Immunogenetic studies of soluble Fc receptor (CD16) antigens in human seminal plasma C. J. Thaler, J. A. McIntyre, W. P. Faulk. Immunogenetic studies of soluble Fc receptor (CD16) antigens in human seminal plasma. Tissue Antigens 1991: 37: 121-126. Abstract: Two forms of CD16 Fc receptors (FcRIII) have been defined on human leukocytes. Type 1, present on polymorphonuclear neutrophils (PMN), expresses the N A alloantigen system and type 2, present on natural-killer (NK) cells and macrophages, has no known polymorphic variations. We have described the presence of soluble FcRIII antigens in human seminal plasma (SP) (see Ref. 9). These SP antigens retain an affinity for IgG-Fc and are biochemically distinct from leukocytic FcRIII. Their origin within the male reproductive tract and .how they relate to FcRIII types 1 and 2 were not known. By using monoclonal antibodies CLB/Gran 11 and Leu 1l c that differentially react with FcRIII type 1 and type 2 from different individuals, we studied how SP FcRIII antigens relate to leukocytic FcRIII. CLB/Gran 11 and Leu 1l c reactivities with PMN and NK cells from a selected panel of male donors were compared with enzyme-linked immunosorbent assay reactivities of CLB/Gran 1 1 and Leu l l c with SP samples from these donors. The reactivity pattern with SP samples was shown to be different from findings with donors’ PMN, but it corresponded with their NK cells. Our results indicate that SP FcRIII antigens do not manifest the polymorphic variations that are detected by CLB/Gran 11 and Leu 1lc on PMN. These findings suggest that SP FcRIII antigens do not originate from PMN.
Two different forms of CD16 Fc receptor have been defined on human leukocytes (1). Type 1 (FcRIII-1), present on neutrophils (PMN), expresses the biallelic NA alloantigen system (2-4) and is anchored to the membrane via a phosphatidylinositol glycan bond (5-8). Type 2 (FcRIII2), present on natural-killer (NK) cells and macrophages, is not polymorphic and has a transmembrane region with a cytopiasmic tail (G8). We have reported the presence of soluble FcRIII antigens in seminal plasma (SP) (9). These antigens retain their affinity for IgG-Fc and it has been proposed that they regulate maternal immunity at insemination and pregnancy (9, 10). It also was suggested that SP FcRIII antigens, if allotypic, could induce allogeneic reactions in the female. SP FcRIII antigens are biochemically distinct from leukocytic FcRIII (9). Their origin within the male reproductive tract and how they relate to FcRIII types 1 and 2 were not known. We have studied how SP FcRIII antigens relate to FcRIII types 1 and 2 by using 2 monoclonal antibodies (mAb) that
Christian J. Thalsr, John A. Mclntyrs and W. Page Faulk Center for Reproduction and Transplantation Immunology, Methodist Hospital of Indiana, Indianapolis, IN, USA
Key words: IgG - Fc receptors - CD16 antigens - NA alloantigens seminal plasma - human
-
Received 24 September, revised, accepted for publication 15 November 1990
differentially react with FcRIII- 1 and FcRIII-2 from different individuals. The mAb Leu l l c reacts with NK cells from all individuals but is negative with PMN from about 50% of donors (11). The mAb CLB/Gran 11 reacts with an epitope that is closely associated with the NAl alloantigen present on FcRIII-1 (2). This mAb reacts with PMN-bound as well as with soluble FcRIII-1 from homozygous NA1 and from heterozygous NAlNA2 individuals. However, CLB/ Gran 11 reacts neither with FcRIII-1 from homozygous NA2 individuals nor with NK cell FcRIII type 2 antigens (2, 12, 13). We have analyzed both Leu 1lc and CLB/Gran 11 reactivities with PMN and NK cells from healthy male volunteers by using flow cytometry. A panel of donors was selected to represent the different patterns of Leu l l c and CLB/Gran 11 reactivities with PMN and NK cells. CLB/Gran 11 and Leu l l c reactivities with SP were determined by using the enzyme-linked immunosorbent assay (ELISA) and results were compared to reactivities with PMN and NK cells. Our findings show that SP FcRIII antigens do not manifest polymorphic
re1
Thaler et al.
variations as detected by Leu l l c and CLB/Gran 11 on FcRIII type 1. Material and Methods Preparations of peripheral blood lymphocytes (PEL) and PMN
Blood from 7 healthy male volunteers was collected by venipuncture into acid citrate dextrose and centrifuged at 700 g for 10 min. Buffy coats were obtained, layered on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and centrifuged at 700 g for 18 min. PBL were removed from the Ficoll Hypaque interface and pellets containing red blood cells (RBC) and PMN were pooled. PMN were purified by hypotonic lysis of RBC as described by Thaler et al. (9). Cells were exposed to distilled H,O for 20 seconds and isotonicity was restored by adding 10% (v/v) of 0.1 M phosphate-buffered 1.5 M NaCl, pH 7.4. Cells were centrifuged at 700 g for 10 min and hypotonic lysis was repeated. After two washes with 0.01 M phosphate-buffered 0.15 M NaCl, pH 7.4, (PBS) this procedure yielded more than 95% pure PMN with greater than 95% viability as shown by exclusion of trypan blue.
