Inhibition of mucosal and systemic Th2-type immune responses by intranasal peptides containing a dominant T cell epitope of the allergen Der p 1
Andrew G. Jarnicki,
Takao Tsuji1, and
Wayne R. Thomas
University of Western Australia Centre for Child Health Research and Department of Microbiology, TVW Telethon Institute for Child Health Research, PO Box 855, West Perth, WA 6872, Australia
1 Department of Microbiology, Fujita Health University, School of Medicine Toyoake, Aichi, Japan
Correspondence to:
Correspondence to: W. R. Thomas
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Abstract
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Although the intranasal administration of peptides containing T cell epitopes has been shown to be a potent method of inhibiting responses to the allergen Der p 1, the experiments to date have concentrated on their ability to regulate immune responses to the injection of antigen in a Th1-type adjuvant. Their ability to regulate responses to a Th2-type immunization and to sensitization via the respiratory mucosa has not been examined. Here it is shown that peptide used in doses required to block delayed-type hypersensitivity can also readily inhibit IgE responses to Der p 1 injected in alum. To examine responses induced in the respiratory mucosa, mice pretreated with intranasal peptide were sensitized with an intranasal dose of Der p 1 in conjunction with a mutated enterotoxin adjuvant. Intranasal peptide even in very high doses did not reduce IgE titers, but the ability of cells from the draining lymph nodes to release IL-4 and IL-13 but not IL-2, IL-5, IL-10 or IFN-
was reduced. These are the first reports on the effect of intranasal peptides containing T cell epitopes on IgE in Th2 immunization and on responses to respiratory immunization. Thus the effect of the peptide-induced mucosal tolerance differs depending on the type of immunization used for sensitization, but the potential to inhibit Th2 responses and responses to respiratory sensitization as well as Th1 responses has been demonstrated.
Keywords: inhibition, Der p 1, IgE, peptides, intranasal
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Introduction
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The intranasal administration of proteins or peptides containing T cell epitopes can be a potent inhibitor of immune responses. This phenomena has been extensively studied in models of autoimmunity where the induction of disease and Th1-type responses can be markedly reduced (1). It has sometimes but not always been associated with increased Th2 cytokine production (25). Studies with the house dust mite allergen Der p 1 have shown that responses to exogenous antigens can also be inhibited by intranasal peptides and have raised the possibility that peptides can be used to treat or prevent allergic disease. The initial studies with the allergen reported that a peptide containing an individual T cell epitope could block the induction of T cell responses to all the epitopes on the allergen as measured by IL-2 release in in vitro T cell stimulation assays (6). The peptides could also inhibit the induction of delayed-type hypersensitivity (DTH) showing their ability to inhibit responses in the whole animal (7). Although these experiments showed that Th1-responses were inhibited by a mechanism involving intramolecular suppression, the regulatory effect on IgE in a Th2-biased response and responses to respiratory exposure have not been reported. This question needs to be resolved because although it has been reported that intranasal peptides did not affect IgE titers, the responses examined were Th1-biased responses induced by the injection of Der p 1 in complete Freund's adjuvant (CFA) (7). The regulatory mechanism in Th2-biased responses, however, can be different. For example, any inhibitory effect on Th2 responses could be equally matched by a decrease in cross-regulatory Th1 cytokines. It is equally necessary to examine responses induced by sensitization via the respiratory mucosa, particularly since it has been found that a secondary intranasal challenge with peptide can re-stimulate T cell responses in the draining lymph nodes and spleen at a time when the mice are tolerant to injected antigen (8). Similar responses have been found after multiple feeding (9).
Here we report that the intranasal administration of peptides containing the dominant T cell epitope of Der p 1 can markedly and selectively decrease IgE antibody isotype produced in response to the allergen injected in alum, a regimen which stimulates Th2 responses. The effect on sensitization by the respiratory route was different where the intranasal peptides did not decrease IgE but reduced the production of the Th2 cytokines IL-4 and IL-13, but not the Th1 cytokines IL-2 or IFN-
or the regulatory IL-10.
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Methods
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Mice
C57BL/6J mice (68 weeks old) were obtained from the Animal Resource Centre (Murdoch, Australia) and kept under specific pathogen free conditions.
Antigens and peptides
Der p 1 was isolated from a 20% spent mite medium dissolved in PBS (gift from CSL, Melbourne, Australia) by affinity chromatography using the mAb 4C1 provided by Professor M. D. Chapman (University of Virginia, Charlottesville, VA). Ovalbumin (OVA) crystalline grade V was purchased from Sigma (St Louis, MO). Peptides were synthesized by Chiron Mimotopes (Clayton, Australia) with a free C-terminus, and analyzed by the manufacturer for purity and yield by reverse-phase HPLC and verified by mass spectrometry (mol. wt = 2151.4 for p114128).
