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Immunosuppression in Chickens by Passive Transfer of Preparations of Specific Immunoglobulins1,2
Immunosuppression in Chickens by Passive Transfer of Preparations of Specific Immunoglobulins1 ' 2 PATRICIA Y. HESTER3,4, PAUL THAXTON, G. WALLACE MORGAN and JOHN BRAKE Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication April 29, 1977) ABSTRACT The immunosuppresive effects of passive transfers of specific immunoglobulin preparations obtained from immune or hyperimmune sera were evaluated in chickens which were challenged with sheep red blood cells. The data indicate that IgY, fractionated from immune sera, and IgY and IgM, fractionated from hyperimmune sera, when passively administered at the doses employed in this study resulted in antibody-mediated suppression. Poultry Science 56:2091-2097, 1977 INTRODUCTION The immunoregulatory roles of specific immunoglobulin (Ig) classes, which were purified from both immune and hyperimmune sera, have been studied extensively, especially in mammals (Clarke et ah, 1963; Finkelstein and Uhr, 1964; Sahiar and Schwartz, 1964; 1965; Moller and Wigzell, 1965; Wigzell, 1966; Henry and Jerne, 1968; Dennert, 1971). In general, humoral immune responses are enhanced by passively transferred IgM and suppressed by IgG. This generalization does not mean that IgM functions solely as an enhancer or that IgG acts exclusively as a suppressor. For instance, the quantity, affinity, and avidity of a particular Ig obtained early during a primary immune response, was less avid than IgG from hyperimmune sera. Gordon and Murgita (1975) demonstrated that IgG 2 and IgGi, which are subclasses of IgG, oppose each other as immunoregulators; IgG 2 enhanced, while IgGi suppressed humoral immunity. Thus, these workers proposed a check and balance system for feedback inhibition of antibody production.
1 Paper No. 5250 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27607. 2 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the products named, nor criticism of similar ones not mentioned. 3 Present address of senior author: Department of Animal Science, Purdue University, West Lafayette, Indiana 27907. 4 A portion of a dissertation presented in partial fulfillment of the requirements for the Ph.D. at North Carolina State University.
Unlike mammalian species, the regulatory role of Ig classes in chickens is unclear. A single report by Kermani-Arab et ah (1975) indicated that IgY antibodies against Marek's disease virus, when passively transferred, resulted in suppression of the ensuing response to Marek's virus. The present study was conducted to clarify the immunoregulatory roles of Ig in chickens. The specific objective was to investigate the immunoregulatory roles of immune IgY and IgM in the chicken. MATERIALS AND METHODS Two trials were conducted using commercial broiler cockerels which were obtained from the NCSU breeding flock. The chicks were housed in non-heated metal batteries in an environmentally controlled room. A brooding source of heat was not provided; however, the room temperature was maintained at 29.4°C. for the first three weeks and then reduced to 25.6 C. for the remainder of each trial. A non-medicated starter-grower ration which met or exceeded the minimum nutritional requirements of the birds was fed. Feed and water were available ad libitum. Each recipient' bird was immunized with 1 ml. of a 15% saline suspension of sheep red blood cells (SRBC). All injections and bleedings were made via the brachial veins unless otherwise stated and sera were collected from the blood samples as described by Hester (1977). The serum samples were stored at —20 C. until used. Immediately prior to the determinations of anti-SRBC agglutinins by the microtitration technique (Thaxton et ah, 1971), the samples were thawed and heat inactivated at 56°C. to
2091
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P. Y. HESTER, P. THAXTON, G. W. MORGAN AND J. BRAKE
destroy the activity of the complement components. Hyperimmune (HIS), immune (IS), and nonimmune (NIS), sera were collected from chickens which were eight weeks of age and not included in the two trials of this study. HIS were prepared by giving birds weekly injections of the SRBC antigen for four consecutive weeks. Seven days following the final SRBC injection approximately 25 ml. of blood were collected from each donor by cardiac stab. IS were collected in the same manner, except that the donors received a single SRBC challenge and blood was collected 4 days post-immunization. NIS were collected from non-immunized donors. The serum samples from each donor were serologically assayed for anti-SRBC agglutinins, then pooled according to a previously described technique (Thaxton and Young, 1974). Preparations of specific Ig from both IS and HIS were separated by the method of Williams and Chase (1967) using Sephadex G-200 gel filtration (Pharmacia, Piscataway, New Jersey). The fractions of IgM and IgY were collected and then characterized by immunoelectrophoresis (Williams and Grabor, 1955). IgY is used to connote the 7S Ig of chickens, since it differs physically and chemically from mammalian IgG (Leslie and Clem, 1969). All fractions were lyophilized and stored at —20°C. until needed. In trial 1, three groups of three chickens each were given 3 ml. of NIS, HIS, or 15 mg. of IgY which was fractionated from HIS and dissolved in 0.85% saline. A fourth group of three birds was not injected and served as handled controls. The SRBC antigen was administered 24 hours following the passive transfer of antibody. The birds were bled immediately prior to and at 2 and 4 days following antigenic challenge. Microtitrations were performed on the serum samples to determine the levels of anti-SRBC antibodies. Four days after antigenic challenge the birds were killed by cervical dislocation and the numbers of plaqueforming cells (PFC) per 10 6 spleen cells were determined. PFC determinations were made by removing the spleens, separating the splenic leucocytes by the albumin-flotation method (Parker, 1961), and directly quantitating the antibody producing cells of the splenic leucocytes by the antibody plaque-forming assay of Cunningham and Szenberg (1968). Trial 2 consisted of six groups of three chickens each. Each group received one of the
