RESUMO
We studied the origin of the neutrophil-activating peptide NAP-2, a presumed 70 amino acid cleavage product of platelet basic protein (PBP) and connective tissue-activating peptide III (CTAP-III). Purified human blood monocytes or lymphocytes were cultured with or without stimuli (LPS or PHA) in the presence or absence of platelet-release supernatant, and the formation of NAP-2 and other neutrophil-activating peptides was monitored. NAP-2 was generated whenever monocytes and platelet release supernatant were present. When a monocyte stimulus was added, NAF/NAP-1 was also formed, and in the presence of LPS a third, less potent neutrophil-stimulating fraction, consisting of NAP-2 variants with 73, 74, and 75 residues, also appeared. Monocytes alone did not yield NAP-2 and no neutrophil-activating peptide was generated by lymphocytes. The monocyte-conditioned medium was found to cleave purified CTAP-III into NAP-2 through proteinases that were highly sensitive to PMSF, moderately sensitive to leupeptin and insensitive to EDTA.
Assuntos
Quimiocinas , Monócitos/enzimologia , Biossíntese Peptídica , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Humanos , Interleucina-8 , Dados de Sequência Molecular , beta-TromboglobulinaRESUMO
Peritoneal macrophages were obtained from untreated mice and from mice treated with thioglycollate medium (TA), proteose peptone medium (PP), or a suspension of streptococcus A cell wall material (SA). The biochemical and secretory properties of these cells in long term cultures (up to 2 wk) were compared. TA-elicited macrophages contained more protein, lactate dehydrogenase, lysosomal hydrolases, and in particular, more plasminogen activator than the other cells studied. All types of macrophages studied were found to release considerable amounts of lysosomal hydrolases (beta-glucuronidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and acid phosphatase) into the medium. Release was independent of phagocytosis and must, therefore, be regarded as true secretion. In both elicited and nonelicited macrophages, the rates of lysosomal enzyme secretion were virtually identical in the presence and in the absence of serum, and they were not enhanced by increasing serum concentrations. Lysosomal enzyme secretion in macrophages appears to depend on protein synthesis, since it was blocked by low concentrations of cycloheximide which neither affected cell viability nor lowered the intracellular enzyme levels. The amounts of lysosomal hydrolases secreted were highest in TA-elicited macrophages. The rates of secretion of PP- or SA-elicited and of nonelicited macrophages were about one-fourth of that of the TA-elicited cells. This difference, although significant, is much smaller than that observed for the secretion of plasminogen activator which was 20-50 times higher in TA-elicited cells. Acid glycosidases were also found in the peritoneal lavage media used for cell harvesting from both treated and nontreated mice. This indicates that active secretion of lysosomal hydrolases may be an in vivo property of the macrophage.
Assuntos
Lisossomos/enzimologia , Macrófagos/enzimologia , Animais , Líquido Ascítico/citologia , Meios de Cultura , Cicloeximida/farmacologia , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , L-Lactato Desidrogenase/metabolismo , Macrófagos/ultraestrutura , Masculino , Camundongos , Tioglicolatos/farmacologiaRESUMO
Macrophages were obtained by peritoneal lavage from untreated mice or from mice which had received either Brewer's thioglycollate broth or a suspension of streptococcus A cell walls intraperitoneally 4 days before. 3 h after harvesting, adherent cells from untreated mice were allowed to phagocytose zymosan, formaldehyde-treated sheep erythrocytes, or latex beads. Phagocytosis was stopped after 1 h and culture was continued for up to 10 days. Phagocytosis of zymosan or sheep erythrocytes triggered the immediate release of lysosomal glycosidases, stimulated the synthesis of cellular lactate dehydrogenase, and induced the delayed production and secretion of plasminogen activator . No such changes were observed upon phagocytosis of latex. Although all three particles used were phagocytosed, only zymosan and sheep erythrocytes stimulated glucose oxidation via the hexose monophosphate shunt. Similar findings were obtained in macrophages elicited with streptococcus A cell walls after zymosan phagocytosis. Thioglycollate-elicited macrophages, however, which were already secreting lysosomal hydrolases and plasminogen activator, could not be activated further by zymosan. The results of this study show that macrophages become activated after phagocytosis of particles that stimulate the activity of their hexose monophosphate shunt. The triggering event appears to be the burst of shunt activity itself or shunt-related biochemical reactions rather than phagocytic uptake per se or particle-dependent complement activation by the alternative pathway. Once initiated, macrophage activation proceeds independently of the intracellular fate of the ingested material .
