Immunology 1990 70 458-464

Arachidonic acid metabolism in heat-shock treated human leucocytes M. KOLLER & W. KON IG Lehrstuhlfiir Med. Mikrobiologie und Immunologie, Arbeitsgruppe Infektabwehrmechanismen, Ruhr-Universitit Bochum, Bochum, FRG

Acceptedfor publication 2 Mayt 1990

SUMMARY fractions (PMN) and lymphocytes/monocytes/basophils (LMB) Human neutrophil granulocyte were stimulated with A23 187 (7 3 yM), opsonized zymosan (1 mg) or FM LP (I 0 - I M) after heat-shock treatment. We observed a temperature- (pretreatment over 40 ) and time-dependent (incubation periods longer than 20 min) suppression in the generation of LTB4, LTB4 metabolites and isomers, as well as LTC4 and 5-HETE. These effects were not reversed after the addition of exogenous arachidonic acid (AA;50 jM). In contrast, heat-shock treatment alone triggered platelets to generate 12-HETE. After 1 hr at 42c, 135 + 24 ng of 12-HETE were generated from 1 x 108 cells. The 12-HETE generation was not dependent on extracellular Ca2 +. Conversion of '4C-AA (2 nmol) revealed an enhanced metabolism of AA to 12-HETE by platelets after heat-shock treatment without exogenous Ca2+. PMN and LMB labelled with 35S-methionine led to heat-shock protein (HSP; 65,000, 83,000 MW) expression after heat-shock treatment at 42° or in the presence of NDGA (1 x 10-5 M) at 37°. These results suggest a regulatory interaction between the generation of lipo-oxygenase products, cellular stress responses and the expression of HSP.

leukotriene generation from granulocytes of severely burned patients has been reported (Kdller et al., 1988; Koller et al., 1989a). Recent observations have shown that leukotriene B4 (LTB4) exerts immunoregulatory activity on human lymphocytes, serving as a positive signal in the intercellular network (Rola-Pleszczynski et al., 1987; Yamaoka, Claesson & Rosen, 1989). In previous studies we have demonstrated that granulocyte functions are suppressed after heat-shock treatment (Kdller et al., 1989b). It is well established that heat-shock treatment leads to the expression of specific but highly conserved subsets of cell proteins that are called heat-shock proteins (HSP) (Lindquist, 1986). Recently, it was also reported that even PMN, with their limited capacity for protein synthesis, are able to express typical HSP after incubation at 42' (Eid, Kravath & Lanks, 1987). The heat-shock response is also induced during infection and inflammatory processes (Polla, 1988) and possibly exerts protective functions after various cellular stress conditions (Lindqvist, 1986). More recently, it was reported that HSP are involved in specific immunological functions, e.g. in the regulation of growth processes in human lymphocytes or as immunodominant antigens in autoimmune diseases (Haire, Peterson & O'Leary, 1988; Ottenhoffet al., 1988; Spector et al., 1989). However, the consequences of a heat-shock treatment for different cellular functions and the role of HSP still remain unclear. Thus, a regulatory interaction between arachidonic acid (AA) metabolites, the stress response of cells, and the expression of HSP might exist. It was the purpose of this study to investigate the cellular responses to stress by analysing the metabolism of AA in stimulated and unstimulated leucocytes.

INTRODUCTION Polymorphonuclear granulocytes (PMN), mononuclear peripheral blood cells (monocytes/lymphocytes) as well as platelets participate as effector cells in the regulation of normal immune functions and are involved in inflammatory processes. The phagocytes (PMN and monocytes) play a key role in host defence against bacterial infections. They exert this function by a variety of properties, e.g. the generation of different inflammatory mediators such as lipo-oxygenase products (leukotrienes and HETE), which have the capacity to induce the classical signs of inflammation, augment exocrine secretions, stimulate platelets and activate different cell types (Samuelsson, 1983; Parker, 1987; Bray, 1986). The generation of lipo-oxygenase products has been observed during pathophysiological events induced by inflammation, shock or allergic reactions (Samuelsson, 1983; Kdnig et al., 1984; Lefer, 1986). In addition, a decreased Abbreviations: AA, arachidonic acid; FCS, fetal calf serum; FMLP, formyl methionyl leucyl phenylalanine; HETE, hydroxy-eicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; HSP heat-shock protein; IL interleukin; LDH, lactate dehydrogenase; LMB, lymphocytes, monocytes, basophils; LT, leukotriene; PBS, phosphate-buffered saline; PG prostaglandin PMN, polymorphonuclear granulocyte; RPHPLC, reversed-phase high-performance liquid chromatography; TCA trichloroacetic acid. Correspondence: Professor W. Kdnig, Med. Mikrobiologie u. Immunologie, AG Infektabwehrmechanismen, Ruhr-Universitit Bochum, Postfach 102148, D-4630 Bochum 1, FRG.

