Molecular basis for the immunosuppressive action of

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1 Immunology Moleular basis for the immunosuppressive ation of steari aid on T ells P. W. TEBBEY & T. M. BUTTKE Department of Mirobiology and Immunology, East Carolina University Shool of Mediine, Greenville, North Carolina, U.S.A. Aeptedfor publiation 6 Marh 199 SUMMARY Studies were performed to determine the mehanism by whih steari aid (18: ) seletively inhibits T-dependent immune responses in vitro. Inubation of mitogen-ativated B and T ells with 18: resulted in dissimilar patterns of inorporation of the saturated fatty aid into their membranes. High-performane liquid hromatography (HPLC) analyses of T ells showed an aumulation of desaturated 18:-ontaining phosphatidylholine (PC) that replaed normal ellular PC. Less signifiant quantities of the same PC speies were seen to aumulate in B-ell membranes; rather, they inreased their proportion of olei aid (18: 1)-ontaining PC. The different lipid ompositions of the lymphoyte ell membranes after exposure to 18: were orrelated with their plasma membrane potentials. In T ells, the aumulation ofdesaturated, 18: -ontaining PC oinided with a rapid (within 8 hr) ollapse of membrane integrity, as determined by flow ytometry. The ollapse of membrane integrity was found to be time and dose dependent. No suh depolarization was observed in B ells whih, by virtue of their desaturating ability, were able to avoid inorporating large amounts of desaturated 18: -ontaining phospholipids into their membranes. It is proposed that a lak of stearoyl-coa desaturase in T ells preludes them from desaturating exogenously derived 18:, thus leading to inreased proportions of 18:-ontaining desaturated PC in their ell membranes. The inreased abundane of this PC speies may enhane membrane rigidity to an extent that plasma membrane integrity is signifiantly impaired, leading to a loss of membrane potential and ultimately ell funtion and viability. INTRODUCTION Various saturated and unsaturated fatty aids have been reported to modulate the immunologial role of lymphoytes both in vitro and in vivo (Erikson, 1986; Gurr, 1983; Meade & Mertin, 1978). Of partiular interest is the finding that some fatty aids an seletively inhibit T-ell-mediated funtions in the relative absene of effets on B ells. For example, steari aid (18: ) has been shown to be a potent inhibitor of phytohaemagglutinin (PHA)-dependent T-lymphoyte proliferation while having little effet on lipopolysaharide (LPS)- indued B-ell proliferation (Buttke, 1984). Further, 18: suppresses primary in vitro antibody responses to T-dependent but not T-independent antigens (Pourbohloul, Mallett & Buttke, 1985). T-helper ells are suggested to be the priniple target of suh immunosuppression, wherein interleukin-2 (IL-2) prodution is inhibited (Pourbohloul & Buttke, 199). Lastly, 18: has been shown to be ytotoxi for T ells but not B ells (Buttke & Cuhens, 1984). Correspondene: Dr T. M. Buttke, Dept. of Mirobiology and Immunology, East Carolina University Shool of Mediine, Greenville, NC , U.S.A. Previous workers have proposed that saturated fatty aids may, via their inorporation into phospholipids, perturb the membrane struture, thus adversely affeting membrane-bound enzymes (Stubbs & Smith, 1984; Mahoney et al., 1977; Tsang, Weyman & Smith, 1977). However, the differential effets of 18: on T and B ells suggest a fundamental differene between the two lymphoyte types in either lipid metabolism or membrane lipid omposition. Indeed, further investigation has revealed that B ells an desaturate 18: to 18: 1, whereas T ells an not (Buttke et al., 1989). This finding suggests that B ells are better able to maintain a funtional balane between saturated and unsaturated fatty aid levels in their membranes by virtue of their having higher levels of stearoyl-coa desaturase, the enzyme responsible for onverting 18: to 18: 1 (Buttke et al., 1989). Suh results may signify an important differene in the regulation of lipid metabolism during B- and T-lymphoyte maturation, whih may be related to their in vivo funtion. The present studies attempted to define the biohemial basis for the seletive ytotoxi effet of 18: on T ells. Sine it has been shown previously that the inability oft ells to onvert 18: to 18: 1 oinides with an aumulation of desaturated 379