Assuntos
Macrófagos/fisiologia , Acetilglucosaminidase/metabolismo , Animais , Meios de Cultura , Eritrócitos , Glucuronidase/metabolismo , Hexosefosfatos/metabolismo , L-Lactato Desidrogenase/metabolismo , Látex , Lisossomos/enzimologia , Masculino , Camundongos , Microesferas , Fagocitose , Ativadores de Plasminogênio/metabolismo , ZimosanRESUMO
The biological properties of a neutrophil-activating factor (NAF), which was recently identified as a novel peptide of approximately 6,000 mol wt, are described. NAF is produced de novo by human blood monocytes upon stimulation with LPS, PHA, and Con A. It induces two main responses in human neutrophils, i.e., exocytosis (release from specific granules in normal, and from specific and azurophil granules in cytochalasin B-treated cells) and the respiratory burst (formation of superoxide and hydrogen peroxide). The action of NAF appears to be mediated by a surface receptor as shown by the following observations. (a) NAF induces a rapid and transient rise in cytosolic free Ca2+; (b) interaction with NAF results in desensitization, since the cells do not respond to a second NAF challenge; and (c) the respiratory burst elicited by NAF is similar in onset, and time course to that induced by C5a or FMLP. The NAF receptor can be distinguished from the receptors of C5a, FMLP, platelet-activating factor, and leukotriene B4 by the lack of cross-desensitization. Unlike C5a, the other host-derived neutrophil-activating peptide, NAF is not inactivated by serum and thus presumably accumulates in inflamed tissue.
Assuntos
Leucócitos Mononucleares/análise , Neutrófilos/efeitos dos fármacos , Peptídeos/isolamento & purificação , Complemento C5/farmacologia , Complemento C5a , Exocitose/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/biossíntese , Interleucina-8 , Leucócitos Mononucleares/efeitos dos fármacos , Mitógenos/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Peptídeos/farmacologia , Receptores de Superfície Celular/análise , Superóxidos/biossínteseRESUMO
Several studies have shown that CC chemokines attract T lymphocytes, and that CD45RO+, memory phenotype cells are considered to be the main responders. The results, however, have often been contradictory and the role of lymphocyte activation and proliferation has remained unclear. Using CD45RO+ blood lymphocytes cultured under different stimulatory conditions, we have now studied chemotaxis as well as chemokine receptor expression. Expression of the RANTES/MIP-1 alpha receptor (CC-CKR1) and the MCP-1 receptor (CC-CKR2) was highly correlated with migration toward RANTES, MCP-1, and other CC chemokines, and was strictly dependent on the presence of IL-2 in the culture medium. Migration and receptor expression were rapidly downregulated when IL-2 was withdrawn, but were fully restored when IL-2 was added again. The effect of IL-2 could be partially mimicked by IL-4, IL-10, or IL-12, but not by IL-13, IFN gamma, IL-1 beta, TNF-alpha, or by exposure to anti-CD3, anti-CD28 or phytohemagglutinin. Activation of fully responsive lymphocytes through the TCR/CD3 complex and CD28 antigen actually had the opposite effect. It rapidly downregulated receptor expression and consequent migration even in the presence of IL-2. In contrast to the effects on CC chemokine receptors, stimulation of CD45RO+ T lymphocytes with IL-2 neither induced the expression of the CXC chemokine receptors, IL8-R1 and IL8-R2, nor chemotaxis to IL-8. The prominent role of IL-2 in CC chemokine responsiveness of lymphocytes suggests that IL-2-mediated expansion is a prerequisite for the recruitment of antigen-activated T cells into sites of immune and inflammatory reactions.
