32 results on '"O'Shea J"'
Search Results
2. Memory Stem T Cells in Autoimmune Disease: High Frequency of Circulating CD8+ Memory Stem Cells in Acquired Aplastic Anemia.
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Hosokawa K, Muranski P, Feng X, Townsley DM, Liu B, Knickelbein J, Keyvanfar K, Dumitriu B, Ito S, Kajigaya S, Taylor JG 6th, Kaplan MJ, Nussenblatt RB, Barrett AJ, O'Shea J, and Young NS
- Subjects
- Adult, Aged, Anemia, Aplastic blood, Anemia, Aplastic diagnosis, Anemia, Sickle Cell diagnosis, Anemia, Sickle Cell immunology, Autoimmune Diseases immunology, Biomarkers blood, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes classification, Female, Humans, Interferon-gamma biosynthesis, Interleukin-2 biosynthesis, Lupus Erythematosus, Systemic diagnosis, Lupus Erythematosus, Systemic immunology, Lymphocyte Activation, Lymphocyte Count, Male, Middle Aged, Recurrence, T-Lymphocyte Subsets, Treatment Failure, Uveitis diagnosis, Uveitis immunology, Anemia, Aplastic immunology, Anemia, Aplastic therapy, CD8-Positive T-Lymphocytes immunology, Immunologic Memory, Precursor Cells, T-Lymphoid immunology
- Abstract
Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4(+) and CD8(+) T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8(+) TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8(+) TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8(+) TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8(+) and CD4(+) T cell subsets in AA patients, including CD8(+) and CD4(+) TSCMs. CD8(+) TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4(+) and CD8(+) TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8(+) TSCM subset is a novel biomarker and a potential therapeutic target for AA.
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- 2016
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3. Ablation of tumor progression locus 2 promotes a type 2 Th cell response in Ovalbumin-immunized mice.
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Watford WT, Wang CC, Tsatsanis C, Mielke LA, Eliopoulos AG, Daskalakis C, Charles N, Odom S, Rivera J, O'Shea J, and Tsichlis PN
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- Animals, Antigen Presentation immunology, Antigen-Presenting Cells immunology, Blotting, Western, Cell Differentiation, Cytokines biosynthesis, Cytokines immunology, Gene Expression, Gene Expression Regulation immunology, Immunoglobulin E biosynthesis, Lymphocyte Activation immunology, MAP Kinase Kinase Kinases genetics, Mice, Mice, Knockout, Pneumonia chemically induced, Pneumonia immunology, Proto-Oncogene Proteins genetics, Th1 Cells immunology, Th2 Cells cytology, MAP Kinase Kinase Kinases immunology, Ovalbumin immunology, Proto-Oncogene Proteins immunology, Th2 Cells immunology
- Abstract
The protein kinase encoded by the Tpl2 proto-oncogene regulates ERK activation and cytokine gene expression in macrophages in response to LPS and TNF-alpha. In this study we show that OVA-immunized Tpl2(-/-) mice express high levels of IgE and develop more severe bronchoalveolar eosinophilic inflammation than Tpl2(+/+) controls, when challenged with OVA intranasally. Bronchoalveolar exudates and supernatants of OVA-stimulated splenocytes from immunized Tpl2(-/-) mice express elevated levels of IL-4 and IL-5, suggesting that Tpl2 ablation promotes the Th2 polarization of the T cell response. Anti-CD3 stimulation of CD4(+) T cells of wild-type and Tpl2 knockout mice revealed that Tpl2 ablation gives rise to a cell autonomous T cell defect that is primarily responsible for the Th2 polarization of the T cell response to Ag. This observation was further supported by experiments addressing the expression of Th1 and Th2 cytokines in OVA-stimulated mixed cultures of CD4(+) T cells from Tpl2(+/+)/OT2 or Tpl2(-/-)/OT2 mice and dendritic cells from Tpl2(+/+) or Tpl2(-/-) mice. Further studies revealed that Th1 cells express significantly higher levels of Tpl2 than Th2 cells. As a result, Tpl2(-/-) Th1 cells exhibit a stronger defect in ERK activation by anti-CD3 than Th2 cells and express low levels of T-bet. Given that the development of Th1 and Th2 cells depends on positive feedback signals from the T cells, themselves, the functional defect of the Tpl2(-/-) Th1 cells provides a mechanistic explanation for the T cell autonomous Th2 polarization in Tpl2(-/-) mice.
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- 2010
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4. Viral FLIP impairs survival of activated T cells and generation of CD8+ T cell memory.
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Wu Z, Roberts M, Porter M, Walker F, Wherry EJ, Kelly J, Gadina M, Silva EM, DosReis GA, Lopes MF, O'Shea J, Leonard WJ, Ahmed R, and Siegel RM
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- Animals, Apoptosis genetics, Apoptosis immunology, CD8-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes pathology, CD8-Positive T-Lymphocytes transplantation, Cell Division genetics, Cell Division immunology, Cell Survival genetics, Cell Survival immunology, Chagas Disease genetics, Chagas Disease immunology, DNA-Binding Proteins physiology, Epitopes, T-Lymphocyte administration & dosage, Epitopes, T-Lymphocyte immunology, Immunophenotyping, Lymphocyte Activation genetics, Lymphopenia genetics, Lymphopenia immunology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Molluscum contagiosum virus immunology, Receptors, Tumor Necrosis Factor physiology, STAT5 Transcription Factor, Signal Transduction genetics, Signal Transduction immunology, T-Lymphocyte Subsets metabolism, T-Lymphocyte Subsets pathology, Trans-Activators physiology, Trypanosoma cruzi immunology, Viral Proteins biosynthesis, Viral Proteins genetics, fas Receptor genetics, CD8-Positive T-Lymphocytes immunology, Immunologic Memory genetics, Lymphocyte Activation immunology, Milk Proteins, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets virology, Viral Proteins toxicity
- Abstract
Viral FLIPs (vFLIPs) interfere with apoptosis signaling by death-domain-containing receptors in the TNFR superfamily (death receptors). In this study, we show that T cell-specific transgenic expression of MC159-vFLIP from the human Molluscum contagiosum virus blocks CD95-induced apoptosis in thymocytes and peripheral T cells, but also impairs postactivation survival of in vitro activated primary T cells despite normal early activation parameters. MC159 vFLIP impairs T cell development to a lesser extent than does Fas-associated death domain protein deficiency or another viral FLIP, E8. In the periphery, vFLIP expression leads to a specific deficit of functional memory CD8(+) T cells. After immunization with a protein Ag, Ag-specific CD8(+) T cells initially proliferate, but quickly disappear and fail to produce Ag-specific memory CD8(+) T cells. Viral FLIP transgenic mice exhibit impaired CD8(+) T cell responses to lymphocytic choriomeningitis virus and Trypanosoma cruzi infections, and a specific defect in CD8(+) T cell recall responses to influenza virus was seen. These results suggest that vFLIP expression in T cells blocks signals necessary for the sustained survival of CD8(+) T cells and the generation of CD8(+) T cell memory. Through this mechanism, vFLIP proteins expressed by T cell tropic viruses may impair the CD8(+) T cell immune responses directed against them.
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- 2004
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5. Inducible expression of Stat4 in dendritic cells and macrophages and its critical role in innate and adaptive immune responses.
