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(The FASEB Journal. 1999;13:823-832.)
© 1999 FASEB

Research Communications

Disruption of filamentous actin inhibits human macrophage fusion

KRISTIN M. DeFIFE, CHRISTOPHER R. JENNEY, ERICA COLTON and JAMES M. ANDERSON1

Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA

1Correspondence: Institute of Pathology, Case Western Reserve University, 2085 Adelbert Rd., Cleveland, OH 44106, USA.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The foreign body reaction to implanted biomaterials, characterized by the presence of macrophages and foreign body giant cells (FBGC), can result in structural and functional failure of the implant. Recently, we have shown that interleukin-4 and interleukin-13 can independently induce human macrophage fusion to form FBGC via a macrophage mannose receptor (MR) -mediated pathway. The MR is believed to mediate both endocytosis of glycoproteins and phagocytosis of microorganisms, which bear terminal mannose, fucose, N-acetylglucosamine, or glucose residues. Polarization of microfilaments to closely apposed macrophage membranes as observed with fluorescence confocal microscopy led us to ask whether MR-mediated fusion occurred via a filamentous actin-dependent pathway. Cytochalasins B and D and latrunculin-A, agents that disrupt microfilaments, inhibited macrophage fusion in a concentration-dependent manner. The concentrations of cytochalasins D and B that inhibited fusion did not significantly decrease macrophage adhesion, spreading, or motility but did inhibit internalization of Candida albicans during interleukin-13-enhanced, MR-mediated phagocytosis. Very low concentrations of cytochalasin B (< 2 µM) induced a slight enhancement of macrophage fusion. Taken together, the results of this study suggest that cytokine-induced, MR-mediated macrophage fusion requires an intact F-actin cytoskeleton and that the mechanism of fusion is similar to phagocytosis.—DeFife, K. M., Jenney, C. R., Colton, E., Anderson, J. M. Disruption of filamentous actin inhibits human macrophage fusion.


Key Words: microfilaments • foreign body giant cells • cytochalasins • macrophage mannose receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FOREIGN BODY GIANT cells (FBGC)2 are hallmark histological features of the foreign body reaction to implanted biomaterials, such as the fabric materials of vascular prostheses (1) . These multinucleated cells form from the fusion of macrophages (2 3 4 5) , which are believed to be the central cellular mediators of the chronic inflammatory response to foreign materials (1) . The presence of these critical cell types has been associated with structural and functional failure of biomedical implants (6, 7) . The role played by FBGC in the inflammatory response is not clear, but analyses of retrieved biomaterials have revealed material surface cracks directly (and only) under adherent FBGC, which suggests that FBGC contribute to biomaterial degradation in vivo (8) . This significant clinical problem has stimulated investigation into the mechanism of macrophage fusion leading to the formation of FBGC.

Recently, we demonstrated that the closely related cytokines interleukin 4 (IL-4) and IL-13 can each induce macrophage fusion and FBGC formation in vitro (9, 10) and, further, that IL-4 may participate in FBGC formation on biomaterials in vivo (11) . That the fusion-promoting ability of IL-13 was not additive to or synergistic with that of IL-4 is consistent with reports of overlapping pathways of signal transduction that are stimulated by these cytokines (12 13 14 15 16) . The IL-4 and IL-13 receptor signaling that initiates events leading to human macrophage fusion has yet to be elucidated, but IL-4 and IL-13 each induce up-regulation of the macrophage mannose receptor (MR; 10, 17, 18 ), which possesses several features favorable for a potential macrophage fusion-mediating receptor. MR are expressed on cells of the macrophage lineage (19) , are not detectable on blood monocytes (19) , and have their expression modulated by inflammatory mediators (20, 21) . Indeed, an essential role for MR in the mechanism of IL-4- and IL-13-induced macrophage fusion is suggested by the prevention of fusion in vitro by inhibitors of MR activity (18, 22) . However, definitive proof that the MR, and not an as yet undescribed MR-like molecule, mediates macrophage fusion awaits the development of antibodies that specifically block MR activity. Nevertheless, the implication of MR in the mechanism of macrophage fusion may reveal additional participants in the mechanism.

The MR is believed to function in innate immunity by recognizing unopsonized microorganisms bearing terminal mannose, fucose, N-acetylglucosamine, or glucose residues (23) . Although most mammalian cells and serum glycoproteins do not express terminal mannose-bearing oligosaccharides, select endogenous proteins that do contain terminal mannose residues, such as lysosomal hydrolases, are bound by the MR (23, 24) . MR-mediated internalization of microorganisms occurs via phagocytosis whereas internalization of glycoproteins occurs via endocytosis (pinocytosis).

