Antisense Oligonucleotide to Cofilin Enhances Respiratory Burst and Phagocytosis in Opsonized Zymosan-stimulated Mouse Macrophage J774.1 Cells

Abstract

Phagocytes play a central role in the host defense system, and the relationship between the mechanism of their activation and cytoskeletal reorganization has been studied. We have previously reported a possible involvement of cofilin, an actin-binding protein, in phagocyte functions through its phosphorylation/dephosphorylation and translocation to the plasma membrane regions. In this work, we have obtained a new line of evidence showing an important role of cofilin in phagocyte functions using the mouse macrophage cell line J774.1 and an antisense oligonucleotide to cofilin. Upon stimulation with opsonized zymosan (OZ), cofilin was phosphorylated, and it accumulated around phagocytic vesicles. As the antisense oligonucleotide to cofilin, a 20-mer S-oligo corresponding to the sequence including the AUG translational initiation site was found to be effective. In the cells treated with the antisense oligonucleotide, the amount of cofilin was less than 30% of that in the control cells, and the level of F-actin was two or three times higher than that in the control cells before and throughout the cell activation. In the antisense oligonucleotide-treated cells, OZ-triggered superoxide production was three times faster than that in the control cells. Furthermore, phagocytosis of OZ was enhanced by the antisense. These results show that cofilin plays an essential role in the control of phagocyte function through regulation of actin filament dynamics.Phagocytes, including neutrophils and macrophages, are at rest in the absence of any stimulant; but, when activated by invading microorganisms or harmful substances, they play a central role in host defense systems through chemotaxis, adhesion, phagocytosis, superoxide production, degranulation, and the release of cytokines or lipid mediators. The mechanism of phagocyte activation has been studied in terms of protein phosphorylation and cytoskeletal reorganization (1). Originally we found that cofilin, an actin-and phosphatidylinositol 4,5-bisphosphate-binding protein, is rapidly dephosphorylated upon cell activation and translocated to plasma membrane regions in neutrophil-like HL-60 cells (2). These findings have been confirmed by other groups (3-5). Cofilin is a widely distributed protein highly conserved among various species and plays an essential role in the control of actin filament dynamics (6 -8). It has been well investigated in, for example, Dictyostelium (9 -11), yeast (12-14), Xenopus egg (15-17), mammalian neural cells (18 -20), and immune cells (21-23). Cofilin can be phosphorylated at Ser-3, and only the unphosphorylated form of cofilin can bind actin and depolymerize or sever the actin filament. It is also proposed that cofilin is involved in the turnover of actin filament.To date we have studied the relation between cofilin and phagocyte functions. We used opsonized zymosan (OZ) 1 as a cell stimulant, a complement C3bi-coated insoluble polysaccharide, the receptor for which is a member of the ␤ 2 integrin family (CR3, CD11b/CD18), and have reported that co...

Ras-related GTPases Rap1 and RhoA Collectively Induce the Phagocytosis of Serum-opsonized Zymosan Particles in Macrophages

“…Indeed, the Rho-ROCK-myosin II pathway is required for CR3-mediated phagocytosis (39). Cofilin antisense enhances the phagocytosis of SOZs in J774.1 cells, which indicates that cofilin is inactivated during phagocytosis (40) and is consistent with our results. However, Rap1 activation by 8CPT-2Me-cAMP did not recover the phosphorylation of cofilin that is suppressed by the Tat-C3 toxin-mediated inactivation of RhoA (Fig.…”

Leukotrienes Target F-actin/Cofilin-1 to Enhance Alveolar Macrophage Anti-fungal Activity

“…Actin polymerization is regulated by phosphorylation/dephosphorylation of actin depolymerizing factors such as cofilin-1 (48). Cofilin-1 is a major actin-binding protein in leukocytes and plays an important role in restraining the respiratory burst and phagocytosis (49,76), and its activity is decreased by phosphorylation at serine-3 by LIMKs. LIMKs can be phosphorylated by Rho-family GTPases via the activation of Ϫ/Ϫ and WT mice were incubated with live C. albicans at a ratio of 1:5 for 60 min to allow ingestion of the fungi.…”

“…Cofilin-1 causes depolymerization at the minus end of filaments, thereby preventing their reassembly (48), and inhibits FcR-mediated phagocytosis (49). However, the importance of this protein in C. albicans phagocytosis is unknown.…”

Lyn, one of the Src‐family tyrosine kinases expressed in phagocytes, plays an important role in β2 integrin‐signalling pathways in opsonized zymosan‐activated macrophage‐like U937 cells

“…[41][42][43][44] In addition, we have previously reported the close relationship between a regulating factor of the actin cytoskeleton and oxidative burst. 11,45 Therefore, the signalling to regulate the actin cytoskeletal system could be one of the important roles that c-Cbl plays in phagocytes, and probably Lyn is a key factor in phosphorylating c-Cbl and in transducing signals from c-Cbl to downstream factors.…”

“…After pre-incubation at 378C for 5 min, the cells were activated with OZ (28 ml of 10 mg ml À1 HBSS suspension for a final concentration of 1 mg ml À1 ) and incubated at 378C. Then, the incubation was stopped and the cells were lysed as described previously 11 with lysis buffer containing 1 mM sodium orthovanadate. c-Cbl and its Tyr774-phosphorylated form were detected by Western blotting as described above.…”