Antigens of immunoglobulin G-Fc receptor III in human male reproductive tract accessory glands*†

Vol. 55, No.3, March 1991

FERTILITY AND STERILITY Copyright<> 1991 The American Fertility Society

Printed on acid-free paper in U.S.A.

Antigens of immunoglobulin G-Fc receptor Ill in human male reproductive tract accessory glands*t

Antonin Bukovsky, M.D.:j: Christian J. Thaler, M.D. John A. Mcintyre, Ph.D. Center for Reproduction and Transplantation Immunology, Methodist Hospital of Indiana, Indianapolis, Indiana

We have documented the presence of soluble antigens of immunoglobulin (lg)G-Fe receptor type III (FcRIII) in human seminal plasma that retain an affinity for the Fe fragment of lgG. The origin of these FcRIII antigens within the male reproductive tract was not known. By using polyclonal and monoclonal antibodies directed against different epitopes on FcRIII molecules, we demonstrated FcRIII reactivity in human prostate and seminal vesicle epithelial cells as well as in their glandular secretions. The FcRIII monoclonal antibody reactivity was removed by absorption with either seminal plasma or polymorphonuclear leukocytes that express FcRIII. Absorption of FcRIII monoclonal antibody with polymorphonuclear leukocytes also removed the reactivity with seminal plasma and vice versa. These data show for the first time that male accessory glands are a source of soluble FcRIII antigens. Fertil Steril 55:595, 1991

Human semen may manifest mechanisms that prevent sperm alteration by immune responses within the female genital tract. 1 Human seminal plasma contains molecules that specifically bind the Fe domain of immunoglobulin (Ig)G. 2 Immunoglobulin G Fe binding by these molecules may be involved in regulation of female responses to inseminated spermatozoa by protecting spermatozoa from IgG-mediated destruction. 2 Immunoglobulin G Fe binding molecules also could protect sperm from antibody-mediated cellular cytotoxicity by competition for membrane-bound Fe receptors on matemal effector cells. Membrane receptors for the Fe domain of IgG facilitate immune responses by targeting immune

Received September 10, 1990; revised and accepted November 7, 1990. * Presented in part at the lOth Annual Meeting of the American Society for the Immunology of Reproduction, Chicago, Illinois, June 20 to 23, 1990. t Sponsored in part by the Methodist Health Foundation Methodist Hospital, Indianapolis, Indiana. :j: Reprint requests: Antonin Bukovsky, M.D., Center for Reproduction and Transplantation Immunology, Methodist Hospital of Indiana, 1701 North Senate Boulevard, Indianapolis, Indiana 46202. Vol. 55, No.3, March 1991

complexes to effector cells. Human leukocytes have at least three different receptors for IgG. 3 Recently, we have shown that Fe binding activity of human seminal plasma is associated with soluble antigens of IgG Fe receptor type III (FcRIII) that retain their binding affinity for IgG-Fc. 4 Membrane-bound FcRIII antigens were designated as cluster of differentiation 16 (CD16). Cluster of differentiation 16 antigens have also been reported on polymorphonuclear neutrophils (PMN), natural killer (NK) cells, and macrophages. 5 •6 It is not known if soluble seminal plasma FcRIII antigens originate from these cells within the male reproductive tract or if they originate from cells not known to bear FcRIII. By using monoclonal and polyclonal antibodies to FcRIII and immunohistochemistry, we have looked for the origin of seminal plasma FcRIII antigens in human male accessory gland tissues. Our results show for the first time that FcRIII antigens are present in prostate and seminal vesicle (SV) epithelia. MATERIALS AND METHODS

Tissues Autopsy samples of prostate and SVs from four individuals (ages 28 to 66 years) were obtained, Bukovsky et al. IgG-FcRIII in male accessory glands

595

cleaned, dissected into tissue blocks of 125 mm 3 , embedded in Tissue-Tek (Miles Inc., Elkhart, IN) optimal cutting temperature compound, snap frozen in liquid nitrogen and stored at -20°C until use. Epididymis and lymph node samples were similarly processed. Immunohistochemistry