Several key factors differentiate endocytosis from phagocytosis. First, most cells can ingest particles of less than 1 µm in diameter by receptor-mediated endocytosis. Larger particles are engulfed specifically by phagocytic cells (25) . Second, most endocytosis is mediated by clathrin-coated pits. Clathrin is not required for phagocytosis (26) . Third, phagocytosis, but not endocytosis, is inhibited at temperatures below 18°C (27, 28) . Fourth, actin filaments are required only for phagocytosis (25) . Phagocytosis mediated by either complement or Fc receptors is sensitive to treatment with cytochalasins (29) , drugs that disrupt actin polymerization (30) , but endocytosis is cytochalasin insensitive (27) . Although its cytoskeletal requirements are unclear, MR-mediated phagocytosis appears to occur in a manner similar to Fc receptor-mediated phagocytosis in that pseudopodia extend around the bound particles (31) . This observation suggests that filamentous actin (F-actin) is required for MR-mediated phagocytosis. Collectively, these contradictions indicate that phagocytosis and endocytosis are mechanistically quite distinct.

An endocytic/phagocytic pathway of macrophage fusion was proposed over 15 years ago (32) . In this model, multinucleated giant cell formation results from macrophages ingesting particles in close apposition to other macrophages. In conjunction with cytokine mediators in the inflammatory exudate that may serve to up-regulate components essential to the mechanism of fusion, a biomedical material that is too large to engulf may induce frustrated phagocytosis in the adherent macrophages (33) , enabling their fusion to form FBGC. Therefore, in this study we asked whether inhibitors of filamentous actin would prevent IL-13-induced macrophage fusion and FBGC formation on a model biomedical material in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Monocyte isolation
Human blood monocytes were isolated from the venous blood of unmedicated donors by a nonadherent, density centrifugation method as described previously (34) . Isolated monocytes were judged >97% viable by trypan blue exclusion and >80% pure by staining for nonspecific esterase and peroxidase. Monocytes were suspended in a medium of RPMI 1640 (Life Technologies, Inc., Grand Island, N.Y.) containing 25% autologous serum and antibiotic/antimycotic mixture (Life Technologies, Inc.). Monocytes were then cultured on dimethyldichlorosilane- (Alfa Aesar, Ward Hill, Mass.) (35, 36) treated coverslips (Thomas Scientific, Swedesboro, N.J.) that had been secured in the wells of 24-well tissue culture polystyrene plates (Falcon 3047, Becton Dickinson and Co., Lincoln Park, N.J.) with silicone rubber rings (Cole-Parmer Instrument Co., Niles, Ill.) (10) . Monocytes were added at a concentration of 5 x 105 per well in 0.5 ml medium to culture plates containing the coverslips and were allowed to adhere for 2 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. Nonadherent cells were removed by aspirating the medium and rinsing the wells with warmed (37°C) Dulbecco's phosphate-buffered saline (PBS; Life Technologies, Inc.) containing calcium and magnesium, and the remaining adherent monocytes were covered with 1 ml per well of fresh medium.

Monocyte/macrophage culture for inhibition of macrophage fusion
On day 3, medium was replaced with fresh RPMI containing heat-treated (56°C for 1 h) autologous serum (25% v/v). At this time, recombinant human IL-13 (R&D Systems, Minneapolis, Minn.) that had been reconstituted per manufacturer's instructions in PBS containing 0.5% bovine serum albumin (BSA; low endotoxin; Sigma Chemical Co., St. Louis, Mo.) was added at a final concentration of 10 ng/ml. Cytochalasin D (Sigma), cytochalasin B (Sigma), and latrunculin-A (Biomol, Plymouth Meeting, Pa.) were also added as indicated. The procedure followed on day 3 was repeated on day 7, and cultures were terminated on day 10. The wells were then rinsed twice for 5 min each with warmed PBS and fixed with methanol for 5 min for light microscopic evaluation or with 3.7% formaldehyde in PBS for 20 min for fluorescence confocal imaging. For measurements of macrophage viability, cultures were rinsed twice for 1 min each with warmed PBS and stained with trypan blue (Sigma) for 1 min. Those cells that excluded trypan blue were counted as viable, and results were averaged from three 20x fields of view from each of four experiments.

