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The Journal of Immunology, 2002, 169: 5028-5035.
Copyright © 2002 by The American Association of Immunologists

The {beta}2-Adrenergic Agonist Salbutamol Potentiates Oral Induction of Tolerance, Suppressing Adjuvant Arthritis and Antigen-Specific Immunity1

Pieter M. Cobelens*, Annemieke Kavelaars*, Anne Vroon*, Marion Ringeling*, Ruurd van der Zee{dagger}, Willem van Eden{dagger} and Cobi J. Heijnen2,*

* Department of Immunology, Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, and {dagger} Department of Infectious Disease and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Therapeutic protocols for treating autoimmune diseases by feeding autoantigens during the disease process have not been very successful to date. In vitro it has been shown that {beta}-adrenergic agonists inhibit pro-inflammatory cytokine production and up-regulate anti-inflammatory cytokine production. We hypothesized that the protective effect of oral administration of Ag would be enhanced by oral coadministration of the {beta}2-adrenergic agonist salbutamol. Here we demonstrate that oral administration of salbutamol in combination with the Ag mycobacterial 65-kDa heat shock protein increased the efficacy of disease-suppressive tolerance induction in rat adjuvant arthritis. To study the mechanism of salbutamol in more detail, we also tested oral administration of salbutamol in an OVA tolerance model in BALB/c mice. Oral coadministration of OVA/salbutamol after immunization with OVA efficiently suppressed both cellular and humoral responses to OVA. Coadministration of salbutamol was associated with an immediate increase in IL-10, TGF-{beta}, and IL-1R antagonist in the intestine. The tolerizing effect of salbutamol/OVA was maintained for at least 12 wk. At this time point IFN-{gamma} production in Ag-stimulated splenocytes was increased in the OVA/salbutamol-treated animals. In conclusion, salbutamol can be of great clinical benefit for the treatment of autoimmune diseases by promoting oral tolerance induction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigen-specific oral tolerance induction has been suggested as a novel approach to treat autoimmune diseases such as rheumatoid arthritis and multiple sclerosis (1, 2). However, clinical trials using soluble autoantigens or related Ags have not been very successful to date. In animal models of arthritis and experimental autoimmune encephalomyelitis it has been shown to be difficult to achieve tolerance by feeding Ag during the disease process, whereas feeding the Ag before disease induction was shown to be successful in decreasing the disease symptoms (3, 4, 5, 6, 7, 8, 9). There are only a few reports describing tolerance induction during disease or in previously primed animals despite the fact that this is the relevant clinical set-up to investigate the efficiency of therapeutic strategies for the treatment of inflammatory autoimmune diseases. Animals can be rendered tolerant only when high doses of Ag are administered orally immediately after immunization, before the onset of clinical signs. Similarly, multiple feedings of low doses of Ag during disease result in only a very modest suppression of immune responses or disease activity (10, 11, 12, 13). In summary, we can conclude that feeding soluble Ag alone is not sufficient to suppress an ongoing immune response or disease process. We propose that oral feeding of Ag in the context of an activated immune system can only be induced when an additional therapeutic strategy is applied to modulate the local cytokine milieu in the gut.

{beta}2-Adrenergic agonists, such as salbutamol, have a broad spectrum of immune regulatory activities. The {beta}2-adrenergic receptor ({beta}2-AR)3 is expressed on T cells, B cells, and macrophages and signals through a G protein of the Gs subtype that couples to adenylate cyclase (14, 15). Triggering of this neurotransmitter receptor on immune cells results in increased intracellular levels of cAMP, which will influence immune functioning (16, 17). {beta}2-Adrenergic agonists are known to inhibit the mitogenic responsiveness of lymphocytes mainly by decreasing IL-2 production (18, 19, 20). In addition, they have an important effect on the production of regulatory cytokines. {beta}2-AR stimulation of monocytes and macrophages leads to a decrease in IL-12 production and an increase in IL-10 production, thereby favoring an anti-inflammatory response (21, 22, 23). Moreover, it has been shown that in vitro administration of salbutamol to Ag-stimulated cultures results in an inhibition of IFN-{gamma} production and increased IL-4 production. This effect is probably due to an inhibitory effect of salbutamol on IL-12 production, which results in a shift in the Th1/Th2 cytokine balance (23).

