Marcus Diamant

Anti-interleukin-6 antibodies in normal human serum.

High‐avidity IgG antibodies to the cytokine inlerleukin‐6 (IL‐6) were found in sera of apparently healthy adult individuals. These antibodies specifically interfered with an FLISA (enzyme‐linked immunosorbent assay) for IL‐6 in which specific polyclonal rabbit antibodies to human recombinant IL‐6 (rlL‐6) were used. Furthermore. using precipitation of 125‐I‐rIL‐6 with rabbit antibodies to human immunoglobulins (Ig). the sera of 7 out of 6S Danish blood donors were found to contain specific antibodies in substantial amounts. Judged by ELISA interference, gel filtration of sera incubated with 125I‐rIL‐6 and second antibody precipitation of 125I‐rIL‐6. IgG seemed to be the dominant IL‐6 binding protein in these normal sera. Using specific antibodies to human in light chains, it was found that the anti‐lL‐6 antibodies were of polyclonal origin. Moreover, there are at least two epitopes on the IL‐6 molecule, because more than one IgG bound lo some IL‐6 molecules at the same time, The anti‐IL‐6 antibodies did not cross‐react with a number of other human recombinant‐derived and native cytokines. The antibodies recognized native as well as rlL‐6. but preferentially monomeric lL‐6.

Inhibition of production and function of interleukin-6 by 1,25-dihydroxyvitamin D3

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to interfere with immunoglobulin production and lymphocyte proliferation in vitro. These lymphocyte functions are influenced by interleukin (IL)-6 produced by antigen presenting cells. Hence, the ability of 1,25-(OH)2D3 to interfere with the production and function of IL-6 was investigated. 1,25-(OH)2D3 and the analogue MC 903 inhibited IL-6 production by LPS-stimulated human mononuclear cells. The precursor 25-OH D3 was ineffective. Likewise, 1,25-(OH)2D3 but not 25-OH D3 inhibited rIL-6-driven as well as rIL-1 alpha/beta-driven proliferation of murine thymocytes. This effect of 1,25-(OH)2D3 was partially or totally overcome by larger concentrations of rIL-6 as well as by rIL-2 and ionomycin. Consistently, the production of IL-6 and IL-2 in rIL-1 driven thymocyte cultures were found to be reduced by 1,25-(OH)2D3. Inhibition of production and function of IL-6 may therefore be involved in 1,25-(OH)2D3-mediated regulation of lymphocyte functions in vitro.

High-affinity IgG autoantibodies to IL-6 in sera of normal individuals are competitive inhibitors of IL-6 in vitro.

Nano- to picomolar concentrations of high affinity IgG antibody to interleukin 6 (IL-6ab) were detected in sera of 71 of 467 normal Danish blood donors (15%). IL-6 bound to the Fab fragments of IL-6ab, and the antibody specifically and competitively interfered with enzyme-linked immunosorbent assays for human IL-6. Only IL-6ab-positive sera interfered with these ELISAs. A statistically positive correlation was found between total IL-6 binding and specific IL-6 binding to serum (P < 0.0001), suggesting that IL-6ab dominates as IL-6 binding factors in normal human serum. The IL-6ab also inhibited binding of IL-6 to receptors on the human U-937 macrophage-like cell line, the human U-266 myeloma cell line and the mouse hybridoma cell line B13.29, clone B9 (B9 cells). IL-6-induced proliferation of the B9 cells was competitively inhibited by IL-6ab. We conclude that sera of normal individuals may contain appreciable levels of autoantibodies to IL-6. These IgG autoantibodies may be physiologically and pathophysiologically important because they are potent inhibitors of IL-6 in vitro.

