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Renal Erythropoietic Factor (REF). Neutralization of in vitro generated erythropoietic activity with anti-erythropoietin
Life Sciences Vol. 7, Part I, pp . 505-509, 1988, Printed in Great Britain.
RENAL ERYTHROPOIETIC FACTOR (REF) .
Pergamon Press
NE[7TRALIZATIOR OF IN VITRO
GENERATED ERYTHROPOIETIC ACTIVITY WITH ANTI-EItYTffftOPOIETIN* Esmail D . Zat~jani, John C . Schooley and Albert S . Gordan Laboratory of Experimental Hematology, Department of Biology, Graduate School of Arts and Science, New York University, New York City and the Lawrence Radiation Laboratory, University of California, Berkeley, California
(Received 17 January 1988; in final form 19 February 1988)
When normal serum and a recently identified (1,2) renal erythropoietic factor (REF), each erythropoietically inactive when tested separately, are incubated in vitm, erythropoiesis stimulating activity appears in the mixture. It has been stated that this observed activity represents generated erythropoietin (erythropoiesis-stimulating factor, ESF)
(3,4) .
Kinetic studies oY
v the REF-serum interaction suggest that the REF is an enzyme acting on a serum substrate to produce the ESF (5) .
However, since the erythropoietic activity
of the incubation mixture is normally determined in vivo ( 6 ), the question may still be raised as to whether this activity is due to ESF generated in the reaction vessel or to another factor that indaices the production of ESF in the assay animal . Since serum from rabbits immunized with ESF (7,8,9) has been shown to neutralize specifically the ESF in vitro and to abolish ite biological effects in vivo , anti-ESF serum was used in the present atndy to provide ilu~ther evid ence that the factor generated during in vitro incubation of REF with serum is actually the ESF. Methods Preparation of Antisera. -- The anti-ESF serum was obtained Pram rabbits *Supported by USPHS research grant 2-RO1-HE03357-11, by the U8 Ata®i.c Energy Commission and by Cancer Research Funds of The University of California .
506
REF
508
Vol. 7, No. 9
immunized with human urinary erythropoietin as previously described (9) .
The
gamma globulin, containing the anti-ESF activity, was extracted frown the immune serum by ammonium sulphate fractionation and was found to be specific against ESF.
Serum was also obtained from goats immunized with a gamma globu-
lin fraction of normal rabbit serum .+
This was used for neutralization of the
anti-ESF globulin as indicated in Groups
6
and 7 (Table 1) .
REF-Serum Incubations . -- Serum for the incubation experiments with the REF was obtained from normal adult rats and dialyzed against 0 .005 M disodium ethylene diaminetetra-acetate (EDTA)
for 24 hours .
This procedure removes
cations required for a phenomenon involving loss of erythropoietic activity that develops in the reaction mixture (4) .
Two hundred and fifty ml of REF-
containing extract were obtained from 125 g of kidneys from male Long-Evens rata (250-280 g) rendered hypoxic by exposure to 0.42 atm. of air for hours (10) .
19
The REF was lyophilized and dissolved in 120 ml of saline .
The
incubation procedure involved addition of 12 ml of the REF solution to 12 ml of EDTA-dialyzed serum .
REF solution and serum incubated separately served as
All incubations were carried out for 30 minutes in a water bath and
controls .
shaken at 37° C .
All reaction vessels were left open to the air.
Whenever
necessary, the incubation mixture was adjusted to its original volume by the addition of saline . In one series of incubations, the anti-ESF globulin (0 .5 ml) was added to the mixture of 12 ml of REF solution and 12 ml of EDTA-dialyzed serum ât the end of the incubation period and e.7lowed to react for 5 minutes (Group 5) . In group
6,
2 ml of goat anti-rabbit gamma globulin serum was added to 0.5 ml
of anti-ESF globulin for 10 minutes at roam temperature .
