Cellular Immunology 266 (2011) 180–186

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Cellular Immunology

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CD4+ T-cells are important in regulating macrophage polarizationin C57BL/6 wild-type mice

Tiffany Chan 1, Elisabeth A. Pek 1, Kathleen Huth, Ali A. Ashkar ⇑Center for Gene Therapeutics and Institute for Infectious Disease Research, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 August 2010Accepted 4 October 2010Available online 30 October 2010

Keywords:CD4+ T-cellsMacrophagePolarization

0008-8749/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.cellimm.2010.10.002

⇑ Corresponding author. Address: Department oMedicine, Institute of Molecular Medicine and HealtInfectious Disease Research, McMaster University, 12MDCL-4015, Hamilton, Ontario, Canada L8N 3Z5. Fax

E-mail address: ashkara@mcmaster.ca (A.A. Ashka1 Both authors contributed equally to the preparatio

During activation, macrophages undergo physiological changes affecting their surface protein expressionand cytokine production and have been subsequently categorized into M1 (classically-activated) and M2(alternatively-activated) macrophages. It remains unclear which lymphocyte population provides theimmune microenvironment to regulate macrophage polarization. In this study, we establish a functionaland phenotypic profile of peritoneal macrophages from C57BL/6 wild-type mice. We also showed thatRag1�/� and Rag2�/�cc�/� mice have similar, exaggerated M1 characteristics in comparison to controlmice, suggesting that NK and/or NK-T cells may not be essential in this process. By controlling for envi-ronmental factors, we determine that lymphocyte-derived cytokines, rather than inherent properties ofmacrophages themselves, are crucial for their regulation. Lastly, we report that macrophages from CD4�/�

mice display an M1 profile, suggesting that CD4+ T-cells play a dominant role over other lymphocyte pop-ulations in providing the cytokine environment for regulating macrophages towards an M2 profile undernormal wild-type conditions.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Macrophages play an essential role in the shift from innate toadaptive immune response through cell-to-cell interactions, andthe release of various cytokines, chemokines, and enzymes [1–3].Once activated, they are engaged in a myriad of polarized functions,from tissue cell destruction to tissue remodelling, pro-inflammatoryto anti-inflammatory responses, and tumor growth prevention toenhanced angiogenesis [4–6]. This paradoxical plasticity of macro-phage functioning has been correlated to their polarized activation.Analogous to the TH1 and TH2 nomenclature, macrophages canmount an M1 (classical) or M2 (alternative) response, respectively[7,8]. In the presence of LPS or IFN-c, macrophages undergo M1polarization and are characterized by increased levels of IL-6,IL-12, IL-15, IL-23, and NO [1,8–10]. This cell-mediated responseallows for enhanced microbicidal and tumoricidal capacities. Onthe other hand, IL-4, IL-10, and IL-13 induce M2 macrophages[11,12]. Unlike M1 macrophages, alternative activation leads toincreased levels of IL-10, TGF-b, arginase, the macrophage mannosereceptor (MMR), and CD23 [6–8,11–13]. Expression of arginase has

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: +1 (905) 522 6750.r).n of this manuscript.

been shown to down-regulate production of NO [14,15], compro-mising their ability to clear pathogens. Instead, they promoteangiogenesis, tissue remodelling, and tissue repair [6,15].

While macrophage polarization is a widely accepted view, thedetails of its underlying mechanism are not clearly understood.Their activation appears to be regulated by their immune microen-vironment, affecting the expression of their surface proteins andproduction of effector cytokines [10,16]. For example, it has beensuggested that the release of IFN-c from Th1 cells, cytotoxic CD8+

lymphocytes, and innate immune NK cells are essential for primingmacrophages towards an M1 phenotype [17–19]. On the otherhand, M2 activation occurs through IL-4 and IL-13 stimulationfrom Th2 cells, mast cells, and basophils [12]. Another study byMills et al. [8] proposed that polarization was independent ofT or B-lymphocytes, as macrophage polarization in NUDE and SCIDmice could still be distinguished in C57BL/6 and Balb/c wild-typemice. Finally, a recent study done be DeNardo et al. [20] demon-strated the role of CD4+ T-cells in regulating pulmonary metastasisby influencing the cytokine environment that alters macrophagephenotype and effector function.