This was done according to Bukovsky et al. 7 with slight modifications. Briefly, 8-#tm cryostat sections were air dried and stored at -80°C in airtight boxes with grains of Drierite (W. A. Hammond Drierite Co., Xenia, OH) to retard water condensation on tissue samples. The slide boxes were opened after equilibration to room temperature (RT), and the sections were fixed with acetone 5 minutes, air dried, and rinsed in 0.01 M phosphate-buffered saline (PBS), pH 7.2. First antibody incubations were done for 60 minutes at RT in a PBS-humidified chamber. After washing three times for 10 minutes in PBS, peroxidase-conjugated swine antimouse 7S Ig (Sevac, Prague, Czechoslovakia) or swine antirabbit Ig (Dakopatts, Glostrup, Denmark), diluted 1:20 in PBS, was added and incubated for 30 minutes. To remove nonspecific background activity, conjugated second antibodies were diluted as above and absorbed with a hamster testis homogenate (300 mg/mL) for 20 minutes and spun for 10 minutes at 10,000 X g. After incubation with second antibodies, the slides were washed two times for 10 minutes in PBS and 10 minutes in 0.05 M Tris buffer pH 7.6. The slides were developed by incubating in substrate buffer containing 3,3'-diaminobenzidine tetrahydrochloride {product No. D-5637; Sigma Chemical Company, St. Louis, MO) 1 mg/mL, 0.002% H 2 0 2 in 0.05 M Tris buffer pH 7 .6. All antibodies were diluted in PBS, pH 7.2. Incubations were performed at RT. The slides were lightly counterstained with Harris' hematoxylin, dehydrated, and mounted in Canada balsam. Controls consisted of isotypematched first antibodies at the same Ig concentrations and dilutions. Conjugate controls were done by substituting first antibodies with PBS. In some experiments the Vectastain ABC-GO Kit (Vector Laboratories Inc., Burlingame, CA) with glucose oxidase substrate was used according to the technical bulletin provided and without hematoxylin counterstain. The influence of acetone fixation on the antigen was studied by omitting the fixation step. Instead, the samples were washed with PBS for 60 minutes before primary antibody application. Alternatively, air-dried slides were fixed by radiation in a 700-watt 596

Bukovsky et al.

IgG-FcRIII in male accessory glands

microwave oven (General Electric Co., Louisville, KY) at full power for 20 seconds. Dual color immunocytochemistry8 experiments were performed by using two unlabeled primary antibodies and the combined peroxidase (PX)/fluorescein isothiocyanate (FITC) technique. This was done by visualization of the first unlabeled antibodies by the PX-conjugated antibody method as described, without hematoxylin counterstain and dehydration. The slides were then incubated for 60 minutes with the second unlabeled antibody, washed three times for 10 minutes in PBS, incubated 30 minutes with FITC-conjugated goat F(ab') 2 antimouse or antirabbit IgG (H +L) (Protos ImmunoResearch, San Francisco, CA), washed three times for 10 minutes in PBS, and mounted in ImmuMount aqueous nonfluorescing mounting medium (Shandon Inc., Pittsburgh, PA). The antibody reagents in the second labeling sequence do not interact with those of the first sequence because the latter are altered by the reaction product of diaminobenzidine.8 Evaluation and photomicrography were performed on an Orthoplan 2 (Ernst Leitz Wetzlar GmbH, Germany) fluorescence photomicroscope. Kodak TMAX 100 film (Eastman Kodak Co., Rochester, NY) for black-and-white prints was used. Antibodies

