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J Leukoc Biol. Author manuscript; available in PMC Feb 13, 2009.
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PMCID: PMC2642590
NIHMSID: NIHMS84396

Biochemical and functional characterization of three activated macrophage populations

Abstract

We generated three populations of macrophages (M[var phi]) in vitro and characterized each. Classically activated M[var phi] (Ca-M[var phi]) were primed with IFN-γ and stimulated with LPS. Type II-activated M[var phi] (M[var phi]-II) were similarly primed but stimulated with LPS plus immune complexes. Alternatively activated M[var phi] (AA-M[var phi]) were primed overnight with IL-4. Here, we present a side-by-side comparison of the three cell types. We focus primarily on differences between M[var phi]-II and AA-M[var phi], as both have been classified as M2 M[var phi], distinct from Ca-M[var phi]. We show that M[var phi]-II more closely resemble Ca-M[var phi] than they are to AA-M[var phi]. M[var phi]-II and Ca-M[var phi], but not AA-M[var phi], produce high levels of NO and have low arginase activity. AA-M[var phi] express FIZZ1, whereas neither M[var phi]-II nor Ca-M[var phi] do. M[var phi]-II and Ca-M[var phi] express relatively high levels of CD86, whereas AA-M[var phi] are virtually devoid of this costimulatory molecule. Ca-M[var phi] and M[var phi]-II are efficient APC, whereas AA-M[var phi] fail to stimulate efficient T cell proliferation. The differences between Ca-M[var phi] and M[var phi]-II are more subtle. Ca-M[var phi] produce IL-12 and give rise to Th1 cells, whereas M[var phi]-II produce high levels of IL-10 and thus, give rise to Th2 cells secreting IL-4 and IL-10. M[var phi]-II express two markers that may be used to identify them in tissue. These are sphingosine kinase-1 and LIGHT (TNF superfamily 14). Thus, Ca-M[var phi], M-II, and AA-M[var phi] represent three populations of cells with different biological functions. J. Leukoc. Biol. 80: 1298–1307; 2006.

Keywords: IL-10, IL-12, sphingosine kinase, LIGHT

INTRODUCTION

Resident tissue macrophages (M[var phi]) can rapidly respond to external stimuli with dramatic alterations in gene expression [1]. This rapid response to stimuli represents the process of M[var phi] activation [2]. M[var phi] respond to a variety of stimuli, including microbial ligands for TLRs [3], endogenous “danger” signals [4], and cytokines. There is an increasing body of evidence to support the idea that the nature of the stimulus, or the combination of stimuli, can exert a profound effect on the type of M[var phi] activation response that occurs. The corollary to this is that different populations of activated M[var phi] can arise in response to different stimuli. The classically activated M[var phi] (Ca-M[var phi]) is generated in response to the cytokine IFN-γ in combination with TNF or stimuli that induce TNF. These cells are prototypical immune effector cells, which kill intracellular pathogens via the production of oxygen and nitrogen radicals [5]. These cells also secrete a battery of inflammatory cytokines, which help to orchestrate and amplify Th1 immune responses. The Ca-M[var phi] is an important component of host defense, but it can also be a potent mediator of inflammation.

A second population of activated M[var phi] was identified more than a decade ago [6] and termed alternatively activated M[var phi] (AA-M[var phi]). These cells arise in response to the Th2 cytokines IL-4 and/or IL-13. These cells are functionally and biochemically distinct from Ca-M[var phi]. They fail to produce NO, but they up-regulate mannose receptor expression [6]. They express several unique “markers”, which are not found on Ca-M[var phi] [7]. These cells are associated with parasitic diseases [8] and may contribute to the production of the extra-cellular matrix (ECM) [9].

A third population of activated M[var phi] was generated by activating M[var phi] in the presence of immune complexes (IC). These activation conditions resulted in an unexpected alteration in cytokine production by these cells. The cross-linking of FcγRs during activation resulted in an abrogation of IL-12 production and strong induction of IL-10 [10, 11]. The IL-10 secreted by these cells made them potent, anti-inflammatory cells [10]. When these M[var phi] were used to present antigen to naïve T cells, they stimulated the production of Th2-like T cells, which produced high levels of IL-4. Consequently, these M[var phi] were termed Type II-activated M[var phi] (M[var phi]-II) [12]. These cells may play a role in the exacerbation of visceral leishmaniasis, where the presence of IgG-coated parasites can induce the production of IL-10 from M[var phi], allowing for disease progression [13]. Several other M[var phi] have also been reported to preferentially produce IL-10 in response to stimulation. These include M[var phi] isolated from tumors [14] or M[var phi] exposed to glucocorticoids [15].