In this report, we investigate various lymphocyte populationsand their role in inducing a cytokine environment, leading to M1and M2 macrophage polarization. To accomplish this, we utilizedseveral strains of mice deficient in various lymphocyte popula-tions. Mice that lack the RAG gene are unable to develop matureT and B-cells [21], while a deficiency in the common gamma chainleads to inadequate functional receptors for many cytokines

T. Chan et al. / Cellular Immunology 266 (2011) 180–186 181

(IL-2, -4, -7, -9, -15, and -21), and a consequent absence of NK andNK-T cells [22–25]. Thus, we use Rag1�/�, Rag2�/�cc�/�, and CD4�/�

mice to rule out specific lymphocyte population, thereby proposingthe mechanism by which macrophage polarization is regulated.

2. Materials and methods

2.1. Mice

Male C57BL/6 wild-type mice, 6–8 weeks of age, were pur-chased from Charles River Laboratory (Montreal, Quebec, Canada).Rag2�/�cc�/� mice were purchased from Taconic Farms (German-town, NY), and Rag1�/� mice were bred in our facility. B6.129S6-Cd4tm1Knw/J (CD4�/�) mice were purchased from The JacksonLaboratory (Bar Harbor, ME). All mice were age and sex-matched,and maintained under specific pathogen-free conditions in theCentral Animal Facility at McMaster University Medical Center. Allanimal experiments were approved by the Animal Research EthicsBoard at McMaster University. Mice were anaesthetized with inject-able Xylocaine/Ketamine and sacrificed after tissue collection.

2.2. Cell processing, culture, and stimulation

Peritoneal macrophages were collected by injecting 3 mL of coldPBS into the peritoneal cavity, followed by removal after suffi-ciently massaging the cavity. Bone marrow and spleen were re-moved and processed into a single cell suspension. Briefly, bonemarrow cells from the femurs and tibias were flushed with coldPBS and splenic tissue was homogenized using the stopper of asterile syringe. Cells were treated with ACK lysis buffer to removered blood cells. All cells were cultured in complete RPMI (contain-ing 1% L-Glut, 1% P/S, 1% Hepes, 10% FBS, and 500 lL 2-Mercap-toethanol). In a 96-well round bottom plate, 2 � 105 peritonealmacrophages were plated per well and stimulated with 100 ng/mL of lipopolysaccharide (LPS) (Sigma, Oakville, Ontario, Canada)or RPMI as a control. Cells were incubated at 37 �C, and 24 h super-natants were collected for IL-12 and IL-10 ELISAs, and 48 h super-natants were collected for a NO Assay. Remaining cells was washedwith cold PBS and cells lysates were used for an Arginase Assay.Splenic and bone marrow cells were plated in petri dishes andincubated at 37 �C for 3 h. Adherent cells were then collected,and re-plated and stimulated in the same manner as peritonealmacrophages.

2.3. Bone-Marrow Derived Macrophage (BMDM) cell cultures

Bone marrow-derived macrophages were derived from bonemarrow cells in vitro according to a previously established protocol[26]. Briefly, bone marrow cells were plated in petri dishes withDMEM (containing 10% FCS and 0.1% Cipro) complete, and incu-bated at 37 �C for 3 h. Non-adherent cells were collected and platedin 6-well plates at 5 million cells per well. Cells were stimulatedwith 10 ng/mL of M-CSF containing media (secreted from L929cells, provided by Dr. Brian Lichty, McMaster University, Hamilton,Ontario, Canada) and allowed to differentiate for 8–10 days. Theremaining adherent cells were harvested by scraping gently afterwashing with PBS. Cells were plated and stimulated as previouslydescribed.

