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Activation of Murine Macrophages

UNIT 14.2

David M. Mosser1 and Ricardo Gonc¸alves2 1

Cell Biology and Molecular Genetics and the Maryland Pathogen Research Institute University of Maryland College Park, Maryland 2 Department of General Pathology, Institute of Biological Sciences Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil

Our understanding of cell-mediated immunity (CMI) has revealed the importance of activated macrophages as key immune effector cells. Over the past decade, we have come to realize that macrophages exhibit remarkable plasticity, and different populations of macrophages with distinct physiologies can develop in response to different stimuli. In fact, it is likely that the number of different macrophage populations that can arise may be as diverse as the activating stimuli that induce them. Some of these stimuli can instruct macrophages to kill microbes (classical activation), lay down extracellular matrix components to promote wound healing (alternative activation), or secrete anti-inflammatory cytokines to terminate inflammation (regulatory macrophages). New ways to biochemically identify these cells have led to a better understanding of the heterogeneity of activated macrophages. As our understanding of the various macrophage populations increases, so does the potential for therapeutic intervention based on targeting specific populations of activated macrophages.  C 2015 by John Wiley & Sons, Inc. Keywords: macrophages r toll-like receptors r lipopolysaccharide r immune complexes r interferon-γ r tumor necrosis factor r IL-4 r IL-10 r IL-12 r mannose receptor

How to cite this article: Mosser D.M. and Gonc¸alves R. 2015. Activation of murine macrophages. Curr. Protoc. Immunol. 111:14.2.1-14.2.10. doi: 10.1002/0471142735.im1402s111

INTRODUCTION One of the hallmarks of macrophages is their ability to respond to environmental stimuli and dramatically change their form and physiology. These responses have collectively been referred to as the “activation” response (Cohn, 1978). Because macrophages have often been studied in the context of host defense, these activation responses have been closely associated with host immune effector functions (Celada and Nathan, 1994). One of the earliest assays for classical macrophage activation was the ability of classically activated macrophages to kill intracellular microbes better than their tissue-resident quiescent counterparts, and this characteristic remains one of the best criteria for classical macrophage activation. We now know, however, that not all macrophage activation responses are the same, and that some forms of “activated” macrophages may actually be more susceptible to infection than their resident counterparts (Mosser, 2003). Because there are so many ways to stimulate macrophages and because these cells are so adept at responding to different environmental cues (Gordon, 2003), the nomenclature of macrophage activation has gotten somewhat confusing. For simplicity’s sake, we will describe three populations of macrophages, each with distinct physiologies, that develop in response to three different sets of stimuli. We think, in fact, that it is likely that these three populations may represent the “tip of the iceberg,” and that there may be as many different populations of macrophages as there are distinct stimuli (Mosser and Edwards, 2008). Innate Immunity Current Protocols in Immunology 14.2.1-14.2.10, November 2015 Published online November 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1402s111 C 2015 John Wiley & Sons, Inc. Copyright 

