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Contents lists available at ScienceDirect

www.ashi-hla.org

journal homepage: www.elsevier .com/locate /humimm

Attenuated expression of interferon-b and interferon-k1 by humanalternatively activated macrophages

0198-8859/$36.00 - see front matter � 2013 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.http://dx.doi.org/10.1016/j.humimm.2013.08.267

Abbreviations: AAM, alternatively activated macrophages; BAL, bronchoalveolarlavage; CAM, classically activated macrophages; IFN, interferon; ISGs, interferonstimulated genes; LPS, lipopolysaccharide; PMA, phorbol-12-myristate-13-acetate;MD, primary human monocyte derived macrophages; RV, rhinovirus.⇑ Corresponding author. Address: Laboratory of Immunobiochemistry, Division of

Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review,Center for Biologics Evaluation and Research, Building 29, Room 203A, 29 LincolnDrive MSC 4555, Bethesda, MD 20892-4555, USA. Fax: +1 301 480 4103.

E-mail address: ronald.rabin@fda.hhs.gov (R.L. Rabin).

Please cite this article in press as: Fiky AE et al. Attenuated expression of interferon-b and interferon-k1 by human alternatively activated macroHum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.08.267

Ashraf El Fiky, Roger Perreault, Gwendolyn J. McGinnis, Ronald L. Rabin ⇑Laboratory of Immunobiochemistry, Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research,US Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892-4555, USA

a r t i c l e i n f o

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Article history:Received 14 January 2013Accepted 10 August 2013Available online xxxx

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a b s t r a c t

Macrophages can be polarized into classically (CAM) or alternatively (AAM) activated macrophages withIFN-c or IL-4, respectively. CAM are associated with type 1 immune responses and are implicated in auto-immunity; AAM are associated with type 2 responses and are implicated in allergic diseases. An imped-iment in investigating macrophage biology using primary human monocyte derived macrophages is thewide inter-donor heterogeneity and the limited quantity of cells that survive in vitro polarization. Toovercome this impediment, we established a protocol to generate CAM and AAM cultures derived fromthe THP-1 human promonocytic cell line. In this report, we demonstrate that THP-CAM and -AAM expressgene and protein markers that define their primary human monocyte derived counterparts, such as IL-1b,CXCL10, and CXCL11 for CAM, and MRC1, IL-4 and CCL22 for AAM. In addition, we demonstrate thatSTAT6 is selectively activated in THP-AAM and, upon LPS stimulation, have an attenuated or delayedexpression of IFN-b, IFN-k1, and IFN a/b pathway genes compared to their CAM counterparts. Takentogether, these findings may help further investigate human diseases associated with the alternativelyactivated macrophage phenotype using this reproducible in vitro macrophage model.

� 2013 Published by Elsevier Inc. on behalf of American Society for Histocompatibility andImmunogenetics.

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1. Introduction

Macrophages are antigen-presenting phagocytes that secretepro-inflammatory mediators and antimicrobial factors inresponse to challenge by intracellular or extracellular pathogens.Over the past decade, it has become clear that macrophages haveremarkable plasticity with which, in response to combinations ofcytokines, pathogens, or both, they are phenotypically shaped [1].Two phenotypes were first described about twenty years ago aseither ‘‘classically’’ (CAM) or ‘‘alternatively’’ activated macrophages(AAM) [2]. CAM are macrophages that are polarized by IFN-c anddisplay a Th1-like phenotype that enhances inflammation, extracellular matrix destruction, apoptosis, and tends to participate inchronic inflammation and tissue injury. AAM, by contrast, arepolarized by IL-4 and/or IL-13, and display a Th2-like phenotypethat limits inflammation, promotes extra cellular matrix formation,

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cell proliferation, angiogenesis, and participates in the resolution ofinflammation and in wound healing [3]. Furthermore, AAMparticipate in the pathogenesis of chronic allergic inflammationand atopic asthma because their polarization and function isdependent on STAT6 [4], and expression of IL-4, IL-10, or IL-13 [3].