2.4. Quantification of cytokines, NO production and arginase activity

Mouse IL-12 total p40 ELISA kit was purchased from eBio-science (San Diego, CA) and IL-10 ELISA kit was purchased fromR&D Systems (Burlington, Ontario, Canada). NO production wasmeasured using the Griess Reagent System. In brief, 100 lL of

48 h culture supernatants were mixed with 100 lL of GriessReagent (Sigma, Oakville, Ontario, Canada). Absorbance was mea-sured at 450 nm. Concentration of NO was determined accordingto a standard curve of sodium nitrite solution. Arginase activitywas measured according to a previously established protocol[27]. Briefly, 2 � 105 cells were lysed with 100 lL of 0.1% TritonX-100 in PBS, supplemented with protease inhibitors (CompleteMinis; Roche, Mississauga, Ontario, Canada). Following a 30 minroom-temperature incubation with shaking, 100 lL of 25 mMTris–HCl (pH 7.5) was added. Another 10 lL of 10 mM MnCl2 wasadded to 100 lL of this lysate. The enzyme was activated by heat-ing samples to 56 �C for 10 min. L-Arginine hydrolysis was con-ducted by incubating 100 lL of this lysate with 100 lL of 0.5 ML-arginine (pH 9.7) at 37 �C for 60 min. The reaction was thenstopped with 800 lL of stop solution, H2SO4 (96%)/H3PO4 (85%)/H2O [1:3:7, v/v/v], and 40 lL of 9% a-isonitrosopropiophenone.Following heating to 99 �C for 30 min, 200 lL of each sample wasread on a microplate reader at 540 nm. A standard curve of ureasolution, from 20 mM to 0 mM, was used to determine final con-centrations. One unit of arginase activity was defined as theamount of enzyme that catalyzed the formation of 1 lmol of ureaper minute at 37 �C.

2.5. Antibodies and flow cytometry

Peritoneal and bone marrow macrophages collected ex vivo, andcultured BMDM were stained for flow cytometric analysis. Briefly,1 � 106 cells were plated in a 96-well round bottom plate, butfewer cells were used if this amount was unavailable. Cells werewashed with FACS buffer (PBS containing 0.1% BSA) and incubatedwith Fc-block (anti-mouse CD16/32; eBioscience, San Diego, CA)for 20 min on ice. Following another wash with FACS buffer, cellswere stained with anti-mouse F4/80 (BM8; APC-conjugated;eBioscience, San Diego, CA), anti-mouse CD23 (B3B4; PE-Cy7-con-jugated; BioLegend, San Diego, CA), and anti-mouse MMR (MR5D3;FITC-conjugate; BioLegend, San Diego, CA) for 30 min on ice in thedark. Cells were washed with FACS buffer and either fixed with 1%PFA in PBS (and run the following day) or immediately run on a BDCanto or BD LSRII flow cytometer (BD Biosciences, San Jose, CA).Data was analyzed using FlowJo 8.8 (Tree Star, Ashland, OR). Datawas analyzed according to fluorescence minus one (FMO) and iso-type controls for each separate experiment. Macrophage-positivepopulations were identified as F4/80+ and all subsequent markerswere gated off this population.

2.6. Statistical analysis

Data was graphed and analyzed in GraphPad Prism 4.0(GraphPad Software, Inc., San Diego, CA) using Student’s t-test orone-way ANOVA with Post-Tukey test, as specified. p 6 0.05 wasconsidered statistically significant. Unless otherwise stated, graphsshow mean ± SEM.

