RESUMO
A natural leukocyte chemoattractant was isolated from bovine serum by an established 4-step purification procedure. Based on its relative molecular mass of 7287 and NH2-terminal sequence, the protein was identified as a carboxy-terminal peptide of the acute phase protein serum amyloid A1 (SAA1). This SAA1(46-112) fragment and its human equivalent SAA1(47-104) were chemically synthesized. Unlike intact SAA1α, these SAA fragments failed to directly chemoattract neutrophils and monocytes, to induce chemokines, and to stimulate downstream extracellular signal-regulated kinase signaling in monocytes. However, the SAA fragments potently synergized with CCL3 to induce monocyte migration and with CXCL8 to stimulate neutrophil shape changes and chemotaxis. Unlike intact SAA1α, SAA1(46-112) did not induce CXCL6 ex vivo but provoked a cooperative intraperitoneal neutrophil recruitment in mice when coinjected with CXCL6 into the peritoneal cavity. Moreover, SAA1(47-104) desensitized the synergy between intact SAA1α and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds formyl peptide receptor 2 (FPR2). This was evidenced by a complete blockade of synergy between the COOH-terminal SAA1 fragments and CXCL8 or CCL3 in neutrophil and monocyte chemotaxis, respectively, by the FPR2 antagonist WRW4 Thus, SAA1 is degraded into fragments lacking chemokine-inducing capacity, while keeping synergy with cytokine-induced chemokines to sustain limited inflammation.
Assuntos
Quimiocina CCL3/imunologia , Quimiocinas/imunologia , Interleucina-8/imunologia , Leucócitos/efeitos dos fármacos , Receptores de Formil Peptídeo/imunologia , Receptores de Lipoxinas/imunologia , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/farmacologia , Animais , Bovinos , Quimiotaxia/efeitos dos fármacos , Feminino , Humanos , Leucócitos/imunologia , Camundongos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Proteína Amiloide A Sérica/síntese químicaRESUMO
The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.
Assuntos
Carcinoma Pulmonar de Lewis/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animais , Carcinoma Pulmonar de Lewis/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Quimiocina CXCL6/metabolismo , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Citometria de Fluxo , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Lipoproteínas/metabolismo , Camundongos , Células RAW 264.7 , Proteína Amiloide A Sérica/genéticaRESUMO
Serum amyloid A (SAA) is an acute phase protein that is upregulated in inflammatory diseases and chemoattracts monocytes, lymphocytes, and granulocytes via its G protein-coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPRL1/FPR2). Here, we demonstrated that the SAA1α isoform also chemoattracts monocyte-derived immature dendritic cells (DCs) in the Boyden and µ-slide chemotaxis assay and that its chemotactic activity for monocytes and DCs was indirectly mediated via rapid chemokine induction. Indeed, SAA1 induced significant amounts (≥5 ng/mL) of macrophage inflammatory protein-1α/CC chemokine ligand 3 (MIP-1α/CCL3) and interleukin-8/CXC chemokine ligand 8 (IL-8/CXCL8) in monocytes and DCs in a dose-dependent manner within 3 h. However, SAA1 also directly activated monocytes and DCs for signaling and chemotaxis without chemokine interference. SAA1-induced monocyte migration was nevertheless significantly prevented (60-80% inhibition) in the constant presence of desensitizing exogenous MIP-1α/CCL3, neutralizing anti-MIP-1α/CCL3 antibody, or a combination of CC chemokine receptor 1 (CCR1) and CCR5 antagonists, indicating that this endogenously produced CC chemokine was indirectly contributing to SAA1-mediated chemotaxis. Further, anti-IL-8/CXCL8 antibody neutralized SAA1-induced monocyte migration, suggesting that endogenous IL-8/CXCL8 acted in concert with MIP-1α/CCL3. This explained why SAA1 failed to synergize with exogenously added MIP-1α/CCL3 or stromal cell-derived factor-1α (SDF-1α)/CXCL12 in monocyte and DC chemotaxis. In addition to direct leukocyte activation, SAA1 induces a chemotactic cascade mediated by expression of cooperating chemokines to prolong leukocyte recruitment to the inflammatory site.
