Biomedical subjects

Bruce D Freedman

Publications and source records attributed to Bruce D Freedman.

16 recordsLinked to original sources

PubMed2006

Host cell Ca2+ and protein kinase C regulate innate recognition of Toxoplasma gondii.

In healthy hosts, acute infection with the opportunistic pathogen Toxoplasma gondii is controlled by innate production of IL-12, a key cytokine crucial for the development of protective immunity. Previous work has established that the mitogen-activated protein kinases (MAPK), particularly p38 and ERK1/2, are important regulators of T. gondii-induced IL-12 synthesis. Here we report that host cell Ca(2+) is required for activation of MAPK by T. gondii, as well as LPS and CpG, and for parasite-induced synthesis of IL-12. In addition, pharmacological mobilization of Ca(2+) stores in macrophages treated with parasites or LPS enhanced MAPK phosphorylation initiated by these stimuli. Investigation of the upstream mechanism by which Ca(2+) regulates MAPK activation revealed that T. gondii induced acute activation of conventional, Ca(2+)-dependent PKCalpha and PKCbeta, which are required for infection-induced MAPK activation and production of IL-12. Despite these findings, neither acute parasite infection nor LPS initiated a measurable Ca(2+) response in macrophages, suggesting that low levels of Ca(2+) are permissive for initiation of pro-inflammatory signaling. Together these data identify host cell Ca(2+) and PKC as crucial regulators of the innate immune response to microbial stimuli, including T. gondii.

Animals↗

PubMed2006

Adhesion- and degranulation-promoting adapter protein is required for efficient thymocyte development and selection.

Adhesion- and degranulation-promoting adapter protein (ADAP) is required in TCR-induced activation and proliferation of peripheral T cells. Loss of ADAP also impairs TCR-initiated inside-out activation of the integrin LFA-1 (CD11a/CD18, alphaLbeta2). In this study, we demonstrate that ADAP-deficient CD4/CD8 double-positive (DP) cells have a diminished ability to proliferate, and that these DP thymocytes up-regulate CD69 poorly in vivo. Moreover, in both MHC class I- and class II-restricted TCR transgenic models, loss of ADAP interferes with both positive and negative selection. ADAP deficiency also impairs the ability of transgene-bearing DP thymocytes to form conjugates with Ag-loaded presenting cells. These findings suggest that ADAP is critical for thymocyte development and selection.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Developmental alterations in thymocyte sensitivity are actively regulated by MHC class II expression in the thymic medulla.

Developing thymocytes are positively selected if they respond to self-MHC-peptide complexes, yet mature T cells are not activated by those same self-complexes. To avoid autoimmunity, positive selection must be followed by a period of maturation when the cellular response to TCR signals is altered. The mechanisms that mediate this postselection developmental tuning remain largely unknown. Specifically, it is unknown whether developmental tuning is a preprogrammed outcome of positive selection or if it is sensitive to ongoing interactions between the thymocyte and the thymic stroma. We probed the requirement for MHC class II-TCR interactions in postselection maturation by studying single positive (SP) CD4 thymocytes from K14/A(beta)(b) mice, in which CD4 T cells cannot interact with MHC class II in the thymic medulla. We report here that SP CD4 thymocytes must receive MHC class II signals to avoid hyperactive responses to TCR signals. This hyperactivity correlates with decreased expression of CD5; however, developmental tuning can occur independently of CD5, correlating instead with differences in the distribution of Lck. Thus, the maturation of postselection SP CD4 thymocytes is an active process mediated by ongoing interactions between the T cell and MHC class II molecules. This represents a novel mechanism by which the thymic medulla prevents autoreactivity.

Animals↗

PubMed2006

The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation.

BACKGROUND: The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. RESULTS: By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. CONCLUSIONS: These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

Actin Cytoskeleton↗

PubMed2006

HS1 functions as an essential actin-regulatory adaptor protein at the immune synapse.