following treatments: 2.7 ml. of NIS, 2.7 ml. of HIS, 13.2 mg. of IgY collected from IS (designated as early IgY), 13.5 mg. of IgY collected from HIS (designated as late IgY), 12.6 mg. of IgM collected from HIS, or non-injected controls. The fractionated Ig preparations were dissolved in 0.85% saline and administered as a single 2.7 ml. intravenous injection per bird at 24 hours prior to antigenic challenge. Serum samples were collected from each bird immediately prior to and at 2 day intervals for 12 days following SRBC challenge. These samples were evaluated serologically for anti-SRBC agglutinins, as well as mercaptoethanol (ME) resistant and sensitive antibody titers (Delhanty and Solomon, 1966). Antibody titers were analyzed as log 2 values (Ambrose and Donner, 1973). Comparisons among treatment means were made by Duncan's new multiple range test (Steel and Torrie, 1960). Statements of significance are based on P<0.01 unless otherwise stated.
RESULTS
Table 1 illustrates the effects of HIS and IgY on the primary immune responses of the four week old birds of trial 1. Using the PFC response and circulating levels of antibody as indicators of immune responsiveness, it was apparent that both HIS and IgY were effective immunosuppressors. At 4 days following SRBC injection, the birds treated with HIS or IgY lacked PFC responses and possessed approximately half the amount of circulating antibodies of the control or NIS-treated birds. The mean primary hemagglutinin titers of the chickens of trial 2 are presented in table 2. Those birds treated with HIS, IgM, or late IgY exhibited significant reductions in anti-SRBC antibody levels throughout the primary immune response (P<0.05 at 2 and 10 days post-antigenic challenge). Suppression of the primary immune response of birds treated with early IgY was evident numerically by 6 days and was significant at 8, 10 (P<0.05), and 12 days following SRBC challenge. Table 3 depicts the ME-sensitive antibody titers of the birds of trial 2. At 0 day, i.e. 24 hours following the passive transfer of antibody, birds treated with HIS and IgM showed significant increases in ME-sensitive antibody titers. Birds given passive transfers of HIS, IgM, or late IgY showed significant reductions in ME-sensitive antibody titers at 2 (P<0.05), 4, 6,
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and 8 (P<0.05) days following antigenic challenge. Those birds treated with early IgY showed a decrease in ME-sensitive antibody titers at 6, 8, and 10 days after the administration of SRBC; however, this decrease was not significant when compared to the ME-sensitive antibody titers of control birds. Birds treated with HIS demonstrated significant reductions in ME-sensitive antibody titers 10 days following antigen (P<0.05). By 12 days following SRBC challenge, no significant differences in MEsensitive antibody titers occurred among the treatment groups. The ME-sensitive antibody titers of the birds of trial 2 indicate that passively administered antibody blocked IgM antibody synthesis. These results were not unexpected, since in the control birds the data indicated that IgM was the predominant Ig synthesized during the early part of the primary immune response. The effects of HIS and preparations of specific Ig on the ME-resistant antibody titers of the chickens of trial 2 are presented in table 4. No significant differences in the ME-resistant antibody titers occurred among the treatment groups at 0, 2, 4, 6, or 10 days after SRBC challenge. Birds given early IgY exhibited significantly reduced ME-resistant antibody titers at 8 days following immunization (P<0.05). Birds which received passive transfers of IgM, early IgY, or late IgY showed ME-resistant antibody titers at 12 days which were reduced significantly when compared with the controls. The ME-resistant antibody titers indicate that the control birds produced IgY by 6 days following antigenic challenge. However, a significant suppression of IgY synthesis by passively administered immunoglobulin was not apparent until 12 days following SRBC challenge.
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The theoretical mechanisms of suppression of humoral immune responses by passively transferred antibody have been compared to the mode of action of actively synthesized antibody during a normal immune response. For example, Neiders et al. (1962) demonstrated that repeated injections of antigen following a single injection of antiserum resulted in suppression of the respective immune response for as long as antigen was administered. These investigators proposed that suppression was a result of proper amounts of actively synthesized antibody feeding back and
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Means (± S.E.M.) which possess different superscripts in a column differ significantly at P<0.01 unless indicated by *
See table 2 for identification of abbreviations.
