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| Author(s): |
Isabel Daufenback Machado
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2013-12-17 |
| Examining board members: |
Sandra Helena Poliselli Farsky;
Ricardo Ambrosio Fock;
Cristiane Damas Gil;
Wothan Tavares de Lima;
Sonia Maria Oliani
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| Advisor: | Sandra Helena Poliselli Farsky |
| Abstract | |
The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1α/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 µg/animal, i.p.) or LPS injection (100 µg/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 µg/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1α and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 µg/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1α/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1α were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils. (AU) | |
| FAPESP's process: | 10/08402-2 - Role of endogenous glucocorticoids and annexin A1 on neutrophil migration: involvement of SDF1 alpha/CXCR-4 and IL-17/IL-23 |
| Grantee: | Isabel Daufenback Machado |
| Support Opportunities: | Scholarships in Brazil - Doctorate |