vine serum albumin (BSA) (Sigma Chemical Company, St. Louis, MO) and 0.2 mM colchicine (Sigma) (RPMI-Colchicine). Aliquots (100 pl) containing 1 x lo6 cells were incubated for 30 min with 20 pl of CLB/Gran 11 (diluted 150 in PBS), Balb/c control ascites fluid (diIuted 150 in PBS), phycoerythrin (PE)-conjugated Leu 1lc (50 pg1/ ml IgGl) or isotope-matched control mAb. Incubations were stopped by washing cells twice with RPMI-colchicine. Two-color fluorescence analyses were done simultaneously by using fluorescein (FITC) and PE-conjugated mAb. For unconjugated primary mAb, cells were washed, incubated for 30 min with FITC-conjugated goat F(ab’), antimouse IgG and washed twice. The cells were examined in a Beckton Dickinson FACStar Plus flow cytometer with a 3 watt argon laser set at 200 mW and electronic gates were set for side scatter and forward scatter of PBL or PMN, respectively. 10000 events were collected and logarithmically acquired FLl and/ or FL2 intensities were displayed on linear scales.
Donor selection Antibodies
Details of the monoclonal and polyclonal antibodies used in this study are listed in Table 1. Control incubations for the ascites antibody CLB/ Gran 11 were done with identical dilutions of Balb/ c control ascites fluid (Cedar Lane Laboratories, Ltd., Hornby, Canada). Flow cytometry
Freshly prepared PBL and PMN were washed twice with RPMI 1640 containing 20 mg/ml bo-
7 healthy male volunteers were studied for Leu 1lc and CLB/Gran 11 reactivities with PMN and NK cells. The donors whose PMN were Leu 1lc-negative were chosen for an evaluation of their SP samples, because in such donors Leu 1lc reactivity is restricted to FcRIII type 2 (1 1). One donor, with Leu llc-positive PMN was included for comparison. In addition, 3 donors were chosen so that the reported (12) high, intermediate and negative CLB/ Gran 11 reactivities with PMN were represented. SP samples from these donors were studied for ELISA reactivities with CLB/Gran 11.
Table 1 List of antibodies used in this study Antibody
Specificity
Description
Specificity Determined
Supplier
CLBiGran 11
FcRllt from NAl-positive individuals (CD16)
lmmunoprecipitation Flow cytometry
Leu l l c (PE-conjugated)
gift from Dr. Kr. A. E. 6. von dem Bome, Amsterdam, Netherlands Beckon Dickinson, Mountain View, CA
Leu 19 (FITCconjugated) Anti-Transferrin receptor Anti-mouse IgG
FCRIII-1 from 50% of individuals Fc RllC2 from all individuals (CD16) NK cell differentation antigen (CD56) Transferrin receptor (Trf-R) mouse IgG
mouse monoclonal l9G21, mouse monoclonal lgG, mouse monoclonal lgGi monoclonal
Immunoprecipitation, Flow cytometry lmmunochemical lmmunohistology affinity purified
Beckon Dickinson, Mountain View, CA DAKOPATTS Copenhagen, Denmark Protos San Francisco, CA
Anti-mouse lg
mouse lg
crossed immuncelectrophoresis
DAKDPATTS Copenhagen, Denmark
122
goat F(ab’), antiserum FlTG conjugated rabbit antibody, peroxidaseconjugated
Irnmunoprecipitation, Flow cytometry
CD16 antigens in seminal plasma Net OD (450 nml 0.5 7
0.4 0.3 -
!Xl-\Flwrescmce GK Height
Figure 1. Leu l l c reactivity with peripheral blood lymphocytes from a representative individual. A subsest of lymphocytes was positive with PE-conjugated Leu l l c (vertical axis). By using two-color fluorescence, these cells were shown to also react with the FITC-conjugated NK cell marker Leu 19. NK cells from all 7 individuals studied were positive with Leu 1lc.
=
Leu l l c
0mAb anti Trf-R
Figure 3. ELISA studies with Leu 1lc and mAb anti-transfemn receptor (background control) Leu l l c showed positive reactivities (mean net OD values+SEM) with seminal plasma samples from all donors. Differences in SP FcRIII antigen expression that were previously reported (9) were independent of Leu 1l c reactivities with the donor's PMN.
Seminal plasma
SP from male volunteers was allowed to undergo spontaneous liquefaction for 30 min at room temperature (RT). Samples were centrifuged at 10000 g for 5 min and the cell- and spermatozoa-free supernatants were aliquoted and stored at -20°C until use. Serial donor samples were collected at least 3 days apart. PMN sonicates
PMNs were washed twice in PBS and 5 x lo6 cells were resuspended in 2 ml of PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma) and 1 mM of TPCK (n-tosyl-1-phenylalanine chlo-
romethyl ketone) (Sigma) as protease inhibitors. Cells were disrupted with a continuous sonication burst of 5 s with the use of a Sonicator W-375 (Heat Systems-Ultrasonics, Inc., Plainview, NY) at number 2.5. Sonicates were centrifuged for 5 min at 10000 g and the supernatants were retained. Pellets were resuspended in 2 ml PBS and the sonication and centrifugation steps were repeated. The supernatants were pooled, aliquoted and stored at - 20°C. ELISA
The ELISA was done as modified from Thaler et al. (9). SP samples (50 pl) diluted in PBS (1:20)were coated overnight at 4°C onto wells of Immulon I1
Leu 1lc - PMN 0
128
-m - -
' ''
' ' 1488' '
pBQ'
I
'Rp9 I
'
1001
PMN-CLB/Gran 11
4
a
-
NA1 NAl NA1 NA2
I
'
''rr-lrr
Figure 2. Leu 1l c reactivity studied with P M N from 4 individuals. PMN from donors 1-3 are negative and PMN from donor 4 are positive with Leu 1lc.