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TABLE 3.—The effect of HIS and preparations of specific imtnunoglo,bulins on the mean ME-sen 1,2 of chickens of trial 2 , 3
Each mean represents 3 birds.
Means (± S.E.M.) which possess different superscripts in a column differ significantly at P<0.01 unless indicated by *
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inhibiting the synthesis of further antibody. The concept of an inhibitory role for actively synthesized antibody does not exclude the possibility that it may also be stimulatory. The question a priori is whether endogenous concentrations of actively synthesized antibody can enhance its own production during the early part of a primary immune response? The dosimetry results obtained from the passive transfers of purified and specific Ig's in mammals have provided support for the concept that actively synthesized antibody can be either inhibitory or stimulatory. The results of the present study, employing the passive transfer of HIS or designated concentrations of prepared Ig, support an inhibitory role of passively administered antibody in chickens. The finding that the passive transfer of preparations of specific Ig to birds at the dosages employed in this study suppressed primary immunity is a significant contribution to the understanding of the immunoregulatory role of avian Ig's. Kermani-Arab et al. (1975) reported that 27 mg. of IgY against Marek's disease virus per day for 3 days resulted in the suppression of Ig's, as well as specific antibody production. The present data support the contention that specific IgY, when passively transferred, is effective in suppressing a humoral immune response. Passively transferred mammalian IgM resulted in either enhancement or suppression of humoral immunity depending on the dosage used (Pearlman, 1967). The results of this study indicate that IgM was an effective suppressor of primary immunity at the dose level of 12.6 mg. per bird. Immunosuppression by the passive administration of IgM, early IgY, or late IgY is further verified by the ME-antibody titers of trial 2. All Ig preparations used in this study, when passively transferred, suppressed both IgM and IgY synthesis. The inhibitory roles of these classes of Ig's suggest that proper concentrations of actively synthesized antibody may also be inhibitory. Specifically, actively produced IgM may possess the immunoregulatory function of not only inhibiting its own synthesis, but also that of IgY. Likewise, actively produced IgY may possess the same regulatory function. The data of trial 2 indicate that the passive transfer of late IgY was more suppressive than early IgY. This difference may be attributed to antibody subclass, affinity, avidity, specificity, and/or half-life. Mammalian subclases of IgG can be either suppressors or enhancers (Gordon
2096
P. Y. HESTER, P. THAXTON, G. W. MORGAN AND J. BRAKE
and Murgita, 1975). T h e avian subclasses of IgY have n o t been characterized; however, t h e distribution of these subclasses m a y differ between early and late IgY. Additionally, Walker and Siskind ( 1 9 6 8 ) r e p o r t e d t h a t a n t i b o d y synthesized later in t h e i m m u n e response possessed a greater affinity, and therefore a greater suppressive ability, t h a n a n t i b o d y p r o d u c e d early in an i m m u n e response. T h e affinity of specific IgY for antigen or a n t i b o d y receptors on B-cells (Basten et al., 1 9 7 2 ) was n o t evaluated in this s t u d y . However, a n t i b o d y affinity should be considered as a possible e x p l a n a t i o n for t h e differences of these respective Ig's in suppressing h u m o r a l i m m u n i t y . T h e specificity of IgY for antigenic d e t e r m i n a n t g r o u p s of SRBC m a y differ between t h e early and late fractions. Likewise, differences in suppression m a y be related t o t h e half-lives of early and late IgY. A s t u d y of t h e half-life of a specific Ig class, as it is synthesized during an i m m u n e response, has n o t been reported. It is possible t h a t all of these factors interact t o cause differences in suppression b e t w e e n early and late IgY or one factor m a y c o n t r i b u t e m o r e t h a n another. T h e results of this s t u d y d o n o t define t h e i m m u n o r e g u l a t o r y role of a n t i b o d y in t h e chicken; however, t h e y c o n t r i b u t e t o t h e overall u n d e r s t a n d i n g of i m m u n o r e g u l a t i o n . Specifically, t h e suppression of avian i m m u n e responses b y t h e passive transfers of IgM and t h e comparative s t u d y of t h e suppression caused b y early and late IgY are n e w c o n t r i b u t i o n s t o t h e u n d e r s t a n d i n g of a n t i b o d y - m e d i a t e d i m m u n o regulation in t h e chicken. ACKNOWLEDGEMENTS Appreciation is e x t e n d e d t o Dee F a b a c h e r and R o b e r t H u n t e r for excellent technical assistance.
