RESUMO
We have recently described a novel glycoprotein, Kp43, expressed on the surface of human natural killer (NK) cells that appears to regulate their functional activity. In this report, signaling mechanisms through the Kp43 surface antigen have been studied. Incubation of interleukin 2 (IL-2)-treated NK cells with anti-Kp43 monoclonal antibody F(ab')2 fragments resulted in the time- and dose-dependent stimulation of NK cell phospholipase D. Phospholipase D activation through the Kp43 surface antigen was found to take place in the absence of polyphosphoinositide turnover and appeared not to depend on the presence of Ca2+ in the extracellular medium. On the other hand, signaling mechanisms through the CD16 receptor (FcR-III) on NK cells were comparatively studied. Stimulation of IL-2-treated NK cells with anti-CD16 monoclonal antibody F(ab')2 fragment also resulted in time- and dose-dependent activation of phospholipase D. However, CD16-triggered phospholipase D activation took place concomitant to phospholipase C-mediated polyphosphoinositide breakdown and showed a strong dependence on extracellular Ca2+. These results provide, to our knowledge, the first evidence for the presence of activatable phospholipase D in NK cells, as well as the first indication that distinct receptor-modulated pathways exist for activation of phospholipase D within the same cell type. On the other hand, phosphatidic acid, the physiologic product of phospholipase D action on phospholipids, was found to mimic the effect of anti-Kp43 monoclonal antibody regarding tumor necrosis factor alpha (TNF-alpha) biosynthesis and secretion by NK cells. Addition of phosphatidic acid vesicles to IL-2-treated NK cell cultures stimulated a TNF-alpha production that was abolished when the cells were previously treated with actinomycin D. Other phospholipids, including lysophosphatidic acid, were ineffective. However, phosphatidic acid-induced TNF-alpha production was strongly inhibited by the presence of propranolol, an inhibitor of phosphatidic acid phosphohydrolase. Moreover, in cells responding to phorbol myristate acetate, a compound that triggers activation of phospholipase D, TNF-alpha synthesis was also inhibited by propranolol. Thus, these data suggest a second messenger role for phosphatidic acid-derived diradylglycerol in the induction of TNF-alpha gene expression.
Assuntos
Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Células Matadoras Naturais/metabolismo , Fosfolipase D/fisiologia , Receptores Fc/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Cálcio/fisiologia , Células Cultivadas , Diglicerídeos/biossíntese , Ativação Enzimática , Humanos , Interleucina-2/farmacologia , Ácidos Fosfatídicos/biossíntese , Receptores de IgGRESUMO
We present in this work the synthesis and cardiovascular effects of new methylthiomorpholine compounds and they were compared with cardiovascular drugs such as captopril, losartan and omapatrilat.
Assuntos
Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacologia , Morfolinas/síntese química , Morfolinas/farmacologia , Fenóis/síntese química , Fenóis/farmacologia , Animais , Anti-Hipertensivos/química , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Frequência Cardíaca/efeitos dos fármacos , Morfolinas/química , Fenóis/química , RatosRESUMO
We have used confocal laser scanning microscopy to determine the dynamics of distribution of activated protein kinase C (PKC) in living Madin Darby canine kidney (MDCK) cells. Using fluorescently tagged phorbol myristate acetate (PMA) as a probe for PKC we have demonstrated its distribution in association with the cell periphery and with the nucleus. Dual labeling experiments using PKC alpha and PKC beta II specific antisera indicate that activated PKC alpha is found in association with the periphery whereas activated PKC beta II is translocated to the nucleus. We have demonstrated increased activity of PKC in nuclear fractions isolated from cells treated with PMA and other PKC activators. These data indicate that upon activation individual isoforms of PKC translocate to different subcellular locations where they are likely to mediate different actions.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Animais , Western Blotting , Compartimento Celular , Núcleo Celular/enzimologia , Polaridade Celular , Células Cultivadas , Cães , Ativação Enzimática , Corantes Fluorescentes , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Acetato de TetradecanoilforbolRESUMO
Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid identified in yeast, bacteria, and plants, but not yet in mammalian cells. Given its structural resemblance to other phospholipid-activating molecules, such as lysophosphatidate, phosphatidate, and diacylglycerol, it was questioned whether DGPP was capable of activating macrophages to release arachidonic acid (AA) metabolites such as the prostaglandins. It has been found that DGPP is able to potently stimulate prostaglandin production in the murine cell line P388D1 by a mechanism that involves activation of the cytosolic Group IV phospholipase A2 (cPLA2). Our results demonstrate that DGPP possesses macrophage-activating-factor properties and suggest a role for this novel compound in the inflammatory response.
Assuntos
Citosol/enzimologia , Difosfatos/metabolismo , Glicerol/análogos & derivados , Macrófagos/enzimologia , Fosfolipases A/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Glicerol/metabolismo , Camundongos , Fosfolipases A2RESUMO
Lipid mediators play a crucial role in human parturition and phospholipase A(2) (PLA(2)) is a key regulator of the production of these compounds. We have investigated by PCR the expression of different groups of PLA(2) and COX enzymes in human fetal membranes (amnion and chorion), placenta and three chorionic cell lines (JEG-3, Jar, BeWo). Our data show that the cytosolic Group IV PLA(2) and COX-1 are expressed in all of them, whereas the secretory forms of PLA(2), (Groups IIA, and V), have a more restricted expression. Group IIA mRNA is most abundant in placenta and chorion, whereas Group V PLA(2) mRNA is most abundant in placenta and amnion. On the other hand, COX-2 is present in placenta, chorion and amnion, but was not detected in any of the chorionic cell lines. These results suggest that both cytosolic and distinct secreted forms of PLA(2) could be involved in arachidonic acid (AA) release preceding prostaglandin production at the fetal/maternal interface.
Assuntos
Membranas Extraembrionárias/enzimologia , Fosfolipases A/metabolismo , Placenta/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Coriocarcinoma , Córion/enzimologia , Desenvolvimento Embrionário e Fetal , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Fosfolipases A/genética , Reação em Cadeia da Polimerase , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Neoplasias UterinasAssuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos alfa 1/fisiologia , Transdução de Sinais , Animais , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Membrana Celular/fisiologia , Diglicerídeos/metabolismo , Cães , Rim , Modelos Biológicos , Fosfatidilinositóis/metabolismo , Fosfolipases A/metabolismo , Canais de Potássio/fisiologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
We have demonstrated previously that protein kinase Calpha (PKCalpha) plays a key role in regulating phospholipase D (PLD) activation by nucleotides and the phorbol ester phorbol-12-myristate-13-acetate in Madin-Darby canine kidney (MDCK-D1) cells. In the current work, we investigated PLD activation in MDCK-D1 cells triggered by the adrenergic receptor agonist epinephrine and its mechanism of activation. Epinephrine, acting through the alpha1-adrenergic receptor subtype, promoted transient translocation of PKCalpha and more prolonged translocation of PKCepsilon to the membrane fraction, indicating activation of these two isoforms. In addition, epinephrine promoted activation of PLD, as shown by a sustained accumulation of phosphatidylethanol. All of these events were blocked by pretreatment of cells with the alpha1-adrenergic antagonist prazosin. D609, an inhibitor of phosphatidylcholine hydrolysis, blocked translocation of PKCalpha and PKCepsilon but did not inhibit PLD activation. Unlike results with PMA, or with the P2 purinergic receptor agonist ATP, epinephrine-stimulated PLD activity was not inhibited in MDCK-D1 cells in which PKCalpha expression is attenuated by an antisense cDNA construct or in cells in which PKC activity was inhibited by 1 microM GF 109203X. However, PLD activation by epinephrine was abolished by concomitant incubation of cells with the calcium chelator EGTA. These data, together with previous results, are consistent with the hypothesis that in MDCK-D1 cells, epinephrine acting on alpha1-adrenergic receptors, promotes a rapid increase in cytosolic Ca2+ that promotes activation of PLD through an as-yet poorly defined mechanism. The data demonstrate that different types of G protein-linked receptors that activate PLD can mediate this activation in either a PKC activation-dependent or -independent manner within a single cell type.