These are summarized in Table 1. The antibodies were used in the highest dilutions producing optimal reactivity. Monoclonal antibody to FcRIII Leu llb was used at 25 and 12.5 #tg/mL. The polyclonal rabbit-anti-FcRIII9 (R-anti-FcRIII) (1:25 or 1:50) and monoclonal antibodies to FcRIII B73.1 10 (1:200) and 3G86 (1:50) were also used. Isotype-matched control monoclonal antibody consisted of Dako-DRC1 specific to dendritic cells, anti-Fct:R2 specific for low affinity IgE Fe receptor and Leu 6 specific for CD1 of thymocytes and Langerhans cells in the skin. Normal rabbit serum (NRS) was used as a control for polyclonal R-anti-FcRIII antibody. The Leu M1 (1:2.5) (reacting with PMN and monocytes, MAB1223 (1:40), reacting with PMN and Leu 19 (1:2.5) recognizing CD56 ofNK cells and neural cell adhesion molecule, 11 were also included. Seminal Plasma

Seminal plasma from two male volunteers was allowed to undergo spontaneous liquefaction for 30 minutes at RT. Samples were spun at 10,000 X g for 5 minutes, and the cell and spermatozoa-free supernatants were aliquoted and stored at -20°C until Fertility and Sterility

Table 1

List of Antibodies

Antibody

Antigen

Primary antibodies Leu llb

FcRIII

R-anti-FcRIII

Description

Specificity

Supplier

FcRIII

Monoclonal lgM-kappa Polyclonal

Functional and inhibition studies, competition studies Immunochemical

B73.1

FcRIII

Monoclonal lgG 1

lmmunochemical

3G8

FcRIII

Monoclonal lgG1

Immunochemical

DRC1

Dendritic cells

Monoclonal lgM

Immunohistochemistry

FcER2

FcERII

Monoclonal lgM

Immunochemical, functional

Leu6

CDI

Monoclonal lgG1

Immunohistochemistry

LeuM1

CD15 (PMN monocytes) CD15 (PMN)

Monoclonal lgG1

Immunocytochemistry

Monoclonal lgM

Immunocytochemistry

CD 56 (NK cells)

Monoclonal lgG1

Immunocytochemistry

Conjugates Antimouse lg

Mouse lg

Immunoelectrophoresis, Ouchterlony

Sevac, Prague, Czechoslovakia

Antirabbit lg

Rabbit lg

Crossed immunoelectrophoresis

Dako Corporation, Santa Barbara, CA

Antimouse lgG (H+L) Antirabbit IgG (H + L) Vectastain ABC-GO kit

Mouse lg

Swine antiserum peroxidase labeled Swine antiserum peroxidase labeled Goat F(ab'h FITC labeled GoatF(ab'h FITC labeled Horse antiserum, biotinated

Crossed immunoelectrophoresis

Protos Laboratories, San Francisco, CA Protos Laboratories, San Francisco, CA Protos Laboratories, San Francisco, CA

MAB1223 Leu 19

Rabbit lg Mouse IgG (H + L)

use. Donors were negative for antibodies to human immunodeficiency virus and hepatitis B surface antigens. Semen samples were monitored according to Thaler et al. 4 to exclude genitourethral infections. Absorption Studies

Specificity of Leu llb reactivity was assessed by absorption with PMN prepared as described previously.4 Briefly, blood from a healthy male volunteer was collected by venipuncture into acid citrate dextrose and centrifuged at 700 X g for 10 minutes. Buffy coats were obtained and centrifuged on FicollHypaque (Pharmacia, Uppsala, Sweden) at 700 X g for 18 minutes. Pellets containing red blood cells (RBC) and PMN were pooled, and RBC were lysed by hypotonic shock in distilled water. After 20 seconds, isotonicity was restored, samples were spun at 700 X g for 10 minutes, and hypotonic lysis was repeated. After two washes with PBS, this procedure yielded preparations consisting of >95% PMN exVol. 55, No. 3, March 1991

