RESUMEN
OBJECTIVE: Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis (OA), we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1(-/-)). DESIGN: Femoral groove cartilage of WT and Has1(-/-) mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. RESULTS: Acute responses (1-2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1(-/-) mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. CONCLUSIONS: We conclude that the ineffective repair of injured cartilage in Has1(-/-) joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.
Asunto(s)
Cartílago Articular/patología , Regulación de la Expresión Génica , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/enzimología , ARN/genética , Animales , Cartílago Articular/enzimología , Cartílago Articular/lesiones , Enfermedad Crónica , Modelos Animales de Enfermedad , Fibrosis/enzimología , Fibrosis/patología , Glucuronosiltransferasa/biosíntesis , Hialuronano Sintasas , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Articulación de la Rodilla/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/patología , Reacción en Cadena de la PolimerasaRESUMEN
Acute and chronic rejection impact distinct compartments of cardiac allografts. Intramyocardial mononuclear cell infiltrates define acute rejection, whereas chronic rejection affects large arteries. Hearts transplanted from male to female C57BL/6 mice undergo acute rejection with interstitial infiltrates at 2 weeks that resolve by 6 weeks when large arteries develop arteriopathy. These processes are dependent on T cells because no infiltrates developed in T cell-deficient mice and transfer of CD4 T cells restored T cell as well as macrophage infiltrates and ultimately neointima formation. Markers of inflammatory macrophages were up-regulated in the interstitium acutely and decreased as markers of wound healing macrophages increased chronically. Programmed cell death protein, a negative costimulator, and its ligand PDL1 were up-regulated in the interstitium during resolution of acute rejection. Blocking PDL1:PD1 interactions in the acute phase increased interstitial T cell infiltrates. Toll-like receptor (TLR) 4 and its endogenous ligand hyaluronan were increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative costimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by the innate TLRs may support the progression of arteriopathy.
Asunto(s)
Rechazo de Injerto/fisiopatología , Trasplante de Corazón , Miocardio/metabolismo , Miocardio/patología , Transducción de Señal/fisiología , Animales , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Quimiocina CCL2/metabolismo , Quimiocina CXCL9/metabolismo , Femenino , Ácido Hialurónico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Receptor Toll-Like 4/metabolismoRESUMEN
Alteration in the glycosaminoglycan hyaluronan (HA) has been demonstrated in numerous renal diseases. We have demonstrated that renal proximal tubular epithelial cells (PTCs) surround themselves in vitro with HA in an organized pericellular matrix or 'coat', which is associated with cell migration, and also form pericellular HA cable-like structures which modulate PTC-mononuclear leukocytes interactions. The aim of this study was to characterize potential regulatory mechanism in the assembly of PTC-HA into pericellular cables. HA cables are generated by PTCs in the absence of serum. Immunohistochemical analysis demonstrates the incorporation of components of the inter-alpha-inhibitor (IalphaI) family of proteins and versican into HA cables. Addition of an antibody to IalphaI/PalphaI (pre-alpha-inhibitor) inhibits cable formation. In contrast, inhibition of tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) has no effect on cable formation, suggesting that their generation is independent of the known heavy-chain transfer activity of TSG-6. Overexpression of HAS3 is associated with induction of HA cable formation, and also increased incorporation of HA into pericellular coats. Functionally, this resulted in enhanced HA-dependent monocyte binding and cell migration, respectively. Cell surface expression of CD44 and trypsin-released cell-associated HA were increased in HAS3-overexpressing cells. In addition, hyaluronidase (hyal1 and hyal2) and bikunin mRNA expression were increased, whereas PalphaI HC3 mRNA expression was unchanged in the transfected cells. The data demonstrate the importance of IalphaI/PalphaI in cable formation and suggest that expression of HAS3 may be critical for HA cable assembly.