Assuntos
Glicerofosfolipídeos , Isoenzimas/fisiologia , Fosfolipase D/metabolismo , Proteína Quinase C/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Cálcio/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Diglicerídeos/metabolismo , Cães , Ativação Enzimática/efeitos dos fármacos , Epinefrina/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Indóis/farmacologia , Maleimidas/farmacologia , Neomicina/farmacologia , Norbornanos , Ácidos Fosfatídicos/metabolismo , Prazosina/farmacologia , Proteína Quinase C-alfa , Proteína Quinase C-épsilon , Transdução de Sinais , Tiocarbamatos , Tionas/farmacologiaRESUMO
We have recently demonstrated the existence of an ATP-activated phospholipase D (PLD) in the nuclei of MDCK-D1 cells (Balboa, M. A., Balsinde, J., Dennis, E. A., and Insel, P. A. (1995) J. Biol. Chem. 270, 11738-11740). We have now found that nuclear PLD is synergistically activated by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and ATP in a time- and concentration-dependent manner, but these compounds do not alter the sensitivity of the enzyme to activation by Ca2+. The synergistic stimulation of PLD activity could be blocked by addition of the protein kinase C inhibitors chelerythrine and calphostin C. Stimulation by GTP gamma S was abolished by guanosine 5'-O-(2-thiodiphosphate). Incubation of isolated nuclei with Clostridium botulinum C3 exoenzyme inhibited the potentiating effect of GTP gamma S on ATP-dependent nuclear PLD activity. Moreover, use of the Rho GDP dissociation inhibitor to extract Rho family G proteins from cell nuclei also inhibits PLD activity. Western blot analyses of isolated nuclei revealed the presence of the small G protein RhoA, but not of RhoB or the ADP-ribosylation factor. GTP gamma S-stimulated ATP-dependent PLD activity could be reconstituted in Rho GDP dissociation inhibitor-washed nuclei by addition of recombinant prenylated RhoA, but not by addition of non-prenylated RhoA. Taken together, these results indicate that nuclear PLD activity is modulated via a RhoA-dependent activation that occurs downstream of protein kinase C. Nuclear PLD, which appears to be a previously unrecognized effector regulated by protein kinase C and G proteins, may be involved in the regulation of nuclear function or structure.
Assuntos
Toxinas Botulínicas , Núcleo Celular/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , ADP Ribose Transferases/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Alcaloides , Animais , Benzofenantridinas , Cálcio/farmacologia , Fracionamento Celular , Linhagem Celular , Clostridium botulinum/enzimologia , Cães , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Rim , Cinética , Naftalenos/farmacologia , Fenantridinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína rhoA de Ligação ao GTPRESUMO
The murine cell line MMGT-16 is of microglial origin and capable of releasing immunoinflammatory cytokines. When stimulated by the proinflammatory stimulus lipopolysaccharide (LPS), MMGT-16 cells secrete large amounts of prostaglandin E(2) (PGE(2)). This PGE(2) production is nearly abolished if amyloid beta-peptide (Abeta (1-40)) is present in the incubation medium. In addition, Abeta (1-40) inhibits cyclooxygenase-2 (COX-2) induction by LPS. Since these effects are not reproduced by the reverse control Abeta (40-1), these results suggest a novel, intriguing modulatory role for amyloid beta peptide in the inflammatory response of microglial cells.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Dinoprostona/biossíntese , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fragmentos de Peptídeos/farmacologia , Animais , Western Blotting , Linhagem Celular , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/análise , Indução Enzimática/efeitos dos fármacos , Inflamação/induzido quimicamente , Isoenzimas/análise , Isoenzimas/biossíntese , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Proteínas de Membrana , Camundongos , Microglia/citologia , Prostaglandina-Endoperóxido Sintases/análise , Prostaglandina-Endoperóxido Sintases/biossíntese , RadioimunoensaioRESUMO
Ceramide has emerged as an important lipid messenger for many cellular processes triggered via surface receptors. In the present study, inflammatory activation of P388D1 macrophages with bacterial lipopolysaccharide (LPS) and platelet-activating factor (PAF) stimulated a transient accumulation of ceramide. Moreover, cell-permeable ceramide mimicked LPS/PAF in triggering arachidonate mobilization in these cells. LPS/PAF-induced ceramide synthesis did not result from sphingomyelinase activation but from increased de novo synthesis. Participation of this pathway in arachidonate signaling was detected since fumonisin B1, an inhibitor of de novo ceramide synthesis, was able to inhibit the LPS/PAF-induced response. These studies have uncovered a new role for sphingolipid metabolism in cellular signaling and constitute evidence that products of the sphingomyelin biosynthetic pathway may serve a specific role in signal transduction by influencing the activity of the novel Group V secretory phospholipase A2.
