FEMS microbiology immunology最新文献

Subcutaneous transplantation of EL4 lymphoma cells within C57BL10 mice evoked an oedematous inflammatory response involving increased leukopoiesis within the bone marrow, a blood leukocytosis, an influx of leukocytes into the transplants and surrounding host connective tissues, and extensive remodelling of surrounding host connective tissues involving fibroplasia and angiogenesis. Dexamethasone not only significantly reduced the numbers of circulating blood leukocytes within C57BL10 mice bearing the subcutaneous EL4 lymphoma transplants, but also reduced the oedematous inflammatory response to the transplants. The decreased influx of inflammatory leukocytes into the site of EL4 lymphoma cell transplantation within the dexamethasone-treated mice, was accompanied by reduced growth of the transplants. Although the EL4 lymphoma cells produce factors with Colony Stimulating Factor activity and with chemotactic activity for cells of the monocyte-macrophage lineage, they do not appear to produce fibroblast growth factors directly but can induce (or stimulate) macrophages to generate fibroblast growth factors in vitro. While not directly inhibiting the growth of subcutaneous fibroblasts in vitro, dexamethasone does suppress the production and/or activity of fibroblast growth factors generated through macrophage-EL4 cell interactions in vitro. The inhibitory effects of dexamethasone on macrophage influx, fibroplasia and angiogenesis within the connective tissue surrounding the EL4 lymphoma transplants appear to be casually related events and would account for the inhibitory effect of dexamethasone on the growth of the lymphoma transplants.

Macrophages elaborate both effector and regulatory immune functions. It was hypothesised that tumours can exert a local alteration of macrophage function. Murine peritoneal macrophage-derived cytokines were assayed in the presence and absence of cells, cytosol fractions or conditioned media (TCCM) from established murine tumour lines. Interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha activities were significantly inhibited by tumour cells or their products, as were the corresponding recombinant human cytokines. Intracellular protein kinase C activation was also measured and was significantly inhibited by murine TCCM, thus suggesting one possible site of inhibitor action. Data analyses indicate that the inhibitory factor(s) is probably not an already well-characterised macrophage inhibitor.

The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E2 release was studied in monocytes (M phi). Both IL-1 alpha and IL-1 beta increased the release of PGE2 in a concentration-dependent manner, with EC50s of 0.48 nM and 0.12 nM, respectively. Intact M phi were prelabelled with [3H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [3H]phosphatidylinositol-4,5-bisphosphate to aqueous soluble radioactivity by M phi homogenates. IL-1 alpha (5.8 nM) increased the accumulation of IPs within 1-4 minutes and increases in IP3 and IP4 occurred before the increase in IP1+2 whereas LPS only increased the IPs level after at least 30 min. IL-1 alpha increased PIC activity in M phi homogenates within 15 min with an EC50 of 0.58 nM and IL-1 beta (0.1 nM) also increased activity. Neither IL-1 alpha nor IL-1 beta affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in M phi.

Recent studies suggest that granulocytes (PMNs) play a role in the pathogenesis of acute and chronic myocardial ischemia and extension of myocardial injury. Granulocytes can release a variety of molecules mediating tissue injury which act synergistically with other molecules and cells. The aim of our investigation was to evaluate the granulocyte function in patients affected by coronary artery disease (CAD) and during coronary angioplasty (PTCA). We studied 20 patients suffering from CAD. The PMN's aggregating activity was greater in the coronary sinus than in the aorta (P < 0.01). The increase in aggregating activity was evident in patients who were smokers: their cells release significantly lower quantities of leukotriene C4 (P < 0.025). In the 20 patients who underwent coronary angioplasty we analyzed superoxide release after stimulation with phorbol-myristate-acetate (PMA). The results showed a greater decrease of PMN's superoxide production in the coronary sinus than in the aorta (P < 0.05). In all patients affected by CAD we evaluated the PMN's expression of CD11b/CD18 membrane integrins. In these patients the increase in expression of CD11b/CD18 was statistically significant in comparison with the controls (P < 0.01). This increase in expression correlates with a higher aggregation (r = 0.87, P < 0.001). The potential role of leukocytes, oxygen radicals, leukotrienes and granulocyte enzymes in the pathophysiology of myocardial injury due to regional ischemia and reperfusion is an area of intense investigation. This paper presents studies carried out in vivo which have been instrumental in demonstrating the role of granulocytes as mediators of myocardial ischemia.

