Bernd Zanker

Evidence that glucocorticosteroids block expression of the human interleukin-6 gene by accessory cells.

The mode of action of glucocorticosteroids as immunosuppressive and antiinflammatory agents is not fully understood. Glucocorticosteroids block synthesis of interleukin 1 by interfering with the transcription of the IL-1 beta gene. Glucocorticosteroids may also induce rapid degradation of IL-1 mRNA. In the presence of antigen, IL-1 is a potent accessory-cell-derived growth and differentiation co-factor for stimulating resting T lymphocytes. The recently defined interleukin 6 protein is even more powerful than IL-1 in promoting T cell growth and differentiation and acts synergistically with IL-1. Like IL-1, IL-6 is produced by accessory cells and exhibits pleiotropic functions. We herein describe the effects of glucocorticosteroids on IL-6 synthesis. We provide evidence that glucocorticosteroids prevent IL-6 gene transcription in human peripheral blood mononuclear cells.

Possible association of the immunosuppressive and B cell lymphoma-promoting properties of cyclosporine.

Central to the immunosuppressive properties of cyclosporine is a drug imposed blockade of the interleukin-2 gene activation. As IL-6 stimulates antigen-activated T cells to release IL-2, we examined the influence of CsA on IL-6 gene expression and IL-6-supported T cell proliferation. Northern blot analysis revealed that CsA failed to abolish IL-6 gene expression in mitogen-activated peripheral blood mononuclear cells. In fact, increased IL-6 gene transcription and increased release of IL-6 bioactivity were detected using mitogen-activated PBMCs cultured with CsA doses (200-800 ng/ml) only slightly in excess of the minimal antiproliferative dose. CsA completely abrogated the IL-6-stimulated proliferative responses of macrophage-depleted T cells stimulated with polyvalent anti-CD3 monoclonal antibodies. It is interesting that CsA-treated patients evidence an increased incidence of polyclonal lymphoproliferative disorders and B cell lymphomas. As IL-6 fosters B cell activation and growth of EBV-transformed B cells, excessive CsA doses may support development of EBV-transformed B cell lymphomas via superinduction of the IL-6 gene.

The immunosuppressive effects of verapamil upon mitogen activated and allo-antigen inducible human cytotoxic T-lymphocytes

In this study, the effect of verapamil, a phenylalkylamine-type Ca2+ antagonist, upon the activation of human mononuclear cells was investigated and a detailed analysis of the kinetics and dose related effects of verapamil upon alloreactive cytotoxic T-cells (CTL) was undertaken. Verapamil suppressed the release of interleukin-2, proliferation and generation of CTL activity in mitogen and alloantigen stimulated human T-lymphocytes in a dose related fashion. Verapamil suppressed the steady state levels of several T-cell activation-associated gene transcripts, i.e. the mRNA encoding for interleukin-2, and a cytotoxic T-cell specific serine esterase. Verapamil exerted a novel immune-suppressive effect, i.e. the inhibition of mature alloantigen-inducible cytolytic T-cells, thus rendering verapamil a progenitor of potent and clinically useful immunosuppressive drugs.

A kinetic analysis of the effects of interleukin-2 diphtheria toxin fusion protein upon activated T cells

The interleukin-2 diphtheria toxin-related fusion protein (IL-2 toxin) inhibits protein and DNA synthesis IN rIL-2 (10(-10) M) stimulated T lymphoblasts in a dose-dependent fashion. However, prior to target cell death very low concentrations of rIL-2 and IL-2 toxin synergistically stimulate [3H] thymidine incorporation despite inhibition of [14C] leucine uptake. A sequential analysis of [3H] thymidine incorporation shows that high IL-2 toxin concentration (10(-9)-10(-7) M) stimulates DNA synthesis at 18 hr of culture and inhibits [3H] thymidine uptake after 24 hr, while low concentrations of IL-2 toxin (10(-12)-10(-10) M) exhibits stimulatory effects only after 24 hr of culture. Anti-Tac a monoclonal antibody directed against the p55 chain of the high affinity IL-2 receptor (IL-2R) blocks the stimulatory effects of high-dose IL-2 toxin, thereby proving that these effects are mediated through the IL-2 domain of the fusion protein. At 7 hr following interaction with IL-2R receptor (IL-2R)+ T cells, IL-2 toxin-treated cells evidence augmented transcription of the heat shock protein gene, an effect indistinguishable from those mediated by rIL-2. We conclude that interaction of IL-2 toxin with IL-2R+ T cells initially mimicks the stimulatory effects of IL-2 upon gene transcription and DNA synthesis yet concomitant inhibition of protein synthesis is evident.

