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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC 2012 November 15.
Published in final edited form as:
PMCID: PMC3208068

A cytosolic phospholipase A2 (cPLA2)-Initiated Lipid Mediator Pathway Induces Autophagy in Macrophages


Autophagy delivers cytoplasmic constituents to autophagosomes and is involved in innate and adaptive immunity. Cytosolic phospholipase (cPLA2) initiated pro-inflammatory lipid mediator pathways play a critical role in host defense and inflammation. The crosstalk between the two pathways remains unclear. Here, we report that cPLA2 and its metabolite lipid mediators induced autophagy in the RAW246.7 macrophage cell line and in primary monocytes. IFN-γ triggered autophagy involves activation of cPLA2. Cysteinyl leukotrienes (CysLTs) D4 and E4 and Prostagladin D2 (PGD2) also induced these effects. The autophagy is independent of changes in mTOR or autophagic flux. cPLA2 and lipid mediator-induced autophagy is ATG5 dependent. These data suggest that lipid mediators play a role in the regulation of autophagy, demonstrating a connection between the two seemingly separate innate immune responses, induction of autophagy and lipid mediator generation.


Recently published studies have strongly indicated that autophagy is a host defense mechanism by which cells respond to microbial invasion and promote cell survival (1-4). Autophagy plays an important role in innate and adaptive immunity (5-8). The signals that activate autophagy and molecular tags guide the formation of double-membrane cytosolic vesicles. These vesicles, designated autophagosomes, sequester invading pathogens and their products, portions of the cytosol and damaged organelles. The autophagosomes ultimately fuse with other vesicles in the endolysosomal pathway to deliver microbial ligands for adaptive or innate immune activation, or with the lysosome for subsequent degradation in autolysosomes (9,10).

After an inflammatory stimulus, cells also may produce lipid mediators, such as leukotrienes or prostaglandins (11). Those lipid messengers are derived from the poly-unstatuated fatty acid, arachidonic acid (AA). As the common precursor, AA is in turn converted to prostaglandins by cyclooxygenase (COX) pathway enzymes or to leukotrienes by the 5-lipoxygenase pathway (5-LO) (11,12). The enzyme which hydrolyzes AA release from membrane phospholipids is cytosolic phospholipase A2 (cPLA2), which is a rate-limiting enzyme that plays a key role in initiating and regulating the multistage biosynthetic process of eicosanoid production. cPLA2 activation is involved in Toll-like Receptor (TLR)-induced innate immune signaling (13). Therefore, a cPLA2-initiated pro-inflammatory lipid mediator pathway may play a pivotal role in the regulation of immune and inflammatory responses (14,15).

Since autophagy is a cellular defense mechanism and the cPLA2-initiated lipid mediator pathway is important for the production of lipid mediators and the promotion of the inflammatory response, we investigated the role of cPLA2 and its lipid products in the induction of autophagy. Further, we studied whether they may participate in IFN-γ–induced autophagy in the macrophage. Here, we report that cPLA2 and its lipid metabolites induce autophagy in the RAW264.7 macrophage cell line and in primary human peripheral blood monocytes. This pathway may also be important in IFN-γ–induced autophagy in macrophages. The induction of autophagy may be via an ATG5-dependent pathway. Therefore, cPLA2 initiated lipid mediator generation may play a role in the autophagy response.


Reagents and antibodies

Earle’s Balanced Salt Solution (EBSS medium) was purchased from Thermo Scientific (Waltham, Mass). Murine and human interferon-γ were purchased from PeproTech Inc (Rock Hill, NJ). cPLA2 inhibitor, N-{(2S,4R)-4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide HCl, a 1,2,4-trisubstituted pyrrolidine derivative, was from EMD Chemicals Inc. (San Diego, CA). MK866, indomethacin, 5-HETE, PGE2, PGD2, Leukotriene B4, Leukotriene D4, Arachdonic acid(AA) and Leukotriene E4 were purchased from Cayman Chemical Co (Ann Arbor, MI). Rabbit polyclonal antibodies against LC3 and actin were from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies against beclin-1 and ATG5 were purchased from Novus (Littleton, CO). A rabbit polyclonal antibody against cPLA2, rabbit anti-phosph S6K monoclonal antibody, and mouse anti-S6K monoclonal antibody were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal antibody against GST was from Santa Cruz Biotechnology (Santa Cruz, CA). E64d and Pepstatin A were from Sigma-Aldrich (St. Louis, MO).

