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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We have previously demonstrated that rats fed ovalbumin (OVA) develop a tolerogenic activity in serum, which upon transfer induces tolerance to OVA and suppression of the immune response to a bystander antigen. Here, we have extended these studies and analysed if the tolerogenic activity in serum could suppress an established immune response in the recipients. Rats were immunized with OVA, 4 and 1 week prior to the transfer of serum from either OVA-fed or control animals.

Rats that received serum from OVA-fed donors had significantly lower delayed-type hypersensitivity (DTH) reaction against OVA 1 week after the serum transfer compared with the controls, and the levels of immunoglobulin (IgG) anti-OVA antibodies were significantly lower 2 and 4 weeks after serum transfer. Monomeric OVA in amounts corresponding to the OVA transferred with serum did not induce the reduction of DTH response or IgG anti-OVA antibody levels. In vitro, the proliferation of OVA-stimulated spleen cells, taken from recipients of tolerogenic serum, was significantly lower compared with spleen cells from the controls. The in vitro suppression seemed to be mediated by a population of CD25+ cells, because the removal of such cells from OVA-stimulated spleen cell suspensions resulted in increased proliferation in cultures from rats receiving tolerogenic serum. Our results showed that the tolerogenic serum factor can suppress an established immune response in recipient animals, possibly through induction of regulatory CD25+ cells. Whether this capacity might be used to influence chronic inflammatory conditions needs to be investigated.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Gut processing of antigen induces a tolerogenic circulating factor as first observed in 1983 by Strobel et al. [1]. This observation has been confirmed in several other studies [2–8]. Attempts have been made to modify ovalbumin (OVA) by chemical alteration, by denaturation or by deaggregation, but none of these procedures induced the same hyporesponsiveness as intestinally processed OVA [2, 3]. Accordingly, it has been shown that the tolerogenic activity in serum is not directly related to the amounts of circulating immunoreactive OVA after feeding [4–6]. We recently showed that the serum factor partly consists of major histocompatibility complex (MHC) class-II positive exosome-like structures assembled in and released from the small intestinal epithelial cell [9], which transfer antigen-specific tolerance to recipient animals. In agreement with this finding, severe combined immunodeficiency (SCID) mice which are unable to produce the tolerogenic serum factor do not produce vesicles expressing MHC class-II molecules and lysosome-associated membrane protein-1 (LAMP-1) inside their small intestinal epithelial cells [10]. Our hypothesis is that this exosome-like structure, named tolerosome, is responsible for the initial events leading to the development of active suppression mediated by regulatory lymphocytes. We know that the tolerogenic serum factor transfers bystander suppression and induces regulatory CD25+ cells which are of importance for the tolerance in the recipients [11]. Accumulating data demonstrate that a population of CD25+ T cells has immunosuppressive properties. The CD25+ T cells downregulate the immune responses mainly via cell contact, probably with the involvement of cytotoxic T-lymphocyte-associated molecule (CTLA)-4 and membrane-bound tumour growth factor (TGF)-β[12], but also the release of suppressive cytokines such as TGF-β and interleukin (IL)-10 has been documented [13–19]. There is a possibility that the initial events after feeding, resulting in the production of a tolerogenic serum factor and the subsequent development of oral tolerance, might be used not only to prevent but also to suppress an ongoing immune response such as a chronic inflammation. Studies have indicated that an established immune response can be suppressed by feeding the animal with the relevant protein [20–22]. An advantage with the utilization of a tolerogenic serum factor instead of feeding with whole protein might be that only regulatory cells are activated by the exosome-like structure produced by the intestinal epithelium, reducing the risk of undesired immune response owing to nontolerogenic antigenic epitopes of the native protein.

We show in this study that gut processing of a protein forms a specific tolerogenic serum factor which can suppress an established immune response in the recipients. The suppression involved regulatory CD25+ lymphocytes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Eight-week-old inbred male Wistar rats (recipients) and 12-week-old outbred male Wistar rats (donors) were obtained from BK Universal (Stockholm, Sweden) and kept in our animal facilities (Göteborg University, Department of Clinical Immunology, Göteborg, Sweden) under standard conditions.

