Effect Of CD40 Ligand Research Articles

Lycorine sensitizes CD40 ligand-protected chronic lymphocytic leukemia cells to bezafibrate- and medroxyprogesterone acetate-induced apoptosis but dasatanib does not overcome reported CD40-mediated drug resistance

Tumor cells in chronic lymphocytic leukemia accumulate in the periphery through the proliferation of a minority of cells in lymph nodes. The proliferative and survival signals in these proliferation centers include interactions with T lymphocytes expressing CD40 ligand. We have demonstrated that the low toxicity combination of bezafibrate and medroxyprogesterone acetate induces mitochondrial superoxide-mediated apoptosis of non-CD40-liganded cells but not of cells exposed to CD40 ligand. Here, we assessed the ability of dasatinib and lycorine to restore bezafibrate- and medroxyprogesterone acetate- induced apoptosis in cells exposed to CD40 ligand. In parallel experiments we compared the ability of dasatinib to induce apoptosis of cells co-treated with fludarabine. Primary chronic lymphocytic leukemia and peripheral blood mononuclear cells were exposed to drug combinations for 72 hours on control and CD40 ligand-expressing fibroblast monolayers. Cells were harvested and analyzed for apoptosis and levels of mitochondrial superoxide using flow cytometry. In some experiments cells were removed from CD40 ligand at 48 hours, retreated and analyzed after a further 24 hours. The effect of CD40 ligand and drug treatments on mitochondrial superoxide levels were assessed. As previously described, dasatinib rendered cells sensitive to fludarabine but only when CD40 ligand was removed for the last 24 hours of culture. In contrast, lycorine restored the bezafibrate- and medroxyprogesterone acetate-induced apoptosis associated with mitochondrial superoxide even during continuous exposure to CD40 ligand. Furthermore, combined bezafibrate, medroxyprogesterone acetate and lycorine had little effect against normal peripheral blood mononuclear cells, whereas dasatinib with fludarabine induced high levels of apoptosis. Our data indicate the potential of bezafibrate, medroxyprogesterone acetate and lycorine as novel therapy in chronic lymphocytic leukemia and have important implications for the reported potential of c-abl kinase inhibitors in this disease.

IL-21 Enhances Antitumor Responses without Stimulating Proliferation of Malignant T Cells of Patients with Sézary Syndrome

IL-21, a common gamma-chain cytokine secreted by activated CD4+ T cells, influences both humoral and cell-mediated immune responses through the regulation of T, B, dendritic, and natural killer (NK) cells. Sézary syndrome is an advanced form of cutaneous T-cell lymphoma, a clonally derived malignancy of CD4+ T cells that is characterized by profound defects in host cellular immune function. As a modulator of both innate and adaptive immune responses, IL-21 could play an important role in augmenting cell-mediated immunity in these patients. Normal donor and Sézary syndrome patient peripheral blood mononuclear cells were cultured with IL-21 and tested for CD8+ T- and NK-cell activation, NK-cell cytotoxicity, and tumor cell proliferation and apoptosis. IL-21 resulted in a modest increase in CD8+ T- and NK-cell activation, associated with a marked increase in cytolytic activity against both K562 and malignant CD4+ T-cell targets. Although IL-21 failed to demonstrate pro-apoptotic effects on the malignant CD4+ T cells, it is noteworthy that it had no demonstrable proliferative effects on these cells. Thus, IL-21 may play an important role in enhancing the host immune response of Sézary syndrome patients through the increased cytolytic activity of T and NK cells.

P-selectin, tissue factor and CD40 ligand expression on platelet-leucocyte conjugates in the presence of a GPIIb/IIIa antagonist

This study was to investigate the appearance of P-selectin, tissue factor (TF) and CD40 ligand (CD40L) on platelet-leucocyte conjugates in the absence and presence of a GPIIb/IIIa antagonist, MK-852, and the effect of adding EDTA to pre-formed conjugates. The purpose was to find out whether these antigens are displaced from the conjugates along with the platelets, thus providing information on their location. Hirudinized blood was stirred with collagen ((2 μg/mL) in the absence and presence of MK-852 (10 μmol/mL). P-selectin, TF and CD40L were measured on platelet-leucocyte conjugates (CD42a positive monocytes and neutrophils) and on single platelets by flow cytometry. Measurements were also made after subsequent addition of EDTA (4 mmol/L). Platelet-leucocyte conjugate formation was markedly enhanced in the presence of MK-852. P-selectin, TF and CD40L expression on the conjugates was also enhanced. Monocytes bound more platelets and expressed more P-selectin, TF and CD40L than neutrophils. EDTA displaced the majority of platelets from the conjugates and also the P-selectin, TF and CD40L, whereas it did not displaced P-selectin or CD40 ligand from platelets themselves. It is concluded that a GPIIb/IIIa antagonist promotes formation of platelet-leucocyte conjugates, which display P-selectin, TF and CD40L that appears to be associated with the adherent platelets. Platelet-monocyte conjugates are prime candidates for arterial inflammation and thrombosis. Pro-inflammatory and pro-thrombotic effects of CD40L and tissue factor may be an explanation of the negative clinical effects using GPIIb/IIIa antagonists.