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Fukao T, Frucht DM, Yap G, Gadina M, O'Shea JJ, and Koyasu S
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- Animals, Antigen-Presenting Cells immunology, Antigen-Presenting Cells metabolism, Autocrine Communication genetics, Autocrine Communication immunology, CD8 Antigens biosynthesis, Cell Differentiation genetics, Cell Differentiation immunology, Cells, Cultured, Cytokines physiology, DNA-Binding Proteins genetics, Dendritic Cells cytology, Dendritic Cells immunology, Immunity, Cellular genetics, Immunity, Innate genetics, Injections, Intravenous, Interferon-gamma biosynthesis, Interleukin-12 physiology, Lipopolysaccharides administration & dosage, Macrophage Activation genetics, Macrophages immunology, Macrophages parasitology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Knockout, STAT4 Transcription Factor, Signal Transduction genetics, Signal Transduction immunology, Th2 Cells immunology, Th2 Cells metabolism, Toxoplasma immunology, Toxoplasma pathogenicity, Trans-Activators genetics, Up-Regulation genetics, Up-Regulation immunology, DNA-Binding Proteins biosynthesis, DNA-Binding Proteins physiology, Dendritic Cells metabolism, Macrophages metabolism, Trans-Activators biosynthesis, Trans-Activators physiology
- Abstract
Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-gamma production by APC as well as IFN-gamma production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-gamma production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4(-/-) DC and macrophages, which fail to produce IFN-gamma. Furthermore, Stat4(-/-) macrophages are defective in NO production in response to IL-12 and are susceptible to TOXOPLASMA: Autocrine IL-12 signaling is required for high-level IFN-gamma production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.
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- 2001
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6. IL-12 receptor beta 2 (IL-12R beta 2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites.
- Author
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Wu C, Wang X, Gadina M, O'Shea JJ, Presky DH, and Magram J
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- Animals, Binding Sites genetics, Binding Sites immunology, Concanavalin A pharmacology, DNA-Binding Proteins metabolism, Female, Gene Targeting, Immune Tolerance genetics, Interferon-gamma biosynthesis, Interferon-gamma metabolism, Interleukin-12 pharmacology, Lymphocyte Activation genetics, Lymphocyte Subsets immunology, Lymphocyte Subsets metabolism, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Knockout, Phosphorylation, Protein Isoforms deficiency, Protein Isoforms genetics, Protein Isoforms physiology, Receptors, Interleukin physiology, Receptors, Interleukin-12, STAT4 Transcription Factor, Spleen cytology, Spleen immunology, Spleen metabolism, Th1 Cells immunology, Th1 Cells metabolism, Trans-Activators metabolism, Interleukin-12 metabolism, Receptors, Interleukin deficiency, Receptors, Interleukin genetics, Signal Transduction genetics, Signal Transduction immunology
- Abstract
Two subunits of the IL-12 receptor (IL-12R), IL-12R beta 1 and IL-12R beta 2, have been identified and cloned. Previous studies demonstrated that the IL-12R beta 1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12R beta 2 in IL-12 signaling, we have generated IL-12R beta 2-deficient (IL-12R beta 2(-/-)) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12R beta 2(-/-) mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12R beta 2(-/-) mice failed to produce IFN-gamma or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12R beta 2(-/-) splenocytes was not induced when incubated with IL-12. IL-12R beta 2(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12R beta 2(-/-) mice were deficient in vivo in their ability to produce IFN-gamma following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12R beta 1 is the subunit primarily responsible for binding IL-12, IL-12R beta 2 plays an essential role in mediating the biological functions of IL-12 in mice.
- Published
- 2000
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7. Stat4 is expressed in activated peripheral blood monocytes, dendritic cells, and macrophages at sites of Th1-mediated inflammation.
- Author
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Frucht DM, Aringer M, Galon J, Danning C, Brown M, Fan S, Centola M, Wu CY, Yamada N, El Gabalawy H, and O'Shea JJ
- Subjects
- Arthritis, Rheumatoid immunology, Arthritis, Rheumatoid metabolism, Cytokines physiology, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins blood, DNA-Binding Proteins genetics, Dendritic Cells immunology, Down-Regulation immunology, Humans, Interferon-alpha pharmacology, Macrophages immunology, Macrophages pathology, Monocytes immunology, Phosphorylation, RNA, Messenger biosynthesis, RNA, Messenger blood, STAT4 Transcription Factor, Synovial Membrane immunology, Synovial Membrane metabolism, Synovial Membrane pathology, Th1 Cells metabolism, Th2 Cells immunology, Trans-Activators antagonists & inhibitors, Trans-Activators blood, Trans-Activators genetics, Arthritis, Rheumatoid pathology, DNA-Binding Proteins biosynthesis, Dendritic Cells metabolism, Macrophage Activation, Macrophages metabolism, Monocytes metabolism, Th1 Cells immunology, Trans-Activators biosynthesis
- Abstract
Stat4 is a key transcription factor involved in promoting cell-mediated immunity, whose expression in mature cells has been reported to be restricted to T and NK cells. We demonstrate here, however, that Stat4 expression is not restricted to lymphoid cells. In their basal state, monocytes do not express Stat4. Upon activation, however, IFN-gamma- and LPS-treated monocytes and dendritic cells express high levels of Stat4. Monocyte-expressed Stat4 in humans is phosphorylated in response to IFN-alpha, but not IL-12. In contrast, the Th2 cytokines, IL-4 and IL-10, specifically down-regulate Stat4 expression in activated monocytes, while having little effect on Stat6 expression. Moreover, macrophages in synovial tissue obtained from patients with rheumatoid arthritis express Stat4 in vivo, suggesting a potential role in a prototypical Th1-mediated human disease. IFN-alpha-induced Stat4 activation in human monocytes represents a previously unrecognized signaling pathway at sites of Th1 inflammation.
- Published
- 2000
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8. Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes.
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Franchimont D, Galon J, Gadina M, Visconti R, Zhou Y, Aringer M, Frucht DM, Chrousos GP, and O'Shea JJ
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- 3T3 Cells, Animals, DNA-Binding Proteins biosynthesis, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Humans, Interferon Regulatory Factor-1, Interferon-gamma antagonists & inhibitors, Interferon-gamma biosynthesis, Interleukin-12 antagonists & inhibitors, Interleukin-12 metabolism, Janus Kinase 2, Mice, Phosphoproteins antagonists & inhibitors, Phosphoproteins biosynthesis, Phosphoproteins genetics, Phosphorylation drug effects, Promoter Regions, Genetic drug effects, Promoter Regions, Genetic immunology, Protein-Tyrosine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases metabolism, Proteins antagonists & inhibitors, Proteins metabolism, Receptors, Interleukin metabolism, Receptors, Interleukin-12, STAT4 Transcription Factor, Signal Transduction drug effects, Signal Transduction immunology, T-Lymphocytes metabolism, TYK2 Kinase, Th1 Cells immunology, Trans-Activators metabolism, Transfection, DNA-Binding Proteins antagonists & inhibitors, Dexamethasone pharmacology, Immunosuppressive Agents pharmacology, Interleukin-12 physiology, Proto-Oncogene Proteins, T-Lymphocytes drug effects, Th1 Cells drug effects, Trans-Activators antagonists & inhibitors
- Abstract
Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.
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- 2000
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9. IL-12 induces IFN regulating factor-1 (IRF-1) gene expression in human NK and T cells.
