Flow cytometric approach to the study of erythrophagocytosis: evidence for an alternative immunoglobulin-independent pathway in agammaglobulinemic mice

Abstract

Neuraminidase treatment of red blood cells (RBCs) is believed to induce changes similar to RBC senescence, and leads to a rapid clearance of RBCs from the circulation in vivo. The objective of this study using immunodeficient SCID mice and the lipophilic fluorescent probe PKH-26 was to ascertain whether antibodies are required as the final signal allowing the phagocytosis of neuraminidase-treated murine RBCs. All of the methods we applied are based on flow cytometry analysis using fluorescent probes: fluoresceinyl isothiocyanate (FITC)-labeled lectins for membrane carbohydrate identification and PKH-26-labeled RBCs for in vitro phagocytosis and in vivo clearance studies. The results can be summarized as follows: (i) the rate of neuraminidase-induced desialylation of RBCs from normal and immunodeficient mice is identical as ascertained with FITC-labeled lectins (wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA 120)); (ii) the rate of clearance of enzyme-treated RBCs from both types of mice is also similar, as is their localization in spleen, liver and lung; (iii) the rates of in vitro phagocytosis of untreated and neuraminidase-treated PKH-26-labeled RBCs from both species of mice are very similar in the presence of homologous sera. In the absence of serum or in the presence of heterologous sera, the rate of phagocytosis is markedly decreased but not totally abolished. These data suggest that neuraminidase-treated RBCs can be cleared via an alternative pathway that is antibody-independent. This pathway exists in immunocompetent mice but with a very low activity and is the only one active in immunodeficient mice. In accordance with results reported by Connor et al . [J. Biol. Chem. 269 (1994) 2399], it is possible that this antibody-independent mechanism is involved in the clearance of circulating senescent RBCs. Finally, the methods described here may also be of interest for the investigation of the mechanisms involved in the phagocytosis of apoptotic cells.