REFERENCES Ambrose, C. T., and A. Donner, 1973. Application of the analysis of variance to hemagglutination titrations. J. Immunol. Meth. 3:165-210. Basten, A., N. L. Warner and T. Mandel, 1972. A receptor for antibody on B lymphocytes. 2. Immunochemical and electron microscopy characteristics. J. Exp. Med. 135:627-642. Clarke, C. A., W. T. A. Donohue, R. B. McConell, W. W. Kulke, D. Lehane and P. M. Shepard, 1963. Further experimental studies on the prevention of Rh haemolytic disease. Brit. Med. J. 2:979-984. Cunningham, A. J., and A. Szenberg, 1968. Further improvements in the plaque technique for detect-
ing single antibody-forming cells. Immunol. 14:599-601. Delhanty, J. J., and J. B. Solomon, 1966. The nature of antibodies to goat erythrocytes in the developing chicken. Immunol. 11:103—113. Dennert, G., 1971. The mechanism of antibodyinduced stimulation and inhibition of the immune response. J. Immunol. 106:951—955. Finkelstein, M. S., and J. W. Uhr, 1964. Specific inhibition of antibody formation by passively administered 19S and 7S antibody. Science 146:67-69. Gordon, J., and R. A. Murgita, 1975. Suppression and augmentation of the primary in vitro immune response by different classes of antibody. Cell. Immunol. 15:392-402. Henry, C , and N. K. Jerne, 1968. Competition of 19S and 7S antigen receptors in the regulation of the p r i m a r y immune response. J. Exp. Med. 128:133-152. Hester, P. Y., 1977. Specific and nonspecific immunosuppression in the domestic chicken. Ph.D. Dissertation, N.C. State University, Raleigh, N.C. 27607. Kermani-Arab, V., T. Moll, W. C. Davis, B. R. Cho, Y. S. Lu and G. A. Leslie, 1975. Immunoglobulins and anti-Marek's disease virus antibody synthesis in chickens after passive immunizations with immunoglobulin Y anti-Marek's disease virus antibody. Amer. J. Vet. Res. 36:1655-1658. Leslie, G. A., and L. W. Clem, 1969. Phylogeny of immunoglobulin structure and function. 3. Immunoglobulins of the chicken. J. Exp. Med. 30:1337-1352. Moller, G., and H. Wigzell, 1965. Antibody synthesis at the cellular level: Antibody-induced suppression of 19S and 7S antibody response. J. Exp. Med. 121:969-989. Neiders, M. E., D. A. Rowley and F. W. Fitch, 1962. The sustained suppression of hemolysin response in passively i m m u n i z e d r a t s . J. Immunol. 88:718-724. Parker, R. C , 1961. Methods of Tissue Culture, 3rd ed., Paul B. Hoeber, Inc., Med. Div. of Harper and Brothers, N.Y., p. 135-137. Pearlman, D. S., 1967. The influence of antibodies on immunologic responses. 1. The effect on the response to particulate antigen in the rabbit. J. Exp. Med. 126:127-148. Sahiar, K., and R. S. Schwartz, 1964. Inhibition of 19S antibody synthesis by 7S antibody. Science 145:395-396. Sahiar, K., and R. S. Schwartz, 1965. The immunoglobulin sequence. 1. Arrest by 6-mercaptopurine and restitution by antibody, antigen or splenectomy. J. Immunol. 95:345-354. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, New York. p. 107-109. Thaxton, P., J. E. Williams and H. S. Siegel, 1971. Microtitration of Salmonella pullorum agglutinins. Avian Dis. 14:813-816. Thaxton, P., and P. S. Young, 1974. Antibodymediated suppression of the primary hemagglutination response in young chickens. Poultry Sci. 53:1839-1842. Walker, J. G., and G. W. Siskind, 1968. Studies on the control of antibody synthesis. Effect of antibody
IMMUNOSUPPRESSION IN CHICKENS affinity upon its ability to suppress antibody formation. Immunol. 14:21—28. Wigzell, H., 1966. Antibody synthesis at the cellular level: Antibody induced suppression of 7S antibody synthesis. J. Exp. Med. 124:953-969. Williams, C. A., and M. W. Chase, 1967. Methods in
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Immunology and Immunochemistry. vol. 1, Academic Press, New York. Williams, C. A., Jr., and P. Grabor, 1955. Immunoelectrophoretic studies on serum proteins. 1. The a n t i g e n s of h u m a n serum. J. Immunol. 74:158-168.
1 Paper No. 5250 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27607. 2 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the products named, nor criticism of similar ones not mentioned. 3 Present address of senior author: Department of Animal Science, Purdue University, West Lafayette, Indiana 27907. 4 A portion of a dissertation presented in partial fulfillment of the requirements for the Ph.D. at North Carolina State University.