- Author
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Galon J, Sudarshan C, Ito S, Finbloom D, and O'Shea JJ
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- 3T3 Cells, Animals, Cell Line, Cells, Cultured, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins biosynthesis, DNA-Binding Proteins physiology, Humans, Interferon Regulatory Factor-1, Interferon-alpha physiology, Interleukin-12 antagonists & inhibitors, Mice, Phosphoproteins antagonists & inhibitors, Phosphoproteins biosynthesis, STAT4 Transcription Factor, Signal Transduction genetics, Signal Transduction immunology, Trans-Activators physiology, Up-Regulation genetics, Up-Regulation immunology, DNA-Binding Proteins genetics, Gene Expression Regulation immunology, Interferons physiology, Interleukin-12 physiology, Killer Cells, Natural metabolism, Phosphoproteins genetics, T-Lymphocytes metabolism
- Abstract
IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive CD4+ cells to Th1 cells; however, relatively few IL-12 target genes have been identified. To better clarify the molecular basis of IL-12 action, we set out to characterize genes up-regulated by IL-12, first by contrasting IL-12- and IFN-alpha-inducible genes. We identified several genes up-regulated by IL-12, namely, MIP-1alpha, MIP-1beta, IL-1RA, and IFN regulatory factor-1 (IRF-1). IRF-1 is a transcription factor regulated by IFNs that is also essential for Th1 responses. We demonstrated that IL-12 directly up-regulates IRF-1 to the same extent as IFN-alpha in normal human T cells and in NK cells. We showed that IL-12 had a direct effect on IRF-1, an effect not mediated indirectly by the induction of IFN-gamma production. Furthermore, IL-2 and IL-12 synergistically induced IRF-1, whereas IFN-alpha and IL-12 did not. The participation of STAT4 in the regulation of IRF-1 was demonstrated in two ways. First, STAT4 was required for the IL-12-dependent transactivation of an IRF-1 reporter construct, and second, STAT4 binding to the IRF-1 promoter was shown using EMSA. In contrast to IL-12, no up-regulation of IRF-1 was found in IL-4-stimulated cells, and IL-4 did not block IL-12-dependent up-regulation of IRF-1. Therefore, IRF-1 may be an important contributor to IL-12 signaling, and we speculate that the defective IL-12 responses seen in IRF-1-/- mice might be attributable, in part, to the absence of this transcription factor.
- Published
- 1999
10. TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs.
- Author
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Sudarshan C, Galon J, Zhou Y, and O'Shea JJ
- Subjects
- Cell Line, Humans, Interferon-gamma biosynthesis, Interleukin-12 antagonists & inhibitors, Interleukin-2 antagonists & inhibitors, Janus Kinase 1, Janus Kinase 2, Mitogen-Activated Protein Kinase 12, Phosphorylation, STAT4 Transcription Factor, STAT5 Transcription Factor, Tumor Suppressor Proteins, DNA-Binding Proteins metabolism, Interleukin-12 pharmacology, Interleukin-2 pharmacology, Milk Proteins, Mitogen-Activated Protein Kinases, Protein Kinases metabolism, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins, Trans-Activators metabolism, Transforming Growth Factor beta pharmacology
- Abstract
The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta.
- Published
- 1999
11. IL-2, but not IL-4 and other cytokines, induces phosphorylation of a 98-kDa protein associated with SHP-2, phosphatidylinositol 3'-kinase, and Grb2.
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Gadina M, Sudarshan C, and O'Shea JJ
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- Cells, Cultured, Cytokines physiology, GRB2 Adaptor Protein, Humans, Intracellular Signaling Peptides and Proteins, Janus Kinase 3, Killer Cells, Natural enzymology, Killer Cells, Natural metabolism, Macromolecular Substances, Membrane Glycoproteins metabolism, Molecular Weight, Neural Cell Adhesion Molecules metabolism, Phosphoproteins biosynthesis, Phosphoproteins metabolism, Phosphorylation, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatase, Non-Receptor Type 6, Protein Tyrosine Phosphatases biosynthesis, Protein-Tyrosine Kinases physiology, SH2 Domain-Containing Protein Tyrosine Phosphatases, Signal Transduction immunology, Substrate Specificity immunology, T-Lymphocytes enzymology, T-Lymphocytes metabolism, Adaptor Proteins, Signal Transducing, Antigens, Differentiation, Interleukin-2 physiology, Interleukin-4 physiology, Neural Cell Adhesion Molecule L1, Phosphatidylinositol 3-Kinases metabolism, Protein Tyrosine Phosphatases metabolism, Proteins metabolism, Receptors, Immunologic, src Homology Domains immunology
- Abstract
Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3'-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3'-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3'-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.
- Published
- 1999
12. Involvement of SHP-2 in multiple aspects of IL-2 signaling: evidence for a positive regulatory role.
- Author
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Gadina M, Stancato LM, Bacon CM, Larner AC, and O'Shea JJ
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- Calcium-Calmodulin-Dependent Protein Kinases metabolism, Cells, Cultured, Enzyme Activation, GRB2 Adaptor Protein, Humans, Intracellular Signaling Peptides and Proteins, Janus Kinase 3, Phosphatidylinositol 3-Kinases metabolism, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatase, Non-Receptor Type 6, Protein-Tyrosine Kinases metabolism, Proteins metabolism, SH2 Domain-Containing Protein Tyrosine Phosphatases, Transcriptional Activation, Adaptor Proteins, Signal Transducing, Interleukin-2 pharmacology, Protein Tyrosine Phosphatases physiology
- Abstract
Binding of IL-2 to its receptor activates several biochemical pathways, but precisely how these pathways are linked is incompletely understood. Here, we report that SHP-2, an SH2-domain containing tyrosine phosphatase, associates with different molecules of the IL-2 signaling cascade. Upon IL-2 stimulation, SHP-2 was coimmunoprecipitated with Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. In contrast, SHP-2 was constitutively associated with JAK1 and JAK3. Finally, SHP-2 expression amplified STAT-dependent transcriptional activation whereas a dominant negative allele inhibited transactivation and the IL-2-induced activation of MAPK (mitogen-activated protein kinase). These results demonstrate the involvement of SHP-2 in multiple pathways of the IL-2 signaling cascade and provide evidence for its positive regulatory role.
- Published
- 1998
13. Tumor-induced suppression of T lymphocyte proliferation coincides with inhibition of Jak3 expression and IL-2 receptor signaling: role of soluble products from human renal cell carcinomas.
- Author
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Kolenko V, Wang Q, Riedy MC, O'Shea J, Ritz J, Cathcart MK, Rayman P, Tubbs R, Edinger M, Novick A, Bukowski R, and Finke J
- Subjects
- Cell Division immunology, Cytotoxicity, Immunologic, Humans, Janus Kinase 3, Lymphocyte Activation immunology, Protein-Tyrosine Kinases antagonists & inhibitors, Proto-Oncogene Mas, Receptors, Interleukin-2 antagonists & inhibitors, Tumor Cells, Cultured, Carcinoma, Renal Cell immunology, Kidney Neoplasms immunology, Protein-Tyrosine Kinases immunology, Receptors, Interleukin-2 immunology, Signal Transduction immunology, T-Lymphocytes immunology, Tumor Escape
- Abstract
The proliferative capacity of T cells infiltrating human tumors is known to be impaired, possibly through their interaction with tumor. Here we demonstrate that soluble products derived from renal cell carcinoma (RCC-S) explants but not normal kidney can inhibit an IL-2-dependent signaling pathway that is critical to T cell proliferation. A major target of the immunosuppression was the IL-2R-associated protein tyrosine kinase, Janus kinase 3 (Jak3). RCC-S suppressed basal expression of Jak3 and its increase following stimulation with anti-CD3/IL-2. Jak3 was most sensitive to suppression by RCC-S; however, reduction in expression of p56(lck), p59(fyn), and ZAP-70 was observed in some experiments. Expression of other signaling elements linked to the IL-2R (Jak1) and the TCR (TCR-zeta, CD3-epsilon, and phospholipase C-gamma) were minimally affected. In naive T cells, RCC-S also partially blocked induction of IL-2R alpha-, beta- and gamma-chain expression when stimulating via the TCR/CD3 complex with anti-CD3 Ab. To determine whether RCC-S suppressed IL-2-dependent signaling, primed T cells were employed since RCC-S had no effect on IL-2R expression but did down-regulate Jak3 expression and, to a lesser degree, p56(lck) and p59(fyn). Reduction in Jak3 correlated with impaired IL-2-dependent proliferation and signal transduction. This included loss of Jak1 kinase tyrosine phosphorylation and no induction of the proto-oncogene, c-Myc. These findings suggest that soluble products from tumors may suppress T cell proliferation through a mechanism that involves down-regulation of Jak3 expression and inhibition of IL-2-dependent signaling pathways.