Assuntos
Quimiocinas/fisiologia , Quimiotaxia de Leucócito , Interleucina-2/fisiologia , Receptores de Quimiocinas , Receptores de Citocinas/metabolismo , Receptores de Citocinas/fisiologia , Linfócitos T/fisiologia , Antígenos CD28/fisiologia , Complexo CD3/fisiologia , Células Cultivadas , Humanos , Interleucina-2/farmacologia , Ativação Linfocitária , Receptores CCR2RESUMO
Two neutral proteinases from human polymorphonuclear leukocytes (PMN), an elastase and the chymotrypsin-like cathepsin G, were purified, and their actions on lymphocytes in culture were studied. Both PMN proteinases stimulate lymphocytes from human peripheral blood and from mouse spleen in vitro, but do not affect thymic cells from either normal or hydrocortisone-treated mice. In stimulated mouse spleen cell cultures, most of the developing blast cells bear surface immunoglobulins, and subsequently appear to engage in antibody synthesis. In their stimulatory action, the two PMN proteinases thus resemble the classic B-cell mitogen LPS and neutral pancreatic proteinases such as trypsin, chymotrypsin, and elastase. The effects of proteinase inhibitors indicate that lymphocyte stimulation is dependent on the proteolytic activity of the enzymes. This work suggests that PMN proteinases, which are released at sites of inflammation, may modulate the function of lymphocytes.
Assuntos
Catepsinas/farmacologia , Ativação Linfocitária , Neutrófilos/enzimologia , Elastase Pancreática/farmacologia , Animais , Quimotripsina/farmacologia , Humanos , Técnicas In Vitro , Lectinas/farmacologia , Linfócitos/imunologia , Camundongos , Camundongos Nus , Receptores de Antígenos de Linfócitos B/análise , Baço/citologia , Timo/citologia , Tripsina/farmacologiaRESUMO
Melanoma growth-stimulatory activity (MGSA), a peptide reported to be mitogenic for Hs294T human melanoma cells, has extensive sequence similarity to the neutrophil-activating peptide NAP-1/IL-8, suggesting functional similarities. To test this hypothesis, MGSA was chemically synthesized and tested for its effects on human neutrophils. It was found to induce chemotaxis, exocytosis of elastase, and changes in cytosolic-free calcium to an extent and at concentrations similar to NAP-1/IL-8. However, MGSA was considerably less potent than NAP-1/IL-8 in inducing the respiratory burst. Intradermal injections in rats of MGSA resulted in a massive accumulation of neutrophils. Our data demonstrate that, apart from its growth-stimulatory activity, MGSA is a potent inflammatory agonist with neutrophil-stimulating properties.
Assuntos
Quimiocinas CXC , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Neoplasias/farmacologia , Neutrófilos/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/sangue , Quimiocina CXCL1 , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Citosol/metabolismo , Humanos , Peróxido de Hidrogênio/sangue , Técnicas In Vitro , Interleucina-8 , Interleucinas/genética , Interleucinas/farmacologia , Cinética , Masculino , Dados de Sequência Molecular , Proteínas de Neoplasias/síntese química , Proteínas de Neoplasias/genética , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/sangue , Ratos , Ratos Endogâmicos , Homologia de Sequência do Ácido Nucleico , Pele/efeitos dos fármacos , Pele/patologiaRESUMO
Platelet basic protein (PBP), connective tissue-activating peptide III (CTAP-III), and platelet factor 4 (PF-4) were purified from human platelet release supernatants by heparin-Sepharose ion-exchange and reversed-phase HPLC, and their neutrophil-activating effects were compared with those of NAP-2, a peptide of 70 amino acids corresponding to part of the sequence of PBP (1) and with sequence homology to NAF/NAP-1. NAP-2-induced elastase release and a rise in cytosolic free Ca2+ at concentrations between 0.3 and 100 nM, and neutrophil chemotaxis at concentrations between 0.03 and 10 nM. It was half as potent as NAF/NAP-1 in inducing exocytosis but showed the same activity in the other responses. By contrast, only minimal if any effects were obtained with PBP, CTAP-III, and PF-4 up to 100 nM. NAP-2 thus appears to behave like a typical chemotactic receptor agonist. It could be generated from PBP and/or CTAP-III released from activated platelets and lead to the accumulation of neutrophils in platelet aggregates.