Assuntos
Ácido Araquidônico/metabolismo , Fumonisinas , Macrófagos/metabolismo , Esfingomielinas/biossíntese , Animais , Ácidos Carboxílicos/farmacologia , Diglicerídeos/biossíntese , Leucemia P388 , Lipopolissacarídeos/farmacologia , Camundongos , Fator de Ativação de Plaquetas/farmacologia , Células Tumorais CultivadasRESUMO
Prostaglandins are known to play a central role in the initiation of labor in humans, and amnionic cells constitute a major source of these compounds. Prostaglandin synthesis and release by amnion cells in response to hormones and ligands takes place after a characteristic 4-5 h lag. However, we report herein that free arachidonic acid (AA), the metabolic precursor of prostaglandins, can be induced at much shorter times (1 h) in human amnionic WISH cells by phorbol 12-myristate 13-acetate (PMA) through activation of protein kinase Calpha (PKCalpha). WISH cells were found to possess both cytosolic group IV phospholipase A2 (cPLA2) and Group VI Ca2+-independent phospholipase A2 (iPLA2). Of these, the cPLA2 was found to be the likely mediator of AA mobilization in PMA-activated WISH cells. PMA also activates phospholipase D (PLD) in these cells and ethanol, a compound that inhibits PLD-mediated phosphatidic acid (PA) formation, blocked AA release. Moreover, prevention of PA dephosphorylation by the PA phosphohydrolase inhibitors propranolol and bromoenol lactone, resulted in inhibition of AA release by PMA-treated WISH cells. Collectively, these data suggest that activation of cPLA2 and attendant AA release by phorbol esters in WISH cells requires prior generation of DAG by phosphatidate phosphohydrolase.
Assuntos
Âmnio/metabolismo , Ácido Araquidônico/metabolismo , Fosfatidato Fosfatase/metabolismo , Ácidos Araquidônicos/farmacologia , Células Cultivadas , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Organofosfonatos , Fosfolipase D/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosforilação , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Previous studies have demonstrated that P388D(1) macrophages are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in two temporally distinct phases. The first phase is triggered by platelet-activating factor within minutes, but needs the cells to be previously exposed to bacterial lipopolysaccharide (LPS) for periods up to 1 h. It is thus a primed immediate phase. The second, delayed phase occurs in response to LPS alone over long incubation periods spanning several hours. Strikingly, the effector enzymes involved in both of these phases are the same, namely the cytosolic group IV phospholipase A(2) (cPLA(2)), the secretory group V phospholipase A(2), and cyclooxygenase-2, although the regulatory mechanisms differ. Here we report that P388D(1) macrophages mobilize AA and produce prostaglandins in response to zymosan particles in a manner that is clearly different from the two described above. Zymosan triggers an immediate AA mobilization response from the macrophages that neither involves the group v phospholipase A(2) nor requires the cells to be primed by LPS. The group VI Ca(2+)-independent phospholipase A(2) is also not involved. Zymosan appears to signal exclusively through activation of the cPLA(2), which is coupled to the cyclooxygenase-2. These results define a secretory PLA(2)-independent pathway for AA mobilization in the P388D(1) macrophages, and demonstrate that, under certain experimental settings, stimulation of the cPLA(2) is sufficient to generate a prostaglandin biosynthetic response in the P388D(1) macrophages.
Assuntos
Ácido Araquidônico/metabolismo , Macrófagos/metabolismo , Fosfolipases A/metabolismo , Prostaglandinas/metabolismo , Animais , Células Clonais , Ativação de Macrófagos , Transdução de SinaisRESUMO
Activation of the cytosolic Group IV phospholipase A(2) (cPLA(2)) by agonists has been correlated with the direct phosphorylation of the enzyme by members of the mitogen-activated protein kinase (MAPK) cascade. Phosphorylation of the cPLA(2) increases the specific activity of the enzyme, thereby stimulating the arachidonic acid release. We show here, however, that conditions that lead to full phosphorylation of the cPLA(2) do not lead to enhanced AA release. As the above observations were made under both Ca(2+)-dependent and Ca(2+)-independent conditions, they emphasize that the current paradigm for activation of the cPLA(2) in cells involving both phosphorylation and Ca(2+) is incomplete and that other factors should be taken into account.