Unlike mammalian species, the regulatory role of Ig classes in chickens is unclear. A single report by Kermani-Arab et ah (1975) indicated that IgY antibodies against Marek's disease virus, when passively transferred, resulted in suppression of the ensuing response to Marek's virus. The present study was conducted to clarify the immunoregulatory roles of Ig in chickens. The specific objective was to investigate the immunoregulatory roles of immune IgY and IgM in the chicken. MATERIALS AND METHODS Two trials were conducted using commercial broiler cockerels which were obtained from the NCSU breeding flock. The chicks were housed in non-heated metal batteries in an environmentally controlled room. A brooding source of heat was not provided; however, the room temperature was maintained at 29.4°C. for the first three weeks and then reduced to 25.6 C. for the remainder of each trial. A non-medicated starter-grower ration which met or exceeded the minimum nutritional requirements of the birds was fed. Feed and water were available ad libitum. Each recipient' bird was immunized with 1 ml. of a 15% saline suspension of sheep red blood cells (SRBC). All injections and bleedings were made via the brachial veins unless otherwise stated and sera were collected from the blood samples as described by Hester (1977). The serum samples were stored at —20 C. until used. Immediately prior to the determinations of anti-SRBC agglutinins by the microtitration technique (Thaxton et ah, 1971), the samples were thawed and heat inactivated at 56°C. to
2091
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P. Y. HESTER, P. THAXTON, G. W. MORGAN AND J. BRAKE
destroy the activity of the complement components. Hyperimmune (HIS), immune (IS), and nonimmune (NIS), sera were collected from chickens which were eight weeks of age and not included in the two trials of this study. HIS were prepared by giving birds weekly injections of the SRBC antigen for four consecutive weeks. Seven days following the final SRBC injection approximately 25 ml. of blood were collected from each donor by cardiac stab. IS were collected in the same manner, except that the donors received a single SRBC challenge and blood was collected 4 days post-immunization. NIS were collected from non-immunized donors. The serum samples from each donor were serologically assayed for anti-SRBC agglutinins, then pooled according to a previously described technique (Thaxton and Young, 1974). Preparations of specific Ig from both IS and HIS were separated by the method of Williams and Chase (1967) using Sephadex G-200 gel filtration (Pharmacia, Piscataway, New Jersey). The fractions of IgM and IgY were collected and then characterized by immunoelectrophoresis (Williams and Grabor, 1955). IgY is used to connote the 7S Ig of chickens, since it differs physically and chemically from mammalian IgG (Leslie and Clem, 1969). All fractions were lyophilized and stored at —20°C. until needed. In trial 1, three groups of three chickens each were given 3 ml. of NIS, HIS, or 15 mg. of IgY which was fractionated from HIS and dissolved in 0.85% saline. A fourth group of three birds was not injected and served as handled controls. The SRBC antigen was administered 24 hours following the passive transfer of antibody. The birds were bled immediately prior to and at 2 and 4 days following antigenic challenge. Microtitrations were performed on the serum samples to determine the levels of anti-SRBC antibodies. Four days after antigenic challenge the birds were killed by cervical dislocation and the numbers of plaqueforming cells (PFC) per 10 6 spleen cells were determined. PFC determinations were made by removing the spleens, separating the splenic leucocytes by the albumin-flotation method (Parker, 1961), and directly quantitating the antibody producing cells of the splenic leucocytes by the antibody plaque-forming assay of Cunningham and Szenberg (1968). Trial 2 consisted of six groups of three chickens each. Each group received one of the
following treatments: 2.7 ml. of NIS, 2.7 ml. of HIS, 13.2 mg. of IgY collected from IS (designated as early IgY), 13.5 mg. of IgY collected from HIS (designated as late IgY), 12.6 mg. of IgM collected from HIS, or non-injected controls. The fractionated Ig preparations were dissolved in 0.85% saline and administered as a single 2.7 ml. intravenous injection per bird at 24 hours prior to antigenic challenge. Serum samples were collected from each bird immediately prior to and at 2 day intervals for 12 days following SRBC challenge. These samples were evaluated serologically for anti-SRBC agglutinins, as well as mercaptoethanol (ME) resistant and sensitive antibody titers (Delhanty and Solomon, 1966). Antibody titers were analyzed as log 2 values (Ambrose and Donner, 1973). Comparisons among treatment means were made by Duncan's new multiple range test (Steel and Torrie, 1960). Statements of significance are based on P<0.01 unless otherwise stated.
