Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases. / May 2005

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases.

Objetivo: Avaliar as concentrações séricas do CA-125 e do CD-23 solúvel, durante dois períodos do ciclo menstrual (primeiro, segundo ou terceiro dia e o sétimo, oitavo ou nono dia), correlacionando-as a queixas clínicas, local de doença, estadiamento pela American Society for Reproductive Medicine e classificação histológica da endometriose pélvica. Pacientes e Métodos: No período de junho de 2007 a outubro de 2009, em estudo transversal, 102 mulheres, com queixas de infertilidade, dor pélvica refratária às terapêuticas clínicas, ou desejando laqueadura tubária, foram avaliadas por videolaparoscopia e divididas em um grupo caso, com endometriose (n=44), e outro controle, sem endometriose (n=58). Todas as pacientes foram submetidas a anamnese para investigação dos sintomas associados a endometriose e, antecedendo em até três meses da videolaparoscopia, a duas coletas de sangue venoso periférico para determinação da concentração do CA-125 e do CD-23 solúvel, por enzima-imunoensaio. Foram empregados os testes de Qui quadrado ou exato de Fisher para comparação de variáveis qualitativas. O teste t de Student, com análise de variância de Levene para amostras independentes, foi usado para diferença das médias de idade, intensidade dolorosa aferida pela escala visual analógica e concentrações dos marcadores, admitindo-se nível de significância igual a 0,05, para todos os testes. Resultados: Observamos que as concentrações médias do CA-125 foram significantemente maiores no grupo caso do que no grupo controle, em ambas as coletas na presença de dismenorréia severa e na ausência de infertilidade e de queixas urinárias cíclicas, mas apenas na segunda coleta naquelas com queixa de dor pélvica crônica e dispareunia de profundidade, permitindo diferenciação quando da presença de endometriose ovariana, grau III ou IV e padrões histológicos estromal ou glandular bem diferenciado. O CD-23 solúvel apresentou concentrações médias menores em pacientes com endometriose quando comparadas a mulheres sem a doença, porém sem atingir significância estatística, bem como essas concentrações foram menores na segunda coleta em pacientes sem endometriose com queixa de alterações intestinais cíclicas. Conclusões: Maiores concentrações do CA-125 em pacientes com endometriose reforçaram a afirmação de que esse marcador pode ser útil no diagnóstico e no manejo dessa doença. No entanto não se identificaram diferenças significantes na concentração desse marcador, em ambas as fases do ciclo menstrual, entre pacientes com endometriose em estádio inicial ou avançado, assim como entre aquelas com lesões histologicamente classificadas como diferenciadas ou indiferenciadas. O presente estudo trouxe uma análise diferenciada das concentrações médias do CD-23 solúvel e de suas associações com sintomas e local de doença / Objective: To evaluate serum concentrations of CA-125 and soluble CD-23, during two periods of the menstrual cycle (first, second or third day and the seventh, eighth or ninth day), correlating them to clinical complaints, site of disease, staging by the American Society for Reproductive Medicine and histological classification of pelvic endometriosis. Patients and Methods: From June 2007 to October 2009, within a cross-sectional study, 102 women with complaints of infertility, pelvic pain refractory to clinical therapies, or desiring for tubal ligation, were evaluated by videolaparoscopy and were divided into a case group, with endometriosis (n = 44), and a control group without endometriosis (n = 58). All patients underwent anamnesis for investigation of symptoms associated with endometriosis and, prior to three months to videolaparoscopy, they were submitted to two peripheral venous blood samples to measure CA-125 and soluble CD-23 concentrations by enzyme-linked immunosorbent assay. We utilized Chi square or Fisher exact test to compare qualitative variables. The t Student test, with Levene\'s variance analysis for independent samples, was used for mean difference in age, pain intensity measured by visual analogue scale and concentrations of markers, assuming a significance level of 0.05, for all tests. Results: We observed that average concentrations of CA-125 were significantly higher in case group than in control group in both samples, in the presence of severe dysmenorrhea and absence of infertility and cyclical urinary complaints, but only in the second collection, for women with chronic pelvic pain and deep dyspareunia, enabling differentiation in the presence of ovarian endometriosis grade III or IV and stromal or glandular well differentiated histological patterns. The soluble CD-23 showed lower average concentrations in patients with endometriosis compared to women without this disease, without statistical significance, and these concentrations were lower in the second collection for patients without endometriosis with complains of cyclical intestinal disorders. Conclusions: Higher concentrations of CA-125 in patients with endometriosis have reinforced the statement that this marker may be useful in the diagnosis and management of this disease. However we did not identify significant differences in concentration of this marker, in both phases of the menstrual cycle, between patients with endometriosis in early or advanced stage, and between those with lesions histologically classified as differentiated or undifferentiated. This study brought a differentiated analysis of the average concentrations of soluble CD-23, and of their associations with symptoms and site of disease.