2 38 P. W. Tebbey & T. M. Buttke phospholipid speies (Buttke et al., 1989), it seemed possible that suh hanges in membrane lipid omposition might lead to impaired membrane integrity (Kuypers et al., 1984; Lange et al., 198). Indeed, the aumulation ofdesaturated 18: -ontaining phospholipid speies results in the loss of membrane potential in a signifiant proportion of T ells, implying perturbation of membrane integrity and loss of viability. METHODS AND MATERIALS Isolation and ulture of lymphoytes Male and female BALB/ mie, 8-12 weeks of age, were obtained from Charles River Breeding Laboratories (Raleigh NC). Spleen ell suspensions were depleted of erythroytes and marophages by NH4C1 lysis and passage of ells through glasswool olumns, respetively (Buttke, Mallett & Cuhens, 1983). The lymphoyte-enrihed suspension was further purified into immunoglobulin-positive (Ig+) (B ells) and Ig- ell populations by inubation on rabbit anti-mouse Ig (Aurate Chemial Co.)-oated Petri plates (Buttke et al., 1983). The non-adherent Ig- population was subsequently inubated in suspension with a monolonal mouse alloantisera direted against the Thy-1 2 antigen, followed by a seond round of seletion on the rabbit anti-mouse Ig-oated Petri plates (Buttke et al., 1983). The adherent populations reovered from the two rounds of positive seletion were > 95% viable, and onsidered to be highly enrihed populations of B and T ells based on several riteria (Buttke et al., 1983). Lymphoytes were ultured in 96-well, round-bottomed, mirotitre plates (Linbro, Flow Laboratories In., MLean, VA) at a onentration of 2-5 x 16 ells per ml of ulture medium. The ulture medium onsisted of RPMI- 164 ontaining 2% NaHCO3, peniillin (5 U/ml), streptomyin (5 pg/ ml) and 5% fetal bovine serum. T lymphoytes were stimulated with phytohaemagglutinin (PHA; Difo) at a onentration of 5 pg/ml and B lymphoytes with lipopolysaharide (LPS; Difo) at 5 pg/ml. Cell ultures were inubated at 37 in a humidified atmosphere ontaining 6% CO2 94% air. Albumin-omplexedfatty aids Steari aid (18: ), bovine serum albumin (BSA) and diatomaeous earth were obtained from Sigma Chemial Co. (St Louis, MO). The fatty aid was adsorbed onto diatomaeous earth and subsequently omplexed to BSA to yield fatty aid: BSA ratios of (Buttke, 1984). Phospholipid extration and analysis Aliquots of 5 x I7 B or T lymphoytes were olleted by entrifugation at 25 g for 1 min, and the pellets were washed one with ie-old ulture medium. Lipids were extrated by the addition of I ml of methanol and 2 ml of hloroform (Folh, Lees & Sloan-Stanley, 1957). After 1 hr, -6 ml of - I M KCI was added and the suspension was entrifuged at 2 g for 5 min to separate the aqueous and organi phases. The lower hloroform layer was reovered and the solvent evaporated under nitrogen in a 37 water bath. Reovered lipids were dissolved in - I ml of 2: 1 (v/v) hloroform-methanol prior to analysis. Total lymphoyte phospholipids were separated by thinlayer hromatography (TLC) on silia gel 6 plates developed with hloroform-methanol-aeti aid-h2 (75:45:12:5-5). TLC plates were predeveloped with aetone followed by heating at 11 for at least 1 hr to overome adverse effets of humidity on phospholipid separation. One spotted with sample, the TLC plates were inubated for a seond time at 11 for 3 min. The plate was then transfered diretly to a pre-equilibrated (approximately 1 hr) TLC hamber for development. Separated phospholipids were loalized using 12 vapour. Phosphatidylholine (PC) frations were eluted from the silia gel with 1 ml of methanol, onverted to diaylglyerides by phospholipase C digestion (Mavis, Bell & Vagelos, 1972), and dinitrobenzylated (Takamura et al., 1986). The resultant diaylglyerobenzoates were separated into moleular speies using a Bekman System Gold HPLC fitted with a 4-6 x 45 mm preolumn and a Bekman 4-6 x 25 mm analytial olumn, both paked with S ym C- 18 Ultrasphere (Bekman). Elution with I ml/min aetonitrile: 2-propanol (7: 3 v/v for the first 5 min, followed by 75:25) allowed separation of 11 moleular speies. Quantifiation of individual moleular speies was arried out by monitoring UV absorption at 23 nm. Moleular speies were identified by omparison of their retention times to