Camundongos AIRmax e AIRmin exibem diferenças significativas no número médio de leucócitos migrantes ao exudato inflamatório produzido por partículas de poliacrilamida. Um dos fatores preponderantes para a maior capacidade inflamatória da linhagem AIRmax é a maior produção de neutrófilos maduros pela medula óssea. No presente estudo avaliamos os fatores celulares e moleculares que conferem diferente potencial hematopoético entre as linhagens AIRmax e AIRmin e a associação destes com a intensidade da resposta inflamatória aguda (AIR). Nossos resultados demonstraram que apenas os animais AIRmax desenvolvem uma mielopoese emergencial induzida pelo Biogel com expansão de progenitores mielóides e produção de citocinas nas primeiras horas da inflamação. Desta forma, concluímos que a capacidade dos animais AIRmax de desenvolver uma alta AIR, em relação aos animais AIRmin, esta associada com o potencial de expansão das células progenitoras mielóides, que por sua vez, pode ser determinada pelos fatores solúveis locais produzidos nas primeiras horas da inflamação. / AIRmax and AIRmin exhibit significant differences in the average number of migrant leukocytes and protein content of the inflammatory exudate produced by polyacrylamide beads. One of the most important factors for the inflammatory capacity of AIRmax is the higher production of mature neutrophils by the bone marrow. In the present study we aimed to evaluate the cellular and molecular factors that confer different hematopoietic capacity between AIRmax and AIRmin and the association of these factors with the intensity of the acute inflammatory response (AIR). Our results showed that only animals AIRmax are able to develop an emergency myelopoiesis induced by Biogel presenting expansion of myeloid progenitors and cytokine production in the early hours of inflammation. Thus, we conclude that the ability of the AIRmax mice for developing a high AIR, compared to AIRmin mice, it is associated with the potential of expansion of myeloid progenitor cells, which in turn might be determined by the local soluble factors produced during the first hours of inflammation.

A leishmaniose visceral (LV) é uma doença crônica e potencialmente fatal causada, no Brasil, pelo protozoário Leishmania infantum. A resistência à LV é relacionada ao desenvolvimento de uma resposta imune celular eficiente. Para isso, células dendríticas (DCs) reconhecem produtos antigênicos do parasito, através dos Toll-like Receptors (TLRs), e, após ativação, produzem fatores, que irão orquestrar o recrutamento celular e desenvolvimento da resposta imune protetora. Dentre os vários TLRs, tem sido demonstrado que o TLR9 está relacionado com a resistência à infecção em modelo de leishmaniose cutânea. No presente estudo, nosso objetivo foi determinar o papel do TLR9 durante a infecção por L. infantum. Nossos resultados demonstram que a expressão de TLR9 é modulada positivamente durante a infecção in vivo e in vitro pela L. infantum. O TLR9 é essencial para o controle da infecção contra L. infantum, uma vez que camundongos TLR9-/- são mais susceptíveis à infecção, apresentando maior número de parasitos no baço e fígado na 4ª e 6ª semana pós-infecção. A fenotipagem de leucócitos presente no baço demonstrou que camundongos TLR9-/- possuem menor número de neutrófilos. De maneira semelhante, a análise imuno-histoquímica mostrou uma reduzida marcação de células 7/4+ (específica para neutrófilos) no fígado de animais TLR9-/-. A falha na migração de neutrófilos observada em animais TLR9-/- não está associada a uma incapacidade de ativação dos polimorfonucleares, mas sim a deficiências na produção de fatores quimiotáticos, como CXCL1 (KC) e CXCL2 (MIP-2) nos órgãos-alvo. Investigando o mecanismo de ação, as células dendríticas oriundas de animais TLR9-/- falham em seu processo de ativação frente à infecção in vitro e in vivo com L. infantum, apresentando um fenótipo semi-maduro, com redução de expressão de MHC-II e moléculas coestimuladoras. Interessante que essas células apresentaram deficiência na produção de mediadores quimiotáticos de neutrófilos como KC e MIP-2, e consequentemente, induzindo o menor recrutamento de neutrófilos in vitro. Em conjunto, nossos resultados demonstram que o TLR9 é