Objetivo: Avaliar as concentrações séricas do CA-125 e do CD-23 solúvel, durante dois períodos do ciclo menstrual (primeiro, segundo ou terceiro dia e o sétimo, oitavo ou nono dia), correlacionando-as a queixas clínicas, local de doença, estadiamento pela American Society for Reproductive Medicine e classificação histológica da endometriose pélvica. Pacientes e Métodos: No período de junho de 2007 a outubro de 2009, em estudo transversal, 102 mulheres, com queixas de infertilidade, dor pélvica refratária às terapêuticas clínicas, ou desejando laqueadura tubária, foram avaliadas por videolaparoscopia e divididas em um grupo caso, com endometriose (n=44), e outro controle, sem endometriose (n=58). Todas as pacientes foram submetidas a anamnese para investigação dos sintomas associados a endometriose e, antecedendo em até três meses da videolaparoscopia, a duas coletas de sangue venoso periférico para determinação da concentração do CA-125 e do CD-23 solúvel, por enzima-imunoensaio. Foram empregados os testes de Qui quadrado ou exato de Fisher para comparação de variáveis qualitativas. O teste t de Student, com análise de variância de Levene para amostras independentes, foi usado para diferença das médias de idade, intensidade dolorosa aferida pela escala visual analógica e concentrações dos marcadores, admitindo-se nível de significância igual a 0,05, para todos os testes. Resultados: Observamos que as concentrações médias do CA-125 foram significantemente maiores no grupo caso do que no grupo controle, em ambas as coletas na presença de dismenorréia severa e na ausência de infertilidade e de queixas urinárias cíclicas, mas apenas na segunda coleta naquelas com queixa de dor pélvica crônica e dispareunia de profundidade, permitindo diferenciação quando da presença de endometriose ovariana, grau III ou IV e padrões histológicos estromal ou glandular bem diferenciado. O CD-23 solúvel apresentou concentrações médias menores em pacientes com endometriose quando comparadas a mulheres sem a doença, porém sem atingir significância estatística, bem como essas concentrações foram menores na segunda coleta em pacientes sem endometriose com queixa de alterações intestinais cíclicas. Conclusões: Maiores concentrações do CA-125 em pacientes com endometriose reforçaram a afirmação de que esse marcador pode ser útil no diagnóstico e no manejo dessa doença. No entanto não se identificaram diferenças significantes na concentração desse marcador, em ambas as fases do ciclo menstrual, entre pacientes com endometriose em estádio inicial ou avançado, assim como entre aquelas com lesões histologicamente classificadas como diferenciadas ou indiferenciadas. O presente estudo trouxe uma análise diferenciada das concentrações médias do CD-23 solúvel e de suas associações com sintomas e local de doença / Objective: To evaluate serum concentrations of CA-125 and soluble CD-23, during two periods of the menstrual cycle (first, second or third day and the seventh, eighth or ninth day), correlating them to clinical complaints, site of disease, staging by the American Society for Reproductive Medicine and histological classification of pelvic endometriosis. Patients and Methods: From June 2007 to October 2009, within a cross-sectional study, 102 women with complaints of infertility, pelvic pain refractory to clinical therapies, or desiring for tubal ligation, were evaluated by videolaparoscopy and were divided into a case group, with endometriosis (n = 44), and a control group without endometriosis (n = 58). All patients underwent anamnesis for investigation of symptoms associated with endometriosis and, prior to three months to videolaparoscopy, they were submitted to two peripheral venous blood samples to measure CA-125 and soluble CD-23 concentrations by enzyme-linked immunosorbent assay. We utilized Chi square or Fisher exact test to compare qualitative variables. The t Student test, with Levene\'s variance analysis for independent samples, was used for mean difference in age, pain intensity measured by visual analogue scale and concentrations of markers, assuming a significance level of 0.05, for all tests. Results: We observed that average concentrations of CA-125 were significantly higher in case group than in control group in both samples, in the presence of severe dysmenorrhea and absence of infertility and cyclical urinary complaints, but only in the second collection, for women with chronic pelvic pain and deep dyspareunia, enabling differentiation in the presence of ovarian endometriosis grade III or IV and stromal or glandular well differentiated histological patterns. The soluble CD-23 showed lower average concentrations in patients with endometriosis compared to women without this disease, without statistical significance, and these concentrations were lower in the second collection for patients without endometriosis with complains of cyclical intestinal disorders. Conclusions: Higher concentrations of CA-125 in patients with endometriosis have reinforced the statement that this marker may be useful in the diagnosis and management of this disease. However we did not identify significant differences in concentration of this marker, in both phases of the menstrual cycle, between patients with endometriosis in early or advanced stage, and between those with lesions histologically classified as differentiated or undifferentiated. This study brought a differentiated analysis of the average concentrations of soluble CD-23, and of their associations with symptoms and site of disease.

