To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% ؎ 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 com- IntroductionIdiopathic, or immune, thrombocytopenic purpura (ITP) is an autoimmune disease characterized by isolated thrombocytopenia in an otherwise healthy person. The thrombocytopenia in ITP is caused by accelerated platelet destruction due to the action of antiplatelet immunoglobulin G (IgG) autoantibodies that bind to antigens on the platelet cell membrane. The platelets are subsequently destroyed by tissue macrophages, predominantly in the spleen. 1 As a result of the accelerated destruction, platelet survival is usually greatly shortened and platelet production is thought to be compensatorily increased. 2,3 However, there is also evidence that platelet production can be impaired in ITP. This was demonstrated in platelet kinetic studies using radiolabeled platelets. [4][5][6] The reduced platelet production rate might be mediated by the action of antiplatelet antibodies, which can bind to megakaryocytes in the bone marrow. [7][8][9] Recent in vitro studies support this concept showing that human megakaryocyte colony formation and proplatelet formation is inhibited 10 and that a reduced expansion of megakaryocytic progenitors can be observed especially in the presence of certain antiplatelet glycoprotein antibodies. 11 However, despite the evidence of a reduced platelet production in several ITP patients, numbers of megakaryocytes in the bone marrow are usually normal or increased. 6 This is compatible with the finding that plasma thrombopoietin (TPO) levels in ITP patients are not significantly different from healthy controls, indicating that the total megakaryocytic mass has not been changed in ITP. Investigating the relationship between thrombokinetic parameters and the glycocalicin index (GCI), a parameter of platelet destruction, 12 we recently demonstrated that there is an inverse correlation between the platelet production rate and the GCI. 13 These results suggest that despite the normal number of megakaryocytes in the bone marrow an increased destruction of platelets and/or megakaryocytes might occur. These findings support the concept of ineffective thrombopoiesis in the bone marrow. To investigate whether apoptosis or other forms of programmed cell death are responsible for this ineffective thrombopoiesis, we examined the ultrastructure of bone marrow megakaryocytes from ITP patients with electron microscopy. The results demonstrate that, independent of the refractoriness of ITP to therapy, in all patients most bone ma...

Cytokine gene expression in T lymphocytes is a strictly regulated process, involving both stimulatory and inhibitory signals. beta-Adrenoceptor (betaAR) agonists are widely used in the treatment of asthma and are able to induce an inhibitory signal on immunological responses after binding to their specific receptors. In this study, the characterization of betaAR subtype(s) (beta1, beta2, and beta3) involved in the regulation of interleukin (IL)-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma (IFN-gamma) mRNA accumulation was studied by using various betaAR agonists and antagonists. Concanavalin A (Con A)-induced IFN-gamma, GM-CSF, and IL-3 mRNAs are dose-dependently inhibited by the nonselective betaAR agonist isoproterenol and by the selective beta2AR agonist fenoterol. IL-4 mRNA accumulation was not susceptible to betaAR stimulation. The observed inhibition on IFN-gamma, GM-CSF, and IL-3 mRNA was blocked by the selective beta2AR antagonist ICI 118,551 (10(-6) M) and by timolol (10(-6) M), a nonselective antagonist. The selective beta1AR antagonist atenolol (0.3 x 10(-6) M) did not have any effect. Secretion of GM-CSF protein in the presence of increasing concentrations of isoproterenol followed a similar pattern as observed for GM-CSF mRNA. In addition, the betaAR-mediated inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation and GM-CSF protein secretion were related to the accumulation of intracellular cyclic adenosine monophosphate (cAMP) levels. Although beta3AR mRNA was detectable in Con A-activated T lymphocytes, we could not demonstrate a functional activity in the regulation of cytokine expression: the beta3AR agonist BRL 37344 had no effect on the accumulation of the studied cytokine mRNAs, and did not significantly affect cellular cAMP levels. These data demonstrate that beta-agonist-induced inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation is solely mediated by beta2-adrenoceptors.

The influence of antibiotic-induced release of endotoxin from in-vitro grown Escherichia coli on the production of tumour necrosis factor-alpha (TNF) by human monocytes was studied. Antibiotics tested were: cefuroxime (7.5 and 75 mg/L); ceftazidime (10 and 100 mg/L); aztreonam (10 and 100 mg/L); imipenem (10 and 100 mg/L); and tobramycin (8 mg/L). The effect of the combination of cefuroxime plus tobramycin, and the effect of taurolidine, an endotoxin-binding agent, on TNF production was also tested. After incubation for 4 h, all antibiotic-treated cultures (high-dose) induced a similar rise in extracellular TNF production when compared to the controls. However, after incubation for 24 h, a significant rise in TNF production was noticed in the cefuroxime and aztreonam-treated cultures (6440 and 5969 ng/L, respectively) compared to the ceftazidime and imipenem-treated cultures (846 and 381 ng/L, respectively). The cefuroxime-induced release of TNF could be reduced by addition of tobramycin (from 6440 to 1615 ng/L). Similar differences in TNF production were noticed in cell-associated TNF. Dose-response curves did not demonstrate differences in TNF production in aztreonam or imipenem-treated cultures. However, for both cefuroxime and ceftazidime-treated cultures, low-dose treatment resulted in significantly higher production of TNF. The differences in TNF production between these antibiotics could be explained by the production of filaments following treatment with cefuroxime, aztreonam and low-dose ceftazidime, resulting in late bacterial lysis with high levels of endotoxin, whereas treatment with imipenem or high-dose ceftazidime resulted in the formation of spheroplasts, resulting in early lysis of the bacteria and much lower levels of endotoxin. The addition of taurolidine to either imipenem or aztreonam-treated cultures prevented a rise in TNF production as a result of nearly complete neutralization of the released endotoxin. It was concluded that the observed differences in TNF production by human monocytes in vitro were related to differences in the mechanisms and amount of antibiotic-induced release of endotoxin.

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-B (NF-B) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-B, might determine the final cellular response.