Subcellular mobility of the calpain/calpastatin network: an organelle transient.

Calpain (Cp) is a calcium (Ca(2+))-dependent cysteine protease. Activation of the major isoforms of Cp, CpI and CpII, are required for a number of important cellular processes including adherence, shape change and migration. The current concept that cytoplasmic Cp locates and associates with its regulatory subunit (Rs) and substrates as well as translocates throughout the cell via random diffusion is not compatible with the spatial and temporal constraints of cellular metabolism.

Tubulin polymerization modulates interleukin-2 receptor signal transduction in human T cells.

Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals.

Atorvastatin affects interleukin-2 signaling by altering the lipid raft enrichment of the interleukin-2 receptor beta chain.

Although the immunomodulatory properties of statins are in part independent of their lipid-lowering effects, cholesterol is a major component of lipid rafts. We therefore studied the effects of atorvastatin (AS) on the raft enrichment of the interleukin-2 receptor (IL-2R) beta chain previously described by us and on early IL-2R signaling events in activated human T cells. We found that concomitant AS exposure during a 3-day stimulation with phytohemagglutinin (PHA) attenuates activation-associated events, such as the enhanced surface expression of the raft marker GM-1 and the induced expression of the activation marker CD25 (the IL-2R alpha chain).

Daclizumab therapy in kidney transplantation-different mechanisms of action in- versus ex-vivo?

Stimulated human T cells from healthy volunteers demonstrate attenuated early interleukin (IL)-2 receptor (R) signaling in the presence of daclizumab (Dac). Aiming to confirm that this ex-vivo effect of Dac is also observed in-vivo, we studied T cells from 3 kidney transplant recipients before and 2-3 weeks and 4-6 months after transplantation. We found by flow cytometry that T cells obtained pre-transplant and stimulated ex-vivo with phytohemeagglutinine upregulated the IL-2R alpha-(CD25) and beta-(CD122) chains as expected.

Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus.

Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit.

Lipid rafts in cytokine signaling.

Lipid rafts are established as critical structures for a variety of cellular processes, including immune cell activation. Beyond their importance for initial immune cell activation at the immunological synapse, lipid rafts are now also being recognized as important sites for cytokine and growth factor signal transduction, both in immune cells as part of secondary regulatory processes, and in non-immune cells. This review summarizes current knowledge regarding the roles of rafts in cytokine signaling and emphasizes the need for measures to better standardize the study of rafts.

Association of the calpain/calpastatin network with subcellular organelles.

The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus.

Lipid rafts, major histocompatibility complex molecules, and immune regulation.

Glycolipid-enriched membrane microdomains ("rafts") are critical sites for signal transduction and other processes such as intracellular transport. While the participation of T-cell rafts in the formation of the immunological synapse is well established, the role of rafts on antigen-presenting cells (APCs) as well as the relationship between these domains and major histocompatibility complex (MHC) molecules is less clearly defined. We therefore investigated whether MHC class I or II molecules are found in rafts of the human macrophage-monocytic cell line U937.

Calpain II colocalizes with detergent-insoluble rafts on human and Jurkat T-cells.

Calpain, a calcium-dependent cysteine protease, is known to associate with the T-cell plasma membrane and subsequently cleave a number of cytoskeletal-associated proteins. In this study, we report the novel observation that calpain II, but not calpain I, associates with membrane lipid rafts on human peripheral blood T-cells and Jurkat cells. Raft-associated calpain activity is enhanced with exogenous calcium and inhibited with calpeptin, a specific inhibitor of calpain activity.

Differential localization of IL-2- and -15 receptor chains in membrane rafts of human T cells.

We studied whether cytokine receptors (Rs) on T cells associate with lipid microdomains ("rafts"). Low-dose phytohemagglutinin (PHA)-stimulated human T cells were separated into cytoplasmic, membrane, and raft fractions by buoyant density centrifugation. Examination of these fractions for the presence of interleukin (IL)-2- and -15R chains and associated signaling molecules by Western blotting revealed marked, selective enrichment of the IL-2/15R beta-chain in rafts before IL-2 stimulation.

Interstitial nephritis, hepatic failure, and systemic eosinophilia after minocycline treatment.

This report describes a 15-year-old white boy who presented with fever, back pain, a disseminated exanthematous rash, renal failure, and hepatopathy 3 weeks after the initiation of oral minocycline therapy for facial acne. Marked peripheral and urine eosinophilia were noted. A bone marrow aspiration showed more than 50% eosinophils without any evidence of malignancy, and a simultaneous kidney biopsy showed acute interstitial nephritis (AIN).

Daclizumab (Zenapax) inhibits early interleukin-2 receptor signal transduction events.

Daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor (R) alpha-chain, is a promising new immunosuppressant in transplantation. As its exact mechanism of action has remained unclear, we examined its short-term effects on primary human T lymphocytes expressing the high-affinity IL-2R. Daclizumab exposure for 20 min neither affected T cell viability nor their surface expression of the IL-2R alpha-, beta-, or gamma-chains.

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain.

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology.

