Natural killer cells regulate T-cell proliferation during human parainfluenza virus type 3 infection

Human parainfluenza virus type 3 (HPIV3) is a major respiratory pathogen in humans. Failure to induce immunological memory associated with HPIV3 infection has been attributed to inhibition of lymphocyte proliferation. We demonstrate that the inability of mixed lymphocytes to respond to virally infected antigen-presenting cells is due to an interleukin-2-dependent, nonapoptotic mechanism involving natural killer (NK) cells and their influence is exerted in a contact-dependent manner. These results suggest a novel regulatory mechanism for NK cells during HPIV3 infection, offering an explanation for viral persistence and poor memory responses.     

Helper T-cell subsets in mouse leishmaniasis: induction,expansion and effector function

It has been a surprise to find that the two distinct subsets of mouse CD4⁺ T cells identified from clones cultured in vitro also occur during Leishmania infection. The spectrum of disease encountered during these infections ranges from successful resolution to fatal dissemination and in mice these outcomes are accompanied by expansion of T_H1 or T_H2 CD4⁺ cells, respectively. This review focuses on the mechanisms that cause such disparate responses to the parasite.     

Sinomenine inhibits primary CD4+ T-cell proliferation via apoptosis

Sinomenine is an active component isolated from Sinomenium acutum and is widely used as an immunosuppressive drug for treating autoimmune diseases. CD4(+) T-cell population plays a key role in adaptive immune response and is related to some autoimmune diseases. In this study, we investigated the possible immunosuppressive effect of sinomenine on CD4(+) T cells and its underlying mechanism. Our data demonstrated that sinomenine remarkably suppressed the proliferation of CD4(+) T cells, blocked the cell cycle progression from G0/G1 phase to S plusG2/M phases. Finally, the immunosuppressive activity elicited by sinomenine in CD4(+) primary lymphocytes was found to be largely accounted for by caspase 3-dependent cells apoptosis. Sinomenine did not significantly alter the expression of bcl-2 in activated CD4(+) primary T cells, suggesting that bcl-2 might not be involved in sinomenine-induced T cells apoptosis. In sum, this study proposes a novel mechanism for the immunosuppressive function of sinomenine on primary mouse CD4(+) T cells.     

Beta 3-adrenergic receptors regulate retinal endothelial cell migration and proliferation

Sympathetic nerves may play a role in vascular disorders of the eye. In the present study, we hypothesized that activation of beta3-adrenergic receptors on retinal endothelial cells would promote migration and proliferation of these cells, two markers of an angiogenic phenotype. We show, for the first time, expression of beta3-adrenergic receptors on cultured retinal endothelial cells. Activation of these receptors with BRL37344, a specific beta3-adrenergic receptor agonist, promoted migration that was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K), the mitogen activated protein kinase component MEK, and matrix metalloproteinases (MMPs) 2 and 9. BRL37344 stimulated proliferation, which could be blocked by inhibitors of Src, PI3K, and MEK. These cells also express the beta1-adrenergic receptor with no beta2-adrenergic receptor expression observed. Stimulation of the beta1-adrenergic receptor with xamoterol, a specific partial agonist, did not promote proliferation or migration. These results support the hypothesis that beta3-adrenergic receptors play a role in proliferation and migration of cultured human retinal endothelial cells.     

Kinase-independent function of RIP1, critical for mature T-cell survival and proliferation