3. Results

3.1. NK and NK-T cells may not be crucial in regulating macrophagestowards an M2 profile

Given that alternatively activated macrophages express anIL-12lowIL-10high cytokine profile and have an increased expressionof CD23 and MMR [12–13,27], we characterized peritoneal macro-phages along an M1/M2 spectrum through these functional andphenotypic readouts. To determine whether NK, NK-T, T-cells, orB-cells had a regulatory role in macrophage polarization,

Fig. 1. Phenotypic and effector function of C57BL/6 wild-type, Rag1�/�, and Rag2�/�cc�/� mice. (A) Peritoneal macrophages were isolated from all strains of adult male miceand stained with mAb against CD23, MMR, and F4/80. Gates were set on F4/80 positive cells prior to further analysis. Data is presented as the mean percentage of CD23 andMMR positive cells ± SEM. ***p < 0.001 (using One-Way ANOVA Post-Tukey Test). Histograms are representative flow cytometry data of one mouse from each group. (B)Peritoneal macrophages from all strains of adult male mice were plated and stimulated with LPS or left untreated. Supernatants were collected at 24 h (for IL-12 and IL-10ELISAs) and 48 h (for NO Assay). Cell lysates were collected after 48 h for Arginase Assay. Data is presented as the mean ± SEM. n.s., not significant; **p < 0.01; ***p < 0.001(using One-Way ANOVA Post-Tukey Test).

182 T. Chan et al. / Cellular Immunology 266 (2011) 180–186

peritoneal macrophages were isolated from wild-type, Rag2�/�cc�/�,and Rag1�/� adult male mice on a C57BL/6 background. Cells werestained and analyzed by flow cytometry for expression of CD23and MMR (Fig. 1A). Interestingly, all strains of macrophages ex-pressed a similar percentage of F4/80+ monocytes (�90%) (datanot shown), suggesting that subsequent marker expression wasfrom a comparative cell population. Compared to wild-type cells,there was a significantly lower percentage of CD23 expression inboth Rag2�/�cc�/� (33.1% versus 9.9%, respectively; p < 0.001)and Rag1�/� (33.1% versus 8.7%, respectively; p < 0.001) mice. Sim-ilar results were found for MMR expression in both Rag2�/�cc�/�

(31.5% versus 11.2%, respectively; p < 0.001) and Rag1�/� (31.5%versus 8%, respectively; p < 0.001) mice (Fig. 1A). Expression ofCD23 and MMR was unremarkable between Rag2�/�cc�/� andRag1�/� macrophages, suggesting that differences were not dueto the absence of NK and NK-T cells as seen in the Rag2�/�cc�/�

mice but rather, the regulatory role of T-cells and/or B-cells.Trends observed in the phenotype of macrophages were

paralleled in their functional activity, as determined by cytokineproduction and enzyme activity (Fig. 1B). When compared towild-type macrophages, levels of IL-12 were greater in Rag1�/�

cultures (36.9 pg/mL versus 91.5 pg/mL, respectively; p = 0.09)and significantly higher in Rag2�/�cc�/� samples (36.9 pg/mL

versus 136.8 pg/mL, respectively; p < 0.01) upon activation withLPS. Again, while IL-12 production was higher in Rag2�/�cc�/�

and Rag1�/� cultures when compared to wild-type samples, levelswere not significantly different between the two strains of knock-out mice. Furthermore, NO levels appeared lower in wild-typemice in comparison to both Rag1�/� (29.5 lM versus 41 lM,respectively; p = 0.09) and Rag2�/�cc�/� cultures (29.5 lM versus38.8 lM, respectively; p = 0.07), although no statistical signifi-cance was observed. Similar levels of NO activity were observedbetween both strains of knock-out mice. The knock-out micenot only displayed a marked increase of M1 functionality, theyalso exhibited a reduced M2 profile. Upon stimulation with LPS,there were significantly lower levels of IL-10 production inRag1�/� compared to wild-type macrophages (376.8 pg/mL versus874.3 pg/mL, respectively; p < 0.001) and Rag2�/�cc�/� comparedto wild-type macrophages (384.5 pg/mL versus 874.3 pg/mL,respectively; p < 0.01). Arginase activity in untreated cultureswas also lower in Rag1�/� (3.1 mM versus 6.2 mM, respectively;p = 0.06) and Rag2�/�cc�/� samples (2.8 mM versus 6.2 mM,respectively; p = 0.11) when compared to wild-type mice,although these levels were not statistically significant. Differencesin IL-10 production and arginase activity were similar in bothstrains of knock-out mice, supporting our earlier hypothesis that