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Arachidonic acid metabolism MATERIALS AND METHODS

Materials Synthetic leukotrienes (LT) B4, C4, D4 and E4, as well as the omega-oxidation products 20-OH-LTB4 and 20-COOH-LTB4, were a generous gift from Dr J. Rokach (Merck Frosst, PointeClaire/Dorval, Quebec, Canada). The leukotriene B4 isomers 6trans-LTB4 and 12-epi-6-trans-LTB4 were obtained from Dr P. Borgeat (University Laval, Quebec, Canada). '4C-arachidonic acid ('4C-AA; specific activity 1 48-2-22 GBq/mmol), [3H]LTB4 and OH-LTB4 (1 1 1-2 22 TBq/mmol), [3H]LTC4 and E4 (0 742 22 TBq/mmol), [3H]PGE2 (3 7-74 TBq/mmol) and L-35Smethionine (> 29-6 TBq/mmol) were purchased from New England Nuclear (NEN, Dreieich). AA, the Ca2+ ionophore A23187, FMLP and zymosan A were obtained from Sigma, Deisenhofen. All other chemicals were from Merck, Darmstadt. Buffer The phosphate-buffered saline (PBS) comprised 120 mm NaCl/ 10 mM Na2PO4/3 mm KH2PO4 (pH 74). RPMI-1640 Select Amine Kit was from Gibco-BRL, Eggenstein. Preparation and stimulation of cells PMN and LMB were separated from heparinized blood (15 U/ml) on a Ficoll-metrizoate gradient followed by dextran sedimentation (Bdyum, 1968). After the preincubation periods at different temperatures the cells were left on ice for 5 min before the stimulation at 37°. The stimulation of the cells was performed as has been described elsewhere (K6ller et al., 1989b). Platelets were isolated from platelet-rich plasma (PRP). PRP was obtained by centrifugation of peripheral blood (aliquots of 9 ml) supplemented with PBS containing 1-5% EDTA (1 ml) at 200 g (25 min at 20°). The supernatant was mixed with an equal volume of PBS (1 5% EDTA) and centrifuged at 1285 g (20 min at 40). The pellet was washed in 10 ml PBS (1-5% EDTA). After centrifugation the pellet was suspended in PBS.

Platelet aggregation Platelet (2 x 108 cells/ml PBS) aggregation was measured optically using a Whole-Blood Aggro-Meter 550 (Chrono-Log Corp., Havertown, PA). Platelets were constantly stirred within the cuvette. Platelet aggregation was quantified as the increase of light transmission. Heat-shock treatment 1 x I07 PMN (LMB) or 1 x 108 platelets in 500 jyl PBS or RPMI1640 medium were exposed in a waterbath in polyethylene tubes (5 ml) up to 420. Control incubations were made at 370. The temperature within the tubes reached that of the waterbath after 8 min (Polla, Bonventre & Krane, 1988) so that the duration of heat shock was not corrected for the time of equilibration. After heat-shock treatment all incubations were left on ice for 5 min to obtain thermal synchronization. Cell viability The trypan blue exclusion test demonstrated a cell viability of 98% for the preincubated cells (preincubation at 370 as well as at 420). To ascertain a possible degranulation or cytotoxic reaction due to the elevated temperatures, the release of lactate dehydrogenase (LDH; as a marker for cytoxic effects), lysozyme and /3glucuronidase (as markers for the azuropilic and/or specific

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granules) was measured. The release of LDH never exceeded 6% of the total cellular LDH content. The percentage corresponds always to the total content of cellular enzymes, which was measured after cell sonication. The release of /3-glucuronidase (PMN) did not exceed 2%; the release of lysozyme was 18 3 + 3-6 when the cells were unstimulated (pretreatment at 37° or 420, respectively). The degranulation of the PMN was reduced after pretreatment at 420 for 60 min when the PMN were subsequently stimulated with the Ca2+ ionophore (lysozyme: 44-4 + 12 1% at 370, 24-9 + 7.8% at 420; /3-glucuronidase: 65+22% at 37, 59+1-9% at 42°).