In the mouse model, greater numbers of AAM in the lungs offemale, compared to male, mice correlated with greater severityof asthmatic inflammation [5]. In humans, asthmatics have moreIL-13 + macrophages in bronchoalveolar lavage fluid than healthycontrols, and MRC1, an AAM signature gene encoding for themannose receptor, is expressed at higher levels by monocytes fromchildren with viral induced asthma exacerbations [6].

Studies of primary human monocyte derived macrophage func-tion are often complicated by limitations in the number of primarymonocytes available from each donor, in addition to donor-to-donor variability among a heterogeneous population, both ofwhich severely impact experimental consistency and reproducibil-ity. To add to these impediments, human and mouse AAM genesmay differ. For example, ARG1 is expressed only by mouse AAM[1,7], and three mouse AAM signature genes, FIZZ1, YM, and YM2have no human homologues [8].

To address these impediments towards the investigation ofpolarized macrophages, we have developed a model using thehuman promonocytic cell line THP-1 and validated it with primaryhuman monocyte derived macrophages that were polarized into

phages.

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CAM and AAM (MD-CAM and MD-AAM) [9]. In this report, wedemonstrate that STAT6 is activated only in THP-AAM followedby a signature expression pattern of pro- and anti-inflammatorymediators by CAM and AAM, respectively, upon polarization andstimulation with LPS. In addition, we demonstrate qualitative,quantitative, and kinetic differences in the expression of types Iand III IFN and IFN a/b pathway genes between these two macro-phage phenotypes.

2. Materials and methods

2.1. Cell culture

The THP-1 monocyte cell line was obtained from the AmericanType Culture Collection (Manassas, VA) and was cultured at aconcentration of 1 � 106 cells/mL, in RPMI 1640 supplemented with2 mM L-glutamine (Invitrogen, Carlsbad, CA) and 10% FBS (QualityBiological, Gaithersburg, MD). For differentiation, THP-1 cells weretreated with PMA (20 ng/lL, Sigma, St. Louis, MO) for 48 h, afterwhich the media was aspirated, and the cells were washed twicein pre-warmed HBSS (Invitrogen). For polarization towards CAM orAAM phenotypes, the cells were cultured in media containingIFN-c (500 IU/mL, Sigma) or IL-4 (100 U/mL, Peprotech, Rocky Hill,NJ) for 96 h, respectively. Escherichia coli O111:B4 lipopolysaccharide(Calbiochem, Bellerica, MA), and used at a concentration of 100 ng/mL.

Human elutriated monocytes were obtained from healthydonors by centrifugal countercurrent elutriation at the Departmentof Transfusion Medicine at the Clinical Center of the NationalInstitutes of Health (NIH). The monocytes were cultured in RPMI1640 supplemented with 2 mM L-glutamine, 10% FBS and M-CSF(100 ng/mL). Media was replaced every 48 h, and after 7 days,the cells were cultured in fresh RPMI 1640 supplemented with10% FBS containing

IFN-c (500 IU/mL), or IL-4 (100 IU/mL) for 18 h, to polarizetowards the CAM or AAM phenotype [9], respectively. The studyhas been approved by the FDA Risks in Human Subjects Committeeand the NIH Human Protections Research Program.

2.2. Quantitative RT-PCR

Cells were washed twice in pre-warmed HBSS (Invitrogen) andtotal RNA was extracted from cellular lysates using the RNeasyMini Kit (Qiagen, Valencia, CA) according to the manufacturer’sinstructions. RNA concentrations were determined with aND2000 spectrophotometer and NanoDrop 3.0.1 software (Nano-Drop Technologies, Wilmington, DE). First-strand cDNA was gener-ated by reverse transcription of 0.5 lg total RNA per sample withrandom hexamers using Verso cDNA synthesis kit (Thermo Scien-tific, Rockford, IL) according to the manufacturer’s instructions ina final reaction volume of 10 lL.

To measure expression of types I and III IFN, the primer/probesets were used as previously described [10]. Otherwise, primer/probe sets were obtained from Applied Biosystems (Foster City,CA) and used according to the manufacturer’s instructions. RT-PCR was performed using either a 7900HT or ViiA7 Real-TimePCR System (Applied Biosystems). Unless indicated otherwise,transcript levels of target genes were normalized according tothe DCq method to respective mRNA levels of the housekeepinggene (HKG) ubiquitin (UBQ) [11]. RT-PCR Ct data were collectedand analyzed with SDS 2.3 or ViiA7 software (Applied Biosystems).

