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Detection of H-Y antigen on human male leukocytes
Journal oflmmunological Methods, 23 (1978) 203--206
203
© Elsevier/North-Holland Biomedical Press
DETECTION OF H-Y ANTIGEN ON HUMAN MALE LEUKOCYTES
ELLEN H. GOLDBERG, TERECITA ARRINGTON and SEI TOKUDA Department of Microbiology, University of New Mexico, School of Medicine, Albuquerque, NM 87131, U.S.A.
(Received 28 December 1977, accepted 24 April 1978)
Using the Staphylococcus aureus binding technique, we have demonstrated H-Y ('male') antigen on human male leukocytes.
INTRODUCTION Using the sperm c y t o t o x i c i t y test and the 'mixed hemadsorption hybrid a n t i b o d y ' (MHA.HA) assay (Wachtel, 1977), we have detected H-Y {male) antigen in several mammalian species. Because both these methods present difficult technical problems, we recently developed a simplified serological assay for the detection of H-Y on mouse lymphocytes (Tokuda et al., 1977). This assay is based on the observation that the protein A c o m p o n e n t of S t a p h y l o c o c c u s aureus (SA) binds to the Fc portion o f IgG molecules, which was adapted by Kearney et al. (1975) to measure SA rosettes. The only limitation of this procedure is the requirement for antibodies of the IgG class, which means that its efficiency depends on the concentration of IgG a n t i b o d i e s i n the anti-H-Y serum. But antibody of this class is in fact present in mouse H-Y antiserum, and we report here that we can use this technique to detect H-Y antigen on human leukocytes, confirming the work of Wachtel et al. (1974). MATERIALS AND METHODS Anti-H-Y serum was produced in C57BL/6 (B6) female mice (purchased from the Jackson Laboratory) by inoculation with 25 X 106 B6 male spleen cells every two weeks. Serum was obtained on days 7, 10, and 14 after the 7th inoculation. Anti-H-Y activity was determined by the c y t o t o x i c i t y test with mouse spermatozoa as target cells according to Goldberg et al. (1971), and by the SA binding procedure described for the detection of H-Y on mouse lymphocytes (Tokuda et al., 1977). The main difficulty in using antiserum raised in mice against h u m a n cells is the presence of heteroantibody (i.e., naturally occurring mouse anti-human antibody). For this reason, the H-Y antisera were tested individually against h u m a n female leukocytes
204 by the SA binding technique, and those sera which gave a background level of less than 30% positive cells were used in the tests report ed below. In all cases, leukocytes frbm female donors of blood G roup 0 gave a lower percent positive background level than did cells from female donors o f bl ood Groups A and B. However, female donors of blood Groups A, B and AB were used in the experiments r epor t e d here. The Cowan I strain of SA containing protein A was cultured in ToddHewitt b r o th (Difco) at 37°C and collected after an 18-h incubation on a shaker. Th e cells were washed 3 times in phosphate-buffered saline (PBS; 0.02 M PO4, 0.15 M NaC1, pH 7.2) containing 0.2% NaN3 and then incubated for 3 h at r o o m t e m p e r a t u r e (RT) in PBS containing 0.5% formaldehyde. T h e cells were then washed 4 times in PBS-NaN3 and placed in a boiling water bath (94°C) f or 2.5 rain at a 10% (v/v) cell concentration. This stock suspension was stored at 4°C in the presence of 0.1% NAN3. Before their use, cells were washed and resuspended in PBS. Five millilitres of venous blood were drawn into syringes and placed immediately into tubes containing 1 ml of dextran solution (20 ml NaC1; 1.0 g dextran o f average molecular weight 264,000 from Sigma Biochemical; 0.1 ml Heparin). After 1 h at 37°C, the leukocyte-rich supernatant was removed and the cells washed twice in 10 ml of Medium 199 (M 199, GIBCO), centrifugation at 200 Xg for 10 min. To remove the remaining e r y t h r o c y t e s , th e cells were resuspended in 5 ml o f M 199 and furt her purified on a Ficoll-Hypaque gradient (density = 1.077 ± 0.001 g/cm3). T he gradients were centrifuged at 4°C at 40 × g, 150 × g and 300 × g for 5 min at each speed. The remaining leukocytes were removed from the interface and washed 3 times at 4°C in 10 ml of M 199 at 100 X g. Th e cells were c o u n t e d and