INVOLVEMENT OF CD44 AND THE CYTOSKELETAL LINKER PROTEIN ANKYRIN IN HUMAN NEUTROPHIL BACTERIAL PHAGOCYTOSIS

The leukocyte CD44 and CD45 cell surface receptors are associated via the linker proteins ankyrin and fodrin with the cytoskeleton, which it self is important in immune cell functions such as adherence, chemotax is, and phagocytosis. The effects of rat antihuman CD44 and CD45 monoc lonal antibodies on phagocytosis of fluoresceinated heat-killed Staphy lococcus aureus 502A by normal human neutrophils (PMNs) during 2 hr in cubation in RPMI-1640 was studied via flow cytometry and confocal micr oscopy. Flow cytometry was performed using an excitation wavelength of 488 nm, fluorescence being measured at 515-560 nm on 50,000 PMNs per sample. Confocal microscopy was performed on samples after further inc ubation with rhodamine-conjugated antiankyrin. Anti-CD44 resulted in a n increase of 27-31% compared to control (P = 0.004) in the proportion of PMNs fluorescing, an increase of 17-24% (P = 0.001) in mean intrac ellular fluorescence per PMN, and an increase in total PMN fluorescenc e of 50-58% compared to control (P < 0.001). In contrast, anti-CD45 ha d little effect on phagocytosis. Colchicine (a microtubule-disrupting agent) enhanced, whereas cytochalasin-D (a microfilament inhibitor) in hibited bacterial phagocytosis; cytochalasin-D completely abrogated th e effect of anti-CD44 on this PMN function. Hyaluronic acid augmented phagocytosis by an increment similar to that observed with anti-CD44. Two-color flow cytometry and confocal microscopy demonstrated that ank yrin always colocalized with ingested fluorescein isothiocyanate (FITC )-labeled bacteria. These data strongly suggest that CD44 is involved in bacterial phagocytosis, provide further evidence of CD44 receptor l inkage to cytoskeletal elements in human leukocytes, and suggest that ankyrin has a significant role in the transport of phagosomes. (C) 199 6 Wiley-Liss, Inc.