Mitochondrial kinetics during amphibian erythropoiesis related to haeme synthesis.

Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation-reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.

Ultrastructural, immunocytochemical and flow cytometry study of mouse peritoneal cells stimulated with carrageenan.

In the present paper we performed a morphological characterization of mouse peritoneal cells stimulated in vivo for 24 h with carrageenan (CAR) and lipopolysaccharide (LPS) by ultrastructural and flow cytometry analysis. In all samples, the flow cytometry studies showed the presence of three major populations consisting of monocytes, macrophages and lymphocytes. A special recruitment of monocytes was detected in CAR-injected mice. Macrophages and monocytes from CAR-treated mice displayed a characteristic phenotype, with a larger number of cytoplasmic vacuoles and numerous membrane projections, as compared to the cells collected from LPS- and PBS-injected mice. The induction of vacuolization was also confirmed upon in vitro treatment with CAR for 15 min to 24 h. The in vivo CAR-induced vacuoles were not related to lipid storage as judged by the lack of lipidic labeling after imidazole treatment at the ultrastructural level. In order to investigate the acidic nature of the vacuoles we used acidothropic probes, Lysotracker Yellow (LY) and Acridine Orange (AO). CAR injection activated the ability of peritoneal cells to incorporate LY around 2-5 times higher than control cells. However, the AO incorporation was 10-fold lower in CAR-stimulated cells than in LPS-stimulated ones. It is possible that the increase in intracellular vacuolization observed in CAR-stimulated cells could be related to exocytosis, since in most vacuoles the inflammatory protein MRP-14 was immunolocalized. The presence of MRP-14 in the culture supernatant of adherent peritoneal cells from CAR-injected mice was further comfirmed by ELISA, suggesting the discharge of MRP-14 enriched vacuole contents in the extracellular medium. We concluded that the morphological characteristics of activated monocytes and macrophages may depend on the nature of the triggering stimuli. Our observations reflect different functional phenotypes of monocytes/macrophages after in vivo stimulation with inflammatory agents such as CAR and LPS.