The Study On Macrophages Phagocytosis Mechanism Regulated By CRH And Urocortin Via Rho GTPases

The mammalian neuroendocrine system and immune system broadly interact and permeate with each other, and both form a highly efficient cellular and molecular network, which plays decisive roles for maintaining organism homeostasis at different conditions, especially in the pathologic status of trauma or sepsis. The interrelation between neuroendocrine system and immune system recently has been become the important hot spot of neuroimmun-endocrinology. The neuropeptides, corticotropin- releasing hormone (CRH) and its structurally related peptide urocortin (UCN) are released under stress. The hypothalamus CRH and UCN modulate indirectly the inflammatory response via the hypothalamus-pituitary-adrenal (HPA) axis and locally, have been shown to participate in an autocrine/paracrine stimulation of inflammation. Previous studies have confirmed that severe trauma or infection can cause organismal immune functional disorder. Moreover, the change of macrophage immunity is an important causative factor of subseguent immunosuppression, resulting in the susceptivity of systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). While the mechanism of macrophage functional disorder is complicated, and it possibly relate to neuroendocrine hormone in microenvironment. Therefore, investigating the molecular mechanism of the functionality of CRH and UCN on macrophages could conduce to maintain organism homeostasis and was affluent in the regulation strategies to modulate the balance immunologic about post trauma immunity disorder.CRH and its structurally related peptides, their receptors (CRHR1 and CRHR2), distribute very extensive in central nervous system and peripheral organs and tissues. CRH receptors, like other G-protein-coupled receptors (GPCRs) can activate multiple G proteins, suggesting that CRH may regulate different signaling pathways. A great deal of information about CRH peptides has already been accumulated in regulating the behavioral, endocrine, autonomic, and immune responses to stress. Increasing evidences show that periphery CRH andUCN play an important role in regulating the immune/inflammatory response. As a main function in the innate immunity of macrophages, the phagocytosis process was always suppressed in the pathologic status of trauma or sepsis. Most of our knowledge of the molecular mechanisms of phagocytosis had been derived from studies in macrophages. However, much less is known about the regulation and its mechanisms of endogenous neuropeptides CRH and UCN on macrophages phagocytosis in the innate immunity.Thus, the present study aims to investigate the roles and mechanisms of CRH and UCN on macrophages phagocytosis. To employ the primary culture of the rat peritoneal macrophage as study object, firstly we studied the function of CRH and UCN on macrophages phagocytosis of fluorescence microballoons by means of the flow cytometry technology. Then the cytoskeletal remodeling during macrophages phagocytosis influenced by CRH and UCN was studied, and F-actin was showed by TRITC labeled by phalloidin. Subsequently, we elucidated the dose-effect and time-effect relationship of CRH and UCN on the phosphorylation of RhoA, Rac1, two members of the Rho family of GTPases that regulates the actin and microtubule cytoskeleton, in order to elaborate the role of Rho GTPases in translating CRH stimuli to cytoskeleton remodeling. In another parts of emperiment, the roles of CRH 1/2 type receptor and PKA, PKC, ERK1/2 signal molecule were studied on the activation of primary cultured peritoneal macrophages in order to interpret the signaling pathways of CRH and UCN to activate RhoA, Rac1.Main results:1. After the stimulation of CRH at 10-9~10-8M or UCN at 10-10~10-7M, the phagosytosis of rat peritoneal macrophages to polystyrene fluorescent microballoons enhanced significantly above in the normal condition by means of the quantitative flow cytometry.2. Under normal condition, F-actin filaments in macrophages homogeneously distributed through the cytoplasm. After CRH stimulaiton, F-actin in macrophages accumulated to the margin of the cells and pseudopodia increased around the cell membrane. Co-incubation of macrophages with UCN induced similar effects on F-actin distribution as CRH. Especially, those changes were all the more obviously at 30min, but actin filaments began to depolymerize after coincubation 60min. These F-actin dynamic changes were consistent with our observations that CRH /UCN could promote macrophages phagocytosis.3. After stimulation of CRH at the concentration of 10-8M, Rac1 phosphorylation increased markedly after co-incubation 30min, and RhoA phosphorylation reached its peak after co-incubation 