RESUMO
Previous studies in vitro have shown an important role for intercellular adhesion molecule-1 (ICAM-1) in adherence interactions of canine neutrophils with canine jugular vein endothelial cells and in cytotoxicity of canine neutrophils for adult cardiac myocytes. To evaluate the regulation of ICAM-1 in myocardial inflammation and its role in the pathogenesis of myocardial ischemia and reperfusion, a series of in vivo and ex vivo studies were performed in canine animals. Systemic administration of LPS elicited ICAM-1 mRNA in several tissues, including myocardium, which demonstrated increasing ICAM-1 staining on intercalated discs of cardiac myocytes. In ischemia and reperfusion protocols: (a) ICAM-1 mRNA was found in ischemic segments within 1 h of reperfusion and in both ischemic and normally perfused segments by 24 h of reperfusion; (b) expression of ICAM-1 was detected in cardiac myocytes in the ischemic region by 6 h of reperfusion; increased expression was seen thereafter as a function of time; (c) post-ischemic (but not preischemic) cardiac lymph collected at intervals from 1 to 24 h after reperfusion elicited ICAM-1 mRNA, ICAM-1 expression, and ICAM-1-dependent neutrophil adhesion in canine jugular vein endothelial cells and in cardiac myocytes with peak cytokine activity seen by 1 h; (d) extravascular localization of neutrophils was detected in ischemic areas only, and was associated with endothelium bearing high levels of ICAM-1 within 1 h of reperfusion; infiltration increased thereafter in association with increasing levels of ICAM-1 mRNA in myocardial segments and increasing levels of ICAM-1 expression on cardiac myocytes. These findings provide the first direct evidence for inflammatory regulation of ICAM-1 in ischemic and reperfused canine myocardium. They support the hypothesis that ICAM-1 participates in neutrophil-mediated myocardial damage.
Assuntos
Moléculas de Adesão Celular/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Adesão Celular , Moléculas de Adesão Celular/genética , Cães , Endotélio Vascular/metabolismo , Feminino , Regulação da Expressão Gênica , Molécula 1 de Adesão Intercelular , Linfa/fisiologia , Masculino , Neutrófilos/metabolismo , RNA Mensageiro/genética , Fatores de TempoRESUMO
Neutrophil adhesion and direct cytotoxicity for cardiac myocytes require chemotactic stimulation and are dependent upon CD18-ICAM-1 binding. To characterize the potential role of IL-8 in this interaction, canine IL-8 cDNA was cloned and the mature recombinant protein expressed in Escherichia coli BL21 cells. Recombinant canine IL-8 markedly increased adhesion of neutrophils to isolated canine cardiac myocytes. This adhesion resulted in direct cytotoxicity for cardiac myocytes. Both processes were specifically blocked by antibodies directed against CD18 and IL-8. In vivo, after 1 h of coronary occlusion, IL-8 mRNA was markedly and consistently induced in reperfused segments of myocardium. IL-8 mRNA was not induced in control (normally perfused) myocardial segments. Minimal amounts of IL-8 mRNA were detected after 3 or 4 h of ischemia without reperfusion. Highest levels of induction were evident in the most ischemic myocardial segments. IL-8 mRNA peaked in the first 3 h of reperfusion and persisted at high levels beyond 24 h. IL-8 staining was present in the inflammatory infiltrate near the border between necrotic and viable myocardium, as well as in small veins in the same area. These findings provide the first direct evidence for regulation of IL-8 in ischemic and reperfused canine myocardium and support the hypothesis that IL-8 participates in neutrophil-mediated myocardial injury.