RESULTS
Table 1 illustrates the effects of HIS and IgY on the primary immune responses of the four week old birds of trial 1. Using the PFC response and circulating levels of antibody as indicators of immune responsiveness, it was apparent that both HIS and IgY were effective immunosuppressors. At 4 days following SRBC injection, the birds treated with HIS or IgY lacked PFC responses and possessed approximately half the amount of circulating antibodies of the control or NIS-treated birds. The mean primary hemagglutinin titers of the chickens of trial 2 are presented in table 2. Those birds treated with HIS, IgM, or late IgY exhibited significant reductions in anti-SRBC antibody levels throughout the primary immune response (P<0.05 at 2 and 10 days post-antigenic challenge). Suppression of the primary immune response of birds treated with early IgY was evident numerically by 6 days and was significant at 8, 10 (P<0.05), and 12 days following SRBC challenge. Table 3 depicts the ME-sensitive antibody titers of the birds of trial 2. At 0 day, i.e. 24 hours following the passive transfer of antibody, birds treated with HIS and IgM showed significant increases in ME-sensitive antibody titers. Birds given passive transfers of HIS, IgM, or late IgY showed significant reductions in ME-sensitive antibody titers at 2 (P<0.05), 4, 6,
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and 8 (P<0.05) days following antigenic challenge. Those birds treated with early IgY showed a decrease in ME-sensitive antibody titers at 6, 8, and 10 days after the administration of SRBC; however, this decrease was not significant when compared to the ME-sensitive antibody titers of control birds. Birds treated with HIS demonstrated significant reductions in ME-sensitive antibody titers 10 days following antigen (P<0.05). By 12 days following SRBC challenge, no significant differences in MEsensitive antibody titers occurred among the treatment groups. The ME-sensitive antibody titers of the birds of trial 2 indicate that passively administered antibody blocked IgM antibody synthesis. These results were not unexpected, since in the control birds the data indicated that IgM was the predominant Ig synthesized during the early part of the primary immune response. The effects of HIS and preparations of specific Ig on the ME-resistant antibody titers of the chickens of trial 2 are presented in table 4. No significant differences in the ME-resistant antibody titers occurred among the treatment groups at 0, 2, 4, 6, or 10 days after SRBC challenge. Birds given early IgY exhibited significantly reduced ME-resistant antibody titers at 8 days following immunization (P<0.05). Birds which received passive transfers of IgM, early IgY, or late IgY showed ME-resistant antibody titers at 12 days which were reduced significantly when compared with the controls. The ME-resistant antibody titers indicate that the control birds produced IgY by 6 days following antigenic challenge. However, a significant suppression of IgY synthesis by passively administered immunoglobulin was not apparent until 12 days following SRBC challenge.
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The theoretical mechanisms of suppression of humoral immune responses by passively transferred antibody have been compared to the mode of action of actively synthesized antibody during a normal immune response. For example, Neiders et al. (1962) demonstrated that repeated injections of antigen following a single injection of antiserum resulted in suppression of the respective immune response for as long as antigen was administered. These investigators proposed that suppression was a result of proper amounts of actively synthesized antibody feeding back and
NIS HIS IgM Early IgY Late IgY
2.7 2.7 2.7 2.7 2.7
0 5 10 1 5
Titer
0.0 ± 0.0 a 0.0 ± 0.0 a 1.7±0.3a 2.7 ± 0 . 3 a 0.0 ± 0.0* 0.3 ± 0 . 3 a
0 2.3 ± 0.33 2.0 ± 0.0 a 0 . 3 ± 0 . 3 b "' 0.3 ± 0.3 b * 1.3 ± 0.8at' 0.3 ± 0.3 b *
2 6.3 4.3 1.0 2.0 4.3 1.3
± 1.3 a ± 0.9 at> + 0.0 b ± 0.6 b ± 0.3ab + 0.3b
4 7.3 ± 0.9 a 5.7 ± 0 . 7 a b 1.3 ± 0 . 3 C 2.7±0.3C 3.3 ± 0 . 9 b c 1.3 ± 0 . 3 C
6 6 5 3 2 2 1
Bleeding times (days post-SRBC ch
on the mean he
3
2
0.0 + 0.0C 0.0 + 0.0 C 1.3 + 0 . 3 b 2.7±0.3a 0.0 ± 0.0C 0.3 ± 0 . 3 b c
0
2.0 ± 0.0 a 2.0 ± 0.0 a 0.3±0.3b* 0.3±0.3b* 1.3 + 0 . 9 a b 0.0 + 0.0 b *
2 6.3 4.3 1.0 2.0 4.3 1.3
±1.3a ± 0.9ab ± 0.0 b + 0.6 b + 0.3ab ±0.3b
4
5.3 + 0.9 a 4.0 + 0 . 6 a b 0.7 ±0.3 C 1.7±0.7 b c 2.7 + 0 . 9 a b c 0.7 ± 0.3 C
6 3.7 3.0 0.7 1.3 2.3 0.3
Bleeding times (days post-SRBC challeng
Each mean represents 3 birds.
Means (± S.E.M.) which possess different superscripts in a column differ significantly at P<0.01 unless indicated by *
See table 2 for identification of abbreviations.
