RESUMO
Monocyte chemoattractant protein (MCP)-1 analogues were designed to determine the role of the NH2-terminal region in structure and function. The NH2-terminal residue was important for function and receptor binding, as it could not be deleted or extended. However the NH2-terminal pyroglutamate residue of the wild type was not essential as it could be replaced by several other noncyclic amino acids without loss of activity. Residues 7-10 were essential for receptor desensitization, but were not sufficient for function, and the integrity of residues 1-6 were required for functional activity. A peptide corresponding to MCP-1, 1-10 lacked detectable receptor-binding activities, indicating that residues 1-10 are essential for MCP-1 function, but that other residues are also involved. Several truncated analogues, including 8-76, 9-76, and 10-76, desensitized MCP-1-induced Ca2+ induction, but were not significantly active. These analogues were antagonists of MCP-1 activity with the most potent being the 9-76 analogue (IC50 = 20 nM) The 9-76 specifically bound to MCP-1 receptors with a Kd of 8.3 nM, which was three-fold higher than MCP-1 (Kd 2.8 nM). The 9-76 analogue desensitized the Ca2+ response to MCP-1 and MCP-3, but not to other CC chemokines, suggesting that it is MCP receptor specific. The availability of these compounds will be helpful in evaluating MCP receptor antagonists as anti-inflammatory therapeutics.
Assuntos
Fatores Quimiotáticos/antagonistas & inibidores , Citocinas/metabolismo , Receptores de Quimiocinas , Amidas/química , Sequência de Aminoácidos , Cálcio/metabolismo , Células Cultivadas , Quimiocina CCL2 , Fatores Quimiotáticos/química , Humanos , Dados de Sequência Molecular , Ligação Proteica , Receptores CCR2 , Receptores de Citocinas/metabolismoRESUMO
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
Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [(3)H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-beta was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered G(o) and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.
Assuntos
Quimiocinas CXC/farmacologia , Células-Tronco Hematopoéticas/citologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Ciclo Celular , Divisão Celular , Senescência Celular , Quimiocina CXCL12 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fatores de Tempo , Fator de Crescimento Transformador beta1RESUMO
We report that stromal cell-derived factor (SDF)-1 has the remarkable capacity to induce sustained signaling through CXC chemokine receptor 4 (CXCR4). In contrast to other chemokines, such as monocyte chemotactic protein 1 (CC chemokine receptor 2 [CCR2]), macrophage inflammatory protein 1beta (CCR5), liver and activation-regulated chemokine (LARC [CCR6]), Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC [CCR7]), and IP10 (CXCR3), SDF-1 stimulates the prolonged activation of protein kinase B and extracellular signal-regulated kinase (ERK)-2. Activation of protein kinase B is reversed by displacement of SDF-1 from CXCR4 or inhibition of phosphatidylinositol 3-kinase. Although increasing concentrations of SDF-1 enhance CXCR4 internalization, kinase activation is prolonged. In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized. Furthermore, activation is prolonged by inhibiting SDF-1 degradation. The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.
Assuntos
Quimiocinas CXC/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Androstadienos/farmacologia , Animais , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt , Linfócitos T/citologia , Fatores de Tempo , WortmaninaRESUMO
Although numerous chemokines act on monocytes, none of them is specific for these cells. Here, we show that breast and kidney-expressed chemokine (BRAK) is a highly selective monocyte chemoattractant. Migration efficacy and Bordetella pertussis toxin-sensitive Ca(2+) mobilization responses to BRAK were strongly enhanced after treatment of monocytes with the cyclic AMP-elevating agents prostaglandin E(2) and forskolin. BRAK is the first monocyte-selective chemokine, as other types of blood leukocytes or monocyte-derived dendritic cells and macrophages did not respond. Expression in normal skin keratinocytes and dermal fibroblasts as well as lamina propria cells in normal intestinal tissues suggests a homeostatic rather than an inflammatory function for this chemokine. In addition, macrophages were frequently found to colocalize with BRAK-producing fibroblasts. We propose that BRAK is involved in the generation of tissue macrophages by recruiting extravasated precursors to fibroblasts, which are known to secrete essential cytokines for macrophage development.