HS1, the leukocyte-specific homolog of cortactin, regulates F-actin in vitro and is phosphorylated in response to TCR ligation, but its role in lymphocyte activation has not been addressed. We demonstrate that HS1-deficient T cells fail to accumulate F-actin at the immune synapse (IS) and, upon TCR ligation, form actin-rich structures that are disordered and unstable. Early TCR activation events are intact in these cells, but Ca2+ influx and IL-2 gene transcription are defective. Importantly, HS1 tyrosine phosphorylation is required for its targeting to the IS and for its function in regulating actin dynamics and IL-2 promoter activity. Phosphorylation also links HS1 to multiple signaling proteins, including Lck, PLCgamma1, and Vav1, and is essential for the stable recruitment of Vav1 to the IS. Taken together, our studies show that HS1 is indispensable for signaling events leading to actin assembly and IL-2 production during T cell activation.

Actins↗

PubMed2006

Mechanisms of calcium signaling and function in lymphocytes.

The multifunctional role for calcium during lymphocyte activation and differentiation is well established; yet how fate-specific changes in calcium concentration are produced, and how these signals are translated into distinct cellular responses, is not known. A critical role has been identified for store-operated calcium release-activated calcium (CRAC) channels; however, the lack of information concerning the structure and mechanism of store-dependent activation has limited our ability to define CRAC's role in the range of calcium-dependent responses of lymphocytes. Moreover, additional calcium-permeant cation channels have been recently identified in lymphocytes. These channels regulate functions distinct from those attributed to CRAC channels. Consequently, this review summarizes well-established mechanisms of calcium signaling and also considers recent findings which suggest that diverse and interacting pathways encode functionally specific calcium signals in lymphocytes. Speculation is also offered about how distinct patterns of calcium signaling are translated into diverse transcriptional responses.

Animals↗

PubMed2005

Hypoxia inducible factor 1 alpha regulates T cell receptor signal transduction.

Low oxygen pressures exist in many solid tissues, including primary and secondary lymphoid organs. One key element in cellular adaptation to hypoxia is induced expression of hypoxia inducible factor (Hif) 1alpha. Here, we have examined the effect of Hif-1alpha, isolated from the myriad other effects of hypoxia, on T cell receptor (TCR) signaling in thymocytes. Because pVHL (von Hippel-Lindau protein) directs the proteolysis of Hif-1alpha under "normoxic" conditions, we achieved constitutive stabilization of Hif-1alpha through thymic deletion of Vhlh and reversed Hif-1alpha stabilization with double deletion of Vhlh and Hif-1alpha. We found that constitutive activity of Hif-1alpha resulted in diminished Ca(2+) response upon TCR crosslinking despite equivalent activation of phospholipase C(gamma1), normal intracellular Ca(2+) stores, and normal entry of Ca(2+) across the plasma membrane. Altered Ca(2+) response was instead due to accelerated removal of Ca(2+) from the cytoplasm into intracellular compartments, which occurred in association with Hif-1alpha-dependent overexpression of the calcium pump SERCA2 (sarcoplasmic/endoplasmic reticulum calcium ATPase 2). These data suggest a unique mechanism for control of TCR signaling through Hif-1alpha, which may be operative at the physiologic oxygen tensions seen in solid lymphoid organs.

Animals↗

PubMed2005

Multiple eicosanoid-activated nonselective cation channels regulate B-lymphocyte adhesion to integrin ligands.

Arachidonic acid (AA) is a substrate for a variety of proinflammatory mediators, which are generated by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P-450 (CYP450) enzymes. COX (e.g., PGs and prostacyclins) and LOX (e.g., leukotrienes) products have well-established proinflammatory roles; however, little is known about the functions of CYP450 products in leukocytes. We previously found that mechanical strain generated by subjecting lymphocytes to hypotonic challenge triggered AA production and that two CYP450 products of AA, 5,6-epoxyeicosatrienoic acid (5,6-EET) and 20-hydroxyeicosatetraenoic acid (20-HETE), as well as a product of LOX, 5-(S)-hydroperoxyeicosatetrenoic acid (5-HPETE), induced Ca(2+) entry into primary B cells. The main goal of the present studies, therefore, was to define the biophysically properties of eicosanoid-activated channels responsible for Ca(2+) entry and the physiological consequences of activating these channels, including their role in mechanical signaling. We found that 5,6-EET, 20-HETE, and 5-HPETE each activated distinct Ca(2+)-permeant nonselective cation channels (NSCCs) in primary B cells. These NSCCs each regulate plasma membrane potential and B-cell adhesion to integrin ligands ICAM-1 and VCAM-1. Thus our data demonstrate that proinflammatory mediators produced in response to osmotic and/or physical stress play a direct role in regulating the B-cell membrane potential and their adhesion to specific ECM proteins. These results not only have important implications for understanding normal mechanisms of B-cell activation, differentiation, and trafficking but also point to novel targets for modulating the pathogenesis of B-cell-mediated inflammatory diseases.

