Int. J. Exp. Path. (1992) 73, 783-791

Interferon-y increases macrophage phospholipidpolyunsaturation: a possible mechanism of endotoxin sensitivity

S.K. Jackson*, H. Darmani*, J.M. Stark* and J.L. HarwoodtDepartments of *Medical Microbiology, University of Wales College of Medicine, and tBiochemistry,

University of Wales College of Cardiff, Cardiff, UK

Received for publication 19 March 1992Accepted for publication 20 July 1992

Summary. Incubation of murine macrophages or the macrophage-like cell line P388D withinterferon-y in vitro induced a significant increase in the polyunsaturated fatty acid content ofphosphatidylethanolamine. These increases were time and dose-dependent, being maximal at12 hours and with 5000 U/mi interferon and were inhibited in the presence of anti-interferon-y monoclonal antibody. Interferon-y induced a significant increase in linoleate in peritonealmacrophages while in the cell line arachidonate was significantly increased. These results areof interest because such increases in the polyunsaturated fatty acid content of phosphatidyl-ethanolamine were previously shown by us to be associated with increased sensitivity toendotoxin in mice in vivo. The implications for interferon-y sensitizing to endotoxin arediscussed.

Keywords: interferon-y, phospholipid, fatty acid, macrophage, endotoxin

Endotoxin, the lipopolysaccharide (LPS)present in the outer membrane of Gram-negative bacteria, is responsible for much ofthe mortality and morbidity in seriously illhospitalized patients (Hinshaw 1984). Thedetailed mechanisms of endotoxin actionand the factors that predispose to it remainpoorly understood. It is known, however,that in experimental animals concomitantbacterial, viral or fungal infections greatlyincrease the sensitivity to endotoxin (Suter &Kirsanow 1961). In mice, for example, infec-tion with Bacille Calmette Guerin (BCG) canincrease their susceptibility to endotoxinmany thousandfold (Peavy et al. 1979).Recently, we showed that macrophages fromBCG-infected mice had an increased content

of polyunsaturated fatty acids (PUFA) intheir membrane phospholipids (Jackson et al.1989). We developed a hypothesis whichsuggested that conditions leading toincreased PUFA content would predispose toendotoxin injury (Stark & Jackson 1990).Furthermore, we showed that endotoxindepletes such phospholipids of PUFA (Starket al. 1990), suggesting that PUFA and theirmetabolites, such as eicosanoids, are impor-tant mediators of the pathophysiology ofendotoxin shock.

It has been suggested that a basis for theincreased sensitivity to LPS in mice infectedwith BCG is the production of interferon-y(Matsumura & Nakano 1988). Interferon-yis a key mediator in inflammatory reactions,

Correspondence: S.K. Jackson, Department of Medical Microbiology, University of Wales College ofMedicine, Heath Park, Cardiff CF4 4XN, UK.

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and is known to stimulate monocyte/macro-phage cells to become activated (Murray1988). Furthermore, antibodies to inter-feron-y can protect animals from the general-ized Shwartzman reaction, in which cyto-kines may induce sensitivity to a subsequentprovoking dose of LPS (Billiau et al. 1987).Cytokines have been shown to affect phos-pholipid metabolism (Meager 1990) and itseemed possible, therefore, that interferon-yand other cytokines might cause the changesin membrane phospholipid composition inBCG and other infections.

This study was undertaken to discoverwhether interferon-y could influence thelipid composition of macrophages in vitro,and so produce changes similar to those seenin vivo under conditions which sensitize toendotoxin.

Materials and methods

Reagents

All reagents were purchased from SigmaChemical Company, Poole, Dorset, UK unlessotherwise stated. The solvents and thin layerchromatography plates were from Merck-BDH Poole, UK. Tissue culture plastics andmedia were obtained from ICN Biomedicals,High Wycombe, UK.

Mice

Adult female BALB/c mice (25-30 g) pur-

chased from Jackson Laboratories were thesource for peritoneal macrophages whichwere collected by lavage with a total of 5 mlsterile phosphate buffered saline (PBS).

