PURPOSE: In the mouse, the macrophage adhesion G protein-coupled receptor (ad-GPCR) molecule, F4/80, is required for the development of regulatory T cells in two models of tolerance, the eye and gut. Since F4/80 is not expressed in humans, the purpose of this research is to determine the human analog of F4/80. F4/80 belongs to a novel family of Epidermal growth factor-seven transmembrane (EGF-TM7) molecules, which include the EGF module-containing mucin-like hormone receptor (EMR) molecules. In the human, EMR1 has sequential homology with F4/80 and EMR2 has shown immune suppressing function in tumor cells. Thus, we investigate the possible suppressor role of the EMR family in human ocular tolerance. METHODS: Human peripheral blood mononuclear cells (huPBMC) were treated with porcine TGFβ2 and LPS or an antigenic stimulant for at least six hours to generate tolerogenic antigen presenting cells (APC). Cells were characterized by flow cytometric analysis for expression of CD14, CD40, PDL1, ILT3, and EMR2. Later, T regulatory cells were generated by incubating tolerogenic APCs with autologous huPBMC for five to seven days. Post culture, the T cells were stained and characterized for expression of CD4, CD25, and FoxP3. RESULTS: Post treatment of huPBMC with TGFβ2 and antigen, the resulting tolerogenic APCs expressed PDL1, ILT3, and EMR2. CD40 remained unchanged and CD14 was constitutively expressed. Post five to seven day culture, tolerogenic APCs treated with TGFβ2 increased the CD4+ CD25+ FoxP3+ lymphocyte populations. CONCLUSIONS: The upregulation of EMR2 on human tolerogenic APCs suggests that EMR2 may have a role in inducing tolerance in humans. Much like its mouse counterpart, F4/80, EMR2 is an adhesion molecule that may facilitate the induction of naïve T lymphocytes to regulatory T lymphocytes. Once the F4/80 analog is established for humans, novel therapies may be developed to interfere or encourage signaling in the treatment of tumors or immune inflammatory diseases, respectively.

A cavidade peritoneal de camundongos abriga uma variedade de células do sistema imune. Inicialmente, devido as limitações metodológicas, acreditava-se que, aproximadamente, 90% das células peritoneais era representada por macrófagos. Em seguida, graças aos extensos estudos com células peritoneais, observou-se que, além dos macrófagos, o peritônio abrigava muitos linfócitos B, principalmente do subtipo B-1. Utilizando metodologias contemporâneas de FACS - citometria de fluxo, este trabalho mostra que, aproximadamente, 30% das células peritoneais são macrófagos, 55% são linfócitos B-1, dos quais 40% pertencem ao subtipo B-1a e 15% ao subtipo B-1b. Os 15%-20% restantes representam outros subtipos celulares, como linfócitos T, linfócitos B-2, linfócitos NK, eosinófilos, além da presença de outras populações de células que não foram possíveis de ser identificadas com os marcadores de superfície utilizados neste trabalho. Em contraste com a literatura, nossos estudos mostraram que os macrófagos da cavidade peritoneal de camundongos representam uma população heterogênea. Por meio da co-expressão de CD11b e F4/80, foi possível descrever dois subtipos funcionais de macrófagos, denominados aqui de PM-1 (CD11bhigh, F4/80high) e PM-2 (F4/8010W, CD11bint). As células PM-2 encontram-se em menor abundância (-10% do total de macrófagos) no peritônio não estimulado e, diferentemente de células PM-1, espraiam-se de forma alongada (bipolar) quando colocadas em cultura. Curiosamente, PM-2 são os únicos macrófagos peritoneais que expressam MHC-II e, ainda, 30% dessa população expressa CD11c, o marcador típico de células dendríticas. Já as células PM-1 possuem morfologia arredondada, estão em abundância no peritônio e produzem altas doses de NO após estímulo com LPS. Ambas as células são capazes de fagocitar bactérias in vivo, no entanto, as células PM-2 parecem mais eficientes, sendo capazes de internalizar quantidades maiores de bactérias. Após infecção in vivo, o número absoluto e a porcentagem de PM-2 aumenta muito no peritônio, chegando a representar metade dos fagócitos da cavidade peritoneal. Os macrófagos PM-1 e PM-2 parecem não representar monócitos já que não compartilham do fenótipo de monócitos presentes no sangue periférico. Deve-se ressaltar que, além das células PM-1 e PM-2, outras subpopulações do peritônio também expressam CD11b e F4/80. Com base na literatura atual, acredita-se que todas as células B-1 do peritônio expressam CD11b, uma molécula que é co-expressa com CD18 para formar o receptor de complemento e a molécula de adesão Mac-1. Entretanto, os estudos, apresentados neste trabalho, mostram que metade de cada subtipo de B-1 (B-1a e B-1b) não expressa CD11b. As células B-1 CD11b+ são maiores, mais granulosas e expressam quantidades maiores de IgM de superfície. As células CD11b+ possuem uma curiosa tendência de se juntar e formar duplas (doublets) fortemente ligadas