Phospholipase A2, an in vivo immunomodulator

PmstaglandinrLeukotrienes and Essential Fatty Acids (1990) 40.31-38 @ Longman Group UK Ltd 1990

0952.327E&WWO-0031/$10.00

Phospholipase AZ, an in vivo Immunomodulator A. Bravo Cuellar,

F. Homo-Delarche*

and S. Orbach-Arbouys

lnstitut de Can&rologie et d’lmmunogtnttique (Univ. Paris&d, Ass. Cl. Bernard & ARC), Hhpital Paul-Brousse, 94804-Villejuif, France. *INSERM V25, CNRS VA-122, H6pital Necker, 75015-Paris, France (Correspondence to A B C at following address; ICIG, Hbpital Paul-Brousse 12-16 av. Paul-Vaillant-Couturier 94804 Villejuif Cidex, France) ABSTRACT.

Arachidonic acid (AA) can be released from membrane phospholipids by the action of phospholipase AZ (PLA2). There is evidence that unsaturated fatty acids, particularly AA, released from membrane phospholipids are required to activate the respiratory burst of macrophages. The data reported here indicate that peritoneal macrophages harvested 30 min after i.p. injection of PLAl can phagocytose Candida albicans more efficiently and emit more chemoluminescence (CL) than normal cells when stimulated by zymosan. PLAz injection also enhances the CL of peritoneal cells from mice already stimulated by immunomodulators such as trehalose dimycolate (TDM), bestatin, or oncostatic drugs such as aclacinomycin (ACM). CL is not sensitive to potassium cyanide (KCN), but is inhibited by catalase, superoxide dismutase (SOD), nordihydroguaiaretic acid (NDGA) and high doses of indomethacin (Hi-%I). In vivo PLAz treatment stimulates the synthesis of both cyclooxygenase and lipoxygenase derivatives of AA metabolism (PGE2, 6-keto, PGFt, TXBz and LTQ. Inhibitors of AA metabolism (NDGA, indomethacin) modulate the production of free oxidizing radicals in this experimental model, partly because of their effect on AA metabolism, as determined by the measuring immtinoreactive products. However, this work indicates that the effects of these inhibitors, which have been extensively used in CL studies, should be interpreted with caution, since their specificity for AA metabolism is relative.

pholipids regulates several functions (2, 3). Alterations in the content of polyunsaturated fatty acids, such as arachidonic acid (AA), thus play a major regulatory role (4), and the availability of AA may be the limiting factor. PLA2 is the principal enzyme responsible for liberating AA, which is then metabolized via the lipoxygenase and/or cyclooxygenase pathways (5). ’ Phagocytic cells can emit chemoluminescence (CL) when they are stimulated by phagocytic and soluble stimuli (6). The CL produced by macrophages depends on the liberation of free oxygen radicals such as 0~ and H202 (7). These radicals have a microbicidal activity and are associated with the destruction of tumoral cells (8, 9, 10). Recent evidence also suggests that CL emission is dependent on AA metabolism. Bromberg and Pick (11) reported that 8 out of 10 unrelated stimulants of the respiratory burst in peritoneal macrophages induced the release of AA from membrane phospholipids as a consequence of phospholipase activation, The unsaturated fatty acid thus liberated could be a second messenger in the stimulus response-coupling leading

Abbreviations AA ACM CL IP KCN LTC4 NDGA PG, PGS PLA2 SOD TDM

arachidonic acid aclacinomycin chemoluminescence intraperitoneally potassium cyanide leukotriene Cd nordihydroguaiaretic acid prostaglandin, prostaglandins phospholipase A2 superoxide dismutase trehalose dimycolate

INTRODUCTION Membrane phospholipids not only constitute defense system for the cell, but are also actively

a in-

volved in signal transmission across the membrane in response to external stimuli (1). The composition of the fatty moieties of plasma membrane phos-

