Cooperative Prosurvival Activity by ERK and Akt in Human Alveolar Macrophages is Dependent on High Levels of Acid Ceramidase Activity This information is current as of September 23, 2021. Martha M. Monick, Rama K. Mallampalli, Mary Bradford, Diann McCoy, Thomas J. Gross, Dawn M. Flaherty, Linda S. Powers, Kelli Cameron, Samuel Kelly, Alfred H. Merrill, Jr. and Gary W. Hunninghake

J Immunol 2004; 173:123-135; ; Downloaded from doi: 10.4049/jimmunol.173.1.123 http://www.jimmunol.org/content/173/1/123 http://www.jimmunol.org/ References This article cites 76 articles, 47 of which you can access for free at: http://www.jimmunol.org/content/173/1/123.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cooperative Prosurvival Activity by ERK and Akt in Human Alveolar Macrophages is Dependent on High Levels of Acid Ceramidase Activity1

Martha M. Monick,2* Rama K. Mallampalli,* Mary Bradford,* Diann McCoy,* Thomas J. Gross,* Dawn M. Flaherty,* Linda S. Powers,* Kelli Cameron,* Samuel Kelly,† Alfred H. Merrill, Jr.,† and Gary W. Hunninghake*

Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of in alveolar macrophage survival, we found high levels of , but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on , and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity. The high levels of acid ceramidase were specific to alveolar macrophages, because Downloaded from there was little ceramidase protein or activity (or sphingosine) in monocytes from matching donors. In evaluating prolonged survival of alveolar macrophages, we observed a requirement for constitutive activity of ERK MAPK and the PI3K downstream effector Akt. Blocking acid ceramidase but not activity in alveolar macrophages led to decreased ERK and Akt activity and induction of cell death. These studies suggest an important role for sphingolipids in prolonging survival of human alveolar macrophages via distinct survival pathways. The Journal of Immunology, 2004, 173: 123–135. http://www.jimmunol.org/ uman alveolar macrophages can survive for long periods membrane (9Ð13). Sphingomyelinases can be activated by proin- in the lung (1). Survival occurs even in the face of expo- flammatory cytokines, growth factors, and environmental stress H sure to chemical pollutants, reactive oxygen species, in- (14). Once generated, ceramide can be deacylated by ceramidases. flammatory mediators, and infectious agents (2). Multiple factors con- This is the rate-limiting step in determining intracellular levels of tribute to that survival. We have shown previously that alveolar sphingosine (15). In turn, sphingosine can be phosphorylated by macrophage survival is decreased by blocking generation of the sphingosine kinase yielding sphingosine-1-phosphate (S1P)3 (16). ceramide (3). In that same study, we found that exog- Ceramide has been shown to be antiproliferative and proapoptotic enously added ceramide activated the prosurvival kinase Akt. Studies (17). In contrast, S1P has been implicated in cell proliferation and by guest on September 23, 2021 in other systems have shown a strong link between ceramide and survival (17Ð19). The function of the intermediate sphingolipid, apoptosis, including a study demonstrating direct dephosphorylation sphingosine, is less clear. In some systems, it serves an antiapop- (inactivation) of Akt by ceramide-activated phosphatases (4). These totic role, whereas in others, it serves a proapoptotic role (20, 21). observations suggest that, in alveolar macrophages, ceramide either Given exogenously, it is a strong inhibitor of many protein kinase has opposite effects on Akt activity or is rapidly converted to another C (PKC) isoforms, but whether endogenous sphingosine has the Akt-activating sphingolipid species. same function is still unclear (22). Sphingosine and its N-acyl derivatives () are the back- The importance of ceramidase activity in maintaining the correct bones of most sphingolipids and participants in many cell-signal- sphingolipid balance is demonstrated in , an inherited ing pathways (5, 6). They are produced by de novo synthesis ini- deficiency in acid ceramidase that is fatal, resulting from lysosomal tiated with the condensation of and palmitoyl-CoA and accumulation of ceramide (7). In cell models, overexpression of acid completed by a series of modifications (7, 8). Ceramide ceramidase can protect from TNF-induced death (15). There are at can also be generated by the action of acid and neutral sphingo- least two types of ceramidases in mammalian cells, an acid cerami- myelinases on in the outer leaflet of the plasma dase that is localized primarily in lysosomes (the site of lipid degra- dation) and a recently cloned neutral/alkaline form about which little is known (14, 23). These two ceramidases are the products of two *University of Iowa Roy J. and Lucille A. Carver College of Medicine and Veterans Administration Medical Center, Iowa City, IA 52242; and †School of Biology, Geor- different genes (24, 25) and have distinct pH optima (acid, pH 4.5; gia Institute of Technology, Atlanta, GA 30332 neutral/alkaline, pH 7.2) (26). A high level of ceramidase activity in Received for publication January 12, 2004. Accepted for publication April 26, 2004. the cell would limit any accumulation of ceramide, even after ceram- ␣ The costs of publication of this article were defrayed in part by the payment of page ide-inducing stimuli such as TNF- . In view of our earlier data that charges. This article must therefore be hereby marked advertisement in accordance short-chain cell-permeable ceramides induced activation of a prosur- with 18 U.S.C. Section 1734 solely to indicate this fact. vival pathway, we pursued studies investigating the biochemical basis 1 This study was supported by a Veterans Affairs Merit Review grant, National In- for these observations within human alveolar macrophages. We were stitutes of Health Grants HL-60316 and ES-09607, and Environmental Protection Agency Grant R826711 (to G.W.H.); National Institutes of Health Grants HL68135, specifically interested in evaluating the role of ceramidase in pro- HL71040, and HL55584 (to R.K.M.); and National Center for Research Resources, Na- longed alveolar macrophage survival. tional Institutes of Health Grant RR00059 from the General Clinical Research Centers Program, and National Institutes of Health Grant ES09204 and GM067877 (to A.H.M.). 2 Address correspondence and reprint requests to Dr. Martha M. Monick, Division of Pulmonary, Critical Care, and Occupational Medicine, Room 100, Eckstein Medical 3 Abbreviations used in this paper: S1P, sphingosine-1-phosphate; PKC, protein ki- Research Building, University of Iowa Roy J. and Lucille A. Carver College of Med- nase C; NOE, N-oleoylethanolamine; DMS, D-erythro-N,N-dimethylsphingosine; icine, Iowa City, IA 52242. E-mail address: [email protected] SDK, sphingosine-dependent kinase.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 124 HUMAN ALVEOLAR MACROPHAGES AND SPHINGOSINE