entre si que estão presentes em abundância no peritônio de camundongos adultos. Além de ligarem entre si, as células B-1 podem formar duplas com macrófagos PM-1 e PM-2. Durante a ontogenia, as células CD11b- surgem primeiro no peritônio e são as progenitoras das células B-1 CD11b+. Após estímulo inflamatório (LPS í. v.) os linfócitos B-1 CD11b+ migram do peritônio para o baço, onde proliferam e transformam-se em células secretoras de anticorpos (plasmócitos). No peritônio, as células B-1 não são capazes de se diferenciar em plasmócitos (células CD138high). Os resultados apresentados aqui mostraram que, diferentemente dos macrófagos PM-2, os PM-1 são os responsáveis por inibir a formação de células secretoras de anticorpos derivadas de B-1. Em suma, nossos dados sugerem que, mediante estímulo inflamatório, as células B-1 maduras (CD11b+) migram do peritônio para o baço, afastandose de macrófagos PM-1. Já no baço, as células B-1 encontram um micro-ambiente favorável para se proliferarem e diferenciarem-se em plasmócitos, secretando a maioria dos anticorpos naturais vistos logo nas primeiras horas pós-estímulo. / Mouse peritoneal cavity (PerC) represents the source for a variety of cellular subsets of the immune system. In early studies, marred by Iimited methodological tools, it was thought that macrophages comprise roughly 90% of total PerC cells. Shortly thereafter, it was recognized that, beside macrophages, the mouse peritoneal cavity shelters large amount of B-lymphocytes, specially the B-1 subset. In essence, by applying contemporary technology, studies presented here show that, roughly 30% of PerC cells comprise macrophages, 55% comprise B-1 cells, which 40% represents the B-1 a subset and roughly 15% are B-1b cells. The remaining 15%-20% of PerC cells comprise a variety of known immune cells, including B-2, T, NK and eosinophils. In addition, there were some other cellular subsets that could not be identified in these studies, probably due to limited cell surface molecules analyzed. Surprisingly, the data presented here show that peritoneal macrophages represent a highly heterogeneous population. Based on the co-expression of both CD11b and F4/80, we have identified two functionally distinct subsets of peritoneal macrophages, named here as PM-1 (CD11bhigh, F480high) and PM-2 (CD11bint, F4/80low). PM-2 are less abundant (-10% within total macrophages) in the PerC of unstimulated mouse, however, unlike PM-1, these cells are able to spread in spindle (bipolar) morphology upon sorting and in vitro culture. Curiously, PM-2 is the only PerC macrophage that expresses surface MHC-II. In addition, one third of PM-2 population expresses CD11c, a universal marker for dendritic cells. In turn, PM-1 cells are round shaped cells, represent the majority of PerC macrophages (-90%) and secrete high amounts of NO upon LPS stimulation. In vivo phagocytosis assay revealed that both cells could internalize bacteria, however, PM-2 cells showed to be more efficient, in that it was able to phagocyte higher amounts of bacteria when compared to PM-1. After i.p. in vivo stimulation, the absolute number and percentage of PM-2 cells increase drastically in the peritoneum, reaching almost half of the total PerC macrophages. Importantly, PM-1 and PM-2 macrophages seem not to represent PerC monocytes since it does not share any of the phenotype expressed by blood monocytes. Interestingly, in addition to PM-1 and PM-2, some other cellular subsets in the PerC, such as eosinophils, are able to express CD11b and F4/80. PerC B-1 cells have long been known to express CD11b, which is co-expressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b+ cells in each B-1 subset are larger, more granular and express higher levels of surface IgM than the CD11b- B-1 cells. Surprisingly, the CD11b+ B-1 subset has the curious tendency to initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. In addition to binding to each other, B-1 cells can also form doublets with PM-1 and PM-2. During ontogeny, CD11b- B-1 cells arise earlier in the mouse PerC and are the progenitors of CD11b+ B-1 cells. Upon LPS stimulation, PerC CD11b+ B-1 cells migrate to the spleen where they proliferate and differentiate into antibody-secreting cells (plasma cells). Within the PerC, B-1 cells are not able to differentiate into plasma cells (CD138high cells). The data shown in here reveal that, unlike PM-2, PM-1 macrophages are the cellular subset responsible for inhibiting the formation of B-1-derived antibody-secreting cells. Altogether, our data suggest that, upon inflammatory stimuli, mature CD11b+ B-1 cells migrate from the PerC to the spleen, avoiding the inhibitory effect of PM-1 cells. In the spleen, B-1 cells find an appropriate environment to proliferate and terminally differentiate into antibody-secreting cells, thus, secreting the majority of immunoglobulin seen in few hours after in vivo stimulation.