Date received 29 September 1989 Date accepted 20 November 1989 31

32 Prostaglandins Leukotrienes and Essential Fatty Acids

to 02- production. On the other hand, AA has recently been shown to act as an intracellular activator of NADPH-oxidase in Fey receptormediated superoxide generation in macrophages (12). In addition, CL emission can be modulated by inhibitors of PLA2 as well as of cyclooxygenase and lipoxygenase enzymes (13, 14, 15). Moreover, some lipoxygenase derivatives of AA metabolism, such as leukotriene Cd (LTQ, directly stimulate free oxygen radical production, while prostaglandins (PGS), the cyclooxygenase derivatives, have an inhibitory effect (15, 16). PLA2 has not, to our knowledge, been tested in vivo as a modifier of the biological activity of peritoneal cells. It has, however, been injected into mice, rats and monkeys for other non-immunological purposes (17, 18, 19). These considerations prompted us to study whether the phagocytic activity and the intensity of the respiratory burst of peritoneal cells can be modified, in vivo, by exogenous PLA2 either alone, or in association with other immunodulatory agents [bestatin, treha-lose dimycolate (TDM) or aclacinomycin (ACM)], which are known to stimulate macrophage functions ((20, 21, 22). The role of derivatives of AA metabolism in exogenous PLA;! action has also been assessed by using cyclooxygenase and lipoxygenase inhibitors and by measuring the different AA metabolites produced by peritoneal macrophages under these circumstances.

MATERIAL AND METHODS Animals Specific pathogen-free C57Bl/6 (B6) 6-week-old male mice were obtained from the breeding centre of the Institute de Recherches Scientifiques sur le Cancer (IRSC, Villejuif, France). They were used within two weeks of delivery.

J. F. Petit (Institute de Biochimie, Orsay, France) was dissolved in 0.9% NaCl and injected into mice (0.05 mg/mouse, i.p.) 4 days before cell harvest. Aclacinomycin (ACM), a cytotoxic antibiotic isolated from cultures of Streptomyces galileus, was kindly supplied by Laboratoires Roger Bellon (Neuilly-sur-Seine, France). The drug was dissolved in 0.9% NaCl (1 mg/ml), stored at -2O”C, and used within 8 days. The mice were injected (4 mgtkg, i.p.) 4 days before cell harvest. Preparation of peritoneal cells Mice were sacrificed by cervical dislocation and peritoneal cells were obtained by washing the peritoneal cavity with 6 ml of Hanks’ solution without phenol red at 4°C (pH 7.2). Smears were routinely prepared by cytocentrifugation and stained with Giemsa and non-specific esterase substrates for differential counts. Live cells were counted by trypan blue exlusion (98% live cells). Phagocytic assay The phagocytic activity of peritoneal macrophages was measured by ingestion of Candida albicans under the light microscope, as previously described (23). Three hundred cells were examined and the percentage of cells having phagocytosed at least one Cundida was calculated. The number of Cundida phagocytosed per cell was also determined. A phagocytic index was calculated as being the average number of Cundida per macrophage, i.e., the total number of ingested Cundida divided by the number of macrophages. The values presented in this work are the means and + standard deviation of five mice individually assayed. Each test was performed twice with similar results. Measurement of peritoneal cell chemoluminescence

Treatments PLA2 (from Nu@ nuju venom) was purchased from Sigma Chemicals (Batch 124F-8010), dissolved in 0.9% NaCl (111 units/ml) and divided into 0.5 ml aliquots, which were kept frozen at -20°C and used within a week. For in vivo administration, appropriate dilutions were made in the same medium, so that mice were injected with 0.2 ml intraperitoneally (i.p.) or with 0.5 ml i.v. 30 minutes before harvesting the cells. Bestatin, isolated from a culture filtrate of Streptomyces olivoreticuli (kindly donated by Laboratoires Roger Bellon, Neuilly-sur-Seine, France), was dissolved in 0.9% NaCl and ip injected (5pg/mouse, i.p.), 4 days before harvesting the cells. Trehalose dimycolate (TDM), kindly donated by

Peritoneal cells were suspended in phenol red-free Hank’s solution ( lo6 cells/ml) . This suspension contained 26-30% macrophages, and 5% neutrophils. CL measurements were performed in a LKB 1250 luminometer at 37°C in a light-tight chamber containing one ml of peritoneal cell suspension. Twenty ~1 of Luminol (Sigma chemicals) were added for a final concentration of 5 x 10 -6M. When background light emission became constant, 100 ~1 of a suspension of opsonized Zymosan (15 mg/ml) (Sigma chemicals) was added, and photoemission was recorded for 60 min. The emitted CL, in (millivolts) was plotted against incubation time (min) (24). The results of some experiments were expressed as the area under the curve (mV.min). Groups of lo-12 mice were used and each in-

Phospholipase

dividual standard formed normal ditions, repeated

was tested. The results are mean values f deviation. Other experiments were perusing pools of peritoneal cells from 10 or PLA2-treated mice. Under these coneach test was performed in triplicate and 3 times with similar results.

