Down-regulation of Th2 responses by Brucella abortus, a strong Th1 stimulus, correlates with alterations in the B7.2-CD28 pathway.

Down-regulation of the Th2-like response induced by ovalbumin-alum (OVA/alum) immunization by heat-killed Brucella abortus was not reversed by anti-IL-12 antibody treatment or in gamma interferon (IFN-gamma) knockout mice, suggesting that induction of Th1 cytokines was not the only mechanism involved in the B. abortus-mediated inhibition of the Th2 response to OVA/alum. The focus of this study was to determine whether an alternative pathway involves alteration in expression of costimulatory molecules. First we show that the Th2-like response to OVA/alum is dependent on B7.2 interaction with ligand since it can be abrogated by anti-B7.2 treatment. Expression of costimulatory molecules was then studied in mice immunized with OVA/alum in the absence or presence of B. abortus. B7.2, but not B7.1, was up-regulated on mouse non-T and T cells following immunization with B. abortus. Surprisingly, B. abortus induced down-regulation of CD28 and up-regulation of B7.2 on murine CD4(+) and CD8(+) T cells. These effects on T cells were maximal for CD28 and B7.2 at 40 to 48 h and were not dependent on interleukin-12 (IL-12) or IFN-gamma. On the basis of these results, we propose that the IL-12/IFN-gamma-independent inhibition of Th2 responses to OVA/alum is secondary to the effects of B. abortus on expression of costimulatory molecules on T cells. We suggest that down-regulation of CD28 following activation inhibits subsequent differentiation of Th0 into Th2 cells. In addition, decreased expression of CD28 and increased expression of B7.2 on T cells would favor B7.2 interaction with CTLA-4 on T cells, and this could provide a negative signal to developing Th2 cells.

Hemoglobin A0 and alpha-crosslinked hemoglobin (alpha-DBBF) potentiate agonist-induced platelet aggregation through the platelet thromboxane receptor.

Chemically modified hemoglobins are potential oxygen-carrying blood substitutes, but their in vivo administration has been associated with a variety of unexpected side events, including increased platelet reactivity. We studied the effects of hemoglobin A0 (HbA0) and alpha-crosslinked hemoglobin (alpha-DBBF) on platelets in vitro. Neither hemoglobin A0 nor alpha-DBBF activated platelets when added alone, but both proteins potentiated submaximal agonist-induced platelet aggregation without increasing other markers of platelet activation such as serotonin secretion. Only agonists that are known to cause release of platelet arachidonic acid (AA) were potentiated while aggregation induced by ADP, which does not release AA, was not potentiated. Blockade of the thromboxane receptor with SQ-29,548 prevented the HbA0-induced and the alpha-DBBF-induced potentiation suggesting that the AA/thromboxane signaling pathway mediates the interaction of platelets with hemoglobin.

Amphotericin B induced abnormalities in human platelets.

Aims-To investigate in vitro the effect of amphotericin B on platelets in order to understand poor platelet recovery in patients receiving platelet transfusions and amphotericin B simultaneously.Methods-Washed platelets were isolated from platelet concentrates and exposed to amphotericin B (4 mug/ml) for one hour. Platelet function was assessed by aggregation response to thrombin (0-0.6 U/ml), serotonin release, response to hypotonic stress, and mean platelet volume. The expression of surface membrane glycoprotein (GP) Ib-IX complex, GPIIb-IIIa complex and CD62P (P-selectin) was examined by flow cytometry using fluorescence labelled monoclonal antibodies. Heterotypic cell adhesion was measured in amphotericin B treated platelets coincubated with isolated, autologous polymorphonuclear leucocytes (PMN) by flow cytometric analysis.Results-Amphotericin B induced platelet dysfunction. The rate of aggregation by thrombin, serotonin uptake and thrombin induced release of serotonin, and the response of platelets to hypotonic stress were inhibited. There was up to a two-fold increase in the mean platelet volume. The expression of platelet surface GPIb-IX and GPIIb-IIIa was not affected. P-selectin, normally expressed only on the surface of activated platelets, was also expressed on unactivated platelets. Amphotericin B increased platelet adherence to PMN and the number of platelets bound per PMN.Conclusions-In vitro, amphotericin B induces P-selectin expression on the surface of unactivated platelets and increases platelet adhesion to PMN, which is exacerbated by storage. Platelet dysfunction resulting from exposure to amphotericin B may contribute to poor platelet recovery in vivo when amphotericin B is administered concomitantly with platelet transfusion.

Effects of gamma-IFN and NGF on subpopulations in a human neuroblastoma cell line: flow cytometric and morphological analysis.

