C. L. Verweij

The influence of tetracyclines on T cell activation

Minocycline has been shown to have an anti‐inflammatory effect in patients with rheumatoid arthritis (RA). Since there is evidence that RA is a T cell‐mediated disease, we investigated the effect of minocycline on human T cell clones derived from the synovium of an RA patient. The T cells, when activated via the T cell receptor (TCR)/CD3 complex, were suppressed functionally by minocycline, resulting in a dose‐dependent inhibition of T cell proliferation and reduction in production of lL‐2. interferon‐gamma (IFN‐γ) and tumour necrosis faetor‐alpha (TNF‐α). Besides an inhibition of IL‐2 production, mitiocycline exerted its effect on T cell proliferation by induction of a decreased IL‐2 responsiveness. We showed that the chelating capacity of minocycline plays a crucial role in the inhibitory effect on T cell function, since the inhibitory effect on T cell proliferation could be annulled by addition of exogenous Ca2+. However, minocycline did not markedly influence the typical TCR/CD3‐induced intracellular Ca2+ mobilization. Taken together. the results clearly indicate that minocycline has immunomodulating effects on human T cells.

Polymorphism of tumour necrosis factor‐alpha (TNF‐α) at position –308 in relation to 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 localization, in the proximity of the HLA‐B locus, and the biological activities of TNF‐α, we investigated the association between AS and a single base polymorphism located at position –308 of the TNF‐α gene. An allele‐specific polymerase chain reaction was developed to monitor this polymorphism. The frequency of the TNF‐α alleles was determined in 66 AS patients and 37 healthy controls. The TNF‐α allele frequency was not significantly different between AS patients and controls.

Regulation and production of IL‐8 by human proximal tubular epithelial cells in vitro

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL‐8. In the present study we have analysed the production of IL‐8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum‐free conditions, were found to produce IL‐8 to different degrees from not detectable levels up to 10.8±1.5 ng IL‐8 per 1×105 cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL‐8 of PTEC is 15.1 and 8.1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0.5 ng/ml rIL‐1α or 1000 U/ml recombinant tumour necrosis factor‐alpha (TNF‐α) to the culture media of PTEC induced an up‐regulation of IL‐8 production up to 6.3‐fold and 3.0‐fold, respectively. The up‐regulation by IL‐1α and TNF‐α was dose‐ and time‐dependent. In contrast, 500 U/ml recombinant interferon‐gamma (rIFN‐γ) down‐regulated the production of IL‐8 3.4‐fold. Northern blot analysis showed that IL‐1α and TNF‐α increased the expression of IL‐8 mRNA, whereas IFN‐γ reduced IL‐8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL‐8 in the kidney, and that IL‐8 produced in the proximal tubule can be induced by various proinflammatory cytokines.

Regulation of C3 and factor H synthesis of human glomerular mesangial cells by IL-1 and interferon-gamma.

Previous reports have shown production of complement components C4. C2 and factor B by renal tissue. We have shown recently that human proximal tubular epithelial cells (PTEC) synthesize C3 in vitro, and that IL‐2 enhances this production. In the present study we demonstrate that human mesangial cells (MC) in culture produce factor H and that supernatants of activated peripheral blood mononuclear cells (T cell growth factor (TCGF)) induce C3 production and enhance factor H synthesis in both a time‐ and dose‐dependent manner. To investigate whether certain defined cytokines from TCGE were responsible for the observed effect., we tested various cytokines for their effect on complement production by MC. It is shown that IL‐1 induces C3 synthesis whereas factor H production is up‐regulated by TFN‐γ, in both a dose‐ and time‐dependent manner. Antibody blocking experiments revealed that C3 synthesis induced by both TCGF and IL‐I could be blocked with antibodies specific for IL‐I, and also that TCGF and IFN‐γ enhanced factor H synthesis could both be blocked with antibodies specific for IFN‐γ. Cycloheximide was able to inhibit C3 and factor H production, suggesting de novo synthesis of the proteins. mRNA‐polymerase chain reaction (PCR) analysis revealed mRNA encoding for C3 after stimulation with TCG Fand IL‐I. Factor H genes are constitutively expressed in cultured mesangial cells and its expression is up‐regulated by TCGF and IFN‐γ. Northern blot analysis with specific probes for C3 and factor H revealed bands which support the results obtained by PCR analysis.

Chloroquine inhibits T cell proliferation by interfering with IL‐2 production and responsiveness

Chloroquine (Chi) is an anti‐rheumatic drug that is widely used in the treatment of rheumatoid arthritis (RA). It seems that T ceils are important in the pathogenesis of RA, but it is not known whether Chl acts via inhibition of T cell function. We here present evidence that Chl, just like cyclosporine A (CsA), inhibits Tcell proliferation as induced with immobilized αCD3 MoAb in a concentration‐dependent manner, at least partly through interfering with the production of IL‐2 protein and the induction of IL‐2 inRNA. Furthermore, Chi impedes the responsiveness of T cell clones to IL‐2 since (1) the inhibition of αCD3 MoAb‐induced proliferation by Chi could not be reversed by rlL‐2 and (2) Chi directly blocks IL‐2‐driven proliferation of cloned T cells. Chi appeared to interfere with the internalization (50% inhibition) and degradation (total blockade) of rIL‐2. Finally, the combination of Chi and CsA synergistically inhibited T ceil proliferation. We conclude that Chi may inhibit functional properties of human T cells. although the drug is 100‐to 1000‐folds less potent than CsA in inhibiting T cell proliferation and IL‐2 production, respectively. It is speculated that the in vitro. effects of Chl might be relevant in explaining the anti‐rheumatic effect of this drug in patients with RA.

