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Humoral immune factors in the paddlefish, Polyodon spathula
Comp. Biochem. Physiol., 1971, Vol. 38B, pp. 523 to 527. Pergamon Press. Printed in Great Britain
HUMORAL IMMUNE FACTORS IN THE PADDLEFISH, POL YODON SPA THULA D. W. L E G L E R , P. F. W E I N H E I M E R , R. T. A C T O N , H. K. D U P R E E , and T. R. R U S S E L L Department of Microbiology, University of Alabama Medical Center, Birmingham, Alabama 35233 (Received 4 Scptentber 1970)
Abstract--1. Natural agglutinin titers to Salmonella typhosa "O" (STO), human group A erythrocytes (HuA) and sheep erythrocytes (SRBC) were studied in twenty-three paddlefish. 2. Natural agglutinin titers to SRBC were low, with high titers observed to STO and HuA. Cold agglutinin titers (4°C) to STO markedly exceeded titers recorded at room temperature. 3. Serum immunoglobulins were shown to account for over forty per cent of the total serum proteins in this species. INTRODUCTION THE PADDLEFISH, a chondrostean fish, is among the more primitive of surviving vertebrates (Romer, 1962). It is a key species in the phylogenesis of immune mechanisms. T h e paddlefish responds vigorously to antigenic stimulation (Finstad & Good, 1966), and possesses a serum complement system similar to that of mammals (Gewurz et al., 1966; Legler et al., 1967). Antibody produced in the paddlefish is of a single macroglobulin type with a sedimentation constant of 14.2S and a molecular weight of 630,000 (Acton et al., 1970). T h e present study reports the serum levels of i m m u n e globulin in this species. Natural antibodies to certain cellular antigens are considered in relation to optimal temperatures. MATERIALS AND METHODS Animals Twenty-three mature paddlefish, Polyodon spathula, were collected from the Osage River near Columbus, Missouri and maintained in quarter-acre outdoor ponds. Animal weights ranged from 6 to 15 kg. Ambient temperature ranged from 18 ° to 24°C. Blood was obtained by cardiac or haemal arch puncture, allowed to clot at refrigerator temperatures, and centrifuged within 24 hr. Antigens Sheep erythrocytes (SRBC) were obtained from Colorado Serum Company, Denver, Colorado. Cells were washed and suspended in saline to a concentration of 1 x 109 ml. Human blood group A (HuA) erythrocytes were obtained fresh from donors and prepared in the same manner. Salmonella typhosa "O" antigen (STO) was prepared as described by Evans (1957), and suspended to a cell concentration of 2 x 10a ml. Immunizations were by intraperitoneal (IP) injection of 1"0 ml antigen suspension.
523
524
D.W. LEGLER, P. F. WEINHEIMER, R. T. ACTON, H. K. Dtn'REEANDT. R. RIIK~LL
Agglutination and hemolytic titrations Agglutination titres to STO, SRBC, and HuA erythrocytes were assayed in Linbro trays (Linbro Chemical Company, New Haven, Connecticut) and results were expressed as the reciprocal of the final dilution showing visible agglutination. Each well contained 0.2 ml of diluted serum and 0"2 ml of a 2% suspension of erythrocytes or a 2 x 10~ suspension of STO in phosphate buffered saline. Agglutination assays were performed in duplicate and incubated for 24 hr at 4°C and at room temperature. Complement levels were quantitated in CHs0 units/ml utilizing paddlefish antibody in a modified standard SRBC hemolytic system (Kabat & Mayer, 1961). The details of these modifications have been discussed (Legler et al., 1967). The reaction system contained 5 x 10s SRBC and one ml of diluted serum in 7.5 ml total volume of barbital buffer containing 0"00015 M Ca ~+ and 0"001 M Mg ~+. Reaction tubes were incubated at 25°C for 4 hours. Immunoglobulin quantitation Purified paddlefish immunoglobulin was prepared according to Acton et al. (1970). Rabbit antisera to the purified macroglobulin was prepared using conventional procedures. The radial immunodiffusion method as described by Alford et al. (1967) was used to estimate serum immunoglobulin levels and serum protein concentrations were determined by the biuret method.
