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The Journal of Immunology, 2007, 179, 3780 -3791
Copyright © 2007 by The American Association of Immunologists, Inc.
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Cytoplasmic Linker Protein-170 Enhances Spreading and Phagocytosis in Activated Macrophages by Stabilizing Microtubules1

Marcelo G. Binker*, Dorothy Y. Zhao*, Sophie J. Y. Pang{dagger} and Rene E. Harrison2,*,{dagger}

* Department of Biological Sciences, University of Toronto at Scarborough, Toronto, Ontario, Canada; and {dagger} Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-{gamma} and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated {alpha}-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-{gamma}/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-{gamma}/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages and neutrophils are the major effector cells of the innate immune response, and are specialized to recognize and respond to pathogens as well as host-derived factors. Activation of macrophages involves complex levels of signal transduction appropriate to the immunological challenge. However, macrophage activation can become dysregulated and prolonged, or excessive activation can lead to inflammatory conditions, including rheumatoid arthritis, inflammatory bowel disease, and septic shock (1). IFN-{gamma} is a proinflammatory cytokine that is produced by the host in response to intracellular pathogens. IFN-{gamma} binds to IFN-{gamma} receptors on macrophages, and IFN-{gamma} signaling enhances Ag processing and presentation capabilities, up-regulates microbicidal effector functions, and inhibits cell proliferation (2, 3). Importantly, IFN-{gamma} sensitizes macrophages to activation from challenge with pathogen products such as LPS (LPS/endotoxin). IFN-{gamma} priming, in combination with LPS engagement on TLRs, causes full classical activation of microbicidal activities in macrophages (4). Through the activation process, resting macrophages undergo several morphological and functional changes, including increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15).

Cell morphology and secretory events are often regulated by the microtubule (MT)3 cytoskeleton (16, 17, 18, 19, 20, 21). MTs are highly polarized filamentous structures, formed from the head-to-tail association of {alpha}- and beta-tubulin heterodimers (22). This arrangement results in two structurally distinct ends, a minus-end anchored into the MT organizing center, and a plus-end oriented toward the cell cortex. MT plus-ends exhibit dynamic instability, constantly switching between growth and shrinkage phases, connected by transitions of catastrophe and rescue (23, 24). MT plus-end tracking proteins (+TIPs) are a group of proteins that target the plus-ends of MTs (25). Deciphering the role of +TIPs in MT dynamics and function has been the subject of intensive research in recent years (26, 27). The cytoplasmic linker protein 170 (CLIP-170) is a member of the +TIP family and is believed to adopt two different conformations, folded and inactive or extended and active, which are dependent on phosphorylation/dephosphorylation events (26, 27, 28). Functionally, the CLIP-170 molecule possesses a coiled-coil domain that separates the MT-binding head domain located at its N terminus, from the C terminus, which has been shown to interact with endocytic vesicles and the MT-associated motor protein, dynein (29). The expression of a dominant-negative (DN) form of CLIP-170 competes endogenous CLIP-170 from MT plus-ends and reduces the rescue frequency, resulting in prolonged shrinkage phases (30). Additional studies have implicated CLIP-170 in MT stabilization by acting as a rescue factor and allowing MT capture at specialized cortical sites (26, 27).

Recently, we showed that MT-depolymerizing agents reduce Fc{gamma}R-mediated phagocytosis in RAW 264.7 cells and resident peritoneal macrophages (31). Interestingly, Fc{gamma}R-mediated phagocytosis proceeds normally in colchicine-treated RAW 264.7 cells activated with PMA or IFN-{gamma} as well as thioglycolate-primed peritoneal macrophages (31). Because stabilized MTs are known to be resistant to MT-depolymerizing agents, these results suggest that MTs become stabilized as a consequence of the activation process. To analyze this possibility, we decided to investigate the presence of stable cytoplasmic MTs in RAW 264.7 cells following full activation with a combination of IFN-{gamma} and LPS. We have documented and quantified stabilized MTs in activated RAW 264.7 cells. Furthermore, using immunostaining and live fluorescence imaging, we identify CLIP-170 as a major regulator of MT stabilization during macrophage activation, necessary for both enhanced cell spreading and phagocytosis in activated macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and Abs

DMEM, FBS, and HEPES-buffered RPMI 1640 were obtained from Wisent. LPS from Salmonella enterica serotype Typhimurium, okadaic acid (OA), and human IgG were purchased from Sigma-Aldrich. BSA, DMSO, EGTA, and Triton X-100 were from BioShop Canada. IFN-{gamma} from Mus musculus was from PeproTech. Alexa Fluor 488-phalloidin was from Molecular Probes. Latex beads (8.31 µm diameter) were from Bangs Laboratories. Mouse anti-{alpha}-tubulin and anti-acetylated {alpha}-tubulin mAbs were from Sigma-Aldrich. Polyclonal rabbit anti-{alpha}-tubulin Ab was from Abcam. Polyclonal rabbit anti-CLIP-170 (No. 2360) Ab was provided by N. Galjart (Erasmus University Medical Center, Rotterdam, The Netherlands). Fluorochrome-conjugated secondary anti-mouse, anti-rabbit, and anti-human Ab were from Jackson ImmunoResearch Laboratories.

Mouse macrophage isolation, cell culture, plasmids, and transfections

Primary macrophages were isolated from the peritoneal cavities of 3-mo-old C57BL/6 mice. Briefly, resident peritoneal macrophages were harvested from sacrificed mice by peritoneal lavage by injecting 5 ml of ice-cold PBS (without Ca2+/Mg2+) containing 5% heat-inactivated FBS. To prevent activation by glass adherence, macrophages from each mouse were plated separately on poly(L-lysine)-coated coverslips (0.2 mg/ml, overnight, before blocking with 0.1% BSA), and grown in DMEM supplemented with 10% FBS and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin) at 37°C in 5% CO2 for 2 h before washing away nonadherent cells.