Assuntos
Quimiocinas CXC/fisiologia , Macrófagos/citologia , Monócitos/metabolismo , Diferenciação Celular , Movimento Celular , Células Cultivadas , Quimiocinas CXC/genética , AMP Cíclico/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Rim/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/fisiologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Toxina Pertussis , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Distribuição Tecidual , Fatores de Virulência de Bordetella/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
An antagonist of human monocyte chemoattractant protein (MCP)-1, which consists of MCP-1(9-76), had previously been characterized and shown to inhibit MCP-1 activity in vitro. To test the hypothesis that, by inhibiting endogenous MCP-1, the antagonist has antiinflammatory activity in vivo, we examined its effect in the MRL-lpr mouse model of arthritis. This strain spontaneously develops a chronic inflammatory arthritis that is similar to human rheumatoid arthritis. Daily injection of the antagonist, MCP-1(9-76), prevented the onset of arthritis as monitored by measuring joint swelling and by histopathological evaluation of the joints. In contrast, controls treated with native MCP-1 had enhanced arthritis symptoms, indicating that the inhibitory effect is specific to the antagonist. In experiments where the antagonist was given only after the disease had already developed, there was a marked reduction in symptoms and histopathology, although individuals varied in the magnitude of the response. The mechanism of inhibition of disease is not known, although the results suggest that it could be more complex than the competitive inhibition of ligand binding that is observed in vitro. The demonstration of the beneficial effects of an MCP-1 antagonist in arthritis suggests that chemokine receptor antagonists could have therapeutic application in inflammatory diseases.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Feminino , Humanos , Articulações/efeitos dos fármacos , Articulações/patologia , Masculino , Camundongos , Camundongos MutantesRESUMO
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
A G protein-coupled receptor (GPCR) is encoded within the genome of Kaposi's sarcoma- associated herpesvirus (KSHV)/human herpesvirus 8, a virus that may be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas. KSHV-GPCR exhibits constitutive signaling activity that causes oncogenic transformation. We report that human interferon (IFN)-gamma-inducible protein 10 (HuIP-10), a C-X-C chemokine, specifically inhibits signaling of KSHV-GPCR. In contrast, monokine induced by IFN-gamma (HuMig), which like HuIP-10 is an agonist of C-X-C chemokine receptor 3, does not inhibit KSHV-GPCR signaling. Moreover, HuIP-10, but not HuMig, inhibits KSHV-GPCR-induced proliferation of NIH 3T3 cells. These results show that HuIP-10 is an inverse agonist that converts KSHV-GPCR from an active to an inactive state. Thus, a human chemokine inhibits constitutive signaling and cellular proliferation that is mediated by a receptor encoded by a human disease-associated herpesvirus.
Assuntos
Quimiocinas CXC/farmacologia , Herpesvirus Humano 8/genética , Receptores de Quimiocinas/fisiologia , Receptores Virais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células 3T3 , Animais , Quimiocina CXCL10 , Quimiocinas CXC/fisiologia , Técnicas de Transferência de Genes , Genes Virais , Humanos , CamundongosRESUMO
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
The T helper (Th) cell pool is composed of specialized cells with heterogeneous effector functions. Apart from Th1 and 2 cells, CXCR5+ T cells have been suggested to be another type of effector T cell specialized for B cell help. We show here that CXCR5+ T cells are heterogeneous, and we identify subsets of CXCR5+ CD4 T cells that differ in function and microenvironmental localization in secondary lymphoid tissues. CD57+CXCR5 T cells, hereafter termed germinal center Th (GC-Th) cells, are localized only in GCs, lack CCR7, and are highly responsive to the follicular chemokine B lymphocyte chemoattractant but not to the T cell zone EBI1-ligand chemokine. Importantly, GC-Th cells are much more efficient than CD57-CXCR5+ T cells or CXCR5- T cells in inducing antibody production from B cells. Consistent with their function, GC-Th cells produce elevated levels of interleukin 10 upon stimulation which, with other cytokines and costimulatory molecules, may help confer their B cell helper activity. Our results demonstrate that CXCR5+ T cells are functionally heterogeneous and that the GC-Th cells, a small subset of CXCR5+ T cells, are the key helpers for B cell differentiation and antibody production in lymphoid tissues.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Cooperação Linfocítica , Receptores de Citocinas/análise , Linfócitos T Auxiliares-Indutores/imunologia , Biomarcadores/análise , Sangue/imunologia , Antígenos CD57/análise , Células Cultivadas , Quimiocina CCL19 , Quimiocina CXCL13 , Quimiocinas CC/farmacologia , Quimiocinas CXC/farmacologia , Quimiotaxia de Leucócito , Criança , Pré-Escolar , Citocinas/biossíntese , Humanos , Imunoglobulinas/biossíntese , Imunofenotipagem , Tonsila Palatina/imunologia , Receptores CXCR5 , Receptores de Quimiocinas , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacosRESUMO
A human receptor that is selective for the CXC chemokines IP10 and Mig was cloned and characterized. The receptor cDNA has an open reading frame of 1104-bp encoding a protein of 368 amino acids with a molecular mass of 40,659 dalton. The sequence includes seven putative transmembrane segments characteristic of G-protein coupled receptors. It shares 40.9 and 40.3% identical amino acids with the two IL-8 receptors, and 34.2-36.9% identity with the five known CC chemokine receptors. The IP10/Mig receptor is highly expressed in IL-2-activated T lymphocytes, but is not detectable in resting T lymphocytes. B lymphocytes, monocytes and granulocytes. It mediates Ca2+ mobilization and chemotaxis in response to IP10 and Mig, but does not recognize the CXC-chemokines IL-8, GRO alpha, NAP-2, GCP-2. ENA78, PF4, the CC-chemokines MCP-1, MCP-2, MCP-3, MCP-4, MIP-1 alpha, MIP-1 beta. RANTES, 1309, eotaxin, nor lymphotactin. The exclusive expression in activated T-lymphocytes is of high interest since the receptors for chemokines which have been shown so far to attract lymphocytes, e.g., MCP-1, MCP-2, MCP-3, MIP-1 alpha, MIP-1 beta, and RANTES, are also found in monocytes and granulocytes. The present observations suggest that the IP10/Mig receptor is involved in the selective recruitment of effector T cells.
Assuntos
Linfócitos T CD4-Positivos/química , Quimiocinas CXC , Quimiocinas/metabolismo , Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Receptores de Citocinas/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/metabolismo , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiotaxia de Leucócito , DNA Complementar/química , Biblioteca Gênica , Humanos , Células Jurkat , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Receptores de Citocinas/química , Alinhamento de Sequência , Relação Estrutura-Atividade , TransfecçãoRESUMO
Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3alpha or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues. MIP-3alpha produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34(+)-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3alpha was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3alpha acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3alpha receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3alpha, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
Assuntos
Quimiocinas CC , Células Dendríticas/imunologia , Pulmão/imunologia , Proteínas Inflamatórias de Macrófagos/metabolismo , Receptores de Quimiocinas , Receptores de Citocinas/genética , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Cálcio/metabolismo , Linhagem Celular , Quimiocina CCL20 , Clonagem Molecular , Primers do DNA/genética , Células Dendríticas/metabolismo , Expressão Gênica , Humanos , Pulmão/metabolismo , Proteínas Inflamatórias de Macrófagos/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR6 , Receptores de Citocinas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
HIV-1 actively replicates in dendritic cell (DC)-T cell cocultures, but it has been difficult to demonstrate substantial infection of purified mature DCs. We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells, even though T cells have higher levels of CD4 and gp120 binding. DCs isolated from skin or from blood precursors behave similarly. Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently, as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37 degrees C. However, few late gag-containing sequences are detected, so that active viral replication does not occur. The formation of these early transcripts seems to follow entry of HIV-1, rather than binding of virions that contain viral DNA. Early transcripts are scarce if DCs are exposed to virus on ice for 4 h, or for 90 min at 37 degrees C, conditions which allow virus binding. Also the early transcripts once formed are insensitive to trypsin. The entry of a M-tropic isolates is blocked by the chemokine RANTES, and the entry of IIIB by SDF-1. RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors. Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor. DCs express more CCR5 and CXCR4 mRNA than T cells. Therefore, while HIV-1 does not replicate efficiently in mature DCs, viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus.