A cavidade peritoneal de camundongos abriga uma variedade de células do sistema imune. Inicialmente, devido as limitações metodológicas, acreditava-se que, aproximadamente, 90% das células peritoneais era representada por macrófagos. Em seguida, graças aos extensos estudos com células peritoneais, observou-se que, além dos macrófagos, o peritônio abrigava muitos linfócitos B, principalmente do subtipo B-1. Utilizando metodologias contemporâneas de FACS - citometria de fluxo, este trabalho mostra que, aproximadamente, 30% das células peritoneais são macrófagos, 55% são linfócitos B-1, dos quais 40% pertencem ao subtipo B-1a e 15% ao subtipo B-1b. Os 15%-20% restantes representam outros subtipos celulares, como linfócitos T, linfócitos B-2, linfócitos NK, eosinófilos, além da presença de outras populações de células que não foram possíveis de ser identificadas com os marcadores de superfície utilizados neste trabalho. Em contraste com a literatura, nossos estudos mostraram que os macrófagos da cavidade peritoneal de camundongos representam uma população heterogênea. Por meio da co-expressão de CD11b e F4/80, foi possível descrever dois subtipos funcionais de macrófagos, denominados aqui de PM-1 (CD11bhigh, F4/80high) e PM-2 (F4/8010W, CD11bint). As células PM-2 encontram-se em menor abundância (-10% do total de macrófagos) no peritônio não estimulado e, diferentemente de células PM-1, espraiam-se de forma alongada (bipolar) quando colocadas em cultura. Curiosamente, PM-2 são os únicos macrófagos peritoneais que expressam MHC-II e, ainda, 30% dessa população expressa CD11c, o marcador típico de células dendríticas. Já as células PM-1 possuem morfologia arredondada, estão em abundância no peritônio e produzem altas doses de NO após estímulo com LPS. Ambas as células são capazes de fagocitar bactérias in vivo, no entanto, as células PM-2 parecem mais eficientes, sendo capazes de internalizar quantidades maiores de bactérias. Após infecção in vivo, o número absoluto e a porcentagem de PM-2 aumenta muito no peritônio, chegando a representar metade dos fagócitos da cavidade peritoneal. Os macrófagos PM-1 e PM-2 parecem não representar monócitos já que não compartilham do fenótipo de monócitos presentes no sangue periférico. Deve-se ressaltar que, além das células PM-1 e PM-2, outras subpopulações do peritônio também expressam CD11b e F4/80. Com base na literatura atual, acredita-se que todas as células B-1 do peritônio expressam CD11b, uma molécula que é co-expressa com CD18 para formar o receptor de complemento e a molécula de adesão Mac-1. Entretanto, os estudos, apresentados neste trabalho, mostram que metade de cada subtipo de B-1 (B-1a e B-1b) não expressa CD11b. As células B-1 CD11b+ são maiores, mais granulosas e expressam quantidades maiores de IgM de superfície. As células CD11b+ possuem uma curiosa tendência de se juntar e formar duplas (doublets) fortemente ligadas entre si que estão presentes em abundância no peritônio de camundongos adultos. Além de ligarem entre si, as células B-1 podem formar duplas com macrófagos PM-1 e PM-2. Durante a ontogenia, as células CD11b- surgem primeiro no peritônio e são as progenitoras das células B-1 CD11b+. Após estímulo inflamatório (LPS í. v.) os linfócitos B-1 CD11b+ migram do peritônio para o baço, onde proliferam e transformam-se em células secretoras de anticorpos (plasmócitos). No peritônio, as células B-1 não são capazes de se diferenciar em plasmócitos (células CD138high). Os resultados apresentados aqui mostraram que, diferentemente dos macrófagos PM-2, os PM-1 são os responsáveis por inibir a formação de células secretoras de anticorpos derivadas de B-1. Em suma, nossos dados sugerem que, mediante estímulo inflamatório, as células B-1 maduras (CD11b+) migram do peritônio para o baço, afastandose de macrófagos PM-1. Já no baço, as células B-1 encontram um micro-ambiente favorável para se proliferarem e