- Published
- 1997
14. Activation of STAT4 by IL-12 and IFN-alpha: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation.
- Author
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Cho SS, Bacon CM, Sudarshan C, Rees RC, Finbloom D, Pine R, and O'Shea JJ
- Subjects
- Animals, Base Sequence, COS Cells, Cells, Cultured, DNA metabolism, DNA-Binding Proteins chemistry, Humans, Killer Cells, Natural immunology, Killer Cells, Natural metabolism, Ligands, Oligodeoxyribonucleotides genetics, Oligodeoxyribonucleotides metabolism, Phosphorylation, Protein Binding, Recombinant Proteins pharmacology, STAT4 Transcription Factor, Serine metabolism, T-Lymphocytes immunology, T-Lymphocytes metabolism, Trans-Activators chemistry, Tyrosine metabolism, DNA-Binding Proteins metabolism, Interferon Type I pharmacology, Interleukin-12 pharmacology, Trans-Activators metabolism
- Abstract
The immunoregulatory cytokine IL-12 plays a central role in cell-mediated immune responses through its effects on NK cells and T lymphocytes. While IL-12 is known to share some functions with other cytokines, such as IFN-alpha, it also maintains distinct roles, such as its ability to induce Th1 differentiation. The molecular basis for these unique and overlapping functions is not well understood. IL-12 has previously been shown to induce tyrosine phosphorylation and DNA-binding of STAT3 and STAT4, members of the signal transducers and activators of transcription (STAT) family. Because STAT4 has only been shown to be activated in response to IL-12, this specificity has been suggested to be a basis for the unique actions of IL-12. In this study, we demonstrated that STAT4 activation by IL-12 is not unique; IL-12 and IFN-alpha, but not IFN-gamma, induced tyrosine phosphorylation and DNA binding of STAT4. Since tyrosine and serine phosphorylation of STAT1 have previously been shown to be important in IFN-alpha-mediated signaling, we also investigated IL-12- and IFN-alpha-induced serine phosphorylation of STAT4. We demonstrated that both cytokines induced serine phosphorylation. This modification was not required for DNA binding, but may be important in STAT-mediated transcription. Thus, STAT4 activation was not IL-12 specific, and IL-12 and IFN-alpha activated STAT4 through both tyrosine and serine phosphorylation. These findings have significant implications for understanding the role of STAT4 in mediating biologic functions; specifically, the data argue that the unique effects of IL-12 cannot be solely explained by STAT4 activation.
- Published
- 1996
15. Regulation of JAK3 expression and activation in human B cells and B cell malignancies.
- Author
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Tortolani PJ, Lal BK, Riva A, Johnston JA, Chen YQ, Reaman GH, Beckwith M, Longo D, Ortaldo JR, Bhatia K, McGrath I, Kehrl J, Tuscano J, McVicar DW, and O'Shea JJ
- Subjects
- B-Lymphocytes drug effects, B-Lymphocytes immunology, Enzyme Activation, Humans, Interleukin-2 immunology, Interleukin-4 immunology, Interleukin-7 immunology, Janus Kinase 1, Janus Kinase 3, Leukemia enzymology, Lymphocyte Activation, Phosphorylation, Tumor Cells, Cultured, B-Lymphocytes enzymology, Interleukins immunology, Leukemia immunology, Protein-Tyrosine Kinases biosynthesis
- Abstract
Members of the Janus family (JAK) of protein tyrosine kinases are critical enzymes in signaling pathways via hematopoietin receptors. We have cloned JAK3, which unlike other known family members (JAK1, JAK2, and TYK2) is preferentially expressed in hematopoietic cells but not in a variety of other cells. Functionally, JAK3 and JAK1 are coupled to the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 in T cells and NK cells. Because of the importance of IL-2, IL-4, and IL-7 in B cell physiology, we sought to determine whether JAK3 was also present in B lymphocytes and whether it was involved in signaling via cytokines that are important for B cell development and function. In this report, we demonstrate that JAK3 is expressed in normal human peripheral blood B cells at levels that are comparable to those in T cells. In addition, the levels were found to be markedly up-regulated following stimulation with staphylococcal protein A Cowan and anti-CD40 Abs. In addition, IL-4 and IL-7 induced the rapid tyrosine phosphorylation of JAK3 and JAK1, and IL-4 activated both JAK3 and JAK1 phosphotransferase activity. JAK3 protein was also detected in immature B cell lines, but not in more well differentiated cell lines. Additionally, JAK3 was detected in lysates from bone marrow lymphoblasts of patients with B cell precursor acute lymphocytic leukemia and cell lines derived from human B cell lymphomas. Together, these data suggest that the regulation of JAK3 expression and activity is likely to be important in B cell development and function.
- Published
- 1995
16. Induction of T cell differentiation and lymphomagenesis in the thymus of mice with severe combined immune deficiency (SCID).
- Author
-
Murphy WJ, Durum SK, Anver MR, Ferris DK, McVicar DW, O'Shea JJ, Ruscetti SK, Smith MR, Young HA, and Longo DL
- Subjects
- Animals, Apoptosis, CD3 Complex analysis, CD4-Positive T-Lymphocytes cytology, CD8 Antigens analysis, Calcium metabolism, Cell Differentiation, Gamma Rays, Gene Rearrangement, beta-Chain T-Cell Antigen Receptor, Mice, Mice, Inbred BALB C, T-Lymphocyte Subsets cytology, Thymus Gland microbiology, Thymus Gland pathology, Time Factors, Whole-Body Irradiation, Mice, SCID immunology, T-Lymphocyte Subsets immunology, Thymoma etiology, Thymus Gland cytology
- Abstract
Severe combined immune deficiency (SCID) mice have a defect in their recombinase system and cannot productively rearrange their immune receptor genes. Thus, SCID thymocytes are arrested at the immature "triple negative" phase, not expressing CD3, CD4, or CD8 surface markers. Whole body irradiation of SCID mice induced maturation of their thymocytes to the CD4+/CD8+ double positive, CD3+low stage of differentiation, and resulted in the generation of a thymic cortical region on histologic examination. No mature single positive T cells were detected in the thymus or the periphery. VDJ rearrangements of TCR-beta with restricted clonality were observed in the double positive cells from a given individual. The CD3 complex was expressed on some of these cells, but the cells failed to mobilize intracellular calcium after cross-linking with CD3 Abs. The double positive cells appeared several weeks after irradiation, persisted for many months in the thymus, and by 6 mo generally developed into metastatic lymphoma. Retroviral activation was undetectable in both the preneoplastic and transformed thymocytes. Thus, it appears that the earliest steps in T cell development can be induced in SCID mice by inducing DNA breaks with radiation. This system represents a model of early thymic development, preneoplasia, and neoplasia.
- Published
- 1994
17. Expression of v-src in T cells correlates with nuclear expression of NF-kappa B.
- Author
-
Eicher DM, Tan TH, Rice NR, O'Shea JJ, and Kennedy IC
- Subjects
- Base Sequence, Gene Expression, HIV Long Terminal Repeat, Humans, Molecular Sequence Data, Transcriptional Activation, Cell Nucleus chemistry, Genes, src, NF-kappa B analysis, T-Lymphocytes chemistry
- Abstract
NF-kappa B is a rapidly inducible transcriptional activator that responds to a variety of signals and influences the expression of many genes involved in the immune response. Protein tyrosine kinases transmit signals from cytokine and immune receptors. Very little information exists linking these two important classes of signaling molecules. We now demonstrate that v-src expression correlates with nuclear expression of a kappa B binding complex similar to that induced by phorbol ester and ionomycin, as detected by electrophoretic mobility shift assay using a variety of kappa B sites. This complex was blocked by the tyrosine kinase inhibitor, herbimycin A. The v-src-induced complex comprised the p50 and p65 components of NF-kappa B, as determined by supershift and immunoblot analysis. As a functional correlate of this finding, transient co-transfection of HIV-1 LTR reporter constructs in a different T cell line demonstrated that v-src activated this promoter in a kappa B-dependent manner. We found that transactivation of the HIV-1 LTR by v-src was more sensitive to mutations of the proximal, rather than the distal, kappa B element. The implications for T cell receptor signaling and HIV-1 gene expression are considered.
