Macrophage Polarization in Response to Wear Particles in Vitro

RESEARCH ARTICLE

Macrophage polarization in response to wear particles in vitro

Joseph K Antonios, Zhenyu Yao, Chenguang Li, Allison J Rao and Stuart B Goodman

Total joint replacement is a highly successful surgical procedure for treatment of patients with disabling arthritis and joint dysfunction. However, over time, with high levels of activity and usage of the joint, implant wear particles are generated from the articulating surfaces. These wear particles can lead to activation of an inflammatory reaction, and subsequent bone resorption around the implant (periprosthetic osteolysis). Cells of the monocyte/macrophage lineage orchestrate this chronic inflammatory response, which is dominated by a pro-inflammatory (M1) macrophage phenotype rather than an anti-inflammatory pro-tissue healing (M2) macrophage phenotype. While it has been shown that interleukin-4 (IL-4) selectively polarizes macrophages towards an M2 anti-inflammatory phenotype which promotes bone healing, rather than inflammation, little is known about the time course in which this occurs or conditions in which repolarization through IL-4 is most effective. The goal of this work was to study the time course of murine macrophage polarization and cytokine release in response to challenge with combinations of polymethyl methacrylate (PMMA) particles, lipopolysaccharide (LPS) and IL-4 in vitro. Treatment of particle-challenged monocyte/macrophages with IL-4 led to an initial suppression of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) production and subsequent polarization into an M2 anti-inflammatory phenotype. This result was optimized when IL-4 was delivered before PMMA particle challenge, to an M1 phenotype rather than to uncommitted (M0) macrophages. The effects of this polarization were sustained over a 5-day time course. Polarization of M1 macrophages into an M2 phenotype may be a strategy to mitigate wear particle associated periprosthetic osteolysis. Cellular & Molecular Immunology (2013) 10, 471–482; doi:10.1038/cmi.2013.39; published online 9 September 2013

Keywords: IL-4; macrophage polarization; osteolysis; PMMA particles; total joint replacement

INTRODUCTION in the periprosthetic tissues is of a predominantly M1 pheno- A common and effective treatment for severe end-stage arth- type.22 The induction and activation of these macrophages leads ritis and joint dysfunction is total joint replacement. Usage of a to an acute inflammatory reaction, tipping the balance against total joint replacement leads to wear and the generation of the functions of the alternatively activated M2 macrophages, particulate debris from the articulating surfaces. Phagocytosis which include bone and wound healing, and angiogenesis.23–27 of this wear debris by monocyte/macrophages leads to their While macrophage diversity includes a large spectrum of phe- activation, proliferation, and differentiation into pro-inflam- notypes, rather than distinctly pro-inflammatory or anti-inflammatory M1 macrophages in the periprosthetic tissues. This matory populations, differing cytokine production profiles have chronic inflammatory reaction reduces bone formation and been described for M1 and M2 macrophages.28 The cytokine increases bone resorption, a process known as periprosthetic release profile of M1 pro-inflammatory macrophages includes osteolysis.1–17 Periprosthetic bone loss leads to subsequent increased tumor-necrosis factor (TNF)-a, interleukin (IL)-1, IL- implant loosening, often requiring revision surgery. 6, IL-12, IL-23 and type 1 interferon, as well as expression of The ingestion of polymer wear particles such as polymethyl inducible NO synthase (iNOS), CCR7 and HLA-DR.29–34 The methacrylate (PMMA) and polyethylene particles leads to the cytokine release profile of M2 anti-inflammatory macrophages production of pro-inflammatory cytokines and chemokines, includes increased IL-4, IL-10, IL-13, and IL-1ra production, as and the systemic migration of monocyte/macrophage precur- well as expression of CD206, Ym1, CD163, CCL1, CCL18, sors to the particle location.18–21 The presence of macrophages FIZZ1, Arginase 1 and chitotriosidase.29–38 These distinctions

Orthopaedic Research Laboratories, Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, CA, USA Correspondence: Dr SB Goodman, MD PhD, Department of Orthopaedic Surgery, 450 Broadway Street, Mailcode 6342, Redwood City, CA 94063, USA. E-mail: [email protected] Received: 10 March 2013; Received: 22 June 2013; Accepted: 15 July 2013 Macrophage polarization in response to wear particles in vitro JK Antonios et al 472