<p>Circulating immune complexes play an important role in the modulation of antibody responses and in the pathogenesis of immune diseases. This thesis deals with the <i>in vivo </i>regulatory properties of antibodies and their specific Fc receptors.</p><p>The immunosuppressive function of IgG is used clinically, to prevent rhesus-negative women from becoming sensitized to rhesus-positive erythrocytes from the fetus. The mechanism behind this regulation is poorly understood but involvement of a receptor for IgG, FcγRII, has been suggested. It is shown in this thesis that IgG and also IgE induce immunosuppression against sheep erythrocytes to a similar extent both in mice lacking all the known Fc receptors as in wild-type animals. These findings imply that antibody-mediated suppression of humoral responses against particulate antigens is Fc-independent and that the major operating mechanism is masking of epitopes.</p><p>Immunization with soluble antigens in complex with specific IgG leads to an augmentation of antibody production. The cellular mechanism behind this control is examined here and it is found that the capture of IgG2a immune complexes by a bone marrow-derived cell expressing FcγRI (and FcγRIII) is essential. An analysis of the ability of IgG3 to mediate this regulation indicated that, in contrast, this subclass of IgG augments antibody responses independently of FcγRI (and FcγRIII). These findings suggest that distinct mechanisms mediate the enhancing effect of different subclasses of antibodies.</p><p>Finally, the contribution of FcγRIII was studied in the development of collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis in humans. It was discovered that while DBA/1 wild-type control mice frequently developed severe CIA, with high incidence, FcγRIII-deficient mice were almost completely protected, indicating a crucial role for FcγRIII in CIA.</p><p>The results presented here help to understand how immune complexes regulate immune responses <i>in vivo</i> and show that Fc receptors for IgG, if involved, could be new targets for the treatment of immune complex-related disorders.</p>

Circulating immune complexes play an important role in the modulation of antibody responses and in the pathogenesis of immune diseases. This thesis deals with the in vivo regulatory properties of antibodies and their specific Fc receptors. The immunosuppressive function of IgG is used clinically, to prevent rhesus-negative women from becoming sensitized to rhesus-positive erythrocytes from the fetus. The mechanism behind this regulation is poorly understood but involvement of a receptor for IgG, FcγRII, has been suggested. It is shown in this thesis that IgG and also IgE induce immunosuppression against sheep erythrocytes to a similar extent both in mice lacking all the known Fc receptors as in wild-type animals. These findings imply that antibody-mediated suppression of humoral responses against particulate antigens is Fc-independent and that the major operating mechanism is masking of epitopes. Immunization with soluble antigens in complex with specific IgG leads to an augmentation of antibody production. The cellular mechanism behind this control is examined here and it is found that the capture of IgG2a immune complexes by a bone marrow-derived cell expressing FcγRI (and FcγRIII) is essential. An analysis of the ability of IgG3 to mediate this regulation indicated that, in contrast, this subclass of IgG augments antibody responses independently of FcγRI (and FcγRIII). These findings suggest that distinct mechanisms mediate the enhancing effect of different subclasses of antibodies. Finally, the contribution of FcγRIII was studied in the development of collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis in humans. It was discovered that while DBA/1 wild-type control mice frequently developed severe CIA, with high incidence, FcγRIII-deficient mice were almost completely protected, indicating a crucial role for FcγRIII in CIA. The results presented here help to understand how immune complexes regulate immune responses in vivo and show that Fc receptors for IgG, if involved, could be new targets for the treatment of immune complex-related disorders.