Metabolism of progesterone by human leukocytes

CLINICAL

IMMUNOLOGY

AND

BRIEF Metabolism C. *Utziversity

SCULLY,*

Department 378 Sauchiehall

22, 147-151 (1982)

IMMUNOPATHOLOGY

COMMUNICATION

of Progesterone M. M. FERGUSON,*

by Human D. SIRRETT,t

Leukocytes

AND J. K. GRANTt

of Oral Medicine and Pathology. Glasgow, Dental Hospital Street, Glasgow, and iDepartmen? of Steroid Biochemistry, Royal Infirmaty, Glasgow. Scotland

and School.

Human mononuclear and polymorphonuclear leukocytes were examined for their ability to metabolize progesterone. Both mononuclear and polymorphonuclear leukocytes metabolized progesterone to three main products, 20a-hydroxy-4-pregnen-3-one, 5 a-pregnan-3,20-dione, and a pregnanolone-like metabolite. The results indicate that 2Oo-hydroxy-4-pregnen-3-one is produced not only by lymphocytes but also by polymorphonuclear leukocytes.

INTRODUCTION

The relationship between sex hormones and immune mechanisms is unclear but there is increasing evidence that these hormones may sometimes have an immunoregulatory role. The enzyme 20a+hydroxysteroid dehydrogenase, responsible for the interconversion of progesterone and 20a-hydroxy-4-pregnen-3-one (20a-hydroxyprogesterone), has been detected in murine T lymphocytes and it has been suggested that this enzyme is a specific T-lymphocyte marker (1, 2). However, progesterone metabolism does not appear to have been examined in leukocytes other than mononuclear cells, or in leukocytes from humans. 20cr-Hydroxysteroid dehydrogenase is involved in both the synthesis and inactivation of progestogens. In tissues such as the corpus luteum, which synthesize progestogens, 20a-hydroxysteroid dehydrogenase coexists with A5-3phydroxysteroid dehydrogenase, whereas at sites of progestogen inactivation 2Oahydroxysteroid dehydrogenase is found along with 5a- and SD-steroid reductases and 3cu-hydroxysteroid dehydrogenase (3, 4). We have therefore examined different human leukocyte populations for their ability to metabolize progesterone, in order to establish the specificity of 200~hydroxysteroid dehydrogenase as an enzyme marker of human T lymphocytes. MATERIALS

AND METHODS

Heparinized human venous blood was obtained from healthy volunteers, leukocytes were separated by gelatin sedimentation for 30 min at 37°C (Plasmagel, Laboratoire R. Bellon, Neuilly Sur Seine, France), and residual erythrocytes were lysed by exposure to hypotonic 0.83% ammonium chloride solution. Leukocytes were isolated by centrifugation at 1OOg for 15 min and then washed by resuspension in medium 199 (Gibco, Glasgow). Mononuclear cells and polymorphonuclear leukocytes were separated by density gradient centrifugation at 450g on a Ficoll-Hypaque gradient, and identified by conventional techniques (5). 147 0090-1229/82/010147-05$01.00/O Copyright All rights

@ 1982 by Academic Ress, Inc. of reprodwtion in any form reserved.

148

BRIEF

COMMUNICATION

The cell populations were incubated at 37°C for 2 hr with [3H]progesterone (0.5 &i, Radiochemical Centre, Amersham, U.K.), nonradioactive progesterone (1 PM), and 1 m&4 NADPH in phosphate-buffered saline. After incubation, steroids were extracted in diethyl ether, the solvent was removed by evaporation, and the residues were dissolved in ethanol containing progesterone and 2Oa-hydroxy-C pregnen-3-one as carriers. In some experiments, carrier 5a-pregnan-3,20-dione was also included. Progesterone and its metabolites were separated by thin-layer chromatography (silica 60F,,), using either diethyl ether: chloroform (3: 1, v/v) or benzene:ethyl acetate ( 1: 1, v/v) for development. Radioactive compounds were located by scanning with a gas-flow counter (Panax, RTLS lA), and carrier steroids by quenching of fluorescence under ultraviolet illumination. The zones of radioactivity detected were scraped off and eluted with diethyl ether, and the eluates were evaporated in vials prior to the addition of scintillant and measurement of radioactivity in a liquid scintillation counter (Packard 3380). In some experiments, portions of the eluates were used for product identification by recrystallization to constant spe-

