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Notizen: Based upon in vivo rat experiments it was recently suggested that interleukin I in the circulation may be implicated in the initial events of β-cell destruction leading to insulin-dependent diabetes mellitus (IDDM) in humans. The aim of the present study was to estimate half-lives of distribution and elimination phases (T1/2β) of human recombinant interleukin 1β(rIL-1β), and its tissue distribution and cellular localization by means of mono-labelled, biologically active 125I-rIL-1β. After intravenous (iv.) injection, 125I-rIL-1β was eliminated from the circulation with a T1/2α of 2.9 min and a T1/2β of 41.1 min. The central and peripheral volume of distribution was 20.7 and 19.1 ml/rat, respectively, and the metabolic clearance rate was 16.9 ml/min/kg. The kidney and liver showed the highest accumulation of tracer, and autoradiography demonstrated that 125I-rIL-1β was localized 10 the proximal tubules in the kidney and to the hepatocytes in the liver. Furthermore, grains were localized to the islets of Langerhans in the pancreas. Tracer-bound proteins corresponding to intact 125I-rIL-1β were found in the circulation after i.v., intraperitoneal (i,p.) and subcutaneous (s.c.) injections, as demonstrated by high performance size exclusion chromatography. trichloracetic acid precipitation and SDS PAGE until 5h after tracer injection. Pre-treatment with “cold” rIL-1β enhanced degradation of a subsequent injection of tracer. The route of administration was of importance for the biological effects of rIL-1β as demonstrated by a reduced food intake, increased rectal temperature and blood glucose after s.c. injection of rIL-1β compared with i.p. The present demonstration of intact rIL-1β in the circulation and the islets of Langerhans supports the hypothesis that systemic IL-1β may be involved in the initial 1β-cell destruction leading to IDDM in humans.

Notizen: Interleukin l (1L-I) exerts both stimulatory and inhibitor) (cytotoxic) effects on insulin producing βcells in isolated pancreatic islets. Since alteration in ion fluxes is crucial for endocrine cell activation and is a denominator of cell death, and since IL-1 was recently shown to increase the total sodium content in a murine pre-B-lymphocyte cell line, we investigated the effect of recombinant human IL-lβ (rhIL-1β) on the cytosolic tree sodium concentration (Na +) in rat islets, furthermore, long-term rhIL-1βeffects on islet cell function were studied during exposure of islets to amiloride. a blocker of the plasma membrane Na+/H+ exchange. One hour of islet exposure to 60 U/ml of rhIL-lβ caused a threefold increase in I Na + . in islet cells, and this effect was abolished by deplelion of extracellular sodium. Blockade of Na+/H+ exchange with amiloride abolished the inhibitory effect of rhIL-1β Son insulin release. In conclusion. rhIL-lβwas found to increase sodium influx in pancreatic islet cells. This might underlie the widespread effects of rhIL-lβ on β-cell function and morphology, possibly related to IL-l-mediated toxic free radical formation.

Notizen: HLA-class III region genes may be associated with susceptibility to insulin-dependent diabetes mellitus (IDDM). In this study an Ncol polymorphism of the tumour necrosis factor beta (TNF-β) gene, which is positioned next lo the tumour necrosis factor alpha (TNF-α) gene in the HLA class 111 region, was detected by restriction fragment length polymorphism (RFLP). This polymorphism has previously been reported lo be located in the TNF-α 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-β 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-β 5.5-kb allele to BS, DR3 haplotypes, and of the TNF-β 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 〈 0.0079), In HLA-DR3,4-atid DQw8-positive persons, the DR3 haplotypes carried the 10.5-kb allele ihrcL- times more frequently in IDDM patients than in controls, suggesting that the 10.5-kb allele when present on DR3 haplotyes may contribute lo susceplibility to IDDM in DR3.4 heterozygous individuals, A contributory role of the Hl.5-kballele in genetic IDDM susceptibility was supported by the sibpair analysis, in which all were TNF-/1 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 bela (IL-1/J)and TNb-α in relation lo the Ncol TNF-/f gene polymorphism. The LPS- and PHA stimulated Mo IL-l/f and TNF-a: secretions were significantly lower for the TNF-β 5.5.10,5 kb heterozygous individuals than for TNF-β 10.5 kb homozygous individuals. Furthermore, the Mo IL-1β and TNF-a secretions of IDDM patients were significantly higher than the Mo secretions of TNF-β genolype-matched healthy controls.This study suggests an association between the 10.5 kb TNF-β allele and IDDM, and demonstrates an association between monokine responses and TNF-β genotypes. These observations may have implications for understanding the pathogenesis of HLA-associated autoimmune diseases including IDDM.