Crossed immunoelectrophoresis Crossed immunoelectrophoresis

Becton Dickinson, Mountain View, CA Gift from H. B. Fleit, Ph.D., Stony Brook, NY Gift from G. Trinchieri, M.D., Philadelphia, PA Gift from G. Trinchieri, M.D., Philadelphia, PA Dako, Copenhagen, Denmark AMAC, Inc., Westbrook, ME Becton Dickinson, Mountain View, CA Becton Dickinson, Mountain View, CA Chemicon, El Segundo, CA Becton Dickinson, Mountain View, CA

hibiting 98% viability by using the trypan blue exclusion test. Two hundred microliters of antibody diluted 1:2 in PBS were absorbed with 6.5 X 106 PMN three times for 20 minutes. Control absorptions were done by using the same voljvol ratio of RBC from the same donor washed three times with PBS. The absorptions were monitored by automated flow cytometry of PMN. Polymorphonuclear neutrophil- and RBC-absorbed antibodies were immediately used for immunohistochemical analysis of lymph node, prostate, and SV tissues. Absorbed antibodies also were tested for enzyme-linked immunosorbent assay (ELISA) reactivities with seminal plasma. Absorptions were performed with lyophilized seminal plasma or bovine serum albumin (BSA, product No. A-7030; Sigma Chemical Company). Three aliquots of 50 ~L seminal plasma were lyophilized in polycarbonate centrifuge tubes (Beckman Instruments Inc., Palo Alto, CA) by using a freeze Bukovsky et al.

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597

dryer (Labconco, Kansas City, KS) overnight. One hundred forty microliters of Leu llb (diluted 1:4 in PBS) were absorbed sequentially for 20 minutes with seminal plasma lyophilizates and ultracentrifuged (100,000 X g for 30 minutes) to remove aggregates. Lyophilizates from three 50 ~L aliquots of BSA (50 mg/mL in PBS) were used as controls. The supernatants were retained. The positive control consisted of unabsorbed Leu llb (diluted 1:4 in PBS) and negative controls consisted of DRC1 and FcfR2 IgM isotypes used at identical antibody concentrations and ultracentrifuged three times. The supernatants were used without further dilution for the indirect immunoperoxidase technique on tissue sections of male accessory glands. Supernatants diluted 1:4 were also examined by fluorescence-activated cell sorter (FACS) for reactivity with PMN. Fluid phase absorptions were performed with seminal plasma, normal human sera, goat sera (Sigma), mouse ascites (Cedarlane Lab. Ltd, Hornby, Ontario, Canada), or NRS. One hundred microliters ofR-anti-FcRIII (diluted 1:25) antibody were incubated for 20 minutes with 35 ~L of the different fluids followed by ultracentrifugation (100,000 X g for 30 minutes) to remove aggregates. The supernatant was retained and the absorption repeated two times. Absorbed antibody was tested by immunohistochemistry on lymph node, prostate, and SV tissues. Flow Cytometry

logarithmically acquired fluorescence intensity was displayed on a linear scale. Enzyme-Linked Immunosorbent Assay

Done as described by Thaler et al.,4 Wells of Immulon II plates (Sigma) were coated overnight at 4°C with 50 ~L of seminal plasma (diluted 1:20 in PBS). Coated wells were washed two times with PBS containing 0.05% Tween 20 (PBS-Tween; Bio Rad Laboratories, Richmond, CA) blocked with 2% BSA and incubated with PMN- or RBC-absorbed Leu llb or isotype-matched control antibody (DRC1) used at identical Ig concentrations. After 30 minutes' incubation at RT, the wells were washed three times with PBS-Tween and incubated 30 minutes with peroxidase-conjugated rabbit antimouse Ig (1:2,000). Plates were washed five times, and color reactions were developed by adding 100 ~L of substrate buffer containing 0.42 mM tetramethylbenzidine (Miles Co., Naperville, IL) and 0.0045% H 20 2 in 0.1 M sodium acetate buffer pH 6.0. The color reactions were stopped by adding 35 ~L of 2 M H 2 S04 to each well. Optical density (OD) readings were measured at 450 nm by using the automated Biomek 1000 workstation (Beckman Instruments Inc., Arlington Heights, IL). Mean OD values were determined from triplicate wells, and net ODs were calculated by subtracting background readings. The difference in ELISA reactivities for PMN -absorbed and RBC-absorbed Leu llb was calculated by using a two-tailed Student's t-testP

