RESUMO
BACKGROUND: To recruit leucocytes to an inflammatory site, chemokine binding to glycosaminoglycans (GAGs) is critical. Therefore, strategies to interfere with this interaction, aiming at the production of anti-inflammatory agents, were developed. These include production of modified chemokines without affinity for G protein-coupled receptors but with enhanced affinity for GAGs. Such modified chemokines compete with functional chemokines for GAG binding, prevent chemokine immobilization and presentation, and inhibit leucocyte migration. In addition to modified chemokines, a GAG-binding peptide consisting of the 30 COOH-terminal residues of CXCL9, that is CXCL9(74-103), inhibited CXCL8- and monosodium urate crystal-induced neutrophil migration. OBJECTIVE: We wanted to explore whether interference with chemokine-GAG interactions by CXCL9(74-103) reduces inflammation in neutrophil-dependent dinitrofluorobenzene-induced contact hypersensitivity. METHODS: For this study, we evaluated several inflammatory parameters, including ear swelling and the levels of chemokines, cytokines, proteases and neutrophils in the ears of dinitrofluorobenzene-induced mice treated with CXCL9(74-103) or buffer. RESULTS: One intravenous injection of CXCL9(74-103), just before painting with dinitrofluorobenzene on the ear, did not affect protein levels of the major murine neutrophil attractant, that is CXCL6, in this contact hypersensitivity model. However, IL-6, CXCL1, CCL2 and matrix metalloproteinase-9 (MMP-9) protein concentrations and peroxidase activity in challenged ears were reduced. In addition, intravenous injection of the CXCL9-derived peptide led to a reduced ear swelling response, indicating that the locally produced chemokines were hindered to attract leucocytes. The inhibiting potential of CXCL9(74-103) was explained by its competition for GAG binding with CXCL1, CXCL6 and CCL3 and inhibition of transendothelial migration of neutrophils to CXCL6. CONCLUSIONS AND CLINICAL RELEVANCE: The CXCL9(74-103) peptide inhibited dinitrofluorobenzene-induced infiltration of neutrophils and neutrophil-dependent inflammation in ears. Therefore, CXCL9(74-103) may be a lead molecule for the development of therapeutic peptides or peptide derivatives that compete with functional chemokines for GAG binding.
Assuntos
Anti-Inflamatórios/farmacologia , Quimiocina CXCL9/química , Dermatite de Contato/etiologia , Dermatite de Contato/metabolismo , Dinitrofluorbenzeno/efeitos adversos , Glicosaminoglicanos/metabolismo , Peptídeos/farmacologia , Animais , Citocinas/metabolismo , Dermatite de Contato/tratamento farmacológico , Feminino , Leucócitos/imunologia , Leucócitos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Ligação Proteica , Pele/imunologia , Pele/metabolismo , Pele/patologia , Migração Transendotelial e TransepitelialRESUMO
The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.
Assuntos
Anti-Inflamatórios/uso terapêutico , Quimiocina CXCL9/uso terapêutico , Gota/tratamento farmacológico , Interleucina-8/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Inibição de Migração Celular/efeitos dos fármacos , Quimiocina CXCL9/química , Quimiotaxia de Leucócito/efeitos dos fármacos , Glicosaminoglicanos/imunologia , Gota/induzido quimicamente , Gota/imunologia , Humanos , Interleucina-8/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neutrófilos/citologia , Neutrófilos/imunologia , Peptídeos/química , Ácido ÚricoRESUMO
Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1ß and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1ß, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.