Two signaling pathways have been closely linked to cell sur- (DMS) was obtained from Biomol Research Laboratories (Plymouth Meet- vival. The PI3K/Akt pathway affects cell survival through a num- ing, PA). The MEK inhibitor U0126 and PI3K inhibitor LY294002 were ber of mechanisms, including inactivation of apoptosis-relevant obtained from Calbiochem. Protease inhibitors were obtained from Roche Diagnostics (Mannheim, Germany). Nitrocellulose and ECL Plus were ob- factors (glycogen synthase kinase 3, Bad, caspase-9, and Forkhead tained from Amersham Biosciences (Piscataway, NJ). Abs were obtained transcription factors) and activation of NF-␬B leading to transcrip- from various sources: Abs to ERK (threonine 202/tyrosine 204), Akt tion of the inducer of apoptosis family of antiapoptotic factors (serine 473), and ␤-actin were from Santa Cruz Biotechnology (Santa (27Ð29). Akt is a PH domain-containing kinase that is activated Cruz, CA), and phosphorylation-specific Abs for ERK and Akt were from Technology (Beverly, MA). The Ab to acid ceramidase was after membrane recruitment to 3-phosphorylated phosphatidyli- obtained from BD Biosciences (San Jose, CA; catalog no. A24520). De- nositols. After activation, Akt phosphorylates a diverse array of veloping Abs (HRP-conjugated anti-rabbit or -mouse Ig) were from Santa substrates. Phosphorylation of Akt substrates results in either ac- Cruz Biotechnology. tivation or, in the case of the apoptosis-linked proteins, inactiva- Isolation of human alveolar macrophages tion. Inhibition of Akt has been strongly linked to decreased cell survival (3, 30Ð35). Alveolar macrophages were obtained from normal nonsmoking volunteers, as previously described (51). Briefly, normal volunteers with a lifetime The MAPKs (ERK, p38, and JNK) are an evolutionarily con- nonsmoking history, no acute or chronic illness, and no current medica- served family of that signal to regulatory targets both in tions, underwent bronchoalveolar lavage. The cell pellet was washed twice the cytoplasm and nucleus (36). The ERK MAPK pathway has in HBSS without Ca2ϩ and Mg2ϩ and suspended in complete medium been linked to transcription of c-fos, activation of the transcription (RPMI 1640 tissue culture medium (Invitrogen Life Technologies, Carls- ␮ factor Elk-1 and Sp-1 and phosphorylation of the AP-1 subunits fra bad, CA) with added gentamicin (80 g/ml)). Differential cell counts were determined using a Wright-Giemsa-stained cytocentrifuge preparation. All 1 and 2 (37Ð39). Relevant to the present study, recent data have cell preparations had between 90 and 100% alveolar macrophages. This Downloaded from supported a cell type-specific role for ERK in cell survival (40Ð study was approved by the Committee for Investigations Involving Human 43). In osteoclasts, TNF-induced survival requires ERK activity Subjects at the University of Iowa. (44). In fibroblasts, ERK activity prevents anchorage and serum Isolation of human blood monocytes removal-induced apoptosis (45). In squamous carcinoma cells, he- patocyte growth factor blocks suspension-induced apoptosis via A volume of 180 ml of heparinized blood was obtained by venipuncture of the same volunteers who underwent bronchoscopy. Monocytes were then activation of ERK (46). It is not known exactly how ERK con- separated from the remaining mononuclear cells (T cells, NK cells, etc.) by http://www.jimmunol.org/ tributes to cell survival. One possibility is a positive effect on removal of all nonmonocytes with an Ab mixture, which cross-links the Elk-1- and AP-1-driven genes. Other possibilities include the fol- targeted cells to RBC before using a Ficoll-Hypaque gradient (Sigma- lowing: the ERK substrate immediate early gene X-1 protein, an Aldrich) (RosetteSep mixture from StemCell Technologies (Vancouver, BC, Canada)). Monocyte purity was evaluated using Wright-Giemsa stain- inhibitor of cell death (40), and direct ERK phosphorylation and ing and was Ͼ95%. inactivation of caspase-9 (47). We believe it is likely that both pathways (Akt and ERK) play a role in the prolonged survival of Cell culture alveolar macrophages. Alveolar macrophages or blood monocytes were cultured in