1 Department of Immunology, 2 Department of Otorhinolaryngology, Head and Neck Surgery and 3 Department of Dermatology, Faculty of Medicine, University of Yamanashi, 1110, Shimokato, Tamaho, Yamanashi 409-3898, Japan
4 Atopy Research Center and 5 Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan
Correspondence to: A. Nakao; E-mail: anakao{at}yamanashi.ac.jp
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Abstract |
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Keywords: food allergy, IgE, Th2
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Introduction |
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Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that regulates cell growth, differentiation and survival and is also involved in the regulation of the immune responses (9, 10). Human milk contains abundant TGF-ß although there is considerable variability in the concentration of TGF-ß in human milk ranging from 200 to 3500 pg ml1 (11, 12). Previous studies have suggested that TGF-ß in human milk is associated with prevention of the development of allergic disease during infancy (13, 14). For instance, Oddy et al. reported in their multivariate analyses that the risk of wheezing at 1 year of age was significantly decreased with increasing TGF-ß1 dose received from breast-feeding (14). Although some immunoregulatory activities of TGF-ß such as induction of oral tolerance may explain such an anti-allergic property of TGF-ß in human milk (9), the mechanisms underlying those observations remain obscure. More importantly, although there is numerous evidence that TGF-ß, when applied systemically, affects several immune responses (9), it is not clear whether TGF-ß, when taken orally, can modulate systemic immune responses or not.
The primary aim of this study is therefore to investigate whether orally administered TGF-ß can affect systemic immune responses and prevent the development of allergic disease. Shida et al. previously showed that oral administration of high-dose ovalbumin peptide (OVA) to OVA-specific TCR transgenic mice led to an increase in the levels of OVA-specific IgE in the sera and the subsequent intravenous challenge of OVA-fed transgenic mice with OVA resulted in anaphylactic reaction, which might be a useful tool for studies of the cellular and molecular mechanisms of the T cell and IgE responses to orally ingested antigen (15). By using this model, we tested whether oral administration of TGF-ß affected the food allergy-related immune reactions.
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Methods |
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Mice
OVA-specific TCR transgenic mice on the BALB/c background, clone DO11.10, which recognizes the 323339 peptide fragment of OVA, were originally obtained from K. Murphy (16) and housed under specific pathogen-free conditions in our facility. These mice were kept in conventional surroundings. Animal experiments were approved by the Institutional Review Board of Juntendo University and University of Yamanashi.
Study design
The DO11.10 mice (610 weeks old) were fed with 100 mg OVA and 5 µg TGF-ß1 or with 100 mg OVA plus control vehicle by gavaging every other day for a total of six times (day 1, 3, 5, 7, 9, 11). For some experiments, anti-TGF-ß-neutralizing antibody or isotype-matched control mouse IgG was intra-peritoneally injected (100 µg per mouse) on the same days as OVA and/or TGF-ß gavaging for a total of six times. To investigate the effects of systemic administration of TGF-ß1 on immune responses induced by OVA feeding in DO11.10 mice, TGF-ß1 or control PBS was subcutaneously injected (5 µg per mouse) on the same days as OVA gavaging for a total of six times.
Flow cytometric analysis
Spleen cells (1 x 106) were stained with FITC-conjugated anti-CD4 mAb, PE-conjugated anti-clonotype-specific mAb KJ1-26 (these were purchased from BD Pharmingen, San Diego, CA, USA), Annexin VFITC (BioVision, Research Products, Mountain View, CA, USA) or 7-aminoactinomycin D (7-AAD) (Sigma Chemical Co.) for 30 min. After washing with PBS, the cells were analyzed on FACSCalibur (BD, San Jose, CA, USA), and the data were analyzed by using WinMDI program (Scripps Research Institute, La Jola, CA, USA).
