Raja S. Mahidhara

Carbon Monoxide Inhibits T Lymphocyte Proliferation via Caspase-Dependent Pathway

T lymphocyte activation and proliferation is involved in many pathological processes. We have recently shown that carbon monoxide (CO), an enzymatic product of heme oxyenase-1 (HO-1), confers potent antiproliferative effects in airway and vascular smooth muscle cells. The purpose of this study was to determine whether CO can inhibit T lymphocyte proliferation and then to determine the mechanism by which CO can modulate T lymphocyte proliferation. In the presence of 250 parts per million CO, CD3-activated T lymphocyte proliferation was, remarkably, inhibited by 80% when compared with controls. We observed that the antiproliferative effect of CO in T lymphocytes was independent of the mitogen-activated protein kinase or cGMP signaling pathways, unlike what we demonstrated previously in smooth muscle cells. We demonstrate that CO inhibited caspase-3 and caspase-8 expression and activity, and caspase inhibition with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK pan-caspase inhibitor) blocked T lymphocyte proliferation. Furthermore, in caspase-8-deficient lymphocytes, the antiproliferative effect of CO was markedly attenuated, further supporting the involvement of caspase-8 in the antiproliferative effects of CO. CO also increased the protein level of p21Cip1, and CO-mediated inhibition of caspase activity is partially regulated by p21Cip1. Taken together, these data suggest that CO confers potent antiproliferative effects in CD3-activated T lymphocytes and that these antiproliferative effects in T lymphocytes are mediated by p21Cip1-dependent caspase activity, in particular caspase-8, independent of cGMP and mitogen-activated protein kinase signaling pathways.

Apoptosis in sepsis.

Sepsis demonstrates a marked dysregulation of the immune system in its ability to fight infection. Previous models have focused on the mechanisms which upregulate and sustain the heightened immune response without addressing the role of down-regulation effectors. Attention has been drawn to these down-regulating mechanisms and their precise role in the pathophysiology of sepsis. Apoptosis is an evolutionarily conserved, energy-dependent mode of cell death requiring the initiation and regulation of complex genetic programs. It is the bodys main method of getting rid of cells which are in excess, damaged, or no longer needed in a controlled manner. The role of this cellular phenomenon in physiology and pathophysiology has been the subject of intense scrutiny over the last decade. Much work has demonstrated that dysregulation of apoptosis does occur in immune and nonimmune cells in in vitro and in vivo models of sepsis. The difficulty has been in tying the phenomenology of apoptosis into the pathophysiology of sepsis. Further work is needed to make these connections to elucidate rational approaches for clinical applications of immunomodulation in sepsis.

Overexpression of mutated IκBα inhibits vascular smooth muscle cell proliferation and intimal hyperplasia formation

Abstract Purpose Vascular injury and inflammation are associated with elaboration of a number of cytokines that signal through multiple pathways to act as smooth muscle cell (SMC) mitogens. Activation of the nuclear factor–kappa B (NF-κB) transcription factor is essential for SMC proliferation in vitro and is activated by vascular injury in vivo. Activation of NF-κB is controlled by several upstream regulators, including the inhibitors of kappa B (IκB). These proteins bind to and keep NF-κB inactivated. The purpose of this study was to determine whether adenoviral gene transfer of a mutated IκBα super-repressor (AdIκBα SR ) could inhibit development of intimal hyperplasia in vivo and to investigate how over-expression of this construct influences in vitro SMC proliferation and cell cycle regulatory proteins. Methods A rat carotid injury model was used to study prevention of intimal hyperplasia. Arteries were assayed 14 days after injury and infection with AdIκBαSR or adenoviral β-galactosidase (AdLacZ). Untreated SMC or SMC infected with AdLacZ or AdIκBαSR were stimulated with 10% fetal bovine serum, interleukin-1β, or tumor necrosis factor-α. Electrophoretic mobility shift assays were used to assay for NF-κB activation. Protein levels of IκBα and cyclin-dependent kinase inhibitors p21 Cip1/Waf1 and p27 Kip1 were determined with Western blot analysis. Proliferation was measured with 3 H-thymidine incorporation assays. Results AdIκBαSR inhibited the development of intimal hyperplasia by 49% ( P Cip1/Waf1 and p27 Kip1 protein levels. Conclusions Gene transfer of IκBα super-repressor inhibited development of intimal hyperplasia in vivo and SMC proliferation in vitro. The antiproliferative activity may be related to cell cycle arrest through upregulation of the cyclin-dependent kinase inhibitors p21 and p27. Overexpression of IκBα may be a future therapeutic option in treatment of vascular diseases.