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Classically Activated Macrophages (Ca-M) Classically activated macrophages are the most thoroughly characterized and welldescribed activated macrophage population (Gordon, 2007; Mosser and Edwards, 2008). These cells are important immune effector cells that are vital to host defense and can also propagate inflammatory responses. Classically activated macrophages are formed in response to a combination of two signals. The first signal is often called a ‘priming’ step, and the prototypical primer of macrophages is IFN-γ. IFN-γ can be produced by innate immune cells such as NK cells, but sustained high levels of IFN-γ production usually come from the (antigen-specific) activation of TH 1 cells during cell-mediated immunity. IFN-γ activates the transcription factors, STAT1/2, which bind to gammaactivated sequences (GAS) in several immune effector genes. The second stimulus for classical activation is typically a “danger signal,” which can be a ligand for any one of the Toll-like receptors (TLRs) or many of the cytosolic pattern recognition receptors. These ligands are primarily expressed on microbial organisms, and have been termed Pathogen Associated Molecular Patterns (PAMPs; Bianchi, 2007). Danger signals derived from damaged host cells can also serve as the second stimulus, and these so-called DamageAssociated Molecular Patterns (DAMPs) can also activate NF-κB and drive cytokine transcription. Activation of these receptors induces the production of TNF, and this cytokine acts in an autocrine fashion to stimulate macrophages. Thus, the two signals that give rise to classically activated macrophages are IFN-γ and TNF. For in vitro activation, macrophages are typically ‘primed’ with IFN-γ overnight and the next morning stimulated with a TLR ligand, such as LPS. The stimulation step can also be the phagocytosis of bacteria that contain TLR ligands that stimulate macrophages during the process of phagocytosis. One of the most reliable ways to measure classical activation of murine macrophages is by their production of nitric oxide (NO). The assay to detect NO, based on the Griess reagent, is simple and convenient, taking only 30 min to complete. Only macrophages activated by the combination of stimuli described above produce NO. If priming with IFN-γ alone results in macrophage NO production, then it is likely that the cytokine is contaminated with LPS (a frequent problem), or that there is some other source of LPS contamination in the reagents used to isolate macrophages, or an infection in the mouse colony. In addition to secreting NO, classically activated macrophages up-regulate MHC and co-stimulatory molecules, and produce a myriad of inflammatory cytokines and mediators. Classically activated macrophages are prodigious secretory cells (Nathan, 1987).

Activation of Murine Macrophages

Alternatively Activated Macrophages (AA-M) The term alternatively activated macrophage was originally described by Stein and Gordon to describe macrophages that were exposed to the cytokine IL-4 (Stein et al., 1992). These cells exhibit characteristics distinct from classically activated macrophages, and instead up-regulate expression of lectin-like receptors, such as the mannose receptor, and chitinase-like molecules (Gordon, 2003). The observation that IL-4 induced the up-regulation of the mannose receptor prompted the name ‘alternatively activated macrophage.’ In some ways, this name is a misnomer because it implies that this form of activation is the only alternative to classical activation, and that is not the case. Unlike classically activated macrophages, these alternatively activated cells are not more adept at killing microbes, and in fact may actually be more susceptible to intracellular infections (H¨olscher et al., 2006). One of the defining characteristics of these cells is an alteration in their metabolism of arginine. Classically activated macrophages express iNOS, an enzyme that allows them to metabolize arginine into NO for microbial killing. In alternatively activated macrophages, IL-4 induces arginase activity, which converts arginine to ornithine, a precursor of polyamines and

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collagen (Murray and Wynn, 2011). This allows these cells to participate in wound healing. Alternatively activated macrophages are produced in vitro by adding IL-4, IL-13, or both cytokines together, overnight (Stein et al., 1992; Wynn, 2003). Some labs consider IL4/13 treatment to be sufficient to generate alternatively activated macrophages, but other labs parallel the protocol used for classically activated macrophages and consider IL-4 to be the priming step. These labs generally add LPS or other TLR ligand to stimulate macrophages the following morning. There are several good markers that have been developed to identify AA-Mφ, and therefore RT-PCR or flow cytometry is the preferred method for identifying AA-M. Importantly, although arg1 is rapidly induced by IL-4 treatment, the presence of this enzyme is not considered to be a reliable marker for AA-Mφ because arg1 can also be induced in many other macrophages (El Kasmi et al., 2008).