Cell culture

The murine macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Biosource, Camarrillo, CA) supplemented with 10% fetal calf serum and 1% antibiotics PenStrep (GIBCO, Carlsbad, CA). Elutriated human peripheral blood monocytes were received from the Department of Transfusion Medicine, Clinical Center, under an institutional review board approved protocol. Cells were washed with PBS and maintained in human monocyte medium (Amaxa Inc, Gaithersburg, MD). For RAW264.7 cell starvation, cells were washed for once, cells were grown in EBSS for 2 hrs and harvested for S6K immunobloting.

Expression Plasmids and transient transfection

pEGFP-LC3 was a gift from Dr. Noboru Mizushima (The Tokyo Metropolitan Institute of Medical Science. Japan) (16). For the cPLA2 plasmid construct, the cPLA2 gene insert was prepared by PCR from pEGFP-cPLA2 (a gift from Dr. J. Evans and Dr. C. Leslie, National Jewish Medical Center, Denver, Co) (17). The insert was then cloned into the mammalian expression GST plasmid. The correct sequence of the positive clones was confirmed by nucleotide sequencing based on the sequence of access number P47712. The plasmids were then transiently transfected into the RAW264.7 cells using FuGENE HD Transfection Reagent from Roche (Nutley, NJ) according to the manufacturer’s protocol.

RNA Interference

The siRNAs of cPLA2, beclin-1, ATG5 and control small inhibitory RNAs (siRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cells were transfected with 30 pmol/ml siRNA by using Nucleofection electroporating transfection (Amaxa Inc, Gaithersburg, MD) following the manufacturer’s directions. The interference of cPLA2, Beclin-1 or ATG5 protein expression was compared to control non-targeting siRNA and confirmed by immunoblotting.

Real-time PCR

Total RNA was extracted from cells using QIA Shredder columns and RNeasy mini kit and was treated with DNase (Qiagen, Valencia, CA). mRNA expression for IRG-47 (IFI-47) was measured using an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA) and IFI-47 probe and primers sets (Applied Biosystems). Reverse transcription and PCR were performed using an RT kit and TaqMan Universal PCR master mix (Applied Biosystems), according to the manufacturer’s directions. Relative gene expression was normalized to GAPDH transcripts, calculated as a fold-change compared with control.

[3H]Arachidonic Acid Release

RAW264.7 cells were incubated overnight with 0.5 μCi of [5,6,8,9,11,12,14,15-3H]arachidonic acid (150-230 Ci/mmol/ml; GE Healthcare, Pascataway, NJ). Media was removed, the cells washed and incubated in control medium or in medium with interferon gamma for 6 hours. Media was harvested and an aliquot assayed in an LS 6500 scintilation counter (Beckman Instruments, Miami, Fla.).

Western Blot Analysis of RNAi-mediated knockdown of cPLA2, beclin-1 or ATG5 in RAW264.7 cells

RAW264.7 cells and primary monocytes were grown in 6-well plates with stimuli or other treatment as indicated. The procedure of western blot analysis was carried out as previously described (13). The primary antibodies were as indicated. For LC3 western blot, the cell lysis buffer was changed to PBS and 2% Triton X-100 (18). Actin was used as a loading control.

Induction of autophagy

Cells were transfected with GST-cPLA2 (1μg /well) for 24 h, or incubated with lipids (100nM) for 6 h, or alternatively, with IFN-γ (0.1μg/ml) for 6 h in nutrient medium.

Fluorescence laser scanning confocal microscopy

RAW264.7 cells were grown on 35 mm glass bottom microwell dishes and transfected by pEGFP-LC3 and with the cPLA2 expression plasmid or siRNA for 24 hrs, or different treatments as described. Cells were then fixed with −20° C methanol overnight in the dishes. The fixed cells were washed with PBS twice and kept in PBS. The green fluorescence labeled cells were observed and the numbers of LC3 positive autophagosomes in cells were quantified using a Leica DMIRB fluorescence inverted microscope with an 63x oil objective and TGS Sl confocal system. For the GFP-LC3 assay, a minimum of 100 GFP-positive cells per sample were counted and the number of GFP-LC3 aggregates were enumerated. Cells were scored as positive if they had more than three large GFP-LC3 puncta and the data were presented as a percentage of the total number of GFP-positive cells. The results are shown as the means ± S.D. from three independent experiments. Laser scanning images were cropped using Adobe Photoshop 5.0.