Analytical HPLC system HPLC were performed on reversed-phase columns (4 x 250 mm) packed with Nucleosil (C18), 5-tim particles (MachereyNagel, Diiren), using a CM 4000 pump (LDC-Laboratory Data Control/Milton Roy, Hasselroth) and the automatic sample injection module WISP 710B (Waters, Eschborn). The column temperature was constantly maintained at 40°. The absorbance of the column effluent was monitored using a variable ultraviolet detector (LDC-SM 4000) adjusted to 280 nm or 235 nm. The peak areas were calculated using a chromatography data system (Nelson Analytical, Mannheim). The composition of the solvent system used has been described elsewhere (Kbller et al., 1989a). The flow rate was maintained at 1 ml/min. All solvents were degassed before use and constantly stirred during HPLC analysis. Identification and quantification of leukotrienes were performed as has been described elsewhere (K6ller et al., 1988). Conversion of '4C-AA To analyse the metabolism of arachidonic acid either PMN or LMB (1 x I07 cells) or platelets (1 x 108 cells) were preincubated for 60 min at 370 or 42' followed by incubation with '4C-AA acid (2 nmol) for a further 40 min at 370. Ca2+/Mg2+ (1 mM/2 5 mM) was added when the Ca2+ ionophore (1 yM) was used as a stimulus. All reactions were terminated with two volumes of methanol-acetonitrile (1:1, v/v) and subsequently centrifuged (2000 g for 15 min). The resulting supernatants were evaporated under a gentle stream of nitrogen dissolved in 500 kl methanolwater (30:70, v/v) and applied to RP-HPLC (see above) connected to a radioactivity detector (RAMONA; Isomess, Straubenhardt) which was continuously (14 ml) supported with scintillator liquid (Rotiszint 2200; Roth, Karlsruhe). The solvent system consisted of a gradient prepared from system A (water/methanol/acetonitrile 60:20:20, v/v), system B (water/ methanol/acetonitrile 40:30:30, v/v) and system C (water/ methanol 5: 95, v/v) as follows: 0-20 min, linear from 100% A to 100% B; 20-33 min, 100%B; 33-52 min, linear from 100% B to 100% C; 52-80 min 100% C. The flow was maintained at 1 ml/ min and elevated to 1 5 ml/min after 55 min in each run. The peak areas were calculated using a chromatography data system (cf. analytical HPLC system). Peak identification was performed using radiolabelled eicosanoids (cf. materials) as reference substances. Protein labelling conditions After termination of heat exposure, the cells (PMN or LMB) were centrifuged at 400 g and washed twice in RPMI-1640 medium containing 10% heat-inactivated FCS. The cell pellets were then suspended in methionine-free RPMI-1640 medium containing 1% FCS, which was additionally supplemented with

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Figure 1. Generation of lipo-oxygenase products from heat-shock treated leucocytes. Cells (1 x 107/500 p1 PBS) were stimulated for 20 min at 37' after pretreatment for different periods at 37° (open bars), 420 (shaded bars) or left on ice for 60 min (control). The data were expressed as mean values + SD from experiments (n = 3) with different donor cells (* P < 0-05). (a) Leukotriene generation from PMN after stimulation with the Ca2+ ionophore A23187 (7 3 jM). The combined amounts of identified leukotrienes are shown. (b) 5-HETE generation from PMN after stimulation with FMLP (10-5 M) in the presence of 50 pM exogenous AA. (c) 12-HETE generation within LMB fractions after stimulation with FMLP (10-5 M) in the presence of 50 pM exogenous AA.