123
Thaler et al.
plates (Dynatech Lab Inc, Alexandria, VA). PMN sonicates were coated at protein concentrations of 0.1 mg/ml. Wells were washed twice with PBS containing 0.05% Tween 20 (Bio Rad Laboratories, Richmond, CA) (PBS-Tween) and incubated with mAb Leu 1lc, CLB/Gran 11 and background controls for 30 min at RT. All antibodies were diluted in PBS-Tween containing 0.5% BSA. mAb Leu 1lc and anti-transferrin receptor, an isotope-matched negative control, were used at IgG concentrations of 20 pg/ml. CLBIGran 11 and control ascites fluid were used at 1:200 dilutions. Plates were washed three times and incubated for 30 min with peroxidase-conjugated rabbit antimouse Ig (1:2000). Plates were washed five times and color reactions were developed by adding 100 pl of substrate buffer containing 0.42 mM tetramethylbenzidine (Miles CO, Napersville, IL) and 0.0045% H,O, in 0.1 M sodium acetate buffer, pH 6.0. The color reaction was stopped by adding 35 pi of 2 M H2S04.Optical density (OD) of each well was measured at 450 nm by using an automated Biomek 1000 workstation (Beckman Instruments Inc., Arlington Heights, IL). Mean OD values were determined from triplicate determinations. Net OD values were calculated by subtracting background ODs. Patterns of reactivity were confirmed with at least 2 separate SP samples from each individual and each sample was tested two or more times on different days. Results Reactivities with Leu 11c
PBL and PMN from 7 donors were screened for Leu l l c reactivities by using flow cytometry. Leu l l c reacted with a small percentage of PBL from N e t OD 1450nrnI
0.35i
0.25
all individuals. By using the FITC-conjugated NK cell marker Leu 19 and two-color analysis, Leu 1lc-positive PBL were shown to be NK cells (Fig. 1). In contrast to reactivity with NK cells from all individuals, Leu 1l c was negative with PMN from donors 1-3 (Fig. 2). Thus, in these individuals Leu l l c reacted selectively with FcRIII type 2. SP samples from donors 1-3 and from donor 4 whose PMN were Leu 1lc-positive were analyzed for Leu l l c reactivity by using the ELISA. SP samples from all donors regardless of the Leu 1lc reactivity with PMN were found to be strongly positive with Leu l l c (Fig. 3). Individual differences that previously had been reported for SP-FcRIII antigen expression (9) were independent of Leu l l c reactivity with PMN. The reactivity of Leu l l c with SP from all individuals indicates that SP FcRIII antigens share immunogenetic characteristics with FcRIII type 2. Reactivities with CLB/Gran 11
Flow cytometric analysis of PMN from different individuals with CLB/Gran 11 showed strongly positive, intermediate and negative reactivities. These differences are thought to result from a gene dose effect of NAl antigen expression (12). Strong, intermediate and negative reactivities with CLB/ Gran 11 are associated with genotypes NAlNAl , NAlNA2 and NA2NA2, respectively (12). CLB/ Gran 11 was negative with PBL from all individuals (data not shown). 3 donors were selected so that one of each represented NA1 homozygous, NA2 homozygous and NAlNA2 heterozygous individuals (Fig. 4). ELISA studies with CLB/Gran 11 were done with SP samples and sonicated PMN membranes. CLB/Gran 11 reacted with PMN sonicates from NAl-positive individuals. In contrast, SP samples from all individuals showed background reactivities with CLB/Gran 11. All SP samples were positive with Leu 1lc (Fig. 5). These results indicate that FcRIII antigens in SP lack polymorphic variations of FcRIII type 1 as detected by mAb CLB/Gran 11. Discussion
"
SP (NA1 NA1)
-
SP (NA1 NA2) CLE gran 11
SP (NA2 NA2) 0Leu 11c
PMN (NAI NA2)
Figure 5. ELISA studies with CLB/Gran 11 showed positive reactivities (mean net OD valuesfSEM) with PMN sonicates from NAI positive individuals. SP samples from all donors were negative with CLB/Gran 11. These results were independent of the donors NA allotype. SP from all individuals were positive, with Leu llc.
