PGE2 and PGF2α biosynthesis in stimulated and nonstimulated peritoneal preparations containing macrophages

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PGE2 AND PGF BIOSYNTHESIS IN STIMULATED AND NONSTIMULATED PERITON i AL PREPARATIONS CONTAINING MACROPHAGEST Alam Farzad Neal S. Penneys2 Abdul Ghaffar Vincent A. Ziboh Jean Schlossberg Departments University

of of

Dermatology, Miami School

Microbiology of Medicine,

and Biochemistry Miami, FL, 99152

Abstract The biosynthesis of PGE2 and PGF2, was measured in intact peritoneal exudate preparations obtained from C_. parvum-treated and control C3H mice. Although both the control and stimulated preparations biosynthesized PGF2c and PGE2 from [l-T%] arachidonic acid, the stimulated preparations generated more of both prostaglandins than did nonstimulated preparations, probably as a result of increased synthesis within macrophages. Increased transformation of PGE2 into PGF2o by PGE2 9-ketoreductase was noted in stimulated preparations when compared to that in control cells. The data suggest that stimulated macrophages are capable of generatlng increased quantities of PGF2a and therefore might function as one source of this substance in resolving inflammatory reactions.

‘This work was supported in from the National Institutes Foundation of Miami. 2Neal Award

S. Penneys is AM 00203 from

the the

part by grants AM 14941 and AM 19079 of Health and by the Dermatology

recipient National

NOVEMBER 1977 VOL. 14 NO. 5

of Research Career Development Institutes of Health.

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Introduction Changes in the ratio of PGE2 to PGF e occur in tissue during the development and resolution of the inf t ammatory process. Velo et al. in a time sequence study of the distribution of prostaglandins in inflammatory exudates observed shifts in this ratio such that marked increases in the amount of PGF2a were observed in deflorescing inflammatory reactions (1). Macrophage cell types are known to arrive late in the evolution of an inflannnatory reaction (2) and might be one source of PGF2a biosynthesis. In this report, we have studied the PGE2 and PGF2e synthesizing capabilities of stimulated and nonstimulated peritoneal preparations containing macrophages. Materials

and

Methods

Materials. PGE2 and PGF2a were the generous gifts from Dr. Udo Axen of Upjohn Co., Kalamazoo, Michigan. Arachidonic acid (20:4, n-6) was obtained from Sigma, St. Louis, MO., and was greater than 33% authentic as determined by a gas liquid chromatographic Commercial arachidonic acid was stored in benzene at technique. -20 C until use. [l-14CJArachidonic acid (55 mCi/mmol), purchased from New England Nuclear, Boston, Mass., was evaporated as described [3H]PGE2 (117 Ci/mmol) was obtained above and stored in benzene. from New England Nuclear, Boston, Mass. Radiopurity was established by thin layer chromatography in the solvent system, chloroform: Silica gel G (type 60) was obtained methanol:acetic acid, 180:10:10. from Brinkman Instruments, Inc., Westbury, N.Y. Corvnebacterium parvum (lot number CA380) was kindly provided by Dr. J. Whishant of Burroughs Wellcome, Research Triangle, North Carolina, as a formalin-killed suspension. Indomethacin and p-nitrophenylphosphate RPMI-1640 medium with L-glutamine were from Sigma, St. Louis, MO. was purchased from Grand Island Biological Co., Grant Island, N.Y. Reagents were of analytical grade and solvents were redistilled before use.