Electron microscopy

Cultures grown in Permanox tissue culture dishes (Nunc Nalgene) were fixed in 2.5% glutaraldehyde, 1% paraformaldehyde, 0.12M sodium cacodylate buffer, pH 7.3, postfixed in 1% OsO4 and en bloc stained with 1% uranyl acetate. The cultures were then dehydrated in graded ethanol and propylene oxide, infiltrated with Epon (Embed-812, Electron Microscopy Sciences) and the polymerized blocks were sectioned parallel to the culture substrate. Thin sections were stained with uranyl acetate and lead citrate, then viewed with a JEM-1200EX electron microscope (JEOL, USA) equipped with an AMT XR-60 digital camera (Advanced Microscopy Techniques). Images for quantification were taken with a 5000X electron microscope magnification setting from sections cut approximately midway between the adherent surface and the upper surface of the cell, such that cytoplasm occupied at least 2/3 of the image area. Structures of interest were counted in 44-47 digitally enlarged images corresponding to ~ half that number of cells each for experimentally treated and control cultures.

Statistical analysis

All statistical analyses were performed in Excel using a two-tailed T test. Where appropriate a Bonferroni adjustment was applied for multiple comparisons. A p value of <0.05 was considered significant.


Over-expression of cPLA2 induces an autophagic response in macrophages

To detect autphagosome formation, green fluorescent protein fused to LC3 (GFP-LC3) was used as a marker of autophagy (5). Microtubule-associated protein light chain (LC3) is a homologue of ATG8 protein in yeast (16). LC3 exists in two forms: the non-lipid form, cytosolic species LC3-I corresponding to the relative molecular mass (Mr) of 18 kD, and its membrane-associated form, LC3-II, conjugated C-terminally to phosphatidylethanolamine, with an apparent Mr of 16 kD. The latter form, LC3-II is found both inside and outside the autophagosome. It can be used to document induction of autophagy (16) with increased levels of the autophagosome protein LC3-II on immunoblots or with the appearance of the cytoplasmic fluorescent puncta formed by inserted-GFP-LC3-II into the membrane of the autophagosome. To test whether cPLA2 can induce autophagy, GFP-LC3 was co-transfected with the GST-vector or the GST-cPLA2-vector into RAW 264.7 macrophages for 24 h. Expression of the indicated proteins is shown on the immunoblot (Figure 1A). Overexpression of cPLA2 induced autophagy in about 60 percent of GFP positive cells as compared to approximately 18 percent of GST-vector control cells (Figure 1B). The confocal images of cells containing GFP-LC3-marked autophagosomes are shown in Figure 1 C. To further confirm this observation, we detected endogenous LC3-I and LC3-II in lysates from GST-vector control cells or GST-cPLA2 treated cells by immunoblotting with anti-LC3 antibodies. Overexpression of cPLA2 increased the amount of LC3-II, which indicates formation of autophagosomes (Figure 1D). The above results suggest that overexpression of cPLA2 may facilitate autophagy in macrophages.

Figure 1
cPLA2 over-expression induces an autophagic response in macrophages. (A) RAW264.7 cells were transfected with GST-cPLA2 for 24 h. A monoclonal antibody against GST was used for immunoblotting. (B) Quantification of cells with GFP-LC3 autophagic organelles ...

