Os neutrófilos estão entre as principais células da imunidade inata e são as primeiras células a migrarem para o sítio de infecção. O BAY 41-2272, estimulador de guanilato ciclase solúvel, é capaz de ativar fagócitos mononucleares e em neutrófilos diminuir migração in vivo e in vitro. O presente trabalho teve como objetivo avaliar o potencial do BAY 41-2272, e sua via, como uma ferramenta farmacológica para modulação da função dos neutrófilos. Para isso foi realizado o tratamento in vitro dos PMNs com o BAY 41-2272. A viabilidade celular, quimiotaxia e funções efetoras como burst oxidativo e produção de citocinas foram avaliadas, observando-se que o BAY 41-2272, como ativador direto, não altera estas funções. No entanto, o pré-tratamento com BAY 41-2272 nas doses de 3 M e 30M por uma hora, com subsequente ativação com PMA, mostrou perfil inibitório na quimiotaxia, produção da citocina IL-8 e no burst oxidativo . Foram avaliados também a expressão de moléculas como CD15, CD31, CD35, CD49d, CD63, CD66b, CD162 e a produção de GMPc. As moléculas de superfícies não mostraram alterações após tratamento direto com BAY 41-2272. A produção de GMPc foi induzida na dose de 30 M de BAY 41-2272 e pelo SNAP (doador de NO). Esses dados sugerem um potencial inibitório do BAY 41-2272 sobre a ação dos neutrófilos, e uma possível alternativa a ser explorada em seus aspectos translacionais na busca por novas terapias destinadas ao controle de doenças ligadas a inflamações crônicas e doenças auto-imunes. / Neutrophils are the major cells of innate immunity and are the first cells to migrate to the site of infection. BAY 41-2272, a soluble guanylate cyclase stimulator, is capable of activating mononuclear phagocytes and in neutrophils decreasing migration in vivo and in vitro. The present work aimed to evaluate the potential of BAY 41-2272, and its pathway, as a pharmacological tool for modulating neutrophil function. For this, the in vitro treatment of PMNs with BAY 41-2272 was performed. Cell viability, chemotaxis and effector functions such as oxidative burst and cytokine production were evaluated, observing that BAY 41-2272, as a direct activator, does not alter these functions. However, pretreatment with BAY 41-2272 at the doses of 3 M and 30 M for one hour, with subsequent activation with PMA, showed an inhibitory profile in chemotaxis, IL-8 cytokine production and in the \"oxidative burst\". We also evaluated the expression of molecules such as CD15, CD31, CD35, CD49d, CD63, CD66b, CD162 and cGMP production. Surface molecules did not show changes after direct treatment with BAY 41-2272. The cGMP production was induced at the dose of 30 M BAY 41-2272 and for SNAP (NO donor). These data suggest an inhibitory potential of BAY 41-2272 on the action of neutrophils, and a possible alternative to be explored in its translational aspects in the search for new therapies aimed at the control of diseases linked to chronic inflammation and autoimmune diseases.

During hematopoiesis, multilineage progenitor cells and the precursors are committed to individual hematopoietic lineages. In normal myelopoiesis, the immature myeloid cells (IMCs) differentiate into macrophages, neutrophils or dendritic cells. However, under tumor burden, these IMCs differentiate into myeloid derived suppressor cells (MDSCs) result in an up-regulation of immune suppressive factors and pro-tumor effect. The development of normal or malignant is tightly controlled by endogenous signals such as transcription factors and epigenetic regulations. HDAC11 is the newest identified members of the histone deacetylase (HDAC) family. Previous study in our group had identified HDAC11 as a negative regulator of interleukin 10 (IL-10) production in antigen-presenting cells (APCs). However, the mechanisms of HDAC11 in regulating myeloid cells differentiation and function remained unclear. We have uncovered for the first time that in the absence of HDAC11, upon LPS stimulation, neutrophils isolated form mice displays an over-production of pro-inflammatory cytokines such as TNF-alpha and IL-6. Strikingly, these HDAC11KO neutrophils showed a significantly higher migratory and phagocytosis activity, resulting from an overexpression of the migratory receptor and cytokine CXCR/L2. We have performed Chromatin Immunoprecipitation (ChIP) analysis on the neutrophils and discovered that HDAC11 was recruited to the promoter regulatory region of these genes we have identified. This part of data will be discussed mainly in chapter 2. Not only does HDAC11 plays a crucial role in the neutrophil function, our group have also found out that lacking of HDAC11 result in an increased suppressive activity of the Myeloid-derived Suppressor Cells (MDSCs). The previous publication of our group had shown that the tumor bearing mice experienced a much more aggressive growth pattern in the HDAC11 KO mice compare with C57BL/6 wild type control. MDSCs isolated from mice lacking HDAC11 appeared to gain increased capability to suppress the function of antigen-specific CD8+ T cells in vitro. Followed by this initial study, in chapter 3, we observed an up-regulation of both expression and enzymatic activity of arginase 1 and Nos2, two enzymes that are crucial in regulating MDSCs suppressive function. The aberrant enzymatic activity of Arg1 and Nos2 in HDAC11KO MDSCs is possibly result from an over-expression of the lineage-specific transcription factor C/EBPβ, which is previously proved to be essential for the differentiation of functional MDSCs. Furthermore, our ChIP data confirmed that HDAC11 may play as an negative regulator of C/EBPβ. Recently, our lab had demonstrated that T cells lacking HDAC11 gained a hyperactive phenotype and anti-tumor effect, indicating that HDAC11 may play a dual role in the host immune system. We further performed an adoptive transfer therapy to C57BL/6 tumor bearing mice. Our data showed that the additional administration of HDAC11KO MDSCs could eliminate, at least partially, the anti-tumor effect by adoptive transfer of HDAC11KO T cells. Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator in the myeloid cells differentiation and function. Based on our data and previous work from our lab, we propose a dual role of HDAC11 played in the host immune system. In the absence of HDAC11, host defenders such as neutrophils and T cells are functionally more aggressive against intruders such as pathogen and cancer. However, the immune suppressors such as MDSCs became more suppressive. The contradictory role HDAC11 played in the immune system may provide some insights for the assessment of the pharmacological value of HDAC11 and contribute to the development of novel immunotherapeutic strategies.

