Browsing by keyword "Antigens, CD11b"
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Cellular activation, phagocytosis, and bactericidal activity against group B streptococcus involve parallel myeloid differentiation factor 88-dependent and independent signaling pathwaysGroup B streptococci (GBS) vigorously activate inflammatory responses. We reported previously that a secreted GBS "factor" activates phagocytes via Toll-like receptor (TLR)2 and TLR6, but that GBS cell walls activate cells independently of these receptors. We hypothesized that the phagocytic immune functions in response to GBS, such as inflammation, uptake, and elimination of bacteria, occur through a coordinated engagement of TLRs, along with the coreceptors CD14 and CD11b/CD18. Using various knockout mice we show that GBS-induced activation of p38 and NF-kappaB depends upon the expression of the cytoplasmic TLR adapter protein, myeloid differentiation factor 88 (MyD88), but not TLR2 and/or TLR4. Macrophages with deletions of CD14 and complement receptor 3 had a normal cytokine response to whole bacteria, although the response to GBS factor was abrogated in CD14-null cells. The intracellular formation of bactericidal oxygen species proved to be MyD88 dependent; however, uptake of GBS, a prerequisite for intracellular killing by O(2) radicals, occurred independently of MyD88. While deletion of complement receptor 3 greatly diminished the uptake of opsonized GBS, it did not affect the formation of bactericidal O(2) radicals or inflammatory signaling intermediates. We conclude that the inflammatory, bactericidal, and phagocytic responses to GBS occur via parallel but independent processes.
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Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activityBACKGROUND: Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P-selectin with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac-1 (integrin alphaMbeta2, CD11b/CD18). The activation-dependent conformational change in monocyte surface Mac-1 results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1. The aim of this study was to develop whole blood flow cytometry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. METHODS: Citrate or D-Phe-Pro-Arg-chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5), CD42a (PE), FITC-conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BD FACSCalibur was used to analyze monocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90 degrees light scatter. These populations were further gated into CD42a-positive (platelet-bound) and CD42a-negative (platelet-free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac-1, and CD11b. Compensation controls were prepared on six normal donors prior to the study and these settings were used throughout the 10 donor study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. RESULTS: The physiologic agonists collagen and ADP increased monocyte-platelet and neutrophil-platelet aggregates and increased leukocyte surface Mac-1/CD11b and surface-bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac-1/CD11b were mainly independent of leukocyte-platelet binding, the increases in surface-bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte-platelet binding. CONCLUSIONS: (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. (ii) The monocyte and neutrophil surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to monocytes and neutrophils.
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Inflammatory cytokine-mediated evasion of virus-induced tumors from NK cell controlInfections with DNA tumor viruses, including members of the polyomavirus family, often result in tumor formation in immune-deficient hosts. The complex control involved in antiviral and antitumor immune responses during these infections can be studied in murine polyomavirus (PyV)-infected mice as a model. We found that NK cells efficiently kill cells derived from PyV-induced salivary gland tumors in vitro in an NKG2D (effector cell)-RAE-1 (target cell)-dependent manner; but in T cell-deficient mice, NK cells only delay but do not prevent the development of PyV-induced tumors. In this article, we show that the PyV-induced tumors have infiltrating functional NK cells. The freshly removed tumors, however, lack surface RAE-1 expression, and the tumor tissues produce soluble factors that downregulate RAE-1. These factors include the proinflammatory cytokines IL-1alpha, IL-1beta, IL-33, and TNF. Each of these cytokines downregulates RAE-1 expression and susceptibility to NK cell-mediated cytotoxicity. CD11b(+)F4/80(+) macrophages infiltrating the PyV-induced tumors produce high amounts of IL-1beta and TNF. Thus, our data suggest a new mechanism whereby inflammatory cytokines generated in the tumor environment lead to evasion of NK cell-mediated control of virus-induced tumors.