A*, an in vivo Immunomodulatot

33

chased from Biosys (France). The organic solvents used in the prostanoid extractions were obtained from Baker (Netherlands). Statistics The results were analysed, using Student’s t-test.

Extraction of the PG fraction and radioimmunoassay (RIA) of PG and LTC Briefly, 2 x lo5 peritoneal macrophages from normal or PLA2 treated mice were incubated in 0.6 ml of Hanks’ solution without phenol red at 37°C for 60 min either with or without 60~1 zymosan. Additional samples were incubated with zymosan and either indomethacin (10 -6M final concentration) or NDGA (10 -5M final concentration). After incubation, the suspension were centrifuged at 400 xg for 5 min and the supernatants were harvested (0.5 ml). The methods used for extraction and the RIA for PGEz, 6 keto PGFi, and TXB;! have been described previously (25). Aliquots of the culture medium (0.5 ml) were mixed with acetone (1: 2, v/v) to precipitate proteins, centrifuged and the supernatants treated with 2 volumes of hexane. After acidification to pH 3.5 with 70% citric acid, the lower phases were mixed with 2 volumes chloroform and incubated, with stirring, at 4°C overnight. The lower or phases were evaporated under nitrogen. Yields were assessed using tritiated prostaglandins. Each RIA was corrected according to the recovery ratio of the eicosanoid measured (25). After extraction, the PG-containing fractions were suspended in O.lM sodium chloride/phosphate buffer, pH 7.4, containing 0.1% gelatin. PGE;!, 6 keto PGFr, and TXB2 were assayed according to the method of Dray et al. (26, 27). LTCJ was measured directly in cell supernatants, without extraction, using the LTC4-specific [ 3H] assay system provided by Amersham Centre, U.K. Other reagents Potassium cyanide (KCN), indomethacin, nordihydroguaiaretic acid (NDGA), superoxide dismutase (SOD) and catalase were purchased from Sigma Chemicals. Indomethacin and NDGA were dissolved in ethanol and diluted in Hanks’ solution with phenol red. The enzymes and KCN were dissolved in phenol red-free Hanks’ solution at pH 7.2 before use. Tritiated protaglandins (F2OOMmlOO Ci/mmole) were purchased from Amersham Centre (United Kingdom) and nonradioactive prostaglandins were purchased from Seragen, inc. (Boston, MA). Antisera to prostaglandins E2 and Fdol, and thromboxane B2 were obtained from Pasteur Institute (France), and anti-6-keto PGF1, antiserum was pur-

RESULTS PLA2 effect on phagocytosis Phagocytosis is a complex activity, generally including an initial adhesion followed by ingestion of microorganisms and, secondly, their destruction. The Candida albicarw ingestion test was used to determine whether treatment with PLA2 could alter the first step of phagocytosis. It was performed in PLAztreated normal mice. Table 1 indicates that 30 min after i.p. injection, 0.7 PLA;? (0.7 units/ml) significantly increased both the number of macrophages able to phagocytose at least one Candida (p=O.O3), and the average number of Candida phagocytosed by these cells (p=O.OOl). Table 1 Phagocytosis of candida albicans after PL&-injection Treatment

Phagocytosis index

% of macrophages having ingested at least one candida

CONTROL

1.91 + .ll

84.9 f 1.92 P = 0.03 95.8

P = 0.001 PLAz

2.87 + .I6

The peritoneal macrophages from control or PLA,-injected mice (0.7 units/mouse i.p.) were incubated with Candida albicans for 30 min. The phagocytic index indicates the average number of Candida phagocytosed per macrophage. The results are the average + SD of 5 individual experiments. Statistical analysis: Student’s t-test.