those of known authenti standards, by gas-liquid hromatography of their ayl hains, and in some ases by onversion to diayglyeroaetates and subsequent argentation TLC (Buttke et al., 1989). Flow ytometri measurements of relative membrane potential Membrane potential measurements were made using the ationi potential-sensitive dye dihexyloxaarboyanine iodide [DiOC6(3)], obtained from Moleular Probes In. (Eugene, OR). A stok solution of DiOC6(3) was prepared in dimethyl sulphoxide and stored at - 2. Just prior to use, aliquots were thawed, diluted to 5 ym with phosphate-buffered saline (PBS) and added to I x 16 ells in 1 ml PBS. The final DiOC6(3) onentration attained (125 nm) is reported to be non-toxi for lymphoytes (Damjanovih et al., 1987). After a 15-min inubation at room temperature, ells were analysed using a Beton- Dikinson FACS 44 flow ytometer. The laser was tuned to an exitation wavelength of 488 nm at an output of 4 mw. Emission of fluoresene was assayed within a band ranging from nm as a measure of relative membrane potential (Shapiro, Natale & Kamentsky, 1979). Control and gramiidintreated (2 pg/ml) lymphoyte populations were used to determine fluoresene values for fully polarized and totally depolarized ells, respetively. At eah time-point, the number of ells in the polarized region for eah ulture ondition was divided by the total number of ells analysed (_ 14) to derive the relative proportion of polarized ells. The ratio obtained at eah time-point was multiplied by 1 to obtain the perentage of polarized ells in eah sample. RESULTS PC moleular speies of mitogen-ativated B and T ells It has been shown previously that inubation of mitogenstimulated B and T ells in the presene of 5 MmM 18: leads to substantial differenes in the PC moleular speies (Buttke et al., 1989). The present studies were therefore performed to determine if suh lipid hanges ould be orrelated with dereased lymphoyte viability. In the initial phase of this study, purified B and T ells were analysed separately for their PC moleular speies. To aount for any anomalies of membrane omposition whih might have ourred as a result of ulture, B and T

3 Immunosuppression by steari aid a_ i in E 1 Moleular speies Figure 1. PC moleular speies of mitogen-ativated B and T lymphoytes. Purified B and T ells were stimulated with LPS or PHA, respetively. Cells were harvested and analysed for their PC moleular speies by HPLC. Quantities of eah moleular speies are shown as a perentage of total PC. Data shown represent the mean (± SD) of three separate experiments. Moleular speies: (a) 18:-22:6 and 18: 1-18:2; (b) 16:-18:2; () 18:-2:4; (d) 18:-22-5; (e) 18:1-18:1; (f) 16:- 18:1 and 18:-18:2; (g) 16:-16:; (h) 18:-18:1; (i) 16:-18:; (j) 16:-2: 1; (k) 18:-18:. ells were first stimulated for 6 hr with PHA or LPS, respetively, in the absene of 18:. Mitogen-ativated B and T ells ontained 11 separable PC moleular speies, with the proportions of individual speies being nearly idential within the two ell types. The results for eah lymphoyte type were therefore ombined and in Fig. 1 the level of eah individual PC speies is shown as the proportion of total PC. A omparison of the results shown in Fig. 1 with the data previously reported for mouse B and T ells (Buttke et al., 1989) reveals a muh higher level of dipalmitoyl (I16:-16: ) PC in the present study. Based on additional observations (T. M. Buttke and S. Van Cleave, unpublished data), the differing levels of 16:-16: may have resulted from a hange in murine diet. It is interesting to note that 16:-16: PC has also been observed in human tonsil lymphoytes (Morimoto & Kanoh, 198). In addition to the desaturated moleular speies, lymphoytes also ontained several moleular speies having the more expeted omposition of one saturated and one unsaturated fatty aid per PC moleule. PC speies ontaining at least one moleule of 16: omprised the majority (-.8%) of PC moleular speies, the remainder ontaining primarily 18: as their saturated moiety. Modulation of PC moleular speies in response to 18: supplementation Next the PC moleular speies of mitogen-stimulated B and T ells that had been inubated for 6 hr in the presene of 6, 12 or 18 gm albumin-omplexed 18: were examined. Seletion of the lowest dose was based on previous studies showing that 5 gm 18: irreversibly inhibited T-ell proliferation by >9% in 1 hr while having muh less effet on B-ell proliferation (Buttke & Cuhens, 1984). The higher doses of 12 and 18 gm 18: were used in an attempt to amplify 18: -indued hanges in membrane lipid omposition. The major effets of 18: supplementation on B- and T-ell PC moleular speies are shown in Fig O 2.I' eln I *5 5 a) CL ' E ~ (a) If - () 2 E i _ 15 ( e (-) B ells (o) TelIls 4k b) 3 _F 2 1 _ o Of t(d) ( h) _ Il Ai-n~~~~~ -~~~~~ e% : added (Q.M) Figure 2. Effet of 18: addition on PC moleular speies of B and T lymphoytes. Purified B and T lymphoytes were stimulated with PHA or LPS, respetively, in the absene or presene of 6 pm, 12 pm or 18 pm 18:. After 6 hr the ells were harvested and analysed for their PC moleular speies. Data are shown for the major moleular speies of PC only and are represented as the mean (± SD) of three separate experiments. (a) 16:-18:1 and 18:-18:2; (b) 16:-16:; () 16:- 18:2; (d) 16:-18:;(e) 18:-18:1;(f) 18:-18:;(g) 18:-2:4;(h) total desaturates. The addition of 18: was aompanied by hanges in the levels of six of the seven major PC moleular speies, with 16:- 18:2 being the singular exeption (Fig. 2). Beause of their low levels (1-2%), it was not possible to reliably test for 18:- indued hanges in the four moleular speies not shown in Fig. 2. In both B and T ells, levels of 16:-16: PC delined with inreasing doses of 18: added (Fig. 2b). However, whereas the proportion of 16:-16: dereased from 4% to 25% in T ells exposed to 18 gim 18:, a further redution to 15% was observed in similarly treated B ells. The deline in 16:-16: PC in T ells was largely aompanied by inreased levels of 16:-18: (Fig. 2d) and 18:-18: (Fig. 2f), whih resulted in the overall preservation of similar levels of total desaturated moleular speies (Fig. 2h). By ontrast, in B ells 16:-16: was replaed by 18:-18:1 (Fig. 2e) and 16:-18:1 and 18:- 18:2 (Fig. 2a), resulting in a signifiant deline in total desaturated PC (Fig. 2h). Overall, with inreasing doses of 18:, T ells were found to substitute 18: for 16: with little hange in the total amount of desaturated speies. B ells, however, showed a paradoxial derease in desaturated speies as a onsequene of 18: exposure. ~~~~~~~11 (g)

4 382 P. W. Tebbey & T. M. Buttke TelIs B el Is (a) (b) 4 3 U E amr jib!% a a). - I,. E a) (-) N ) ) IL Time (hr) Figure 4. Disruption of T-ell membrane potential by 18: is time and dose dependent. Purified B (b) and T (a) ells were mitogen stimulated and ultured in the absene or presene of various doses of 18:. Aliquots were removed at eah hourly time-point and the ells were stained with DiOC6 (3) prior to flow ytometri analysis. Fluoresene intensity values were assigned to depolarized and polarized ells based upon Gramiidin-treated and untreated ell populations. Data are expressed as the perentage of ells displaying fluoresene intensities omparable to ontrol (polarized) ells; eah data point is based upon the analysis of I4 ells. Symbols: () no 18: ; (A) 6 pm 18:; (U) 12 ym 18:; and () 18 tim 18: Fluoresene intensity Figure 3. Relative membrane potential of B and T ells exposed to 18 :. Purified B and T ells were stimulated with PHA or LPS, respetively, and ultured in the absene (solid lines) or presene (dashed lines) of 18 ym 18:. Aliquots were removed after hr (a, b), 4 hr (, d) and 8 hr (e, f), the ells stained with DiOC6(3), and analysed by flow ytometry. Gramidiin S was also used to determine the fluoresene intensity of totally depolarized ells (dotted lines, a, b). Effet of 18: supplementation on lymphoyte membrane potential Previous studies with erythroytes have determined that ell haemolysis results after replaement of only 25% of native PC with 18:-18: PC (Lange et al., 198; Kuypers et al., 1984). An aumulation of the desaturated lipid speies was thought to inrease membrane rigidity and leakiness, and thus promote haemolysis. To test the effet of 18: exposure on the integrity of B- and T-ell membranes, experiments were undertaken to orrelate 18: supplementation with hanges in membrane potential, this being a marker for a funtionally intat plasma membrane. Upon inubation with the membrane potential-sensitive dye, DiOC6(3), both B and T lymphoytes displayed a level of fluoresene intensity expeted for ells having an