provide a useful tool to identify the phenotype of the different followed by the addition of basal medium. The cells were spun macrophage population in tissue specimens or in vitro studies. down again and resuspended in augmented basal medium sup- In addition, exogenous delivery of specific cytokines, may pro- plemented with 30% leukocyte-conditioned medium (LCM) vide the potential to stimulate a targeted response, both for (v/v) and 10 ng/ml macrophage colony-stimulating factor. purposes of study, and for future clinical intervention.39 LCM, an important source of multiple forms of colony stimu- The effects of LPS in inducing macrophage differentiation lating factors, was harvested as supernatant from the culture of into an M1 phenotype, and the effect of IL-4 and IL-13 in L929 cells, to promote macrophage proliferation and differ- inducing macrophage differentiation into an M2 phenotype entiation.47,48 The cells were counted using a hemocytometer, have been well established.40–44 Recent studies have begun to and plated into 175 cm2 culture flasks (BD, Franklin Lakes, NJ, examined the effects of these factors in the presence of wear USA) at a concentration of 503106 cells per flask. Culture particles. Rao et al.22 have shown the ability to alter the pheno- medium was changed every 3 days for 1 week, to remove unat- type of wear particle challenged macrophages through the tached cells, as the cells were allowed to reach confluency. Cells addition of IL-4. The addition of the anti-inflammatory cyto- were lifted with 0.25% Trypsin/EDTA (Invitrogen), washed and kine IL-4 led to an increase in M2 macrophage polarization by 7 resuspended in augmented basal media. Seven groups were days after PMMA particle challenge in vitro. IL-4 mitigated defined, each with specific pre-treatment and treatment condi- wear particle induced osteolysis through the preferential polari- tions. For subsequent ELISA and RT-PCR experiments, cells zation of macrophages to an M2 phenotype using the murine were plated in 24-well culture plates (BD, Franklin, Lakes, NJ, calvarial model.45 This work suggests the possibility for direct USA) at an initial concentration of 13105 cells per well, into clinical translational application, however the conditions and their appropriate pre-treatment condition. For subsequent flow time course in which IL-4 is most effective in polarizing macro- cytometry experiments, cells were plated in 10 cm culture dishes phages to an M2 phenotype, even in the presence of wear at an initial concentration of 13106 cells per dish, into their particles, remain unclear. To address this question in this appropriate pre-treatment condition. study, IL-4 was administered to uncommitted (M0) monocyte/ macrophages, as well as macrophages pre-treated to promote M1 PMMA particle preparation or M2 polarization before particle challenge. The kinetics of the Phagocytosable PMMA particles (Polysciences, Warrington, effects of IL-4 administration on macrophage polarization was PA, USA), 1–10 mm in diameter, were sterilized in 70% ethanol assessed at multiple time points through 5 days in vitro. three times for 10 min each and incubated overnight in 70% The ability to minimize the inflammatory immune response ethanol with shaking. The following day, the particles were and enhance bone healing and repair may prove valuable in washed three times with phosphate-buffered saline, and main- maximizing the longevity of artificial joints. This may be pos- tained at a stock solution of 5% (v/v). The particles tested sible, through the manipulation of macrophage polarization, negative for the presence of endotoxin using the Limulus by stimulating the conversion of monocytes/macrophages into Amoebocyte Lysate Assay (Cat# QCL-1000; Biowhittaker, the M2 phenotype using IL-4. The goal of this work was to Walkersville, MD, USA). A final does of 0.15% (v/v) of challenge murine macrophages in vitro with polymethyl PMMA particles was used, as per previous studies.16,22 methacrylate particles, lipopolysaccharide (LPS) and IL-4 and to characterize the cytokine release profile and macrophage Macrophage stimulation polarization over time using enzyme-linked immunosorbent The plated cells underwent 6 days of pre-treatment, with media assays (ELISA), real-time PCR and flow cytometry. changed on day 3, followed by up to 5 days of treatment. Use of a 5-day time course allowed for the detailed analysis of gene MATERIALS AND METHODS expression and cytokine release over time without the need for Macrophage cell culture media changes or repeated treatment administrations.49–52 The All experimental protocols have been approved by the Stanford time course and concentrations of LPS and IL-4 used to induce Administrative Panel for Laboratory Animal Care. To harvest M1 or M2 macrophage polarization were chosen according to bone marrow macrophages, bone marrow cells were collected previous studies.22,32,53–59 Seven pre-treatment and treatment from the femora and tibiae of 15 Jackson C57BL/6J mice aged groups were defined (Table 1). Group I served as a negative 46 8–10 weeks old. The mice were killed with CO2 gas, and control, with the macrophages receiving no stimuli. Group II sterilized in 70% ethanol three times for 5 min each. Under tested the effects of stimulation with PMMA particles. Groups sterile conditions, the femora and tibiae of the mice were sur- IIIandIVwereusedtostudytheroleofLPSandIL-4,respec- gically removed. Using a 25-gauge needle, the bone marrow tively, on undifferentiated macrophages. Group V tested the was flushed into a 50 ml centrifuge tube by injecting 5 ml of ability of IL-4 to alter the polarization of undifferentiated macro- basal medium (RPMI Medium 1640 supplemented with 10% phages, even in the presence of two pro-inflammatory stimuli: fetal bovine serum, 1% Antibiotic-Antimycotic (Invitrogen, PMMA particles and LPS. Similarly, Group VI tested whether Grand Island, NY, USA)) into the marrow cavity. The cells were pre-treatment of macrophages toward an M1 phenotype with filtered through a 100 um cell strainer into another 50 ml centri- LPS would optimize or hinder macrophage repolarization using fuge tube. The cells were spun down and resuspended in ice IL-4, even in the presence of PMMA particles and LPS. Group cold red blood cell lysis buffer (Invitrogen) for 2 min at 4 uC, VII tested whether pre-treatment of macrophages towards an M2