Evaluation of FBGC formation
Cells were stained sequentially with May-Grünwald stain (Sigma) for 1 min, phosphate buffer (pH 7.4) for 1 min, Giemsa stain (34) diluted 1:14 in deionized distilled water for 5 min, and two brief distilled water rinses. Percent fusion was determined as the number of nuclei in FBGC (cells with >2 nuclei) divided by the number of nuclei contained in all adherent cells counted (5) . Four to five experiments using different blood donors were conducted per culture condition, and nuclei were counted in three 20x objective fields for each condition. FBGC areas were measured from 30 cells per condition from each experiment using the morphometric software SigmaScan Pro (Jandel Scientific Software, San Rafael, Calif.).

Fluorescence confocal scanning laser microscopy of microfilaments and MR
Cells were permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 30 s and the samples were then rinsed three times with PBS. Nonspecific sites were blocked with 1:100 donkey serum (Jackson ImmunoResearch, West Grove, Pa.) in PBS for 30 min at 37°C. Blocking serum was removed, and anti-MR antiserum (the generous gift of P. Stahl, University of Washington, St. Louis, Mo.) or goat immunoglobulin G (IgG; R&D Systems), each diluted 1:100 in PBS containing 3% BSA, was incubated with cells for 1 h at 37°C. The cultures were then rinsed four times for 5 min each with PBS. Rhodamine phalloidin (Molecular Probes, Eugene, Oreg.), used at a final dilution of 1.25 U/ml, was reconstituted per manufacturer's instructions and added to 15 µg/ml FITC-conjugated donkey anti-goat IgG (Jackson ImmunoResearch) in PBS. The secondary antibody/rhodamine phalloidin solution was incubated with the samples for 30 min at room temperature. After three rinses of 10 min each with PBS, samples were removed from the culture plates and mounted on glass slides with Gel/Mount (Biomedia Corp., Foster City, Calif.). Confocal scanning laser microscopy (MRC-600, Bio-Rad, Hercules, Calif.) was used to image cells, and optical slices of ~1 µm in thickness were taken less than 2 µm from the coverslip surface. Confocal parameters were set so that control IgG images were black; the same settings were used to collect all MR images.

Evaluation of MR-mediated phagocytosis
Candida albicans yeast, the generous gift of Dr. Mahmoud Ghannoum (Case Western Reserve University, Cleveland, Ohio), were incubated overnight at 37°C in yeast nitrogen base broth (Difco Laboratories, Detroit, Mich.). The yeasts were washed in PBS by centrifugation and heat-killed at 100°C for 30 min (37) . Yeast were counted on a hemacytometer and added to the monocyte/macrophage cultures at a concentration of 6 x 105 per well.

Monocytes/macrophages were cultured as described above with medium change and addition of IL-13 on day 3. On day 4, the cultures were rinsed with PBS containing cations and recovered with RPMI 1640 medium without serum. Mannose-BSA (Man-BSA; EY Laboratories) and galactosylated BSA (Gal-BSA; EY Laboratories) were added at final concentrations of 500 µg/ml. Cytochalasins D and B were added at final concentrations of 50 nM and 50 µM, respectively. Cultures were incubated with these compounds for 30 min, then the yeasts were added to all wells.

After an additional 1 h incubation, cultures were treated with tannic acid (1% w/v in water) for 1 min and stained with May-Grunwald/Giemsa (38) . Samples were viewed by brightfield microscopy to count the number of pink (or internalized) and violet (or extracellular) yeast for 100 macrophages for each culture condition from each of three monocyte donors. Results are expressed as mean ± SE.

Quantification of macrophage motility and morphology
Monocytes were plated at 1.5 x 106 per well as above in Attofluor cell chambers (Molecular Probes). After 3 days of culture, monocytes/macrophages were stained with Cell Tracker Green CMFDA (Molecular Probes) per manufacturer's instructions. Optical slices were taken in 3 µM increments with a 40x objective lens for each sample beginning at the coverslip surface and ending at the apical surface of the adherent cells. Extended focus images were created by projecting the optical slices into one image using COMOS software (Bio-Rad). Extended focus confocal images were recorded every 10 min for 3 h to create a time-lapse movie. Movies of the confocal images were made using Confocal Assistant software (T. Brelje, University of Minnesota Medical School, Minneapolis, Minn.), and adherent cells were categorized as either motile or nonmotile. Cells that moved beyond their original position were designated motile. Those that remained stationary or had minor cytoplasmic oscillations were designated nonmotile. Three experiments were conducted for each of the following culture conditions: untreated, 20 nM cytochalasin D, 50 nM cytochalasin D, and 50 µM cytochalasin B.