RESULTS The paddlefish possesses high levels of natural agglutinins to certain cellular antigens (Table I). Mean STO agglutinintiters of 2048 recordedat 4°C exceeded STO agglutinintiters at 25°C despite an ambientwatertemperatureof 24°C at the time of serum collection. Agglutinintiters against HuA and SRBC were also higher when incubated at 4°C as comparedto readings at 25°C. TABLE 1--ANTIBODY AND CHs0 LEVELS IN NORMAL PADDLEFISH SERA
Incubation temperature 4°C
25°C
Antigen titered
Mean
Range
STO HuA SRBC
2048 512 32
512-8192 64-8192 < 8-256
Mean 256 128 16
CH60 Range
Mean
Range
128-1024 32-1024 < 8-1024
16"1
2-57
Primary immunization with SRBC resulted in an increase in SRBC agglutinin titers (Table 2). T h e fact that accompanying change was observed in hemolytic activity following SRBC challenge suggests that complement might be the limiting factor in the paddlefish natural hemolytic system. Immunization with S T O resulted in an increase in mean S T O cold agglutinin titer to 8192, but no effects were noted on anti-SRBC agglutinin production or on CH60 titer. T h e adjuvant effect of S T O previously noted in the gar (Legler et al., 1970) was not observed in the paddlefish.
HUMORAL IMMUNE FACTORS IN THE PADDLEFISH TABLE 2--EFFECTS OF PRIMARY IMMUNIZATION WITH S T O AGGLUTININ AND c n s 0 LEVELS
Pre-immunization titer
No. of animals 5 10
Antigen
OR S R B C
525 ON MEAN S R B C
Post-immunization titer
CHs0
Agglutinin
CHs0
Agglutinin
15"8 16"5
32 32 (2048)*
18"1 16"0
128 32 (8192)
SRBC STO
Agglutinin titers at 4°C, hemolytic titers at 25°C. Serum was harvested at 28 days post-immunization. * Agglutinin titers to STO in parentheses. I m m u n e macroglobulin levels in S T O i m m u n e serum were estimated at 1-7 g/100 ml as derived f r o m the graph calculations shown in Fig. 1. Total serum protein levels were 3.7 g/100 ml serum, indicating that the macroglobulin comprised in excess of 4 0 % of the total s e r u m proteins.
I
I
I
I
I 6
I 8
I0
8.0 6.0 4.0
I/5
O I L U T I O N OF WHOLE
O~
E z rn O
2.0
1.0
0.8 o 0.6 re" (.9
L) ,~ 0.4
0.2
0.1 0
I 2
I 4 DIAMETER
I0
mm
FIo. 1. A standard graph of the precipitin ring diameters of purified immune macroglobulin plotted against varying protein concentrations of the macroglobulin expressed in mg/ml. Each point on the standard curve ( ) represents the average of duplicate determinations at each concentration. The test serum (- - -) was from a pool of twenty-three paddlefish.
526
W. LEGLER,P. F. WEINHEIMER,R. T. ACTON,H. K. DUeRF2ANDT. R. RUSSELL
DISCUSSION Our observation that immune macroglobulin comprises over 40% of the total serum protein in the paddlefish compares favourably with values reported in the nurse shark. Approximately one-half of the total serum protein of the nurse shark was reported to consist of immunoglobulin (Fidler et al., 1969). These results suggest that immunoglobulin formation represents a major physiological process quite early in the phylogenesis of protective immunity in the vertebrates. This study has also shown that the paddlefish preferentially synthesizes cold agglutinins to certain cellular antigens. Observations in the carp (Avtalion, 1969) and the Japanese eel (Muroga & Egusa, 1969) indicate that antibody synthesis continues in these animals with an elevation of titer at low temperature, providing that the animals were first antigenically stimulated at higher temperatures. Preferential synthesis of cold agglutinins to cellular antigens was also noted in the gar, a holeost (Legler et al., 1971). These findings support the speculation that several populations of antibodies may be synthesized, each having differing affinity or avidity for a specific antigen at different temperatures. Such an immune capability would provide survival benefit in an environment subject to marked changes in ambient temperature.