Assuntos
Anticorpos Monoclonais/metabolismo , Hidrolases/metabolismo , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Apoptose , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linhagem Celular , Citrulina/metabolismo , Feminino , Fibroblastos , Humanos , Infliximab , Leucócitos Mononucleares/metabolismo , Camundongos , Ativação de Neutrófilo , Neutrófilos/imunologia , Ligação Proteica , Desiminases de Arginina em ProteínasRESUMO
Posttranslational modifications, e.g. proteolysis, glycosylation, and citrullination regulate chemokine function, affecting leukocyte migration during inflammatory responses. Here, modification of CXCL5/epithelial cell-derived neutrophil-activating protein-78 (ENA-78) by proteases or peptidylarginine deiminases (PAD) was evaluated. Slow CXCL5(1-78) processing by the myeloid cell marker aminopeptidase N/CD13 into CXCL5(2-78) hardly affected its in vitro activity, but slowed down the activation of CXCL5 by the neutrophil protease cathepsin G. PAD, an enzyme with a potentially important function in autoimmune diseases, site-specifically deiminated Arg(9) in CXCL5 to citrulline, generating [Cit(9)]CXCL5(1-78). Compared with CXCL5(1-78), [Cit(9)]CXCL5(1-78) less efficiently induced intracellular calcium signaling, phosphorylation of extracellular signal-regulated kinase, internalization of CXCR2, and in vitro neutrophil chemotaxis. In contrast, conversion of CXCL5 into the previously reported natural isoform CXCL5(8-78) provided at least 3-fold enhanced biological activity in these tests. Citrullination, but not NH(2)-terminal truncation, reduced the capacity of CXCL5 to up-regulate the expression of the integrin α-chain CD11b on neutrophils. Truncation nor citrullination significantly affected the ability of CXCL5 to up-regulate CD11a expression or shedding of CD62L. In line with the in vitro results, CXCL5(8-78) and CXCL5(9-78) induced a more pronounced neutrophil influx in vivo compared with CXCL5(1-78). Administration of 300 pmol of either CXCL5(1-78) or [Cit(9)]CXCL5(1-78) failed to attract neutrophils to the peritoneal cavity. Citrullination of the more potent CXCL5(9-78) lowers its chemotactic potency in vivo and confirms the tempering effect of citrullination in vitro. The highly divergent effects of modifications of CXCL5 on neutrophil influx underline the potential importance of tissue-specific interactions between chemokines and PAD or proteases.
Assuntos
Antígenos CD13/metabolismo , Catepsina G/metabolismo , Quimiocina CXCL5/metabolismo , Hidrolases/metabolismo , Neutrófilos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Antígenos CD13/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Catepsina G/genética , Linhagem Celular , Quimiocina CXCL5/genética , Quimiocina CXCL5/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Hidrolases/genética , Camundongos , Neutrófilos/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Desiminases de Arginina em Proteínas , CoelhosRESUMO
Objectives: To explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis. Methods: Latent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines. Results: All MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort. Conclusion: Synovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.
Assuntos
Artrite Reumatoide/metabolismo , Citrulinação , Metaloproteinase 9 da Matriz/metabolismo , Líquido Sinovial/metabolismo , Animais , Citrulina/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-TraducionalRESUMO
Interactions between chemokines and enzymes are vital in immunoregulation. Structural protein citrullination by peptidylarginine deiminase (PAD) has been associated with autoimmunity. In this report, we identified a novel naturally occurring posttranslational modification of chemokines, that is, the deimination of arginine at position 5 into citrulline of CXC chemokine ligand 10 (CXCL10) by rabbit PAD and human PAD2. Citrullination reduced (>/= 10-fold) the chemoattracting and signaling capacity of CXCL10 for CXC chemokine receptor 3 (CXCR3) transfectants; however, it did not affect CXCR3 binding. On T lymphocytes, though, citrullinated CXCL10 remained active but was again weaker than authentic CXCL10. PAD was also able to convert CXCL11, causing an impairment of CXCR3 signaling and T-cell activation, though less pronounced than for CXCL10. Similarly, receptor binding properties of CXCL11 were not altered by citrullination. However, deimination decreased heparin binding properties of both CXCL10 and CXCL11. Overall, chemokines are the first immune modulators reported of being functionally modified by citrullination. These data provide new structure-function dimensions for chemokines in leukocyte mobilization, disclosing an anti-inflammatory role for PAD. Additionally because citrullination has severe consequences for chemokine biology, this invites to reassess the involvement and impact of PAD and citrullinated peptides in inflammation, autoimmunity, and hematologic disorders.
