How hydrogen peroxide and CXCL8 modulate neutrophil recruitment "in vivo"

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2014

Pulmonary neutrophil recruitment and bronchial reactivity in formaldehyde-exposed rats are modulated by mast cells and differentially by neuropeptides and nitric oxide

We have used a pharmacological approach to study the mechanisms underlying the rat lung injury and the airway reactivity changes induced by inhalation of formaldehyde (FA) (1% formalin solution, 90 min once a day, 4 days). The reactivity of isolated tracheae and intrapulmonary bronchi were assessed in dose-response curves to methacholine (MCh). Local and systemic inflammatory phenomena were evaluated in terms of leukocyte countings in bronchoalveolar lavage (BAL) fluid, blood, bone marrow lavage and spleen. Whereas the tracheal reactivity to MCh did not change, a significant bronchial hyporesponsiveness (BHR) was found after FA inhalation as compared with naive rats. Also, FA exposure significantly increased the total cell numbers in BAL, in peripheral blood and in the spleen, but did not modify the counts in bone marrow. Capsaicin hindered the increase of leukocyte number recovered in BAL fluid after FA exposure. Both compound 48/80 and indomethacin were able to prevent the lung neutrophil influx after FA, but indomethacin had no effect on that of mononuclear cells. Following FA inhalation, the treatment with sodium cromoglycate (SCG), but not with the nitric oxide (NO) synthase inhibitor L-NAME, significantly reduced the total cell number in BAL. Compound 48/80, L-NAME and SCG significantly prevented BHR to MCh after FA inhalation, whereas capsaicin was inactive in this regard. on the other hand, indomethacin exacerbated BHR. These data suggest that after FA inhalation, the resulting lung leukocyte influx and BHR may involve nitric oxide, airway sensory fibers and mast cell-derived mediators. The effect of NO seemed to be largely restricted to the bronchial tonus, whereas neuropeptides appeared to be linked to the inflammatory response, therefore indicating that the mechanisms responsible for the changes of airway responsiveness caused by FA may be separate from those underlying its inflammatory lung effects. (c) 2005 Elsevier B.V. All rights reserved.

MTA-induced neutrophil recruitment: a mechanism dependent on IL-1β, MIP-2, and LTB4

Objective: The objective of this study was to investigate the mediators and the resident peritoneal cells involved in the neutrophil migration (NM) induced by mineral trioxide aggregate (MTA) in mice. Study design: MTA (25 mg/cavity) was injected into normal and pretreated peritoneal cavities (PC) with indomethacin (IND), dexamethasone (DEX), BWA4C, U75302, antimacrophage inflammatory protein-2 (MIP-2), and anti-interleukin-1β (IL-1β) antibodies and the NM was determined. The role of macrophage (MO) and mast cells (MAST) was determined by administration of thioglycollate 3% or 48/80 compound, respectively. The concentration of IL-1β and MIP-2 exudates was measured by ELISA. Results: MTA induced dose- and time-dependent NM into mice PC, with the participation of MO and MAST. NM was inhibited by DEX, BWA4C, and U75302, as well as anti-MIP-2 and anti-IL-1β antibodies. In the exudates, IL-1β and MIP-2 were detected. Conclusions: This study suggests that MTA induces NM via a mechanism dependent on MAST and MO mediated by IL-1β, MIP-2, and LTB4. © 2008 Mosby, Inc. All rights reserved.

NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B4 in a murine model of gout

Objective Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)derived leukotriene B4 (LTB4) in driving tissue inflammation and hypernociception in a murine model of gout. Methods. Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1 beta (IL-1 beta), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB4 activity, cytokine (IL-1 beta, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. Results. Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophildependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1 beta/MyD88-dependent manner. LTB4 was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1 beta production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB4 after MSU crystal injection, and LTB4 was relevant in the MSU crystalinduced maturation of IL-1 beta. Mechanistically, LTB4 drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. Conclusion. These results reveal the role of the NLRP3 inflammasome in mediating MSU crystalinduced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB4 in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNF alpha and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor a (TNF alpha), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ETA/ETB receptor antagonist bosentan, and selective ETA or ETB receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFa and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c+ markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ETA-and ETB-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNF alpha and CXCL1/CXCR2-dependent mechanism.

Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury.

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital Microscopic Studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression Of L-selectin and beta(2)-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to NO, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. (C) 2009 Elsevier Inc. All rights reserved.

Low intensity laser therapy (LILT) in vivo acts on the neutrophils recruitment and chemokines/cytokines levels in a model of acute pulmonary inflammation induced by aerosol of lipopolysaccharide from Escherichia coli in rat

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Annexin-A1 peptide down-regulates the leukocyte recruitment and up-regulates interleukin-10 release into lung after intestinal ischemia-reperfusion in mice