Regulatory Macrophages (R-Mφ) A third population of macrophages has potent anti-inflammatory characteristics that distinguish them from Ca-Mφ and AA-Mφ (Mosser and Edwards, 2008; Sica et al., 2008). These immunoregulatory macrophages can be generated in response to a variety of different stimuli, and consequently there is valid concern that these cells should not all be grouped together into one so-called regulatory macrophage population (Murray et al., 2014). However, all of these macrophages share the characteristics of producing high levels of IL-10 and growth and angiogenic factors, and decreased amounts of some key inflammatory cytokines; therefore, for simplicity, we are taking the liberty of grouping them together into a single category called R-Mφ. Although there are many different ways to induce these cells, in all cases there seems to be a “two-signal” requirement, with the first signal being any number of unique “reprogramming” signals (see below), and the second signal being stimulation via TLRs or other pattern recognition receptors. The two signals are generally applied simultaneously to most efficiently generate regulatory macrophages. The “reprogramming” signal, which can include high-density immune complexes, prostaglandin E2 , adenine nucleotides, apoptotic cells, or even IL10 itself, generally does not induce a secretory phenotype on its own. However, these reprogrammers have a dramatic effect when paired with TLR stimuli. The combination of these signals gives rise to a population of macrophages that are committed to IL10 production and consequently have potent anti-inflammatory activity. Because these cells also produce growth and angiogenic factors, there is speculation that they may be generated at the end of an immune response to repair the damage associated with the inflammatory M1 response (Cohen and Mosser, 2013). This idea would be consistent with our contention that the main role of macrophages is to maintain homeostasis in the host. To date, there are no protein biomarkers that are stably expressed on the surface of regulatory macrophages. Therefore, in the murine system, their identification relies on an analysis of multiple transcripts that are predictably altered in R-Mφ, or the use of an ELISA to measure differential cytokine secretion. The overproduction of IL-10 and the decreased production of the p40 subunit of IL-12/23 have been previously used to identify R-Mφ (Gerber and Mosser, 2001). The identification of human R-Mφ relies on the reciprocal secretion of IL-10 and IL-12/23, because transcripts associated with human R-Mφ have not yet been defined. NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly. NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Innate Immunity

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BASIC PROTOCOL

THE GENERATION OF CLASSICALLY ACTIVATED MACROPHAGES (Ca-M) The generally accepted procedure for classically activating macrophages consists of priming monolayers of macrophages for 6 to 24 hr with IFN-γ and then stimulating the cells with LPS or other TLR ligands. The two signals can be applied simultaneously with similar results if the second stimulus persists in culture. Macrophages isolated from the peritoneal cavity of mice, or macrophages derived from bone-marrow precursors, are the two most frequently used sources of cells for in vitro activation (see UNIT 14.1; Zhang et al., 2008). The two-step activation protocol will result in an increase in the production of many cytokines, an increase in the secretion of reactive nitrogen and oxygen intermediates, an up-regulation of co-stimulatory molecule expression, and the production of inflammatory mediators. These cells exhibit cytotoxicity against tumors and are better able to kill intracellular microbes. NOTE: All reagents and equipment in direct contact with live cells must be sterile and endotoxin-free. Macrophages are extremely sensitive to the biological effects of endotoxin, and therefore control values will not be at baseline if endotoxin is anywhere in the system. Furthermore, the phenomenon of “endotoxin tolerance” can confound analysis of results if macrophages are inadvertently exposed to endotoxin prior to stimulation. Consequently, sterile (single-use) plasticware is the preferred storage choice for anything coming into contact with macrophages.

Materials Murine bone marrow–derived macrophages or resident peritoneal cells (UNIT 14.1; Zhang et al., 2008) DMEM/F12-10 medium (see recipe) Murine recombinant interferon γ (IFN-γ; R&D Systems, cat. no. 485-MI) in DMEM/F12-10 Serum-free DMEM/F12 medium (Life Technologies, cat. no. 10565) Lipopolysaccharide (LPS; 10 to 100 ng/ml Ultrapure LPS, E. coli 0111:B4; Invivogen, cat no. tlrl-3pelps) in DMEM/F12-10 Flat-bottom 24- or 48-well tissue culture plates (Thermo Scientific Nunc, cat. no. 142485) 1a. For bone marrow–derived macrophages: Add a total of 0.2 × 106 (for 48-well plate) or 0.4 × 106 (for 24-well plate) bone marrow–derived macrophages per well in a total volume of 0.25 or 0.5 ml of DMEM/F12-10. Culture monolayers of macrophages overnight at 37°C in a humidified 37°C, 5% CO2 incubator. Add fresh DMEM/F12-10 to monolayers 2 hr before experimentation. Experimental controls should include cells that are not primed or stimulated, and these cells should not exhibit the activation phenotype.

