On-line version ISSN 1414-431X
Braz J Med Biol Res vol. 31 n. 11 Ribeirão Preto Nov. 1998
Braz J Med Biol Res, November 1998, Volume 31(11) 1421-1424 (Short Communication)
F.M. Oliveira, A.F. Silva-Neto, C.A. Silva and C.M. Gontijo
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil
Correspondence and Footnotes
Oral tolerance is a phenomenon that may occur in animals exposed to protein antigens for the first time by the oral route. They become unable to produce immune responses at the levels normally observed when they are immunized parenterally with antigen in the presence of adjuvants. Lipids have been used as adjuvants for both parenteral and oral immunization. In the present study we coupled ovalbumin with palmitate residues by incubating the protein with the N-hydroxysuccinimide palmitate ester and tested the preparation for its ability to induce oral tolerance. This was performed by giving 20 mg of antigen to mice by the oral route 7 days prior to parenteral immunization in the presence of Al(OH)3. Mice were bled one week after receiving a booster that was given 2 weeks after primary immunization. Specific antibodies were detected by ELISA. Despite the fact that the conjugates are as immunogenic as the unmodified protein when parenterally injected in mice, they failed to induce oral tolerance. This discrepancy could be explained by differences in the intestinal absorption of the two forms of the antigen. In fact, when compared to the non-conjugated ovalbumin, a fast and high absorption of the lipid-conjugated form of ovalbumin was observed by "sandwich" ELISA.
Key words: oral tolerance, lipid-protein conjugates, antigen absorption, gut mucosa, ovalbumin
The gut mucosa represents the principal region through which most of the antigens penetrate the body. This is mainly due to its extensive area, as well as to the presence of different substances derived from food and of the intestinal flora. The absorption of such antigens is mainly due to the presence and activity of M cells which are in close association with the Peyer's patches (1,2). In the local lymphoid tissue, antigens may induce the production of secretory IgA, which also regulates their absorption, as well as a phenomenon called oral tolerance (3-5). In this case, animals that have been orally treated with antigen usually do not produce humoral or cellular immune responses when they are later parenterally immunized, even in the presence of adjuvants (6-8). Many factors can interfere with the occurrence of oral tolerance, such as the nature of the antigen as well as the dose, frequency and time interval between doses (9). There are also other host-related factors, such as its immunological history, age and genetic background, that interfere with the phenomenon (8-13).
In most of our studies on the induction of oral tolerance (5,8,10,13) we have chosen B6D2F1 mice (an abbreviation for (C57/Bl6 x DBA/2) F1 mice) as hosts and ovalbumin (Ova) as antigen. The possibility of coupling hydrophobic palmitate residues to proteins, which interested us when studying antigen processing and presentation by B cells carrying surrogate antigen receptors (14-17), opened new perspectives for the study of the mechanism of oral tolerance induction. Lipids have been used as adjuvants in many protocols for both parenteral and oral immunization.
The aim of the present study was to determine whether the coupling of palmityl residues to ovalbumin would affect oral tolerance induction in B6D2F1 mice of both sexes, obtained from our breeding unit at the Federal University of Minas Gerais, and used when approximately 8 to 12 weeks old. The lipid-ovalbumin conjugates (Ova-palm) were prepared according to a technique previously described by our group (17), although some small modifications were introduced. Briefly, 1 g of the N-hydroxysuccinimide palmitate ester (Sigma Chemical Co., St. Louis, MO) dissolved in 100 ml of absolute ethanol (55oC) was mixed with 4 g chicken egg albumin (ovalbumin) grade V (Sigma) dissolved in 800 ml of phosphate-buffered saline (PBS) with 0.1% NaHCO3, pH 8, in the presence of 0.6% deoxycholate (DOC; Sigma). After overnight incubation at room temperature, the mixture was centrifuged and the precipitated conjugate (Ova-palm) was collected and washed in 50 mM NaHCO3. After freezing in liquid nitrogen, the precipitate was lyophilized.