the c o n c e n t r a t i o n was adjusted to 107 cells/ml. 0.05 ml anti-H-Y (diluted to 1/2) serum was added to 0.25 ml o f male l e u k o cy tes and incubated for 30 min at 4°C. To test for specificity of the reaction for H-Y, leukocytes from females were also treated with anti-H-Y serum. Af ter washing twice with 5 ml (20 volumes) of M 199 containing IgG free fetal calf serum (FCS; previously heated to 56°C for 30 min), the cell pellet was mixed with 0.05 ml o f SA suspension and incubated for 15 min at 4°C. To reduce the background n u m b e r of unattached SA, the m i xt ure was washed twice in 10 ml o f M 199, containing 2% FCS, centrifugation at 200 Xg for 10 min at 4°C. A f t er the final wash, the pellet was resusp e n d ed in 0.25 ml o f M 199. Smears were prepared on microscope slides, air dried, fixed in methanol and stained by the Giemsa m e t h o d for 5 min. T h e slides were observed by light microscopy at 400 × and 1000 × magnification. A cell was scored as positive if 5 or m o re SA were attached. RESULTS The results presented in Table 1 clearly show that there is a high degree o f binding of mouse H-Y a n t i b o d y to h u m a n male leukocytes. In order to
205 TABLE 1 REACTION OF MOUSE H-Y ANTISERUM WITH MALE AND FEMALE HUMAN LEUKOCYTES USING THE S T A P H Y L O C O C C U S A U R E U S (SA) BINDING TECHNIQUE Tested leukocytes from: Male Female
Unabsorbed
Absorbed with male leukocytes
Absorbed with female leukocytes
Percent positive cells (binding > 5 SA) 71.3 a 6.1 61.5 26.6 22.4 21.6
a Summary of 7 experiments. Variation from one male to another was 55--86%; from one female to another was 12--38%. However, the variation for any one individual in experiments performed on different days was not more than 10%.
test f o r its specific r e a c t i v i t y against t h e H-Y antigen, a l i q u o t s o f m o u s e antiH-Y s e r u m ( d i l u t e d 1/2 in M 199) w e r e a b s o r b e d w i t h equal n u m b e r s o f e i t h e r m a l e o r f e m a l e h u m a n l e u k o c y t e s (4 v o l u m e s s e r u m t o 1 v o l u m e p a c k e d cells f o r 30 m i n in an ice b a t h ) , and t h e a b s o r b e d sera w e r e t h e n t e s t e d o n m a l e a n d f e m a l e h u m a n l e u k o c y t e s . Results s h o w n in T a b l e 1 d e m o n s t r a t e essentially c o m p l e t e loss o f a c t i v i t y a f t e r a b s o r p t i o n with m a l e cells with o n l y a slight loss o f activity a f t e r a b s o r p t i o n w i t h f e m a l e cells, no m o r e t h a n can b e e x p e c t e d f r o m n o n - s p e c i f i c causes. In a n o t h e r series o f e x p e r i m e n t s , a l i q u o t s o f H-Y antisera w e r e a b s o r b e d t w i c e w i t h m a l e a n d f e m a l e h u m a n l e u k o c y t e s a n d t h e n t e s t e d on B6 m a l e l y m p h n o d e cells. In no case were we able to e l i m i n a t e c o m p l e t e l y t h e H-Y r e a c t i o n against l y m p h o c y t e s f r o m m a l e mice. DISCUSSION T h e s e results suggest t h a t H-Y antigens o f h u m a n a n d m o u s e are crossr e a c t i n g b u t n o t identical. We had p r e v i o u s l y r e p o r t e d a d i f f e r e n t i a l susc e p t i b i l i t y o f m o u s e s p e r m f r o m d i f f e r e n t strains t o t h e c y t o l y t i c a c t i o n o f H-Y a n t i s e r u m suggesting e i t h e r (1) strain d i f f e r e n c e s in t h e a m o u n t o f H-Y antigen on s p e r m , o r (2) H - Y p o l y m o r p h i s m , as r e p o r t e d b y H i l d e m a n n a n d C o o p e r ( 1 9 6 7 ) . A l t e r n a t i v e l y , m o u s e H-Y is a c o m p l e x o f antigens, s o m e o f w h i c h are a b s e n t in h u m a n s . V a r i a t i o n o f t h e p e r c e n t positive cells f r o m o n e m a l e to a n o t h e r ( 5 5 - 86%) p r o b a b l y reflects d i f f e r e n t genetic b a c k g r o u n d s o f t h e s e individuals. T h e r e is e v i d e n c e in t h e m o u s e t h a t t h e e x p r e s s i o n o f H-Y is r e l a t e d t o t h e H-2 t y p e o f t h e animal (Wachtel, 1 9 7 7 ) . P e r h a p s in m a n also t h e e x p r e s s i o n o f H-Y antigen is r e l a t e d t o t h e m a j o r h i s t o c o m p a t i b i l i t y p h e n o t y p e o f t h e cell, i.e., H L A ( O h n o , 1977).
