Brief Definitive Report
E V I D E N C E FOR D I F F E R E N C E S
IN E R Y T H R O C Y T E
SURFACE RECEPTORS FOR THE MALARIAL PARASITES, PLASMODIUM FALCIPARUM AND
PLASMODIUM KNOWLESI BY LOUIS H. MILLER, J. DAVID HAYNES, FLORENCE M. McAULIFFE, TSUGIYE SHIROISHI, J O H N R. DUROCHER, ANY MARY H. McGINNISS
(From the Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, and the Blood Bank, Clinical Center, National Institutes of Health, Bethesda, Maryland 20014; and the Department of Immunology, Walter Reed Army Institute of Research, Washington, D. C. 20012; and the Pennsylvania Hospital, Philadelphia, Pennsylvania 19107)
The asexual erythrocytic cycle responsible for the clinical manifestations of malaria is perpetuated by merozoites which rupture out of infected erythrocytes and invade uninfected ones. Interaction between the merozoites and receptors on the erythrocyte surface initiate invasion (1-3). Chemical or immunologic interference between the merozoite and its receptor could block the infection at this point in the life cycle. Previously, we studied the characteristics of the erythroycte surface that are required for interaction and subsequent invasion by Plasmodium knowlesi merozoites (3, 4). Erythrocytes which lacked Duffy blood group determinants (Duffy negative erythrocytes) (3) or had been treated with chymotrypsin (4) were resistant to invasion. With the recent advances in in vitro cultivation ofPlasmodium falciparum (5, 6), it was possible to compare the erythrocyte surface determinants required for invasion by P. falciparum, the major malaria of man, and P. knowlesi, a monkey malaria that infects man.
Materials and Methods H u m a n erythrocytes (test cells) which e i t h e r lacked a blood group d e t e r m i n a n t (Table I) or had been treated with various enzymes (Table II) were exposed to P. falciparum and P. knowlesi merozoites in vitro, and the percentage of test cells infected was determined. Chimpanzee erythrocytes infected with P. falciparum (Camp strain) and frozen in liquid nitrogen were thawed (7) on the day of the experiment. They contained approximately 50% infected erythroeytes and varied in viability from experiment to experiment. The infected cells were centrifuged in microhematocrit tubes. Cells from the upper h a l f of the tubes, containing about 90% infected erythrocytes were mixed with test erythrocytes in a ratio of 1 to 25 and cultured as previously described (6). Erythrocytes were removed from culture 48 h later, spread in a t h i n film on a slide, stained with Giemsa, and the percent of erythrocytes infected with young trophozoites (ring forms) determined. Rhesus erythrocytes t h a t are refractory to invasion by P. falciparum were included in each experiment (Table I). In parallel experiments, samples of test erythrocytes were cultured with P. knowlesi-infected cells (3). Since P. falciparum merozoites were released after 48 h in culture and P. knowlesi within 1 h, test erythrocytes were incubated at 35°C for 48 h before addition of P. knowlesi-infected cells. Source of Human Erythrocytes. Erythrocytes were collected in acid citrate dextrose (ACD) and m a i n t a i n e d at 4 C. Erythrocytes from the F i n n i s h E n ( a - ) (G.W.) and two controls arrived in ice THE JOURNAL OF EXPERIMENTAL MEDICINE • VOLUME
146,
1977
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MILLER ET AL.
