LPS-Induced Migration of Peritoneal B-1 Cells is Associated with Upregulation of CXCR4 and Increased Migratory Sensitivity to CXCL12

B-1 cells, which constitute a predominant lymphocyte subset in serosal cavities and produce most of natural antibodies, are subdivided into the CD5+ B-1a and CD5- B-1b cell subpopulations, but the differential roles of B-1a and B-1b cells are not well understood. We report that B-1a cells preferentially migrate out of the peritoneal cavity and upregulate the expression of CXCR4 with heightened sensitivity to CXCL12 and CXCL13 upon LPS treatment compared to B-1b and B-2 cells. Whereas B-1a cells were slightly more abundant than B-1b and B-2 cells in the homeostatic condition, the number of B-1a cells preferentially decreased 48 hr after LPS treatment. The decrease in the peritoneal B-1a cell number was accompanied with increased migration of B-1a cells toward CXCL-12 and CXCL-13 in in vitro transmigration assay using peritoneal B cells from LPS treated mice. The expression level of CXCR4, but not of CXCR5, was also more prominently increased in B-1a cells upon LPS stimulation. LPS-stimulated B-1a cells did not accumulate in omental milky spots in contrast to B-2 cells. These results suggest that B-1a cells actively migrate out of the peritoneal cavity through the regulation of the migratory responsiveness to chemokines and actively participate in systemic immune responses.

Revisitando a cavidade peritoneal de camundongos: identificação de novos subtipos funcionais de linfócitos B-1 e macrófagos / Revisiting the mouse peritoneal cavity: identification of new functionally distinct subsets of macrophages and B-1 lymphocytes

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 Iinfócitos 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...

Pharmacological study of anti-allergic activity of Syzygium cumini (L) Skeels

Myrtaceae is a plant family widely used in folk medicine and Syzygium and Eugenia are among the most important genera. We investigated the anti-allergic properties of an aqueous leaf extract of Syzygium cumini (L.) Skeels (SC). HPLC analysis revealed that hydrolyzable tannins and flavonoids are the major components of the extract. Oral administration of SC (25-100 mg/kg) in Swiss mice (20-25 g; N = 7/group) inhibited paw edema induced by compound 48/80 (50 percent inhibition, 100 mg/kg; P <= 0.05) and, to a lesser extent, the allergic paw edema (23 percent inhibition, 100 mg/kg; P <= 0.05). SC treatment also inhibited the edema induced by histamine (58 percent inhibition; P <= 0.05) and 5-HT (52 percent inhibition; P <= 0.05) but had no effect on platelet-aggregating factor-induced paw edema. SC prevented mast cell degranulation and the consequent histamine release in Wistar rat (180-200 g; N = 7/group) peritoneal mast cells (50 percent inhibition, 1 æg/mL; P <= 0.05) induced by compound 48/80. Pre-treatment of BALB/c mice (18-20 g; N = 7/group) with 100 mg/kg of the extract significantly inhibited eosinophil accumulation in allergic pleurisy (from 7.662 ± 1.524 to 1.89 ± 0.336 x 10(6)/cavity; P <= 0.001). This effect was related to the inhibition of IL-5 (from 70.9 ± 25.2 to 12.05 ± 7.165 pg/mL) and CCL11/eotaxin levels (from 60.4 ± 8.54 to 32.8 ± 8.4 ng/mL) in pleural lavage fluid, using ELISA. These findings demonstrate an anti-allergic effect of SC, and indicate that its anti-edematogenic effect is due to the inhibition of mast cell degranulation and of histamine and serotonin effects, whereas the inhibition of eosinophil accumulation in the allergic pleurisy model is probably due to an impairment of CCL11/eotaxin and IL-5 production.

Efeito adjuvante da Propionibacterium acnes e de seu componente polissacarídico solúvel purificado sobre a migração e atividade citotóxica de células do exsudato peritoneal de camundongos C57BI/6 / Modulatory effect of killed-Propionibacterium acnes and its purified soluble polysaccharide on peritoneal exudate cells from C57BI/6 mice: major NKT cell recruitment and increased cytotoxicity