Assuntos
Quimiocina CCL3/imunologia , Células Dendríticas/efeitos dos fármacos , Interleucina-8/imunologia , Monócitos/efeitos dos fármacos , Proteína Amiloide A Sérica/farmacologia , Anticorpos Neutralizantes/farmacologia , Linhagem Celular , Quimiocina CCL3/antagonistas & inibidores , Quimiocina CCL3/genética , Quimiocina CXCL12/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Cultura em Câmaras de Difusão , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica , Humanos , Interleucina-8/agonistas , Interleucina-8/antagonistas & inibidores , Interleucina-8/genética , Monócitos/citologia , Monócitos/imunologia , Cultura Primária de Células , Receptores CCR1/antagonistas & inibidores , Receptores CCR1/genética , Receptores CCR1/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
Infection, sterile injury, and chronic inflammation trigger the acute phase response in order to re-establish homeostasis. This response includes production of positive acute phase proteins in the liver, such as members of the serum amyloid A (SAA) family. In humans the major acute phase SAAs comprise a group of closely related variants of SAA1 and SAA2. SAA1 was proven to be chemotactic for several leukocyte subtypes through activation of the G protein-coupled receptor FPRL1/FPR2. Several other biological activities of SAA1, such as cytokine induction, reported to be mediated via TLRs, have been debated recently. Especially commercial SAA1, recombinantly produced in Escherichia coli, was found to be contaminated with bacterial products confounding biological assays performed with this rSAA1. We purified rSAA1 by RP-HPLC to homogeneity, removing contaminants such as lipopolysaccharides, lipoproteins and formylated peptides, and re-assessed several biological activities attributed to SAA1 (chemotaxis, cytokine induction, MMP-9 release, ROS generation, and macrophage differentiation). The homogeneous rSAA1 (hrSAA1) lacked most cell-activating properties, but its leukocyte-recruiting capacity in vivo and it's in vitro synergy with other leukocyte attractants remained preserved. Furthermore, hrSAA1 maintained the ability to promote monocyte survival. This indicates that pure hrSAA1 retains its potential to activate FPR2, whereas TLR-mediated effects seem to be related to traces of bacterial TLR ligands in the E. coli-produced human rSAA1.
Assuntos
Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Proteína Amiloide A Sérica/farmacologia , Doadores de Sangue , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Citocinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , TransfecçãoRESUMO
Chronic hepatitis C virus (HCV) infection accounts for a large proportion of hepatic fibrosis and carcinoma cases observed worldwide. Mechanisms involved in HCV-induced hepatic injury have yet to be fully elucidated. Of particular interest is the capacity of HCV to regulate inflammatory responses. Here, we reveal modulation of cytokine activity by the HCV proteins non-structural protein 3 (NS3), glycoprotein E2, and core protein for their ability to induce chemokine expression in various liver bystander cells. Chemokines sustain chronic liver inflammation and relay multiple fibrogenic effects. CCL2, CCL3, CCL20, CXCL8, and CXCL10 were differentially expressed after treatment of monocytes, fibroblasts, or liver sinusoidal microvascular endothelial cells (LSECs) with HCV proteins. In comparison to NS3 and glycoprotein E2, core protein was a stronger inducer of chemokines in liver bystander cells. Interferon-γ (IFN-γ) and interleukin-1ß (IL-1ß) synergized with core protein to induce CCL2, CCL20, CXCL8, or CXCL10 in fibroblasts or LSECs. These findings reveal new mechanisms of hepatic injury caused by HCV.