Acknowledgements--One of us (T. R. R.) is Fisheries Biologist, Missouri Department of Conservation, Columbia, Missouri. We wish to thank the Missouri Department of Conservation for their cooperation. Partial support was provided by Federal Aid in Fish Restoration Funds under Missouri Projects F-1-R. Additional support was provided in part by United States Public Health Service grants AI-08068 (D. W. L.), AI-02693, AI-09153, and AM-0355. Dr. H. K. Dupree is Fisheries Biologist, U.S.D.I., Bureau of Sport Fisheries and Wildlife, Southeastern Fish Culture Laboratory, Marion, Alabama. REFERENCES ACTON R. T., WEINHEIMER P. F., WOLCOTT M., EVANS E. E., & BENNETTJ. C. (1970) N-terminal sequences of immunoglobulin heavy and light chains from three species of lower vertebrates. Nature, Lond. 228, 991-992. ALFORD C. A., SHAEFERJ., BLANKENSHIPJ., STRAUMFJORDJ. V., & CASSADYG. (1967) A correlated immunologic, microbiologic, and clinical approach to the diagnosis of acute and chronic infections in newborn infants. New Eng..7. Med. 277, 437-44-9. AVTALION R. R. (1969) Temperature effect on antibody production and immunological memory in carp (Cyprinus carpio) immunized against bovine serum albumin (BSA). Immunol. 17, 927-931. EVANS E. E. (1957) Manual of Microbiological Methods (Edited by CONN H. J. & PELCZAR M. J.), p. 199. McGraw-Hill, New York. FIDLERJ. E., CLEML. W., & SMALLP. A., JR. (1969) Irnmunoglobulin synthesis in neonatal nurse sharks. Comp. Biochem. Physiol. 31,365-371. FINSTADJ. & GOOD R. A. (1966) Phylogenetic studies of adaptive immune responses in the lower vertebrates. In Phylogeny of Immunity (edited by SMITH R. T., MIESCHERP. A., & GOOD R. A.), pp. 173-189. University of Florida Press, Gainesville. GEWURZ H., FINSTADJ., MUSCHELL. H., & GOOD R. A. (1966) Phylogenetic inquiry into the origins of the complement system. In Phylogeny of Immunity (edited by SMXTHR. T., MIESCHERP. A., & GOOD R. A.), pp. 105-117. University of Florida Press, Gainesville. KABAT E. A. & MAYER M. M. (1961) Experimental Immunochemistry, p. 149. Thomas, Springfield.
HUMORAL IMMUNE FACTORS IN THE PADDLEFISH
527
LEGLERD. W., ACTONR. T., WEINHEIMERP. F. & DUPREEH. K. (1971) Humoral antibody responses in the gar, Lepisosteus osseus. Immunol. (In press.) LEGLER D. W., EVANS E. E. & DUPREE H. K. (1967) Comparative immunology: serum complement of freshwater fishes. Trans. Amer. Fish. Soc. 96(3), 237-242. MUROCAK., & ECUSAS. (1969) Immune response of the Japanese eel to Vibrio anguillarum-Effects of temperature on agglutinating antibody production in starved eels. Bull. Jap. Soc. Scientific Fisheries 39(9), 868-874. Rorcma A. S. (1962) The Vertebrate Body, p. 43. Saunders, Philadelphia. Key Word Index--Antibodies; complement; natural antibodies; serum immunoglobulins; cold aggiutinins. Polyodon spathula, paddlefish.