BRIEF D E F I N I T I V E REPORT
from Finland and were cultured on 2 separate days with each parasite. Some erythrocytes (Kell null, J k [ a - b - ] , Lu[a-b-], Ge[a-], S-s-U-, En[a-] [A.P., England], and their controls) were obtained as frozen droplets in liquid nitrogen. Control erythrocytes were from healthy individuals with a common blood group phenotype. Erythrocytes were washed three times in culture media before use. Enzyme Treatmentof Human Erythrocytes. Erythrocytes were treated with chymotrypsin and trypsin, and the enzymes were inhibited after treatment as described previously (3, 4, 8). For neuraminidase treatment, a 20% suspension of erythrocytes in Tris-buffered saline with 3.6 mM KCI, 1.6 mM CaCl2, and 1.2 mM MgSO4, pH 7.4, was incubated with 25 U/ml of neuraminidase (Vibrio cholerae, B grade, Calbiochem, San Diego, Calif.) at 37°C for 30 min. Control erythrocytes from the same individual were treated identically except that no enzyme was used. The enzyme effect on the erythrocytes was monitored in each experiment: (a) chymotrypsin, removal of Duffy blood group activity; (b) neuraminidase, 76 -+ 1.4 (SEM) percent reduction in electrophoretic mobility; (c) trypsin, 34 +_ 1.4 (SEM) percent reduction in electrophoretic mobility and removal of MN blood ~rouD activity. Results H u m a n e r y t h r o c y t e s l a c k i n g specific blood g r o u p d e t e r m i n a n t s w e r e all susceptible to invasion by P. falciparum, including e r y t h r o c y t e s w i t h blood g r o u p p h e n o t y p e s ( F y [ a - b - ] a n d S-s-U-) c h a r a c t e r i s t i c of b l a c k African p o p u l a t i o n s (Table I). T h e two e r y t h r o c y t e s a m p l e s of the E n ( a - ) p h e n o t y p e showed a reduction in invasion by P. falciparum. Since a twofold decrease in i n v a s i o n is w i t h i n the r a n g e of v a r i a t i o n b e t w e e n different individuals, it is not k n o w n if the reduced invasion was caused b y the E n ( a - ) p h e n o t y p e or some o t h e r u n r e l a t e d e r y t h r o c y t e factor. In c o n t r a s t to t h e s m a l l reduction in susceptibility of E n ( a - ) cells to P. falciparum, Duffy n e g a t i v e cells ( F y [ a - b - ] ) a r e r e f r a c t o r y to infection b y P. knowlesi a n d r h e s u s cells are r e f r a c t o r y to infection by P. falciparum (Table I). T h e p a t t e r n of r e s i s t a n c e of e n z y m e - t r e a t e d h u m a n e r y t h r o c y t e s to infection differed for P. falciparum a n d P. knowlesi (Table II). C h y m o t r y p s i n (0.1 m g / m l ) t r e a t m e n t blocked infection b y P. knowlesi b u t h a d no influence on infection by P. falciparum. Conversely, t r y p s i n or t r y p s i n plus n e u r a m i n i d a s e caused a m a r k e d reduction in infection b y P. falciparum b u t little or no c h a n g e in susceptibility to P. knowlesi. N e u r a m i n i d a s e also caused a significant reduction in infection by P. falciparum, a l t h o u g h t h e effect w a s less t h a n t h a t of t r y p s i n or t r y p s i n plus n e u r a m i n i d a s e . N e u r a m i n i d a s e t r e a t m e n t did not block infection by P. knowlesi. Discussion In a previous s t u d y we o b s e r v e d directly the i n t e r a c t i o n b e t w e e n e r y t h r o c y t e s a n d P. knowlesi merozoites (9). I t consisted of a t t a c h m e n t of the merozoite to t h e e r y t h r o c y t e m e m b r a n e followed b y d e f o r m a t i o n a n d invasion. N o n e of t h e s e e v e n t s occurred w h e n susceptible h u m a n e r y t h r o c y t e s w e r e t r e a t e d w i t h chymot r y p s i n (Mason, S. J., L. H. Miller, a n d J. A. D v o r a k . U n p u b l i s h e d data.), indicating t h a t t h e receptor n e c e s s a r y for a t t a c h m e n t of P. knowlesi w a s removed. I n t h e p r e s e n t s t u d y c h y m o t r y p s i n t r e a t m e n t (0.1 m g / m l ) t h a t blocks invasion b y P . knowlesi h a d no effect on i n v a s i o n b y P . falciparum. On t h e o t h e r h a n d , t r e a t m e n t of h u m a n e r y t h r o c y t e s w i t h t r y p s i n or n e u r a m i n i d a s e reduces invasion b y P. falciparum b u t not b y P. knowlesi. I f P. falciparum i n v a d e s e r y t h r o c y t e s b y a s i m i l a r m e c h a n i s m to P. knowlesi, t h e n infection of e n z y m e -
MILLER ET AL.