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Table 1 Pre-treatment and treatment conditions for the seven Mini Kit (Qiagen). The purified RNA was reverse transcribed into defined experimental groups complementary DNA using a high-capacity complementary DNA Pre-treatment Treatment reverse transcription kit (Applied Biosystems, Grand Island, NY, USA). Predesigned probes for 18S (Mm03928990_g1), TNF-a Day 6 until collection after (Mm00443258_m1), IL-1ra (Mm00446186_m1), IL-1b (Mm00 Days 0–3 Days 3–6 6–120 h 434228_m1), IL-10 (Mm00439614_m1), RANK (Mm0043 Group I 7135_m1) and osteoprotegerin (OPG) (Mm01205928_m1) were Group II PMMA purchased from Applied Biosystems (Grand Island, NY, USA). Group III PMMA1LPS 18S was used as the housekeeping gene. Real-time PCR was per- Group IV PMMA1IL-4 formed using an ABI Prism 7900HT Sequence Detection System Group V PMMA1LPS1IL-4 (Applied Biosystems). Reactions were run in triplicate, using the 18S Group VI LPS PMMA1LPS1IL-4 rRNA housekeeping gene as an internal control. Expression data Group VII LPS IL-4 PMMA was normalized to the geometric mean of 18S, and relative gene { Abbreviations: IL-4, Interleukin-4 at (20 ng/ml); LPS, lipopolysaccharide expression levels analyzed using the 2 DDCT method.60 at (1 mg/ml); PMMA, polymethyl methacrylate at 0.15% (v/v). LCM media was changed on days 3 and 6, supplemented with the Flow cytometry appropriate reagents shown. Group I received only LCM media throughout Fluorescence-activated cell sorting was used to quantitatively the entire experiment. assess the change macrophage populations in response to phenotype with IL-4 after LPS administration would mitigate the PMMA particles, LPS and IL-4 over time. F4/80 and CD11b pro-inflammatory effects of PMMA particles. were used as pan-macrophage markers, iNOS as a marker of M1 macrophages, CD206 as a marker of M2 macrophages, and Groups I–V received no pre-treatment defined as only LCM 55,61–74 media for 6 days. Group VI received only LCM media for days ethidium monoazide for exclusion of dead cells. The cells 0–3, followed by LCM media1LPS (1 mg/ml) for days 3–6. were trypsinized and scraped from the 10 cm dishes, centrifuged Group VII received LCM media1LPS (1 mg/ml) for days 0– and resuspended in fluorescence-activated cell sorting staining 3, followed by LCM media1IL-4 (20 ng/ml) for days 3–6. After buffer (phosphate-buffered saline supplemented with 2% fetal completion of pre-treatment on day 6, seven treatment groups bovine serum). The cells were incubated in mouse Fc block (rat were the defined as follows, to study the effect of IL-4 in the anti-mouse CD16/C32; BD Pharmigen, San Diego, CA, USA) at presence of the pro-inflammatory stimulus PMMA particles: a concentration of 1 : 50 for 15 min. The cells were then incu- (i) LCM media only; (ii) LCM media1PMMA particles (0.15% bated with APC-conjugated anti-F4/80 (eBioscience, San Diego, v/v); (iii) LCM media1PMMA particles (0.15% v/v)1LPS CA, USA) at a concentration of 1 : 100, PE-Cy7 conjugated anti- (1 mg/ml); (iv) LCM media1PMMA particles (0.15% v/ CD11b (eBioscience) at a concentration of 1 : 50, PE-conjugated v)1IL-4 (20 ng/ml); (v) LCM media1PMMA particles anti-CD206 (Biolegend, San Diego, CA, USA) at a concentration (0.15% v/v)1LPS (1 mg/ml)1IL-4 (20 ng/ml); (vi) LCM of 1 : 100, and ethidium monoazide at a concentration of media1PMMA particles (0.15% v/v)1LPS (1 mg/ml)1IL-4 1 : 2500, or their appropriate isotype controls, for 10 min in (20 ng/ml); and (vii) LCM media1PMMA particles (0.15% the dark followed by 10 min under direct light. The cells were v/v). The culture media were collected at 6, 16, 24, 48, 72 and centrifuged, and resuspended with fixation/permeabilization 120 h after treatment, and analyzed for cytokine release using solution (BD) for 20 min at 4 uC. The cells were washed twice ELISA. At these time points, RNA was extracted by the TRIzol in Perm/Wash buffer (BD), then incubated at 4 uCwithFITC- method and quantified by real-time PCR as described below. conjugated anti-iNOS (BD) at a concentration of 1 : 50 for 30 min. The cells were washed twice more with Perm/Wash ELISA buffer, then resuspended in fluorescence-activated cell sorting Cytokine production, reflective of the pro-inflammatory and staining buffer. Forward and side scatter gating was set to anti-inflammatory functions of M1 and M2 macrophages, include viable cells. Viable cells were then gated to include only respectively, was assessed using ELISA. ELISA kits for TNF-a macrophages, defined as F4/80-positive and CD11b-positive were purchased from Biolegend (San, Diego, CA, USA) and cells. Macrophages were gated to distinguish M1 and M2 posi- ELISA kits from IL-1ra were purchased from R&D Systems tive and negative populations. M1 macrophages were defined as (Minneapolis, MN, USA). Protocols followed those provided iNOS-positive, CD206-negative cells. M2 macrophages were by each manufacturer. ELISAs were run in duplicate. defined as iNOS-negative, CD206-positive cells. The absolute number of macrophages of various populations was calculated, RNA extraction and RT-PCR and the ratio of M2/M1 macrophages taken. Sorting was done After harvesting the cell culture media, 200 ml of TRIzol reagent using a BD LSR II flow cytometer, and analyzed by FlowJo (Invitrogen) was used per well to extract RNA from the macro- software (TreeStar, Ashland, OR, USA). phages according to the manufacturer’s protocol. Due to low RNA quantities per well, the TRIzol extractions from three Statistical analysis identically prepared wells were pooled prior to RNA purifica- ELISA data was analyzed using a two-way analysis of variance tion. The extracted RNA was then purified using the RNeasy to test for significance followed by Tukey multiple comparisons