Asunto(s)
Células Epiteliales/metabolismo , Ácido Hialurónico/metabolismo , Túbulos Renales Proximales/metabolismo , alfa-Globulinas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Movimiento Celular , Medios de Cultivo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Ácido Hialurónico/análisis , Ácido Hialurónico/fisiología , Hialuronoglucosaminidasa/farmacología , Inmunohistoquímica , Túbulos Renales Proximales/citología , Leucocitos Mononucleares/metabolismo , Masculino , Microscopía Confocal , Precursores de Proteínas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Coloración y Etiquetado , Testículo/enzimología , Factores de Tiempo , Inhibidores de Tripsina/farmacología , Células U937 , Versicanos/análisis , Versicanos/metabolismoRESUMEN
BACKGROUND: Activated T cells are more susceptible to apoptosis than resting T cells. As intestinal T cells normally exhibit a higher state of activation, increased apoptosis may be necessary to maintain immune homeostasis in the specialised microenvironment of the mucosa. On the other hand, in Crohn's disease (CD) mucosal T cells are resistant to apoptosis, suggesting abnormal regulation of cell death mechanisms. AIMS: To investigate differences in expression of anti- and proapoptotic Bcl-2 family proteins, key regulators of apoptosis, between circulating and mucosal T cells, and possible alterations in CD. PATIENTS AND METHODS: Lamina propria T cells (LPT) were isolated from 10 control, seven CD, and eight ulcerative colitis (UC) patients, and peripheral blood T cells (PBT) from healthy volunteers. Purified T cells were stained intracellularly for Bcl-2, Bcl-x(L), and Bax, and mean fluorescence intensity measured by flow cytometry. RESULTS: Compared with PBT, the expression level of Bcl-2 and Bax, but not Bcl-x(L), was significantly greater in LPT, resulting in lower Bcl-x(L)/Bax ratios. In PBT, Bax expression was highly and significantly correlated with both Bcl-2 and Bcl-x(L), but correlation with Bcl-2 was absent in LPT. Bax expression in CD, but not UC, LPT was significantly lower than in control LPT, resulting in a significantly higher Bcl-x(L)/Bax ratio. The significant correlation of Bcl-x(L) to Bax was preserved in CD, but not UC, LPT. CONCLUSIONS: Regulation of Bcl-2 family protein expression differs between circulating and mucosal T cells, probably underlying diverse survival potentials. In CD LPT, a low Bax expression and a high Bcl-x(L)/Bax ratio favour resistance to apoptosis and may contribute to the chronicity of inflammation.
Asunto(s)
Apoptosis/fisiología , Enfermedad de Crohn/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Femenino , Citometría de Flujo , Humanos , Inmunidad Celular , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Proteína X Asociada a bcl-2RESUMEN
NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.
Asunto(s)
Virus de la Influenza A/fisiología , Óxido Nítrico Sintasa/biosíntesis , ARN Bicatenario/fisiología , eIF-2 Quinasa/fisiología , Animales , Bronquios/citología , Bronquios/enzimología , Bronquios/metabolismo , Bronquios/virología , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Activación Enzimática , Inducción Enzimática , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Factor 1 Regulador del Interferón , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II , Fosfoproteínas/biosíntesis , ARN Bicatenario/síntesis química , ARN Viral/síntesis química , ARN Viral/farmacología , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
The adherence of leukocytes on the endothelium is mediated in part by the transient expression of the E-selectin adhesion molecule. Because we have previously shown that the dsRNA-activated kinase PKR mediates dsRNA induction of NF-kappaB, we used murine aortic endothelial (MuAE) cells isolated from wild-type and PKR-null mice to investigate the role of PKR in the induction of E-selectin expression by dsRNA (pIC) and TNF-alpha. E-selectin mRNA and protein expression was inducible by both pIC and TNF-alpha in wild-type MuAE cells, whereas induction of E-selectin expression by these agents was defective in PKR-null MuAE cells. Induction of E-selectin promoter activity and NF-kappaB DNA binding activity were substantially reduced in pIC- or TNF-alpha-treated PKR-null cells, indicating a role for PKR in both pIC and TNF-alpha induction of E-selectin via an NF-kappaB-dependent pathway. In PKR-null cells, pIC-mediated degradation of IkappaBbeta is deficient. Activation of this pathway requires the PKR-dependent degradation of the IkappaBbeta protein. Moreover, both phosphorylated and unphosphorylated activating transcription factor 2 DNA-binding activities were reduced in PKR-null aortic endothelial cells. These results indicate that the PKR is required for full activation of E-selectin expression by pIC and TNF-alpha in primary mouse aortic endothelial cells identifying activating transcription factor 2 as a new target for PKR-dependent regulation and suggest a role for PKR in leukocyte adhesion.