In order to test the tolerogenic properties of the conjugate, we performed the following protocol for the induction of oral tolerance (by gavage) which we had described previously for ovalbumin (5). Briefly, after being lightly anesthetized with ether, mice were intubated with a urethral polyvinyl catheter calibrated to reach the stomach, and given 20 mg of antigen (Ova or Ova-palm) in 0.3 ml of saline (0.15 M NaCl) intragastrically (ig). Gavages were performed 7 days prior to parenteral immunization, when mice were then injected intraperitoneally (ip) with 0.5 ml saline containing 10 µg Ova or Ova-palm mixed with 1 mg Al(OH)3 as adjuvant. Two weeks later, mice were submitted to the same protocol as a booster, but without Al(OH)3. Mice were bled one week after secondary immunization, and after clotting and centrifugation the serum samples were collected and stored at -20oC. The presence of anti-Ova antibodies in the samples was assayed by ELISA procedures. Briefly, polystyrene plates (Nunc, Copenhagen, Denmark) were coated overnight at 4oC with 2 µg Ova diluted in 100 µl coating buffer per well, washed with saline containing 0.05% (w/v) Tween-20, saturated with 0.25% (w/v) casein in PBS, washed again and then coated with serial dilution of mouse antiserum starting at 1:40. After 1 h at 37oC, plates were washed, incubated for 1 h at 37oC with goat anti-mouse globulin antiserum conjugated to peroxidase (Southern Biotechnology, Birmingham, AL), washed and developed by the addition of H2O2 and ortho-phenylene-diamine (OPD; Sigma); the reaction was interrupted at 20 min by the addition of H2SO4 at 1/20, and read at 492 nm in EIA-reader (Bio-Rad, Hercules, CA). The absorbance values obtained in the assays were reported as a score (ELISA*), which represents the mean ± SEM (N = 4-6) of the sums of the absorbance values of ELISA tests run with serum dilutions of 1/40 to 1/5120. This way of expressing the results was equivalent to reporting the area under the titration curves or electing absorbance at one particular serum dilution as representative. The significance of the difference between experimental and control groups was assessed by two-tailed Student t-tests. Positive and negative control samples were run on every plate to standardize the assays.
As shown in Figure 1, mice produced the same amount of anti-Ova antibodies after being orally treated with saline and immunized with either Ova-palm or unmodified Ova. This happened despite the presence of hydrophobic residues in Ova-palm conjugates, which might modify the shape of the protein and induce the display of a different set of epitopes. This view is supported by the fact that the conjugates are not as soluble as ovalbumin. As expected, mice orally pretreated with Ova displayed significantly lower levels of anti-Ova antibodies after being parenterally immunized with either form of the antigen. On the other hand, oral pretreatment with Ova-palm did not induce tolerance. Both forms of the antigen were able to elicit the production of anti-Ova antibodies at the same level as controls.
Since Ova and Ova-palm conjugates exhibited similar immunogenic characteristics but different tolerogenic properties, we designed a protocol to evaluate the kinetics of their intestinal absorption. Mice were treated orally with both forms and, at different times, 100 µl of blood samples were collected from the retro-orbital plexus with calibrated micropipettes (H.E. Pedersen, Copenhagen, Denmark). Samples were immediately diluted in 0.4 µl saline and, after clotting and centrifugation, the supernatant was collected for detection of the antigen by "sandwich" ELISA. Briefly, rabbit anti-Ova antibodies were purified by affinity chromatography using sepharose-4B columns (Pharmacia, Uppsala, Sweden) coupled with Ova. These antibodies were used to coat polystyrene plates as described above. After blocking with PBS/casein, washing and addition of supernatants, plates were incubated for 1 h. The same rabbit anti-ova antibodies were biotinylated. Briefly, 2 mg of antibodies and 50 µg of biotinyl-N-hydroxysuccinimide ester (Sigma) were dissolved and mixed in 1.0 ml 0.1 M NaHCO3. After a 4-h incubation at room temperature, the solution was dialyzed against PBS. The biotinylated antibodies were allowed to react with streptavidin coupled with peroxidase (Sigma) for 1 h and the reaction was completed as described above. A standard solution of ovalbumin was run in parallel for quantification of the antigen in the samples. As we can see in Figure 2, when compared to mice orally treated with Ova, the antigen was found earlier and at higher levels in the blood of mice that were orally treated with the same amount of Ova-palm.
In the present communication, we have shown that ovalbumin coupled with palmitate residues is unable to induce oral tolerance. On the other hand, its immunogenic property remains the same when administered parenterally. We also noticed that antibodies produced by mice immunized with both forms of the antigen can be equally detected by ELISA using plates coated with either unmodified or palmitate-conjugated ovalbumin (data not shown). These data indicate that the antigenic characteristics, as well as the immunogenic properties, remain the same.
An explanation for the lack of tolerance induction by Ova-palm conjugates may be related to excess antigen. Antigen doses are crucial for oral tolerance induction (9). Since we observed high levels of intestinal absorption with the conjugates, we tried to induce oral tolerance with the conjugate using lower amounts of the antigen (10-100 times less), but again we were unable to induce oral tolerance (data not shown).
As mentioned earlier in this paper, although little is known concerning their mechanisms of action, lipids have been used as adjuvants for both parenteral and oral immunization. Their use includes the preparation of liposome vesicles, immunostimulating complexes (ISCOMs) and water-in-oil emulsions like Freund's complete and incomplete adjuvants (18-20). We suggest that ovalbumin coupled with lipid residues might induce immune responses instead of tolerance when administered by the oral route.