Immunization Four weeks before serum transfer, recipient rats were immunized subcutaneously (SC) in the hind legs with 100 µg of OVA (Grade V; Sigma Chemical Co., St. Louis, MO, USA) or 100 µg of human serum albumin (HSA) (Grade V; Sigma) mixed with complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). Two booster immunizations were carried out in the same way and with the same doses but in Freund's incomplete adjuvant (Difco Laboratories) 1 week prior to and 6 weeks after serum transfer. One week after the last immunization, the rats were killed by CO2 exposure. Blood samples were collected from the tip of the tail at different time points during the experiment.

In parallel, five donor rats were immunized, 1 week after 2 h of OVA-feeding, with 100 µg of OVA mixed in CFA (Difco Laboratories).

Serum transfer procedure Sixty 12-week-old rats were starved overnight and then fed an OVA-containing diet or a standard diet lacking OVA (controls) (AnalyCen, Lidköping, Sweden) for 2 h. The estimated intake of OVA during feeding was 400 mg/rat. Two hours after feeding, blood was collected by heart puncture. The obtained sera were pooled in one OVA-fed pool (30 rats) and one control-fed pool (30 rats). Recipient rats were injected with 3.5 ml of serum intraperitoneally (IP) on the same day as the serum was collected, with the OVA concentration in serum being 300 ng/ml (see below).

Determination of OVA concentration in serum after 2 h of feeding Round bottom microtitre plates (Greiner Labortechnik Ltd., Cambridge, UK) were coated overnight with 5 µg/ml of polyclonal affinity purified goat immunoglobulin (IgG) anti-OVA antibodies (Cappel, West Chester, PA, USA). After washing, 100 µl of serum was added to the wells and serially diluted in eight 2-fold steps and incubated for 3 h at room temperature. The plates were washed three times with phosphate-buffered saline (PBS)-Tween before alkaline phosphatase-conjugated goat IgG anti-OVA antibodies (Cappel) were added overnight. The enzyme activity was visualized by p-nitrophenyl phosphate (1 mg/ml; Sigma) dissolved in diethanolamine buffer, pH 9.8. The absorbance values (405 nm; Titertek multiscan, VA, USA) were compared with those obtained for an OVA standard. The sensitivity was 3 ng/ml.

Preparation of monomeric OVA OVA (Sigma) at 5 mg/ml in PBS was ultracentrifuged at 100 000 × g for 3 h, and the absorbance (280 nm) was measured before and after the centrifugation in an ultraviolet (UV)-spectrophotometer. The upper one-third was collected as monomeric OVA, diluted in PBS to 300 ng/ml, and 3.5 ml/rat was immediately injected IP into preimmunized recipients.

As a second control, 3.5 ml of nonultracentrifuged OVA (Grade V; Sigma) at a concentration of 300 ng/ml in PBS was injected IP 1 week after the secondary immunization into recipient rats.

Delayed-type hypersensitivity (DTH) reaction One week after serum transfer and 2 weeks after the first booster immunization, the rats were intracutaneously challenged with 50 µg of OVA or HSA in 20 µl of PBS in each ear. The ear thickness was measured in a blinded fashion before and 24 h after challenge with a micrometer caliper (Oditest, Kroplin, Hessen, Germany). In a preliminary experiment, donor rats fed OVA for 2 h were intracutaneously challenged with 50 µg of OVA in 20 µl of PBS in each ear 2 weeks after the immunization with OVA.

Determination of antibody levels IgG antibodies against OVA and HSA were measured by an enzyme-linked immunosorbent assay (ELISA). Round bottom microtitre plates (Greiner Labortechnik) were coated with OVA (5 µg/ml) or HSA (5 µg/ml) (no blocking procedures were necessary) in PBS and incubated overnight. The serum samples were diluted in PBS-Tween (0.05%) in eight 5-fold steps and incubated overnight. Next, the plates were incubated with rabbit anti-rat IgG antibodies, diluted 1/5000 (Zymed Laboratories Inc, San Francisco, CA, USA) for 2–3 h at room temperature. Alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies, diluted 1/10 000 (Sigma), were then added to the plates and incubated for 1 h. The enzyme activity in the wells was visualized by the substrate p-nitrophenyl phosphate (1 mg/ml; Sigma) dissolved in diethanolamine buffer, pH 9.8. The absorbance was measured in a spectrophotometer at 405 nm (Titertek multiscan). The incubations were performed in humid atmosphere at room temperature. Between the incubations, the plates were washed with 3 × 200 µl of PBS-Tween in an ELISA washer (Titertek plus; ICN Biomedicals Inc., Costa Mesa, CA, USA).