Includes bibliographical references. / xvii, 228 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Methods were developed to observe and quantify neutrophil transendothelial migration, both together with or independently of neutrophil-endothelial adhesion. Two mechanicisms of neutrophil transmigration were identified, and the role of one of the major mediators of this process, IL-8, was described. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine 1994

Copies of author's previously published articles inserted. / Includes bibliographical references. / vii, 39 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Shows that the cytokine tumour necrosis factor [alpha] (TNF-[alpha]) enhances the adhesion of neutrophils to the endothelium by an action both on the neutrophil and on the endothelial cell. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

<p>Granulocyte adhesion and release of their granule proteins are key steps during selective accumulation of a certain cell to an inflammatory site. Eosinophils are specifically recruited to sites of allergic inflammation and parasitic infection, whereas neutrophil influx predominates in bacterial infection and rheumatoid arthritis. </p><p>A simple, reliable and convenient method was developed for the measurement of granulocyte adhesion and release of granule proteins by using the normal population of granulocytes. The design allows simultaneous quantitative assessment of eosinophil and neutrophil adhesion to proteins and degranulation. </p><p>Using this method, manganese ions (Mn²⁺) induced a higher level of eosinophil adhesion to fibronectin, fibrinogen and albumin as compared with neutrophils. PMA induced comparable levels of eosinophil and neutrophil adhesion. F-MLP stimulated a rapid, short-term adhesion of neutrophils to fibrinogen. </p><p>In the same conditions PMA alone stimulated a dose-dependent release of ECP from cells that adhered to both fibronectin and fibrinogen. Meanwhile, Mn²⁺ amplified the release of ECP induced by PMA. Furthermore, release of ECP was shown to be associated with cell death.</p><p>PMA, in combination with Mn²⁺, induced a marked release of ~ 80%of the intracellular content of lactoferrin and HNL in neutrophils. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL. A maximal release of MPO of 15-20% was obtained from neutrophils stimulated by PMA and Mn²⁺. Release of lactoferrin and HNL showed a significant negative relationship to the viability of cells.</p><p>Stimulated by PMA, eosinophils from pollen-atopic patients during early pollen season displayed a markedly enhanced adhesion and release of ECP of eosinophils compared with eosinophils from the references. Priming with IL-5 caused a significantly higher adhesion and release of ECP by eosinophils in response to PMA. GM-CSF priming enhanced eosinophil adhesion in response to PAF and PMA plus Mn²⁺, but did not enhance the release of ECP.</p><p>In conclusion, the assay allows a simple quantification of eosinophil and neutrophil adhesion, as well as degranulation by using the normal population of granulocytes. Cellular adhesion plays an important role in the regulation of both eosinophil and neutrophil degranulation, but adhesion and degranulation can be induced separately.</p>