Asunto(s)
Selectina E/biosíntesis , Endotelio Vascular/metabolismo , ARN Bicatenario/farmacología , Factor de Necrosis Tumoral alfa/farmacología , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genética , Animales , Aorta , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Selectina E/genética , Selectina E/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Activación Enzimática/inmunología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/genética , Inducción Enzimática/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Luciferasas/biosíntesis , Luciferasas/genética , Sustancias Macromoleculares , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/inmunología , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , eIF-2 Quinasa/fisiologíaRESUMEN
Pathological changes in inflammatory bowel disease include an increase in intestinal mucosal mononuclear leukocytes and hyperplasia of the muscularis mucosae smooth muscle cells (M-SMCs). Because virus infections have correlated with disease flare, we tested the response of cultured M-SMCs to respiratory syncytial virus, measles virus, and the viral analogue, poly(I.C). Adhesion of U937 cells and peripheral blood mononuclear cells was used to measure the leukocyte-interactive potential of M-SMCs. Untreated M-SMCs, only minimally adhesive for leukocytes, bound U937 cells after treatment with respiratory syncytial virus or measles virus. Mononuclear leukocytes also bound to poly(I.C)-treated M-SMCs. Although both vascular cell adhesion molecule-1 mRNA and protein increased 3-4-fold in poly(I.C)-treated M-SMC cultures, U937 cell adhesion was not blocked by an anti-vascular cell adhesion molecule-1 monoclonal antibody. However, hyaluronidase digestion of poly(I.C)- or virus-treated M-SMCs dramatically reduced leukocyte adhesion ( approximately 75%). Fluorophore-assisted carbohydrate electrophoresis demonstrated a approximately 3-fold increase in surface-bound hyaluronan on poly(I.C)-treated M-SMCs compared with untreated controls. In addition, pretreatment of mononuclear cells with a blocking anti-CD44 antibody, greatly decreased adhesion to poly(I.C)-treated M-SMCs. Recognition of this virus-induced hyaluronan/CD44 mechanism of mesenchymal cell/leukocyte interaction introduces a new avenue in the research of gut inflammation.
Asunto(s)
Receptores de Hialuranos/fisiología , Ácido Hialurónico/metabolismo , Mucosa Intestinal/fisiología , Leucocitos Mononucleares/fisiología , Leucocitos Mononucleares/virología , Virus del Sarampión/fisiología , Poli I-C/farmacología , Virus Sincitiales Respiratorios/fisiología , Molécula 1 de Adhesión Celular Vascular/genética , Antígenos CD/fisiología , Adhesión Celular , Células Cultivadas , Colon , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hialuronoglucosaminidasa/farmacología , Interferón-alfa/farmacología , Interferón gamma/farmacología , Mucosa Intestinal/citología , Músculo Liso/fisiología , ARN Mensajero/análisis , Transcripción Genética , Factor de Necrosis Tumoral alfa/farmacología , Células U937 , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
BACKGROUND & AIMS: Immune-nonimmune cell interactions modulate mucosal immunity. We investigated the expression of adhesion molecules by intestinal fibroblasts, the effect of immune cell-derived factor on fibroblast binding of T cells, and the consequences of interfering with adhesion molecule expression on fibroblast-T cell interaction. METHODS: Expression of fibroblast intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 surface and messenger RNA (mRNA) was measured before and after exposure to immune cell-derived supernatants. Fibroblasts were treated with antibodies to ICAM-1 or VCAM-1, or ICAM-1 antisense oligonucleotide Isis 2302, before a T-cell adhesion assay. RESULTS: Fibroblast activation by immune cell-derived cytokines enhanced ICAM-1 and VCAM-1 surface expression and mRNA as well as adhesiveness for T cells. Blockade with neutralizing antibodies showed that binding was almost exclusively dependent on ICAM-1. Isis 2302 specifically reduced fibroblast ICAM-1 mRNA and dose-dependently inhibited ICAM-1 surface expression and T-cell binding. CONCLUSIONS: ICAM-1 is essential for intestinal fibroblast binding of T cells, a phenomenon that is efficiently and specifically disrupted by ICAM-1 antisense oligonucleotides. These observations emphasize the crucial regulatory role of fibroblasts in mucosal immunity and their potential as targets for therapeutic intervention in intestinal inflammation.
Asunto(s)
Fibroblastos/patología , Molécula 1 de Adhesión Intercelular/fisiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Linfocitos T/fisiología , Northern Blotting , Adhesión Celular , Células Cultivadas , Medios de Cultivo Condicionados , Fibroblastos/inmunología , Humanos , Inflamación , Molécula 1 de Adhesión Intercelular/biosíntesis , Leucocitos Mononucleares/inmunología , Oligonucleótidos Antisentido , ARN Mensajero/análisis , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.