essencial para o controle da infecção por L. infantum, através de um mecanismo dependente do recrutamento de neutrófilos para o sítio inflamatório mediado pela ativação apropriada de fatores quimiotáticos de neutrófilos pelas DCs. / Visceral leishmaniasis (VL) is a chronic and potentially fatal disease caused by protozoan Leishmania infantum, in Brazil. The resistance against VL is related to the development of cellular immune response. During infection, the dendritic cells (DCs) recognize antigenic products through Toll-like Receptors (TLRs) and then orchestrate the cellular recruitment and immune response development. Among several TLRs, it has been showed that TLR9 is related to resistance to cutaneous leishmaniasis. In the present study, our aim was to determinate the role of TLR9 during L. infantum infection. Our results demonstrated that TLR9 is up regulated during in vitro and in vivo L. infantum infection. TLR9 is critical for protective immunity against L. infantum, since TLR9-/- mice infected were more susceptible to infection, displaying high amounts of parasites in spleen and liver, at 4th and 6th weeks post-infection. Phenotyping the leukocytes into the spleen, TLR9-/- mice presented reduced neutrophils when compared to WT. Likewise, imunohistochemistry analyses showed the reduced of 7/4+ cells (specific to neutrophils) staining into the TLR-9-/- liver. The failure of neutrophil migration is not associated to their stage of activation impaired, but due the reduced levels of CXCL1 (KC) and CXCL2 (MIP-2) (neutrophil chemoatracttant) produced into into the spleen cells culture from infected TLR9-/-. Furthermore, DCs from TLR9-/- presented a semi-mature stage during in vitro and in vivo L. infantum infection, showing reduced expression of MHC-II and co-stimulatory molecule. Interestingly, the ability of DC to produce the neutrophil chemotact mediators (KC and MIP-2) was completely reduced by that derived from TLR9-/- mice, affecting neutrophil migration into Boyden chamber. Altogether, we demonstrated that TLR9 presents a critical role in the protective response against L.infantu through the mechanism dependent of crosstalk between neutrophil recruitment and DC activation.

Os neutrófilos representam a primeira linha de defesa do hospedeiro, atuando na contenção e eliminação de patógenos. Contudo, alterações na vida média, no excessivo recrutamento e ativação dos neutrófilos estão associados a danos teciduais e a doenças inflamatórias e autoimunes. A modulação das funções efetoras dos neutrófilos pode auxiliar no tratamento de tais patologias. Neste sentido, os produtos naturais constituem uma importante fonte de novas substâncias imunomoduladoras. Um estudo recente demonstrou que, a inibição do metabolismo oxidativo de neutrófilos pelo extrato etanólico bruto das folhas de Baccharis dracunculifolia (EEBBd) correlaciona-se com a proporção entre ácido cafeico (CaA) e outros compostos fenólicos contidos nesta amostra. Para dar prosseguimento à investigação do potencial imunomodulador do EEBBd e do CaA, os objetivos do presente estudo foram avaliar o efeito modulador desses produtos naturais: (i) em três funções efetoras de neutrófilos humanos - fagocitose, atividade microbicida e metabolismo oxidativo estimulado por agentes independentes de receptores (forbol-12-miristato-13-acetato; PMA) e dependentes apenas de receptores Fcgama (imunocomplexos não-opsonizados; IC) ou de receptores Fcgama associados a receptores do complemento (imunocomplexos opsonizados com complemento; IC-SHN); (ii) na atividade da mieloperoxidase (MPO); (iii) na expressão de receptores de membrana; (iv) e na captura (scavenger) de H2O2 e HOCl. O CaA foi mais efetivo do que o EEBBd em inibir a atividade da MPO e em capturar H2O2 e HOCl. A análise in silico revelou que o CaA bloqueia a entrada do sítio ativo da MPO através da interação com os resíduos Gln-91, His-95 e Arg-239; os dois últimos resíduos são essenciais para clivar o H2O2 e para estabilizar o sítio ativo, respectivamente. A eficiência dos agentes utilizados para estimular o metabolismo oxidativo, medido por quimioluminescência dependente de lucigenina e de luminol, ocorreu na seguinte ordem: PMA > IC-SHN > IC. Embora o PMA tenha sido o agente mais efetivo