Monocytes from different individuals show variable cytokine production in response to a variety of stimuli. We wished to determine the sets of conditions (cytokine combinations) that would enable us to demonstrate stable inter-individual differences in the production of IL-1 alpha, IL-1 beta, IL-1Ra, on-6 and tumour necrosis factor-alpha (TNF-alpha) by monocytes. We assessed the ability of a number of recombinant human cytokines (granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), TNF-alpha, IL-4, IL-6, transforming growth factor-beta (TGF-beta), IL-10 and IL-1Ra)) to stimulate or inhibit the production of one or more of these monocyte products. GM-CSF was found to stimulate the production of all five of these cytokines in a highly reproducible manner. TNF-alpha also up-regulated production of IL-1 alpha, IL-1 beta, IL-1Ra and IL-6 by monocytes, but the variability in the results of cells cultured from the same individuals on different occasions was greater. Other cytokines either stimulated production of only some of the five cytokine products tested, or stimulated the production of some cytokine products while inhibiting production of others. This was especially evident when cytokines were used in combination with GM-CSF: IFN-gamma down-regulated production of IL-1Ra while up-regulating the production of IL-1 alpha/beta, IL-6 and TNF-alpha, while IL-4 had the exact opposite effect. Polymorphisms in regions of cytokine genes that affect transcription may account for some of the interindividual variation in cytokine production. We have shown that a stable estimate of cytokine production phenotype can be obtained when monocytes collected on at least two separate occasions are stimulated by GM-CSF in vitro. We have looked for a relationship between IL-1 production and an 86-bp variable repeat polymorphism in intron 2 of the IL-1Ra gene. A less common allele of this polymorphism (allele 2) was associated with increased production of IL-1Ra protein, and also reduced production of IL-1 alpha protein by monocytes.

The role of the Apo-1/Fas gene promoter MvaI polymorphism in RA and SLE is unclear and further substantiation in larger patient samples is needed.

The frequency of the uncommon allele (TNF2) of a polymorphism in the promoter region of the tumour necrosis factor alpha (TN Fα) gene in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was found to be 3 times that of the normal anglo-saxon population. In SLE patients, this allele was strongly associated with HLA-DR3 expression and was also more frequent in patients who did not have malar rash. Functional studies of normal monocyte cytokine production in vitro showed that this genotype was associated with increased IL-1α protein production but there were no differences in the production of TNFα protein.

No abstract

Interleukin 1 (IL-1), IL-6, and tumour necrosis factor (TNF) a are pleiotropic cytokines produced predominantly by macrophages which have been implicated in the pathogenesis of rheumatoid arthritis (RA). Sulphasalazine has been shown to have disease modifying properties and to inhibit the production of cytokines in vitro. To evaluate the effect of sulphasalazine on cytokine production in vivo, serum cytokine levels were measured in a group of patients with RA entered into a randomised controlled trial. Serum levels of IL-la, IL-1,B, IL-6, and TNF a were measured at baseline and at two monthly intervals for six months in 17 patients receiving sulphasalazine and in 22 patients treated with placebo. The two groups of patients had a similar age and sex distribution, had had RA for less than a year, had no joint erosions, and had not been treated previously with any other disease modifying drugs.In the 39 patients studied IL-la was detected (>0.1 ng/ml) at baseline in 14 patients (median 0-24 ng/ml), IL-1,B in 25 patients (median 1-0 ng/ml), TNF a in 27 patients (median 1.2 ng/ml), and IL-6 in 33 patients (median 0.44 ng/ml). In the group treated with sulphasalazine there was a progressive and signficant decline in senrm IL-la, IL-1,B, and TNF a levels over the six month period (median levels at six months were <0-1, 0-12, and 0.44 ng/ml respectively). Interleukin 6 levels were significantly reduced only at the four month time point (median level of 0-23 ng/ml). These reductions were associated with improvements in clinical and laboratory measures of disease activity. In contrast patients receiving the placebo showed no changes in serum cytokine levels and no improvement in clinical and laboratory indices of disease activity. These results suggest that sulphalazne may exert its disease modifying effect partly by suppressing cytokine production in vivo. (Ann Rheum Dis 1992; 51: 946-950)

Human cytomegalovirus (HCMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in HCMV infection is well documented. Traditionally laboratory strains of HCMV have been used in experimental investigations in vitro; however the continuous propagation of these strains in fibroblasts have attenuated the virus making it unsuitable for infecting other cell systems such as endothelial cells. In this study a recent clinical isolate of HCMV was propagated through several passages in endothelial cells and was used to investigate the effect of HCMV infection of human umbilical vein endothelial cells (HUVEC) on IL-1 production and cell proliferation. Infection of HUVEC with the clinical isolate of HCMV (at multiplicity of infection 5:1) suppressed the production of IL-1 alpha (82%) and IL-1 beta (99%) at 5 h post infection; the levels returned to that of the control within 24h post infection. Ultraviolet inactivated (but not heat killed) virus produced similar suppression confirming that a thermolabile viral structural protein or intact virion were responsible for this suppression. Infection of HUVEC with the clinical isolate increased the number of these cells and the rate of their proliferation. An increase of infected HUVEC number under quiescent growth conditions continued as the infection progressed (6-10 days post infection), exhibiting, at 3 days post infection, 5 times the number of uninfected HUVEC (control) which did not tolerate the quiescent culture conditions for more than 4 days. Live virus is responsible for this increase because UV-inactivated virus did not maintain the proliferation of HUVEC. These studies suggest that while infection of HUVEC with a recent clinical isolate of HCMV suppressed the production of IL-1 at early hours after infection, it increased the proliferation of these cells at later stages of infection.

Immune complexes (ICs) in the serum of 43 patients with chronic idiopathic thrombocytopenic purpura (ITP) were measured by the C1q deviation assay during the active and inactive phases of the disease. An inverse relationship between platelet count and levels of ICs was demonstrated in all but one patient. To test whether this phenomenon was specific for chronic ITP, ICs were assayed in sera from two groups of control patients with thrombocytopenia. Goup 1 had thrombocytopenia due to recognized immune mechanisms while group 2 had thrombocytopenia secondary to non-immune mechanisms. In both these groups the degree of thrombocytopenia proved to be inversely proportional to IC levels, which was similar to the pattern observed in chronic ITP. The specificity of the assay for detection of ICs was confirmed by demonstrating a positivity rate of 65% in sera of patients with systemic lupus erythematosus, a known IC disease. On analysis the ICs were shown to have molecular weights in excess of 500,000 daltons and contain variable immunoglobulin classes. The findings implicate ICs in immune destruction of platelets both in chronic ITP (as has been suggested previously) and also in thrombocytopenia secondary to known immune mechanisms. In addition the association of ICs with non-immune thrombocytopenias is consistent with the hypothesis that platelets play an important role in clearance of ICs from the circulation, thereby protecting the vascular endothelium from damage.

SUMMARY IL-1 production (secreted and cell-associated) was measured in monocyte cultures stimulated by a variety of agents in vitro. Monocytes either adherent to conventional plastic culture plates in serumfree conditions, or in suspension in culture medium containing serum were stimulated to produce IL-I during culture. In non-adherent, serum-free conditions, monocytes produced very low or undetectable amounts of IL