antibody response to different concentrations of theantigen (BSA). In contrast, in animals given BSA alone, antigen concentrations above a certain dose resulted ...
INFECTION AND IMMUNITY, Aug. 1977, p. 361-365 Copyright 0 1977 American Society for Microbiology
Vol. 17, No. 2
Printed in U.S.A.
Effect of Vitamin A on the Systemic and Local Antibody Responses to Intragastrically Administered Bovine Serum Albumin KENNETH R. FALCHUK,* W. ALLAN WALKER, JOSEPH L. PERROTTO,
AND
KURT J.
ISSELBACHER Departments of Medicine and Pediatrics, Harvard Medical School, the Pediatric Gastrointestinal and Gastrointestinal Units, Massachusetts General Hospital, Boston, Massachusetts 02114, and the Gastrointestinal Unit, Peter Bent Brigham Hospital,* Boston, Massachusetts 02115 Received for publication 10 August 1976
The effects of vitamin A on the immune response to bovine serum albumin (BSA) were studied in adult mice. Treatment with vitamin A by the intragastric or parenteral routes markedly increased the local as well as the systemic antibody response to different concentrations of the antigen (BSA). In contrast, in animals given BSA alone, antigen concentrations above a certain dose resulted in a decreased or even absent anti-BSA response. These studies suggest that vitamin A may be an appropriate adjuvant in oral immunization.
Orally administered macromolecules, such as bovine serum albumin (BSA), are absorbed by both the neonatal and adult mammalian intestine (21) and have been demonstrated to induce a local (22) as well as a systemic immune response (15). It is also known that the administration of adjuvants can result in an enhanced immune response to parenterally administered antigen (13). The present investigation was undertaken to study the adjuvant effect of vitamin A on the local and systemic immune responses after the intragastric (i.g.) administration of different concentrations of BSA.
per animal. Simultaneously, other animals (group II) received, by the same route, 3,000 USP units of vitamin A palmitate (Aquasol A; USV Pharmaceutical Corp., Tuckahoe, N.Y.) per day for 2 days before and on the day of antigen (BSA) administration. Other mice (group III) were given 10 mg of BSA i.g. and 3,000 USP units of vitamin A palmitate as a subcutaneous (s.c.) injection, following the same schedule as that for mice immunized by the i.g. route. All animals were subsequently boosted twice, at 2-week intervals, by the i.g. route with the same dose of BSA. In addition, animals in groups II and III were also given 3,000 USP units of vitamin A (i.g. or s.c., respectively) with each dose of BSA. (ii) Parenteral immunization. As a control, mice (group IV) were immunized with 10 mg of BSA dissolved in 0.2 ml of phosphate-buffered saline, MATERIALS AND METHODS given by intraperitoneal (i.p.) injection, and were Animals. Three-week-old female Swiss albino subsequently boosted twice with 100 ,ug of BSA at 2mice, obtained from Charles River Breeding Co. week intervals. Simultaneously, other mice (group (Wilmington, Mass.), were fed on a BSA-free diet V) were given 3,000 USP units of vitamin A palmi(Purina rat chow; Ralston Purina Co., St. Louis, tate by the same route for 2 consecutive days before and on the day of antigen administration, according Mo.) for 4 weeks. Immunization. Groups of 8 to 10 mice, 7 to 8 to the schedule of Cohen and Cohen (2), and later weeks old (Table 1), were immunized by various challenged with 100 ,ug of BSA at 2-week intervals. routes with BSA (fraction V; Nutritional Biochemi- As an additional control for the vitamin A palmitate cals Corp. Cleveland, Ohio) according to the follow- preparation used, another vitamin A preparation, vitamin A alcohol (retinol; St. Louis, Mo.), solubiing schedule. (i) Oral Immunization. BSA was cleared of aggre- lized in an equal volume of peanut oil, was used in gates and polymers by ultracentrifugation at 40,000 animals given 10 mg of BSA i.g. To evaluate immurpm for 30 min. The BSA, as determined by Sepha- nological memory, BSA was withheld for 7.5 weeks. dex (G-100) gel filtration, was greater than 90% After this period of time all mice were again boosted monomers under these conditions. Mice (group I) with BSA. Animals were bled by puncture of the were fed i.g. via polyethylene tubing (PE-50) with retro-orbital sinus 14 days after the initial immuni0.2 ml of 1.3% sodium bicarbonate (NaHCO3), fol- zation (bleed 1), 10 days after each boost (bleeds 2, 3, lowed 30 min later by the administration of mono- and 5), and on the day of the final boost with BSA meric BSA dissolved in 0.2 ml of phosphate-buffered (bleed 4). Sera from all animals in each group were saline (0.01 M phosphate buffer, pH 7.4, in 0.14 M pooled and stored at -20°C. All experiments deNaCl) in concentrations ranging from 0.5 to 50 mg scribed were repeated a minimum of three times. 361
INFIECT. IMMUN.