The precipitate was
removed by centrifugation end the supernatant added to the mixture of l2 ml of REF and 12 ml of EDTA-dialyzed serum which had been incubated for 30 minutes .
In group 7, 0.5 ml of anti-ESF globulin was added to the system containing
+Supplied by Antibodies, Inc ., Devis, California
Vol. 7, No. 9
REF
507
12 ml of the REF and 12 ml of EDTA-dialyzed serum at the end of the standard 30-minute incubation period .
5
After
minutes, 2 ml of goat anti-rabbit gamma
globulin serum was pipetted into this mixture and allowed to react for 10 minutes at room temperature .
The precipitate was removed by centrifugation
and the supernatant retained for testing . Assay of Erythropoietic Activity . -- Erythropoietic activity of the incubation mixtures and supernatants was determined in the transfusion-induced plethoric mouse (8) .
Each mouse received a single 2-ml intraperitoneal
injection of the mixture or supernatant fluid following the schedule described earlier (8) .
Results are expressed as ~~ of the injected dose of 59Fe in the
calculated total blood volume per 2 ml of the fluid assayed . Results and Discussion The results are su.~nmari~ed in Table 1 .
Pieither the REF (Group 2) nor
serum (Group 3) when incubated alone showed arty detectable erythropoietic activity .
Incubation of the REF and serum together for 30 minutes resulted
in the appearance of considerable erythropoietic activity in the mixture (Group 4) .
Addition of
G.5
ml of anti-ESF globulin to this mixture at the
termination of the 3G-minute incubation period neutralized the erythropoietic activity (Group 5) .
This result shows that the erythropoietic response
observed in the mice is actually caused by the ESF .
It has been established
that any anti-ESF injected into assay mouse would neutralize the ESF irrespective of the site of origin of the hormone (7) .
To prevent injection of anti-
ESF into assay mouse, the rabbit gamma globulin (anti-ESF) was precipitated with boat anti-rabbit gamma globulin .
When this precipitated anti-ESF was
removed before addition of the supernatant to the REF-serum mixture (Group 6), the ESF that was produced was not neutralized indicating that complete removal of anti-ESF had been achieved .
However, if the anti-ESF was precipitated and
re^ :oved from the REF-serum mixture after the initial 30-minute incubation, no erythropoietic response occurred (Group 7), thus eliminating the possibility
508
REF
Vol . 7, No. 9
that the effect in the assay animal was due to administered anti-ESF . TABLE 1 Effect of Anti-ESF Globulin on Erythropoietic Activity of REF-Serum Incubation Mixtures Group
Material Tested
No . Assa
72 hr `~, RHC- 5 9fe Uptake
1
Saline
10
C .10 ± C .O11
2
REF alone
10
0 .12 ± O .G1
3
NRS 2 alone
l0
G .11 ± 0 .01
4
REF + NRS
10
2 .30 ± 0 .24
5
(REF + NRS) + Anti-ESF3
10
O .16 ± 0 .01
6
(REF + NRS) + (Anti-ESF + 2 ml Goat antibody) 4
12
1 .6_? ± G .CS
7
( (REF + NRS) + Anti-ESF) + 2 ml Goat antiboc~y5
12
x .22 ± ~ .C4
1 . Standard error of the mean . 2 . FIRS-Normal rat serum dialysed against EDTA . 3 . Anti-ESF added at the end of incubation period . 4 . Anti-ESF ora : first neutralized with goat antibody and added to the REF-serum :Rirture at the end of incubation period . 5 . Anti-ESF was added to the REF-serum mixture at the end of incubation period . The remaining, anti-ESF was then re:~oved :with goat antibody before injection of the mixture . The present stüc~yr supports our earlier contention (3,5,11) that the erythropoiesis stimulatinE activity of the REF-serum .:3rture i~ hte to _GF generated in this in vitro system . Sueu~ary When the renal erythropoietic factor and normal serum, each erythropoieticelly inactive when tested separately, are incubated in vitro , a principle is generated which stimulates erythropoiesis in plethorized assay mice .