One hundred-microliter aliquots containing 1 X 106 PMN, used for absorptions of Leu llb mono-

clonal antibody, were washed two times with PBS, incubated for 30 minutes with 20 ~L of FITC-conjugated goat F(ab') 2 antimouse or antirabbit IgG (H+L) (Protos ImmunoResearch), and washed two times. In another experiment, 100-~L aliquots containing 1 X 106 PMN, freshly prepared and washed two times with RPMI 1640 (Sigma) containing 2% (vol/vol) BSA (Sigma) and 0.2 mM colchicine (Sigma) were incubated for 30 minutes with 20 ~L of PMN-, seminal plasma-, or control-absorbed antibodies. Cells were washed two times and incubated with FITC conjugates as above. The supernatants from absorptions with lyophilized seminal plasma or BSA were tested undiluted or further diluted 1:4 for reactivity with PMN. The fluorochrome-stained cells were examined in a F ACStar PLUS (Becton Dickinson, Mountain View, CA) flow cytometer with a 3-watt argon laser set at 200mW. Electronic gates were set for PMN by using side and forward light scatter. A total of 10,000 events were collected and 598

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lgG-FcRIII in male accessory glands

RESULTS Anti-FcRIII Reactivity to Prostate, SV, and Lymph Node Tissues

The monoclonal antibody Leu llb specific for FcRIII reacted with SV and prostatic epithelial cells (Fig. 1A; Fig. 2A and C) showing some variability among glandular tissue within the samples. Some glands exhibited distinct cytoplasmic and perinuclear reactivity in epithelial cells particularly in those adjacent to the basal lamina (Fig. 2A), whereas in other glands the surface reactivity of epithelial cells was most prominent (Fig. 2C). Seminal vesicle colloid also was reactive in some glands. A similar pattern of reactivity was found with B73.1, a monoclonal antibody specific for a different epitope of FcRIIIP Another distinct CD16 monoclonal antibody, 3G8, reacted with SV epithelial cells close to the lumen and only in some areas of the glandular tissue (Fig. 1C and D). The 3G8 monoclonal antibody did not react with epithelial cells of the prostate Fertility and Sterility

was also enhanced with SVs if unfixed and PBSpretreated sections were used. Reactivities of all other antibodies were not changed by this procedure. This indicates that 3G8 reactive FcRIII epitopes in prostatic and SV epithelium are more accessible in unfixed tissues. No reactivity of Leu llb was noted with the epididymal epithelium used as a control tissue (not shown). Polyclonal R-anti-FcRIII reacted with both SV and prostatic epithelium. A prominent reactivity was noted with basal epithelial cells (Fig. 3A and B). Seminal vesicle reactivity varied within a given individual. The R-anti-FcRIII also reacted intensely with intravascular leukocytes. By using a doubleantibody labeling technique, these cells also reacted with monoclonal antibody MAB1223, which detects PMN. The R-anti-FcRIII-positive PMN were found closely adjacent to both the prostatic and SV basal lamina (Fig. 3A and B). The presence of NK cells, which also bear FcRIII, was studied in male accessory gland tissues by using monoclonal antibody Leu 19 against the NK cell marker CD56. We did not detect CD56 mononuclear cells in the lamina propria or within the epithelium of the prostate or SVs. There was, however, strong reactivity of the Leu 19 monoclonal antibody with neural tissue within the stroma and the lamina propria of the epithelium (not shown). Control immunohistology studies were done by including lymph node sections on each slide. All anti-CD16 antibodies reacted with PMN in lymph nodes. The 3G8 also reacted with macrophages in the lymph nodes as detected by the combined PX/ FITC technique using anti-CD16 with MAB1223 (anti-PMN) or Leu M5 (anti-macrophage) antibodies (not shown). In isotype- or serum-matched control experiments and in PBS conjugate controls, there was no reactivity of the stroma or epithelium (Fig. 1B; Fig. 3C). Background reactivity was also negligible in absorption experiments (Fig. 2D). Figure 1 Seminal vesicle sections reacted with anti-FcRIII monoclonal antibodies (S, stroma; E, epithelium; L, lumen). (A), Leu llb shows strong reactivity with luminal epithelium. (B), Isotype-matched control monoclonal antibody anti-DRC1 is negative. Monoclonal antibody 3G8 directed to a different FcRIII epitope shows variable expression in different glandular regions. Although some epithelia are positive with 3G8 (C), other areas only react with stromal mononuclear cells (arrows in D). (ABCGO technique; magnification X200).