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tests to compare treatment groups within time points using Effects of IL-4 administration on cytokine gene expression Prism 6.0 (Graphpad Software, La Jolla, CA, USA). P values profiles less than 0.05 were considered significant. Data were reported After pre-treatment, macrophages were treated with the appro- as mean6standard error. priate combination of PMMA particles, LPS and IL-4, and their mRNA gene expression profiles analyzed over a time course RESULTS ranging from 6 h to 5 days using real-time PCR. mRNA expression is calculated relative to Group I mRNA expression. Effects of pre-treatment conditions Expression levels of the pro-inflammatory cytokines TNF-a To study the effects of IL-4 administration on macrophage and IL-1b peaked at 6 and 16 h post treatment, respectively, response to wear particles, macrophages were first pre-treated followed by a large and sustained decrease in all treatment according to their group designation (Table 1), and analyzed conditions (Figure 2a and c). The expression of both TNF-a for the relative expression of multiple cytokines and the release and IL-1b increased with PMMA administration alone in of TNF-a and IL-1ra using real-time PCR and ELISA. All Group II, and was greatest when stimulated with both groups were analyzed on day 6, after pre-treatment was com- PMMA particles and LPS in Group III. IL-4 administration plete, and before treatment conditions. Group VI cells, which in the presence of both PMMA and LPS, as in Group V, was received 3 days pre-treatment with LPS, showed greatly ele- not sufficient to mitigate a strong pro-inflammatory state. vated expression levels of the pro-inflammatory cytokine IL- After pre-treatment to M1 macrophages in Group VI, however, 1b mRNA relative to control cells receiving only LCM media IL-4 further reduced TNF-a and IL-1b expression. IL-4 was (Figure 1a). These cells also showed increases in TNF-a, IL-1ra, most effective in suppressing the expression of pro-inflamma- and IL-10 mRNA expression. Macrophages that received pre- tory cytokines when undifferentiated or pre-treated M2 macro- treatment with LPS followed by IL-4 showed increased mRNA phages were challenged with PMMA particles alone, as in expression levels of IL-1ra and OPG compared to control, and a Group VII (Figure 2a and c). reduction in IL-1b and TNF-a expression compared to LPS Gene expression levels of the anti-inflammatory cytokines treatment alone (Figure 1a). The dramatic increase in IL-1b IL-1ra and IL-10 also showed early peaks, at 6 and 24 h post mRNA expression following LPS exposure suggests increased treatment, respectively, although with slightly different differentiation into M1 macrophages in Group VI, with expression patterns (Figure 2b and d). As seen with the pre- increases in anti-inflammatory cytokines IL-1ra and Il-10 likely treatment gene expressions, pro-inflammatory treatment due to increased feedback regulatory mechanisms. The large groups such as Groups III and Group V, also increased anti- reduction in IL-1b mRNA expression after IL-4 administra- inflammatory cytokine expression, likely as part of a regulatory tion, while maintaining some increased expression of IL-1ra process. IL-4 administration to undifferentiated macrophages may suggest a move away from a predominant M1 phenotype, in Group IV, did not lead to a large increase in IL-1ra or IL-10 towards an M2 phenotype. expression. The effective suppression of an inflammatory state Cell culture supernatants were analyzed using ELISA for the may not have necessitated a large increase in anti-inflammatory release of the pro-inflammatory cytokine TNF-a, and the anti- cytokine expression. Similarly, in Group VII, the addition of inflammatory cytokine IL-1ra. Pre-treatment with LPS led to PMMA particles to M2 macrophages led to a modest increase significant increases in TNF-a release suggestive of M1 polari- in IL-1ra expression but low IL-10 expression (Figure 2b and zation, which was suppressed by subsequent addition of IL-4 in d). Group VII (Figure 1b). Similar to the results analyzing gene RANK-L, through interaction with RANK, stimulates osteo- expression, IL-1ra release was significantly increased in both clast formation from monocyte/macrophage precursors lead- pre-treatment conditions, more so in Group VI (Figure 1c). ing to increased bone resorption. OPG acts as a RANK-L decoy Within a dynamic system macrophages are receiving a ba- receptor, serving to prevent interaction of RANK-L and RANK, lance of both pro-inflammatory and anti-inflammatory signals, thereby decreasing osteoclast formation. Evaluation of RANK with the dominant signals determining the primary response and OPG mRNA expression in the various treatment condi- and other signals maintained for regulation of that response. tions varied notably over the 5-day time course. No clear pat- As a reflection of this balance, we analyzed the effects of these tern for the effects of IL-4 administration on RANK or OPG pre-treatment conditions on the ratio of IL-1ra to TNF-a expression emerged. release. While LPS pre-treatment increased both TNF-a and IL-1ra release individually, the IL-1ra/TNF-a ratio did not Effects of IL-4 administration on cytokine release change significantly (Figure 1d). The decrease in the ratio is After treatment, the cell culture media were analyzed for TNF- reflective of the large increase in TNF-a release, and a more a and IL-1ra release using ELISA. Treatment with LPS and pro-inflammatory environment. Pre-treatment with IL-4 after PMMA particles in Group III led to significant increases in LPS, however, led to a significant increase in IL-1ra/TNF-a TNF-a release relative to Group I and Group VII within 6 h ratio suggestive of anti-inflammatory macrophage polariza- (Figure 3a). By 16 h, Group III LPS and PMMA particle treat- tion. Therefore, Group VI pre-treatment led to increased M1 ment led to significantly higher TNF-a release than all other macrophage polarization, and Group VII pre-treatment to treatment conditions. As seen in Group V, concurrent admin- increased M2 macrophage polarization. istration of IL-4 with LPS and PMMA without pre-treatment