PLA2 effect on chemoluminescence of peritoneal cells Dose-response tion route

and the influence of the administra-

The CL emitted by phagocytic cells after stimulation gives an adequate overall evaluation of free oxygen radical production. Mice were injected i.p. with 0.35, 0.7 or 1.4 units PLA2 per mouse. Thirty minutes later, the peritoneal cells were harvested, and CL was elicited with zymosan. Figure 1 shows the development of the response as a function of time. The control curve reached its maximum after 10 min and remained at that value for 60 min. The amplitude was minimal in all the groups. The responses of all the injected mice were higher than those of the controls. Maximum enhancement was obtained in the

34 Prostaglandins Leukotrienes and Essential Fatty Acids

15,

PLAP 0.7 UNITS /MOUSE PLA2 1.4 UNITS

$

10

g A f

PLA,+KCN PLA2 5’

PLA2 0.35

/,+

_- f-+-t-)--_r lo

2030405050

PLA2+!%i rPLA2+CATALASE

CONTROL MIN

Dose-dependent effects of PLA, on CL. Groups of 10 to 15 mice were iniected with PLA, harvest (1.4. 0.7, 0.35 units/mouse, i.p. 36 min before cell*harvest).‘Each peritoneal cell suspension was tested individually. CL was induced by addition of Zymosan. The curves are drawn with the average values f SD. Fig. 1

group injected with 0.7 units PLA2. This dose was used as a standard dose in all the subsequent experiments. Groups of 10 mice were injected, either i.p. or i.v., with 0.7 units PLA:! determine whether PLA2 has a localized or systemic effect. Zymosanenhanced CL was observed only with cells from i.p.-injected mice. The CL of peritoneal cells from i.v.-injected mice did’not increase, even with doses as high as 2.1 units per mouse (not shown). This experiment indicates that PLA2 acts locally. Sensitivity of the response oxidants

PLA2 *ETHANOL

UNITS

to KCN

and to anti-

The peritoneal cells were treated with KCN, which inhibits mitochondria respiration without affecting CL in normal cells (29) to determine whether mitochondrial respiration participates in the CL response of treated cells. The zymosan-enhanced CL was not changed by 100 mM KCN in either PLAz-treated (0.7 units/mouse) or control groups (data non shown), indicating that the enhancement was not due to modification of the mitochondrial respiratory chain. The amount of luminal-dependent CL is an overall measure of intracellular and myelopeoxidasedependent oxidative free radicals. Various specific scavengers were added to the cells in vitro, to dissect the response and evaluate their respective contributions (Fig. 2). Each test was carried out on 1 X lo6 peritoneal cells taken from a pool of 10 PLA;?-treated (0.7 units/mouse) or control mice. The response was evaluated from the area under each CL-time curve (millivolts minutes). The CL of PLATtreated cells was slightly inhibited when the cells were incubated with zymosan plus 20 mM

10

20

30

40

50

60 MIN

Fig. 2 Sensitivity ot chemoluminescence to KCN, SOD, catalase and ethanol. Grou s of 10 mice were injected i.p. with PLA2 (0.7 unit Yp mouse i.p.) 30 min before cell harvest. Peritoneal cells were pooled and CL elicited with zymosan in the presence of either KCN 1OODM final concentration, SOD (300 units/ml) catalase 2750 units/ml) or ethanol (20DM final concentration). Each experiment was repeated three times with similar results.

ethanol which eliminates OH. In similar conditions, SOD (300 units/ml), which eliminates the initially formed 02- radical and leads to the formation of H202, inhibited 56% of the PLA2-induced response. Catalase (2750 units/ml), which transforms H202 into H20, entirely inhibited the response. Sensitivity of the response to arachidonic acid (AA) metabolism

inhibitors

of

Peritoneal cells from 10 PLA2-injected (0.7 units/mouse) or normal mice were pooled and the zymosan-elicited was measured in the presence of either indomethacin or NDGA, added in vitro (Fig, 3). The area under each curve was calculated (millivoltsminutes). The emission of CL PLA2-injected by mouse macrophage was not modified by 10-w indomethacin, whereas that of control macrophages was slightly enhanced. 10-3M indomethacin inhibited CL in both list and control cells (Fig. 3). NDGA (lo-‘M or 10-8M markedly reduced the CL of both list and control macrophages (Fig. 3). The. inhibitors were not cytotoxic as judged by trypan blue exclusion, even at doses which inhibit 90% of the response. Potentiation of the activity of various compounds