Differential modulation of CD4 and CD8 T-cell proliferation by induction of nitric oxide synthesis in antigen presenting cells

Background. On antigenic stimulation, CD4 T cells generally proliferate more readily than CD8 T cells. The purpose of the present experiments was to determine whether nitric oxide (NO) might differentially modulate CD4 vs. CD8 T-cell proliferation. Methods. Various concentrations of C57BL/6 iNOS +/+ and −/− bone marrow (BM)-derived antigen presenting cells (APC) (obtained by culture in granulocyte-macrophage colony-stimulating factor [GM-CSF] and interleukin [IL]-4) were cultured with purified BALB/c CD4 or CD8 T cells. Results. Proliferation of CD4 T cells was similar in the presence of both NO synthase (iNOS) +/+ and −/− APC, whereas CD8 T cell proliferation was inhibited at the higher concentrations of iNOS +/+ dendritic cells (DC), coincident with increased levels of NO2 in the culture supernatant. Analysis of cytokine levels revealed that more interferon (IFN)-&ggr;, a potent inducer of NO synthesis in many cell types, was present in CD8 T cell than in CD4 T-cell–APC cultures. Addition of IFN-&ggr; to CD4 T-cell–APC cultures resulted in induction of NO synthesis and inhibition of proliferation at higher levels of NO than that required to inhibit CD8 T cell proliferation. However, CD4 T-cell proliferation was moderately inhibited in the presence of lipopolysaccharide (LPS)-stimulated CD11c+ DC, coincident with production of IFN-&ggr; and induction of NO synthesis. Conclusions. These findings indicate that CD8 T-cell proliferation can be inhibited by lesser amounts of APC-derived NO than is necessary to inhibit CD4 T cell proliferation. NO synthesis was not initiated in CD4 T cell–DC cultures unless costimulatory molecules were up-regulated and IFN-&ggr; was produced.

Nitric oxide-mediated inhibition of caspase-dependent T lymphocyte proliferation

Nitric oxide (NO), a pleiotropic signaling molecule produced at sites of inflammaion, is a powerful inhibitor of lymphocyte proliferation. Caspases, central effector proteases in apoptosis, have recently been implicated as critical mediators of T cell activation. We and others have shown that NO can inhibit caspases by S‐nitrosylation, which is reversible by the reducing agent dithiothreitol (DTT). The purpose of the present study was to determine whether NO inhibits lymphocyte proliferation by modulating caspase activity. Caspase inhibition with z‐VAD‐fmk blocked T cell proliferation. NO‐dependent inhibition of T cell proliferation was associated with an inhibition of caspase activity and activation, and this effect was reversible by DTT. Previous studies demonstrated inhibition of apoptosis through S‐nitrosylation of caspases; the present studies extend this effect to inhibition of caspase‐dependent T cell proliferation.

Interferon Regulatory Factor 1 (IRF-1) induces p21WAF1/CIP1 dependent cell cycle arrest and p21WAF1/CIP1 independent modulation of survivin in cancer cells

We have shown that the ectopic expression of Interferon Regulatory Factor 1 (IRF-1) results in human cancer cell death accompanied by the down-regulation of the Inhibitor of Apoptosis Protein (IAP) survivin and the induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). In this report, we investigated the direct role of p21 in the suppression of survivin. We show that IRF-1 down-regulates cyclin B1, cdc-2, cyclin E, E2F1, Cdk2, Cdk4, and results in p21-mediated G1 cell cycle arrest. Interestingly, while p21 directly mediates G1 cell cycle arrest, IRF-1 or other IRF-1 signaling pathways may directly regulate survivin in human cancer cells.

Pan-Caspase Inhibitor Prevents Anti-CD3 Mediated T-Lymphocyte Proliferation

BACKGROUND. Host defense requires the ability for upregulation and downregulation of lymphocyte responses. It is well known that proliferative responses can be seen in conjunction with subsequent cell death suggesting that the two processes are linked.(1) Caspases are constitutively expressed zymogens which, upon activation, participate in receptor and nonreceptor mediated apoptosis.(2) The purpose of this study was to assess whether caspase activity was required for the proliferation of splenic-derived T lymphocytes by using the nonspecific, irreversible inhibitor z-VAD-fmk. METHODS. Splenocytes from C57Bl/6 mice were passed over an antibody coated column (R&D) to obtain a >90% CD3+ population. Cells were stimulated to proliferate with immobilized monoclonal anti-CD3 (BD Pharmingen, 100µg/ml) in 10% MLC media. Proliferation was assessed by 3 H thymidine incorporation at time points with and without zVAD-fmk (Alexis, 100µM). Western Blot analysis was performed by SDS-PAGE and probed with polyclonal Caspase 3 antibody H-277 (Santa Cruz, 1:1000). Caspase 3-like activity was assessed by 100µM Ac-DEVD-pNA (Alexis) substrate followed by quantitation by spectrophotometry at 405nm. RESULTS. TCR stimulation of purified T lymphocytes at 72 hours resulted in proliferation 500 fold greater than unstimulated controls. Treatment of lymphocytes with zVAD inhibited CD3 mediated proliferation by greater than 99%. Delay in the administration of inhibitor after stimulation decreased the effect on proliferation (see Figure 1).