Assuntos
Ácido Araquidônico/metabolismo , Macrófagos/fisiologia , Fosfolipases A/metabolismo , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Citosol/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Fosfolipases A2 do Grupo IV , Cinética , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
A major issue in lipid signaling relates to the role of particular phospholipase A2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527-8531). We have now further evaluated the role of the iPLA2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA2 plays a major role in regulating phospholipid fatty acyl turnover in P388D1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA2 antisense oligonucleotide on PAF-stimulated arachidonate release.
Assuntos
Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Leucemia P388/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipídeos/metabolismo , Animais , Lipídeos de Membrana/metabolismo , Camundongos , Naftalenos/farmacologia , Fosfolipases A/metabolismo , Fosfolipases A2 , Pironas/farmacologiaRESUMO
Secretory phospholipase A2 (sPLA2) is the major effector involved in arachidonic acid (AA) mobilization and prostaglandin E2 (PGE2) production during stimulation of P388D1 macrophages with the inflammatory stimuli bacterial lipopolysaccharide and platelet-activating factor. We herein demonstrate that PGE2 in stimulated P388D1 cells is accounted for by the inducible cyclooxygenase (COX)-2. COX-1, though present, appears not to participate significantly in stimulus-induced PGE2 production in P388D1 macrophages. Reconstitution experiments utilizing exogenous recombinant sPLA2 demonstrate that activation of the sPLA2 at the plasma membrane is highly dependent on previous activation of the cytosolic phospholipase A2 (cPLA2). Collectively these results demonstrate (i) that functional coupling exists between sPLA2 and COX-2 in activated cells, (ii) the critical role that cPLA2 plays in lipid mediator production, and (iii) that there is crosstalk between cPLA2 and sPLA2 in the cell.
Assuntos
Isoenzimas/metabolismo , Macrófagos/metabolismo , Fosfolipases A/química , Fosfolipases A/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Linhagem Celular , Reagentes de Ligações Cruzadas , Ciclo-Oxigenase 2 , Camundongos , Fosfolipases A2 , Ligação ProteicaRESUMO
In the present study we have comparatively analysed the specific activity of a panel of immunotoxins (ITs) prepared by coupling Ricin to several monoclonal antibodies (MoAbs) directed against the CD3, CD4 and CD8 T cell membrane molecules. Peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) were incubated with the different ITs for 2 h, in the presence of 0.1 M lactose, washed and subsequently stimulated with either phytohemagglutinin (PHA) or an anti-CD3 MoAb. Our results indicate that the proliferative response of PBMC to both stimuli was specifically inhibited (greater than 95%) by either anti-CD3 IT or a combination of anti-CD4 and CD8 ITs, at concentrations comparable to those previously used for ex vivo T cell depletion (300 ng/ml). When used individually at the same dose, anti-CD8 and anti-CD4 ITs inhibited the PHA-induced PBMC proliferative response 40 and 70% respectively. When either anti-CD4 or anti-CD8 IT-treated cells were activated with PHA and cultured for 14 days in the presence of IL-2, less than 2% of the blasts expressed the corresponding antigens as assessed by flow cytometry analysis. Similar results were observed when BMMC were treated with the different ITs. In contrast, the growth of CFU-GM was minimally affected (0-25% inhibition). Our data indicate that ITs directed against T cell subsets are highly active and specific reagents that may be potentially useful for pre-transplant bone marrow purging.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Imunotoxinas/farmacologia , Ricina/farmacologia , Linfócitos T/imunologia , Medula Óssea/imunologia , Ensaio de Unidades Formadoras de Colônias , Eletroforese em Gel de Poliacrilamida , Humanos , Ativação Linfocitária/efeitos dos fármacos , Mitose/efeitos dos fármacos , Monócitos/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
