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Plaque forming cells to 2,-4 dinitrophenyl in spleens of immunized and nonimmunized mice
Immunochemistry. Pergamon Press 1970. Vol.7, pp. 989-992. Printedin Great Britain
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THE
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Plaque forming cells to 2,-4 dinitrophenyl in spleens of immunized and nonimmunized mice* (Received lOJuly 1970) Among defined haptenic determinants, the 2,-4-dinitrophenyl group (DNP) has been used extensively for a variety of immunological studies [ 1]. Most recently, a number of interesting but puzzling observations have been made using DNP. A relatively high number of immunoglobulins from sera of humans with macroglobulinemia and multiple myeloma and from mice in which the tumor has been induced have binding activity with nitrophenyl ligands[2]. Anti-DNP antibodies have also been found in the sera of nonimmunized individuals [3]. The present report demonstrates that mice immunized to DNP-carrier respond vigorously to the hapten, confirming the data of Yamada and Yamada [4]. This response occurs to a much greater degree than would be anticipated on the basis of stimulation by a single haptenic determinant. Furthermore, it is shown that nonimmunized mice possess a very high number of cells capable of forming immunoglobulins to the DNP ligand. The frequence of such 'background' anti-DNP plaque-forming cells (PFC) is also far in excess of the expected expression of random immunological clones and suggests that DNP as a determinant is chemically unique in that it can bind with a wide spectrum of immunoglobulin-combining sites. A/J female mice were obtained from The Jackson Laboratory, Bar Harbor, Maine and ranged in age from 10 to 15 weeks. Bovine serum albumin (BSA) (Armour and Co.) and human y globulin (HyG, obtained through the courtesy of the American Red Cross as Cohn Fraction II and further chromatographed on DEAE-cellulose) were used as carrier proteins. These were conjugated with DNP according to the method of Eisen[5] to yield complexes of DNP34BSA and DNPGI-HyG where the subscript indicates the number of haptenic residues per molecule of protein. Plaque-forming cells (PFC) against BSA and H y G were assayed using a modification[6] of the Jerne hemolytic assay[7]. This assay was also adapted for the detection of anti-DNP PFC's. Hapten conjugated erythrocytes were prepared by mixing 0.4 ml packed goat red cells with 10 ml of DNP34-BSA (8 mg/ml) and 2 ml carbodiimide (100mg/ml) for 60 rain at 4°C. The cells were then washed 4 times and a 6 per cent suspension was used in the plaque system. Direct and indirect plaques were quantitated, the latter using goat antimouse IgG serum at a dilution previously determined to be optimal in this assay. The expression of the number of plaques against DNP, H y G or BSA was corrected for background PFC against goat erythrocytes. *This is publication No. 406 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation. The work was supported by United States Public Health Service Grant A1-07007, Atomic Energy Commission Contract AT (04-3)410 and American Cancer Society Grant T-519. 989
990
Communications to the Editors
Two observations were made demonstrating the intensity of the response of mice to DNP. Firstly, animals immunized intraperitoneally with 0"2 mg DNPHyG in complete Freund's adjuvant were capable of forming as many as 1000 PFC/106 spleen cells (357,000 PFC/spleen) on the ninth day following immunization (Table 1). Secondly, lethally irradiated (950r) recipients of 100× 106 normal syngeneic spleen cells similarly challenged and tested formed over 1,600 PFC/106 spleen cells plated (145,900 PFC/spleen) (Table l). These PFC seemed specifically directly toward DNP since they were assayed against DNP on a carrier (BSA) immunologically by noncross-reactive with the immunizing carrier (HyG). This specificity was substantiated further by inhibition experiments where plaque formation was inhibited only in the presence of DNP conjugates, regardless of carrier (Table 2). Table 1. 2,-4-Dinitrophenyl plaque-forming cells (PFC) from spleens of DNP-HyG immunized or irradiated, reconstituted and immunized or normal A/J mice
Treatment
No. Plaque-formingcells/10 ~spleen cells to: of DNP BSA HyG Goat RBC mice D"
DNP-HTG a 950r+ 100× 10~NSCb+DNP-HTG None
6 6 21
I '~
D I