radiolabelled methionine (0-74 MBq/ml) and incubated for 90 min at 37°. After centrifugation at 400g for 15 min at 40, the pellets were resuspended and precipitated in 1 ml TCA (30%) at 4° over night. Centrifugation was carried out at 2000 g at 40 and the precipitated protein was washed twice with cold PBS and solubilized in 200-500 p1 of Laemmli's sample buffer (Laemmli, 1970) containing the thiol-protecting agent dithiothreitol (0-1 M). Aliquots of 150 pl were taken from the solubilized samples left for 3 min in a boiling waterbath and were applied to

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Influence of heat shock (42°) on the generation of lipo-oxygenase products from stimulated human leucocytes Human peripheral PMN and LMB (each 1 x 107/500 yl PBS) platelets (1 x 108/500 yl PBS) were incubated at 42° for different time periods, subsequently stimulated at 370 and analysed for their capacity to generate lipo-oxygenase products. The viability of cells was not affected in the course of the incubation procedures at 42° (cf. the Materials and Methods). As shown (Fig. 1), a significant reduction in leukotriene generation from PMN and LMB was observed after preincubation periods which exceeded 20 min. No differences in the formation of the individual leukotrienes were observed. Thus, the total amounts of generated leukotrienes were calculated (LTC4, LTB4, LTB4 isomers and omega-oxidation products of LTB4). The stimulation of PMN with the Ca2+ ionophore A23 187 (7 3 pM) is shown as an example (Fig. la). Similar results were obtained when FMLP (10-5) or opsonized zymosan (1 mg) were used as stimuli. The generation of 5-HETE from PMN after heat-shock treatment showed a parallel pattern compared to leukotriene generation (Fig. 1 a). When PMN were directly stimulated at 420 with the Ca2+ ionophore A23187 (7 3 pM), no reduction in the generation of lipo-oxygenase products was observed compared to stimulation at 370 (data not shown). In Fig. Ib, a stimulation of PMN with FMLP (10-5 M) in the presence of 50 pM exogenous AA

Vertical gel electrophoresis and autoradiography SDS-PAGE of 35S-methionine-labelled proteins was performed in a 10% or 12-5% linear gel, according to the method of Laemmli (1970), starting with a stacking gel (3% polyacrylamide). Each gel was stained with Coomassie brilliant blue (0 01 %) for the calculation of molecular weight (MW) markers. The gels were dried immediately after staining and autoradiographed using a Kodak XAR-5 film. Statistics Data from different experiments with different donor cells were combined and expressed as mean + SD. The Student's t-test for independent means was used to provide statistical analysis.

is demonstrated. The addition of exogenous AA did not restore the capacity to generate leukotrienes (data not shown) and 5-HETE from PMN. When LMB were stimulated under the same conditions, the major mono-HETE was identified as 12-HETE, probably generated from contaminating platelets which cannot be totally removed from PMN or LMB fractions using the Ficoll-Hypaque technique. However, neutrophils, erythocytes and lymphocytes are known to generate 12-HETE (Spector, Gordon & Moore, 1988). Unexpectedly, the generation of 12-HETE was not decreased after heat-shock treatment at 420, as was shown for other lipo-oxygenase products (Fig. la, b). In contrast, a significant increase was obtained after preincubation for 60 min at elevated temperatures (Fig. 1c).

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Figure 3. Conversion of 14C-AA by LMB. LMB were preincubated for 60 min at 37° (a) or 420 (b). 1, unstimulated cells; 2, stimulation with the Ca2+ ionophore (1 tM) for a further 45 min at 370, 3, as 2, cells were pretreated with indometacin (10-5 M) for 15 min at 370; 4, cells were pretreated with NDGA (l0-5 M) for 15 min at 37°. Peak 1, not identified; peaks 2, 3 and 4, cyclo-oxygenase products; peak 5, 12-HETE; peak 6, arachidonic acid. Detection of radioactivity was performed as described in the Materials and Methods.

12-HETE generation from platelets after heat-shock treatment Obviously, the major source of 12-HETE is the platelet (Spector et al., 1988). Thus, it appeared likely that the 12-lipo-oxygenase is activated during heat-shock treatment. To confirm this assumption, isolated platelets were exposed to heat shock (42°) without a subsequent stimulation and without addition of exogenous Ca2+. As is shown (Fig. 2), a spontaneous generation of 12-HETE was obtained from platelets during heat-shock treatment in a time-dependent manner compared to cells which were left at 37°. After 20 min of hyperthermic treatment, the 12HETE generation was significantly elevated. After 60 min at 420, 134+21 ng of 12-HETE were generated from 1 x 108 cells. An aggregation of platelets under the chosen experimental conditions was not observed, as was determined by platelet aggregometry and light microscopy of stained cell smears (data not shown).

