Michael Stoeck

INDUCTION OF IL-15 MRNA AND PROTEIN IN A549 CELLS BY PRO-INFLAMMATORY CYTOKINES

Interleukin-15 is a recently discovered cytokine which is functionally similar to IL-2. In order to learn more about possible targets for modulation of the expression of IL-15 we investigated the expression of IL-15 mRNA and protein in the A549 (human lung carcinoma) cell line. Constitutive expression of IL-15 mRNA was detected in A549 cells. Treatment with TNF-alpha or IL-1 beta (10 ng/ml each) induced an about 2-fold increase of IL-15 mRNA; IFN-gamma induced significant effects only at 100 ng/ml. Stimulation with a combination of TNF-alpha and IFN-gamma was not superior to stimulation with TNF-alpha alone. EGF, KGF and the combination thereof were without effects. IL-15 protein was detected in cellular lysates of unstimulated cells and was increased by stimulation with TNF-alpha or IL-1 beta. No significant amounts of IL-15 protein were detected in cellular supernatants.

A sensitive, IL-2-independent, assay for IL-1

The thymocyte costimulator (LAF) assay, the standard biological test used for IL-1 titration, has a low sensitivity and lacks specificity since it can be potentiated by the IL-2 which is frequently present in IL-1-containing biological fluids. We describe here a new IL-1 titration method which takes advantage of the capacity of a thymoma line, EL4-6.1, to differentiate and express IL-2 receptors upon stimulation by IL-1 in the presence of a suboptimal dose of phorbol diester. Membrane IL-2R measurement on this indicator cell line permits the detection of 1-2 X 10(-4) ng/ml IL-1, compared to 5 X 10(-2) ng/ml in the LAF assay. In addition, rIL-2 up to 250 U/ml has no effect on IL-1 measurement by this assay, which also exhibits a 100-fold lower sensitivity to inhibitory effects of prostaglandin, compared to the LAF assay. Finally, tumor necrosis factor alpha only exerts a weak costimulation effect at very high doses. A flow cytometry technique and an ELISA are described for IL-2 receptor detection. Due to its high sensitivity and specificity, this novel assay should now permit reliable IL-1 titration in biological fluids such as IL-2-rich lymphocyte culture supernatants.

Transforming growth factors β1 and β2 as well as milk growth factor decrease anti‐CD3‐induced proliferation of human lymphocytes without inhibiting the anti‐CD3‐mediated increase of [Ca2+]i and the activation of protein kinase C

Porcine transforming growth factor 1 and 2 (pTGF‐β1 and ‐β2) and milk growth factor (MGF) at 1 ng/ml significantly inhibited the proliferation of human lymphocytes induced by anti‐CD3 antibodies. In contrast, the anti‐CD3‐mediated increase of intracellular Ca2+ and the activation and translocation of protein kinase C were not affected by the transforming growth factors.

Effect of Transforming Growth Factor Beta on the EL4 Thymoma Variant EL4/6.1: Dissociation of Inhibition of Proliferation from Expression of IL-1 and IL-2 Receptors

In order to further characterize the action of transforming growth factor beta (TGF-beta) on lymphoid cells, we investigated the effects of porcine TGF-beta 1 and -2 on the IL-1 sensitive EL4/6.1 thymoma cell line. The proliferation of EL4/6.1 thymoma cells was inhibited by TGF-beta 1 and TGF-beta 2 (1 ng/ml) to a similar degree, the population doubling time was increased by 50-60%, total inhibition was not achieved. This decrease of proliferation was associated with an increase of the number of cells in the G0/G1 compartment of the cell cycle. TGF-beta-mediated inhibition could not be overcome by adding exogenous rIL-1 nor was the binding capacity for IL-1 reduced. In addition, TGF-beta did not interfere with the induction of IL-2 receptors by a combination of Ionomycin+PMA+IL-1. The data suggest that TGF-beta mediated inhibition of thymocyte/lymphocyte proliferation is not associated with an inhibition of the expression or the induction of expression of IL-2 or IL-1 receptors.

Studies on the mechanism whereby cyclosporin A inhibits T-lymphocyte activation.

The immunosuppressive drug Cyclosporin A (CyA) inhibited the ConA-induced DNA synthesis in C57B1/6 spleen cells at a concentration of 40 ng/ml totally; this inhibition could not be overcome by the addition of highly purified interleukin-1. ConA-induced RNA synthesis was also inhibited by concentrations of 40 or 200 ng/ml CyA, although total inhibition could not be achieved. In contrast, lipopolysaccharide-induced proliferation could not be inhibited. CyA at a concentration of 40 ng/ml also inhibited the ConA-induced production of interleukin-2 by mouse spleen cells, this inhibition was not due to a toxic mechanism. On the contrary, the proliferative response of T cell blasts from a long-term T cell line (M2) to interleukin-2 containing supernatants was not inhibited by concentrations of 40 or 200 ng/ml CyA; only at 20-100-fold higher concentrations partial inhibition could be observed. One of the earliest events in the course of lymphocyte activation, the enhanced incorporation of unsaturated fatty acids into the lymphocyte plasma membranes; was also inhibited by concentrations of CyA, which abrogated the ConA-induced DNA synthesis. The inhibition of the enhanced incorporation of 14C-oleic acid and 14C-linoleic acid, which are incorporated by the membrane-bound lysolecithin-acyltransferase, thus suggests a molecular site of action for CyA.

Stimulation of FACS-analysed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin + phorbol-12,13-dibutyrate does not overcome their proliferative deficit.

Human tumour‐infiltrating lymphocytes (TIL) were prepared by enzyme digestion from a series of different tumours and were purified on a fluorescence‐activated cell sorter (FACS II) according to their CD4+ and CD8+ phenotype. CD4+ and CD8+ TIL were stimulated separately in a low density microculture system with phytohacmagglutinin (PHA) or with ionomycin plus phorbol‐12, 13‐dibutyrate (PDBu). The PHA‐induced proliferation of TIL was highly decreased when compared with control peripheral blood lymphocytes. A decreased proliferation of TIL was also observed when cells were stimulated with ionomycin plus PDBu, a combination which is thought to circumvent early events associated with lymphocyte activation. Some TIL were also plated in limiting dilution where they showed decreased frequencies of proliferating T cell precursors. The data suggest that one component of the inhibition of TIL must be acting ‘ownstream’ of the early events of lymphocyte activation.