PubMed · 1612285
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J Salánki. 1992. Monitoring the environment.. https://doi.org/10.1096/fasebj.6.9.1612285
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Interleukin-1 (IL-1) is an important mediator in inflammation and immunological processes. The findings of native IL-1 inhibitors suggest a negative feedback mechanism to down-regulate IL-1 mediated acute inflammation. IL-1 inhibitors were also found elevated in disease states associated with high IL-1 levels. We have previously described one such IL-1 inhibitor derived from the human M20 myelomonocytic cell line. In this paper we present several biological and biochemical characteristics of the M20 IL-1 inhibitor. Various in vitro activities of the inhibitor are described and its IL-1 specificity in these assays is demonstrated. Purification of the inhibitor was performed by DEAE-high performance liquid chromatography, isoelectric focusing, gel filtration and dye ligand chromatography column. This protein factor has a MW of 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. The inhibitor has no cross-reactivity against a panel of known cytokines (IL-1 alpha, IL-1 beta, IL-2, sIL-2R, IL-6, tumor necrosis factor (TNF), interferon-gamma (IFN-gamma)) and is distinct from the IL-1 receptor antagonist (IL-1ra). The purified IL-1 inhibitor was destroyed by trypsin, 2-mercaptoethanol, sodium dodecyl sulfate and extremes in pH and in temperature. Only IL-1 induced (but not the IL-2, IL-6 or TNF induced) thymocyte proliferation and PGE2 production by fibroblasts were inhibited by the inhibitor, thus showing specificity to IL-1 in these assays.
Cloned CD4 T cells of the Th2 type make IL-4 and related cytokines upon receptor cross-linking, whereas cloned CD4 T cells of the Th1 type make IL-2, IFN-gamma, and TNF-beta. These two types of CD4 T cell are also reported to use distinct mechanisms of signal transduction. It has been reported that Th1 cells flux Ca2+ upon receptor cross-linking, whereas Th2 cells do not. We have noted that when cloned Th2 cells are exposed to high levels (20 U/ml) of IL-2, they show an altered phenotype. Such cells are much more sensitive to activation by certain antireceptor antibodies, they flux calcium upon receptor ligation without additional cross-linking with anti-Ig antibodies, and they make much larger amounts of IL-4. In addition, the organization of their TCR is altered, with increased levels of the TCR-eta chain and an increase in the extent of association of CD4 with CD3 and CD45, changes similar to those found in Th1 cells. These results suggest that there is no fundamental difference in the signal transduction apparatus of Th1 and Th2 cells; rather, the IL-2 made by Th1 cells may create similar phenotypic changes in these cells and thus create the impression of altered signal transduction mechanisms. These results do show that exposure to high levels of IL-2 can profoundly affect signal transduction in T cells. Furthermore, we found that the Ca2+ signal caused by CD3 antibodies seemed to differ in character from that caused by TCR antibodies suggesting that the use of CD3 antibodies is not always a good model for activation through the TCR.
In recent years there has been an increasing interest in measuring the levels of TGF beta produced by peripheral blood mononuclear cells (PBMC), since its abnormal regulation seems to be involved in several pathological states. Platelet-contamination, a common feature in PBMC populations isolated by the standard Ficoll-Paque method, would theoretically disturb the measurement of the levels of TGF beta produced by mononuclear cells, since platelets represent an important source of this cytokine. In this study, supernatants of PBMC cultures from healthy subjects, either platelet-contaminated or uncontaminated, were assayed for TGF beta activity in three different bioassays. We report that the presence of platelets led in most cases to an important overestimation of the TGF beta levels produced by MNC in the Swiss-3T3 bioassay and in a PBMC proliferation assay. In contrast, in the Mv1Lu bioassay these levels were significantly underestimated, an effect which we attribute to the presence of other platelet-derived growth factors. These results suggest that the elimination of platelets from PBMC cultures is essential if TGF beta production by mononuclear cells is to be studied.