Asunto(s)
Acetilcisteína/farmacología , Citocinas/antagonistas & inhibidores , Citocinas/farmacología , Selectina E/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adhesión Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Selectina E/genética , Endotelio Vascular/citología , Regulación de la Expresión Génica/efectos de los fármacos , Genes/efectos de los fármacos , Glutatión/análogos & derivados , Glutatión/metabolismo , Disulfuro de Glutatión , Humanos , Interleucina-1/farmacología , Monocitos/efectos de los fármacos , Monocitos/fisiología , FN-kappa B/metabolismo , FN-kappa B/farmacología , Transcripción Genética/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
In this study, we investigate whether human inflammatory bowel disease (IBD) (ulcerative colitis and Crohn's disease) is associated with altered lymphokine secretion profiles, as recently found in various animal models of chronic intestinal inflammation. In initial studies, we determined the proliferative responses of purified lamina propria (LP) CD4+ T cells from patients with IBD under defined conditions of T cell stimulation. We found that IBD LP CD4+ T cells in comparison with control LP CD4+ T cells have diminished TCR/CD3 pathway proliferative responses, whereas CD2/CD28 accessory pathway proliferative responses are relatively preserved. In further studies centering on lymphokine production, we showed that LP T cells from inflamed Crohn's disease mucosa manifest increased IFN-gamma secretion compared with control LP T cells, particularly when stimulated via the CD2/CD28 pathway. Subsequent ELISPOT analysis indicated that this was due to an increased number of IFN-gamma-secreting CD4+ T cells. In contrast, IL-4 and IL-5 production by Crohn's disease LP T cells was decreased compared with that of control LP T cells. Of interest, IL-2 production by Crohn's disease LP T cells was also reduced, as was IL-2 production by peripheral blood T cells. In parallel studies, LP T cells from inflamed ulcerative colitis mucosa stimulated via either the TCR/CD3/CD28 or CD2/CD28 produced increased amounts of IL-5, again when measured either as secreted IL-5 or by ELISPOT analysis. Such increased IL-5 production was not associated with increased IL-4 secretion and, in contrast to Crohn's disease, ulcerative colitis LP T cell production of IL-2 and IFN-gamma was normal. Taken together, these studies provide strong evidence that the immunopathologic process characteristic of the two major forms of IBD is associated with very different cytokine secretion patterns. These different patterns may determine the type of inflammatory process present.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Interferón gamma/biosíntesis , Interleucina-5/biosíntesis , Antígenos CD2/inmunología , Complejo CD3/inmunología , Antígenos CD4/inmunología , Diferenciación Celular , División Celular , Citometría de Flujo , Humanos , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Membrana Mucosa/inmunología , Transducción de SeñalRESUMEN
Circulating monocytes and vascular endothelial cells (EC) interact in a complex and dynamic manner that varies between vascular beds. The objective of this study was twofold: to ascertain if monocytic cell adhesion to vascular endothelium differed between specific anatomic regions of the canine aorta, and to investigate the effect of known EC stimulators on monocytic cell adhesion to cells from these regions. Initial in vitro studies measuring adherence of U937 cells, a human monocytic cell line, to canine jugular vein and aortic EC monolayers revealed a dose-dependent increase in adhesion to EC stimulated with interleukin-1 (IL-1), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or thrombin. While there was no regional difference in monocytic cell adherence to unstimulated EC in tissue culture, studies demonstrated greater monocytic cell adhesion to stimulated EC cultured from the distal versus proximal aorta. In organ culture, unstimulated adhesion of U937 cells or autologous monocytes was significantly greater to the distal aorta than the proximal aorta. Although monocytic cell adhesion to both the proximal and distal aorta increased with stimulation, the percentage increase in the proximal aorta, 1,086% with IL-1, 237% with PMA, 209% with LPS, and 174% with thrombin, was greater than in the distal aorta, demonstrating a significant functional difference in the endothelium from separate anatomic regions of a single vessel. This may have a direct relevance to the regional specificity of vascular disease.