Fig. 2. Lymphocyte-induced cytokine environment is essential for regulating macrophage polarization. (A) Bone marrow-derived macrophages (BMDMs) were derived fromC57BL/6 wild-type and Rag1�/�male mice bone marrow and stained with mAb against CD23, MMR, and F4/80. Gates were set on F4/80 positive cells prior to further analysis.Data is presented as the mean percentage of CD23 and MMR positive cells ± SEM. n.s., not significant (using unpaired t test). Histograms are representative flow cytometrydata of one mouse from each group. (B) BMDMs were plated and stimulated with LPS or left untreated. Supernatants were collected at 24 h (for IL-12 and IL-10 ELISAs) and48 h (for NO Assay). Data is presented as the mean ± SEM. (C) BM was stained ex vivo and compared to BMDM for C57BL/6 wild-type and (D) Rag1�/� mice, as previouslydescribed. Data is presented as the mean percentage of CD23 and MMR positive cells ± SEM, gated off a F4/80+ macrophage population. Histograms are representative flowcytometry data of one mouse from each group. *p < 0.05; ***p < 0.001 (using unpaired t test).

T. Chan et al. / Cellular Immunology 266 (2011) 180–186 183

macrophage phenotype and functionality may not be regulatedby NK or NK-T cells. Subsequently, all further experiments wereconducted using Rag1�/� mice.

3.2. Macrophage polarization is dependent on a lymphocyte-inducedcytokine environment

Our data suggests that differences in macrophage phenotypeand functionality between knock-out and wild-type mice are dueto their deficient lymphocyte populations. To further test thishypothesis, we sought to determine whether these differenceswere due to the absence of lymphocyte derived cytokines orwhether they could be a result of inherent macrophage defects.We controlled for environmental factors by culturing naïve bone-marrow cells with M-CSF to create purified bone-marrow derivedmacrophages (BMDM) from wild-type and Rag1�/� mice. Bothstrains of BMDMs expressed a similar percentage of F4/80+ cells(�80%) (data not shown). In accordance with our hypothesis,CD23 and MMR expression in wild-type and Rag1�/� BMDMs were

not significantly different (Fig. 2A). This confirms our earlier find-ings, where the loss of a lymphocyte-induced cytokine environ-ment in wild-type mice shifts macrophages from a M2-likephenotype towards M1, creating a similar profile to the one ob-served earlier in peritoneal macrophages from Rag1�/� mice(Fig. 1A).

Our phenotypic data was further supported by the functionalassays of these BMDM cultures (Fig. 2B). Upon stimulation withLPS, both wild-type and Rag1�/� cells produced similar levels ofIL-12 (700.2 pg/mL and 558 pg/mL, respectively) and IL-10(701.9 pg/mL and 813.6 pg/mL, respectively), with no significantdifference in NO production (15.3 lM and 13.5 lM, respectively).To ensure that our findings were not due to inherent differencesbetween the two strains of mice, we compared the expression ofCD23 and MMR in bone-marrow (BM) macrophages stainedex vivo and BMDM cultures from each strain of mice. As expected,in comparison to wild-type BM macrophages, wild-type BMDMsdisplayed a significant decrease in expression of CD23 (11.9% ver-sus 4.4%, respectively; p < 0.001) and MMR (10.8% versus 4.4%,

Fig. 3. CD4+ T-cells are essential for providing a cytokine environment to regulate macrophage polarization. (A) Peritoneal macrophages were isolated from C57BL/6 andCD4�/�mice and stained with mAb against CD23, MMR, and F4/80. Gates were set on F4/80 positive cells prior to further analysis. Data is presented as the mean percentage ofCD23 and MMR positive cells ± SEM, gated off a F4/80+ macrophage population. *p < 0.05; **p < 0.01 (using unpaired t test). Histograms are representative flow cytometrydata of one mouse from each group. (B) Peritoneal macrophages were plated and stimulated with LPS or left untreated. Supernatants were collected at 24 h (for IL-12 and IL-10 ELISAs) and 48 h (for NO Assay). Cell lysates were collected after 48 h for Arginase Assay. Data is presented as the mean ± SEM. *p < 0.05 (using unpaired t test).

