CELLULAR

IMMUNOLOGY

136,41-53

(199 1)

Phorbol Ester Plus Calcium lonophore Induces Release of Arachidonic Acid from Membrane Phospholipids of a Human B Cell Line’ ELLEN B. GILLIAM, PETER G. SCHULAM, J. PATRICK WHELAN, HOWARD M. ROSENBLATT, AND WILLIAM T. SHEARER’ Departments

of Pediatrics, and Texas Received

Microbiology Children’s

November

and Immunology, Hospital, Houston,

15, 1990; accepted

Baylor College Texas 77030

February

of Medicine,

21, 1991

Binding of LA350, a lymphoblastoid human B cell line, by phorbol my&ate acetate (PMA) plus a calcium ionophore, either ionomycin or A23 187, produced unique alterations in the release of arachidonic acid (AA) from cellular phospholipids. After equilibrium labeling of cells with radioactive fatty acids, [‘%&A demonstrated a selective enhanced release from the cells in response to the binding of PMA plus calcium ionophore as compared to the release of [‘%]stearic acid (STE), [3H]oleic acid (OLE) and [3H]palmitic acid (PAL). The major phospholipid sources of the released [%]AA were shown to be phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The participation of protein kinase C (PKC) in the enhanced synergistic release of [14C]AA was demonstrated by the inhibition of the release by the PKC inhibitor, staurosporine. Approximately 2-6s of the labeled AA liberated was converted to S-hydroxyeicosatetraenoic acid by an endogenous 5-lipoxygenase. Therefore during cell activation the B cell is capable of liberating AA via a PKC-dependent mechanism, implicating AA and/or its metabolites in signal transduction. 0 1991 Academic press, Inc.

INTRODUCTION sIg3 expressed on the B lymphocyte acts as a receptor for the Ag to which the cell can mount an immune response. Interaction of sIg with ligand (i.e., Ag or anti-p Ig) leads to the activation of PLC ( l-4). PLC hydrolyzes PI-bisphosphate to IP3 and DAG (4, 6) as well as PC into CP and DAG (7). IP3 induces a rise of intracellular calcium from intracellular stores and DAG allows for the translocation and activation of PKC ’ Supported in part by NIH Grant SPOI-AI2 1289 and by a special fund of Texas Children’s Hospital. 2 To whom correspondence and reprint requests should be addressed at Section of Allergy and Immunology, Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. 3 Abbreviations used: PMA, phorbol myristate acetate; PKC, protein kinase C; PC, phosphatidylcholine; PI, phosphatidylinositol; PA, phosphatidic acid; PE, phosphatidylethanolamine; AA, arachidonic acid; PAL, palmitic acid; STE, stearic acid, OLE, oleic acid; FFA, free fatty acid; NL, neutral lipid pool; TxB,, thromboxane BZ; LTC,, leukotriene C,; HETE, hydroxyeicosatetraenoic acid; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; IN, ionomycin; sIg, surface immunoglobulin; PLC, phospholipase C; IP,, inositol-1,4,5&sphosphate; DAG, diacylglycerol; CP, choline phosphate; PG, prostaglandin; TX, thromboxane; CO, cyclooxygenase; LO, lipoxygenase; LT, leukotriene.

41 0008-8749/91

$3.00

Copyright Q 199 I by Academic Press, Inc. All rights of reproduchon in any form reserved.