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ab2500 ** **

9460 2000 3 days LPS 9440 pretreatment 40 3 days LPS 1500 pretreatment, 30 3 days IL-4 pretreatment 1000 20 Concentration (pg/mL) a

Fold Expression 10 500 TNF

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Figure 1 Pre-treatment with LPS or LPS then IL-4 resulted in M1 and M2 macrophage populations, respectively. (a) Pre-treatment for 3 days with LPS (1 mg/ml) resulted in increased mRNA expression of IL-1b relative to no pre-treatment, and to a lesser extent anti-inflammatory cytokines, as analyzed by real-time PCR. Subsequent 3-day pre-treatment with IL-4 greatly reduced the mRNA expression of both pro-inflammatory cytokines TNF-a and IL-1b, and anti-inflammatory cytokines IL-1ra and IL-10, while increasing OPG expression. (b, c) TNF-a and IL-1ra secretion were analyzed by ELISA. LPS pre-treatment led to increased TNF-a secretion suggestive of M1 phenotype polarization, which was suppressed after IL-4 administration. Both LPS and IL-4 pre-treatment led to increased production of IL-1ra. (d) IL-4 administration after LPS pre-treatment led to increased IL-1ra/TNF-a ratio showing an increase in the release of anti-inflammatory cytokines relative to pro-inflammatory cytokines beyond that expected solely for regulatory purposes (n53 per group). *Pf0.05, **Pf0.01, ***Pf0.001. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; LPS, lipopolysaccharide; OPG, osteoprotegerin; PCR, polymerase chain reaction; TNF, tumor-necrosis factor. was again unable to significantly suppress the effects of pro- show high early IL-1ra release, it was suppressed by 48 h, and inflammatory stimuli. However, with prior pre-treatment to maintained at a lower concentration. IL-4 treatment groups M1 macrophages in Group VI, TNF-a release was significantly following M1 pre-treatment, however, showed steady decreased. As seen previously, the lowest TNF-a levels after increases in IL-1ra concentration, with Group VII reaching particle stimulation were achieved when the wear particles were significantly higher release than Groups I, II and IV by 48 h placed into an already anti-inflammatory environment in post treatment, and maintained that increase through the 5- Group VII (Figure 3a). day time course (Figure 3b). Macrophages pre-treated with a Unlike the gene expression results, the greatest IL-1ra pro-inflammatory stimulus and converted to an M1 pheno- release is seen with IL-4 administration, particularly as type before IL-4 administration and particle challenge in pre-treatment to particle challenge (Figure 3b). While pro- Group VII showed the greatest increase in the ratio of IL- inflammatory experimental groups such as Groups III and V 1ra to TNF-a secretion. The levels approached or surpassed

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abTNFa IL-1ra 40 800 Group II Group II 600 Group III Group III 400 30 Group IV Group IV 250 Group V Group V 200 20 Group VI Group VI 150 Group VII Group VII Fold Expression Fold Expression 100 10 50

0 0 6 6 4 8 2 6 6 4 8 2 1 2 4 7 20 1 2 4 7 20 1 1 Time (hrs) Time (hrs)

c IL-1b d IL-10 15000 800 Group II Group II Group III Group III 600 Group IV Group IV 10000 Group V Group V Group VI 400 Group VI Group VII 5000 Group VII Fold Expression Fold Expression 200

0 0 6 6 4 8 2 1 2 4 7 20 6 6 4 8 2 0 1 1 2 4 7 2 1 Time (hrs) Time (hrs)

e RANK f OPG 1.0 40 Group II Group II

0.8 Group III Group III 30 Group IV Group IV 0.6 Group V Group V Group VI 20 Group VI 0.4 Group VII Group VII Fold Expression Fold Expression 10 0.2

0.0 0 6 6 4 8 2 6 6 4 8 2 1 2 4 7 20 1 2 4 7 20 1 1 Time (hrs) Time (hrs)

Figure 2 The cytokine mRNA expression profiles of macrophages in the presence of wear particles, as analyzed by real-time PCR, is influenced by IL-4 administration. Fold expression is calculated relative to Group I mRNA expression. (a, c) Treatment with PMMA particles, with or without LPS led to upregulation of pro-inflammatory cytokines TNF-a and IL-1b relative to Group I, peaking at 6 and 16 h post treatment, respectively. This increase in expression was maintained in groups stimulated with LPS through 48 h. IL-4 effectively suppressed expression of TNF-a and IL-1b whether administered to undifferentiated or pre-treated macrophages. (b, d) IL-1ra and IL-10 expressions were upregulated in macrophages activated with LPS, peaking at 24 and 6 h, respectively. Macrophages treated with IL-4 after LPS pre-treatment also showed increased IL-1ra and IL-10 expression. (e, f) RANK expression was downregulated, and OPG expression upregulated in all treatment conditions relative to control at relatively steady levels over the 5-day time course. IL, interleukin; LPS, lipopolysaccharide; OPG, osteoprotegerin; PCR, polymerase chain reaction; PMMA, polymethyl methacrylate; TNF, tumor-necrosis factor. the normal and fluctuating balance evident in control Group Notable differences were seen between the mRNA and pro- I, suggesting a suppression of the inflammatory response tein expression of TNF-a and IL-1ra including differences in (Figure 3c). peak expression during the time course. This may be explained