Cell culture

Growth medium. Cells were cultivated inDulbecco's modification of Eagle's medium(DMEM) which had been supplemented with10% fetal calf serum (FCS) and an antibioticantimycotic solution containing 100 units/ml penicillin, 100 jg/ml streptomycin and0.25 ,ug/ml amphotericin-B.

Primary cultures. Freshly collected macro-phages were washed twice with sterile PBSby centrifugation at 1000 g for 5 minutes.Red blood cells were removed by hypotoniclysis (distilled water for 20 seconds followedby addition of tenfold excess PBS). The cellswere collected by centrifugation at 1000 gfor 5 minutes and resuspended in DMEM.Cells for lipid analyses were plated on culturedishes (2 x 106 per 90 mm dish) for 12 h in a3 7°C humidified C02 incubator.

Cell lines. The cell lines used in the course ofthis investigation were the murine macro-phage-like DBA/2 lymphoid neoplasmP388D and the Chinese hamster ovary lineCH021 IA. The latter cell line was usedprimarily for production of murine inter-feron-y.

Interferon-y.

Murine interferon-y was obtained from Gen-zyme and from supernatant from theCH02 11A cell line (see below). All experi-mental results obtained were identical withinterferon from either source, but due toeconomic considerations, interferon-y fromthe cell line was used routinely.

Preparation of murine interferon-y

The CH02 1lA cell line, a transfectantexpressing the interferon-y gene, was main-tained in DMEM for 72 h at 3 7°C in ahumidified atmosphere of 5% C02. Thesecells constitutively produce murine inter-feron-y (Morris & Ward 1987) and theculture supernate was used to stimulate cellswith this cytokine. The activity of interferon-y in the culture supernates was determinedby means of a virus inhibition assay andfound to be comparabl with commerciallyavailable interferon-y (Genzyme) (1 mlsupernatant from CHO ,ell line had approxi-mately 5000 U (0.5 jug) interferon-y acti-vity). The supernatant was assayed fortumour necrosis factor (TNF) (Matthews &Neale 198 7), and the results confirmed there

784

Interferon-y increases macrophage phospholipid polyunsaturationwas no TNF activity (< 5 pg/mi). Both therecombinant murine interferon-y and theCHO cell supernatant were assayed for thepresence of endotoxin by a chromogenicLimulus amebocyte lysate assay supplied byQuadratech, Epsom. Neither source of inter-feron-y was contaminated with endotoxin(< 10 pg/ml).

Exposure of cells to interferon-y

Exposure of primary murine macrophagesand P388D cell monolayers to mouse inter-feron-y (100-1000 ng/ml; 1000-10000 U/ml; 0.2-2.0 ml ofCHO cell supernatant) wascarried out for 3-24 h in a 3 7°C, humidified5% CO2 incubator. At the end of the incuba-tion period the cells were scraped off with arubber policeman into centrifuge tubes andwashed three times with PBS by centrifuga-tion at 1000 g for 5 minutes. The cells werefinally resuspended in 1 ml deionized waterand sonicated for 30 minutes in an ultra-sonic water bath at maximum power. Oncecomplete lysis of the cells had been achieved(verified by light microscopy) phospholipidextraction was performed.

Anti-interferon-y monoclonal antibody

Experiments with interferon-y outlinedabove were also performed after incubationof the interferon-y-containing supernatantwith a monoclonal hamster anti-murineinterferon-y antibody (Genzyme). 50 Mug ofthis antibody was found to cause 80% inhibi-tion of the effect of 5000 U of interferon-y.Therefore in inhibition experiments, 50 jug ofthe monoclonal antibody was incubatedwith 1 ml (5000 U) of CHO cell supernatantfor 2 h at 22°C before addition of thissupernatant to macrophage cells.