The response to PLA2 of peritoneal cells from mice previously stimulated by compounds described as macrophage activators (bestatin, TDM and ACM) was examined (Fig. 4). Each group contained 10 CL of their mice, and the zymosan-elicited

Phospholipase TaMe 2 Prostanoid

and leukotriene

Treatment

AZ, an in vivo Immunomodulator

cq production by peritoneal phagocytic cells from normal and partreated

Experimental Spontaneous

conditions of cell incubation Zymosan

Zymosan + Indomethacin

mice

Zymosan + NDGA 10-b IO-‘rn

PGE, CONTROL PLAz

85 + 11 I78 + 37+

613 + 123 880 + 132

240 + 11 50 f 2**

85 I? 25 109 f 56

TXB, CONTROL PLA2

90 + 20 120 +_ 41

344 + 79 293 +_ 151

61 f 8 92 + 52

121 f 58 103 +_ 53

861 + 173 2445 + 274**

8162 + 621 9007 f 1956

304 + 115 886 f 55**

1017 f 427 1943 * 997

252 rt 14 455 + 100*

3837 f 415 1559 + 786*

7609 * 553 4104 + 1046**

6-keto PGF,+CONTROL PLA, LTC, CONTROL PLA,

35

463 + 160 497 + 135

PG were measured by RIA in the supernatant of peritoneal macro hages either triggered or not triggered with zymosan (60Dl) for 60 min at 37°C in 5% CO,. Results are expressed as pg 2 ~1 $ cells and are the means + SD of 3 independent experiments. Statistical analysis by Student’s test, (*) p
CONTNOL

Fig. 3 Sensitivity of chemoluminescence to indomethacin and NDGA. Peritoneal cells from 10 PLAZinjected mice (0.7 units/mouse i.p., 30 min before cell harvest) or control mice were pooled and CL was measured in the presence of different doses of either indomethacin (lob6 or lOb3M final concentration) or NDGA (lo-8 or lo-5M final concentration). CL was elicited with zymozan. The results are expressed in millivolts minutes RD. Each experiment has been done at least in triplicate and repeated 3 times with comparable results.

peritoneal cells was measured for each individual cell suspension. The peritoneal cells of mice treated with bestatin, TDM or ACM four days before sacrifice emitted more CL that cells from untreated mice. Injection of PLAz 30 min before cell harvest leads to a further increase in CL emission over that of peritoneal cells from mice given bestatine, TDM or ACM alone (fig 4 A, B, C, respectively). Effect of PLAl on the production of AA metabolism The effects of PLA2 on the generation of PGE2, 6-

keto-PGF,,, TXB,? and LTC4 are shown in Table 2. In the absence of zymosan stimulation, in vivo

Fig. 4 Chemoluminescence response to PLA2 injection of peritoneal cells from immunostimulated mice Groups of 12 mice received i.p. on day-4 either TDM (O.OSDg.mouse), bestatin (SDg/mouse) or aclacinomycin (4 mg/kg) with or without the i.p. injection of 0.7 units/mouse PLA, 30 min before cell harvest. Each peritoneal cell suspension was tested individually. The results are expressed as millivolts minutes f SD.

PLA2 treatment significantly increased PGE2, 6keto-PGF,. and LTC4 production, and had no effect on TXB2 synthesis. Zymosan markedly stimulated the production of all the derivatives of AA metabolism measured by control untreated cells. The synthesis of AA products by cells retreated in vivo with PLA2 and stimulated in vitro with zymosan was comparable to that of zymosan-treated control cells, except that LTC4 was significantly decreased (p=O.Ol). Indomethacin inhibited the production of cyciooxygenase pathway derivatives (PGE2, TXB2 and 6-keto-PGFiJ in zymosan treated control and PLATtreated cells, while significantly increasing that of LTC4 (p
36