We hypothesized that the protective effect of oral administration of Ag would be enhanced by oral coadministration of the {beta}2-adrenergic agonist salbutamol. To study the mechanism involved in the potentiating effect of salbutamol on oral tolerance induction, we analyzed intestinal and systemic cytokine production as well as specific Ab responses in an OVA-specific delayed-type hypersensitivity (DTH) model. The clinical relevance of oral coadministration of salbutamol for tolerance induction was tested in a rat model of adjuvant arthritis (AA).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Lewis rats (6–8 wk old; University of Limburg, Maastricht, The Netherlands) and BALB/c mice (6–8 wk old; Charles River, Sulzfeld, Germany) were kept at the Utrecht University animal facility and fed a standard diet (Hope Farms, Woerden, The Netherlands) and water ad libitum.

Induction and clinical evaluation of AA

AA was induced in male Lewis rats by a single intradermal injection of 100 µl of CFA (5 mg Mycobacterium tuberculosis (strain H37Ra)/ml IFA (Difco, Detroit, MI) at the base of the tail. The severity of arthritis was assessed daily by standard methods in a blinded protocol (24). A cumulative score was calculated for each individual animal by summation of the individual disease scores from day 11 until day 20.

Mycobacterial 65-kDa heat shock protein (HSP65) from Mycobacterium bovis was expressed in Escherichia coli and isolated as previously described (25). Rats were treated orally with 450 µg of salbutamol (Sigma, St. Louis, MO) together with 30 µg of HSP65 or superoxide dismutase (SOD; Sigma) in 1 ml of PBS using an 18-gauge animal-feeding needle (Popper & Sons, New York, NY). Treatment was started when most of the animals lost weight, that is, at the onset of clinical arthritis. Oral administration of HSP65 or SOD and salbutamol was repeated every other day for an additional four doses.

Induction and assessment of DTH

To test the OVA-specific immune response, a modified protocol from Chung et al. (10) was used. Female BALB/c mice were immunized i.p. with 20 µg of OVA (grade V; Sigma) emulsified in CFA (Difco). Eighteen days after immunization, mice were boosted i.p. with 20 µg of OVA in IFA (Difco). One week later mice were challenged by an s.c. injection into the footpad of 50 µl PBS containing 400 µg/ml OVA and 1 mg/ml AlOH3. After 24 h we measured the thickness of the footpad. The increase in paw thickness in vehicle-treated animals was <1%.

Mice were treated orally with 10 µg of salbutamol and 250 µg of OVA dissolved in 0.5 ml PBS using a 20-gauge stainless steel animal-feeding needle (Popper & Sons). Treatment was started 5 days after the first immunization. Oral administration of the drugs was repeated five times every other day. To analyze long term effects of the tolerance protocol, mice were boosted again with OVA in IFA on days 43 and 68 after immunization, followed by a challenge 7 days later.

In the pretreatment protocol, mice were fed six times 10 µg of salbutamol and 250 µg of OVA dissolved in PBS every other day from 13 days before immunization with OVA in CFA.

OVA-specific Ab production

Microtiter plates (Maxisorp F96; Nunc, Roskilde, Denmark) were coated with OVA (5 µg/ml) in PBS. After washing the plates with PBS/Tween (0.05%), the plates were blocked with 200 µl/well PBS/1% BSA (Sigma). Diluted serum samples were added to the wells and incubated for 2 h at room temperature. Biotin-conjugated goat anti-mouse IgG (Zymed, South San Francisco, CA) diluted in PBS/1% BSA/0.005% Tween 20 (IgGtotal, 1/10,000; IgG1 and IgG2a, 1/1,000) was added, and incubation was performed. Plates were developed with HRP-labeled streptavidin (1/10,000) and 3,3',5,5'-tetramethylbenzidine substrate (ICN Biomedicals, Zoetermeer, The Netherlands). Ab concentrations in units per milliliter were determined from standard curves constructed from a serially diluted serum pool. The maximal detectable level (top of the S-curve) in the standard serum pool was designated as containing 4000 U/ml.

Splenocyte culture

Splenocytes were cultured serum-free in quadruplicate in 200 µl at 1.5 x 105 cells/well in presence or the absence of OVA (125 µg/ml, final concentration).

Preparation of intestinal samples

Five days after immunization with OVA in CFA mice were treated orally with 10 µg of salbutamol. In some experiments naive mice were treated orally with 10 µg of salbutamol. Eight hours later mice were sacrificed, and 2 cm of distal ileum was collected, homogenized in PBS, and used for determining cytokine production.

Cytokine mRNA analysis

RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD), and cytokine mRNA expression was analyzed by an RNase protection kit (BD PharMingen, San Diego, CA). RNA expression was visualized by phosporimaging and Molecular Analyst software (Bio-Rad, Richmond, CA). Results are expressed as the percentage of household gene (L32) expression.

Cytokine ELISA

IFN-{gamma} and TNF-{alpha} were determined using ELISA kits from U-Cytech (Utrecht, The Netherlands). IL-10 and TGF-{beta} were determined using OptEIA kits from BD PharMingen.