Neste trabalho. analisamos o efeito modulador da Propionibacterium acnes (P. acnes) morta pelo calor e de seu componente polissacarídico solúvel purificado (PS) sobre células do exsudato peritoneal de camundongos C57BI/6 tratados intraperitonealmente. Tanto a bactéria como o PS aumentaram o número de macrófagos, células dendríticas e linfócitos NKT para a cavidade peritoneal dos animais. A P. acnes aumentou principalmente o número de linfócitos NKT CD3+CD4-CDS-, e em menor grau a subpopulação CD3+CDS+, sendo o componente polissacarídico responsável pelo aumento desta última subpopulação. Células dendríticas do exsudato peritoneal de animais tratados com P. acnes, sem receber nenhum estímulo in vitro diferenciarem-se mais precocemente em células dendríticas maduras. Analisamos também o efeito da P. acnes ou de seu componente polissacarídico purificado sobre a função citotóxica in vitro de macrófagos e linfócitos NKT. Macrófagos obtidos de animais tratados tanto com a bactéria, como com seu componente polissacarídico foram citotóxicos para células de melanoma (B16F10) em co-cultura. Esta atividade foi mediada principalmente pela produção de NO, uma vez que a ausência deste fator praticamente aboliu tal função. Além do NO, a citotoxicidade mediada por macrófagos obtidos dos animais tratados somente com a bactéria também tem a participação de TNF-a e IFN-y. Somente os linfócitos NKT obtidos dos animais tratados com P. acnes foram citotóxicos para células de melanoma murino, após serem estimulados in vitro com IL-12, sendo esta função independente de IFN-y. ° estímulo intraperitoneal com P. acnes, dependente principalmente do seu componente polissacarídico, modula a função de células da resposta imune inata responsáveis por induzir uma resposta imune adaptativa eficaz e protetora.

Origen y desarrollo de linfocitos B1: una población celular involucrada en defensa y autoinmunidad / Origin and development of B1 lymphocytes: a cell population involved in defence and autoimmunity

Las células B1, responsables de la producción de IgM sérica en ausencia de aparente estimulaciónantigénica, son linfocitos B maduros con ubicación anatómica y características fenotípicas y funcionalesparticulares. Los linfocitos B1 se ubican mayoritariamente en cavidad peritoneal y pleural, presentancaracterísticas de células activadas y son de mayor tamaño y complejidad citoplasmática que las células B convencionales.Mientras que estos últimos deben diferenciarse a células plasmáticas para poder secretarinmunoglobulinas, los linfocitos B1 liberan espontáneamente anticuerpos al medio extracelular operando bajoun programa de diferenciación particular. Los anticuerpos producidos por los linfocitos B1 tendrían un rol protector,ya que están implicados en la remoción de células envejecidas y apoptóticas, en mecanismos deinmunomodulación y en resistencia a infecciones, sin embargo su participación en procesos autoinmunes tambiénha sido sugerida. Muchos estudios han aportado información sobre el origen, desarrollo y diferenciaciónde los linfocitos B1, los cuales son analizados en esta revisión.

B1 lymphocytes are an anatomically, phenotypically, and functionally distinct subset ofB cells producing the bulk of natural serum IgM in the absence of any apparent stimulation by specific antigens.These cells are a dominant population of B cells in peritoneal and pleural cavities and they have characteristicsof activated cells and higher cell size and cytoplasmic complexity than conventional B cells. B1 cells spontaneouslysecrete antibodies and operate under a differentiation program that is unique and differs from the paradigmassociated with Ig-secreting B-2 cells. The antibodies produced by B1 cells may participate in a variety ofphysiological activities since they are involve in immune regulation, clearance of senescent and apoptotic cellsand resistance to infection. However, it has been suggested that they are also involved in autoimmunity. Manyadvances have been made to describe the origin, development and differentiation of B1 cells, which will beexamined here.

IgE expression on the surface of B1 and B2 lymphocytes in experimental murine schistosomiasis

In a previous study we monitored the distribution and phenotype expression of B1 cells during the evolution of experimental murine schistosomiasis mansoni and we proposed that the B1 cells were heterogeneous: a fraction which originated in the spleen and followed the migratory pathway to mesenteric ganglia, while the other was the resident peritoneal B1-cell pool. In the present study, we have addressed the question of whether these two B1-lymphocyte populations are involved in the production of the late Ig isotype IgE, which is present in high levels in schistosomal infection. Lymphocyte expression of surface markers and immunoglobulins were monitored by immunofluorescence flow cytometry. Both in the spleen and mesenteric ganglia, the B1 and B2 cells were induced to switch from IgM to IgE in the early Th2-dominated phase of the disease, with an increase of IgE in its later phases. Conversely, peritoneal B1-IgM+ switched to the remaining IgE+ present in high numbers in the peritoneal cavity throughout the disease. We correlated the efficient induction of the expression of late Ig isotypes by B1 cells with high levels of inflammatory cytokines due to the intense host response to the presence of worms and their eggs in the abdominal cavity. In conclusion, B1 cells have a different switch behavior from IgM to IgE indicating that these cell sub-populations depend on the microenvironment.