Assuntos
Quimiocina CXCL10/imunologia , Quimiocina CXCL11/imunologia , Citrulina/metabolismo , Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocina CXCL10/química , Quimiocina CXCL10/isolamento & purificação , Quimiocina CXCL10/farmacologia , Quimiocina CXCL11/farmacologia , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Dados de Sequência Molecular , Fito-Hemaglutininas/farmacologia , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Coelhos , Receptores CXCR/metabolismo , Receptores CXCR3/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , TransfecçãoRESUMO
CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH2-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH2-terminal amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH2-terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.
Assuntos
Plaquetas/metabolismo , Células Endoteliais/fisiologia , Fator Plaquetário 4/metabolismo , Adulto , Idoso , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fator Plaquetário 4/genética , Fator Plaquetário 4/farmacologia , Isoformas de Proteínas , Proteínas RecombinantesRESUMO
Application of oncolytic viruses is a valuable option to broaden the armament of anticancer therapies, as these combine specific cytotoxic effects and immune-stimulating properties. The self-replicating H-1 parvovirus (H-1PV) is a prototypical oncolytic virus that, besides targeting tumor cells, also infects endothelial cells, thus combining oncolytic and angiostatic traits. To increase its therapeutic value, H-1PV can be armed with cytokines or chemokines to enhance the immunological response. Some chemokines-more specifically, the CXCR3 ligands CXCL4L1 and CXCL10-combine immune-stimulating properties with angiostatic activity. This study explores the therapeutic value of recombinant parvoviruses carrying CXCL4L1 or CXCL10 transgenes (Chi-H1/CXCL4L1 or Chi-H1/CXCL10, respectively) to inhibit the growth of the human Kaposi sarcoma cell line KS-IMM. KS-IMM cells infected by Chi-H1/CXCL4L1 or Chi-H1/CXCL10 released the corresponding chemokine and showed reduced migratory capacity. Therefore, the antitumoral capacity of Chi-H1/CXCL4L1 or Chi-H1/CXCL10 was tested in mice. Either in vitro infected KS-IMM cells were injected or subcutaneously growing KS-IMM xenografts were treated by peritumoral injections of the different viruses. Surprisingly, the transgenes did not increase the antitumoral effect of natural H-1PV. Further experiments indicated that CXCL4L1 and CXCL10 interfered with the expression of the viral NS1 protein in KS-IMM cells. These results indicate that the outcome of parvovirus-based delivery of CXCR3 ligands might be tumor cell type dependent, and hence its application must be considered carefully.
Assuntos
Quimiocina CXCL10/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Parvovirus/genética , Fator Plaquetário 4/genética , Sarcoma de Kaposi/terapia , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Sarcoma de Kaposi/irrigação sanguínea , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologiaRESUMO
Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and gelatinase B are coexpressed at sites of inflammation, where an intense interaction occurs between leukocytes and endothelial cells. To investigate whether a functional link exists between PECAM-1 activation and gelatinase B production, the regulatory role of PECAM-1, IFN-gamma, IFN-beta, LPS, and PMA on the production of gelatinase B (MMP-9) was studied in vitro in normal human umbilical vein endothelial cells (HUVECs), human peripheral blood mononuclear cells (PBMCs), and in a human monocytic leukemia cell line. In THP-1 cells, progelatinase B levels were slightly up-regulated by immobilized PECAM-1-specific monoclonal antibody (mAb) and soluble recombinant PECAM-1 when compared with strong induction by LPS and PMA. IFN-beta inhibited the induced and basal gelatinase B production but had no modulating effect on the expression of PECAM-1. HUVECs mainly produced progelatinase A (proMMP-2). Treatment with LPS and triggering of the endothelial cells with PECAM-1 mAb or recombinant PECAM-1 had no effect on gelatinase A or B production, whereas PMA stimulated the production of progelatinase B. IFN-beta significantly up-regulated the expression of PECAM-1 in HUVECs but did not affect gelatinase secretion. Finally, in PBMCs, progelatinase B production was increased by soluble PECAM-1 mAb, recombinant PECAM-1, LPS, and PMA, whereas IFN-beta reduced gelatinase B secretion. IFN-beta did not alter PECAM-1 expression on PBMCs. Thus, PECAM-1 and gelatinase B are differently regulated in leukocytes and endothelial cells.