Morphological data were compiled from the first image collected for each culture condition using SigmaScan Pro. Macrophages were manually outlined to measure surface area, perimeter, and major axis length, which is defined as the distance between the two pixels on the perimeter that are furthest from each other. Shape factor describes how circular an object is and is defined as (4{pi} x area)/perimeter. A perfect circle has a shape factor of 1.0, and a line has a shape factor approaching zero. Compactness, defined perimeter2/area, describes the shape of an object as it moves from a circle to a line. The minimum compactness of a perfect circle is 4{pi}, and as an object tends toward the shape of a line, compactness approaches infinity. A minimum of 145 macrophages were included for each measurement; results for morphological data are each presented as mean ± SE.

The unpaired Student's t test was used for all statistical analyses (StatView, Abacus Concepts, Inc., Berkeley, Calif.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Filamentous actin and mannose receptors accumulate at closely apposed macrophage membranes
Phagocytosis requires intact microfilaments whereas endocytic processes occur without direct involvement of the cytoskeleton (27, 29) . Because the MR can mediate both processes, IL-13-treated macrophage cultures were stained for F-actin and MR in order to investigate cellular localization during fusion to form FBGC. Strong F-actin and MR fluorescence was concentrated along the peripheral cytoplasm of cells that were extending filopodia (Fig. 1 A) and were polarized to areas where membranes were in close apposition (Fig. 1B ). When cultures were treated with cytochalasin D and IL-13, MR expression was observed on the membranes of closely apposed cells, but F-actin fluorescence was not as strong (Fig. 1C ). Similar results were observed with cytochalasin B treatment (not shown). MR expression was very weak in cultures that were not treated with any exogenous agents (Fig. 1D ).



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  		Figure 1. Concentration of F-actin and MR along apposed macrophage membranes. Filopodia extended from cell membranes that also had concentrations of MR (A). F-actin and MR were concentrated near juxtaposed macrophage membranes (B). When the cells were treated with 20 µM cytochalasin D, MR fluorescence was strong but F-actin was disrupted (C). Untreated cultures had normal F-actin staining but very weak MR fluorescence (D). Scale bar = 10 µM.

Disruption of filamentous actin inhibits macrophage fusion
We previously demonstrated that inhibitors of MR activity prevent macrophage fusion (18) . Inasmuch as the MR mediates the mechanistically different processes of endocytosis and phagocytosis, microfilament-disrupting drugs were tested for their ability to attenuate FBGC formation. IL-13-induced macrophage fusion was inhibited in a concentration dependent manner with cytochalasin D (Fig. 2 A). Significant inhibition of 48% was achieved with 20 nM cytochalasin D (P=0.026). Although not statistically significant, macrophage fusion was attenuated by 18–32% at lower concentrations. Cytochalasin D was added to otherwise untreated cultures to measure the effect on macrophage adhesion. A gradual decrease in macrophage adhesion was observed with increasing cytochalasin D concentrations. A statistically significant (P=0.026) degree of cell loss was measured when 50 nM CD was added; however, 79% of macrophages remained adherent compared with untreated wells. Similar effects on fusion and adhesion were observed with cytochalasin B treatment (Fig. 2B ). Cytochalasin B caused a concentration-dependent attenuation of IL-13-induced macrophage fusion that became statistically significant at doses of 5 µM and higher (P<0.05). Inhibition of fusion approached 100% at the highest dose tested (50 µM) without a significant decrease in the number of adherent macrophages.



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  		Figure 2. Effect of cytochalasins on macrophage adhesion and fusion. Cytochalasin D (A) or cytochalasin B (B) was added at the indicated concentrations to monocyte/macrophage cultures also treated with 10 ng/ml IL-13. On day 10, cultures were fixed and stained with May-Grünwald/Giemsa. Percent adhesion from samples treated only with cytochalasins and percent fusion from samples treated with IL-13 and cytochalasins are expressed as mean ± SE. *P<0.05 compared with untreated.