Background: Intestinal ischemia/reperfusion (IR) injury is a serious and triggering event in the development of remote organ dysfunction, from which the lung is the main target. This condition is characterized by intense neutrophil recruitment, increased microvascular permeability. Intestinal IR is also responsible for induction of adult respiratory distress syndrome, the most serious and life-threatening form of acute lung injury. The purpose of this study was to investigate the effect of annexin-A1 protein as an endogenous regulator of the organ remote injury induced by intestinal ischemia/reperfusion. Male C57bl/6 mice were subjected to intestinal ischemia, induced by 45 min occlusion of the superior mesenteric artery, followed by reperfusion. Results: The intestinal ischemia/reperfusion evoked a high intensity lung inflammation as indicated by the number of neutrophils as compared to control group. Treatment with annexin-A1 peptidomimetic Ac2-26, reduced the number of neutrophils in the lung tissue and increased its number in the blood vessels, which suggests a regulatory effect of the peptide Ac2-26 in the neutrophil migration. Moreover, the peptide Ac2-26 treatment was associated with higher levels of plasma IL-10. Conclusion: Our data suggest that the annexin-A1 peptidomimetic Ac2-26 treatment has a regulatory and protective effect in the intestinal ischemia/reperfusion by attenuation of the leukocyte migration to the lung and induction of the anti-inflammatory cytokine IL-10 release into the plasma. The anti-inflammatory action of annexin-A1 and its peptidomimetic described here may serve as a basis for future therapeutic approach in mitigating inflammatory processes due to intestinal ischemia/reperfusion. © 2013 Guido et al.; licensee BioMed Central Ltd.

M-ficolin and leukosialin (CD43): new partners in neutrophil adhesion

M-ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM-ficolin bound CD43 and prevented the access of anti-CD43 mAb. Moreover, rM-ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M-ficolin is secreted by fMLP-activated neutrophils, and this endogenous M-ficolin also binds to CD43 and competes with anti-CD43 mAb. Anti-CD43 antibody cross-linking or fMLP resulted in M-ficolin and CD43 colocalization on polarized neutrophils. The binding of rM-ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti-CD43 mAb. The M-ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM-ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M-ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M-ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual-adhesive/antiadhesive roles of CD43 in neutrophil recruitment. J. Leukoc. Biol. 91: 469-474; 2012.

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling

The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response. (C) 2011 Elsevier Inc. All rights reserved.

Annexin-A1 peptide down-regulates the leukocyte recruitment and up-regulates interleukin-10 release into lung after intestinal ischemia-reperfusion in mice

Abstract Background Intestinal ischemia/reperfusion (IR) injury is a serious and triggering event in the development of remote organ dysfunction, from which the lung is the main target. This condition is characterized by intense neutrophil recruitment, increased microvascular permeability. Intestinal IR is also responsible for induction of adult respiratory distress syndrome, the most serious and life-threatening form of acute lung injury. The purpose of this study was to investigate the effect of annexin-A1 protein as an endogenous regulator of the organ remote injury induced by intestinal ischemia/reperfusion. Male C57bl/6 mice were subjected to intestinal ischemia, induced by 45 min occlusion of the superior mesenteric artery, followed by reperfusion. Results The intestinal ischemia/reperfusion evoked a high intensity lung inflammation as indicated by the number of neutrophils as compared to control group. Treatment with annexin-A1 peptidomimetic Ac2-26, reduced the number of neutrophils in the lung tissue and increased its number in the blood vessels, which suggests a regulatory effect of the peptide Ac2-26 in the neutrophil migration. Moreover, the peptide Ac2-26 treatment was associated with higher levels of plasma IL-10. Conclusion Our data suggest that the annexin-A1 peptidomimetic Ac2-26 treatment has a regulatory and protective effect in the intestinal ischemia/reperfusion by attenuation of the leukocyte migration to the lung and induction of the anti-inflammatory cytokine IL-10 release into the plasma. The anti-inflammatory action of annexin-A1 and its peptidomimetic described here may serve as a basis for future therapeutic approach in mitigating inflammatory processes due to intestinal ischemia/reperfusion.

Annexin-A1 peptide down-regulates the leukocyte recruitment and up-regulates interleukin-10 release into lung after intestinal ischemia-reperfusion in mice

BACKGROUND: Intestinal ischemia/reperfusion (IR) injury is a serious and triggering event in the development of remote organ dysfunction, from which the lung is the main target. This condition is characterized by intense neutrophil recruitment, increased microvascular permeability. Intestinal IR is also responsible for induction of adult respiratory distress syndrome, the most serious and life-threatening form of acute lung injury. The purpose of this study was to investigate the effect of annexin-A1 protein as an endogenous regulator of the organ remote injury induced by intestinal ischemia/reperfusion. Male C57bl/6 mice were subjected to intestinal ischemia, induced by 45 min occlusion of the superior mesenteric artery, followed by reperfusion. RESULTS: The intestinal ischemia/reperfusion evoked a high intensity lung inflammation as indicated by the number of neutrophils as compared to control group. Treatment with annexin-A1 peptidomimetic Ac2-26, reduced the number of neutrophils in the lung tissue and increased its number in the blood vessels, which suggests a regulatory effect of the peptide Ac2-26 in the neutrophil migration. Moreover, the peptide Ac2-26 treatment was associated with higher levels of plasma IL-10. CONCLUSION: Our data suggest that the annexin-A1 peptidomimetic Ac2-26 treatment has a regulatory and protective effect in the intestinal ischemia/reperfusion by attenuation of the leukocyte migration to the lung and induction of the anti-inflammatory cytokine IL-10 release into the plasma. The anti-inflammatory action of annexin-A1 and its peptidomimetic described here may serve as a basis for future therapeutic approach in mitigating inflammatory processes due to intestinal

Regulation of leukocyte recruitment by the long pentraxin PTX3

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.