(5. 6, 8). Stimulation of the PC and Pi cycles by cell membrane ligands in lymphoblastoid human B cell lines has been shown to cause an increase in the secretion of immunogiobulin (4, 9, IO). The DAG generated from both the PC and PI cycles is either phosphorylated to produce phosphatidic acid that can be recycled back into the phospholipid pool. or it can be hydrolyzed by DAG lipase into glycerol plus the two fatty acids esterified to the number 1 and 2 carbons of the glycerol backbone. The predominant fatty acid at the sn-2 position of mammalian phospholipids is AA, a 20carbon polyunsaturated fatty acid. AA can be alternatively liberated from membrane phospholipids directly by the calcium-dependent enzyme PLAz . AA can be converted by CO into PG and TX and by LO into LT. lipoxins, and HETE. We have demonstrated in several human lymphoblastoid cell lines, including LA3.50, an endogenous 5-LO which when stimulated with calcium ionophore generated the AA metabolite 5-HETE (I 1). To provide the groundwork for additional, ongoing studies of AA metabolites in this ligand-activated B cell line LA350, it would be important to examine the release of [‘4C]AA in cells stimulated by PMA plus calcium ionophore. These ligands, which respectively lead to the activation of PKC and a rise in intracellular calcium, emulate the messengers induced by PI and PC hydrolysis. These two stimuli are also capable of producing selective release of AA in diverse types of cells ( 12- 1.4). Similar investigations in other simulated cell systems have suggested roles for AA metabolites in gene activation for subsequent tumor necrosis factor production ( 15 ). Thus, signal transduction pathways involving release of AA may produce cell activation by modification of expression of genes which code for enzyme or cytokine production. Accordingly, we have looked at the selectivity of [r4C]AA release from cellular phospholipids and the mechanism of enzymatic release of radioactivity from cells prelabeled with [“C]AA and stimulated with PMA plus ionophore. Also. we have studied the conversion of AA into metabolic products capable of participation in ceil signal transduction events. The highly selective pattern of AA release by PMA plus ionophore-stimulated B cell lines suggests that AA and its 5-lipoxygenase-produced metabolites may play importantbiological roles in cell regulatory events and, possibly, in immunoglobulin production. Preliminary reports of these findings have been made (16. 17). MATERIALS

AND

METHODS

Cell line. LA350 is an Epstein-Barr virus-transformed human lymphoblastoid Bcell line which contains surface IgM and secretes IgM. LA350 was maintained in tissue culture as described previously (18). Reagents. The following is a list of reagents used and their sources: RPM1 1640, Medium 199, penicillin/streptomycin, glutamine? Hepes buffer, Waymouth’s MB752f 1 medium, gentamycin, trypan blue, and fetal calf serum (FCS) (GIBCO, Grand Island. NY); ionomycin (IN), PMA, and staurosporin (Calbiochem, La Jolla, CA); A23 187. fatty acid-free bovine serum albumin (BSA), and d&odium ethylenediaminetetraacetic acid (EDTA) (Sigma, St. L,ouis, MO); tritiated eicosanoid standards of thromboxane B2 (TXB& leukotriene C, (LTC,& %hydroxyeicosatetraenoic acid (5-HETE), and AA (180-240 Ci/mmol) (NEN-DuPont, Boston, MA); [‘4C]AA (50-60 mCi/mmol) (NENDuPont and Amersham, Arlington Heights, IL); [‘“CISTE (50-60 mCi/mmol), [3H]PAL (40-60 Ci/mmole) and [3H]OLE (2-10 Ci/mmol) (Amersham); phospholipid