Assuntos
Quimiocinas , Neutrófilos/efeitos dos fármacos , Peptídeos/farmacologia , Fator Plaquetário 4/farmacologia , Proteínas/farmacologia , Sequência de Aminoácidos , Cálcio/fisiologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Citosol/fisiologia , Exocitose/efeitos dos fármacos , Humanos , Técnicas In Vitro , Interleucina-8 , Dados de Sequência Molecular , Elastase Pancreática/metabolismo , beta-TromboglobulinaRESUMO
Lactoferrin has been identified in rabbit heterophil leukocytes on the basis of its immunological reactivity, electrophoretic mobility, acid-resistant iron-binding properties, and spectral characteristics. Leukocyte lactoferrin was found to be exclusively localized in the specific (secondary) granules, which have been resolved from other subcellular components by zonal differential centrifugation and by isopycnic equilibration.
Assuntos
Grânulos Citoplasmáticos/análise , Ferro/metabolismo , Leucócitos/análise , Ligação Proteica , Proteínas/análise , Animais , Eletroforese , Leucócitos/metabolismo , CoelhosRESUMO
The subcellular localization of elastase and of neutral proteases hydrolyzing histone and casein was determined in human and rabbit polymorphonuclear leukocytes using fractionation by isopycnic centrifugation. Granule-rich fractions obtained by this technique were extracted and analyzed by acrylamide gel electrophoresis, and proteolytic activity on the gels was demonstrated by staining with either N-acetyl-D,L-alanine alpha-naphthyl ester or naphthol AS-D acetate as substrate. In both species, all neutral proteases assayed were found to be localized exclusively in the azurophil granules. Specific activities were about 10-30 times higher in human than in rabbit preparations. In extracts of human azurophil granules up to 10 proteins exhibiting esterolytic activity could be demonstrated after electrophoretic separation. Three major and two or three minor components of these esterases were shown to possess elastase activity. Similar zymograms prepared with extracts from rabbit azurophil granules revealed only one major elastase band. The electrophoretic analysis further showed that the most strongly cationic proteins of both human and rabbit PMNs were also confined to the azurophil granules.
Assuntos
Grânulos Citoplasmáticos/enzimologia , Neutrófilos/ultraestrutura , Elastase Pancreática/análise , Peptídeo Hidrolases/análise , Frações Subcelulares/enzimologia , Fosfatase Alcalina/análise , Animais , Caseínas , Catálise , Centrifugação Isopícnica , Eletroforese em Gel de Poliacrilamida , Hexosaminidases/análise , Histonas , Humanos , Concentração de Íons de Hidrogênio , Muramidase/análise , Neutrófilos/enzimologia , Peroxidases/análise , Coelhos , Fatores de TempoRESUMO
IgE-independent mediator release from basophils is considered an important event in inflammation, particularly in nonallergic immediate hypersensitivity and in allergic late-phase reactions. This study demonstrates that after exposure to IL-3, basophils release histamine and leukotrienes in response to the neutrophil-activating peptide NAF/NAP-1. Thus, the sequential action of two pure cytokines can promote basophils mediator release. In the presence of IL-3, NAF/NAP-1 functions like a "histamine-releasing factor" and may therefore not only induce cellular infiltration but also provoke symptoms of hypersensitivity reactions.
