Morphometric evaluation of the fetal rat liver after maternal dexamethasone treatment: effect on the maturation of erythroid and megakaryocytic cells.

BACKGROUND: During pregnancy, glucocorticoids are frequently used to accelerate fetal lung maturation in preterm delivery. However, prenatal administration of glucocorticoids has been shown to affect organs such as fetal liver, an important hematopoietic organ during fetal development. OBJECTIVE: The aim of this study was to document the qualitative and quantitative changes in erythroid and megakaryocytic cell populations found in fetal livers as well as the hematology profile in neonates after maternal glucocorticoid treatment in rats. METHODS: Pregnant female Wistar rats were treated with dexamethasone 21-phosphate from days 13 to 16 of gestation. On the 17th day of pregnancy, the fetuses were collected and their livers processed for light and transmission electron microscopy. Glycol methacrylate-embedded sections were stained with PAS to determine the erythroblast and megakaryocytic cell frequencies. Fetal liver pieces embedded in Spurr resin were analyzed by transmission electron microscopy for morphologic changes. A standard hematology profile was evaluated in neonatal rats. RESULTS: In the fetuses from treated dams, the total cell number of erythroid cells in livers was significantly reduced compared to control fetuses (P < .001), but erythroblasts did not present ultrastructural abnormalities. The degree of maturation in the megakaryocyte series tended to be increased. In neonates, there were elevated numbers of nucleated RBCs (P = .002), along with a higher HCT and HGB (P = .02). In addition, the platelet concentration was also significantly increased (P < .007). CONCLUSION: These results suggest that maternal dexamethasone treatment has quantitative effects on erythroid and megakaryocytic cells in fetal liver and the neonatal hematology profile in rats.

TGF-ß-mediated sustained ERK1/2 activity promotes the inhibition of intracellular growth of Mycobacterium avium in epithelioid cells surrogates.

Transforming growth factor beta (TGF-ß) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-ß production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-ß and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-ß produced by ECs are sufficient to induce a self-sustaining autocrine TGF-ß signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-ß to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-ß receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-ß produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-ß receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-ß production. In summary, our findings indicate that the amplitude of TGF-ß signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.

Recombinant interleukin-4-treated macrophages, epithelioid cell surrogates, harbor and arrest Mycobacterium avium multiplication in vitro.

Our group has previously described that murine peritoneal macrophages treated in vitro for 7 days with recombinant interleukin-4 (rIL-4) acquire morphological and functional characteristics of epithelioid cells (ECs) found in granulomatous lesions. Although EC function has not been clarified so far, it has been suggested that these cells could present antigens and control multiplication of mycobacteria. These aspects have been addressed here using in vitro EC surrogates. Using immunocytochemistry and immunofluorescence methods, we have observed an increased expression of CD11b, CD54, CD86 and CD40 molecules on rIL-4-treated macrophages when compared to untreated ones. Cytokine-treated cells were less phagocytic for latex beads (P<0.03) and more pinocytic for dextran particles than untreated macrophages. T-cell lymphoproliferation assays using ovalbumin (OVA) and Mycobacterium avium as antigens showed that both cultured macrophages were equally efficient as antigen presenting cells (APCs). However, M. avium antigens were better presented in vivo by EC surrogates (P<0.01). Both macrophage cultures were similarly infected by M. avium. However, while the infection level was maintained in the cytokine-treated population, untreated macrophages showed a progressive increase in the number of bacilli/cell with time (P<0.01) and a reduction of about 65% in cell population. After 96 h of M. avium infection, untreated cells secreted higher amounts of tumor necrosis factor-alpha (P<0.005) while rIL-4-treated macrophages showed higher, although not significant, transforming growth factor-beta production. Also, EC surrogates produced less nitric oxide than control macrophages (P<0.05). Hence, EC surrogates restrain M. avium growth and act as APCs in vitro and in vivo.

Murine macrophages cultured with IL-4 acquire a phenotype similar to that of epithelioid cells from granulomatous inflammation.

Epithelioid cells (ECs) found in granulomas are thought to derive from mononuclear phagocytes. Although GM-CSF and/or IL-4 are known to promote cell differentiation their role in the development of ECs has never been demonstrated. Here we showed that mouse macrophages treated exclusively with recombinant IL-4 (rIL-4) differentiate into epithelioid-like cells. Macrophages cultivated with rIL-4 presented a fried-egg shape, and ultrastructural studies revealed membrane interdigitations, cytoplasmic vesicles, prominent Golgi complex, and rough endoplasmic reticulum. Compared with controls, rIL-4 treated cells displayed increased expression of MHC class II molecules and of Migration Inhibitory Factor-Related Protein-14. Whereas mannose receptor-mediated phagocytosis was increased, Fcgamma-receptor mediated phagocytosis and the production of nitric oxide were decreased in treated cultures. All these features overlap those reported for ECs from granulomatous lesions. In conclusion, treatment of mouse peritoneal macrophages with rIL-4 drives their in vitro differentiation to an epithelioid phenotype and provides a tool to investigate the biology of ECs.

Envolvimento da interleucina-4 na transformaçäo epitelióide de macrófagos peritoneais residentes / Interleukin-4 involvement in the epithelioid transformation o resident peritoneal macrophages

A diferenciação de macrófagos em células epitelióides parece estar associada à secreção local de citocinas inflamatórias tais como fator estimulador de colônias de macrófagos e granulócitos (GM-CSF), interleucina-4 (IL-4) e fator de necrose tumoral (TNF-a). O mecanismo que dirige este tipo de diferenciação não está completamente entendido embora a formação de células multinucleadas tenha sido obtida in vitro com o emprego de GM-CSF e IL-4. Assim, estas citocinas foram utilizadas em experimentos visando reproduzir in vitro o mecanismo de formação de células epitelióides a partir de macrófagos residentes. Nossos resultados mostraram que apenas macrófagos tratados com IL-4 apresentaram morfologia característica de células epitelióides. Estudos ultra-estruturais revelaram que estas células possuem retículo endoplasmático rugoso e complexo de Golgi proeminentes, além de numerosas vesículas e mitocôndrias. Interdigitações entre macrófagos adjacentes também foram observadas. O tratamento com IL-4 aumentou a expressão da proteína MRP-14, de moléculas MHC de classe II e de receptores de manose. Por outro lado, houve uma acentuada redução na produção de óxido nítrico e na fagocitose mediada por receptores Fcg. O conjunto de características morfológicas e funcionais de macrófagos tratados durante 7 dias com IL-4 é semelhante ao observado em células epitelióides de lesões granulomatosas. Isto sugere um provável envolvimento desta citocina no mecanismo de transformação epitelióide de macrófagos