In the present study we searched for restriction fragment length polymorphisms (RFLP) in the human interleukin-1 beta (IL-1 beta) gene and for correlations to monocyte (Mo) function in non-related healthy donors and insulin-dependent diabetic patients. We demonstrated a diallelic polymorphism with the restriction enzyme TaqI consisting of fragments of 9.4 kb and 13.4 kb. No differences in allele or genotype frequencies of this RFLP were observed between randomly selected controls and randomly selected patients with insulin-dependent diabetes mellitus (IDDM). However, when analysing IDDM patients negative for HLA-DR3 and -DR4, our data demonstrate that the 13.4 kb allele is more frequent in this group compared to a matched control group. The functional impact of this RFLP was studied by analysing in vitro stimulated Mo IL-1 beta response. An IL-1 beta allele dosage effect on secretory capacity was observed after LPS-stimulation: 13.4/13.4 kb homozygous individuals secreted significantly more IL-1 beta than 9.4/13.4 kb heterozygous individuals, who secreted significantly more than 9.4/9.4 kb homozygous individuals. Analyses of supernatants from LPS-stimulated Mo cultures from individuals with each TaqI IL-1 beta genotype revealed no differences in the mouse thymocyte co-stimulatory assay when compared on a molar basis, indicating that the TaqI polymorphism gave rise only to quantitative differences in expression levels and probably not to a mutant IL-1 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the correlation between different tumor necrosis factor (TNF) and class II major histocompatibility complex alleles in the lipopolysaccharide- or phytohemagglutinin-induced secretion of TNF-alpha and TNF-beta by human monocytes and peripheral blood mononuclear cells in 87 unrelated Danish male individuals. Significant differences in TNF-alpha secretory capacity between TNF NcoI restriction fragment length polymorphisms, TNFa and TNFc microsatellite alleles and DR alleles were identified. No correlation with TNF-beta secretory capacity was found for any of the markers studied. TNF genotyping allowed us to define four extended HLA haplotypes which correlate with TNF-alpha secretory capacity. Two of these are DR4 positive: DQw8, DR4, TNFB*1, TNFa6, B44, A2 and DQw8, DR4, TNFB*2, TNFa2, B15, A2. Individuals carrying the TNFB*2, TNFa2 haplotype had a higher TNF-alpha secretory capacity than those carrying the TNFB*1, TNFa6 haplotype. In a group of DR3/DR4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM), the frequency of the TNFa2 allele was higher than in HLA-DR matched controls, whereas the TNFa6 allele was more frequent in control individuals. In the DR3/DR4 heterozygous diabetic group 12/26 had the alleles combination DQw8, DR4 (Dw4), C4A3, TNFB*2, TNFa2, B15, whereas only 1/18 controls had this haplotype. This diabetogenic haplotype is identical to the DR4 haplotype which correlates with a higher TNF-alpha response. These observations suggest a direct role for the TNF locus in the pathogenesis of IDDM.

The effects of dietary supplementation with omega-3-polyunsaturated fatty acids (omega-3-PUFA) on the proliferative response of PBMC and on the secretion of monokines and arachidonic acid metabolites from PBMC and monocytes (Mo) from healthy subjects and patients with recent-onset insulin-dependent diabetes mellitus (IDDM) were examined. Three groups of eight to nine healthy individuals were randomized to either 2.0 g/day or 4.0 g/day of omega-3-PUFA devoid of vitamins A and D, or an isocaloric amount of placebo. Furthermore, eight patients with recent-onset IDDM received 4.0 g/day of omega-3-PUFA. IL-1 beta production and TNF-alpha secretion was determined before and after 7 weeks of treatment, and 10 weeks after withdrawal of treatment. Significant increases in platelet and PBMC membrane eicosapentaenoic acid was found in omega-3-PUFA-treated individuals. omega-3-PUFA treatment significantly reduced the content of IL-1 beta in lysates of PBMC, but did not affect PBMC or Mo secretion of IL-1 beta, TNF-alpha or prostaglandin E2 (PGE2) or PBMC leukotriene B4 (LTB4) secretion in healthy subjects or in IDDM patients. A significant inhibition of the PHA-stimulated, but not the spontaneous or PPD-stimulated, proliferative response of PBMC was observed in healthy and diabetic subjects treated with omega-3-PUFA. No correlation was found between PHA-stimulated PBMC proliferation and PBMC secretion of TNF-alpha and IL-1 beta. There were no significant differences in the spontaneous or the PPD- or PHA-stimulated proliferative responses of PBMC between diabetic and healthy individuals at entry. We conclude that although dietary supplementation with 4.0 g/day of omega-3-PUFA inhibits the proliferation of PBMC and reduces IL-1 beta immunoreactivity in PBMC and Mo, it does not alter monokine, PGE2 or LTB4, secretion in healthy or IDDM subjects.