Assuntos
Endotélio Vascular/enzimologia , Fatores Imunológicos/farmacologia , Interferon beta/farmacologia , Metaloproteinase 9 da Matriz/biossíntese , Monócitos/enzimologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Células Cultivadas , Regulação Enzimológica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/genética , Especificidade de Órgãos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Proteínas Recombinantes/farmacologia , Regulação para CimaRESUMO
The synovial cavity constitutes the ideal stage to study the interplay between microbial Toll-like receptor (TLR) ligands and cytokines. Infiltrated leukocytes and synovial fibroblasts produce cytokine- and chemokine-induced proteases for remodeling the extracellular matrix. The regulation of chemokine function for attraction and activation of leukocytes constitutes a key feature in host immunity and resolution of inflammation after infection. Enhanced levels of the CXC chemokine ligand (CXCL9)/monokine induced by interferon-gamma (IFN-gamma) and CXCL11/IFN-inducible T cell alpha chemoattractant, two chemoattractants for activated T cells and natural killer cells, and ligands for CXC chemokine receptor 3 (CXCR3) were detected in the synovial fluid of septic arthritis compared with osteo- and crystal arthritis patients. In vitro, IFN-gamma and TLR3 ligation by double-stranded RNA (dsRNA) induced the expression of CXCL9 and CXCL11 in leukocytes and skin-muscle fibroblasts, whereas ligation of TLR2, TLR4, TLR5, and TLR9 by peptidoglycan (PGN), lipopolysaccharide (LPS), flagellin, and unmethylated CpG oligonucleotides, respectively, did not. PGN and LPS, but not unmethylated CpG oligonucleotides, even inhibited IFN-gamma-induced CXCL9 and CXCL11 expression in leukocytes. In sharp contrast, in fibroblasts, the TLR ligands PGN, dsRNA, LPS, and flagellin synergized with IFN-gamma for the production of CXCL9 and CXCL11. Although TLR ligands stimulate leukocytes to produce CXCL8/interleukin-8 during the early innate defense, they contribute less to the production of CXCR3 ligands, whereas fibroblasts are important sources of CXCR3 ligands. These results illustrate the complex interaction between cytokines and TLR ligands in infection.
Assuntos
Artrite Infecciosa/metabolismo , Quimiocinas CXC/biossíntese , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Líquido Sinovial/imunologia , Artrite Infecciosa/imunologia , Artrite Infecciosa/patologia , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiocinas CXC/análise , Sinergismo Farmacológico , Endotoxinas/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Interferon gama/imunologia , Ligantes , Glicoproteínas de Membrana/imunologia , Osteoartrite/imunologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Receptores CXCR3 , Receptores de Superfície Celular/imunologia , Receptores de Quimiocinas/metabolismo , Líquido Sinovial/química , Receptor 2 Toll-Like , Receptor 3 Toll-Like , Receptor 4 Toll-Like , Receptor 5 Toll-Like , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that - after first-line in vitro assays - characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.