1b. For peritoneal cells: Plate 2.5 × 105 (for 48-well plate) or 5 × 105 (for 24-well plate) total mononuclear cells (resident peritoneal cells which are the product of Basic Protocol 1 in UNIT 14.1) in a total volume of 250 or 500 μl DMEM/F1210, respectively. Wash monolayers at 2 hr with warm DMEM/F12 to remove B and T cells, and then culture overnight as described above for bone marrow–derived macrophages (step 1a). To wash monolayers, gently aspirate supernatant and quickly add 0.5 ml warm DMEM/F12. Repeat wash step. Approximately 30% to 50% of the total mononuclear cells that are added to wells will result in adherent macrophages. Activation of Murine Macrophages

3. Add IFN-γ to prime macrophages and incubate cells 6 to 18 hr before adding the stimulus.

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Each laboratory should establish optimal concentrations needed to prime and trigger macrophages. Initially, a dose-response for IFN-γ priming signal should be established using a range from 50 to 250 U/ml. In our hands, 100 to 200 U/ml is optimal for priming macrophages.

4. Following priming and before stimulation, wash monolayers in serum-free DMEM/F12 (see step 1b). 5. Add LPS to the appropriate wells to trigger stimulation. An optimal range of LPS should be determined. The range to consider is 1 ng/ml to 100 ng/ml. In our hands 10 ng/ml is optimal, but this concentration will vary depending on the source of LPS and its purity. Stimulated cells are incubated in a humidified 37°C, 5% CO2 incubator for various times depending on the assay.

6. Measure mRNA at (typically) 4 to 6 hr. Cytokine production is generally measured at 24 hr, and the killing of intracellular microbes is generally determined over a 48 to 72 hr period. One of the easiest ways to assay for classical macrophage activation is to measure NO production using the Griess reagent. This simple assay typically takes less than 30 min to complete. Activated macrophages can be investigated for their ability to display cytotoxic function against pathogens [e.g., parasites or bacteria; see UNITS 14.5 (Green et al., 1994) and 14.6 (Drevets et al., 2015)] or production of RNI (UNIT 14.5; Green et al., 1994), or tumor cells (UNIT 14.7; Cox, 1994).

THE GENERATION OF ALTERNATIVELY ACTIVATED MACROPHAGES (AA-M)

ALTERNATE PROTOCOL 1

All materials are the same as described for classically activated macrophages, except that IFN-γ is replaced by IL-4 or IL-13.

Additional Materials (also see Basic Protocol) IL-4: murine recombinant interleukin-4 (R&D Systems, cat. no. 404-mL) in DMEM/F12-10 (see recipe for DMEM/F1-10) IL-13: murine recombinant interleukin-13 (R&D Systems, cat. no. 413-mL in DMEM/F12-10 (see recipe for DMEM/F1-10) 1. Add peritoneal or bone marrow derived macrophages to 24- or 48 well plates in DMEM/F12-10 and cultivate overnight as described in the Basic Protocol. The following morning, add fresh DMEM/F12-10 to monolayers prior to priming. 2. Add IL-4 or IL-13 are added to macrophages overnight (12 to 16 hr). The optimal concentration of each is 10 to 20 U/ml. Some (most) laboratories consider the addition of IL-4 or IL-13 to macrophages to be sufficient to generate AA-MΦ. This treatment will induce the expression of Ym1 and Relmα, two well-established biomarkers of murine AA-Mφ. Others, however, combine this IL-4 step with a subsequent stimulation step using LPS or other TLR ligand, similar to what was described in the Basic Protocol for Ca-MΦ. Cells are stimulated with LPS (10 ng/ml) and incubated in a humidified 37°C, 5% CO2 incubator for various times, depending on the assay. Reverse transcriptase PCR (or real time-PCR) and western blotting are typically used to identify markers that are unique to AA-MΦ. The two most well-established markers for these cells are Relmα (Fizz1) and YM1. Neither of these antigens is surface-bound, so RT-PCR or western blotting are typically used to identify them. Other markers include AMAC-1 (CCL18), stabilin-1, and C-type lectins in the CLEC family. Since arginase is rapidly induced by IL-4, the early production of urea by macrophage lysates is also suggestive of alternative activation; however, caution should be exercised when using this as the only measure of alternative activation.