Statistical analysis

Group differences were analyzed by one-way ANOVA, followed by Fisher’s least significance difference test or Student’s t test for unpaired data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oral administration of salbutamol potentiates HSP65-induced disease suppression in AA

We tested the possible clinical use of salbutamol to potentiate oral tolerance induction during a disease process in the AA model in Lewis rats. In an earlier study we had shown that ongoing AA in Lewis rats cannot be suppressed by feeding of cross-reactive Ag mycobacterial HSP65 alone. Therefore, we now coadministered salbutamol and HSP65. We started treatment at the onset of clinical signs of arthritis (on day 11 after immunization). From this time point rats received 450 µg of salbutamol and 30 µg of HSP65 orally every other day for a total of five doses. Control rats received salbutamol or HSP65 alone or 30 µg of SOD as an irrelevant Ag. Feeding HSP65 in combination with salbutamol significantly suppressed the clinical signs of arthritis (mean cumulative arthritis score, 40.2 ± 2.5 in SOD-treated rats vs 18.3 ± 2.4 in HSP65/salbutamol-treated animals; Fig. 1Go). Feeding irrelevant Ag SOD in combination with salbutamol had no significant effect on the arthritis score. In addition, treatment with salbutamol alone did not affect disease severity.



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  		FIGURE 1. Oral coadministration of mycobacterial HSP65 and salbutamol (Sal) reduces AA scores. From day 11 after induction of AA we treated rats (n = 8/group) orally every other day with 30 µg of HSP65 with or without 450 µg of salbutamol. Control rats received SOD with or without salbutamol. Similar results were seen in two separate experiments. **, p < 0.01, HSP65/salbutamol vs SOD and vs HSP65.

 
Oral coadministration of salbutamol and OVA after immunization with OVA-CFA induces tolerance

Since AA in rats is a monophasic disease, the model cannot be used to determine whether salbutamol modulates long term tolerance induction during an ongoing immune process. Therefore, we examined the effect of salbutamol on oral tolerance induction in a murine model of OVA-induced DTH. We immunized mice i.p. with OVA in CFA, and 18 days later animals received a booster with OVA in IFA. One week after boosting, we challenged the animals with OVA in the footpad and measured paw swelling 24 h later. Five days after immunization with OVA in CFA we started oral tolerance induction. From this time point onward mice received orally 10 µg of salbutamol and 250 µg of OVA every other day for a total of five doses. Control mice received PBS, OVA alone, or salbutamol alone. Oral administration of OVA or salbutamol alone had no significant effect on the DTH response (Fig. 2GoA). However, when we coadministered OVA and salbutamol the DTH response was reduced by 70% (p < 0.01; Fig. 2GoA).



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  		FIGURE 2. Oral OVA/salbutamol (Sal) after immunization reduces cellular immune responses. On day 0 we immunized the mice and on day 5 we started treatment every other day with oral administration of OVA and salbutamol. We boosted the mice with OVA in IFA on day 18 (A), day 43 (B), and day 68 (C). One week later, we determined the DTH response. All data represent the mean ± SEM of eight individual animals in each group and are representative of two independent experiments. **, p < 0.01, OVA/salbutamol vs PBS; ##, p < 0.01 vs OVA.

 
From a clinical perspective it is crucial to test the long term efficacy of oral coadministration of OVA and salbutamol. To this end mice that had been treated with one regimen of OVA/salbutamol from days 5–10 after immunization received a second and a third booster (i.p. injection of OVA in IFA) on days 43 and 68 after immunization. One week after each booster we determined the OVA-specific DTH response. OVA/salbutamol-treated animals were still tolerant after the second booster and even after a third booster (Fig. 2Go, B and C). The DTH response of mice that had been fed only OVA or only salbutamol did not differ significantly from the response in PBS-treated mice after two or three boosters (Fig. 2Go, B and C).

We also analyzed sera after the first booster (day 35 after immunization) from each group of mice for anti-OVA IgGtotal and the isotypes IgG1 and IgG2a to get an impression of the contribution of Th1-driven and Th2-driven Ab responses. As shown in Fig. 3Go, A–C, the Ab responses were lower in both the OVA- and salbutamol-treated groups than in PBS-treated mice for all isotypes. However, the responses were suppressed more profoundly in the OVA/salbutamol-treated group. Furthermore, the most dramatic decrease in Ab response after OVA/salbutamol treatment was found in the Th1-driven IgG2a isotype (>90% reduction, vs 70% in OVA-specific IgG1).