- Published
- 1994
18. A role for protein tyrosine kinase activity in natural cytotoxicity as well as antibody-dependent cellular cytotoxicity. Effects of herbimycin A.
- Author
-
O'Shea JJ, McVicar DW, Kuhns DB, and Ortaldo JR
- Subjects
- Antigens, Differentiation physiology, Benzoquinones, Humans, Interferon-gamma metabolism, Interleukin-2 pharmacology, Killer Cells, Natural drug effects, Lactams, Macrocyclic, Protein-Tyrosine Kinases antagonists & inhibitors, Receptors, Fc physiology, Receptors, IgG, Receptors, Interleukin-2 analysis, Rifabutin analogs & derivatives, Antibody-Dependent Cell Cytotoxicity drug effects, Cytotoxicity, Immunologic drug effects, Protein-Tyrosine Kinases physiology, Quinones pharmacology
- Abstract
NK cells, CD3- large granular lymphocytes, have diverse means by which they lyse targets, including antibody-dependent cellular cytotoxicity. The low affinity receptor for the Fc portion of Ig (Fc gamma RIIIA), like the TCR, is a multimeric receptor complex coupled to a protein tyrosine kinase. In the present study, we observed that inhibition of tyrosine kinase activity by herbimycin A interferes with receptor-mediated phosphorylation of a variety of substrates and mobilization of intracellular calcium. Fc gamma RIIIA induced IL-2R alpha-chain expression was also extremely sensitive to herbimycin A as was antibody-dependent cellular cytotoxicity, in fact more so than receptor-mediated phosphorylation and calcium mobilization. In contrast to Fc gamma RIIIA, the surface molecules and biochemical mechanisms involved in NK cytotoxicity and lymphokine-activated killing are not well characterized. Interestingly, however, herbimycin A also blocks these modes of cytolysis, implicating a role for tyrosine kinase function in these processes. Whether FcR-mediated signaling and receptor-mediated signaling involved in NK activity share specific biochemical intermediates is not known, but the involvement of tyrosine kinase function in the latter means of cytotoxicity may provide novel avenues for understanding the biochemical basis of this perplexing cellular function.
- Published
- 1992
19. Characterization of the family of dimers associated with Fc receptors (Fc epsilon RI and Fc gamma RIII).
- Author
-
Letourneur O, Kennedy IC, Brini AT, Ortaldo JR, O'Shea JJ, and Kinet JP
- Subjects
- Animals, Cell Line, Humans, Killer Cells, Natural immunology, Leukemia, Basophilic, Acute immunology, Rats, Receptors, Antigen, T-Cell analysis, Receptors, IgE, Receptors, IgG, Antigens, Differentiation analysis, Antigens, Differentiation, B-Lymphocyte analysis, Immunoglobulin E metabolism, Immunoglobulin G metabolism, Receptors, Fc analysis
- Abstract
The receptor for IgE (Fc epsilon RI) is a multimeric complex containing one alpha chain, one beta chain with four transmembrane domains and one homodimer of disulfide-linked gamma-chains. The Fc epsilon RI gamma-chains form additional disulfide-linked dimers with the homologous zeta- and eta-chains, as part of the TCR complex. The low affinity receptor for IgG (Fc gamma RIII)2 on NK cells is also associated with zeta-chains. Here we show that the gamma-chain is expressed in NK cells both as a group of heterogenous gamma gamma homodimers and also as a heterodimer bound to zeta. Fc gamma RIIIA is associated with three types of dimers zeta zeta, gamma zeta, and notably gamma gamma as well. In fact, gamma gamma appears to be the predominant species associating with Fc gamma RIIIA. The surface expressed Fc epsilon RI also associates with the same group of heterogenous gamma gamma homodimers. We also show that there is no C-terminal posttranslational cleavage of gamma occurring before its insertion into the plasma membrane as previously suggested. Thus, like the TCR, Fc gamma RIIIA may form a variety of receptor isoforms, though at present we do not understand the functional implications of these structures.
- Published
- 1991
20. Mechanistic studies of transforming growth factor-beta inhibition of IL-2-dependent activation of CD3- large granular lymphocyte functions. Regulation of IL-2R beta (p75) signal transduction.
- Author
-
Ortaldo JR, Mason AT, O'Shea JJ, Smyth MJ, Falk LA, Kennedy IC, Longo DL, and Ruscetti FW
- Subjects
- CD3 Complex, Gene Expression Regulation drug effects, Humans, Interleukin-2 metabolism, Killer Cells, Natural immunology, Phosphorylation, RNA, Messenger analysis, Receptors, Cell Surface physiology, Receptors, Interleukin-2 genetics, Receptors, Transforming Growth Factor beta, Tyrosine metabolism, Antigens, Differentiation, T-Lymphocyte analysis, Interleukin-2 pharmacology, Killer Cells, Natural drug effects, Lymphocyte Activation drug effects, Receptors, Antigen, T-Cell analysis, Receptors, Interleukin-2 analysis, Signal Transduction, Transforming Growth Factor beta pharmacology
- Abstract
CD3- large granular lymphocyte (LGL) express constitutive levels of functional IL-2R beta. TGF-beta inhibited several IL-2R beta-mediated events in LGL, including IL-2-induced NK and lymphokine-activating factor activities, IFN-gamma gene expression and secretion, and IL-2R alpha expression. TGF-beta inhibited these IL-2-induced LGL functions in a dose-dependent and reversible manner. By contrast, TGF-beta had little effect on LGL IL-2R beta expression and TGF-beta receptors were not induced by IL-2. Studies were performed to examine binding and internalization of radiolabeled IL-2. These experiments demonstrated that the rapid binding and internalization of [125I]IL-2 was not altered in CD3- LGL pretreated with TGF-beta. These internalization studies indicated that the TGF-beta inhibition represented postreceptor-binding events in NK cells. Further studies were initiated to examine signaling events in CD3- LGL. When IL-2-induced tyrosine phosphorylation events were examined, significant inhibition was seen of selected phosphoproteins in TGF-beta-pretreated cells. In addition, the ability of TGF-beta to also inhibit IL-2 induction of LGL IL-2R alpha and IFN-gamma mRNA expression was consistent with the hypothesis that posttranscriptional mechanisms were unlikely to be affected by TGF-beta. Collectively, these data indicated that TGF-beta inhibited IL-2-induced CD3- LGL functions and suggested that TGF-beta inhibition occurs either at the level of specific tyrosine phosphorylation and/or IL-2-induced transcriptional control factors.
- Published
- 1991
21. Tumor-promoting phorbol esters induce rapid internalization of the C3b receptor via a cytoskeleton-dependent mechanism.
