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Published online before print June 7, 2007
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From the Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, and the Departments of Pathology and Biomedical Engineering,* Yale University School of Medicine, New Haven, Connecticut
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Abstract |
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We have recently described a requirement for the chemokine CCL2/MCP-1 in the formation of FBGCs.9 Specifically, we have shown limited FBGC formation in vivo in MCP-1-null mice and after the addition of MCP-1 blocking antibody and peptides to interleukin (IL)-4-treated human peripheral blood monocytes in vitro. However, we were unable to determine whether MCP-1-null macrophages were inherently deficient in fusion. In the present study, we describe the preparation of murine bone marrow-derived macrophages and the establishment of the IL-4 fusion assay using these cells. Furthermore, we confirmed the relevance of the assay by demonstrating an inherent fusion defect in MCP-1-null macrophages.
Previous studies using human peripheral blood monocytes have shown that these cells undergo significant cytoskeletal rearrangement during IL-13-induced fusion.10 Specifically, it was shown that, in comparison to untreated macrophages, more F-actin was located in the immediate proximity of the ventral plasma membrane of FBGCs. Formation of podosome-like structures has also been reported in the murine macrophage cell line 264.7 exhibiting FBGC formation on highly polished pure titanium.2 Limited fusion has also been reported in the murine macrophage cell line IC-21.11 However, these and several other macrophage cell lines were found to be poorly fusogenic in response to IL-4 (S.M.J., E.S., and T.R.K., manuscript in preparation). Changes in macrophage shape and formation of cell protrusions have also been reported in vivo after the implantation of cellophane films in rats.12
Although the critical signaling events leading to FBGC formation via macrophage-macrophage fusion are unknown, there are shared features with phagocytosis.13
Specifically, the involvement of the phagocytic cellular machinery, including vacuolar-type ATPase and calcium-independent phospholipase A2, has recently been shown to be critical for fusion.13
In phagocytosis, macrophages use opsonic (Fc
R, C3) and nonopsonic (C-type lectins) receptors to phagocytose microbes and particles.14,15
Engulfment of particles through the C3 receptor occurs through a direct sinking of the plasma membrane and is morphologically distinct from FcR-mediated phagocytosis, which involves pseudopod extension. Interestingly, IL-4-induced FBGC formation has been shown to involve pseudopod formation as well as direct sinking of the plasma membrane of one cell to another.13,16
In addition, similar to phagocytosis, actin polymerization and reorganization are observed during macrophage fusion and, when inhibited, prevent fusion and subsequent FBGC formation.17
In the present study, we expanded on these observations by identifying lamellipodia formation as a key feature of the IL-4-induced cytoskeletal changes in macrophages. Consistent with the role of Rac1 in this process, we detected IL-4-induced Rac1 activation before changes in cell shape. By using pharmacological inhibitors of small GTPases and a Rac1-specific short interfering RNA (siRNA), we show that inhibition of Rac activation or Rac1 knockdown can prevent lamellipodia formation and fusion without interfering with the ability of macrophages to uptake polystyrene microspheres. Previous studies have shown the efficient knockdown of Rac1 via siRNA.18 In addition, the specificity and efficacy of NSC23766 in preventing the activation of Rac1 in vitro and in vivo has been established.19,20 Here, we also demonstrated that localized release of NSC23766 attenuated FBGC formation in vivo.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Mouse macrophages were obtained from the bone marrow of C57BL6 mice (The Jackson Laboratories, Bar Harbor, ME). Selected studies were performed with macrophages derived from mice lacking MCP-1 (C57BL6). Marrow was flushed from the femurs of mice and collected in Iscove’s modified Dulbecco’s medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum and penicillin/streptomycin/fungizone. Cells were layered over Lympholyte-M (Accurate Chemical, Westbury, NY) and centrifuged according to the supplier’s instructions. The fraction containing mononuclear cells was collected and plated (2.5 x 106 cells/ml) in expansion medium [Iscove’s modified Dulbecco’s medium supplemented with 20% fetal bovine serum, L-glutamine, penicillin/streptomycin/fungizone, and 1.5 ng/ml huM-CSF (R&D Systems, Minneapolis, MN), and 100 ng/ml flt-3 ligand (R&D Systems)]. Cultures were fed on day 5, and cells were collected by scraping on day 10 and used for analysis. In general, three mice per genotype were used in each isolation procedure.