Inflammatory and immunological parameters of disease activity in rheumatoid arthritis patients treated with minocycline

The objective of this study was to analyze the anti-inflammatory effect of minocycline in rheumatoid arthritis. Serum samples of 65 RA patients who completed a 26-week randomized double-blind trial of minocycline (100 mg twice a day) versus placebo were studied. In this trial some clinical parameters and in particular the acute phase response decreased significantly in the minocycline-treated group. Serum levels of albumin and interleukin-6 (IL-6) were compared with CRP levels in order to study the acute phase response. Furthermore, rheumatoid factor (RF) and total immunoglobulin isotypes as well as serum levels of soluble interleukin-2 receptor (sIL2-2R) were determined in order to study immunological parameters of the disease. Immunoglobulins and cytokines were measured by ELISA. Serum levels of albumin remained stable, whereas serum CRP levels decreased both in the minocycline- and in the placebo-treated group. Serum levels of IL-6 decreased in the minocycline-treated group only and this decrease was positively correlated with the decrease in CRP levels. Minocycline significantly decreased serum IgM-RF, IgA-RF, total IgM and total IgA levels. In addition the ratio of IgM-RF/total IgM decreased in the minocycline-treated group. No such changes were observed in the placebo-treated group. The anti-inflammatory effect of minocycline in RA patients may be due to the reduction in the synthesis of IL-6 and rheumatoid factor.

Characterization of complement C6 deficiency in a PVG/c rat strain

Complement C6 plays an important role in the effector phase of complement‐mediated cell lysis. Recently, a PVG/c rat strain deficient in haemolytic C6 activity was discovered. In the present study we show that these rats lack both antigenic and functional C6, and that repetitive immunization of these rats with PVG/c+ serum results in generation of specific anti‐rat C6 antibodies. The observed absence of rat C6 was further investigated at the genomic and transcriptional level using a 492‐bp cDNA of rat C6, cloned from a rat liver cDNA library using full length human C6 as a probe. Northern blot analysis revealed the presence of C6 mRNA in livers of both PVG/c− and PVG/c+ rats, corresponding to a size of ≈ 3.3 kb, although the level of C6 mRNA expression was ≈ 100‐fold less in PVG/c− rats. In addition, using rat C6‐specific primers, positive signals were obtained in kidneys of both rat strains by reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis. Southern blot analysis of digested genomic DNA did not reveal evidence for large C6 gene deletions. We conclude that the lack of C6 protein in the PVG/c− rat strain is not due to a (large) C6 gene deletion, but presumably is caused by an unstable mRNA or a point mutation in the C6 gene resulting in an aberrant transcription of the C6 gene. Alternatively, a gene coding for a product involved in C6 biosynthesis that acts in trans may carry a mutation.

Cytokine-regulated production of the major histocompatibility complex class-III-encoded complement proteins factor B and C4 by human glomerular mesangial cells

Local production of complement within normal or diseased kidneys could be of importance during local inflammatory reactions. In the present study, we demonstrate that human MCs are able to synthesize the MHC class-III-encoded complement proteins factor B and C4 in vitro. This synthesis is strongly upregulated following stimulation with cytokine-containing supernatants of activated peripheral blood mononuclear cells. All primary cell lines tested so far are able to synthesize factor B and C4 after stimulation. To determine more specifically whether defined cytokines are able to enhance factor B and C4 complement production, MCs were stimulated with IL-1 alpha, IFN-gamma, and TNF-alpha. Factor B synthesis was increased in a dose-dependent fashion by IL-1 alpha, TNF-alpha, and IFN-gamma, whereas C4 synthesis was only upregulated by IFN-gamma. Furthermore, factor B synthesis was upregulated after stimulation with IFN-alpha, -beta, and -gamma and C4 synthesis only by IFN-gamma. The synthesis of factor B and C4 was inhibited by cycloheximide, suggesting de novo protein synthesis. The cytoplasmic localization of both components was shown by immunofluorescence studies. Northern and dot blot analysis revealed induction of factor B and C4 mRNA after stimulation with cytokines.

Alternative splicing of IgA Fc receptor (CD89) transcripts

An alternatively spliced CD89 transcript is present in peripheral blood mononuclear cells (PBMC) and U937 cells. The alternatively spliced CD89 mRNA species lacks the exon 4 sequence, encompassing 288 nucleotides, that encodes the extracellular membrane-proximal immunoglobulin-like domain (EC2).

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.