- Author
-
O'Shea JJ, Brown EJ, Gaither TA, Takahashi T, and Frank MM
- Subjects
- Antibodies, Monoclonal, Binding, Competitive, Cytochalasin B pharmacology, Fibronectins pharmacology, Humans, Immunoglobulin Fab Fragments metabolism, Neutrophils metabolism, Phorbol 12,13-Dibutyrate, Receptors, Complement immunology, Receptors, Complement 3b, Temperature, Tetradecanoylphorbol Acetate pharmacology, Cytoskeleton metabolism, Phagocytosis drug effects, Phorbol Esters pharmacology, Phorbols pharmacology, Receptors, Complement drug effects
- Abstract
Plasma membrane expression as well as phagocytic capability of the C3b receptor (CR1) are under regulatory control. Phorbol esters are one class of agents which have been shown to influence both of these events. In this study, by using radiolabeled Fab fragments of a monoclonal anti-CR1 antibody to tag the receptor and acid elution of surface-bound Fab, we showed that both phorbol myristate acetate and phorbol dibutyrate induced internalization of the C3b receptor; this occurred in a dose- and time-dependent manner in the absence of occupancy of the receptor by ligand. This was shown to occur in neutrophils, monocytes, and macrophages. We also showed that phorbol esters enhanced CR1-dependent phagocytosis despite the presence of two-thirds fewer receptors present on the plasma membrane. However, fibronectin, another agent that influences phagocytosis, had no effect on receptor internalization. Phorbol ester internalization was temperature-dependent and was inhibitable by cytochalasins B and D. Inhibition of internalization was reversible when cytochalasin B was removed. Phorbol esters also induced increased detergent insolubility of CR1 with kinetics similar to those of receptor internalization. It is possible that association of CR1 with the cytoskeleton is important to the process of "activation" of CR1 in phagocytosis.
- Published
- 1985
22. Calcium requirements for increased complement receptor expression during neutrophil activation.
- Author
-
Berger M, Birx DL, Wetzler EM, O'Shea JJ, Brown EJ, and Cross AS
- Subjects
- Calcimycin pharmacology, Calcium antagonists & inhibitors, Calmodulin antagonists & inhibitors, Chlorpromazine pharmacology, Cytoplasmic Granules enzymology, Edetic Acid pharmacology, Gallic Acid analogs & derivatives, Gallic Acid pharmacology, Humans, Neutrophils enzymology, Neutrophils immunology, Receptors, Complement drug effects, Receptors, Complement 3b, Trifluoperazine pharmacology, Calcium physiology, Neutrophils metabolism, Phagocytosis drug effects, Receptors, Complement biosynthesis
- Abstract
It has recently been shown that human neutrophils rapidly increase surface expression of membrane receptors for C3b (CR1) and C3bi (CR3) in response to chemoattractants and other stimuli. In the present studies, we used monoclonal antibodies and flow cytometry to assess the role of Ca2+ in this process. Stimulation with ionophore A23187 in the presence of 1.2 mM Ca2+ increased CR1 330% and CR3 650% compared with unstimulated cells at 37 degrees C. Because this indicated that increasing the cytosolic free Ca2+ caused increased receptor expression, we examined the role of Ca2+ in the response to other stimuli as well. Adding 1.2 mM Ca2+ or 5 mM EDTA to the media in which the polymorphonuclear leukocytes were suspended had no effect on the CR1 response to f-MLP or LTB4, whereas Ca2+ slightly enhanced and EDTA markedly inhibited the CR3 response to these stimuli. The effects of Mg2+-EGTA and EDTA were identical. TMB-8 (200 microM), which inhibits the release of Ca2+ from intracellular stores, completely blocked the increased expression of both receptors induced by fMLP or LTB4. Increased expression of both receptors was also prevented by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (10 microM), but not by chlorpromazine sulfoxide. Phorbol myristate acetate (0.1 ng/ml) increased CR1 230% and CR3 265%. Again Ca2+ and EDTA did not alter the CR1 response, whereas Ca2+ increased CR3 to 287% and EDTA reduced CR3 to 187% of control. TMB-8 (250 microM) completely blocked both CR1 and CR3 responses to this stimulus as well. Thus, release of intracellular Ca2+ is necessary and sufficient for increased CR1 expression in response to diverse stimuli, but maximal increases in CR3 expression require an additional influx of extracellular Ca2+. These results indicate that the mechanisms by which the surface expression of the two different complement receptors increase are different in their requirements for extracellular Ca2+. In comparison with the work of others, we suggest that the processes of increased complement receptor expression and secretion of granular enzymes may also differ.
- Published
- 1985
23. Aluminum fluoride induces phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells.
- Author
-
O'Shea JJ, Urdahl KB, Luong HT, Chused TM, Samelson LE, and Klausner RD
- Subjects
- Animals, Antigens immunology, Colforsin pharmacology, Cyclic AMP metabolism, Cytosol metabolism, Egtazic Acid pharmacology, Mice, Phosphatidylinositol Phosphates, Phosphorylation, Protein Kinase C metabolism, Protein-Tyrosine Kinases metabolism, Sodium Fluoride pharmacology, T-Lymphocytes metabolism, Tetradecanoylphorbol Acetate pharmacology, Aluminum pharmacology, Aluminum Compounds, Calcium metabolism, Fluorides pharmacology, Membrane Lipids metabolism, Phosphatidylinositols metabolism, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes drug effects
- Abstract
Antigen activation of murine T lymphocytes leads to phosphorylation of three subunits of the murine T cell antigen receptor (L.E. Samelson, M.D. Patel, A.M. Weissman, J.B. Harford, and R.D. Klausner. 1986. Cell 46:1083). Two kinases are activated in this process: protein kinase C which leads to phosphorylation of the gamma and, to a lesser extent, the epsilon subunits on serine residues and a tyrosine kinase which phosphorylates the p21 subunit (M.D. Patel, L.E. Samelson, and R.D. Klausner. 1987. J. Biol Chem. 262:5831). We sought to determine whether treatment of these cells with NaF could simulate any of these antigen-induced events. Indeed NaF treatment resulted in breakdown of polyphosphoinositides and production of phosphoinositols. This treatment also resulted in a rise in cytosolic free Ca2+. EGTA failed to block this rise suggesting that NaF liberated intracellular stores of Ca2+. Finally NaF treatment resulted in phosphorylation of the gamma and epsilon chains of the T cell receptor indistinguishable from the effects of phorbol esters. The NaF effect was potentiated by addition of A1Cl3 consistent with the view that the active moiety is A1F4-. The A1F4--induced phosphorylations were abolished in cells in which protein kinase C was depleted by prior treatment with phorbol myristate acetate. All of these observations are compatible with the interpretation that the A1F4- phosphorylation is mediated by protein kinase C. Antigen and anti-receptor antibody-induced receptor serine phosphorylation and phophatidylinositol turnover are blocked by raising intracellular levels of cyclic adenosine monophosphate. In contrast, A1F4--induced effects were insensitive to cyclic adenosine monophosphate.