Notizen: The particular susceptibility to insulin-dependent diabetes mellitus (IDDM) conferred by HLA-DR3,4 heterozygosity has been suggested to be an effect of transcomplementation of HLA class II molecules. To test this hypothesis of special IDDM-specific hybrid determinants and to evaluate the T-cell repertoire towards a specific antigen in IDDM patients we generated a total of 352 PPD-specific T-cell lines by the soft-agar cloning technique and studied their restriction by HLA class II molecules. Of these lines, 227 were from nine IDDM patients, of whom six were DR3,4 heterozygotes, and 125 from 10 healthy controls Forty-six T-cell lines elicited specific responses in at least two experiments and in addition to T-cell lines demonstration class-II-restricted PPD specificity, lines with an alloreactivity occurred. HLA-DQ-restricted PPD-specific T-cell line were not identified and a possible DP restriction (DPw2) was only observed with one line. These data indicate that PPD is preferentially presented to T cells in the context of HLA-DR/Dw, Presentation of PPD by hybrid molecules in IDDM patients or by IDDM-specific class II epitopes recognized by the T-cell lines was not demonstrated. By restriction fragment length polymorphism analysis using a probe for the joining region of the T-cell receptor gamma gene, T-cell lines generated by the soft-agar cloning technique were found to be oligoclonal. It is concluded that soft-agar cloning should be followed by subsequent limiting dilution in order to assure monoclonality. Different preparations of antigen-presenting cells (APC) were tested. In several cases the T-cell lines were not able to respond to PPD presenting by Epstein -Barr-virus transformed lymphoblastoid cell lines (LCL). It was demonstrated that lipopolysaccharides (LPS) of E. coli potently reduce the proliferative response of antigen-specific and alloreactive T cells when T-cell-depleted peripheral blood mononuclear cells (E− cells) were used as APC, whereas only limited inhibition was observed when LCL were used as APC in the presence of LPS. This effect of LPS is suggested to be mediated by increased prostaglandin secretion by monocytes among the E− cells since indomethacin abolished the effect of LPS. This observation may have implications for T cell cloning procedures since we have found that most commercially available culture media are heavily contaminated with endotoxin.

Notizen: Leucocyte migration cultures for detection of cellular hypersensitivity in man were prepared for light and electron microscopy 2 and 4 hours after the onset of migration. Antigen-containing as well as control cultures were studied. The cell types found were polymorphonuclear granulocytes (PMN), eosinophilic granulocytes (EO) and lymphocytes (LYM). In the central part of the cultures differential countings showed values comparable to those of peripheral blood. The peripheral monolayer of the migration area contained a large proportion of EO and comparatively few LYM. The cell number of the monolayer increased between the second and fourth hours of migration. Ultrastructurally the PMN presented a more or less disintegrated cytoplasm, but their ability to phagocytize and hydrolyse the corpuscular antigen (brucella bacteria) seemed undisturbed. The EO and LYM appeared less active, although their cytoplasm also contained signs of phagocytic activity. The LYM contained sparse elements of granular endoplasmic reticulum. Apart from the bacterial phagocytosis in the PMN no morphological differences were noted between the cells in the control and antigen-containing cultures.

Notizen: The secretions of interleukin 1 (IL-1), tumour necrosis factor α (TNF), and prostaglandin E2 (PGE2) of low-dose E. coli lipopolysaccharide (LPSJ-stimulated human monocytes (Mø) were investigated in an endotoxin (ET)-free milieu (〈1.6 pg LPS/ml). Human Mø cultures from nine healthy men were stimulated with 0, 12.5–500, and 250,000 pg LPS/ml as measured by a very sensitive Limulus test. The IL-1 activity was tested by the mouse costimulatory thymocyte (LAF) assay, which was thoroughly standardized and characterized (interassay variation 22–24%, intra-assay variation 3–7%). Spontaneous Mø secretions of IL-1, TNF, and PGE2 were negligible, but 12.5 pg LPS/ml significantly stimulated the secretions of these Mø products and the monokine responses to 500 and 250,000 pg LPS/ml were almost in the same range. It was demonstrated that the secretions of IL-1-TNF and TNF-PGE2 were strongly correlated. Pronounced interindividual differences in LPS responsiveness were demonstrated, and two low-responders, one of whom was HLA-DR 1,2-positive, were identified. Three first-degree relatives of the DR1.2-positive low-responder had similar low responses. Furthermore. Mø cultures were prepared weekly for 4 weeks from four HLA-DR different men and the only DR2.2 homozygous individual had low monokine responses. In conclusion, stable interindividual differences in in vitro monokine and PGE2 secretions of LPS-stimulated Mø were demonstrated. It is suggested that HLA-DR2-positive individuals may be low responders.