The involvement of the VLA-4 integrin in an alternative leukocyte homotypic adhesion mechanism that is LFA-1/ICAM-1 independent, has been previously reported. We describe here the localization of beta 1 and alpha 4 integrin subunits at sites of cell-cell contact, on both beta 1- and alpha 4-induced aggregates of B lymphoblastoid Ramos cells. Moreover, the distribution of different VLA-alpha subunits was also examined on beta 1-induced cell aggregates of alpha 2- and alpha 4-transfected K-562 cells. Both alpha 2 and alpha 4 integrin subunits were mainly localized at sites of intercellular boundaries, suggesting a possible role for these integrins in leukocyte intercellular adhesion. The fibronectin receptor alpha 5 subunit was either diffuse throughout the plasma membrane, or displayed some accumulation at sites of cell-cell contact. Even though homotypic aggregation of U-937 cells was induced with the anti-alpha 5 P1D6 mAb, the alpha 5 subunit showed only partial redistribution to regions of cell-cell contact, compared with the complete redistribution of the alpha 4 subunit in the alpha 4-induced aggregates. The reorganization of the actin-cytoskeleton was observed at sites of intercellular boundaries in both the anti-beta 1- and anti-alpha 4-induced cell aggregates. Hence, F-actin and the cytoskeletal protein talin co-localized with beta 1 and alpha 4 integrin clusters at sites of cell-cell contact. Signal transduction during VLA-mediated homotypic cell adhesion has also been investigated. We found co-localization of beta 1 and alpha 4 subunits with tyrosine-phosphorylated proteins at cell-cell contact regions during cell aggregation. These data indicate that VLA integrin-mediated leukocyte aggregation results in clustering of beta 1-integrins at sites of cell-cell contact, together with co-localization of cytoskeletal proteins. These results also suggest that protein tyrosine phosphorylation is an important signal transduction mechanism when VLA integrins participate in intercellular contacts.
Assuntos
Comunicação Celular , Proteínas do Citoesqueleto/fisiologia , Integrinas/fisiologia , Leucócitos/fisiologia , Tirosina/metabolismo , Actinas/análise , Adesão Celular , Agregação Celular , Humanos , Integrina beta1 , Fosforilação , Receptores de Fibronectina/análise , Receptores de Antígeno muito Tardio/análise , Células Tumorais CultivadasRESUMO
Leukocyte adhesion to and subsequent spreading on the endothelium are the initial steps in the migration of these cells to the surrounding tissues. We have investigated the participation of different VLA heterodimers in cell spreading by using the anti-beta 1 TS2/16 monoclonal antibody (mAb) which induces a conformational change of different VLA integrin receptors, enabling a high-affinity interaction with their ligands. Both VLA-4 and VLA-5 fibronectin (FN), as well as VLA-2 collagen (COL) receptors mediated cell spreading and morphological changes. The spreading of U-937 and alpha 4-transfected K-562 cells was induced in both FN and VCAM-1, suggesting that the morphological changes may be induced by cell-cell as well as cell-extracellular matrix (ECM) interactions. Furthermore, the beta 1-regulated cell spreading on VCAM-1 and COL took place independently of the VLA-5 FN receptor function. The enhancing effect on cell attachment induced by anti-beta 1 TS2/16 mAb was observed in the presence of different doses of cytochalasin D, whereas cell spreading was abolished. Signal transduction during beta 1-stimulated integrin-ligand interaction was also investigated. We have found the co-localization of beta 1 integrins and tyrosine-phosphorylated proteins during the spreading of U-937 and alpha 2- and alpha 4-transfected K-562 cells on both ECM (FN and COL) and cellular (VCAM-1) ligands. Kinetic studies showed that tyrosine phosphorylation was almost coincident with cellular spreading. The tyrosine phosphorylation of polypeptides of 130 kDa and 77 kDa was triggered in U-937 cells by the interaction of FN with the VLA-5 receptor in a high-affinity conformation. However, no signaling was observed by inducing the high-affinity state of receptor in the absence of appropriate ligand. These data suggest that tyrosine kinase activation is a post-receptor occupancy event that might be critical in regulating the adhesive properties of integrins.