In vitro immunomodulatory effects of pentoxifylline

Pentoxifylline (PTX), a methylxanthine derivative and phosphodiesterase inhibitor, is known to influence production and/or function of some cytokines. We examined the effect of PTX on the in vitro expression of cytokine genes using endotoxin- or phytohaemagglutinin (PHA)-stimulated human blood mononuclear cells. The expression of tumour necrosis factor (TNF)alpha, TNF beta interleukin (IL)-2 and interferon (IFN)gamma was inhibited by PTX in a dose-dependent manner, whereas expression of IL-1 alpha, IL-1 beta, and IL-6 was unaffected at concentrations up to 300 microM of PTX. The amount of TNF beta mRNA in PHA-stimulated blood mononuclear cells was reduced by PTX. Finally, PTX stimulated PHA-induced cell proliferation whereas antigen-induced cell proliferation was inhibited in the presence of PTX. The PTX analogues HWA-138 and A-802715 inhibited TNF alpha mRNA expression from endotoxin-stimulated mononuclear cells. These data suggest that PTX-analogues affect the in vitro immune response at different target points and that the response depends upon the respective triggering mechanism(s).

Fusidic acid, an immunosuppressive drug with functions similar to cyclosporin A.

Fusidic acid, a tetracyclic triterpenoic acid, is used for local and systemic treatment of bacterial infections. Its in vitro effects on the human immune response were tested. Activated blood mononuclear cells released lower levels of interleukin (IL) 1 in the presence of nontoxic and clinically attainable levels of fuscidic acid (15 to 50 micrograms/mL). In contrast, the drug failed to affect the production of two other monocyte-derived cytokines, tumor necrosis factor (TNF)-alpha and IL 6. The production of the T-cell-derived cytokines, IL 2 and interferon-gamma (IFN-gamma), were also suppressed (IC50: 5 to 15 micrograms/mL). The early costimulatory effects of IL 1 and IL 6 on mouse thymocytes and human T cells were suppressed by similar levels of the drug, as was the hybridoma growth-promoting function of IL 6. T-cell proliferation induced by phytohemagglutinin or allogeneic cells was reversibly inhibited (IC50: 15 micrograms/mL). These functions of fusidic acid were strikingly similar to those of cyclosporin A. Because of the low toxicity of the former, it may have a role as a clinically useful suppressor of immunoinflammatory processes.

INTERLEUKIN-6 PRODUCTION BY THYROID EPITHELIAL CELLS. ENHANCEMENT BY INTERLEUKIN-1

Interleukin-1 is a potent inhibitor of thyroglobulin and cAMP production in human thyroid cells and the inhibitory effect is enhanced by tumor necrosis factor-alpha and interferon-gamma. In the present study secondary cultures of human thyroid cells produced interleukin-6 and the production was significantly increased after exposure of the cells to recombinant interleukin-1 alpha and -1 beta. This increase was dose-dependent and concomitant of the IL-1 induced decrease in cAMP and thyroglobulin production. Both tumor necrosis factor-alpha and -beta also augmented interleukin-6 production, but less potently than interleukin-1. Interferon-gamma did not affect the production of interleukin-6. The rat thyroid cell line FRTL-5 produced interleukin-6 spontaneously, and the production was enhanced after addition of recombinant interleukin-1 beta. A pathogenetic role of interleukin-6 in autoimmune thyroid disease is suggested.

Cytokine vaccination: neutralising IL-1α autoantibodies induced by immunisation with homologous IL-1α

Abstract High-affinity IgG autoantibodies (aAb) to IL-1α are among the most frequently found aAb to cytokines in humans. To establish an animal model with aAb to IL-1α, we immunised mice with recombinant murine IL-1α. Unprimed and Bacille Calmette-Guerin (BCG)-primed BALB/cA mice were vaccinated with IL-1α coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1α. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1β. The induced anti-IL-1α aAb inhibited binding of IL-1α to the murine T-cell line NOB-1 by simple competition and neutralised IL-1α, but not IL-1β-induced IL-6 in vivo. The aAb did not induce visible discomfort in the animals. In conclusion, long-lasting and high levels of neutralising and specific IgG aAb to IL-1α can be induced in mice by vaccination with recombinant murine IL-1α conjugated to PPD. Studies of the effects of IL-1α aAb in such animals may help clarify the importance of naturally occurring IL-1α aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients with immunoinflammatory diseases and cytokine-dependent tumours.

