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* Department of Internal Medicine III, Clinical Division of Endocrinology and Metabolism and
Institute of Immunology, University of Vienna Medical School, Vienna, Austria; and
CeMM–Center of Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
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Abstract |
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1/calcium signaling has been shown so far. In this study, we investigated PUFA-mediated changes in downstream T cell signal transduction. We show that among the mitogen-activated protein kinase families activation of c-Jun NH2-terminal kinase, but not phosphorylation of extracellular signal-regulated kinase-1/-2 or p38 is inhibited. CD3/CD28-induced activity of NF-AT was markedly reduced by PUFA treatment, while activation of other nuclear receptors (AP-1 and NF-
B) remained unaltered. Furthermore, IL-2 promoter activity, IL-2 and IL-13 mRNA levels, IL-2 secretion, and IL-2R 
-chain expression were significantly diminished by PUFA treatment, whereas the expression of IFN-, IL-4, IL-10, and CD69 remained essentially unaffected by PUFAs. In conclusion, PUFA treatment of T cells inhibits selectively c-Jun NH2-terminal kinase and NF-AT activation, resulting in diminished production of IL-2 and IL-13. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) have been shown (6, 7, 8, 9, 10). Because of the central importance of T cell activation for the immune response, PUFA effects on T lymphocyte signal transduction are of particular interest.
T cell activation is mediated via the TCR-CD3 complex. Stimulation of the TCR by Ag presented by the MHC rapidly leads to tyrosine phosphorylation and activation of adapter molecules and enzymes, including the linker for activation of T cells (LAT) and phospholipase C (PLC) (11). PLC activity elicits a rise in cytoplasmic calcium concentration, a key event of T cell activation (12). Activation of further signaling mediators is partially dependent on costimulatory signals that are triggered via costimulatory cell surface receptors such as CD28 or CD59 (13, 14). In consequence to early protein phosphorylation steps and calcium response, mitogen-activated protein kinases (MAPKs) are activated by phosphorylation. The three major families of MAPKs, extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), and p38 MAPK, are regulated by distinct but cross-talking signaling cascades (15). Such signals culminate in the activation of transcription factors such as NF-AT, AP-1, and NF-B (16, 17, 18). These transcription factors bind recognition sites within promoter sequences to induce transcription of cytokines, including IL-2, the major T lymphocyte proliferation factor (19). Thus, T cell stimulation leads to IL production and proliferation, thereby promoting the adaptive immune response.
PUFA treatment of human T cells diminishes TCR/CD3-induced calcium response and proliferation (20, 21, 22, 23). Furthermore, a decrease in IL-2 production in Jurkat T cells treated with PUFAs has recently been reported (24). However, the particular events of T cell signal transduction downstream of the calcium response that are affected by PUFAs are still unresolved. In this study, we show that PUFAs inhibit T cell activation on the MAPK and the nuclear factor level in a highly selective manner, specifically blocking JNK and NF-AT activation. Furthermore, expression of IL-2, IL-2R -chain, as well as IL-13 were significantly diminished in PUFA-treated peripheral blood T lymphocytes (PBTLs), while the expression of other cytokines and activation markers remained unaffected. By providing detailed insight into the influence of PUFAs on human T cell activation, these data considerably enhance our understanding of how these substances exert their immunomodulatory effects.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs were obtained as follows: OKT3 (anti-CD3
) from Ortho Pharmaceuticals (Raritan, NJ); anti-ERK and anti-phospho-ERK, anti-JNK1, and anti-p38 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-p38 and anti-phospho-c-Jun from New England Biolabs (Beverly, MA); MEM-43 (anti-CD59) from Monosan (Uden, The Netherlands); HRP-labeled 4G10 (anti-phosphotyrosine) and anti-phospho-JNK from Upstate Biotechnology (Lake Placid, NY); Leu28 (anti-CD28), and PE-labeled anti-CD25 and anti-CD69 from BD PharMingen (San Jose, CA); goat anti-mouse (GAM) and F(ab')2 of GAM IgG from Sigma-Aldrich (St. Louis, MO); HRP-labeled GAM IgG from Bio-Rad (Hercules, CA); and HRP-labeled goat anti-rabbit IgG from Accurate (Westbury, NY).