B-Lymphocytes↗

PubMed2005

Mechanisms of hypotonicity-induced calcium signaling and integrin activation by arachidonic acid-derived inflammatory mediators in B cells.

We previously characterized the initial steps in the activation of novel (calcium-permeant) nonselective cation channels (NSCCs) and calcium release-activated calcium channels in primary murine B lymphocytes. Phospholipase C products, namely diacylglycerol and d-myo-inositol 1,4,5-trisphosphate, were identified as proximal intracellular agonists of these respective channels following mechanical stimulation of B cells. However, neither the distal steps in NSCC activation nor the contribution of these channels to sustained mechanical signaling were defined in these previous studies. In this study, single cell measurements of intracellular Ca(2+) were used to define the mechanisms of NSCC activation and demonstrate a requirement for arachidonic acid liberated from diacylglycerol. Several arachidonic acid-derived derivatives were identified that trigger Ca(2+) entry into B cells, including the lipoxygenase product 5-hydroperoxyeicosatetranenoic acid and the cytochrome P450 hydroxylase product 20-hydroxyeicosatetraenoic; however, the cytochrome P450 epoxygenase product 5,6-epoxyeicosatrienoic acid is primarily responsible for hypotonicity-induced responses. In addition to regulating calcium entry, our data suggest that eicosanoid-activated NSCCs have a separate and direct role in regulating the avidity of integrins on B cells for extracellular matrix proteins, including ICAM-1 and VCAM-1. Thus, in addition to defining a novel osmotically activated signal transduction pathway in B cells, our results have broad implications for understanding how inflammatory mediators dynamically and rapidly regulate B cell adhesion and trafficking.

Animals↗

PubMed2005

HIV-1 gp120-induced TNF-{alpha} production by primary human macrophages is mediated by phosphatidylinositol-3 (PI-3) kinase and mitogen-activated protein (MAP) kinase pathways.

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein gp120 to CD4 followed by a chemokine receptor, but these interactions may also take place independently from infection. gp120 stimulation of primary human macrophages is known to trigger production of cytokines implicated in pathogenesis, particularly tumor necrosis factor alpha (TNF-alpha), but the mechanisms have not been determined. We sought to define the pathways responsible for TNF-alpha secretion by monocyte-derived macrophages (MDM) following HIV-1 gp120 stimulation. MDM exposure to recombinant macrophage-tropic (R5) gp120 led to dose- and donor-dependent release of TNF-alpha, which was cyclohexamide-sensitive and associated with up-regulated message. Pretreatment with specific inhibitors of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1/2 (ERK-1/2; PD98059, U0126) and p38 (SB202190, PD169316) inhibited the secretion of TNF-alpha. gp120-elicited TNF-alpha production was also blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors (wortmannin, LY294002). Moreover, PI-3K inhibition ablated gp120-induced phosphorylation of p38 and ERK-1/2. The response was inhibited by a CC chemokine receptor 5 (CCR5)-specific antagonist, indicating that CCR5 was in large part responsible. These results indicate that gp120-elicited TNF-alpha production by macrophages involves chemokine receptor-mediated PI-3K and MAPK activation, that PI-3K is an upstream regulator of MAPK in this pathway, and that p38 and ERK-1/2 independently regulate TNF-alpha production. These gp120-triggered signaling pathways may be responsible for inappropriate production of proinflammatory cytokines by macrophages, which are believed to play a role in immunopathogenesis and in neurological sequelae of AIDS.

Androstadienes↗

PubMed2005

Distinct calcium channels regulate responses of primary B lymphocytes to B cell receptor engagement and mechanical stimuli.