HUMORAL IMMUNE FACTORS IN THE PADDLEFISH, POL YODON SPA THULA D. W. L E G L E R , P. F. W E I N H E I M E R , R. T. A C T O N , H. K. D U P R E E , and T. R. R U S S E L L Department of Microbiology, University of Alabama Medical Center, Birmingham, Alabama 35233 (Received 4 Scptentber 1970)
Abstract--1. Natural agglutinin titers to Salmonella typhosa "O" (STO), human group A erythrocytes (HuA) and sheep erythrocytes (SRBC) were studied in twenty-three paddlefish. 2. Natural agglutinin titers to SRBC were low, with high titers observed to STO and HuA. Cold agglutinin titers (4°C) to STO markedly exceeded titers recorded at room temperature. 3. Serum immunoglobulins were shown to account for over forty per cent of the total serum proteins in this species. INTRODUCTION THE PADDLEFISH, a chondrostean fish, is among the more primitive of surviving vertebrates (Romer, 1962). It is a key species in the phylogenesis of immune mechanisms. T h e paddlefish responds vigorously to antigenic stimulation (Finstad & Good, 1966), and possesses a serum complement system similar to that of mammals (Gewurz et al., 1966; Legler et al., 1967). Antibody produced in the paddlefish is of a single macroglobulin type with a sedimentation constant of 14.2S and a molecular weight of 630,000 (Acton et al., 1970). T h e present study reports the serum levels of i m m u n e globulin in this species. Natural antibodies to certain cellular antigens are considered in relation to optimal temperatures. MATERIALS AND METHODS Animals Twenty-three mature paddlefish, Polyodon spathula, were collected from the Osage River near Columbus, Missouri and maintained in quarter-acre outdoor ponds. Animal weights ranged from 6 to 15 kg. Ambient temperature ranged from 18 ° to 24°C. Blood was obtained by cardiac or haemal arch puncture, allowed to clot at refrigerator temperatures, and centrifuged within 24 hr. Antigens Sheep erythrocytes (SRBC) were obtained from Colorado Serum Company, Denver, Colorado. Cells were washed and suspended in saline to a concentration of 1 x 109 ml. Human blood group A (HuA) erythrocytes were obtained fresh from donors and prepared in the same manner. Salmonella typhosa "O" antigen (STO) was prepared as described by Evans (1957), and suspended to a cell concentration of 2 x 10a ml. Immunizations were by intraperitoneal (IP) injection of 1"0 ml antigen suspension.
523
524
D.W. LEGLER, P. F. WEINHEIMER, R. T. ACTON, H. K. Dtn'REEANDT. R. RIIK~LL
Agglutination and hemolytic titrations Agglutination titres to STO, SRBC, and HuA erythrocytes were assayed in Linbro trays (Linbro Chemical Company, New Haven, Connecticut) and results were expressed as the reciprocal of the final dilution showing visible agglutination. Each well contained 0.2 ml of diluted serum and 0"2 ml of a 2% suspension of erythrocytes or a 2 x 10~ suspension of STO in phosphate buffered saline. Agglutination assays were performed in duplicate and incubated for 24 hr at 4°C and at room temperature. Complement levels were quantitated in CHs0 units/ml utilizing paddlefish antibody in a modified standard SRBC hemolytic system (Kabat & Mayer, 1961). The details of these modifications have been discussed (Legler et al., 1967). The reaction system contained 5 x 10s SRBC and one ml of diluted serum in 7.5 ml total volume of barbital buffer containing 0"00015 M Ca ~+ and 0"001 M Mg ~+. Reaction tubes were incubated at 25°C for 4 hours. Immunoglobulin quantitation Purified paddlefish immunoglobulin was prepared according to Acton et al. (1970). Rabbit antisera to the purified macroglobulin was prepared using conventional procedures. The radial immunodiffusion method as described by Alford et al. (1967) was used to estimate serum immunoglobulin levels and serum protein concentrations were determined by the biuret method.