complete me- In these studies, we found that alveolar macrophages had high dium at 1Ð5 ϫ 106 cells/ml depending on the experiment. Cultures were sphingosine and low S1P levels compared with monocytes. High done in 1.8-ml microfuge tubes or six-well tissue culture plates at 37¡C and by guest on September 23, 2021 5% CO . levels of sphingosine, in alveolar macrophages, unlike monocytes, 2 was attributed to acid ceramidase mRNA, protein, and activity. Isolation of whole-cell extracts Alveolar macrophage survival required constitutive levels of both Alveolar macrophages were cultured in various conditions. Whole-cell pro- ERK and Akt pathways. A link between the acid ceramidase ac- tein was obtained by lysing the cells on ice for 20 min, in 500 ␮l of lysis tivity and Akt and ERK was found by inhibiting ceramidase ac- buffer (0.05 M Tris (pH 7.4), 0.15 M NaCl, 1% Nonidet P-40, with added tivity with the compound N-oleoylethanolamine (NOE). NOE protease and phosphatase inhibitors: 1 protease minitab (Roche Biochemi- cals, Indianapolis, IN)/10 ml and 1ϫ phosphatase inhibitor mixture (no. blocked both Akt and ERK constitutive activity and induced al- 524625; Calbiochem)). The lysates were then sonicated for 20 s, kept at veolar macrophage death. This was in contrast to the lack of sur- 4¡C for 30 min, and spun at 15,000 ϫ g for 10 min, and the supernatant was vival effects of inhibiting sphingosine kinase. These data suggest saved. Protein determinations were made using a protein measurement kit that the conversion of ceramide to sphingosine (due to high acid (protein assay, no. 500-0006; Bio-Rad, Hercules, CA). Cell lysates were Ϫ ceramidase activity) is a critical step leading to sphingosine-de- stored at 70¡C until use. pendent activation of Akt and ERK in human alveolar macro- Cell survival analysis phages. In contrast to alveolar macrophages, blood monocytes For analysis of cell survival, alveolar macrophages or blood monocytes demonstrate low levels of sphingosine, high levels of S1P, and a were cultured alone or with pathway inhibitors (ceramidase, NOE at 1 mM; requirement for sphingosine kinase activity in cell survival. The sphingosine kinase, DMS at 10 ␮M; ERK, U0126 at 10 ␮M; PI3K/Akt, prosurvival effect of sphingosine in alveolar macrophages is con- LY294002 at 10 ␮M) for the described times. Duplicate cultures were 6 sistent with a number of recent studies demonstrating sphingosine- performed on all experiments (10 cells/ml in RPMI 1640 with added gen- tamicin). After the incubation period, one sample was analyzed by the dependent inactivation of 14-3-3, a ubiquitous adaptor protein with trypan blue exclusion method, and the percentage of dead cells was cal- roles in multiple signaling pathways including inactivation of both culated. At least 300 cells were counted for each sample from a minimum ERK and Akt (48Ð50). The data also demonstrate that, during the of six fields. The other sample was stained with ethidium homodimer process of differentiation from blood monocytes, human alveolar (EthD-1; Molecular Probes, Eugene, OR) at 8 ␮M, and images were ob- tained of both bright-field and fluorescence using a Leica (Deerfield, IL) macrophages develop high-level expression of acid ceramidase as DMRB microscope equipped with a Qimaging RETICA 1300 digital cam- a cellular marker that is integrally linked to extended survival of era and imaging system. In some instances, live cells were stained with these cells in the lung. calcein AM (1 ␮M; Molecular Probes), and the number of dead cells es- timated by manually counting the EthD-1 positive cells in comparison to Materials and Methods the calcein-AM-positive cells. These counts established a linear relation- ship between the fluorescence images and the trypan blue data. Materials Sphingolipid analysis Chemicals were obtained from Sigma-Aldrich (St. Louis, MO). NOE (in- hibits acid ceramidase) was obtained from Calbiochem (San Diego, CA). For sphingolipid analysis, freshly isolated alveolar macrophages and blood The sphingosine kinase inhibitor D-erythro-N,N-dimethylsphingosine monocytes from the same individual were cultured for1hat37¡C, frozen The Journal of Immunology 125