The
antibody specifically directed against erythropoietin neutralizes this activity in the incubation mixture .
The 3ata support our contention that the principle
generated in vitro is actually erythropoietin . References 1.
J .F . CONTRERA and A,S, GORDON, Science ~, 653 (1?66) .
509
REF
Vol . ?, No. 9
28, 330 (1966) .
2.
J,F, CONTRERA, A,S, GORDON and A,H, WEINTRAUB, Blood
3.
A,S, GORDON, E,D, ZANJANI, J,F, CONTRERA, G,W, COOPER, K.K, WONG and R, KATZ, Blood
4.
E,D, ZANJANI, J,F, CONTRERA, G,W, COOPER, A,S, GORDON and K,K, WONG, Science ~,
5.
28, 977 (1966) "
1367 (1967) .
E,D, ZANJANI, J.F, CONTRERA, A,S, GORDON, G,W, COOPER, K,K, WONG and R, KATZ, Proc . Soc . Exp . Biol . and Med.
6.
A,S, GORDON and A,H, WEINTRAUB, Erythropoieais , p. 1 Grane and Stratton, N,Y,
7.
(1962) .
J,C, SCHOOIEY and J,F, GARCIA, Proc . Soc . Exp. Hiol . an d Med.
8.. J,F, GARCIA and J;C
9.
~, 505 (1967) .
SCHOOIEY, Pros . Soc. Exp. Biol . and Med.
(1963) " J,C, SCHOOLEY and J,F, GARCIA, Blood ~,
~, 325 ].]2, 712
204 (1965) .
10 . A,S, GORDON, R, KATZ, E,D, ZANJANI and E,A; MIRAND, Proc . Soc. Exp. $iol . ana Mea.
~, 475 (1966) .
11 . A,S, GORDON, G,W. COOPER sud E,D, ZANJANI, Seminars in Hematolo®r ~4, Grime and Stratton, N.Y,
(1967) .
337
RENAL ERYTHROPOIETIC FACTOR (REF) .
Pergamon Press
NE[7TRALIZATIOR OF IN VITRO
GENERATED ERYTHROPOIETIC ACTIVITY WITH ANTI-EItYTffftOPOIETIN* Esmail D . Zat~jani, John C . Schooley and Albert S . Gordan Laboratory of Experimental Hematology, Department of Biology, Graduate School of Arts and Science, New York University, New York City and the Lawrence Radiation Laboratory, University of California, Berkeley, California
(Received 17 January 1988; in final form 19 February 1988)
When normal serum and a recently identified (1,2) renal erythropoietic factor (REF), each erythropoietically inactive when tested separately, are incubated in vitm, erythropoiesis stimulating activity appears in the mixture. It has been stated that this observed activity represents generated erythropoietin (erythropoiesis-stimulating factor, ESF)
(3,4) .
Kinetic studies oY
v the REF-serum interaction suggest that the REF is an enzyme acting on a serum substrate to produce the ESF (5) .
However, since the erythropoietic activity
of the incubation mixture is normally determined in vivo ( 6 ), the question may still be raised as to whether this activity is due to ESF generated in the reaction vessel or to another factor that indaices the production of ESF in the assay animal . Since serum from rabbits immunized with ESF (7,8,9) has been shown to neutralize specifically the ESF in vitro and to abolish ite biological effects in vivo , anti-ESF serum was used in the present atndy to provide ilu~ther evid ence that the factor generated during in vitro incubation of REF with serum is actually the ESF. Methods Preparation of Antisera. -- The anti-ESF serum was obtained Pram rabbits *Supported by USPHS research grant 2-RO1-HE03357-11, by the U8 Ata®i.c Energy Commission and by Cancer Research Funds of The University of California .