Conversion of exogenous 14C-AA In order to confirm the enhanced 12-HETE generation during heat-shock treatment (Fig. lc, Fig. 2), either PMN or the LMB fraction or platelets were incubated with "4C-labelled AA (2 nmol) after heat-shock treatment (1 hr, 420) for 45 min under different incubation conditions (stimulated and unstimulated). When Ca2+ ionophore A23187 (1 gM) was used as stimulus, Ca2+ (1 mM) was added to the incubation mixture. In addition, pretreatment of the cells with indomethacin (10-5 M) or nordihydroguaiaretic acid (NDGA) (10-5 M) was partly carried out 15 min before the stimulus was added. Figure 3 shows the results obtained with the LMB fraction in a series of representative radio-chromatograms. Chromatograms obtained after pre-

treatment of cells at 370 were indicated as A and those obtained after pretreatment of cells at 420 were indicated as B. The labelled AA metabolites were numbered according to their elution time and were identified by respective radiolabelled standards (cf. the Materials and Methods) or classified due to their sensitivity against indomethacin or NDGA. As is shown in Fig. 3 (a and b), the cells predominantly metabolized exogenous AA into 12-HETE (peak 5). The 12HETE generation was enhanced after pretreatment of the cells at 42°, especially under unstimulatory conditions. The 12HETE generation was enhanced by 57% after heat-shock treatment (Fig. 3, Bl). This enhancement also occurred in platelets and PMN fractions by 103% and 480%, respectively (data not shown). Furthermore, in addition to an unidentified polar AA metabolite (peak 1), two other metabolites were released from the unstimulated heat-shock treated LMB fraction (peaks 2 and 4). Among them peak 4 was increased in comparison to the pretreatment of cells at 370 (Fig. 3, Al). Under stimulating conditions (Ca2+ ionophore A23 187, 1 pM) a further metabolite (peak 3) was detected (Fig. 3, A2). In contrast to unstimulated conditions, the generation of the metabolites 2, 3, and 4 was decreased when cells were stimulated after heatshock treatment (Fig. 3, B2). Similar results were obtained with platelets. The arachidonic acid metabolites were products of the cyclo-oxygenase pathway, as was concluded by their sensitivity to indometacin pretreatment, whereas 1 2-HETE was not affected (Fig 3, A3 and B3). However, none of these peaks comigrated with synthetic PGE2. Peaks 2 and 4 were also generated from purified platelets, suggesting that they are platelet products (possibly thromboxanes). Pretreatment of cells with NDGA (10-5 M) abolished the generation of 12-HETE; in contrast, the release of free AA was clearly increased (Fig. 3, A4 and B4).

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Figure 4. Synthesis of proteins in PMN. Human PMN (1 x IO' cells/ml RPMI-1640 medium) were labelled and autoradiography was performed as described in the Materials and Methods. Pretreatment of cells: lane 1, 60 min at 370; lane 2, 60 min at 420; lane 3, cells were stimulated with the Ca2+ ionophore (1 pM) for 45 min at 37°; lane 4, as lane 3 cells were pretreated with indometacin (I0-5 M) for 15 min at 370; lane 5, as lane 3, cells were pretreated with NDGA ( 10 - 5 M) for 15 min at 37°; lane 6, cells were left on ice.

Figure 5. Synthesis of proteins in LMB. Human LMB (1 x 107 cells/ml RPMI-1640 medium) were labelled and autoradiography was performed as described in the Materials and Methods. Pretreatment of cells: lane 1, 60 min at 370; lane 2, 60 min at 42°; lane 3, cells were stimulated with the Ca2+ ionophore (1 pM) for 45 min at 37°; lane 4, as lane 3 cells were pretreated with indometacin (10-5 M) for 15 min at 37°; lane 5, as lane 3, cells were pretreated with NDGA (10-5 M) for 15 min at 370; lane 6, cells were left on ice.