25 1003 0 0 -- 1684 0 0 6-4 21"4 0 0
D
I
0 0 0 I-9 0 0
D
I
1).3 0.1 2.1 1.4 1.6 0.4
"0.2 mg DNP-HTG in complete Freund's adjuvant given intraperitoneally 9 days before assay. bNSC: Normal syngeneic spleen cells. CDire~t PFC. aIndirect PFC. The unexpected magnitude of such a specific response suggested that a substantial number of precursor cells was specifically stimulated by DNP and that this might be reflected by a relatively high number of anti-DNP PFC in spleens of nonimnmnized A/J mice. The data shown in Table 1 depict that (1) a high number of anti-DNP PFC were in fact present in nonimmunized animals, (2) these were present in much greater number than the number of PFC against goat RBC and (3) there were no PFC to either carrier protein used in the present experiments. The specificity of anti-DNP PFC in non-immunized mice was also demonstrable by inhibition tests (Table 2). It can be seen that inhibition was total when DNP was present on any carrier (DNP-RSA, DNP-BSA, DNP-HyG), but absent with nonsubstituted carriers (BSA, HyG) or a carrier substituted with noncross-reacting haptens (sulphanyl BSA). Such specific DNP-PFC were also found in the spleens of other mouse strains (BALB/c, CBA, C57BL/Ks). These data emphasize the uniqueness of DNP as a haptenic determinant. A high level of background anti-DNP PFC had previously been seen by Yamada and Yamada [4] and was suggestive by the data of Brandriss [5], who observed agglutinating activity toward DNP coupled cells in the sera of normal humans. In a recent report, describing a primary response against DNP in vitro using
Communcations to the Editors
991
Table 2. Specificity of DNP plaque-forming cells (PFC) obtained from spleens of DNP-HyG immunized or normal A/J mice Indirect PFC/106 spleen cells Inhibitors a None DNP~4-RSAc DNPa4-HyG DNPnrBSA Su~9-BSAd BSA HyG
Immunized miceb
Nonimmunized mice
802 0 4 0 798 790 794
28-2 0 0 0 29.0 28.1 28.1
aAdded to the cell-agarose mixture at a final concentration of 80 ~g/ml. blmmunized 9 days before with a single intraperitoneal injection of 0"2 mg DNPa4-HyG in CFA. eDNP-RSA: dinitrophenyl rabbit serum albumin. dSu-BSA: sulphanyl-bovine serum albumin. normal mouse spleen explant, Segal et a/.[8] found that a number of cultures not treated with antigen in vitro nevertheless had anti-DNP activity. It is probable that this activity was that of immunoglobulins secreted by cells similar to those assayed by the plaque technique. This raises the question whether such an in vitro response is indeed a primary antibody response or rather reflects the stimulation of any of a large number of cells reactive with DNP. The existence of such a high frequency of cells which can bind DNP might be the result of either specific immunologic interaction or nonspecific binding between DNP with a cell or its products. The chemistry of DNP may be such that, as a haptenic determinant, it can react with a wide spectrum of antibody specificities, or as an uncharged aromatic derivative it can bind independently of antibody specificity with immunoglobulins possessing certain hydrophobic regions [9]. An interaction between precursor cells and DNP could be based on similar alternatives since there is increasing evidence that lymphocytes have antigen binding receptors on their surface[10, 11] and that these receptors may be immunoglobulin in nature [12]. Since it appears that a combination of antigen with specific receptors on precursor cells is necessary to the induction of antibody formation, the existence of a large number of such cells would be expected to give results presently reported. The frequency of DNP-specific myeloma proteins[2] is far in excess of what might be expected on the basis of random selection of immunological clones, unless the frequency of such DNP binding clones is also very high. The present data support such a hypothesis and emphasize that great care should be given in the interpretation of experimental findings using nitrophenyl ligands.
992
Comnmnication to the Editors
Acknowledgment-The authors gratefully acknowledge the technic,d assistance of Miss Sandra Templin. Department of Experimental Pathology Scrippts Clinic & Research Foundation 476 Prospect Street LaJoUa, Calif 92037, U.S.A.
.I.M. CHILLER* W.O. WEI(;LE?
*Supported by United States Public Health Service Training (;rant GM 00683. ?Research Career Awardee of the United States Public Health Service (5-K6-GM6936).