Cytochalasin B appeared to have a biphasic effect on IL-13-induced macrophage fusion. At higher concentrations (>=5 µM), fusion was inhibited by more than 45% whereas cytochalasin B enhanced fusion at lower concentrations (0.5 to 1 µM) (Fig. 3 ). Although there is a slight increase in the number of macrophages participating in fusion shown by a higher percent fusion, the increase is not statistically significant. To more clearly observe the enhancing effects of cytochalasin B, a suboptimal concentration of IL-13 was added to induce fusion. With 5 ng/ml IL-13 added per well, a slightly greater increase in fusion was observed from 0 to 1 µM cytochalasin B added (Fig. 3) . However, this increase was also not statistically significant. The enhancement is most clearly manifested by an increase in FBGC surface area (Fig. 4 ). The increase in area may be due to a combination of increased cytoplasmic spreading and increased macrophage fusion because there was also a small increase in the number of nuclei per giant cell (Fig. 5 ). Table 1 shows estimates of the surface area contributed by each macrophage and demonstrates that at low concentrations of cytochalasin B, each macrophage accounts for significantly more surface area when compared with cultures treated with higher concentrations or no cytochalasin B (P<0.05).



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  		Figure 3. Effect of cytochalasin B on macrophage fusion. Cytochalasin B was added at the indicated concentrations to monocyte/macrophage cultures also treated with 10 ng/ml IL-13. On day 10, cultures were fixed and stained with May-Grünwald/Giemsa. Percent fusion is expressed as mean ± SE.



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  		Figure 4. Effect of cytochalasin B on FBGC size. Cytochalasin B was added at the indicated concentrations to monocyte/macrophage cultures also treated with 10 ng/ml IL-13. On day 10, cultures were fixed and stained with May-Grünwald/Giemsa. Areas are expressed as mean surface area ± SE.



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  		Figure 5. Effect of cytochalasin B on the number of nuclei per FBGC. Cytochalasin B was added at the indicated concentrations to monocyte/macrophage cultures also treated with 10 ng/ml IL-13. On day 10, cultures were fixed and stained with May-Grünwald/Giemsa. Results are expressed as mean ± SE.


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  		Table 1. Average surface area in FBGC contributed by each macrophage

We wished to confirm a role for F-actin in the mechanism of macrophage fusion by preventing fusion with latrunculin-A, a toxin from a Red Sea sponge that disrupts actin polymerization by a different mechanism than the cytochalasins. As shown in Fig. 6 , a concentration-dependent inhibition of fusion was achieved without a significant decrease in macrophage adhesion.



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  		Figure 6. Effect of latrunculin-A on macrophage adhesion and fusion. Latrunculin-A was added at the indicated concentrations to cultures treated as described for Fig. 2 . Percent adhesion from samples treated only with latrunculin-A and percent fusion from samples treated with IL-13 and latrunculin-A are expressed as mean ± SE. *P<0.05 compared with untreated.

None of the added drugs was toxic to cells at the highest tested concentrations, as measured by trypan blue exclusion (P>0.06). Treatment with 20 and 50 nM cytochalasin D resulted in macrophage viabilities of 91.5 ± 2.2% and 85.8 ± 2.9%, respectively, compared with 93.5 ± 1.8% viability of untreated cultures. Viability levels of 94.8 ± 0.9% and 80.5 ± 11.9% were measured when 20 and 50 µM cytochalasin B, respectively, were added to cultures. Treatment with 50 and 100 µM latrunculin-A resulted in cultures of macrophages with 89.3 ± 1.9% and 82.0 ± 6.5% viability.

IL-13 up-regulates MR-mediated phagocytosis
A conventional assay for macrophage phagocytosis of Candida albicans was conducted to confirm up-regulation of MR activity by IL-13. In this assay, yeast can be discriminated as intracellular or extracellular by color. Table 2 shows that IL-13 greatly increased the binding and internalization of yeast compared with untreated macrophages. Further, the binding was blocked by the competitive MR inhibitor, Man-BSA but not by a nonspecific neoglycoprotein, Gal-BSA. Uptake, but not binding, was inhibited by 50 µM cytochalasin B. When 50 nM cytochalasin D was added in one experiment, 436 violet yeast and 82 pink yeast were counted.