NIS HIS IgM Early IgY Late IgY
2.7 2.7 2.7 2.7 2.7
1
Fraction
Antisera
TABLE 3.—The effect of HIS and preparations of specific imtnunoglo,bulins on the mean ME-sen 1,2 of chickens of trial 2 , 3
Each mean represents 3 birds.
Means (± S.E.M.) which possess different superscripts in a column differ significantly at P<0.01 unless indicated by *
(ml.)
3
2
1 NIS = nonimmune sera; HIS = hyperimmune sera; IgM = fraction collected from HIS with each bird receiving 12.6 days post antigenic challenge with each bird receiving 13.2 mg. ;late IgY = fraction collected from HIS with each bird recei
Fraction
(ml.)
Antisera
TABLE 2.-Tbe effect of HIS and preparatio ns of specific immunoglobulins 1 2 of,:hickens of trial 2 ' ,3
IMMUNOSUPPRESSION IN CHICKENS
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2095
inhibiting the synthesis of further antibody. The concept of an inhibitory role for actively synthesized antibody does not exclude the possibility that it may also be stimulatory. The question a priori is whether endogenous concentrations of actively synthesized antibody can enhance its own production during the early part of a primary immune response? The dosimetry results obtained from the passive transfers of purified and specific Ig's in mammals have provided support for the concept that actively synthesized antibody can be either inhibitory or stimulatory. The results of the present study, employing the passive transfer of HIS or designated concentrations of prepared Ig, support an inhibitory role of passively administered antibody in chickens. The finding that the passive transfer of preparations of specific Ig to birds at the dosages employed in this study suppressed primary immunity is a significant contribution to the understanding of the immunoregulatory role of avian Ig's. Kermani-Arab et al. (1975) reported that 27 mg. of IgY against Marek's disease virus per day for 3 days resulted in the suppression of Ig's, as well as specific antibody production. The present data support the contention that specific IgY, when passively transferred, is effective in suppressing a humoral immune response. Passively transferred mammalian IgM resulted in either enhancement or suppression of humoral immunity depending on the dosage used (Pearlman, 1967). The results of this study indicate that IgM was an effective suppressor of primary immunity at the dose level of 12.6 mg. per bird. Immunosuppression by the passive administration of IgM, early IgY, or late IgY is further verified by the ME-antibody titers of trial 2. All Ig preparations used in this study, when passively transferred, suppressed both IgM and IgY synthesis. The inhibitory roles of these classes of Ig's suggest that proper concentrations of actively synthesized antibody may also be inhibitory. Specifically, actively produced IgM may possess the immunoregulatory function of not only inhibiting its own synthesis, but also that of IgY. Likewise, actively produced IgY may possess the same regulatory function. The data of trial 2 indicate that the passive transfer of late IgY was more suppressive than early IgY. This difference may be attributed to antibody subclass, affinity, avidity, specificity, and/or half-life. Mammalian subclases of IgG can be either suppressors or enhancers (Gordon
2096
P. Y. HESTER, P. THAXTON, G. W. MORGAN AND J. BRAKE
and Murgita, 1975). T h e avian subclasses of IgY have n o t been characterized; however, t h e distribution of these subclasses m a y differ between early and late IgY. Additionally, Walker and Siskind ( 1 9 6 8 ) r e p o r t e d t h a t a n t i b o d y synthesized later in t h e i m m u n e response possessed a greater affinity, and therefore a greater suppressive ability, t h a n a n t i b o d y p r o d u c e d early in an i m m u n e response. T h e affinity of specific IgY for antigen or a n t i b o d y receptors on B-cells (Basten et al., 1 9 7 2 ) was n o t evaluated in this s t u d y . However, a n t i b o d y affinity should be considered as a possible e x p l a n a t i o n for t h e differences of these respective Ig's in suppressing h u m o r a l i m m u n i t y . T h e specificity of IgY for antigenic d e t e r m i n a n t g r o u p s of SRBC m a y differ between t h e early and late fractions. Likewise, differences in suppression m a y be related t o t h e half-lives of early and late IgY. A s t u d y of t h e half-life of a specific Ig class, as it is synthesized during an i m m u n e response, has n o t been reported. It is possible t h a t all of these factors interact t o cause differences in suppression b e t w e e n early and late IgY or one factor m a y c o n t r i b u t e m o r e t h a n another. T h e results of this s t u d y d o n o t define t h e i m m u n o r e g u l a t o r y role of a n t i b o d y in t h e chicken; however, t h e y c o n t r i b u t e t o t h e overall u n d e r s t a n d i n g of i m m u n o r e g u l a t i o n . Specifically, t h e suppression of avian i m m u n e responses b y t h e passive transfers of IgM and t h e comparative s t u d y of t h e suppression caused b y early and late IgY are n e w c o n t r i b u t i o n s t o t h e u n d e r s t a n d i n g of a n t i b o d y - m e d i a t e d i m m u n o regulation in t h e chicken. ACKNOWLEDGEMENTS Appreciation is e x t e n d e d t o Dee F a b a c h e r and R o b e r t H u n t e r for excellent technical assistance.