at Ϫ70¡C, and then lyophilized. The lyophilized cells were then analyzed Results for complex sphingolipids, sphingoid bases, and sphingoid base phosphates Human alveolar macrophages have high sphingosine levels according to previously described methods (52). compared with blood monocytes Western analysis Matching sets of blood monocytes and alveolar macrophages were obtained from normal volunteers. Fig. 1A shows a Wright-Giemsa Western analysis for the presence of particular proteins or for phosphory- Ͼ lated forms of proteins was performed on whole-cell proteins. A total of stain of alveolar macrophages ( 95% pure following bronchos- 30Ð80 ␮g of protein was mixed 1:1 with 2ϫ sample buffer (20% glycerol, copy) compared with blood monocytes (Ͼ95% pure following a 4% SDS, 10% 2-ME, 0.05% bromophenol blue, and 1.25 M Tris (pH 6.8), negative selection procedure (see Materials and Methods)). Ini- all chemicals from Sigma-Aldrich) heated to 95¡C for 5 min and loaded tially, unstimulated cell pellets were analyzed for sphingomyelin, onto a 10% SDS-PAGE gel and run at 100 V for 90 min. Cell proteins were transferred to nitrocellulose (ECL; Amersham Biosciences) by semidry transfer (Bio-Rad) at 25 V for 45 min. Equal loading of the protein groups on the blots was evaluated by staining reprobed or replicate blots for ␤-ac- tin or in the case of phosphorylation specific blots by stripping and rep- robing with Abs to the total protein. The nitrocellulose was blocked with 5% milk in TTBS (TBS with 0.1% Tween 20) for 1 h, washed, and then incubated with the primary Ab overnight. The blots were washed four times with TTBS and incubated for 1 h with HRP-conjugated anti-rabbit or -mouse IgG Ab. Immunoreactive bands were developed using a chemilu- minescent substrate (ECL Plus; Amersham Biosciences; or SuperSignal West Femto; Pierce, Rockford, IL). An autoradiograph was obtained, with Downloaded from exposure times of 10 s to 2 min. Protein levels were quantitated using a FluorS scanner and Quantity One software for analysis (Bio-Rad). The data were analyzed, and statistics were performed using GraphPad (San Diego, CA) software.