BRIEFDEFINITIVE REPORT
279
TABLE I The Susceptibility of Human Erythrocytes Lacking Various Blood Group Antigens to Invasion by P. falciparum and P. knowlesi Percent infected erythrocytes
Blood group of (number studied) Test cells
ABO null (1)* Rh null (1)* Kell null (1)* J k ( a - b - ) (1)* Lu ( a - b - ) (1)* Tn (1) Ge ( a - ) (1) S-s-U- (3) En ( a - ) (G.W., Finland) En ( a - ) (A.P., England) Fy ( a - b - ) (4) Rhesus (Macuc~ mulatta) (5)
P. falciparum
P. knowlesi
Test cell
Control
Test cell
2.1 2.0 1.8 2.6 1.9 5.7 10.1 7.4 4.8 4.9 8.4 -
E V I D E N C E FOR D I F F E R E N C E S
IN E R Y T H R O C Y T E
SURFACE RECEPTORS FOR THE MALARIAL PARASITES, PLASMODIUM FALCIPARUM AND
PLASMODIUM KNOWLESI BY LOUIS H. MILLER, J. DAVID HAYNES, FLORENCE M. McAULIFFE, TSUGIYE SHIROISHI, J O H N R. DUROCHER, ANY MARY H. McGINNISS
(From the Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, and the Blood Bank, Clinical Center, National Institutes of Health, Bethesda, Maryland 20014; and the Department of Immunology, Walter Reed Army Institute of Research, Washington, D. C. 20012; and the Pennsylvania Hospital, Philadelphia, Pennsylvania 19107)
The asexual erythrocytic cycle responsible for the clinical manifestations of malaria is perpetuated by merozoites which rupture out of infected erythrocytes and invade uninfected ones. Interaction between the merozoites and receptors on the erythrocyte surface initiate invasion (1-3). Chemical or immunologic interference between the merozoite and its receptor could block the infection at this point in the life cycle. Previously, we studied the characteristics of the erythroycte surface that are required for interaction and subsequent invasion by Plasmodium knowlesi merozoites (3, 4). Erythrocytes which lacked Duffy blood group determinants (Duffy negative erythrocytes) (3) or had been treated with chymotrypsin (4) were resistant to invasion. With the recent advances in in vitro cultivation ofPlasmodium falciparum (5, 6), it was possible to compare the erythrocyte surface determinants required for invasion by P. falciparum, the major malaria of man, and P. knowlesi, a monkey malaria that infects man.
Materials and Methods H u m a n erythrocytes (test cells) which e i t h e r lacked a blood group d e t e r m i n a n t (Table I) or had been treated with various enzymes (Table II) were exposed to P. falciparum and P. knowlesi merozoites in vitro, and the percentage of test cells infected was determined. Chimpanzee erythrocytes infected with P. falciparum (Camp strain) and frozen in liquid nitrogen were thawed (7) on the day of the experiment. They contained approximately 50% infected erythroeytes and varied in viability from experiment to experiment. The infected cells were centrifuged in microhematocrit tubes. Cells from the upper h a l f of the tubes, containing about 90% infected erythrocytes were mixed with test erythrocytes in a ratio of 1 to 25 and cultured as previously described (6). Erythrocytes were removed from culture 48 h later, spread in a t h i n film on a slide, stained with Giemsa, and the percent of erythrocytes infected with young trophozoites (ring forms) determined. Rhesus erythrocytes t h a t are refractory to invasion by P. falciparum were included in each experiment (Table I). In parallel experiments, samples of test erythrocytes were cultured with P. knowlesi-infected cells (3). Since P. falciparum merozoites were released after 48 h in culture and P. knowlesi within 1 h, test erythrocytes were incubated at 35°C for 48 h before addition of P. knowlesi-infected cells. Source of Human Erythrocytes. Erythrocytes were collected in acid citrate dextrose (ACD) and m a i n t a i n e d at 4 C. Erythrocytes from the F i n n i s h E n ( a - ) (G.W.) and two controls arrived in ice THE JOURNAL OF EXPERIMENTAL MEDICINE • VOLUME
146,
1977
277
278
MILLER ET AL.