Cells and fatty acid treatment
Human Jurkat T cell lines E6-1 and J7.7 were purchased from American Type Culture Collection (Manassas, VA). To obtain PBTLs, mononuclear cells were purified from buffy coats of peripheral blood given by healthy volunteers using Ficoll-Paque (Amersham, Uppsala, Sweden) density gradient centrifugation. Subsequently, PBTLs were isolated by rosetting with neuraminidase-treated sheep erythrocytes (Dade Behring, Marburg, Germany). The obtained population contained >90% CD3-positive cells. Cells were treated with PUFAs as described in detail elsewhere (20). Briefly, PBTLs and Jurkat cells were incubated for 1–3 days in serum-free IMDM (Invitrogen, Groningen, The Netherlands) containing 0.4% (w/v) BSA (fraction V; Sigma-Aldrich) supplemented with free fatty acids at a concentration of 6.25/50 µM (final concentration of solvent ethanol 
0.05% (v/v)). Viability of cells after PUFA treatment was >95% as estimated by trypan blue exclusion (data not shown). Notably, cells were washed three times before stimulation, thereby eliminating any free fatty acid during stimulation and subsequent determinations.
Analysis of MAPKs
Cells were washed and resuspended to 2 x 107 Jurkat cells/ml or 5 x 107 PBTLs/ml in HBSS (Invitrogen) including 10 mM HEPES (pH 7.4). Phosphorylation of ERK was induced by incubation of cells with 5 µg/ml OKT3 for 1 min followed by cross-linking with 10 µg/ml F(ab')2 of GAM IgG. For stimulation via CD59, cells were preincubated on ice with 5 µg/ml anti-CD59 mAb and equilibrated to 37°C for 2 min before addition of cross-linker. For activation of JNK and p38, cells were preincubated with anti-CD59 or anti-CD28 mAbs (5 µg/ml for 15 min on ice) and OKT3 (1 min at 37°C) and subsequently stimulated by cross-linking. Alternatively, cells were stimulated by 25 ng/ml PMA (Alexis, San Diego, CA) plus 1 µM ionomycin (Sigma-Aldrich). Stimulation was stopped by addition of ice-cold washing buffer. Cells were pelleted by short centrifugation (12,000 x g for 20 s) and lysed on ice for 30 min in TBS (pH 7.4) containing 1% Nonidet P-40 (Pierce, Rockford, IL), phosphatase (1 mM sodium orthovanadate, 10 mM NaF, 5 mM sodium pyrophosphate, 25 mM 
-glycerophosphate, and 5 mM EDTA), and protease inhibitors. Nuclei were removed by short centrifugation; supernatants are referred to as whole cell lysates. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Hybond ECL, Amersham, U.K.). Phosphorylation of JNK, ERK, and p38 was assessed using Abs directed against phosphorylated isoforms. Phosphorylated p42 ERK-2 was additionally distinguished from the unphosphorylated form by a shift in electromobility. Ags were detected by HRP-labeled secondary Abs. Chemiluminescence was generated using the BM chemiluminescence blotting substrate kit (Roche, Mannheim, Germany) and quantified on a Lumi-Imager (Roche). JNK of 5 x 106 cells/sample was precipitated and assayed for its activity to phosphorylate c-Jun using a stress-activated protein kinase/JNK Assay kit (New England Biolabs) following the provided protocol.
Luciferase reporter assays
Jurkat cells (clone 41-19) stably transfected with an IL-2 promoter (position -583 to +40) luciferase reporter construct (25) were generously provided by T. Baumruker (Novartis Research Institute, Vienna, Austria), cultured in standard medium including G418-sulfate (0.8 mg/ml; Alexis, Laeuflingen, Switzerland), and fatty acid-treated for 2 days as described above. Cells were stimulated in triplicates in HBSS without phenol red (Invitrogen) supplemented with 10 mM HEPES (pH 7.4) in 96-well flat-bottom polystyrene OptiPlates (Packard, Groningen, The Netherlands) precoated with anti-CD3 plus anti-CD59 or anti-CD28 mAbs for 4 h at 37°C. Subsequently, cells were lysed by the addition of 5x lysis buffer (Promega, Madison, WI). After automatic addition of luciferin substrate solution (Promega), fluorescence attributable to luciferase activity was determined on a Luminoskan RS (Lab Systems, Helsinki, Finland).