B CELLS

AND

ARACHIDONIC

ACID

RELEASE

43

reference standards (Avanti Polar Lipids, Alabaster, AL); neutral lipid and fatty acid reference standards (NuChek, Elysian, MN); silica gel H thin layer chromatography (TLC) plates (Analtech, Newark, DE); Sep-Pak Cl8 cartridge (Rainin, Woburn, MA); glass distilled chloroform, methanol, and acetone (Burdick and Jackson, Muskegan, MI); high-performance liquid chromatography (HPLC) grade water (Fisher, Pittsburgh, PA). Determination of equilibrium labeling. Cells from tissue culture were centrifuged 3 min (6OOg), washed once, and resuspended at 0-4°C in labeling medium (Medium 199, 40 mM in Hepes, pH 7.4, containing 15% heat-inactivated FCS). Cell viability (:-90%) was assessed by exclusion of trypan blue. Final cultures containing 1 X lo5 cells and 0.5 &i of [14C]AA or 0.4 &i of [14C]STE in 1 ml of labeling medium were incubated at 37°C. The reaction was terminated by placing the cultures on ice; the cells were washed in cold 0.15 M NaCl; and the lipids were extracted by the method of Bligh and Dyer (19,20). For determination of total lipid radioactivity, the chloroform phase was transferred to glass liquid scintillation vials. After evaporation of the chloroform, the residues were assessedfor radioactivity by liquid scintillation spectrometry (Tri-Carb 2000CA Packard Instrument Co., Laguna Hills, CA). For determination of radioactivity in isolated lipid fractions the chloroform phase was dried under nitrogen and redissolved in chloroform, and the lipids were separated by TLC as described below. Analysis of cellular lipids by TLC. Individual phospholipids were isolated by the following adaptation of the method of Rouser (2 1): first dimension, chloroform:methanol:ammonium hydroxide:water (65:25:2:3); second dimension, chloroform:acetone: methanol:acetic acid:water (3:4: 1: 1:0.5). Samples were chromatographed with authentic phospholipid and neutral lipid standards. This system was developed for the separation of phospholipids. The neutral lipids (NL) including mono-, di-, and triglycerides migrated with the solvent fronts. Free fatty acids (FFA) migrated separately from both the NL and the separated phospholipids. Lipids were visualized by I2 staining. The resulting spots were scraped into vials and assessed for radioactivity by liquid scintillation spectrometry. Separation of mono-, di-, and triglycerides was accomplished by redissolving the dried chloroform phase in hexane and performing one-dimensional TLC in ethyl ether:hexane:acetic acid (70:30:1). This is the first dimension in the system described by Prescott and Majerus (22). Enhancement offatty acid release. Cells were prelabeled in labeling medium as above for 4 or 24 hr. Four-hour labeling conditions included 2 X lo6 cells/ml plus the following concentrations of isotopes: 0.4 &i/ml [14C]AA or [14C]STE with or without 2 &i/ml [3H]PAL or [3H]OLE. Twenty-four-hour labeling incubations contained 1 X lo6 cells/ml and 0.2 &i/ml [14C]AA or [14C]STE with or without 1 &i/ ml of [3H]PAL or [3H]OLE. Labeled cells were washed twice at 0-4”C and resuspended at 4 X lo6 viable cells/ml in incubation medium (RPM1 1640 with rglutamine, 20 mA4 in Hepes, pH 7.4, containing 15% heat inactivated FCS). Total dpm was determined by counting aliquots of this final cell suspension. 0.25 ml (1 X lo6 cells) was plated per culture; PMA or DMSO carrier control and A23 187 or DMSO carrier control and, where applicable, staurosporine or DMSO carrier control were diluted in the incubation medium and added. The final volume per culture was adjusted to 0.6 or 1.2 ml; stimulation was carried out at 37°C. The reaction was stopped by

44

C;lt.l.IAM

t.‘l’

41

cooling to 0-4°C; the cells were centrifuged 5 min (12OOg); and ahquots of the supernatant were taken for liquid scintillation counting. In some experiments the cellular phosphohpidswere analyzed for loss of [?ZY]AA label. For this, the cell pellets were washed and the lipids extracted and analyzed as described above. When the composition of the radioactivity detected in the supernatants was to be analyzed, 6 X IQ6 cells/ml were labeled for 4 hr with 1 &i/ml of high specific activity [“HIAA. Test incubations (2 X IO6 cells/ml) were carried out as above. After centrifugation of the stimulated cells, 0.9/l .2 ml aliquots of the supematant from triplicate cultures were combined and extracted for analysis by HPLC. Extruction qf supernatuntsfk HPLC analysis. The methodology of Powell (23) as reported by Shak (24) was adapted to remove excess protein from the supernatants. Cold methanol was added to a fmal concentration of 80%; samples were left at --20°C.’ at least 1 hr; then, centrifuged IO min ( 12OOg).The resulting supematant was adjusted to a concentration of I 5% methanol by the addition of HPLC grade water. This sample was extracted on a Sep-Pak C ,x cartridge using the modification of the protocol reported by Powell et ~11.(25) as described by Schulam and Shearer (11). Recovery of [14C]AA and tritiated TX&, LTC4, and 5-HETE standards from incubation medium by this method was >80%$. Analysis o/‘supernatanr extracts /).I reverse-phuse HPLC‘ Reverse-phase chromatography was performed on a S-pm C 1X. 4.6 X 250 mm Microsorb column (Rainin) using the protocol adapted from Henke r’t ul. (26) by Schulam and Shearer (I 1) Retention times for standards were as follows: TXB?. 25 min: LTCJ. 43 minutes: 5-Hete. 78 min: AA, 85 min. Statistical analysis. Statistical comparison were made with the Student’s 1 test for unpaired data. RESULTS Equilibrium lubeling ~~‘cel1.sMitt? [ “13].4A and [““CJSIZ’. To determine whether labeling with [14C]AA and [14C]STE for various time periods would be adequate to achieve equilibrium, we determined the incorporation of [14C]AA and [14C]STE into the neutral lipid and phospholipid fractions of cells over time (Fig. 1). There was a very rapid Labeling of the neutral lipid fraction by [ 14C]AA which fell rapidly to an equilibrium level by 24 hr. Separate analyses revealed that >90% of the f14C]AA in the NL pool at 4 and 24 hr was incorporated into triglycerides (data not shown). The label appearance in the major individual phospholipids (PC, PE, PI), however, demonstrated a gradual rise to near-equilibrium levels by 24 hr. [14C]STE also demonstrated a similar labeling pattern except that the pronounced early labehng of NL was not observed and considerable free STE remained unmetabolized. Because these studies demonstrated that the intracellular pools of radioactive fatty acids were at equilibrmm after 24 hr of incorporation. we assume that release of radioaetivity was a valid measure of the release of mass. Release qf [‘“Cl by cells preiubeled with [‘“Cjuruchidonic ucidc Ejfects of PMA plm ionophores. We performed isotope release studies in cells prelabeled for 24 hr with [14C]AA, washed, resuspended in incubation medium, and then stimuiated for 4 hr with various concentrations of ionomycin and PMA (Fig. 2A). .A concentration of