RAW 264.7 cells were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% FBS at 37°C in 5% CO2. For all experiments, RAW 264.7 cells were grown to 60–80% confluence in tissue culture 6-well plates, with or without glass coverslips. Transient transfections of cells were performed using FuGENE 6 (Roche Molecular Biochemicals), according to the manufacturer’s instructions, with overnight incubations for plasmid expression. The wild-type (WT) and DN-CLIP-170-GFP constructs were obtained from Y. Komarova (Northwestern University Medical School, Chicago, IL).

Activation and pharmacological treatment

Resident peritoneal macrophages (RPMs) or resting RAW 264.7 cells were activated by a combination of 100 U/ml IFN-{gamma} and 1 µg/ml LPS for 3, 6, or 24 h. Inhibition of protein phosphatase 2A (PP2A) was performed by incubating resting RAW 264.7 cells with 80 nM OA in DMSO for 24 h.

Cell spreading

The cell area of resting and activated or pharmacologically treated RAW 264.7 cells was measured using differential interference contrast (DIC) imaging and ImageJ 1.32j software (National Institutes of Health). For each condition, at least 150 cells corresponding to at least 50 randomly selected cells from three coverslips were analyzed for each of three independent experiments.

Phagocytosis assays

Latex beads (8.31 µm diameter) were opsonized with 1 mg/ml human IgG for 1 h at 37°C. After three washes with PBS, 100 µl of opsonized latex beads was added to resting and activated or pharmacologically treated RAW 264.7 cells plated on coverslips. Samples were subjected to centrifugation for 1 min at 300 x g to synchronize phagocytosis, followed by incubation at 37°C for the indicated time points. Binding indexes represent the number of bound latex beads per 100 cells. Phagocytic indexes represent the number of internalized latex beads per 100 cells. For each condition, at least 150 cells corresponding to at least 50 randomly selected cells from three coverslips were analyzed for each of three independent experiments.

Fluorescence imaging

After completion of activation or activation followed by phagocytosis, cells were washed twice with PBS and fixed in 4% paraformaldehyde in PBS at room temperature for 30 min. For CLIP-170 immunostaining, cells were fixed in 1 mM EGTA in methanol at –20°C for 10 min, followed by 15 min in 4% paraformaldehyde in PBS at room temperature, to preserve MT plus-ends (32). Cells were permeabilized with 0.15% Triton X-100 in PBS for 20 min and then blocked in 1% BSA in PBS for 1 h. Incubation with primary Ab was followed by incubation with the corresponding fluorochrome-conjugated secondary Ab. Where indicated, secondary Ab were incubated together with Alexa Fluor 488-phalloidin to label F-actin. To identify external latex beads, cells were incubated with fluorochrome-conjugated secondary anti-human Ab prior to permeabilization. Each incubation with Ab was preceded by three washes in PBS and lasted for 1 h at room temperature, in the presence of 1% BSA. The stained samples were washed three times in PBS and then mounted using Dako mounting medium (DakoCytomation). For live imaging experiments, cells plated on round 25-mm glass coverslips were placed in thermostatted holder chamber for microscopy, allowing the temperature to be maintained at 37°C throughout the experiment. Cells were incubated in HEPES-buffered RPMI 1640 while fluorescence images were recorded. For time-lapse epifluorescent microscopy, images were collected at 3-s intervals for the indicated time periods.

Both live and fixed cells were analyzed by confocal microscopy using a Zeiss Axioplan 2 imaging laser-scanning confocal microscope (Zeiss) with a x63 oil immersion objective and the conventional laser excitation and filter sets, or by epifluorescence microscopy using a Zeiss AxioVert microscope (Zeiss) equipped with an AxioCam MRm camera.

To determine the distance of endogenous CLIP-170 staining on MTs, Zeiss LSM software was used to measure the length of CLIP-170 staining that colocalized with MTs. The length of CLIP-170 along MTs was measured for at least 300 MTs. Cells were chosen randomly for measurements from three independent experiments. To characterize CLIP-170 dynamics in live cells, at least 15 cells expressing WT-CLIP-170-GFP were analyzed per condition for each of three independent experiments. The analyzed cells were scored as a cell solely presenting WT-CLIP-170-GFP plus-end distribution with CLIP-170-GFP moving as punctate spots most likely representing the plus-ends of MTs. Conversely, cells were scored as presenting WT-CLIP-170-GFP extended distribution when one or more long, stationary tracks of CLIP-170-GFP were observed, presumably along MTs. To determine the relative fluorescence intensity of acetylated {alpha}-tubulin in each DN-CLIP-170-GFP-expressing cell or -nonexpressing activated cells, at least 15 cells were analyzed per condition for each of three independent experiments, using ImageJ 1.32j software (National Institutes of Health).

Immunoblotting

Cell lysates were dissolved in Laemmli buffer and boiled for 5 min. Equal amount of protein was separated on 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Blots were blocked for 1 h in TBS containing 5% BSA and then incubated with the corresponding primary Ab. The bound Ab was visualized by relevant peroxidase-coupled secondary Ab, using the ECL method (Pierce). Densitometry of three independent experiments was performed using Scion Image software (National Institutes of Health) to calculate the corresponding fold increases compared with control cell lysates.