There have been suggestions that the ratio of IL-12 to IL-10 can be used as a simple way to classify activated M[var phi] into two categories, M1 (classical) or M2 (alternative or nonclassical) [16, 17]. This simplified classification is based on the well-established tenet that Ca-M[var phi] are an important source of IL-12 [18]. M[var phi] in the M2 category produce reduced amounts of IL-12 but higher levels of IL-10. The implication from this classification is that the cells in the latter category (M2) would share functional and physiological properties. We undertook the present studies to determine how similar two of the M2 M[var phi], AA-M[var phi] and M[var phi]-II, were. To our surprise, we found that these cells are quite distinct functionally and biochemically. In fact, M[var phi]-II more closely resembled Ca-M[var phi] than AA-M[var phi]. We conclude that each of the three populations of M[var phi] studied here has distinct characteristics by which it can be identified. Thus, we suggest that the simple designation of all non-Ca-M[var phi] as M2 may be a misleading oversimplification.

MATERIALS AND METHODS

Mice

Six- to 8-week-old BALB/c mice were purchased from Taconic Farms (Germantown, NY). Mice were used at 6 –10 weeks of age as a source of bone marrow-derived M[var phi] (BMM[var phi]). Breeding pairs of mice transgenic for OVA323–339/Ad-specific, DO11.10 [19] TCR-αβ were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in high-efficiency particulate air-filtered Thoren units (Thoren Caging Systems, Hazleton, PA) at the University of Maryland (College Park, MD). All procedures were reviewed and approved by the University of Maryland Institutional Animal Care and Use Committee.

M[var phi] activation

BMM[var phi] were prepared as described previously [20]. Briefly, BM was flushed from the femurs and tibias of mice, and cells were plated in petri dishes in DMEM/F12 supplemented with 10% FBS, penicillin/streptomycin-glutamine, and 20% conditioned medium from the supernatants of M-CSF-secreting L929 (LC14) fibroblasts. Cells were fed on Day 2, and complete medium was replaced on Day 6. Cells were used at 7–10 days for experiments.

Ca-M[var phi] and M[var phi]-II were prepared by priming BMM[var phi] overnight with 100 U/ml recombinant IFN-γ (R&D Systems, Minneapolis, MN). Ca-M[var phi] were washed and stimulated with l0 ng/ml Ultra-Pure LPS (Escherichia coli, K12, Invivogen, San Diego, CA). M[var phi]-II received LPS along with IC consisting of IgG-OVA. IC were made by mixing a tenfold molar excess of rabbit anti-OVA IgG (Cappel, Durham, NC) to OVA (Worthington, Lake-wood, NJ) for 30 min at room temperature, as described [11]. AA-M[var phi] were prepared by stimulating BMM[var phi] with 10 U/ml recombinant IL-4 (R&D Systems) as described previously [8].

RT-PCR and real-time PCR

PCR was performed after cDNA synthesis using Platinum PCR SuperMix (Invitrogen, Carlsbad, CA) and primer pairs specific for inducible NO synthase (iNOS), arginase-1 (Arg-1), sphingosine kinase-1 (SPHK1), FIZZ1, IL-10, IL12p40, and GAPDH. Real-time PCR was performed on GAPDH, TNF superfamily 14 LIGHT (homologous to lymphotoxins, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM)/TNF-related 2), and SPHK1. These primer pairs are presented in Table 1.

TABLE 1
Primers Used in PCR Analysis

Real-time PCR was conducted with the ABI Prism 7700 sequence detection system using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hurcules, CA) following the manufacturer’s instructions. For data analysis, the comparative threshold cycle (CT) value for GAPDH was used to normalize loading variations in the real-time PCRs. A ΔΔ CT value was then obtained by subtracting control Δ CT values from the corresponding experimental ΔCT. The ΔΔCT values were converted to fold difference compared with the control by raising two to the ΔΔ CT power.