1. Sneller MC & Strober W (1986). M cells and host defense. Journal of Infectious Diseases, 154: 737-741. [ Links ]
2. Neutra MR & Kraehenbuhl JP (1994). Mucosal immunization via M cells for production of protective secretory IgA antibodies. American Journal of Tropical Medicine and Hygiene, 50: 10-13. [ Links ]
3. Husband AL & Gowans JL (1978). The origin and antigen-dependent distribution of IgA-containing cells in the intestine. Journal of Experimental Medicine, 148: 1146-1160. [ Links ]
4. Challacombe SJ & Tomasi TB (1980). Systemic tolerance and secretory immunity after oral immunization. Journal of Experimental Medicine, 152: 1459-1472. [ Links ]
5. Rios MJC, Pereira MAC, Lopes LM, Gontijo CM, Castanheira EB & Vaz NM (1988). Tolerance and priming initiated by mucosal contact with protein antigen in inbreed strains of mice. Brazilian Journal of Medical and Biological Research, 21: 825-836. [ Links ]
6. Wells HG (1911). Studies on the chemistry of anaphylaxis. III. Experiments with isolated proteins, especially those of the hen's egg. Journal of Infectious Diseases, 9: 147-171. [ Links ]
7. Thomas HC & Parrot MV (1974). The induction of tolerance to a soluble protein antigen by oral administration. Immunology, 27: 631-639. [ Links ]
8. Vaz NM, Maia LCS, Hanson DG & Lynch JM (1977). Inhibition of homocytotropic antibody responses in adult mice by previous feeding with the specific antigen. Journal of Allergy and Clinical Immunology, 60: 110-115. [ Links ]
9. Peng HJ, Turner MW & Strobel S (1989). The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration. Immunology, 67: 425-430. [ Links ]
10. Hanson DG, Vaz NM, Maia LCS & Lynch JL (1979). Inhibition of specific immune responses by feeding protein antigens. III. Evidence against maintenance of tolerance to ovalbumin by orally-induced antibodies. Journal of Immunology, 123: 2337-2344. [ Links ]
11. Strobel S & Ferguson A (1984). Immune response to fed protein antigens in mice. 3. Systemic tolerance or priming is related to age at which antigen is first encountered. Pediatric Research, 18: 588-594. [ Links ]
12. Mowat AM, Lamont AG & Bruce MG (1987). A genetically determined lack of oral tolerance to ovalbumin is due to failure of the immune system to respond to intestinally-derived tolerogen. European Journal of Immunology, 17: 1673-1676. [ Links ]
13. Vaz NM, Rios MJC, Lopes LM, Gontijo CM, Castanheira EB, Jacquemart F & Andrade LAB (1987). Genetics of susceptibility to oral tolerance to ovalbumin. Brazilian Journal of Medical and Biological Research, 20: 785-790. [ Links ]
14. Gontijo CM & Möller G (1991). Membrane-incorporated immunoglobulin receptors increase the antigen-presenting ability of B cells. Scandinavian Journal of Immunology, 34: 577-584. [ Links ]
15. Gontijo CM & Möller G (1992). Increasing the efficiency of lipid-conjugated antibodies incorporation into the membrane of antigen presenting B cells. Brazilian Journal of Medical and Biological Research, 25: 909-912. [ Links ]
16. Gontijo CM & Möller G (1993). Membrane-incorporated surrogate receptors increase antigen presentation by resting B cells, but not by LPS-activated blasts. Scandinavian Journal of Immunology, 37: 59-63. [ Links ]
17. Gontijo CM & Möller G (1993). B cells carrying surrogate receptors in their membranes process and present antigen to specific murine T cells. Scandinavian Journal of Immunology, 38: 565-569. [ Links ]
18. Davis SS, Hunneyball IM, Illum L, Ratcliffe JH, Smith A & Wilson CG (1985). Recent advances in the use of microspheres for targeted therapy. Drugs under Experimental and Clinical Research, 11: 633-640. [ Links ]
19. Lovgren K & Morein B (1988). The requirement of lipids for the formation of immunostimulating complexes (iscoms). Biotechnology and Applied Biochemistry, 10: 161-172. [ Links ]
20. Davis SS, Washington C, West P, Illum L, Liversidge G, Sternson L & Kirsh R (1987). Lipid emulsions as drug delivery systems. Annals of the New York Academy of Sciences, 507: 75-88. [ Links ]
Address for correspondence: C.M. Gontijo, Departamento de Bioquímica e Imunologia, ICB-UFMG, Caixa Postal 486, 30161-970 Belo Horizonte, MG, Brasil. Fax: +55-31-441-5963. E-mail: firstname.lastname@example.org
Presented at the XIII Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, Brasil, August 26-29, 1998. Research supported by CNPq (No. 400421/93.3), FAPEMIG (No. 59/93) and PRPq-UFMG (No. 23072049103/9393). F.M. Oliveira was supported by the PIBIC scholarship program (CNPq). A.F. Silva-Neto and C.A. Silva were supported by the PROBIC scholarship program (FAPEMIG). Received April 9, 1998. Accepted August 3, 1998.