506
REF
508
Vol. 7, No. 9
immunized with human urinary erythropoietin as previously described (9) .
The
gamma globulin, containing the anti-ESF activity, was extracted frown the immune serum by ammonium sulphate fractionation and was found to be specific against ESF.
Serum was also obtained from goats immunized with a gamma globu-
lin fraction of normal rabbit serum .+
This was used for neutralization of the
anti-ESF globulin as indicated in Groups
6
and 7 (Table 1) .
REF-Serum Incubations . -- Serum for the incubation experiments with the REF was obtained from normal adult rats and dialyzed against 0 .005 M disodium ethylene diaminetetra-acetate (EDTA)
for 24 hours .
This procedure removes
cations required for a phenomenon involving loss of erythropoietic activity that develops in the reaction mixture (4) .
Two hundred and fifty ml of REF-
containing extract were obtained from 125 g of kidneys from male Long-Evens rata (250-280 g) rendered hypoxic by exposure to 0.42 atm. of air for hours (10) .
19
The REF was lyophilized and dissolved in 120 ml of saline .
The
incubation procedure involved addition of 12 ml of the REF solution to 12 ml of EDTA-dialyzed serum .
REF solution and serum incubated separately served as
All incubations were carried out for 30 minutes in a water bath and
controls .
shaken at 37° C .
All reaction vessels were left open to the air.
Whenever
necessary, the incubation mixture was adjusted to its original volume by the addition of saline . In one series of incubations, the anti-ESF globulin (0 .5 ml) was added to the mixture of 12 ml of REF solution and 12 ml of EDTA-dialyzed serum ât the end of the incubation period and e.7lowed to react for 5 minutes (Group 5) . In group
6,
2 ml of goat anti-rabbit gamma globulin serum was added to 0.5 ml
of anti-ESF globulin for 10 minutes at roam temperature .
The precipitate was
removed by centrifugation end the supernatant added to the mixture of l2 ml of REF and 12 ml of EDTA-dialyzed serum which had been incubated for 30 minutes .
In group 7, 0.5 ml of anti-ESF globulin was added to the system containing
+Supplied by Antibodies, Inc ., Devis, California
Vol. 7, No. 9
REF
507
12 ml of the REF and 12 ml of EDTA-dialyzed serum at the end of the standard 30-minute incubation period .
5
After
minutes, 2 ml of goat anti-rabbit gamma
globulin serum was pipetted into this mixture and allowed to react for 10 minutes at room temperature .
The precipitate was removed by centrifugation
and the supernatant retained for testing . Assay of Erythropoietic Activity . -- Erythropoietic activity of the incubation mixtures and supernatants was determined in the transfusion-induced plethoric mouse (8) .
Each mouse received a single 2-ml intraperitoneal
injection of the mixture or supernatant fluid following the schedule described earlier (8) .
Results are expressed as ~~ of the injected dose of 59Fe in the
calculated total blood volume per 2 ml of the fluid assayed . Results and Discussion The results are su.~nmari~ed in Table 1 .
Pieither the REF (Group 2) nor
serum (Group 3) when incubated alone showed arty detectable erythropoietic activity .
Incubation of the REF and serum together for 30 minutes resulted
in the appearance of considerable erythropoietic activity in the mixture (Group 4) .
Addition of
G.5
ml of anti-ESF globulin to this mixture at the
termination of the 3G-minute incubation period neutralized the erythropoietic activity (Group 5) .
This result shows that the erythropoietic response
observed in the mice is actually caused by the ESF .
It has been established
that any anti-ESF injected into assay mouse would neutralize the ESF irrespective of the site of origin of the hormone (7) .
To prevent injection of anti-
ESF into assay mouse, the rabbit gamma globulin (anti-ESF) was precipitated with boat anti-rabbit gamma globulin .
When this precipitated anti-ESF was
removed before addition of the supernatant to the REF-serum mixture (Group 6), the ESF that was produced was not neutralized indicating that complete removal of anti-ESF had been achieved .