2.3. IFN a/b pathway PCR gene expression array

The Human Interferon a, b pathway Response RT2 Profiler PCRArray and RT2 Real-Time SyBR Green/ROX PCR Mix were purchasedfrom SuperArray Bioscience (Frederick, MD) and were used

Please cite this article in press as: Fiky AE et al. Attenuated expression of interHum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.08.267

according to the manufacturer’s instructions. Raw Ct values wereanalyzed using the RT2 Profiler PCR Array Data Analysis based onalgorithms provided by the manufacturer including normalizationof gene expression to the arithmetic mean of the Ct values of fiveHKG: B2M, HPRT1, RPL13A, GAPDH and ACTB.

2.4. ELISA measurement of cytokine proteins

Protein expression in the cell supernatants was measured withthe human cytokine ultra-sensitive 10-plex panel (Invitrogen,Carlsbad, CA) according to the manufacturer’s instructions.

2.5. Western blot

Cells (2 � 106 per sample) were lysed with 250 lL RIPA lysisbuffer system (Santa Cruz Biotechnology, Santa Cruz, CA). Celllysates were centrifuged at 5500 rpm for 15 min. Supernatant wastested for protein concentration by BCA Protein Assay (Pierce-Thermoscientific, Rockford, IL). NuPAGE� LDS Sample Buffer(Invitrogen) was then added to samples before boiling for 10 minat 100 �C. Lysates were separated on 10% NuPAGE� Novex 10%Bis–Tris gels at 170 V for 1 h in NuPAGE� MOPS SDS Running Buffer(Invitrogen). Proteins were transferred to nitrocellulose blots usingthe iBlot Blotting system (Invitrogen). Membranes were blocked in2% BSA Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBST,Sigma) for 2 h at 4 �C followed by overnight incubation withprimary antibodies for STAT6 and p-STAT6 at 1:2500, or Tubulinat 1:5000 (Santa Cruz Biotechnology) then washed and re-probedwith secondary antibody (1:5000) in 2% BSA–TBST (Cell SignalingTechnology, Boston, MA). Protein bands were visualized using theSuperSignal West Pico ECL system (Pierce-Thermoscientific).

2.6. Statistical analysis

Statistical analyses were preformed with the Mann whitneynon-paired test with PRISM 5.0 software (Graphpad, San Diego,CA). p values equal to or less than 0.05 were considered significant.

3. Results

3.1. Differential gene and protein expression by THP-1 derivedclassically (THP-CAM) and alternatively activated (THP-AAM)macrophages

To produce THP-1 derived classically or alternatively-activatedmacrophage populations, serial dose and time optimization exper-iments were conducted where THP-1 promonocytes were differen-tiated subsequent to PMA treatment for 48 h followed bypolarization with either IFN-c or IL-4 for additional 96 h (Fig. 1A).As shown in Fig. 1B, THP-CAM and -AAM are adherent with typicalflat, elongated and branching macrophage morphology.

To validate the THP-CAM and -AAM experimental model, wemeasured the expression of a representative set of pro- and anti-inflammatory genes using qRT-PCR. After polarization (Fig. 2A),THP-CAM preferentially expressed pro-inflammatory genes encod-ing CXCL10 and CXCL11, while THP-AAM preferentially expressedanti-inflammatory genes encoding the mannose receptor (MRC1)and the chemokine CCL22. After stimulating the polarized cellswith LPS (Fig. 2B), THP-CAM preferentially secreted IFN-c and IL-1b, while THP-AAM preferentially secreted IL-10 and IL-4 intothe supernatant.

STAT6 has been shown to be activated via phosphorylation atTyr641 in response to IL-4 before translocating to the nucleus toregulate cytokine-induced gene expression [12]. Indeed, Fig. 3shows that while STAT6 is expressed equally in both THP-CAMand -AAM, it is only activated in THP-AAM.

feron-b and interferon-k1 by human alternatively activated macrophages.

