RESUMO
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
Assuntos
Armadilhas Extracelulares/metabolismo , Neoplasias Experimentais/terapia , Receptores de Quimiocinas/agonistas , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Células HT29 , Humanos , Microscopia Intravital/métodos , Células Matadoras Naturais/imunologia , Ligantes , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/imunologia , Receptores de Interleucina-8B/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Immune suppression in the tumor microenvironment (TME) is an intractable issue in anti-cancer immunotherapy. The chemokine receptors CXCR1 and CXCR2 recruit immune suppressive cells such as the myeloid derived suppressor cells (MDSCs) to the TME. Therefore, CXCR1/2 antagonists have aroused pharmaceutical interest in recent years. In this review, the medicinal chemistry of CXCR1/2 antagonists and their relevance in cancer immunotherapy have been summarized. The development of the drug candidates, along with their design rationale, clinical status and current challenges have also been discussed.
Assuntos
Fatores Imunológicos/farmacologia , Imunoterapia , Neoplasias/terapia , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Animais , Relação Dose-Resposta a Droga , Humanos , Fatores Imunológicos/química , Estrutura Molecular , Neoplasias/imunologia , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8B/imunologia , Relação Estrutura-AtividadeRESUMO
Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.
Assuntos
Endotélio Vascular/virologia , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Herpesvirus Humano 8/fisiologia , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , RNA Viral/metabolismo , Internalização do Vírus , Movimento Celular , Células Cultivadas , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Repressão Enzimática , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Deleção de Genes , Herpesvirus Humano 8/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Mutação , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA/metabolismo , Interferência de RNA , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Transdução de SinaisRESUMO
The CXCL1/CXCR2 axis plays a crucial role in recruiting neutrophils in response to microbial infection and tissue injury, and dysfunction in this process has been implicated in various inflammatory diseases. Chemokines exist as monomers and dimers, and compelling evidence now exists that both forms regulate in vivo function. Therefore, knowledge of the receptor activities of both CXCL1 monomer and dimer is essential to describe the molecular mechanisms by which they orchestrate neutrophil function. The monomer-dimer equilibrium constant (~20 µm) and the CXCR2 binding constant (1 nm) indicate that WT CXCL1 is active as a monomer. To characterize dimer activity, we generated a trapped dimer by introducing a disulfide across the dimer interface. This disulfide-linked CXCL1 dimer binds CXCR2 with nanomolar affinity and shows potent agonist activity in various cellular assays. We also compared the receptor binding mechanism of this dimer with that of a CXCL1 monomer, generated by deleting the C-terminal residues that stabilize the dimer interface. We observe that the binding interactions of the dimer and monomer to the CXCR2 N-terminal domain, which plays an important role in determining affinity and activity, are essentially conserved. The potent activity of the CXCL1 dimer is novel: dimers of the CC chemokines CCL2 and CCL4 are inactive, and the dimer of the CXC chemokine CXCL8 (which is closely related to CXCL1) is marginally active for CXCR1 but shows variable activity for CXCR2. We conclude that large differences in dimer activity among different chemokine-receptor pairs have evolved for fine-tuned leukocyte function.
Assuntos
Quimiocina CXCL1/metabolismo , Leucócitos/metabolismo , Multimerização Proteica/fisiologia , Receptores de Interleucina-8B/agonistas , Quimiocina CXCL1/química , Quimiocina CXCL1/genética , Células HL-60 , Humanos , Leucócitos/citologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
The innate immune system is a crucial component of inflammatory reactions, while the central nervous system (CNS) is the most vulnerable site of the body to inflammatory tissue injury. Neuroinflammatory brain pathologies are disorders in which the CNS is threatened by its own immune system. Chemokine receptor CXCR2 and its ligands have been implicated in several neuroinflammatory brain pathologies, as well as in neutrophil recruitment and in the developmental positioning of neural cells. This review focuses on the basics of CXCR2, its regulating role in bone marrow neutrophil recruitment, oligodendrocyte progenitor cell positioning and neural repair mechanisms, as well as its diverse roles in neuroinflammatory brain pathologies.
