Biomedical subjects

K Mark Coggeshall

Publications and source records attributed to K Mark Coggeshall.

14 recordsLinked to original sources

PubMed2006

Calcium-dependent viral internalization is required for adenovirus type 7 induction of IL-8 protein.

The host response to adenovirus (Ad) infection involves induction of cytokines in lung epithelia. We have demonstrated induction of the lung neutrophil chemokine interleukin-8 (IL-8) by Ad7, a major lung pathogen, in A549 lung epithelial cells and lung tissue through activation of the Erk signaling pathway. However, the mechanism of IL-8 induction is still unclear. In this paper, we first showed that Ad7 viral gene expression is not essential for IL-8 induction as psoralen-UV inactivation of Ad7 did not affect IL-8 mRNA induction or IL-8 protein induction in A549 cells. We then inhibited internalization of Ad7 by treatment of A549 cells with EGTA in calcium-free medium during exposure to Ad7. We verified that this treatment inhibited Ad internalization by confocal microscopy, FACS analysis and Ad E1A and fiber mRNA expression. Preventing internalization by calcium depletion did not inhibit Erk activation by Ad7. However, calcium-dependent internalization was required for IL-8 protein production in Ad7 exposed cells. This is not likely due to an effect of calcium depletion on downstream Erk signaling or IL-8 protein production since calcium depletion did not block IL-8 protein production stimulated by PMA, and because addition of EGTA subsequent to Ad7 internalization also did not prevent Ad induction of IL-8. These studies indicate that Ad7 internalization is calcium-dependent and is required for IL-8 protein induction upon Ad7 infection. Ad7 induction of Erk is independent of calcium and does not require virus internalization.

Adenoviruses, Human↗

PubMed2006

A unique CD72 epitope suggests a potential interaction with Fc gamma RII/CD32 on B lineage lymphocytes.

It has long been known that ligation of the transmembrane CD72 glycoprotein delivers signals to B lymphocytes, with the outcome depending on context. Of particular interest is its ability to function as a counter-receptor/ ligand for the CD100 semaphorin protein. We have now obtained evidence that CD72 physically interacts on the lymphocyte membrane with Fcgamma receptor II (CD32). The association was first revealed with a new monoclonal antibody that recognizes polymorphic determinants on murine CD72. Although the specificity for CD72 was clear from immunoblotting, transfection and other experiments, staining with this reagent was inhibited when cells were pretreated with an Fc receptor-blocking antibody (CD16/CD32 specific). Furthermore, confocal microscopy revealed that the two molecules co-distributed on viable B cells. We also used the antibody to determine when CD72 becomes available to maturing lymphocytes. The marker is first acquired as large pre-B cells and enter the IL-7 independent phase of maturation within bone marrow. Subsequent interactions between CD72 and CD32 may cooperatively deliver negative signals that modulate humoral immune responses.

Animals↗

PubMed2006

Bacillus anthracis spores stimulate cytokine and chemokine innate immune responses in human alveolar macrophages through multiple mitogen-activated protein kinase pathways.

Contact with the human alveolar macrophage plays a key role in the innate immune response to Bacillus anthracis spores. Because there is a significant delay between the initial contact of the spore with the host and clinical evidence of disease, there appears to be temporary containment of the pathogen by the innate immune system. Therefore, the early macrophage response to Bacillus anthracis exposure is important in understanding the pathogenesis of this disease. In this paper, we studied the initial events after exposure to spores, beginning with the rapid internalization of spores by the macrophages. Spore exposure rapidly activated the mitogen-activated protein kinase signaling pathways extracellular signal-regulated kinase, c-Jun-NH2-terminal kinase, and p38. This was followed by the transcriptional activation of cytokine and primarily monocyte chemokine genes as determined by RNase protection assays. Transcriptional induction is reflected at the translational level, as interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) cytokine protein levels were markedly elevated as determined by enzyme-linked immunosorbent assay. Induction of IL-6 and TNF-alpha, and, to a lesser extent, IL-1alpha and IL-1beta, was partially inhibited by the blockade of individual mitogen-activated protein kinases, while the complete inhibition of cytokine induction was achieved when multiple signaling pathway inhibitors were used. Taken together, these data clearly show activation of the innate immune system in human alveolar macrophages by Bacillus anthracis spores. The data also show that multiple signaling pathways are involved in this cytokine response. This report is the first comprehensive examination of this process in primary human alveolar macrophages.

Bacillus anthracis↗

PubMed2005

IL-6 blocks a discrete early step in lymphopoiesis.