124
We have reported the presence of soluble FcRIII antigens in human SP (9). The origin of these FcR antigens in SP is under study. PMN that are stimulated (14) or treated with phospholipase C (5, 6) can liberate membrane-bound FcRIII molecules. Cultured NK cells also have been shown to release FcRIII (1, 5, 6). Thus, soluble SP FcRIII antigens could originate from leukocytes that are present within the male reproductive tract (15, 16). By using mAb Leu 1l c and CLB/Gran 11 that differ-
CD16 antigens in seminal plasma entially react with FcRIII type 1 and type 2 from different individuals (2, 11, 12, 13), we investigated how the pattern of PMN and NK cell reactivities relate to findings with SP FcRIII antigens. Our studies indicate that phenotypic differences identified on PMN from different individuals are not expressed on FcRIII antigens in SP. CLB/Gran 11 and anti-NA1 alloantibodies have been shown to mutually crossblock, so the epitope recognized by CLB/Gran 11 appears to be closely related to or identical with the NAl alloantigen (2). The lack of CLB/Gran 11 reactivity with SP from NAl-positive individuals suggests that NA1 alloantigens are absent from SP FcRIII. This could be biologically significant in preventing intravaginal sensitization to paternal NA alloantigens, as maternal IgG alloantibodies transported across placentae have been associated with neonatal neutropenia (17). Positive reactions of Leu l l c with SP samples from donors whose PMN were Leu l l c negative, and negative reactions of CLB/Gran 11 with SP from NAl-positive individuals indicate that PMN are not a main source of SP FcRIII antigens. The uniform pattern of Leu l l c and CLB/Gran 11 reactivity with SP samples from different individuals corresponds with donors’ NK cells. FcRIII type 2 that is present on NK cells and macrophages has no known variations between different individuals (1). In spite of these shared characteristics with FcRIII-2, SP FcRIII antigens might not originate from NK cells or macrophages. SP FcRIII antigens could represent molecules that are distinct from leukocytic FcRIII. This possibility has been suggested as FcRIII antigens in SP and on leukocytes differ in electrophoretic mobilities (9). Recent immunohistological studies in our laboratory indicate that FcRIII antigens are expressed and released into SP by secretory epithelia of male reproductive tract accessory glands (18). Differences of FcRIII antigens from leukocytes and SP could result from tissue-specific modifications of FcRIII-2-gene expression. Alternatively, SP FcRIII antigens could be encoded by a different gene and share specific epitopes with FcRIII-2. The presence of these antigens in SP and their deposition into the vagina during insemination could indicate a novel mechanism of regulating maternal immunity for normal conception and pregnancy.
Acknowledgments
This work was supported in part by the Methodist Health Foundation. We thank Harold D. Boldt for flow cytometric analyses and Sandra L. Hollowell for secretarial assistance. We thank Dr. A. E. G.
Kr. von dem Borne for monoclonal antibody CLB/ Gran 11. References 1. Ravetech JV, Perussia B. Alternative membrane forms of FcyRIII (CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in a single nucleotide substitution. J Exp Med 1989: 170: 481-97. 2. Werner G, Kr von dem Borne AEG, Bos MJE, et al. Localization of the human NAI alloantigen on neutrophil Fc receptors. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte typing I1 Vol 3 Human Myeloid and Hematopoietic Cells. New York: Springer Verlag, 1986 109-21. 3. Ory PA, Goldstein IM, Kwoh EE, Clarkson SB. Characterization of polymorphic forms of Fc receptor I11 on human neutrophils. J CIin Invest 1989: 83: 1671-81. 4. Ory PA, Clark MR, Kwoh EE, Clarkson SB, Goldstein IM. Sequence of complementary DNAs that encode the NAl and NA2 forms of Fc receptor I11 on human neutrophils. J CIin Invest 1989: 84: 1688-91. 5. Selvaraj P, Rosse WF, Silber R, Springer TA. The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal nocturnal hemoglobinuria. Nature 1988: 333:565-7. 6. Ueda E, Kinoshita T, Nojima I, Inoue K, Kitani T. Different membrane anchors of FcyRIII (CD16) on K/NK lymphocytes and neutrophils. Protein-vs Lipid-Anchor. J Immunol 1989: 143: 1274-7. 7. Edberg JC, Redecha PB, Salmon JE, Kimberly RP. Human FcyRIII (CD 16). Isoforms with distinct allelic expression, extracellular domains, and membrane linkages on polymorphonuclear and natural killer cells. J Immunol 1989: 143: 1642-9. 8. Lanier L, Phillips JH, Testi R. Membrane anchoring and spontaneous release of CD 16 (FcRIII) by natural killer cells and granulocytes. Eur J Immunol 1989: 19 7758. 9. Thaler CJ, Faulk WP, McIntyre JA. Soluble antigens of IgG receptor FcyRIII in human seminal plasma. J Immunol 1989: 143 1937-42. 10. Thaler CJ. Immunologic role for seminal plasma in insemimation and pregnancy. Am J Reprod Immunol 1989: 21: 147-50. 11. Perussia B, Starr S, Abraham S, Fanning V, Trinchieri G. Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. 1. Characterization of the lymphocyte subset reactive with B73.1 JImmunoZ 1983: 130: 213341. 12. Salmon JE, Edberg JC, Kimberly RP. Fcy receptor I11 on human neutrophils allelic variants have functionally distinct capacities. J Clin Invest 1989: 8 5 1287-95. 13. Huizinga TWJ,de Haas M, Kleijer M, Nuijens JH, Roos D, Kr von dem Borne AEG. Soluble Fcy receptor I11 in human plasma originates from release by neutrophils. J Clin Invest 1990: 86: 416-23. 14. Huizinga TWJ, van der Schoott CE, Jost C, et al. The P1linked receptor FcRIII is released on stimulation of neutrophils. Nature 1988: 333:667-9. 15. el Demiry MI, Hargreave TB, Busuttil A, James K, Chisholm GD. Identifying leukocytes and leukocyte subpopulations in semen using monoclonal antibody probes. Urology 1986: 28: 492-6. 16. Wolff H, Anderson DJ. Immunohistologic characterization and quantitation of leucocyte subpopulations in human semen. Fertil Steril 1988: 49:497-504.