Assuntos
Basófilos/fisiologia , Liberação de Histamina/efeitos dos fármacos , Interleucina-3/farmacologia , Peptídeos/farmacologia , SRS-A/metabolismo , Relação Dose-Resposta a Droga , Glucuronidase/metabolismo , Humanos , Imunoglobulina E/fisiologia , Técnicas In Vitro , Interleucina-8 , Neutrófilos/fisiologiaRESUMO
A new neutrophil-activating peptide, termed ENA-78, was identified in the conditioned media of stimulated human type II epithelial cell line A549. In response to stimulation with either interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha), ENA-78 was produced and secreted concomitantly with IL-8, GRO alpha, and GRO gamma. ENA-78 consists of 78 amino acids [sequence; see text] and has a molecular weight of 8,357. It has four cysteines positioned identically to those of IL-8 and analogues, and thus belongs to the CXC family of peptides. ENA-78 is related to neutrophil-activating peptide 2 (NAP-2) and GRO alpha (sequence identity, 53% and 52%, respectively) and IL-8 (22% identity). Like NAP-2 and GRO alpha, ENA-78 stimulates neutrophils, inducing chemotaxis, a rise in intracellular free calcium and exocytosis. Cross-desensitization experiments indicate that ENA-78 acts through the same type of receptors as IL-8, NAP-2, and GRO alpha.
Assuntos
Quimiocinas CXC , Interleucina-8/análogos & derivados , Interleucina-8/farmacologia , Neutrófilos/efeitos dos fármacos , Peptídeos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Quimiocina CXCL5 , Humanos , Interleucina-8/química , Dados de Sequência Molecular , Peptídeos/química , beta-TromboglobulinaRESUMO
After phagocytosis of yeast opsonized with IgG, neutrophil leukocytes (polymorphonuclear leukocytes [PMN]) expressed high levels of neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) mRNA, which peaked after 3-5 h and were still elevated after 18 h. A similar but quantitatively less prominent effect was obtained with lipopolysaccharide (LPS). After phagocytosis, but not after exposure to LPS, the PMN progressively released considerable amounts of NAP-1/IL-8 into the culture medium (18.6-50 ng/ml in 18 h). The peptide released was biologically active, as indicated by the transient elevation of cytosolic-free calcium in PMN exposed to aliquots of the culture supernatants, and desensitization by prestimulation of the cells with recombinant NAP-1/IL-8. By producing NAP-1/IL-8 at sites where they phagocytose invading microorganisms, PMN could enhance the recruitment of new defense cells.
Assuntos
Interleucina-8/sangue , Neutrófilos/fisiologia , Fagocitose , Cálcio/sangue , Células Cultivadas , Citosol/metabolismo , Humanos , Interleucina-8/biossíntese , Interleucina-8/genética , Cinética , RNA Mensageiro/sangue , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificaçãoRESUMO
A novel human CC chemokine consisting of 78 amino acids and having a molecular mass of 8,778.3 daltons (VVIPSPCCMF FVSKRIPENR VVSYQLSSRS TCLKAGVIFT TKKGQQ SCGD PKQEWVQRYM KNLDAKQKKA SPRARAVA) was isolated together with three minor COOH-terminally truncated variants with 73, 75, and 76 residues. The new chemokine was termed eotaxin-2 because it is functionally very similar to eotaxin. In terms of structure, however, eotaxin and eotaxin-2 are rather distant, they share only 39% identical amino acids and differ almost completely in the NH2-terminal region. Eotaxin-2 induced chemotaxis of eosinophils as well as basophils, with a typically bimodal concentration dependence, and the release of histamine and leukotriene C4 from basophils that had been primed with IL-3. In all assays, eotaxin-2 had the same efficacy as eotaxin, but was somewhat less potent. The migration and the release responses were abrogated in the presence of a monoclonal antibody that selectively blocks the eotaxin receptor, CCR3, indicating that eotaxin-2, like eotaxin, acts exclusively via CCR3. Receptor usage was also studied in desensitization experiments by measuring [Ca2+]i changes in eosinophils. Complete cross-desensitization was observed between eotaxin-2, eotaxin and MCP-4 confirming activation via CCR3. No Ca2+ mobilization was obtained in neutrophils, monocytes and lymphocytes, in agreement with the lack of chemotactic responsiveness. Intradermal injection of eotaxin-2 in a rhesus monkey (100 or 1,000 pmol per site) induced a marked local infiltration of eosinophils, which was most pronounced in the vicinity of postcapillary venules and was comparable to the effect of eotaxin.