B CELLS AND ARACHIDONIC 60

1

A. Arachidonic Acid

B. StearicAcid

I 0 l

NL

v n v

PI FFA PS

PC 0 PE

t I.

I.

I.

I,

I.

0

8

16

24

32

45

ACID RELEASE

4

40

10

II.

480

I

I.

I.

I,

I,

I,

8

16

24

32

40

48

HOURS

I. Incorporation of [14C]AA and [14C]STE into neutral lipids (NL) and phospholipids of B cells. Etfect of Length of Labeling Interval. Cells (1 X 105) in l-ml labeling medium containing 0.5 rCi [14C]AA or 0.4 PCi [14C]STE were incubated at 37°C. Results are expressed as the mean f the standard error (SEM) of the percentage of total radioactivity in the chloroform fraction; i.e., FIG.

% Total Radioactivity in Chloroform =

dpm in Isolated Lipid X ,oo dpm in Chloroform Phase

The total radioactivity in the chloroform fraction per I X IO5 cells was 4 hr

24 hr

48 hr

dpm

SEM

dpm

SEM

dpm

SEM

AA

18,416

STE

14,121

694 389

31,225 24,869

889 1053

30,23 1 25,886

906 1670

-

The results are taken from two experiments performed in triplicate.

10M9 M PMA produced no additional release of isotope over that due to increasing concentrations of ionomycin, up to 1.4 pLM. Significant increases in the release of isotope (primarily [14C]AA, see below) occurred in the presence of lop8 and 1O-7 A4 PMA and increasing concentrations of ionomycin (P < 0.0005). A virtually identical pattern of release of isotope was produced when A23 187 (0 to 1.O PM) ionophore was substituted for ionomycin (Fig. 2B). In these studies a concentration of 1.4 +V ionomycin produced an effect equivalent to that seen with 0.5 WV A23 187. Specificity offatty acid in enhanced isotope release studies. To examine the specificity of the fatty acid in the isotope release studies, we compared the release of isotope by cells prelabeled for 24 hr with [14C]AA, [14C]STE, [3H]OLE, or [3H]PAL (Fig. 3). For

46

.. E

CdLlLiAM

i-,7‘ 4L

/

FIG. 2. Kelease of ‘“C by cells prelabeled with [‘“CJAA and bound by PMA and ionophores. Cells (I X lob/ml) were labeled with [‘%JAA (0.2 &‘i/ml) for 24 hr at 37°C. washed, and incubated (I X lOh cells, culture) in incubation medium (0.6 ml) with the indicated test reagents for 4 hr at 37°C. Results are expressed as the mean -+ the standard error of the percentage of total incorporated radioactivity found in the external medium: i.e.. ‘i> Radioactlvitl