125
Thaler et al. 17. Lalezari P. Granulocyte antigen systems. In: Engelfriet CP, van Loghem JJ, Kr von dem Borne AEG, eds, Immunohematology. Amsterdam: Elsevier Science Publishers, 1984 334. 18. Bukovsky A, Thaler CJ, McIntyre JA. Antigens of IgGFc receptor 111 (CD16) in human male reproductive tract accessory glands. Fertil Steril 1991: 55 (in press).
Address: Chrisliun J. Thuler Center for Reproduction and Transplantation Immunology Methodist Hospital of Indiana, Inc 1701 N. Senate Blvd Indianapolis, IN 46202 USA
Two different forms of CD16 Fc receptor have been defined on human leukocytes (1). Type 1 (FcRIII-1), present on neutrophils (PMN), expresses the biallelic NA alloantigen system (2-4) and is anchored to the membrane via a phosphatidylinositol glycan bond (5-8). Type 2 (FcRIII2), present on natural-killer (NK) cells and macrophages, is not polymorphic and has a transmembrane region with a cytopiasmic tail (G8). We have reported the presence of soluble FcRIII antigens in seminal plasma (SP) (9). These antigens retain their affinity for IgG-Fc and it has been proposed that they regulate maternal immunity at insemination and pregnancy (9, 10). It also was suggested that SP FcRIII antigens, if allotypic, could induce allogeneic reactions in the female. SP FcRIII antigens are biochemically distinct from leukocytic FcRIII (9). Their origin within the male reproductive tract and how they relate to FcRIII types 1 and 2 were not known. We have studied how SP FcRIII antigens relate to FcRIII types 1 and 2 by using 2 monoclonal antibodies (mAb) that
Christian J. Thalsr, John A. Mclntyrs and W. Page Faulk Center for Reproduction and Transplantation Immunology, Methodist Hospital of Indiana, Indianapolis, IN, USA
Key words: IgG - Fc receptors - CD16 antigens - NA alloantigens seminal plasma - human
-
Received 24 September, revised, accepted for publication 15 November 1990
differentially react with FcRIII- 1 and FcRIII-2 from different individuals. The mAb Leu l l c reacts with NK cells from all individuals but is negative with PMN from about 50% of donors (11). The mAb CLB/Gran 11 reacts with an epitope that is closely associated with the NAl alloantigen present on FcRIII-1 (2). This mAb reacts with PMN-bound as well as with soluble FcRIII-1 from homozygous NA1 and from heterozygous NAlNA2 individuals. However, CLB/ Gran 11 reacts neither with FcRIII-1 from homozygous NA2 individuals nor with NK cell FcRIII type 2 antigens (2, 12, 13). We have analyzed both Leu 1lc and CLB/Gran 11 reactivities with PMN and NK cells from healthy male volunteers by using flow cytometry. A panel of donors was selected to represent the different patterns of Leu l l c and CLB/Gran 11 reactivities with PMN and NK cells. CLB/Gran 11 and Leu l l c reactivities with SP were determined by using the enzyme-linked immunosorbent assay (ELISA) and results were compared to reactivities with PMN and NK cells. Our findings show that SP FcRIII antigens do not manifest polymorphic
re1
Thaler et al.
variations as detected by Leu l l c and CLB/Gran 11 on FcRIII type 1. Material and Methods Preparations of peripheral blood lymphocytes (PEL) and PMN
Blood from 7 healthy male volunteers was collected by venipuncture into acid citrate dextrose and centrifuged at 700 g for 10 min. Buffy coats were obtained, layered on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and centrifuged at 700 g for 18 min. PBL were removed from the Ficoll Hypaque interface and pellets containing red blood cells (RBC) and PMN were pooled. PMN were purified by hypotonic lysis of RBC as described by Thaler et al. (9). Cells were exposed to distilled H,O for 20 seconds and isotonicity was restored by adding 10% (v/v) of 0.1 M phosphate-buffered 1.5 M NaCl, pH 7.4. Cells were centrifuged at 700 g for 10 min and hypotonic lysis was repeated. After two washes with 0.01 M phosphate-buffered 0.15 M NaCl, pH 7.4, (PBS) this procedure yielded more than 95% pure PMN with greater than 95% viability as shown by exclusion of trypan blue.
vine serum albumin (BSA) (Sigma Chemical Company, St. Louis, MO) and 0.2 mM colchicine (Sigma) (RPMI-Colchicine). Aliquots (100 pl) containing 1 x lo6 cells were incubated for 30 min with 20 pl of CLB/Gran 11 (diluted 150 in PBS), Balb/c control ascites fluid (diIuted 150 in PBS), phycoerythrin (PE)-conjugated Leu 1lc (50 pg1/ ml IgGl) or isotope-matched control mAb. Incubations were stopped by washing cells twice with RPMI-colchicine. Two-color fluorescence analyses were done simultaneously by using fluorescein (FITC) and PE-conjugated mAb. For unconjugated primary mAb, cells were washed, incubated for 30 min with FITC-conjugated goat F(ab’), antimouse IgG and washed twice. The cells were examined in a Beckton Dickinson FACStar Plus flow cytometer with a 3 watt argon laser set at 200 mW and electronic gates were set for side scatter and forward scatter of PBL or PMN, respectively. 10000 events were collected and logarithmically acquired FLl and/ or FL2 intensities were displayed on linear scales.