Extraction of lipidsThe method of Garbus et al. (196 3) was used.To 1 ml of the lysed cell suspension 3.75 mlchloroform/methanol (1:2 v/v) was added,mixed thoroughly and left at room tempera-ture for 30 minutes. Then chloroform (1.25

ml) and 2 M KCl in 0.5 M KPO4 buffer, pH 7.4(1.2 5 ml) were added and the solution mixedthoroughly again. The chloroform phasecontaining extracted phospholipids wasdried down in a stream of nitrogen and thensubjected to thin layer chromatography.

Thin layer chromatography (TLC)

The dried phospholipids were dissolved in 30Mul chloroform, spotted onto Silica Gel plates(BDH) and chromatographed in a solventsystem consisting of chloroform: meth-anol: acetic acid: water (50: 30: 8: 1, v/v).Phospholipid standards were chromato-graphed on separate lanes. The resolvedphospholipids were located under ultravioletlight after staining with 8-anilino-1-naph-thalenesulphonic acid, scraped off into seal-able tubes and processed for gas liquidchromatography.

Analysis of lipid fatty acids by gas-liquidchromatography

Production of fatty acid methyl esters wascarried out using sulphuric acid (2.5%) inanhydrous methanol (1 ml). As an internalstandard, an appropriate quantity of henei-cosanoic acid (21:0) was added. After seal-ing, the tubes were heated for 2 h at 70°C.The tubes were then cooled and 2.5 ml 5%NaCl was added. The methyl esters wereextracted three times with 3 ml petroleumether (60-80°C) and then dried under astream of nitrogen and redissolved in chro-matographically pure petroleum ether.The methyl esters were analysed with a

Perkin Elmer 8600 gas chromatographequipped with a flame ionization detector. Afused silica WCOT 50 m x 0.25 mm i.d.column coated with CP-Sil 88 was used. Thecolumn temperature was ramped from 205to 25 5sC and the injector and detectortemperatures were 275 and 305°C respect-ively. Carrier gas (nitrogen) was used at 20p.s.i. Peak areas, retention times and re-sponse factors were automatically com-puted, the yields being calculated from the21:0 internal standard.

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Statistical analysis

Statistical significance was assessed by Stu-dent's t-test calculated with the Minitabstatistics program on an IBM PC.

Results

The dose of interferon-y used (5000 U/ml)was based on reported values for the biologi-cal activity of this agent (Sechler et al. 1988).In an assay of interferon-induced superoxideproduction from human monocytes, theseauthors reported a significant response with100 U/ml interferon which increased up toover 10000 U/ml. We tested a similar rangeof concentrations of IFN-y and subsequentlyused a value of 5000 U/ml for routineexperiments. Dose-response measurements(Fig. 1) showed that this concentrationinduced a significant change in the percent-age of polyunsaturated fatty acids in phos-pholipids from peritoneal macrophages. Thetime of interferon exposure was also impor-tant for the expression of increased unsatu-ration of the phospholipids. It was found tobe maximal after 12 hours (data not shown),and this time was used for all the incubationsreported.The effect of interferon-y on the growth

rate of P388D cells in culture was also

0._

40

0r,C.

o

0v

1.400 ** **

1.200 *

1.000 -

0.800 -

06000.400

0.0000. 0000 1 2 3 4

Interferon-y (log10 U/mi)

-j

5

Fig. 1. Dose-response curve for polyunsaturatedfatty acid incorporation into phosphatidylethano-lamine in murine peritoneal macrophages withinterferon-y. Values shown are means + standarddeviations for 4 experiments. *P< 0.05;**P<0.01.

determined to establish if any changes infatty acid incorporation were due simply toincreased growth. Interferon treatment at5000 units/ml did not alter the growth curve

for these cells (results not shown).The results from the analysis of phospholi-

pid fatty acid compositions for control andinterferon-treated cells are summarized inTables 1-4. From Table 1 it can be seen thatthe influence of interferon-y is to cause an

increase in the ratio of polyunsaturated tosaturated fatty acids in peritoneal macro-

Table 1. Phospholipid fatty acid composition (as percentage by weight) for murine peritonealmacrophages incubated for 12 hours with 5000 U/ml of interferon-y (IFN-y) and in untreatedmacrophages (Control). Results are expressed as mean± standard deviation of at least 5 experiments