However, if the anti-ESF was precipitated and
re^ :oved from the REF-serum mixture after the initial 30-minute incubation, no erythropoietic response occurred (Group 7), thus eliminating the possibility
508
REF
Vol . 7, No. 9
that the effect in the assay animal was due to administered anti-ESF . TABLE 1 Effect of Anti-ESF Globulin on Erythropoietic Activity of REF-Serum Incubation Mixtures Group
Material Tested
No . Assa
72 hr `~, RHC- 5 9fe Uptake
1
Saline
10
C .10 ± C .O11
2
REF alone
10
0 .12 ± O .G1
3
NRS 2 alone
l0
G .11 ± 0 .01
4
REF + NRS
10
2 .30 ± 0 .24
5
(REF + NRS) + Anti-ESF3
10
O .16 ± 0 .01
6
(REF + NRS) + (Anti-ESF + 2 ml Goat antibody) 4
12
1 .6_? ± G .CS
7
( (REF + NRS) + Anti-ESF) + 2 ml Goat antiboc~y5
12
x .22 ± ~ .C4
1 . Standard error of the mean . 2 . FIRS-Normal rat serum dialysed against EDTA . 3 . Anti-ESF added at the end of incubation period . 4 . Anti-ESF ora : first neutralized with goat antibody and added to the REF-serum :Rirture at the end of incubation period . 5 . Anti-ESF was added to the REF-serum mixture at the end of incubation period . The remaining, anti-ESF was then re:~oved :with goat antibody before injection of the mixture . The present stüc~yr supports our earlier contention (3,5,11) that the erythropoiesis stimulatinE activity of the REF-serum .:3rture i~ hte to _GF generated in this in vitro system . Sueu~ary When the renal erythropoietic factor and normal serum, each erythropoieticelly inactive when tested separately, are incubated in vitro , a principle is generated which stimulates erythropoiesis in plethorized assay mice .
The
antibody specifically directed against erythropoietin neutralizes this activity in the incubation mixture .
The 3ata support our contention that the principle
generated in vitro is actually erythropoietin . References 1.
J .F . CONTRERA and A,S, GORDON, Science ~, 653 (1?66) .
509
REF
Vol . ?, No. 9
28, 330 (1966) .
2.
J,F, CONTRERA, A,S, GORDON and A,H, WEINTRAUB, Blood
3.
A,S, GORDON, E,D, ZANJANI, J,F, CONTRERA, G,W, COOPER, K.K, WONG and R, KATZ, Blood
4.
E,D, ZANJANI, J,F, CONTRERA, G,W, COOPER, A,S, GORDON and K,K, WONG, Science ~,
5.
28, 977 (1966) "
1367 (1967) .
E,D, ZANJANI, J.F, CONTRERA, A,S, GORDON, G,W, COOPER, K,K, WONG and R, KATZ, Proc . Soc . Exp . Biol . and Med.
6.
A,S, GORDON and A,H, WEINTRAUB, Erythropoieais , p. 1 Grane and Stratton, N,Y,
7.
(1962) .
J,C, SCHOOIEY and J,F, GARCIA, Proc . Soc . Exp. Hiol . an d Med.
8.. J,F, GARCIA and J;C
9.
~, 505 (1967) .
SCHOOIEY, Pros . Soc. Exp. Biol . and Med.
(1963) " J,C, SCHOOLEY and J,F, GARCIA, Blood ~,
~, 325 ].]2, 712
204 (1965) .
10 . A,S, GORDON, R, KATZ, E,D, ZANJANI and E,A; MIRAND, Proc . Soc. Exp. $iol . ana Mea.
~, 475 (1966) .
11 . A,S, GORDON, G,W. COOPER sud E,D, ZANJANI, Seminars in Hematolo®r ~4, Grime and Stratton, N.Y,
(1967) .
337