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  		Table 2. Phagocytosis of Candida albicans

Cytochalasin D does not decrease macrophage motility but does alter morphology
Microfilament participation has been demonstrated in several monocyte/macrophage functions that may influence macrophage fusion, including migration (39) , attachment and adhesion (39 40 41 42) , spreading (40, 43) , and secretion (44) . To ask whether cytochalasin D inhibits macrophage fusion by preventing motility, live cultures were examined by fluorescence confocal microscopy after treatment with concentrations of cytochalasin D sufficient to prevent fusion. Cytochalasin D did not inhibit macrophage motility, but treatment with 50 nM cytochalasin D resulted in a slight increase in the number of motile cells (Table 3 ).


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  		Table 3. Effect of cytochalasin D on macrophage motility and morphology

However, some morphological alterations were observed (Table 3) . Cytochalasin D treatment significantly decreased the calculated shape factor and significantly increased the calculated compactness of macrophages, indicating that treated cells are more elongated than untreated cells. The cells were not rounded, as the overall surface areas of treated macrophages were slightly greater than untreated macrophages.

In contrast, the highest concentration of cytochalasin B used to inhibit fusion (50 µM) did reduce the motility, although the difference was not statistically significant. In combination with the slight rounding of macrophages, these results may account for the greater suppression of macrophage fusion with the highest concentration of cytochalasin B compared with cytochalasin D.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An essential role for F-actin in the mechanism of macrophage fusion is proposed by this study's novel observation that microfilament-disrupting agents prevented IL-13-induced macrophage fusion and FBGC formation. This information further elucidates a cytokine-induced, receptor-mediated mechanism of macrophage fusion, which has been demonstrated by our previous studies. We have shown that the lymphokines IL-4 and IL-13 up-regulate MR surface expression and induce FBGC formation (9, 10) , and demonstrate here that IL-13 up-regulates MR activity. The MR is believed to hold an essential role in macrophage fusion, because inhibitors of its activity prevent the formation of FBGC (18) . The implication of the MR led to other questions regarding the structural support for macrophage fusion because of its dual roles in inflammation and innate immunity. The MR has been shown to mediate both the endocytosis of certain glycoproteins, a process that does not require intact microfilaments, and the phagocytosis of unopsonized microorganisms, a process that requires microfilaments (25) . Inasmuch as the present results show that F-actin is necessary for MR-mediated membrane fusion, it is suggested that MR-mediated macrophage fusion is a microfilament-dependent process similar to phagocytosis.

However, the ligand for the MR that initiates fusion is unknown. It is possible that lysosomal hydrolases, which accumulate at sites of inflammation, ligate the MR to initiate fusion. Macrophages secrete several enzymes that are natural ligands for the MR, including acid phosphatase, ß-glucuronidase, and ß-glucosaminidase (45) . Therefore, the mechanism leading to FBGC formation may involve recapture of macrophage-derived lysosomal enzymes by the MR. Glycoprotein binding by the MR appears to suggest an endocytic over a phagocytic pathway. However, appropriate ligands may be simultaneously engaged by concentrated MR along the cell membranes of closely situated cells. As shown in this study, the interactions between the closely apposed membranes occur along a length several orders of magnitude longer that the 0.1–0.2 µM expected for endocytosis (46) . Osteoclast progenitors express cell surface mannose residues prior to fusion to form multinucleated cells (47) . The ligand for MR that leads to macrophage fusion may therefore, alternatively, be a cell surface glycoprotein that bears appropriate oligosaccharides and may function like an opsonin for MR engagement, leading to membrane fusion. It is also possible that fusion may result from an as yet undescribed interaction with a ligand that is noncarbohydrate in nature, because a structurally similar member of this multilectin family (48) , a phospholipase A2 receptor, binds unglycosylated secretory phospholipases A2 (49) .

Electron microscopic studies have demonstrated that MR-mediated phagocytosis morphologically resembles Fc receptor-mediated phagocytosis (31) . As Fc receptors bind to an IgG-coated particle, actin polymerization and cross-linking occurs subjacent to clustering receptors. Actin assembly drives pseudopod extension around the particle, promoting the interaction of additional receptors with ligands on the particle. The formation of the phagosome is completed by this `zipper' mechanism, and the F-actin support structures are remodeled and disassembled (25) . The pseudopodial extension observed during MR-mediated phagocytosis suggests that a similar microfilament-mediated `zippering' occurs during particle engulfment. A similar mechanism can be envisioned for macrophage-macrophage membrane fusion, with each macrophage appearing to the other as a large particle. F-actin polarization subjacent to macrophage membranes that appear to be undergoing fusion supports this model of cytoskeleton-supported receptor interactions. In addition, we have observed enhancement of MR fluorescence in regions of macrophage membranes in close contact, suggesting receptor clustering (18) .