RESULTS The paddlefish possesses high levels of natural agglutinins to certain cellular antigens (Table I). Mean STO agglutinintiters of 2048 recordedat 4°C exceeded STO agglutinintiters at 25°C despite an ambientwatertemperatureof 24°C at the time of serum collection. Agglutinintiters against HuA and SRBC were also higher when incubated at 4°C as comparedto readings at 25°C. TABLE 1--ANTIBODY AND CHs0 LEVELS IN NORMAL PADDLEFISH SERA
Incubation temperature 4°C
25°C
Antigen titered
Mean
Range
STO HuA SRBC
2048 512 32
512-8192 64-8192 < 8-256
Mean 256 128 16
CH60 Range
Mean
Range
128-1024 32-1024 < 8-1024
16"1
2-57
Primary immunization with SRBC resulted in an increase in SRBC agglutinin titers (Table 2). T h e fact that accompanying change was observed in hemolytic activity following SRBC challenge suggests that complement might be the limiting factor in the paddlefish natural hemolytic system. Immunization with S T O resulted in an increase in mean S T O cold agglutinin titer to 8192, but no effects were noted on anti-SRBC agglutinin production or on CH60 titer. T h e adjuvant effect of S T O previously noted in the gar (Legler et al., 1970) was not observed in the paddlefish.
HUMORAL IMMUNE FACTORS IN THE PADDLEFISH TABLE 2--EFFECTS OF PRIMARY IMMUNIZATION WITH S T O AGGLUTININ AND c n s 0 LEVELS
Pre-immunization titer
No. of animals 5 10
Antigen
OR S R B C
525 ON MEAN S R B C
Post-immunization titer
CHs0
Agglutinin
CHs0
Agglutinin
15"8 16"5
32 32 (2048)*
18"1 16"0
128 32 (8192)
SRBC STO
Agglutinin titers at 4°C, hemolytic titers at 25°C. Serum was harvested at 28 days post-immunization. * Agglutinin titers to STO in parentheses. I m m u n e macroglobulin levels in S T O i m m u n e serum were estimated at 1-7 g/100 ml as derived f r o m the graph calculations shown in Fig. 1. Total serum protein levels were 3.7 g/100 ml serum, indicating that the macroglobulin comprised in excess of 4 0 % of the total s e r u m proteins.
I
I
I
I
I 6
I 8
I0
8.0 6.0 4.0
I/5
O I L U T I O N OF WHOLE
O~
E z rn O
2.0
1.0
0.8 o 0.6 re" (.9
L) ,~ 0.4
0.2
0.1 0
I 2
I 4 DIAMETER
I0
mm
FIo. 1. A standard graph of the precipitin ring diameters of purified immune macroglobulin plotted against varying protein concentrations of the macroglobulin expressed in mg/ml. Each point on the standard curve ( ) represents the average of duplicate determinations at each concentration. The test serum (- - -) was from a pool of twenty-three paddlefish.
526
W. LEGLER,P. F. WEINHEIMER,R. T. ACTON,H. K. DUeRF2ANDT. R. RUSSELL
DISCUSSION Our observation that immune macroglobulin comprises over 40% of the total serum protein in the paddlefish compares favourably with values reported in the nurse shark. Approximately one-half of the total serum protein of the nurse shark was reported to consist of immunoglobulin (Fidler et al., 1969). These results suggest that immunoglobulin formation represents a major physiological process quite early in the phylogenesis of protective immunity in the vertebrates. This study has also shown that the paddlefish preferentially synthesizes cold agglutinins to certain cellular antigens. Observations in the carp (Avtalion, 1969) and the Japanese eel (Muroga & Egusa, 1969) indicate that antibody synthesis continues in these animals with an elevation of titer at low temperature, providing that the animals were first antigenically stimulated at higher temperatures. Preferential synthesis of cold agglutinins to cellular antigens was also noted in the gar, a holeost (Legler et al., 1971). These findings support the speculation that several populations of antibodies may be synthesized, each having differing affinity or avidity for a specific antigen at different temperatures. Such an immune capability would provide survival benefit in an environment subject to marked changes in ambient temperature.