Animals lacking Src homology 2 domain-containing inositol 5-phosphatase (SHIP) display a reduction in lymphopoiesis and a corresponding enhancement of myelopoiesis. These effects are mediated at least in part by elevated levels of interleukin 6 (IL-6). Here, we show the lymphopoiesis block in SHIP-/- mice is due to suppression of the lymphoid lineage choice by uncommitted progenitors. The suppression can be reproduced in vitro with recombinant IL-6, and IL-6 acts directly on hematopoietic progenitors. The block is partially overcome in SHIP-/- IL-6-/- double-deficient animals. IL-6 does not suppress but actually enhances proliferation of lymphoid-committed progenitors, indicating the IL-6 target cells are hematopoietic stem cells or multipotent progenitors. The findings suggest a mechanism for the lymphopenia that accompanies proinflammatory diseases.

Animals↗

PubMed2005

Vav activation and function as a rac guanine nucleotide exchange factor in macrophage colony-stimulating factor-induced macrophage chemotaxis.

Signal transduction mediated by phosphatidylinositol 3-kinase (PI 3-kinase) is regulated by hydrolysis of its products, a function performed by the 145-kDa SH2 domain-containing inositol phosphatase (SHIP). Here, we show that bone marrow macrophages of SHIP(-/-) animals have elevated levels of phosphatidylinositol 3,4,5-trisphosphate [PI (3,4,5)P(3)] and displayed higher and more prolonged chemotactic responses to macrophage colony-stimulating factor (M-CSF) and elevated levels of F-actin relative to wild-type macrophages. We also found that the small GTPase Rac was constitutively active and its upstream activator Vav was constitutively phosphorylated in SHIP(-/-) macrophages. Furthermore, we show that Vav in wild-type macrophages is recruited to the membrane in a PI 3-kinase-dependent manner through the Vav pleckstrin homology domain upon M-CSF stimulation. Dominant inhibitory mutants of both Rac and Vav blocked chemotaxis. We conclude that Vav acts as a PI 3-kinase-dependent activator for Rac activation in macrophages stimulated with M-CSF and that SHIP regulates macrophage M-CSF-triggered chemotaxis by hydrolysis of PI (3,4,5)P(3).

Animals↗

PubMed2004

Loss of SHIP and CIS recruitment to the granulocyte colony-stimulating factor receptor contribute to hyperproliferative responses in severe congenital neutropenia/acute myelogenous leukemia.

Mutations in the G-CSF receptor (G-CSFR) in patients with severe congenital neutropenia (SCN) are postulated to contribute to transformation to acute myelogenous leukemia (AML). These mutations result in defective receptor internalization and sustained cellular activation, suggesting a loss of negative signaling by the G-CSFR. In this paper we investigated the roles of SHIP and cytokine-inducible Src homology 2 protein (CIS) in down-modulating G-CSFR signals and demonstrate that loss of their recruitment as a consequence of receptor mutations leads to aberrant signaling. We show that SHIP binds to phosphopeptides corresponding to Tyr744 and Tyr764 in the G-CSFR and that Tyr764 is required for in vivo phosphorylation of SHIP and the formation of SHIP/Shc complexes. Cells expressing a G-CSFR form lacking Tyr764 exhibited hypersensitivity to G-CSF and enhanced proliferation, but to a lesser degree than observed with the most common mutant G-CSFR form in patients with SCN/AML, prompting us to investigate whether suppressor of cytokine signaling proteins also down-modulate G-CSFR signals. G-CSF was found to induce the expression of CIS and of CIS bound to phosphopeptides corresponding to Tyr729 and Tyr744 of the G-CSFR. The expression of CIS was prolonged in cells with the SCN/AML mutant G-CSFR lacking Tyr729 and Tyr744, which also correlated with increased G-CSFR expression. These findings suggest that SHIP and CIS interact with distal phosphotyrosine residues in the G-CSFR to negatively regulate G-CSFR signaling by limiting proliferation and modulating surface expression of the G-CSFR, respectively. Novel therapeutic approaches targeting inhibitory pathways that limit G-CSFR signaling may have promise in the treatment of patients with SCN/AML.

Carrier Proteins↗

PubMed2004

Src homology 2-containing 5-inositol phosphatase (SHIP) suppresses an early stage of lymphoid cell development through elevated interleukin-6 production by myeloid cells in bone marrow.