em estimular o metabolismo oxidativo, ambas as amostras (EEBBd e CaA) inibiram com maior intensidade esta função celular estimulada por PMA do que a mesma função estimulada por IC-SHN e IC. Além disso, ambas, EEBBd e CaA, não alteraram os níveis de expressão dos receptores TLR2, TLR4, CD16, CD32, e CD11b/CD18. Nas maiores concentrações avaliadas, EEBBd (50 ug/mL) e CaA (90 ug/mL) não foram citotóxicos para os neutrófilos. O EEBBd inibiu intensamente a capacidade fagocítica e reduziu discretamente a capacidade microbicida dos neutrófilos frente à Candida albicans. Portanto, o CaA contribui para ação inibitória do EEBBd no metabolismo oxidativo e na atividade da MPO, mas não na capacidade fagocítica e microbicida de neutrófilos. Por fim, o efeito imunomodulador do CaA e do EEBBd não é mediado por alterações na viabilidade celular ou na expressão de receptores de membrana em neutrófilos. O conjunto de resultados obtidos pode auxiliar na elucidação do mecanismo de ação destes produtos naturais sobre as funções efetoras de neutrófilos, bem como no desenvolvimento de novos fármacos para o tratamento de doenças inflamatórias mediadas pela ativação exacerbada de neutrófilos. / Neutrophils represent the first line of host defense that acts in the containment and clearance of pathogens. However, changes in life span and excessive recruitment and activation of neutrophils are associated with tissue damage and inflammatory and autoimmune diseases. Modulation of the effector functions of neutrophils can help to treat these pathologies. In this sense, natural products constitute an important source of novel immunomodulating compounds. A recent study has demonstrated that the neutrophil oxidative metabolism inhibition by the crude ethanol extract of Baccharis dracunculifolia (EEBBd) leaves correlates with the ratio of caffeic acid (CaA) to other phenolic compounds that exist in it. To continue investigating the immunomodulating potential of EEBBd and CaA, the present study aimed to examine whether these natural products modulate: (i) three effector functions of human neutrophils phagocytosis, microbial killing, and oxidative metabolism elicited by a receptor-independent stimulus (phorbol-12-myristate-13-acetate; PMA) and by receptor-dependent stimuli that bind Fcgama receptors alone (non-opsonized immune complexes; IC) or in combination with complement receptors (complement-opsonized immune complexes; IC-SHN); (ii) myeloperoxidase (MPO) activity; (iii) expression of membrane receptors; (iv) H2O2 e HOCl scavenging. CaA was more effective than EEBBd in inhibiting MPO activity and scavenging H2O2 and HOCl. The in silico analysis revealed that CaA blocks the entrance of the active site of MPO through the interaction with Gln-91, His-95, and Arg-239; the two last residues are essential to cleave H2O2 and stabilize the active site, respectively. The agents used to stimulate the oxidative metabolism, as measured by the lucigenin- and luminol-dependent chemiluminescence assays, acted in the following ranking order of efficiency: PMA > IC-SHN > IC. Although PMA was the most efficient agent at stimulating the neutrophil oxidative metabolism, both samples (EEBBd and CaA) suppressed the PMA-induced oxidative metabolism more effectively than they suppressed the same cell function elicited by IC-SHN and IC. Both EEBBd and CaA did not alter the levels of TLR2, TLR4, CD16, CD32, and CD11b/CD18 receptors expression. At the highest concentrations tested, EEBBd (50 ug/mL) and CaA (90 ug/mL) were not toxic towards neutrophils. EEBBd strongly diminished the phagocytic capacity and slightly reduced the Candida albicans killing ability of neutrophils. In conclusion, CaA contributes to the EEBBd inhibitory action on the oxidative metabolism and MPO activity but not on the phagocytic capacity and microbial killing ability of neutrophils. Furthermore, the immunomodulating effect of CaA and EEBBd is not mediated by alterations in either cell viability or expression of membrane receptors in neutrophils. Together, the results can help to unravel the mechanism of action of these natural products on the effector functions of neutrophils, and to develop new drugs to treat inflammatory diseases mediated by neutrophil overactivation.