Expression of HSP

As was demonstrated, heat-shock treatment of leucocytes led to significant alterations in the generation of AA metabolites (Figs 1, 2 and 3). However, the generation of HSP is known as a typical response of cells due to heat-shock or stress treatment (Lindquist, 1986; Craig, 1985). Therefore, the expression of heat-shock proteins was additionally analysed in PMN and LMB fractions. As is evident, the patterns of newly synthesized proteins from PMN (Fig. 4) and LMB (Fig. 5) were altered after different preincubation temperatures. Cells which were left on ice served as a control (lanes 6). Heat-shock treated cells (lanes 2) expressed a protein with a relative MW of 65,000 (arrows), which was not detected in the control incubations. In heat-shock treated cells, the expression of other proteins was suppressed, indicating the 65,000 MW protein to be a HSP. However, in LMB this protein was already expressed after pretreatment at 370 (Fig. 5, lane 1). A similar pattern was also obtained for a quantitatively less expressed protein with a calculated relative

MW of 83,000 (small arrow, Fig. 5). Pretreatment of the cells with the Ca ionophore A23187 (1 gM) in the presence of 1 mM Ca2+ at 370 (lanes 3) revealed no changes in the expression of proteins compared to incubations without the stimulus (lanes 2). Cyclo-oxygenase inhibition by indometacin (10-5 M) did not alter protein expression in stimulated cells (lanes 4). In contrast, in the presence of NDGA (10-5 M) the expression of the 65,000 MW protein was induced in PMN (Fig. 4, lane 5) or increased (also valid for the 83,000 MW protein) in LMB (Fig. 5, lane 5). Furthermore, in both cell fractions the expression of a protein with a calculated relative MW weight of 33,000 (small arrow Fig. 4) was decreased under these conditions.

DISCUSSION A large amount of information regarding the regulation at transcriptional and translational levels after heat-shock treatment has been collected and reviewed in detail (Lindquist, 1986; Craig, 1985). However, the consequences of heat-shock treatment for different cellular functions and the role of HSP still remain unclear. The presented results extend recent observations that demonstrated that effector functions of PMN and basophils, like chemotaxis, the generation of oxygen radicals or mediator release (leukotrienes, histamine), were decreased after heat-shock treatment (Kdller et al., 1989a). In this study the generation of mono-HETES was analysed and the experiments included LMB and platelets. The heat-shock response of human peripheral blood cells is different. Whereas the 5-lipo-oxygenase pathway in PMN and LMB is deactivated, after heat-shock platelets showed an activation of the 12-lipo-oxygenase pathway, even in the absence of extracellular Ca2+. However, we could not rule out that the 5-lipo-oxygenase itself is heat sensitive. On the other hand, Calderwood et al. (1989) recently described an increase in AA release and LTB4 generation from Chinese hamster fibroblasts after heat shock treatment up to 450 The release of arachidonic acid from phospholipids and its metabolism into cyclo-oxygenase products (especially TxA2) are considered to be key events that shift the platelet response from reversible to irreversible platelet aggregation (Sies, 1989). However, during heat-shock treatment the release of 12-HETE was not correlated with irreversible platelet aggregation. In addition, it has been reported that 12-HPETE and 12-HETE are inhibitors of platelet aggregation. (Sies, 1989; Croset & Lagarde, 1983). Thus, the release of 12-HETE may provide antiaggregatory signals during cellular stress responses. The biological significance of the 12-lipo-oxygenase pathway in platelets is still unclear. However, 12-HETE is involved in transcellular interactions with PMN and monocytes (Marcus et al., 1987; Bigby & Meslier, 1989). In addition to the formation of 12oxygenated products (e.g. 12,20 DiHETE or 5S,12S-DiHETE) via cellular interactions, 12-HETE also produces functional effects through incorporation into membrane phospholipids and thus alters the structures of lipid bilayers (Spector et al., 1988). The heat-shock response is a complex reprogramming of cellular activities in which different factors may be involved. There is accumulating evidence for a link between lipid mediator generation and the heat-shock response. In this context we previously described the decreased leukotriene release and impairment of the chemiluminescence response from granulo-