The antibody activity was expressed in arbitrary ELISA units calculated from a standard curve obtained with a hyperimmune serum.

Cell preparations and cell cultures Spleens were removed 1 week after the last immunization and squeezed through a fine wire grid and filtered through a nylon filter (Falcon; Becton-Dickinson, NJ, USA). The cells were suspended in Hank's medium and centrifuged in Ficoll (Ficoll-Paque Research Grade; Pharmacia Biotech AB, Uppsala, Sweden) for 30 min at 400 × g without break at room temperature. The mononuclear cell layer was collected and resuspended in Hank's solution. After repeated washes in Hank's solution, the cells were suspended in Iscove's medium supplemented with 10% foetal calf serum, 1%l-glutamine, 1% sodium pyruvate and gentamicin 100 µg/ml (Garamycin; Schering-Plough Co., Ireland).

The cells were finally cultured at a concentration of 4 × 105 viable cells/well in 200 µl of Iscove's supplemented medium in flat bottom 96-well plates (Nunc, Roskilde, Denmark). The cultures were stimulated with: (1) concanavalin A, 2 µg/well (ConA, Sigma), and (2) OVA, 80 µg/well (Sigma). OVA and ConA were sterile filtered (0.22 µm; Millipore S.A 67, Molheims, France) and diluted in Iscove's supplemented medium. The cells were incubated in 5% CO2 atmosphere for 72 h and pulsed with 1 µCi/well [3H]-thymidine (Amersham International, Amersham, UK) for 12–16 h. The cells were then harvested and dried (Inotech; Ninolab AB, Upplands Väsby, Sweden), and the incorporated [3H]-thymidine was measured in a β-counter (Matrix96; Canberra Packard, Uppsala, Sweden).

Depletion of CD25+ cells and fluorescence-activated cell sorter (FACS) analyses Spleen cells from the group receiving serum from OVA-fed animals were pooled into three separate cell suspensions. The controls were also pooled into three separate cell fractions. The cells were incubated with an optimal dilution of mouse anti-rat CD25 (OX-39; the clone was obtained from the European collection of animal cell cultures, Salisbury, UK) or control antibody of the same isotype. A second incubation was done with a goat anti-mouse IgG fluorescein isothiocyanate (FITC)-conjugated antibody (Star 70; Serotec, Oxford, UK) and 1% rat serum. A final incubation with Mini Macs mouse anti-FITC beads (Miltenyi Biotec GmbH, Bergish Gladbach, Germany) was performed. All incubations were done on ice for 20 min, and the cells were washed with FACS-buffer (5 mm ethylenediaminetetraacetic acid (EDTA), 0.25% bovine serum albumin, 0.02% sodium azide, pH 7.2) between the incubations. The cell suspensions were then added to Mini Macs separation columns (Miltenyi). The cells not retained in the columns were seeded out for proliferation. A FACScan (Becton-Dickinson) was gated for lymphocytes, and the percentage of CD25+ cells in the different cell fractions was determined

Statistics The Mann–Whitney U-test was used for statistical analyses.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

No detection of a mixed lymphocyte reaction between spleen cells from donor and recipient rats

We investigated if there was a mixed lymphocyte reaction (MLR) between spleen cells from donor (Wistar) and recipient (Wistar furth) rats. Spleen cells from the rat strains were mixed, and the in vitro proliferation was tested. As demonstrated in Fig. 1, no MLR was detected between spleen cells from the outbred and inbred rat strain. In contrast, mixing spleen cells from two noncongenic rat strains induced an MLR.

image

Figure 1. Mixed lymphocyte reaction. Spleens were removed and prepared into single cell suspensions and mixed 1 : 1. The bars represent five different animals, and the standard error of the mean is indicated. Statistical analyses were done using the Mann–Whitney U-test.