206 ACKNOWLEDGEMENT This w o r k was s u p p o r t e d b y N a t i o n a l I n s t i t u t e s o f Health Research G r a n t s Nos. A I 1 1 5 6 0 , CA 1 0 1 2 9 and R R 0 8 1 3 9 . REFERENCES Wachtel, S.S., 1977, Immunol. Rev. 33, 33. Tokuda, S., T. Arrington, E.H. Goldberg and J. Richey, 1977, Nature 267,433. Kearney, R., E. Chia and A. Basten, 1975, J. Immunol. 114, 1143. Wachtel, S.S., G.C. Koo, E.E. Zuckerman, U. Hammerling, M.P. Scheid and E.A. Boyse, 1974, Proc. Natl. Acad. Sci. USA 71, 1215. Goldberg, E.H., E.A. Boyse, D. Bennett, M. Scheid and E.A. Carswell, 1971, Nature 232, 478. Hildemann, W.H. and E.L. Cooper, 1967, Transplantation 5,707. Ohno, S., 1977, Immunol. Rev. 33, 59.
203
© Elsevier/North-Holland Biomedical Press
DETECTION OF H-Y ANTIGEN ON HUMAN MALE LEUKOCYTES
ELLEN H. GOLDBERG, TERECITA ARRINGTON and SEI TOKUDA Department of Microbiology, University of New Mexico, School of Medicine, Albuquerque, NM 87131, U.S.A.
(Received 28 December 1977, accepted 24 April 1978)
Using the Staphylococcus aureus binding technique, we have demonstrated H-Y ('male') antigen on human male leukocytes.
INTRODUCTION Using the sperm c y t o t o x i c i t y test and the 'mixed hemadsorption hybrid a n t i b o d y ' (MHA.HA) assay (Wachtel, 1977), we have detected H-Y {male) antigen in several mammalian species. Because both these methods present difficult technical problems, we recently developed a simplified serological assay for the detection of H-Y on mouse lymphocytes (Tokuda et al., 1977). This assay is based on the observation that the protein A c o m p o n e n t of S t a p h y l o c o c c u s aureus (SA) binds to the Fc portion o f IgG molecules, which was adapted by Kearney et al. (1975) to measure SA rosettes. The only limitation of this procedure is the requirement for antibodies of the IgG class, which means that its efficiency depends on the concentration of IgG a n t i b o d i e s i n the anti-H-Y serum. But antibody of this class is in fact present in mouse H-Y antiserum, and we report here that we can use this technique to detect H-Y antigen on human leukocytes, confirming the work of Wachtel et al. (1974). MATERIALS AND METHODS Anti-H-Y serum was produced in C57BL/6 (B6) female mice (purchased from the Jackson Laboratory) by inoculation with 25 X 106 B6 male spleen cells every two weeks. Serum was obtained on days 7, 10, and 14 after the 7th inoculation. Anti-H-Y activity was determined by the c y t o t o x i c i t y test with mouse spermatozoa as target cells according to Goldberg et al. (1971), and by the SA binding procedure described for the detection of H-Y on mouse lymphocytes (Tokuda et al., 1977). The main difficulty in using antiserum raised in mice against h u m a n cells is the presence of heteroantibody (i.e., naturally occurring mouse anti-human antibody). For this reason, the H-Y antisera were tested individually against h u m a n female leukocytes