The ability of human polymorphonuclear leukocytes (PMNL; neutrophils), to sense and move to sites of infection is essential for our defense against pathogens. Cell motility is critically dependent on a dynamic remodeling of morphology. The morphological polarization toward chemoattractants, such as N-formyl-Met-Leu-Phe (fMLF), is associated with temporary extension and stabilization of lamellipodia in the direction of movement. The underlying mechanisms of cell motility are, however, still not entirely elucidated. It is therefore an urgent task to extend the present experimental evidence to give solid basis for a comprehensive model. Here it is shown that nitric oxide (NO) stimulates the morphological response of neutrophils, most likely due to transient increases in [Ca2+]i, following addition of NO-donors. This will, hypothetically, activate gelsolin and other actin filament severing proteins, leading to a subsequent decrease in filamentous actin. The incapability to efficiently turnover the actin filament network then blocks all motile activity. It is also shown that N-formyl peptide receptors on polarized neutrophils accumulate non-uniformly towards regions involved in motility. It is suggested that neutrophils use the asymmetric receptor distribution for directional sensing and sustained migration. A model for lamellipodium extension, where water fluxes play a pivotal role is presented. It is suggested that water fluxes through water-selective aquaporin (AQP) channels, contribute to the propulsive force for formation of various membrane protrusions and, thus, cell motility. It is well known that small G proteins of the Rho family GTPases play important roles in the intracellular signaling underlying cell motility. In morphologically polarized neutrophils it is shown that Cdc42, Rac2 and RhoA display spatially distinct distributions, which allows for sequential chemoattractant stimulation of neutrophil motility. The specific localizations of Rac2, Cdc42 and RhoA relative to each other and filamentous actin and fMLF receptors support the hypothesized order of activation and regulation of neutrophil cell motility. In conclusion, the detailed analysis of motility-related issues presented here provide new data allowing further refinement of previous models of neutrophil motility.

Granulocyte adhesion and release of their granule proteins are key steps during selective accumulation of a certain cell to an inflammatory site. Eosinophils are specifically recruited to sites of allergic inflammation and parasitic infection, whereas neutrophil influx predominates in bacterial infection and rheumatoid arthritis. A simple, reliable and convenient method was developed for the measurement of granulocyte adhesion and release of granule proteins by using the normal population of granulocytes. The design allows simultaneous quantitative assessment of eosinophil and neutrophil adhesion to proteins and degranulation. Using this method, manganese ions (Mn2+) induced a higher level of eosinophil adhesion to fibronectin, fibrinogen and albumin as compared with neutrophils. PMA induced comparable levels of eosinophil and neutrophil adhesion. F-MLP stimulated a rapid, short-term adhesion of neutrophils to fibrinogen. In the same conditions PMA alone stimulated a dose-dependent release of ECP from cells that adhered to both fibronectin and fibrinogen. Meanwhile, Mn2+ amplified the release of ECP induced by PMA. Furthermore, release of ECP was shown to be associated with cell death. PMA, in combination with Mn2+, induced a marked release of ~ 80%of the intracellular content of lactoferrin and HNL in neutrophils. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL. A maximal release of MPO of 15-20% was obtained from neutrophils stimulated by PMA and Mn2+. Release of lactoferrin and HNL showed a significant negative relationship to the viability of cells. Stimulated by PMA, eosinophils from pollen-atopic patients during early pollen season displayed a markedly enhanced adhesion and release of ECP of eosinophils compared with eosinophils from the references. Priming with IL-5 caused a significantly higher adhesion and release of ECP by eosinophils in response to PMA. GM-CSF priming enhanced eosinophil adhesion in response to PAF and PMA plus Mn2+, but did not enhance the release of ECP. In conclusion, the assay allows a simple quantification of eosinophil and neutrophil adhesion, as well as degranulation by using the normal population of granulocytes. Cellular adhesion plays an important role in the regulation of both eosinophil and neutrophil degranulation, but adhesion and degranulation can be induced separately.

Background: A multitude of studies suggest an association between periodontal disease and adverse birth outcomes. Although the evidence is controversial, it is biologically plausible, and the key link maybe inflammation. Purpose: To correlate levels of periodontal disease as measured by conventional methods with PMN counts from an oral rinse in pregnant women. Methods: Sixty-three pregnant women were recruited. Periodontal examinations were carried out. Fifteen-second saline rinses were collected. ABTS, a colour changing redox agent was added to each rinse. The intensity of the colour reaction was measured by absorbance to count the oral PMNs. Results: A statistically significant difference in oral PMN counts was observed between those with periodontal disease and the healthy patients (P < .05). The sensitivity and specificity using the bleeding index was 0.94 and 0.76 respectively. Conclusions: The rinse assay can be used as a screening tool for periodontal disease and oral inflammation in pregnant women.

Background: A multitude of studies suggest an association between periodontal disease and adverse birth outcomes. Although the evidence is controversial, it is biologically plausible, and the key link maybe inflammation. Purpose: To correlate levels of periodontal disease as measured by conventional methods with PMN counts from an oral rinse in pregnant women. Methods: Sixty-three pregnant women were recruited. Periodontal examinations were carried out. Fifteen-second saline rinses were collected. ABTS, a colour changing redox agent was added to each rinse. The intensity of the colour reaction was measured by absorbance to count the oral PMNs. Results: A statistically significant difference in oral PMN counts was observed between those with periodontal disease and the healthy patients (P < .05). The sensitivity and specificity using the bleeding index was 0.94 and 0.76 respectively. Conclusions: The rinse assay can be used as a screening tool for periodontal disease and oral inflammation in pregnant women.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.