Asunto(s)
Adhesión Celular/fisiología , Endotelio Vascular/citología , Monocitos/citología , Animales , Aorta/citología , Adhesión Celular/efectos de los fármacos , Línea Celular , Perros , Endotoxinas/farmacología , Interleucina-1/farmacología , Microscopía Electrónica de Rastreo , Técnicas de Cultivo de Órganos , Porcinos , Acetato de Tetradecanoilforbol/farmacología , Trombina/farmacologíaRESUMEN
Antioxidants have been proposed to be anti-atherosclerotic agents; however, the mechanisms underlying their beneficial effects are poorly understood. We have examined the effect of alpha-tocopherol (alpha-tcp) on one cellular event in atherosclerotic plaque development, monocyte adhesion to stimulated endothelial cells (ECs). Human umbilical vein ECs were pretreated with alpha-tcp before stimulation with known agonists of monocyte adhesion: IL-1 (10 ng/ml), LPS (10 ng/ml), thrombin (30 U/ml), or PMA (10 nM). Agonist-induced monocytic cell adhesion, but not basal adhesion, was inhibited in a time- and concentration-dependent manner by alpha-tcp. The IC50 of alpha-tcp on an IL-1-induced response was 45 microM. The inhibition correlated with a decrease in steady state levels of E-selectin mRNA and cell surface expression of E-selectin which is consistent with the ability of a monoclonal antibody to E-selectin to inhibit monocytic cell adhesion in this system. Probucol (50 microM) and N-acetylcysteine (20 mM) also inhibited agonist-induced monocytic cell adhesion; whereas, several other antioxidants had no significant effect. Protein kinase C (PKC) does not appear to play a role in the alpha-tcp effect since no suppression of phosphorylation of PKC substrates was observed. Activation of the transcription factor NF-kappa B is reported to be necessary but not sufficient for E-selectin expression in EC. Electrophoretic mobility shift assays failed to show an alpha-tcp-induced decrease in activation of this transcription factor after cytokine stimulation. It has been hypothesized that alpha-tcp acts as an anti-atherosclerotic molecule by inhibiting generation of oxidized LDL--a putative triggering molecule in the atherosclerotic process. Our results point to a novel alternative mechanism of action of alpha-tcp.
Asunto(s)
Endotelio Vascular/fisiología , Monocitos/efectos de los fármacos , Vitamina E/farmacología , Secuencia de Bases , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/genética , Células Cultivadas , Selectina E , Endotelio Vascular/citología , Humanos , Interleucina-1/farmacología , Datos de Secuencia Molecular , Monocitos/fisiología , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/análisisRESUMEN
Recent studies have shown that the synthetic peptides SFL LRN and SFL LRN PND KYEPF (thrombin receptor-activating peptides (TRAP)) derived from the deduced sequence of the new amino terminus of the cleaved thrombin receptor can mimic thrombin receptor activation, act as full agonists for platelet activation, and induce prostaglandin I2 production as well as cytosolic Ca2+ increase in human umbilical vein endothelial cells (HUVEC). Here, we have compared the ability of these synthetic peptide ligands and thrombin to stimulate platelet-derived growth factor (PDGF) production by, and monocyte adhesion to, HUVEC. Thrombin (50 units/ml) and TRAP (25 microM) maximally stimulated monocyte adhesion. Furthermore, the stimulation of E-selectin cell surface expression and the steady-state E-selectin mRNA levels by thrombin and TRAP were comparable. Thrombin (50 units/ml) stimulated PDGF production 400% above the basal level in 24 h, whereas the 6-mer and 14-mer TRAP, even at 200 microM, did not significantly stimulate PDGF production. Northern analysis, however, revealed that TRAP at 100 microM stimulated PDGF-A and -B chain mRNA expression to a level similar to that induced by thrombin. These results suggest that activation of cell signaling by TRAP can mimic thrombin and is sufficient for the stimulation of monocyte adhesion to HUVEC; however, thrombin-stimulated PDGF production by HUVEC may require mechanisms in addition to the signaling events initiated by TRAP or may require the participation of a novel thrombin receptor.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Endotelio Vascular/metabolismo , Monocitos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptores de Trombina/efectos de los fármacos , Secuencia de Aminoácidos , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Selectina E , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Monocitos/citología , Receptores de Trombina/metabolismoRESUMEN
Injury to the vascular endothelium and the subsequent inflammatory response are considered prerequisites for the development of atherosclerosis. Platelet-derived growth factor (PDGF) production by and monocyte adhesion to aortic endothelial cells (EC) may participate in this inflammatory process and therefore are two potential targets for control by anti-inflammatory agents. Our previous studies have demonstrated that monocyte adhesion and PDGF production are stimulated by thrombin in EC. Here, we provide evidence that treatment of EC with the anti-inflammatory agent 3-deazaadenosine (c3Ado) effectively abolished thrombin-stimulated PDGF production and monocyte adhesion. c3Ado had no significant effect on either basal monocyte adhesion or constitutive PDGF production. c3Ado was also effective in negating monocyte adhesion induced by other agonists, such as interleukin-1, phorbol 12-myristate 13-acetate (PMA), and lipopolysaccharide. Northern analysis demonstrated that c3Ado significantly reduced thrombin- and PMA-stimulated steady-state levels of PDGF-A chain, PDGF-B chain, and endothelial-leukocyte adhesion molecule-1 (ELAM-1) mRNAs. Nuclear run-on studies demonstrated that a marked transcriptional activation of these genes by thrombin and PMA was abrogated by c3Ado treatment. The transcriptional rate of the alpha-tubulin gene was unaffected by the drug. Antibody binding studies with an anti-ELAM-1 monoclonal antibody 7A9 revealed that thrombin-stimulated EC expression of ELAM-1 was abolished by c3Ado, indicating that the suppression of ELAM-1 expression on EC surface may be a mechanism by which c3Ado interferes with monocyte adhesion. Experiments with the nucleoside transport inhibitor nitrobenzylthioinosine suggested that the transport of c3Ado into EC was required for its inhibitory activity. In addition, L-homocysteine thiolactone was found to potentiate the inhibitory activity of c3Ado, suggesting that the accumulation of intracellular c3Ado homocysteine may be the underlying mechanism by which c3Ado inhibits thrombin-induced EC function. Taken together, these results indicate that c3Ado may prove effective against vascular injury and inflammation through its ability to inhibit induction of both monocyte adhesion and PDGF production.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Trombina/farmacología , Tubercidina/farmacología , Nucleótidos de Adenina/farmacología , Northern Blotting , Moléculas de Adhesión Celular/genética , Células Cultivadas , Selectina E , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Interleucina-1/farmacología , Isomerismo , Lipopolisacáridos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Factor de Crecimiento Derivado de Plaquetas/genética , Acetato de Tetradecanoilforbol/farmacología , Trombina/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Thrombin stimulates multiple functions in cultured endothelial cells (EC), including an increase in cell surface adhesion sites for monocytes and the production of platelet-derived growth factor (PDGF). We have initiated studies to define the intracellular signaling pathways involved in these two thrombin-induced EC functions by focusing on the possible roles of the Na(+)-H+ antiporter and guanine nucleotide-binding proteins (G proteins). Amiloride suppressed thrombin-stimulated PDGF production by human aortic EC without affecting either basal PDGF production or overall protein synthesis. The steady-state mRNA levels of PDGF-A and PDGF-B chain were not reduced by amiloride. In replicate EC cultures, amiloride had no effect on thrombin-stimulated monocyte adhesion. In addition, thrombin induction of PDGF production, but not monocyte adhesion, was abrogated in the absence of extracellular sodium. Thrombin stimulation of both monocyte adhesion and PDGF production appeared to involve a pertussis toxin-insensitive G protein. Thrombin induced an increase in [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to human EC membranes. GTP gamma S, in the presence of a suboptimal concentration of thrombin, caused maximal stimulation of both monocyte adhesion and PDGF production. The effect of GTP gamma S on PDGF production was at the level of transcription. These results indicate that the EC is capable of responding to a pluripotent agonist such as thrombin through multiple signaling pathways, which converge and diverge to achieve differential cellular responses.
Asunto(s)
Endotelio Vascular/metabolismo , Membranas Intracelulares/fisiología , Monocitos/fisiología , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Trombina/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Proteínas Portadoras/fisiología , Adhesión Celular , Endotelio Vascular/citología , Proteínas de Unión al GTP/fisiología , Humanos , Membranas Intracelulares/metabolismo , Intercambiadores de Sodio-HidrógenoRESUMEN
Monocyte adhesion to endothelium represents the first step in the emigration of this leukocyte from blood to tissue during such pathologic and physiologic processes as atherosclerotic plaque development, wound healing, and inflammation. We have examined the role of carbohydrate moieties in the binding of mononuclear cells to endothelium in vitro. Wheat germ agglutinin (WGA) completely inhibited binding of the human monocytic cell line U937 to pig or human endothelial cells (EC). The inhibition was abolished by the presence of N-acetyl glucosamine, a preferred ligand for WGA. This sugar itself, however, had no effect on monocytic cell binding to EC, suggesting that WGA is inhibiting the cell-cell interaction by binding to a distinct sugar moiety. We tested a series of simple and phosphorylated sugars for the ability to inhibit U937 cell binding to EC. Two phosphorylated disaccharides, lactose-1-phosphate and maltose-1-phosphate, but not 14 other sugars, caused complete suppression of monocyte adhesion to EC. Among the inactive sugars were mannose-6-phosphate and fructose-1-phosphate, which have been shown by others to markedly suppress lymphocyte adhesion to EC. A nonionic detergent, n-octyl-beta-D-glucopyranoside (octyl glucoside), which contains a sugar group as a hydrophilic moiety, also inhibited U937 cell or human monocyte binding to human or porcine EC. The inhibition was observed at a nontoxic concentration of octyl glucoside and appeared to be due to an effect on the monocytic cell rather than the EC. When suboptimal doses of WGA and octyl glucoside were added in combination to the U937 cell-EC adhesion assay, the level of inhibition was greatly reduced when compared with either of the inhibitors alone, suggesting an interaction between these two blocking agents. Lactose-1-phosphate, but not octyl glucoside or WGA, blocked neutrophil adhesion to EC. In summary, our results indicate that specific cell surface carbohydrate groups are required for the adhesion of monocytes to the endothelium.