Assuntos
Basófilos/efeitos dos fármacos , Quimiocinas CC , Fatores Quimiotáticos de Eosinófilos/farmacologia , Citocinas/farmacologia , Eosinófilos/efeitos dos fármacos , Receptores de Quimiocinas , Receptores de Citocinas/fisiologia , Sequência de Aminoácidos , Animais , Quimiocina CCL11 , Quimiotaxia , Citocinas/química , Liberação de Histamina/efeitos dos fármacos , Humanos , Leucotrieno C4/metabolismo , Macaca mulatta , Masculino , Dados de Sequência Molecular , Receptores CCR3RESUMO
Although most leukocytes, T lymphocytes in particular, respond to several different chemokines, there is virtually no information on chemokine activities and chemokine receptors in B lymphocytes. A putative chemokine receptor, BLR1, that is expressed in Burkitt's lymphoma cells and B lymphocytes was cloned a few years ago. Deletion of the gene for BLR1 yielded mice with abnormal primary follicles and germinal centers of the spleen and Peyer's patches, reflecting the inability of B lymphocytes to migrate into B cell areas. By screening expressed sequence tag DNA sequences, we have identified a CXC chemokine, termed B cell-attracting chemokine 1 (BCA-1), that is chemotactic for human B lymphocytes. BCA-1 cDNA encodes a protein of 109 amino acids with a leader sequence of 22 residues. The mature protein shares 23-34% identical amino acids with known CXC chemokines and is constitutively expressed in secondary lymphoid organs. BCA-1 was chemically synthesized and tested for activity on murine pre-B cells 300-19 transfected with BLR1 and on human blood B lymphocytes. In transfected cells, BCA-1 induced chemotaxis and Ca2+ mobilization demonstrating that it acts via BLR1. Under the same conditions, no activity was obtained with 10 CXC and 19 CC chemokines, lymphotactin, neurotactin/fractalkine and several other peptide ligands. BCA-1 was also a highly effective attractant for human blood B lymphocytes (which express BLR1), but was inactive on freshly isolated or IL-2-stimulated T lymphocytes, monocytes, and neutrophils. In agreement with the nomenclature rules for chemokine receptors, we propose the term CXCR5 for BLR1. Together with the observed disturbance of B cell colonization in BLR1/ CXCR5-deficient mice, the present results indicate that chemotactic recruitment by locally produced BCA-1 is important for the development of B cell areas of secondary lymphoid tissues.
Assuntos
Linfócitos B/imunologia , Quimiocinas CC/imunologia , Quimiocinas CXC , Proteínas de Ligação ao GTP/imunologia , Receptores de Citocinas/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Quimiocina CXCL13 , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Receptores CXCR5 , Receptores de Quimiocinas , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismoRESUMO
Chemotactic cytokines of the CC subfamily (CC chemokines) are considered as major mediators of allergic inflammation owing their actions on basophil and eosinophil leukocytes. The monocyte chemotactic protein (MCP) 1 is a potent inducer of mediator release from basophils but is inactive on eosinophils. To obtain information on the structural determinants of the activities of MCP-1, we have synthesized several NH2-terminally truncated analogues and tested their effects on basophils and eosinophils. Through deletion of the NH2-terminal residue, MCP-1(2-76) was obtained, which was a potent activator of eosinophils, as assessed by chemotaxis, cytosolic free Ca2+ changes, actin polymerization, and that induction of the respiratory burst. In contrast, the activity of MCP-1(2-76) on basophil leukocytes was dramatically decreased (50-fold) compared with that of full-length MCP-1. Deletion of the next residue led to total loss of activity on eosinophil and basophil leukocytes. Analogues with three or four residue deletions, MCP-1(4-76) and MCP-1(5-76), were again active on both cells, whereas all further truncation analogues, MCP-1(6-76) through MCP-1(10-76), were inactive. Thus, a minimal structural modification can change receptor and target cell selectivity of MCP-1. Our observations indicate that the recognition sites of CC chemokine receptors on eosinophils and basophils are similar, although they discriminate between MCP-1 and MCP-1(2-76) and suggest NH2-terminal processing as a potential mechanism for the regulation of CC chemokine activities.