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a 6000 Group I

Group II

Group III 4000 Group IV

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0 6 6 6 6 6 6 6 16 24 48 72 16 24 48 72 16 24 48 72 16 24 48 72 16 24 48 72 16 24 48 72 16 24 48 72 120 120 120 120 120 120 120

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Figure 3 IL-4 administration leads to a preferential increase in IL-1ra secretion relative to TNF-a, suggestive of polarization toward an M2 phenotype. TNF-a and IL-1ra secretion was measured by ELISA. (a) Stimulation with LPS led to significant increases in TNF-a production (* at 6 and 120 h; *** at 16, 24, 48 and 72 h). IL-4 delivered concurrent with PMMA particle and LPS stimulation without pre-treatment in Group V was unable to suppress TNF-a levels, secreting elevated levels of TNF-a relative to all groups except Group III (*** at 16 h; ** at 24 h; * at 72 h). IL-4 administered to undifferentiated macrophages in Group IV, or macrophages pre-treated with LPS in Groups VI–VII, effectively lowered TNF-a production for the duration of the time course relative to LPS-treated macrophages (*** at 16 and 48 h; ** at 24 and 72 h; * at 120 h). (b) Pre- treatment of macrophages with IL-4 prior to particle challenge in Group VII led to a steady and significant increase in IL-1ra production relative to all groups without pre-treatment by 5 days post treatment (***). (c) By 5 days after treatment, IL-4 pre-treatment in Group VII led to increased IL-1ra/ TNF-a ratio relative to all particle-treated groups without pre-treatment (**). This finding suggests an increase in the release of anti-inflammatory

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cytokines relative to pro-inflammatory cytokines suggestive of increased polarization towards an M2 phenotype. *Pf0.05, **Pf0.01, ***Pf0.001. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; LPS, lipopolysaccharide; PMMA, polymethyl methacrylate; TNF, tumor-necrosis factor.

by intrinsic differences between the testing methods. The higher M2 macrophages relative to other treatment groups results of mRNA expression show the transcripts present at (Figure 4e). As seen by ELISA analysis of cytokine release, the specific moment of collection, while analysis of cytokine treatment of undifferentiated macrophages with IL-4 in secretion by ELISA measures the total concentration of the Groups IV and V did not lead to sustained increases in M2 individual cytokine, whether the protein was secreted imme- populations, suggesting repolarization using IL-4 may be most diately before collection or hours earlier. Although the half-life effective if administered to previously activated macrophages. of cytokines is thought to be extremely short in vivo, cytokines Group III macrophages showed a large rise in CD206 expres- in cell culture experiments have been shown to last far longer, sion at 24 h that was not sustained. This parallels the pattern of on the order of hours.75 Therefore results of in vitro studies IL-1ra expression as detected by ELISA and real-time PCR, suggest recent rather than immediate cell activity. Similarly, as with a peak at 24 h with a subsequent decline, possibly serving mentioned, the groups showing the highest IL-1ra expression to activate regulatory mechanisms to control the pro-inflam- differed between the mRNA and protein levels. It is thought matory response (Figure 4e). Many macrophages that were that IL-1ra, TNF-a and other cytokines are regulated not only positive for both iNOS and CD206 were detected, possibly at the transcriptional level, but also through post-transcrip- representing intermediate populations, or macrophages tran- tional modifications regulated by a 39-untranslated region.76,77 sitioning through M1 and M2 phenotypes (Figure 4F). Changes in post-transcriptional modification due to differing Similarly, IL-4 treatment after LPS pre-treatment led to pro-inflammatory or anti-inflammatory environments may increased levels of macrophages not expressing either marker impact the efficiency or extent of translation to functional of differentiated after 72 h, suggesting a possible return towards protein. a non-inflammatory and undifferentiated state (Figure 4g).

Analysis of macrophage populations by flow cytometry DISCUSSION In addition to their differing cytokine release profile, M1 and This study investigated the time course of macrophage polari- M2 macrophages are characterized by the regulation of intra- zation in response to wear particles and IL-4 treatment in vitro. cellular and surface proteins. To further assess the ability of IL- To accomplish this, bone marrow-derived macrophages were 4 to repolarize macrophages toward an M2 phenotype in the cultured for different lengths of time under various pre-treat- presence of wear particles, flow cytometry was used to analyze ment and treatment conditions. To test for optimal conditions the macrophage populations present in the various treatment in which macrophages are polarized toward an M2 phenotype, groups at 6, 24 and 72 h. Macrophages were defined as cells select groups of macrophages received pre-treatments leading positive for pan-macrophage markers F4/80 and CD11b. to M1 or M2 macrophage populations. Treatment of macro- Within the macrophage population, M1 macrophages were phages with PMMA wear particles, with or without LPS, led to defined as iNOS-positive, CD206-negative cells, and M2 rapid increases in the expression of pro-inflammatory cyto- macrophages as iNOS-negative, CD206-positive cells. Group kines TNF-a and IL-1b. Costimulation of undifferentiated II macrophages challenged with PMMA particles without pre- macrophages with PMMA particles and LPS was too powerful treatment showed higher levels of iNOS expression after 6 h of a pro-inflammatory stimulus to allow repolarization using IL- treatment relative to Group VII cells pre-treated with LPS then 4. Addition of IL-4 to pre-treated M1 or M2 macrophages IL-4 before particle challenge (Figure 4a). By 72 h, total levels of before or during particle challenge, however, was sufficient to iNOS expression were similar between the treatment groups markedly reduce expression of these pro-inflammatory cyto- (Figure 4b). Alternatively, 6 h post treatment, the proportion of kines. This inhibition was effective both initially, and through- CD206-positive cells was similar across both treatment groups out the 5-day time course. The greatest increases in the gene (Figure 4c). By 72 h, macrophages pre-treated with IL-4 expression of the anti-inflammatory cytokines IL-1ra and IL- showed greater expression of CD206 (Figure 4d). These find- 10 did not come from macrophages treated with IL-4, however, ings suggest IL-4 may lead to a rapid suppression of pro- but those receiving the strongest activation signals. The inflammatory factors, followed by a subsequent increase in increase in IL-1ra and IL-10 expression may serve as a regula- anti-inflammatory markers. tory mechanism to control the extent of the overall pro- As with the analysis of pro-inflammatory and anti-inflam- inflammatory process. In addition, effective suppression of matory cytokines, it is important to consider shifts in the bal- pro-inflammatory cytokines may reduce the signals that pro- ance between M1 and M2 markers. When the ratio of the mote an anti-inflammatory response. Increases in IL-10 absolute numbers of M2 to M1 macrophages was taken, groups expression in macrophages stimulated with LPS has been with IL-4 administration after pre-treatment again showed no reported in other studies.78 Similarly, analysis of cytokine particular increases in M2 macrophages at 6 h post treatment. release showed increased TNF-a in groups treated with particles By 72 h after treatment, Groups VI and VII showed increasingly and LPS, even in the presence of concurrently administered