In Vitro Fusion Assay, Rac1 Activation, and Small GTPase Inhibition
Expanded monocytes/macrophages were plated at 1 x 106 cells per well in nontissue culture-treated polystyrene 24-well plates in expansion media lacking M-CSF and flt-3 ligand and supplemented with 10 ng/ml recombinant mouse GM-CSF (R&D Systems) and 10 ng/ml recombinant mouse IL-4 (R&D Systems). Media were changed at days 3 and 5, and fusion was analyzed on day 7. Three parameters of fusion were quantified including percent fusion, number of FBGCs per high-power field, and number of nuclei per FBGC. Three 24-wells per group were analyzed and the experiments were performed at least in triplicate. Each experiment was repeated with a newly expanded monocyte preparation. Three to six images per well were collected and analyzed allowing for the analysis of a total of 25 to 50 images per group as indicated in the figure legends.
Rac1 activity was determined in protein lysates using the luminescence-based Rac1 G-LISA activation assay (cytoskeleton) according to the manufacturer’s instructions. Rac1 inhibitor NSC23766 (Calbiochem, La Jolla, CA) was added to wells at a concentrations varying from 50 to 200 µmol/L, at the initiation of fusion and during scheduled feedings. A concentration of 100 µmol/L was found to be optimal for inhibition of Rac1 activation. Rho kinase inhibitor, Y-32885 also known as Y-compound (Sigma, St. Louis, MO), was added to wells at concentrations varying from 5 to 50 µmol/L, at the initiation of fusion and during scheduled feedings. A concentration of 10 to 30 µmol/L was found to be optimal for Rho kinase inhibition. Higher doses had a deleterious effect on cells. siRNA transfections of expanded monocytes were performed using Lipofectamine (Invitrogen, Carlsbad, CA) and 50 or 100 nmol/L mRac1 (5'-CAGACAGACGUGUUCUUAAUUUGCU-3') or a 100 nmol/L scrambled control siRNA with no known target sequence (both from Invitrogen). Knockdown efficiency was determined 24 hours after transfection by Western blot using anti-Rac1 antibody (clone 23A8; Upstate Technology, Lake Placid, NY).
Phagocytosis and Rac1 Inhibition
Phagocytosis was induced by the addition of Fluoresbrite YG microspheres with a 3-µm diameter (Polysciences, Warrington, PA) at sphere:cell ratios of 1:1, and 10:1, as noted. Some cultures were treated with IL-4 and exposed to beads in the presence or absence of 10 µmol/L NSC23766 to evaluate the effect of the inhibitor on phagocytosis and fusion. To evaluate whether there was a higher threshold for inhibition of phagocytosis by NSC23766, the inhibitor was added to wells at various concentrations as described above. Uptake was evaluated at 1 to 3 hours by phase microscopy or at 7 days by fluorescence microscopy. To facilitate comparisons, the results are expressed as number of microspheres per nucleus by dividing the number of beads by the number of nuclei in multinucleated cells.
Cell Staining
For counting and morphological analysis, cells were double-stained with May-Grunwald stain (Sigma) and Wright-Giemsa stain (Sigma) according to standard protocols. All experiments were done in triplicate, and a total of 25 to 50 high-power fields were analyzed for each group. For visualization of the acting cytoskeleton and nuclei, cells were fixed in 4% paraformaldehyde (J.T. Baker, Phillipsburg, NJ) for 20 minutes at room temperature and stained with rhodamine-phalloidin and 4,6-diamidino-2-phenylindole according to standard protocols. All wells were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and examined with the aid of an Axiovert 200M microscope (Carl Zeiss, Thornwood, NY) equipped with fluorescent optics. To confirm the uptake of polystyrene spheres in the presence of the Rac1 inhibitor, 0.9-µm-thick optical sections were obtained and analyzed. To analyze the effect of Rac inhibition on the uptake of microspheres, the percentage of cells containing microspheres per high-power field and the number of microspheres per cell were counted.