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Adult C3H mice (18-22 g) were Activation of macrophages. Peritoneal innoculated i.p. with 1.4 mg of C. parvum in 0.2 volume. exudate cells were harvested (14 days after injection) by lavage with 3 ml of RPHI-1640 medium containing 10 tU/ml heparin, 100 U/ml penicillin and 100 ug/ml streptomycin. Cells were washed 3 times and Activation of macrophages was suspended in Dulbecco A solution. ascertained by increased acid phosphatase activity per cell assayed as previously described with paranitrophenyl phosphate as the substrate (3). Non-injected mice of similar age and sex were used as control cell donors. Protein determinations were as described by Lowrv et al. (4) with bovine serum albumin as the standard. Peritoneal exudate ‘Removal and recovery of adherent cells. cells from test and control mice were susoended in ~~~1-1640 medium containing 10% fetal calf serum at a concentration of 2.5 x lo6 cells per ml. and 2.5 ml of this suspension was incubated at 37 C in plastic tissue culture grade petri dishes. After 30 minutes of incubation, the nonadherent cells were removed by gently rocking the petri dish and decanting the medium. The adherent cells were lifted off the plastic by vigorous washing with Dulbecco’s solution devoid of Ca* and Mg*. The non-adherent and adherent cells were centrifuged at 150 g and resuspended in normal saline. On the basis of previous observations and the acid phosphatase content, it was presumed that the malority of adherent cells were macrophages. Biosynthesis of-PGEp.and PGF,, from arachidonic acid.The 1.2 ml incubation mixture contained: a.25 !.tCi jl-14Cjarachidonic acid, 100 ug unlabeled arachidonic acid and varyrng numbers of intact cells obtained as described above. Arachidonic acid was sonicated into normal saline and the incubations were started by addition of intact cells. Reactions were carried at 37 C for varying times in a shaking water bath. The reactions were terminated by addition of 5 ml of chloroform-methanol (2: 1) and vortexing. Separation of PGE2 and PGF2a was accomplished by thin layer chromatography before and after treatment of the extract by 50% methanolic KOH as previously described (5). Zero time controls received 5 ml of chloroform-methanol (2:l) imnediatelv after addition of macroohaaes. Conversion of PGE7 into PGF,, by Gacrophages. Aliquots of stimulated and non-stimulated macroohaae nrenarations (obtained as described above) were incubated aerbbicaliy ;n 2 ml of‘KrebsRinger phosphate buffer, pH 7.4, containing 10 mM glucose, 100 PM indomethacin, and [3H]PGE2 (0.5 pCi, 5 pmole) in a Dubnoff shaking incubator at 37 C for 15 minutes. Control experiments contained PGE2 in buffer with glucose and indomethacin but with no cells. PGF2, was separated from radioactive substrate by thin layer chromatography and conversion of unreacted PGE into PGB2 followed b a second chromatographic step, as previousl$ described (6). [SH-JPGF2u was eluted from the silica gel and counted in a toluenebased scintillant. Results Ten-fold

or

greater

increases

NOVEMBER 1977 VOL. 14 NO. 5

in acid

phosphatase

activity

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were observed in whole peritoneal preparations and in adherent cell subpopulations obtained from C. parvum-treated animals when compared to those obtained from non-stTmulated controls (representative experiSpecific activity of acid phosphatase showed ment in Figure I). similar increases in stimulated populations when expressed as either 1ncreased change in optical densfty per mg protein or per cell. acid phosphatase activity has been described previously as a concomiy tant of macrophage activation (7,8).

-95

I

10

1

I

IS

20

VOLUtlE(YL)

Fig.

832

1.

Acid Phosphatase Activity in a Stimulated and NonStimulated Peritoneal Exudate Preparation. The upper line represents activity in stimulated homogenate and the lower in control homogenate.

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Initial experiments indicated that the optimal number of cells in a reaction mixture differed depending on whether the cells had been obtained from control donors or from C.parvum-treated animals. Unless otherwise noted, incubations Oith r&-stimulated peritoneal preparations contained 2 x 106 cells per reaction vessel while those with stimulated peritoneal cells, 1 x 106. Incubations of non-stimulated peritoneal preparations for 15 and 30 minutes resulted in linear generation of PGE2 and PGFgcr. Similar experiments with stimulated preparations revealed that PGE2 and PGF2a. generation approached a plateau by 30 minutes. Arachidonic acid in a micellar form may be ingested at a more rapid rate by stimulated peritoneal exudate cells and thus be available as a substrate for prostaglandin biosynthesis more quickly than in nonstimulated cells. At both time points, the amount of radioactive PGE2 and PGFge in stimulated preparations was greater than that in control preparations (Figure 2A and 28).

6.4 586 4,8 is z s 8 H

4,o 3.2 2 AI la6

-

1 -

0.E I-

15’ INCUBATION

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TIM

(MINI

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B

PGE2 2.4I -

2.0

1 0.8

0.4

INCUBATION TIME (HI&

Fig.

834

2.

