TNF-alpha promoter polymorphisms, production and susceptibility to multiple sclerosis in different groups of patients.

TNF-alpha production in whole blood cultures upon stimulation with LPS was determined in 179 individuals from 61 families in order to characterise the magnitude of inherited differences in TNF-alpha production. The three families characterised by highest TNF production showed 7.1 +/- 0.3 ng TNF/ml upon culture with 10 ng LPS and 10.2 +/- 0.2 ng TNF/ml upon culture with 1000 ng LPS. in contrast to the three families characterised by the lowest TNF production that showed a production of 1.6 +/- 0.1 ng TNF upon culture with 10 ng and 2.5 +/- 0.2 ng/ml upon culture with 1000 ng LPS/ml. This difference could not be attributed to the promoter polymorphisms -308 G to A. -238 G to A or -376 G to A, although the -238 GA donors produced 2.1 +/- 0.9 ng TNF upon culture with 10 ng endotoxin compared to 3.2 +/- 2.2 ng TNF for the -238 GG donors. In line with these results the frequency of the -238 GG genotype was increased in hospitalized MS patients in a nursing home (100% 238GG, n = 57) compared to MS patients in an outpatient's clinic (94% 238GG, n = 98) or Dutch controls (90% 238GG, n = 180). These results suggest that the -238 GG genotype is differently distributed in hospitalized MS patients in a nursing home.

Microsatellite alleles and single nucleotide polymorphisms (SNP) combine to form four major haplotype families at the human interleukin-10 (IL-10) locus.

Interleukin-10 (IL-10) is a pivotal immunoregulatory cytokine, influencing many aspects of the immune response. The IL-10 gene is located on chromosome 1 at 1q31-32 and is highly polymorphic. One microsatellite and three single nucleotide polymorphisms (SNPs) have been recorded within the 1.2 kb immediately upstream of the gene, with an additional microsatellite present at 4 kb upstream. The relationship between these two classes of polymorphism is poorly defined in the IL-10 gene. Haplotypes have been presented comprising alleles from the two microsatellite loci, and independently from the three SNPs, but these have not yet been brought together to define unified halpotypes. In the present report we describe the 29 IL-10 haplotypes found in 56 Dutch European families and show that they fall into four major haplotype groups, each of which spans the 4 kb upstream of the IL-10 gene and has a different distribution of IL10.G alleles. In addition, we describe three novel single nucleotide polymorphisms in the human IL-10 gene and suggest how they relate to these four haplotype families.

Site alpha is crucial for two routes of IFN gamma-induced MHC class I transactivation: the ISRE-mediated route and a novel pathway involving CIITA.

The constitutive and cytokine-induced levels of major histocompatibility (MHC) class I expression are tightly controlled at the transcriptional level. In this study, it is shown that the cis-acting regulatory element site alpha of the MHC class I promoter is essential for the IFN gamma-induced transactivation of MHC class I gene expression through the ISRE. Moreover, it was discovered that the class II transactivator (CIITA), which is itself under the control of the IFN gamma induction pathway, strongly transactivates MHC class I gene expression and exerts its activity through site alpha. Therefore, site alpha is a crucial regulatory element, mediating the classic route of IFN gamma induction via the ISRE as well as a novel route of MHC class I transactivation involving CIITA.

Antigen processing and presentation by human trophoblast-derived cell lines.

The trophoblast-derived choriocarcinoma cell lines JEG-3 and JAR express the nonclassical MHC class I molecules HLA-G (JEG-3) or, at a low level, HLA-E (JAR), but lack expression of the classical MHC class I molecules HLA-A and HLA-B. Expression of these nonclassical MHC class I genes was found to coincide with expression of the genes encoding the peptide transporter associated with Ag processing (TAP). In immunoprecipitation studies, a physical interaction between the TAP complex and HLA-G or HLA-E could be demonstrated. To investigate whether trophoblast-derived cell lines were capable of peptide processing, transport, and loading of MHC class I molecules, HLA-A*0201-expressing transfectants of JEG-3 and JAR were used for functional studies. These transfectants were recognized by both allospecific cytotoxic T cell clones and, after viral infection, by an influenza A matrix peptide-specific cytotoxic T cell clone, indicating that these trophoblast-derived cell lines were capable of presenting endogenously derived peptides in the context of HLA-A*0201. From these observations, it can be inferred that the TAP complex and other molecules involved in Ag processing and presentation by MHC class I molecules are functionally active in these trophoblast-derived cell lines. This implies that trophoblasts are able to provide antigenic peptides for presentation by nonclassical MHC class I molecules that are naturally expressed by this cell type.

Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production.

A protocol to test foetal calf serum (FCS) for contamination with bovine viral diarrhoea virus (BVDV) is described. Following this protocol, which combines cell culture methods and detection of pestivirus RNA, seven batches of FCS were tested. Infectious BVDV was detected in four of those batches. One of the remaining batches contained a relatively high number of non-infectious BVDV particles. A sample of this batch was formulated with aluminium hydroxide and aluminium phosphate as adjuvant into an experimental vaccine preparation. This product was injected twice into BVDV seronegative cattle with a 4 week interval. Blood samples taken 4 weeks after the second application were negative for BVDV specific antibodies. Our data stress that detection of BVDV RNA is not sufficient for a complete risk assessment on FCS. Discrimination between infectious and non-infectious BVDV is essential. This can only be achieved by cell culture methods.

The role of enhancer A in the locus-specific transactivation of classical and nonclassical HLA class I genes by nuclear factor kappa B.

HLA class I expression is tightly controlled at the transcriptional level by several conserved regulatory elements in the proximal promoter region. In this study, the two putative kappa B motifs of enhancer A (kappa B1 and kappa B2) of the classical and nonclassical HLA class I genes were investigated for their binding properties of transcription factors and tested for their contribution to the NF-kappa B-induced route of transactivation. It was shown that NF-kappa B-induced transactivation through enhancer A is most important for the HLA-A locus, which contains two NF-kappa B binding sites. Although the enhancer A of HLA-B contains only one NF-kappa B binding site (kappa B1), there was still a moderate transactivation by NF-kappa B. Since HLA-F, which also possesses one NF-kappa B binding site but lacks protein binding to its KB2 site, was not transactivated by NF-kappa B, the NF-kappa B-mediated transactivation through the kappa B1 motif in HLA-B is most probably facilitated by binding of the transcription factor Spl to the upstream kappa B2 site. Thus, transcriptional regulation of HLA class I genes by NF-kappa B is restricted to the HLA-A and HLA-B loci.

Interleukin 10 secretion in relation to human IL-10 locus haplotypes.

Stimulation of human blood cultures with bacterial lipopolysaccharide (LPS) shows large inter-individual variation in interleukin 10 (IL-10) secretion, which has been shown to have a genetic component of over 70%. Alleles at two microsatellite loci in the 4 kb immediately upstream of the human IL-10 transcription initiation site in 132 individuals from 56 Dutch families were defined and assigned as haplotypes. LPS-induced IL-10 secretion was measured by ELISA and related to the IL-10 promoter haplotypes present in 78 unrelated individuals obtained from these families. Analysis showed that LPS-induced IL-10 secretion from unrelated individuals varied with IL-10 promoter haplotypes (P = 0.024; Kruskal-Wallis test). Two observations were made in relation to secreted IL-10 levels and promoter haplotypes; first, those haplotypes containing the allele IL10.R3 were associated with lower IL-10 secretion than haplotypes containing any other IL10.R allele. Second, the haplotype IL10.R2/IL10.G14 was associated with highest IL-10 secretion overall, whereas the haplotype IL10.R3/IL10.G7 was associated with lowest IL-10 secretion. These data demonstrate that the ability to secrete IL-10 can vary in man according to the genetic composition of the IL-10 locus.

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.

Relevance of Fcgamma receptor and interleukin-10 polymorphisms for meningococcal disease.

The contribution of individual Fcgamma receptor (FcgammaR) subclasses to meningococcal phagocytosis was studied. In addition, functional FcgammaR polymorphisms were determined in 50 patients with meningococcal disease (MD), in 183 first-degree relatives of MD patients, and in 239 healthy control subjects, to study the association of FcgammaR genotypes with disease. Efficient internalization of opsonized Neisseria meningitidis serogroup B was mediated via multiple FcgammaR subclasses on phagocytes. Accordingly, a low-efficiency combination of FcgammaRIIa-R/R131, FcgammaRIIIa-F/F158, and FcgammaRIIIb-NA2/2 genotypes was increased significantly in relatives of patients with MD, compared with healthy control subjects (P<.05; odds ratio, 2.6; 95% confidence interval, 1.1-6.3). FcgammaRIIa and FcgammaRIIIa genotype distributions differed between patients with sepsis and those with meningitis. Combined genotypes of FcgammaRIIa and interleukin-10 -1082, which was previously reported as being associated with MD outcome, were distributed randomly in control subjects but not in relatives of patients with MD (P<.01). These data provide further evidence for the association of polymorphic genes on chromosome 1 and MD.

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.

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.