FALCHUK ET AL.
362
Quantitation of anti-BSA in serum and mucosal extracts. The antigen-binding activity (ABC) of serum and extracts of intestinal mucosal scrapings were determined according to a modification (23) of the ammonium sulfate method of Minden and Farr (12). Crystalline BSA (Nutritional Biochemicals) was iodinated with carrier-free Nal25I (New England Nuclear, Boston, Mass.) by using a modification of the method of Greenwood et al. (9). Mucosal extracts were obtained as previously described (23), and the protein concentrations of these extracts were determined by the method of Lowry et al. (11). The small intestine was everted over a glass rod and subsequently scraped with a glass slide. The mucosal scrapings were homogenized in a tissue grinder (Potter-Elvehjem homogenizer) and then suspended in 10 ml of saline. This suspension was centrifuged at 40,000 rpm for 30 min at 4°C, and the supernatant was saved. This step was repeated once and the combined supernatants from all animals were pooled as "mucosal extracts." After dialysis in water and lyophilization, stored fractions were reconstituted in 2 ml of a 1:10 dilution of normal mouse serum in borate buffer (0.1 M, pH 8.4).
RESULTS
Serum ABC. (i) i.g. BSA alone. The serum ABC (as micrograms per milliliter) in mice given different concentrations of BSA by the i.g. route is shown in Table 1. In these animals the serum anti-BSA activity could not be detected until after a second dose of BSA and subsequently increased considerably after the third antigen boost (bleed 3). The highest serum ABC was found in those mice immunized with the 10-mg dose of BSA. No anti-BSA activity was demonstrated in animals given the lowest (0.5 mg) or the highest (50 mg) dose of BSA. Moreover, an increase in the dose of BSA above 10 mg per animal did not result in a greater
BSA (mg)
ABC. After withholding the antigen (BSA) for 7.5 weeks, the ABC decreased (bleed
serum
4). (ii) Parenteral BSA alone. In mice immunized by the i.p. route, the serum ABC was undetectable on day 14, was 4.8 ,ug/ml on day 25, and increased to 28 ,g/ml on day 38. As in previous groups, the serum anti-BSA activity decreased to 8 ,ug/ml on day 80 and increased to 42 ,tg/ml 10 days after the final dose of BSA (day 90). i.g. BSA and vitamin A. Table 2 shows the serum ABC (as micrograms per milliliter) in mice given vitamin A and different concentrations of BSA by the i.g. route. Antibodies to BSA were demonstrated in all groups. In mice immunized with the lowest concentration of BSA, antibodies to BSA were found only after the third dose of BSA, whereas, in those given the highest dose of antigen, the anti-BSA response was demonstrated after the initial dose of BSA. In the remaining groups, antibodies to BSA were found after the second dose of BSA. In contrast to mice immunized only with BSA, the serum ABC of animals given vitamin A and BSA increased as the concentration of the antigen increased from 0.5 to 50 mg per animal. No difference in antibody response was noted in animals orally immunized with 10 mg of BSA and given vitamin A alcohol, orally or parenterally, rather than vitamin A palmitate. No difference was observed in the ABC of mice given BSA and vitamin A by the i.g. route and those given BSA i.g. and vitamin A by s.c. injection (Fig. 1). i.p. BSA and vitamin A. In mice immunized with BSA and vitamin A by the i.p. route, the serum anti-BSA response was demonstrated
TABLE 1. ABC in serum of mice immunized by i.g. and parenteral routes ABC (Ag/ml) on bleed no:a 2 (day 25)
1 (day 14)
4 (day 80)
3 (day 38)
i.g.
5 (day 90)
0.5 1 10 20
0 0 0 0
0.0 0.4 (0.3-0.5) 0.6 (0.4-0.7) 0.1 (0.08-0.2)
0.0 1.0 (0.7-1.3) 5.0 (3.2-6.4) 0.8 (0.5-0.9)
0.0 0.13 (0.05-0.2) 0.3 (0.22-0.38) 0.0
0.0 0.8 (0.7-0.94) 3.6 (2.4-4.5) 1.8 (0.9-2.4)
50b
0
0.0
0.0
0.0
0.0
Parenteral 42 (32-56) 8.0 (6.4-9.2) 28 (15-38) 4.8 (3-6.4) 0 10 a Data are expressed as micrograms of 125I-labeled BSA bound per milliliter of serum at an ABC-33 of 0.2 zg of 1251-labeled BSA protein. ABC-33 refers to the reciprocal dilution of antiserum that will precipitate 33% of the labeled antigen added to the test system (23). Average value of pooled serum of three groups of 8 to 10 mice. Ranges of values are shown in parentheses. Mice were bled 14 days after the initial dose of BSA (bleed 1), 10 days after each boost (bleeds 2, 3, and 5), and after withholding BSA for 7.5 weeks (bleed 4). b No ABC could be demonstrated in the serum of these mice 10 days after the i.p. challenge with BSA given the day after bleed 5.