BRIEF D E F I N I T I V E REPORT
from Finland and were cultured on 2 separate days with each parasite. Some erythrocytes (Kell null, J k [ a - b - ] , Lu[a-b-], Ge[a-], S-s-U-, En[a-] [A.P., England], and their controls) were obtained as frozen droplets in liquid nitrogen. Control erythrocytes were from healthy individuals with a common blood group phenotype. Erythrocytes were washed three times in culture media before use. Enzyme Treatmentof Human Erythrocytes. Erythrocytes were treated with chymotrypsin and trypsin, and the enzymes were inhibited after treatment as described previously (3, 4, 8). For neuraminidase treatment, a 20% suspension of erythrocytes in Tris-buffered saline with 3.6 mM KCI, 1.6 mM CaCl2, and 1.2 mM MgSO4, pH 7.4, was incubated with 25 U/ml of neuraminidase (Vibrio cholerae, B grade, Calbiochem, San Diego, Calif.) at 37°C for 30 min. Control erythrocytes from the same individual were treated identically except that no enzyme was used. The enzyme effect on the erythrocytes was monitored in each experiment: (a) chymotrypsin, removal of Duffy blood group activity; (b) neuraminidase, 76 -+ 1.4 (SEM) percent reduction in electrophoretic mobility; (c) trypsin, 34 +_ 1.4 (SEM) percent reduction in electrophoretic mobility and removal of MN blood ~rouD activity. Results H u m a n e r y t h r o c y t e s l a c k i n g specific blood g r o u p d e t e r m i n a n t s w e r e all susceptible to invasion by P. falciparum, including e r y t h r o c y t e s w i t h blood g r o u p p h e n o t y p e s ( F y [ a - b - ] a n d S-s-U-) c h a r a c t e r i s t i c of b l a c k African p o p u l a t i o n s (Table I). T h e two e r y t h r o c y t e s a m p l e s of the E n ( a - ) p h e n o t y p e showed a reduction in invasion by P. falciparum. Since a twofold decrease in i n v a s i o n is w i t h i n the r a n g e of v a r i a t i o n b e t w e e n different individuals, it is not k n o w n if the reduced invasion was caused b y the E n ( a - ) p h e n o t y p e or some o t h e r u n r e l a t e d e r y t h r o c y t e factor. In c o n t r a s t to t h e s m a l l reduction in susceptibility of E n ( a - ) cells to P. falciparum, Duffy n e g a t i v e cells ( F y [ a - b - ] ) a r e r e f r a c t o r y to infection b y P. knowlesi a n d r h e s u s cells are r e f r a c t o r y to infection by P. falciparum (Table I). T h e p a t t e r n of r e s i s t a n c e of e n z y m e - t r e a t e d h u m a n e r y t h r o c y t e s to infection differed for P. falciparum a n d P. knowlesi (Table II). C h y m o t r y p s i n (0.1 m g / m l ) t r e a t m e n t blocked infection b y P. knowlesi b u t h a d no influence on infection by P. falciparum. Conversely, t r y p s i n or t r y p s i n plus n e u r a m i n i d a s e caused a m a r k e d reduction in infection b y P. falciparum b u t little or no c h a n g e in susceptibility to P. knowlesi. N e u r a m i n i d a s e also caused a significant reduction in infection by P. falciparum, a l t h o u g h t h e effect w a s less t h a n t h a t of t r y p s i n or t r y p s i n plus n e u r a m i n i d a s e . N e u r a m i n i d a s e t r e a t m e n t did not block infection by P. knowlesi. Discussion In a previous s t u d y we o b s e r v e d directly the i n t e r a c t i o n b e t w e e n e r y t h r o c y t e s a n d P. knowlesi merozoites (9). I t consisted of a t t a c h m e n t of the merozoite to t h e e r y t h r o c y t e m e m b r a n e followed b y d e f o r m a t i o n a n d invasion. N o n e of t h e s e e v e n t s occurred w h e n susceptible h u m a n e r y t h r o c y t e s w e r e t r e a t e d w i t h chymot r y p s i n (Mason, S. J., L. H. Miller, a n d J. A. D v o r a k . U n p u b l i s h e d data.), indicating t h a t t h e receptor n e c e s s a r y for a t t a c h m e n t of P. knowlesi w a s removed. I n t h e p r e s e n t s t u d y c h y m o t r y p s i n t r e a t m e n t (0.1 m g / m l ) t h a t blocks invasion b y P . knowlesi h a d no effect on i n v a s i o n b y P . falciparum. On t h e o t h e r h a n d , t r e a t m e n t of h u m a n e r y t h r o c y t e s w i t h t r y p s i n or n e u r a m i n i d a s e reduces invasion b y P. falciparum b u t not b y P. knowlesi. I f P. falciparum i n v a d e s e r y t h r o c y t e s b y a s i m i l a r m e c h a n i s m to P. knowlesi, t h e n infection of e n z y m e -
MILLER ET AL.
BRIEFDEFINITIVE REPORT
279
TABLE I The Susceptibility of Human Erythrocytes Lacking Various Blood Group Antigens to Invasion by P. falciparum and P. knowlesi Percent infected erythrocytes
Blood group of (number studied) Test cells
ABO null (1)* Rh null (1)* Kell null (1)* J k ( a - b - ) (1)* Lu ( a - b - ) (1)* Tn (1) Ge ( a - ) (1) S-s-U- (3) En ( a - ) (G.W., Finland) En ( a - ) (A.P., England) Fy ( a - b - ) (4) Rhesus (Macuc~ mulatta) (5)
P. falciparum
P. knowlesi
Test cell
Control
Test cell
2.1 2.0 1.8 2.6 1.9 5.7 10.1 7.4 4.8 4.9 8.4 -
