the capacity of the distinct CD28 response elements to function in the context of the IL-6 promoter in response to T-cell activation. Domain swapping experiments revealed that the IL-8 CD28RE and HIV-1 NF-kappaB motif conferred similar responsiveness as the genuine IL-6 NF-kappaB motif in the transcriptional activation of the IL-6 promoter upon CD28 costimulation. In contrast, replacement of the IL-6 NF-kappaB sequence by the IL-2 CD28RE motif strongly reduced the responsiveness of the IL-6 promoter. These data indicate that despite the sequence similarity, two different classes of CD28 responsive elements exist that differ in their NF-kappaB binding capacity and the ability to confer CD28 costimulatory responsiveness toward a heterologous promoter.
Antibodies↗
Treatment with monoclonal anti-tumor necrosis factor alpha antibody results in an accumulation of Th1 CD4+ T cells in the peripheral blood of patients with rheumatoid arthritis.
OBJECTIVE: In 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. METHODS: Phenotypic analysis of peripheral blood T cell subsets was performed on blood from RA patients before and after treatment with an anti-TNFalpha mAb. RESULTS: An 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.
Adult↗
Expression of the thioredoxin-thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis.
OBJECTIVE: To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases. METHODS: Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied. RESULTS: Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX. CONCLUSION: The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.
Aged↗
Decreased interleukin-10 and increased interleukin-12p40 mRNA are associated with disease activity and characterize different disease stages in multiple sclerosis.
It has been shown that proinflammatory and antiinflammatory cytokines correlate with disease activity in multiple sclerosis (MS). To establish whether such correlations depend on the disease stage, we assessed in a longitudinal fashion the expression of interleukin (IL)-12 (p40 and p35), tumor necrosis factor-alpha, interferon-gamma, and IL-10 mRNA by competitive polymerase chain reaction in unstimulated peripheral blood mononuclear cells of relapsing-remitting (RR) and secondary progressive (SP) MS patients, in relation to monthly clinical and magnetic resonance imaging monitoring. MS patients had increased levels of IL-12p40 and decreased levels of IL-10 mRNA compared with controls; this difference was most pronounced in SP patients. Both RR and SP patients had increased levels of IL-12p40 mRNA compared with controls during the development of active lesions. Moreover, in RR MS an increase was found before relapse. IL-12p35 mRNA was decreased in both groups, and in relation to disease activity it showed a pattern different from IL-12p40 mRNA. In RR MS, IL-10 mRNA was low 4 weeks before magnetic resonance imaging activity and 6 weeks before relapse; a significant increase to normal levels was noted when active lesions became apparent. In contrast, SP patients showed low IL-10 mRNA levels constitutively, suggesting that IL-10 plays an important role in the control of disease progression.
Adult↗
Genetic risk factors for autoimmune diseases.
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Animals↗
Polymorphism within the tumor necrosis factor alpha (TNF) promoter region in patients with ankylosing spondylitis.
In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the location of the TNF gene in the vicinity of the HLA-B locus, and the prominent role in inflammation of its product, we investigated the association between AS and two G to A transition polymorphisms located at position -238 and -376 in the promoter region of the TNF gene. The distribution of the TNF alleles was determined in 86 HLA-B27+ AS patients and 163 healthy controls. From the 86 AS patients, 33 suffered from acute anterior uveitis (AAU). No significant difference for the TNF-376 polymorphism in AS and healthy controls was observed. The frequency of the TNF-238A allele in HLA-B27+ AS patients was significantly decreased compared to random controls (p = 0.021). However, the frequency of the TNF-238A allele in HLA-B27+ AS patients was not significantly different from that observed in HLA-B27+ healthy individuals (p = 0.6). Assessment of association showed that the TNF-238G allele is in linkage disequilibrium with the HLA-B27 allele (delta = 0.053; P = 0.008). Therefore, we conclude that the association between TNF-238G and AS is secondary to the HLA-B27 gene and that TNF-238 and-TNF-376 alleles are not likely to be involved in the susceptibility to AS.
Alleles↗
An IL-13 promoter polymorphism associated with increased risk of allergic asthma.
IL-13 plays a crucial role in the development of allergic asthma by several mechanisms, including induction of IgE antibodies, airway eosinophilia and hyper-reactivity. We previously established a deregulated production of IL-13 by T cells from allergic asthma patients. In this report we describe the identification of a novel IL-13 promoter polymorphism (C to T exchange) at position -1055. The IL-13 -1055 TT genotype is associated with allergic asthma (P = 0.002), altered regulation of IL-13 production (P < 0.002), and increased binding of nuclear proteins to this region. We postulate that the presence of this polymorphism predisposes to the development of allergic asthma.
Asthma↗
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.
Adult↗
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.
Adult↗
Tumour necrosis factor gene polymorphisms as severity markers in rheumatoid arthritis.
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Arthritis, Rheumatoid↗