The role of interleukin-6 in mitogenic T-cell activation: detection of interleukin-2 heteronuclear RNA by polymerase chain reaction.

It has been documented that interleukin-6 (IL-6) supports the proliferation of purified, anti-CD3-stimulated murine T cells. We found that stimulation of human peripheral blood mononuclear cells (PBMCs) with anti-CD3 induced a significant accumulation of IL-6 mRNA, indicating that antigen-mediated T-cell activation may involve IL-6 release from accessory cells. Phytohemagglutinin (PHA) had little effect upon IL-6 gene expression. In keeping with these findings, anti-IL-6 reduced but did not abolish anti-CD3-mediated proliferation of PBMCs, but had no significant effect upon PHA-stimulated proliferation. The addition of recombinant (r) IL-6 enhanced the proliferation of anti-CD3-stimulated PBMCs and increased the accumulation of IL-2 mRNA in PHA-stimulated PBMCs during the first 5 hr of culture. Nuclear run-off experiments did not reveal significant changes in IL-2 transcription in PHA plus rIL-6-treated PBMCs attempting to assume that IL-6 mediates stabilization of IL-2 mRNA. However, monitoring of partially spliced IL-2 mRNA by polymerase chain reaction revealed a clear increase in IL-2 heteronuclear RNA. Thus IL-6 increases the rate of IL-2 transcription which was not detectable by conventional in vitro transcription assays. We conclude that anti-CD3 triggers T-cell proliferation through a process that is partially but not entirely dependent upon release of IL-6. IL-6, in turn, supports IL-2 transcription. Insofar as anti-CD3 mimics antigen-triggered activation of the T-cell receptor complex, IL-6 appears to support the early immune response by augmenting antigen-triggered IL-2 gene expression.

Influence of Macrophage Activation on the Synthesis of Complement Components C2, C3, C4

Macrophages are a major site of complement synthesis. In the guinea pig production of complement components C1, C2, C4, C3, D, B, and P by peritoneal macrophages has been demonstrated (reviewed in Ref. 1). Whereas marked differences exist in biological activity between resident, elicited and activated macrophages (2), we investigated whether this holds true for the synthesis and secretion of complement components C2, C3, and C4.

Similar effects of cyclosporine and verapamil on lymphokine, interleukin 2 receptor, and proto-oncogene expression.

Since calcium channel blocking agents and CsA exert an antiproliferative effect upon T cell mitogenesis, we have compared and characterized their immunosuppressive properties at the level of gene activation. Verapamil (greater than or equal to 30 microM), which blocks T cell mitogenesis and a rise in cytosolic calcium, was added to cultures of peripheral blood mononuclear cells stimulated with phytohemagglutinin (5 micrograms/ml) and phorbol myristate acetate (5 ng/ml). Northern blot analysis was performed using cDNA probes for the p55 interleukin 2 receptor (Tac; IL-2R), interleukin 2 and c-myc at 20 hr of culture. Accumulation of IL-2 encoding mRNA within the cytoplasm was completely abrogated by verapamil. However, IL-2R and c-myc encoding mRNA were clearly detectable in verapamil-treated cell cultures. Surface expression of the Tac protein in mitogen-activated T cells was also not blocked by verapamil as shown by FACS analysis. In companion experiments with CsA, verapamil only partially inhibits the intracellular processes leading to T cell activation. A calcium-independent pathway may exist for the expression of IL-2R and c-myc, while an increase of intracellular Ca2+ may provide the additional signal for IL-2 gene expression. Although the in vitro concentrations of verapamil used in these experiments are in excess of common clinically therapeutic levels, the results help clarify the mode of CsA action and may provide a new tool to dissect the early events of T cell activation.