Jurkat E6-1 cells were transiently transfected with NF-AT, AP-1, and NF-B firefly luciferase reporter constructs (Stratagene, La Jolla, CA) plus Renilla luciferase expression vector (Promega) using Lipofectamine transfection (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were treated for 24 h with 50 µM fatty acids and subsequently stimulated by plate-bound anti-CD3 plus anti-CD28 mAbs on 24-well plates. Luciferase luminescence was detected in a luminometer (Berthold, Bad Wildbad, Germany) applying dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions. To quantify transcription factor activation (arbitrary units) firefly luciferase light units were divided by Renilla luciferase light units that served as a control for transfection efficiency. Renilla luciferase light units were similar in all investigated samples (data not shown).
mRNA levels and secretion of ILs, surface protein expression
Fatty acid-treated (50 µM for 3 days) PBTLs were stimulated in 24-well plates by immobilized anti-CD3 plus anti-CD28 mAbs for 14 h for mRNA quantitation and for 24 h for determination of IL secretion and surface protein expression. Total RNA was isolated using TRI reagent (Sigma-Aldrich). Hybrids were generated by incubating 10 µg of total mRNA with 32P-labeled ribonucleotide antisense probes generated with an in vitro transcription kit and the human cytokine multiprobe template set hCK-1, and subsequently treated with RNase A+T1 from the RNase protection kit (all BD PharMingen). Denatured samples were separated on a 6% polyacrylamide-urea gel, dried and exposed to a storage phosphor screen, recorded by a PhosphorImager, and analyzed using the ImageQuant program (all Molecular Dynamics, Sunnyvale, CA). Secreted cytokines (IL-2, IL-4, IL-10, and IFN-1) and expression of surface activation markers (CD25, CD69) were determined by standard ELISA techniques and by surface immunofluorescence and flow cytometry, respectively.
Statistics
Data are presented in means ± SEM. Comparisons were performed by two-tail unpaired Student’s t test and a p < 0.05 was considered to be statistically significant.
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Results |
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The influence of PUFAs on early protein tyrosine phosphorylation leading to diminished calcium response has been shown previously (20, 21). To study the impact of PUFA treatment on downstream T cell signaling, we first analyzed effects on the level of MAPK activation. Activation of MAPKs relies on phosphorylation of specific tyrosine and threonine residues. In Jurkat T cells, treatment with polyunsaturated eicosapentaenoic acid (20:5,3) caused a striking inhibition of JNK activity, assayed by phosphorylation of c-Jun, when stimulated for 15 min via CD3 plus different costimulatory signals (CD59, CD28, PMA) compared with cells treated with saturated stearic acid (18:0; Fig. 1A) that served as a control. Quantification of the JNK activity assays revealed an inhibition of JNK activity in PUFA-treated T cells when stimulated via CD3 costimulation (CD3/CD59, 22 ± 10% of control; CD3/CD28 31, ± 6; CD3/PMA 27, ± 15). However, JNK activity in PUFA-treated cells was comparable to controls when stimulated by PMA/ionomycin (92 ± 9% of control). Time-resolved analysis of CD3/CD28-induced JNK phosphorylation in control-treated Jurkat T cells revealed successively increasing phosphorylation of pp46 (corresponding to JNK1) and pp54 (corresponding to JNK2), with a peak at 10 min of stimulation followed by a minimal decrease until 20 min. In contrast, phosphorylation of both JNK1 and JNK2 was markedly diminished in PUFA-treated cells compared with controls starting at 5 min of stimulation and thereafter (Fig. 1A). In contrast to JNK, PUFA treatment did not affect phosphorylation of p38 in Jurkat T cells, independent of the activating stimuli and the investigated time points (Fig. 1B). Phosphorylation of ERK-1 (pp44) and ERK-2 (pp42), induced via CD3 or CD59, was not altered by PUFA treatment, as demonstrated by a phospho-ERK-specific Ab as well as by the altered electromobility of phosphorylated ERK-2 (Fig. 1C). In addition to Jurkat T cells, investigation of MAPK phosphorylation in PBTLs also revealed predominant inhibition of JNK by PUFA treatment (Fig. 2). The difference between controls and PUFA-treated cells was most striking at 5 and 10 min of CD3/CD28 stimulation. (Fig. 2A). Hence, PUFA treatment of T cells selectively inhibits JNK activation but does not interfere with phosphorylation of other MAPKs.