Microarray analysis

RNA was prepared from 5 × 106 Ca-M[var phi] or M[var phi]-II 2 h after activation with LPS or LPS + IC, respectively. High-quality RNA was first purified using the TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. RNA was then DNase (Roche Diagnostics, Indianapolis, IN)-treated and purified using the RNeasy Mini kit (Qiagen, Valencia, CA), following the RNA cleanup protocol. RNA quality assessment and microarray analysis were performed at the University of Maryland Biotechnology Institute’s Microarray Core Facility. Microarray analysis was performed using the Affymetrix GeneChip Mouse Genome 430 2.0 (Santa Clara, CA), according to the manufacturer’s instructions. This array allowed for the assessment for changes in expression of ~39,000 transcripts. Robust changes in expression were determined according to the GeneChip expression analysis data analysis manual (Affymetrix), selecting for statistically significant changes in expression from Ca-M[var phi] (control) to M[var phi]-II (experimental). Briefly, robust changes from Ca-M[var phi] to M[var phi]-II had a signal call of “P” or present and had statistically significant increases or decreases based on the detection and change algorithm, respectively, as determined by the Affymetrix Microarray Suite software. Robust changes also had a signal log ratio greater than 1 for increases. Two sets of biological replicates were compared for consistent, robust changes. Details of these arrays as well as raw data have been deposited in the National Center for Biotechnology Information (NCBI; Bethesda, MD) Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series Accession Number GSE4811.

Immunoprecipitation and Western blot analyses

LIGHT/TNFSF14 was immunoprecipitated using 5 μg α-mouse LIGHT mAb (R&D Systems, Clone 261639) per ml cell culture supernatant. Samples were subject to SDS-PAGE on 15% resolving gels and transferred to polyvinylidene difluoride membrane. Membranes were blotted with α-mouse LIGHT mAb and HRP-conjugated goat α-rat IgG secondary antibody (Santa Cruz Biotechnology, CA). Membranes were developed using Lumi-LightPLUS Western blotting substrate (Roche Diagnostics) according to the manufacturer’s instructions.

NO production/arginase activity

NO production was estimated from the accumulation of NO2 in the medium after 24 h of M[var phi] activation using the Greiss reagent, as described previously [21]. Briefly, equal volumes of culture supernatant and Greiss reagent (100 μl) were mixed for 10 min at room temperature. Absorbance at 540λ was measured with a Labsystems Multiscan Ascent assay plate reader. A solution of NO2 was used to construct a standard curve.

Arginase activity was measured in cell lysates as described previously [22]. Briefly, 5 × 105 cells were washed and lysed with 100 μl 0.1% Triton X-100, 16 h after activation. Lysates were combined with 25 mM Tris-HC1 and 1 mM MnCl2, and enzyme was activated by heating for 10 mm at 55°C. The hydrolysis of arginine to ornithine and urea was conducted by incubating the lysates with 100 μl 0.5 M L-arginine (pH 9.7) at 37°C for 60 min. The reaction was stopped with 800 μl H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). The urea concentration was measured at 550 nm after addition of 40 μl α-isoni-trosopropiophenone (9% solution in ethanol), followed by heating at 100°C for 30 min.

T cell stimulation assays

CD3+ T cells were prepared from the spleens of DO11.10 mice by negative selection using the SpinSep mouse T cell enrichment kit (StemCell Technologies Inc., Vancouver, BC.) according to the manufacturer’s instructions. For primary stimulation assays, 2 × 105 M[var phi] were plated per well in 48-well plates and activated as described above. Two hours following stimulation of M[var phi], 5 ×105 T cells were added to each well in a total volume of 0.550 ml RPMI 1640 (Cellgro, Herndon, VA) supplemented with 10% FCS, HEPES, glutamine, Pen/Strep, and 50 μM 2- ME. For proliferation assays, cells were CFSE (Invitrogen/Molecular Probes, Eugene, OR)-stained. Briefly, T cells were washed with PBS and resuspended at l × 107 cells/ml in 5 μM CFSE in PBS. Cells were stained while rocking for 7 min, and then labeling was quenched by adding an equal volume of FBS. Cells were washed an additional two times in complete media. In secondary stimulation assays, 4 days following the primary stimulation, fresh RPMI was added with 10 U/ml IL-2 (R&D Systems) to maintain cell viability. Seven days following the primary stimulation, cells were removed from culture, washed, counted, and added to immobilized anti-CD3 (eBioscience, San Diego, CA), which was prepared by adding 5 μg/ml anti-CD3 to tissue-culture dishes in PBS overnight. Cytokines were measured 24 h later by ELISA or after 6 h by intracellular staining.

Cytokine measurement by ELISA

Cytokines were measured by ELISA using the following antibody pairs from BD PharMingen (San Diego, CA): IL-12p40, C15.6 and Cl7.8; IL-10, JES5-2A5 and JES5-16E3; IFN-γ, R4-6A2 and XMG1.2; IL-4, 11B11 and BVD6-24G2, according to the manufacturer’s instructions.

