Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils.

To address the question whether leukocyte integrins are able to generate signals activating neutrophil functions, we investigated the capability of mAbs against the common beta chain (CD18), or the distinct alpha chains of CR3, LFA-1, or gp150/95, to activate neutrophil respiratory burst. These investigations were performed with mAbs bound to protein A immobilized to tissue culture polystyrene. Neutrophils plated in wells coated with the anti-CD18 mAbs IB4 and 60.3 released H2O2; H2O2 release did not occur when neutrophils were plated in wells coated with an irrelevant, isotype-matched mAb (OKDR), or with mAbs against other molecules (CD16, beta 2-microglobulin) expressed on the neutrophil surface at the same density of CD18. Four different mAbs, OKM1, OKM9, OKM10, 60.1, which recognize distinct epitopes of CR3 were unable to trigger H2O2 or O2- release from neutrophils. However, mAbs against LFA-1 or gp150/95 triggered both H2O2 and O2- release from neutrophils. Stimulation of neutrophils respiratory burst by both anti-CD18, and anti-LFA-1 or gp150/95 mAbs was totally inhibited by the microfilaments disrupting agent, cytochalasin B, and by a permeable cAMP analogue. While the capability to activate neutrophil respiratory burst was restricted to anti-LFA-1 and gp150/95 mAbs, we observed that mAbs against all members of leukocyte integrins, including CR3, were able to trigger neutrophil spreading. These findings indicate that, in neutrophils, all three leukocyte integrins can generate signals activating spreading, but only LFA-1 and gp150/95 can generate signals involved in activation of the respiratory burst. This observation can be relevant to understand the mechanisms responsible for the activation of neutrophil respiratory burst by tumor necrosis factor-alpha, which has been shown to be strictly dependent on expression of leukocyte integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. Wright. 1989. J. Cell Biol. 109:13411349.

Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils.

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti-Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12-myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.

Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions.

Cross-linking of the neutrophil-beta 2- or beta 3-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosin phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61hck and p58c-fgr, we examined adhesion-dependent respiratory burst in PMNs isolated from hck -/-, fgr -/-, and hck -/- fgr -/- knockout mice. Purified bone marrow PMNS from wild-type mice released significant amounts of O2- when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl-methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck-/-fgr-/- double-mutant mic, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck-/-fgr-/-PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMN plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck-/-fgr-/- PMNs, failed to respond. Direct cross-linking of the subunits of beta 2 and beta 2 integrins by surface-bound mAbs was elicited O2- production by wild-type PMNs, while the double-mutant hck-/-fgr-/- cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck-/-fgr-/- PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck-/-or fgr-/-single mutant cells produced O2- at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61 hck and p 58c-fgr is required for signaling through leukocyte beta 2 and beta 3 integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.

Atrial fibrillation during acute myocardial infarction: association with all-cause mortality and sudden death after 7-year of follow-up.

AIMS: Atrial fibrillation/flutter (AF/FL) is a common complication of acute myocardial infarction (AMI). Indeed, the determinants of AF/FL in AMI-patients and the association of AF/FL with mortality are not well-known. The purpose of the present study was to investigate the relationship between presence of AF/FL and mortality in patients with AMI and to report on predictors of AF/FL. METHODS: We studied 505 patients enrolled in three intensive care units with definite AMI and followed up for 7 years. No patient was lost to follow-up. Patients with AF/FL during the 1st week of hospitalisation were compared with those with steady sinus rhythm. End-points were all-cause mortality and modes of death. RESULTS: At multivariable logistic regression analysis, elderly, body mass index, congestive heart failure (CHF), history of hypertension and plasma cholesterol (in a negative fashion) were independently associated with the presence of AF/FL. At survival analysis, after full adjustment, AF/FL was not associated with in-hospital mortality. After 7 years of follow-up, AF/FL was found to be associated with all-cause mortality [adjusted odds ratio (OR) = 1.6; 95% confidence interval (CI) = 1.2-2.3], together with age, diabetes mellitus, creatine kinase-MB isoenzyme (CK-MB) peak, CHF, estimated glomerular filtration rate and thrombolysis. At adjusted logistic polynomial regression analysis, AF/FL was found to be associated with an excess of mortality for reasons of sudden death (SD) (adjusted OR = 2.7; 95% CI = 1.2-6.4). No interaction was observed between AF/FL and medications on in-hospital mortality. For 7-year mortality, angiotensin-converting enzyme (ACE)-inhibitors and digitalis showed an independent negative (protective) interaction chiefly on SD (adjusted OR = 0.06; 95% CI = 0.01-0.74, and RR = 0.10; 95% CI = 0.02-0.58, respectively). CONCLUSIONS: Patients with AMI and AF/FL portend a poor prognosis in the long-term chiefly because of an excess of SD. Treatment with ACE-inhibitors and digitalis may have long-term beneficial effects on SD.