Assuntos
Leucócitos/imunologia , Receptores de Antígeno muito Tardio/metabolismo , Anticorpos Monoclonais , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Colágeno/metabolismo , Citocalasina D/farmacologia , Fibronectinas/metabolismo , Humanos , Leucócitos/citologia , Leucócitos/metabolismo , Ligantes , Fosforilação , Tirosina/metabolismo , Molécula 1 de Adesão de Célula VascularRESUMO
Phosphatidate (PA) is synthesized by a variety of cells in response to physiological agonists. Addition of PA vesicles to [3H]arachidonic acid (AA)-labeled macrophages was found to induce the release of radiolabel in a dose- and time-dependent manner. This effect correlated with the uptake of PA by the macrophages and appeared to be attributable to PA itself and not to a PA metabolite. In parallel with AA release, PA induced a rapid increase in lysophosphatidylcholine in cells prelabeled with [14C]glycerol. Down-regulation of protein kinase C by long term exposure of the cells to phorbol myristate acetate or cell treatment with the protein kinase C inhibitor staurosporine did not affect the PA response. Also, removal of external calcium or cell treatment with the calmodulin antagonist trifluoperazine did not affect PA-induced AA release, while inhibiting the responses to zymosan, phorbol 12-myristate 13-acetate, and ionophore A23187. PA-induced AA release was not affected by intracellular calcium depletion by treatment with quin2/AM in the presence of EGTA. When assayed toward an AA-containing substrate, PA was able to enhance phospholipase A2 activity from cell homogenates in the absence of calcium. The dose dependence and magnitude of the PA effect correlated with those observed for PA-induced AA release in whole cells. Inclusion of ATP in the assay mixture did not affect the activity of the PA-stimulated phospholipase A2. These findings suggest a role for PA in the cascade of events leading to AA release in macrophages through Ca(2+)-independent stimulation of an AA-selective phospholipase A2.
Assuntos
Ácido Araquidônico/metabolismo , Macrófagos Peritoneais/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfolipases A/metabolismo , Trifosfato de Adenosina/farmacologia , Alcaloides/farmacologia , Animais , Cálcio/metabolismo , Sistema Livre de Células , Relação Dose-Resposta a Droga , Regulação para Baixo , Ativação Enzimática , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Fosfolipases A/agonistas , Fosfolipases A2 , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transdução de Sinais , Estaurosporina , Trifluoperazina/farmacologiaRESUMO
We have raised a mAb, termed HP-3E4 (IgM), by immunizing BALB/c mice against human IL2-activated NK cells. The HP-3E4 mAb recognized in different donors variable proportions (< 2-70%) of either fresh or activated NK cells. A small population of T cells (alpha/beta and gamma/delta) appeared HP-3E4+ in PBL. No reactivity was detected on other leukocytes and a panel of cell lines from different lineages. By immunohistochemical staining of different tissues, few HP-3E4+ cells were detected only in lymphoid organs. Analysis of CD56+CD16+CD3- clones (n = 167) from unrelated donors (n = 6), showed that the Ag was stably expressed on 8 to 70%, moreover it was detected on some gamma/delta + T cell clones, whereas all CD3+ alpha/beta +(CD4+ and CD8+) clones analyzed (n = 90) were HP-3E4-. As assessed by SDS-PAGE analysis, the HP-3E4 mAb immunoprecipitated a 58-kDa surface structure. When compared with two mAbs (GL183 and EB6) previously reported to bind also a clonally distributed 54- to 58-kDa Ag, the HP-3E4 mAb appeared to recognize a distinct epitope, thus allowing to further define NK cell subsets. Stimulation of IL2-activated NK cells with the mAb triggered TNF-alpha and IFN-gamma production, which was enhanced by using the mAb attached to plastic or in the presence of suboptimal concentrations of phorbol esters. Although the HP-3E4 mAb did not significantly modify NK cell-mediated cytotoxicity against different targets, with the exception of the Hmy-C1R cell line, it activated BLT esterase secretion. Remarkably, the HP-3E4 mAb triggered phosphoinositide turnover and an early increase of [Ca2+]i, as well as tyrosine phosphorylation of several cellular substrates including CD3 zeta; inhibition of tyrosine kinase activity with genistein hampered the HP-3E4-mediated stimulation of cytokine production. Our data provide further support for the structural diversity of a 58-kDa surface Ag, whose expression is restricted to discrete NK and T cell subsets. Moreover, the results support the fact that the molecule plays an active role in regulating NK cell functions through signal transduction mechanisms comparable to those triggered via Fc gamma RIII.