eletronegative intraellular milieu (solid line, Fig. 3a,b). Gramiidin S, an antibioti known to depolarize ells (Damjanovih et al., 1987), was used to define ells in a totally depolarized state. At a onentration of 2 pg/ml, a maximum redution in fluoresene intensity of both ell types was observed (dotted line, Fig. 3a,b). The proportion of viable lymphoytes has previously been reported to derease with time even under optimal ell ulture onditions (Buttke & Cuhens, 1984). This effet is also seen in the histograms shown in Fig. 3b-f at time-points of, 4 and 8 hr, respetively. Inubation of B and T ells with 18 gm 18: (dashed line, Fig. 3) resulted in a further time-dependent derease in the proportion of polarized T ells to an extent omparable to that seen with Gramiidin S. By omparison, 18: had no signifiant effet on the proportion of polarized B ells. A more detailed analysis of the effets of varying doses of 18: on T and B ell-membrane potential were subsequently arried out. Purified B and T ells were separately stimulated with LPS or PHA, respetively, in the absene or presene of 6-18 /M 18:. Following inubation at 37, aliquots were removed from eah of the eight ultures at hourly intervals and the ells were stained with DiOC6(3) prior to analysis by flow ytometry. The proportion of polarized lymphoytes was subsequently determined as a funtion of both time and 18: dose (Fig. 4). The relative membrane potential of B ells was not altered by the addition of 18: to the ulture medium, but T ells displayed a redued membrane potential as early as 5-6 hr after exposure to 6 UMm 18:. Higher doses of 18: had more pronouned effets, reduing the number of polarized ells to <3% at the 8 hr time-point. The results presented are in agreement with previous findings that the inhibitory effets of a similar dose (5 gm) of 18: on mitogen-indued DNA synthesis were manifest within 4-1 hr after exposure to 18: (Buttke & Cuhens, 1984). It was attempted to disern a orrelation between membrane lipid omposition and membrane integrity by analysing data obtained from Fig. 2 as a funtion of data derived from Fig. 4. Although the level of total desaturated moleular speies in B and T ells ould not be orrelated with the redued membrane potential, an exellent orrelation (r= --85) was observed

5 Immunosuppression by steari aid Q- 2 1 ( r = N;,, ~4 6 8 % polarized ells Figure 5. Correlation between membrane lipid omposition and membrane integrity. The sum of 18:-18: plus 16:-18: PC moleular speies in B (open irles) and T (solid irles) ells exposed to -18 PM 18: for 6 hr (derived from Fig. 2) was plotted as a funtion of the proportion of polarized ells at the 6 hr time-point (derived from Fig. 4). A best fit line was determined by linear regression analysis. when the perentage of polarized ells was plotted versus the sum of 16:-18: and 18:-18: PC (Fig. 5). This suggests that the replaement of 16:-16: with desaturates ontaining one or more 18: moiety per moleule are primarily responsible for 18: -indued T-ell death. DISCUSSION The studies desribed in this report were designed to determine the mehanism by whih 18: seletively kills murine T ells (Buttke & Cuhens, 1984). To this end, a model system for the study of saturated fatty aid uptake and subsequent metabolism by B and T lymphoytes has been developed. The system uses highly enrihed populations of B and T lymphoytes (> 95%) and albumin-omplexed fatty aids. Both onditions were essential for defining the basis of 18: inhibition. Previous workers have used heterogenous leuoyte populations in whih omparative studies of lipid metabolism between B and T lymphoytes were preluded. Further, in studies wherein the fatty aids were delivered as ethanoli solutions, large intraellular pools of non-esterified fatty aids were shown to aumulate (Klausner et al., 198). By ontrast, other studies have shown that 18: provided as an albumin omplex is effiiently taken up and esterified into ellular phospholipids, with < 1% remaining unesterified (Yang, Cuhens & Buttke, 1986; Buttke et al., 1989). Furthermore, using ['4C]18: it has been shown that the majority of exogenously supplied albumin-omplexed 18: is inorporated into PC (Buttke et al., 1989). Thus analyses of PC moleular speies serves as a useful parameter for assessing the overall effets of 18: on B- and T-ell membranes. As shown previously (Buttke et al., 1989) and in this study, inubation of B and T ells with albumin-omplexed 18: results in marked hanges in the membrane lipid ompositions of both ell types. Although both B and T ells take up similar quantities of 18: into PC (Buttke et al., 1989), following 18: exposure the two ell types display substantial differenes in their PC moleular speies. The addition of 18: to B ells leads to an unexpeted inrease in unsaturated speies at the expense of desaturates. Presumably, this shift in unsaturation is due to their ability to desaturate the exogenously supplied 18: using the stearoyl-coa desaturase enzyme. Conversely, T ells, whih are uniquely defiient in stearoyl-coa desaturase (Buttke et al., ) annot similarly avoid the inorporation of 18: into their membrane phospholipids. As a result, the T ells are fored into inreasing their levels of phospholipid speies ontaining 18:, in partiular 16:-18: and 18:-18: PC. Importantly, the demonstrated hanges were dose-dependent and ourred within 6 hr, a time suffiient to indue T-ell death (Buttke & Cuhens, 1984). The ultimate effet of the observed hanges in the membrane omposition of 18:-treated T ells may have been indiated previously. In studies with erythroytes, Lange et al. (198) and Kuypers et al. (1984) showed that stohiometri replaement of native erythroyte PC with 18:-18: was aompanied by inreased osmoti fragility, resulting in haemolysis. Thus it was determined whether the previously observed aumulation of 18:-18: PC in T ells (Buttke et al., 1989) ould also be assoiated with inreased membrane leakiness. The ability of B and T ells to maintain a membrane potential was assayed as an indiator of plasma membrane integrity. It was found that 18: exposure led to a dose-dependent deline in the proportion of T ells apable of maintaining a membrane potential. A similar ollapse of the plasma membrane permeability barrier was not observed in B ells. The onept that 18:-indued depolarization is responsible for redued T-ell viability is in agreement with the relatively rapid (4-1 hr) ytotoxi effet of the fatty aid. These studies do not prove a ause-and-effet relationship between membrane lipid hanges and disruption of the membrane potential. Nevertheless, they do show a strong orrelation (r= - 85) between the proportion of polarized B and T ells and their levels of 16:-18: plus 18:-18: PC. Data obtained in this study and elsewhere (Buttke & Cuhens, 1984; Buttke et al., 1989) olletively suggest the following paradigm for the inhibition of T-dependent immune responses by 18: : B ells inorporate 18:, desaturate a portion of it to yield 18: 1, and insert both fatty aids into phospholipids to maintain a funtional level of membrane fluidity. T ells, however, due to their lak of stearoyl-coa desaturase, annot onvert 18: to 18: 1. Consequently, the T ells replae muh of their 16: and olefini moieties with 18:, and aumulate signifiant amounts of both 16:-18: and 18:-18: PC. Replaement of 16: -16:, and perhaps other speies, by 16: - 18: and 18:-18: would be expeted to derease membrane fluidity and promote the formation of gel-like membrane domains. Suh alterations in the physial properties of T-ell membranes may lead to a ollapse of plasma membrane potential and, ultimately, ell death. Lastly, the seletive toxiity of 18: for T ells and its rapid mehanism of ation may have linial relevane in allograft or autoimmune situations. Immunosuppressive agents suh as ylosporin A are widely used for delaying the onset of allograft rejetion (Borel et al., 1976). The therapeuti value of ylosporin derives from its marked seletivity toward T ells (Cohen et al., 1984). Like 18:, ylosporin has also been shown to depolarize T ells (Damjanovih et al., 1987), and both agents blok IL-2 prodution (Kronke, Leonard & Depper, 1984; Pourbohloul & Buttke, 199). Sine ylosporin has numerous potential side effets, inluding nephrotoxiity, hepatotoxiity and malignant lesions (Kahan et al., 1986), alternative immunosuppressive agents are required. If the effets of 18 : on T ells ould be retained in vivo, the fatty aid ould effetively and rapidly immunosuppress ell-mediated responses, but without the serious side-effets of ylosporin.

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