Variability of endometrial glandular opening count in infertile patients prior to first IVF treatment.

The aim of the present study was to evaluate the number of endometrial glandular openings, using previously reported software that provides an objective count, and to assess the variability of this parameter during the luteal phase in a population of women who had no hormonal abnormalities presenting with tubal infertility or male factor infertility. A cross-sectional study was performed comprising 561 patients selected for a diagnostic hysteroscopy for the investigation of infertility. Hysteroscopy was performed during the mid-secretory phase prior to the first IVF treatment cycle. A total of 561 image frames from all patients were analysed. All images were automatically selected by the software, which also evaluated the number of endometrial glandular openings. The mean +/- SD glandular opening count was 53.2 +/- 30 (range 4-158). The analysis of variation showed a significant difference (P = 0.001) among all video frames. In conclusion, endometrial glandular opening count, as measured by the method described, can be used in investigations during the luteal phase. Although a lack of pattern was observed in endometrial maturation, this feature should be explored further in this subgroup of patients.

Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport.

Src-family kinases play a central role in regulation of hematopoietic cell functions. We found that mouse erythrocytes express the Src-family kinases Fgr and Hck, as well as Lyn. To directly test whether Fgr and Hck play any role in erythrocyte function, we analyzed red cells isolated from fgr-/-, hck-/-, and fgr-/- hck-/- knock-out mice. Mean corpuscular hemoglobin concentration and median density are increased, while K content is decreased, in fgr-/- hck-/- double-mutant erythrocytes compared with wild-type, fgr-/-, or hck-/- erythrocytes. Na/K pump and Na/K/Cl cotransport were not altered, but K/Cl cotransport activity was significantly and substantially higher (approximately three-fold) in fgr-/- hck-/- double-mutant erythrocytes. This enhanced K/Cl cotransport activity did not depend on cell age. In fact, in response to bleeding, K/Cl cotransport activity increased in parallel with reticulocytosis in wild-type erythrocytes, while abnormal K/Cl cotransport did not change as a consequence of reticulocytosis in fgr-/- hck-/- double-mutant erythrocytes. Okadaic acid, an inhibitor of a phosphatase that has been implicated in activation of the K/Cl cotransporter, inhibited K/Cl cotransport in wild-type and fgr-/- hck-/- double-mutant erythrocytes to a comparable extent. In contrast, staurosporine, an inhibitor of a kinase that has been suggested to negatively regulate this same phosphatase enhanced K/Cl cotransport in wild-type but not in fgr-/- hck-/- double-mutant erythrocytes. On the basis of these findings, we propose that Fgr and Hck are the kinases involved in the negative regulation of the K/Cl cotransporter-activating phosphatase. Abnormality of erythrocyte K/Cl cotransport in fgr-/- hck-/- double-mutant animals represents the first demonstration that Src-family kinases may be involved in regulation of membrane transport.

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes: evidence for the involvement of three mechanisms.

1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.

Inhibition by quercetin of activation of polymorphonuclear leucocyte functions. Stimulus-specific effects.

The effect of some bioflavonoids on the activation of polymorphonuclear leucocyte respiration and exocytosis was examined. At 10-5-10-4 M concentration, quercetin, but not morin and rutin, was found to inhibit the concanavalin A-induced enhancement of oxygen consumption markedly, without impairing leucocyte viability and concanavalin A binding. The inhibition could be reversed by either washing the leucocytes or adding a 10-fold molar excess of 1-anilino-8-naphthalene sulphonate. Concanavalin A-dependent cell secretion of lysozyme was also totally inhibited by 30 muM quercetin. The effect of quercetin on the activation of leucocyte respiration appeared to be stimulus specific. In fact, at a concentration of the flavonoid (75 muM) which provided a 95% inhibition of the concanavalin A-induced stimulation, the respiratory activation produced by phospholipase C was inhibited by about 50% and that caused by myristic acid and by the antibiotic Br-X537A by less than 25%. These data suggest that quercetin exerts its activity at specific sites of the plasma membrane of the leucocytes, and that this compound might be used to identify the membrane domain whereon different stimuli act to originate the initial stimulatory signal.

Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (-247 mV) cytochrome b.

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be -247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low Em7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.

Activation of human monocyte-derived macrophages by interferon gamma is accompanied by increase of poly(ADP-ribose) polymerase activity.