Assuntos
Inflamação/imunologia , Inflamação/patologia , Neurônios/imunologia , Neurônios/patologia , Receptores de Interleucina-8B/fisiologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Humanos , Inflamação/etiologia , Neurônios/metabolismo , Infiltração de Neutrófilos/imunologia , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/uso terapêuticoRESUMO
This study evaluated the relevance of CXCR2 chemokine receptors in oral squamous cell carcinoma, by means of in vitro and in vivo approaches. The in vitro incubation of the selective and non-peptide CXCR2 receptor antagonist N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl) Urea (SB225002; 25 to 800 nM) produced a time- and concentration-dependent inhibition of SCC158 (rat) and HN30 (human) cell lines viability. Conversely, this antagonist did not significantly affect the viability of the immortalized keratinocyte lineage, HaCaT. Additionally, the incubation of human IL-8 and rat CINC-1 CXCR2 agonists produced a concentration-related increase on HN30 and SCC158 proliferation. The submucosal injection of SCC158 cells (5 × 10(6) cells) into the tongue of Fischer 344 rats induced tumor development, which displayed typical clinical features. Immunohistochemical analysis of rat tongue biopsies revealed a marked increase of CXCR2 receptor immunoreactivity, which was accompanied by augumented expression of VEGF and caspase-3. Our data suggests an important role for CXCR2 receptors in oral squamous cell carcinoma.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Receptores de Interleucina-8B/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Masculino , Neoplasias Bucais/patologia , Compostos de Fenilureia/farmacologia , Ratos , Ratos Endogâmicos F344 , Receptores de Interleucina-8B/agonistasRESUMO
To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria-derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo-monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td-RFP611 (Arr-td-RFP611) or enhanced green fluorescent protein (EGFP; Arr-EGFP), were found to colocalize with internalized fluorescently labeled Gro-alpha a few minutes after Gro-alpha addition. Intriguingly, however, Arr-td-RFP611 and Arr-EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2-activating ligand. Under these conditions, Arr-td-RFP611 showed a largely homogeneous cytosolic distribution, whereas Arr-EGFP segregated, to a large degree, into granular spots. These observations indicate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr-EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC(50) value similar to that observed for Arr-EGFP redistribution. Thus, the redistribution assay, when based on Arr-EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr-td-RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism.
Assuntos
Arrestina/metabolismo , Benzamidas/farmacologia , Ciclobutanos/farmacologia , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Arrestina/genética , Bovinos , Linhagem Celular , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Endocitose/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Interleucina-8B/genética , Proteínas Recombinantes de Fusão/genética , Proteína Vermelha FluorescenteRESUMO
We reported that PARP-1 exhibits differential roles in expression of inflammatory factors. Here, we show that PARP-1 deletion was associated with a significant reduction in inflammatory cell recruitment to mouse airways upon intratracheal administration of LPS. However, PARP-1 deletion exerted little effect in response to TNF exposure. LPS induced massive neutrophilia and moderate recruitment of macrophages, and TNF induced recruitment of primarily macrophages with smaller numbers of neutrophils in the lungs. Following either exposure, macrophage recruitment was blocked severely in PARP-1(-/-) mice, and this was associated with a marked reduction in MCP-1 and MIP-1alpha. This association was corroborated partly by macrophage recruitment in response to intratracheal administration of MCP-1 in PARP-1(-/-) mice. Surprisingly, although neutrophil recruitment was reduced significantly in LPS-treated PARP-1(-/-) mice, neutrophil numbers increased in TNF-treated mice, suggesting that PARP-1 deletion may promote a macrophagic-to-neutrophilic shift in the inflammatory response upon TNF exposure. Neutrophil-specific chemokines mKC and MIP-2 were reduced significantly in lungs of LPS-treated but only partially reduced in TNF-treated PARP-1(-/-) mice. Furthermore, the MIP-2 antagonist abrogated the shift to a neutrophilic response in TNF-exposed PARP-1(-/-) mice. Although CXCR2 expression increased in response to either stimulus in PARP-1(+/+) mice, the DARC increased only in lungs of TNF-treated PARP-1(+/+) mice; both receptors were reduced to basal levels in treated PARP-1(-/-) mice. Our results show that the balance of pro-neutrophilic or pro-macrophagic stimulatory factors and the differential influence of PARP-1 on these factors are critical determinants for the nature of the airway inflammatory response.