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Fig. 1. Generation of THP-CAM and -AAM. (A) Protocol for generation of THP-CAM and THP-AAM. The concentrations of PMA, IFN-c, and IL-4 were 20 ng/mL, 500 IU/mL,100 IU/mL, respectively and (B) morphology of THP-1 monocytes as they proceed through in vitro differentiation into macrophages and polarization into THP-CAM and -AAM.Images are 20�.

Fig. 2. Polarized THP-CAM and -AAM differentially express phenotype-specific genes. THP-CAM and AAM were harvested after polarization with IFN-c and IL-4, respectively,for qRT-PCR analysis relative to the HKG UBQ (DCq) (A), and after stimulation with LPS (100 ng/mL) for analysis of cytokine secretion by ELISA (B). Each symbol represents anindependent experiment. ⁄p 6 0.05, ⁄⁄p 6 0.01, ns = not shown, because exogenously added IFN-c and IL-4 could not be efficiently washed out at the zero time point.

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3.2. Attenuated expression of IFN a/b pathway genes by THP-AAM

Having demonstrated that THP-1 monocytes can be polarized toexpress CAM- or AAM-oriented markers, we used the THP-CAMand AAM model to investigate differential expression of a panel

Please cite this article in press as: Fiky AE et al. Attenuated expression of interHum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.08.267

of 84 IFN a/b pathway genes after polarization, and after 1 and4 h of stimulation with LPS. As shown in Fig. 4, a subset of key ISGs(e.g., Mx1, ISG15, etc.) were preferentially expressed by THP-CAMwhile their expression in THP-AAM was markedly impaired at bothtime points. None of the 84 IFN a/b pathway genes measured in

feron-b and interferon-k1 by human alternatively activated macrophages.

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Fig. 3. STAT6 is phosphorylated only in THP-AAM. THP-CAM and -AAM wereharvested after polarization, lysed, and analyzed for STAT6 expression andphosphorylation. Tubulin was probed as a loading control. Figure shown is arepresentative of 3 experiments.

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this assay were preferentially enhanced by the THP-AAM eitherafter polarization or in response to LPS stimulation (data notshown).

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3.3. Attenuated expression of IFN-b and k1 by THP-AAM

Having demonstrated that THP-CAM and -AAM differentiallyexpressed IFN a/b pathway genes, we then used a sensitive andspecific qRT-PCR assay developed in our laboratory to measureexpression of the 12 subtypes IFN-a, IFN-b, and the three subtypesof IFN-k [10]. Analysis of types I and III IFN expression in THP-CAM

Fig. 4. Attenuated expression of IFN a/b pathway genes by LPS-stimulated THP-AAM. THafter 0, 1, and 4 h, for analysis of IFN a/b pathway gene expression by qRT-PCR. Three indshown. Each target gene is normalized to the mean of the expression (Ct value) of five h

Please cite this article in press as: Fiky AE et al. Attenuated expression of interHum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.08.267

and -AAM after 1 and 4 h of LPS stimulation revealed that out ofthe 16 genes we measured, five were consistently expressed inthe THP-CAM and -AAM: IFN-b, -k1, a4, a10, and -a14. Specifically,IFN-b and -k1 were expressed after 4 h of LPS stimulation only inTHP-CAM (Fig. 5, top). Both THP-CAM and -AAM expressed IFN-asubtypes, there was a trend towards weighted expression by theTHP-CAM of IFN-a4 and -a10 (Fig. 5, bottom).

3.4. Differential gene and protein expression by primary humanmonocyte derived classically (MD-CAM) and alternatively-activated(MD-AAM) macrophages

We then sought to demonstrate that the patterns of expressionof CAM and AAM-related markers and IFN-b and k1 genes in theTHP-CAM and -AAM model reflect expression in primary humanmonocyte derived CAM and AAM counterparts. Therefore, accord-ing to established protocols [9], we polarized primary humanmonocytes to generate monocyte-derived CAM and AAM (MD-CAM and -AAM, respectively) from aggregate total of 12 humandonors. As previously reported [13], MD-CAM preferentially ex-pressed genes for CXCL10 and CXCL11, while the MD-AAM prefer-entially expressed MRC1 and CCL22 (Fig. 6A), and only the MD-AAM expressed IL-4 protein after stimulation with LPS (Fig. 6B,bottom left). The trends were towards greater expression by theLPS-stimulated MD-CAM of IFN-c and IL-1b, and by the MD-AAMof IL-10.