Flow cytometry and intracellular staining

M[var phi] were stained with FITC-conjugated α-I-Ad (AMS-32.1) or PE-conjugated α-CD86 (GL1; BD PharMingen). T cells were stained with FITC-conjugated antibodies against CD25 (PC61), CD69 (H1.2F3), or CD62 ligand (CD62L; MEL-14) and a PE-conjugated antibody against Thyl.2 (53-2.1; eBioscience). CFSE-stained T cells were also stained against Thy1.2 for gating. Intracellular staining was performed on secondarily stimulated T cells using the PharMingen Cytofix/Cytoperm kit with GolgiStop, according to the manufacturer’s instructions with some modifications. Briefly, cells were incubated in the presence of GolgiStop for 6 h after reactivation. Cells were harvested, washed, and resuspended in Cytofix/Cytoperm solution for 20′ at 4°C and then washed in Perm/Wash solution. Cells were stained with a PE-conjugated antibody against IL-4 (11B11) and a FITC-conjugated antibody against IL-10 (JES5-16E3, BD PharMingen). Cells were analyzed on a Becton Dickinson FACS flow cytometer (San Jose, CA). Quadrants were set to an appropriate isotype control antibody for analysis.

RESULTS

Activated M[var phi] differ in cytokine production

We generated three distinct populations of activated M[var phi] in vitro from murine BMM[var phi] and performed a series of parallel comparisons between them. As a first step, we measured cytokine production from each of these cells. Ca-M[var phi] have long been associated with the secretion of IL-12 [18], whereas M[var phi]-II were previously shown to produce high amounts of IL-10 [23]. Others have also suggested that the AA-M[var phi] were also a good source of IL-10 [24]. Cytokine production from these three populations of M[var phi] was compared. As expected, Ca-M[var phi] produced relatively high levels of IL-12 but low levels of IL-l0 (Fig. 1A). Coupling M[var phi] activation with FcγR ligation by the addition of IC resulted in a population of M[var phi] (M[var phi]-II), which produced high levels of IL- 10 and low levels of IL-12 (Fig. 1A), as previously reported [10]. Priming of these cells with IFN-γ was not necessary to see this reciprocal alteration in cytokine production caused by the addition of IC (Fig. 1B).

Fig. 1
Cytokine profiles of activated M[var phi]. (A) IL-10 (hatched bars, left axis) and IL-12 (solid bars, right axis) were measured by ELISA 16 h after M[var phi] activation. AA-M[var phi] were prepared by treating M[var phi] overnight with IL-4 (10 U/ml). ...

IL-4-treated M[var phi] (AA-M[var phi]) failed to secrete detectable levels of IL-12 or IL-10 (Fig. 1A). Cytokine production by AA-M[var phi] was not significantly different from unstimulated M[var phi]. As previous studies reported that M[var phi] isolated from infections in which IL-4 predominated were a rich source of IL-10 [25], we primed M[var phi] with IL-4 overnight and then stimulated them with LPS the next morning and examined cytokine production. The stimulation of IL-4-primed cells with LPS resulted in a modest increase in the production of IL-10 (Fig. 1C). However, the level of this cytokine remained substantially below that of M[var phi]-II. IL-4-primed cells were also stimulated with LPS + IC. These IL-4-primed cells responded to the addition of IC by secreting high levels of IL-10 (Fig. 1C). Thus, all three populations of activated M[var phi] display distinct profiles of cytokine production, and yet, these cells exhibit functional plasticity. Thus, the nature, order, and/or duration of stimulation can influence cytokine production, as reported previously [26].

Activated M[var phi] exhibit different patterns of arginine metabolism

Arginine metabolism has been described previously as one of the defining characteristics of murine AA-M[var phi] [8]. Therefore, we examined iNOS and arginase production in each of the three M[var phi] populations. First, we measured the accumulation of NO2 in culture supernatants by the Greiss assay [21]. Ca-M[var phi] and M[var phi]-II produced relatively high levels of NO, whereas the AA-M[var phi] produced virtually no NO (Fig. 2A). We also measured the amount of urea generated by these three populations of M[var phi]. Ca-M[var phi] and M[var phi]-II possessed virtually no arginase activity and therefore, were unable to produce urea when lysates were incubated with L-arginine. The opposite was true of the AA-M[var phi], which produced high levels of urea (Fig. 2B). Thus, with regard to arginine metabolism, AA-M[var phi] were distinct from the other two populations of M[var phi]. AA-M[var phi] failed to produce NO but readily catabolized arginine to urea, whereas the converse was true for Ca-M[var phi] and M[var phi]-II (Fig. 2).