Sézary syndrome patients demonstrate a defect in dendritic cell populations: effects of CD40 ligand and treatment with GM-CSF on dendritic cell numbers and the production of cytokines

AbstractSézary syndrome (SzS) is an advanced form of cutaneous T-cell lymphoma associated with involvement of the peripheral blood by malignant T cells. The disease is defined by impaired cell-mediated immunity and the production of interferon-γ (IFN-γ) and interleukin-2 (IL-2), possibly as a result of deficient IL-12 production. To understand the mechanism of this impairment, we examined the composition and function of dendritic cells and monocytes in the blood of SzS patients with different levels of peripheral blood tumor burden. Consistent with our previous observations, numbers of monocytes in SzS patients were comparable to numbers observed in healthy donors. In contrast, decreased IL-12 production correlated with a decrease in the numbers of CD11c+ dendritic cells, which was particularly profound among patients with medium (20%-50% circulating malignant T cells) and high (more than 50% circulating malignant T cells) tumor burden. Furthermore, CD123+ dendritic cells, major producers of IFN-α, were significantly diminished in SzS patients, regardless of the level of tumor burden. Granulocyte macrophage–colony-stimulating factor–treated patients experienced an increase in the number of dendritic cells but not in IFN-α or IL-12 production. However, in vitro stimulation of peripheral blood mononuclear cells from SzS patients with rCD40L and IFN-γ significantly increased the production of IL-12. Thus, our results demonstrate a profound defect in circulating dendritic cells in SzS patients that may contribute to the pathogenesis of the cytokine disorders and to the depressed cellular immunity. Importantly, the ability of rCD40L to potently induce IL-12 production from monocytes and residual dendritic cells of SzS patients could potentially serve as an immune-restorative therapeutic agent.

Effect of CD40 ligand on the course of murine histoplasmosis

Effect of CD40 ligand on the course of murine histoplasmosis

Effect of CD40 ligand on the course of murine histoplasmosis.

CD40 ligand-CD40 ligation is important in the development of T-cell-mediated immune responses. The purpose of this study was to examine the role of CD40L in recovery from histoplasmosis using a murine model of intratracheally induced infection. B6C3F1 mice were infected intratracheally with Histoplasma capsulatum yeast and monitored for clearance of the organism from the lungs and spleen. CD40L treatment was begun on either day -2 or +2 post inoculation and continued until day 14 in CD4-depleted animals and from day -2 to day +4 in non-immunosuppressed animals. Amphotericin B treatment was begun four days following inoculation and given every other day for 10 days. CD40L reduced fungal burden by less than one log when started two days before infection but did not act synergistically with low-dosage amphotericin B (0.2 mg kg(-1) qod) in CD4 depleted mice. Low-dose amphotericin B, CD40L, and the combination of the two failed to lower the fungal burden in a second experiment using a more virulent isolate of the same strain of H. capsulatum in CD4-depleted mice. Furthermore, CD40L did not increase the concentrations of IFN-gamma, IL-12 or IL-10 in the lungs or spleens of infected animals. In summary, CD40L had minimal or no effect on the course of infection in this murine model of histoplasmosis.

Effect of CD40 ligand and other immunomodulators on Pneumocystis carinii infection in rat model

The corticosteroid-treated animal is well established as an experimental model for the study ofPneumocystis carinii pneumonitis (PCP). Latent or acquired infection with P. carinii in the murine lung progresses to fatal pneumonitis when the host is profoundly immunocompromized. In this study the effects of five immunomodulators; recombinant CD40 ligand (CD40L), bryostatin 1, recombinant FLT3 ligand (FLT3L), recombinant granulocyte colony-stimulating factor (G-CSF) and recombinant interleukin-15 (IL-15) were investigated against PCP in a dexamethasone immunosuppressed Sprague–Dawley rat model. The majority of rats (70%) treated with CD40L at the onset of dexamethasone immunosuppression were protected against PCP. When CD40L was given after 10 days of immunosuppression, only 40% of the rats resolved the infection. However, 95% of the control animals developed PCP. Immunosuppressed rats treated with bryostatin 1, an immune activator had a partial (50%) protection against P. carinii infection. In contrast, daily administration of FLT3L, IL-15 or G-CSF provided no protection against P. carinii infection.

Maintenance of nuclear factor-kappa B/Rel and c-myc expression during CD40 ligand rescue of WEHI 231 early B cells from receptor-mediated apoptosis through modulation of I kappa B proteins.

Engagement of surface IgM (sIgM) on WEHI 231 murine B lymphoma cells leads to abortive activation and apoptosis, suggesting that this cell line may represent a model for tolerance. Loss of viability in these cells is preceded by an early increase in c-myc RNA levels followed by a decline below control values, implicating c-myc in the control of apoptosis. Costimulation with CD40 ligand (CD40L) has been shown to rescue WEHI 231 cells from anti-sIgM-induced apoptosis, and therefore, the effects of CD40L on c-myc RNA and protein levels were measured. Treatment of these cells with the combination of CD40L and anti-sIgM led to induction and maintenance of elevated levels of c-myc RNA and protein. Since transcriptional regulation of c-myc is mediated through two nuclear factor-kappa B (NF-kappa B) sites in WEHI 231, the effects of CD40L on DNA binding by this family of transcription factors were evaluated. CD40L induced and sustained the levels of NF-kappa B binding to both of these sites, paralleling the changes in c-myc RNA levels. Elevated levels of NF-kappa B were partially achieved through a sustained decrease in the steady state amount of the NF-kappa B/Rel-specific inhibitory protein, I kappa B alpha, and a transient decrease in I kappa B beta. These data lend support to the hypothesis that anti-sIgM-induced apoptosis is caused by a drop in c-myc expression and that an appropriate second signal, such as that provided by CD40L, is able to rescue these cells by inducing NF-kappa B and thereby maintaining c-myc RNA levels.