DOCK180 Is Induced with Differentiation from CD14 Positive Monocytes and It Regulates Phagocytosis in Both Macrophages and Dendritic Cells.

A guanine nucleoside exchange factor (GEF), DOCK180, which is an archetype of the CDM family protein, plays a pivotal role in the regulation of cell motility and phagocytosis through the activation of small GTPase Rac. The expression of DOCK180 is mostly ubiquitous except in hematopoietic cells, while another CDM family protein, DOCK2 is strictly found in hematopoietic cells. Although a knockout study against DOCK2 demonstrated the essential role of DOCK2 regarding rodent T- and B-cell motility and chemotaxisis, no involvement of DOCK2 in the action of phagocytes, such as macrophages or dendritic cells (DCs), has yet been observed. We, therefore, investigated the possible role of DOCK180 in the phagocytosis of hematopoietic cells. CD14 positive monocytes, as well as T- or B lymphocytes, isolated from healthy human donors did not express DOCK180 observed by Western blotting. Consistent with this finding, a real time PCR study revealed near negligible levels of DOCK180 mRNA in monocytes. On the other hand, macrophages or DCs, differentiated from monocytes in vitro in the presence of M-CSF or IL-4 and GM-CSF for seven days, were found to express extremely high levels of DOCK180 protein and mRNA. Time course studies showed that DOCK180 mRNA rapidly increased by 6 hours after the induction of CD14 positive monocytes into both macrophages and DCs and its protein expression was clearly detected by Western blotting after Day 2. While the expression of DOCK180 on DCs gradually increased until it reached a plateu level on Day 4, the expression of macrophages showed a linear increase up to Day 7. In contrast to DOCK180, the expression of DOCK2 remained unchanged throughout this study. Furthermore, a phagocytosis assay using Zymosan A particles demonstrated an increase in the phagocyte activity accompanying the expression levels of DOCK180 during both types of differentiation. Protein knockdown assays against DOCK180 using siRNA also confirmed the significance of DOCK180 in the phagocytosis of differentiated macrophages and DCs. Finally, an immunohistochemical study with anti-DOCK180 monoclonal antibody showed that macrophages exhibit the phagocytosis in the front edge of inflammatory granulation tissue, and expressed a high level of DOCK180. Taken together, DOCK180 was thus found to play a role in the induction of phagocyte activity regarding the differentiation from monocytes into either macrophages or DCs, thereby demonstrating the biological significance of DOCK180 in the phagocytosis of human hematopoietic cells.