Intracellular Ca(2+) plays a central role in controlling lymphocyte function. Nonetheless, critical gaps remain in our understanding of the mechanisms that regulate its concentration. Although Ca(2+)-release-activated calcium (CRAC) channels are the primary Ca(2+) entry pathways in T cells, additional pathways appear to be operative in B cells. Our efforts to delineate these pathways in primary murine B cells reveal that Ca(2+)-permeant nonselective cation channels (NSCCs) operate in a cooperative fashion with CRAC. Interestingly, these non-CRAC channels are selectively activated by mechanical stress, although the mechanism overlaps with BCR-activated pathways, suggesting that they may operate in concert to produce functionally diverse Ca(2+) signals. NSCCs also regulate the membrane potential, which activates integrin-dependent binding of B cells to extracellular matrix elements involved in their trafficking and localization within secondary lymphoid organs. Thus, CRAC and distinct Ca(2+) permeant NSCCs are differentially activated by the BCR and mechanical stimuli and regulate distinct aspects of B cell physiology.

Animals↗

PubMed2004

Chemokine receptor utilization and macrophage signaling by human immunodeficiency virus type 1 gp120: Implications for neuropathogenesis.

Human immunodeficiency virus type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 for entry. Macrophages and microglia (M/M) are the principal productively infected brain cells in HIV encephalopathy (HIVE), and neuronal injury is believed to result both from direct effects of viral proteins and indirect effects mediated by macrophage activation and secretion of neurotoxic products. In vitro, direct injury by the viral envelope glycoprotein gp120 can be mediated by neuronal CXCR4, but most HIV-1 isolates from the central nervous system (CNS) studied to date use CCR5 (R5 strains) rather than CXCR4 (X4 or R5X4 strains). Additionally, it remains unknown how HIV induces M/M activation and neurotoxin secretion. To address these issues, the authors analyzed a CNS-derived primary isolate, TYBE, and showed that it uses CXCR4 only and replicates efficiently in macrophages through CXCR4-mediated entry. The authors also showed that both R5 and X4 gp120 activate intracellular signals in macrophages through CCR5 and CXCR4, including calcium elevations; K+, Cl- and nonselective cation channel activation; phosphorylation of the nonreceptor tyrosine kinase Pyk2; and activation of p38 and SAPK/JNK mitogen-activated protein kinases (MAPKs). Finally, the authors showed that macrophages stimulated with gp120 produce soluble factors through MAPK-dependent pathways, including beta-chemokines implicated in HIVE pathogenesis. The findings emphasize that both X4 and R5 HIV-1 isolates may contribute to HIVE pathogenesis, and that gp120/chemokine receptor interactions in M/M trigger specific signal transduction pathways that may affect M/M function and provide a mechanism underlying CNS injury.

AIDS Dementia Complex↗

PubMed2003

Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signaling pathways.

Macrophages are major targets for infection by human immunodeficiency virus type 1 (HIV-1). In addition to their role as productive viral reservoirs, inappropriate activation of infected and uninfected macrophages appears to contribute to pathogenesis. HIV-1 infection requires initial interactions between the viral envelope surface glycoprotein gp120, the cell-surface protein CD4, and a chemokine receptor CCR5 or CXCR4. Besides their role in HIV-1 entry, CCR5 and CXCR4 are G protein-coupled receptors that can activate multiple intracellular signaling pathways. HIV-1 gp120 has been shown to activate signaling pathways through the chemokine receptors in several cell types including lymphocytes, neurons, and astrocytes. In some cell types, these consequences may cause cellular injury. In this review, we highlight our data demonstrating diverse signaling events that occur in primary human macrophages in response to gp120/chemokine receptor interactions. These responses include K+, Cl-, and nonselective cation currents, intracellular Ca2+ increases, and activation of several kinases including the focal adhesion-related tyrosine kinase Pyk2, mitogen-activated protein kinases (MAPK), and phosphoinositol-3 kinase. Activation of the MAPK leads to gp120-induced expression of chemokines such as monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1beta and the proinflammatory cytokine tumor necrosis factor alpha. These responses establish a complex cytokine network, which may enhance or suppress HIV-1 replication. In addition, dysregulation of macrophage function by gp120/chemokine receptor signaling may contribute to local inflammation and injury and further recruit additional inflammatory and/or target cells. Targeting these cellular signaling pathways may have benefit in controlling inflammatory sequelae of HIV infection such as in neurological disease.