Asunto(s)
Carbohidratos/fisiología , Adhesión Celular , Endotelio Vascular/fisiología , Monocitos/fisiología , Animales , Carbohidratos/farmacología , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Membrana Celular/fisiología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Glucósidos/farmacología , Humanos , Interleucina-1/farmacología , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Fosfatos de Azúcar/farmacología , Porcinos , Aglutininas del Germen de Trigo/farmacologíaRESUMEN
The coagulation protein thrombin has been shown to stimulate multiple endothelial-cell (EC) functions, including production of platelet-derived growth factor and of platelet-activating factor (PAF), and neutrophil adhesion. We have found that thrombin causes increased binding of monocytic cells (U937 cells and normal human monocytes) to cultured EC of various species. Maximum adhesion of monocytes to pig aortic EC occurred 6 h after thrombin treatment and remained elevated through 24 h. Stimulation of adherence by bovine alpha-thrombin was half-maximal at 15 units/ml, and reached a plateau at 50 units/ml. Catalytically inactive thrombin (phenylmethanesulphonyl fluoride-treated) had no effect on monocyte adhesion to EC. Heparin, but not the endotoxin antagonist polymyxin B, suppressed the stimulation of adhesion by thrombin without altering basal adhesion. Two lines of evidence suggested that protein kinase C (PKC) was involved in the intracellular signalling to increase monocyte adhesion to EC. First the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated monocytic-cell adhesion to EC at a dose consistent with stimulation of PKC (half-maximal response at 1-3 nM) and with a time course similar to that for thrombin stimulation (maximal by 4 h). Diacylglycerol, a physiological activator of PKC, also stimulated U937-cell adhesion to EC. Secondly, H7, a PKC inhibitor, completely blocked stimulation of monocyte adhesion to EC by thrombin or PMA. The structural analogue of H7, HA1004, which preferentially inhibits cyclic-AMP- and cyclic-GMP-dependent protein kinases, had no effect on stimulated monocyte adhesion. The PKC inhibitor also blocked the stimulation of monocyte adhesion to EC by interleukin-1 and endotoxin, but did not alter the basal level of monocyte binding to unstimulated EC. Thrombin stimulation of monocyte adhesion differed from the reported stimulation of neutrophil adhesion by thrombin in that the latter process reached a maximum in minutes rather than hours. In addition, neither PAF itself nor agents known to stimulate PAF production by EC, such as arachidonate and the Ca2+ ionophore A23187, had any effect on monocyte adhesion. These results demonstrate a PKC-dependent cytokine-like action of the coagulation protein thrombin in modulating monocytic-cell adhesion to EC, a phenomenon of potential importance in many pathological and physiological processes.
Asunto(s)
Endotelio Vascular/citología , Monocitos/citología , Proteína Quinasa C/fisiología , Trombina/farmacología , Animales , Aspirina/farmacología , Calcimicina/farmacología , Bovinos , Adhesión Celular/efectos de los fármacos , Heparina/farmacología , Humanos , Técnicas In Vitro , Factor de Activación Plaquetaria/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Porcinos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Neonatal infection of mice with Moloney murine leukemia virus (MuLV-M) results in the establishment of a chronic virus-carrier state. Such MuLV-carrier mice exhibit several immunologic abnormalities including generalized immunosuppression and autoimmunity. Previously, we found thymocytes from MuLV-M-carrier mice to be cytotoxic for normal syngeneic and allogeneic fibroblasts but not for xenogeneic (hamster) target cells. However, when the same syngeneic or allogeneic target cells were infected with MuLV-M, they were "spared" from the autoreactivity, leading us to speculate that the MuLV receptor on the target cell membrane was involved in the autoreactivity. To address this question, we tested MuLV-carrier thymocytes for their ability to lyse hamster/mouse-hybrid target cells; some of which possessed chromosome 5 (which codes for the ecotropic MuLV receptor). Of the nine hybrid cell lines initially tested, only the five clones that carried chromosome 5 were killed by the autoreactive thymocytes. In additional experiments, we noted that the cytotoxic reaction was inhibited in the presence of a monoclonal antibody that reacts with an MuLV-M gp70 epitope. The results suggest that the autoreactive cytotoxicity is mediated, at least in part, through the formation of a "bridge" between MuLV budding from the membrane of the thymocytes and the ecotropic MuLV receptor on the target cells.