Real-time RT-PCR detection of acid ceramidase mRNA http://www.jimmunol.org/ A total of 1 ␮g of total RNA was reversed transcribed to cDNA using RETROscript RT-PCR kit (Ambion, Austin, TX). The resulting cDNA was subjected to PCR in a Bio-Rad iCycler iQ system as follows: in a 0.2-ml PCR tube (Bio-Rad), 2 ␮l of cDNA (10% of synthesis reaction) was added to 48 ␮l of PCR mixture containing 160 ␮M each dNTP (Invitrogen Life

Technologies), 3.0 mM MgCl2 (Invitrogen Life Technologies), 1:15,000 SYBR Green I DNA Dye (Molecular Probes), 0.2 ␮M each sense and antisense primers (IDT, Coralville, IA), and 2.5 U of Platinum TaqDNA (Invitrogen Life Technologies). Amplification and data collection were performed as previously described (53). Primers for human acid cerami- dase and HPRT genes are as follows (5Ј to 3Ј): acid ceramidase, forward, by guest on September 23, 2021 AGT CTG GGG AAG GTT GTG TG; acid ceramidase, reverse, CAG TCC CGC AGG TAA GTT TC; HPRT, sense, CCT CAT GGA CTG ATT ATG GAC; and HPRT, antisense, CAG ATT CAA CTT GCG CTC ATC.

Quantitation of acid ceramidase mRNA Relative quantitative gene expression was calculated as follows. For each

sample assayed, the threshold cycles (Ct) for reactions amplifying acid ceramidase and HPRT were determined. Acid ceramidase mRNA abun- dance, relative to HPRT mRNA abundance, was calculated by the formula 2Ϫ(⌬⌬Ct). Validity of this approach was confirmed by using serial 10-fold dilutions of template containing acid ceramidase and HPRT genes. Using this set of template mixtures, the amplification efficiencies for acid cer- and HPRT amplimers were found to be identical.

Acidic and neutral ceramidase activity assay Alveolar macrophages were treated and homogenized in lysis buffer con-

taining 50 mM sodium acetate (pH 4.5), 0.5% Triton X-100, 5 mM MgCl2, 1 mM EDTA, and 5 mM D-galactonic acid-lactone for the acidic cerami-

dase, and 50 mM Tris (pH 8.0), 0.5% Triton X-100, 5 mM MgCl2,1mM EDTA, and 5 mM D-galactonic acid-lactone for the neutral ceramidase. Cell homogenates were centrifuged for 10 min at 14,000 ϫ g, and the supernatant was taken for an in vitro assay. A total of 100 ␮g of protein in a total volume of 100 ␮l was incubated for 20 h at 37¡C with 20 nCi of FIGURE 1. Human alveolar macrophages have high sphingosine levels 14 [ C]ceramide. Thereafter, the reaction was stopped by the addition of 200 compared with blood monocytes. Matching sets of human alveolar mac- ␮ l of water, and lipid extraction was performed by addition of 2 ml of rophages and blood monocytes were obtained from normal volunteers as chloroform/methanol (2:1; v/v). The lower phase was concentrated, and described in Materials and Methods. A, Freshly isolated cells were lyoph- lipids were resolved by TLC using chloroform/methanol/ammonia (90:20: ilized, and sphingolipid analysis was done by tandem mass spectrometry. 0.5; v/v) as a solvent. Spots corresponding to ceramide and stearic acid were analyzed and quantitated using an imaging analyzer. A Wright-Giemsa stain of isolated monocytes and alveolar macrophages is shown. The sphingolipid data are shown as picomoles (p/moles) per mil- lion cells. The data are a composite of three separate experiments. B, Statistical analysis Freshly isolated alveolar macrophages were treated with C2 ceramide (10 Statistical analysis was performed on densitometry data, ELISA results, ␮M) for 1 h. Cells were frozen and lyophilized, and sphingolipid analysis and real-time PCR data. Significance was determined by Student’s t test. was performed. The data are a composite of three separate experiments. 126 HUMAN ALVEOLAR MACROPHAGES AND SPHINGOSINE ceramide, sphingosine, and S1P by electrospray tandem mass spectrometry (52). Fig. 1A demonstrates that alveolar macrophages at baseline have significant levels of sphingosine (comparable or slightly greater than the amounts of ceramide). This is in compar- ison to blood monocytes, which have significant levels of ceramide but virtually no sphingosine (154 pM per million macrophages compared with 3 pM per million monocytes). There was essen- tially no S1P in the alveolar macrophages. We next evaluated the effect of adding exogenous C2 ceramide, because this was shown previously to promote the survival of alveolar macrophages. Fig. 1B demonstrates that the addition of ceramide to alveolar macro- phages causes an increase in sphingosine. The data also suggest that sphingosine kinase activity in these cells is low, because there is no S1P either at baseline or after an increase in sphingosine after the exogenous addition of C2 ceramide. As a composite, these data demonstrate that sphingolipid profiles in baseline alveolar macro- phages compared with blood monocytes differ significantly in the

amount of sphingosine. Alveolar macrophages have high sphin- Downloaded from gosine levels that increase rapidly if ceramide levels are increased.