60min. From 10-10M to 10-7M, UCN could highly significantly enhance RhoA,Rac1 phosphorylation. Especially, after stimulation of UCN at the concentration of 10-8M, Rac1 phosphorylation increased likewise markedly after co-incubation 30min; but RhoA phosphorylation took on bimodal changes in 60~120min after UCN stimulation.4. After W56 as a specific inhibitor of RhoA-ROCK, Y27632 as a specific inhibitor of Rac1 pre-incubation with macrophages, CRH or UCN stimulated phagocytosis enhancement was obviously inhibited. And blockade of Rho signals by the inhibitor W56 or Y27632 prevented CRH or UCN-triggered actin reorganization in macrophages, leading to round cells with incomplete development of cytoskeletal extensions.5. Pre-incubation of antalarmin, a specificity CRHR1 antagonist or astressin, a non-specificity CRHR1/2 antagonist with macrophages all could obviously inhibit phagocytosis enhancement stimulated by CRH or UCN. After antalarmin stimulation in advance, RhoA phosphorylation induced by CRH was markedly suppressed, and the inhibiting extent of Rac1 phosphorylation was nearly to that after astressin pre-incubation. Antalarmin stimulation in advance did not affect RhoA phosphorylation induced by UCN, and induced a minor suppression to Rac1 phosphorylation. While after astressin pre-incubation, the phosphorylation level of RhoA and Rac1 induced by UCN was greatly inhibited.6. Co-incubation of CRH or UCN with rest primary peritoneal macrophages induced cAMP-dependen PKA activity increase significantly. PKA activity reached its peak at 15min after CRH stimulation, and PKA activity showed a gradual increase tendency from 10min to 60min. The activity of PKC increased quickly in macrophages stimulated by CRH or UCN, and reached its peak at 15min. After MDL-12330A was used to selectly block PKA, RhoA phosphorylation stimulated by CRH or UCN was inhibited greatly. When we used chelerythrine chloride, a select blocker to PKC preincubated with macrophages 30min, RhoA phosphorylation induced by CRH increased, and Rac1 phosphorylation has no changes. While chelerythrine chloride preincubation to UCN induced RhoA phosphorylation descent by 63%, meanwhile Rac1 phosphorylation descent obvious, moreover lower to in control.7. After CRH stimulation at the concentration of 10-8M, the level of ERK1/2 phosphorylation in macrophages enhancd guickly, significantly at 5min and keeped on to 15min. While, co-incubation of UCN with macrophages induced ERK1/2 phosphorylation level to a gradual increase tendency from 5min to 30min. When PD98059 incubated 30min in advance, RhoA phosphorylation induced by CRH was completely suppressed. And when pre-incubated to UCN, the response of RhoA phosphorylation reversed completely, the suppressing effect of Rac1 phosphorylation was 47.7%.Conclusions:1. CRH and UCN can enhance macrophages phagocytosis, and the promoting phagocytosis of UCN is better than that of CRH. The role of CRH on macrophages phagocytosis has a concentration dependent characteristic, after CRH at 10-8M stimulation the effect of phagocytosis is markedly elevated. From 10-10M to 10-7M concentration, UCN even can obviously promote macrophages phaocytosis to fluorescent microsphere.2. CRH and UCN can induce cytoskeletal remodeling in macrophages, preparing for augmented phaphagocytosis. Stimulation with CRH or UCN triggered a shape change with accumulation of F-actin on the margin of the cells, the formation of lamellipodia and focal intensities across the cytoplasm, and a profusion of elongated pseudopodia.3. Rho GTPases plays an important role in modulating innate phagocytosis immunity through endogenous neuropeptide CRH and UCN. CRH and UCN can promote RhoA/Rac1 phosphorylation in macrophages, then trigger cytoskeletal remodeling, leading to the enhancement of macrophages phagocytosis.4. CRH and UCN regulate macrophages phagocytosis in rat peritoneal macrophage through different signal transduction pathways in a CRH receptor type-specific manner, eventually determining actin rearrangement by Rho specific pathways. CRH has been demonstrated to promote Rac1 and RhoA phosphorylation through bingding to CRHR1, via the cAMP-PKA/ERK1/2 pathways. The effects of UCN on Rac1 and RhoA phosphorylation are through CRHR2 and activation the PKC/ERK1/2 signal pathways.5. CRH and its structurally related peptide UCN, play similar roles on promoting macrophages phagocytosis. But their signal transduction pathways are different, which reflects that their difference in regulating the phagotrophic ability and the cytoskeletal remodeling during macrophage phagocytosis. By regulating the cytoskeletal remodeling, these peptides may determine the degree to macrophage phagocytosis influenced by exogenous stimula in organismal microenvironment.