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  		FIGURE 3. Oral OVA/salbutamol (Sal) after immunization reduces humoral immune responses. On day 0 we immunized the mice and on day 5 we started treatment every other day with oral OVA and salbutamol. We boosted the mice with OVA in IFA on day 18 (A–C) and days 43 and 68 (D–F). On day 35 (A–C) and day 82 (D–F) we analyzed sera for OVA-specific Ab production. All data represent the mean ± SEM of eight individual animals in each group and are representative of two independent experiments. *, p < 0.05 vs PBS; **, p < 0.01 vs PBS; #, p < 0.05 vs OVA; ##, p < 0.01 vs OVA.

 
Even after the third booster (day 82 after immunization) the effect of one treatment regimen with OVA and salbutamol on Ab production was maintained (Fig. 3Go, D–F). Similar to what was observed for the DTH response, after the third booster there was no significant difference in IgG levels between the PBS group and the groups that received OVA alone or salbutamol alone (Fig. 3Go. D–F).

Effect of oral coadministration of salbutamol on OVA-specific tolerance induction before immunization

To address the question of when salbutamol can exert its immunomodulatory effect, we also examined the effects of salbutamol in a protocol in which oral tolerance was induced before induction of the immune response. Therefore, mice were first treated every other day with 250 µg of OVA in the absence or the presence of 10 µg of salbutamol six times. Three days after the last feeding animals were immunized with OVA in CFA. The DTH response was measured on day 25 after immunization with OVA-CFA. The results show that although oral feeding of OVA alone led to suppression of the observed DTH response, no additional effect of salbutamol on OVA-induced tolerance induction was observed (Fig. 4GoA).



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  		FIGURE 4. Oral OVA/salbutamol (Sal) before immunization does not potentiate tolerance induction. Naive mice were treated every other day with oral administration of OVA and salbutamol six times. Three days after the final treatment, mice were immunized with OVA in CFA. We boosted the mice with OVA in IFA on day 18. One week later we determined the DTH response (A) and on day 35 we analyzed sera for OVA-specific Ab production (B–D). All data represent the mean ± SEM of eight individual animals in each group and are representative of two independent experiments. *, p < 0.05 vs PBS; **, p < 0.01 vs PBS.

 
In addition, OVA-specific Ab titers were determined. OVA-treated animals showed reduced levels of OVA-specific IgGs, but coadministration of salbutamol did not lead to a more effective tolerance induction of the humoral response to OVA (Fig. 4Go, B–D).

Salbutamol modulates intestinal cytokine production

We next investigated the effect of salbutamol on the expression of regulatory cytokines in the small intestine. Five days after immunization with OVA in CFA, mice received a single dose of salbutamol orally. Eight hours later we prepared a homogenate of whole intestinal samples and determined pro- and anti-inflammatory cytokine expressions at both the mRNA and the protein level. Oral salbutamol treatment resulted in down-regulation of IFN-{gamma} and up-regulation of IL-10 mRNA expression in the small intestine (Fig. 5GoA). The expression of IL-12 mRNA was below the detection limit. Salbutamol did not affect mRNA expression of the pro-inflammatory cytokine IL-1{beta}. However, mRNA encoding the anti-inflammatory mediator IL-1R antagonist (IL-1Ra) was significantly augmented in mice given oral salbutamol compared with those given oral PBS (Fig. 5GoA). In a pilot study we also examined the effect of multiple treatments with salbutamol on intestinal cytokine production and obtained similar alterations in cytokine mRNA expression in the intestine (data not shown).



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  		FIGURE 5. Oral salbutamol changes cytokine in the small intestine of immunized animals. mRNA (A) and protein cytokine expression (B and C) of homogenates from the small intestine from PBS-treated ({square}) and salbutamol-treated ({blacksquare}) mice. mRNA cytokine expression is expressed as a percentage of household gene expression (L32). All data represent the mean ± SEM of 8–10 individual animals in each group. *, p < 0.05, salbutamol vs PBS; **, p < 0.01 salbutamol vs PBS.

 
We confirmed some of our mRNA data by measuring IFN-{gamma} and IL-10 at the protein level in intestinal samples by ELISA. In addition, we measured the inflammatory cytokine TNF-{alpha} and the anti-inflammatory cytokine TGF-{beta} by ELISA. The production of the inflammatory cytokines IFN-{gamma} and TNF-{alpha} was down-regulated in salbutamol-fed mice, whereas that of the anti-inflammatory cytokines IL-10 and TGF-{beta} was up-regulated compared with PBS-treated mice (Fig. 5GoB). Feeding OVA alone did not have any effect on intestinal IFN-{gamma} or IL-10 levels (IFN-{gamma}, 22 ± 2 pg/ml; IL-10, 135 ± 15 pg/ml). Moreover, the effect of oral administration of OVA plus salbutamol was similar to the effect of salbutamol alone on intestinal IFN-{gamma} and IL-10 levels (IFN-{gamma}, 12 ± 1 pg/ml; IL-10, 310 ± 30 pg/ml).