The death receptor, Fas, triggers apoptotic death and is essential for maintaining homeostasis in the peripheral lymphoid organs. RIP1 was originally cloned when searching for Fas-binding proteins and was later shown to associate also with the signaling complex of TNFR1. Although Fas exclusively induces apoptosis, TNFR1 primarily activates the pro-survival/pro-inflammatory NF-κB pathway. Mutations in Fas lead to lymphoproliferative (lpr) diseases, and deletion of TNFR1 results in defective innate immune responses. However, the function of RIP1 in the adult lymphoid system has not been well understood, primarily owing to perinatal lethality in mice lacking the entire RIP1 protein in germ cells. This current study investigated the requirement for RIP1 in the T lineage using viable RIP1 mutant mice containing a conditional and kinase-dead RIP1 allele. Disabling the kinase activity of RIP1 had no obvious impact on the T-cell compartment. However, T-cell-specific deletion of RIP1 led to a severe T-lymphopenic condition, owing to a dramatically reduced mature T-cell pool in the periphery. Interestingly, the immature T-cell compartment in the thymus appeared intact. Further analysis showed that mature RIP1^−/− T cells were severely defective in antigen receptor-induced proliferative responses. Moreover, the RIP1^−/− T cells displayed greatly increased death and contained elevated caspase activities, an indication of apoptosis. In total, these results revealed a novel, kinase-independent function of RIP1, which is essential for not only promoting TCR-induced proliferative responses but also in blocking apoptosis in mature T cells.The pro-survival signaling pathways provide protection against cell death responses at various stages during T lymphopoiesis as well as maintenance of the mature population.^{1, 2} Apoptosis is a major programmed cell death pathway, which can be induced through either intrinsic or extrinsic signals.³ Under normal circumstances, the pro-survival and apoptosis signaling pathways are tightly regulated, which ensures generation of diverse T-cell repertoires, while avoiding autoimmunity. For instance, the Bcl-2 and Bcl-X_L genes, which inhibit the intrinsic apoptotic pathway, are essential for both T-cell development and peripheral maintenance.^{4, 5} However, lack of cell death, as in the case of inactivation of Bim, a pro-apoptotic protein of the Bcl-2 family, results in lymphoproliferative and autoimmune diseases.⁶ The extrinsic pathway of apoptosis is triggered through cell receptors, including Fas/Apo-1 and tumor necrosis factor receptor 1 (TNFR1). Whereas Fas is a professional death receptor, TNFR1 primarily signals the pro-survival pathway by activating NF-κB, which also promotes inflammation.^{7, 8}Receptor-interacting protein (RIP or RIP1) was originally cloned as a potential Fas-interacting protein.⁹ However, later studies found that lack of RIP1 has no effect on Fas-induced apoptosis.^{10, 11} Subsequently, RIP1 was also found to associate with the signaling complex of TNFR1.¹² It was shown that RIP1 deficiency disrupts NF-κB activation induced by TNFR1 in primary mouse embryonic fibroblast cells or human Jurkat T lymphoma cells.^{10, 11} Several functional domains of RIP1 have been defined. In particular, RIP1 contains a serine/threonine kinase domain (KD) at the amino-terminus and a death domain (DD) at the carboxy-terminus. The intermediate domain, but not the protein serine/threonine KD of RIP1, is required for the activation of NF-κB.¹³ The DD of RIP1 interacts with the DD of TNFR1-associated death domain (TRADD) protein, a signaling adaptor, leading to both apoptosis and NF-κB activation.¹² Therefore, RIP1 may serve as a scaffold protein in addition to being a protein serine/threonine kinase.The function of the KD of RIP1 remained unknown until the landmark work by Holler et al.,¹⁴ implicating a novel function for RIP1 in a caspase-independent cell death process with certain characteristics of necrosis, namely necroptosis. Importantly, mutations targeting the kinase activity of RIP1 abolish necroptotic cell death induced by TNFR1. The in vivo role of RIP1-mediated necroptosis was first revealed by analysis of the embryonic defect displayed by mice lacking the Fas-associated death domain (FADD) protein.¹⁵ The FADD adaptor protein relays exclusively apoptotic signals in the pathways triggered by Fas, TNFR1, and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs or DR4/5).^{16, 17, 18} Whereas none of the DRs are essential for mouse development, FADD deficiency resulted in midgestation death of mouse embryos.^{19, 20} Interestingly, when RIP1 is absent, normal embryonic development is restored in FADD^−/− mice,¹⁵ indicating that FADD^−/− embryonic lethality is caused by RIP1-dependent necroptosis.Although normal during embryogenesis, RIP1^−/− FADD^−/− double knockout (DKO) mice display perinatal lethality,¹⁵ similar to the phenotype of RIP1^−/− single knockout mice.¹⁰ In contrast, deletion of a RIP1-related protein kinase, RIP3, fully restores normal embryonic as well as postnatal development in FADD^−/− mice.²¹ Recent studies demonstrated that RIP1^−/− mice can only reach adulthood when both FADD and RIP3 are absent, indicating that RIP1 protects neonatal cells from FADD-mediated apoptosis and RIP3-dependent necroptosis.^{22, 23, 24, 25} Importantly, FADD^−/− RIP3^−/− DKO mice and RIP1^−/− FADD^−/− RIP3^−/− triple knockout mice develop age-dependent lymphadenopathy and splenomegaly, reminiscent of the lymphoproliferative (lpr) disease displayed by Fas^−/− mice. Therefore, both apoptosis and necroptosis are essential for homeostasis in the peripheral lymphoid organs.Previous studies have indicated that RIP1 is essential for T-cell development, because RIP1-deficient fetal liver cells fail to reconstitute the T-cell compartment in immunodeficient recipient mice.^{15, 26} A recent study showed that lack of RIP1 in hematopoietic stem cells and progenitors (HSCs/Ps) leads to a severe defect in hematopoiesis.²⁷ However, the temporal requirement for RIP1, particularly at postlineage commitment stages, remains unclear. In the current study, T lineage-specific deletion of RIP1 revealed a novel stage-specific requirement for RIP1 to protect T cells from apoptosis as well as to allow normal proliferative responses.     