We have investigated poly(ADP-ribosyl)ation processes in human monocyte-derived macrophages and the effect of the activating cytokine, interferon gamma (IFN-gamma) on these processes. IFN-gamma was shown to increase the activity of poly(ADP-ribose) polymerase in human macrophages. A 2-3-fold enhancement of poly(ADP-ribose) polymerase activity was observed after 3-4 h of incubation with IFN-gamma, whose effects were dose-dependent and maximal at 20-50 U/ml. Staining with anti-poly(ADP-ribose) antibodies and purification of ADP-ribosylated nuclear proteins by affinity chromatography over boronate agarose showed that enhancement of poly(ADP-ribose) polymerase activity by IFN-gamma was accompanied by accumulation of poly(ADP-ribose) polymers in nuclear proteins. The effects of IFN-gamma on poly(ADP-ribose) polymerase activity were not due to an enhanced accumulation of the message for the enzyme, indicating that the activation of the enzyme activity was due to post-transcriptional modifications. IFN-gamma was shown to induce DNA strand breaks in human macrophages. This phenomenon followed the same time-course and was evident with the same doses of IFN-gamma that increased poly(ADP-ribose) polymerase activity. Since poly(ADP-ribose) polymerase is known to require DNA nicks for its activity, the capability of IFN-gamma to induce DNA strand breaks can explain its effects on poly(ADP-ribosyl)ation processes.

Integrin signalling in neutrophils and macrophages.

Integrins have been characterized extensively as adhesion receptors capable of transducing signals inside the cell. In myelomonocytic cells, integrin-mediated adhesive interactions regulate different selective cell responses, such as transmigration into the inflammatory site, cytokine secretion, production or reactive oxygen intermediates, degranulation and phagocytosis. In the last few years, great progress has been made in elucidating mechanisms of signal transduction by integrins in neutrophils and macrophages. This review summarises the current information on the role of integrins in regulating myelomonocytic cell functions and highlights the signalling pathways activated by integrin engagement in these cells. Also, exploiting the current knowledge of mechanisms of integrin signal transduction in other cell types, we propose a model to explain how integrins transduce signals inside neutrophils and macrophages, and how signaling pathways leading to regulation of selective cell functions may be coordinated.

The CD4 molecule belongs to the phenotypic repertoire of most cases of acute myeloid leukemia.

In the present study fresh leukemic cells obtained from 23 patients with acute myeloid leukemia (AML; FAB subtypes: three M1, five M2, two M3, five M4, eight M5) were investigated for the membrane expression of the CD4 molecule by cytofluorimetric analysis with an anti-CD4 monoclonal antibody (mAb). In 15 cases the presence of the CD4 mRNA was also investigated using Northern blot analysis. Membrane expression of the CD4 molecule was demonstrated in 19 out of 23 cases, and it was found to be weaker than in CD4+ lymphocytes and monocytes obtained from normal controls. Full-length CD4 mRNA was detected in 12 out of 15 (80%) cases, and AML cells positive for CD4 mRNA expression also expressed the CD4 antigen. Since the CD4 molecule expressed by T cells is associated with p56lck, a member of the src family of intracellular tyrosine kinases, we investigated whether the CD4 molecule expressed by myeloid blasts is also associated with a tyrosine kinase activity. In vitro kinase assays performed on anti-CD4 immunoprecipitates from lysates of myeloid leukemia cells from four CD4+ cases were negative for the presence of a tyrosine kinase activity. This finding was not due to the lack of expression of members of the src family since we were able to detect at least p60src and p59fyn in myeloid leukemia cells. According to our results, the CD4 molecule seems to belong to the phenotypic repertoire of most AML, irrespective of their FAB subtypes. However, in myeloid blasts this molecule is not associated with a tyrosine kinase activity as it occurs in T lymphocytes.

Integrin signal transduction in myeloid leukocytes.

Integrin-mediated adhesion serves as a powerful costimulus for neutrophil activation. Clustering of integrins at the leukocyte membrane by interaction with surface-bound ligands (extracellular matrix proteins or endothelial cell counter-receptors) leads to a number of signaling events that culminate in actin cytoskeletal rearrangement and neutrophil functional responses such as migration, degranulation, and respiratory burst. Although the signaling reactions elicited by integrin ligation are complex and the relative contribution of each pathway to neutrophil function is unclear, a large body of evidence suggests that activation of tyrosine kinases is a very proximal event in these signaling cascades. This review summarizes the role of adhesion in activating neutrophil functional properties and the contribution of leukocyte tyrosine kinases to regulation of integrin signaling in myeloid cells. Significant advances in our understanding of leukocyte integrin signaling have been afforded by studies using knockout mice lacking members of the Src-family of tyrosine kinases normally expressed in myeloid cells. These studies have demonstrated that these kinases (Hck, Fgr, and Lyn) are not required for myeloid cell development or for many of the functional properties of myeloid cells but are critical in controlling integrin-mediated signaling events. Absence of these kinases results in impaired adhesion-dependent neutrophil activation both in vivo and in vitro. These studies suggest that leukocyte-specific tyrosine kinases may be good therapeutic targets for controlling myeloid cell-dependent inflammatory disease.