Assuntos
Ar , Bioensaio/métodos , Leucocitose/induzido quimicamente , Teste de Materiais/métodos , Nanopartículas/toxicidade , Testes de Toxicidade/métodos , Animais , Bioensaio/instrumentação , Teste de Materiais/instrumentação , Camundongos , Tamanho da Partícula , Poliestirenos , Testes de Toxicidade/instrumentaçãoRESUMO
BACKGROUND: Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH(2)-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others. METHODOLOGY/PRINCIPAL FINDINGS: Aminopeptidase-cleaved CXCL8(2-77) and CXCL8(3-77), the product of alternative cleavage of the signal peptide CXCL8(-2-77) and the previously studied forms containing 77 and 72 amino acids, CXCL8(1-77) and CXCL8(6-77), were prepared by solid-phase peptide synthesis, purified and folded into active proteins. No differences in binding and calcium signaling potency were detected between CXCL8(1-77), CXCL8(-2-77), CXCL8(2-77) and CXCL8(3-77) on cells transfected with one of the human CXCL8 receptors, i.e. CXCR1 and CXCR2. However, CXCL8(-2-77) was more potent compared to CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) in signaling and in vitro chemotaxis of peripheral blood-derived human neutrophils. Moreover, CXCL8(-2-77) was less efficiently processed by plasmin into the more potent CXCL8(6-77). The truncated forms CXCL8(2-77) and CXCL8(3-77) had higher affinity for heparin than CXCL8(1-77), a property important for the presentation of CXCL8 on endothelial layers. Upon intraperitoneal injection in mice, elongated, truncated and intact CXCL8 were equally potent to recruit neutrophils to the peritoneal cavity. CONCLUSIONS: In terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH(2)-terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.
Assuntos
Aminopeptidases/metabolismo , Interleucina-8/metabolismo , Interleucina-8/farmacologia , Sinais Direcionadores de Proteínas/fisiologia , Animais , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fibrinolisina/metabolismo , Heparina/metabolismo , Humanos , Interleucina-8/química , Camundongos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Ligação Proteica , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Trombina/metabolismoRESUMO
Chemokines influence tumor growth directly or indirectly via both angiogenesis and tumor-leukocyte interactions. Platelet factor-4 (CXCL4/PF-4), which is released from alpha-granules of activated platelets, is the first described angiostatic chemokine. Recently, it was found that the variant of CXCL4/PF-4 (CXCL4L1/PF-4var) could exert a more pronounced angiostatic and antitumoral effect than CXCL4/PF-4. However, the molecular mechanisms of the angiostatic activities of the PF-4 forms remain partially elusive. Here, we studied the biological properties of the chemically synthesized COOH-terminal peptides of CXCL4/PF-4 (CXCL4/PF-4(47-70)) and CXCL4L1/PF-4var (CXCL4L1/PF-4var(47-70)). Both PF-4 peptides lacked monocyte and lymphocyte chemotactic activity but equally well inhibited (25 nmol/L) endothelial cell motility and proliferation in the presence of a single stimulus (i.e., exogenous recombinant fibroblast growth factor-2). In contrast, when assayed in more complex angiogenesis test systems characterized by the presence of multiple mediators, including in vitro wound-healing (2.5 nmol/L versus 12.5 nmol/L), Matrigel (60 nmol/L versus 300 nmol/L), and chorioallantoic membrane assays, CXCL4L1/PF-4var(47-70) was found to be significantly (5-fold) more angiostatic than CXCL4/PF-4(47-70). In addition, low (7 microg total) doses of intratumoral CXCL4L1/PF-4var(47-70) inhibited B16 melanoma growth in mice more extensively than CXCL4/PF-4(47-70). This antitumoral activity was predominantly mediated through inhibition of angiogenesis (without affecting blood vessel stability) and induction of apoptosis, as evidenced by immunohistochemical and fluorescent staining of B16 tumor tissue. In conclusion, CXCL4L1/PF-4var(47-70) is a potent antitumoral and antiangiogenic peptide. These results may represent the basis for the design of CXCL4L1/PF-4var COOH-terminal-derived peptidomimetic anticancer drugs.