Released =

dpm in External Medium x loo. Total dpm in 0.6 ml Culture

(A) Effects of IN. Total dpm per 0.6 ml tinai suspension was 234,290. The results are from four experiments performed in triplicate. (B) Effects of A23187. Total dpm per 0.6 ml fmal suspension was 350.525. The results are taken from a representative experiment performed in triplicate.

convenience of expression we term the release of isotope as the release of the labeling fatty acid. Only in ceils prelabeled with [14C]AA did ionophore (0.5 pA4A23 187) plus lo-* A4 PMA produce a significant increased release of isotope over baseline release as early as 0.5 hr. (P < 0.0005). This enhanced release of [14C]AA was much greater than any enhanced release of [3H]PAL, [3H]OLE, or [14C]STE at 1 and 2 hr, (P < 0.0005). Virtually identical results were obtained when the cells were prelabeIed for 4 rather than 24 hr (data not shown). Identification qfphospholipids servmgas source ofreleased [‘JC’]A.4. We attempted to identify the phospholipids which were releasing [14C] isotope in response to cell stimulation with lo-* M PMA plus 0.5 PM A23 187 (Fig. 4A). Highly synergistic decreases in residual phospholipid radioactivity were produced by the binding of PMA plus A23187 to cells (P < 0.005). The data suggested that PC, PE, and PI served as sources for the released [ 14C] isotope although complex mechanisms involving transfer of AA between phospholipids could not be ruled out (see discussion). There was no ligand-enhanced release of label from the neutral lipid fraction of the cell. These data were obtained with cells prelakled for 24 hr; we performed similar experiments with cells prelabeled for 4 hr and obtained the same results (data not shown). Inhibition of PMA plus A23181-enhanced release oJ’If4C]AA by PKC inhibitor, staurosporine. To examine the participation ofPKC in the PMA plus A23 187-enhanced

B CELLS AND ARACHIDONIC A.1’4C]ARACHlDONIC

ACID

+ CC”TRCL 8 Pwl lo-% + 1\23,e,0.5rY 8

ACID RELEASE

B.fk]STEARIC

ACID

’ D.f%l]PALMITIC

ACID

47

5-

PYA . A23W7 ,$ -

‘0’ C.ftiIOLEIC

ACID

Jr

c T I ” : I R E L E A

2-

2-

1-

1-

;oi*L----0

0.5

Y

1

1.5

2

TIME (HRS)

2.5

0

0.5

1

13

2

2.5

TIME (HRS)

FIG. 3. Specificity of release of radioactive fatty acids from prelabeled cells bound by PMA and A23 187. Effect of time. Cells (1 X 106/ml) were labeled with [%]AA or [“‘CISTE (0.2 &i/ml) plus [3H]PAL or [‘HIOLE (I &i/ml) for 24 hr at 37°C washed and incubated (1 X IO6cells/culture) in incubation medium II (1.2 ml) with the indicated test reagents for various time intervals at 37°C. Results are expressed as the mean f the standard error of the percentage of total incorporated radioactivity found in the external medium: i.e., % Radioactivity Released =

dpm in External Medium x 1oo, Total dpm in 1.2 ml Culture

Total dpm for I X lo6 cells were [14C]AA 232,200 dpm; [“‘CISTE 169,294 dpm; [3H]OLE 1,200,132 dpm; and [3H]PAL 1,19 1,269 dpm. The results presented for STE, PAL, and OLE are the mean + standard errors of two experiments performed in triplicate. The results for AA represent three experiments performed in triplicate.