cPLA2 is involved in IFN-gamma induced macrophage autophagy

IFN-gamma induces autophagy in macrophages (1), a process that is important for eliminating intracellular bacterial infection and cell defense. However, the mechanism or pathway by which this occurs is not clear. Previous reports suggest that IFN-γ induces cPLA2 activity in human epithelial cells and in HL-60 cells(19, 20). Therefore, we next examined whether cPLA2 participates in IFN-gamma-induced autophagy in macrophages. We first reduced the expression of endogenous cPLA2 by transfecting two siRNAs directed against cPLA2 into RAW264.7 macrophages and then treated the cells with or without IFN-γ. As demonstrated by immunoblotting, two cPLA2 siRNAs reduced cPLA2 expression in macrophages (Figure 2A) and the reduction of cPLA2 expression impaired IFN-γ -induced autophagy based on the amount of endogenous LC3-II detected (Figure 2B). Further, RAW 264.7 macrophages were co-transfected with GFP-LC3 and cPLA2 siRNA or the control scrambled siRNA. The formation of GFP-LC3 autophagosomes was analyzed with or without IFN-γ stimulation by confocal fluorescence microscopy. These images are shown in Figure 2C. Interferon-gamma induced autophagic vesicle formation. This response was reduced in cells treated with siRNA against cPLA2 compared to cells treated with a control siRNA (Figure 2C and 2D). To further confirm that cPLA2 is involved in IFN-γ-induced autophagy in macropahges, we utilized a pyrrolidine derivative cPLA2 inhibitor, N-{(2S,4R)-4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide HCl, to perform the above experiments (Figure 3). Treatment of cells with this cPLA2 inhibitor blocked IFN-gamma-induced AA release (Figure 3B). Consistent with the siRNA data, cPLA2 inhibition partially blocked IFN-γ-induced autophagy as measured by localization of LC3 to autophagosomes or changes in LC3-II by immunoblot (Figure 3D). These results indicate that cPLA2 at least in part regulates IFN-γ-induced autophagy in macrophages. In order to determine if the cPLA2 inhibitor altered IFN-γ-induced transcription, the effect of the cPLA2 inhibitor was studied on an IFN-gamma inducible gene. IRG-47 (IFI-47) is IFN-γ reducible gene that has distinct roles in immune defense against protozoan infections(21). RAW264.7 cells were treated with or without cPLA2 inhibitor (10uM) for 2 h before treatment of IFN-γ for 6 h. Cells were harvested and IRG-47 mRNA levels were determined by real-time PCR (RT-PCR) (Figure 3E). IFN-γ induction of IRG-47 was not altered by treatment with the cPLA2 inhibitor, suggesting that cPLA2 does not regulate IFN-γ gene induction.

Figure 2
cPLA2 is involved in IFN-γ-induced macrophage autophagy. (A) RAW264.7 cells were transfected with two cPLA2-specific siRNAs or nonspecific siRNA (NC siRNA) for 24 h and immunoblots were performed with polyclonal antibodies against cPLA2 and actin. ...
Figure 3
cPLA2 inhibition impairs IFN-gamma induced autophagy. (A,C) RAW264.7 cells were transfected with GFP-LC3 for 24 hrs. Cells were treated with or without a cPLA2 inhibitor (10 μM) for two h followed by incubation with or without IFN-γ (0.1μg/ml) ...

cPLA2 related eicosanoid mediators induce autophagy

Inflammatory lipid mediators (eicosanoids) are final metabolites of cPLA2 activation. We examined the hypothesis that inflammatory lipid mediators are important in the induction of autophagy. A COX inhibitor, indomethacin or 5-Lo inhibitor, MK866, was used to treat RAW264.7 macrophages transfected with GFP-LC3 prior to IFN-γ stimulation. Either inhibition of COX or 5LO partially inhibited IFN-γ-induced GFP-LC3 autophagosome formation as compared with IFN-γ stimulation alone (Figure 4A), suggesting that the lipid mediators from either pathway may be involved in the induction of autophagy. We tested whether eicosanoids that are known to be produced by macrophages might induce these changes. cysLTD4 and cysLTE4 from the 5-Lo pathway and PGD2 from the COX pathway induced an increase in the level of GFP-LC3 autophagosome formation (Figure 4B and 4C) and endogenous LC3-II formation (Figure 4D). LTB4, 5-HETE and PGE2 had no significant effect. Arachidonic acid added to the media induced LC3-II (Figure 4F) suggesting that arachidonic acid may be metabolized to an active eicosanoid capable of inducing the autophagic response.

Figure 4
Lipid mediators induce autophagy. (A) RAW264.7 cells were transiently transfected with GFP-LC3 for 24 h. Cells were treated with either indomethacin (10μM) or MK866 inhibitors (10μM) for 2 h before incubation with IFN-γ (0.1μg/ml) ...

We next examined the autophagic response in primary cells, using monocytes separated from human peripheral blood. Human monocyte expression of endogenous LC3-II was assayed by immunobloting (Figure 4E). While no LC3-II was detected in untreated monocytes, IFN-gamma-induced an increase in LC3-II, which was partially inhibited by pretreatment with a cPLA2 inhibitor. When monocytes were stimulated with the lipid mediators cysLTD4, cysLTE4 or PGD2, LC3-II formation was again noted. Thus, a cPLA2-initiated lipid mediator pathway for the induction of autophagy is present in human monocytes.