Cellular & Molecular Immunology Macrophage polarization in response to wear particles in vitro JK Antonios et al 479 a 6 hours b 72 hours Group II Group VII Group II Group VII 0 0 0 0 90.5 36.6 92.6 96.3 iNOS iNOS iNOS iNOS

0 9.55 0 63.4 0 7.36 0 3.69 F4/80 F4/80 F4/80 F4/80

6 hours 72 hours c Group II Group VII d Group II Group VII 0 0 0 0 14.8 9.61 32.3 53.2 CD206 CD206 CD206 CD206

0 85.2 0 90.4 0 67.7 0 46.8 F4/80 F4/80 F4/80 F4/80 efg

0.03 400000 400000 Group I Group I Group I Group II Group II Group II 300000 Group III 300000 Group III Group III 0.02 Group IV Group IV Group IV Group V 200000 Group V 200000 Group V

Group VI Group VI Group VI 0.01 Group VII Group VII 100000 100000 Group VII Ratio of M2/M1 Cells Number of M1-M2- Cells Number of M1+M2+ Cells 0 0.00 0 6 6 24 72 6 24 72 24 72 Time (hrs) Time (hrs) Time (hrs)

Figure 4 The effect of IL-4 on populations of M1 and M2 macrophages. Cells positive for both pan-macrophage markers F4/80 and CD11b by flow cytometry were defined as macrophages. Within that population, macrophages were analyzed using iNOS as a marker of M1 phenotype, and CD206 as a marker of M2 phenotype. (a, c) By 6 h post particle challenge, Group VII macrophages pre-treated with IL-4 showed smaller populations of iNOS-positive cells, but similar populations of CD206-positive cells. (b, d) By 72 h post particle challenge, Group VII macrophages pre-treated with IL-4 showed similar populations of iNOS-positive cells, but increased populations of CD206-positive cells. (e) Over the 3-day time course, cells treated with IL-4 after LPS pre-treatment trended toward a higher ratio of M2 (iNOS2CD2061) to M1 (iNOS1CD2062) macrophages. (f, g) Cells positive for both M1 and M2 markers, and negative for both markers were quantified. IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide.

IL-4. The effects of IL-4 were greatly enhanced when adminis- in CD206 levels in IL-4 treated conditions after pre-treatment. tered to pre-treated M1 macrophages. TNF-a concentrations Examination of the relative numbers of M1 and M2 macro- decreased, and IL-1ra progressively increased over the 5-day phages showed no clear pattern initially, but by 72 h, the popu- time course in these conditions. While macrophages receiving lation in IL-4 pre-treated macrophages trended toward activation signals again increased IL-1ra levels initially, macro- increased macrophages expressing the M2 phenotype. phages receiving IL-4 pre-treatment before particles challenge While it serves as a useful framework to consider the various resulted in statistically significant increases in the ratio of anti- roles of macrophages throughout various tissues, the division inflammatory to pro-inflammatory signals. of macrophage phenotypes strictly into M0, M1 and M2 is We show an initial decrease in the prevalence of M1 macro- overly simplistic. In our study, a considerable population of phage markers with the addition of IL-4 using flow cytometry, macrophages was identified which coexpressed both M1 and suggesting suppression of pro-inflammatory functions consis- M2 macrophage markers. Such populations has also been tent with the gene expression and cytokine release results. At reported in other studies; however, the role of these macro- these early stages, we did not observe a change in the propor- phages have yet to be well understood.64 While previous studies tion of M2 phenotype markers within the macrophage popu- have examined the changes in macrophage markers and mor- lation. After 3 days of treatment, iNOS levels rose in all phology during activation, these cells may represent previously treatment conditions, while we also observed a greater increase unidentified macrophage phenotypes that should be studied