Comparative functional characterization of mouse bone marrow-derived mast cells and peritoneal mast cells in response to non-immunological stimuli.

The cultured mouse mast cells that are dependent on spleen-derived factor for their proliferation and maintenance and have been shown to be similar to mucosal mast cells in terms of their T-cell dependence and histochemical staining characteristics. Mast cell heterogeneity has been confirmed by functional characterization of mouse bone marrow-derived mast cells (MBMMC) and mouse peritoneal mast cells (MPMCs). MPMCs released around 30% of histamine when stimulated with compound 48/80 whereas MBMMC were almost unresponsive to the same stimulus. Calcium Ionophore A23187 on the other hand, released histamine in dose-dependent manner from MBMMC. The study was undertaken to investigate the effect of antiallergic drug, disodium cromoglycate (DSCG), a synthetic cromone and quercetin, a plant-derived flavonoid on Ca ionophore A23187 induced histamine release from MBMMC. MBMMCs were almost unresponsive to DSCG whereas Ca Ionophore induced histamine release was blocked by Quercetin. The results indicate that response of mast cells at one anatomic site to a given stimulus does not necessarily predict the response of mast cells at a different anatomic location to the same stimulus. It shows functional heterogeneity within a single species. So, it cannot be assumed that antiallergic compounds stabilizing mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites.

Atrial natriuretic peptide induces rat peritoneal mast cell activation by cGMP-independent and calcium uptake-dependent mechanism

Atrial natriuretic peptide (ANP), a 28 amino acid basic polypeptide, is known to induce histamine release from human and rat mast cells in vitro and cause a wheel formation in rat skin. However, cellular events associated with histamine release are not clearly understood. In this study, we have examined the calcium flux and cGMP formation associated with histamine release in the ANP-treated mast cells. ANP, in vitro, induced mast cell degranulation and histamine release in a dose-dependent manner. ANP also induced an enhanced calcium uptake into cells and increased the cellular level of cGMP in mast cells. A high level of calcium in the media caused an inhibition of ANP-dependent histamine release but enhanced the level of intracellular cGMP of mast cells. ANP inducing a dose-dependent increase in vascular permeability of rat skin was confirmed by the extravasation of the circulating Evans blue. The results indicate ANP induced the histamine release and an increase in vascular permeability through mast cell degranulation in cGMP-independent and calcium uptake-dependent manner.

Effect of Crotalus durissus terrificus (Laurenti, 1768) venom on the evolution of Ehrlich ascites tumor

The effect of Crotalus durissus terrificus (LAURENTI, 1768) venom on the evolution of Erlich ascites tumor cells was evaluated. Thus, 30-day-old male mice of the Swiss strain were inoculated intraperitoneally with 1x103 tumor cells. Then, 7 groups of animals were formed: 3 control groups (physiological, venom and tumor) and 4 experimental groups that received different doses of venom. The experimental groups received 5 intraperitoneal venom injections on the 1st, 4th, 7th, 10th and 13th days after tumor implantation. On the 14th day, 5 animals from each one og the groups were sacrificed, and the varibles such as the total and differential counts of cells in the peritoneal cavity and functional state of peritoneal macrophages by macrophage spreading were evaluated. The other 5 remaining animals were kept in the laboratory for 60 days for observation of their survival percentage. The results obtained were statistically analysed by the Kruskal-Wallis test at 5 per cent significance level. It was observed that Crotalus durissis terrificus venom increases survival time of mice, but does not increase mortality percentage. This venom also increases the percentage of macrophage spreading. We suggest that snake venoms can cause inhibition of tumor growth by activating the inflammatory reaction, mainly the macrophages, stimulating the production of TNF-alpha, IL-1, IL-6 and IL-8. These cytokines may act on tumor cells by different mechanisms, inducing its complete elimination.

Effect of Leishmania donovani antigens on superoxide anion generation by mice polymorph in vitro.