Reverse transcriptionPCR
Spleen cells were prepared into single-cell suspensions and CD4+ T cells were isolated using T cell subset columns (R&D Inc.) according to the suggested protocol. Total RNA was then extracted by using the Isogen solution (Nippon Gene, Toyama, Japan) as recommended by the manufacture's instructions. Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using the first strand cDNA synthesis kit (Ready To Go; Amersham Biosciences Corp., Piscataway, NJ, USA). PCR amplification (Fas ligand and Fas; 94°C for 45 s, 55°C for 45 s and 72°C for 45 s; 35 cycles, T-bet and GATA-3; 94°C for 45 s, 60°C for 45 s and 72°C for 45 s; 30 cycles, ß-actin; 94°C for 0.5 min, 56°C for 0.5 min and 72°C for 1 min; 35 cycles) was performed in a DNA engine cycler (MJ Research, Inc., Waltham, MA, USA). The PCR products were separated by 2.0% agarose gel electrophoresis and stained with 0.5 µg ml1 ethidium bromide. The primer pairs (Fas ligand and Fas) used were purchased from R&D Inc. Mouse T-bet, GATA-3 andß-actin primers are as followsT-bet: forward, 5'-AACCAGTATCCTGTTCCCAGC-3', reverse, 5'-TGTCGCCACTGGAAGGATAG-3'; GATA-3: forward, 5'-CTCCTTTTTGCTCTCCTTTTC-3', reverse, 5'-AAGAGATGAGGACTGGAGTG-3' and ß-actin: forward, 5'-TGGAATCCTGTGGCATCCATGAAAC-3', reverse, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'.
Determination of antigen-specific IgE antibody in serum
Two days after the last feeding (day 13), the titer of OVA-specific IgE antibody in mouse serum was assessed by a 24-h passive cutaneous anaphylaxis reaction as described by Ovary (17).
Determination of antigen-specific IgG1 and IgG2a antibody in serum
The amount of OVA-specific IgG1 or IgG2a in mouse serum was measured by ELISA as described elsewhere (18). In brief, ELISA plates were coated with OVA (250 µg ml1), washed three times with PBS containing 0.05% Tween20 (PBST) and blocked with blocking buffer (PBS containing 2% BSA; Sigma Chemical Co.). Serum samples were added to the wells after 1 : 1000 or 1 : 3000 dilution in blocking buffer. As a control, serial dilutions of pooled serum from OVA-sensitized wild-type BALB/c mice were analyzed in each plate. After 1 h incubation, wells were washed with PBST, added either biotinylated anti-mouse IgG1 or IgG2a (Pharmingen) at 2 µg ml1 in blocking buffer and incubated for 1 h. After washing, wells were incubated with 100 µl of ExtrAvidin alkaline phosphatase (1 : 2000 dilution, Sigma Chemical Co.) for 45 min, washed with PBST and the reaction was developed with p-Nitrophenyl Phosphate Liquid Substrate System (pNPP) (Sigma Chemical Co.).
Induction of an anaphylactic reaction
Anaphylactic reaction was induced as previously described (15). Briefly, 1 mg of OVA dissolved in 0.1 ml PBS was injected into the tail vein of mice fed with OVA plus TGF-ß1 or OVA plus control vehicle. The mice were observed for a 30-min period after OVA injection. The anaphylactic reaction was evaluated and rated according to four scores as follows: , no reaction observed; +, the mice are slow and mobile if provoked; ++, the mice are stationary even if provoked and +++, the mice lie down and have severe anaphylactic shock.
Cytokine ELISA
Spleen cells (5 x 106 cells) isolated from mice fed with OVA plus TGF-ß1 or OVA plus control vehicle were re-stimulated with OVA (100 µg ml1). Seventy-two hours after the culture, supernatants were collected for measurement of cytokines. The amount of IL-4, IL-12 and IFN- in the culture supernatant of spleen cells was determined using murine IL-4, IL-12 and IFN-
ELISA kits (R&D Inc.).
Serum IgE levels
Blood was collected from DO11.10 mice at indicated times and serum samples were obtained by centrifugation and stored at 80°C until use. Total IgE levels were measured by a sandwich murine IgE ELISA kit (Yamasa Co. Ltd, Chiba, Japan).
Data analysis
Data are summarized as mean ± SD. An unpaired Student's t-test was used for the statistical analysis of the results. P < 0.05 was considered to be significant.
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Results |
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According to their protocol with some modification, OVA-specific TCR transgenic mice DO11.10 were fed with 100 mg OVA and 5 µg of TGF-ß1 or with 100 mg OVA plus control vehicle every other day for a total of six times (day 1, 3, 5, 7, 9, 11). Two days after the last feeding (day 13), the mice were analyzed for antibody responses and anaphylactic reaction. This protocol induced total and OVA-specific serum IgE elevation and an anaphylactic reaction following intravenous OVA challenge in DO11.10 mice (Fig. 2 and Table 1, see below).