FIG. -IA. ldenttfication ofphosphohpid source ol’released “‘C’ by cells preiabelcd with [“C]AA and bound by PMA and 423 187. Cells (1 S: IO’/ml) were labeled with [‘4C]AA (0.2 pfi/ml) for 24 hr at 37°C. washed. and incubated (I x 10’ cells/culture~ m incubation medium I I.? ml) with DMSOcontrol or iWx M PM4 and/or with 0.5 ,u:LI A23 I g? for 3 hr at 37°C. ‘The lipids were extracted and separated by TLC to assess loss of labeled AA from selected phosphohpid and the neutral lipid pools. The data. representing two experiments performed in triplicate. is expressed as the mean t the standard error of DPM.

release of [ ‘“C]AA from phospholipids, we performed release experiments in the presence of 1 X IO- ’ M staurosporjne, a known PKC inhibitor (27) (Fig. 4B). Highly significant inhibition of release of [14C]AA from PC, PE, and PI (P < 0.01) occurred in the presence of IO-’ M staurosporine. The neutral lipid fraction was not affected. Cell viability in this experiment was not affected by the presence of staurosporine as assessed by trypan blue.

d

40

FIG. 4B. Inhibitron of release 01 ‘Y‘ by staurosporine in cells prelabeled with [‘Y&44 and hound by PMA and ,423 187. Cultures contained IO 7 .II staurosporine to assess the role of PKC in the synergistic release of the label from these lipids. They were part of the experiments described in the legend to Fig. 4A.

B CELLS

AND

ARACHIDONIC

ACID

RELEASE

49

Demonstration that the isotope released by cells prelabeled with AA is AA and its 5-lipoxygenase-derived product, 5-HETE. To identify the major components of the radioactivity released by PMA plus A23 187~stimulated cells which had been prelabeled with [3H]AA, we performed HPLC on medium from cells that had been prelabeled, washed, resuspended in fresh medium (nonradioactive), and stimulated for 2 hr (Fig. 5). The chromatographic profiles clearly described that the principal released labeled compound was unmodified AA and that a significant amount of 5-HETE was also released (note scale difference for 5-HETE and AA). There was a synergistic effect present when the cells were bound by both PMA and A23187, suggesting enzymatic conversion of AA to 5-HETE. DISCUSSION There are several publications that suggest that AA metabolites influence B cell metabolism and function. Prostaglandin Ez, for example, has been shown to either inhibit (28,29) or stimulate immunoglobulin production (30). Leukotrienes also have been shown to modulate immunoglobulin production by B cells; for example, Ambrus et al. demonstrated an inhibitory effect due to leukotriene C4 (31). Yamaoka et al. have shown that leukotriene B4 stimulates proliferation and differentiation of B cells (32). The ability of exogenous eicosanoids to influence B cell function leads one to suspect the existence of lipoxygenase and cyclooxygenase activity in B lymphocytes. Kecently, Behrens et al. have demonstrated that lipoxygenase inhibitors enhanced the proliferative response of mitogen-stimulated human B cells, suggesting potential endogenous lipoxygenase activity (33). And as mentioned in the introduction, we have demonstrated endogenous 5-LO activity in several human B cell lines (11). In developing a theory for AA metabolism it is crucial to establish that the parent molecule, AA, has a unique metabolism and is released in response to cell membrane ligands that initiate signal transduction and cell activation and secretion of immunoglobulin in the case of B cells. Thus, in our present series of investigations of the role of signal transduction and subsequent immunoglobulin secretion by human lymphoblastoid B cells, we clearly show here that, in terms of release, AA possesses unique properties. Moreover, our findings are consistent with the partial conversion of AA into 5-HETE by 5-lipoxygenase (11). Our assumption that AA and/or its metabolites play some role in signal transduction has been borne out by the many recent findings that suggest a role for eicosanoids as second messengers. Farrar and Humes have shown that the metabolites of the LO pathway may be involved in IL- 1, IL-2, and phorbol ester-induced signal transduction in the T cell lines, EL-4 and BFS (34). Russell, Torres, and Johnson, in a series of papers, demonstrated that AA and its LO products play a central role in IL-Zinduced IFN-7 production (35-37). Piomelli et al. demonstrated that the eicosanoid 12-HPETE, acts as a second messenger for a neuroactive peptide, FMRFamide, during inhibition of serotonin-induced depolarization (38). Two groups have published reports suggesting t.hat the AA metabolites are the intracellular messengers for the G-protein-gated K’ channels in cardiac excitation (39, 40). Moreover, Horiguchi et al. have shown that the AA metabolites act as messengers in phorbol ester-induced expression of the TNF gene in HL-60 human promyelocytic leukemia cells ( 15). Several factors may play a role in the selective release of AA from phospholipids of stimulated cells. First, enzymatic cleavage by phospholipase A2 and/or diglyceride

IiILLlAM

I?I 41

A. Control 14,000

14,000

12,000

12,000 10,000

10,000

DPhl

8,000

8,000

6,000

6,000

4,660

4.000

zoo0

2,000

500

500

400

400

300

300

200

200

100

100 0

0 0

12.5

25.0

37.5

50.0 Time

62.5

75.0

67.5

100.0

(Min.)