Human alveolar macrophages contain high (compared with blood monocytes) constitutive acid ceramidase activity

We have recently shown that generation of ceramide induces ac- http://www.jimmunol.org/ tivation of the prosurvival kinase Akt in alveolar macrophages (3). These data are inconsistent with multiple studies in other systems that have linked ceramide generation to apoptosis (12, 13, 54Ð56). One possible means of reconciling our data with other studies is the hypothesis that alveolar macrophages have high ceramidase activity that would rapidly convert ceramide to sphingosine. The presence of high ceramidase activity in alveolar macrophages

would also explain the high sphingosine levels found in the sphin- by guest on September 23, 2021 golipid analysis. Alveolar macrophages and blood monocytes from FIGURE 2. Human alveolar macrophages contain high (compared with matched donors were lysed and used in a ceramidase activity as- blood monocytes) constitutive acid ceramidase activity. Matching sets of say. The lysates were made in both an acid buffer (consistent with human alveolar macrophages and blood monocytes were obtained from acid ceramidase) and a neutral buffer (consistent with neutral or normal volunteers as described in Materials and Methods. Cell pellets were basic ceramidase). We then performed ceramidase activity assays lysed in acid pH buffer or neutral pH buffer (as described), and a cerami- as described in Materials and Methods. Fig. 2 demonstrates that dase assay was performed using [14C]ceramide as a substrate. Ceramidase alveolar macrophages have baseline acid and not neutral cerami- activity is measured by the degree of formation of a stearic acid conversion dase activity, and that this activity is ϳ9-fold greater in compar- product. The TLC plate shows three separate experiments. Quantitation of the TLC plate was by a phosphor imager, and the data are shown as a ison to blood monocytes. There are low levels of ceramidase ac- percentage of conversion product compared with the assay control lane (no tivity in the monocytes that may provide the sphingosine substrate cell lysate). The assay control lane is from a sample containing [14C]cer- for the generation of S1P seen in monocytes (Fig. 1). amide and assay buffer with no cell lysates.

Human alveolar macrophages contain high amounts of acid ceramidase mRNA and protein nations for this discrepancy include a possible artifact induced by the Ab affinity and the possibility that monocyte levels do not To determine the mechanisms for high-level acid ceramidase ac- reach a threshold level. A more likely explanation is that there are tivity in alveolar macrophages, we assayed ceramidase mRNA and differences in acid ceramidase translation and protein stability be- protein levels. We isolated RNA and protein from alveolar mac- tween the alveolar macrophage and the blood monocyte that ex- rophages and blood monocytes (obtained from matching donors) plain the lack of detection of acid ceramidase protein in the context and compared acid ceramidase levels. Fig. 3A demonstrates sig- of measurable amounts of mRNA in blood monocytes. The com- nificantly higher constitutive levels of acid ceramidase protein in posite of the data found in Figs. 2 and 3 is that alveolar macro- alveolar macrophages compared with blood monocytes. We next phages contain significantly elevated amounts of acid ceramidase analyzed mRNA levels in the two cell types. Consistent with the activity, protein, and mRNA compared with their precursor cell, increased protein, alveolar macrophages also expressed high levels the blood monocyte. of acid ceramidase mRNA compared with blood monocytes (Fig. 3B). Thus, high levels of acid ceramidase activity are likely due to Inhibition of acid ceramidase activity, but not sphingosine increased biosynthesis of this regulatory enzyme within alveolar kinase activity, induces alveolar macrophage cell death macrophages. The differences between the protein amounts are The compound NOE is a well-described inhibitor of acid cerami- significantly greater than the amounts of mRNA. Possible expla- dase (57). In the next set of experiments, we made use of this The Journal of Immunology 127 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Human alveolar macrophages contain high amounts of acid ceramidase mRNA and protein. Matching sets of human alveolar macrophages and blood monocytes were obtained from normal volunteers as described in Materials and Methods. A, Whole-cell protein was obtained shortly after isolation, and Western analysis was performed for the small a subunit of acid ceramidase. Densitometry was obtained on the film, and the graph represents the three separate experiments. Equal loading is demonstrated by probing an identical blot for ␤-actin. B, mRNA was isolated from matching pairs of alveolar macrophages and blood monocytes by real-time RT-PCR. Acid ceramidase levels (three separate experiments) are shown as a comparison with the housekeeping gene HPRT. HPRT levels were comparable between alveolar macrophages and blood monocytes. compound to evaluate the role of acid ceramidase in alveolar mac- kinase had only minimal effect on cell viability. In the experiment rophage survival. We also treated cells with DMS, a known in- shown in Fig. 4B, we analyzed acid ceramidase activity in control hibitor of sphingosine kinase (58). Alveolar macrophages were cells compared with NOE-treated cells and found significantly re- treated with NOE or DMS, and cell survival was evaluated using duced activity in the NOE-treated cells (ϳ80%). To evaluate ethidium homodimer and trypan blue staining of dead cells (59, whether the prosurvival role of sphingosine was an alveolar mac- 60). Fig. 4A demonstrates that blocking acid ceramidase activity rophage-specific effect, we also treated blood monocytes with the causes significant alveolar macrophage cell death. Increases are same two inhibitors (NOE and DMS) and evaluated cell survival. seen as soon as 3 h, and ϳ50% of the cells are dead after6hof In contrast to the alveolar macrophages, blood monocytes were acid ceramidase inhibition. In contrast, inhibition of sphingosine sensitive to killing by the inhibition of sphingosine kinase (78% 128 HUMAN ALVEOLAR MACROPHAGES AND SPHINGOSINE Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Inhibition of acid ceramidase activity induces alveolar macrophage cell death. A, Human alveolar macrophages were cultured with the acid ceramidase inhibitor NOE (1 mM), or a sphingosine kinase inhibitor, DMS (10 ␮M), for 6 h. The photomicrographs demonstrate increased entry of EthD-1 (a marker of cell death) with NOE exposure. Also shown is percent dead by trypan blue exclusion (each sample is the composite of 3 ϫ 100 cells assayed for trypan blue staining). The trypan blue data are from three separate experiments. B, Human alveolar macrophages were cultured with and without NOE (1 mM) for 2 h, and acid ceramidase activity was assayed. The amount of stearic acid conversion product is a measure of ceramidase activity in the cell lysate. The figure shows the results from three separate sets of alveolar macrophages, and each sample was run in duplicate. Percentage of [14C]ceramide converted to a stearic acid conversion product is shown at the bottom of the figure (14C counts in conversion product/total counts of [14C]ceramide plus conversion product) times 100 for each experimental group. C, Blood monocytes were cultured as described for the alveolar macrophages (Control; NOE (1 mM); DMS (10 ␮M)), and cell viability was analyzed by EthD-1 staining and trypan blue exclusion. The data (trypan blue) are from three separate experiments. The EthD-1 photomicrographs are representative of three separate experiments. dead at 6 h). This is consistent with the higher levels of S1P cell survival. In addition, the relatively low levels of ceramidase found in blood monocytes (see Fig. 1 for sphingolipid analysis). activity in blood monocytes may make them more susceptible to These data suggest that the high constitutive activity of acid killing after exogenous C2 ceramide. The high levels of cer- ceramidase in alveolar macrophages plays a significant role in amidase activity in alveolar macrophages, in contrast, would The Journal of Immunology 129