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Arachidonic acid metabolism cytes of severely burned patients (K6ller et al., 1988, 1989a). Extended burns and severe pathophysiological events obviously result in multiple different cellular stress conditions (Demling, 1985). The involvement of HSP in these functional alterations of leucocytes after severe traumata is under our current investigation. However, HSP induction in vivo is known to occur under heat, ischemia and reperfusion injury, wounding and inflammation (Polla, 1988). Both the biological correlates of the altered generation of lipo-oxygenase products and the physiological significance of HSP expression are connected by the following observation. Activation of B lymphocytes by crosslinking the Bcell antigen receptors (anti-p beads to Staphylococcus aureus strain Cowan I; SAC) results in the selective expression of HSP70 mRNA and the respective protein (Spector et al., 1989). Heat-shock genes (HSP70, HSP90) are also activated by IL-2mediated signalling mechanisms in T lymphocytes (Ferris et al., 1988). The immunomodulatory activity of leukotrienes on lymphocytes has been described. It was suggested that LTB4 regulates immune cell functions by inducing interferon-gamma production from T cells or further affects the production of cytokines such as IL-1 or IL-2 (Rola-Pleszczynski et al., 1987; Rola Pleszczynski, Gagnon & Chavaillaz, 1988; Johnson, Russel & Torres, 1988; Russel, Torres & Johnson, 1987). More recently, LTB4 was shown to amplify the lymphokine-driven activation, replication and differentiation of human B lymphocytes (Yamaoka et al., 1989). It was demonstrated that cyclopentenone prostaglandins induce HSP (Ohno et al., 1988; Santoro, Garcia & Amici, 1989). In contrast to the generation of LTB4, which was suppressed after heat-shock treatment, PGE2 generation of PMN was reported to be unaffected (Maridonneau-Parini, Clerc & Polla, 1988), indicating a different activation of the AA cascade after hyperthermic treatment. Whereas PGE2 is known as an anti-proliferative substance, probably due to the elevation of cAMP levels, recent evidence indicates that LTB4 serves as a positive signal by enhancing cGMP levels (Hadden & Coffey 1982). Moreover, glucocorticosteroids suppress the release of AA via the phospholipase A2 pathway by the induction of intracellular PLA2 inhibitors (Blackwell & Flower, 1983). The mechanisms whereby glucocorticosteroids are immunosuppressive are unknown; however, the inhibition of IL-2 production from lymphocytes by glucocorticosteroids was completely reversed in the presence of exogenous LTB4 (Goodwin et al., 1986). In this regard, it is of particular relevance that the glucocorticosteroid receptor was identified as an asymmetric hetero-oligomeric complex, which consists of a hormone-binding subunit and two HSP90 molecules. It was suggested that, in the absence of the hormone, the HSP molecules interact and cap the positively charged DNAbinding domain of the steroid receptor (Lefebvre et al., 1989; Binart et al., 1989; Denis, Gustafsson & Wikstrom, 1988). NDGA is known as a lipo-oxygenase inhibitor. We demonstrated here the induction of HSP under NDGA (10-l M) treatment of PMN and LMB at physiological temperatures. However, we could not rule out that other enzymes involved in the cellular signal transduction (e.g. phospholipases A2 or C) were affected by the high-dose NDGA or that NDGA itself induces HSP expression. The HSP are usually classified and named according to their apparent molecular mass. The calculated molecular mass of the less-expressed HSP83 in this study correlates well with previous reports (Eid et al., 1987;

Maridonneau-Parini et al., 1988). Differences were obtained in the calculation of the predominent HSP in this study (65,000 MW) to reported values of 70,000 MW, which may be due to different methodological procedures. At present we have no final explanations for the different responses of heat-shock treated human leucocytes, which may be reflected by their different capacity in the release of lipooxygenase products. Further studies are required to link the expression of HSP to the components of the signal transduction cascade. Whether these results imply that leukotrienes exert inhibitory functions in HSP expression has to be investigated. In addition, it has to be clarified whether the depressed LTB4 generation in burned patients (K6ller et al., 1988, 1989a) is due to HSP expression of the cells. ACKNOWLEDGMENTS M. Kdller was supported by the Fraunhofer-Gesellschaft, and W. K6nig by the Deutsche Forschungsgemeinschaft.

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