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Two hours of feeding (donor rats) induces tolerance against OVA

Five animals were fed OVA-diet for 2 h (estimated intake of OVA was 400 mg/rat) and immunized with OVA in CFA 1 week later. The OVA-fed rats had a significantly lower DTH response against OVA 2 weeks after the immunization compared with control animals receiving the standard diet for 2 h (Fig. 2).

image

Figure 2. Delayed-type hypersensitivity (DTH) reaction against ovalbumin (OVA) in serum donor animals. Animals were fed OVA or control diet (control-fed) for 2 h and immunized with 100 µg of OVA mixed with complete Freund's adjuvant 1 week later. Ear challenge was done 2 weeks after the immunization. The DTH reaction was measured as increase in ear thickness 24 h after the challenge with 50 µg of OVA in 20 µl of phosphate-buffered saline (PBS). The bars represent the mean value of five different animals in each group, and the standard error of the mean is indicated. Statistical analyses were done using the Mann–Whitney U-test.

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An established T-cell-mediated immune response can be suppressed with a tolerogenic serum factor

Rats were immunized with OVA SC in the hind legs 4 weeks and 1 week prior to serum transfer. The concentration of OVA in the donor serum after 2 h of feeding was approximately 300 ng/ml. Preimmunized rats that received serum from OVA-fed donors had a significantly lower DTH reaction against OVA than animals receiving control serum (Fig. 3).

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Figure 3. Delayed-type hypersensitivity (DTH) reaction against ovalbumin (OVA). Rats were subcutaneously immunized two times with 100 µg of OVA in Freund's adjuvant. One week after the last immunization, they were given either serum from OVA-fed rats (n = 8), serum from control-fed rats (n = 8), monomeric OVA (n = 5) or native OVA (n = 5). The amounts of native OVA and monomeric OVA were the same as in the serum from OVA-fed animals. Ear challenge was performed 1 week after the serum transfer with 50 µg of OVA in 20 µl of phosphate-buffered saline (PBS). The DTH reaction was measured as increase in ear thickness 24 h after challenge. The bars represent the mean value, and the standard error of the mean is indicated. Statistical analyses were done using the Mann–Whitney U-test.

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Rats injected with the corresponding amounts of native (nonultracentrifuged) or monomeric OVA as that present in the serum from OVA-fed animals showed no suppression of their immune response against OVA (Fig. 3), indicating that the tolerogenic activity found in serum was modified by OVA.

The serum factor did not have a general immune suppressive activity, because animals immunized with HSA two times and then given serum from OVA-fed animals had the same magnitude of DTH reaction against HSA as animals only immunized with HSA (Fig. 4A).

image

Figure 4. (A) Delayed-type hypersensitivity (DTH) reaction against human serum albumin (HSA). Rats were subcutaneously (SC) immunized with 100 µg of HSA in Freund's adjuvant 4 weeks and 1 week prior to injection of serum from ovalbumin (OVA)-fed animals. The control group was treated in the same manner but did not receive serum. Ear challenge with 50 µg of HSA in 20 µl of PBS was performed 1 week after the serum transfer. The DTH reaction was measured as increase in ear thickness 24 h after challenge. The bars represent the mean value of eight animals, and the standard error of mean is indicated. Statistical analyses were done using the Mann–Whitney U-test. (B) Serum immunoglobulin (IgG) anti-HSA antibodies in rats, which were treated in the same way as in A. The bars represent the mean value of eight animals, and the standard error of the mean is indicated. Statistical analyses were done using the Mann–Whitney U-test.

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The effect of the tolerogenic serum factor on IgG anti-OVA antibody levels in preimmunized rats

The levels of IgG anti-OVA antibodies were similar in both groups prior to the transfer of serum (Fig. 5). Two weeks after the serum transfer, the group that received serum from OVA-fed animals had significantly lower levels of IgG anti-OVA antibodies in serum than control animals given serum from rats fed on standard diet. The level of IgG anti-OVA antibodies continued to be reduced throughout the experiment in the group receiving tolerogenic serum. The injection of monomeric or native OVA in the same amount as in serum from OVA-fed animals did not downregulate the IgG anti-OVA antibody levels in serum compared with controls (Fig. 6). Suppression of the IgG anti-OVA antibody production was antigen-specific as the level of IgG anti-HSA antibodies in rats immunized with HSA was unaffected by the transfer of serum from OVA-fed animals (Fig. 4B). In addition, the total IgG antibody levels in serum were similar in both groups (not shown).