Fig. 2
Activated M[var phi] exhibit different patterns of arginine metabolism. (A) NO2 accumulation after 24 h of M[var phi] activation measuring iNOS activity. Equal volumes of cell supernatants were mixed with Greiss reagent for 10 min, and the absorbance ...

Biochemical markers to discriminate between populations of M[var phi]

RNA was isolated from the three populations of activated M[var phi] 4 h after stimulation and analyzed by RT-PCR (Fig. 3). We first examined iNOS and Arg-1 mRNA in each of the three populations. As previously reported and consistent with the protein activity data above, M[var phi] exposed to the Th2-associated cytokine IL-4 produced mRNA for arginase but failed to produce iNOS mRNA [27]. Conversely, Ca-M[var phi] and M[var phi]-II expressed iNOS mRNA but failed to express arginase (Fig. 3). Ca-M[var phi] produced high levels of IL-12 but little detectable IL-10, whereas M[var phi]-II expressed high levels of IL-10 and less IL-12. Only the AA-M[var phi] expressed the marker FIZZ1 (found in inflammatory zone 1) described previously, a secreted protein that has been associated with allergic and pulmonary inflammation [7, 28], confirming that FIZZ1 represents a reliable marker for murine AA-M[var phi] (Fig. 3).

Fig. 3
Activated M[var phi] have different mRNA expression profiles. RT-PCR was performed to examine the expression of iNOS, Arg-1, SPHK1 (SK-1), FIZZ1, IL-10, and IL-12 (p40) mRNA levels in four different M[var phi] populations. cDNA from unstimulated M[var phi] ...

As there have been no reported markers for M[var phi]-II, we performed an initial microarray analysis using the Affimetrix GeneChip Mouse Genome 430 2.0. The analysis included a comparison between Ca-M[var phi] and M[var phi]-II, as these two populations of cells exhibited several biochemical similarities (Fig. 3). Microarray analysis revealed a limited number of changes in gene expression between these two cell types. When a twofold difference in transcript levels was used as the cut-off, only 184 of the 39,000 possible transcripts represented on this chip (<0.5%) were increased consistently in M[var phi]-II relative to Ca-M[var phi]. Table 2 lists 29 genes of known biological function, which were increased by threefold or more in M[var phi]-II and had a signal greater than 2000. These 29 genes are the only ones that fit these criteria. The data discussed in this publication have been deposited in the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series Accession Number GSE4811.

TABLE 2
Increases in Gene Expression in M[var phi]-II Relative to Ca-M[var phi]a

From this analysis, several genes that were up-regulated in M[var phi]-II were examined. SPHK1 was shown to be up-regulated by more than threefold in M[var phi]-II, relative to Ca-M[var phi]at 2 h poststimulation. We therefore compared SPHK1 mRNA levels in the three M[var phi] populations. Only M[var phi]-II expressed detectable levels of SPHK1 mRNA by conventional PCR 4 h after stimulation (Fig. 3). This observation was extended by quantitative real-time PCR (QRT-PCR; Fig. 4A). This analysis revealed that SPHK1 mRNA was induced robustly in M[var phi]-II, and mRNA levels increased substantially over time, relative to unstimulated cells (Fig. 4A). There was some induction of SPHK1 mRNA in Ca-M[var phi]; however, this induction was modest relative to M[var phi]-II. Thus, high SPHK1 expression may be useful to identify M[var phi]-II in tissue.

Fig. 4
M[var phi]-II up-regulate SPHK1 and TNFSF14/LIGHT. (A) Relative SPHK1 mRNA as measured by real-time PCR after M[var phi] activation by LPS (●), LPS + IgG-OVA IC (▲), or IL-4 (○). (B) Relative LIGHT mRNA as measured by real-time ...