Neuroblastomas are neural crest-derived tumors that contain neuronal, melanocyte, and Schwann cell precursors. We examined the effects of treatment with gamma-interferon (gamma-IFN) and nerve growth factor (NGF), alone, and in combination, on these progenitor subpopulations in the human neuroblastoma cell line, SH-SY5Y. Using fluorescence-activated flow cytometry (FACS), changes in expression of three differentiation-specific or -associated marker proteins, the 200 kD neurofilament protein, the myelin basic protein, and the S-100 protein, were analyzed. Growth rates and morphological changes associated with each treatment over the 2-wk incubation period were noted. The greatest effects were observed with combined IFN + NGF treatment. These were significant increases in expression of all three proteins, distinctive morphological signs of differentiation, and extensive inhibition of proliferation compared to control cultures. Treatment with NGF alone resulted in increased neurofilament protein expression and in the length and number of neurite extensions, but there was no effect on the growth rate. IFN induced striking morphological changes, significant inhibition of growth, and changes in protein expression that correlated with neuronal to non-neuronal subpopulation shifts due to the death of differentiated cells. When treatment was discontinued after 15 d, the morphological changes induced by NGF were reversed within 2-3 d, while those induced by IFN +/- NGF were present up to 4 wk post-treatment. Small, neuroblastic colonies were observed throughout the treatment period and within 4-6 wk after the cessation of treatment this cell-type fully reconstituted the cultures suggesting the presence of a stem cell. Our results indicate that treatment with gamma-IFN +/- NGF can regulate growth and induce, either stem cells or progenitor neuronal, Schwann and melanocyte subpopulations in the SH-SY5Y cell line to irreversibly differentiate.

Adhesion of platelets to chemotactically responsive and non-responsive neutrophils.

Aims-To investigate the heterotypic adhesion of unactivated platelets to chemotactically responsive (migrated) and non-responsive (non-migrated) polymorphonuclear neutrophils (PMN).Methods-Platelets and PMN were isolated from autologous, normal blood. Migrated and non-migrated PMN were separated after N-formylmethionyl-leucylphenylalanine (FMLP) stimulation. Platelets were labelled with a fluorescent monoclonal antibody directed against CD41 (GPIIb-IIIa). Platelets (3 x 10(8)/ml) and PMN (3 x 10(6)/ml) were incubated together. Heterotypic cell adhesion was measured in isolated PMN and PMN co-incubated with platelets by flow cytometric analysis of platelet marker fluorescence in PMN gated events. Platelet-PMN adhesion was also visualised by fluorescence microscopy.Results-In studies of isolated PMN, contaminating platelets were bound to 16-34% of unstimulated PMN, 7-22% of stimulated PMN, 2-4% of migrated PMN, and 17-24% of non-migrated PMN. When platelets were co-incubated with migrated or non-migrated PMN, 15-78% of PMN bound one or two platelets.Conclusions-Unactivated platelets adhere to isolated PMN in vitro. Fewer unactivated platelets were adhered to migrated PMN than to non-migrated PMN in isolated PMN preparations. These results indicate that platelets adhering to PMN are removed during PMN migration.

Penicillin-induced dysfunction of platelet membrane glycoproteins.

We examined the effects of the beta-lactam antibiotic penicillin G on platelet function and on specific membrane glycoproteins in vitro. Platelet concentrates exposed to 3 to 10 mmol/L penicillin for 48 hours showed irreversible inhibition of aggregation by thrombin in washed platelets after removal of the antibiotic. Although a brief 15-minute exposure to similar doses of penicillin also inhibited thrombin aggregation, the inhibition was reversed on removal of the penicillin by washing. Aggregation activity was also restored to normal levels by stimulation with high thrombin concentrations (> 0.4 U/ml). Results of the aggregation studies led us to examine how penicillin affects platelet membrane proteins. Membranes isolated after 48 hours of exposure of platelet concentrates to penicillin showed no differences from the control in total protein profiles on sodium dodecylsulfate--polyacrylamide gel electrophoresis or in the glycoprotein Ib or IIb content on immunoblotting. However, flow cytometric analysis with fluorescently labeled monoclonal antibodies revealed that exposure of platelets to penicillin for 15 minutes inhibited thrombin-induced modulations in the glycoproteins Ib, Ib-IX, IIb-IIIa, and P-selectin. These effects were observed with washed platelets and platelets in plasma. Penicillin also inhibited the regulation of expression of glycoproteins Ib-IX and IIb-IIIa in adenosine diphosphate--activated platelets. The inhibitory effects were partially reversed at high agonist concentrations.