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
REFERENCES Siskind G. W. and Benacerraf B., Adv. lmmunol. 10, 1 (1969). Metzger H., Potter M. and Terry W., lmmunochemist~ 6, 831 (1969). Brandriss M. W., Nature, Lond. 221,960 (1969). Yamada H. and Yamada A.,J. lmmun. 103, 357 (1969). Eisen H. N., Methods in Medical Research. Yearbook Medical Publishers, Inc., Chicago (1964). Golub E. S., Mishell R. 1., Weigle W. O. and Dutton R. W.,J. lmmun. 100, 133 (1968). Jerne N. K. and Nordin A. A., Science 140, 405 (1963). Segal S., Globerson A., Feldman M., Harmovich J. and Sela M.,.[. exp. Med. 131, 93 (1970). Parker C. W. and Osterland C. L., Biochemistry9, 1{)74(1970). Naor D. and Sulitzeanu D+, Nature, Lond. 214, 687 (1967). Ada G. L. and Byrt P. N., Nature, Lond. 222, 1291 (1969). Raft+M. C., Sternberg M. and Tayh)r R, B., Nature. Lond. 225, 553 (1970).
lmmunochemistrv. Pergamon Press 1970. Vol. 7, pp+ 992-996. Printed in (;feat Britain
A simple method for quantitation of hemolytic antibodies* (Received5June 1970) W h e n an u n k n o w n a m o u n t o f hemolytic anti-SRBC s e r u m is allowed to diffuse radially f r o m a well in a layer o f a g a r containing s h e e p erythrocytes, the final area o f lysis r e a c h e d was f o u n d to be directly p r o p o r t i o n a l to the square root o f the s e r u m dilution a n d inversely p r o p o r t i o n a l to the concentration o f erythrocytes in the agar. By s t a n d a r d i z i n g the technical conditions o f the e x p e r i m e n t it is possible to use this principle for the quantitative d e t e r m i n a t i o n o f hemolytic antibodies. A d u l t Swiss Webster mice w e r e used fl)r the p r e p a r a t i o n o f hemolytic antisera. Several mice were injected intraperitoneally with 0-1 ml o f a 50 p e r cent suspension o f thrice-washed s h e e p erythrocytes. F o u r days following injection, *This work was supported by Grant GB 6860 from the National Science Foundation.
COMMUNICATIONS
TO
THE
EDITORS
Plaque forming cells to 2,-4 dinitrophenyl in spleens of immunized and nonimmunized mice* (Received lOJuly 1970) Among defined haptenic determinants, the 2,-4-dinitrophenyl group (DNP) has been used extensively for a variety of immunological studies [ 1]. Most recently, a number of interesting but puzzling observations have been made using DNP. A relatively high number of immunoglobulins from sera of humans with macroglobulinemia and multiple myeloma and from mice in which the tumor has been induced have binding activity with nitrophenyl ligands[2]. Anti-DNP antibodies have also been found in the sera of nonimmunized individuals [3]. The present report demonstrates that mice immunized to DNP-carrier respond vigorously to the hapten, confirming the data of Yamada and Yamada [4]. This response occurs to a much greater degree than would be anticipated on the basis of stimulation by a single haptenic determinant. Furthermore, it is shown that nonimmunized mice possess a very high number of cells capable of forming immunoglobulins to the DNP ligand. The frequence of such 'background' anti-DNP plaque-forming cells (PFC) is also far in excess of the expected expression of random immunological clones and suggests that DNP as a determinant is chemically unique in that it can bind with a wide spectrum of immunoglobulin-combining sites. A/J female mice were obtained from The Jackson Laboratory, Bar Harbor, Maine and ranged in age from 10 to 15 weeks. Bovine serum albumin (BSA) (Armour and Co.) and human y globulin (HyG, obtained through the courtesy of the American Red Cross as Cohn Fraction II and further chromatographed on DEAE-cellulose) were used as carrier proteins. These were conjugated with DNP according to the method of Eisen[5] to yield complexes of DNP34BSA and DNPGI-HyG where the subscript indicates the number of haptenic residues per molecule of protein. Plaque-forming cells (PFC) against BSA and H y G were assayed using a modification[6] of the Jerne hemolytic assay[7]. This assay was also adapted for the detection of anti-DNP PFC's. Hapten conjugated erythrocytes were prepared by mixing 0.4 ml packed goat red cells with 10 