M[var phi]-II express LIGHT, a member of the TNFSF

From the microarray analysis, another marker for M[var phi]-II emerged. This was murine LIGHT or TNFSF14. It was found to be present at more than 8.5-fold higher levels in the M[var phi]-II relative to Ca-M[var phi]. To confirm LIGHT mRNA induction in M[var phi]-II, QRT-PCR was performed to compare mRNA levels in the three M[var phi] populations. In the M[var phi]-II, LIGHT mRNA increased by ~200-fold over unstimulated cells, peaking at 2 h poststimulation (Fig. 4B). Ca-M[var phi] had a slight induction of LIGHT mRNA, but these levels were always at least tenfold lower than these observed in M[var phi]-II (Fig. 4B). There was no evidence for LIGHT induction in AA-M[var phi], and in fact, the addition of IL-4 marginally decreased LIGHT mRNA levels in AA-M[var phi].

It has been reported previously that LIGHT may be secreted or cleaved from the surface of cells in a soluble form [29]. To address this possibility, M[var phi] were primed with IFN-γ, and then stimulated for 6 h with LPS or LPS + IC. IL-4-treated M[var phi] were not addressed, as the expression of LIGHT was not induced in this population (Fig. 4B). LIGHT was then immunoprecipitated from the supernatants of stimulated cells with a mAb specific for the extracellular domain of LIGHT. A 20- to 25-kD band, which corresponds to the molecular mass of sLIGHT, was detected only in the supernatants of M[var phi]-II (Fig. 4C). This suggests that M[var phi]-II are the primary M[var phi] producers of LIGHT and that this molecule is rapidly cleaved from the surface of these cells or secreted as sLIGHT by M[var phi]-II.

Antigen presentation by activated M[var phi]

Activated M[var phi] are able to present antigen to T cells [11]. We wished to examine the antigen-presenting potential of each of the three M[var phi] populations. One of the defining characteristics of an efficient APC is the expression of MHC Class II and B7 costimulatory molecules. BMM[var phi] were primed with IFN-γ overnight or left unprimed. Primed cells were then stimulated for 24 h with LPS (Ca-M[var phi]) or LPS + IgG-OVA IC (M[var phi]-II). AA-M[var phi] were stimulated with IL-4. These cells were then stained with FITC-conjugated α-I-Ad or PE-conjugated α-CD86. Expression in activated cells was compared with that of unstimulated M[var phi]. M[var phi]-II had the highest expression of MHC Class II (Fig. 5A) and CD86 (Fig. 5B). Ca-M[var phi] also up-regulated MHC Class II and CD86, although not to the level of M[var phi]-II. In contrast, AA-M[var phi] only minimally up-regulated MHC Class II and CD86 expression. Thus, the three M[var phi] populations express distinct levels of these molecules and therefore, may have different potentials to present antigen to T cells.

Fig. 5
Activated M[var phi] express different levels of MHC Class II and B7 costimulatory molecules. Flow cytometry profiles for (A) MHC Class II and (B) B7.2 (CD86) on activated M[var phi] 24 h after stimulation. M[var phi] were primed with IFN-γ and ...

Each of the M[var phi] populations was analyzed for its ability to present OVA antigen to naive OVA-specific DO11.10 T cells [19]. For these studies, BMM[var phi] were primed overnight with IFN-γ (100 U/ml) or IL-4 (10 U/ml, AA-M[var phi]). Cells primed with IL-4 overnight were confirmed to have high arginase and FIZZ1 expression (data not shown). Cells were washed and stimulated with LPS + OVA (Ca-M[var phi]), LPS + IgG-OVA (M[var phi]-II), or OVA alone (AA-M[var phi] and unstimulated M[var phi]). CD3+ T cells were isolated from total splenocytes from DO11.10 ice and added to M[var phi] 2 h after activation. T cells and M[var phi] were cocultured for 24 h and then stained for CD25, CD69, and CD62L expression. CD25 and CD69 typically increase with T cell activation, whereas CD62L decreases [3032]. The expression of each marker was assessed by flow cytometry, gating on Thyl.2+ cells. As expected, unstimulated M[var phi] drove minimal up-regulation of CD25 (Fig. 6, left panels) and CD69 (Fig. 6, center panels) on T cells. They also induce only minimal down-regulation of CD62L (Fig. 6, right panels). T cells cocultured with M[var phi]-II showed the greatest signs of activation, expressing the highest levels of CD25 and CD69 expression and the lowest expression of CD62L (Fig. 6). Thus, despite the potent production of IL-10 from M[var phi]-II, these cells are effective activators of naïve T cells. The cocultivation of T cells with Ca-M[var phi] was also able to induce T cell activation markers, albeit slightly less than M[var phi]-II. There was a slight decrease in the up-regulation of CD25 and CD69 and less of a down-regulation of CD62L (Fig. 6). AA-M[var phi] induced only minimal signs of T cell activation, which were similar to unstimulated M[var phi]. Thus, M[var phi]-II appear to be efficient APC, and this is in stark contrast to AA-M[var phi], which were poor at inducing T cell activation.