The polarization of F-actin to areas of close cell–cell membrane apposition and inhibition of fusion by cytochalasins suggest that F-actin functions during the fusion process itself. However, F-actin participation has been demonstrated for many monocyte/macrophage functions critical during the inflammatory response that may influence macrophage competency to participate in FBGC formation, including migration (39) , attachment and adhesion (39 40 41 42) , spreading (40, 43) , and secretion (44) . Therefore, macrophage adherence and motility were examined to ensure that microfilament disruption specifically prevented macrophage fusion instead of the necessary, but peripheral, events of monocyte/macrophage adhesion and locomotion. The present study demonstrates that, at doses sufficient to prevent fusion, cytochalasins did not significantly inhibit monocyte/macrophage adhesion or viability. A period of adherence and monocyte morphological differentiation to the macrophage phenotype, including cell spreading, is required prior to macrophage fusion (9) . Cytochalasins did not induce a loss of cell spreading. Similar surface areas were measured for both treated and untreated macrophages. Cytochalasin D-treated macrophages had a more elongated morphology that is suggestive of a motile phenotype. Together with the small increase in the number of motile cells, these results appear to predict increased opportunities for membrane–membrane contact for fusion rather than a decrease in fusion, as was observed. Therefore, it is unlikely that the small morphological alterations induced by cytochalasin D account for the inhibition of macrophage fusion. This finding is consistent with a recent study demonstrating that macrophage motility was not significantly altered by culture surface chemistry or cytokine treatment and that motility could not be used to predict surface-dependent modulations of macrophage fusion (50) . Conversely, the highest concentration of cytochalasin B used to inhibit fusion did slightly reduce motility and cell elongation, which may account for the stronger inhibition of fusion compared with cytochalasin D.

The actions of cytochalasins on actin are quite complex and incompletely understood. Cytochalasins bind to the barbed ends of actin filaments and cause net inhibition of polymerization and depolymerization (30) . It is generally believed that cytochalasin D specifically binds actin, especially when used at low concentrations. The low concentrations of cytochalasin D that inhibited fusion are expected to minimize any potential nonspecific interactions. In addition, the concentrations of cytochalasin D used in this study are lower than those used to inhibit phagocytosis (29) , membrane ruffling (51) , and stress fiber formation (51) in other studies. Therefore, the inhibition of macrophage fusion by cytochalasin D indicates a microfilament-dependent mechanism of MR-mediated fusion.

The slight enhancement of macrophage fusion observed with low doses of cytochalasin B was not observed with cytochalasin D treatment. Cytochalasins have multiple effects on actin that are concentration dependent (30) , and interaction between cytochalasin B and actin differs from cytochalasin D and actin. It is important to note that cytochalasin B, unlike cytochalasin D, binds the glucose transporter protein (52, 53) . However, the concentrations of cytochalasin B that enhanced fusion are ~100-fold lower than those used to investigate glucose transporter binding in macrophages (53) . Therefore, it is unlikely that the observed effects of cytochalasin B at such low concentrations are not microfilament mediated. It is possible that slight disruption of the actin cytoskeleton increased MR mobility in the membrane, which has been postulated to accelerate receptor–ligand bond formation (54) . Additional experiments are necessary to elucidate this interesting finding, including treatment with glucose transport inhibitors.

In conclusion, when taken together with the observations that MR mediates macrophage fusion, that the MR can mediate both phagocytosis and endocytosis, and that F-actin is required for phagocytosis, the present findings provide support for a phagocytic model of macrophage fusion. This study enhances our understanding of the mechanism of multinucleated giant cell formation during the pathophysiology of chronic inflammation.


   ACKNOWLEDGMENTS
 
This research was supported by the National Heart, Lung, and Blood Institute, Devices and Technology Branch, Grant HL 55714; The Whitaker Foundation; and The Center for Cardiovascular Biomaterials at Case Western Reserve University.


   FOOTNOTES
 
2 Abbreviations: BSA, bovine serum albumin; F-actin, filamentous actin; FBGC, foreign body giant cells; IgG, immunoglobulin G; IL, interleukin; MR, macrophage mannose receptor; PBS, phosphate-buffered saline.

Received for publication June 2, 1998. Revision received December 14, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Anderson, J. M. (1993) Mechanisms of inflammation and infection with implanted devices. Cardiovasc. Pathol. 2,33S-41S
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