Donor selection Antibodies
Details of the monoclonal and polyclonal antibodies used in this study are listed in Table 1. Control incubations for the ascites antibody CLB/ Gran 11 were done with identical dilutions of Balb/ c control ascites fluid (Cedar Lane Laboratories, Ltd., Hornby, Canada). Flow cytometry
Freshly prepared PBL and PMN were washed twice with RPMI 1640 containing 20 mg/ml bo-
7 healthy male volunteers were studied for Leu 1lc and CLB/Gran 11 reactivities with PMN and NK cells. The donors whose PMN were Leu 1lc-negative were chosen for an evaluation of their SP samples, because in such donors Leu 1lc reactivity is restricted to FcRIII type 2 (1 1). One donor, with Leu llc-positive PMN was included for comparison. In addition, 3 donors were chosen so that the reported (12) high, intermediate and negative CLB/ Gran 11 reactivities with PMN were represented. SP samples from these donors were studied for ELISA reactivities with CLB/Gran 11.
Table 1 List of antibodies used in this study Antibody
Specificity
Description
Specificity Determined
Supplier
CLBiGran 11
FcRllt from NAl-positive individuals (CD16)
lmmunoprecipitation Flow cytometry
Leu l l c (PE-conjugated)
gift from Dr. Kr. A. E. 6. von dem Bome, Amsterdam, Netherlands Beckon Dickinson, Mountain View, CA
Leu 19 (FITCconjugated) Anti-Transferrin receptor Anti-mouse IgG
FCRIII-1 from 50% of individuals Fc RllC2 from all individuals (CD16) NK cell differentation antigen (CD56) Transferrin receptor (Trf-R) mouse IgG
mouse monoclonal l9G21, mouse monoclonal lgG, mouse monoclonal lgGi monoclonal
Immunoprecipitation, Flow cytometry lmmunochemical lmmunohistology affinity purified
Beckon Dickinson, Mountain View, CA DAKOPATTS Copenhagen, Denmark Protos San Francisco, CA
Anti-mouse lg
mouse lg
crossed immuncelectrophoresis
DAKDPATTS Copenhagen, Denmark
122
goat F(ab’), antiserum FlTG conjugated rabbit antibody, peroxidaseconjugated
Irnmunoprecipitation, Flow cytometry
CD16 antigens in seminal plasma Net OD (450 nml 0.5 7
0.4 0.3 -
!Xl-\Flwrescmce GK Height
Figure 1. Leu l l c reactivity with peripheral blood lymphocytes from a representative individual. A subsest of lymphocytes was positive with PE-conjugated Leu l l c (vertical axis). By using two-color fluorescence, these cells were shown to also react with the FITC-conjugated NK cell marker Leu 19. NK cells from all 7 individuals studied were positive with Leu 1lc.
=
Leu l l c
0mAb anti Trf-R
Figure 3. ELISA studies with Leu 1lc and mAb anti-transfemn receptor (background control) Leu l l c showed positive reactivities (mean net OD values+SEM) with seminal plasma samples from all donors. Differences in SP FcRIII antigen expression that were previously reported (9) were independent of Leu 1l c reactivities with the donor's PMN.
Seminal plasma
SP from male volunteers was allowed to undergo spontaneous liquefaction for 30 min at room temperature (RT). Samples were centrifuged at 10000 g for 5 min and the cell- and spermatozoa-free supernatants were aliquoted and stored at -20°C until use. Serial donor samples were collected at least 3 days apart. PMN sonicates
PMNs were washed twice in PBS and 5 x lo6 cells were resuspended in 2 ml of PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma) and 1 mM of TPCK (n-tosyl-1-phenylalanine chlo-
romethyl ketone) (Sigma) as protease inhibitors. Cells were disrupted with a continuous sonication burst of 5 s with the use of a Sonicator W-375 (Heat Systems-Ultrasonics, Inc., Plainview, NY) at number 2.5. Sonicates were centrifuged for 5 min at 10000 g and the supernatants were retained. Pellets were resuspended in 2 ml PBS and the sonication and centrifugation steps were repeated. The supernatants were pooled, aliquoted and stored at - 20°C. ELISA
The ELISA was done as modified from Thaler et al. (9). SP samples (50 pl) diluted in PBS (1:20)were coated overnight at 4°C onto wells of Immulon I1
Leu 1lc - PMN 0
128
-m - -
' ''
' ' 1488' '
pBQ'
I
'Rp9 I
'
1001
PMN-CLB/Gran 11
4
a
-
NA1 NAl NA1 NA2
I
'
''rr-lrr
Figure 2. Leu 1l c reactivity studied with P M N from 4 individuals. PMN from donors 1-3 are negative and PMN from donor 4 are positive with Leu 1lc.