Phospholipid 16:0 18:0 18:1 18:2 20:4 Ratio*

Control PC 26.5+11.5 27.4+10.8 24.3+3.2 16.5+3.7 5.1+5.2 0.4+0.09IFN-7PC 30.3+4.2 16.2+2.3 26.1 +2.3 20.9+5.6 3.4+2.3 G.6+0.15Control PE 17.8 +4.3 28.2 + 8.9 31.3 + 11.9 13.8 +4.4 8.6+4.4 0.48+0.16IFN-yPE 20.0+13.5 25.2 +3.9 25.5+10.4 21.4+7.6** 10.7+6.1 0.91 +0.25**Control PI 28.0+4.6 17.8+1.4 24.8+6.7 23.5+12.0 5.7+3.9 0.62+0.23IFN-y PI 20.1+7.7 18.9+2.6 30.8+8.8 24.7+12.3 5.3+4.7 0.78+0.32

16:0, Palmitate; 18:0, stearate; 18:1, oleate; 18:2, linoleate; 20:4, arachidonate. PC, Phosphatidyl-choline; PE, phosphatidylethanolamine; PI, phosphatidylinositol.

*Ratio, Ratio of polyunsaturated: saturated fatty acids.**P< 0.05 vs control.

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Interferon-y increases macrophage phospholipid polyunsaturationTable 2. Phospholipid fatty acid composition (as percentage by weight) for murine peritonealmacrophages incubated without fetal calf serum for 12 hours with 5000 U/ml of interferon-y (IFN-y) andfor untreated macrophages (Control). Results are expressed as mean ± standard deviation of at least 5experiments

Phospholipid 16:0 18:0 18:1 18:2 20:4 Ratio*

Control PC 37.5±8.6 23.7±2.1 16.8+4.2 13.6±4.5 8.1±2.4 0.36±0.11IFN-yPC 32.5±4.8 27.7±6.1 18.0±5.1 15.1+3.8 6.4±1.6 0.36±0.07Control PE 19.7±4.5 41.7±5.3 17.9±6.39 9.2±3.8 10.6±4.3 0.33±0.11IFN-yPE 21.0±6.25 39.7±2.8 17.3±2.14 11.4±5.6 10.6±5.0 0.36±0.05Control PI 27.1±7.5 41.1 ±6.1 19.1 +2.7 11.1±5.1 4.3±3.4 0.22±0.05IFN-y PI 20.4±4.4 46.9±6.2 17.8±3.7 10.5±3.13 4.2±2.3 0.22±0.05

Abbreviations as for Table 1.*Ratio, Ratio of polyunsaturated: saturated fatty acids.

Table 3. Phospholipid fatty acid composition (as percentage by weight) for the murine cell line P388Dincubated for 12 hours with 5000 U/ml of interferon-y (IFN-y) and for untreated cells (Control). Resultsare expressed as mean± standard deviation of at least 5 experiments

Phospholipid 16:0 18:0 18:1 18:2 20:4 Ratio*

Control PC 23.9±2.7 16.5±2.9 48.4±3.7 10.3±0.7 0.65±0.75 0.27±0.02IFN-yPC 25.1±2.3 17.6±4.5 44.7±10.6 11.5±1.8 0.96±1.5 0.28±0.14Control PE 19.3±6.4 26.7±3.6 37.3±6.99 7.4±3.8 9.2±3.7 0.38±0.12IFN-yPE 12.7±2.0 27.1 ±2.1 40.0±2.34 4.7± 1.1 15.4±3.6*** 0.51 ±0.01***Control PI 27.8±7.4 19.9±6.1 43.0±7.0 8.4±3.4 1.0±0.5 0.19±0.01IFN-y PI 25.6±5.6 17.9±4.3 49.0±5.7 5.4±2.5 1.9±1.1 0.16±0.007

Abbreviations as for Table 1.*Ratio, Ratio of polyunsaturated: saturated fatty acids.***P< 0.01 vs control.