Assuntos
Células Dendríticas/virologia , HIV-1/fisiologia , Receptores de Citocinas/imunologia , Linfócitos T/virologia , Replicação Viral , Células Cultivadas , Quimiocina CCL5/farmacologia , Técnicas de Cocultura , Células Dendríticas/imunologia , Produtos do Gene gag/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Interleucina-4/farmacologia , Reação em Cadeia da Polimerase , Pele/imunologia , Linfócitos T/imunologia , Transcrição Gênica , Vírion/imunologia , Vírion/fisiologiaRESUMO
The human immunodeficiency virus-1 (HIV-1) protein Tat binds to cell surface antigens and can regulate cellular responses. Tat has similar immunosuppressive effects as transforming growth factor-beta (TGF beta) and both inhibit lymphocyte proliferation. TGF beta is expressed by primary human articular chondrocytes and is their most potent growth factor. The present study analyzed the interactions of TGF beta and HIV Tat in the regulation of human articular chondrocytes. Synthetic or recombinant full-length Tat (1-86) induced chondrocyte proliferation and this was of similar magnitude as the response to TGF beta. Tat peptides that did not contain the RGD motif had similar chondrocyte stimulatory activity as full-length Tat. Among a series of Tat peptides, peptide 38-62 which contains the basic domain was the only one active, suggesting that this region is responsible for the effects on chondrocyte proliferation. Full-length Tat and peptide 38-62 synergized with TGF beta and induced proliferative responses that were greater than those obtained with any combination of the known chondrocyte growth factors. Further characterization of the interactions between Tat and TGF beta showed that Tat increased synthesis and TGF beta activity and TGF beta 1 mRNA levels. The stimulatory effects of Tat and peptide 38-62 on chondrocyte proliferation were reduced by neutralizing antibodies to TGF beta and by TGF beta antisense oligonucleotides. These results identify a virally encoded protein and a synthetic peptide derived from it as novel and potent chondrocyte growth stimuli which act at least in part through the induction of TGF beta.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Produtos do Gene tat/farmacologia , Fator de Crescimento Transformador beta/biossíntese , Sequência de Bases , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.
Assuntos
Interleucina-8/síntese química , Dobramento de Proteína , Proteínas/síntese química , Sequência de Aminoácidos , Humanos , Interleucina-8/química , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Proteínas/químicaRESUMO
Interleukin-3 (IL-3), a protein of 140 amino acids, was chemically synthesized by means of an automated peptide synthesizer and was shown to have the biological activities attributed to native IL-3. Assays of synthetic analogues established that an amino terminal fragment has detectable IL-3 activity, but that the stable tertiary structure of the complete molecule was required for full activity. The results demonstrate that automated peptide synthesis can be applied to the study of the structure and function of proteins.
Assuntos
Linfocinas/síntese química , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Interleucina-3 , Linfocinas/farmacologia , Mastócitos/efeitos dos fármacos , Megacariócitos/efeitos dos fármacos , Camundongos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Interleukin-8 (IL-8), a pro-inflammatory protein, has been shown by nuclear magnetic resonance (NMR) and x-ray techniques to exist as a homodimer. An IL-8 analog was chemically synthesized, with the amide nitrogen of leucine-25 methylated to selectivity block formation of hydrogen bonds between monomers and thereby prevent dimerization. This analog was shown to be a monomer, as assessed by analytical ultracentrifugation and NMR. Nevertheless, it was equivalent to IL-8 in assays of neutrophil activation, which indicates that the monomer is a functional form of IL-8.
Assuntos
Interleucina-8/farmacologia , Neutrófilos/fisiologia , Cálcio/metabolismo , Quimiotaxia de Leucócito , Humanos , Ligação de Hidrogênio , Interleucina-8/análogos & derivados , Interleucina-8/química , Interleucina-8/metabolismo , Elastase de Leucócito , Modelos Químicos , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8ARESUMO
Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases. Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system. Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A. Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation. This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response.
Assuntos
Citocinas , Inflamação/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Quimioatraentes de Monócitos/metabolismo , Animais , Cálcio/metabolismo , Domínio Catalítico , Linhagem Celular , Quimiocina CCL7 , Quimiocinas/antagonistas & inibidores , Quimiocinas/metabolismo , Quimiotaxia de Leucócito , Colágeno/metabolismo , Ativação Enzimática , Biblioteca Gênica , Hemopexina/química , Hemopexina/metabolismo , Humanos , Inflamação/patologia , Espectrometria de Massas , Metaloproteinase 2 da Matriz/química , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/metabolismo , Proteínas Recombinantes/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.