184 T. Chan et al. / Cellular Immunology 266 (2011) 180–186

respectively; p < 0.001) (Fig. 2C). This difference was not found inRag1�/� samples, as BM macrophages and BMDMs expressed sim-ilar levels of CD23 (3.4% and 5.8%, respectively) and MMR (2.9% and3.1%, respectively) markers (Fig. 2D). These findings suggest thatwild-type macrophages developed in the absence of lymphocyte-derived cytokines acquire a polarized M1 phenotype, while cultur-ing does not have a significant effect on Rag1�/� macrophagessince they are already deficient in the crucial lymphocyte-inducedcytokine environment.

3.3. CD4+ T-cells mediate macrophage polarization

Our previous results suggest that T and/or B-cells are the essen-tial lymphocyte population in regulating macrophage polarization.It has been previously shown the CD4+ T-cells can promote invasionand metastasis of mammary adenocarcinoma by regulating thephenotypic and effector function of tumor-associated macrophages[20]. Thus, we sought to compare macrophages from CD4�/� towild-type mice on a C57BL/6 background. As expected, when com-pared to wild-type cells, CD4�/� macrophages displayed a signifi-cantly lower expression of CD23 (33.1% versus 17.1%,respectively; p < 0.01) and MMR markers (31.5% versus 15.7%,respectively; p < 0.05) (Fig. 3A). Our functional data supports these

findings, as demonstrated in the exaggerated M1 profile and down-regulation of M2 characteristics in CD4�/� macrophage cultures(Fig. 3B). Upon stimulation with LPS, CD4�/� macrophages pro-duced a significantly higher amount of IL-12 compared to wild-typemacrophages (78.2 pg/mL versus 46.9 pg/mL, respectively;p < 0.05), in addition to a slightly greater amount of NO production(45.6 lM versus 29.5 lM, respectively; p = 0.26). Furthermore,when compared to wild-type cells, CD4�/� macrophages producedsignificant less IL-10 (874.3 pg/mL versus 533.5 pg/mL, respec-tively; p < 0.05) when stimulated with LPS. CD4�/� cells also hadsignificantly lower levels of arginase activity when untreated incomparison to wild-type samples (2.3 mM versus 6.2 mM, respec-tively; p < 0.05). It is interesting to note that with LPS, cytokine pro-duction and NO levels of CD4�/�macrophages were similar to levelsfound earlier in Rag1�/� mice (Fig. 1B). This suggests that CD4+

T-cells, rather than CD8+ T-cells or B-cells, are crucial in providinga cytokine environment that regulates macrophage polarization.

4. Discussion

It is well-established that mononuclear phagocytes are highlyversatile in response to microenvironmental signals [10,16].Cytokines and microbial products influence changes in terms of

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macrophage receptor expression, effector function, and cytokineand chemokine production. Ultimately, their polarized activatedstates represent two extremes of a continuum: the M1 (classi-cally-activated) macrophage that is engaged in anti-microbialactivity and the M2 (alternatively-activated) macrophage that pro-motes tumor progression [7–8]. However, while there may be dis-tinct populations of activated macrophages, their classificationshould not be over-simplified. Even after activation, macrophagesmay evolve to exhibit characteristics that are shared by more thanone macrophage population and they can continuously adapt suchthat changes in their microenvironment could progressively altertheir functional capabilities [17,28–29]. Here, we characterizemacrophages along an M1/M2 spectrum, where wild-type macro-phages exhibit a significantly increased expression of CD23 andMMR markers, and significantly higher levels of IL-10 and arginaseactivity when compared to Rag1�/� and Rag2�/�cc�/� samples.This exaggerated M2 profile is also complemented by a reducedM1 readout, as seen in their lower levels of IL-12 and NO expres-sion compared to knock-out samples. Our findings are supportedby a previous study that suggested an M2 profile to be the defaultpathway for macrophage differentiation under normal conditions[9]. Such a significant difference in macrophage profile betweenwild-type and knock-out mice likely reflects the role of a specificlymphocyte population in regulating macrophage polarization.