FIGURE 5. Exogenous sphingosine or safingol reverses acid ceramidase in- Downloaded from hibition-induced alveolar macrophage death. Alveolar macrophages were cul- tured in the following conditions for 6 h: 1) Control, 2) NOE (1 mM), 3) sphin- gosine (5 ␮M), 4) safingol (5 ␮M), 5) NOE (10 ␮M) and sphingosine (5 ␮M), http://www.jimmunol.org/ 6) NOE (1 mM) and sphingosine (5 ␮M) and DMS (10 ␮M), 7) NOE (1 mM) and safingol (5 ␮M), and 8) NOE (1 mM) and safingol (5 ␮M) and DMS (10 ␮M). Cell viability was analyzed by EthD-1 staining and trypan blue exclusion. The trypan blue data are shown in a graph of positive-staining cells (300 cells were counted for each experimental condi- tion). The data are a composite of three by guest on September 23, 2021 separate experiments.

make them relatively immune to killing by exogenous C2 cer- gosine that cannot be phosphorylated by sphingosine kinase (61). The amide, as we have demonstrated previously (3). original studies on safingol showed that all four stereoisomers of sphingosine and sphinganine acted alike in inhibiting PKC (62). The Sphingosine and L-threo-dihydrosphingosine reverse the study proposed two models to explain this—one that regarded the antisurvival effects of acid ceramidase inhibition in alveolar charge of the sphingoid base to be the most important feature and macrophages the other noting that all four stereoisomers can present a similar con- To further link the survival effects of acid ceramidase activity to formation at the membrane surface if the major determinate of the sphingosine, we treated alveolar macrophages with NOE (ceramidase interaction is the two hydroxyls and an amine. The authors suggest inhibition) with or without the addition of either sphingosine or L- that the position of the alkyl chain is less critical. The combined effect threo-dihydrosphingosine (safingol). Safingol is an analog of sphin- of these data suggests that safingol may be used as a sphingosine-like 130 HUMAN ALVEOLAR MACROPHAGES AND SPHINGOSINE Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. Akt and ERK activity is required for alveolar macrophage survival. A, Alveolar macrophages were cultured for 1 h with or without LY294002 (PI3K, 10 ␮M) or U0126 (MEK inhibitor, 10 ␮M). Whole-cell lysates were obtained, and Western analysis was performed for active Akt (phosphorylated on serine 473) or active ERK (phosphorylated on threonine 202/tyrosine 204). Equal loading was determined by staining identical blots for ␤-actin. The graphs represent densitometry from three separate donors. B, Human alveolar macrophages were treated as described above with the addition of one group, which received both LY294002 (10 ␮M) and U0126 (10 ␮M). At the end of 6 h, EthD-1 entry was evaluated (photomicrograph). At the end of 6 or 24 h, trypan blue exclusion was evaluated. The data are graphed as percentage of cells that are trypan blue positive. The graph represents data from three separate experiments. Also shown is a Western blot demonstrating the effect of LY294002 and U0126 on ERK and Akt activity. The Journal of Immunology 131 molecule that cannot be phosphorylated by sphingosine kinase. Fig. 5 no effect on Akt and ERK activity (Fig. 7B). In contrast, acid demonstrates that the decreased cell survival seen with NOE treat- ceramidase inhibition blocked the constitutive Akt and ERK acti- ment was reversed by maintaining sphingosine levels by adding back vation (Fig. 7C). This occurred after as little as 30 min of NOE exogenous sphingosine or safingol. The addition of sphingosine or treatment at a time point when NOE had not yet induced any cell safingol alone had no effect on alveolar macrophage viability. In some killing, suggesting a specific effect on signaling. We have shown in cases, we also added a sphingosine kinase inhibitor with the sphin- Fig. 1 that blood monocytes have significantly less sphingosine gosine or safingol. This did not alter the increased viability generated than alveolar macrophages. We next asked whether baseline ac- by both sphingosine or safingol. These data demonstrate that the de- tivities of Akt and ERK differed between the two cell types. In Fig. creased survival seen with acid ceramidase inhibition is due to a lack 7D, we show that, in unstimulated cell lysates from alveolar mac- of sphingosine and not S1P. These data also confirm the specificity of rophages and blood monocytes from the same donor, there is sig- the inhibition by NOE. The reversal of NOE’s effect on survival by nificantly more Akt and ERK activity in the alveolar macrophages. the addition of sphingosine or a nonphosphorylatable sphingosine an- This is consistent with an important role for sphingosine in con- alog demonstrates that the cell death seen with NOE is due to a de- stitutive activation of Akt and ERK. As a composite, these data crease in the levels of sphingosine. suggest that alveolar macrophage survival is linked to Akt and ERK activation, which in turn is dependent on constitutive acid Akt and ERK activity is required for alveolar macrophage ceramidase activity. survival We next addressed the mechanism of acid ceramidase-induced sur- Downloaded from vival in alveolar macrophages. We have recently shown that there Discussion are high baseline levels of Akt activity in alveolar macrophages This study demonstrates for the first time that human alveolar mac- (60). Akt kinase is linked to cell survival via the inhibition of a rophages selectively express high levels of acid ceramidase, which number of apoptotic molecules including Bad, caspase-9, and mediates cell survival. Endogenous levels of sphingosine were Forkhead transcription factor. In addition, Akt has a well-de- much greater within alveolar macrophages compared with blood scribed link to NF-␬B activation either via an effect on I␬B kinase monocytes secondary to high constitutive levels of acid cerami- ␤ or via transactivating phosphorylation of p65 (34, 63Ð65). Re- dase. The sphingosine levels rapidly increased with the addition of http://www.jimmunol.org/ cent studies have attributed strong antiapoptotic function to NF-␬B exogenous ceramide (C2 ceramide), consistent with high baseline (66, 67). Activation of the ERK MAPK has also been linked to cell acid ceramidase activity. One defining feature of the alveolar mac- survival in a number of systems (43, 47, 68). We first evaluated rophage is its long life span (1, 69). A noteworthy observation is baseline levels of Akt and ERK activity in unstimulated alveolar that the extended life span occurs even in the setting of constant macrophages. We did this by treating newly isolated alveolar mac- exposure to inhaled bacterial and other, potentially injurious, en- rophages with Akt and ERK inhibitors for 30 min. Whole-cell vironmental stimuli. Interestingly, these stimuli (including endo- lysates were obtained, and kinase activity was evaluated using Abs toxin, oxidative stress, and cytokines such as TNF-␣) can also