diferenciarem-se em plasmócitos, secretando a maioria dos anticorpos naturais vistos logo nas primeiras horas pós-estímulo. / Mouse peritoneal cavity (PerC) represents the source for a variety of cellular subsets of the immune system. In early studies, marred by Iimited methodological tools, it was thought that macrophages comprise roughly 90% of total PerC cells. Shortly thereafter, it was recognized that, beside macrophages, the mouse peritoneal cavity shelters large amount of B-lymphocytes, specially the B-1 subset. In essence, by applying contemporary technology, studies presented here show that, roughly 30% of PerC cells comprise macrophages, 55% comprise B-1 cells, which 40% represents the B-1 a subset and roughly 15% are B-1b cells. The remaining 15%-20% of PerC cells comprise a variety of known immune cells, including B-2, T, NK and eosinophils. In addition, there were some other cellular subsets that could not be identified in these studies, probably due to limited cell surface molecules analyzed. Surprisingly, the data presented here show that peritoneal macrophages represent a highly heterogeneous population. Based on the co-expression of both CD11b and F4/80, we have identified two functionally distinct subsets of peritoneal macrophages, named here as PM-1 (CD11bhigh, F480high) and PM-2 (CD11bint, F4/80low). PM-2 are less abundant (-10% within total macrophages) in the PerC of unstimulated mouse, however, unlike PM-1, these cells are able to spread in spindle (bipolar) morphology upon sorting and in vitro culture. Curiously, PM-2 is the only PerC macrophage that expresses surface MHC-II. In addition, one third of PM-2 population expresses CD11c, a universal marker for dendritic cells. In turn, PM-1 cells are round shaped cells, represent the majority of PerC macrophages (-90%) and secrete high amounts of NO upon LPS stimulation. In vivo phagocytosis assay revealed that both cells could internalize bacteria, however, PM-2 cells showed to be more efficient, in that it was able to phagocyte higher amounts of bacteria when compared to PM-1. After i.p. in vivo stimulation, the absolute number and percentage of PM-2 cells increase drastically in the peritoneum, reaching almost half of the total PerC macrophages. Importantly, PM-1 and PM-2 macrophages seem not to represent PerC monocytes since it does not share any of the phenotype expressed by blood monocytes. Interestingly, in addition to PM-1 and PM-2, some other cellular subsets in the PerC, such as eosinophils, are able to express CD11b and F4/80. PerC B-1 cells have long been known to express CD11b, which is co-expressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b+ cells in each B-1 subset are larger, more granular and express higher levels of surface IgM than the CD11b- B-1 cells. Surprisingly, the CD11b+ B-1 subset has the curious tendency to initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. In addition to binding to each other, B-1 cells can also form doublets with PM-1 and PM-2. During ontogeny, CD11b- B-1 cells arise earlier in the mouse PerC and are the progenitors of CD11b+ B-1 cells. Upon LPS stimulation, PerC CD11b+ B-1 cells migrate to the spleen where they proliferate and differentiate into antibody-secreting cells (plasma cells). Within the PerC, B-1 cells are not able to differentiate into plasma cells (CD138high cells). The data shown in here reveal that, unlike PM-2, PM-1 macrophages are the cellular subset responsible for inhibiting the formation of B-1-derived antibody-secreting cells. Altogether, our data suggest that, upon inflammatory stimuli, mature CD11b+ B-1 cells migrate from the PerC to the spleen, avoiding the inhibitory effect of PM-1 cells. In the spleen, B-1 cells find an appropriate environment to proliferate and terminally differentiate into antibody-secreting cells, thus, secreting the majority of immunoglobulin seen in few hours after in vivo stimulation.