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.