Prostaglandins Leukotrienes and Essential Fatty Acids

DISCUSSION The data reported here clearly indicate that peritoneal cells from PLA2 injected mice show enhanced phagocytic activity and produce more free oxidizing radicals after stimulation than do normal cells. However, peritoneal cells are not an homogeneous population. The enhanced phagocytosis of C.albicans is measured in macrophage cells, but the CL-emission in our experimental conditions is produced by macrophages and, in spite of their low concentration, neutrophils which can secret free oxygen radicals (8). Unfractionated cells were generally used in order to stay close to physiological conditions, and similar mechanisms trigger light emission by both neutrophils and macrophages after zymosan stimulation. We have also shown that activation of peritoneal cells is a membrane phenomenon, because KCN, which impairs the respiratory chain in mitochondria, does not affect CL in PLAz-injected mice (similar results were obtained with rotenone -data not shown). The inhibiting effect of catalase and SOD on CL suggests that the main radicals implicated were H202 and 02-, respectively. These radicals have been linked to bacteridical activity and cytotoxic effects on tumoral cells (8, 9, 10). It should be noted that PLA2 may enhance the activity of peritoneal cells that have already been optimally stimulated by bestatin, TDM or ACM. There compounds stimulate macrophage functions (21, 22, 23), suggesting that the mechanisms of action of these compounds, are different from that of PLA2. The studies on the possible dependence of CL emission on AA metabolism in this system showed that. CL emission by both normal or PLAz-treated cells is inhibited by NDGA and by high concentrations of indomethacin (10-3M). These findings indicate that the lipoxygenase pathway of AA metabolism may participate in the development of CL, as was reported for normal cells (13-15). In contrast, low concentrations of indomethacin (10-6M) did not modify the response of PLAztreated cells, but stimulated the CL of normal cells. This may be attributed to inhibition of PG production, as PG are known to inhibit CL emission, and/or to a better availability of the lipoxygenase pathway (30). The in vivo-effect of PLAz on AA metabolism, the degree of inhibitor specificity and any relationship between AA metabolism and CL in this experimental model, were assessed by measuring the production of the principal macrophage products of AA metabolism in the presence of the agents previously tested on CL. In vivo PLA;! treatment induces a significant increase in peritoneal cell PGE2, 6 keto-PGFI, and LTG, while having no effect on TXB2 production. Thromboxane synthetase is generally considered to be less

modulated than other PG synthetases (31). Neverthese results suggest that in vivo theless, administered PLA,! can increase the availability of free AA to peritoneal cells, which is therefore available for the enzymes of both the cyclooxygenase and lipoxygenase pathways. As expected, zymosan greatly stimulated the production of all the AA metabolites measured by untreated cells. The ‘absolute values’ were not statistically significant when the cells were pretreated in vivo by PLA2, except for LTC4. These results may be compared to those of Schade (32), who found that, following preincubation of peritoneal macrophages with LPS, the amount of LTC4 released during phagocytosis of zymosan was substantially decreased, where the levels of PGE* and PG12, were the same in LPStreated and control cells. Indomethacin (1O-6 M) reduced the production of all cyclooxygenase derivatives of AA metabolism, while increasing the production of LTC4, probably because of better availability of AA to-the lipoxygenase pathway in both untreated and PLAztreated cells. The level of LTC4 released from PLAz-treated cells by zymosan in the presence of indomethacin are approximately one half that of untreated cells. The LTC4 production of PLA*treated cells was similarly lower than the untreated cells, when these cells were stimulated by zymosan in the absence of indomethacin. Moreover, the amounts of LTC4 produced, in the presence of zymosan, by control cells from untreated mice without indomethacin and from PLArtreated mice with indomethacin were comparable while there was a significant increase in LTCz production by cells from untreated animals in the presence of indomethacin. This difference is probably due to changes in AA metabolism induced by PLA2-treatment. Thus, the increased CL emission observed over the control values obtained after zymosan triggering min the presence of indomethacin may be related to the increase in LTC4 production, since exogeneous LTC4 has been reported to stimulate CL (16), and to the concomitant decrease in PG which inhibits CL (15, 24) production. NDGA, which inhibits CL emission in all cases, also reduces the cyclooxygenase and lipoxygenese derivatives of AA, accounting for its poor specificity. The effect of NDGA on CL is probably not related only to the inhibition of lypoxygenase products, since NDGA also scavenges free radicals and inhibits NADPH oxidase activity by acting at the level of the electron transport (34). In conclusion, i.p. injection of PLAZ into mice increased the ability of the peritoneal macrophages to phagocytize particles and to liberate active oxygen. It also enhances the CL of peritoneal cells from mice already stimulated by immunoregulators such