when incubated with either acetone or microwave fixed samples. However, reactivity was detected in the epithelium of unfixed sections pretreated with PBS. The reactivity of monoclonal antibody 3G8 Vol. 55, No.3, March 1991

Absorption Studies

The specificity of Leu llb reactivity in the male accessory glands was investigated and compared with its reactivity against seminal plasma and PMN. Individual absorption studies were done with seminal plasma and PMN. Leu llb reactivity to prostatic and SV epithelia was markedly decreased after absorption with seminal plasma, whereas control absorptions with BSA had no effect (Fig. 2A and B). In support of the decreased reactivity with male accessory gland tissues, F ACS analysis of the seminal plasma-absorbed Leu 11b showed reduced reactivities with PMN (Fig. 4A). Bukovsky et al.

lgG-FcRIII in male accessory glands

599

absorbed Leu llb also was significantly reduced with PMN as shown by FACS (Fig. 4B). No reduction of reactivity was noted with identical control absorptions done with RBC (Fig. 2C). Polymorphonuclear neutrophil- and control RBC-absorbed Leu llb was compared for ELISA reactivity with seminal plasma. Leu llb reactivity was significantly decreased by PMN absorption (Table 2). Dilution of R-anti-FcRIII into seminal plasma and normal human serum, but not goat serum or mouse ascites, removed the reactivity of R-antiFcRIII to PMN in prostate, SV s, and lymph nodes. This treatment, however, did not significantly affect reactivities with prostatic epithelium (not shown). DISCUSSION

Figure 2 Prostatic tissue sections (S, stroma; E, epithelium). Reaction with Leu llb, control absorbed with BSA (A), Leu llb absorbed with seminal plasma (B), Leu llb control absorbed with RBC (C), and Leu llb absorbed with PMN (D). Note marked decrease of Leu llb reactivity in (B) with only weak reaction of basal epithelial cells (arrowheads in B) and complete absorption in (D). Strong staining of some epithelial cell nuclei (arrows and arrowheads in A) or cell surfaces (small arrowheads in B) demonstrate variability within and among the glandular tissue. (Indirect immunoperoxidase technique, hematoxylin counterstain; magnification X200).

Absorptions of Leu llb with PMN consistently abolished Leu llb reactivity with both the prostatic and SV epithelial cells (Fig. 2D). Reactivity ofPMN600

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lgG-FcRIII in male accessory glands