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Production of the central T cell growth factor IL-2 critically depends on activation of the transcription factors NF-AT, AP-1, and NF-B (19). To investigate which of these transcription factors is affected by PUFA enrichment of T cells, we transiently transfected Jurkat T cells with plasmids containing a reporter (luciferase) gene controlled by respective nuclear factor binding sites. Stimulation of these cells via CD3/CD28 revealed that NF-AT activation was markedly reduced in 20:5,3-treated cells compared with 18:0-treated controls (Fig. 3). AP-1 activation was not reduced in PUFA-treated cells, despite the diminished JNK activity described above. NF-B activity remained unchanged as well (Fig. 3). When early T cell signaling was bypassed by stimulation with PMA/ionomycin, no differences of NF-AT and AP-1 activation between PUFA (20:5) and control(18:0)-treated cells were detected. NF-B activity tended to be diminished in PUFA-treated T cells when induced via PMA/ionomycin, but this inhibition failed to reach statistical significance (p = 0.08 vs 18:0, Fig. 3).
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To investigate the effects of PUFAs on cytokine expression, we stimulated PBTLs via CD3 plus coreceptors and analyzed mRNA expression and secretion of a variety of cytokines. The amount of IL-2 mRNA was markedly reduced in PUFA (20:5,3)-treated PBTLs compared with control(18:0)-treated cells, stimulated via CD3 and CD28. Also IL-13 mRNA was reduced but none of the other analyzed cytokine mRNAs (IL-4, IL-9, IL-10, IFN-; Fig. 4). In accordance to the data on the mRNA level, the secretion of IL-2 but none of the other investigated cytokines (IL-4, IL-10, IFN-) was markedly inhibited in PUFA (20:5,3; 20:4,6)-treated compared with control-treated PBTLs (18:0; Fig. 5) when stimulated via CD3 plus CD59 or CD3 plus CD28. The extent of the relative inhibition was very similar in CD3/CD28- and in CD3/CD59-stimulated cells, demonstrating that the PUFA effect does not depend on a distinct costimulatory signal.
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-chain
Production of cytokines regulates proliferation of T cells in concert with induced expression of high-affinity cytokine receptors. TCR or cytokine-induced IL-2R -chain (CD25) assembles with and subunits to form the high-affinity IL-2R (26). Induction of CD25 is controlled on transcriptional level by an array of nuclear factors, including NF-AT and AP-1 (27). CD3-induced surface expression of CD25 was markedly reduced by PUFA treatment, independent of the triggered costimulatory receptor (Fig. 7). No significant difference in the expression of the surface activation marker CD69 was detected between control and PUFA-treated cells (Fig. 7), demonstrating a selective inhibition by PUFAs also concerning activation marker expression.
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Discussion |
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PUFAs of the n-3 and n-6 series inhibit TCR/CD3-induced calcium response and diminish IL-2 production of T cells (20, 21, 22, 23, 24) but the signaling events mediating the reduced IL-2 expression in PUFA-treated T cells have remained unknown so far. PUFA treatment selectively inhibited CD3/costimulation-induced JNK activation on the MAPK level (Figs. 1 and 2). There were no differences in JNK activity between PUFA and control-treated T cells when cells were stimulated with PMA/ionomycin, indicating that very proximal signaling events are responsible for PUFA-mediated inhibition. PUFA treatment did not influence p38 MAPK or ERK-1/-2 phosphorylation in Jurkat T cells in our study in contrast to published data on ERK (28). Differences in culture conditions during fatty acid treatment could contribute to this discrepancy. When using dialyzed FBS and defatted BSA as in Ref. 28 , only the added species of free fatty acids are present during the treatment. In contrast, mainly saturated and monounsaturated fatty acids are bound to nonstripped BSA preparations as used in our studies, giving a "natural" background of fatty acids during the experiment. Our data on unchanged ERK-1/-2 phosphorylation are strengthened by analysis of two different stimuli (CD3 and CD59) in Jurkat T cells and by data on ERK phosphorylation in PBTLs that were both analyzed in a time-dependent manner. All of these experiments did not reveal alterations in ERK-1/-2 phosphorylation between PUFA and control-treated T cells (Figs. 1C and 2C). Moreover, our results on ERK phosphorylation correspond to data on stimulated CD69 surface expression, which critically depends on the Ras/ERK pathway activation (29) and remained unaltered by PUFA treatment as well (Fig. 7).