3.5. Attenuated expression of IFN-b and k1 by MD-AAM

Analysis of types I and III IFN expression in MD-CAM and -AAMafter 1 and 4 h of LPS stimulation revealed that out of the 16 genes

P-CAM and -AAM were stimulated with LPS (100 ng/mL), and cells were harvestedependent results of the most regulated genes (of a total of 84 in the microarray) areousekeeping genes, B2M, HPRT1, RPL13A, GAPDH and ACTB and designated as DCq.

feron-b and interferon-k1 by human alternatively activated macrophages.

Fig. 5. Attenuated expression of types I and III IFN by LPS-stimulated THP-AAM. THP-CAM and -AAM were stimulated with LPS (100 ng/mL), and cells were harvested after 1and 4 h for analysis of IFN expression by qRT-PCR. Types I and III IFN genes that were expressed are shown as copy number/lg of total RNA. Each symbol represents anindependent experiment. ⁄p 6 0.05, ⁄⁄p 6 0.01.

Fig. 6. Phenotypic gene expression by classically and alternatively activated primary human monocyte-derived macrophages (MD-CAM and -AAM, respectively) validates theTHP-1 modelQ3 . MD-AAM and -CAM were harvested after polarization with IFN-c and IL-4, respectively, for qRT-PCR analysis relative to the HKG UBQ (DCq) (A), and afterstimulation with LPS (100 ng/mL) for analysis of cytokine secretion by ELISA (B). Each symbol represents an independent experiment. ⁄p 6 0.05, ⁄⁄p 6 0.01, and ⁄⁄⁄p 6 0.001,ns = not shown, because exogenously added IFN-c and IL-4 could not be efficiently washed out at the zero time point.

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Fig. 7. Attenuated expression of types I and III IFN by LPS-stimulated MD-AAM. MD-CAM and -AAM were stimulated with LPS (100 ng/mL), and cells were harvested after 1and 4 h for analysis of IFN expression by qRT-PCR. Types I and III IFN genes that were expressed are shown as copy number/lg of total RNA. Each symbol represents anindependent experiment. ⁄p 6 0.05, ⁄⁄p 6 0.01.

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we measured, four were consistently expressed in the MD-CAMand -AAM: IFN-b, -k1, a1, and -a10. Similar to THP-CAM, IFN-band -k1 were expressed after 4 h of LPS stimulation only inMD-CAM (Fig. 7, top). Of the IFN-a subtypes, both MD-CAM and-AAM expressed IFN-a1, and similar to the THP-1 derived model,there was a trend towards weighted expression by the MD-CAMof IFN-a10 (Fig. 7, bottom). Unlike the THP-1 derived model, how-ever, the MD-CAM and -AAM did not express IFN-a4 or -a14.

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4. Discussion

The THP-1 human promonocytic cell line was derived byTsuchiya et al. from the peripheral blood of a 1 year old male withacute monocytic leukemia [14]. We used this cell line to generateAAM and CAM that share morphology and phenotypic gene andprotein expression patterns with their primary human derivedcounterparts. For example, THP-AAM preferentially expressMRC1, the gene for the mannose receptor, and CCL22, whileTHP-CAM preferentially express CXCL10 and CXCL11. Upon LPSstimulation, THP-AAM preferentially express IL-4 and IL-10, whileTHP-CAM preferentially express IL-1b and IFN-c. IL-4 responsivetranscription factor, STAT6, was only activated in THP-AAM. Inaddition, THP-AAM show qualitative, quantitative, and kinetic dif-ferences in the expression of types I and III IFN and IFN a/b path-way genes compared to THP-CAM that extend to their primaryhuman monocyte derived AAM and CAM, respectively.

THP-1 cells are often used to investigate the pro-inflammatorynature of macrophages [15–18] rather than to compare pro- andanti-inflammatory polarized states. Differentiation protocols have

Please cite this article in press as: Fiky AE et al. Attenuated expression of interHum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.08.267

been developed treating monocytic cell lines such as U937,HL-60 or THP-1, often using PMA at high concentrations for a pro-longed period of time [19]. In this report, we used a relatively lowdose of PMA (20 ng/mL) for a limited period of time (48 h) tostimulate differentiation while minimizing toxicity.