Table 4. Phospholipid fatty acid composition (as percentage by weight) for murine cell line P388Dincubated without fetal calf serum for 12 hours with 5000 U/ml of interferon-y (IFN-y) and for untreatedcells (Control). Results are expressed as mean± standard deviation of at least 5 experiments

Phospholipid 16:0 18:0 18:1 18:2 20:4 Ratio*

Control PC 26.7±1.1 11.2±0.9 59.9±2.6 1.7±1.1 0.7±0.9 0.06±0.04IFN-yPC 27.4±1.2 11.8±0.8 58.4±1.8 1.4±0.4 1.0±0.6 0.05±0.03Control PE 18.6±0.9 27.2±2.1 40.3±2.2 6.1±1.7 7.7±1.2 0.3±0.04IFN-yPE 16.8±3.25 23.5±1.7 44.8±4.3 5.5±1.5 10.3±1.1** 0.4±0.07**Control PI 17.6±6.2 25.7±11.0 49.2±4.7 5.2±4.1 trace 0.17±0.08IFN-y PI 14.9 ± 6.8 30.3 ±9.1 43.9 ± 3.8 5.5 ±4.3 trace 0.25 ±0.16

Abbreviations as for Table 1.*Ratio, Ratio of polyunsaturated: saturated fatty acids.**P< 0.05 vs control.

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S.K. Jackson et al.

phages, which is statistically significant forphosphatidylethanolamine. When the cellsare cultured in the absence of calf serum,however, this change is reduced, indicatingthat the cells may be utilizing fatty acids inthe serum (Table 2). If the macrophage-likecell line P388D is substituted for mouse

peritoneal macrophages, a similar pattern ofincreases in polyunsaturated fatty acids isobserved in the presence or absence of calfserum (Tables 3 and 4). Again, this change issignificant for phosphatidylethanolamine.However, there are detailed differences in theresponse for each cell type, as might beexpected considering the distinct patterns ofthe fatty acids of individual phospholipids.Thus, the effect of interferon-y in macro-

phages is to increase the linoleate of phos-phatidylethanolamine (Table 1) while inP388D cells (Table 3) it increases the arachi-donate content significantly.

In addition, for the P388D cells thechanges induced in the fatty acid pattern ofphosphatidylethanolamine by interferon-ywere seen in both the presence and absenceof fetal calf serum. Although mammaliancells cannot synthesize linoleate, and formarachidonate only when linoleate is avail-able as a precursor, the increase in theproportions of these acids in phosphatidyl-ethanolamine could have been due to redis-tribution from other cellular lipids or, in thecase of arachidonate, because the cells con-

vert endogenous linoleate to arachidonate.Alternatively, and bearing in mind that theeffects of interferon-y were much largerwhen cells were cultivated in fetal calfserum, then at least part of the increases inlinoleate and arachidonate could have beendue to the availability of these acids from thecalf serum. Indeed, an examination of thecalf serum revealed that appreciableamounts of linoleate and arachidonate were

available for uptake. Fatty acids in serum are

carried as albumin-bound conjugates (Spec-tor 19 75) and, therefore, we tested the effectof fatty acid supply by culturing cells in theabsence of fetal calf serum but in the pres-

ence of BSA-linoleate or BSA-arachidonateconjugates.Such cells were then tested in the presence

of interferon-y and their fatty acid composi-tions examined. The results (not shown)indicated that increased availability of eitherlinoleate or arachidonate, in the form ofalbumin complexes, had no effect on the totalfatty acid composition of either macrophageor P388D cells, respectively. This suggeststhat the differences in response to interferon-y seen when cells are incubated in thepresence or absence of bovine calf serum

(Table 1 vs 2 and Table 3 vs 4) are not duesimply to the increased availability ofpolyunsaturated fatty acids in the serum;

instead, they suggest that some other factoror factors in bovine calf serum may enhance

Table 5. Phospholipid fatty acid composition for the cell line P388D incubated without fetal calf serum for12 hours with 5000 U/ml of interferon-y (IFN-y), without interferon (Control) and with interferon-y inthe presence of monoclonal antibody to interferon-y (IFN-y + mAB). Results shown are the means of 3experiments

16:0 18:0 18:1 18:2 20:4 Ratio*

Control 19.1 26.7 40.3 5.7 8.2 0.30IFN-y 16.9 22.2 43.6 5.6 11.7** 0.40**IFN-y+mAB 21.9 25.9 41.3 5.9 7.0 0.27

Abbreviations as for Table 1.*Ratio, Ratio of polyunsaturated: saturated fatty acids.**P< 0.05 vs control.

