Inhibition of p38 mitogen-activated protein kinase unmasks a CD30-triggered apoptotic pathway in anaplastic large cell lymphoma cells.

CD30, a non-death domain-containing member of the tumor necrosis factor receptor superfamily, triggers apoptosis in anaplastic large cell lymphoma cells. The CD30 signaling pathways that lead to the induction of apoptosis are poorly defined. Here, we show that the induction of apoptosis by CD30 requires concurrent inhibition of p38 mitogen-activated protein kinase, which itself is activated by engagement of CD30 with CD30 ligand. Treatment of anaplastic large cell lymphoma cells with CD30 ligand and pharmacologic inhibitors of p38 mitogen-activated protein kinase, but not with CD30 ligand or inhibitors alone, triggered the activation of caspase-8 and the induction of apoptosis. Caspase-8 activation occurred within a few hours (2.5-4 h) after receptor triggering, was unaffected by the neutralization of ligands for the death domain-containing receptors TNFR1, Fas, DR3, DR4, or DR5, but was abolished by the expression of a dominant-negative form of the adaptor protein FADD. Importantly, we show that expression of the caspase-8 inhibitor c-FLIP(S) is strongly induced by the CD30 ligand, and that this is dependent on the activation of p38 mitogen-activated protein kinase. Thus, we provide evidence that the induction of apoptosis by CD30 in anaplastic large cell lymphoma cells is normally circumvented by the activation of p38 mitogen-activated protein kinase. These findings have implications for CD30-targeted immunotherapy of anaplastic large cell lymphoma.

Production of hemo- and immunoregulatory cytokines by erythroblast antigen+ and glycophorin A+ cells from human bone marrow.

BACKGROUND: Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM. RESULTS: We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl A+ or AG-EB+) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukin (IL)-1beta, IL-2, IL-4, IL-6, interferon (IFN)-gamma, transforming growth factor (TGF)-beta1, tumor necrosis factor (TNF)-alpha and IL-10. They can be sub-divided into glycophorin A positive (Gl A+) and erythroblast antigen positive (AG-EB+) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-beta1 and TNF-alpha production were found between Gl A+ and AG-EB+ BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-gamma and IL-2 production specifically by the AG-EB+ ENC. Thus, Gl A+ and AG-EB+ ENC produce IL-1beta, IL-2, IL-4, IL-6, IFN-gamma, TGF-beta1 and TNF-alpha. Gl A+ ENC also produce IL-10. CONCLUSION: Cytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM.

Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method.

Quantitative analysis of human immunoregulatory cytokines in physiological media and cell cultures plays an important role in fundamental and clinical research. Here we describe the quantification of interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) in human serum and peripheral blood mononuclear cell (PBMC)-conditioned medium by electrochemiluminescence method (ECL). We demonstrate that this approach allows to detect cytokine concentration from 1 pg/ml. The high sensitivity in combination with accuracy and wide range of determined concentration indicates that ECL meets the standards of quantitative analysis of cytokines. Simplicity and short time of procedure, small assay volume and high reproducibility make ECL method competitive in practical use with conventional quantitative methods of cytokine detection.

Cytokine-Synthesizing Activity of Erythroid Cells.

The presence of mRNA of cytokines IL-1alpha, IL-1beta, IL-4, IL-6, IFN-gamma, TGF-beta, GM-CSF and the absence of mRNA of IL-2, IL-3 and IL-5 were identified in erythroid cells isolated from spleen of mice subjected to erythropoiesis-stimulating actions (treated with phenylhydrazine, acute hypoxia) and from spleen of newborn mice. Gene expression of cytokines in erythroid cells therewith differed both qualitatively and quantitatively depending on erythropoiesis-stimulating action. Erythroid cells of newborn mice contained mRNA of GM-CSF, but did not contain mRNA of IFN-gamma, whereas erythroid cells of mice under acute hypoxia or treated with phenylhydrazine had mRNA of IFN-gamma, but no mRNA of GM-CSF. After cell cultivation with erythropoietin, qualitative and quantitative changes in gene expression of cytokines were observed including the appearance of mRNA of IL-2, IL-3 but not of IL-5. The fact of gene expression of cytokines was confirmed on cells from single erythroid colonies. The level of mRNA expression of cytokines in erythroid cells was comparable with, and for a number of cytokines even higher than in splenocytes of mice immunized with T-dependent antigen. Moreover, it was demonstrated that erythroid cells are capable of translating proteins themselves. The production of GM-CSF and IFN-gamma by erythroid cells extracted from spleen of newborn mice, as well as from mice after erythropoiesis-stimulating actions (treatment with phenylhydrazine, acute hypoxia, acute blood loss) was detected. The level of IFN-gamma production was comparable with the level of production of this cytokine by mitogen-stimulated splenocytes. Thus, erythroid cells, as well as the cells of practically all hemopoietic lineages are capable to express and produce a number of hemo- and immunoregulatory cytokines by means of which they can participate in the regulation of hemo- and immunopoiesis, as well as exert a self-regulatory lineage restricted effect on cell proliferation and differentiation.