Antibody-induced engagement of beta2 integrins in human neutrophils causes a rapid redistribution of cytoskeletal proteins, Src-family tyrosine kinases, and p72syk that precedes de novo actin polymerization.

Beta2 integrins mediate rearrangement of the cytoskeleton and activation of selective cell functions in neutrophils. To elucidate early events following beta2 integrin ligation, we analyzed redistribution of cytoskeletal and signaling proteins as a consequence of antibody-induced integrin clustering. Incubation of neutrophils on surface-bound anti-beta2 subunit antibodies induced a very rapid (within 1 min) redistribution of the cytoskeletal proteins talin, alpha-actinin, and paxillin, and the tyrosine kinases p58(c-fgr), p53/56(lyn), and p72(syk) to a cell fraction insoluble in Triton X-100. Cytoskeletal and signaling proteins redistribution preceded de novo actin polymerization because cytochalasin B did not inhibit this redistribution. Antibody engagement of all the three distinct beta2 integrins (CD11a, CD11b, CD11c) expressed by neutrophils induced redistribution of cytoskeletal proteins and tyrosine kinases. Several tyrosine phosphorylated proteins were also rapidly redistributed as a consequence of beta2 integrin engagement and this was not affected by blocking de novo actin polymerization with cytochalasin B. In addition, genistein, an inhibitor of tyrosine kinase activities which strongly reduced protein tyrosine phosphorylation, had no effect on redistribution of cytoskeletal proteins, Src-family kinases, and p72(syk). These findings suggest that integrin-dependent cytoskeleton rearrangement in neutrophils occurs in at least two distinct steps and nucleation of some cytoskeletal proteins together with tyrosine kinases precedes rearrangement of the actin-based cytoskeleton and tyrosine kinases activation. On the basis of these and previous findings we propose a model explaining mechanisms of integrin signaling in neutrophils.

Tumor necrosis factor triggers redistribution to a Triton X-100-insoluble, cytoskeletal fraction of beta 2 integrins, NADPH oxidase components, tyrosine phosphorylated proteins, and the protein tyrosine kinase p58fgr in human neutrophils adherent to fibrinogen.

Tumor necrosis factor (TNF) triggers cell spreading, release of granule constituents, and production of toxic oxygen derivatives in human neutrophils adherent to fibrinogen. This response requires cytoskeleton reorganization and is dependent on expression of beta 2 integrins. We analyzed distribution of distinct proteins in Triton X-100-soluble and insoluble fractions in neutrophils adherent to fibrinogen. We found that stimulation of adherent neutrophils with TNF causes the redistribution to a Triton-insoluble fraction of alpha-actinin, beta 2 integrins, and the four components whose assembly constitutes an active NADPH oxidase: the gp91-phox, p22-phox, p47-phox, and p67-phox proteins. Redistribution of these different proteins to a Triton-insoluble fraction took relatively long times and was maximal after about 30 min of stimulation with TNF. Prevention of actin polymerization with cytochalasin B hampered the TNF-induced redistribution of these proteins from a Triton-soluble to an insoluble fraction. In addition, tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr were recovered in this Triton-insoluble fraction. These findings show that stimulated, beta 2 integrin-dependent adhesion of neutrophils to fibrinogen is accompanied by redistribution to cytoskeletal structures of (1) beta 2 integrins, that is, neutrophil receptors for fibrinogen; (2) proteins involved in neutrophil effector functions, that is, components of NADPH oxidase; and (3) tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr, molecules that are potentially involved in the formation of a submembranous signaling complex.

Role of Src kinases and Syk in Fcgamma receptor-mediated phagocytosis and phagosome-lysosome fusion.