Assuntos
Quimiocina CCL2/análogos & derivados , Quimiotaxia de Leucócito , Eosinófilos/efeitos dos fármacos , Actinas/metabolismo , Basófilos/efeitos dos fármacos , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Liberação de Histamina , Humanos , Leucotrieno C4/metabolismo , Explosão Respiratória , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
CC chemokines produced by CD8(+) T cells are known to act as HIV-suppressive factors. We studied the possible role of these chemokines in HIV-1-specific killing of target cells. We found that the activity of cytotoxic T lymphocytes (CTLs) in CTL lines or freshly isolated peripheral blood mononuclear cells from HIV-1-infected individuals is markedly enhanced by RANTES (regulated on activation, normal T cell expressed and secreted) and virtually abolished by an antibody neutralizing RANTES or the RANTES receptor antagonist RANTES(9-68). Lysis was mediated by CD8(+) major histocompatibility complex class I-restricted T cells and was obtained with target cells expressing epitopes of the HIV-1LAI proteins Gag, Pol, Env, and Nef. The cytolytic activity observed in the presence or absence of added RANTES could be abolished by pretreatment of the CTLs with pertussis toxin, indicating that the effect is mediated by a G protein-coupled receptor. The chemokines monocyte chemotactic protein (MCP)-3, MCP-4, and eotaxin acted like RANTES, whereas macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MCP-1, and stromal cell-derived factor 1 were inactive, suggesting a role for the eotaxin receptor, CCR3, and ruling out the involvement of CCR1, CCR2, CCR5, and CXCR4. CTL activity was abrogated by an antibody that blocks CCR3, further indicating that specific lysis is triggered via this chemokine receptor. These observations reveal a novel mechanism for the induction of HIV-1-specific cytotoxicity that depends on RANTES acting via CCR3.
Assuntos
Quimiocina CCL5/imunologia , Citotoxicidade Imunológica , Infecções por HIV/imunologia , HIV-1/imunologia , Receptores de Quimiocinas/imunologia , Linhagem Celular , Quimiocina CCL5/farmacologia , Humanos , Receptores CCR3RESUMO
CC chemokines constitute a novel class of cytokines that attract and activate monocytes and lymphocytes, as well as basophil and eosinophil leukocytes, with distinct target cell profiles, and are believed to be involved in the regulation of different types of inflammation. The action of the recently identified monocyte chemotactic protein 3 (MCP-3) on human basophil and eosinophil function was studied and compared with that of other CC chemokines. In basophils, MCP-3, MCP-1, RANTES, and macrophage inflammatory protein (MIP)-1 alpha all induced cytosolic-free calcium concentration ([Ca2+]i) changes and, with different efficacies, chemotaxis (RANTES = MCP-3 >> MCP-1 > MIP-1 alpha), histamine release (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha), and leukotriene C4 formation, after IL-3 pretreatment (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha). Thus, MCP-3 was as effective as MCP-1 as an inducer of mediator release, and as effective as RANTES as a stimulus of basophil migration. In contrast to MCP-1, MCP-3 was also a stimulus for eosinophils, and induced [Ca2+]i changes and chemotaxis as effectively as RANTES, which is the most potent chemotactic cytokine for these cells. Desensitization of the transient changes in [Ca2+]i was used to assess receptor usage. In basophils, stimulation with MCP-3 prevented responsiveness to MCP-1 and RANTES, but not to MIP-1 alpha. No single CC chemokine (except for MCP-3 itself) affected the response to MCP-3, however, which was prevented only when the cells were prestimulated with both MCP-1 and RANTES. In eosinophils, by contrast, cross-desensitization between RANTES and MCP-3 was obtained. RANTES and to a lesser extent MCP-3 also desensitized eosinophils toward MIP-1 alpha. The desensitization data suggest the existence of three chemokine receptors: (a) a MCP-1 receptor expressed on basophils but not eosinophils that is activated by MCP-1 and MCP-3; (b) a RANTES receptor in basophils and eosinophils that is activated by RANTES and MCP-3; and (c) a MIP-1 alpha receptor that is activated by MIP-1 alpha, RANTES and, more weakly, by MCP-3. This study shows that MCP-3 combines the properties of RANTES, a powerful chemoattractant, and MCP-1, a highly effective stimulus of mediator release, and thus has a particularly broad range of activities toward both human basophil and eosinophil leukocytes.