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THE GENERATION OF REGULATORY MACROPHAGES (R-M) During the later stages of immune responses, the production of high-affinity antibody and the resulting antibody-antigen immune complexes that are formed can act on macrophages and induce these cells to be potent anti-inflammatory cells that secrete high levels of IL-10, as well as growth and angiogenic factors. These cells also cease to produce the p40 subunit of IL-12 and IL-23. This reciprocal alteration in the production of these two cytokines has been used to define a number of different regulatory macrophage populations that are similar to the ones generated in response to immune complexes. These in vitro–generated R-Mφ may be similar to macrophages that have been reported to reside in the uterine deciduum and suppress immune responses to prevent fetal rejection (Gustafsson et al., 2008). We describe the induction of regulatory macrophages by immune complexes as an example of regulatory macrophage generation. Other stimuli can generally be substituted for immune complexes with similar (though not exact) results. The generation of these regulatory macrophage cells closely parallels that of CaMφ (described above), except that at the time of (TLR) stimulation, the reprogramming signal (IgG-containing immune complexes) is also added. We have used many particulate and soluble ICs to generate these cells, but in this protocol we will describe the use of particulate SRBS/anti-SRBC and soluble OVA/anti-OVA immune complexes. ALTERNATE PROTOCOL 2

Generation of Regulatory Macrophages Using IgG-Opsonized Sheep Red Blood Cells IgG-opsonized sheep red blood cells (IgG-SRBC) can be used as insoluble immune complexes.

Materials 10% washed sheep red blood cells (SRBC; Lampire Biological Laboratories, cat. no. 7249008; SRBC should be used within 2 weeks of receiving them) Anti-SRBC IgG (MP Biomedicals, cat. no. 0855806; we typically store anti-SRBC frozen at a concentration of 33 μg/ml) Dulbecco’s phosphate-buffered saline without Ca or Mg Mini-rotator (Glas-Col or equivalent) 24-well culture plate Additional reagents and equipment for ELISA (UNIT 2.1; Hornbeck, 2015) 1. Opsonize SRBC with sub-agglutinating concentrations of IgG. To determine the amount of antibody to add to SRBC, add increasing dilutions of antibody to parallel tubes containing 2 × 108 SRBC in 0.5 ml phosphate buffered saline saline without Ca or Mg in sterile polypropylene microcentrifuge tubes. A 1:200 dilution of antibody stock is usually sufficient to generate high-density IgG-SRBC needed to generate regulatory macrophages.

2. Gently rotate tubes containing IgG and SRBC on a Mini-Rotator or equivalent for 45 min at room temperature. After 45 min, it is likely that the highest concentrations of IgG will induce SRBC agglutination, and these tubes are discarded. The concentration just below this amount is used to generate R-Mφ. Small clusters of agglutinated IgG-SRBC will not adversely affect the results and need not be removed from the mixture.

3. Prepare the IgG-SRBC using the antibody dilution determined. Activation of Murine Macrophages

4. Add a total of 1.0 × 106 IgG-SRBC to a macrophage monolayer of 1 × 105 LPSstimulated macrophages in a well of a 24-well plate to induce R-M.

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5. Measure regulatory transcripts by RT-PCR 4 hr post-stimulation. Measure cytokines by ELISA (UNIT 2.1; Hornbeck, 2015) 12 to 24 hr post-stimulation.

Generation of Regulatory Macrophages Using Soluble OVA/Anti-OVA Ova-anti-OVA immune complexes can be used as a source of soluble immune complexes.

