Kathy Forrest

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. However, after IL-2 stimulation (200 U/ml, 20 min), immunoblots of cell lysates demonstrated attenuation of the IL-2-induced tyrosine phosphorylation of 65-75 kDa proteins by Daclizumab, but not by isotype controls. Since this is the molecular weight of the IL-2R beta- and gamma-chains, which are both tyrosine-phosphorylated by IL-2, we next examined the effect of Daclizumab on their IL-2-induced tyrosine phosphorylation. In immunoblots of IL-2R beta- and gamma-chain-immunoprecipitates the tyrosine phosphorylation of both chains by IL-2, but not by IL-15, was attenuated in the presence of Daclizumab. Furthermore, co-immunoprecipitation experiments showed that Daclizumab inhibited the IL-2-induced association of these chains, a prerequisite for their mutual tyrosine phosphorylation. Lastly, we demonstrated that Daclizumab inhibits the receptor-downstream induction of the IL-2-activated DNA-binding protein STAT5 in gel shift assays. We conclude that Daclizumab directly and specifically interferes with IL-2 signaling at the receptor level by inhibiting the association and subsequent phosphorylation of the IL-2R beta- and gamma-chains induced by ligand binding. Under our experimental conditions, Daclizumab had no effects on cell viability, and it did not modulate the surface expression of the IL-2R alpha-, beta-, or gamma-chains.

The capacity of Group V sPLA2 to increase atherogenicity of apoE-/- and LDLR-/- mouse LDL in vitro predicts its atherogenic role in vivo

Objective—In vitro data indicate that human LDL modified by Group V secretory phospholipase A2 (GV sPLA2) is proatherogenic. Consistent with this, gain and loss of function studies demonstrated that GV sPLA2 promotes atherosclerosis in LDLR−/− mice. The current study investigates whether GV sPLA2 promotes atherosclerotic processes in apoE−/− mice. Methods and Results—LDL (d=1.019 to 1.063) from apoE−/− and LDLR−/− mice fed chow or Western diet were hydrolyzed by GV sPLA2. Phosphatidylcholine on LDL from LDLR−/− mice fed either a chow or Western diet was hydrolyzed to a greater extent (61.1±0.4% and 45.3±4.6%) than the corresponding fractions from apoE−/− mice (41.7±3.6% and 39.4±1.2%). ApoE−/− LDL induced macrophage foam cell formation in vitro without modification by GV sPLA2, whereas hydrolysis of LDLR−/− LDL was a prerequisite for foam cell formation. In contrast to findings in LDLR−/− mice, GV sPLA2 deficiency did not significantly reduce atherosclerosis in apoE−/− mice, although collagen content was significantly reduced in lesions of apoE−/− mice lacking GV sPLA2. Conclusions—The ability of GV sPLA2 to promote atherosclerotic lipid deposition in apoE−/− and LDLR−/− mice may be related to its ability to increase the atherogenic potential of LDL from these mice as assessed in vitro.

Group X secretory phospholipase A2 augments angiotensin II-induced inflammatory responses and abdominal aortic aneurysm formation in apoE-deficient mice

OBJECTIVE Abdominal aortic aneurysm (AAA) is a complex vascular disease characterized by matrix degradation and inflammation and is a major cause of mortality in older men. Specific interventions that prevent AAA progression remain to be identified. In this study, we tested the hypothesis that Group X secretory phospholipase A(2) (GX sPLA(2)), an enzyme implicated in inflammatory processes, mediates AAA. METHODS AND RESULTS GX sPLA(2) was detected by immunostaining in human aneurysmal tissue and in angiotensin II (Ang II)-induced AAAs in apolipoprotein E-deficient (apoE(-/-)) mice. GX sPLA(2) mRNA was increased significantly (11-fold) in abdominal aortas of apoE(-/-) mice in response to Ang II infusion. To define the role of GX sPLA(2) in experimental AAAs, apoE(-/-) and apoE(-/-) x GX sPLA(2)(-/-) (GX DKO) mice were infused with Ang II for either 10 (n=7) or 28 (n=24-26) days. Deficiency of GX sPLA(2) significantly reduced the incidence and severity of AAAs, as assessed by ultrasound measurements in vivo of aortic lumens and by computer-assisted morphometric analyses ex vivo of external diameter. Results from gene expression profiling indicated that the expression of specific matrix metalloproteinases and inflammatory mediators was blunted in aortas from GX DKO mice compared to apoE(-/-) mice after 10-day Ang II infusion. Ang II induction of cyclooxygenase-2, interleukin-6, matrix metalloproteinase (MMP)-2, MMP-13 and MMP-14 was reduced significantly in GX DKO mice compared to apoE(-/-) mice. CONCLUSION GX sPLA(2) promotes Ang II-induced pathological responses leading to AAA formation.

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 β‐chain in rafts before IL‐2 stimulation. After IL‐2 stimulation, a substantial amount of the β‐chain was found in the membrane fraction. This partial translocation was also observed for the β‐chain‐associated molecules JAK‐1, p56lck, and grb‐2. Finally, raft disruption with methyl‐β‐cyclodextrin (MBCD) attenuated IL‐2‐induced tyrosine phosphorylation events and selectively decreased the surface expression of the IL‐2/15R β‐chain detected by flow cytometry. These results show that the IL‐2/15R β‐chain is enriched in rafts obtained from low‐dose, PHA‐stimulated T cells, that IL‐2 binding alters this enrichment, and that this enrichment may be functionally relevant as a possible mechanism to ensure cytokine selectivity and specificity.