Assuntos
Quimiocinas/metabolismo , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células Cultivadas , Quimiocinas/genética , Hepacivirus/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologiaRESUMO
During an inflammatory response, a large number of distinct mediators appears in the affected tissues or in the blood circulation. These include acute phase proteins such as serum amyloid A (SAA), cytokines and chemokines and proteolytic enzymes. Although these molecules are generated within a cascade sequence in specific body compartments allowing for independent action, their co-appearance in space and time during acute or chronic inflammation points toward important mutual interactions. Pathogen-associated molecular patterns lead to fast induction of the pro-inflammatory endogenous pyrogens, which are evoking the acute phase response. Interleukin-1, tumor necrosis factor-α and interferons simultaneously trigger different cell types, including leukocytes, endothelial cells and fibroblasts for tissue-specific or systemic production of chemokines and matrix metalloproteinases (MMPs). In addition, SAA induces chemokines and both stimulate secretion of MMPs from multiple cell types. As a consequence, these mediators may cooperate to enhance the inflammatory response. Indeed, SAA synergizes with chemokines to increase chemoattraction of monocytes and granulocytes. On the other hand, MMPs post-translationally modify chemokines and SAA to reduce their activity. Indeed, MMPs internally cleave SAA with loss of its cytokine-inducing and direct chemotactic potential whilst retaining its capacity to synergize with chemokines in leukocyte migration. Finally, MMPs truncate chemokines at their NH2- or COOH-terminal end, resulting in reduced or enhanced chemotactic activity. Therefore, the complex interactions between chemokines, SAA and MMPs either maintain or dampen the inflammatory response.
Assuntos
Quimiocinas/metabolismo , Inflamação/metabolismo , Metaloproteinases da Matriz/metabolismo , Proteína Amiloide A Sérica/metabolismo , Transdução de Sinais/imunologia , Animais , Quimiotaxia/imunologia , Citocinas/metabolismo , Espaço Extracelular/metabolismo , HumanosRESUMO
Expression of the acute phase protein serum amyloid A (SAA) is dependent on the release of the pro-inflammatory cytokines IL-1, IL-6 and TNF-α during infection and inflammation. Hepatitis C virus (HCV) upregulates SAA-inducing cytokines. In line with this, a segment of chronically infected individuals display increased circulating levels of SAA. SAA has even been proposed to be a potential biomarker to evaluate treatment efficiency and the course of disease. SAA possesses antiviral activity against HCV via direct interaction with the viral particle, but might also divert infectivity through its function as an apolipoprotein. On the other hand, SAA shares inflammatory and angiogenic activity with chemotactic cytokines by activating the G protein-coupled receptor, formyl peptide receptor 2. These latter properties might promote chronic inflammation and hepatic injury. Indeed, up to 80 % of infected individuals develop chronic disease because they cannot completely clear the infection, due to diversion of the immune response. In this review, we summarize the interconnection between SAA and cytokines in the context of HCV infection and highlight the dual role SAA could play in this disease. Nevertheless, more research is needed to establish whether the balance between those opposing activities can be tilted in favor of the host defense.
Assuntos
Citocinas/imunologia , Hepatite C/imunologia , Hepatite C/fisiopatologia , Proteína Amiloide A Sérica/imunologia , Animais , Citocinas/genética , Hepacivirus , Humanos , Inflamação , Fígado/imunologia , Fígado/patologia , Camundongos , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/imunologia , Receptores de Lipoxinas/genética , Receptores de Lipoxinas/imunologia , Proteína Amiloide A Sérica/análiseRESUMO
Serum amyloid A1 (SAA1) is a prototypic acute phase protein, induced to extremely high levels by physical insults, including inflammation and infection. Human SAA and its NH2-terminal part have been studied extensively in the context of amyloidosis. By contrast, little is known about COOH-terminal fragments of SAA. Intact SAA1 chemoattracts leukocytes via the G protein-coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPR2). In addition to direct leukocyte activation, SAA1 induces chemokine production by signaling through toll-like receptor 2. We recently discovered that these induced chemokines synergize with intact SAA1 to chemoattract leukocytes in vitro and in vivo. Gelatinase B or matrix metalloproteinase-9 (MMP-9) is also induced by SAA1 during infection and inflammation and processes many substrates in the immune system. We demonstrate here that MMP-9 rapidly cleaves SAA1 at a known consensus sequence that is also present in gelatins. Processing of SAA1 by MMP-9 at an accessible loop between two alpha helices yielded predominantly three COOH-terminal fragments: SAA1(52-104), SAA1(57-104), and SAA1(58-104), with a relative molecular mass of 5,884.4, 5,327.3, and 5,256.3, respectively. To investigate the effect of proteolytic processing on the biological activity of SAA1, we chemically synthesized the COOH-terminal SAA fragments SAA1(52-104) and SAA1(58-104) and the complementary NH2-terminal peptide SAA1(1-51). In contrast to intact SAA1, the synthesized SAA1 peptides did not induce interleukin-8/CXCL8 in monocytes or fibroblasts. Moreover, these fragments possessed no direct chemotactic activity for neutrophils, as observed for intact SAA1. However, comparable to intact SAA1, SAA1(58-104) cooperated with CXCL8 in neutrophil activation and migration, whereas SAA1(1-51) lacked this potentiating activity. This cooperative interaction between the COOH-terminal SAA1 fragment and CXCL8 in neutrophil chemotaxis was mediated by FPR2. Hence, proteolytic cleavage of SAA1 by MMP-9 fine tunes the inflammatory capacity of this acute phase protein in that only the synergistic interactions with chemokines remain to prolong the duration of inflammation.