8. PMA

DPM

14,000

14,000

12,000

12,000

10,OOa

1o,ow

8,000

6,000

6,000

6,000

4,ooo

4,000

2,000

2.000

500

500

400

400

300

300

200

200

100

100 0

0 0

12.5

25.0

37.5

50.0 Time

62.5

76.0

87.5

100.0

(Min.)

FIG. 5. HPLC‘ analysis of released radioactivity from LA350 cells prelabeled with [“H]AA. Cells 6 K IO”/ ml were labeled with [3H]AA (1 pCi/mI) for 4 hr at 37°C. washed, and incubated (2 X lo6 cells/culture) in incubation medium (1.2 ml) with DMSO control or lo-* FM PMA and with DMSO control or 0.5 & A23 187 for 2 hr. Analysis of the supernatant by HPLC demonstrated a small peak eluting at 78 min. the retention time for 5-BETE and a large peak eluting at 85 min, the retention time for AA (note scale difference for 5-HETE and AA). Actual dpm in the peaks were as follows: Control: AA 27.986 S-HETE 2389; PMA: AA 29,842 5-HETE 2 113: A23 187: AA 363 15 S-HETE 2767: PMA + A23 187: .4A 94,77 1 5-HETE 4347. For supernatants from cells treated with PMA 4~ A23 187 these figures extrapolate to 76.78~ and 2.5% of the increase in dpm over supernatants from control cells being in AA and 5-HETE.

lipase is suggested by this selective release of [‘4C]AA but other effects of PMA and calcium ionophore, such as inhibition of the reacylation of liberated AA into phospholipids, are possible (41). Second, the availability of phospholipid-bound AA to perturbation by cell membrane ligands has been clearly demonstrated in the work of Garcia-Gil and Siraganian in antigen and A23 187~stimulated rat basophilic leukemia

B CELLS

AND

ARACHIDONIC

ACID

51

RELEASE

C. A23187

DPM

14,000

-

12,000

-

-

12,060

10,000

-

-

10,000

6,000

-

-

6,000

6,000

-

-

6,000

4,000

-

-

4,000

P

2,000

- 14,000

2,000 p 500 s

500

0

12.5

25.0

37.5

50.0 Time

62.5

75.0

67.5

100.0

(Min.)

D. PMA t A23187 14,000

DPY

14,000

12,000

-

-

10,000

-

-

10,000

8,MH)

-

-

6,000

6,000

-

-

6,000

4,000

-

-

4,000

2,000

7 I

:

2,000

12,000

-‘5w

500’400

-

300

-

I

-

300

200

-

-

200

iw

-

-

1w

0 0

12.5

26.0

37.5

I 60.0 Time

FIG.

I 62.5

-

rL&) 75.0

400

0 67.5

lW.O

(Min.)

5--Continued

cells ( 12). Third, the phospholipid source of liberated AA in cell activation systems depends upon the type of cell involved, the time of prelabeling, and the nature of the stimulant (42). Our findings are that PC, PE, and PI release AA in response to stimulation by PMA plus A23187. In molar terms, PC is the most important source of released AA. Although our studies do not address the issue, it is possible that transacylation reactions exchange AA between phospholipids (43) e.g., PC to PI, in a complex pattern of release. Our recent investigations with LA350 and several other B cell lines demonstrating evidence of Slipoxygenase activity are made more pertinent by this report documenting that the parent compound of SHETE, AA, is uniquely released by transmembrane cell signals. It is highly likely that 5-HETE and other AA-derived metabolites, by I hemselves or in concert with cell activation ligands, modulate B cell function, including

52

~;ll.I.i,AM

El

41..

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