Involvement of CD44 and the cytoskeletal linker protein ankyrin in human neutrophil bacterial phagocytosis

Abstract

The leukocyte CD44 and CD45 cell surface receptors are associated via the linker proteins ankyrin and fodrin with the cytoskeleton, which itself is important in immune cell functions such as adherence, chemotaxis, and phagocytosis. The effects of rat antihuman CD44 and CD45 monoclonal antibodies on phagocytosis of fluoresceinated heat-killed Staphylococcus aureus 502A by normal human neutrophils (PMNs) during 2 hr incubation in RPMI-1640 was studied via flow cytometry and confocal microscopy. 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 incubation with rhodamine-conjugated antiankyrin. Anti-CD44 resulted in an 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 intracellular fluorescence per PMN, and an increase in total PMN fluorescence of 50-58% compared to control (P < 0.001). In contrast, anti-CD45 had little effect on phagocytosis. Colchicine (a microtubule-disrupting agent) enhanced, whereas cytochalasin-D (a microfilament inhibitor) inhibited bacterial phagocytosis; cytochalasin-D completely abrogated the 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 ankyrin 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 linkage to cytoskeletal elements in human leukocytes, and suggest that ankyrin has a significant role in the transport of phagosomes.

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils

“…Redistribution of PAK1 to the cytoskeleton in response to cell surface receptor activation is inhibited by wortmannin and cytochalasin D Wortmannin and cytochalasin D have both been reported to modulate the functional responses of leukocytes through effects on the actin cytoskeleton [12,[34][35][36]. We observed that 10 µM cytochalasin D, a potent inhibitor of actin polymerization, added to neutrophils 10 min before stimulation with fMLP blocked cell polarization and the incorporation of F-actin into cell surface extensions.…”

“…The PI 3-kinase inhibitor wortmannin blocks chemotactic peptide-induced development of cell polarity, increases in cytoskeletal actin, and phagocytosis in human neutrophils [8,10,12,[34][35][36][37][38]41]. We examined the effect of nanomolar (i.e., PI 3-kinase-specific) concentrations of wortmannin on PAK1 localization.…”

Hyaluronan-dependent pericellular matrix☆

“…Both CD44 and RHAMM associate with the cytoskeleton [81,82], control tumor cell invasiveness, and transduce signals involved in proliferation, locomotion, focal adhesion turnover [83,84], indicating that both receptors may be responsible for hyaluronan traction. CD44 has been demonstrated to mediate phagocytosis [85,86], bringing up the idea that hyaluronan in the pericellular matrix may by used by cells to draw in other matrix components, cellular debris or other particles, thus facilitating phagocytosis. The full role of cellular traction on hyaluronan organization is not yet clear.…”

Inflammation-Associated Autophagy-Related Programmed Necrotic Death of Human Neutrophils Characterized by Organelle Fusion Events

“…CD44 has a large number of isoforms generated by alternative RNA splicing (10). The most common standard isoform is highly expressed on neutrophils (11). The glycosaminoglycan disaccharide hyaluronan (HA) is the main ligand of CD44, although other extracellular matrix proteins seem also able to interact with CD44 (10).…”