Statistical analysis

Experimental values are presented as mean ± SEM of the indicated number of determinations. Student’s t test was used to evaluate data, with values of p < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Enhanced level of stable cytoplasmic MTs in activated macrophages

We previously observed enhanced MT stabilization in macrophages primed with IFN-{gamma} (31). We examined whether MTs are stabilized in RPMs fully activated with IFN-{gamma} and LPS (100 U/ml and 1 µg/ml, respectively). We immunostained cells with an anti-acetylated {alpha}-tubulin Ab to monitor MT stabilization. After formation, MTs can experience several different posttranslational modifications. Acetylation of the lysine 40 residue of {alpha}-tubulin is a posttranslational modification that occurs in long-lived MTs and serves as a marker of stable MT subsets (22). Immunostaining and confocal imaging of unactivated RPMs revealed few acetylated MTs (Fig. 1A), compared with the total population of MTs, detected using a pan-{alpha}-tubulin Ab (Fig. 1A, insets). In contrast, cells stimulated with IFN-{gamma} and LPS for 24 h showed increased levels of acetylated MTs (Fig. 1A). We next investigated this phenomenon in RAW 264.7 cells. Unactivated, resting RAW 264.7 cells also showed few acetylated cytoplasmic MTs (Fig. 1B), compared with the total population of MTs (Fig. 1B, insets). In resting cells, acetylated MTs were largely confined to the midbodies of cells in division (Fig. 1B, arrow). In contrast, cells stimulated with IFN-{gamma} and LPS for 3, 6, and 24 h exhibited increasing levels of acetylated cytoplasmic MTs (Fig. 1B). For a more quantitative assessment of stable MT subpopulations in RAW 264.7 cells during activation, Western blotting for acetylated {alpha}-tubulin and total {alpha}-tubulin of whole cell lysates was performed. Using equal protein loading, we normalized for the increase in cell size observed in IFN-{gamma}/LPS-activated RAW 264.7 cells. The extent of tubulin modification, as expressed by the relative ratio of modified to total tubulin, was 3.7-fold higher in IFN-{gamma}/LPS-activated cells compared with nonactivated cultures after 24-h activation (Fig. 1C). Loading lysates according to cell number also revealed an increase in total tubulin in activated cells compared with control, indicating both an increase in MT polymer as well as an enhanced proportion of modified tubulin (data not shown).


Figure 1
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  		FIGURE 1. Macrophages activated with IFN-{gamma} plus LPS exhibit increased levels of stable cytoplasmic MTs. RPMs were stimulated with IFN-{gamma} plus LPS (100 U/ml and 1 µg/ml, respectively) or left unstimulated for 24 h (A), or resting RAW 264.7 cells were stimulated with IFN-{gamma} plus LPS (100 U/ml and 1 µg/ml, respectively) or left unstimulated for 3, 6, and 24 h (B and C). After the indicated time periods, cells were processed for immunofluorescence (A and B) or were lysed, and cell lysates were subjected to immunoblotting (C). A and B, Representative merged confocal Z-stack images of the acetylated {alpha}-tubulin staining revealing stable MTs in macrophages. Arrow indicates midbody of cells in division. Insets show the {alpha}-tubulin labeling. Scale bars = 10 µm. C, Representative immunoblot showing the acetylated {alpha}-tubulin and {alpha}-tubulin levels in resting and activated RAW 264.7 cells.

 
We also analyzed the effects of individual macrophage stimulators on MT stabilization. Immunostaining and confocal imaging of RAW 264.7 cells treated with either IFN-{gamma} or LPS alone, or in combination for 24 h, showed an increase in acetylated {alpha}-tubulin staining, compared with resting cells (Fig. 2A). Consistent with our fluorescence microscopy observations, Western blotting for acetylated {alpha}-tubulin and total {alpha}-tubulin of whole cell lysates revealed an enhancement in stabilized MTs for individual or combined activation treatments, in comparison with unactivated, control resting macrophages (Fig. 2B). The most pronounced MT stabilization was observed when macrophages were activated with IFN-{gamma} and LPS together (Fig. 2). Macrophage activation by IFN-{gamma} and LPS appears to change the composition of cytoplasmic MTs to create a less dynamic and more stable MT network.


Figure 2
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  		FIGURE 2. Macrophages activated with IFN-{gamma} and/or LPS exhibit increased levels of stable MTs. Resting RAW 264.7 cells were stimulated with 0.01 µg/ml LPS, 0.1 µg/ml LPS, 1 µg/ml LPS, 100 IU/ml IFN-{gamma}, or 100 U/ml IFN-{gamma} plus 1 µg/ml LPS or left unstimulated for 24 h. After the indicated time periods, cells were processed for immunofluorescence (A) or were lysed, and cell lysates were subjected to immunoblotting (B). A, Representative merged confocal Z-stack images of the acetylated {alpha}-tubulin staining revealing stable MTs in macrophages. Arrow indicates midbody of cells in division. Insets show the {alpha}-tubulin labeling. Scale bars = 10 µm. B, Representative immunoblot showing the acetylated {alpha}-tubulin and {alpha}-tubulin levels in resting and activated RAW 264.7 cells.

 
Extended distribution of CLIP-170 on MTs in activated macrophages

We sought to identify mediators of the MT stabilization that we observed in activated macrophages by examining +TIPs. CLIP-170 was the first +TIP discovered, and its expression and activity have not been examined in immune cells to date. Using a fixation technique that preserves MT plus-ends (32) and immunostaining for CLIP-170 (using a specific anti-CLIP-170 Ab, No. 2360) and MTs, we found by confocal microscopy that CLIP-170 localizes to MT plus-ends in RPMs (Fig. 3A). This MT plus-end distribution is similar to that which has been observed in all other cell types examined to date (28, 33, 34, 35). Interestingly, in RPMs activated with IFN-{gamma} and LPS, a notable difference in CLIP-170 distribution was observed, with CLIP-170 showing extended staining from the plus-end toward the middle of MTs (Fig. 3A). We quantified the percentage of endogenous CLIP-170 showing short punctate staining on MT plus-ends vs extended staining along the MT (>3 µm) in resting and activated macrophages (Fig. 3, B and C). In resting macrophages, only 3.4 ± 1.1% of CLIP-170 extended longer than 3 µm from the MT plus-end, with an average length of 0.9 ± 0.34 µm. In contrast, in activated macrophages, a significant 23.3 ± 3.2% of CLIP-170 extended longer than 3 µm along the length of the MT, with an average length of 2.3 ± 0.8 µm. Similar to our acetylated {alpha}-tubulin results (Fig. 2), we observed the most pronounced redistribution of CLIP-170 to the length of the MTs when macrophages were treated with IFN-{gamma} and LPS together; however, LPS alone at lower concentrations also induced this phenomena (data not shown).