ALTERNATE PROTOCOL 3

Materials OVA: 1 mg/ml Chicken egg albumin (OVA, Worthington Biochemical Corporation, cat. no. LS003049) in DMEM/F12 medium Anti-OVA IgG: 4 mg/ml of rabbit polyclonal IgG to chicken egg albumin (Cappel anti-OVA IgG, MP Biomedicals, cat. no. 0855304) in DMEM/F12 medium Endotoxin removal buffer for protein solution: EndClean (BioVintage, cat. no.18602) DMEM/F12 medium (e.g., Life Technologies, cat. no. 10565) DMEM/F12-10 medium (see recipe) Lipopolysaccharide (LPS; 10 to 100 ng/ml Ultrapure LPS, E. coli 0111:B4; Invivogen, cat no. tlrl-3pelps) in DMEM/F12-10 Detoxi-gel endotoxin- removal column (Life Technologies, cat. no. 20339) Mini-rotator (Glas-Col or equivalent) Additional reagents and equipment for ELISA (UNIT 2.1; Hornbeck, 2015) 1. Generate endotoxin-free OVA by using a Detoxi-Gel Endotoxin Removing Column according to the manufacturer’s instructions. Use End°Clean according to the manufacturer’s instructions to remove endotoxin from the anti-OVA IgG preparation. To generate R-Mφ, immune complexes are usually added to macrophages simultaneously with a TLR ligand, such as LPS, in which case the removal of LPS from OVA and anti-OVA may not be necessary.

2. Dispense a 14-μl aliquot of OVA (14 μg) into a 1.5-ml microcentrifuge tube into which 250 μl DMEM/F12 medium has been added. Slowly add 50 μl anti-OVA IgG preparation from step 1 into the tube, bringing the final volume to 0.314 ml or 314 μl. Rotate the final mixture on a Mini-Rotator for 30 min at room temperature. Each laboratory should establish baselines for the optimal ratio of anti-OVA IgG to OVA necessary to achieve optimal IL-10 induction. Typically, a 5- to 10-fold molar excess of anti-OVA IgG to OVA is used to make high-density immune complex. The addition of a small volume of immune complexes to macrophages will induce the regulatory phenotype (see Gallo et al., 2010).

3. Seed macrophages in DMEM/F12-10 in 24- or 48-well plates, as described in the Basic Protocol. 4. Stimulate macrophages with 2 to 50 ng/ml LPS along with a 1:10 dilution of IgGcontaining immune complexes (OVA/anti-OVA from step 1). Incubate plates in a humidified 37°C, 5% CO2 incubator for 8 to 24 hr. Collect supernatants for the measurement of TNF, IL-12/23(p40), and IL-10, which are determined by ELISA (UNIT 2.1; Hornbeck, 2015). Cultures should always include parallel wells of cells that are exposed to LPS alone. This comparison will reveal the reciprocal alteration in IL-10 and IL-12/23 that is typical of regulatory macrophages. Other controls should include cells that receive no stimulation, cells that are exposed to (LPS-free) OVA alone, or cells exposed to anti-OVA IgG alone. Priming cells with IFN-γ is optional. One can still achieve the reciprocal alterations in cytokines following IFN-γ priming and stimulation in the presence of immune complexes, but now the IL-12 p70 subunit can be measured instead of the p40 subunit.

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TNF is a cytokine that may not be influenced by the presence/absence of immune complexes, and therefore it is a good control to ensure that the viability of both populations is comparable.

REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A.