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Figure 5. Serum immunoglobulin (IgG) anti-ovalbumin (OVA) antibodies in rats immunized with 100 µg of OVA in Freund's adjuvant, 4 weeks and 1 week before transfer of serum from OVA-fed or control-fed animals. The dots represent the mean value of eight different animals in each group, and the standard error of the mean is indicated. Statistical analyses were done using the Mann–Whitney U-test.

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image

Figure 6. Serum immunoglobulin (IgG) anti-ovalbumin (OVA) antibodies. Rats were immunized with 100 µg of OVA in Freund's adjuvant 4 weeks and 1 week before the injection of either phosphate-buffered saline (PBS), monomeric or native OVA. The amount of native OVA and monomeric OVA was the same as in the serum from OVA-fed animals. The bars represent the mean value of five different animals in each group, and the standard error of the mean is indicated. Statistical analyses were done using the Mann–Whitney U-test.

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In vitro proliferation against OVA and the effect of depleting CD25+ cells

OVA-stimulated spleen cell cultures from rats receiving serum from OVA-fed animals proliferated significantly less in vitro compared with control cultures (Fig. 7A). The ConA-stimulated proliferation was approximately the same in both groups. Depletion of CD25+ cells from the spleen suspensions made from rats receiving tolerogenic serum resulted in increased proliferation against OVA (Fig. 6). The number of CD25+ cells in the spleen cell cultures was before depletion approximately 7% and after depletion 1.5%.

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Figure 7. (A) In vitro proliferation of spleen cells after stimulation with ovalbumin (OVA), and (B) the effect of depletion of CD25+ cells from the spleens. Rats were immunized with 100 µg of OVA in complete Freund's adjuvant 4 weeks before transfer of serum from either OVA-fed or control-fed rats. Booster immunizations were performed 1 week prior to and 6 weeks after the serum transfer in the same way as the primary immunization but with incomplete Freund's adjuvant. The spleen cells were prepared 1 week after the last booster immunization. The bars represent the mean value of cultures from eight different animals in each group, and the standard error of the mean is indicated. Statistical analyses were done using the Mann–Whitney U-test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study demonstrates that the transferable tolerogenic serum factor induced by feeding a protein can suppress an established immune response to the same antigen in recipient rats. Such in vivo downregulation was related to reduced levels of IgG anti-OVA antibodies in serum and decreased DTH reaction against OVA. We also showed that the proliferation of OVA-stimulated spleen cells was lower in vitro if the rats had received serum from OVA-fed donors. Cells expressing CD25 at least partly mediated the suppression because it was abolished by the removal of such cells. The time point for serum transfer (1 week after a secondary immunization) was based on the fact that we, in earlier studies, had noticed that rats developed a marked antibody production against OVA at this particular point.

The transfer of serum did not induce nonspecific immune suppression in the recipients, as the group that received serum from OVA-fed animals had a normal immune response against HSA. This indicated that potential allogenic antibodies in the transferred serum did not interfere with the parameters that we used to detect the induction of tolerance in the recipients.

It has earlier been shown that an ongoing immune response to a protein can be suppressed by concomitant feeding of the protein [20–23]. Thus, Leishman et al. demonstrated that it was possible with one single feed of OVA (25 mg) to suppress the DTH reaction and the in vitro proliferation of OVA-stimulated lymph node cells in preimmunized animals [22]. The antibody production against OVA was not affected by feeding though. This was in contrast to our result where we also noted decreased specific antibody production after serum transfer. The discrepancy between direct feeding of protein and injection of the serum factor on specific antibody levels in preimmunized animals might suggest that the tolerogenic serum factor preferentially induces downregulatory events. Feeding to preimmunized animals might initially stimulate both suppression and a more ‘aggressive’ immune response to the antigen. We have previously noted that animals fed OVA had higher levels of IgG anti-OVA antibodies 5 days after immunization with OVA than control-fed animals [24]. This early priming effect in the OVA-fed group was then replaced by the suppression of the IgG anti-OVA antibody levels compared with control-fed rats.