Notizen: Recombinant human interleukin 1β (rhIL-1β) and supernatants of Escherichia coli lipopolysaccharides-stimulated human monocyte (Mo) cultures, containing native human IL-lβ (nhIL-1β), 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-1β and Mo culture supernatants (Mo supernatants), and to compare the biological and the immunological activity of preparations of rhlL-lβ and nhlL-1β during each step of an identical purification procedure. The biological activity of rhIL-1β/nhIL-lβ preparations was characterized by the use of the LAF assay and the rat islet insulin release assay. An IL-1β enzyme-linked immunosorbent assay (ELISA) was established in order to compare the biological and immunological responses of the IL-lβ preparations.We report that the significant difference between rhIL-lβ 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 nhlL-lβ and lL-6 of Mo supernatants. The highly purified rhIL-β possessing the correct amino-terminal sequence and nhIL-lβ have identical biological and immunological activities demonstrating a specific biological activity (SBA) of 3x102 U/ng IL-lβ, Thus, we have no indications of secondary or tertiary structural differences between rhIL-1β and purified nhIL-lβ.In contrast, both in the LAF assay and in the rat islet insulin release assay the SBA of anaminoextended rhIL-lβ form, Met-Glu-Ala-Glu-rhIL-lβ, was only 1-2% of the SBA of rhIL-lβ, 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-lβ 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-1β preparations purified by the use of exactly the same procedures.

Notizen: Interleukin 1β(IL-1β) and tumour necrosis factor alpha (TNF-α) 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 (DR 1,2, DR 1,3, DR 1,4, DR 3,4). Monokine assays were established and evaluated and the secretions of IL-1β, TNF-α, and PGE2 measured in Mo cultures (2 h, 6 h, 20 h) 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-β gene Ncol 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-1β and TNF-α responses of Mo from TNF-β 10.5 kb homozygous healthy individuals were significantly higher than for TNF-β 5.5/10.5 kb heterozygotes.IL-1β and TNF-α 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-α-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-α secretion and reduced the levels of IL-β immunoreactivity in Mo lysates. IFN and TNF-α did not have any effects on LPS-stimulated Mo secretion of IL-1 β immunoreactivity.We conclude that Mo IL-1β and TNF-α 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-β gene polymorphism rather than by HLA class II genes. This observation may be important for understanding the association of certain H LA haplotypes with autoimmune phenomena and disease.

Notizen: The effects of dietary supplementation with ω-3-polyunsaturated fatty acids (ω-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 ω-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 ω-3-PUFA. IL-lβ production and TNF-α 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 ω-3-PUFA-treated individuals. ω-3-PUFA treatment significantly reduced the content of IL-Ib in lysates of PBMC, but did not affect PBMC or Mo secretion of IL-1β, TNF-α 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 (o-3-PUFA. No correlation was found between PHA-stimulated PBMC proliferation and PBMC secretion of TNF-α and IL-1β. 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 ω-3-PUFA inhibits the proliferation of PBMC and reduces IL-I immunoreactivity in PBMC and Mo, it does not alter monokine, PGE2 or LTB4, secretion in healthy or IDDM subjects.

Notizen: Supernatants of peripheral blood mononuclear cells from healthy human donors stimulated with recall antigen (purified protein derivative of tuberculin) or lectin (phytohaemagglutinin) markedly inhibited the insulin release from isolated human and rat islets of Langerhans, and decreased rat islet contents of insulin and glucagons in a dose-dependent manner. A maximal effect on islet function was obtained with supernatant concentrations down to 5%. Supernatants of mononuclear cells stimulated with tuberculin were more potent than supernatants produced by lectin stimulation. Culture medium reconstituted with tuberculin or phytohaemagglutinin did not impair islet function. Electron microscopy demonstrated that supernatants were cytotoxic to islet cell. The cytotoxic mononuclear cell mediator(s) was non-dialysable, sensitive to heating to 56°C, labile even when stored at -70°C, but stable when lyophilised.