Beta1 and beta2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation - PubMed (original) (raw)

Beta1 and beta2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation

Amy K McNally et al. Am J Pathol. 2002 Feb.

Abstract

An in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion was used to investigate the cell/substrate adhesive mechanisms that support multinucleated foreign body giant cell (FBGC) formation. Monocytes were cultured for 3 days and IL-4 was added to induce macrophage fusion and FBGC formation by day 7. Functionally defined anti-integrin antibodies demonstrated that initial monocyte adhesion is mediated by beta2 integrins, whereas during the induction of macrophage fusion by IL-4, an additional dependence on beta1 integrins is acquired. The combination of anti-beta1 plus anti-beta2 was most effective, reducing macrophage/FBGC adhesion to 10% of controls. Consistent with integrin-mediated signaling, the tyrosine kinase inhibitor genistein and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002 also attenuated macrophage/FBGC adhesion. Confocal microscopic analysis revealed that beta2 integrins are present on monocytes after initial adhesion and are strongly expressed on fusing macrophages, particularly in peripheral cell areas, and on FBGCs. In contrast, beta1 integrins are not detected on monocytes but begin to appear during macrophage development and are strongly expressed on fusing macrophages and FBGCs. For the first time, these results demonstrate the IL-4-induced acquisition of cooperation between beta1 and beta2 integrins in the cell/substrate adhesive interactions that are required for multinucleated FBGC formation.

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Figures

Figure 1.

Figure 1.

The effects of EDTA or EGTA and various inhibitors of nonintegrin receptors on initial monocyte adhesion (A) and macrophage/FBGC adhesion (B). Freshly isolated monocytes or macrophages/FBGCs (day 3 to day 7) were treated with the indicated inhibitors as described in Materials and Methods. Results are expressed as the percent adhesion of untreated control cells, n = 3 monocyte donors. From left to right: EDTA, 10 mmol/L; EGTA, 10 mmol/L; heparin, 2 μmol/L; dextran sulfate, 25 μg/ml; lipopolysaccharide (LPS), 50 μg/ml; chondroitin sulfate A (CS-A), 1 mg/ml; chondroitin sulfate B (CS-B), 1 mg/ml.

Figure 2.

Figure 2.

Effects of soluble fibronectin or its proteolytic fragments on initial monocyte adhesion (A) and macrophage/FBGC adhesion (B). Freshly isolated monocytes or macrophages/FBGCs (day 3 to day 7) were treated with 0.05 mg/ml (solid bars), 0.1 mg/ml (shaded bars), or 0.25 mg/ml (open bars) of purified plasma fibronectin (FN), the 120-kd FN fragment, the 40-kd FN fragment, or human serum albumin as described in Materials and Methods. Results are expressed as the percent adhesion of untreated control cells, n = 3 monocyte donors.

Figure 3.

Figure 3.

The effects of anti-integrin antibodies on initial monocyte adhesion (A) or macrophage/FBGC adhesion (B). Freshly isolated monocytes or macrophages/FBGCs (day 3 to day 7) were treated with 50 μg/ml each of the indicated anti-integrin antibodies (b1, β1; b2, β2; b3, β3; aM, αM) individually or in various combinations or with nonspecific control IgGs as described in Materials and Methods. Results are expressed as the percent adhesion of untreated control cells, n = 3 monocyte donors.

Figure 4.

Figure 4.

The effects of inhibitors of integrin-mediated cell signaling. Increasing concentrations of the tyrosine kinase inhibitor genistein (A) or the phosphatidylinositol-3-kinase inhibitors LY294002 (B) and wortmannin (C) were added during the induction of macrophage fusion/FBGC formation (day 3 to day 7) as described in Materials and Methods. Results are expressed as the percent adhesion of untreated control cells, n = 3 monocyte donors.

Figure 5.

Figure 5.

Expression of integrins on monocytes or monocyte-derived macrophages. Integrin expression was analyzed on monocytes after initial adhesion after 1.5 hours (A, B, and C) or on day 3 of culture (D, E, and F). Permeabilized cells were stained with mouse monoclonal anti-integrin detecting antibodies (Table 1) ▶ and anti-mouse IgG conjugated to Cy-5. A and D, β1 integrin; B and E, β2 integrin; and C and F, β3 integrin. Representative images are of single 2-μm slices at the cell/substrate interface. Scale bars, 50 μm.

Figure 6.

Figure 6.

Expression of integrins on fusing macrophages/FBGCs. A, β1 integrin; B, β2 integrin; and C, β3 integrin. Cells were fixed and permeabilized on day 7 after the induction of macrophage fusion by IL-4. Fusing macrophages/FBGCs were then stained with mouse monoclonal anti-integrin detecting antibodies (Table 1) ▶ followed by a mixture of anti-mouse IgG conjugated to Cy-5 (green, integrins), rhodamine phalloidin (red, F-actin), and YO-YO-1 (blue, nuclei). Representative micrographs are shown of merged Z-series images. Scale bars, 50 μm.

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