HLA-class III region genes may be associated with susceptibility to insulin-dependent diabetes mellitus (IDDM). In this study an NcoI polymorphism of the tumour necrosis factor beta (TNF-beta) gene, which is positioned next to the tumour necrosis factor alpha (TNF-alpha) gene in the HLA class III region, was detected by restriction fragment length polymorphism (RFLP). This polymorphism has previously been reported to be located in the TNF-alpha gene. Caucasian HLA-DR3,4 heterozygous IDDM patients (n = 26) and DR-matched healthy controls (n = 19), as well as randomly selected IDDM patients (n = 27) and controls (n = 25) were studied. In addition four multiplex families (49 individuals) and eight HLA-non-identical sibpairs concordant for IDDM were analysed. The TNF-beta gene RFLP analysis showed fragments of 5.5 kb and 10.5 kb, which behaved as alleles. In all groups there was a haplotype assignment of the TNF-beta 5.5-kb allele to B8,DR3 haplotypes, and of the TNF-beta 10.5-kb allele to B15,DR4-positive haplotypes. The allelic and genotypic frequencies differed between DR3,4 IDDM patients and DR3,4 controls, and the DR3,4 control group differed significantly from the randomly selected control group (P less than 0.0079). In HLA-DR3,4- and DQw8-positive persons, the DR3 haplotypes carried the 10.5-kb allele three times more frequently in IDDM patients than in controls, suggesting that the 10.5-kb allele when present on DR3 haplotypes may contribute to susceptibility to IDDM in DR3,4 heterozygous individuals. A contributory role of the 10.5-kb allele in genetic IDDM susceptibility was supported by the sibpair analysis, in which all were TNF-beta identical. Five were 10.5 kb homozygous, and the remaining three pairs were 5.5/10.5 kb heterozygous. Twenty-five healthy and eight newly diagnosed IDDM patients were randomly selected to study the Escherichia coli lipopolysaccharides (LPS)-purified protein derivate (tuberculin) (PPD)-, and phytohaemagglutinin (PHA)-stimulated monocyte (Mo) secretions of interleukin 1 beta (IL-1 beta) and TNF-alpha in relation to the NcoI TNF-beta gene polymorphism. The LPS- and PHA-stimulated Mo IL-1 beta and TNF-alpha secretions were significantly lower for the TNF-beta 5.5/10.5 kb heterozygous individuals than for TNF-beta 10.5 kb homozygous individuals. Furthermore, the Mo IL-1 beta and TNF-alpha secretions of IDDM patients were significantly higher than the Mo secretions of TNF-beta genotype-matched healthy controls. This study suggests an association between the 10.5 kb TNF-beta allele and IDDM, and demonstrates an association between monokine responses and TNF-beta genotypes.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) may be pathogenetically important in insulin-dependent diabetes mellitus (IDDM), which is associated with genes of the HLA region. Since a regulatory role of HLA region genes on monokine production may exist, we looked for an association between the monokine and prostaglandin E2 (PGE2) responses of monocytes (Mo) from 20 healthy males (18-50 years) with HLA-DR types relevant for IDDM susceptibility and resistance (DR1,2, DR1,3, DR1,4, DR3,4). Monokine assays were established and evaluated and the secretions of IL-1 beta, TNF-alpha, and PGE2 measured in Mo cultures (2h, 6h, 20h) prepared by endotoxin-free techniques and stimulated by low-dose E. coli lipopolysaccharides (LPS). There were no significant associations between Mo responses and HLA-DR phenotype. Likewise, Mo from DR2 (n = 5) and DR4 (n = 5) homozygous healthy males demonstrated no significant differences in monokine and PGE2 responses of Mo. In the HLA class III region a diallelic TNF-beta gene NcoI polymorphism consisting of alleles of 5.5 kb and 10.5 kb was recently described and associated with susceptibility to autoimmune diseases including IDDM. We report that IL-1 beta and TNF-alpha responses of Mo from TNF-beta 10.5 kb homozygous healthy individuals were significantly higher than for TNF-beta 5.5/10.5 kb heterozygotes. IL-1 beta and TNF-alpha responses of Mo from males (18-35 years) with newly diagnosed (n = 10) and long-standing IDDM (n = 10) and from age- and HLA-DR-matched healthy males (n = 10) were studied. LPS, gamma interferon (IFN), and TNF-alpha-stimulated Mo cultures were investigated. No significant differences were found between Mo responses of IDDM patients and controls. IFN (1000 U/ml) in the presence of LPS significantly potentiated LPS-stimulated Mo TNF-alpha secretion and reduced the levels of IL-1 beta immunoreactivity in Mo lysates. IFN and TNF-alpha did not have any effects on LPS-stimulated Mo secretion of IL-1 beta immunoreactivity. We conclude that Mo IL-1 beta and TNF-alpha production is normal in patients with recent-onset and long-standing IDDM. The interindividual differences in monokine responses may be accounted for by the diallelic human TNF-beta gene polymorphism rather than by HLA class II genes. This observation may be important for understanding the association of certain HLA haplotypes with autoimmune phenomena and disease.