Despite extensive profiling of macrophage populations, thespecific regulatory process by which macrophages polarize hasnot been fully established. Previous studies have confirmed thatthe plasticity of macrophages is dependent on environmentalcues [9–11,17,30–31] and our study reveals similar findings.Using purified bone-marrow derived macrophages, we found thatenvironmental cytokines were responsible for the differences thatwe observed between wild-type and Rag1�/� macrophages. In theabsence of a lymphocyte-induced cytokine environment, macro-phage profiling could no longer be distinguished between thetwo strains. There were no apparent differences between IL-12,NO, and IL-10 levels, and CD23 and MMR expression was compa-rable. Further comparison of BM and BMDM cultures of eachstrain of mice allowed us to confirm the role of the cytokine envi-ronment. As expected, bone-marrow cells from wild-type miceexpressed a significantly greater percentage of CD23 and MMRmarkers when compared to bone-marrow cells from Rag1�/�

mice, supporting our earlier findings where an M2 profiledevelops under normal conditions. However, upon culturing withM-CSF, whereby bone-marrow cells are grown in the absence oftheir in vivo cytokine environment, differences between BMDMCD23 and MMR expression in wild-type and Rag1�/� mice is sig-nificantly reduced. It is interesting to note that this similarity isdue to the significant decrease of M2 markers in wild-typeBMDM, while the expression of M2 markers in Rag1�/� do notchange with culture. These results are expected – we hypothesizethat bone-marrow cells from Rag1�/� mice are already deficientin the essential lymphocyte-induced cytokine environment andsubsequent culture in similar cytokine-deficient conditions wouldnot have a significant effect. We subsequently aimed to identifythe specific lymphocyte population that was responsible for pro-viding this cytokine environment.

While Rag1�/� and Rag2�/�cc�/� macrophages displayed a dis-tinct M1 profile in comparison to their wild-type controls, therewas no significant difference when comparing Rag1�/� andRag2�/�cc�/� macrophage phenotype or functionality. The c-chainmutation renders mice completely deficient in NK and NK-T cells[22–25], and we would have expected significant differences inmacrophage profiling between the two mice strains if the afore-mentioned cell type was essential. RAG transcripts are not detectedin mature NK cells [32] and another study by Shinkai et al. [33]found that the lack of RAG-2 function in mice did not effect the

development of other cell types. In fact, these mice had greater lev-els of NK cell activity, presumably to compensate for the void leftby the lack of mature T and B-lymphocyte populations. However,it has been shown that the tissue distribution of various NK cellsubsets are altered in RAG-1 deficient mice when compared towild-type mice, although this was not be attributed to the absenceof T and B-cells [34]. Thus, although our results suggest macro-phage polarization to be NK and/or NK-T cell independent, weacknowledge that their function could be underdeveloped due tothe deficient RAG-1 gene, thus masking their regulatory effect onmacrophages. Furthermore, recent studies have demonstrated animportant role for cross-talk between NK cells and macrophagesin certain settings. Nedvetzki et al. [31] found that macrophagesstimulated with high doses of LPS were directly killed throughNK cell cytotoxicity. Similarly, Salmonella-infected macrophageswere shown to prime NK cells by producing IL-12/IL-15, which inturn led to the destruction of macrophages via NK cell degranula-tion and IFN-c production [35]. Thus, although cross-talk betweenNK cells and macrophages are important for clearing infections,our data suggests that NK cells may not be as crucial in macro-phage regulation in the naïve state.