Assuntos
Basófilos/imunologia , Fatores Quimiotáticos/fisiologia , Eosinófilos/imunologia , Proteínas Quimioatraentes de Monócitos , Quimiocina CCL4 , Quimiocina CCL5 , Quimiocina CCL7 , Citocinas/farmacologia , Humanos , Técnicas In Vitro , Linfocinas/farmacologia , Proteínas Inflamatórias de Macrófagos , Monocinas/farmacologiaRESUMO
The cytokine NAP-1/IL-8 is produced by a variety of different cells in response to inflammatory stimuli and elicits several biological responses from PMN. Experiments presented here demonstrate that PMN exposed to NAP-1/IL-8 expressed increased amounts of CD11b/CD18, as well as CD11c/CD18 and CR1, on their cell surface, while expression of Fc gamma RIII and HLA-A,B,C remained essentially unchanged. Increased CD11b/CD18 and CD11c/CD18 appears to correspond with the release of specific granules by NAP-1/IL-8. NAP-1/IL-8 was also a potent stimulator of several of the binding activities of CD11b/CD18. Ligation of EC3bi by CD11b/CD18 was rapidly enhanced by NAP-1/IL-8, but phagocytosis of the ligated particles was not induced by the agonist. In addition, enhanced binding of EC3bi was observed in the absence of an increase in receptor expression as shown with PMN cytoplasts. NAP-1/IL-8 promoted additional adhesive interactions between CD11b/CD18 and the biosynthetic precursor of LPS, lipid IVa, fibrinogen, and endothelial cells, suggesting that NAP-1/IL-8 may promote leukocyte adhesion in vivo that could lead to recruitment of PMN to sites of tissue inflammation.
Assuntos
Antígenos CD/imunologia , Fatores Quimiotáticos/farmacologia , Interleucinas/farmacologia , Neutrófilos/imunologia , Receptores de Adesão de Leucócito/imunologia , Anticorpos Monoclonais , Antígenos CD18 , Adesão Celular , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Interleucina-8 , Cinética , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Proteínas Recombinantes/farmacologiaRESUMO
Ligation of CCR5 by the CC chemokines RANTES, MIP-1alpha or MIP-1beta, and of CXCR4 by the CXC chemokine SDF-1alpha, profoundly inhibits the replication of HIV strains that use these coreceptors for entry into CD4(+) T lymphocytes. The mechanism of entry inhibition is not known. We found a rapid and extensive downregulation of CXCR4 by SDF-1alpha and of CCR5 by RANTES or the antagonist RANTES(9-68). Confocal laser scanning microscopy showed that CCR5 and CXCR4, after binding to their ligands, are internalized into vesicles that qualify as early endosomes as indicated by colocalization with transferrin receptors. Internalization was not affected by treatment with Bordetella pertussis toxin, showing that it is independent of signaling via Gi-proteins. Removal of SDF-1alpha led to rapid, but incomplete surface reexpression of CXCR4, a process that was not inhibited by cycloheximide, suggesting that the coreceptor is recycling from the internalization pool. Deletion of the COOH-terminal, cytoplasmic domain of CXCR4 did not affect HIV entry, but prevented SDF-1alpha-induced receptor downregulation and decreased the potency of SDF-1alpha as inhibitor of HIV replication. Our results indicate that the ability of the coreceptor to internalize is not required for HIV entry, but contributes to the HIV suppressive effect of CXC and CC chemokines.