From these data, the male reproductive tract accessory glands appear to represent a major source of the FcRIII antigens that appear in the ejaculate. The reactivity of monoclonal antibody Leu llb was detected in prostatic and SV epithelia and also in the intraluminal colloid of the SVs. Leu llb binds a functional epitope of FcRIII on NK cells and PMN. 5 It also reacts with seminal plasma.4 We have shown that Leu llb reactivity with prostate and SV epithelium is removed by either PMN or seminal plasma absorption. Polymorphonuclear neutrophils also removed the reactivity of Leu llb with seminal plasma and vice versa. This indicates that determinants recognize<;~ by Leu llb on PMN are also represented in seminal plasma as well as in the prostatic and SV secretory epithelium. Monoclonal antibodies B73.1 and 3G8 recognize FcRIII epitopes distinct from Leu llb as do R-antiFcRIII polyclonal antibodies. All reacted with human male accessory gland epithelia, although different antibodies showed distinctive patterns within prostate and SV. For example, 3G8 exhibited no reactivity with the prostate in fixed sections and in SVs not all glandular tissue consistently reacted. However, if fixation was omitted and the sections were placed in PBS before incubation with antibody, 3G8 reacted. This indicates that either the 3G8 epitope is present but altered by fixation or it becomes accessible by enzymatic processes occurring in unfixed tissue. We favor the latter possibility because some epithelial cells in SVs reacted with 3G8 in fixed sections. This observation may relate to the reported changes in FcRIII epitope expression13 in which the presence of certain cryptic epitopes on the FcRIII molecule was suggested. In contrast to 3G8, polyclonal R-anti-FcRIII antibodies reacted consistently with the basal epitheFertility and Sterility

Figure 3 Prostatic tissue (A) and SV (B, C) sections (S, stroma; E, epithelium; L, lumen) incubated with Ranti-FcRIII (A, B) and NRS (C). Open arrows indicate the interface between the stroma and epithelium. Note strongly reacting PMN (arrowheads in A and B) adjacent to the epithelium that exhibit strong basal reactivity and weak reactivity with other glandular cells; compare with control in C. (Indirect immunoperoxidase technique, hematoxylin counterstain; magnification X200).

lial cells of prostatic epithelium. The reactivity with SVs had a similar pattern to that in the prostate, but it exhibited larger variability within the tissue and among samples from different individuals. There was also strong reactivity of R-anti-FcRIII with PMN that was not observed to such an extent with other anti-CD16 antibodies. This particular reactivity was removed by diluting the antibody in seminal plasma and also normal human serum, which has been shown to contain soluble FcRIIl. 14 Serum from another species and mouse ascites did not affect R-anti-FcRIII reactivities. However, the reactivity of R-anti-FcRIII against the male accessory glands persisted after absorption with seminal plasma or normal human serum. This could indi-

..,

A

'

I

cate a higher density ofFcRIII antigens on prostatic and SV epithelia than expressed by PMN within the tissues. Alternatively, the remaining activity with prostatic and SV epithelia may represent an additional specificity on this polyclonal reagent. This is possible as R-anti-FcRIII was raised to affinity-purified FcRIII that was denaturated in sodium dodecyl sulfate. 9 The prostate and SVs are hormonally dependent tissues responding by epithelial cell differentiation to male sex steroids. 15 It may be worthwhile to investigate if the hormonal milieu can influence the expression of FcRIII antigens and their secretion into the seminal plasma. Expression of FcRIII on epithelial cells of male reproductive accessory glands

I I I

'

_-, ..

_

/ Figure 4 Fluorescence-activated cell sorter analyses of seminal plasma and PMN absorbed Leu llb. (A), PMN reactivity of Leu llb, control absorbed with BSA ( · · · ), Leu llb absorbed with seminal plasma ( • · · · • ), and conjugate control (--- ). (B), Leu llb, control absorbed with RBC ( • • ·) and Leu llb absorbed with PMN ( · • • • ·).The histograms represent: x-axis, intensity of fluorescence (log scale); y-axis, relative number of cells. Vol. 55, No.3, March 1991

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Table 2 Reactivity of Leu llb Absorbed With PMN or RBC As Detected by ELISA in Seminal Plasma of Two Donors• 3. Donor

PMN absorption

RBC absorption

Seminal plasma 03 Seminal plasma 123

0.035 ± 0.02b 0.000 ± 0.01 c

0.182 ± 0.006 0.045 ± 0.02

4.

5.

• Values are net OD ± SD. b p < 0.003. c p < 0.05.