C57BL/6 and Balb/c have been well established to be TH1 andTH2-prone, respectively [36–37], and this difference has beenattributed to the varying levels of IL-2-induced IL-4 productionfrom naïve CD4 T-cells [38]. Specifically, greater levels of IL-4 pro-duction via STAT5 activation in Balb/c mice were found to bedirectly controlled by their genetic background, influencing T-celldifferentiation. In addition to their influence on TH cell differentia-tion, a more recent study by DeNardo et al. [20] demonstrated thatCD4+ T-cells expressing IL-4 could directly regulate the phenotypicand effector function of tumor associated macrophages in responseto mammary adenocarcinoma. The results from our study showsimilar findings. After characterizing the phenotypic and functionalprofile of macrophages in CD4�/� and wild-type control mice, wefound an exaggerated M1 profile in CD4�/� mice with respect tosignificantly greater levels of IL-12 and significantly less IL-10and arginase activity. NO levels were also higher in CD4�/� sam-ples, although not significant. Interestingly, all cytokine and en-zyme activity levels between Rag1�/� and CD4�/� macrophageswere not significant: IL-12 (91.5 pg/mL versus 78.2 pg/mL, respec-tively; p = 0.58), NO (41 lM versus 45.6 lM, respectively; p = 0.69),IL-10 (376.8 pg/mL versus 535.5 pg/mL, respectively; p = 0.10), andarginase activity (3.1 mM versus 2.3 mM, respectively; p = 0.33)(data not shown). Similarly, there was not a significant differencebetween Rag1�/� and CD4�/� MMR expression (8% versus 15.7%,respectively; p = 0.0715), although CD23 expression was signifi-cantly greater in CD4�/� cells (8.7% versus 17.1%, respectively;p < 0.001). This suggests that among the deficient lymphocyte pop-ulations, CD4+ T-cells appear to be the most crucial in regulatingmacrophages towards an M2 profile under normal conditions.

5. Conclusion

In summary, this report is one of the first to demonstrate thatCD4+ T-cells play an important role in macrophage regulation inwild-type mice. Since there were no significant differences betweenphenotype and functionality of Rag1�/� and Rag2�/�cc�/� samples,we hypothesized that NK and NK-T cells may not responsible for reg-ulating macrophages towards an M2 profile. Using BMDMs, we con-firmed that the cytokine environment, rather than differences ingenetic background or type of macrophage population, was impor-tant in mediating this process. Specifically, the cytokine environ-ment from CD4+ T-cells appeared to regulate polarization, as themacrophages from CD4�/� mice display an exaggerated M1 profilein comparison to wild-type macrophages. Future studies should

186 T. Chan et al. / Cellular Immunology 266 (2011) 180–186

confirm its role as the crucial lymphocyte population and themechanism by which it regulates macrophages. Stein et al. [12]reported that treating macrophages with IL-4 induced an M2functional phenotype and they hypothesized that the specificrelease of IL-4 from CD4+ T-cells played a crucial role in regulatingmacrophages. A deficiency of CD4+ T-cells, and subsequent lack ofIL-4 production, may explain the exaggerated M1 profile we foundin Rag1�/�, Rag2�/�cc�/�, and CD4�/� mice. Further experimentsshould be conducted to observe whether the addition of IL-4 inCD4�/�mice could compensate for the inadequate cytokine environ-ment, returning the exaggerated M1 profile to that seen in wild-typemice. Ultimately, our study shows that macrophages are polarizedtowards an M2 profile under normal conditions and this regulatoryprocess is lost in the absence of CD4+ T-cells.

Acknowledgments

This work was supported by grants from the Canadian Institutesof Health Research (CIHR) and the Canadian Breast Cancer Founda-tion (CBCF) Ontario branch to Ali A. Ashkar. A.A.A. is a recipient of aCareer award from Rx&D/CIHR.

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