Annexin A1 mediates the anti‐inflammatory effects during the granulocytic differentiation process in all‐trans retinoic acid‐treated acute promyelocytic leukemic cells

Abstract

Annexin A1 (AnxA1) originating from mature neutrophils and their microparticles (MPs) plays an important anti‐inflammatory role during the resolution phase of inflammation. However, the role of AnxA1 during the process of granulocytic differentiation is still unknown. All‐trans retinoic acid (ATRA) can induce acute promyelocytic leukemic (APL) cells to differentiate along the granulocytic lineage and has been used successfully in treating APL patients. In this study, we investigated whether or not AnxA1 contributed to the anti‐inflammatory properties of ATRA‐treated APL (NB4; ATRA‐NB) cells using the transmigratory and adhesive assays. We found that ATRA was able to enhance the surface expression of AnxA1 and its receptor (FPR2/ALX) and the release of AnxA1‐containing MPs from ATRA‐NB4 cells, while the expression of annexin V was not elevated on the latter cells. Further studies demonstrated that exogenous AnxA1 could inhibit ATRA‐NB4 cells in their transmigratory activity and adhesion to endothelial cells. In addition, the transmigratory activity of ATRA‐NB4 cells can be significantly enhanced by pretreatment with a FPR2/ALX neutralizing antibody, suggesting that endogenous AnxA1 may contribute to the anti‐migratory effects. Finally, ATRA‐NB4‐derived MPs could also inhibit recipient cells in their transmigratory and adhesive activities and these anti‐inflammatory effects could be inhibited by pretreatment of MPs with a specific anti‐AnxA1 antibody. Flowcytometry studies further demonstrated that FITC‐labeled AnxA1 could be transported from MPs to the membrane of recipient ATRA‐NB4 cells. We conclude that biologically active AnxA1 may play a role in the anti‐inflammatory properties of ATRA‐treated APL cells during the process of granulocytic differentiation. J. Cell. Physiol. 227: 3661–3669, 2012. © 2012 Wiley Periodicals, Inc.

CX3CL1(+) Microparticles Mediate the Chemoattraction of Alveolar Macrophages toward Apoptotic Acute Promyelocytic Leukemic Cells

“…We confirmed that apo-MPs released during the apoptotic process differ phenotypically and quantitatively from those MPs released during cellular activation [20,21]. Recent studies indicate that MPs released by activated neutrophils or differentiated ATRA-APL cells are associated with anti-inflammatory property in which the anti-inflammatory mediator annexin A1 is involved [15,16,22]. In contrast to these studies, we demonstrate that apo-MPs released by apoptotic ATRA-NB4 cells induced a higher pro-transmigratory activity on NR8383 cells than those MPs released by living ATRA-NB4 cells (Fig.…”

“…The flow-cytometric analysis of the MP preparations showed the expected heterogeneous populations, with sizes varying approximately between 0.1 and 2µm as verified by the use of control beads (2-µm each; ACBP-20-10; Becton Dickson). The numbers of MPs within the same gated area of MPs in the flow cytometry dot plots, as shown in Figure 3A, were then calculated [15,16]. …”

“…The non-permeablizied ATRA-NB4 cells and NR8383 cells were stained with either rabbit CX3CL1 polyclonal antibody (MAB365; R&D System, Minneapolis, USA) and rabbit CX3CR1 polyclonal antibody (AF5825; R&D System, Minneapolis, USA), respectively, before analysis by FAC Scan as reported previously [4,15]. …”

“…ATRA-NB4 cells were irradiated and then incubated for up to 24 hours at 37°C before performing protein extraction and Western blots as described previously [15]. Mouse polyclonal antibodies against human CX3CL1 (MAB3561; R&D Systems, Minneapolis, USA) and monoclonal antibody against Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma, St. Louis, Missouri, USA) were used for the Western blotting.…”

“…Mouse polyclonal antibodies against human CX3CL1 (MAB3561; R&D Systems, Minneapolis, USA) and monoclonal antibody against Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma, St. Louis, Missouri, USA) were used for the Western blotting. The results were first normalized against the GAPDH present in the same cells and then expressed as a fold increase relative to the UV un-irradiated ATRA-NB4 cells as control [4,15]. …”