proteinase complexes in human seminal fluid*

FERTILITY AND STERILITY

Vol. 57, No.2, February 1992

Copyright © 1992 The American Fertility Society

Printed on acid·free paper in U.S.A.

a2-Macroglobulin and a2-macroglobulinjproteinase complexes in human seminal fluid* Michael D. Kramer, M.D.tt Markus M. Simon, Ph.D.§ Wolfgang Tilgen, M.D.II

Helmut Naher, M.D. II Claus W. E. Justus, M.D.'II Detlef Petzoldt, M.D. II

Institut fur Immurwlogie und Serologie, and Universitiits-Hautklinik, Heidelberg, and Max-Planck-Institut, Freiburg, Germany

Objective: To determine the broad-spectrum proteinase inhibitor lX2-macroglobulin (lX2M) and its functional subforms, i.e., free lX2M and lX2M/proteinase complexes, in human seminal fluid by using specific enzyme immunoassay systems. Setting: The study has been performed in the Andrology Department of the Dermatology Clinics and the Laboratory of Immunopathology of the Institute of Immunology. Patients, Participants: Routine patients attending the Andrology Department. Interventions: The data have been obtained without particular interventions before or after collection of seminal fluid. Main Outcome Measures: The aim of the study was to determine whether lX2M or lX2M/proteinase complexes are present in human seminal fluid. Results: The concentration of total lX2M in human seminal fluid ranged from 1 to >1,000 ng/mL, and between 56% and 85% of the inhibitor was complexed with proteinases. Conclusion: These findings show that lX2M and lX2M/proteinase complexes have to be considered as functionally relevant biomolecules in male genital tract secretions. Fertil Steril 1992;57:417-21 Key Words: lX2-Macroglobulin, protein complexes, human seminal fluid

iX2-Macroglobulin (iX2M) is a broad spectrum proteinase inhibitor inactivating mammalian and even procaryotic endopeptidases of all known classes (serine-, cysteine-, aspartic-, and metallo-type proteases). In human plasma it is present at a concentration of about 2.5 giL; however, approximately 40% of the total body iX2M occurs extravascularly and exchanges slowly with the vascular pool. In its native form, iX2M is a glycoprotein of approximately

Received March 7, 1991; revised and accepted September 25, 1991. * Supported by grant Kr 931/2-2 of the Deutsche Forschungsgemeinschaft, Bonn, Germany. t Institut fiir Immunologie und Serologie. t Reprint requests: Michael D. Kramer, M.D., Institut fiir Immunologie und Serologie, Immunpathologie, 1m Neuenheimer Feld 305, D-W -6900 Heidelberg, Germany. § Max-Planck-Institut fiir Immunbiologie. II U niversitiits-Hautklinik. 'II Present address: Boehringer Ingelheim, Vetmedica GmbH, Ingelheim/Rhein, Germany. Vol. 57, No.2, February 1992

700 kd consisting of four identical subunits each of which has a molecular weight (MW) of approximately 170 kd (for review see Van Leuven 1 ). When iX2M interacts with a proteinase, a peptide bond localized within the so-called "bait region" of the inhibitor molecule is cleaved. This cleavage event is followed by a conformational tightening of the enzyme/inhibitor complex leading to the "trapping" of the enzyme. 2 Because of their different electrophoretic mobilities, the noncomplexed iX2M molecule is termed the "slow" form of iX2M (iX2Ms) and the iX2M/proteinase complex the "fast" form of iX2M. The trapped proteinases retain their enzymatic activity but because of steric hindrance they are only able to cleave substrates of lower «20,000) but not such of higher MW (>20,000).3-5 In view of its interaction with a broad range of endopeptidases and its presence in the extravascular interstitial space, free iX2M, i.e., iX2Ms, has been proposed as a second line regulatory element in extravascular proteolysis. It is thought to counteract inKramer et al.