123
Thaler et al.
plates (Dynatech Lab Inc, Alexandria, VA). PMN sonicates were coated at protein concentrations of 0.1 mg/ml. Wells were washed twice with PBS containing 0.05% Tween 20 (Bio Rad Laboratories, Richmond, CA) (PBS-Tween) and incubated with mAb Leu 1lc, CLB/Gran 11 and background controls for 30 min at RT. All antibodies were diluted in PBS-Tween containing 0.5% BSA. mAb Leu 1lc and anti-transferrin receptor, an isotope-matched negative control, were used at IgG concentrations of 20 pg/ml. CLBIGran 11 and control ascites fluid were used at 1:200 dilutions. Plates were washed three times and incubated for 30 min with peroxidase-conjugated rabbit antimouse Ig (1:2000). Plates were washed five times and color reactions were developed by adding 100 pl of substrate buffer containing 0.42 mM tetramethylbenzidine (Miles CO, Napersville, IL) and 0.0045% H,O, in 0.1 M sodium acetate buffer, pH 6.0. The color reaction was stopped by adding 35 pi of 2 M H2S04.Optical density (OD) of each well was measured at 450 nm by using an automated Biomek 1000 workstation (Beckman Instruments Inc., Arlington Heights, IL). Mean OD values were determined from triplicate determinations. Net OD values were calculated by subtracting background ODs. Patterns of reactivity were confirmed with at least 2 separate SP samples from each individual and each sample was tested two or more times on different days. Results Reactivities with Leu 11c
PBL and PMN from 7 donors were screened for Leu l l c reactivities by using flow cytometry. Leu l l c reacted with a small percentage of PBL from N e t OD 1450nrnI
0.35i
0.25
all individuals. By using the FITC-conjugated NK cell marker Leu 19 and two-color analysis, Leu 1lc-positive PBL were shown to be NK cells (Fig. 1). In contrast to reactivity with NK cells from all individuals, Leu 1l c was negative with PMN from donors 1-3 (Fig. 2). Thus, in these individuals Leu l l c reacted selectively with FcRIII type 2. SP samples from donors 1-3 and from donor 4 whose PMN were Leu 1lc-positive were analyzed for Leu l l c reactivity by using the ELISA. SP samples from all donors regardless of the Leu 1lc reactivity with PMN were found to be strongly positive with Leu l l c (Fig. 3). Individual differences that previously had been reported for SP-FcRIII antigen expression (9) were independent of Leu l l c reactivity with PMN. The reactivity of Leu l l c with SP from all individuals indicates that SP FcRIII antigens share immunogenetic characteristics with FcRIII type 2. Reactivities with CLB/Gran 11
Flow cytometric analysis of PMN from different individuals with CLB/Gran 11 showed strongly positive, intermediate and negative reactivities. These differences are thought to result from a gene dose effect of NAl antigen expression (12). Strong, intermediate and negative reactivities with CLB/ Gran 11 are associated with genotypes NAlNAl , NAlNA2 and NA2NA2, respectively (12). CLB/ Gran 11 was negative with PBL from all individuals (data not shown). 3 donors were selected so that one of each represented NA1 homozygous, NA2 homozygous and NAlNA2 heterozygous individuals (Fig. 4). ELISA studies with CLB/Gran 11 were done with SP samples and sonicated PMN membranes. CLB/Gran 11 reacted with PMN sonicates from NAl-positive individuals. In contrast, SP samples from all individuals showed background reactivities with CLB/Gran 11. All SP samples were positive with Leu 1lc (Fig. 5). These results indicate that FcRIII antigens in SP lack polymorphic variations of FcRIII type 1 as detected by mAb CLB/Gran 11. Discussion
"
SP (NA1 NA1)
-
SP (NA1 NA2) CLE gran 11
SP (NA2 NA2) 0Leu 11c
PMN (NAI NA2)
Figure 5. ELISA studies with CLB/Gran 11 showed positive reactivities (mean net OD valuesfSEM) with PMN sonicates from NAI positive individuals. SP samples from all donors were negative with CLB/Gran 11. These results were independent of the donors NA allotype. SP from all individuals were positive, with Leu llc.
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We have reported the presence of soluble FcRIII antigens in human SP (9). The origin of these FcR antigens in SP is under study. PMN that are stimulated (14) or treated with phospholipase C (5, 6) can liberate membrane-bound FcRIII molecules. Cultured NK cells also have been shown to release FcRIII (1, 5, 6). Thus, soluble SP FcRIII antigens could originate from leukocytes that are present within the male reproductive tract (15, 16). By using mAb Leu 1l c and CLB/Gran 11 that differ-
CD16 antigens in seminal plasma entially react with FcRIII type 1 and type 2 from different individuals (2, 11, 12, 13), we investigated how the pattern of PMN and NK cell reactivities relate to findings with SP FcRIII antigens. Our studies indicate that phenotypic differences identified on PMN from different individuals are not expressed on FcRIII antigens in SP. CLB/Gran 11 and anti-NA1 alloantibodies have been shown to mutually crossblock, so the epitope recognized by CLB/Gran 11 appears to be closely related to or identical with the NAl alloantigen (2). The lack of CLB/Gran 11 reactivity with SP from NAl-positive individuals suggests that NA1 alloantigens are absent from SP FcRIII. This could be biologically significant in preventing intravaginal sensitization to paternal NA alloantigens, as maternal IgG alloantibodies transported across placentae have been associated with neonatal neutropenia (17). Positive reactions of Leu l l c with SP samples from donors whose PMN were Leu l l c negative, and negative reactions of CLB/Gran 11 with SP from NAl-positive individuals indicate that PMN are not a main source of SP FcRIII antigens. The uniform pattern of Leu l l c and CLB/Gran 11 reactivity with SP samples from different individuals corresponds with donors’ NK cells. FcRIII type 2 that is present on NK cells and macrophages has no known variations between different individuals (1). In spite of these shared characteristics with FcRIII-2, SP FcRIII antigens might not originate from NK cells or macrophages. SP FcRIII antigens could represent molecules that are distinct from leukocytic FcRIII. This possibility has been suggested as FcRIII antigens in SP and on leukocytes differ in electrophoretic mobilities (9). Recent immunohistological studies in our laboratory indicate that FcRIII antigens are expressed and released into SP by secretory epithelia of male reproductive tract accessory glands (18). Differences of FcRIII antigens from leukocytes and SP could result from tissue-specific modifications of FcRIII-2-gene expression. Alternatively, SP FcRIII antigens could be encoded by a different gene and share specific epitopes with FcRIII-2. The presence of these antigens in SP and their deposition into the vagina during insemination could indicate a novel mechanism of regulating maternal immunity for normal conception and pregnancy.