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Interferon-y increases macrophage phospholipid polyunsaturationthe uptake of such acids into the cells and/oralter internal metabolism of these com-pounds.The same increases in macrophage or

P388D cell phosphatidylethanolamine lino-leate and arachidonate content were pro-duced from both the recombinant murineinterferon-y and from the interferon-y pro-duced from the CHO cell line. Furthermore,when the interferon from either source waspre-incubated with a monoclonal antibodyto murine interferon-y before addition to thecells there was no increase in the polyun-saturated fatty acid content (representativedata for the P388D cell line is shown in Table5). This confirms that the changes in fattyacid content of the phosphatidylethanola-mine fraction of macrophages and P388Dcells was indeed caused by the interferon-y.

Discussion

This study has demonstrated that interferon-y induces a significant increase in thepolyunsaturated fatty acid content of phos-phatidylethanolamine in murine peritonealmacrophages and in the macrophage-likecell line P388D. This result is interesting for anumber of reasons. First, it suggests that hostsusceptibility to endotoxin, which we haveshown is enhanced by increased macro-phage phospholipid polyunsaturated fattyacid content (Jackson et al. 1989), may bemediated in part through interferon-y.

Secondly, the increases in polyunsatur-ated fatty acids were significant inphosphatidylethanolamine, which was thesame phospholipid class that we found toshow increased polyunsaturated fatty acidcontent in vivo in mice given BCG (Jackson etal. 1989).

Thirdly, because the results presented hereprovide direct evidence that interferon-y caninfluence phospholipid fatty acid composi-tion, this in turn implicates alterations inmembrane composition and function. Thussome of the many effects of interferon-y oncellular function may be mediated via itseffect on membrane composition.

Analysis of the fatty acid composition ofthe phospholipids present in the macro-phages and the P388D cell line shows thatpalmitate (16: 0) and stearate (18: 0) are themajor saturated fatty acids and oleate (18: 1)is the major unsaturated fatty acid. Culturedcells generally utilize the fatty acids of theserum lipids present in the medium forsynthesis of the acyl groups of their phospho-lipids rather than synthesize fatty acids denovo. However, the individual phospholipidsclearly exhibit distinct patterns in the fattyacyl moieties of their phospholipids whichare dependent on cellular metabolism ratherthan simply reflecting fatty acid abundancein the medium (Daniel et al. 1980).

Peritoneal macrophages, which are termi-nally differentiated, exhibited a statisticallysignificant change in phospholipid fatty acidcomposition with interferon only in thepresence of calf serum. This reflects theirdependence on fatty acids derived from theserum. In contrast, the cell line P388Dexhibited similar patterns of phospholipidfatty acid composition with interferon inboth the presence and absence of calf serumin the medium, although the changes weregreater in its presence.The effect of interferon-y on both macro-

phages and the P388D cell line was toincrease the unsaturated nature of the phos-pholipids with a significant increase in thepolyunsaturated fatty acid composition ofphosphatidylethanolamine. The increases inpolyunsaturated fatty acid content could beinhibited by monoclonal antibody to inter-feron-y, confirming that only interferon-ywas mediating these effects. This is in con-trast to a study which found that interferon-/ decreased the unsaturated fatty acids in thephospholipids of the mouse sarcoma cell lineS-180 (Chandrabose & Cuatrecasas 1981).However, these discrepancies may beexplained by considering the different func-tions of these interferons; interferon-# has ananti-viral effect and can inhibit the prolifera-tion of normal and transformed cells. Theinhibition of virus maturation and release ininterferon-# treated cells might be expected

