Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes

Abstract

When cells undergo apoptosis, or programmed cell death, they expose phosphatidylserine (PS) on their surface. Macrophages that efficiently phagocytose apoptotic cells also express PS on their surface, although at a lower level. The PS exposed on both cells is required for phagocytosis, because uptake is inhibited by masking PS on either cell with annexin V, a PS-binding protein. The inhibition is not additive, suggesting that the exposed PS molecules on the two cells participate in a common process. We asked whether this dual requirement reflects bridging of the target cell and macrophage by bivalent, PS-binding annexins. Monoclonal antibodies (mAbs) against annexins I or II stained a variety of live phagocytes. Apoptotic Jurkat T lymphocytes and human peripheral T lymphocytes, but not apoptotic thymocytes, were stained by anti-annexin I but not II. Phagocytosis of apoptotic targets was inhibited by mAbs to annexins I or II, or by pretreatment of macrophages with the same mAbs. Pretreatment of apoptotic thymocytes had no effect, whereas pretreating Jurkat cells with anti-annexin I or removing annexin I with EGTA was inhibitory. Annexin bridging is vectorial, because annexin is bound to PS molecules on targets but not on macrophages, suggesting annexins serve as both ligand and receptor in promoting phagocytosis.

Figure 1.

Binding of anti-annexin mAbs to live J774 and EP macrophages. (A and B) J774 macrophages were stained with anti-annexin I, II, or mouse IgG₁ isotype control mAbs (A), or anti-annexin V or mouse IgG_2a isotype control mAbs (B). (C) EP macrophages were stained with anti-annexin I, II, or mouse IgG₁ isotype control mAbs. (D and E) J774 macrophages were washed twice with buffer containing 10 mM EGTA (thick line) or 2 mM Ca²⁺ (thin line), and then stained with anti-annexin I or mouse IgG₁ isotype control mAbs (D), or anti-annexin II or mouse IgG₁ isotype control mAbs in the same buffer (E). Cells were analyzed by flow cytometry after secondary staining with goat antimouse IgG-FITC.

Figure 2.

Binding of anti-annexin mAbs to live U937 cells. Undifferentiated U937 cells (thin line) or U937 cells treated with PMA for 2 d (thick line) were stained with anti-annexin I or mouse IgG₁ isotype control mAbs (A), anti-annexin II or mouse IgG₁ isotype control mAbs (B), or anti-annexin V or IgG_2a isotype control mAbs (C), and cells were examined by flow cytometry after secondary staining with goat anti-mouse IgG-FITC. (D) Cells treated with PMA for 2 d were stained with FITC-annexin V in the presence (thick line) or absence (thin line) of Ca²⁺ to distinguish Ca²⁺-dependent, PS-specific binding, and analyzed by flow cytometry.

Figure 3.

Phagocytosis of apoptotic mouse thymocytes in the presence of anti-annexin mAbs. Mouse thymocytes, either untreated or treated with 10^-6 M dexamethasone for 6 h to induce apoptosis, were presented to J774 (A and E) or EP (B–D) macrophages in the presence of various anti-annexin and IgG₁ isotype control mAbs (A and B), different concentrations of anti-annexin I mAb (C) or anti-annexin II mAb (D), or a combination of anti-annexin I and II mAbs (E).

Figure 4.

Phagocytosis of apoptotic mouse thymocytes by differentiated U937 macrophages. U937 cells were treated with PMA for 1 d to induce differentiation, and presented with mouse thymocytes, either untreated or treated with 10^-6 M dexamethasone for 6 h to induce apoptosis, in the presence of RGDS or N-acetylglucosamine (GlcNAc) (A) or anti-annexin I, II or IgG₁ isotype control mAbs (B).

Figure 5.

Phagocytosis of PS-expressing or opsonized erythrocytes by EP macrophages in the presence of anti-annexin mAbs. (A) PS expressing, lipid-symmetric (LS), or PS-nonexpressing, lipid-asymmetric (LA) erythrocytes or (B) erythrocytes opsonized with antiglycophorin antibody were presented to EP macrophages in the presence of anti-annexin mAbs.

Figure 6.

Phagocytosis of apoptotic thymocytes or apoptotic Jurkat cells after pretreatment of either macrophages or target cells with anti-annexin mAbs, or after treatment of Jurkat cells with EGTA. (A and B) Mouse thymocytes (T), either untreated or treated with 10^-6 M dexamethasone for 6 h to induce apoptosis, or (C) Jurkat T lymphocytes (T), either untreated or treated with anti-Fas mAb for 15 h at 37°C to induce apoptosis, were presented to J774 macrophages. Either macrophages or target cells were incubated with anti-annexin mAbs for 30 min at 37°C, washed, and then used in phagocytosis assays. Coincubation of macrophages and targets with anti-annexin mAbs, as done in Figure 3, is shown for comparison. One sample of apoptotic Jurkat cells was treated with EGTA to remove annexin I before presenting to macrophages.

Figure 7.

Binding of anti-annexin mAbs to apoptotic Jurkat cells. Jurkat T lymphocytes treated with anti-Fas mAb for 15 h at 37°Cto induce apoptosis were stained with annexin V-FITC (A), anti-annexin I (B) or anti-annexin II mAb (C), followed by staining with PE-conjugated goat anti-mouse IgG. Jurkat T lymphocytes were also double stained with anti-annexin I (D) or IgG₁ isotype control (E) followed by goat anti-mouse IgG-PE staining, and after washing, with annexin V-FITC. Thin line, mAb staining of gated annexin V-negative cells; thick line, mAb staining of gated annexin V-positive cells. (F) Jurkat T lymphocytes were washed with buffer containing 10 mM EGTA before antiannexin I mAb staining.

Figure 8.

Expression of annexins by apoptotic thymocytes and apoptotic Jurkat cells. (A) Mouse thymocytes, either untreated or treated with 10^-6 M dexamethasone for 4 h, were stained with annexin V-FITC to confirm the presence of apoptotic cells. (B) Apoptotic mouse thymocytes were stained with anti-annexin I, II, V, or mouse IgG₁, IgG_2a isotype control mAbs, and analyzed by flow cytometry after secondary staining with goat anti-mouse IgG-FITC. (C) Whole cell lysates of apoptotic Jurkat cells, washed with buffer without EGTA (J) or with EGTA (J+E), or whole cell lysates of apoptotic primary thymocytes (T) were resolved by SDS-PAGE, and the blot of the gel probed with anti-annexin I mAb. I, purified annexin I.

Figure 9.

Binding of anti-annexin mAbs to apoptotic human peripheral T lymphocytes. PHA-activated T lymphocytes treated with camptothecin for 15 h at 37°C to induce apoptosis were stained with annexin V-FITC (A), anti-annexin I mAb (B), anti-annexin II mAb (C), or IgG₁ isotype control mAb (D), followed by staining with PE-conjugated goat anti-mouse IgG. (E) Camptothecin-treated T lymphocytes were washed with buffer containing 10 mM EGTA before anti-annexin I mAb staining.