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Biotechnology and Applied Biochemistry (2001) 34, (143–149) (Printed in Great Britain)

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In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross-linking or diamide treatment

José A. Jordán, F. Javier Alvarez, L. Alfredo Lotero, Angel Herráez, José C. Díez and M. Cristina Tejedor¹

Departamento de Bioquímica y Biología Molecular, Campus Universitario, Universidad de Alcalá, E-28871 Alcalá de Henares (Madrid) SPAIN

Key words: carbonic anhydrase, chemical oxidation, dialysis loading, enzyme delivery, macrophages.

Abbreviations used: BS³, bis(sulphosuccinimidyl) suberate; CA, carbonic anhydrase; diamide, azodicarboxylic acid bis(dimethylamide); DTSSP, 3,3´-dithiobis(sulphosuccinimidyl propionate); FCS, fetal-calf serum; Hct, haematocrit; MFs, peritoneal macrophages; MPS, mononuclear phagocytic system; PBSH, Hanks PBS; RBC, red blood cell.

¹To whom correspondence should be addressed (e-mail cristina.tejedor@uah.es).

Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red-blood-cell membrane surface, namely oxidant compounds, such as ascorbate/Fe⁺² and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3-cross-linking reagents. We monitored the phagocytosis of oxidized or Band 3-cross-linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe³⁺, but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross-linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS³) and 3,3´-dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS³-cross-linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short-term delivery to cells of the mononuclear phagocytic system.

Received 19 June 2001/26 July 2001; accepted 8 August 2001

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