The Src homology (SH)2-containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP(-/-) animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP(-/-) mice. Lineage-negative marrow (Lin(-)) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP(-/-) mice. Furthermore, culture supernatants of SHIP(-/-) Lin(-) cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP(-/-) Lin(-) cells contained more IL-6 transcripts than wild-type Lin(-) cells, and neutralizing anti-IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP(-/-) Lin(-) cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin(-) bone marrow cells reproduced the phenotype of SHIP(-/-) bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.

Animals↗

PubMed2002

The Src homology 2 domain-containing inositol 5-phosphatase negatively regulates Fcgamma receptor-mediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors.

Molecular mechanisms by which the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) negatively regulates phagocytosis in macrophages are unclear. We addressed the issue using bone marrow-derived macrophages from FcgammaR- or SHIP-deficient mice. Phagocytic activities of macrophages from FcgammaRII(b)(-/-) and SHIP(-/-) mice were enhanced to a similar extent, relative to those from wild type. However, calcium influx was only marginally affected in FcgammaRII(b)(-/-), but greatly enhanced in SHIP(-/-) macrophages. Furthermore, SHIP was phosphorylated on tyrosine residues upon FcgammaR aggregation even in macrophages from FcgammaRII(b)(-/-) mice or upon clustering of a chimeric receptor containing CD8 and the immunoreceptor tyrosine-based activation motif (ITAM)-bearing gamma-chain or human-restricted FcgammaRIIa. These findings indicate that, unlike B cells, SHIP is efficiently phosphorylated in the absence of an immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptor. We further demonstrate that SHIP directly bound to phosphorylated peptides derived from FcgammaRIIa with a high affinity, comparable to that of FcgammaRII(b). Lastly, FcgammaRIIa-mediated phagocytosis was significantly enhanced in THP-1 cells overexpressing dominant-negative form of SHIP in the absence of FcgammaRII(b). These results indicate that SHIP negatively regulates FcgammaR-mediated phagocytosis through all ITAM-containing IgG receptors using a molecular mechanism distinct from that in B cells.

Amino Acid Motifs↗

PubMed2002

Convergence of signaling pathways on the activation of ERK in B cells.

The B cell receptor (BCR) initiates three major signaling pathways: the Ras pathway, which leads to extracellular signal-regulated kinase (ERK) activation; the phospholipase C-gamma pathway, which causes calcium mobilization; and the phosphoinositide 3-kinase (PI 3-kinase) pathway. These combine to induce different biological responses depending on the context of the BCR signal. Both the Ras and PI 3-kinase pathways are important for B cell development and activation. Several model systems show evidence of cross-regulation between these pathways. Here we demonstrate through the use of PI 3-kinase inhibitors and a dominant-negative PI 3-kinase construct that the BCR-induced phosphorylation and activation of ERK is dependent on PI 3-kinase. PI 3-kinase feeds into the Ras signaling cascade at multiple points, both upstream and downstream of Ras. We also show that ERK activation is dependent on phospholipase C-gamma, in keeping with its dependence on calcium mobilization. Last, the activation of PI 3-kinase itself is completely dependent on Ras. We conclude that the PI 3-kinase and Ras signaling cascades are intimately connected in B cells and that the activation of ERK is a signal integration point, since it requires simultaneous input from all three major signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2002

C-reactive protein induces signaling through Fc gamma RIIa on HL-60 granulocytes.

Human C-reactive protein (CRP) at acute phase levels of 10-200 microg/ml triggered the phosphorylation of FcgammaRIIa, Syk kinase, and phospholipase Cgamma2 in granulocytic HL-60 cells. CRP also stimulated translocation to the membrane of both phospholipase Cgamma2 and phosphatidylinositol-3-kinase. The signaling response triggered by CRP was a rapid, early event with kinetics similar to the response elicited by human IgG. Both soluble-aggregated CRP and monomeric CRP cross-linked FcgammaRII to generate a signal of the same intensity. The results are consistent with signaling through the intrinsic immunoreceptor tyrosine-based activation motif of the cytoplasmic domain of FcgammaRIIa, the major CRP-receptor on monocytes and neutrophils that is responsible for CRP-mediated phagocytosis. The signaling events driven by CRP have the potential to regulate infiltrating neutrophil activities.

Antigens, CD↗

PubMed2002

Negative signaling.

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Amino Acid Motifs↗

PubMed2002

Activation of nuclear factor of activated T cells by human T-lymphotropic virus type 1 accessory protein p12(I).