204 by the SA binding technique, and those sera which gave a background level of less than 30% positive cells were used in the tests report ed below. In all cases, leukocytes frbm female donors of blood G roup 0 gave a lower percent positive background level than did cells from female donors o f bl ood Groups A and B. However, female donors of blood Groups A, B and AB were used in the experiments r epor t e d here. The Cowan I strain of SA containing protein A was cultured in ToddHewitt b r o th (Difco) at 37°C and collected after an 18-h incubation on a shaker. Th e cells were washed 3 times in phosphate-buffered saline (PBS; 0.02 M PO4, 0.15 M NaC1, pH 7.2) containing 0.2% NaN3 and then incubated for 3 h at r o o m t e m p e r a t u r e (RT) in PBS containing 0.5% formaldehyde. T h e cells were then washed 4 times in PBS-NaN3 and placed in a boiling water bath (94°C) f or 2.5 rain at a 10% (v/v) cell concentration. This stock suspension was stored at 4°C in the presence of 0.1% NAN3. Before their use, cells were washed and resuspended in PBS. Five millilitres of venous blood were drawn into syringes and placed immediately into tubes containing 1 ml of dextran solution (20 ml NaC1; 1.0 g dextran o f average molecular weight 264,000 from Sigma Biochemical; 0.1 ml Heparin). After 1 h at 37°C, the leukocyte-rich supernatant was removed and the cells washed twice in 10 ml of Medium 199 (M 199, GIBCO), centrifugation at 200 Xg for 10 min. To remove the remaining e r y t h r o c y t e s , th e cells were resuspended in 5 ml o f M 199 and furt her purified on a Ficoll-Hypaque gradient (density = 1.077 ± 0.001 g/cm3). T he gradients were centrifuged at 4°C at 40 × g, 150 × g and 300 × g for 5 min at each speed. The remaining leukocytes were removed from the interface and washed 3 times at 4°C in 10 ml of M 199 at 100 X g. Th e cells were c o u n t e d and the c o n c e n t r a t i o n was adjusted to 107 cells/ml. 0.05 ml anti-H-Y (diluted to 1/2) serum was added to 0.25 ml o f male l e u k o cy tes and incubated for 30 min at 4°C. To test for specificity of the reaction for H-Y, leukocytes from females were also treated with anti-H-Y serum. Af ter washing twice with 5 ml (20 volumes) of M 199 containing IgG free fetal calf serum (FCS; previously heated to 56°C for 30 min), the cell pellet was mixed with 0.05 ml o f SA suspension and incubated for 15 min at 4°C. To reduce the background n u m b e r of unattached SA, the m i xt ure was washed twice in 10 ml o f M 199, containing 2% FCS, centrifugation at 200 Xg for 10 min at 4°C. A f t er the final wash, the pellet was resusp e n d ed in 0.25 ml o f M 199. Smears were prepared on microscope slides, air dried, fixed in methanol and stained by the Giemsa m e t h o d for 5 min. T h e slides were observed by light microscopy at 400 × and 1000 × magnification. A cell was scored as positive if 5 or m o re SA were attached. RESULTS The results presented in Table 1 clearly show that there is a high degree o f binding of mouse H-Y a n t i b o d y to h u m a n male leukocytes. In order to
205 TABLE 1 REACTION OF MOUSE H-Y ANTISERUM WITH MALE AND FEMALE HUMAN LEUKOCYTES USING THE S T A P H Y L O C O C C U S A U R E U S (SA) BINDING TECHNIQUE Tested leukocytes from: Male Female
Unabsorbed
Absorbed with male leukocytes
Absorbed with female leukocytes
Percent positive cells (binding > 5 SA) 71.3 a 6.1 61.5 26.6 22.4 21.6
a Summary of 7 experiments. Variation from one male to another was 55--86%; from one female to another was 12--38%. However, the variation for any one individual in experiments performed on different days was not more than 10%.