Finally, we investigated the effect of salbutamol on intestinal cytokine levels in naive animals. The data presented in Fig. 6Go demonstrate that feeding salbutamol to naive animals reduces the levels of IFN-{gamma}, IL-10, and TNF-{alpha} in the intestine and has no effect on intestinal TGF-{beta} levels. Feeding OVA alone to naive animals did not have any effect on intestinal cytokine levels, and addition of OVA to the salbutamol feeding did not further modulate intestinal cytokine levels (data not shown).



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  		FIGURE 6. Oral salbutamol changes cytokine in the small intestine of naive animals. Cytokine levels were determined in homogenized small intestine samples by ELISA after feeding PBS ({square}) or salbutamol ({blacksquare}). All data represent the mean ± SEM of six to eight individual animals in each group. *, p < 0.05, salbutamol vs PBS.

 
Salbutamol modulates Ag-induced cytokine production by splenocytes

One of the important questions is how salbutamol treatment alters the regulatory cytokine production of Ag-specific cells. To address this question we collected spleens on day 9 after immunization, i.e., after two feedings with OVA and salbutamol. Subsequently, we stimulated splenic cells in vitro for 72 h with OVA and determined IFN-{gamma} and IL-10 production. Control cultures without OVA did not contain detectable levels of IFN-{gamma} and IL-10. Feeding OVA or salbutamol alone elevates OVA-induced IFN-{gamma} production by splenocytes in vitro. Coadministration of OVA and salbutamol did not further enhance IFN-{gamma} production (Fig. 7GoA). In contrast, feeding OVA alone or salbutamol alone did not change IL-10 production by splenocytes. Only the coadministration of OVA and salbutamol resulted in an increase in IL-10 production after Ag-specific stimulation of splenocytes in vitro (Fig. 7GoB).



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  		FIGURE 7. Oral OVA/salbutamol (Sal) alters Ag-induced cytokine production by splenocytes. A and B, Animals were immunized with OVA/CFA and fed PBS, OVA, salbutamol, or OVA/salbutamol on days 5 and 7 after immunization. Alternatively, animals were fed PBS, OVA, salbutamol, or OVA/salbutamol according to the pretreatment protocol and subsequently immunized with OVA/CFA. We stimulated splenocytes with OVA on day 9 after immunization and assayed supernatant for IFN-{gamma} and IL-10 production. Data represent the mean ± SEM of six individual animals in each group. *, p < 0.05 vs PBS; **, p < 0.01, OVA/salbutamol vs PBS.

 
To investigate the effects of pretreatment with OVA and salbutamol on cytokine production by splenocytes, animals were fed OVA, salbutamol, and OVA plus salbutamol according to the pretreatment protocol, followed by immunization with OVA in CFA. On day 9 after immunization, splenocytes were collected and stimulated in vitro with OVA to induce cytokine production. The data in Fig. 7GoC show that pretreatment with OVA before immunization of the animals induced a significant increase in in vitro splenocyte IFN-{gamma} production. Pretreatment with salbutamol alone did not have any effect on IFN-{gamma} or IL-10 production. Moreover, in vitro splenocyte IFN-{gamma} production after coadministration of OVA and salbutamol was similar to that after feeding OVA alone (Fig. 7GoC). Splenocyte IL-10 production was not affected by pretreatment with OVA, salbutamol, or OVA/salbutamol (Fig. 7GoD).

Feeding OVA/salbutamol results in long term changes in Ag-induced cytokine production in vitro by splenocytes

To address the question of whether the long term tolerance of the DTH response is reflected by long term changes in the T cell compartment, we investigated Ag-induced cytokine production on day 35 after immunization. We treated mice orally with OVA/salbutamol from day 5 after immunization with OVA in CFA. Mice received a booster on day 18 after immunization. Two weeks later (day 35) we cultured the splenocytes in vitro in the presence of OVA for 72 h. At this time point, IFN-{gamma} production was significantly enhanced in OVA/salbutamol-treated animals compared with PBS-treated animals, whereas treatment with OVA or salbutamol alone did not significantly change IFN-{gamma} production (Fig. 8GoA). In contrast to the enhanced splenic IL-10 production immediately after feeding OVA/salbutamol (day 9), there was no longer a significant change in OVA-induced IL-10 production by splenocytes from OVA/salbutamol-treated animals on day 35 (Fig. 8GoB).



