A rapid evaluation of phagocytosis and killing of Candida albicans by CD13+ leukocytes.

Flow cytometry can be adopted for routine monitoring of the immune functions of human polymorphonuclear leukocytes (PMNs) in several disease states. We recently developed a rapid and reproducible assay for the evaluation of the phagocytosis and killing of Candida albicans blastospores by human PMNs. Whole blood leukocytes were incubated with opsonized fluorescein isothiocyanate-labeled (FITC-labeled) blastospores for phagocytosis and killing assays. To discriminate between ingested, membrane-bound and free C. albicans blastospores, ethidium bromide (EtBr) was added to the samples prior to the flow cytometric analysis. EtBr induces a loss of green fluorescence in non-phagocytized C. albicans blastospores. Phagocytosis is determined by gating the phagocytes and calculating the percentage of phagocyte-associated green fluorescent cells. Intracellular killing is determined by first lysing phagocytes by hypotonic shock and then adding propidium iodide (PI) in order to identify red dead blastospores. Killing is measured in terms of the percentage of double-marked blastospore cells. We suggest that this method is a reliable and inexpensive technique to evaluate the immune reactivity of PMNs and peripheral blood monocytes (PBMs) in cases of immunosuppression.

Increased levels of CD8+ and CD4+ 45RA+ lymphocytes in schizophrenic patients.

Peripheral blood (PB) lymphocyte subpopulations, IgG, IgM, IgA and IgE serum immunoglobulins and C3 and C4 complement fractions were evaluated in 29 schizophrenic patients, 31 of their relatives and 20 healthy subjects. The patients fulfilled DSM-III criteria for schizophrenia, and were unmedicated for 3 months prior to the PB sample collection. When compared to healthy controls and their own relatives, the schizophrenic patients showed a lower level of CD4+ cells, while the CD4+ 45RA+ (naive) subset was significantly higher. Conversely, the number of CD4+ 45RA- (memory) lymphocytes was significantly lower in schizophrenic patients in comparison to their relatives and controls, while the CD8+ supressor/cytotoxic T-cell percentage was significantly higher. No significant differences were observed for the IgG, IgM, IgA, IgE and C3 and C4 complement fraction levels among the three groups. The present data confirm the presence of immunological abnormalities in schizophrenic patients and suggest a possible role of environmental factors in the triggering of an autoimmune pathogenic mechanism.

Different endothelins stimulate cytokine production by peritoneal macrophages and microglial cell line.

Endothelins (ETs), potent vasoconstricting peptides, are produced by macrophages upon stimulation and may participate in the amplification or regulation of the inflammatory response. However, it is not clear whether ETs can act in an autocrine manner on macrophages and which role they play in relationship with other cytokines. To address these issues, we studied the effects of ETs on the production of inflammatory cytokines by mouse peritoneal macrophages or by a retrovirus-transformed microglial cell line. Here, we report that ET-2, but not ET-1 or ET-3, is able to stimulate the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) by peptone-elicited mouse macrophages (pMO). In contrast, ET-3 and ET-1, but not ET-2, are active on microglial cells. No tumour necrosis factor-alpha (TNF-alpha) or nitric oxide (NO) were detected in the supernatants of ET-stimulated cultures. The activity of ET-2 on pMO was time and dose dependent and was inhibited by the addition of ETA and ETB receptor antagonists, BQ123 and IRL1038, respectively. In addition, when pMO were stimulated by interferon-gamma (IFN-gamma) in the presence of ET-2, a significant inhibition of IL-6 and IL-1 production was observed compared with the effects of the same doses of IFN-gamma or ET-2 used separately. The inhibition was specifically due to the activity of ET-2, since it was reversed by the addition of BQ123 or IRL1038. Similar results were seen when the content of NO in the supernatants of pMO stimulated by IFN-gamma plus ET-2 was evaluated. These results suggest that ETs may possess both a pro-inflammatory action on macrophages from different tissues and a regulatory activity on IFN-gamma.