Protein kinase C (PKC) appears to have a central role in the O2- response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP2)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling to PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O2-. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.

Stimulation of human neutrophils with the chemotactic peptide fMet-Leu-Phe results in activation of a rapid, transient burst of oxidant secretion, which reaches a maximal rate by about 1 min after stimulation. This phase of oxidant secretion is then followed by intracellular oxidant production, which is detected by luminol chemiluminescence but not by assays such as cytochrome c reduction or scopoletin oxidation. The rapid phase of oxidant secretion requires increases in intracellular free Ca2+ and phospholipase A2 activity, but not the activities of phospholipase D or protein kinase C. In contrast, intracellular oxidant production requires the activities of phospholipase D and protein kinase C. A model is thus proposed suggesting the sequential activation of different phospholipases which activate oxidase molecules on the plasma membrane or else from the membranes of specific granules.

In contrast to the phorbol ester oxidative response, which only develops during dimethylsulphoxide (DMSO)-induced differentiation of the human leukemic myeloblast HL-60 cell-line, the endotoxin response was observed in undifferentiated and differentiated cells. The Ca2+ response to endotoxin, detected in both differentiated and undifferentiated HL-60 cells, consisted of a transient 10-50 nM increase in intracellular Ca2+. A very slow, irreversible increase in intracellular Ca2+ was detected at high 1-100 micrograms/ml endotoxin concentrations, and this effect, and the inositol phosphate response, correlated with the surfactant activities of various endotoxins and Lipid A. Arachidonic acid and sodium arachidonate 1-50 microM stimulated a large 200-500 nM and transient Ca2+ response in undifferentiated HL-60 cells, which was significantly greater than that elicited by 1-50 microM eicosapentaenoic acid, and was not observed at similar concentrations of arachidonic acid methyl ester or myristic acid. These concentrations (1-50 microM) of arachidonic acid were observed to have surfactant activities on the plasma membrane. At lower arachidonic acid concentrations a marked potentiation of both Ca2+ and oxidative responses to the chemotactic peptide fMet-Leu-Phe was detected. It is possible that the arachidonic acid released during phospholipase A2 activation of neutrophils may be involved in cellular cross-talk and, at higher concentrations, in directly activating Ca2+ and superoxide production. It is also possible that previously reported effects of endotoxin at high concentrations are an in vitro artefact of surfactant properties of endotoxin.

We have investigated transferrin synthesis by human and mouse lymphoid and myeloid cells. It was found that transferrin synthesis is a property of mouse but not human macrophages, whereas in man T lymphocytes synthesised transferrin. Synthesis by mouse macrophages showed a dose-dependent increase in response to gamma-interferon (gamma-IFN), but iron added as ferric nitrilotriacetate had no effect. Macrophage-derived transferrin was found to contain iron already bound to it and was able to support Con A-stimulated mouse lymphocyte proliferation.

Hemolysin (HlyA) and related toxins are secreted across both the cytoplasmic and outer membranes of Escherichia coli and other pathogenic Gram-negative bacteria in a remarkable process which proceeds without a periplasmic intermediate. It is directed by an uncleaved C-terminal targetting signal and the HlyD and HlyB translocator proteins, the latter of which are members of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells. Our mutational analyses of the HlyA targetting signal and definition for the first time of stages and intermediates in the HlyB/HlyD-dependent translocation allow a discussion of the hemolysin export process in the wider context of protein translocation.