Fabrication of NSC23766-Loaded EVAc Scaffolds
Poly(ethylene-co-vinyl acetate) (EVAc) scaffolds were prepared via a solvent evaporation method, as described previously.21
In brief, 92 mg of Ficoll (Mr, 70,000) were mixed with 8 mg of NSC23766 in DI H2O and lyophilized into a powder. The resultant powder was ground via mortar and pestle to facilitate homogenous distribution into the EVAc solution. A 100-mg/ml solution of EVAc in methylene chloride was prepared, of which 1 ml was mixed extensively with the fine powder, with the resulting suspension being poured into a chilled Teflon mold and cooled at –80°C for 30 minutes. The mold was then further chilled at –20°C for 48 hours and dried under a vacuum at 0°C for an additional 48 hours to enable solvent evaporation. The dried EVAc was then cut into small disks (
3-mm diameter, 2-mm thickness, 11.19 ± 1.07 mg) with a cork borer and sterilized under UV light for 20 minutes. Control disks were made with 100 mg of Ficoll.
Implantation of Biomaterials
All procedures were performed in accordance with the regulations adopted by the National Institutes of Health and approved by the Animal Care and Use Committee of Yale University. Subcutaneous implantations were performed essentially as described previously.9 A total of six control mice (C57BL/6), age 3 to 4 months, were used for implantations. Each mouse received one control and one experimental implant that were placed at least 2 cm apart, allowing for six implants per group. Implants were excised en bloc, at 2 weeks after implantation, as described previously and processed for histological and immunohistochemical analysis.9
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Macrophages were expanded from the bone marrow of wild-type mice and induced to fuse into multinucleated giant cells through concurrent addition of GM-CSF and IL-4. Under baseline conditions, fusion was determined as the percentage of nuclei that were part of multinucleated cells and was found to be 75.5 ± 16.7%. To validate the use of marrow-derived macrophages in the fusion assay system we prepared and examined the fusion of cells derived from mice that lack MCP-1. Previously, we have shown that FBGC formation in vivo was compromised in these mice and that inhibition of MCP-1 activity limited the fusion of human monocytes.9
Consistent with our previous findings, the fusion of MCP-1-null macrophages was reduced in comparison to cells prepared from littermate control mice (Figure 1)
. Overall fusion was estimated to be 78.9 ± 12.4 in control macrophages and 31.8 ± 7.36% in MCP-1-null macrophages (P 
0.05). In addition to a reduction in overall fusion, MCP-1-null macrophages formed less and smaller FBGCs containing a reduced number of nuclei (Figure 1)
.
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Changes in cell shape in macrophages that were induced to fuse were evident as early as 24 hours and became prominent at 48 hours after addition of the fusion cocktail (Figure 2B)
. Visualization of the actin cytoskeleton with rhodamine-phalloidin confirmed the loss of peripheral actin rings that were present in control cells (Figure 2, C and D)
. Dramatic elongation was characteristic of cells that appeared in the process of fusion (Figure 2D)
. In addition, assembly of punctate actin in a polarized manner was observed in multinucleated cells (Figure 2D)
. These results are consistent with reported observations on the fusion of human monocytes.10
Mouse macrophages also displayed extensive lamellipodia formation at 48 hours, which was predominantly a feature of single cells in the vicinity of FBGCs, even though occasionally FBGCs were seen to have such structures (Figure 2D)
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Small GTPases have a well-established role in regulating cytoskeletal rearrangements.22
Consistent with this function, we detected an increase in activation of Rac1 after exposure to the fusogenic stimulus (Figure 3A)
. Furthermore, application of the small molecular weight and highly specific inhibitor of Rac activation (NSC23766) for 7 days limited the fusion of macrophages (Figure 3)
. All three parameters of fusion, including percent fusion (Figure 3E)
, the number of FBGCs (Figure 3F)
, and the number of nuclei per FBGC (Figure 3G)
were decreased by treatment with 50 µmol/L NSC23766. Visualization of the cell cytoskeleton by rhodamine-phalloidin at earlier time points, ie, 2 and 4 days, indicated that cells treated with NSC23766 underwent minimal elongation and did not form lamellipodia (Figure 3, B and D)
. In addition, the activation of Rac1 was decreased below baseline levels (Figure 3A)
. The response to the inhibitor seemed to be dose-dependent because cells treated with 50 µmol/L NSC23766 exhibited more fusion than cells treated with 100 µmol/L (Figure 3, E–G)
. Treatment of cells with 200 µmol/L NSC23766 did not reduce fusion further (data not shown).
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. Cell elongation was observed in all IL-4-induced cells treated with Y-32885, suggesting that the process is not dependent on the function of Rho kinase.
