CXCL12 estimula fibroblastos pulmonares a produzir CCL3, CXCL2, LTB4 e LTC4 via p38, MEK1/2, PI-3K e JNK

A quimiocina C-X-X motif ligand 12 (CXCL12) e seu receptor de quimiocina 4 (CXCR4) desenvolvem um papel critico na inflamacao das vias aereas. No entanto, os efeitos da ativacao da via CXCL12/CXCR4 sobre fibroblastos pulmonares ainda sao desconhecidos. Neste estudo, investigamos o efeito da via CXCL12/CXCR4 sobre a quimiocina (C-C motif) ligante 3 (CCL3) e (C-X-C motif) ligante 2 (CXCL2) e sobre os mediadores lipidicos leucotrienos B4 (LTB4) e C4 (LTC4) por fibroblastos pulmonares e a sinalizacao intracelular envolvida neste processo. CXCL12 foi capaz de induzir a producao de CCL3, CXCL2, LTB4 e LTC4; a producao de CCL3 nao e dependente da producao de CXCL2, mas a producao de CXCL2 e dependente da producao de CCL3. A producao de LTB4 pode ser parcialmente regulada por CXCL2 e CCL3 e a producao de LTC4 e dependente da producao de CCL3 e CXCL2. Fibroblastos pulmonares constitutivamente expressam CXCR4 e a estimulacao com CXCL12 induz sua expressao. Analises de Western blot mostraram que CXCL12 aumenta a expressao proteica de CXCR4 e induz a fosforilacao da S339 do CXCR4. A expressao genica constitutiva e induzida de CXCR4 foram inibidas pelo anticorpo anti-CXCL2. No entanto, o anticorpo anti-CCL3 e o inibidor farmacologico MK886 foram capazes de diminuir a expressao genica induzida de CXCR4. Os fibroblastos pulmonares foram pre-tratados com MK886, dexametasona (Dexa) e/ou loratadina (Lor). MK886 e Lor promoveram a diminuicao da producao de LTC4 e LTB4, mas nao a de CCL3 e CXCL2. Dexa diminuiu niveis de CCL3, CXCL2, LTB4 e LTC4, e quando associado com Lor esta diminuicao foi mais eficaz. Identificamos que as vias de sinalizacao intracelular PI-3K e JNK estao envolvidas na producao CCL3; p38, MEK1 / 2, PI-3K e JNK estao envolvidas na producao de CXCL2 e as vias p38 e MEK1/2 estao envolvidas na producao de LTB4 por meio da ativacao do eixo CXCL12/CXCR4 em fibroblastos pulmonares. CXCL12 induz o aumento de CCL3, CXCL2, LTB4 e LTC4 em fibroblastos pulmonares. (AU) C-X-X motif ligand 12 (CXCL12) and its specific receptor Chemokine receptor 4 (CXCR4) play a critical role in airway inflammation. However, the effects of CXCL12/CXCR4 axis on pulmonary fibroblast activation are unknown. In this study, we investigated the effect of CXCL12/CXCR4 axis on chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-X-C motif) ligand 2 (CXCL2), leukotrienes B4 (LTB4) and C4 (LTC4) production by pulmonary fibroblasts and the intracellular signaling involved in the process. CXCL12 induced CCL3, CXCL2, LTB4 and LTC4 production, and CCL3 production is not dependent on CXCL2; but CXCL2 production is dependent on CCL3 production. LTB4 production can be partially down-regulated by CXCL2 and CCL3 production and LTC4 production is dependent on CCL3 and CXCL2 production. Pulmonary fibroblasts constitutively expressed CXCR4, and CXCL12 stimulation up-regulated its expression. Western blot analysis showed that CXCL12 increased protein expression of CXCR4 and induced phosphorylation at S339 of CXCR4. Constitutive CXCR4 expression was decreased by anti-CCL3 antibody or MK 886. Inducible CXCR4 was inhibited by anti-CXCL2 antibody. Indeed pulmonary fibroblasts were pretreated with MK886, dexamethasone (Dexa) and loratadine (Lor). MK886 and loratadine was able to reduced LTB4 and LTC4 production but not CCL3 and CXCL2. Dexa decreased CCL3, CXCL2, LTB4 and LTC4 production, and when associated with Lor this decrease was more effective. We found that PI-3K and JNK intracellular signaling play a role in CCL3 production; p38, MEK1/2, PI-3K and JNK are involved in CXCL2 production and p38 and MEK1/2 pathways are involved in LTB4 production by CXCL12/CXCR4 axis activation in pulmonary fibroblasts. Taken together, our results suggest that CXCL12 increased CCL3, CXCL2, LTB4 and LTC4 production in pulmonary fibroblasts, activating different intracellular mechanisms, and playing an important role in production of pro-inflammatory mediators involved in the regulation. (AU)