Assuntos
Regulação da Expressão Gênica , Interleucina-8/biossíntese , Interleucina-8/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Adesão Celular/fisiologia , Movimento Celular , Doença das Coronárias/metabolismo , Cães , Endotélio Vascular/fisiologia , Feminino , Inflamação/metabolismo , Interleucina-8/farmacologia , Masculino , Dados de Sequência Molecular , Traumatismo por Reperfusão Miocárdica/patologia , Ativação de Neutrófilo/fisiologia , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Distribuição Tecidual , Ativação TranscricionalRESUMO
Canine intercellular adhesion molecule-1 (ICAM-1) plays a primary role in the adherence of canine neutrophils to endothelial cells and in the cytotoxicity of canine neutrophils for adult cardiac myocytes. We have cloned the canine ICAM-1 gene and have analyzed the conservation of ICAM-1 amino acid (aa) sequences in man, chimpanzee, mouse, rat and dog. Canine ICAM-1 displays 61% identity with human ICAM-1. Cys residues critical to the immunoglobulin (Ig) fold structure and four sites of N-linked glycosylation are absolutely conserved in ICAM-1 from all species. Residues in the cytoplasmic tail associated with cytoskeletal alpha-actinin binding are highly conserved, supporting the hypothesis that intracellular attachment is indeed important for ICAM-1 function. Residues critical for human ICAM-1 binding to the beta 2-integrin leukocyte-function-associated antigen 1 (LFA-1) are highly conserved between all species, whereas those residues demonstrated to play an important role in interaction of human ICAM-1 with macrophage activation complex 1 (Mac-1) are not highly conserved. Residues critical for ICAM-1 binding to rhinovirus and malaria-infected red blood cells (IRBC) are not highly conserved.
Assuntos
Molécula 1 de Adesão Intercelular/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sequência Conservada , DNA Complementar/genética , Cães , Endotélio Vascular/citologia , Eritrócitos/parasitologia , Eritrócitos/virologia , Biblioteca Gênica , Humanos , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Malária/imunologia , Dados de Sequência Molecular , Infecções por Picornaviridae/imunologia , Ligação Proteica , Rhinovirus/imunologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
OBJECTIVE(S): Neutrophil sequestration in the lung after cardiopulmonary bypass has been shown to be dependent on the adhesion molecule CD18. Thus we sought to determine whether endothelial expression of intercellular adhesion molecule-1 (a ligand for CD18) in pulmonary capillaries mediates neutrophil adhesion in this setting. METHODS: Seven adult mongrel dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After warming, dogs were reperfused for up to 9 hours and lung biopsy specimens were obtained. Lung tissue was examined by Northern and Western blot analysis and by immunohistologic methods. Three sham-operated dogs served as time-matched controls. RESULTS: Northern blots demonstrated increased expression of intercellular adhesion molecule-1 messenger ribonucleic acid within 5 minutes of cessation of bypass (or approximately 30 minutes after aortic crossclamp release), which persisted at 9 hours of recovery and was not present in controls. Western blots showed intercellular adhesion molecule-1 protein expression before bypass but a measurable increase in intercellular adhesion molecule-1 protein in four of seven dogs in the bypass group by the ninth hour of recovery. Pulmonary neutrophil accumulation 9 hours after cardiopulmonary bypass was greater in those dogs with an increased intercellular adhesion molecule-1 protein expression. Immunoelectron microscopy demonstrated the pulmonary capillary endothelium capable of increased intercellular adhesion molecule-1 protein expression at the 9-hour time point. CONCLUSIONS: Cardiopulmonary bypass resulted in intercellular adhesion molecule-1 induction in the canine lung during recovery. An increased expression of intercellular adhesion molecule-1 protein in the lung was associated with an increased accumulation of neutrophils in affected animals. Thus intercellular adhesion molecule-1 expression may serve as a mechanism that predisposes the lungs to inflammatory cell-mediated injury postoperatively.
Assuntos
Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Pulmão/metabolismo , Ativação de Neutrófilo/fisiologia , Neutrófilos/metabolismo , Animais , Northern Blotting , Western Blotting , Ponte Cardiopulmonar , Cães , Imuno-Histoquímica , Pulmão/irrigação sanguínea , Microcirculação , Período Pós-OperatórioAssuntos
Moléculas de Adesão Celular/metabolismo , Interleucina-6/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/química , Cães , Feminino , Expressão Gênica , Hibridização In Situ , Molécula 1 de Adesão Intercelular , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Suínos , Fatores de TempoRESUMO
Extensive monocyte recruitment is an early phenomenon associated with the development of atherosclerotic lesions, suggesting an active role for the involvement of adhesion receptors expressed by endothelial cells. In this study we describe the contribution of hemodynamic shear forces in regulating the expression of a few of the monocyte adhesion receptors, including intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin on endothelial cells. A parallel plate flow chamber and recirculating flow loop device was used to expose human umbilical vein endothelial cells (HUVECs) to different levels of shear (2-25 dyn/cm2). Subsequently the cells were analyzed either for shear induced changes in the mRNA levels of adhesion receptors by Northern blot analyses or for changes in the surface expression of ICAM-1 using flow cytometry. Results from the fluorescence analysis showed a transient increase in the surface expression of ICAM-1, 12 hr after exposure to 25 dyn/cm2 shear, returning to basal levels within 24 hr. This was quite different from the time dependent response of ICAM-1 to lipopolysaccharide (LPS), where ICAM-1 expression was maximally induced 18-24 hr post-stimulus. ICAM-1 mRNA level appeared slightly elevated after exposure to shear for 1 hr, compared to basal values, but dropped below basal levels within 6 hr. This biphasic response was seen irrespective of the magnitude of applied shear stress. VCAM-1 mRNA expression, in contrast, decreased below the baseline expression within an hour after onset of flow, and appeared to be considerably down-regulated within 6 hr. After exposure to shear for 24 hr, no increase in mRNA levels could be detected for either molecule, at any shear magnitude. E-selectin mRNA was less responsive to shear stress, especially at the lower magnitudes of shear. After an hour of exposure to flow E-selectin mRNA level appeared slightly reduced compared with control levels, but it remained at this level even after 6 hr of flow. These results indicate that the expression of adhesion receptors is sensitive to local shear stresses in a manner that is molecule specific in the short term even though prolonged exposure to flow results in similar down-regulation for both ICAM-1 and VCAM-1.
Assuntos
Moléculas de Adesão Celular/biossíntese , Endotélio Vascular/fisiologia , Molécula 1 de Adesão Intercelular/biossíntese , Células Cultivadas , Selectina E , Humanos , RNA Mensageiro/análise , Estresse Mecânico , Fatores de Tempo , Veias Umbilicais/fisiologia , Molécula 1 de Adesão de Célula VascularRESUMO
Previous studies have demonstrated that alveolar macrophages (AMphis) adherent to plastic release interleukin-8 in response to lipopolysaccharide (LPS). We sought to determine whether LPS could also alter surface adhesion molecule expression and thus modulate additional AMphi adhesive interactions. Canine AMphis obtained by bronchoalveolar lavage of excised lung were adhered onto tissue culture plastic and exposed to LPS (0.01 to 100 ng/ml). Expression of beta 2 integrins and intercellular adhesion molecule-1 (ICAM-1) was subsequently determined by flow cytometry, a cDNA probe to canine ICAM-1 was used to quantify ICAM-1 mRNA, and changes in adhesion molecule function were assessed by evaluating the extent of homotypic aggregation. ICAM-1 and CD11a/CD18 were present on freshly isolated AMphis. CD11b/CD18 and CD11c/CD18 were expressed at lower levels. Nonadherent AMphis expressed a pattern of beta 2 integrin and ICAM-1 comparable to adherent cells. During short-term LPS stimulation (3 h), adherent AMphis increased both the synthesis and expression of ICAM-1. CD18 expression was either decreased or remained unchanged with LPS stimulation. LPS stimulation in vitro (> 0.01 ng/ml) enhanced the homotypic aggregation of adherent AMphis. Aggregation was blocked by monoclonal antibodies to ICAM-1 (CL18/6) and CD11a (R7.1) and CD18 (R15.7). Similar kinetics were found for expression of ICAM-1 and homotypic aggregation, suggesting that up-regulation of ICAM-1 is a major determinant of the LPS-stimulated aggregation of AMphis.
Assuntos
Antígenos CD/biossíntese , Moléculas de Adesão Celular/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/metabolismo , Animais , Anticorpos Monoclonais , Líquido da Lavagem Broncoalveolar/citologia , Antígenos CD11 , Antígenos CD18 , Moléculas de Adesão Celular/genética , Agregação Celular/efeitos dos fármacos , Células Cultivadas , Cães , Integrinas/biossíntese , Molécula 1 de Adesão Intercelular , RNA Mensageiro/biossíntese , Receptores de Adesão de Leucócito/biossíntese , Transcrição GênicaRESUMO
BACKGROUND: Neutrophil-induced injury of myocardial cells requires the expression of intercellular adhesion molecule-1 (ICAM-1) on the myocyte surface and is mediated by ICAM-1-CD11b/CD18 adhesion. We have previously shown that interleukin-6 (IL-6) cytokine activity, present in cardiac lymph, induces ICAM-1 on isolated cardiac myocytes. Furthermore, in previous in vivo studies, we have also shown ICAM-1 mRNA induction in the myocardium within the first hour of reperfusion in the previously ischemic viable zone. We hypothesized that induction of IL-6 synthesis in the myocardium was an integral part of the reaction to injury resulting from ischemia and reperfusion and was associated with induction of ICAM-1 on myocardial cells. METHODS AND RESULTS: In this study, cloned canine IL-6 cDNA was used as a molecular probe to study the regulation of IL-6 in an awake canine model of myocardial ischemia and reperfusion. IL-6 mRNA was induced in ischemic and reperfused segments of myocardium preferentially in segments previously exposed to severe ischemia. Peak levels of IL-6 mRNA were reached within 3 hours of reperfusion. At the same time, IL-6 mRNA and ICAM-1 mRNA were found in the same myocardial segments. In contrast to hearts that were ischemic for 1 hour and reperfused for 3 hours, nonreperfused hearts after 4 hours of persistent ischemia demonstrated minimal induction of ICAM-1 or IL-6 despite similar degrees of injury and blood flow reductions during ischemia. After 24 hours of persistent ischemia, levels of IL-6 mRNA were comparable to those observed in hearts that were ischemic for 1 hour and subsequently reperfused for 24 hours. CONCLUSIONS: Our results demonstrate induction of IL-6 mRNA in the myocardium and that this synthesis is accelerated by reperfusion. Evidence is also provided to show that peak IL-6 mRNA precedes that of ICAM-1 mRNA. These findings are compatible with our hypothesis that IL-6 is important in the induction of ICAM-1 in the area of ischemia. In addition, these studies suggest that the necessary factors to promote adhesive interactions between transmigrated neutrophils and cardiac myocytes are present in reperfused myocardium.
Assuntos
Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-6/biossíntese , Traumatismo por Reperfusão Miocárdica/etiologia , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , DNA Complementar , Cães , Feminino , Expressão Gênica , Humanos , Interleucina-6/genética , Masculino , Dados de Sequência Molecular , Traumatismo por Reperfusão Miocárdica/metabolismo , RNA Mensageiro/genética , Suínos , Fatores de TempoRESUMO
BACKGROUND: Acute inflammation may play a role in injury during reperfusion following myocardial ischemia. Studies in vitro suggest that intracellular adhesion molecule-1 (ICAM-1) mediates neutrophil adherence to cardiac myocytes and neutrophil-mediated injury. We have shown cytokine activity in postischemic cardiac lymph sufficient to maximally express ICAM-1 on myocytes and that ICAM-1 mRNA is found in the previously ischemic myocardium early in reperfusion. METHODS AND RESULTS: In the present study, we used in situ hybridization techniques to detect ICAM-1 mRNA and examine the cells of origin, relation to cell injury, and relation to inflammatory infiltration after 1 hour of ischemia and varying times of reperfusion. By 1 hour of reperfusion, ICAM-1 mRNA was detected in much of the ischemic myocardium, except in areas of contraction band necrosis. At 2 and 3 hours, a clear demarcation of necrotic areas surrounding ischemic areas of viable myocardium with ICAM-1 mRNA staining was present, and ICAM-1 mRNA staining increased with time. Nonischemic areas had no visible ICAM-1 mRNA staining in the first 3 hours. By 24 hours of reperfusion, ICAM-1 mRNA was present in both control and ischemic segments (excluding the necrotic areas) compatible with a generalized circulation of cytokines persistent at 24 hours. In the absence of reperfusion, ICAM-1 mRNA staining was not seen in the first 3 hours and was markedly reduced at 24 hours. The interface of viable and necrotic cells also contained the most extensive inflammatory infiltration. CONCLUSIONS: Evidence is presented that induction of ICAM-1 mRNA has highly specific localization to ischemic but viable myocardium. Induction of ICAM-1 mRNA transcription in early reperfusion may render the viable "border zone" susceptible to neutrophil-induced injury.