lX2-Macroglobulin in human seminal fluid

417

jurious proteolytic tissue degradation, especially when specific inhibitor systems are exhausted, and it has therefore been termed "a wandering scavenger of endopeptidases".1 l¥2- Macroglobulin can influence cellular immune responses in vitro. 6 - 9 In particular, l¥2MF was found to suppress T cell-mediated immune responses, most likely via degradation of the immunostimulatory mediator interleukin-2 (IL 2).8 On the other hand, l¥2M can act as a carrier for immunoregulatory cytokines. lO The aim of the present study was to explore whether l¥2M or l¥2M/proteinase complexes may be envisaged as relevant biomolecules in male genital tract secretions. To this end, we have used solid phase enzyme immunoassays that allow to quantify total l¥2Mll or to selectively quantify either form of l¥2M, i.e., the native inhibitor molecule (l¥2MS) or proteolytic ally modified l¥2M, (l¥2MF) respectively.12 By using these assay systems, we have now analyzed the presence of l¥2M and its distinct subforms, i.e., l¥2Ms and l¥2MF, in human seminal fluid. Moreover, we asked whether the amount of total l¥2M, l¥2Ms, or l¥2MF is correlated with the presence of elastase of polymorphonuclear leukocytes (PMN elastase), a known indicator of male genital tract inflammation. MATERIALS AND METHODS Collection of Human Seminal Fluid

Human seminal fluid specimens were obtained from routine patients of the Andrology Department of the Dermatology Clinics, University of Heidelberg. After liquefication, for 30 minutes at room temperature, samples were centrifuged for 10 minutes at 1,000 X g to obtain cell-free seminal plasma. Afterwards, the samples were stored at -20°C until further use. Before the enzyme immunoassays, the samples were centrifuged again for 10 minutes at 10,000 X g and appropriately diluted (as indicated in the legends to figures or tables) in phosphatebuffered saline (PBS) containing 0.05% (vol/vol) Tween 20. Quantification of Total l¥2M, Free a2M (a2Ms), and a2MjProteinase Complexes (a2MF) in Human Seminal Fluid

Total l¥2M contained in human seminal fluid samples was determined by using an assay system based on polyclonal anti-l¥2M serum and monoclonal anti-l¥2M monoclonal antibody (mAb) clone M2Y The assay was standardized by parallel testing of 418

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human standard plasma samples and conventional radial immunodiffusion assays. By using recently described assay systems based on specific streptococcal l¥2M receptors,12 l¥2MF and l¥2Ms were quantified. Streptococcal extracts containing receptors specific for either form of l¥2M were coated as specific ligands onto flat bottom microtiter plates. After incubation with appropriately diluted human seminal fluid samples and binding of the respective form of l¥2M, bound l¥2Ms or l¥2MF was detected by using mAb clone M2 that recognizes both forms of l¥2M. The assay system was standardized by using either freshly drawn human plasma, which has been found to be devoid of significant amounts of l¥2MF, and by human plasma pretreated with pancreatic trypsin to convert free l¥2M into l¥2M/proteinase complexes, i.e., into l¥2MF. Quantification of PMN Elastase in Human Seminal Fluid

Polymorphonuclear elastase was quantified by using a recently described enzyme-linked immunosorbent assay system based on polyclonal anti-PMN elastase antibodies and anti-PMN elastase mAb clone Ela 6.2.4.13 Immunoprecipitation and Detection by Immunoblotting of a2M Contained in Human Seminal Fluid

Forty-five microliters of freshly drawn human plasma or 50 ttL of human seminal fluid (sample no. 38; see Fig. 1) were mixed with 50 ttL of sepharose

10

15

70

25

30

35

40

45

Sample number

Figure 1 Determination of total a2M in human seminal fluid. Human seminal fluid samples obtained from 45 individual donors attending the Andrology Department at the Dermatology Clinics of Heidelberg University were tested in the enzyme immunoassay for total a2M." The a2M concentration was calculated by curve fitting and regression analysis from a titration curve obtained with human standard plasma with a known concentration of a2M.

Fertility and Sterility

4B coupled with anti-a2M mAb M2. After the addition of 100 ILL of PBS, the samples were mixed end-over-end for 2 hours. The sepharose beads were gently washed three times by using 1 mL of PBS. After suspension in 100 ILL of reducing sodium dodecyl sulfate (SDS) sample buffer, the beads were boiled for 3 minutes and centrifuged at 10,000 X g for 5 minutes. The resulting supernatant was separated by SDS polyacrylamide gel electrophoresis using 8% acrylamide separating gels. The separated proteins were electrophoretically transferred onto nitrocellulose, and a2M was visualized by using polyclonal rabbit anti-a2M immunoglobulin (Ig)G and peroxidase-labeled species-specific antirabbit IgG antibodies. RESULTS Quantification of Total a2M in Human Seminal Fluid

Human seminal fluid samples collected in the Andrology Department of the Dermatology Clinics, University of Heidelberg, were analyzed for the presence of total a2M by using a solid phase enzyme immunoassay recently described in detailY As exemplified for 45 randomly collected samples in Figure 1, the concentration of total a2M in individual samples ranged from 1 to 1,028 mg/L. The latter concentration is in the range of that found in normal human sera (approximately 2.5 giL). To explore whether increased concentrations of a2M are passively transferred into male genital tract secretions under conditions of severe inflammation, the same seminal fluid specimens were also analyzed for the presence ofPMN elastase, a known parameter of male genital tract inflammation. 14 Between 38 and 899 nglmL of total PMN elastase were detected in the human seminal fluid samples. The coefficient of correlation between the amount of a2M and the amount of PMN elastase was found to be 0.218, indicating a lack of correlation between these two parameters. Furthermore, no correlation was found between sperm count or volume of sperm and concentration of a2M (data not shown). When a2M was determined in split ejaculate fractions, we found that highest concentrations were found in the main fraction (data not shown). Immunochemical Characterization of a2M Contained in Human Seminal Fluid

Immunochemical studies on human seminal fluid were performed to explore the molecular identity of Vol. 57, No.2, February 1992

the immunoreactive material being detected in the enzyme immunoassays. As illustrated in Figure 2, immunoprecipitation followed by immunoblotting using anti-a2M mAb M2 revealed a molecular species that was similar in size and immunoreactivity to purified a2M and to a2M contained in normal human plasma. These data indicate that a2M contained in human seminal fluid is molecularly and iI]1munologically identical to plasma-derived a2M. The Relative Distribution of a2Ms and a2MF in Human Seminal Fluid Containing Elevated Levels of Total a2M

In the next experiments, we explored whether a2M contained in human seminal fluid is present in its native (slow form [a2Ms]) form or whether it is complexed to proteolytic enzymes (fast form [a2MF]). For these experiments we employed recently established assay systems that differentiate a2Ms from a2MF. As summarized in Table 1, the majority of a2M (between 56.8% and 87.3%) contained in human seminal fluid specimens was of the fast form. This is in contrast to normal human plasma in which approximately 95% of the a2M is present in its functionally active, i.e., slow form. DISCUSSION

This study demonstrates that human seminal fluid may contain considerable amounts of a2M and

A B 170-'" 97 66-

.. (

0 ~

to Cl ...:lI::

4231-

Figure 2 Immunochemical characterization of lX2M contained in human seminal fluid. Purified lX2M (A), human seminal fluid (C), and plasma (D) were analyzed by immunoprecipitation and immunoblotting. Lane (B) is a control in which instead of lX2Mcontaining sample, PBS was taken for analysis. Kramer et al.

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Table 1 Amount of a2M and a2MF in Human Seminal Fluid Specimens' Total a2M

Human plasma Trypsin -treated human plasma Sample no. 1 3 9 13 14 15 23 25 26 30 35 38 40 43 45 49 79 84

a2 MF

PMN elastase

I'g/mL

%

ng/mL

2,300

3.0

ND b

2,300

98.4

ND

312 263 333 289 574 639 343 261 259 300 419 837 449 1028 272 237 543 447

78.1 71.8 67.0 87.3 75.3 69.4 63.4 83.6 75.6 82.8 81.0 58.2 56.8 75.8 85.0 77.2 67.8 59.8

48 456 118 277 556 67 422 410 251 40 618 386 712 418 576 44 42 195

• Human seminal fluid specimens with a a2MF content> 200 Ilg/ml were retested for a2MF, a2Ms and total PMN elastase. 12,13 Values are means of three replicate determinations for each parameter. From the data obtained for a2MF and a2Ms, the relative amount (percentage) of a2MF was calculated. The performance of the assay systems for a2MF or a2Ms was controlled by testing either native or trypsin-treated human blood plasma as previously described. 11 b ND, not determined.

that the majority of the inhibitor molecules are present as a complex with proteolytic enzymes. The lack of correlation between the level of £X2M -either total, free or complexed (data not shown)-with the level of PMN elastase, a known indicator of male genital tract inflammation,14 indicates that other and/or additional mechanisms than inflammation are responsible for displacement of £X2M into male genital tract secretions. The biological and, in particular, the possible pathogenic role of £X2M in seminal fluid is also not known at present. At least two possibilities have to be considered. First, £X2M could control the activity of physiologically relevant proteolytic enzymes normally operative in seminal fluid. It is known that proteolytic enzymes contained in male genital secretions participate in various, physiologically relevant functions, including liquefaction of the ejaculate and the acrosomal reaction. 15- 17 Complex formation of the respective proteinases with the broad-specific inhibitor £X2M could interfere with these processes and could thus be one factor adding to male infertility. 420

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Secondly, £x2M/proteinase complexes might interfere with local immune responses in the male genital tract. This possibility is impelled by the earlier findings that £x2M/proteinase complexes can suppress cellular immune responses,6-9 most likely via proteolytic inactivation of the cytokine IL2.8 Because cellular immunity plays an important role in the control of genital infections induced, e.g., by Treponema pallidum,18 Chlamydia trachomatis,19 or human immunodeficiency virus,20 local supression of cellular immune responses by £x2M/proteinase complexes may promote infection with these sexually transmitted pathogens. In more general terms, the capacity of £x2M/proteinase complex to process low MW peptide substrates (MW < 20,000) may cause the processing, i.e., activation or inactivation, of biologically relevant peptides (MW < 20,000) present in sperm. To explore these possibilities, we will in future studies isolate £X2M and its distinct subforms, i.e., free and complexed £X2M, from human seminal fluid. These isolated compounds will be studied for their influence on sperm quality parameters and on cellular immune responses.

Acknowledgments. The authors are indebted to Ms. Eva Schickel, Ms. Margit Miiller-Bardorff, and Ms. Inge Hoffmann (Universitats-Hautklinik, Heidelberg) for help with the experimental work; to Ursula Pekar, M.D., and Sebastian Biltz, M.D., (Universitats-Hautklinik) for obtaining the human seminal fluid samples; and to Ms. Sabine Kaute and Ms. Sabine Preussmann (Fotolaboratorium der Universitats-Hautklinik) for help with the photographic work.

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Fertility and Sterility

7. Petersen CM, Ejlersen E, Moestrup SK, Jensen PH, Sand 0, Sottrup-Jensen L: Immunosuppressive properties of electrophoretically "slow" and "fast" form a2-macroglobulin. Effects on cell-mediated cytotoxicity and (allo-) antigen-induced T cell proliferation. J Immunol 142:629, 1989 8. Borth W, Teodorescu M: Inactivation of human interleukin2 (IL-2) by a2-macroglobulin-trypsin complexes. Immunology 57:367, 1986 9. Mannhalter JW, Borth W, Eibl MM: Modulation of antigeninduced T cell proliferation by a2M-trypsin complexes. J Immunol 136:2792, 1986 10. James K: Interaction between cytokines and a2-macroglobulin. Immunol Today 11:163, 1990 11. Justus C, Muller S, Kramer MD: Application of novel monoclonal antibodies in the purification, quantification, and immunohistological localization of the proteinase inhibitor a2-macroglobulin. Enzyme Microb Technol10:524, 1988 12. Justus CWE, Muller HP, Simon MM, Kramer MD: Quantification of free a2-macroglobulin and a2-macroglobulinprotease complexes by a novel ELISA system based on streptococcal a2-macroglobulin receptors. J Immunol Methods 126:103, 1990 13. Kramer MD, Muller-Bardorff M, Simon MM, Tilgen W, Schickel E, Petzoldt D: Measurement of free human leukocyte elastase and human leukocyte elastase/aI-proteinase inhib-

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