Fig. 6
Activated M[var phi] drive different levels of T cell activation. DO11.10 T cells were stained after 24 h of coculture with unstimulated (top panels), AA-M[var phi] (upper-middle panels), Ca-M[var phi] (lower-middle panels), and M[var phi]-II (bottom ...

Activated M[var phi] drive different levels of antigen-specific T cell proliferation

To directly compare T cell activation by these three populations of M[var phi], CD3+ T cells were CFSE-stained and added to each population of M[var phi] 2 h after stimulation. M[var phi] and T cells were cocultured for 96 h and stained with PE-conjugated α-Thy 1.2. Proliferation of Thyl.2+ cells was assessed by flow cytometry. As expected, M[var phi] given antigen alone were able to drive only minimal levels of T cell proliferation, as shown by minimal dilution of the CFSE stain (Fig. 7A). AA-M[var phi] were equally poor at driving T cell proliferation (Fig. 7A). Ca-M[var phi] and M[var phi]-II induced a robust, primary T cell response characterized by several rounds of T cell proliferation (Fig. 7A). Thus, M[var phi]-II are particularly effective as APC, inducing the rapid expression of early activation markers (Fig. 6), and they support T cell proliferation (Fig. 7). AA-M[var phi], in contrast, are relatively poor at inducing T cell activation markers, and they fail to support the proliferation of naive T cells. Even after stimulation with LPS, IL-4-primed M[var phi] failed to support substantial amounts of T cell proliferation (Fig. 7B).

Fig. 7
Activated M[var phi] drive different levels of T cell proliferation. (A) Naïve T cells were isolated from total splenocytes from DO11.10, CFSE-stained, and co-cultured in the presence of unstimulated (top panel), AA-M[var phi] (upper-middle panel), ...

We also examined secondary T cell responses to antigen and APC. For these assays, T cells were stimulated with each of the M[var phi] populations and antigen for 7 days. T cells were washed and restimulated under nonbiasing conditions with plate-bound anti-CD3 for 24 h. Cytokine levels were measured by ELISA. Although cytokine production from T cells activated by M[var phi]-II has been examined previously [11], a comparison among the three cell types has not been reported. T cells activated by Ca-M[var phi] (LPS+OVA) produced relatively high levels of IFN-γ and less IL-4, whereas T cells activated by M[var phi]-II produced relatively high levels of IL-4 and significantly reduced levels of IFN-γ (Fig. 8A, inset), as previously reported [11]. We measured IL-10 production from these T cells. M[var phi]-II give rise to T cells which produced high levels of IL-10 in the secondary response, whereas Ca-M[var phi] induced a population of T cells that produced only modest levels of IL-10 (Fig. 8A). This is not a result of an overall lack of antigen presentation, as Ca-M[var phi] induced relatively high levels of IFN-γ production from T cells (Fig. 8A, inset). AA-M[var phi] failed to induce IL-10 production in the secondary response, possibly as a result of the lack of significant levels of proliferation in the primary response. Flow cytometry and intracellular cytokine staining were used to measure cytokine production on the single-cell level (Fig. 8B). Following T cell activation by Ca-M[var phi], there was only a small percentage of T cells in the population that produced IL-4 (5.8%) or IL-10 (1%; Fig. 8B, left panel). There were few if any double producers. In contrast, following activation with M[var phi]-II, a substantial portion of the T cell population produced IL-4 (31.6%) or IL-10 (14.7%), and 10% of total T cells produced IL-4 and IL-10 (Fig. 8B, right panel).

Fig. 8
Cytokine profiles of T cells after secondary stimulation. T cells were cocultured with AA-M[var phi] Ca-M[var phi], or M[var phi]-II for 1 week, washed, then restimulated with plate-bound α-CD3 (5 μg/ml). (A) IL-10 from T cells restimulated ...