Phospholipase

as TDM and bestatin, or oncostatic drugs such as ACM. Such a mechanism may be of great interest whenever an additional level of stimulation is required. The increased capacity of peritoneal cells to produce more free oxidizing radicals after PLA2 treatment takes place at the membrane level. In vivo PLA;! treatment increases the production of both cycle-and lipooxygenase derivatives of AA metabolism. Inhibitors of AA metabolism modulate the production of free oxidizing radicals in this experimental model, partly as a result of their effect on AA metabolism, as determined by the measurement of PG and leukotrienes. However, the specificity of these agents for AA metabolism is relative, and the release and metabolism of AA and the secretion of reactive oxygen intermediates appear to be two frequently coincident, but not mutually dependent, pathways in the mouse peritoneal cells (34).

References 1. Trail1 K N, Wick G. Lipid and lymphocyte function. Immunol. Today, 5: 70-76, 1984. 2. Hirata F, Matsuda K, Wano Y, Hattori T. The biochemical mechanism of cellular activation. p. 93 in ‘Advances in lmmunopharmacology 3’ (L Chedid, J W Hadden, F Spreafico, P Dukor, D Willoushby eds.). Pergamon Press, Oxtord, 1986. 3. Scot! J A. Phospholipase activity and plasma membrane homeostasis. J. Theor. Biol. 111: 659-665,1984. 4. Sandermann H j r. Regulation ot membrane enzymes by lipids. Hiochem. Biophus. 515: 209-237.1978. 5. Hirata F, Axelrod J. Phospholipid methylation and biological signal transmission. Science, 209: 1082-1090,198O. 6. Allen R C, Stjemholm R L, Steele R H. Evidence for the generation of an electronic excitation state (s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem. Biophys. Res. Comm. 47: 679-684, 1972. 7. Allen R C, Loose L D. Phagocytic activation of a luminol dependent chemiluminesc&ce in rabbit alveolar and peritoneal macrophages. Hiochem. Biophys. Res. Comm., 169: 245-552, 1976. 8. Fantone J C. Ward P A. Role of oxveen-derived free radicals’and metabolites in ‘leukocyte-dependent inflammatory reactions. Amer. J. Path., 107: 393-418, 1982. 9. Sasada M, Johnston R B j r. Macrophage microbicidal activity. Correlation between phagocytosis-associated oxidative metabolism and the Pilling of candida by macrophages. J. Exp. med., 152: 85-98, 1980. 10. Adams D 0, Johnson W J, Marino P A. Mechanisms of target recognition and destruction in macrophage-medIated rumor cytotoxicity. Fed. Proc. 41: 2212-2221, 1982. 11. Bromberg Y, Pick E. Unsaturated fatty acids as second messengers of superoxide generation by macrophages. Cell. Immunol., 79: 240-252, 1983. 12. Sakata A, Ida E, Tominaga M, Onoue K. Arachidonic acid acts as an intracellular activator of NADPH-oxidase in Fey receptor-mediated superoxide generation in macrophages. J. Immunol., 138: 4353-4359,1987.