The present study was planned to study the effect of priming of BALB/c mice peritoneal polymorphs (PMNLs) by viable, heat killed and formaline fixed promastigotes of L. donovani and soluble extract of proteins of L. donovani promastigotes on their capacity to produce toxic oxygen radicals (TOR). TOR production was measured by superoxide dismutase (SOD), inhibitable cytochrome C reduction and nitroblue tetrazolium reduction tests. No significant stimulation or inhibition in SOD inhibitable cytochrome C reduction and nitroblue tetrazolium reduction was seen when PMNLs were primed by soluble leishmanial protein, and formalin fixed and denatured promastigotes for both 60 and 90 min. Priming of BALB/c mice peritoneal polymorphs with live promastigotes of L. donovani for more than 60 min produced approximately 30 per cent inhibition in superoxide generation as measured by both the tests (P < 0.05). The inhibition in superoxide generation by polymorphs may be one of the important escape mechanisms in the causation of the visceral leishmaniasis.

Schistosoma mansoni: host cell adhesion to the different stages of the parasite, in vivo

The peritoneal cavity of laboratory mice was used to study the phenomenon of host cell adhesion to different evolutive stages of the Schistosoma mansoni (cercaria, adult worm, developing and mature eggs, miracidium, young and mature daughter sporocysts). Material recovered from the peritoneal cavity 30 and 180 min after the inoculation of each evolutive form was examined with the help of a stereomicroscope. The free swimming larvae (cercaria and miracidium), and the evolutive forms producing such larvae (mature egg and mature daughter sporocyst) elicited the host cell adhesion phenomenon. In all forms but cercariae the adherent cells remained as so till 180 minutes after inoculation

Peritonites: papel das celulas mesoteliais e suas implicacoes na terapeutica / Peritonitis: mesothelial cells activity and their terapeutic implications

Entre os complexos mecanismos de defesa do peritonio frente a agressao bacteriana ou quimica, sao de gande importancia aqueles ligados a atividade das celulas mesoteliais, que proporcionam a mais pronta e eficiente resposta para remocao peritoneal destes agentes, suas toxinas e outras particulas. Alem desta acao de limpeza ("clearance"), e reconhecida a grande capacidade fibrinolitica destas celulas, importante na reabsorcao dos materiais aderentes de dentro da cavidade peritoneal. Diversos fatores podem promover o desequilibrio deste sistema; entre eles, encontramos procedimetos terapeuticos adjuvantes e mesmo primarios. Baseados nisto, foram desenvolvidos diversos estudos experimentais que concluiram pela necessidade de maior cuidado na execucao dos procedimentos, e sugerem a utilizacao de determinadas substancias, como a heparina, capazes de evitar prejuizo ao sistema ou de favorecer seu desempenho, o que podera vir a ser de utilidade no tratamento desta afeccao

Analise citogenetica relacionada a idade e ao tratamento com drogas em macrofagos peritoneais e celulas da medula ossea de camundongos femeas Swiss hiperimunes intactos ou ooforectomizados / Citogenetic studies relationships to age and to tretment with drugs in peritoneal macrophages and bone marrow cells in the intact and ovarectomized female mouse Swiss hiperimunes

Estudos citogeneticos relacionados com a idade em camundongos Swiss femeas hiperimunes intactos mostraram aumento da frequencia de macrofgos peritoneais com alteracoes cromossomicas estruturais aos 6 meses e aumento progressivo da frequencia de macrofagos hiperdiploides dos 6 aos 15 meses de idade. Nos animais ooferectomizados, os macrofagos apresentaram estes aumentos aos 2 meses, 30 dias apos a ooferectomia, e dos 6 aos 18 meses, respectivamente. Os resultados evidenciam uma relacao entre alteracoes hormonais e aberracoes cromossomicas nos macrofagos peritoneais. Estas aberracoes cromossomicas nao foram observadas em celulas da medula ossea dos animais intactos e dos ooferectomizados, sugerindo respostas diferentes nos macrofagos peritoneais e seus precurssores na medula ossea.Os antioxidantes acido ascorbico e alfa-tocoferol protegeram os macrofagos peritoneais de camundongos ooforectomizados das aberracoes cromossomicas estruturais, evidenciando papel dos radicais livres no aparecimento das mesmas. Considerando que os estrogenos têm acao antioxidante na peroxidacao lipidica, que o tocoferol é um captador de radicais lipofilicos e que o ácido ascórbico pode ser um regenarador do tocoferol, é provável que as quebras verificadas nos cromossomos dos macrófagos peritoneais de camundongos ooforectomizados resultam de peridoxidacao lipidica. O metotrexato mostrou efeito sinergico com a ooforectomia em relacäo ás alteracöes cromossômicas, e teve este efeito parcialmente suprimido pelo ácido ascórbico, tanto nos animais intactos, como nos ooferectomizados, sugerindo que, além da inibicäo de reparo de DNA, a droga tenha possível acäo através dos radicais livres. O benzonidazol provocou um grande número de delecöes, sobretudo nos macrófagos peritoneais de animais ooferectomizados. Seu efeito foi antagonizado, em grande proporcäo, pelo tocoferol, o que corrobora os dados da literatura em relacäo á atuacäo do benzonidazol através da formacäo de radicais livres. Por serem mais suscetíveis ás alteracöes hormonais e aos tratamentos com drogas que as celulas da medula óssea, conclui-se que os macrófagos säo adequados para estudos dos fenômenos ligados ao envelhecimento, dos mecanismos de inducäo de aberracöes cromossomicas, dos efeitos de drogas e seu modo de acäo, constituindo, assim, importante modelo para investigacöes em citogenetica