Next, we examined the RAW264.7 macrophages by electron microscopy to look for early autophagosomes and autolysosomes (Figure 5). Early autophagosomes were defined by the presence of a double membrane envelope surrounding a region of cytoplasm, whereas autolysosomes, also called late autophagosomes, were defined by a single membrane envelope with cytoplasmic content that appeared degraded or condensed. In addition to autophagosomes and autolysosomes, we observed tubular cytoplasmic structures with two membranes separated by a space of ~ 10 nm with an electron-dense core. Profiles of these tubular structures often occurred in clusters, suggesting that the structures could assume a curled configuration. We also noted many examples of these tubular structures surrounding areas of cytoplasm, which appeared to undergo degradation. Such compound structures were counted as autophagosomes or autolysosomes. We counted the total number of autophagosomes and autolysosomes in 44-47 fields from ~ 20 cells each from control and cysLTD4-treated cultures. cysLTD4-treated cells had 1.8-fold more autophagosomes or autolysosomes per field than control cells. Moreover, 49% of the fields had 3 or more autophagic structures in CysLTD4-treated cells compared to 18% in controls. These results are consistent with the increased abundance of LC3-positive autophagosomes (Figure 4B) in CysLTD4-treated cells. We also counted the number of widely separated individual profiles and clusters of profiles of the tubular dense-core structures. We found 1.7-fold more of these structures in CysLTD4-treated cells than in controls.

Figure 5
Electron micrographs of the cytoplasm of RAW246.7 cells. Both control cells (a) and cells treated with CysLTD4 for 6 h (b and c) contained early autophagosomes with an irregular double membrane envelope (large arrows, not shown in control), late autophagosomes ...

Autophagy induced by cPLA2 over-expression is ATG5-dependent

Beclin-1 and ATG5 are central regulators in autophagy (22,23). Beclin-1 is a component of the class III phosphatidylinositol 3-kinase (PI3KC3) complex, which involves vesicle nucleation in the early stage of autophagy. ATG5 participates in the vesicle elongation of autophagy (24). We reduced the expression of beclin-1 and ATG5 in RAW264.7 macrophages by transfection with beclin-1 or ATG5 siRNA (Figure 6A and 6B). GFP-LC3 autophagosomes were observed with cPLA2, siRNAs targeting beclin-1 or ATG5 and GFP-LC3 co-transfection for 24 hrs. Knockdown of ATG5 appeared to inhibit cPLA2-induced changes in GFP-LC3 autophagosome formation (Figure 6C). Similarly cells were stimulated with cysLTD4 after transfection with siRNAs against beclin-1 or ATG5 and GFP-LC3 co-transfection for 24 h. GFP-LC3 autophagosomes were again increased by cysLTD4 treatment. This increase was reduced in cells treated with siRNA against ATG5 relative to the cells transfected with control non-specific siRNA (NC siRNA) or siRNA against beclin-1 (Figure 6D). Endogenous LC3-II protein was deceased in cells transfected with siRNA against ATG5 (Figure 6E). These data suggest that the cPLA2 initiated pathway induced autophagy is ATG5 dependent.

Figure 6
cPLA2 initiated autophagy is ATG5 dependent. (A-B) RAW264.7 cells were transiently transfected with control siRNA (NC siRNA) or siRNA directed at beclin-1 or ATG5 for 24 h and assessed by immunoblotting for beclin-1 or ATG5. (C) Quantification of LC3 ...

We further studied the effect of LTD4 on cellular processes which might effect autophagosome accumulation. In order to determine if LC3-II accumulation is an effect of increased autophagy or a result of a block in autophagic flux, RAW264.7 cells were treated with or without lysozome inhibitors E64d and Pepstatin A and changes in LC3-II analyzed by western blot (Figure 7A). Pepstatin A and E64d treatment appeared to increase LC3-II accumulation in cells not treated with LTD4 but to a greater degree in cells treated with LTD4. Therefore, LC3-II accumulation in LTD4 treated cells does not appear to be a result of inhibition of autophagic flux. In order to determine if the effect of LTD4 on LC3-II accumulation might be the result of mTOR inhibition, phospho-S6K was determined by western blot. Treatment of RAW264.7 cells with LTD4 was not associated with a decrease in the phosphorylation of the mTOR substrate, S6K. As a control, amino acid starvation of these cells was associated with inhibition of S6K phosphorylation (Figure 7B).