VOL. 17, 1977
IMMUNE RESPONSE TO BSA AND VITAMIN A
363
TABLE 2. ABC in serum of mice immunized i.g. with BSA and treated with vitamin A ABC
BSA (mg)
Day 90
Day 80
Day 28
Day 25
Day 14
(Ag/ml)a
0.0 0.0 0.2 (0.16-0.3) 0.0 0.0 1.6 (0.9-2) 0.0 0.8 (0.6-1) 0.5 (0.3-1.7) 0.0 9.0 (7.4-12) 1.6 (1.3-2.5) 6.6 (5-10.4) 1.8 (1.6-2.2) 0.0 8.5 (6.2-11.4) 3.0 (2.2-3.6) 10.0 (7.8-14) 5.0 (3.8-6.4) 0.0 9.6 (6.4-13) 0.8 (0.6-1.1) 15.4 (11.5-18.2) 6.0 (4-7.6) 0.6 (0.3-0.8) a Data are expressed as micrograms of 125I-labeled BSA bound per milliliter of serum at an ABC-33 of 0.2 ,ug of 125I-labeled BSA protein. ABC-33 refers to the reciprocal dilution of antiserum that will precipitate 33% of labeled antigen added to the text system (23). Average value of pooled serum of three groups of 8 to 10 mice. Ranges of values are shown in parentheses. 0.5 1 10 20 50
12f 10 8f
V/ TAMIN A
0 --OBSA Only
T
IjI.GE ]J.
*-* BSA + Vitamin A
I-SE
SE
160 _
6r
t 120 4
/
80 2
14J~~~~~01
0
25
40
80
100
DA YS
FIG. 1. Comparison of serum ABC (ABC-33 of 0.2
of l25I-labeled BSA protein, as micrograms per milliliter plus or minus standard error [SE] at various time intervals (25, 40, 80, and 100 days) after initial immunization in mice immunized by i.g. administration of BSA (10 mg) and given vitamin A either ig. (group II) or s.c. (group III) as an adjupg
vant.
earlier (bleed 1) and was greater than in mice immunized with BSA without vitamin A (Fig. 2). Intestinal anti-BSA activity. The ABC in mucosal extracts obtained 10 days after the fourth boost is demonstrated in Table 3. Antibodies to BSA were found in all groups of mice immunized by the i.p. and i.g. routes except in those given 50 mg of BSA i.g. Among animals immunized by the i.g. route, the highest mucosal anti-BSA activity was demonstrated in the group given vitamin A and the highest concentration of BSA (50 mg). The ABC of mucosal extracts was higher in all mice immunized with BSA and vitamin A, compared with those given BSA only. As expected from previous studies (23), the highest anti-BSA activity was demonstrated in those animals immunized with BSA and vitamin A by the i.p. route.
(1
0
O
-
10
20
30
40
50
DA YS FIG. 2. Comparison of immunological response in mice immunized i.p. with BSA (10 mg) alone (0, group IV) and with BSA and parenterally administered vitamin A used as an adjuvant (-, group V). Serum ABC (as micrograms per milliliter plus or minus standard error [SE]) were determined at various time intervals after immunization. (See text for specific immunization schedule.)
DISCUSSION It is known that the normal adult mammalian intestine maintains a protective barrier against the absorption of ingested antigens as well as bacteria and endotoxin. Several investigators (3, 20) have demonstrated that macromolecules such as BSA and horseradish peroxidase are absorbed by the adult intestine. Cornell et al. (3) showed that soluble antigens are initially engulfed in membrane-bound vesicles and are then either combined with lysosomes, resulting in breakdown, or passed undigested to the lateral surface of the cell and then extruded by reversed pinocytosis into the interstitial space. These antigens may then come in contact with regional lymphoid tissue or pass
364
IN4FECT. IMMUN.
FALCHUK ET AL.