Ptugastetune

Prejnamtone 50PregMne-3. 20 a oti Pmgastetune

M-dione

J

+ 24

Solvent Ft0nt FIG. I. Chromatogram or polymorphonuclear system ether:chloroform products included hydroxyprogesterone): 3 JO-dione

of the metabolites of progesterone leukocytes separated by thin-layer (3: I. v/v). Both cell populations (from origin to solvent front): a metabolite

with

mobility

similar

produced by human blood mononuclear chromatography on silica 60FzS1 in the produced a similar chromatogram. The 20a-hydroxy-4-pregnen-3-one (20~ to that

of pregnanolone:

and

So-pregnan-

149

BRIEF COMMUNICATION

cific activity. The yield of metabolites was expressed in terms of cellular content as picomoles per hour per milligram of DNA.

DNA

RESULTS

Progesterone was converted to at least three products by both mononuclear and polymorphonuclear leukocyte preparations (Fig. 1). A product with chromatographic mobility identical to that of 20a-hydroxy-4-pregnen-3-one (Rf = 0.3) which was distinct from the residual substrate (R, = 0.6) was found. The bulk of the radioactivity recovered with the 20cr-hydroxy-4-pregnen-Zone fraction from the ether:chloroform thin-layer chromatography subsequently coeluted with the same carrier on thin-layer chromatography with benzene:ethyl acetate (1: 1, v/v) as solvent. This product also recrystallized from acetone/hexane to constant specific activity (Table 1). Progesterone was also reduced to a metabolite that showed chromatographic mobility similar to that of pregnanolone isomers in the systems used (Table 1). Recrystallization of this metabolite from acetone/hexane suggested possible similarity to pregnanolone isomers .5a-pregnan-3a-o 1-20-one and 5/3pregnan-3a-ol-20-one but there was not identity with either isomer. Progesterone was also reduced to a third metabolite, Sol-pregnan-3,20-dione (Table 1). Each of the three metabolites was produced by both mononuclear and polymorphonuclear leukocytes (Table 2). Both leukocyte populations produced 20~~ hydroxy-4-pregnen-3-one and 5a-pregnan-3,20-dione to a comparable extent but polymorphonuclear leukocytes appeared to produce more of the pregnanolonelike metabolite. Progesterone was not metabolized to pregnanolone by either cell population. DISCUSSION

The results indicate that both human peripheral blood mononuclear cells and polymorphonuclear leukocytes metabolized progesterone to 2Ocr-hydroxydTABLE METABOLISM

OF PROGESTERONE

1

BY LEUKOCYTES:

BY RECRYSTALLIZATION

TO CONSTANT

IDENTIFICATION SPECIFIC

OF METABOLITES ACTIVITY

Specific activity at successive recrystallizationb (cpmlwg) Metabolite”

Carrier added

1

2

3

4

5

2Oa-Hydroxy-dpregnen3-one 17Lu-Hydroxyprogesterone

0.97 0.32

0.86 0.14

0.82 0.22

0.83 0.11

0.85 ND

Peak 2

Deoxycorticosterone 17~Hydroxyprogesterone 5/3-Pregnan-3a-ol-20-one 5ru-Pregnan-3a-ol-2@one

0.33 0.11 0.51 0.42

0.07 0.03 0.33 0.16

0.03 0.03 0.26 0.10

0.02 0.02 0.11 0.09

ND ND 0.07 0.05

Peak 3

Sa-Pregnan-3,2@dione

1.90

1.76

1.93

2.05

1.94

Peak 1

n Peak 1, ZOa-OH-progesterone: Peak 2, pregnanolone-like dione. Peaks shown in Fig. 1. h Recrystallization from acetone/hexane mixtures. ND = not done.