CAM are largely regarded as pro-inflammatory because theyhighly express cytokines such as IL-12 and TNF-a, and efficientlykill microorganisms [1,3,20]. In addition, they elicit infiltration ofneutrophils, express proinflammatory mediators and matrixdegrading proteases [21]. The selective expression of CXCL10,CXCL11 and IL1-b by THP-CAM validates their use as a model forCAM.

In the lung, AAM arise from resident macrophages in proximityto respiratory epithelial cells that express the cytokines TSLP, IL-33and IL-25, all of which enhance type 2 immune responses inducedby IL-4 in a STAT6 dependent manner [4]. AAM are regarded asanti-inflammatory because they express IL-10 and scavengerreceptors such as the nonopsonic mannose receptor and thegalactose receptor, and participate in fibrotic remodeling whichis associated with chronic allergic inflammation [22–24]. Theselective expression of CCL22, IL-10, and MRC, as well as activationof STAT6, by THP-AAM validates their use as a model for AAM.

We employed the THP-1 model to compare CAM and AAMexpression patterns of types I and III IFN and ISGs because IFN-band -k1 are known to protect against exacerbations of asthma in-duced by respiratory viruses. Specifically, deficient expression ofIFN-b by asthmatic patients correlates with enhanced replicationof rhinovirus [25,26] and macrophages isolated from their BALfluid produce lower levels of IFN-k after stimulation with LPS,Streptococcus pneumoniae or RV [27]. On the other hand, type I

feron-b and interferon-k1 by human alternatively activated macrophages.

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IFN may exacerbate the asthmatic phenotype, for example, byenhancing the expression of FceRI by pDC from atopic patients,which in the presence of high levels of IgE enhances, rather thansuppresses, expression of IL-4 and IL-13 [28].

Rather than challenging with a virus, we studied for this first re-port LPS stimulation through TLR4 because of their varied roles inthe pathogenesis of allergic asthma. For example, the fusion (F)protein of respiratory syncytial virus is a functional ligand ofTLR4 [29], and Der p 2, an allergenic protein of the house dust miteDermatophagoides pteronyssinus, is a structural and functionalhomolog of the TLR4 co-receptor MD-2 [30]. Perhaps most impor-tantly, the dependency of the risk of asthma associated with TLR4D299G or T399I polymorphisms on the level of endotoxin exposure[31] is a quintessential example of gene-environment interactionscritical to the pathogenesis of allergic asthma.

Upon stimulation with LPS, THP-CAM preferentially expressedIFN-b and IFN-k1, MyD88, STAT1 and the ISGs: IFIT1, Mx1, CXCL10,OAS1 and ISG15. Consistent with an attenuated and delayedexpression of IFN-b and IFN-k1 in THP-AAM, expression of thesame ISGs was either impaired or delayed. Furthermore, therewas a trend in both the THP-1 model and primary human macro-phages towards CAM-specific expression of IFN-a10, which has ahigh affinity for the IFNAR1/2 receptor complex, and high anti-viralpotency [32]. Unlike the primary human macrophages, however,THP-CAM did not express IFN-a1, the lowest affinity IFN- a sub-type. Since IFN-a is expressed by primary macrophages in responseto synthetic ligands and virulent Mycobacterium tuberculosis (MTB),THP-CAM may provide an opportunity to explore the function ofthis uniquely low affinity type I IFN [33].

The role of types I and III IFN in chronic allergic inflammationand exacerbations of asthma have not been fully characterized.Particularly since AAM have an important role in sustainingchronic allergic inflammation, we suggest that our THP-1 derivedAAM model may be a valuable tool to investigate receptor medi-ated responses and dissect signaling mediators governing the IFNexpression in the context of allergic inflammation.

Acknowledgments

This work was supported by funding the Oak Ridge Institute forSciences and Engineering (ORISE) and the FDA medical counter-measure initiative (McMi). We thank Jennifer Reed for criticalreview of this manuscript.

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feron-b and interferon-k1 by human alternatively activated macrophages.