Phagocytosis is increased by Fcgamma receptors (FcgammaRs), and studies with syk(-/-) macrophages demonstrated that Syk kinase is required for FcgammaR phagocytosis. Similar studies with macrophages lacking the Src family kinases Hck, Fgr, and Lyn showed that these kinases are not required for phagocytosis but that they enhance the rate of particle engulfment. In this report we show that both wild-type and hck(-/-)fgr(-/-) macrophages expressed Fyn, Src, and Yes and that these kinases were activated on ingestion of immunoglobulin G (IgG)-coated particles and redistributed, together with Syk, to actin-rich phagocytic cups and the phagosomal membrane. At doses blocking IgG-dependent phagocytosis, the tyrosine kinase inhibitors PP1 and piceatannol inhibited both Src family kinase and Syk activities, as well as their redistribution to actin-rich phagocytic cups. Hck, Fgr, and Lyn were dispensable for lysosome-phagosome fusion (PLF) induced by IgG-coated particles. However, PP1 or piceatannol hampered unopsonized yeast-induced PLF despite the fact that they did not block yeast internalization.

Sulfatides trigger cytokine gene expression and secretion in human monocytes.

We investigated whether sulfatides are able to trigger transmembrane signals and activation of selective cell functions in human monocytes. Sulfatides stimulated an increase in cytosolic free-calcium in monocytes, and this depended on the release of calcium from intracellular stores. Non-sulfated galactocerebrosides had no effect on monocyte cytosolic free calcium. Sulfatides enhanced expression of tumor necrosis factor, interleukin-8, and interleukin-1 beta, but not interleukin-12/natural killer cell stimulating factor mRNAs. Sulfatides also triggered secretion of cytokines into the extracellular medium, although they were much less effective than lipopolysaccharide. Both enhanced expression of cytokine mRNAs and secretion by sulfatides required sulfation of the galactose ring of the glycolipid as non-sulfated galactocerebrosides had no effect. These findings suggest that sulfatides that are released at sites of inflammation can amplify the inflammatory reaction triggering cytokine expression in, and release by, monocytes.

Interferon-gamma and tumor necrosis factor-alpha enhance p60src expression in human macrophages and myelomonocytic cell lines.

We investigated modulation of p60src expression in human mononuclear phagocytes. By analysis of [35S]methionine-labelled cells we found that synthesis of p60src is higher in human monocytes compared to macrophages derived from in vitro cultivation of monocytes. Western blot analysis showed that expression of p60src in monocyte-derived macrophages can be enhanced if monocytes are differentiated into macrophages in the presence of interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha). Enhanced p60src expression caused by IFN-gamma or TNF-alpha correlated with an enhanced autophosphorylating kinase activity assayed in anti-p60src immune precipitates. In vivo phosphorylation of p60src and analysis of phosphopeptides by tryptic digestion showed that treatment with cytokines did not affect the pattern of phosphorylation of distinct phosphopeptides. The human monocytic cell lines, U937 and HL-60, induced to differentiate along the monocytic pathway by IFN-gamma, or a combination of IFN-gamma and TNF-alpha, expressed higher amounts of the p60src, but not of the p59fyn or p62yes, kinase activity. These findings show that p60src is modulated in the course of differentiation of human monocytes to macrophages, and that macrophage-activating cytokines increase p60src expression in human monocyte-derived macrophages.

Activation of SRC family kinases in human neutrophils. Evidence that p58C-FGR and p53/56LYN redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein caveolin, display an enhanced kinase activity.

Protein tyrosine phosphorylation is one of the signals involved in stimulation of neutrophil (PMN) functions. We found that phorbol myristate acetate (PMA) activates the src family tyrosine kinases p58c-fgr and p53/56lyn in suspended PMNs. Moreover, we found that up to about 20% of p58c-fgr and p53/56lyn redistribute to a Triton X-100-insoluble fraction after PMA stimulation, and it is this fraction of the two kinases which diplays an increased activity. These changes of p58c-fgr and p53/56lyn distribution and activity correlate with tyrosine phosphorylation of endogenous substrates. In fact, in PMA-stimulated PMNs tyrosine phosphorylated proteins are mostly recovered in a Triton-insoluble cell fraction. To separate cytoskeletal from caveolar structures, which both display Triton X-100-insolubility, we used the detergent n-octyl beta-D-glucopyranoside (OGP) which solubilises components of caveolae. We found that the caveolae marker protein, caveolin, as well as the cytoskeletal protein alph-actinin and p58c-fgr and p53/56lyn, is insoluble in OGP. These findings suggest that PMA stimulation promotes the formation of multimolecular complexes containing cytoskeletal proteins, caveolin-containing structures and src family protein tyrosine kinases. Moreover, they show that p58c-fgr and p53/56lyn associated with this multimolecular complex display an enhanced kinase activity.

Effect of modulation of protein kinase C on the cAMP-dependent chloride conductance in T84 cells.

The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium. The permeable cAMP analog 8-Br-cAMP (100 microM) and the calcium ionophore ionomycin (1 microM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diacylglycerol analog 1-oleoyl-2-acetyl-glycerol (100 microM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br-cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductance in T84 cells.