fraction; peak 3, 5a-pregnane-3,20-

150

BRIEF

COMMUNICATION TABLE

ME.FABOL

ISM OF PROGESTERONE PRODUCED BY DIFFFREVI

Metabolites ~~~~~~~~~~

~-.-_______ Leukocyte

ZOwHydroxy-Jpregnen-3-one

population

Polymorphonuclear leukocytes Mononuclear ’ Mean

values

cells of five

2 BY Lt-UKOCY I ES: METABOL CEI I POPUL..~IIONS

experiments

I 1 ES

produced”

Pregnanolonelike fraction

Su-Pregnan3.X-dione

136.5

.?I I.9

86.7

174.3

177.3

106. I

expressed

as pmol

hr-’

mg- ’ DNA.

pregnen-3-one (20a-hydroxyprogesterone) and to saturated pregnane derivatives. particularly 5a-pregnan-3,20-dione and a pregnanolone-like derivative (Table 1). These conversions appeared to be properties of mononuclear cells and polymorphonuclear leukocytes and the different leukocytes were remarkably uniform in their conversion of progesterone to 20a-hydroxy-4-pregnen-3-one and 5~ pregnan-3,20-dione although polymorphonuclear leukocytes appeared to be more active in the production of the pregnanolone-like fraction (Table 2). Metabolism of progesterone by murine splenic lymphocytes was recently reported and, in spite of the absence of reports of the investigation of metabolism of progesterone by other leukocytes, 20a-hydroxysteroid dehydrogenase was suggested to be a T-lymphocyte marker enzyme (1). The present results indicate that 20a-hydroxysteroid dehydrogenase is not specific to T lymphocytes but is present in polymorphonuclear as well as mononuclear leukocytes. Many components of the immune response, such as the lymphoproliferative response (6, 7) and the mixed lymphocyte reaction (8), are altered during pregnancy or by sex steroids. 20a-Hydroxysteroid dehydrogenase is found mainly in ovarian tissue where its activity regulates progesterone secretion by the reduction of progesterone to 20a-hydroxy-4-pregnen-3-one (3,4), and it has been suggested that 20a-hydroxysteroid dehydrogenase may influence the inhibitory effect of progesterone on immune responses (2). Mononuclear cells not only metabolize progestogens but also appear to metabolize estradiol- 176 to estriol(9) and estrone to estradiol (10). The identification of these various steroid-metabolizing enzyme activities in leukocytes might imply either that leukocytes represent a form of target tissue for sex steroids or alternatively that they are involved in the biotransformation of sex steroids. The metabolism of progesterone by polymorphonuclear leukocytes is particularly intriguing in view of the accepted relatively restricted metabolic activities in these cells. Although the full significance of these steroid conversions by leukocytes remains unclear, further investigations may elucidate the mechanisms underlying the interactions of the endocrine and immune systems. REFERENCES 1. Weinstein. 2. Weinstein.

Y., Lindner, H. R., and Eckstein, Y., J. I~nmrr~ol. 119, 1223, 1977.

B.,

NNIII~

(L~>/rtlrv~j

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1977

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151

COMMUNICATION

3. Wiest, W. G., and Kidwell, W. R., In “The Gonads” (K. W. McK;ern, Ed.), p. 295, Appleton-Century-Crofts, New York, 1969. 4. Lindner, H. R., and Lamprecht, S. A., Advan. Biosci. 4, 419, 1969. 5. WHO/IARC Special Technical Report, &and. J. Zmrmtnol. 3, 521, 1974. 6. Purtilo, D. T., Hallgren, H. M., and Yunis, E. J., Lancer 1, 769, 1972. 7. Carr, M. C., Sites, D. P., and Fudenberg, H. H., Cell. Irnrnunol. 8, 448, 1973. 8. Kasakura, S., Nature (London) 246, 496, 1973. 9. Mason, W. L., Muijsson-Arnold, I. E., McKenzie, M., Cantrell, E. T., and Busbee, D. L., Steroids 10.

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Received July 29, 1981: accepted August 31, 1981

C., and Machin, D., Cancer

Res.

38,