Acknowledgements--One of us (T. R. R.) is Fisheries Biologist, Missouri Department of Conservation, Columbia, Missouri. We wish to thank the Missouri Department of Conservation for their cooperation. Partial support was provided by Federal Aid in Fish Restoration Funds under Missouri Projects F-1-R. Additional support was provided in part by United States Public Health Service grants AI-08068 (D. W. L.), AI-02693, AI-09153, and AM-0355. Dr. H. K. Dupree is Fisheries Biologist, U.S.D.I., Bureau of Sport Fisheries and Wildlife, Southeastern Fish Culture Laboratory, Marion, Alabama. REFERENCES ACTON R. T., WEINHEIMER P. F., WOLCOTT M., EVANS E. E., & BENNETTJ. C. (1970) N-terminal sequences of immunoglobulin heavy and light chains from three species of lower vertebrates. Nature, Lond. 228, 991-992. ALFORD C. A., SHAEFERJ., BLANKENSHIPJ., STRAUMFJORDJ. V., & CASSADYG. (1967) A correlated immunologic, microbiologic, and clinical approach to the diagnosis of acute and chronic infections in newborn infants. New Eng..7. Med. 277, 437-44-9. AVTALION R. R. (1969) Temperature effect on antibody production and immunological memory in carp (Cyprinus carpio) immunized against bovine serum albumin (BSA). Immunol. 17, 927-931. EVANS E. E. (1957) Manual of Microbiological Methods (Edited by CONN H. J. & PELCZAR M. J.), p. 199. McGraw-Hill, New York. FIDLERJ. E., CLEML. W., & SMALLP. A., JR. (1969) Irnmunoglobulin synthesis in neonatal nurse sharks. Comp. Biochem. Physiol. 31,365-371. FINSTADJ. & GOOD R. A. (1966) Phylogenetic studies of adaptive immune responses in the lower vertebrates. In Phylogeny of Immunity (edited by SMITH R. T., MIESCHERP. A., & GOOD R. A.), pp. 173-189. University of Florida Press, Gainesville. GEWURZ H., FINSTADJ., MUSCHELL. H., & GOOD R. A. (1966) Phylogenetic inquiry into the origins of the complement system. In Phylogeny of Immunity (edited by SMXTHR. T., MIESCHERP. A., & GOOD R. A.), pp. 105-117. University of Florida Press, Gainesville. KABAT E. A. & MAYER M. M. (1961) Experimental Immunochemistry, p. 149. Thomas, Springfield.
HUMORAL IMMUNE FACTORS IN THE PADDLEFISH
527
LEGLERD. W., ACTONR. T., WEINHEIMERP. F. & DUPREEH. K. (1971) Humoral antibody responses in the gar, Lepisosteus osseus. Immunol. (In press.) LEGLER D. W., EVANS E. E. & DUPREE H. K. (1967) Comparative immunology: serum complement of freshwater fishes. Trans. Amer. Fish. Soc. 96(3), 237-242. MUROCAK., & ECUSAS. (1969) Immune response of the Japanese eel to Vibrio anguillarum-Effects of temperature on agglutinating antibody production in starved eels. Bull. Jap. Soc. Scientific Fisheries 39(9), 868-874. Rorcma A. S. (1962) The Vertebrate Body, p. 43. Saunders, Philadelphia. Key Word Index--Antibodies; complement; natural antibodies; serum immunoglobulins; cold aggiutinins. Polyodon spathula, paddlefish.