Orientadores: Pedro Luiz Rosalen, Severino Matias de Alencar, Hyun Koo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-19T19:12:45Z (GMT). No. of bitstreams: 1 Bueno-Silva_Bruno_D.pdf: 2632427 bytes, checksum: b52293c180ef623f98558731cc4ce2fe (MD5) Previous issue date: 2012 / Resumo: A própolis brasileira destaca-se entre os produtos naturais por ser considerada uma rica fonte para a descoberta de novos compostos bioativos. Entre as própolis brasileiras, destaca-se a vermelha (tipo 13), a qual apresentou atividade antimicrobiana, antioxidante e antitumoral, sendo que seu principal constituinte químico são as isoflavonas, diferentemente de qualquer outro tipo de própolis brasileira. Portanto, o objetivo deste trabalho foi avaliar as propriedades antibacteriana, anticárie e antiinflamatória da própolis vermelha, identificando os possíveis compostos ativos responsávelis por estas atividades biológicas. Este objetivo foi atingido por meio das seguintes metodologias: 1- fracionamento bioguiado do extrato etanólico da própolis vermelha (EEP); 2- identificação e isolamento dos compostos ativos, 3- avaliação do potencial anticárie dos compostos ativos utilizando modelos: a) in vitro de inibição de biofilme oral monoespécie nos níveis molecular, estrutural e bioquímico; b) de cárie animal submetido a um severo desafio cariogênico; e 4- avaliação das propriedades anti-inflamatórias do EEP e de seus compostos isolados. Como resultado do fracionamento bioguiado, foram isolados e identificados 2 compostos: neovestitol e vestitol. Para os testes antimicrobianos e anti-cárie foi utilizada a fração 4.2 que continha os dois compostos isolados enquanto que para os testes anti-inflamatórios os compostos foram testados separadamente. A fração inibiu o desenvolvimento de biofilme em discos de hidroxiapatita na concentração de 800 ?g/mL, através da inibição da formação dos polissacarídeos e do teor protéico. Essa inibição foi observada através de: a) análise dos dados bioquímicos (redução de 40% da quantidade de políssacarídeos e 20% da quantidade protéica); b) redução da atividade das enzimas produtoras de políssacarídeos (glucanos), as glucosiltransferases B, C e D em aproximadamente 60%; c) redução da biomassa do biofilme e espessura da matrix de políssacarídeos extracelular sem afetar a viabilidade bacteriana, observada através da análise computadorizada das imagens obtidas com o microscópio confocal laser; d) alteração da expressão de 59 genes (52 dowregulated e 6 upregulated) de S. mutans observadas por microarray, sendo que os resultados dos genes: copY, copA, copZ, sloA, gtfD e amyA foram validados por qRT-PCR. A fração com os dois compostos também foi efetiva em reduzir a incidência e severidade das lesões de cárie em estudo animal sem afetar a viabilidade bacteriana. Em relação à atividade anti-inflamatória, tanto EEP quanto neovestitol e vestitol foram capazes de reduzir a migração leucocitária no processo inflamatório assim como inibiram a produção de citocinas pró-inflamatórias. Portanto, o fracionamento bioguiado revelou-se um processo eficaz para obtenção de novos compostos bioativos e tanto o EEP quanto seus compostos isolados (neovestitol e vestitol) apresentaram atividade anti-cárie e anti-inflamatória, revelando-se compostos promissores para o desenvolvimento de novos medicamentos / Abstract: The Brazilian propolis stands out among the natural products because it is considered a rich source for discovering new bioactive compounds. Among Brazilian propolis, the red one (type 13) exhibited antimicrobial, antitumor and antioxidant activities. Moreover, the main chemical constituents are the isoflavones, which are distinctive from other types of Brazilian propolis. Therefore, this study aimed to evaluate the antibacterial, anticaries and anti-inflammatory properties of red propolis, identifying the putative active(s) compound(s) that could be responsible for the biological activities. This objective was reached through the following methods: 1 - bioassay-guided fractionation of the ethanolic