Human T-lymphotropic virus type 1 (HTLV-1) is the agent of an aggressive malignancy of CD4(+) T lymphocytes, called adult T-cell lymphoma/leukemia, and is associated with numerous immune-mediated diseases. To establish infection, HTLV-1 must activate targeted T cells during early stages of infection. We recently demonstrated that the HTLV-1 accessory protein p12(I) is critical for persistent infection in vivo and for viral infectivity in quiescent primary lymphocytes, suggesting a role for p12(I) in lymphocyte activation. To test whether p12(I) modulates signaling pathways required for T-lymphocyte activation, we examined AP-1-, NF-kappaB-, and nuclear factor of activated T cells (NFAT)-driven reporter gene activity in p12(I)-expressing Jurkat T cells compared to vector-transfected control cells. HTLV-1 p12(I) specifically induced NFAT-mediated transcription approximately 20-fold in synergy with the Ras/mitogen-activated protein kinase pathway, but did not influence AP-1- or NF-kappaB-dependent gene expression. Inhibition of calcium-dependent signals by cyclosporin A, BAPTA-AM [glycine, N,N'-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl]-[bis(acetyloxy)methyl ester], and a dominant negative mutant of NFAT2 abolished the p12(I)-mediated activation of NFAT-dependent transcription. In contrast, inhibition of phospholipase C-gamma and LAT (linker for activation of T cells) did not affect p12(I)-induced NFAT activity. Importantly, p12(I) functionally substituted for thapsigargin, which selectively depletes intracellular calcium stores. Our data are the first to demonstrate a role for HTLV-1 p12(I) in calcium-dependent activation of NFAT-mediated transcription in lymphoid cells. We propose a novel mechanism by which HTLV-1, a virus associated with lymphoproliferative disease, dysregulates common T-cell activation pathways critical for the virus to establish persistent infection.

Calcium↗

PubMed2002

Regulation of signal transduction by the Fc gamma receptor family members and their involvement in autoimmunity.

Recent studies on the nature and mode of action of inhibitory receptors and their intracellular phosphatase effector enzymes have identified a new area of research in the etiology of autoimmune diseases. Myeloid cells play a critical role in autoimmunity through their IgG receptors. A number of recent findings reveal that such cells express inhibitory receptors, including the elusive CD32/Fc gamma RIIB isoform, and express inhibitory phosphatases like SHP-1 and SHIP. Animals lacking the effector phosphatases exhibit a pronounced autoimmune and/or pro-inflammatory phenotype. Animals deficient in the expression of the inhibitory receptors often display a much less severe phenotype, likely due to the fact that hematopoietic cells have numerous and probably redundant inhibitory receptors. Genetic deficiencies in the limited number of effector molecules (SHP-1 and SHIP) lead to more dramatic effects on hematopoietic cells and level of inflammation in such animals. These recent findings in animal models open the intriguing possibility that the human homologues of genes encoding the inhibitory receptors like Fc gamma RIIB or effector phosphatases like SHIP might contribute to autoimmune diseases. However, while identification of genes involved in autoimmunity will greatly aid in diagnosis of human autoimmune disease, it is necessary to understand the biochemical mechanisms of action of the numerous inhibitory receptors and phosphatases in the hematopoietic system. Such information will permit the rational design of more efficient and effective treatments for patients. Additional experiments directed at the role and mechanism(s) of action of the inhibitory phosphatases SHP-1 and SHIP will uncover new candidates for diagnosis and treatment of autoimmune diseases.

Amino Acid Motifs↗

PubMedDate not supplied

CD95 rapidly clusters in cells of diverse origins.

We have shown that CD95-mediated cell death requires a clustering of the receptor in distinct sphingolipid-rich domains of the cell membrane (Grassme et al., 2000, Cremesti et al., 2000). These domains form in response to acid sphingomyelinase (ASM)-induced ceramide generation. However, recent studies challenged the finding of early CD95 clustering (Algeciras-Schimnich et al., 2002). Here, six independent groups tested clustering of CD95 in diverse cell type including primary cells ex vivo and established cell lines. The studies show clustering of CD95 within seconds to minutes in all cell types tested by the different groups. In addition, clustering of CD95 was detected after stimulation of cells using three agonistic anti-CD95 antibodies (CH11, APO-1-3 and JO2), CD95 ligand and stimuli that induce an upregulation and activation of the endogenous CD95/CD95 ligand system. The data confirm our previous studies and suggest rapid, i.e., within seconds to minutes, CD95 clustering as a general phenomenon occurring in many cell types.

Animals↗