A2, an in vivo Immunomodulator

13. Cheung K, Archibald A C, Robinson M F. The origin of chemiluminescence produced by neutrophils stimulated by zymosan. J. Immunol., 130: 2324-2329,1983. 14. Rush D N, Keown P A. Human monocyte chemiluminescence triggered by IgG aggregates. Requirement of phospholipase-a&vat&i &d modulation by Fc receptor ligands. Cell. lmmunol., 87: 252-258.1984. 15. Weideman M J, Smith R, Heaney T, Alaudeen S. On the mechanism of the generation of chemiluminescence by macrophages. Behring Inst. Mitt., 65: 42-54. 1980. 16. Hartung H P. Leukotriene C4 (LTC4) elicits respiratory burst in peritoneal macrophages. Europ. J. Pharmacol.. 91: 154-160, 1983. 17. Bejar R, Curbelo V, Davis C, Gluck L. Premature labor. ii. Bacterial source of phospholipase. Obstetrics & Gynecology 57: 479-482, 1981. 18. Gutierrez J M. Lomonte B, Chaves F, Moreno E. Cuerdas L. Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper. Comp. Hiochem. Physiol. 84: 159-164.1986. 19. Zawalich W, Zawalich K. Effect of exogenous phospholipase A2 on insulin secretion from perfused rat islets. Diabetes 34: 471-476, 1985. 20. Bravo Cuellar A, Balercia G, Levesque J P, Liu X H. Osculati F. Orbach-Arbouvs S. Enhanced activity of peritoneal cells after aclacinomycin injection: effect of pretreatment with sup&oxide dismutase on aclacinomycin induced cytological alterations and antitumoral activity. Cancer Res., 49: 1578-1586, 1989. 21. Bruley-Rosset M, Florentin I, Kiger N, Schulz J. MathC G. Levamisole and bestatin in immunodeficient aged mice. p. 59 in ‘Small molecular immunohodifiers of microbial origin. Fundamental and clinical studies of bestatin’ IH Umezawa ed.), Pergamon Press, Oxford, 198i. 22. Lemaire G, Tenu J P, Petit J F. Natural and synthetic trehalose diesters as immunomodulators. Medical Research Reviews 6: 243-274, 1986. 23. Bravo Cuellar A, Scott Algara D, Metzger G, Orbach-Arbouys S. Enhanced activity of peritoneal cells after aclacinomycin administration. Cancer Res., 47: 3477-3484, 1987. 24. Bravo Cuellar A, Homo-Delarche F. Cabannes J, Orbach-Arbouys S. Enhanced activity of murine peritoneal cells after aclacinomycin injection: characteristics of the enhanced respiratory burst. Cancer Res. 48: 3440-3444, 1988. 25 Homo-Delarche F, Duval D, Papiernik M. Prostaglandin production by phagocytic cells of the mouse thymic reticulum in culture and its modulation by indomethacin and corticosteroids. J. Immunol.. 135: 506-511; 1985. 26 Dray F, Charbonnel B, Maclouf J. Radioimmunoassay of prostaglandins F2, E 1 and E2 in human plasma. Eru. J. Chim. Invest., 5: 311-318,1975. 27. Russo-Marie F, Paing M, Duval D. Involvement of glucocorticoid receptors in steroid-induced inhibition of prostaglandin secretion. J. Biol. Chem., 254: 8498-8504, 1979. 28. Perroteau I. Netchitailo P. Homo-Delarche F. Delarue C, ‘Lihrmann L, ieboulenger F, Vaudry H. Role of exogenous and endogenous prostaglandins in steroidogenesis by isolated frog interrenal glands: evidence for dissociation in adrenocorticotrophin and angiotensin action. Endocrinology, 115: 1765-1773, 1984. 29. Klebanoff S J. Oxvaen-dependent cvtotoxic mechanisms of phagocytes. p. 111 in ‘Advances in host defense mechanisms’. (J I Gallin. A S Fauci eds.). Raven Press, New Y&k, 1982. 30. Weidemann M J, Peskar B A, Wrogemann K,

37

38 Prostaglandins Leukotrienes and Essential Fatty Acids Rietschel E T, Staudinger H, Fisher H. Prostaglandin and thromboxane synthesis in a pure macrophage population and the inhibition by E-type prostaglandines of chemiluminescence: Febs Letters 89: 136-140, 1978. 31. Tripps C S, Leahy K M, Needleman P. Thromboxane synthase is preferentially conserved in activated mouse peritoneal macrophages. J. Clin. Invest., 76: 898-901, 1985. 32. Shade U. F. The effect of endotoxin on the lipoxygenase-mediated conversion of exogenous and

endogenous arachidonic acid in mouse peritoneal macrophages. Prostaglandins 34: 385400,1987. 33. Crawford D R, Schneider D L. Identification of ubiquinone-50 in human neutrophils and its role in microbicidal events. J. Biol. Chem., 257: 6662-6668,1982. 34. Tsunawaki S, Nathan C F. Release of arachidonate and reduction of oxygen. Independent metabolic bursts of the mouse peritoneal macrophage. J. Biol. Chem., 261: 11563-11570,1986.