Assuntos
Proteínas Angiostáticas/farmacologia , Antineoplásicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Fator Plaquetário 4/farmacologia , Proteínas Angiostáticas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Bioensaio , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Modelos Animais de Doenças , Humanos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neovascularização Patológica/fisiopatologia , Neovascularização Patológica/prevenção & controle , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fator Plaquetário 4/agonistas , Fator Plaquetário 4/síntese química , Fator Plaquetário 4/químicaRESUMO
Biological functions of proteins are influenced by posttranslational modifications such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttranslational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1-77), generating CXCL8(1-77)Cit(5). Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD efficiently and site-specifically citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1beta. In comparison with CXCL8(1-77), CXCL8(1-77)Cit(5) had reduced affinity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1-77), CXCL8(1-77)Cit(5) was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6-77). Upon intraperitoneal injection, CXCL8(6-77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1-77). Despite its retained chemotactic activity in vitro, CXCL8(1-77)Cit(5) was unable to attract neutrophils to the peritoneum. Finally, in the rabbit cornea angiogenesis assay, the equally potent CXCL8(1-77) and CXCL8(1-77)Cit(5) were less efficient angiogenic molecules than CXCL8(6-77). This study shows that PAD citrullinates the chemokine CXCL8, and thus may dampen neutrophil extravasation during acute or chronic inflammation.
Assuntos
Citrulina/química , Hidrolases/metabolismo , Interleucina-8/metabolismo , Animais , Arginina , Doenças Autoimunes , Córnea/metabolismo , Glicosilação , Humanos , Inflamação , Neovascularização Fisiológica , Fosforilação , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em Proteínas , CoelhosRESUMO
CXCR3 ligands were secreted by tissue fibroblasts and peripheral blood-derived mononuclear leukocytes in response to interferon-gamma (IFN-gamma) and Toll-like receptor (TLR) ligands. Subsequent purification and identification revealed the presence of truncated CXCL11 variants missing up to 6 amino acids. In combination with CD26/dipeptidyl peptidase IV, the metalloprotease aminopeptidase N (APN), identical to the myeloid cell marker CD13, rapidly processed CXCL11, but not CXCL8, to generate truncated CXCL11 forms. Truncated CXCL11 had reduced binding, signaling, and chemotactic properties for lymphocytes and CXCR3- or CXCR7-transfected cells. CD13/APN-truncated CXCL11 failed to induce an intracellular calcium increase but was still able to bind and desensitize CXCR3 for intact CXCL11 signaling. CXCL11 efficiently bound to CXCR7, but CXCL11 was not able to induce calcium signaling or ERK1/2 or Akt phosphorylation through CXCR7. CD26-truncated CXCL11 failed to attract lymphocytes but still inhibited microvascular endothelial cell (HMVEC) migration. However, further processing of CXCL11 by CD13 resulted in significant reduction of inhibition of HMVEC migration. Taken together, during inflammation or cancer, CXCL11 processing by CD13 may lead to a reduced number of tumor-infiltrating lymphocytes and in a more angiogenic environment.
Assuntos
Antígenos CD13/metabolismo , Movimento Celular , Quimiocinas CXC/metabolismo , Linfócitos/citologia , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Quimiocina CXCL11 , Quimiocinas CXC/genética , Células Endoteliais/citologia , Variação Genética , Humanos , Inflamação/patologia , Linfócitos do Interstício Tumoral/patologia , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Receptores CXCR , Receptores CXCR3 , Transdução de SinaisRESUMO
CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor 3 (CXCR3) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR3 or TLR4 ligands, respectively) synergized with interferon-gamma (IFN-gamma) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-gamma was combined with TLR3 ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-gamma-dependent CXCR3 ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), in combination with IFN-gamma, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG(1)Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-alpha plus IFN-gamma, but not synergy between IFN-gamma and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the physiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.