Tumour necrosis factor (TNF) or cachectin is an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF (rTNF)/100 microliter. There was no interference of medium, serum, plasma, spinal fluid, or urine and no cross-reaction with natural or recombinant IL-1-alpha, IL-1-beta, IL-2, IFN-gamma, or lymphotoxin (TNF-beta). Recovery of TNF added to the media was 85-123% (n = 22). The relative standard deviations within and between assays were 7% and 8%, respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30 microliter or 12.5 pg/30 microliter of rTNF. IL-1-alpha and IL-1-beta slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50-300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100-1000 ng/ml). In contrast, using 0.01-100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine. Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.

Recombinant human interleukin 1 beta (rhIL-1 beta) and supernatants of Escherichia coli lipopolysaccharides-stimulated human monocyte (Mo) cultures, containing native human IL-1 beta (nhIL-1 beta), demonstrate significant differences when tested in the mouse co-stimulatory thymocyte (lymphocyte activating factor [LAF]) assay. The aims of the present study were to investigate this characteristic difference between rhIL-1 beta and Mo culture supernatants (Mo supernatants), and to compare the biological and the immunological activity of preparations of rhIL-1 beta and nhIL-1 beta during each step of an identical purification procedure. The biological activity of rhIL-1 beta/nhIL-1 beta preparations was characterized by the use of the LAF assay and the rat islet insulin release assay. An IL-1 beta enzyme-linked immunosorbent assay (ELISA) was established in order to compare the biological and immunological responses of the IL-1 beta preparations. We report that the significant difference between rhIL-1 beta and supernatants of Mo cultures, which was only demonstrable in the LAF assay, is due to the presence of interleukin 6 (IL-6) in the Mo supernatants. We describe a simple cation exchange chromatography separating nhIL-1 beta and IL-6 of Mo supernatants. The highly purified rhIL-1 beta possessing the correct amino-terminal sequence and nhIL-1 beta have identical biological and immunological activities demonstrating a specific biological activity (SBA) of 3 x 10(2) U/ng IL-1 beta. Thus, we have no indications of secondary or tertiary structural differences between rhIL-1 beta and purified nhIL-1 beta. In contrast, both in the LAF assay and in the rat islet insulin release assay the SBA of an amino-extended rhIL-1 beta form, Met-Glu-Ala-Glu-rhIL-1 beta, was only 1-2% of the SBA of rhIL-1 beta, suggesting that structural changes were introduced into the molecule by the amino-terminal extension. In the present study we have demonstrated that systematic combined testing of IL-1 beta preparations in two different biological assays and an immunological assay is useful for the characterization and comparison of the activity of recombinant and native IL-1 beta preparations purified by the use of exactly the same procedures.

Abstract. Animal experiments demonstrate that interleukin-lfl (IL-Ib) is beta-cell cytotoxic in vitro and inhibits insulin secretion in vivo. However, it is unknown if IL-ID affects beta-cell function in man. Since IL-Ip and other cytokines are main mediators of the acute phase response, the objectives of the present study were to examine beta-cell function in patients with major burn injuries, and to test if changes in betacell function correlated to systemic levels of IL-IS and tumour necrosis factor ci (TNFol). We established and validated an IL-lB assay measuring free and protein bound IL-Ip; protein bound IL-lP was detached from the IL-Ip specific binding protein by acidification, rendering it accessible for the employed antibody. The IL-1 p specific binding protein (43-60 kDa) was found in serum and plasma from all tested patients and normal subjects. Survivors of burn injuries had a stimulated beta-cell function, whereas non-survivors had an impaired beta-cell function as indicated by an increased plasma concentration of proinsulin, and an increased proinsulin/insulin ratio. In addition, nonsurvivors had significantly increased plasma levels of TL-I/?. However, we could not demonstrate any correlation between C-peptide, proinsulin, insulin or proinsulin/insulin ratio and plasma concentration of IL-1fi.In conclusion, beta-cell function abnormalities are evident in patients with major burn injuries, and a high plasma level of IL-Ip correlates with a fatal outcome. However, the present study did not provide evidence for the hypothesis that beta-cell function is influenced by circulating IL-IB or TNFci during the acute phase response.