Among the analyzed nuclear factors, only activation of NF-AT was markedly inhibited by PUFA treatment. Decreased JNK activity could have been expected to decrease activity of AP-1 and NF-B, as well (30), but we did neither detect a decrease in stimulated AP-1 or NF-B activity (Fig. 3) nor in degradation of IB-, the NF-B inhibitory protein (data not shown). The residual JNK activity of 
30% of control values in PUFA-treated T cells (Figs. 1 and 2) appears to be sufficient to phosphorylate some but not all JNK substrates, thereby activating different downstream signaling pathways to a varying extent. Notably, NF-AT has been demonstrated to be the critical mediator of JNK signaling in mature T cells (31, 32). Therefore, the PUFA-mediated block in JNK activation could provoke a selective inhibition of NF-AT activation in T cells as shown here.
Activation of JNK and NF-AT requires a calcium response (12, 16, 33). In contrast to TCR/CD3-triggered stimulation, activation of JNK and NF-AT was not inhibited following stimulation that included the calcium ionophore ionomycin (Figs. 1 and 3). Ionomycin directly induces a rise in cytoplasmic calcium concentration, thereby bypassing any blocked signaling events located further upstream. The rescue from PUFA-mediated inhibition by ionomycin stimulation strongly indicates that decreased calcium response is the primary cause for decreased JNK and NF-AT activation in PUFA-treated T cells. Moreover, impaired calcium response in PUFA-treated T cells could also underlie the reduced IL-2 promoter activity as suggested by results from experiments with various fatty acid species whose effects on calcium response and IL-2 promoter activity were strikingly similar to each other (Fig. 6; cf Ref. 20).
PUFA treatment primarily reduced IL-2 mRNA whereas mRNA expression of other cytokines, including the main Th2 cytokine IL-4, remained unaffected (Fig. 4). This finding is consistent with in vivo effects of PUFAs in mice (34). Since Th2 cells have been shown not to require JNK for activation upon Ag stimulation (35) and NF-AT is of particular importance for expression of Th1 pattern of genes (36), the selective inhibition of JNK and NF-AT could explain why the production of the major Th2 cytokines IL-4 and IL-10 was not altered by PUFA treatment. As an exception to the rule, the Th2 cytokine IL-13 mRNA was also diminished by PUFA treatment. Importantly, IL-13 is a mediator of IgE production in patients with bronchial asthma (37). Thus, reduced IL-13 levels may contribute to the beneficial effects of PUFAs in such patients (38, 39), although recent data demonstrate that IL-13 is of only minor importance for Th2 responses when compared with IL-4 (40). Also IFN- expression may be expected to be affected by PUFA treatment, since its expression is also controlled by NF-AT (36), and Wallace et al. (34) found PUFA-mediated inhibition of Con A-stimulated IFN- production in murine spleen lymphocytes. The varying effect of PUFA treatment on the expression of genes with NF-AT binding sites in their promoters could be due to the fact that PUFAs inhibited activity of a consensus NF-AT binding site only to 50% of control values in our experiments (Fig. 3). This partial inhibition might be relevant for transcription of some NF-AT-regulated genes while expression of others could remain unaffected depending on the relative importance of NF-AT transactivation on gene transcription. Moreover, the selective effect of PUFAs on cytokine expression could also be attributed to the involvement of different NF-AT family members.