Stimulation of the B9 hybridoma cell line by soluble interleukin-6 receptors

Interleukin-6 (IL-6) exerts its effects by binding to specific receptors on the cell surface. The IL-6 receptor consists of at least two components, a ligand binding 80 kDa low-affinity component (IL-6R) and a signal-transducing non-ligand binding 130 kDa component (gp130). The presence of soluble forms of these components has been described in both conditioned media and biological fluids. The soluble (s) IL-6R has been shown to enhance the IL-6 sensitivity of several both murine and human IL-6 sensitive cell types. A sensitive and commonly used method for measuring biological IL-6 activity is based on the IL-6 dependent proliferation of the murine hybridoma cell line B9. In this paper, we demonstrate that recombinant (r) human (h) sIL-6R enhances the sensitivity of B9 cells in a dose-dependent manner. The rhsIL-6R enhanced the binding of 125I-rhIL-6 to B9 cells. The rhsIL-6R induced stimulation of B9 proliferation was maximal at 100 ng/ml, even without addition of rhIL-6 and in the presence of anti-hIL-6 antibodies. This may be due to endogenous IL-6 production by the B9 cells, low levels of IL-6 in the fetal calf serum used, or perhaps an IL-6 independent effect by the rhsIL-6R. In conclusion, this and other reports point to the necessity of confirming measured biological activities through the use of neutralizing specific antibodies or parallel measurements in immunochemical assays.

The Effects of Interleukin-1β (IL-1β) on Human Thyrocyte Functions Are Counteracted by the IL-1 Receptor Antagonist1

The cytokine interleukin-1β (IL-1β) is an important regulator of thyroid cell function. IL-1 receptors are present on normal thyrocytes, but the signaling pathway is not fully clarified. As the adenylate cyclase is presumably not activated, we have in the present study investigated whether the cGMP pathway was involved in the actions of IL-1β, whether the effects of IL-1β on cultured human thyrocytes were reversible, and whether the effects were counteracted by IL-1 receptor antagonist (IL-1ra), a naturally occurring, specific blocker of IL-1 receptors on many cells. TSH-stimulated cultured human thyroid cells exposed for 72 h to IL-1β (0.0002–20 μg/liter = 1–105 IU/liter) exhibited a dose-dependent and reversible inhibition of thyroglobulin and cAMP release and a dose-dependent stimulation of cGMP and IL-6 release. These effects were counteracted by coincubation with 250 or 125 μg/liter, but not with 25 and 2.5μ g/liter, IL-1ra. IL-1ra by itself inhibited the release of cAMP, but did not modulate the rel...

Sulphatide and its precursor galactosylceramide influence the production of cytokines in human mononuclear cells

Sulphatide is expressed in the central and peripheral neural system, in islets of Langerhans, and in tissues affected by late diabetic complications. Autoantibodies to sulphatide are present in patients with insulin‐dependent diabetes and the Guillain‐Barré syndrome. Cytokines influence these disease processes, and we therefore studied whether sulphatide and its precursor galactosylceramide (gal‐cer) influence the in vitro production of cytokines by blood mononuclear cells (MNC) originating from 15 healthy persons. Using lipopolysaccharide (LPS)‐stimulated cells, sulphatide increased the IL‐2 production (163±17% of controls without sulphatide, p=0.02), and gal‐cer increased the IL‐1α production (145±13%, p=0.006), whereas neither gal‐cer nor sulphatide had an effect on the production of IL‐6, IL‐10 or TNFα. When stimulating cells with phytohaemagglutinin (PHA), sulphatide decreased the production of IL‐6 (88±5%, p=0.009), IL‐10 (66±3%, p=0.000003), and TNFα (75±9%, p=0.02). Galcer, however, increased the production of IL‐6 (188±13%, p=0.000006), and decreased the production of TNFp (80±6%, p=0.007). Neither galcer nor sulphatide had an effect on the production of IL‐2 or IFNy from PHA‐stimulated cells. Northern blot analysis using an IL‐6 probe similarly showed an increased amount of IL‐6 mRNA after galcer incubation (range 469%‐150%, n=3) of PHA‐stimulated control. Thus, sulphatide and galcer influence the production of several cytokines thought to be involved in immunoinflammatory disease processes.