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further.79–81 Our study has shown that polarization of results suggest that IL-4 may first serve to suppress the macrophages toward an M2 phenotype through IL-4 is most expression and production of pro-inflammatory products effective when first pre-treated toward an M1 phenotype. As that would normally be produced in response to wear parti- such, a subset of these cells may represent macrophages in cles. Subsequently, macrophages begin to increase the pro- the process of polarizing toward an M1 or M2 phenotype, duction of anti-inflammatory cytokines and M2 markers upregulating CD206 and other anti-inflammatory markers over time, leading to an increase in macrophages of an M2 while downregulating the production of iNOS and other phenotype within the population. These results lend support pro-inflammatory markers. iNOS is predominantly an intra- to the proposed benefits of local IL-4 administration in asso- cellular protein. Recent studies have shown that down-regu- ciation with total joint replacements as a means of slowing lation of iNOS expression and suppression of nitric oxide the progression of periprosthetic osteolysis and promoting production may be accomplished by the formation of aggre- bone healing. Further studies are necessary, however, to pro- somes.82–84 Although flow cytometry separates populations vide greater understanding of the mechanisms by which through the presence or absence of particular cell markers, macrophage populations are altered by IL-4. Recent work the permeabilization required for detection of intracellular by Tang et al.85–87 have shown the ability to detect and markers may provide access to sequestered and inactivated monitor activated macrophages and neutrophils in real time intracellular iNOS. Characterizing the various macrophage in vivo through the use of NIR nanoprobes. This technology, populations using multiple M1 and M2 markers and an along with probes targeted for alternatively activated macro- extended time course of study, is likely to provide informa- phages, may be combined with established in vivo models of tion about the roles of such cells. In addition, histological particle-induced osteolysis.45 These studies may provide analysis of the subcellular location of pro-inflammatory and more detailed insight into the mechanisms by which IL-4 anti-inflammatory markers in double positive cells may dis- affects macrophage populations and the subsequent impact tinguish populations functionally or transiently expressing of these changes on bone loss or repair. markers of M1 and M2 phenotypes. There are several limitations to this study. Due to low ACKNOWLEDGEMENTS quantities of RNA extracted and purified per well, samples We would like to thank Dr Alexander Harris of Stanford University for from three identical experiments were pooled for each assistance in the statistical analysis. We would also like to thank Dr experimental condition. While this allowed for the analysis Stephen Badylak and Brian Sicari for assistance in choosing of gene expression of multiple pro-inflammatory and anti- appropriate macrophage markers. This work was supported by NIH inflammatory cytokines over the time course, it did not grants 2R01AR055650-05 and 1R01AR063717-01; the Ellenburg Chair allow for statistical testing. The trends seen in the gene in Surgery at Stanford University; and the Stanford Medical Scholars Program. expression data are supported by the analysis of cytokine release by ELISA. Protein analysis was done, however, on only one representative pro-inflammatory and one anti- inflammatory cytokine, TNF-a and IL-1ra, respectively. 1 Harris WH. Wear and periprosthetic osteolysis: the problem. Clin To survey the changes in macrophage populations upon Orthop Relat Res 2001; 393: 66–70. particle challenge and IL-4 administration, combinations 2 Amstutz HC, Campbell P, Kossovsky N, Clarke IC. Mechanism and of four markers were used to identify M1 and M2 popula- clinical significance of wear debris-induced osteolysis. Clin Orthop Relat Res 1992; 276: 7–18. tions. While commonly used as pan-macrophage markers, 3 Schmalzried TP, Jasty M, Harris WH. Periprosthetic bone loss in total neither F4/80 nor C11b are exclusively found on macro- hip arthroplasty. Polyethylene wear debris and the concept of the phages. F4/80 can be found on subsets of dendritic cells, effective joint space. J Bone Joint Surg Am 1992; 74: 849–863. and CD11b on granulocytes and subsets of T cells and 4 Hirakawa K, Bauer TW, Stulberg BN, Wilde AH. Comparison and 61,62 quantitation of wear debris of failed total hip and total knee dendritic cells. To control for the unspecific nature of arthroplasty. J Biomed Mater Res 1996; 31: 257–263. these markers, multiple steps were taken. First, cultures 5 Margevicius KJ, Bauer TW, McMahon JT, Brown SA, Merritt K. were grown in leukocyte-conditioned medium and macro- Isolation and characterization of debris in membranes around total phage colony-stimulating factor to promote macrophage joint prostheses. J Bone Joint Surg Am 1994; 76: 1664–1675. 6 Schwarz EM, Benz EB, Lu AP, Goater JJ, Mollano AV, Rosier RN et al. differentiation and proliferation.Inaddition,inflowcyto- Quantitative small animal surrogate to evaluate drug efficacy in metric analysis of the macrophage populations, only cells preventing wear debris-induced osteolysis. J Orthop Res 2000; 18: double positive for both F4/80 and CD11b were considered 849–855. to be macrophages. 7 Merkel KD, Erdmann JM, McHugh KP, Abu-Amer Y, Ross FP, Teitelbaum SL. Tumor necrosis factor-alpha mediates orthopedic In summary, this study demonstrated that the ability to implant osteolysis. Am J Pathol 1999; 154: 203–210. polarize macrophages toward an M2 phenotype in the pres- 8 Shanbhag AS, Hasselman CT, Rubash HE. The John Charnley Award. ence of wear particles is optimized when M1 macrophages Inhibition of wear debris mediated osteolysis in a canine total hip are treated with IL-4 prior to or concurrent with particle arthroplasty model. Clin Orthop Relat Res 1997; 344: 33–43. 9 Wooley PH, Morren R, Andary J, Sud S, Yang SY, Mayton L et al. challenge. Initial changes are seen within the first 6 h, and Inflammatory responses to orthopaedic biomaterials in the murine are sustained or furthered over a 5-day time course. Our air pouch. Biomaterials 2002; 23: 517–526.

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