test f o r its specific r e a c t i v i t y against t h e H-Y antigen, a l i q u o t s o f m o u s e antiH-Y s e r u m ( d i l u t e d 1/2 in M 199) w e r e a b s o r b e d w i t h equal n u m b e r s o f e i t h e r m a l e o r f e m a l e h u m a n l e u k o c y t e s (4 v o l u m e s s e r u m t o 1 v o l u m e p a c k e d cells f o r 30 m i n in an ice b a t h ) , and t h e a b s o r b e d sera w e r e t h e n t e s t e d o n m a l e a n d f e m a l e h u m a n l e u k o c y t e s . Results s h o w n in T a b l e 1 d e m o n s t r a t e essentially c o m p l e t e loss o f a c t i v i t y a f t e r a b s o r p t i o n with m a l e cells with o n l y a slight loss o f activity a f t e r a b s o r p t i o n w i t h f e m a l e cells, no m o r e t h a n can b e e x p e c t e d f r o m n o n - s p e c i f i c causes. In a n o t h e r series o f e x p e r i m e n t s , a l i q u o t s o f H-Y antisera w e r e a b s o r b e d t w i c e w i t h m a l e a n d f e m a l e h u m a n l e u k o c y t e s a n d t h e n t e s t e d on B6 m a l e l y m p h n o d e cells. In no case were we able to e l i m i n a t e c o m p l e t e l y t h e H-Y r e a c t i o n against l y m p h o c y t e s f r o m m a l e mice. DISCUSSION T h e s e results suggest t h a t H-Y antigens o f h u m a n a n d m o u s e are crossr e a c t i n g b u t n o t identical. We had p r e v i o u s l y r e p o r t e d a d i f f e r e n t i a l susc e p t i b i l i t y o f m o u s e s p e r m f r o m d i f f e r e n t strains t o t h e c y t o l y t i c a c t i o n o f H-Y a n t i s e r u m suggesting e i t h e r (1) strain d i f f e r e n c e s in t h e a m o u n t o f H-Y antigen on s p e r m , o r (2) H - Y p o l y m o r p h i s m , as r e p o r t e d b y H i l d e m a n n a n d C o o p e r ( 1 9 6 7 ) . A l t e r n a t i v e l y , m o u s e H-Y is a c o m p l e x o f antigens, s o m e o f w h i c h are a b s e n t in h u m a n s . V a r i a t i o n o f t h e p e r c e n t positive cells f r o m o n e m a l e to a n o t h e r ( 5 5 - 86%) p r o b a b l y reflects d i f f e r e n t genetic b a c k g r o u n d s o f t h e s e individuals. T h e r e is e v i d e n c e in t h e m o u s e t h a t t h e e x p r e s s i o n o f H-Y is r e l a t e d t o t h e H-2 t y p e o f t h e animal (Wachtel, 1 9 7 7 ) . P e r h a p s in m a n also t h e e x p r e s s i o n o f H-Y antigen is r e l a t e d t o t h e m a j o r h i s t o c o m p a t i b i l i t y p h e n o t y p e o f t h e cell, i.e., H L A ( O h n o , 1977).
206 ACKNOWLEDGEMENT This w o r k was s u p p o r t e d b y N a t i o n a l I n s t i t u t e s o f Health Research G r a n t s Nos. A I 1 1 5 6 0 , CA 1 0 1 2 9 and R R 0 8 1 3 9 . REFERENCES Wachtel, S.S., 1977, Immunol. Rev. 33, 33. Tokuda, S., T. Arrington, E.H. Goldberg and J. Richey, 1977, Nature 267,433. Kearney, R., E. Chia and A. Basten, 1975, J. Immunol. 114, 1143. Wachtel, S.S., G.C. Koo, E.E. Zuckerman, U. Hammerling, M.P. Scheid and E.A. Boyse, 1974, Proc. Natl. Acad. Sci. USA 71, 1215. Goldberg, E.H., E.A. Boyse, D. Bennett, M. Scheid and E.A. Carswell, 1971, Nature 232, 478. Hildemann, W.H. and E.L. Cooper, 1967, Transplantation 5,707. Ohno, S., 1977, Immunol. Rev. 33, 59.