Biosynthesis of PGF2e (A) and PGE2 (B) from [14c]Arachidonic Acid by Peritoneal Exudate Preparations. Activated and non-activated cell preparations in this figure are represented by lined and open bars respectively. Mean and standard error of the mean (eight determinations). The per cent conversion is based on 106 cells,

NOVEMBER 1977 VOL. 14 NO. 5

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To determine that increased prostaglandin biosynthesls in peritoneal exudate preparations was due to increased quantities within macrophages and not to increased concentration in other cell types, macrophages were purified by allowing them to adhere to a plastic surface (9). Adherent cells had greater quantities of acid phosphatase activity than did non-adherent cells, 1.75 and 0.73 respectiveFurthermore, activated adherent ly (units, 103 x 0.0.405 per cell). cells had greater quantities of acid phosphatase activity than did nonactivated adherent cells, 2.05 and 1.60, respectively. Nonadherent cell populations fromC. parvum-treated mice showed no differences in the amount of radioactive PGE2 and PGF2a detected at the end of a 13 minute incubation than that detected after incubation with nonadherent control cells, PGE2: 1.75 and 1.95 respectively, and PGF2e : 4.30 and 5.01 respectively (units, per cent conversion of radioactive arachidonic acid/lo6 cells) (mean of 4 determinations). On the other hand, adherent cell populations from c. parvum-treated mice contained more radioactive product than did those cells obtained PGE2: 1.60 and 0.90 respectively, from non-stimulated control animals, and PGF2u :5.70 and 2.65 respectively. This data suggests that the increased quantities of radioactive prostaglandins observed in whole peritoneal exudate preparations reflected changes in the adherent cell component of the exudate. PGE2 g-ketoreductase activity was 2-3 times greater in peritoneal exudates obtained from stimulated animals than that noted in control cells (2.41 and 1.08 respectively expressed as per cent conversion In exudates of PGE~ per 106 cells, mean of 3 determinations). seoarated into adherent and nonadherent cell fractions, nonadherent ceils from 6. parvum-treated mice reduced more PGE2 than did those from contro‘r animals (3.42 and 1.31. mean of 4 determinations). Similarly, adherent ceils from stimulated mice had higher per-cent conversion of PGE2 than did those from controls (4.46 and 3.71, mean of 4 determinations). Adherent cells from control and stimulated animals reduced more PGE2 than did nonadherent cells from the same animals (3.71 and 1.31, 4.46 and 3.42 respectively). Although preliminary, our data suggest that stimulation of mice with L. parvum augmented the reduction of PGE2 by PGE2 3-ketoreductase in both peritoneal exudate subpopulations although the enzyme activity appeared greater in adherent cells. Discussion The mechanisms by which inflammatory reactionsresolveare poorly 1) that factors actively understood. Two possible approaches are: function in the limitation of the inflammatory process and 2) that the absence of inflammatory mediators limit the inflammatory process. More than likely, the resolution of the inflammatory reaction will be defined as a combination of these two approaches and will be as complex as the processes that result in its evolution. PGF2a, a major prostaglandin product, may function in the resolution of the inflammatory process by competitively inhibiting

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1977 VOL. 14 NO. 5

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some of the mediator functions of PGE2 and promoting tissue repair. Crunkhorn et al. demonstrated that injection of PGF2a into guinea pig skin prior to injection of PGE2 suppressed the inflammatory effect of PGE2 (10). Crunkhorn and Will is have also suggested that acute inflammation may involve a shift of synthesis from PGF2u to PGE2 (11). Willoughby found that PGF2a suppressed the inflatmmtory reaction induced by tissue injury and a variety of permeability factors, including histamine and serotonin (12). Murota, et al. observed that PGF2, stimulated fJbroblasts in culture to produce and release hexoseamine-containing substances, suggesting a role for PGF2a in wound repair (13). Why macrophages generate relatively more PGF2u than PGE2 remains unexplained. The release of large quantities of PGF2c by macrophages in culture (14) may reflect in part the activity of PGE2 g-ketoreductase, an enzyme that can reduce PGE2 into PGF2e. Our data demonstrated that PGE2 9-ketoreductase activity was present in peritoneal preparations containing macrophages and that greater enzyme activity was found in stimulated preparations than in control non-stimulated ones. has a function(s) in the cessation We hypothesize that PGF the biosynthesis of of the inflanznatorv orocess. % e assume that PGF2a by macrophagks’is not an artefact produced by an in vitro situation but assists this cell in the oerformance of its-in the inflammatory cascade. As stated above, considerable evidence functions in many systems in such a has accrued that PGFza actively way as to limit the Inflammatory actions of PGE2. Data in this report support the concept that the infiltrating macrophage may be one source of PGF2u in a resolving inflamnatory reaction.

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Morley. Produce

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on

IntraNature

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