Involvement of Annexin A2 in Serum-MAF Dependent Phagocytic Activation of Macrophages

Results

Serum-MAF induces translocation of annexin A2 to the cell cortex. As shown in Figure 1, in untreated macrophages (without serum-MAF), annexin A2 was observed as tiny dots scattered within the cytoplasm (Figure 1A, F). Within 30 s after serum-MAF treatment (Figure 1B, G), annexin A2 accumulated in the cortical region. At 60 s after serum-MAF treatment, thin actin accumulation at the edge of membrane ruffling started to be observed as a bud for a newly formed frill-like structure (Figure 1H). In some ruffles, annexin A2 cortical accumulation was observed around the basal region of the ruffles (Figure 1C). After 180 and 300 s, an evident frill-like structure was formed (Figure 1I and J). Although the total fluorescent intensity of annexin A2 was increased, particularly in some cortical regions, annexin A2 was no longer present in the fully formed frill-like structure (Figure 1D and E).

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Figure 1.

The changes in the localization of annexin A2 and F-actin. Macrophages were fixed at 0 s (A, F; Untreated), 30 s (B, G), 60 s (C, H), 180 s (D, I), and 300 s (E, J) after addition of 8 μg/ml serum macrophage activating factor (MAF) and stained for annexin A2 (red), F-actin (green), and nuclei (blue). Upper images (A-E) show only annexin A2 staining, and lower images (F-J) show merged images. White arrowheads indicate cortical annexin A2 staining. Blank and filled yellow arrowheads indicate the buds and fully formed frill-like structures, respectively. Note that total annexin A2 staining was increased from 180 s concomitant with frill-like structure formation; however, most of the annexin A2 disappeared from the vigorously ruffling membrane. Scale bars; 25 μm.

Relationship between F-actin and annexin-A2 accumulation in the cortical region. In the absence of serum-MAF, only a few small dots of annexin A2 were also observed in the cortex (Figure 2A, small blank arrowheads). The distribution of annexin A2 and F-actin was mutually exclusive. When F-actin resided in the cortex, annexin A2 dots were situated beneath the membrane (Figure 2A). Furthermore, 30 or 60 s after serum-MAF addition, cortical accumulations of annexin A2 became evident and lined up in some cortical areas (Figure 2B and C). F-actin and annexin A2 still showed mutually exclusive localization in some cortical regions (blank arrowheads), although their colocalization was obvious (filled arrowheads). Although annexin A2 accumulation beneath cortical F-actin (small arrowheads) was still observed at 30 s, these two became aligned in the cortical region at 60 s. Annexin A2 was excluded from the flattened membrane ruffling (Figure 2C, yellow arrow).

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Figure 2.

The changes in the localization of annexin A2 and F-actin just after addition of serum-derived macrophage activating factor (MAF). These are enlarged images of cells from the same experiment as in Figure 1, without DAPI staining. A higher magnification of each inserted dotted square is indicated on the left side. Large and small arrowheads indicate the cortical localization of F-actin and annexin A2, respectively. Blank and filled arrowheads indicate areas of localisation of only F-actin or annexin A2 or areas where they are colocalized, respectively. The yellow arrow indicates small-membrane ruffling. Cortical colocalization of annexin A2 and F-actin gradually increased up to 60 s. Scale bars: 10 μm and 1 μm as indicated.

Ca²⁺ chelator inhibited annexin A2 accumulation and frill-like structure formation. Intracellular Ca²⁺ is considered to mediate annexin A2-activation (12). When EGTA (2.5 mM) was added to the culture medium, cortical annexin A2 accumulation within 60 s (data not shown) and increased annexin A2 expression at 5 min (Figure 3) were inhibited. Frill-like structure formation was also suppressed by Ca²⁺ chelation.

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Figure 3.

Effect of Ca²⁺ chelation on annexin A2 and F-actin localization. Macrophages preincubated with (C, D) or without (A, B) EGTA (2.5 mM) for 60 min were treated with (B, D) or without (A, C) 8 μg/ml serum-MAF for 5 min. Cells were stained for annexin A2 (red), F-actin (green), and nuclei (blue). Scale bar; 20 μm.

Ca²⁺ chelation down-regulates phagocytic activity. The phagocytic activity of serum-MAF-treated macrophages showed a rapid and significant increase (10 min, in this experiment), in addition to the later activation (30, and 60 min; Figure 4) (3). Under Ca²⁺-chelated conditions (addition of EGTA (2.5 mM)), the phagocytic activity of serum-MAF-treated macrophages was down-regulated at all time points, as compared to that without EGTA. It was noted that even in Ca²⁺-chelated conditions, serum-MAF up-regulated phagocytic activity similar to that observed under normal conditions.

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Figure 4.

Effect of Ca²⁺ chelation on phagocytic activity. Macrophages were preincubated with or without EGTA (2.5 mM) for 60 min. The phagocytic activities of macrophages treated with (serum-MAF) or without (control) 8 μg/ml serum-MAF for 5 min were measured. Error bars represent standard deviation. *p<0.05, compared to the untreated control at 10 min (t-test; n=3). N.S.: Not significant.