Acknowledgments
This work was supported in part by the Methodist Health Foundation. We thank Harold D. Boldt for flow cytometric analyses and Sandra L. Hollowell for secretarial assistance. We thank Dr. A. E. G.
Kr. von dem Borne for monoclonal antibody CLB/ Gran 11. References 1. Ravetech JV, Perussia B. Alternative membrane forms of FcyRIII (CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in a single nucleotide substitution. J Exp Med 1989: 170: 481-97. 2. Werner G, Kr von dem Borne AEG, Bos MJE, et al. Localization of the human NAI alloantigen on neutrophil Fc receptors. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte typing I1 Vol 3 Human Myeloid and Hematopoietic Cells. New York: Springer Verlag, 1986 109-21. 3. Ory PA, Goldstein IM, Kwoh EE, Clarkson SB. Characterization of polymorphic forms of Fc receptor I11 on human neutrophils. J CIin Invest 1989: 83: 1671-81. 4. Ory PA, Clark MR, Kwoh EE, Clarkson SB, Goldstein IM. Sequence of complementary DNAs that encode the NAl and NA2 forms of Fc receptor I11 on human neutrophils. J CIin Invest 1989: 84: 1688-91. 5. Selvaraj P, Rosse WF, Silber R, Springer TA. The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal nocturnal hemoglobinuria. Nature 1988: 333:565-7. 6. Ueda E, Kinoshita T, Nojima I, Inoue K, Kitani T. Different membrane anchors of FcyRIII (CD16) on K/NK lymphocytes and neutrophils. Protein-vs Lipid-Anchor. J Immunol 1989: 143: 1274-7. 7. Edberg JC, Redecha PB, Salmon JE, Kimberly RP. Human FcyRIII (CD 16). Isoforms with distinct allelic expression, extracellular domains, and membrane linkages on polymorphonuclear and natural killer cells. J Immunol 1989: 143: 1642-9. 8. Lanier L, Phillips JH, Testi R. Membrane anchoring and spontaneous release of CD 16 (FcRIII) by natural killer cells and granulocytes. Eur J Immunol 1989: 19 7758. 9. Thaler CJ, Faulk WP, McIntyre JA. Soluble antigens of IgG receptor FcyRIII in human seminal plasma. J Immunol 1989: 143 1937-42. 10. Thaler CJ. Immunologic role for seminal plasma in insemimation and pregnancy. Am J Reprod Immunol 1989: 21: 147-50. 11. Perussia B, Starr S, Abraham S, Fanning V, Trinchieri G. Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. 1. Characterization of the lymphocyte subset reactive with B73.1 JImmunoZ 1983: 130: 213341. 12. Salmon JE, Edberg JC, Kimberly RP. Fcy receptor I11 on human neutrophils allelic variants have functionally distinct capacities. J Clin Invest 1989: 8 5 1287-95. 13. Huizinga TWJ,de Haas M, Kleijer M, Nuijens JH, Roos D, Kr von dem Borne AEG. Soluble Fcy receptor I11 in human plasma originates from release by neutrophils. J Clin Invest 1990: 86: 416-23. 14. Huizinga TWJ, van der Schoott CE, Jost C, et al. The P1linked receptor FcRIII is released on stimulation of neutrophils. Nature 1988: 333:667-9. 15. el Demiry MI, Hargreave TB, Busuttil A, James K, Chisholm GD. Identifying leukocytes and leukocyte subpopulations in semen using monoclonal antibody probes. Urology 1986: 28: 492-6. 16. Wolff H, Anderson DJ. Immunohistologic characterization and quantitation of leucocyte subpopulations in human semen. Fertil Steril 1988: 49:497-504.
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Thaler et al. 17. Lalezari P. Granulocyte antigen systems. In: Engelfriet CP, van Loghem JJ, Kr von dem Borne AEG, eds, Immunohematology. Amsterdam: Elsevier Science Publishers, 1984 334. 18. Bukovsky A, Thaler CJ, McIntyre JA. Antigens of IgGFc receptor 111 (CD16) in human male reproductive tract accessory glands. Fertil Steril 1991: 55 (in press).
Address: Chrisliun J. Thuler Center for Reproduction and Transplantation Immunology Methodist Hospital of Indiana, Inc 1701 N. Senate Blvd Indianapolis, IN 46202 USA