Vitamin E stimulates endotoxin-inhibited monocyte migration and phagocytosis in vivo

Intravenous administration of E. coli endotoxin (LPS, 30 microng/kg, iv) inhibited thioglycollatestimulated accumulation of monocytes into the rat peritoneal cavity, measured 48 and 72 h later, by about 40%. Pretreatment of animals with vitamin E (50 mg/kg, im) restored thioglycollate-induced monocyte migration in LPS-challenged rats to control levels. Vitamin E also partially reversed LPS-induced inhibition of monocyte phagocytosis in vivo. The results suggest that vitamin E could be beneficial in restoring leukocyte function in septicemic states

Chemotactic effect of PAF-acether on peritoneal eosinophils from normal rats

The chemotactic activity of PAF-acether was compared with that of tetrapeptide eosinophil chemotactic factors of anaphylaxis (ECF-A, Ala-Gly-Ser Glu and Val-Gly-Ser-Glu) using eosinophils obtained from the peritoneal cavity of normal rats. Cells were isolated by separation over discontinuous metrizamide gradients which resulted in eosinophil suspensions of 80 to 90% purity. PAF-acether produced 7-fold greater than the maximal activity obtained with the ECF-A-tetrapeptides. BN 52021 and WEB 2086 inhibited PAF-acether-induced eosinophil chemotaxis in a dose-dependent manner, suggesting that this phenomenon is mediated by specific PAF-acether receptors

Infecçäo em diálise peritoneal intermitente / Peritonitis in intermittent peritoneal dialysis (IPD)

Com o objetivo de verificar a incidência de peritonite em diálise peritoneal intermitente (DPI), assim como morbidade, flora infectante e aspectos clínicos pertinentes, foram estudadas bacteriologicamente 101 sessöes de diálise peritoneal, dentre 547 realizadas no Hospital ana Nery (INAMPS) no período de julho de 1983 a março de 1984. Foram procedidas culturas da prótese de Deane, do dialisado no 1§ e último banhos, cultura da ponta do cateter a cada duas semanas, ou quando havia sinais clínicos de peritonite. Realizou-se também estudo citológico do mesmo material. Vinte e dois pacientes portadores de insuficiência renal crônica foram incluídos no estudo, sendo nove do sexo feminino e 13 do masculino, com idades variando de 16 a 67 anos. Todos eram submetidos a duas sessöes semanais de 20 horas, utilizando prótese de Deane. Staphylococcus epidermidis e Staphylococcus aureus foram as bactérias mais freqüentemente isoladas. Peritonite clínica, no entanto, com febre e dor abdominal e líquido francamente turvo, foi observada em apenas cinco oportuniddades. Com o líquido peritoneal de dez desses pacientes, foram procedidas curvas de crescimento bacteriano, utilizando-se inóculos de ñ 10**5 de Staphylococcus epidermidis, observando-se em alguns pacientes nítido efeito inibitório deste líquido após seis e 24 horas de incubaçäo a 37-C, quando comparado com o controle, que consistia em semelhante inóculo feito em tripticase soja (TSB). Os autores discutem os mecanismos de defesa celulares e humorais do líquido peritoneal e sugerem medidas profiláticas com vistas à reduçäo da infecçäo em DPI