Assuntos
Artrite Psoriásica/metabolismo , Artrite Reumatoide/metabolismo , Quimiocinas CXC/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Toll-Like/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias , Antígenos Virais , Células Cultivadas , Quimiocinas CXC/imunologia , Ilhas de CpG , Células Endoteliais , Feminino , Fibroblastos/metabolismo , Humanos , Interferon gama , Interleucina-1 , Ligantes , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos , Receptores CXCR3 , Líquido Sinovial/metabolismo , Fator de Necrose Tumoral alfaRESUMO
Leukocyte infiltration during acute and chronic inflammation is regulated by exogenous and endogenous factors, including cytokines, chemokines and proteases. Stimulation of fibroblasts and human microvascular endothelial cells with the inflammatory cytokines interleukin-1beta (IL-1beta) or tumour necrosis factor alpha (TNF-alpha) combined with either interferon-alpha (IFN-alpha), IFN-beta or IFN-gamma resulted in a synergistic induction of the CXC chemokine CXCL10, but not of the neutrophil chemoattractant CXCL8. In contrast, simultaneous stimulation with different IFN types did not result in a synergistic CXCL10 protein induction. Purification of natural CXCL10 from the conditioned medium of fibroblasts led to the isolation of CD26/dipeptidyl peptidase IV-processed CXCL10 missing two NH2-terminal residues. In contrast to intact CXCL10, NH2-terminally truncated CXCL10(3-77) did not induce extracellular signal-regulated kinase 1/2 or Akt/protein kinase B phosphorylation in CXC chemokine receptor 3-transfected cells. Together with the expression of CXCL10, the expression of membrane-bound CD26/dipeptidyl peptidase IV was also upregulated in fibroblasts by IFN-gamma, by IFN-gamma plus IL-1beta or by IFN-gamma plus TNF-alpha. This provides a negative feedback for CXCL10-dependent chemotaxis of activated T cells and natural killer cells. Since TNF-alpha and IL-1beta are implicated in arthritis, synovial concentrations of CXCL8 and CXCL10 were compared in patients suffering from crystal arthritis, ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. All three groups of autoimmune arthritis patients (ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis) had significantly increased synovial CXCL10 levels compared with crystal arthritis patients. In contrast, compared with crystal arthritis, only rheumatoid arthritis patients, and not ankylosing spondylitis or psoriatic arthritis patients, had significantly higher synovial CXCL8 concentrations. Synovial concentrations of the neutrophil chemoattractant CXCL8 may therefore be useful to discriminate between autoimmune arthritis types.
Assuntos
Quimiocinas CXC/metabolismo , Citocinas/farmacologia , Dipeptidil Peptidase 4/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Mediadores da Inflamação/farmacologia , Doenças Autoimunes/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/química , Derme/irrigação sanguínea , Combinação de Medicamentos , Humanos , Recém-Nascido , Interleucina-8/metabolismo , Artropatias/metabolismo , Ligantes , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Receptores CXCR3 , Receptores de Quimiocinas/metabolismo , Doenças Reumáticas/metabolismo , Transdução de SinaisRESUMO
Parenteral administration of interferon (IFN)-beta is one of the currently approved therapies for multiple sclerosis. One characteristic of this disease is the increased production of gelatinase B, also called matrix metalloproteinase (MMP) 9. Gelatinase B is capable of destroying the blood-brain barrier, and of cleaving myelin basic protein into immunodominant and encephalitogenic fragments, thus playing a functional role and being a therapeutic target in multiple sclerosis. Here we demonstrate that gelatinase B proteolytically cleaves IFN-beta, kills its activity, and hence counteracts this cytokine as an antiviral and immunotherapeutic agent. This proteolysis is more pronounced with IFN-beta-1b than with IFN-beta-1a. Furthermore, the tetracycline minocycline, which has a known blocking effect in experimental autoimmune encephalomyelitis, an in vivo model of acute inflammation in multiple sclerosis, and other MMP inhibitors prevent the in vitro degradation of IFN-beta by gelatinase B. These data provide a novel mechanism and rationale for the inhibition of gelatinase B in diseases in which IFN-beta has a beneficial effect. The combination of gelatinase B inhibitors with better and lower pharmacological formulations of IFN-beta may reduce the side-effects of treatment with IFN-beta, and is therefore proposed for multiple sclerosis therapy and the immunotherapy of viral infections.