Inhibition of stimulated IL-2R -chain (CD25) expression and thus formation of the high-affinity IL-2R by PUFAs may contribute to the well-established inhibitory effect of PUFAs on T cell proliferation (22, 23). Notably, expression of CD25 is regulated in a manner similar to IL-2 including binding sites for NF-AT in the promoter sequences of the respective genes (27, 41). The inhibitory effects of PUFAs on IL-2 and CD25 expression as well as IL-2 promoter activity were independent of the triggered costimulatory receptor (CD28 or CD59, Figs. 5–7), suggesting that PUFAs primarily impair TCR/CD3-mediated signaling. IL-2 production and CD25 expression was not fully rescued by PMA/ionomycin stimulation (Ref. 24 and data not shown), an observation that points to an additional inhibitory mechanism elicited by PUFA treatment. A possibility could be PUFA-mediated activation of peroxisome proliferator-activated receptors (PPARs). PPARs are nuclear factor receptors that bind fatty acid derivatives but inhibitory effects on T cells have only been demonstrated with high-affinity ligands (10, 42, 43, 44). JNK and NF-AT activity was not inhibited in PUFA-treated T cells stimulated via PMA/ionomycin (Figs. 1 and 3), essentially ruling out a significant impact of PPARs in the PUFA-mediated inhibition of TCR/CD3-induced activation of JNK and NF-AT.
The diminished calcium response of PUFA-treated T cells was recently shown by us to be caused by displacement of the crucial adapter molecule LAT from lipid rafts (21). Thus, previous results along with those of the present study strongly indicate that PUFA-induced alterations of lipid rafts that lead to diminished calcium response also underlie impaired T cell downstream signaling such as JNK and NF-AT activation. Concluding from data of this and previous studies (20, 21, 45), we suggest the following model of how PUFAs affect T cell activation: PUFAs are incorporated into raft lipids, leading to displacement of LAT from rafts (45). Altered LAT membrane subdomain distribution impairs PLC and calcium signaling (21). Reduced calcium response then interferes with JNK and NF-AT activation. The block of these and possibly other signals impairs IL-2 production and cell surface expression of high-affinity IL-2R, thereby providing a basis for reduced T cell proliferation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas M. Stulnig, Department of Internal Medicine III, Division of Endocrinology and Metabolism, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail address: thomas.stulnig{at}akh-wien.ac.at
3 Abbreviations used in this paper; PUFA, polyunsaturated fatty acid; JNK, c-Jun NH2-terminal kinase; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinase; PBTL, peripheral blood T lymphocyte; PLC, phospholipase C; ERK, extracellular signal-regulated kinase; GAM goat anti-mouse; PPAR, peroxisome proliferator-activated receptor.
Received for publication November 13, 2002. Accepted for publication April 3, 2003.
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References |
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B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
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(PPAR) is expressed in resting murine lymphocytes: the PPAR in T and B lymphocytes is both transactivation and transrepression competent. J. Biol. Chem. 277:6838. and immunoregulation: PPAR mediates inhibition of helper T cell responses. J. Immunol. 164:1364.This article has been cited by other articles:
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S. R. Shaikh and M. Edidin Immunosuppressive effects of polyunsaturated fatty acids on antigen presentation by human leukocyte antigen class I molecules J. Lipid Res., January 1, 2007; 48(1): 127 - 138. [Abstract] [Full Text] [PDF]
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was determined from whole cell lysates and served as quantity control. Lower panel, Phospho-JNK was determined by immunoblotting whole cell lysates with anti-phospho-JNK Ab. Phospho-p38 (B) and phospho-ERK-1 (pp44) and phospho-ERK-2 (pp42) (C) were determined by immunoblotting whole cell lysates with phospho-specific Abs. JNK1, p38, and ERK-2, respectively, were detected from stripped membranes. Representative blots from three to five independent experiments are shown.
, p < 0.1; *, p < 0.05; and **, p < 0.01. PUFA- and control-treated unstimulated samples were not distinguishable in amounts of any investigated cytokine.
, •) or polyunsaturated (
, 
, 
) fatty acids for 2 days. Cells were stimulated for 4 h on plates coated with indicated Abs, followed by determination of luciferase activity. Stimulation indices were calculated by dividing luciferase activities of stimulated cells by those of unstimulated cells. Results of lipid-modified cells were related to those of unmodified solvent control cells and expressed in percentage of control (means ± SEM from three independent experiments).