Figure 7
LTD4-induced autophagy is not regulated at the level of autophagic flux or by mTOR. (A) RAW264.7 cells were treated with or without lysozome inhibitors E64d (5 ug/ml) and Pepstatin A (5 ug/ml) and with or without LTD4 (100 nM) for 6 hr. Cells were harvested ...


In this report, we explored whether a cPLA2 initiated lipid mediator pathway participates in the induction of autophagy. We observed that a cPLA2-initiated lipid mediator pathway induces autophagy in both a murine macrophage cell line (RAW264.7) and in human primary monocytes. The induction may be ATG5-dependent and independent of autophagic flux or mTOR inhibition. The autophagy induction may be due to the action of lipid mediators generated downstream of cPLA2. In addition, lipid mediators appear to be involved in IFN-γ-induced autophagy, suggesting that cPLA2 initiated lipid mediators may be downstream effectors of IFN-γ signaling for autophagy.

Autophagy has been implicated previously in both health-promoting and disease-associated states. It has been thought to be a cellular homeostatic mechanism. Autophagy has been described in a variety of processes including neoplasia, neurodegeneration, myopathies, development, aging, innate and adaptive immune responses (5,8). Lipid mediators also may play a role in regulating immune and inflammatory responses (25). Our results have linked two events, autophagy and cPLA2-initiated lipid mediator generation, which may bridge two aspects of the innate immune response. It is suggested that lipid mediator induced inflammation may in part regulate autophagy induction.

Lipid mediators are likely key participants in the pathogenesis of inflammatory diseases (26). Our inhibitor and lipid mediator stimulation data suggest that autophagy was induced by the lipid products from two multi-enzyme pathways downstream of cPLA2. LTB4 and LTC4 are products of the 5-Lo pathway of AA metabolism. LTC4 is converted extracellularly to LTD4 and LTE4. Consequently, LTC4, LTD4, and LTE4 are together referred to as cysteinyl leukotrienes (CysLTs). On the basis of multiple assays, including western blots, immunofluorescence microscopy and electron microscopy, we have shown that a subset of lipid mediators can induce autophagy in murine macrophages. Autophagy induction was observed with CysLT D4/E4 and Prostgladin D2 (PGD2) treatment; but not with LTB4, 5-HETE or PGE2. These data suggest that specific receptor expression signaling is associated with the lipid mediator induction of autophagy in murine macrophages (27).

CysLTs and PGD2 exert their actions through activation of their receptors, such as CysLT subtype 1 receptor (CysLT1) (28), CysLT2 (29) and PGD2 receptor (DP) (30). These are seven transmembrane domain G protein-coupled receptors that bind ligands to mediate intercellular signaling of inflammatory and other cells. The mechanism by which the signaling of lipid mediators may induce autophagy is not clear. Monocytes and macrophages can produce pro-inflammatory eicosanoids and express the CysLT receptors on the cell surface (31). cysLTs modify macrophage functions. For instance, LTD4 primes alveolar macrophages to release macrophage inflammatory protein 1α, TNF-α, and nitric oxide on exposure to inflammatory mediators (31). It may induce autophagy as well.

The autophagy induced by lipid mediators may be an aspect of cellular responses against microbial invasion. Previous research suggested that several lipids could modulate the macrophage innate immune response against mycobacteria and enhance their killing (32). Both NF-κB-dependent and -independent mechanisms are involved in macrophage killing of mycobacteria and both mechanisms can be enhanced by selected lipids.

The cPLA2-initiated lipid mediator pathway that induces autophagy appears to be ATG5-dependent. It may involve autophagic vesicle elongation. Several other proteins have been shown to bind ATG5. For example, in hepatitis C virus (HCV) infection, ATG5 is an interacting protein for the HCV NS5B protein (33). ATG5 may also contribute to autophagic cell death by interacting with Fas-associated protein with death domain (FADD) (34). The mechanism by which ATG5 may participate in the cPLA2-initiated lipid mediator pathway induced autophagy remains to be determined. In conclusion, cPLA2 acting via generation of lipid mediators, appears to be capable of inducing or amplifying an autophagic response in monocytes and macrophages.


We are grateful to Shervin Esfahani for assistance with electron microscopy.

This work was supported by the intramural research program of the Clinical Center, NIH


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