ml of DNP34-BSA (8 mg/ml) and 2 ml carbodiimide (100mg/ml) for 60 rain at 4°C. The cells were then washed 4 times and a 6 per cent suspension was used in the plaque system. Direct and indirect plaques were quantitated, the latter using goat antimouse IgG serum at a dilution previously determined to be optimal in this assay. The expression of the number of plaques against DNP, H y G or BSA was corrected for background PFC against goat erythrocytes. *This is publication No. 406 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation. The work was supported by United States Public Health Service Grant A1-07007, Atomic Energy Commission Contract AT (04-3)410 and American Cancer Society Grant T-519. 989
990
Communications to the Editors
Two observations were made demonstrating the intensity of the response of mice to DNP. Firstly, animals immunized intraperitoneally with 0"2 mg DNPHyG in complete Freund's adjuvant were capable of forming as many as 1000 PFC/106 spleen cells (357,000 PFC/spleen) on the ninth day following immunization (Table 1). Secondly, lethally irradiated (950r) recipients of 100× 106 normal syngeneic spleen cells similarly challenged and tested formed over 1,600 PFC/106 spleen cells plated (145,900 PFC/spleen) (Table l). These PFC seemed specifically directly toward DNP since they were assayed against DNP on a carrier (BSA) immunologically by noncross-reactive with the immunizing carrier (HyG). This specificity was substantiated further by inhibition experiments where plaque formation was inhibited only in the presence of DNP conjugates, regardless of carrier (Table 2). Table 1. 2,-4-Dinitrophenyl plaque-forming cells (PFC) from spleens of DNP-HyG immunized or irradiated, reconstituted and immunized or normal A/J mice
Treatment
No. Plaque-formingcells/10 ~spleen cells to: of DNP BSA HyG Goat RBC mice D"
DNP-HTG a 950r+ 100× 10~NSCb+DNP-HTG None
6 6 21
I '~
D I
25 1003 0 0 -- 1684 0 0 6-4 21"4 0 0
D
I
0 0 0 I-9 0 0
D
I
1).3 0.1 2.1 1.4 1.6 0.4
"0.2 mg DNP-HTG in complete Freund's adjuvant given intraperitoneally 9 days before assay. bNSC: Normal syngeneic spleen cells. CDire~t PFC. aIndirect PFC. The unexpected magnitude of such a specific response suggested that a substantial number of precursor cells was specifically stimulated by DNP and that this might be reflected by a relatively high number of anti-DNP PFC in spleens of nonimnmnized A/J mice. The data shown in Table 1 depict that (1) a high number of anti-DNP PFC were in fact present in nonimmunized animals, (2) these were present in much greater number than the number of PFC against goat RBC and (3) there were no PFC to either carrier protein used in the present experiments. The specificity of anti-DNP PFC in non-immunized mice was also demonstrable by inhibition tests (Table 2). It can be seen that inhibition was total when DNP was present on any carrier (DNP-RSA, DNP-BSA, DNP-HyG), but absent with nonsubstituted carriers (BSA, HyG) or a carrier substituted with noncross-reacting haptens (sulphanyl BSA). Such specific DNP-PFC were also found in the spleens of other mouse strains (BALB/c, CBA, C57BL/Ks). These data emphasize the uniqueness of DNP as a haptenic determinant. A high level of background anti-DNP PFC had previously been seen by Yamada and Yamada [4] and was suggestive by the data of Brandriss [5], who observed agglutinating activity toward DNP coupled cells in the sera of normal humans. In a recent report, describing a primary response against DNP in vitro using
Communcations to the Editors
991
Table 2. Specificity of DNP plaque-forming cells (PFC) obtained from spleens of DNP-HyG immunized or normal A/J mice Indirect PFC/106 spleen cells Inhibitors a None DNP~4-RSAc DNPa4-HyG DNPnrBSA Su~9-BSAd BSA HyG
Immunized miceb
Nonimmunized mice
802 0 4 0 798 790 794
28-2 0 0 0 29.0 28.1 28.1
