RESUMO
OBJECTIVE: To evaluate the effects of anti-CD44 IM7.8.1 antibody, HMW-HA and LMW-HA on leukocyte migration and adhesion, and the induction of proinflammatory mediators, in mouse air-pouch inflammation induced by zymosan. METHODS: Leukocytes were obtained from zymosan-air pouches after the intra-pouch injection of anti-CD44 IM7.8.1, isotype control, HMW-HA, LMW-HA or PBS. TNF-alpha, IL-1beta and iNOS mRNA were estimated in leukocytes by semi-quantitative RT-PCR. Matrix metalloproteinases (MMPs) from exudates were evaluated by zymography and Western Blot. Adhesion and migration of leukocytes were evaluated in HA-coated plates and Boyden chambers respectively. RESULTS: IM7.8.1 decreased iNOS mRNA levels and the activity of both MMP-9 and MMP-2 eight h after injection into zymosan air pouch while IM7.8.1, HMW-HA and LMW-HA had no effect on IL1-beta or TNF-alpha mRNA levels. Leukocytes from air pouch adhered to and migrated in vitro against both HMW-HA and LMW-HA. LMW-HA increased the number of leukocytes in the air pouch and iNOS mRNA levels as compared to PBS injection. In contrast, HMW-HA decreased leukocyte count and reduced iNOS mRNA levels. Paradoxically, the activity of both MMP-9 and MMP-2 was increased by HMW-HA and decreased by LMW-HA. CONCLUSIONS: Both CD44 and HA can modulate leukocyte migration and induction of proinflammatory mediators in mouse zymosan air pouch inflammation. IM7.8.1 had consistent anti-inflammatory effects, reducing iNOS, MMP-9 and MMP-2. HMW-HA and LMW-HA were able to modulate both the induction of proinflammatory mediators and leukocyte count in the air pouch.
Assuntos
Receptores de Hialuronatos/biossíntese , Ácido Hialurônico/biossíntese , Metaloproteinases da Matriz/metabolismo , Óxido Nítrico Sintase/biossíntese , Animais , Anticorpos Monoclonais/química , Western Blotting , Adesão Celular , Movimento Celular , Quimiotaxia , Citocinas/metabolismo , Primers do DNA/química , Heparina de Baixo Peso Molecular/metabolismo , Receptores de Hialuronatos/química , Ácido Hialurônico/química , Inflamação , Interleucina-1/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Reação em Cadeia da Polimerase , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To study local inflammation induced by zymosan in the murine air pouch, considered a model of synovial-like tissue inflammation, we investigated the time-course synthesis of CD44 and tumor necrosis factor-alpha (TNF-alpha) mRNA and established a relationship with leukocyte migration into the air pouch and CD44 expression on the leukocyte membrane. METHODS: Leukocytes from the air pouch exudate were collected and counted at 1, 4, 12, 24, 48, and 72 h after zymosan or saline injection. CD44 and TNF-alpha mRNA were studied by RT-PCR. CD44 variable exon analysis was assessed by Southern blot and CD44 membrane expression by flow cytometry. RESULTS: Leukocyte accumulation after zymosan injection was significantly higher than in saline injected controls. CD44 standard and variable isoforms including at least variable exons v6 and v9 were highly expressed in leukocytes from the zymosan air pouch exudate. In contrast, only the CD44 mRNA standard isoform was present in leukocytes from saline air pouch. Maximal TNF-alpha mRNA level was observed at 48 h after zymosan injection, whereas CD44 mRNA was constantly expressed throughout the whole term of the experiment, although variations in leukocyte count and relative formula were observed. CONCLUSION: Expression of CD44 variable isoform in leukocytes was specifically induced by zymosan, since none was detected in saline controls. TNF-alpha mRNA expression and leukocyte count at every time point served as markers for local inflammation. The presence of variable isoforms, including at least exons v6 and v9, consistently expressed throughout the assay suggests that they could play a role in this arthritis-like inflammation induced under zymosan stimulus.
Assuntos
Artrite Reumatoide/imunologia , Receptores de Hialuronatos/genética , Sinovite/imunologia , Zimosan , Animais , Anticorpos Monoclonais , Artrite Reumatoide/induzido quimicamente , Southern Blotting , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Éxons , Exsudatos e Transudatos/imunologia , Citometria de Fluxo , Expressão Gênica/imunologia , Receptores de Hialuronatos/química , Receptores de Hialuronatos/imunologia , Isomerismo , Leucócitos/química , Leucócitos/citologia , Leucócitos/imunologia , Camundongos , RNA Mensageiro/análise , Sinovite/induzido quimicamente , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.
Assuntos
Adjuvantes Imunológicos/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Leucemia de Células T/patologia , Células Tumorais Cultivadas/patologia , Animais , Divisão Celular , Intervalos de Confiança , Citometria de Fluxo , Leucemia de Células T/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Ligação Proteica , Células Tumorais Cultivadas/metabolismoRESUMO
Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity.
Assuntos
Receptores de Hialuronatos/metabolismo , Enteropatias/imunologia , Traumatismo por Reperfusão/imunologia , Adjuvantes Imunológicos/metabolismo , Animais , Modelos Animais de Doenças , Receptores de Hialuronatos/genética , Enteropatias/metabolismo , Isquemia/imunologia , Isquemia/metabolismo , Fígado/imunologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Traumatismo por Reperfusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.
RESUMO
Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity.