andrologia 20 (5): 404410 (1988) Received May 10, 1988

Fearing Research Laboratory, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts/USA

Male Genital Tract Mammation Associated with Increased Numbers of Potential Human Immunodeficiency Virus Host Cells in Semen* Entziindung im Bereich des mannlichen Genitaltraktes assoziiert mit erhohten Zahlen von potentiellen HIV- Wirtszellen im Sperma H. Wolff and D. J. Anderson

Key Words: HIV, semen - inflammation, genital tract, HIV - CD4 Lymphocytes, HIV ~ genital tract, HIV semen, HIV

Summary: In this study we investigated whether elevated levels of the inflammatory mediator granulocyte elastase in seminal plasma were associated with increased numbers of CD4' T helperlinducer lymphocytes and monocytes/macrophages in semen, the principal host cells of the human immunodeficiency virus 0. Semen samples were obtained from 105 men attending an infertility clinic. CD4' lymphocytes, monocytes/macrophages and cells expressing the common leukocyte antigen (CD45) were identified by monoclonal antibodies (MAb's) in a biotin- streptavidin immunoperoxidase technique. Granulocyte elastase levels in seminal plasma were determined by an enzyme-linked immunosorbent assay. In 17 men, granulocyte elastase levels were higher than 1000 n g / d seminal plasma, indicating male genital tract inflammation. Compared to men with low/normal granulocyte elastase levels in semen (< 250 nglml), these men showed significantly higher mean numbers of total leukocytes, CD4' lymphocytes and monocytes/macro- phages in semen (P <0.001); median cell numbers for the group with high/inflammatory granulocyte elastase levels were increased 38-fold for total leukocytes (19,800,000 versus 520,625 per ejaculate), 19-fold for monocytes/macrophages (2,594,000 versus 134,565), and &fold for CD4+ lymphocytes (82,900 versus 14,100). Because of the increased numbers of potential HIV- host cells in inflammatory semen, male genital tract inflammation may be an important cofactor in the sexual transmission of the human immunodeficiency virus.

Zusammenfassung: Wir untersuchten, ob ein Zusammenhang besteht zwischen erhohten Seminalplasmaspiegeln des Entziindungsparameters Granulozytenelastase und erhohten Zahlen von CD4+-Lymphozyten und Monozyten/Makrophagen im Sperma; letztere sind potentielle Wirtszellen des Human Immunodeficiency Virus 0. In 105 Ejakulaten von Infertilitatspatien- ten m d e n anhand von monoklonalen Antikorpern in einer Immunperoxidasetechnik folgende

* Presented at the IV. International Conference on AIDS, Stockholm/Sweden, 1988.

HIV-Host Cells 405

Zellen quantifiiert CD4-positiveT-HeIferlymphozyten, Monozyten/Makrophagen und alle Zellen mit dem ,,Common Leukocyte Antigen" (CD45), also die Gesamtheit aller weillen Blutzellen im Sperma. Das Vorliegen einer Entziindung im Bereich des mannlichen Genitaltraktes m d e durch enzymimmunologische Messung der Granulozytenelastase im Seminalplasma bestimmt. Bei 17 Mannern lagen Spiegel iiber lOOOng/ml vor, ein starker Hinweis auf einen entziindlichen Vorgang. Im Vergleich zu Mannern mit niedrigen Granulozytenelastasespiegeln (< 250 nglml), zeigten die Manner mit hoch/entziindlichen Werten ( > 1000 ng/ml) signifikant erhohte Zahlen an Gesamtleukozyten, CD4+-Lymphozyten und Monozyten/Makrophagen im Sperma (P <0,001); die Medianwerte in der Entziindungsgruppe waren 38fach erhoht fur Gesamtleukozyten, 19fach erhoht fur Monozyten/Makrophagen und 6fach erhoht fur CD4'- Lymphozyten. Unsere Ergebnisse zeigen, daR beim Vorliegen entziindlicher Veranderungen im mannlichen Genitaltrakt die Zahlen potentieller HIV-Wirtszellen im Sperma stark erhoht sind. Dies konnte ein biologisch relevanter Kofaktor bei der ijbertragung des Human Immunodefi- ciency Virus durch Sperma sein.

Introduction

It is well established that sexual intercourse is the principal mode of transmission of the human immunodeficiency virus, HIV (Peter- man etal. - 1986; Padian etal. - 1987). However, some HIV-seropositive men ap- pear to be more infectious than others, and as yet there is little information on what biologic cofactors might affect the incidence of sexual HIV-transmission (Peterman et al. - 1988). As HIV has been localized in the mononu- clear cell-fraction of semen (Ho et al. - 1984; Zagury et al. ~ 1984), and as it is known that HIV can infect CD4' T helper/inducer ly- mphocytes and monocytes/macrophages (Dalgleish et al. - 1984; Gartner et al. - 1986), we have speculated that increased numbeis of these cells in semen could increase the infect- ivity of an HIV-positive man (Wolff and Anderson - 1988b). This speculation was stimulated by the observation that there are enormous inter-individual variations of CD4' lymphocyte and monocyte/macro- phage numbers in semen from fertile and infertile men (Wolff and Anderson ~ 1988a).

The aim of this study was to evaluate the possibility that male genital tract inflamm- ation is associated with increased numbers of CD4' lymphocytes and monocytes/macro- phages in semen, and thus may be a cofactor in the incidence of sexual HIV-transmission.

Materials and Methods

Semen samples were obtained from 105 ran- domly selected men attending the infertility clinic of the Brigham and Women's Hospital in Boston, Massachusetts for diagnostic and therapeutic reasons. The mean age was 37 f 5 years (range, 22 to 42 years).

Routine semen analysis was performed on a Cell Soft Automated Semen Analyzer (Cryo Resources, Ltd., New York, NY) as described previously (Hill et al. - 1987). The seminal plasma was obtained by centrifug- ation (600 x g, 10 minutes), and was stored at - 70°C until used for granulocyte elastase determinations. The cellular pellet of the ejaculate was washed twice in phosphate- buffered saline (PBS), and 5-ul-drops of the final cell suspension, containing approxi- mately 5 x 105 spermatozoa, were applied to multiwell glass microscope slides (Roboz Surgical, Washington, DC). After air-drying, the slides were fixed in acetone for 10 minutes and stored frozen at - 70°C until used for immunohistology .

Total white blood cells, CD4'lymphocytes and monocytes/macrophages were identified by monoclonal antibodies (MAb's) in a biotin-streptavidin immunoperoxidase tech- nique as recently described (Wolff and Ander- son - 1988a). Total leukocyte numbers in semen were determined by the MAb anti-

andrologia 20, No. 5 (1988)

406 H. Wolff and D. J. Anderson

HLe-1 (Becton Dickinson, Mountain View, CA) which reacts with the common leukocyte antigen (CD45), present on all granulocytes, monocytes/macrophages and lymphocytes. CDCpositive lymphocytes were identified by the MAb anti-Leu-3 a&b (Becton Dickinson) and monocytes/macrophages by the MAb Dako-Macrophage (Dako Corp., Santa Bar- bara, CA).

After 30 minutes incubation with approri- ately diluted primary MAb’s or PBS (nega- tive control), the microscope slides were rinsed in PBS and processed further with reagents from a Histostdin SP-KitR (Zymed Laboratories, South San Francisco, CA). Briefly, biotinylated rabbit anti-mouse anti- body was applied for 10 minutes, followed by the streptavidin-peroxidase complex for 5 minutes. After addition of the substrate- chromogen complex (H202 and aminoeth- ylcarbazole) for 15 minutes, a red-brown precipitate appeared on those cells which were initially reactive with the primary MAb’s. After counterstaining with Mayer’s hematoxylin, the slides were permanently mounted by an aqueous medium (GVA- Mount, Zymed Laboratories). MAb-positive cells relative to spermatozoa were enumerated in an adequate number of fields under a bright-field microscope; MAb- positive cells per ejaculate could be calculated due to the known sperm concentration and the volume of the ejaculate.

Levels of granulocyte elastase in frozen/thawed seminal plasma samples were determined by an enzyme-liked immunosorb- ent assay (PMN-Elastase ELISA, Merck, Darmstadt, FRG) as described previously (Wolff and Anderson - 1988~). In brief, duplicate test standards and 1/100 dilutions of seminal plasma were incubated for 60 minutes in test tubes which had been pre- coated by the manufacturer with sheep anti- elastase antibodies. Bound elastase-a, - inhibitor complexes then reacted with anti-a]- inhibitor antibodies which were linked to alkaline phosphatase. After incubation with the substrate 4-nitrophenylphosphate, the reaction was stopped with 2 N NaOH, and

200 pl of each test tube were transferred to 96- well microtiter plates. The optical density was measured at 405nm in a multiscan plate reader (Bio-Tek Instruments, Inc., Burling- ton, VT). The concentrations of immu- noreactive granulocyte elastase (expressed as ng/ml seminal plasma) were determined by use of a standard curve; samples with more than l000ng/ml were re-tested at a 1/1000 dilution of the seminal plasma.

Data analysis and unpaired student’s t-test were performed on an Apple Macintosh microcomputer (Apple Computer, Inc., Cupertino, CA) with a StatWorks Software programme (Data Metrics, Inc., Philadel- phia, PA).

Results

Potential HIV-host cells in semen as detected by monoclonal antibodies against CD4’ lymphocytes and monocytes/macrophages are shown in Figures 1 and 2. Red-brown MAb-positive cells could be clearly different- iated from hematoxylin-blue MAb-negative cells. Because anti-Leu-3 a&b-reactivity was occasionally observed on some large macro- phages in semen (it is known that mono- cytes/macrophages can express the CD4 anti- gen; Gartner et al. - 1986), only small-sized lymphoid cells with clear membrane reactiv- ity were classified as CD4’ lymphocytes.

When the semen samples were grouped according to a granulocyte elastase classific- ation system proposed by Jochum et al. (1986), three subgroups of ejaculates were established: group A with low/normal granu- locyte elastase levels ( < 250 ng/ml; n = 50), group B with intermediate levels (250-1000 ng/ml; n = 38), and group C with hgh/inflammatory levels ( > 1000 ng/ml; n = 17).

In group C, the mean numbers of total leukocytes, monocytes/macrophages, and CD4’ lymphocytes were significantly higher than in group A and B (P < 0.001 and < 0.05; Table 1). Likewise, as Figure 3 demonstrates, median cell numbers per ejaculate were con-

andrologia 20, No. 5 (1988)

HIV-Host Cells 407

Fig. 1: A CD4+ lymphocyte in human semen (anti-Leu-3 a& 3 b; immunoperoxidase; original magnification, x 1000).

Fig. 2: A macrophage in human semen (Dako-Macrophage; immunoperoxidase; original magnification, x 1000)

andrologia 20, No. 5 (1988)

408 H. Wolff and D. J. Anderson

Table I: Granulocyte elastase levels and numbers of total white blood cells, monocytes/macrophages and CD4' IvmDhocvtes in semen

Elastase Group All White Blood Cells Monocytes/Macrophages CD4' Lymphocytes (ng/ml semen) mean f SD mean f SD mean f SD

A. < 250 ng/ml (n = 50) 1 ,I 99,004 f 1,660,989* 31 6,492 f 46521 3 42,642 f 66,384 B. 25&1000 nglml (n = 38) 4,167,688 f 8,481,922 721,255 f 2,073,860 175,603 f 642,l 32 C. > 1000 ng/ml (n = 17)

~~ ~~~

27,429,l 85 f 29,563,911" 3,289,076 f 2,271,332** 540,752 f 121 0,172" _ _ _ _ _ _ _ _ ~ ~ ~ _ _ _ _ _ _ _ _ (Inflammation) ~~

* Numbers are per ejaculate (mean volume, 3.4 f 1.9 ml). +. Numbers in group C are significantly higher by unpaired student's t-test than in Group A (P < 0.001) and group B (P < 0.05).

30 v1 ELASTASE GROUP 2 25

20

,.. -<250 nglrnl

0 250-1000 ng/rnl 3 v

w

15

2 w 10 d m 5 4

u Fig. 3 Parallel increase of g r & h c y t e elastase, total white

host cells in semen. ALL LEUKOCYTES MONOCYTESIMd CD4 LYMPHOCYTES blood cells and potential HIV-

x 106 lo5 lo3

siderably higher in the inflammatory group C (> 1000 ng granulocyte elastase per ml semen) than in group A (< 250 ng/ml); the increase was 38-fold for total leukocytes (1 9,800,000 versus 520,625 per ejaculate), 19- fold for monocytes/macrophages (2,594,000 versus 134,565), and 6-fold for CD4' lymph- ocytes (82,900 versus 14,100).

Discussion

The cellular mechanisms of HIV-trans- mission by sexual intercourse are not yet completely understood. An early speculation was that leukocytes in semen could serve as vectors in the transmission of the then un- known pathogen of the acquired immunode- ficiency syndrome AIDS (Anderson and Yunis - 1983). This speculation was sub- sequently confirmed by reports on mononu-

clear cells in semen carrying infectious HIV (Ho etal. ~ 1984; Zagury etal. - 1984). However, epidemiologic data indicate that there are considerable variations in the in- cidence of sexual HIV-transmission; as de- scribed in one study, HIV was transmitted from an infected man to a woman through a single heterosexual intercourse, while in other cases no male-to-female transmission occured despite hundreds of unprotected heterosexual intercourses (Peterman et al. - 1988).

We have previously speculated that one possible cofactor in the incidence of HIV- transmission may be the number of potential HIV-host cells in semen because we had observed large variations of CD4+ lymph- ocytes and monocytes/macrophages in semen from different men (Wolff and Anderson - 1988a, b). The aim of this study was to evaluate whether increased numbers of pot-

andrologia 20, No. 5 (1988)

HIV-Host Cells 409

ential HIV-host cells in semen are associated with male genital tract inflammation. Because male genital tract inflammation is often clinically silent, we used granulocyte elastase concentrations in seminal plasma as an objective marker for the presence and extent of inflammation (Jochum et al. - 1986). Our study population was comprised of randomly selected men attending an infert- ility clinic because this was a convenient source of human semen, enabling us to study a large number of semen samples with low/normal and high/inflammatory granu- locyte elastase levels.

We found that ejaculates with high/in- flammatory granulocyte elastase levels con- tained significantly more total leukocytes, CD4’ lymphocytes and monocytes/macro- phages than ejaculates with low/normal granulocyte elastase levels. Concordant with an overall increase of total leukocytes (pre- dominantly granulocytes), potential HIV- host cells such as CD4’ lymphocytes and monocytes/macrophages were also dramati- cally increased in inflammatory semen.

It is possible that cytokines and other soluble factors released at sites of genital tract inflammation induce activation of CD4’ lymphocytes and monocytes/macrophages. Immunoperoxidase-staining of semen smears with high concentrations of granulocyte elastase in some cases revealed substantial numbers of cells expressing leukocyte- activation markers such as HLA-DR. anti- gens, transferrin receptors and interleukin 2 receptors (Wolff and Anderson, unpublished results). This has important implications because it was shown that cells containing HIV-provirus can be induced by cytokines to transcribe and express previously dormant HIV (Fauci etal. - 1988). Therefore, in addition to the numerical increase of potent- ial HIV-host cells in semen, male genital tract inflammation may also increase the number of HIV-copies per host cell. Overall, our data indicate that male genital tract inflammation may be an important biologic cofactor in the transmission of the human immunodefici- ency virus by human semen.

andrologia 20, No. 5 (1988)

Acknowledgements: We appreciate the skill- ful technical assistance of Ms. Adriana Mar- tinez, Ms. Heidi M. P. Faris and Ms. Deirdre Meehan. Routine semen analysis was perfor- med by the staff of the Reproductive Endocri- nology Laboratory of the Brigham and Women’s Hospital, Boston, MA.

Granulocyte elastase test kits were kindly provided by Dr. Hermann Lang, Merck, Darmstadt, FRG.

This work was supported by National Institutes of Health grants A1 23669 and CA 42738. Dr. Wolff was the recipient of a research fellowship from the Max Kade Foundation, New York, NY.

References

Anderson, D.J. and E.J. Yunis. 1983. “Trojan Horse” leukocytes in AIDS. New Engl. J. Med. 309, 984.

Dalgleish, A.G., P.C.L. Beverly, P.R. Clapham, D.H. Crawford, M.F. Greaves and R.A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312, 763-761.

Fauci, A. 1988. The human immunodificiency virus: infectivity and mechanisms of pathogenesis. Science 239, 61 1422.

Gartner, S., P. Markovits, D.M. Markovits, M.H. Kap- Ian, R.C. Gallo and M. Popovic. 1986. The role of mononuclear phagocytes in HTLV-III/LAV infec- tion. Science 233, 215-219.

Hill, J.A., F. Haimovici, J.A. Politch and D. J. Anderson. 1987. Effects of soluble products of activated lymph- ocytes and macrophages (lymphokines and mono- kines) on human sperm motion parameters. Fertil. Steril. 47, 46M65.

Ho, D.D., R.T. Schooley, T.R. Rota, J.C. Kaplan, T. Flynn, S.Z. Salahuddin, M.A. Gonda and M.S. Hirsch. 1984. HTLV-I11 in semen and blood of a healthy homosexual man. Science 226, 451453.

Jochum, M., W. Papst and W.-B. Schill. 1986. Granu- locyte elastase as a sensitive diagnostic parameter of silent male genital tract inflammation. andrologia 18, 413419.

Padian, N., L. Marquis, D.P. Francis, R.E. Anderson, G.W. Rutherford, P.M. O’Malley and W. Winkel- stein. 1987. Male-to female transmission of human immunodeficiency virus. J. Amer. med. Ass. 258, 788-790.

Peterman, T.A., and J.W. Curran. 1986. Sexual trans- mission of human immunodeficiencv virus. J. Amer. med. Ass. 256, 2222-2226.

Peterman. T.A.. R.L. Stoneburner. J.R. Allen, H.W. Jaffe and J.W. Curran. 1988. Risk of human immunode- ficiency virus transmission from heterosexual adults with transfusion-associated infections. J. Amer. med. ASS. 259, 55-58.

410 H. Wolff and D. J. Anderson

Wolff, H. and D.J. Anderson. 1988a. Immunohistologic characterization and quantitation of leukocyte sub- populations in human semen. Fertil. Steril. 49,

Wolff, H. and D.J. Anderson. 1988b. Potential human immunodeficiency virus-host cells in human semen. AIDS Research and Human Retroviruses 4, 1-2.

Wolff, H. and D.J. Anderson. 1988c. Evaluation of granulocyte elastase as a seminal plasma marker for leukocytospermia. Fertil. Steril. 50, 129-132.

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Zagury, D., J. Bernard, J. Leibovitch, B. Safai, J.E. Groopman, M. Feldman, M.G. Sarngadharan and R.C. Gallo. 1984. HTLV-I11 in cells cultured from semen of two patients with AIDS. Science 226, 449451.

Address: Dr. Deborah J. Anderson, Fearing Research Laboratory, Harvard Medical School, Seeley G. Mudd Building-205, 250 Longwood Avenue, Boston, MA 021 15, USA.

Book Review

G. Hirsch/W. Eberbach: Auf dem Weg zum kiinstlichen Leben. Retortenkinder, Leihmutter, programmierte Gene. 592 S., Brinkhauser Verlag, Munchen 1987. DM 29,80

Zwei Juristen bewegen sich ,,auf dem Wege zum kunstlichen Leben" und bemuhen sich, alles zu erortern, was zu diesem Thema bereits gemacht wird b m . sich im Stadium der Entwicklung befindet. Insofern wird hier die beruhmte ,,Lucke" geschlossen, weil die Autoren es verstehen, die doch recht trockene Materie lebendig darzustellen. Dabei mu13 man allerdings beriicksichtigen, daI3 alles durch die juristische Brille gesehen wird. So verstandlich der Drang und wohl auch Zwang der Jurisprudenz sein mag, arztliche Belange zu regeln . . . es ist immer wieder bedauerlich, dalj Arzte das nicht selbst kowen. So z.B. durch ein Moratorium bei der In-vitro-Fertilisation. Aber dann heiljt es, man durfte den Anschlulj an das Ausland nicht verlieren usw. usw. und schon ist ein Moratorium aus der Diskussion.

Der Titel selbst verwirrt etwas, da es ja ein ,,kunstliches Leben" noch nicht gibt; dafur ist man auf dem Wege . . . Bedauerlich ist die Formulierung von dem ,,abgestuften Schutz des Lebens", die juristisch sicher korrekt ist, aber fur den Laien doch eine merkwurdige Resonanz auslosen mu& Kann Schutz fur Leben wirklich abgestuft sein? Hier ist im Zusammenhang mit dem Strafrecht ein Vergleich zur Abtreibung gezogen, was von mir nicht akzeptiert werden kann, da die Abtreibung uberhaupt nichts mit Embryonenschutz zu tun hdben darf. Leider kein Sachregister, was bei diesem Umfang und Thema dringend erforderlich ware. Die Literatur, welche im Text zitiert wird, findet sich nicht immer im Verzeichnis. Das sind Schonheitsfehler, uber die man leider hinwegsehen mu13; der Charakterdes Buches leidet darunter wohl nur fur den, der an Primarliteratur interessiert ist. Das Studium dieses Buches ist eine wesentliche Hilfe bei der Bewaltigung der bedruckenden Problematik, die um die Befruchtungstechnologien angeordnet ist, und es tragt zur Information breitester Schichten durch den flussigen Stil bei.

C. Schirren, Hamburg

andrologia 20, No. 5 (1988)