extract of propolis (EEP), 2 - identification and isolation of active compounds, 3 - evaluation of the anticaries potential of active compounds using the following models: a) in vitro monospecies biofilm and analyzed in the molecular, structural and biochemical levels b) in vivo rodent model of dental caries using a severe cariogenic challenge, and 4 - evaluation of anti-inflammatory properties of EEP and isolated compounds. As a result of bioassay-guided fractionation two compounds were isolated and identified: neovestitol and vestitol. A fraction 4.2 contains mostly these two compounds, was used for antimicrobial and anti-caries evaluations. For anti-inflammatory evaluation, the compounds were tested separately. The bioactive fraction at 800 ?g/mL decreased the development of biofilm on hydroxyapatite discs, by reducing the formation of extracellular polysaccharides (EPS) and the protein content. This inhibition was observed through four different methodologies: a) analysis of biochemical data (reduction of 40% of EPS and 20% of protein content), b) reduction of approximately 60% of activity of enzymes glucosiltransferases B, C and D that produces polysaccharides (ie. Glucans), c) reduction of biofilm biomass and thickness of EPS-matrix without affecting bacterial cell viability, as determined by confocal laser microscopy and computational analysis; d) disruption of expression of 59 genes (52 upregulated and 7 dowregulated) as determined by microarray, copY, copZ, sloA, gtfD and amyA were validated by qRT-PCR. Also, fraction containing vestitol and neovestitol was effective in reducing the incidence and severity of caries in vivo without affect viability of bacterial cells. In relation to anti-inflammatory activity, EEP, neovestitol and vestitol were able to reduce leukocyte migration during inflammation process and also, inhibited pro-inflammatory cytokines release. Therefore, the bioassay-guided fractionation identified bioactive compounds, which along with EEP exhibited anti-caries and anti-inflammatory activities. The agents may be promising compounds for the development of new drugs against oral diseases / Doutorado / Farmacologia, Anestesiologia e Terapeutica / Doutor em Odontologia

Alarmins and damage associated molecular pattern (DAMP) are endogenous proteins with distinct and various intracellular roles that when released extracellularly act as startingsignals for inflammatory immune responses. The endogenous protein High mobility group box 1 (HMGB1) acts as a DAMP and has been shown to drive progression of multiple inflammatory and autoimmune diseases. During homeostasis HMGB1 is localized in the nucleus of almost any cell, where its main function is organization of the DNA and regulation of transcription. Upon cell death or immune cell activation HMGB1 can be translocated into the cytoplasm for subsequent release into the extracellular space. Extracellular HMGB1 can act as a DAMP by activating several receptors of the immune system. Recent studies focus on HMGB1 release and functional regulation due to prost-translational modifications (PTMs) on cysteine residues. However, little is known about enzymatic regulation of HMGB1. The aim of this thesis was to investigate the possibility of proteolytic processing of HMGB1 by enzymes, which play a crucial role in inflammatory diseases and their progression. We utilized an in vitro model that mimics natural conditions of the autoimmune disease arthritis. Enzymatic digestion of HMGB1 was performed in kinetics studies using the neutrophilic enzymes cathepsin G, neutrophil Elastase as well as matrix metalloproteinase-3, which is released from tissues at the site of inflammation. We defined that HMGB1 is a novel substrate of all of the tested enzymes. All enzymes induced different cleavage pattern. In conclusion, my findings open up the possibility for future studies involving the observed fragments of HMGB1 and their functional features. It also demonstrated that HMGB1 is affected by protease modifications in a disease relevant environment.