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. In addition, we demonstrate that calpain cleaves the cytoskeletal-associated protein, talin, during the first 30-min after cell stimulation. We propose that lipid raft associated-calpain II could function in early TCR signaling to facilitate immune synapse formation through cytoskeletal remodeling mechanisms. Hence, we demonstrate that the positioning of calpain II within T-cell lipid rafts strategically places it in close proximity to known calpain substrates that are cleaved during Ag-specific T-cell signaling and immune synapse formation.

Group X secretory phospholipase A2 negatively regulates adipogenesis in murine models

Studies in vitro indicate that group X secretory phospholipase A2 (GX sPLA2) potently releases arachidonic acid (AA) and lysophosphatidylcholine from mammalian cell membranes. To define the function of GX sPLA2 in vivo, our laboratory recently generated C57BL/6 mice with targeted deletion of GX sPLA2 (GX−/− mice). When fed a normal rodent diet, GX−/− mice gained significantly more weight and had increased adiposity compared to GX+/+ mice, which was not attributable to alterations in food consumption or energy expenditure. When treated with adipogenic stimuli ex vivo, stromal vascular cells isolated from adipose tissue of GX−/− mice accumulated significantly more (20%) triglyceride compared to cells from GX+/+ mice. Conversely, overexpression of GX sPLA2, but not catalytically inactive GX sPLA2, resulted in a significant 50% reduction in triglyceride accumulation in OP9 adipocytes. The induction of genes encoding adipogenic proteins (PPARγ, SREBP‐1c, SCD1, and FAS) was also significantly blunted by 50–80% in OP9 cells overexpressing GX sPLA2. Activation of the liver X receptor (LXR), a nuclear receptor known to up‐regulate adipogenic gene expression, was suppressed in 3T3‐L1 and OP9 cells when GX sPLA2 was overexpressed. Thus, hydrolytic products generated by GX sPLA2 negatively regulate adipogenesis, possibly by suppressing LXR activation.—Li, X., Shridas, P., Forrest, K., Bailey, W., Webb, N. R. Group X secretory phospholipase A2 negatively regulates adipogenesis in murine models. FASEB J. 24, 4313–4324 (2010). www.fasebj.org

Group X Secretory Phospholipase A2 Regulates Insulin Secretion through a Cyclooxygenase-2-dependent Mechanism

Background: Arachidonic acid and its metabolites regulate pancreatic glucose-stimulated insulin secretion (GSIS) through multiple mechanisms. Results: Group X secretory phospholipase A2 (GX sPLA2) suppresses GSIS; suppression was abolished when COX-2 activity or PGE2-EP3 receptor signaling were inhibited. Conclusion: GX sPLA2 inhibits GSIS by augmenting PGE2 production. Significance: GX sPLA2 may be targeted for ameliorating beta cell dysfunction in type 2 diabetes. Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA2 is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA2-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA2 overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA2 (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA2 on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.

Atorvastatin Affects Interleukin-2 Signaling by Altering the Lipid Raft Enrichment of the Interleukin-2 Receptor β Chain

ABSTRACT 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) β 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 α chain). In contrast, brief AS treatment after PHA stimulation increased GM-1 surface expression and virtually abolished the selective raft enrichment of the IL-2R β chain. Although this AS-associated increase in GM-1 expression resembled that seen in the presence of the raft-disrupting cholesterol chelator methyl-β-cyclodextrin (MBCD), the two agents had contrasting effects on the tyrosine phosphorylation of the IL-2R β chain by exogenous IL-2: MBCD essentially abolished this event, whereas AS tended to enhance it and shifted its occurrence out of rafts. We conclude that AS affects IL-2R signaling by altering the raft enrichment of the IL-2R β chain and propose that this effect is one mechanism underlying the immunomodulatory properties of statins.

STAT5 pathway: target of anti-CD4 antibody in attenuation of IL-2 receptor signaling.

Background. Anti-CD4 antibodies induce long-term graft survival by incompletely understood mechanisms, and CD4-ligation with HIV gp120-derivatives attenuates interleukin (IL)-2 receptor signaling. We examined the latter in the context of the CD4-modulating antibody 16H5. Materials and Methods. We performed immunoblots to assess the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT)5 and Akt in the presence or absence of 16H5. Furthermore, we documented the effects of 16H5 on the induction of STAT5, activating protein (AP)-1, and myc by IL-2 in DNA-binding assays. 3H-thymidine incorporation of the human lymphoid cell line CMO, which exhibits constitutive activation of the STAT5 pathway and IL-2-independent growth, was also measured during 16H5 treatment. Results. In human T lymphocytes, 16H5 attenuated both the tyrosine phosphorylation of STAT5 by IL-2 and the IL-2-induced DNA-binding of this transcription factor. In contrast, 16H5 had no effect on the serine phosphorylation of Akt by IL-2 or on the IL-2-induced DNA-binding of myc. Signal transduction involving AP-1 was unaffected by 16H5 and IL-2. 16H5 also attenuated CMO cell proliferation. Conclusions. 16H5 targets the STAT5 signaling pathway to attenuate IL-2 receptor signal transduction in human T cells. This observation provides a molecular explanation for the immunomodulatory actions of anti-CD4 antibodies.

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. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R β -chain and enhanced the association of the IL-2R γ -chain with cytoskeletal tubulin. The IL-2R β -chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level.