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  		FIGURE 8. Long term effects of feeding OVA/salbutamol (Sal) to immunized animals on Ag-induced cytokine production by splenocytes. Animals were immunized with OVA/CFA and fed PBS, OVA, salbutamol, or OVA/salbutamol. On day 35 after immunization and on day 82 after immunization, we stimulated splenocytes with OVA and assayed supernatant for IFN-{gamma} and IL-10 production. Data represent the mean ± SEM of six individual animals in each group. *, p < 0.05, vs PBS; **, p < 0.01, OVA/salbutamol vs PBS.

 
Interestingly, we observed the same effects of OVA/salbutamol treatment on OVA-induced cytokine production after three boosters of OVA in IFA (day 82 after immunization). There was no significant difference in IL-10 production but there was significantly elevated IFN-{gamma} production by splenocytes from OVA/salbutamol-treated mice (Fig. 8Go, C and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we show that oral tolerance induction during an ongoing immune response or an inflammatory disease process can be significantly improved by oral coadministration of the {beta}2-adrenergic agonist salbutamol. During AA in the rat as well as in an OVA-specific DTH model in mice, salbutamol potentiates the efficacy of oral tolerance induction ( Figs. 1–3GoGoGo). From a clinical point of view it is interesting that feeding salbutamol together with the Ag OVA from days 5 to 15 after immunization induces a long-lasting tolerance. Even after three boosters with OVA/IFA the cellular immune response in these mice was tolerant, as determined in the DTH model. We conclude that administration of salbutamol as an additional therapeutic strategy in tolerance protocols using (auto)antigens for the treatment of human cell-mediated autoimmune diseases has to be explored.

Investigating the effect of feeding OVA and salbutamol to already immunized animals on the humoral immune response resulted in a number of interesting observations. First of all, we show that repetitive feeding of a low dose of OVA alone after immunization can result in lower levels of anti-OVA IgG1 and IgG2a (Fig. 3Go). In addition, feeding salbutamol alone results in reduced IgG1 and IgG2a levels. The combination of OVA/salbutamol had an additive inhibitory effect on IgG1 levels, whereas the effect on IgG2a is potentiated by the combined treatment.

It should be noted that feeding OVA alone or salbutamol alone to immunized animals did not have any effect on the OVA-specific DTH, while it did decrease Ab production, suggesting that two at least partially independent mechanisms are involved in the modulation of humoral and cellular immune responses after feeding OVA and salbutamol in immunized animals (Figs. 2Go and 3Go). It may be possible that the reduced humoral immunity after feeding salbutamol alone to immunized animals results from direct immunomodulatory effects of salbutamol on the initiation phase of the humoral response. Such a direct immunomodulatory effect of salbutamol alone administered to immunized animals also may be responsible for the enhanced splenocyte IFN-{gamma} production on day 9 after immunization (Fig. 7Go). However, tolerization of DTH in immunized animals requires both OVA and salbutamol (Fig. 2Go). Therefore, we conclude that the reduced DTH response does not result from a direct immunomodulatory effect of salbutamol, but is the result of modulation of the response to oral OVA by salbutamol. This conclusion is further supported by our data on the immediate effects of feeding OVA/salbutamol to immunized animals on Ag-induced IL-10 production by splenocytes on day 9 after immunization; only after feeding the combination of OVA and salbutamol is IL-10 production by splenocytes increased (Fig. 7Go). Feeding OVA or salbutamol alone does not alter IL-10 production. Similarly, when we investigated the long term effects (on days 35 and 82 after immunization) of feeding OVA and salbutamol to immunized animals we observed that splenic IFN-{gamma} production was only increased after feeding OVA plus salbutamol (Fig. 8Go). At these later time points we no longer observed any effect of OVA alone or salbutamol alone on splenic IFN-{gamma} production.

Surprisingly, feeding OVA, salbutamol, or OVA plus salbutamol after immunization resulted in an immediate increase (as determined on day 9 after immunization) in splenocyte IFN-{gamma} production in all groups (Fig. 7Go). It has been reported previously that feeding a high dose of OVA to immunized animals can result in an immediate increase in splenocyte IFN-{gamma} production (26). However, to our knowledge this is the first report describing an immediate increase in splenic IFN-{gamma} by feeding a low dose of OVA.