Please read the abstract in the section 00front of this document / Thesis (PhD (Geneeskundige Immunologie))--University of Pretoria, 2005. / Pharmacology / unrestricted

In alcoholic hepatitis (AH), high neutrophil counts correlate with inflammation and poor clinical outcomes. Here, we sought to elucidate the neutrophil-mediated pathogenesis of AH. We revealed that in vivo neutrophil extracellular trap (NET) formation was significantly increased in AH patients and that alcohol alone is sufficient to induce NET formation in neutrophils; thereby, neutrophils increase liver damage through increased NET formation. Moreover, we identify that alcohol-induced NET formation is vital to NETosis and that high-density neutrophils (HDNs) become low-density neutrophils (LDNs) after NET formation in response to alcohol. Through transcriptome profile analysis, we found that genes related to neutrophil activation and immune responses are significantly upregulated in AH HDNs but significantly downregulated in AH LDNs compared to HDNs from healthy subjects. These data suggest that AH HDNs and LDNs have opposing phenotypes: HDNs are activated and more prone to release NETs, while LDNs are functionally incompetent. Consequently, the increase in activated HDNs and defective LDNs are likely associated with an increase in liver damage through NET formation and enhanced susceptibility to infection in AH patients, respectively. Therefore, we evaluated the therapeutic benefits of preventing NET formation in HDNs using peptidyl arginine deiminase 4 (PAD4) inhibition and granulocyte colony-stimulating factor (G-CSF) treatment as well as neutrophil depletion in mice. We observed that in vivo neutrophil depletion and G-CSF treatment prevent NET formation in the liver, thereby significantly reducing liver damage in alcohol-fed mice. Our work identifies the neutrophil/NET-mediated mechanisms of AH pathogenesis and provides insights into therapeutic interventions for AH.

Neutrophils are major cell type of innate immunity, that can eliminate pathogens by different mechanisms. One of these mechanisms is called NETosis, which leads to release of decondensed chromatin and citrullinated histone proteins. Citrullination is post-translational modification catalysed by peptidylarginine deiminase (PAD) and causing transformation of possitively charged arginin to neutral citrullin and can change expression of cytokine genes. Concetrations of pro-inflammatory cytokines (IL-8, TNF, IL-1) were measured after activation of PAD4 and induction of citrullination. Calcium ionophore was used to induce citrullinaton, Cl-amidine and TDFA were used as inhibitors. Production of cytokines was assessed by ELISA on protein level and by qPCR on mRNA level. It was found that induction of citrullination led to increased concentrations of IL-8 and IL-1. Elevated gene expression of IL-8 was confirmed on mRNA level. Both inhibitors were able to decrease level of histone H3 citrullination and IL-8 and IL-1 concentrations. Expression of TNF was not detected on protein and mRNA level.

abstract: An estimated 267 million women worldwide are HSV-2 seropositive, including roughly 20% of reproductive-aged American women. HSV-2 is a neurotropic virus that establishes a persistent, life-long infection that increases risk for STI acquisition in individuals. The vaginal epithelium represents a critical first line of defense against infection, and during acute infection, underlying immune mechanisms in the epithelium may be critical to protect against disease pathogenesis. The recently identified pro-inflammatory cytokine IL-36gamma has been shown to be expressed at mucosal epithelia, including the female reproductive tract (FRT) and may be an important factor in host defense. Although IL-36gamma has been shown to be induced in the FRT after exposure to microbial products, the contributions of IL-36gamma to host defense mechanisms in response to this clinically relevant STI pathogen are not well understood. This dissertation describes the regulation of IL-36gamma in the FRT and explores its contribution to the host response against genital HSV-2 infection. To test the hypothesis that IL-36gamma is a key regulator of mucosal inflammation and immunity in the FRT, hormonal regulation of IL-36gamma in the FRT was investigated using estrogen- and progesterone-conditioned mice. From this preliminary study, it was shown that progesterone dampens IL36G expression relative to estrogen and may potentially increase susceptibility to infection. Next, the impact of IL-36gamma treatment on HSV-2 infection and replication in human 3-D vaginal epithelial cells was explored. In parallel, the impact of intravaginal IL-36gamma delivery on HSV-2 disease pathogenesis was evaluated using a lethal murine challenge model. IL-36gamma pre-treatment significantly limited HSV-2 replication in vitro and in vivo and was associated with transient neutrophil infiltration that corresponded with decreased disease severity and increased survival in mice. Last, the requirement for IL-36gamma in host defense was investigated utilizing IL-36gamma-/- mice in a lethal HSV-2 murine challenge model. Following infection, IL-36gamma-/- mice exhibited significantly impaired neutrophil recruitment, decreased overall survival time, and significantly increased viral neuroinvasion relative to wild type mice. Collectively, these data indicate that IL-36gamma is a crucial regulator of HSV-2-induced neutrophil infiltration and appears to function in a previously uncharacterized manner to limit viral neuroinvasion in genital HSV-2 disease pathogenesis. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