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Because activation-induced T cell apoptosis via Fas/Fas ligand system was involved in the depletion of antigen-specific T cells by repeated high-dose OVA feeding in DO11.10 mice (19), we performed FACS analysis for splenic mononuclear cells 48 h after the first feeding (day 3) with an early apoptosis marker, Annexin V. As shown in Fig. 1(C), the percentage of Annexin V+7-AAD cells (7-AAD was used to exclude non-specifically stained dead cells) in KJ1-26+ T cells was increased in mice fed with OVA plus control vehicle when compared with non-treated control mice, which was also abrogated by oral administration of OVA plus TGF-ß1. In addition, reverse transcription (RT)PCR analysis showed that expression of Fas ligand, but not Fas, was decreased in CD4+ splenic T cells obtained from mice fed with OVA plus TGF-ß1 when compared with those obtained from mice fed with OVA plus control vehicle (Fig. 1D). Oral administration of TGF-ß1 alone to DO11.10 mice did not affect their spleen size or other parameters examined here (data not shown). These results indicated that oral administration of TGF-ß inhibited activation-induced cell death (AICD) of OVA-specific T cells induced by OVA feeding in DO11.10 mice. The reduction of Fas ligand expression may explain, in part, the reduction of OVA-induced AICD in DO11.10 mice fed with OVA plus TGF-ß.
Oral administration of TGF-ß suppressed food allergy-related immune responses induced by OVA feeding in DO11.10 mice
We then examined the effects of orally administered TGF-ß on food allergy-related immune responses in this model. Total serum IgE elevation was completely suppressed in mice fed with OVA plus TGF-ß1 when compared with mice fed with OVA plus control vehicle 2 and 9 days after the last feeding (Fig. 2A). The levels of OVA-specific IgE and IgG1 in the sera were also decreased, whereas the levels of OVA-specific IgG2a were increased in mice fed with OVA plus TGF-ß1 when compared with mice fed with OVA plus control vehicle 2 days after the last feeding (Fig. 2B).
In addition, OVA-induced IL-4 production by splenocytes was decreased, whereas OVA-induced IFN- or IL-12 productions were increased in mice fed with OVA plus TGF-ß1 when compared with mice fed with OVA plus control vehicle (Fig. 2C). Furthermore, RTPCR analysis showed that expression of T-bet, a marker of Th1-type immune responses (20), was increased, whereas expression of GATA-3, a marker of Th2-type immune responses (20), was decreased in splenic CD4 T cells from mice fed with OVA plus TGF-ß1 when compared with mice fed with OVA plus control vehicle (Fig. 3).
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Anti-TGF-ß antibody abrogated the inhibitory effects of orally administered TGF-ß on serum IgE response and anaphylactic reaction associated with OVA feeding in DO11.10 mice
To investigate the mechanisms underlying the above observations, we asked whether neutralization of systemic TGF-ß activity by anti-TGF-ß-neutralizing antibody affected the inhibitory effects of orally administered TGF-ß on serum IgE response and anaphylactic reaction associated with OVA feeding in DO11.10 mice.
As shown in Fig. 4 and Table 1, intra-peritoneal injection of anti-TGF-ß antibody abrogated the suppression of the total and OVA-specific IgE production and anaphylactic reaction by OVA plus TGF-ß feeding in DO11.10 mice. OVA-specific IgG2a production in mice fed with OVA plus TGF-ß was reduced to the background levels by the treatment with anti-TGF-ß antibody (Fig. 4B). Thus, the inhibitory effects of orally administered TGF-ß on serum IgE response and anaphylactic reaction might be attributed, in part, to its systemic activity possibly appeared after absorption of orally administered TGF-ß from the mouse intestine.
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Discussion |
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Interestingly, the effects of orally administered TGF-ß on a food allergy-related immune response were somewhat different from those of systemically applied TGF-ß. Orally administered TGF-ß suppressed Th2-type immune responses with significant Th1 skewing, whereas systemically administered TGF-ß suppressed both Th1- and Th2-type immune responses induced by high-dose OVA feeding in DO11.10 mice (Figs 2 and 5). Previous in vitro studies suggested that TGF-ß inhibited both Th1- and Th2-type T cell development associated with suppression of T-bet and GATA-3 (21), which was also confirmed by our hands (data not shown). The outcome of TGF-ß effects on T cells was shown to strongly depend on the cellular context such as stage of differentiation and cytokine milieu (22). It is thus possible that administration of TGF-ß through oral route stimulates many types of cells which produce various cytokines in response to TGF-ß, e.g. intestinal epithelial cells (23), and effects of TGF-ß on T cells are affected through the cytokine milieu. Clarification of mechanisms underlying the specific enhancing effects of orally administered TGF-ß on Th1-type immune responses is an important and interesting issue in future studies.