Assuntos
Moléculas de Adesão Celular/biossíntese , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/etiologia , Miocárdio/metabolismo , Animais , Moléculas de Adesão Celular/genética , Cães , Feminino , Molécula 1 de Adesão Intercelular , Masculino , Miocardite/etiologia , RNA Mensageiro/análiseRESUMO
Acute inflammation has been suggested as a potential mechanism for some of the injury associated with reperfusion of the ischemic myocardium. This hypothesis implies that viable myocardial cells adjacent to the lethally injured cells are vulnerable to injury induced by the neutrophil influx observed to attend reperfusion. In our previous work, we demonstrated that the presence of ICAM-1 on the surface of cardiac myocytes is required for neutrophils to directly damage them; blocking monoclonal antibodies to either ICAM-1 on cardiac myocytes or Mac-1 on activated neutrophils completely precluded neutrophil-induced myocyte injury. We also demonstrated that postischemic cardiac lymph (cardiac extracellular fluid) contained leukotactic factors (primarily C5a) and cytokines present in concentrations sufficient to maximally induce Mac-1 on the surface of neutrophils and ICAM-1 on the surface of isolated dog cardiac myocytes. The present study sought to further these observations by examining the site of potential ICAM-1 induction as a function of time of reperfusion, degree of ischemia, and viability of myocardial cells. Our evidence suggests that ICAM-1 mRNA is induced very early after reperfusion only in the previously ischemic myocardium and is not seen in the nonischemic myocardium during the early hours of reperfusion. Moreover, ICAM-1 mRNA induction is seen most intensely in the ischemic area directly bordering the necrotic area (which, after 1-hr reperfusion, does not contain any ICAM-1 mRNA) and immediately abutting the site of maximal influx of neutrophils. Thus, the induction of ICAM-1 and the influx of neutrophils (presumably activated by the chemotactic factors that guided their migration) exists on the border between viable and necrotic cells. This provides the first direct molecular evidence for a jeopardized border zone on the edge of myocardial infarction during reperfusion. As previously demonstrated, this reaction is wholly dependent upon tissue injury of the ischemic myocardium and therefore represents an example of a mechanism of injury extension induced as a reaction to a primary injury. The degree of specificity of this reaction demonstrated by the subendocardial sparing directly adjacent to ischemic cells suggests finely modulated mechanisms by which this process is controlled.
Assuntos
Moléculas de Adesão Celular/metabolismo , Traumatismo por Reperfusão Miocárdica/etiologia , Miocardite/etiologia , Animais , Adesão Celular , Moléculas de Adesão Celular/genética , Cães , Histocitoquímica , Hibridização In Situ , Molécula 1 de Adesão Intercelular , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocardite/genética , Miocardite/metabolismo , Neutrófilos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Our studies in vitro demonstrate that neutrophil mediated injury of isolated cardiac myocytes requires the presence of ICAM-1 on the surface of the myocyte and CD11b/CD18 activation on the neutrophil. In post-ischemic cardiac lymph, there is rapid appearance of C5a activity during the first hours of reperfusion. Interleukin-6 activity is present throughout the first 72 h of reperfusion and is sufficient to induce ICAM-1 on the surface of the cardiac myocyte. In situ hybridization studies suggest that ICAM-1 mRNA is found in viable myocardial cells on the edge of the myocardial infarction within 1 h of reperfusion. ICAM-1 protein expression on cardiac myocytes is seen after 6 h of reperfusion, and increases thereafter. Non-ischemic tissue demonstrates no early induction of ICAM-1 mRNA or ICAM-1 protein on myocardial cells. In our most recent experiments, we have determined that reperfusion is an absolute requirement for the early induction of myocardial ICAM-1 mRNA in previously ischemic myocardial cells. To further assess this, we have cloned and sequenced a canine interleukin-6 (IL-6) cDNA. The data suggest that early induction of IL-6 mRNA is also reperfusion dependent as it could be demonstrated in the same ischemic and reperfused segments in which ICAM-1 mRNA was found. Peak expression of IL-6 mRNA occurred much earlier than that for ICAM-1 mRNA. Similar experiments were then performed with a molecular probe for interleukin-8 (IL-8). This chemokine is a potent neutrophil stimulant and has a higher degree of specificity for neutrophils than classic chemoattractants such as C5a.