Figure 3
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  		FIGURE 3. The distribution of endogenous CLIP-170 changes upon macrophage activation. A–E, Resident peritoneal macrophages (A–C) or resting RAW 264.7 cells (C–E) were stimulated with IFN-{gamma} plus LPS (100 U/ml and 1 µg/ml, respectively) or left unstimulated for 24 h. After the indicated time period, cells were processed for immunofluorescence (A and D). A and D, Representative confocal images showing the costaining of endogenous CLIP-170 (green) and {alpha}-tubulin (red). Open arrowheads indicate short, punctate staining of CLIP-170 on MT plus-ends. Arrows indicate a representative example of extended CLIP-170 staining along the MT. Scale bars = 10 µm. C, Cartoon of a MT with plus (+) and minus (–) ends. CLIP-170 length was measure from the MT plus-end toward the minus-end. B and E, Quantification of the length of endogenous CLIP-170 staining along MTs. Data expressed as means ± SEM. *, p < 0.05.

 
We then proceeded to analyze endogenous CLIP-170 distribution in the RAW 264.7 murine macrophage cell line. As we observed in RPMs, CLIP-170 localized to MT plus-ends in resting, unstimulated RAW 264.7 cells (Fig. 3D). In RAW 264.7 cells activated with IFN-{gamma} and LPS, a similar difference in CLIP-170 distribution was observed, with CLIP-170 immunostaining accumulating along the length of the MTs from the MT plus-ends (Fig. 3D). In resting RAW 264.7 cells, only 3.8 ± 1.8% of CLIP-170 extended longer than 4 µm from the MT plus-end, with an average length of 1.7 ± 0.4 µm. In contrast, in activated macrophages, a significant 25.4 ± 2.9% of CLIP-170 extended longer than 4 µm along the length of the MT, with an average length of 3.6 ± 1.2 µm (Fig. 3E). We also quantified endogenous levels of CLIP-170 in resting and activated RAW 264.7 cells using immunoblotting; however, no significant difference in CLIP-170 protein levels was observed between resting cells and cells activated with IFN-{gamma} and LPS for 24 h (data not shown).

To monitor CLIP-170 dynamics in RAW 264.7 cells, we expressed full-length GFP-tagged CLIP-170 (WT-CLIP-170-GFP). Live epifluorescent imaging revealed typical MT plus-end tracking behavior of WT-CLIP-170-GFP in resting RAW 264.7 cells (Fig. 4A and supplemental Fig. 1).4 In contrast, in IFN-{gamma}/LPS-activated RAW 264.7 cells, WT-CLIP-170-GFP was observed as long tracks, presumably along MTs, that were relatively stationary (Fig. 4A and supplemental Fig. 2).4 Although the majority of resting cells (95.6 ± 1.2%) showed WT-CLIP-170-GFP as moving punctate spots most likely representing the plus-ends of MTs, only 22.2 ± 3.1% of transfected activated cells exhibited this typical MT plus-end tracking behavior (Fig. 4, B and C). Remarkably, a significant 77.8 ± 3.1% of activated cells contained one or more extended tracks of CLIP-170-GFP per cell, compared with 4.4 ± 1.2% of resting cells (Fig. 4, B and C).


Figure 4
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  		FIGURE 4. The distribution of WT-CLIP-170-GFP changes upon macrophage activation. A–C, Resting RAW 264.7 cells were transfected with WT-CLIP-170-GFP and after overnight incubation were stimulated with IFN-{gamma} plus LPS for 24 h, or left unstimulated (resting). After the indicated time periods, cells were imaged using epifluorescent microscopy. A, Representative epifluorescent images of resting and activated macrophages expressing WT-CLIP-170-GFP. Images were acquired every 3 s for 180 s. Time is shown in seconds in the top right corner. Open arrowheads indicate a typical WT-CLIP-170-GFP plus-end distribution. Arrows indicate a representative extended distribution of WT-CLIP-170-GFP. Scale bars = 10 µm. B, Quantification of transfected cells containing plus-end or extended distribution of WT-CLIP-170-GFP. Data expressed as means ± SEM. *, p < 0.05 and **, p < 0.01. C, Drawings of the plus-end and extended distribution of WT-CLIP-170-GFP ({circ}) on MTs. MT plus (+) and minus (–) ends are indicated.

 
CLIP-170 is required to stabilize MTs in activated macrophages

We observed that activated RAW 264.7 cells had enhanced stabilized MTs and a marked redistribution of CLIP-170 from the plus-end of MTs. We next investigated whether CLIP-170 distribution was solely a consequence of MT stabilization or whether CLIP-170 redistribution itself was the causative agent of MT stabilization during macrophage activation. To test this hypothesis, we expressed WT and DN forms of CLIP-170 in resting and activated RAW 264.7 cells and monitored MT stabilization in these cells using immunofluorescence with an anti-acetylated {alpha}-tubulin Ab. WT-CLIP-170-GFP expression showed an expected punctate patterning in resting RAW 264.7 cells without altering the levels of acetylated {alpha}-tubulin, compared with nonexpressing cells (Fig. 5A). Activated macrophages expressing WT-CLIP-170-GFP showed high levels of acetylated {alpha}-tubulin similar to those of untransfected activated cells (Fig. 5B). Thus, exogenous expression of CLIP-170 did not alter acetylated {alpha}-tubulin levels in either resting or activated RAW 264.7 cells. To determine a role for CLIP-170 in producing the stabilized MT array in activated macrophages, we transfected a GFP-tagged DN mutant form of the protein (DN-CLIP-170-GFP) in IFN-{gamma} plus LPS-activated RAW 264.7 cells. The DN construct is lacking the N-terminal head region responsible for CLIP-170 binding to MTs (36). The expression of DN-CLIP-170-GFP in activated RAW 264.7 cells showed an expected cytosolic distribution, as previously described (36). Importantly, transfection of DN-CLIP-170-GFP caused a marked reduction of acetylated {alpha}-tubulin immunostaining in activated cells expressing this construct (Fig. 5C). Statistical analysis of the acetylated {alpha}-tubulin relative fluorescence intensity per cell (Fig. 5D) showed a significant reduction in DN-CLIP-170-GFP-expressing activated RAW 264.7 cells, compared with untransfected activated cells (0.08 ± 0.05 and 0.97 ± 0.04, respectively).