Assuntos
Quimiotaxia/imunologia , Citocinas/metabolismo , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animais , Células Cultivadas , Fibroblastos , Humanos , Metaloproteinase 9 da Matriz/química , Monócitos/imunologia , Monócitos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes , Proteína Amiloide A Sérica/químicaRESUMO
Serum amyloid A (SAA) is, like C-reactive protein (CRP), an acute phase protein and can be used as a diagnostic, prognostic or therapy follow-up marker for many diseases. Increases in serum levels of SAA are triggered by physical insults to the host, including infection, trauma, inflammatory reactions and cancer. The order of magnitude of increase in SAA levels varies considerably, from a 10- to 100- fold during limited inflammatory events to a 1000-fold increase during severe bacterial infections and acute exacerbations of chronic inflammatory diseases. This broad response range is reflected by SAA gene duplications resulting in a cluster encoding several SAA variants and by multiple biological functions of SAA. SAA variants are single-domain proteins with simple structures and few post-translational modifications. SAA1 and SAA2 are inducible by inflammatory cytokines, whereas SAA4 is constitutively produced. We review here the regulated expression of SAA in normal and transformed cells and compare its serum levels in various disease states. At low concentrations (10-100 ng/ml), early in an inflammatory response, SAA induces chemokines or matrix degrading enzymes via Toll-like receptors and functions as an activator and chemoattractant through a G protein-coupled receptor. When an infectious or inflammatory stimulus persists, the liver continues to produce more SAA (≥ 1000 ng/ml) to become an antimicrobial agent by functioning as a direct opsonin of bacteria or by interference with virus infection of host cells. Thus, SAA regulates innate and adaptive immunity and this information may help to design better drugs to treat specific diseases.
Assuntos
Variação Estrutural do Genoma/genética , Proteína Amiloide A Sérica/genética , Humanos , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismoRESUMO
Levels of serum amyloid A (SAA), a major acute phase protein in humans, are increased up to 1000-fold upon infection, trauma, cancer or other inflammatory events. However, the exact role of SAA in host defense is yet not fully understood. Several pro- and anti-inflammatory properties have been ascribed to SAA. Here, the regulated production of SAA by cytokines and glucocorticoids is discussed first. Secondly, the cytokine and chemokine inducing capacity of SAA and its receptor usage are reviewed. Thirdly, the direct (via FPR2) and indirect (via TLR2) chemotactic effects of SAA and its synergy with chemokines are unraveled. Altogether, a complex cytokine-SAA-chemokine network is established, in which SAA plays a key role in regulating the inflammatory response.