- Published
- 1987
24. Human C5a modulates monocyte Fc and C3 receptor expression.
- Author
-
Yancey KB, O'Shea J, Chused T, Brown E, Takahashi T, Frank MM, and Lawley TJ
- Subjects
- Complement C5a, Flow Cytometry, Humans, Monocytes immunology, N-Formylmethionine Leucyl-Phenylalanine pharmacology, Neutrophils metabolism, Receptors, Complement 3b, Receptors, IgG, Rosette Formation, Complement C5 physiology, Monocytes metabolism, Receptors, Complement metabolism, Receptors, Fc metabolism
- Abstract
FcIgG and C3 (CR1 and CR3) receptors are responsible for binding opsonized particles, phagocytosis, and immune adherence reactions by circulating and tissue-fixed mononuclear phagocytes. Alterations in the expression of these receptors may thus significantly influence the function of these cells. Because chemoattractants have been shown to both recruit and modulate the function of monocytes, this study specifically examines the effects of human C5a and N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) on human peripheral blood monocyte FcIgG and C3 receptor expression in vitro. Adherent, elutriator-purified monocytes were incubated with C5a (10(-7) to 10(-10) M) or FMLP (10(-5) to 10(-10) M) for 30 min at 37 degrees C, and FcIgG receptor expression was assessed by rosetting with sheep erythrocytes sensitized with limiting dilutions of IgG. Human C5a caused dose-related increases in Fc rosettes of 28% at 10(-9) M, 63% at 10(-8) M, and 167% at 10(-7) M (p less than 0.01). In contrast, no significant increases in monocyte Fc receptor expression were induced by FMLP. Similar rosetting experiments were performed with sheep erythrocytes opsonized with limiting amounts of human C3b to assess C3b receptor expression on adherent human monocytes stimulated with C5a (10(-7) to 10(-10) M) or FMLP (10(-6) to 10(-9) M) for 30 min at 37 degrees C. Again, human C5a caused dose-related increases in monocyte C3b rosette formation; at 10(-8) M and 10(-7) M concentrations of C5a, these increases equaled 119% and 196%, respectively (p less than 0.05). In these experiments, 10(-6) M FMLP also caused a significant increase of 110% in monocyte C3b rosette formation (p less than 0.05). Modulation of monocyte cell surface receptors by human C5a or FMLP was also examined by measuring cell fluorescence and side scatter by dual channel flow cytometry after staining normal leukocytes in citrated venous blood with receptor-specific monoclonal antibodies. These flow cytometric studies demonstrated that both C5a and FMLP induce dose-related increases in CR1 (C3b receptor) and CR3 (iC3b receptor) expression in both monocytes and neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1985
25. Identification and characterization of the phosphatidylinositol kinase in membranes of murine T lymphocytes.
- Author
-
O'Shea JJ, Harford JB, and Klausner RD
- Subjects
- 1-Phosphatidylinositol 4-Kinase, Adenosine Triphosphate pharmacology, Animals, Calcium pharmacology, Cell Fractionation, Cell Membrane enzymology, Chromatography, Gel, Hot Temperature, Hybridomas enzymology, Magnesium pharmacology, Mice, Phosphatidylinositols pharmacology, Phosphotransferases antagonists & inhibitors, Phosphotransferases isolation & purification, Subcellular Fractions enzymology, Substrate Specificity, Phosphatidylinositol Phosphates, Phosphotransferases metabolism, T-Lymphocytes enzymology
- Abstract
Polyphosphoinositide metabolism appears to be a widely employed mechanism by which membrane receptors transduce the signal to activate cells. This pathway has been implicated in the activation of T lymphocytes. Unlike many receptor systems in which decreased levels of polyphosphoinositides are observed, T lymphocytes demonstrate increased concentrations of polyphosphoinositides as well as phosphoinositols (particularly IP3) on activation by mitogens. To understand the early biochemical events involved in T cell activation, we sought to identify and characterize the enzyme responsible for phosphorylating phosphatidylinositol (PI) to phosphatidylinositol 4-phosphate (PIP) in murine T lymphocytes. This kinase was found to be an integral membrane protein of T lymphocytes. It was found to be Mg2+ (apparent Km = 5 mM)- or Mn2+-dependent, whereas Ca2+ was noted to be a competitive inhibitor of Mg2+. ATP is the preferred substrate over GTP (apparent Km = 0.14 mM and 0.62 mM, respectively). The kinase is inhibited by both PIP and PI 4,5-bisphosphate (PIP2), but not by other phospholipids or angiotensin II (a substrate for tyrosine kinase). The enzyme activity has identical characteristics in membranes derived from a T cell hybridoma, thymocytes, and T cell clones. The enzyme migrated predominantly as a single peak with an apparent m.w. of approximately 60,000 on gel filtration chromatography. The relationship of this enzyme to viral oncogene products such as pp60v-src and p68v-ros is discussed.
- Published
- 1986
26. T cell antigen receptor phosphorylation induced by an anti-receptor antibody.
- Author
-
Samelson LE, O'Shea JJ, Luong H, Ross P, Urdahl KB, Klausner RD, and Bluestone J
- Subjects
- Animals, Antigen-Antibody Reactions, Antigens immunology, Inositol Phosphates biosynthesis, Interleukin-2 biosynthesis, Isoelectric Point, Macromolecular Substances, Mice, Molecular Weight, Phosphorylation, Phosphotyrosine, Protein Kinases metabolism, Protein-Tyrosine Kinases metabolism, Tyrosine analogs & derivatives, Tyrosine metabolism, Antibodies, Monoclonal immunology, Antigens, Differentiation, T-Lymphocyte physiology, Phosphoproteins physiology, Receptors, Antigen, T-Cell physiology, T-Lymphocytes physiology
- Abstract
In previous studies we demonstrated that the antigen receptor complex on murine T cells is phosphorylated after antigen or mitogen activation. After the clonotypic structures bind antigen, the invariant subunits or CD3 molecules are the target of dual kinase activation. The antigen receptor CD3-gamma-chain subunit is phosphorylated on serine residues by activated protein kinase C and the p21 subunit is phosphorylated by a tyrosine kinase. Herein we demonstrate that another mechanism of receptor activation by the stimulatory monoclonal antibody 145-2C11, which binds the CD3-epsilon chain, results in a similar pattern of kinase activation and receptor phosphorylation.
- Published
- 1987
27. Fibronectin-enhanced phagocytosis of an alternative pathway activator by human culture-derived macrophages is mediated by the C4b/C3b complement receptor (CR1).
- Author
-
Bohnsack JF, O'Shea JJ, Takahashi T, and Brown EJ
- Subjects
- Animals, Elapid Venoms pharmacology, Erythrocytes metabolism, Humans, Macrophages metabolism, Monocytes immunology, N-Acetylneuraminic Acid, Receptors, Complement 3b, Sheep immunology, Sialic Acids metabolism, Complement Activation drug effects, Complement Pathway, Alternative drug effects, Erythrocytes immunology, Fibronectins physiology, Macrophages immunology, Phagocytosis drug effects, Receptors, Complement physiology
- Abstract
We examined the ability of human monocytes and culture-derived macrophages under serum-free conditions to phagocytose desialated sheep erythrocytes (E), an activator of the alternative pathway of human complement. Freshly derived monocytes ingested desialated erythrocytes, but the degree of phagocytosis varied among individual donors. However, exposing the phagocyte to intact plasma fibronectin (Fn) had no effect on monocyte phagocytosis. Macrophages derived from monocytes in culture were far more efficient at ingesting desialated E, and the extent of phagocytosis was proportional to the degree of desialation. Although exposure of macrophages to substrate-bound Fn or fluid-phase Fn enhanced the phagocytosis of desialated E, pretreatment of desialated E with Fn did not enhance phagocytosis, demonstrating that Fn acted through an interaction with the macrophages. Fn-enhanced phagocytosis of desialated E was inhibited by treating macrophages with a monoclonal antibody to the C4b/C3b receptor (CR1), but not with a monoclonal antibody to the receptor for C3bi (CR3). Addition of cobra venom factor (CVF) to the macrophages also inhibited Fn-enhanced phagocytosis of desialated E. Phagocytosis of IgG-sensitized E, either in the absence or in the presence of Fn, was not significantly affected by anti-CR1 or CVF, demonstrating that these reagents did not lead to a general inhibition of phagocytosis. These experiments suggest that macrophages may deposit enough C3b onto desialated E to cause CR1-mediated phagocytosis in the presence of Fn. The ability of macrophages to opsonize and ingest foreign particles that activate complement may be critically important in areas of inflammation where concentrations of serum-derived specific opsonins may be inadequate.
- Published
- 1985
28. Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils.
- Author
-
O'Shea JJ, Brown EJ, Seligmann BE, Metcalf JA, Frank MM, and Gallin JI
- Subjects
- Acetone pharmacology, Antibodies, Monoclonal immunology, Binding Sites, Antibody, Cytoplasmic Granules metabolism, Fluorescent Antibody Technique, Granulomatous Disease, Chronic pathology, Humans, Molecular Weight, Neutrophils drug effects, Organoids metabolism, Precipitin Tests, Receptors, Complement drug effects, Receptors, Complement immunology, Receptors, Complement physiology, Receptors, Complement 3b, Subcellular Fractions metabolism, Tetradecanoylphorbol Acetate pharmacology, Cell Compartmentation drug effects, Complement C3b Inactivator Proteins metabolism, Neutrophils metabolism, Receptors, Complement isolation & purification
- Abstract
In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.