specific for the activating phosphorylations for both kinases (Akt, increase ceramide levels (15, 70). Thus, the present results suggest by guest on September 23, 2021 serine 473; ERK, threonine 202/tyrosine 204). To rule out the pos- that deacylation of ceramide to sphingosine via acid ceramidase is sibility of isolation-induced kinase activity, some cells were also an important survival mechanism protecting alveolar macrophages placed in culture for up to 6 h, and then the same assay was per- from repetitive environmental exposures. This effect was linked to formed. The results in these experiments were identical with the sphingosine and not S1P in alveolar macrophages because adding newly isolated macrophages (data not shown). As in our previous back sphingosine or safingol, a sphingosine analog with properties study, we found significant constitutive Akt activation (Fig. 6A). similar to sphingosine that cannot be phosphorylated, reversed the We also found high levels of constitutive ERK activity (Fig. 6A). antisurvival effects of acid ceramidase inhibition. Sphingosine in Because of the described link between these two pathways and cell alveolar macrophages was upstream of two survival factors (ERK survival and the high constitutive activity in alveolar macrophages, and Akt). Sphingosine triggered both ERK and Akt activation, and we investigated the effect of inhibiting these pathways on alveolar inhibition of acid ceramidase or ERK and Akt led to increased cell macrophage survival. We treated alveolar macrophages with in- death. Inhibition of sphingosine kinase, although it decreased hibitors of ERK (U0126) and Akt (LY294002), and measured cell monocyte survival, had no effect on alveolar macrophage survival viability. We found that inhibiting either Akt or ERK induced or constitutive activity of Akt or ERK. The lack of endogenous some cell death (Fig. 6B), but combining the two inhibitors sig- sphingosine in the blood monocytes was reflected in the low levels nificantly increased the amount of cell death. The Western analysis of baseline Akt and ERK activity in the monocytes. Thus, sphin- demonstrates that U0126 and LY294002 are specific for the de- gosine contributes to alveolar macrophage survival via an effect on scribed pathways (ERK and PI3K), and that inhibiting one path- ERK and Akt survival pathways (Fig. 8). way does not significantly inhibit the other. These data suggest that The best-described antiapoptotic sphingolipid is S1P (16, 18). baseline Akt and ERK activity have synergistic effects on alveolar One obvious explanation of our data is that the acid ceramidase- macrophage survival. generated sphingosine is converted to S1P by the actions of a sphingosine kinase. We believe that ERK and Akt activation in Sphingosine regulates Akt and ERK activity in alveolar alveolar macrophages (as distinct from other cell types including macrophages blood monocytes) are directly in response to sphingosine. Observa- To investigate a link between acid ceramidase activity and survival tions in this study that suggest a role for sphingosine in activation of pathways, we first treated alveolar macrophages with sphingosine ERK and Akt include the following: 1) high levels of sphingosine and and measured ERK and Akt activation. We found that, in alveolar low levels of S1P in alveolar macrophages, 2) lack of a S1P increase macrophages, increasing sphingosine can activate both ERK and when sphingosine levels are increased with exogenous C2 ceramide, Akt (Fig. 7A). We asked whether Akt and ERK activity was due to 3) reversal of acid ceramidase inhibition-induced cell death by both sphingosine kinase-dependent S1P as has been found for other sphingosine and a nonphosphorylatable analog, 4) stimulation of both cells. We found that inhibition of sphingosine kinase (DMS) had ERK and Akt activity by exogenous sphingosine, and 5) inhibition of 132 HUMAN ALVEOLAR MACROPHAGES AND SPHINGOSINE Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. Sphingosine activates and acid ceramidase inhibition blocks Akt and ERK activity in alveolar macrophages. A, Human alveolar macrophages were treated with sphingosine (5 ␮M) for various times. Whole-cell lysates were obtained, and ERK and Akt activity was analyzed by Western analysis. Equal loading of the blots was analyzed by reprobing the blot for ␤-actin. B, Human alveolar macrophages were treated with DMS (10 ␮M) for 30 min. Whole-cell lysates were obtained, and ERK and Akt activity was analyzed by Western analysis. Similar data were obtained with longer exposures (1 and 3 h) (data not shown). C, Human alveolar macrophages were treated with NOE (1 mM) for 30 min. Whole-cell lysates were obtained, and ERK and Akt activity was analyzed by Western analysis. The graph represents densitometry of three separate experiments. D, Alveolar macrophages and blood monocytes from the same donor were lysed, and Western analysis was performed for active (phosphorylated) Akt and ERK. The experiment is representative of three separate experiments. sphingosine kinase having no effect on either alveolar macrophage ity (Fig. 4B) and by the fact that its effects can be reversed by adding survival or baseline Akt and ERK activities but killing blood mono- back specific molecules (Fig. 5). cytes. We have established the specificity of the inhibitor used in these Despite a number of studies supporting a role for sphingosine in studies (NOE) by demonstrating that it blocks acid ceramidase activ- inducing apoptosis (21), some recent literature suggests a possible The Journal of Immunology 133

FIGURE 8. This is a diagram of the proposed role of acid ceramidase in blocking the proapop- totic effects of ceramide by rapidly converting cer- amide to sphingosine, resulting in activation of ERK and Akt in human alveolar macrophages. Downloaded from

mechanism for sphingosine-induced antiapoptotic pathways. Me- gosine in the alveolar macrophage. We link acid ceramidase ac- http://www.jimmunol.org/ gidish et al. described sphingosine-dependent kinase (SDK)-1 tivity to activation of the prosurvival pathways, ERK and Akt, and (49), which is activated by sphingosine and not ceramide or S1P. demonstrate that loss of both pathways has a greater negative im- A novel observation by the same group demonstrated that SDK-1 pact on cell survival than loss of either one. Inhibition of acid has sequence homology with the catalytic domain of PKC␦ and ceramidase, and not sphingosine kinase, blocks both Akt and ERK phosphorylates the adaptor protein 14-3-3 (48, 71). Phosphoryla- activities, leading to decreased alveolar macrophage survival. Be- tion of 14-3-3 at serine 58 results in dissociation of the active cause alveolar macrophages play a central role in pulmonary host dimeric form and inhibition of 14-3-3 activity (50). These data defenses and inflammatory responses in the lung, understanding suggest that one possible downstream effect of sphingosine in the signaling pathways that contribute to longevity of these cells is alveolar macrophages is SDK-1 activation with resultant 14-3-3 crucial for understanding lung homeostasis. by guest on September 23, 2021 inactivation. Interestingly, 14-3-3 activity is linked to inhibition of Akt via a negative effect on the kinase upstream of Akt, phosphoi- Acknowledgments nositide-dependent protein kinase 1 (72). 14-3-3 has also recently We thank Elaine Wang for preparing the sphingolipid samples and been shown to inactivate Raf-1, leading to decreased ERK activa- Dave Fultz for graphics assistance. tion (73, 74). It is an intriguing possibility that, in alveolar mac- rophages, high levels of sphingosine result in phosphorylation and References inactivation of 14-3-3, contributing to activation of ERK and Akt 1. Thomas, E. D., R. E. Ramberg, G. E. Sale, R. S. 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