The fact that feeding salbutamol alone to immunized animals also increases splenic OVA-induced IFN-{gamma} production is at odds with the reported effects of salbutamol on IFN-{gamma} production in vitro. In in vitro systems, {beta}2-adrenergic agonists have been shown to reduce IFN-{gamma} production via inhibition of IL-12 production by APC. However, it has also been shown that in conditions where IL-12 is not limiting, {beta}2-adrenergic agonists can enhance the production of IFN-{gamma} by T cells, presumably via enhancement of IL-12R signaling (27). Thus, {beta}2-adrenergic agonists such as salbutamol can either increase or decrease IFN-{gamma} production depending on the presence of additional signals. Our data suggest that in the in vivo situation tested here the enhancing effect of salbutamol on IFN-{gamma} production over-rules a possible inhibitory effect.

Interestingly, our data show that salbutamol only potentiated tolerance induction during an ongoing immune response and not when salbutamol was coadministered with the Ag before immunization (Fig. 4Go). Moreover, coadministration of salbutamol before immunization did not further modulate cytokine production by splenocytes as determined on day 9 after immunization of the animals (Fig. 7Go). Therefore, we conclude that {beta}2-adrenergic stimulation is only effective in potentiating tolerance induction in the context of an activated immune system. One possible explanation for this phenomenon would be that immunization increases the sensitivity of the {beta}2-AR on immune cells.

Indeed, we have shown recently that peripheral blood cells of patients with rheumatoid arthritis produce more cAMP upon triggering of the {beta}2-AR than cells of healthy control donors (28). Moreover, {beta}2-AR agonists could more efficiently suppress LPS-induced TNF-{alpha} production by peripheral blood cells of arthritis patients than the TNF-{alpha} production by cells of healthy donors. The increased receptor efficacy was due to a down-regulation of an intracellular enzyme, G protein-coupled receptor kinase 2 in peripheral blood cells of patients with rheumatoid arthritis and in rats with AA (28, 29). We and others have shown that down-regulation of the enzyme G protein-coupled receptor kinase 2 leads to less desensitization of the {beta}2-adrenergic receptors, resulting in a more efficient receptor coupling (28, 30, 31, 32). It may well be that increased sensitivity of {beta}2-adrenergic receptors observed during rheumatoid arthritis in humans and AA in rats is also present after immunization with OVA/CFA and is responsible for the potentiating effect of salbutamol.

In line with the in vivo observations, we did not observe an effect of feeding salbutamol alone before immunization on splenic OVA-induced IL-10 and IFN-{gamma} production after immunization (Fig. 7Go). Moreover, pretreatment with oral OVA alone or with OVA plus salbutamol leads to an increased IFN-{gamma} production after immunization to the same extent. Finally, there were differences with respect to the immediate effect of salbutamol on intestinal cytokine production in immunized vs nonimmunized animals. As an immediate effect of oral salbutamol or OVA/salbutamol administration in immunized animals, we observed an increase in intestinal production of anti-inflammatory cytokines, such as IL-10, and TGF-{beta}, whereas the production of proinflammatory cytokines, such as IFN-{gamma} and TNF-{alpha}, decreased (Fig. 5Go). In contrast, oral administration of salbutamol or OVA/salbutamol to naive animals before immunization reduced IFN-{gamma}, IL-10, and TNF-{alpha} levels and did not affect TGF-{beta} levels in the intestine (Fig. 6Go). In summary, in immunized animals salbutamol administration alters the intestinal pro-and anti-inflammatory balance, whereas in naive animals both pro- and anti-inflammatory cytokines are inhibited. Thus, our data suggest that changing the balance between pro- and anti-inflammatory cytokines in the gut is associated with potentiation of Ag-induced tolerance by salbutamol.

Although we do not know which cells in the intestine are responsible for the observed changes in cytokine production, it is conceivable that the predominant cellular component of the intestine, i.e., enterocytes, plays a major role. Firstly, we have preliminary evidence that purified enterocytes express {beta}2-adrenergic receptors (P. M. Cobelens, N. Eijkelkamp, A. Kavelaars, and C. J. Heijnen, unpublished observations). Secondly, it is known that enterocytes or enterocytic cell lines can actively produce a number of cytokines, including TNF-{alpha}, TGF-{beta}, IL-10, and IL-1Ra (33, 34, 35, 36, 37, 38, 39). Macrophages also express {beta}2-adrenergic receptors, and in vitro exposure to a {beta}2-agonist results in decreased TNF-{alpha} production as well as increases in TGF-{beta}, IL-1Ra, and IL-10, presumably via accumulation of intracellular cAMP (21, 22, 40, 41, 42, 43). Therefore, we propose that modulation of intestinal cytokine levels by oral salbutamol is the result of a direct effect of the drug on enterocytes. We cannot exclude, however, that effects of salbutamol on intestinal T cells or macrophages also contribute to the changes in cytokine expression in the intestine.