Assuntos
Movimento Celular , Quimiocina CXCL2 , Sistema do Grupo Sanguíneo Duffy , Lipopolissacarídeos , Pulmão , Macrófagos Alveolares , Neutrófilos , Poli(ADP-Ribose) Polimerases , Receptores de Superfície Celular , Receptores de Interleucina-8B , Fator de Necrose Tumoral alfa , Animais , Camundongos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/imunologia , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Quimiocina CXCL2/genética , Quimiocina CXCL2/imunologia , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/imunologia , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/imunologia , Traqueia/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND AND PURPOSE: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. EXPERIMENTAL APPROACH: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [(35)S]-GTPgammaS binding approaches in addition to chemotaxis of human neutrophils. KEY RESULTS: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [(125)I]-interleukin-8 ([(125)I]-IL-8) without affecting the K(d). In contrast, IL-8 was unable to prevent binding of [(3)H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene alpha, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [(35)S]-GTPgammaS binding in this preparation. CONCLUSIONS AND IMPLICATIONS: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling.
Assuntos
Agonismo Inverso de Drogas , Compostos de Fenilureia/farmacologia , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/antagonistas & inibidores , Triazóis/farmacologia , Regulação Alostérica/fisiologia , Animais , Células CHO , Quimiocinas/química , Quimiocinas/metabolismo , Cricetinae , Cricetulus , Humanos , Ligação Proteica/fisiologia , Receptores de Interleucina-8B/metabolismoRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a highly prevalent genetic disease that results in cyst formation in kidney and liver. Cytokines and growth factors secreted by the cyst-lining epithelia are positioned to initiate autocrine/paracrine signaling and promote cyst growth. Comparative analyses of human kidney and liver cyst fluids revealed disparate cytokine/growth factor profiles. CXCR2 agonists, including IL-8, epithelial neutrophil-activating peptide (ENA-78), growth-related oncogene-alpha (GRO-alpha), are potent proliferative agents that were found at high levels in liver but not kidney cyst fluids. Liver cysts are lined by epithelial cells derived from the intrahepatic bile duct (i.e., cholangiocytes). In polarized pkd2(WS25/-) mouse liver cyst epithelial monolayers, CXCR2 agonists were released both apically and basally, indicating that they may act both on the endothelial and epithelial cells within or lining the cyst wall. IL-8 and human liver cyst fluid induced cell proliferation of HMEC-1 cells, a human microvascular endothelial cell line, and Mz-ChA1 cells, a human cholangiocyte cell model. IL-8 expression can be regulated by specific stresses. Hypoxia and mechanical stretch, two likely stressors acting on the liver cyst epithelia, significantly increased IL-8 secretion and promoter activity. AP-1, c/EBP, and NF-kappaB were required but not sufficient to drive the stress-induced increase in IL-8 transcription. An upstream element between -272 and -1,481 bp allowed for the stress-induced increase in IL-8 transcription. These studies support the hypothesis that CXCR2 signaling promotes ADPKD liver cyst growth.
Assuntos
Proliferação de Células , Líquido Cístico/metabolismo , Cistos/metabolismo , Interleucina-8/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , Rim Policístico Autossômico Dominante/complicações , Receptores de Interleucina-8B/metabolismo , Animais , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiocina CXCL5/metabolismo , Cistos/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Interleucina-8/genética , Interleucina-8/farmacologia , Rim/metabolismo , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatias/etiologia , Hepatopatias/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Receptores de Interleucina-8B/agonistas , Transdução de Sinais , Estresse Mecânico , Canais de Cátion TRPP/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Stem cell research is currently focused on totipotent stem cells and their therapeutic potential, however adult stem cells, while restricted to differentiation within their tissue or origin, also have therapeutic utility. Transplantation with bone marrow hematopoietic stem cells (HSC) has been used for curative therapy for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow or mobilize to peripheral blood by G-CSF, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. The chemokine/chemokine receptor axis also mobilizes HSC that occurs more rapidly than with G-CSF. In mice, the HSC and progenitor cells (HPC) mobilized by the CXCR2 receptor agonist GRObeta can be harvested within minutes of administration and show significantly lower levels of apoptosis, enhanced homing to marrow, expression of more activated integrin receptors and superior repopulation kinetics and more competitive engraftment than the equivalent cells mobilized by G-CSF. These characteristics suggest that chemokine axis-mobilized HSC represent a population of adult stem cells distinct from those mobilized by G-CSF, with superior therapeutic potential. It remains to be determined if the chemokine mobilization axis can be harnessed to mobilize other populations of unique adult stem cells with clinical utility.
Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Quimiocinas/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Receptores de Quimiocinas/agonistas , Células-Tronco Adultas/classificação , Células-Tronco Adultas/citologia , Animais , Quimiocina CXCL12/fisiologia , Quimiocina CXCL2/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco de Sangue Periférico , Quimera por Radiação , Receptores de Quimiocinas/fisiologia , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/efeitos dos fármacosRESUMO
Previous studies have shown that neutrophils (PMNs) facilitate melanoma cell extravasation [M.J. Slattery, C. Dong, Neutrophils influence melanoma adhesion and migration under flow conditions, Intl. J. Cancer 106 (2003) 713-722] Little is known, however, about the specific interactions between PMNs, melanoma and the endothelium (EC) or the molecular mechanism involved under flow conditions. The aim of this study is to investigate a "two-step adhesion" hypothesis that involves initial PMN tethering on the EC and subsequent melanoma cells being captured by tethered PMNs. Different effects of hydrodynamic shear stress and shear rate were analyzed using a parallel-plate flow chamber. Results indicate a novel finding that PMN-facilitated melanoma cell arrest on the EC is modulated by shear rate, which is inversely-proportional to cell-cell contact time, rather than by the shear stress, which is proportional to the force exerted on formed bonds. Beta2 integrins/ICAM-1 adhesion mechanisms were examined and the results indicate LFA-1 and Mac-1 cooperate to mediate the PMN-EC-melanoma interactions under shear conditions. In addition, endogenously produced IL-8 contributes to PMN-facilitated melanoma arrest on the EC through the CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) on PMN. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the EC.
Assuntos
Moléculas de Adesão Celular/metabolismo , Melanoma/metabolismo , Neutrófilos/metabolismo , Estresse Mecânico , Adesão Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Dextranos/farmacologia , Endotélio/metabolismo , Endotélio/patologia , Humanos , Interleucina-8/metabolismo , Melanoma/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Resistência ao Cisalhamento , Células Tumorais CultivadasRESUMO
Rat cytokine-induced neutrophil chemoattractant-3 (CINC-3) has neutrophil chemotactic activity comparable with that of CINC-1 and CINC-2, but induces calcium mobilization more potently than CINC-1 and CINC-2. However, only one CINC receptor, CXCR2, has been found in rat neutrophils. Therefore we attempted to determine the biochemical basis for the differences in neutrophil responses to CINC-1/-2 versus CINC-3. Both chemotactic activity and calcium mobilization induced by CINC-3 were desensitized by a 100-fold excess of CINC-1, which was consistent with our previous results showing that CINC-1 has 70-fold lower affinity to the receptor on rat neutrophils than CINC-3. Desensitization appeares to be reflected by the affinity of the ligands to the receptor. CINC-1- and CINC-3-induced chemotaxis was sensitive to inhibition by pertussis toxin, whereas calcium mobilization induced by CINC-1 and CINC-3 was insensitive. These results suggest that CINCs induce neutrophil chemotaxis and calcium mobilization through distinct G-proteins with different efficiency.
Assuntos
Cálcio/metabolismo , Quimiocinas CXC , Quimiotaxia/fisiologia , Receptores de Interleucina-8B/metabolismo , Animais , Quimiocina CXCL1 , Quimiocinas/farmacologia , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ratos , Ratos Wistar , Receptores de Interleucina-8B/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Agonist treatment of cells expressing the chemokine receptor, CXCR2, induces receptor phosphorylation and internalization through a dynamin-dependent mechanism. In the present study, we demonstrate that a carboxyl terminus-truncated mutant of CXCR2 (331T), which no longer undergoes agonist-induced phosphorylation, continues to undergo ligand-induced internalization in HEK293 cells. This mutant receptor exhibits reduced association with beta-arrestin 1 but continues to exhibit association with adaptin 2 alpha and beta subunits. Replacing Leu320-321 and/or Ile323-Leu324 with Ala (LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA) in wild-type CXCR2 or 331T causes little change in ligand binding and signaling through Ca(2+) mobilization but greatly impairs the agonist-induced receptor sequestration and ligand-mediated chemotaxis. The LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA mutants of CXCR2 exhibit normal binding to beta-arrestin 1 but exhibit decreased binding to adaptin 2alpha and beta. These data demonstrate a role for the LLKIL motif in the carboxyl terminus of CXCR2 in receptor internalization and cell chemotaxis and imply a role for adaptin 2 in the endocytosis of CXCR2.