It could be argued that the difference in IgG anti-OVA antibody levels between controls and preimmunized rats given serum from OVA-fed donors was too low to have any biological effect (see Fig. 5). However, the lower level of antibodies in this group correlated with a moderate DTH reaction and a lower in vitro proliferative response against OVA. In addition, it is known from other studies that low concentrations of antibodies can dramatically affect the immune system, probably reflecting the delicate balance within the immune system [25].

Regulatory activity has previously been ascribed to CD25+ cells, both in animal and in human studies [26–30]. Regulatory CD25+ T cells do not proliferate after T-cell receptor stimulation; i.e. they seem to be anergic. Studies on autoimmune and colitis models demonstrate that the CD25+ cells are induced in the thymus, and once activated with the antigen, require no further activation, and that the effector phase is antigen independent [31, 32]. The CD25+ cells suppress the immune response through cell contact, but the release of suppressive cytokines by these regulatory cells has also been demonstrated [33, 34]. If exogenous IL-2 or anti-CD28 antibodies are added to CD25+ T cells, their regulatory capacity is abrogated [18, 19, 35]. In agreement with the present study, the finding that regulatory CD25+ T cells induced by components in the gut mucosa are antigen specific has also recently been shown by Zhang et al. [36]. They showed that feeding OVA transgenic mice with OVA induces CD25+ T cells that have the ability to suppress the proliferation of CD25 T cells after OVA stimulation.

It has not yet been firmly established where and how the serum factor is generated, but ‘gut processing’ of the actual protein antigen has been demonstrated to be necessary [3]. The status of immunocompetent cells in the intestine is also important for the development of the serum factor, as highlighted by the fact that SCID mice and irradiated mice are unable to produce this factor. Furrie et al. showed using immunoblotting techniques covering 2.5–6.8 kDa, two bands at 2.1 and 2.4 kDa in tolerogenic serum but not in control serum [8]. In agreement with this observation, we have seen tolerogenic activity in a fraction of serum containing components smaller than 100 000 Da, but the suppressive activity was more pronounced in a fraction from serum containing structures larger than 100 kDa [11]. This finding suggested that the serum factor is not exclusively monomeric OVA, which has a molecular weight of 43 kDa, and in addition a larger structure. Also in this study we found that monomeric or native OVA was unable to downregulate the established immune response to OVA in the recipients. Altogether, these observations imply that the OVA is processed in the gut to become tolerogenic or that some larger antigen-specific structure is induced. We recently presented a description of MHC class-II positive exosome-like structures in serum, tolerosomes, assembled in and released from the small intestinal epithelial cell with the capacity to induce antigen-specific tolerance in recipients [9].

Two hours of OVA-feeding, with an estimated intake of approximately 400 mg OVA, was sufficient to induce tolerance to OVA in the donors. We would like to stress that the DTH values in Fig. 1 were measured after one immunization following 2 h of feeding and cannot directly be correlated with the DTH values in Fig. 2, which was obtained after two immunizations before the injection of serum.

We believe that the serum factor might be one component in the development of oral tolerance in animals fed on proteins, perhaps acting both locally in the gut and in the periphery. Future work will focus on the characterization of the serum factor or the so-called tolerosomes.

In conclusion, we have demonstrated that rats fed OVA developed a tolerogenic serum factor that was able to suppress an established immune response to OVA in recipient animals. CD25+ regulatory T cells were involved in the suppression, as the removal of such cells from spleen cell suspensions led to increased in vitro proliferation. Further investigations are needed to examine if the serum factor can be used to suppress immune-mediated chronic inflammations.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank the staff in the animal house at Sahlgrenska sjukhuset for their invaluable help with the animals, and Paul Bland and Per Brandtzaeg for critical reading of the manuscript. This research was supported by grants from the Swedish Medical Research Council (No. 215), Vårdal Foundation and Konsul Th C Berghs Foundation in Sweden.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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