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Sequência de Bases , Northern Blotting , Complemento C5a/metabolismo , Sondas de DNA/química , Sondas de DNA/genética , Cães , Feminino , Regulação da Expressão Gênica/genética , Hibridização In Situ , Molécula 1 de Adesão Intercelular/genética , Interleucina-6/química , Interleucina-6/genética , Interleucina-8/química , Interleucina-8/genética , Masculino , Dados de Sequência Molecular , Neutrófilos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The mechanisms by which neutrophils migrate through the alveolar interstitium during acute lung inflammation are unknown. We wished to determine whether platelet-activating factor (PAF) and IL-8, two important mediators in neutrophil transendothelial migration, stimulated neutrophil adherence and motility on lung fibroblasts. Canine fibroblasts grown from lung explants were characterized by light and electron microscopy, and flow cytometry. Unstimulated neutrophils adhered poorly (less than 2%) to cultured fibroblasts. However, neutrophils stimulated with PAF (20-200 nM) showed a dose-dependent increase in adherence that was largely (70%) mediated by the beta 2 (CD11/CD18) integrins; adherence was less dependent (50%) on fibroblast intercellular adhesion molecule-1. Conversely, neutrophils stimulated with canine rIL-8 did not increase their adherence to fibroblasts. PAF-stimulated neutrophils were nonmotile on the surface of the fibroblast, but subsequent addition of rIL-8 (10(-8) M) induced motility that was entirely CD1 8 dependent. Fibroblasts stimulated with human rTNF-alpha or Escherichia coli endotoxin (LPS) were a significant source of IL-8 mRNA. In response to rTNF-alpha (50 U/ml), IL-8 mRNA was detected at 2 h by northern blot analysis; it peaked at 6 h and returned to baseline by 24 h. Fibroblasts stimulated with rTNF-alpha secreted IL-8 protein into the culture medium; secreted IL-8 was chemotactic for neutrophils. These data suggest that fibroblasts can function not only as an adhesive substrate, but also as a source of stimulation for neutrophil migration through the inflamed alveolar interstitium.
Assuntos
Antígenos CD18/efeitos dos fármacos , Antígenos CD18/fisiologia , Movimento Celular/imunologia , Fatores Quimiotáticos/farmacologia , Fibroblastos/imunologia , Pulmão/imunologia , Neutrófilos/imunologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/imunologia , Cães , Feminino , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Interleucina-8/biossíntese , Interleucina-8/genética , Interleucina-8/farmacologia , Pulmão/ultraestrutura , Masculino , Neutrófilos/ultraestrutura , Fator de Ativação de Plaquetas/farmacologia , RNA Mensageiro/biossíntese , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Healing after myocardial infarction is characterized by the presence of macrophages in the infarcted area. Since augmented monocyte influx has been implicated as a potential mechanism for improved healing after reperfusion, we wished to study the induction of monocyte chemoattractant protein-1 (MCP-1) during reperfusion. METHODS AND RESULTS: The cDNA for MCP-1 was cloned from a canine jugular vein endothelial cell (CJVEC) library and exhibited 78% identity with the deduced amino acid sequence of human MCP-1. Samples of myocardium were taken from control and ischemic segments after 1 hour of ischemia and various times of reperfusion; total RNA was isolated from myocardial samples and probed with a cDNA probe for canine MCP-1. Induction of MCP-1 mRNA occurred only in previously ischemic segments within the first hour of reperfusion, peaked at 3 hours, and persisted throughout the first 2 days of reperfusion. In the absence of reperfusion, no significant MCP-1 induction was seen. Both ischemic (but not preischemic) cardiac lymph and human recombinant TNF-alpha induced MCP-1 in CJVECs. MCP-1 was identified by immunostaining on infiltrating cells and venular (but not arterial) endothelium by 3 hours. In contrast, in situ hybridization showed MCP-1 mRNA to be confined to the endothelium of small veins (venules) 10 to 70 microns in diameter. CONCLUSIONS: MCP-1 mRNA is induced in the endothelium of a specific class of small veins immediately after reperfusion. MCP-1 induction is confined to the previously ischemic area that has been reperfused. We suggest a significant role for MCP-1 in monocyte trafficking in the reperfused myocardium.