Because TGF-ß is secreted as a latent form and then activated by extremes of pH, heat, proteases and thrombosopondin-1 extracellulary (24), the latent form of TGF-ß present in human milk is thought to be activated by acidification at low pH of gastric juice in the stomach and maintain its biological activity when milk is taken orally (3).
Consistent with the above-mentioned notion, Letterio et al. previously showed that orally administered TGF-ß was absorbed from the intestine and appeared in systemic circulation in mice (25). We also found in this study that neutralization of systemic TGF-ß activity by intra-peritoneal administration of anti-TGF-ß antibody abrogated the effects of orally administered TGF-ß on serum IgE response and anaphylactic reaction (Fig. 4). Taken together, systemic activity possibly appeared after absorption of orally administered TGF-ß from the mouse intestine might explain, at least in part, the effects of orally administered TGF-ß observed in the current study.
Our preliminary study showed that, when mice were fed with relatively low-dose TGF-ß1 (510 ng per mouse), the effects on serum IgE response and anaphylactic reaction were marginal in contrast to the dose (5 µg per mouse) used in current study (data not shown). Although TGF-ß may be relatively resistant to degradation by acidification of gastric juice or proteolytic enzyme in the intestine when compared with other cytokines, it should undergo such degradation to some extent. Therefore, we speculate that a small portion of high-dose TGF-ß escaped from the degradation might be absorbed from the mouse intestine, enter the circulation keeping its activity and affect systemic immune responses.
TGF-ß has been shown to suppress antigen-induced T cell death, at least in part, through regulation of Fas ligand expression (26), which was consistent with the current results (Fig. 1). For example, Chen et al. reported that activation-induced T cell death induced by anti-CD3 mAb was strikingly enhanced in TGF-ß1 null mice with increase of Fas/Fas ligand expression (27). Our results also suggest that orally administered TGF-ß as well as endogenous TGF-ß can regulate T cell survival via regulation of Fas/Fas ligand system.
The relationship between inhibition of AICD and modulation of Th1/Th2-type immune responses by orally administered TGF-ß remains unclear. Naive, Th1-type and Th2-type T cells have been suggested to have different susceptibility to AICD although it appears to be still controversial (28, 29). We speculate that orally administered TGF-ß might preferentially inhibit AICD of Th1-differentiating, but not Th2-differentiating, T cells, resulting in enhanced Th1-type responses as suggested by the literature (22).
Interestingly, we did not find any significant effects of orally administered TGF-ß on other body systems (e.g. the respiratory system) than the immune system in mice in our experimental protocol (data not shown). This may be due to the short half-life of active TGF-ß in circulation (12 min) (30) or different sensitivity to TGF-ß in different organs. In fact, we failed to detect clear differences in serum concentrations of TGF-ß1 between non-treated mice and TGF-ß-fed mice at least up to 4 h after TGF-ß1 feeding, which might be due to rapid clearance of the cytokine in vivo (data not shown). In anyway, unfavorable consequences that might occur by more long-term or high-dose oral administration of TGF-ß, such as fibrosis, must be investigated carefully in future studies.
In summary, we showed that orally administered TGF-ß could modulate a food allergy-related immune response. Thus, TGF-ß, when taken orally at high dose, appears to have the capacity to affect systemic immune responses. The results may explain, at least in part, the previous observations that TGF-ß in human milk is associated with prevention of the development of allergic disease during infancy (13, 14). However, physiological relevance of our study remains unclear because we cannot say that the total amount of orally administered TGF-ß1 used in the current study (30 µg per mouse) corresponds to the total amount of TGF-ß1 that breast-feeding infants receive due to the difficulty of dosage comparison between mice and humans. Thus, a more direct approach (e.g. an experiment to test if neutralizing antibodies against TGF-ß suppress the ability of breast milk to reduce allergic responses.) is still necessary to clarify the role of TGF-ß in human milk.
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Acknowledgements |
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Abbreviations |
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7-AAD | 7-aminoactinomycin D |
AICD | activation-induced cell death |
cDNA | complementary DNA |
OVA | ovalbumin peptide |
PBST | PBS containing 0.05% Tween20 |
RT | reverse transcription |
TGF-ß | transforming growth factor-ß |
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Notes |
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Received 5 December 2004, accepted 4 March 2005.
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References |
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