Figure 5
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  		FIGURE 5. CLIP-170 is required for MT stabilization in activated macrophages. A–D, Resting RAW 264.7 cells were transfected with WT- or DN-CLIP-170-GFP, and after overnight incubation cells were stimulated with IFN-{gamma} plus LPS for 24 h, or left unstimulated. Following fixation, cells were immunostained with an anti-acetylated {alpha}-tubulin Ab. Representative merged confocal Z-stack images of resting (A) and activated (B) macrophages transfected with WT-CLIP-170-GFP. Arrow indicates midbody of cells in division. C, Representative merged confocal Z-stack image of activated macrophages transfected with DN-CLIP-170-GFP. Insets show the corresponding DIC images. The white dashed line outlines the perimeter of the DN-CLIP-170-GFP-expressing cell. Scale bars = 10 µm. D, Quantification of the relative fluorescence intensity per cell of the acetylated {alpha}-tubulin immunostaining in the DN-CLIP-170-GFP-expressing (DN-CLIP) and untransfected activated cells (Control). *, p < 0.01.

 
Increased accumulation of CLIP-170 along MTs and enhanced level of stable MTs in OA-treated resting macrophages

The ability of CLIP-170 to bind to MTs is believed to depend on its phosphorylation status (26, 27). We next examined potential regulators of CLIP-170 association with MTs in macrophages. We examined the role of the serine/threonine PP2A because it has been shown to directly bind to MTs in several cell types (37). Moreover, OA, a PP2A and protein phosphatase 1 (PP1) inhibitor, causes dissolution of peripheral MTs in endothelial cells (38), and inhibits in vitro binding of CLIP-170 to MTs (39). To inhibit PP2A in RAW 264.7 cells, we incubated resting macrophages with OA (80 nM, 24 h). When we examined endogenous CLIP-170 levels in OA-treated resting RAW 264.7 cells using immunofluorescence, we surprisingly observed extended CLIP-170 tracks along MTs (Fig. 6A). In resting macrophages treated with OA, a significant 32.1 ± 2.1% of CLIP-170 staining extended >4 µm along the MT, with an average length of 3.1 ± 1.1 µm (Fig. 6B). As observed in Fig. 3D, control (vehicle) resting RAW 264.7 cells exhibited punctate MT plus-end staining pattern of CLIP-170 (Fig. 6A). In these cells, only 2.3 ± 2.7% of CLIP-170 showed staining that extended a distance greater than 4 µm along the MT, from the plus-end toward the middle of the MT, with an average length of staining of 1.5 ± 0.4 µm (Fig. 6B).


Figure 6
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  		FIGURE 6. OA increases both the accumulation of CLIP-170 at MT plus-ends and the level of stable MTs in resting macrophages. A and B, Resting RAW 264.7 cells were treated with 80 nM OA or vehicle (DMSO) for 24 h. After fixation, cells were subjected to immunostaining. A, Representative confocal images showing the costaining of endogenous CLIP-170 (green) and {alpha}-tubulin (red). Open arrowheads indicate a representative CLIP-170 short punctate staining on MT plus-ends. Arrows indicate a representative example of extended CLIP-170 staining along the MT. Scale bars = 10 µm. B, Quantification of the length of CLIP-170 staining along MTs. Data expressed as means ± SEM. *, p < 0.05. C and D, Resting RAW 264.7 cells were transfected with WT-CLIP-170-GFP and after overnight incubation were treated with 80 nM OA or vehicle (DMSO) for 24 h. C, Representative epifluorescent images of control (vehicle) and OA-treated resting macrophages expressing WT-CLIP-170-GFP. Images were acquired every 3 s for 180 s. Time is shown in seconds in the top right corner. Open arrowheads indicate a typical WT-CLIP-170-GFP plus-end distribution. Arrows indicate a representative extended distribution of WT-CLIP-170-GFP. Scale bars = 10 µm. D, Quantification of transfected cells containing plus-end or extended distribution of WT-CLIP-170-GFP. Data expressed as means ± SEM. *, p < 0.05 and **, p < 0.01. E, Representative merged confocal Z-stack image of acetylated {alpha}-tubulin staining in control (vehicle) and OA-treated resting macrophages. Insets show {alpha}-tubulin labeling. Scale bars = 10 µm. F, Representative immunoblot of acetylated {alpha}-tubulin and {alpha}-tubulin levels in control (vehicle) and OA-treated resting macrophages.

 
Next, we proceeded to monitor CLIP-170 dynamics by expressing WT-CLIP-170-GFP in control (vehicle) and OA-treated resting RAW 264.7 cells. As previously observed (Fig. 4A), control (vehicle) macrophages exhibited the typical MT plus-end tracking of WT-CLIP-170-GFP (Fig. 6C and supplemental Fig. 3).4 However, OA-treated resting RAW 264.7 cells showed WT-CLIP-170-GFP-decorating filaments (Fig. 6C and supplemental Fig. 4),4 similar to what we observed with IFN-{gamma}/LPS-activated RAW 264.7 cells. The majority of resting cells (97.8 ± 0.9%) showed WT-CLIP-170-GFP as moving punctate spots most likely representing the plus-ends of MTs (Fig. 6D). However, whereas only 2.2 ± 0.9% of control (vehicle) cells had one or more extended tracks of CLIP-170-GFP per cell, a significant 62.2 ± 2.8% of OA-treated cells were found presenting at least one extended track that persisted throughout imaging (Fig. 6D).