REFERENCES Ambrose, C. T., and A. Donner, 1973. Application of the analysis of variance to hemagglutination titrations. J. Immunol. Meth. 3:165-210. Basten, A., N. L. Warner and T. Mandel, 1972. A receptor for antibody on B lymphocytes. 2. Immunochemical and electron microscopy characteristics. J. Exp. Med. 135:627-642. Clarke, C. A., W. T. A. Donohue, R. B. McConell, W. W. Kulke, D. Lehane and P. M. Shepard, 1963. Further experimental studies on the prevention of Rh haemolytic disease. Brit. Med. J. 2:979-984. Cunningham, A. J., and A. Szenberg, 1968. Further improvements in the plaque technique for detect-
ing single antibody-forming cells. Immunol. 14:599-601. Delhanty, J. J., and J. B. Solomon, 1966. The nature of antibodies to goat erythrocytes in the developing chicken. Immunol. 11:103—113. Dennert, G., 1971. The mechanism of antibodyinduced stimulation and inhibition of the immune response. J. Immunol. 106:951—955. Finkelstein, M. S., and J. W. Uhr, 1964. Specific inhibition of antibody formation by passively administered 19S and 7S antibody. Science 146:67-69. Gordon, J., and R. A. Murgita, 1975. Suppression and augmentation of the primary in vitro immune response by different classes of antibody. Cell. Immunol. 15:392-402. Henry, C , and N. K. Jerne, 1968. Competition of 19S and 7S antigen receptors in the regulation of the p r i m a r y immune response. J. Exp. Med. 128:133-152. Hester, P. Y., 1977. Specific and nonspecific immunosuppression in the domestic chicken. Ph.D. Dissertation, N.C. State University, Raleigh, N.C. 27607. Kermani-Arab, V., T. Moll, W. C. Davis, B. R. Cho, Y. S. Lu and G. A. Leslie, 1975. Immunoglobulins and anti-Marek's disease virus antibody synthesis in chickens after passive immunizations with immunoglobulin Y anti-Marek's disease virus antibody. Amer. J. Vet. Res. 36:1655-1658. Leslie, G. A., and L. W. Clem, 1969. Phylogeny of immunoglobulin structure and function. 3. Immunoglobulins of the chicken. J. Exp. Med. 30:1337-1352. Moller, G., and H. Wigzell, 1965. Antibody synthesis at the cellular level: Antibody-induced suppression of 19S and 7S antibody response. J. Exp. Med. 121:969-989. Neiders, M. E., D. A. Rowley and F. W. Fitch, 1962. The sustained suppression of hemolysin response in passively i m m u n i z e d r a t s . J. Immunol. 88:718-724. Parker, R. C , 1961. Methods of Tissue Culture, 3rd ed., Paul B. Hoeber, Inc., Med. Div. of Harper and Brothers, N.Y., p. 135-137. Pearlman, D. S., 1967. The influence of antibodies on immunologic responses. 1. The effect on the response to particulate antigen in the rabbit. J. Exp. Med. 126:127-148. Sahiar, K., and R. S. Schwartz, 1964. Inhibition of 19S antibody synthesis by 7S antibody. Science 145:395-396. Sahiar, K., and R. S. Schwartz, 1965. The immunoglobulin sequence. 1. Arrest by 6-mercaptopurine and restitution by antibody, antigen or splenectomy. J. Immunol. 95:345-354. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, New York. p. 107-109. Thaxton, P., J. E. Williams and H. S. Siegel, 1971. Microtitration of Salmonella pullorum agglutinins. Avian Dis. 14:813-816. Thaxton, P., and P. S. Young, 1974. Antibodymediated suppression of the primary hemagglutination response in young chickens. Poultry Sci. 53:1839-1842. Walker, J. G., and G. W. Siskind, 1968. Studies on the control of antibody synthesis. Effect of antibody
IMMUNOSUPPRESSION IN CHICKENS affinity upon its ability to suppress antibody formation. Immunol. 14:21—28. Wigzell, H., 1966. Antibody synthesis at the cellular level: Antibody induced suppression of 7S antibody synthesis. J. Exp. Med. 124:953-969. Williams, C. A., and M. W. Chase, 1967. Methods in
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Immunology and Immunochemistry. vol. 1, Academic Press, New York. Williams, C. A., Jr., and P. Grabor, 1955. Immunoelectrophoretic studies on serum proteins. 1. The a n t i g e n s of h u m a n serum. J. Immunol. 74:158-168.