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Because lamellipodia formation has been shown to play a role in some aspects of phagocytosis,23
we examined whether NSC23766 could inhibit both processes. Application of NSC23766 to macrophage cultures that were induced to fuse in the presence of polystyrene microspheres indicated that even though fusion was limited, the uptake of spheres was not compromised (Figure 5)
. Examination of cultures shortly after addition of microspheres (1 to 3 hours) revealed similar uptake in control and NSC23766-treated cells (Figure 5, A and B)
. At 7 days, in the absence of the Rac1 inhibitor, giant cell formation and uptake of beads were evident (Figure 5C)
. As expected, limited lamellipodia formation was observed in cells treated with NSC23766, but it did not have an effect on the uptake of microspheres (Figure 5D)
. To confirm the intracellular location of the microspheres in cultures treated with NSC23766, we obtained 0.9-µm-thick optical sections that included both nuclei and spheres confirming the intracellular location of the latter (Figure 5E)
. To exclude the possibility that the lack of an effect on phagocytosis was attributable to a difference in dose sensitivity, we repeated the experiment in the presence of varying concentrations of NSC23766. No differences in microsphere uptake, measured as percentage of cells containing microspheres and number of microspheres per nucleus, were observed (Figure 5, E and F)
. Consistent with the notion that fusion and phagocytosis are distinct processes, we observed that MCP-1-null macrophages were not compromised in their ability to uptake microspheres (Figure 5F)
. Taken together, these observations suggest that monocyte fusion and the uptake of microspheres are mediated by distinct intracellular mechanisms.
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To confirm further the involvement of Rac1 in macrophage fusion, we used a Rac1-specific siRNA approach. Treatment of cells with 50 or 100 nmol/L mRac1 siRNA resulted in significant reduction in total Rac1 in comparison to cells treated with 100 nmol/L scrambled control siRNA (Figure 6A)
. Addition of IL-4 induced robust fusion in control-treated cells, whereas fusion was limited in cells treated with mRac1 siRNA (Figure 6, B and C)
. Analysis of fusion indicated a reduction in all three parameters of fusion in both 50- and 100-nmol/L-treated cells (Figure 6, E–G)
. Overall, fusion was limited to an extent that was similar to that observed in cultures treated with NSC23766. To determine whether Rac1 knockdown influenced phagocytosis, we exposed mRac1 siRNA-treated cultures to microspheres and monitored their uptake from 1 hour to 7 days. Consistent with our findings with NSC23766, we found that phagocytosis was not compromised in mRac1 siRNA-treated cells (Figure 6D)
.
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NSC23766-eluting EVAc scaffolds were prepared and the release of the drug was evaluated in vitro and determined to be in the range of 5 µg/mg of scaffold and was sustained for at least 18 days (Supplemental Figure S1 at http://ajp.amjpathol.org). EVAc scaffolds with or without NSC23766 were implanted subcutaneously in wild-type mice for 2 weeks and the formation of FBGCs was evaluated. A significant reduction in the number of FBGCs surrounding NSC23766-eluting implants in comparison to control implants was observed. Macrophage recruitment, assessed by immunohistochemistry with Mac3 antibody (Figure 7, C and D)
, and the overall foreign body response, assessed by H&E staining (Figure 7, A and B)
, was similar between the two groups. The lack of an association between FBGC formation and the foreign body response is consistent with our previous observations in MCP-1-null mice in which we observed a reduced number of FBGCs but normal biomaterial encapsulation.9
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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20%), and the majority of the cells were shown to have less than five nuclei.25
We believe that the high level of fusion we observe in our cultures is attributable to the fact that we isolated and expanded an enriched monocyte population before inducing fusion. More importantly, the establishment of a murine macrophage fusion assay should allow for the examination of cells derived from genetically modified animals. Such studies can be performed without the need for molecular or pharmacological inhibitors and can lead to the elucidation of additional genetic and molecular determinants of fusion. To verify the utility of the assay, we present evidence for an inherent fusion deficiency in macrophages derived from MCP-1-null mice, previously shown to display reduced FBGC formation in vivo.9
Because multinucleated FBGCs and osteoclasts are formed from the fusion of monocyte-derived cells, it will also be possible and informative to test cells from mice with known osteoclast defects for their ability to form FBGCs. Such an approach has identified a critical role for dendritic cell-specific transmembrane protein (DC-STAMP) in the formation of both cell types.29 Previous studies investigating fusion have noted the occurrence of cytoskeletal changes similar to those observed in phagocytosis.13,30 Although the rearrangement of the cell’s cytoskeleton is an important component of both phagocytosis and fusion, the role of lamellipodia formation in fusion has not been previously reported. Consistent with the vital role of Rac1 in lamellipodia formation,31 we observed Rac1 activation in IL-4-treated cells before changes in cell shape. Furthermore, reduction of total Rac1 via siRNA or the pharmacological inhibition of its activation led to a dose-dependant reduction in fusion, suggesting that lamellipodia formation is critical for this process. Inhibition of Rho kinase, on the other hand, did not have a profound effect on fusion. In addition, there was no effect on cell elongation and no apparent delay in the appearance of fused cells. Thus, we conclude that the cellular changes that precede fusion are not dependent on Rho-mediated processes. Interestingly, inclusion of Y-32885 limited the formation of larger FBGCs suggesting a role for Rho in the growth of FBGCs. We speculate that this might be attributable to a negative effect of the inhibitor on the recruitment of single cells to multinucleated cells.