To investigate whether the extended CLIP-170 distribution along MTs in OA-treated RAW 264.7 cells correlated with MT stabilization, we immunostained these cells for acetylated {alpha}-tubulin. Confocal analysis of OA-treated resting RAW 264.7 cells revealed enhanced levels of stable cytoplasmic MTs compared with control (vehicle) cells (Fig. 6E). Immunoblotting analysis also revealed a marked increase in acetylated {alpha}-tubulin in OA-treated resting RAW 264.7 cells, compared with control cells (Fig. 6F).

Enhanced cell spreading and phagocytosis in IFN-{gamma}/LPS-activated RAW 264.7 cells

Enhanced cell spreading was previously observed in macrophages individually activated with IFN-{gamma} (5) or LPS (6, 7). To evaluate this phenomenon in fully activated macrophages, resting RAW 264.7 were stimulated with IFN-{gamma}/LPS for 24 h (Fig. 7A). Cells were fixed and imaged with DIC microscopy, and cell areas were measured using ImageJ software. Statistical analysis revealed a significant increase in the relative cell areas of activated RAW 264.7 cells (4.28 ± 0.33), compared with resting macrophages (Fig. 7B).


Figure 7
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  		FIGURE 7. Activation of macrophages enhances both cell spreading and phagocytosis. A and B, Resting RAW 264.7 cells were either untreated or activated with IFN-{gamma} and LPS for 24 h, followed by fixation. A, Representative DIC images of resting and activated macrophages. Scale bars = 20 µm. B, Cell area of fixed cells was quantified and presented relative to control resting macrophages. Data expressed as means ± SEM. *, p < 0.01. C–G, Resting and IFN-{gamma}/LPS-activated RAW 264.7 cells were exposed to IgG-opsonized 8-µm latex beads for 3 (C and E) or 10 (F and G) min. After the indicated time periods, cells were fixed and immunostained. C, Representative merged confocal Z-stack image showing the staining of actin (left column) and {alpha}-tubulin (right column). Insets, Show external latex beads. D, Magnifications of selected boxes from C, merged with the corresponding DIC images. Scale bars = 10 µm. E, Quantification of particle binding to macrophages. Data are expressed as means ± SEM. *, p < 0.05. F, Representative DIC images of phagocytosis in resting and activated macrophages. Insets show the presence of stained, external latex beads. Scale bars = 20 µm. G, Statistical analysis of phagocytosis in macrophages. Data expressed as means ± SEM. *, p < 0.05.

 
Because enhanced phagocytosis is observed in macrophages stimulated with either IFN-{gamma} or LPS (13, 14, 15), we proceeded to examine the phagocytic abilities of resting vs IFN-{gamma}/LPS dually activated RAW 264.7 cells. MTs have been reported to penetrate sites of early phagocytosis (31), so we first examined MTs at early times of phagocytosis in resting and IFN-{gamma}/LPS-activated macrophages. Cells were exposed to IgG-opsonized 8-µm latex beads for 3 min, followed by fixation and staining with an {alpha}-tubulin Ab and phalloidin to visualize the actin cups. In both resting and IFN-{gamma}/LPS-activated RAW 264.7 cells, MTs were observed extending deep into the region of phagocytic cup formation (Fig. 7, C and D). The particle-binding index was quantified in these cells, and we observed a significant 1.77-fold increase in the binding abilities of activated RAW 264.7 cells, compared with resting macrophages (132.5.3 ± 7.8 and 74.7 ± 5.1, respectively; Fig. 7E). When cells were exposed to IgG-opsonized 8-µm latex beads for 10 min (Fig. 7F), a significant 2.23-fold increase in the phagocytic index was observed in activated RAW 264.7 cells, compared with resting macrophages (102.3 ± 6.5 and 45.9 ± 3.2, respectively; Fig. 7G).

OA increases both cell spreading and phagocytosis in resting macrophages

CLIP-170 binding to MTs and MT stabilization were positively regulated in resting RAW 264.7 cells by the inhibition of PP2A (Fig. 6). Because we observed similar changes in IFN-{gamma}/LPS-activated RAW 264.7 cells (Figs. 1–4), which also exhibited both enhanced cell spreading and phagocytosis of large particles (Fig. 7), we next investigated these processes in OA-treated resting macrophages. Treating resting macrophages with OA caused enhanced cell spreading, when imaged by DIC microscopy (Fig. 8A). Following quantification, a statistically significant increase in the relative cell area of OA-treated RAW 264.7 cells (4.01 ± 0.39) was observed compared with control (vehicle) resting macrophages (Fig. 8B).


Figure 8
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  		FIGURE 8. OA enhances both cell spreading and phagocytosis in resting macrophages. A–D, Resting RAW 264.7 cells were treated with 80 nM OA or vehicle for 24 h. Cells were then fixed for cell area measurement (A and B), or phagocytosis of IgG-opsonized 8-µm latex beads was performed for 10 min before fixation (C and D). A, Representative DIC images of control (vehicle) and OA-treated resting macrophages. Scale bars = 20 µm. B, Cell area of fixed cells was quantified and presented relative to control resting macrophages. Data expressed as means ± SEM. *, p < 0.01. C, Representative DIC images of phagocytosis in control (vehicle) and OA-treated resting macrophages. Insets show external latex beads. Scale bars = 20 µm. D, Statistical analysis of phagocytosis in macrophages. Data expressed as means ± SEM. *, p < 0.05.

 
When we exposed control (vehicle) and OA-treated resting RAW 264.7 cells to IgG-opsonized 8-µm latex beads for 10 min (Fig. 8C), we found a significant increase in the phagocytic index of OA-treated macrophages, in comparison with control macrophages (97.6 ± 7.8 and 55.4 ± 4.2, respectively; Fig. 8D).

