Search

Human neutrophils respond with an increased phagocytosis when exposed to TNF. Two types of TNF receptors have been identified, namely 55 kDa (TR55) and 75 kDa (TR75). We addressed the problem of the role of these receptors in the priming effect of TNF. By using monoclonal antibodies (MoAbs) directed either against TR55 or TR75, we have shown that 1) only TR55 is the signaling receptor for the potentiation of Fc-mediated phagocytosis and upregulation of β2-integrin CD11b/CD18; 2) TR75 may control the function of TR55 by regulating the binding of TNF to the signaling receptor.Copyright 1995, 1999 Academic Press, Inc.

In this paper, we investigated the subcellular distribution of p190rho guanosine triphosphatase-activating protein (p190 GAP) in human neutrophils stimulated with different agonists. The results show that in neutrophils treated with formyl-methionyl-leucyl-phenylalanine (FMLP) (1) p190 GAP was translocated from the cytosol to the membranes; (2) the translocation of p190 GAP took place only at doses of FMLP that induced the translocation of rac 1 and rac 2 and the activation of the NADPH oxidase; and (3) the kinetic of translocation of p190 GAP paralleled that of rac 1 and rac 2. However, when the agonist was concanavalin A (ConA) or phorbol 12-myristate 13-acetate (PMA), rac 1 and rac 2, but not the p190 GAP, were translocated.

It has been recently suggested that protein tyrosine phosphorylation is involved in FcγRs-mediated signalling. In this paper we have investigated if in human neutrophils a tyrosine phosphorylation of FγRII takes places after the binding with immunocomplexes and if this phosphorylation plays a role in phagocytic signal. The immunoprecipitation with mAb anti-FcγRII of lysates of neutrophils challenged in suspension with insoluble immunocomplexes (IIC) or sheep erythrocytes opsonized with IgG (E-IgG), followed by immunoblotting with anti-phosphotyrosine antibody, demonstrated that FcγRII was tyrosine phosphorylated. When neutrophils were pretreated with different doses of tyrosine kinase inhibitors, genistein or erbstatin, the phosphorylation of FcγRII induced by IIC or E-IgG was dose dependently inhibited. In these conditions both genistein and erbstatin failed to inhibit the phagocytosis of E-IgG. These results demonstrated that in human neutrophils in suspension the binding to Fc of IgG induces a tyrosine phosphorylation of FcγRII but this phosphorylation is not an essential signal for phagocytosis of IgG-opsonized particles.Copyright 1994, 1999 Academic Press, Inc.

Although Interleukin-10 (IL-10) has been recently shown to modulate lipopolysaccharide (LPS)-induced release of cytokines in human granulocytes, the intracellular signalling pathways of LPS have been only partially defined, while those of IL-10 remain unknown. The present study shows that LPS induces an increase in tyrosine phosphorylation of a discrete number of proteins, in a time- and concentration-dependent manner. In addition, IL-10 negatively influenced protein tyrosine phosphorylation in LPS-treated human polymorphonuclear leukocytes (PMN). The effect of IL-10 was evident only after 60 min LPS-stimulation and was detected by analysing either cell lysates or lysates which were previously immunoprecipitated with anti-phosphotyrosine antibodies. Amongst the tyrosine phosphoproteins mostly affected by IL-10 in LPS-stimulated cells were the species with molecular weights ranging from 46 to 49 kDa. The identity and possible function of these proteins remain unknown. Taken together, our results suggest that tyrosine phosphorylation may constitute one of the intracellular events that mediate LPS and IL-10 responses in granulocytes.Copyright 1995, 1999 Academic Press, Inc.

The correlation between the increase of [Ca2+]i and the activation of hydrolysis of phosphoinositide-4,5-bisphosphate and formation of inositol(1,4,5)trisphosphate in neutrophils treated with Fc receptor-binding agonists is still under discussion. In this communication evidence is presented supporting the conclusion that, as it is widely accepted for the activation of other receptors, also upon the activation of Fc receptors the stimulation of the production of inositol(1,4,5) trisphosphate is involved in the increase in [Ca2+]i. In fact: i) treatment of neutrophils with immune complexes induced a very rapid phosphoinositide hydrolysis measured as [3H]inositol phosphates production from [3H]phosphoinositides and as inositol(1,4,5) trisphosphate formation measured with radioreceptor assay; ii) immune complexes caused a dose-dependent increase of [Ca2+]i; iii) the increase of [Ca2+]i correlated with the production of inositol(1,4,5) trisphosphate with respect to time course, dose dependence and pertussis toxin insensitivity.Copyright 1993, 1999 Academic Press

The stimulation of phosphoinositide hydrolysis by a number of agonists (phosphoinositide response) is a ubiquitous transmembrane signalling process for the regulation of several cell functions. Two mechanisms of activation have been identified that involve different phospholipases C: one regulated by G-proteins and another regulated by receptors having an intrinsic tyrosine kinase domain or that stimulate intracellular tyrosine kinase activity. This last mechanism is activated in several immunological cells, including lymphocytes, mastocytes, NK cells and monocytes, in response to agonists that bind antigen receptors, and receptors for IgE and IgG. In the present study, we have investigated the role of tyrosine phosphorylation in the stimulation of phosphoinositide hydrolysis mediated by FcγRs in human neutrophils. The results demonstrated that: 1) the activation of FcγRs with insoluble immune complexes (IIC) induced a tyrosine phosphorylation of several proteins that was dose-dependently inhibited by the tyrosine kinase inhibitor, genistein; 2) the activation of FcγRs caused a stimulation of phosphoinositide hydrolysis measured as [3H]inositol phosphates formation; 3) genistein depressed the activation of phosphoinositide hydrolysis; 4) among the several proteins that became tyrosine phosphorylated upon FcγRs activation by IIC, one 145 kDa protein was identified as PLC-γ2, using a specific antiserum. The phosphorylation of PLC-γ2 was completely inhibited by genistein. These results demonstrate that the phosphoinositide response to activation of FcγRs involves the tyrosine phosphorylation of PLC-γ2.Copyright 1994, 1999 Academic Press, Inc.

In neutrophils of a chronic granulomatous disease (CGD) patient with a lack of p67phox the mRNA for p67phox was present in normal amount and size. This mRNA was reverse transcribed, and the coding region was analyzed by single-strand conformation polymorphism analysis. Direct DNA sequencing allowed the identification of a A479-to-T and A481-to-G substitution in exon 5 of the p67phox gene resulting in a double nonconservative amino acid change160Lys-to-Glu and161Asp-to-Val (D160V-K161E). This defect was found in the genomic DNA of this patient in heterozygous state and does not correspond to those previously found in other cases of CGD lacking the p67phox.