Because of the profound effect of siRNA-mediated knockdown and NSC23766 treatment on fusion and the fact that Rac1 has been shown to be required for certain phagocytic processes,32 we investigated the effect of Rac1 down-regulation or inhibition on the phagocytosis of microspheres. Our primary goal was to examine whether fusion and phagocytosis proceeded through the activation of the same cellular machinery. We considered this to be a critical issue because we believe that strategies that aim to limit FBGC formation should not compromise other macrophage functions that might be beneficial, such as clearance of microscopic debris from implantation sites. Neither NSC23766 nor Rac1 knockdown could block the uptake of microspheres, suggesting that the process is independent of Rac1. We examined the cell cytoskeleton during the uptake of microspheres, and we did not observe pseudopod extensions, implicating a mechanism involving the direct sinking of the particles into cells. The fact that Rac1 activity is not required for this uptake mechanism provides further support for our conclusion.33 To confirm further the involvement of distinct signaling pathways involving phagocytosis and fusion, we observed that MCP-1-null macrophages, which are inherently deficient in fusion, displayed normal engulfment of microspheres.
To expand on these findings and to examine the biological relevance of our in vitro observations, we used a strategy to deliver NSC23766 in vivo during FBGC formation. Specifically, we prepared the drug-loaded polymer EVAc, which has been previously shown to serve as a vehicle for long-term drug release in vivo.34,35 We hypothesized that the polymer itself would induce FBGC formation and allow us to examine the effect of Rac1 inhibition on this process as well as the overall foreign body response. We found that localized release of NSC23766 in vivo resulted in a significant reduction of FBGCs, confirming the importance of Rac1 activation in the formation of FBGCs. Attenuation of FBGC formation did not have an effect on the overall encapsulation of the EVAc scaffold suggesting that the tissue remodeling around biomaterials is independent of FBGCs. However, we have previously shown that reduction of FBGCs is associated with reduced degradation and damage to biodegradable biomaterials.9
We have demonstrated that mouse marrow-derived macrophages can be used efficiently in an in vitro fusion assay. Furthermore, we found that fusion is dependent on early cytoskeletal remodeling, specifically formation of lamellipodia, and can be attenuated by down-regulation or inhibition of Rac1 activation. Prevention of lamellipodia formation did not compromise the ability of macrophages to uptake microparticles. Thus, the processes of fusion and phagocytosis can be partially decoupled. In addition, localized delivery of the Rac1 inhibitor limited FBGC formation in vivo. Therefore, strategies can be developed that aim to prevent damage to biomaterials while preserving phagocytic activity in macrophages.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grant GM 072194-01 to T.R.K.).
Supplemental material for this article can be found on http://ajp.amjpathol.org.
Accepted for publication April 25, 2007.
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References |
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R, phagocytosis. Curr Biol 2002, 12:1413-1418[CrossRef][Medline]This article has been cited by other articles:
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  J. L. Moreno, I. Mikhailenko, M. M. Tondravi, and A. D. Keegan IL-4 promotes the formation of multinucleated giant cells from macrophage precursors by a STAT6-dependent, homotypic mechanism: contribution of E-cadherin J. Leukoc. Biol., December 1, 2007; 82(6): 1542 - 1553. [Abstract] [Full Text] [PDF]
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