CLIP-170 is required for enhanced cell spreading and phagocytosis in activated macrophages

Finally, we directly assayed whether CLIP-170-mediated MT stabilization is important for the increased cell spreading and phagocytosis observed in macrophages activated by IFN-{gamma}/LPS. We expressed WT and DN forms of GFP-tagged CLIP-170 and quantified cell spreading and phagocytosis in transfected resting and activated RAW 264.7 cells. In comparison with resting cells expressing the WT form of CLIP-170-GFP, the expression of DN-CLIP-170-GFP did not cause significant differences in the relative cell area of resting RAW 264.7 cells (1.04 ± 0.12 and 1.09 ± 0.18, respectively; Fig. 9A). The expression of WT-CLIP-170-GFP did not affect the relative cell area of resting macrophages, compared with nontransfected cells (data not shown). However, whereas IFN-{gamma} plus LPS-activated RAW 264.7 cells expressing WT-CLIP-170-GFP had an increased cell area comparable to activated nontransfected cells, the expression of the DN form of CLIP-170 severely attenuated cell spreading in activated macrophages (4.19 ± 0.46 and 1.75 ± 0.39, respectively; Fig. 9A).


Figure 9
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  		FIGURE 9. CLIP-170 is required to increase both cell spreading and phagocytosis in activated macrophages. A–C, Resting RAW 264.7 cells were transfected with WT- or DN-CLIP-170-GFP, and after overnight incubation were stimulated with IFN-{gamma} plus LPS for 24 h, or left untreated. Cells were then fixed for cell area measurement (A) or allowed to internalize IgG-opsonized 8-µm latex beads for 10 min (B and C). A, Cell areas of CLIP-170-GFP-expressing macrophages were visualized by DIC microscopy, quantified, and presented relative to WT-CLIP-170-GFP-expressing resting macrophages. Data are expressed as means ± SEM. *, p < 0.01. B, Merged DIC images with GFP fluorescent images (white) of CLIP-170-GFP-transfected resting and activated macrophages. Arrows indicate the GFP-expressing cells. Insets show external latex beads. Scale bars = 20 µm. C, Statistical analysis of phagocytosis in CLIP-170-GFP-expressing macrophages. Data expressed as means ± SEM. *, p < 0.05.

 
Resting and IFN-{gamma}/LPS-activated RAW 264.7 cells were then transfected with WT- and DN-CLIP-170-GFP and exposed to IgG-opsonized 8-µm latex beads for 10 min before fixation and immunostaining (Fig. 9B). In all conditions, we found that macrophages were able to ingest these large particles, although not with the same capacity. Resting macrophages expressing either WT or DN forms of CLIP-170 did not show a significant difference in their phagocytic indexes (52.6 ± 6.2 and 49.9 ± 5.4, respectively; Fig. 9C). However, whereas the phagocytic index of activated macrophages expressing WT-CLIP-170-GFP doubled in comparison with resting macrophages, the expression of the DN-CLIP-170-GFP in activated macrophages abolished this enhancement, limiting the phagocytic index to similar values observed in resting cells (110.3 ± 6.9 and 54.9 ± 6.1, respectively; Fig. 9C). Together, these data strongly suggest that macrophage activation leads to CLIP-170-mediated MT stabilization that enhances both cell spreading and phagocytosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The process of activation confers to macrophages the increased functional activity necessary for an adequate immune response. Our current work shows that macrophage activation stabilizes cytoplasmic MTs, and this MT stabilization is required to increase cell spreading and Fc{gamma}R-mediated phagocytosis of large particles.

We observed an increase in stable MTs in mouse peritoneal macrophages activated with IFN-{gamma} and LPS, which was also observed in activated RAW 264.7 cells in a time-dependent fashion. We also found that IFN-{gamma} or LPS alone induced stable MTs in RAW cells, although stabilized MTs were most apparent when macrophages were stimulated with IFN-{gamma} and LPS in combination, suggesting a synergistic effect. IFN-{gamma} exposure has been reported to cause MT assembly in vitro (40), whereas similar assays showed that LPS inhibited MT polymerization in a pH-dependent manner (41). LPS induced MT stabilization in human monocytes (42) and increased {alpha}- and beta-tubulin levels in human monocytes and in the monocyte/macrophage cell line THP-1 (43). Although we and others have noted a marked change in MT dynamics in activated macrophages, the mechanism of MT stabilization during macrophage activation has not been elucidated. In this study, we provide evidence that CLIP-170 is necessary for MT stabilization in activated macrophages. The +TIPs have been demonstrated to be regulators of MT dynamics (25, 26, 27). CLIP-170 was shown to interact with the actin scaffolding protein IQGAP1, potentially linking and stabilizing MTs to specialized cortical regions in migrating polarized cells (26, 34). Furthermore, endogenous CLIP-170 was found to accumulate at desmosomal plaques, where a significant fraction of MTs becomes stabilized, suggesting that CLIP-170 controls the changes in MT properties induced by epithelial cell-cell junction formation (44).

Our studies with DN CLIP-170 confirmed that CLIP-170 was a key mediator in MT stabilization during macrophage activation. Interestingly, both endogenous CLIP-170 and WT-CLIP-170-GFP showed a redistribution along the MTs in activated macrophages. CLIP-170 accumulated along the length of the MT and was fairly stationary, unlike the rapid MT plus-end tracking behavior observed in resting macrophages. These results suggest that the function of CLIP-170 in MT stabilization depends on its distribution along MTs. Interestingly, MTs in spermatid manchettes are highly stabilized and GFP-CLIP-170 was observed to be largely immobile in spermatids from GFP-CLIP-170 knockin mice (45). Consistent with a MT stabilization function, the absence of CLIP-170 severely affected the formation and/or maintenance of the spermatid manchette in CLIP-170 knockout mice (45).