TABLE 3. ABC in serum and mucosal extracts of mice immunized with BSA or BSA with vitamin A Mucosal extractsc Animals Serumb (jg/ml) i.g. BSA (1 mg) BSA (1 mg), vitamin A BSA (10 mg) BSA (10 mg), vitamin A BSA (20 mg) BSA (20 mg), vitamin A BSA (50 mg) BSA (50 mg), vitamin A
0.8 1.6 3.6 9.0 1.8 8.5 0.0 9.6
(0.7-0.94) (0.9-2) (2.4-4.5) (7.4-12) (0.9-2.4) (6.2-11.4)
(6.4-13.2)
jg/group
jig/mg
0.6 3.1 1.0 2.8 1.8 2.4 0.0 5.2
0.01 0.05 0.02 0.05 0.03 0.04 0.00 0.09
i.p. BSA (10 mg)
42.0 (32-56) 1.2 0.02 16.0 220 (174-248) 0.3 BSA (10 mg), vitamin A a Animals were sacrificed 10 days after the boost 4. b Data are expressed as average micrograms of 125I-labeled BSA bound by 1 ml of pooled serum at an ABC33 (see legend to Fig. 2) of 0.2 ,ug of l25I-labeled BSA protein. Ranges of values are in parentheses. c Data are expressed as micrograms of 125I-labeled BSA bound by the pooled mucosal extracts of each group as well as micrograms per milligram of mucosal protein. Mucosal extracts were prepared as a single pool from 10 animals and, therefore, are representative rather than averages.
into the systemic circulation and induce either a local or a systemic immune response. Warshaw and Walker (19) demonstrated in rats that a small but antigenically significant fraction of BSA manages to traverse the mature gut, entering the lymphatic effluent and portal circulation. Furthermore, the amount of protein absorbed was proportional to the dose of BSA given. Rothberg et al. (14) demonstrated the presence of antibodies to BSA in the serum of rabbits after ingestion of this protein. These antibodies, although quantitatively different, were qualitatively similar to those of parenterally immunized animals, suggesting absorption of quantities sufficient to reach the systemic circulation and result in systemic immunization. Vitamin A, a lysosomal membrane labilizer (4), has been shown to augment the immune response to various antigens, such as erythrocytes (10), BSA (17), ovalbumin (2), and bovine gamma globulin (5). In the present study we found that the administration of nontoxic doses of two preparations of vitamin A (palmitate and alcohol) before and at the time of sensitization with different concentrations of BSA resulted in an increased production of antibodies to this protein. After the i.g. administration of BSA alone, circulating antibodies to this antigen were also demonstrated, but, contrary to the results observed in mice given BSA and vitamin A, an increase in the dose of BSA above the concentration of 10 mg per animal did not result in an increased serum or intestinal anti-BSA response. Furthermore, in mice given the highest dose of BSA by the i.g. route, no
ABC could be demonstrated, and later, even after an i.p. challenge with BSA (Table 1), these animals failed to respond. This finding suggested to us that a state of immunological unresponsiveness was established in these animals. The observation that i.g. immunization with a higher concentration of BSA may result in a lower secondary immune response or even in a state of immunological unresponsiveness (paralysis) confirms the findings of Sercarz and Coons (16) and Grantham and Fitch (8) of a decreased or even absent antibody response to larger doses of antigen given by the parenteral route. Although we have not elucidated the mechanisms responsible for the inhibition of antibody production by high doses of antigen given by the i.g. route, others have hypothesized that either small quantities of manufactured antibody (7) or a circulating factor may play a role as an inhibitor of antibody production (6). Andre et al. (1) recently demonstrated that the enteric immunization of mice with sheep erythrocytes may result in a state of immunological hyporesponsiveness. Moreover, the serum of these animals was capable of conferring tolerance to nonimmune recipient mice and caused in vitro paralysis in activity of antibody-producing cells. This enterically induced toleragen seems to consist of immune complexes with immunoglobulin A as the antibody. In mice immunized i.g. with the same concentration of BSA, the adjuvant effect of vitamin A given by either the i.g. or the s.c. route was similar. Furthermore, we found that in vitamin A-treated animals the local as well as the systemic antibody response to BSA in-
VOL. 17, 1977
IMMUNE RESPONSE TO BSA AND VITAMIN A
creased as the concentration of BSA increased and was greatest in those animals immunized with the highest dose of BSA. With the administration of vitamin A, we potentiated the immune response to increasing concentrations of free BSA and were able to break the state of immunological unresponsiveness that results from enteric immunization with large doses of BSA. Although our intent was not to distinguish between local and systemic production of antibodies, our findings demonstrate that: (i) i.g. immunization with BSA results in a systemic immune response, (ii) antigen concentrations above a certain level of i.g.-administered BSA (10 mg) can result in a decreased or even absent antibody response, and (iii) these studies confirm the role of vitamin A as an immunological adjuvant on the systemic and possibly on the local (intestinal) response to antigens given by various routes. This adjuvant effect of vitamin A has been attributed to an increased lysosomal membrane permeability (17) or to an increased lymphocyte proliferation in lymph nodes (18), as well as to altered macrophage antigen degradation and processing (2). Further studies need be done to establish the mechanism of enhanced responsiveness in these conditions. ACKNOWLEDGMENTS This research was supported by Public Health Service grant AM-16269 from the National Institute of Arthritis, Metabolism and Digestive Diseases. LITERATURE CITED 1. Andre, C., J. F. Heremans, J. P. Vaerman, and C. L. Cambiaso. 1975. A mechanism for the induction of 2.