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.     

Granulocyte-macrophage colony-stimulating factor enhances macrophage accessory function in con A-stimulated T-cell proliferation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to augment various macrophage (M phi) functions, including antigen presentation in the antibody-producing response. We investigated the augmentative effect of GM-CSF on M phi A-cell activity in concanavalin A-stimulated T-cell proliferation. Pretreatment with GM-CSF of peritoneal M phi enhanced the T-cell proliferative response. This effect of GM-CSF was dose dependent and GM-CSF supplementation was needed at the beginning of M phi culture. We observed that GM-CSF induced M phi spreading and firm attachment accompanied with enlargement of the cytoplasm, but could not induce de novo expression of Ia antigen. GM-CSF treatment enabled M phi to produce more interleukin (IL)-1 and IL-6 upon stimulation with lipopolysaccharides or polyinosinic-polycytidylic acid, but was unable to stimulate M phi directly. This was confirmed by Northern blot analysis. These results indicate that GM-CSF augments M phi A-cell activity through the enhancement of the capacity of M phi to produce IL-1 and IL-6.     

Synthetic prostacyclin analogs differentially regulate macrophage function via distinct analog-receptor binding specificities

PGI(2) (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI(2), in the regulation of both innate and acquired immunity. As PGI(2) is unstable, we sought to define the effects of various PGI(2) analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, carbaprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the G(alphas) protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI(2) analogs and more closely resembled the effects of PGE(2). Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies differences in immunoregulatory properties of clinically administered PGI(2) analogs. These studies are the first to explore the capacity of PGI(2) to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents.     

Oxygen free radicals regulate NMDA receptor function via a redox modulatory site

A novel modulatory site on the N-methyl-D-aspartate (NMDA) receptor that is sensitive to sulfhydryl redox reagents was recently described. Here we report that this redox modulatory site is susceptible to oxidation by reactive oxygen species endogenous to the CNS. Oxygen free radicals generated by xanthine and xanthine oxidase were observed to decrease NMDA-induced changes in intracellular free Ca2+ concentrations and NMDA-evoked cation currents in cortical neurons in culture. Additionally, a sublethal production of free radicals by xanthine and xanthine oxidase reversed a dithiothreitol-induced enhancement of NMDA-mediated neurotoxicity in vitro. These results show that NMDA receptor function is modulated at its redox site by endogenous substances that normally accompany tissue reperfusion following an ischemic event. This novel mechanism for NMDA receptor regulation may have profound implications in the outcome of glutamate neurotoxicity in vivo.     

Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy in Drosophila

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IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms

Some members of the inhibitor of apoptosis (IAP) family suppress apoptosis by neutralizing caspases. The current model suggests that all caspase-regulatory IAPs function as direct enzyme inhibitors, blocking effector caspases by binding to their catalytically active pockets. Here we show that IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Whereas XIAP binds directly to the active-site pockets of effector caspases, we find that regulation of effector caspases by Drosophila IAP1 (DIAP1) requires an evolutionarily conserved IAP-binding motif (IBM) at the neo-amino terminus of the large caspase subunit. Remarkably, unlike XIAP, DIAP1-sequestered effector caspases remain catalytically active, suggesting that DIAP1 does not function as a bona fide enzyme inhibitor. Moreover, we demonstrate that the mammalian IAP c-IAP1 interacts with caspase-7 in an exclusively IBM-dependent, but active site pocket-independent, manner that is mechanistically similar to DIAP1. The importance of IBM-mediated regulation of effector-caspases in vivo is substantiated by the enhanced apoptotic potency of IBM-mutant versions of drICE, DCP-1 and caspase-7.     

Preferred SH3 Domain Partners of ADAM Metalloproteases Include Shared and ADAM-Specific SH3 Interactions

A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior.     

ATF3 and CH25H regulate effector trogocytosis and anti-tumor activities of endogenous and immunotherapeutic cytotoxic T lymphocytes

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