- Published
- 1985
29. Characterization of the C3 receptor induced by herpes simplex virus type 1 infection of human epidermal, endothelial, and A431 cells.
- Author
-
Kubota Y, Gaither TA, Cason J, O'Shea JJ, and Lawley TJ
- Subjects
- Antibodies, Monoclonal immunology, Cell Line, Gene Expression Regulation, Humans, Receptors, Complement biosynthesis, Receptors, Complement immunology, Receptors, Complement 3b, Receptors, Fc analysis, Receptors, Fc biosynthesis, Rosette Formation, Umbilical Veins, Carcinoma, Squamous Cell analysis, Endothelium analysis, Epidermis analysis, Receptors, Complement isolation & purification, Simplexvirus physiology
- Abstract
Herpes simplex virus type 1 (HSV-1) infection induces the appearance of viral analogues of human Fc IgG and C3 receptors on the surface of human cells. The virally induced C3 receptor(s) has been broadly defined as a C3b receptor, but its ligand binding characteristics have not been rigorously defined. In this study, human epidermal cells, A431 cells, and human umbilical vein endothelial cells infected with HSV-1 demonstrated rosetting with sheep erythrocytes (E) coated with IgG (E-IgG) or the complement components C3b (EAC3b) or iC3b (EAC3bi), but not with E-IgM, C4 (EAC14), C3d (EAC3d), or E alone. Rosetting was markedly enhanced by pretreatment of HSV-1-infected cells with neuraminidase. Unlike human C3 receptors, the HSV-1-induced C3 receptor was found to be trypsin resistant. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. EDTA failed to prevent rosetting with EAC3bi. Furthermore, blocking studies using monoclonal antibodies against CR1 and CR3 revealed that the anti-CR1 antibody 5C11 consistently blocked EAC3b and EAC3bi rosetting with HSV-1-infected cells in a dose dependent manner, but monoclonal antibodies against CR3 did not. This study indicates that the HSV-1-induced C3 receptor is an analogue of CR1.
- Published
- 1987
30. Osmotic stress and the freeze-thaw cycle cause shedding of Fc and C3b receptors by human polymorphonuclear leukocytes.
- Author
-
Takahashi T, Inada S, Pommier CG, O'Shea JJ, and Brown EJ
- Subjects
- Absorption, Animals, Blood Group Antigens immunology, Escherichia coli immunology, Humans, Neutrophils immunology, Opsonin Proteins, Osmolar Concentration, Precipitin Tests, Receptors, Complement analysis, Receptors, Complement 3b, Receptors, IgG, Rosette Formation, Sheep, Freezing, Neutrophils metabolism, Phagocytosis, Receptors, Complement metabolism, Receptors, Fc metabolism, Stress, Physiological
- Abstract
A major problem in the cryopreservation of human polymorphonuclear leukocytes (PMN) is the loss of phagocytic function in cryopreserved cells. This is not a problem with cryopreserved monocytes. To study the reasons for this difference in detail, PMN and monocytes were either osmotically stressed in hypertonic media or were frozen to various temperatures. Cells were then returned to conditions of physiologic osmolarity and temperature. All cells remained viable. However, the ability of PMN to phagocytize bacteria and to bind sheep erythrocytes (E) opsonized with IgG, C3b, or C3bi decreased sharply after exposure to media of 600 mOsM or greater and after freezing to -1.5 degrees C. In contrast, monocytes were unaffected until a concentration of 1500 mOsM or a freezing temperature of -5 degrees C was exceeded. To determine whether the functional losses of surface receptor activity in PMN resulted from a loss of receptors from the membranes or from inactivation or internalization of receptors, opsonized E were incubated in the supernatants from stressed PMN. On subsequent incubation with healthy PMN, these E made fewer rosettes than control opsonized E. The inhibitory effect of the supernatants on rosetting of IgG-sensitized E could be removed by preincubation with IgG bound to Sepharose 4B. Immunoprecipitation of C3b and C3bi receptors from surface-iodinated, osmotically stressed, and control PMN suggested that about 50% of cell surface complement receptors were lost from the cell surface during osmotic stress. These experiments suggest that receptors for IgG and C3 are extruded from PMN cell membranes as a result of hyperosmotic stress, which is associated with the freeze-thaw cycle. This may be an early event in the functional damage done to PMN during attempts at cryopreservation.
- Published
- 1985
31. Expression of specific binding sites on Candida with functional and antigenic characteristics of human complement receptors.
- Author
-
Edwards JE Jr, Gaither TA, O'Shea JJ, Rotrosen D, Lawley TJ, Wright SA, Frank MM, and Green I
- Subjects
- Antibodies, Monoclonal, Candida cytology, Candida albicans immunology, Complement C3 metabolism, Complement C3b metabolism, Complement C3d, Humans, Receptors, Complement 3b, Candida immunology, Receptors, Complement immunology
- Abstract
Receptors for C3 degradation fragments (CR1, CR2, and CR3) are present on many human cells including phagocytes and lymphoid cells and may be critical in the attachment of invading microorganisms. In these studies Candida were found to mimic the human CR by binding erythrocytes coated with specific human C3 fragments. Yeast forms of Candida species were adhered to glass slides and were allowed to germinate. Sheep erythrocytes (E) were coated with IgM (EA) and human complement components to prepare EA, EAC14, EAC3b, EAC3bi, and EAC3d. These test cells were then examined for adherence to the organism. Antibodies to human CR1, CR2, and CR3 were used to evaluate their potential for blocking adherence of the test erythrocytes to Candida. Fluorescein-labeled antibodies to human complement receptors were also used to characterize the binding sites. EAC3bi and EAC3d, but not E, EA, or EAC14, bound extensively to the germ tubes and pseudohyphae of Candida albicans and C. stellatoidea. EAC3b bound infrequently. Other Candida species, generally considered less pathogenic, bound significantly fewer specific test erythrocytes than C. albicans. Monoclonal antibodies to human CR1 and CR3 (3D9, 1B4, C511, 2B6, anti-B2, Mo1, and anti-Mac-1), in general, did not block adherence of test erythrocytes. Blocking of adherence of EAC3bi and EAC3d test erythrocytes coated with small quantities of C3 fragments occurred with high concentrations of monoclonal (anti-CR2) HB-5 and polyclonal (anti-CR2) anti-GP 140. Immunofluorescence studies demonstrated binding of Mo-1 to the germinated forms of the organism, whereas binding of the other antibodies was not seen. These studies suggest a surface constituent on the organism similar to CR on human cells. Additional studies are necessary to further define the molecular nature of the binding site. The ability of organisms to mimic human CR may be more generalized than previously known and may serve as a mechanism for modification of the inflammatory and immune response.
- Published
- 1986
32. Activation of the C3b receptor: effect of diacylglycerols and calcium mobilization.
- Author
-
O'Shea JJ, Siwik SA, Gaither TA, and Frank MM
- Subjects
- Calcimycin pharmacology, Calcium physiology, Calcium Channel Blockers pharmacology, Drug Synergism, Gallic Acid analogs & derivatives, Gallic Acid pharmacology, Humans, Neutrophils metabolism, Phagocytosis drug effects, Phorbol Esters pharmacology, Receptors, Complement drug effects, Receptors, Complement 3b, Type C Phospholipases pharmacology, Calcium metabolism, Complement Activation drug effects, Diglycerides pharmacology, Glycerides pharmacology, Receptors, Complement metabolism
- Abstract
The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.
- Published
- 1985
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