Asunto(s)
Citotoxicidad Inmunológica , Leucemia Experimental/inmunología , Linfocitos T Citotóxicos/inmunología , Timo/citología , Animales , Anticuerpos Monoclonales/fisiología , Unión Competitiva , Fibroblastos/metabolismo , Leucemia Experimental/etiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Virus de la Leucemia Murina de Moloney/inmunología , Virus de la Leucemia Murina de Moloney/metabolismo , Receptores Virales/genética , Linfocitos T Citotóxicos/microbiología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de FusiónRESUMEN
Adhesion of blood-borne monocytes to the vascular endothelium is the first step in the infiltration of this leukocyte into the vessel wall or the interstitial space during inflammation. A significant role for the monocyte in both wound healing and atherogenesis is now well accepted. The molecular interactions involved in monocyte attachment to the endothelium are unknown. To study this phenomenon we have developed an in vitro system that uses the human monocytic tumor cell line U937 as a model for the blood-borne monocyte. 51Cr-labeled U937 cells were found to adhere with high affinity to cultured endothelial cells (ECs) from several sources. Much less binding was observed to either smooth muscle cells or fibroblasts from several species. Conditioned medium and cocultivation experiments ruled out the possibility that target cells could affect U937 cell binding by secretion of factors. Binding of U937 cells to porcine aortic ECs reached equilibrium after 30 min at 37 degrees C and 90 min at 4 degrees C with similar extent of binding at the two temperatures. Binding of U937 to the endothelium reached saturation at 9-12 U937 per porcine aortic EC (semi-confluent) with half-maximal binding at 1.5 X 10(6) U937 cells/ml. Bound cells dissociated with a half-life of 20 h at 37 degrees C. Adhesion of U937 cells was blocked by prior incubation of ECs with normal monocytes but not with platelets, lymphocytes, or neutrophils. Trypsin treatment or detergent solubilization of ECs inhibited U937 cell binding. A striking effect of EC density on monocytic cell adhesion was observed with bovine, rat, and porcine ECs. Confluent cultures of these cells exhibited negligible binding of U937, but when plated sparsely, the same cells were excellent targets for U937 cell adhesion. In addition, when confluent cultures of bovine aortic ECs were "wounded" with a cotton swab and then allowed to recover for 24 h at 37 degrees C, U937 cells were found to adhere most readily to the ECs migrating into the wound and neighboring the wound but not to ECs in the confluent monolayer away from the wound edge. These latter results may have implications for the focal adhesion of monocytes to the vessel wall in vivo.
Asunto(s)
Vasos Sanguíneos/citología , Monocitos/citología , Animales , Bovinos , Adhesión Celular , Recuento de Células , Línea Celular , Endotelio/citología , HumanosRESUMEN
Homozygous hr/hr mice of the HRS/J strain have a high incidence of lymphoma not seen in congenic hr/+ littermates. The suggestion has been made that a helper T cell defect in the immune system of the homozygotes may account for this. To further examine the immune status of these mice, we compared the ability of spleen cells of mice of both types to stimulate, as well as to generate, allogeneic cell-mediated cytolytic responses and to produce a soluble mediator of such responses. In addition, both types of mice were tested for their ability to produce interferon (IFN) or to have their splenic natural killer cell (NK) levels augmented by polyriboinosinic-polyribocytidylic acid (poly rI-rC). We found that the spleen cells of young or aging mice of either type were able to stimulate the generation of alloreactive cytotoxic cells, but they did not inappropriately stimulate the generation of syngeneic cytotoxic cells. Similarly, spleen cells of both types of mice produced a T helper cell-derived growth factor (interleukin 2) involved in the proliferation of cytolytic cells and generated a classical cell-mediated lympholysis (CML) response to alloantigen-bearing cells. Finally, spontaneous NK cell levels, as well as poly rI-rC augmented IFN and NK cell levels, were similar in both types of mice. Thus, it appears that if defects in the immune system are solely responsible for the high incidence of lymphoma in hr/hr mice, the defects involve components or regulatory aspects of that system that have yet to be defined.