Montelukast (ML) is primarily an antagonist of type 1 cysteinyl leukotriene receptors (CysLT1R), an activity which underpins its therapeutic efficacy in bronchial asthma. However, ML has also been reported to be useful in the treatment of acute and chronic inflammatory disorders of both infective and non-infective origin in which CysLTs are unlikely to be the predominant mediators of harmful inflammatory responses. These include conditions such as chronic obstructive pulmonary disease and cystic fibrosis in which the neutrophil is believed to be the primary offender, suggesting that ML may possess neutrophil-targeted, CysLT1R-independent mechanisms of anti-inflammatory activity. Accordingly, the laboratory research presented in this thesis was designed with the primary objectives of characterizing possible CysLT1R-dependent and – independent neutrophil-targeted anti-inflammatory activities of ML in vitro, and consisted of 3 phases. These were investigation of: i) the effects of the CysLTs, LTC4 and LTD4 (in the absence and presence of ML) on mobilization of intracellular Ca2+ stores, generation of reactive oxygen species (ROS) and release of primary and secondary granule proteinases; ii) the effects of ML on a series of pro-inflammatory activities of neutrophils following activation of the cells with the chemoattractants FMLP and platelet-activating factor (PAF); and iii) the interactive, anti-inflammatory effects on neutrophils of ML in combination with the long-acting beta-2 agonist, formoterol. In addition to the aforementioned activities, measurement of the production and expression of CR3, as well as generation of inositol triphosphate (IP3), cyclic AMP, and activities of the enzymes cAMP- and cGMP-phosphodiesterases (PDEs) in isolated neutrophil cytosol and membrane fractions, were also included. The following assays were used: i) chemiluminescence procedures for the detection of ROS; ii) a colourimetric procedure for the detection of elastase; iii) ELISA procedures for the detection of the matrix metalloproteinases (MMPs) 8- and -9, LTB4, and cyclic AMP; iv) fura-2-based spectrofluorimetry and a radiometric procedure for monitoring cytosolic Ca2+ fluxes; v) flow cytometry for CR3; and vi) radioassays for IP3 and activity of cAMP- and cGMP-PDEs. Exposure of neutrophils to LTD4, but not LTC4, activated a very modest and transient increase in cytosolic Ca2+, but failed to initiate the generation of ROS or release of elastase or MMP-8. However, brief pre-treatment with either LTC4 or LTD4 sensitized the cells for increased production of ROS and release of granule proteinases following activation with FMLP, which was partially attenuated by inclusion of ML. In the second part of the study, pre-treatment of neutrophils with ML, at therapeutically relevant concentrations, resulted in dose-related inhibition of the FMLP- or PAF-activated generation of ROS and LTB4, as well as the release of elastase, with the former being unaffected by an inhibitor of 5-lipoxygenase (MK886), compatible with a CysLT1R-independent mechanism of anti-inflammatory activity. From a mechanistic perspective, these interactions of ML with neutrophils were associated with accelerated clearance of Ca2+ from the cytosol of the cells which could not be attributed to inhibition of production of IP3, but was, however, associated with increased levels of cAMP, apparently as a consequence of non- specific inhibition of cyclic nucleotide phosphodiesterases. In the third part of the study, combining ML with formoterol caused (in most cases) additive inhibitory effects on the generation of ROS and LTB4, release of granule proteinases, as well as expression of CR3, which again were associated with elevations in cAMP and interference with Ca2+ mobilization. In conclusion, ML appears to attenuate neutrophil activation by CysLT1R-dependent and –independent mechanisms. In the case of the former by interfering with the modest sensitizing (priming) interactions of LTC4 and LTD4 with neutrophils, and in the latter by inhibition of PDEs, leading a to sustained elevation in cAMP, resulting in rapid clearance of Ca2+ from the cytosol and decreased uptake of the cation from the extracellular milieu. / Thesis (PhD)--University of Pretoria, 2011. / Immunology / Unrestricted