DISCUSSION

The classical activation of M[var phi] leads to the production of a variety of lipid mediators, cytokines, and chemokines [16]. These mediators make Ca-M[var phi] important effector cells, which can efficiently kill intracellular microorganisms. These same mediators, however, make these cells potent inflammatory cells, which can mediate autoimmune pathologies. The thorough characterization of Ca-M[var phi] has been aided greatly by the reliable generation of these cells in vitro, simply by priming cultivated M[var phi] with IFN-γ and then stimulating them with TNF or TLR activators. These defined, in vitro studies have told us a great deal about the activation response and the biochemistry of the mediators produced during it. AA-M[var phi] were first identified following the addition of IL-4 to cultures of resident M[var phi]. These cells were shown to express higher levels of the mannose receptor [6]. Subsequent studies have identified a number of reliable markers for the AA-M[var phi], including FIZZ1 and YM1/2 [7]. Many of the studies to characterize the AA-M[var phi] were performed on M[var phi] isolated from mice, following experimental trematode [8] or nematode [33] infections. These studies showed that AA-M[var phi] are physiologically distinct from Ca-M[var phi], in that they up-regulate the ECM-associated proteins fibronectin and βIG-H3 [34], the chemokine alternative M[var phi]-associated chemokine 1 [35], and the receptor for β-glucan, Dectin-1 [36]. The expression of arginase by murine M[var phi] restricts the availablility of L-arginine, a substrate for iNOS. This not only prevents NO production by these cells but also gives them the ability to contribute to the formation of the ECM through the production of prolines [9]. It should be noted that human monocytes may not respond to Th2 cytokines in the same way as murine BMM[var phi] cells, as they fail to up-regulate arginase activity when treated with IL-13 [37]. AA-M[var phi] may provide immunity during helminth infections [13, 38], but they can also contribute to disease pathology in a murine model of experimental schistosomiasis [8]. Despite all we now know about these cells from disease models, a careful parallel comparison with other M[var phi] following defined, in vitro activation conditions has not been performed previously.

The M[var phi]-II remains poorly characterized, relative to these other M[var phi] populations. We previously reported on the alterations in cytokine production by these cells [10, 11] and on functional properties that are distinct from Ca-M[var phi] [12]. These cells develop during some Leishmania infections and contribute to disease progression [13]. In the present work, we show that M[var phi]-II lack arginase and are unable to produce urea from arginine. In this respect, they are similar to Ca-M[var phi] but markedly different from AA-M[var phi]. M[var phi]-II express high levels of costimulatory molecules and are efficient APC, another property that distinguishes them from AA-M[var phi]. Finally, neither M[var phi]-II nor Ca-M[var phi] express FIZZ1 or YM1, two markers for AA-M[var phi]. Thus, there are clear biochemical and functional distinctions between these two populations of M[var phi]. We also compared M[var phi]-II with Ca-M[var phi] by microarray analysis and found that there were only a limited number of transcripts that were different between these two populations.

We identified two potential markers for M[var phi]-II. We show that M[var phi]-II up-regulate LIGHT, which may not only be a marker for M[var phi]-II, but it may also bear functional activity. LIGHT has been shown to costimulate T cell responses through HVEM (TR2) [39], which is expressed on most lymphocyte populations [40]. LIGHT can also transmit co-stimulatory signals into T cells through interactions with the soluble receptor TR6, enhancing activation in response to suboptimal TCR interactions [41, 42]. Thus, LIGHT may play a role as an additional costimulatory molecule, contributing to T cell activation and proliferation in response to M[var phi]-II. In addition to LIGHT, SPHK1 may be another previously undescribed marker for the M[var phi]-II. SPHKs catalyze the production of sphingosine-1 phosphate from sphingosine. Mice have two isoforms, SPHK1a and SPHK1b, which are not distinguished by our PCR analysis. These two proteins can differ with respect to enzymatic activity, stability, and cellular location [43]. SPHK1 has been shown to be necessary for C5a-triggered, intracellular Ca2+ signals [44]. Although this does not necessarily fit our model of M[var phi]-II being antiinflammatory M[var phi], this enzyme may be an important mediator of calcium fluxes in these M[var phi]. It has also been proposed that SPHK1 may play a role in retaining cell viability of endotoxin-stimulated M[var phi] [45]. In T cells, SPHK1 controls the overproduction of the Th1-associated cytokines, IL-2, TNF-α, and IFN-γ [43, 46]. Thus, the induction of SPHK may be an important negative-regulator of inflammatory cytokines and chemokines in the M[var phi]-II.

In summary, we provide a side-by-side comparison of three distinct populations of activated M[var phi]. This comparison has allowed us to characterize each with regard to physiology and functionality. It also puts us in a strong position to identify a panel of markers for each cell population, as a first step toward identifying specific “signatures” for each of the various activated M[var phi] populations associated with different disease states.

Acknowledgments

This research was supported in part by the National Institutes of Health Grant AI49383.

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