Our observed CLIP-170 decoration along MTs may be the consequence of an increased amount of CLIP-170 available for interaction with MTs, or possibly an inhibition of CLIP-170 release from the plus-ends, allowing it to accumulate along the length of the MT. We did not observe an increase in CLIP-170 protein levels in activated macrophages, so the regulation of binding of CLIP-170 to MTs is most likely due to posttranslational modifications of this protein. CLIP-170 association and disassociation from MTs have been shown to be regulated by a phosphorylation/dephosphorylation cycle (46). In vitro studies have shown that a high OA concentration (1 µM) inhibits the binding of CLIP-170 to MTs (39). This apparent contradiction may arise from the concentration-dependent inhibition exhibited by OA on its specific substrates, PP2A and PP1 (47). Because we used concentrations of OA (80 nM) to inhibit PP2A specifically, our results introduce a new level of complexity in the exquisite regulation of CLIP-170, suggesting that perhaps PP2A and PP1 exert opposite effects in the control of CLIP-170. Unfortunately, we cannot corroborate this because the inhibition of both PP2A and PP1 caused detachment of macrophages in our hands (data not shown), which has been reported by others (48).

In addition to CLIP-170 redistribution, OA-treated resting macrophages also exhibited increased stable cytoplasmic MTs. Previous studies reported that OA destabilize MTs in other cell types (48, 49). Again, these analyses used OA at concentrations that additionally inhibit PP1, suggesting that PP1 may be affecting MT dynamics differently than PP2A. Interestingly, an in vitro study recently showed that the CLIP-170-interacting protein Lis1 (32), which plays an essential role in brain development, can inhibit PP2A, but not PP1 (50). Future experiments are required to establish a possible role for Lis1 in regulating PP2A and CLIP-170-MT associations in activated macrophages.

We have also provided evidence that the observed redistribution of CLIP-170 is functionally relevant in immune cells. We found that CLIP-170 is necessary for MT stabilization and is required for enhanced cellular spreading and engulfment of large particles in activated macrophages (see Fig. 10). Although the necessity of intact MTs for cell spreading is well documented (16, 17), the requirement for stabilized MTs in cell spreading is a novel finding. Traditionally, acetylated {alpha}-tubulin was considered a marker of stable MTs, whereas its function was not clear (22). However, MT hyperacetylation was recently associated with an increase in cell spreading (51). Importantly, the MT plus-end-associated motor kinesin interacts preferentially with acetylated MTs in neuronal cells and in vitro (52). Because directional MT-associated cargo transport occurs by MT plus-end and MT minus-end directed motors (53), we hypothesize that the increased levels of acetylated MTs found in activated macrophages are directing the necessary cargo toward the cell cortex to produce and maintain the observed increased cell spreading.


Figure 10
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  		FIGURE 10. A model of MT stabilization in activated macrophages. Resting macrophages possess mainly dynamic MTs, which explore the intracellular space. CLIP-170 binds and localizes at the plus-end of these MTs. Resting macrophages are fairly round and can engulf large particles. Following activation of macrophages with IFN-{gamma} and LPS, CLIP-170 redistributes along the length of MTs, leading to MT stabilization and enhanced cell spreading and phagocytic capacity.

 
The engulfment of large particles has been reported to depend on the amount of available membrane (54), where the plasma membrane appears to be a major source (55). The participation of endomembranes has also been considered for efficient particle engulfment, and MTs are the most prominent candidate to deliver these membranes to the phagocytic sites (56, 57). Cell spreading notably enhances the exposed cell surface, thereby increasing the possibility of contact with external particles. We noted an enhanced particle-binding ability in activated macrophages, indicating that cell size is most likely the major contributing factor to enhanced phagocytosis observed in activated macrophages. Although slight, our observed differences between the fold increase in phagocytosis over binding (26%) in activated macrophages suggest that stabilized MTs, as a platform for motor activity, may also be behind the enhanced phagocytosis observed in activated macrophages. Kinesin has not only been implicated in the control of cell shape, but also in pseudopod formation (18). Characteristically, Fc{gamma}R-mediated phagocytosis is accompanied by pseudopodial extension (58), as well as by focal exocytosis (59). The coordination of both pseudopodial extension and focal exocytic membrane insertion requires PI3K activity, because engulfment of large particles is completely blocked by the PI3K inhibitors wortmannin and LY294002 (58). We recently showed that disruption of MTs by colchicine reduces both the translocation of the p85{alpha} regulatory subunit of PI3K and the accumulation of PI3K products at the phagocytic cups of resting macrophages during Fc{gamma}R-mediated phagocytosis (31). We speculate that the prolonged contacts between stabilized MTs and the cell cortex may be facilitating the delivery of endomembranes, which in coordination with a PI3K-orchestrated membrane insertion and pseudopodial extension enhance both cell spreading and phagocytosis in activated macrophages.


   Acknowledgments
 
We thank Nazeel Qureshi and David Douda for their contributions to this work, Jenny Jongstra-Bilen for her assistance with mouse peritoneal macrophages, and Dr. James Booth for critical reading of the manuscript. We thank Dr. Yulia Komarova (Northwestern University, Chicago, IL) for the CLIP-170-GFP constructs, Dr. Niels Galjart (Erasmus University Medical Center, Rotterdam, The Netherlands), and Dr. Holly Goodson (Notre Dame University, Notre Dame, IN) for providing the anti-CLIP-170 Abs.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Canadian Institutes for Health Research Grant MOP-68992 and a Natural Science and Engineering Research Council grant to R.E.H. D.Y.Z. is the recipient of a Natural Science and Engineering Research Council Undergraduate Student Research Award. S.J.Y.P. is the recipient of a University of Toronto fellowship award. R.E.H. is the recipient of the Ontario Early Researcher Award. Back

2 Address correspondence and reprint requests to Dr. Rene E. Harrison, Department of Biological Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto, Ontario, M1C 1A4, Canada. E-mail address: harrison{at}utsc.utoronto.ca Back

3 Abbreviations used in this paper: MT, microtubule; CLIP-170, cytoplasmic linker protein-170; DN, dominant negative; OA, okadaic acid; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; RPM, resident peritoneal macrophage; +TIP, MT plus-end tracking protein; WT, wild type; DIC, differential interference contrast. Back

4 The online version of this article contains supplemental material. Back

Received for publication April 25, 2007. Accepted for publication July 11, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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