Assuntos
Citocinas/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animais , Humanos , Fígado/metabolismo , Transdução de SinaisRESUMO
Cell migration depends on the ability of leukocytes to sense an external gradient of chemotactic proteins produced during inflammation. These proteins include chemokines, complement factors, and some acute phase proteins, such as serum amyloid A. Serum amyloid A chemoattracts neutrophils, monocytes, and T lymphocytes via its G protein-coupled receptor formyl peptide receptor 2. We demonstrate that serum amyloid A1α more potently chemoattracts neutrophils in vivo than in vitro. In contrast to CD14(+) monocytes, no rapid (within 2 h) induction of interleukin-8/CXC chemokine ligand 8 or macrophage-inflammatory protein-1α/CC chemokine ligand 3 was observed in purified human neutrophils after stimulation of the cells with serum amyloid A1α or lipopolysaccharide. Moreover, interleukin-8/CXC chemokine ligand 8 induction in monocytes by serum amyloid A1α was mediated by toll-like receptor 2 and was inhibited by association of serum amyloid A1α with high density lipoprotein. This indicates that the potent chemotactic response of neutrophils toward intraperitoneally injected serum amyloid A1α is indirectly enhanced by rapid induction of chemokines in peritoneal cells, synergizing in a paracrine manner with serum amyloid A1α. We observed direct synergy between IL-8/CXC chemokine ligand 8 and serum amyloid A1α, but not lipopolysaccharide, in chemotaxis and shape change assays with neutrophils. Furthermore, the selective CXC chemokine receptor 2 and formyl peptide receptor 2 antagonists, SB225002 and WRW4, respectively, blocked the synergy between IL-8/CXC chemokine ligand 8 and serum amyloid A1α in neutrophil chemotaxis in vitro, indicating that for synergy their corresponding G protein-coupled receptors are required. Additionally, SB225002 significantly inhibited serum amyloid A1α-mediated peritoneal neutrophil influx. Taken together, endogenous (e.g., IL-1ß) and exogenous (e.g., lipopolysaccharide) inflammatory mediators induce primary chemoattractants such as serum amyloid A that synergize in an autocrine (monocyte) or a paracrine (neutrophil) fashion with secondary chemokines induced in stromal cells.
Assuntos
Quimiotaxia de Leucócito/imunologia , Interleucina-8/imunologia , Neutrófilos/imunologia , Comunicação Parácrina/imunologia , Receptores de Formil Peptídeo/imunologia , Receptores de Interleucina-8B/imunologia , Receptores de Lipoxinas/imunologia , Proteína Amiloide A Sérica/imunologia , Receptor 2 Toll-Like/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Feminino , Humanos , Masculino , Comunicação Parácrina/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/antagonistas & inibidoresRESUMO
Bovine serum is a rich source of cytokines and growth factors supporting in vitro cell culture. Here, a novel bovine monocyte chemotactic factor (boMCF-1) was isolated from commercial bovine serum by a four step purification procedure including adsorption to silicic acid, heparin affinity and cation-exchange chromatography and reversed phase HPLC. Homogeneous boMCF-1 was characterized as a 7717Da protein by mass spectrometry and identified by Edman degradation as the predicted product of bovine macrophage inflammatory protein-1α gene (boMIP-1α/CCL3) isoform 2 (lacking three NH2-terminal amino acids), belonging to the MIP subfamily of CC chemokines. Monocyte chemotactic activity of boCCL3 isoform 2 was completely desensitized by human CCL3 and CCL5, partially by CCL7 and not by CCL2. Its activity was better inhibited by CCR1 than by CCR5 blockade. BoCCL3 isoform 2, present in bovine serum at about 10 ng/ml, functioned as a most potent chemoattractant for immature (but not mature) dendritic cells with a minimal effective concentration of 0.03 ng/ml and a maximal chemotactic index of 30 at 0.3 ng/ml. Its chemotactic activity on immature dendritic cells was significantly desensitized by human CCL3, CCL5 and CCL7. Blockade of CCR5 rather than CCR1 partially prevented chemotactic activity, whereas blockade of both further enhanced this inhibition. BoCCL3 isoform 2 was not chemokinetic but, like human CCL3, synergized with CXCL12 in monocytic cell chemotaxis. Since it cannot be deduced which is the exact human homolog of boCCL3 isoform 2, further research is required to study the biology of other boCCL3 family members.