6.

could be a marker of hormone-induced differentiation and secretion. The biological role of soluble FcRIII antigens in seminal plasma is not known. Significant differences in CD16 antigen expression in seminal plasma among individuals were reported, and affinity experiments have shown that soluble FcRIII antigens in seminal plasma retained their lgG-binding activity.4 It could be speculated that male infertility is associated with low FcRIII antigen levels in seminal plasma, particularly in the partners of women exhibiting antisperm antibodies. Thus investigation and quantification of seminal plasma FcRIII antigens from sterile males might be of diagnostic importance. The insemination of washed spermatozoa from infertile males with seminal plasma from normal donors might represent a simple and efficacious treatment for such cases of infertility.

7.

8.

9.

10.

11.

12.

13.

Acknowledgments. We thank Howard B. Fleit, Ph.D., State University of New York at Stony Brook, New York, for R-antiFcRIII, and Professor Giorgio Trinchieri, M.D., Wistar Institute, Philadelphia, Pennsylvania, for monoclonal antibodies 3G8 and B73.1. We thank Mr. Harold Boldt for help with flow cytometry. 14. REFERENCES 1. Thaler CJ: Immunological role for seminal plasma in insemination and pregnancy. Am J Reprod Immunol21:147, 1990 2. Witkin SS, Richards JM, Bongiovanni AM, Zelikovski G:

602

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15.

An IgG-Fc binding protein in seminal fluid. Am J Reprod Immunol 3:23, 1983 Unkeless JC, Scigliano E, Freedman VH: Structure and function of human and murine receptors for IgG. Ann Rev Immunol 6:251, 1988 Thaler CJ, Faulk WP, Mcintyre JA: Soluble antigens oflgG receptor Fe gamma Rill in human seminal plasma. J Immunol 143:1937, 1989 Perussia B, Trinchieri G, Jackson A, Warner NL, Faust J, Rumpold H, Kraft D, Lanier LL: The Fe receptor on human natural killer cells: phenotypic, functional and comparative studies with monoclonal antibodies. J Immunol133:180, 1984 Fleit HB, Wright SD, Unkeless JC: Human neutrophil Fe gamma receptor distribution and structure. Proc Natl Acad Sci USA 79:3275, 1982 Bukovsky A, Presl J, Zidovsky J, Mancal P: The localization of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and fallopian tube. Immunology 48:587, 1983 Sternberger LA: The unlabeled antibody method: hormone receptor, Golgi-like and dual color immunocytochemistry. J Histochem Cytochem 27:1658, 1979 Fleit HB, Wright SD, Durie CJ, Valinsky JE, Unkeless JC: Ontogeny of Fe receptors and complement receptor (CR3) during human myeloid differentiation. J Clin Invest 73:516, 1984 Perussia B, Starr S, AbrahamS, Fanning V, Trinchieri G: Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fe receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1. J Immunol 130:2133, 1983 Lanier LL, Testi R, Bindl J, Phillips JH: Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. J Exp Med 169:2233, 1989 Neter J, Wassermann W, Kutner MH: Applied Linear Statistical Models, 2nd edition. Homewood, IL, Richard D Irwin, Inc, 1985 Huizinga TWJ, Kleijer M, Roos D, Kr von dem Borne AEG: Differences between FcRIII of human neutrophils and human K/NK lymphocytes in relation to the NA antigen system. In Leukocyte Typing IV, White Cell Differentiation Antigens, Edited by W Knapp, B Dorken, WR Gilks, EP Rieber, RE Schmidt, H Stein, AEG Kr von dem Borne. Oxford, Oxford University Press, 1989, p 582 Huizinga TWJ, de Haas M, Kleijer M, Nuijens JH, Roos D, Kr von dem Borne AEG: Soluble Fe gamma receptor III in human plasma originates from release by neutrophils. J Clin Invest 86:416, 1990 Teeter KJ, DahlE: The ultrastructure of the accessory sex organs of the male rat. 7. The effect of an anti-androgenic compound, SK and F 22340. Urol Res 2:159, 1975

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