Calcium Signaling↗

PubMed2003

Regulation of T cell activation and tolerance by phospholipase C gamma-1-dependent integrin avidity modulation.

Ag receptor engagement without costimulation induces a tolerant state in CD4(+) T cells termed anergy. Anergic CD4(+) T cells are primarily characterized by the inability to produce IL-2, but the biochemical basis for this functional defect is not completely understood. We demonstrate that primary CD4(+) T cells anergized by costimulatory blockade exhibit impaired TCR-coupled phospholipase C (PLC)gamma-1 activation. This defect is associated with the marked reduction of multiple downstream signaling events required for IL-2 transcription, including mobilization of intracellular Ca(2+) and activation of the mitogen-activated protein kinase cascade. We also found that primary anergic CD4(+) T cells fail entirely to modulate their integrin binding avidity in response to TCR stimulation. Integrin avidity modulation is required for full T cell activation and effector function, and as we show in this study, is completely dependent upon PLCgamma-1 activity. Finally, analogs that mimic the actions of diacylglycerol and inositol 1,4,5-triphosphate, the immediate products of PLCgamma-1 activity, restored integrin avidity modulation and IL-2 production by anergic T cells. Thus, deficient coupling of PLCgamma-1 to the TCR appears to be a central biochemical defect that could potentially account for the failure of multiple functional responses in primary anergic CD4(+) T cells.

Animals↗

PubMed2003

HIV-1 gp120 chemokine receptor-mediated signaling in human macrophages.

The chemokine receptors CCR5 and CXCR4 serve as the cellular receptors in conjunction with CD4 for HIV-1 entry and infection of target cells. Although the virus has subverted these molecules for its own use, their natural function is to respond to activation and migration signals delivered by extracellular chemokines. A principal research objective of our laboratory is to understand the consequences of virus-chemokine receptor interactions for cellular function, as well as for entry and infection. We hypothesized that CXCR4-using (X4) and CCR5-using (R5) HIV-1 strains might elicit signals through the chemokine receptors that result in aberrant function and/or regulate virus entry or postentry steps of infection. We have focused on primary human macrophages, which express both CXCR4 and CCR5, because macrophages are a principal target for HIV-1 in vivo, inappropriate macrophage activation appears to play a major role in the pathogenesis of certain sequelae of AIDS, such as HIV encephalopathy, and macrophage infection is regulated at several steps subsequent to entry in ways that are linked to envelope- receptor interactions. This review summarizes our recent findings regarding the mechanisms of chemokine-receptor signaling in macrophages, the role of viral envelope glycoproteins in eliciting macrophage signals, and how these activation pathways may participate in macrophage infection and affect cell functions apart from infection.

HIV Envelope Protein gp120↗

PubMed2002

Modulation of Kv channel expression and function by TCR and costimulatory signals during peripheral CD4(+) lymphocyte differentiation.

Ionic signaling pathways, including voltage-dependent potassium (Kv) channels, are instrumental in antigen-mediated responses of peripheral T cells. However, how Kv channels cooperate with other signaling pathways involved in T cell activation and differentiation is unknown. We report that multiple Kv channels are expressed by naive CD4(+) lymphocytes, and that the current amplitude and kinetics are modulated by antigen receptor-mediated stimulation and costimulatory signals. Currents expressed in naive CD4(+) lymphocytes are consistent with Kv1.1, Kv1.2, Kv1.3, and Kv1.6. Effector CD4(+) cells generated by optimal TCR and costimulation exhibit only Kv1.3 current, but at approximately sixfold higher levels than naive cells. CD4(+) lymphocytes anergized through partial stimulation exhibit similar Kv1.1, Kv1.2, and/or Kv1.6 currents, but approximately threefold more Kv1.3 current than naive cells. To determine if Kv channels contribute to the distinct functions of naive, effector, and anergized T cells, we tested their role in immunoregulatory cytokine production. Each Kv channel is required for maximal IL-2 production by naive CD4(+) lymphocytes, whereas none appears to play a role in IL-2, IL-4, or IFN-gamma production by effector cells. Interestingly, Kv channels in anergized lymphocytes actively suppress IL-4 production, and these functions are consistent with a role in regulating the membrane potential and calcium signaling.

Animals↗