DMEM/F12-10 Dulbecco’s Modified Eagle medium/F12 (Life Technologies, cat. no. 10565) 10% (v/v) fetal bovine serum (FBS; HyClone) 10 mM L-glutamine 100 IU/ml penicillin 100 μg/ml streptomycin Penicillin/streptomycin can be replaced by 50 μg/ml gentamicin sulfate. COMMENTARY Background Information

Activation of Murine Macrophages

Macrophages have remarkable plasticity, allowing these cells to respond to a variety of different stimuli and undergo distinct physiological changes in response to different environmental cues. The anti-microbial effects of activated macrophages were first detailed by Mackaness (1962). For many years, it was assumed that all activated macrophages shared similar capabilities, with enhanced ability to elaborate immune mediators and kill microorganisms. With acquired recombinant and purified cytokines as tools, the interactions between cells and cytokines have now been more extensively studied, and we now understand that there are several ways to influence the physiology of these cells. In addition to classically activated macrophages that are induced with IFN-γ, macrophages exposed to IL-4 or IL-13 undergo a different series of responses and undoubtedly serve different biological functions (Raes et al., 2002), including the maintenance of healthy adipose tissue (Wu et al., 2011), thermoregulation (Nguyen et al., 2011), and immunity to helminthes (Gordon and Martinez, 2010). The regulatory macrophages described above are distinct from AA-Mφ and Ca-Mφ. Additional studies to assign markers to each macrophage population will allow us to identify these cells in tissue and contribute to better understanding the physiology of each cell type during pathological processes (Edwards et al., 2006). The stimuli described above represent prototypes for macrophage activation. We expect that over the coming years different combinations of cytokines and stimuli may give rise to other populations of macrophages with different phenotypes and physiologies (Wynn

et al., 2013). These studies promise to reveal the enormous differentiative potential of macrophages, and to provide hints about how we can exploit the heterogeneity of these cells to diagnose diseases, and to manipulate immune responses to influence disease progression.

Critical Parameters Macrophages are extremely sensitive to environmental cues, and therefore all reagents must be of high quality and endotoxin-free. The use of pathogen-free mice is especially critical. Macrophages taken from the peritoneal cavity of unmanipulated mice should not produce NO, and they should not present antigen to na¨ıve T cells. They should retain the characteristics identified in Table 14.2.1. Glassware upon which macrophages are cultivated should be acid washed and thoroughly rinsed. Defined or characterized FBS that is low in endotoxin should be used, and high amounts of FBS in the tissue culture medium will influence macrophage function. It is important to utilize peritoneal macrophages immediately following isolation, and bone marrow-derived macrophages that have been maintained in culture for between 7 to 10 days, because these cells become progressively unresponsive over time. Activated macrophages adhere to substrates tenaciously and spread upon them. Consequently, they are very difficult to remove without causing cell damage.

Anticipated Results The classical activation of bone marrow– derived and peritoneal macrophages can be expected to result in the production of relatively high levels of tumor necrosis factor

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Table 14.2.1 Some Characteristics of Activated Macrophages

Resident Mφ

Ca-Mφ

AA-Mφ

R-Mφ

MHC-Class II

Low

High

Low

High

CD80/86

Low to absent

High

Low to absent

High

APC function

Low to absent

Moderate

Low or inhibitory

High

NO production

Absent

High

Absent

High

IL-12/IL-10

Absent/absent

High/low

Absent/low

Low/high

(TNF), IL-12/23p40, and nitric oxide. These cells should produce only modest levels of IL-10, and they should exhibit microbicidal and tumoricidal activity. Alternatively activated macrophages should produce low or undetectable levels of TNF and IL-12/23p40, with only a moderate increase in IL-10 production. These macrophages should display low or no nitric oxide synthase activity and no significant microbicidal activity. They are poor antigen presenting cells, but are frequently more phagocytic than TLR-stimulated macrophages. Regulatory macrophages will produce high levels of IL-10 and low amounts of IL-12/23. These cells also produce growth and angiogenic factors that can facilitate tissue regeneration. With some, but not all regulatory macrophages, TNF production will be similar to that of classically activated macrophages, and these cells present antigen quite efficiently. Comments on preferred activation protocols will be found in subsequent units describing various macrophage functions.

Acknowledgments This work was supported in part by NIH grant GM 102598, FAPEMIG, and CNPq.

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Activation of Murine Macrophages

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Current Protocols in Immunology