789

S.K. Jackson et al.to be facilitated by increased membranephospholipid saturation and rigidity (Chat-terjee et al. 1982). In contrast, interferon-y isa product ofT-lymphocytes and has a centralrole in the immune response (Billiau &Dijkmans 1990). It is a macrophage activat-ing factor and a differentiation stimulus formacrophages and other cell types. One effectof interferon-y stimulation ofmacrophages isto increase the expression of MHC class IIantigens, which are required for recognitionof foreign antigens by TH-cells. In addition toMHC antigen expression, interferon-y modu-lates the synthesis and expression of othercell-surface antigens, enzymes and inflam-matory mediators (Murray 1988). Increasedmembrane phospholipid unsaturation, andhence increased membrane fluidity would beexpected to facilitate these stimulatoryevents (Yeagle 1989).

Moreover, interferon-y can 'prime' macro-phages for subsequent 'triggering' by a

second agent to produce cytotoxic or

tumoricidal activity (Hamilton & Adams1987). Endotoxin (LPS) is one of the mostpotent second 'triggering' agents of inter-feron-primed macrophages (Hamilton &Adams 1987). In this regard, interferon-y isacting to induce the cell to respond toLPS. It has been shown that interferon-yincreases the ability of macrophages to pro-

duce endogenous mediators, such as TNF, inresponse to LPS (Gifford & Lohmann-Matthes1987).

Because the activated macrophage plays acentral role in the development of endotoxicshock (Morrison & Ryan 1987), it is possiblethat increases in interferon-y may induce astate of heightened sensitivity to endotoxin.Furthermore, we showed that conditionswhich sensitized mice to endotoxin in vivo(mycobacterial infection) resulted in in-creased polyunsaturated fatty acid content ofmacrophage phospholipids (Jackson et al.1989). These same conditions are known toinduce interferon-y (Rook et al. 1987).

In this study we have found, in bothcell types, that interferon-y producedmore polyunsaturated acyl groups in

phosphatidylethanolamine than in phospha-tidylcholine. Phosphoglycerides have beenshown to contain predominantly saturatedacyl groups at position 1 and either monoun-saturates or polyunsaturates at position 2(Gurr & Harwood 1991). Therefore, themajor differences in composition from ourresults would be expected at position 2.These differences may be interesting inregard to membrane fluidity since phospho-glycerides are known to possess an asym-metry in the membrane bilayer (Op DenKamp 1979). Interestingly, interferon treat-ment of the macrophages resulted in signifi-cantly increased incorporation of linoleicacid into phosphatidylethanolamine, whilein the cell line arachidonic acid was signifi-cantly increased. It is possible that theP388D cells synthesize arachidonic acidfrom linoleic acid, rather than transfer itfrom other lipids, whereas macrophages donot have as much desaturase activity andcannot modify their arachidonate composi-tion in this way.The present study has shown that inter-

feron-y induces precisely the same fatty acidchanges in macrophage phosphatidyletha-nolamine in vitro as we observed after BCGinfection in vivo. Furthermore, we previouslyshowed the same class of polyunsaturatedfatty acids were utilized and decreased inBCG-primed mice after administration ofendotoxin, indicating that this altered pat-tern of fatty acid contents was indeed impor-tant in the sensitization to endotoxin in vivo(Stark et al. 1990). In addition, we have alsodemonstrated that a significant increase inthe unsaturation of phosphatidylethanola-mine is also produced in a macrophage-likecell line. It has been shown that interferon-ycan restore the lipopolysaccharide binding ofunresponsive mouse lung macrophages(Akagawa & Tokunaga 1985). These resultssuggest that interferon-y might play a role inthe regulation of cellular responses to endo-toxin in vivo, and that at least some of itsstimulatory effects may be induced via itsinfluence on the membrane lipid unsatu-rated fatty acid content.

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Interferon-y increases macrophage phospholipid polyunsaturation 79IAcknowledgement

We would like to thank the Medical ResearchCouncil for the award of a grant.

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