We show that OVA/salbutamol administration to immunized animals results in an immediate increase in Ag-induced IL-10 production by splenocytes, as determined on day 9. It is not clear, however, whether macrophages or T cells are responsible for the effects of salbutamol on IL-10 production, but we can exclude a direct effect on Th2-cells. Sanders et al. (44) have shown that only Th1 cells express {beta}2-adrenergic receptors, whereas mature Th2 cells do not at either the mRNA or the receptor protein level. In view of these data we conclude that increased OVA-induced IL-10 production by cells from mice fed OVA and salbutamol cannot result from a direct effect of salbutamol on Th2 cells. Probably, salbutamol will act by inhibiting IL-12 production or increasing IL-10 production by macrophages, thereby favoring IL-10 production by Ag-specific cells. It may well be possible that IL-10 is produced by Tr1 cells, and it is not known whether they express {beta}2-AR (45, 46).

We also show here that coadministration of OVA/salbutamol results in long-lasting tolerance for at least 12 wk. At this time point, however, cytokine patterns in the intestine are no longer different from those in PBS-treated animals (data not shown). Moreover, Ag-induced IL-10 production has returned to normal levels. In contrast, we still observed a marked increase in OVA-induced IFN-{gamma} production by splenocytes after one booster (at 5 wk) and after three boosters (at 12 wk) in OVA/salbutamol-treated animals (Fig. 8Go). Based on these findings we suggest that IFN-{gamma} plays an important role in the maintenance of tolerance. The latter statement is supported by the results of a recent study in which we could induce tolerance in the adjuvant arthritis model in the rat by feeding HSP65 provided a protease inhibitor (i.e., soybean trypsin inhibitor) was coadministered (24). Using this specific regimen, successful tolerance induction was associated with an increase in the production of HSP65-induced IFN-{gamma} production on day 35 after disease induction (P. M. Cobelens, A. Kavelaars, and C. J. Heijnen, unpublished observations). Moreover, in line with an important role for IFN-{gamma} in tolerance induction, it has been shown that tolerance could not be induced in IFN-{gamma} knockout mice (47). It should be noted, however, that there are also studies that suggest that IFN-{gamma} is not required for induction of tolerance. For example, there are studies showing that tolerance can be induced normally in IFN-{gamma} receptor-/- animals and in animals that do not produce IFN-{gamma} due to the complete absence of IL-12 (26, 48).

Based on our results, we suggest that long-lasting tolerance induction is not simply mediated by a decrease in the balance between pro- and anti-inflammatory cytokines, but will involve immediate up-regulation of anti-inflammatory cytokines during oral Ag administration as well as long-lasting up-regulation of some pro-inflammatory mediators, i.e., IFN-{gamma}, to control the ongoing immune or disease process. In this respect it is of interest that studies using animal models of autoimmune diseases suggest that IFN-{gamma} plays a role in down-regulating tissue inflammation (49, 50, 51, 52). Moreover, a study by Lee et al. (53) suggested that the induction of IFN-{gamma} by orally administered Ag contributes to systemic tolerance by decreasing the migration of T cells to peripheral sites of inflammation.

Oral coadministration of salbutamol to rats during AA has proven to be very effective in down-regulating the clinical symptoms of the disease. In a previous report we documented that no significant tolerance can be induced in AA in the rat by oral administration of multiple doses of the (cross-reactive) Ag mycobacterial HSP65 only (24). We show here that effective tolerance can be achieved with oral HSP 65 treatment by coadministration of salbutamol. Moreover, in the OVA model we showed that a single course of six feedings with salbutamol and Ag induces a long-lasting suppression of the immune response for at least 12 wk. The latter results underline the clinical importance of the present work and suggest that salbutamol or other specific {beta}2-adrenergic agonists already in clinical use for other purposes may be of great value for improving the therapeutic efficacy of oral tolerance induction protocols using autoantigens for the treatment of pro-inflammatory autoimmune diseases in man.


   Acknowledgments
 
We thank Dr. Graham Rook for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by Star-Grant SW07 from the University Medical Center Utrecht. Back

2 Address correspondence and reprint requests to Dr. Cobi J. Heijnen, Laboratory for Psychoneuroimmunology, KC03.068.0 University Medical Center Utrecht, Wilhelmina Children’s Hospital, Lundlaan 6, 3584 EA Utrecht, The Netherlands. E-mail address: c.heijnen{at}wkz.azu.nl Back

3 Abbreviations used in this paper: {beta}2-AR, {beta}2-adrenergic receptor; AA, adjuvant arthritis; DTH, delayed-type hypersensitivity; HSP65, 65-kDa heat shock protein; IL-1Ra, IL-1R antagonist; SOD, superoxide dismutase. Back

Received for publication December 17, 2001. Accepted for publication September 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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