3. 4.
5.
6.
immunological tolerance by antigen feeding: antigenantibody complexes. J. Exp. Med. 142:1509-1519. Cohen, B. E., and I. K. Cohen. 1973. Vitamin A. Adjuvant and steroid antagonist in the immune response. J. Immunol. 111:1376-1380. Cornell, R., W. A. Walker, and K. J. Isselbacher. 1971. Small intestinal absorption of horseradish peroxidase. A cytochemical study. Lab. Invest. 25:42-48. Dingle, J. T. 1961. Studies on the mode of action of excess of vitamin A. Release of a bound protease by the action of vitamin A. Biochem. J. 79:509-512. Dreser, D. W. 1968. Adjuvanticity of vitamin A. Nature (London) 217:527-529. Fauci, A. S., and J. S. Johnson. 1971. Suppression of antibody synthesis. Evidence for a circulating inhibi-
365
tor of antibody synthesis demonstrable at the cellular level. J. Immunol. 107:1052-1062. 7. Grantham, W. G. 1972. The secondary response to high and low dose priming in mice. J. Immunol. 108:562565. 8. Grantham, W. G., and F. W. Fitch. 1975. The role of antibody feedback inhibition in the regulation of the secondary antibody response after high and low dose priming. J. Immunol. 114:394-398. 9. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation of '31I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123. 10. Jurin, M., and I. F. Tannock. 1972. Influence of vitamin A on immunological response. Immunology 23:283-287. 11. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 12. Minden, P., and R. S. Farr. 1967. Handbook of experimental immunology, p. 463-492. Blackwell Inc., Oxford. 13. Nakashima, I., T. Kobayshi, and N. Kato. 1971. Alterations in the antibody response to bovine serum albumin by capsular polysaccharide of Klebsiella pneumoniae. J. Immunol. 107:1112-1121. 14. Rothberg, R. M., S. C. Kraft, and R. S. Farr. 1967. Similarities between rabbit antibodies produced following ingestion of bovine serum albumin and following parenteral immunization. J. Immunol. 98:386416. 15. Rothberg, R. M., S. C. Kraft, and S. M. Michalek. 1973. Systemic immunity after local antigenic stimulation of the lymphoid tissue ofthe gastrointestinal tract. J. Immunol. 111:1906-1913. 16. Sercarz, E. E., and A. H. Coons. 1963. The absence of antibody-producing cells during unresponsiveness to BSA in the mouse. J. Immunol. 90:478-491. 17. Spitznagel, J. K., and A. C. Allison. 1970. Mode of action of adjuvants: retinol and other lysosome-labilizing agents as adjuvants. J. Immunol. 104:119-127. 18. Taub, R. N., R. K. Adrienne, and D. W. Dresser. 1970. The effect of localized injection of adjuvant material on the draining lymph node. Immunology 18:171-186. 19. Warshaw, A. L., and W. A. Walker. 1974. Intestinal absorption of intake antigenic protein. Surgery 76:495-499. 20. Warshaw, A. L., W. A. Walker, and K. J. Isselbacher. 1974. Protein uptake by the intestine. Evidence for absorption of intact micromolecules. Gastroenterology 66:987-992. 21. Walker, W. A., and K. J. Iselbacher. 1974. Uptake and transport of macromolecules by the intestine. Possible role in clinical disorders. Gastroenterology 67:531-550. 22. Walker, W. A., K. J. Isselbacher, and K. J. Bloch. 1972. Intestinal uptake of macromolecules. Effect of oral immunization. Science 177:608-610. 23. Walker, W. A., K. J. Isselbacher, and K. J. Bloch. 1973. Intestinal uptake of macromolecules. Effect of parenteral immunization. J. Immunol. 111:221-226.