aAdded to the cell-agarose mixture at a final concentration of 80 ~g/ml. blmmunized 9 days before with a single intraperitoneal injection of 0"2 mg DNPa4-HyG in CFA. eDNP-RSA: dinitrophenyl rabbit serum albumin. dSu-BSA: sulphanyl-bovine serum albumin. normal mouse spleen explant, Segal et a/.[8] found that a number of cultures not treated with antigen in vitro nevertheless had anti-DNP activity. It is probable that this activity was that of immunoglobulins secreted by cells similar to those assayed by the plaque technique. This raises the question whether such an in vitro response is indeed a primary antibody response or rather reflects the stimulation of any of a large number of cells reactive with DNP. The existence of such a high frequency of cells which can bind DNP might be the result of either specific immunologic interaction or nonspecific binding between DNP with a cell or its products. The chemistry of DNP may be such that, as a haptenic determinant, it can react with a wide spectrum of antibody specificities, or as an uncharged aromatic derivative it can bind independently of antibody specificity with immunoglobulins possessing certain hydrophobic regions [9]. An interaction between precursor cells and DNP could be based on similar alternatives since there is increasing evidence that lymphocytes have antigen binding receptors on their surface[10, 11] and that these receptors may be immunoglobulin in nature [12]. Since it appears that a combination of antigen with specific receptors on precursor cells is necessary to the induction of antibody formation, the existence of a large number of such cells would be expected to give results presently reported. The frequency of DNP-specific myeloma proteins[2] is far in excess of what might be expected on the basis of random selection of immunological clones, unless the frequency of such DNP binding clones is also very high. The present data support such a hypothesis and emphasize that great care should be given in the interpretation of experimental findings using nitrophenyl ligands.
992
Comnmnication to the Editors
Acknowledgment-The authors gratefully acknowledge the technic,d assistance of Miss Sandra Templin. Department of Experimental Pathology Scrippts Clinic & Research Foundation 476 Prospect Street LaJoUa, Calif 92037, U.S.A.
.I.M. CHILLER* W.O. WEI(;LE?
*Supported by United States Public Health Service Training (;rant GM 00683. ?Research Career Awardee of the United States Public Health Service (5-K6-GM6936).
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
REFERENCES Siskind G. W. and Benacerraf B., Adv. lmmunol. 10, 1 (1969). Metzger H., Potter M. and Terry W., lmmunochemist~ 6, 831 (1969). Brandriss M. W., Nature, Lond. 221,960 (1969). Yamada H. and Yamada A.,J. lmmun. 103, 357 (1969). Eisen H. N., Methods in Medical Research. Yearbook Medical Publishers, Inc., Chicago (1964). Golub E. S., Mishell R. 1., Weigle W. O. and Dutton R. W.,J. lmmun. 100, 133 (1968). Jerne N. K. and Nordin A. A., Science 140, 405 (1963). Segal S., Globerson A., Feldman M., Harmovich J. and Sela M.,.[. exp. Med. 131, 93 (1970). Parker C. W. and Osterland C. L., Biochemistry9, 1{)74(1970). Naor D. and Sulitzeanu D+, Nature, Lond. 214, 687 (1967). Ada G. L. and Byrt P. N., Nature, Lond. 222, 1291 (1969). Raft+M. C., Sternberg M. and Tayh)r R, B., Nature. Lond. 225, 553 (1970).
lmmunochemistrv. Pergamon Press 1970. Vol. 7, pp+ 992-996. Printed in (;feat Britain
A simple method for quantitation of hemolytic antibodies* (Received5June 1970) W h e n an u n k n o w n a m o u n t o f hemolytic anti-SRBC s e r u m is allowed to diffuse radially f r o m a well in a layer o f a g a r containing s h e e p erythrocytes, the final area o f lysis r e a c h e d was f o u n d to be directly p r o p o r t i o n a l to the square root o f the s e r u m dilution a n d inversely p r o p o r t i o n a l to the concentration o f erythrocytes in the agar. By s t a n d a r d i z i n g the technical conditions o f the e x p e r i m e n t it is possible to use this principle for the quantitative d e t e r m i n a t i o n o f hemolytic antibodies. A d u l t Swiss Webster mice w e r e used fl)r the p r e p a r a t i o n o f hemolytic antisera. Several mice were injected intraperitoneally with 0-1 ml o f a 50 p e r cent suspension o f thrice-washed s h e e p erythrocytes. F o u r days following injection, *This work was supported by Grant GB 6860 from the National Science Foundation.