Immune defects observed in patients with primary malignant brain tumors.

Malignant glioblastomas (gliomas) account for approximately one third of all diagnosed brain tumors. Yet, a decade of research has made little progress in advancing the treatment of these tumors. In part this lack of progress is linked to the challenge of discovering how glial tumors are capable of both modulating host immune function and neutralizing immune-based therapies.

Apoptotic elimination of peripheral T lymphocytes in patients with primary intracranial tumors.

Object: Patients with gliomas exhibit severe T lymphopenia during the course of the disease. This study was conducted to determine the mechanism(s) responsible for the lymphopenia.

Methods: Using two-color fluorescent staining techniques, the authors show that significant numbers of T cells undergo apoptosis in the peripheral blood of patients with gliomas.

Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers.

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10.

Calcium-dependent signaling pathways in T cells. Potential role of calpain, protein tyrosine phosphatase 1b, and p130Cas in integrin-mediated signaling events.

Engagement of beta1 integrin receptors initiates an increase in intracellular calcium concentrations in T cells, potentially affecting calcium-sensitive signaling pathways. The calcium-activated cysteine protease, calpain, regulates a variety of cell functions by calcium-dependent limited proteolysis. To investigate the function of calpain in T cells, we sought to determine the role of this protease in calcium-dependent signaling events.

T cell receptor-mediated signaling is defective in T cells obtained from patients with primary intracranial tumors.

It has been well established that patients with malignant glioblastomas exhibit T cell anergy. In this report, we further investigate the nature of this T cell anergy. The results demonstrate that tumor size but not location correlates with decreased mitogen or anti-CD3 mAb responsiveness of T cells obtained from patients.

Insulin-like growth factors (IGF) enhance three-dimensional (3D) growth of human glioblastomas.

Human glioblastomas (gliomas) are characterized as rapidly growing brain tumors which are highly invasive but rarely metastatic. Human gliomas synthesize and secrete increased levels of insulin-like growth factors (IGFs) as well as expressing increased numbers of IGF receptors when compared to normal brain tissue. These observations suggest the existence of an IGF-mediated autocrine mechanism for glioma growth regulation.

Proteolytic cleavage of alpha-actinin by calpain in T cells stimulated with anti-CD3 monoclonal antibody.

Stimulation of the TCR/CD3 complex on T cells initiates rearrangement of the actin cytoskeleton. The results presented show that a temporal increase in the appearance of filamentous actin begins immediately after stimulation of T cells with immobilized anti-CD3 mAb. The formation of filamentous actin in these stimulated cells reaches a steady state within 30 min after anti-CD3 mAb stimulation.

Dexamethasone and prostaglandin E2 modulate T-cell receptor signaling through a cAMP-independent mechanism.

One possible explanation for the link between stress and increased incidence of infection can be attributed to concomitant increases in levels of glucocorticoids (GS) and prostaglandin E2 (PGE2), both of which possess potent immunoregulatory activities. We have previously demonstrated that concentrations of PGE2 and the synthetic glucocorticoid, dexamethasone (DEX), which individually do not inhibit human T-cell responsiveness to anti-CD3 monoclonal antibody (mAb), act synergistically to inhibit IL-2 secretion and subsequent T-cell proliferation. In the present paper, we demonstrate that treatment of anti-CD3 mAb-stimulated T-cells with low (10(-8) and 10(-9) M) concentrations of DEX and PGE2 results in the inhibition of steady-state levels of IL-2 mRNA.

Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells.

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind 125I-IGF-I with an affinity of 1.77 nM (Kd) and express approximately 230 receptors/cell.

cAMP accumulation in T-cells inhibits anti-CD3 monoclonal antibody-induced actin polymerization.

The results presented in this report offer a novel explanation for how stimulation of the beta-adrenergic receptor (beta AR) inhibits the ability of T cells to proliferate after interaction with immobilized anti-CD3 monoclonal antibody (mAb). Accordingly, T cells binding to immobilized anti-CD3 mAb but not anti-CD4 mAb undergo time-dependent F-actin assembly with concomitant formation of pseudopodia. This process is completely inhibited in the presence of isoproterenol (ISO) indicating that stimulation of the beta AR on T cells interferes with the biochemical processes responsible for the assembly of actin.

Induction of cAMP-dependent protein kinase (PKA) activity in T cells after stimulation of the prostaglandin E2 or the beta-adrenergic receptors: relationship between PKA activity and inhibition of anti-CD3 monoclonal antibody-induced T cell proliferation.

Recently, we have shown that T cells exposed to concentrations of prostaglandin E2 (PGE2) or the beta-adrenergic receptor agonist isoproterenol (ISO) that elicit equimolar levels of cAMP accumulation do not inhibit anti-CD3 monoclonal antibody-induced T cell proliferation to the same extent. This report extends these studies by investigating the induction of cAMP-dependent protein kinase (PKA) in T cells stimulated with PGE2 or ISO. The kinetics of PKA activity induced by PGE2 or ISO in T cells are similar but PGE2 induces more PKA activity.