Immunization and intranasal administration
Mice were immunized either i.p. with 10 µg of antigen adsorbed to 10 mg aluminum hydroxide gel in 100 µl PBS or s.c. at the base of the tail with 100 µg antigen in 100 µl of PBS emulsified with 100 µl CFA (Difco, Detroit, MI). For intranasal administration, the peptides and antigens were dissolved in PBS and 20 µl administered to the nares of lightly anaesthetized mice with a micropipette. Previous studies have used a peptide consisting of residues 110131 of Der p 1 but most studies here were performed with peptide 114128 (SNYCQIYPPNANKIR) which spans the minimal major CD4 H-2b epitope between residues 118126 (authors' unpublished observations).
Respiratory sensitization was produced by the intranasal administration of 100 µg of Der p 1 with 10 µg of a mutated subunit of Escherichia coli heat-labile enterotoxin in 20 µl of PBS. In some experiments the responses were boosted by the same dose of Der p 1 and mutated toxin. The mutant toxin has been shown to have very little toxicity and enhanced adjuvant activity without inducing cAMP (10). It was a deletion mutant lacking R192, T193 and I194 constructed by PCR from a HindIIIEcoR1 fragment of the LT-A subunit gene. The mutant plasmid was named as pTSU135. After pTSU135 was transformed into the E. coli MV1184 strain, the mutant LT protein was purified with immobilized D-galactose-affinity column chromatography (10).
T cell assays
Lymph node cells were assayed for antigen-induced cytokine production 10 days after immunization. For mice immunized with antigen in CFA the inguinal and periaortic lymph nodes were used and for respiratory immunization the superficial cervical lymph nodes. The nodes were pressed through a stainless steel mesh, washed and cultured at 5x105 cells/well in 96-well round-bottomed 96 microtiter trays (Nunc, Roskilde, Denmark) in 200 µl of DMEM medium (Gibco, Grand Island, NY) supplemented with 2% FCS (CSL, Melbourne, Australia), 50 mM L-glutamine (Gibco) and 50 µM of 2-mercaptoethanol (BDH supplies, Poole, UK) and 20 mM HEPES (ICN Biochemicals Co., OH). Antigen was added to the cultures at various concentrations and the cells incubated at 37°C in a 5% CO2 atmosphere for 24 or 48 h. Supernatants were stored at 20°C until required. IL-2 was assayed by measuring the [3H]thymidine incorporation of CTLL-2 indicator cells (7), and IL-4, -5, -10 and -13 and IFN-
were measured by sandwich ELISA. Maxisorb plates (Nunc) were coated with a monoclonal capture antibody by incubating at 4°C overnight with antibody diluted to the suppliers instructions in 0.1 M Na2HPO4, pH 9.0. The plates were washed 5 times in 0.05% Tween 20 in PBS and blocked with 1% BSA in PBS for 1 h. The plates were washed as above, and 50 µl of the samples to be assayed was added and incubated for 2 h at room temperature with shaking before a further 5 washings and the addition of 50 µl of biotinylated detection antibody diluted in PBS containing 0.05% Tween 20 and 1% BSA. After 1 h the plates were washed 5 times and incubated with 100 µl of streptavidinmulti-horseradish peroxidase (CLB, Amsterdam, The Netherlands) for 30 min and washed 8 times. The reactions were developed with TMB substrate (Graphic Scientific, Brisbane, Australia), stopped with IM H2SO4, and read at 450 nm using a Microplate EL311autoreader (BioTek, Winooski, VT). Cytokine concentrations were determined against a curve of recombinant standards. For IL-5 the results are expressed as units of a standard recombinant supernatant while the others are in pg. The antibodies used to detect IL-4 were 55434 and 18042D (capture and detection respectively), for IL-5, 18051D and 18062D, for IL-10, 18141D and 18152D, for IL-13, MAB413 and BAF413, and for IFN-
, 18112D and 18181D. The anti-IL-13 antibodies were obtained from R & D (Minneapolis, MN) and the others were from PharMingen (La Jolla, CA).
Antibody titration
Sera were assayed for IgG1 and IgG2a by ELISA. Maxisorb plates were coated with Der p 1 at 1 µg/ml in 0.1 M Na2HPO4, pH 9.0 and the assay conducted as for the cytokine ELISAs above. The reactivity was developed with biotinylated goat anti-IgG1 (1070-08; Southern Biotechnology Associates, Birmingham, AL) or anti-IgG2a (1080-08; Southern Biotechnology Associates). Dilutions of a pool of hyperimmune sera were used to construct a standard curve and the titers were calculated from the standard which was given an arbitrary concentration of 1000 U/ml.
IgE antibodies were assayed by passive cutaneous anaphylaxis (PCA) or by ELISA. For PCA 0.05 ml of doubling dilutions of sera were injected intradermally into the shaved back of adult male Sprague-Dawley rats. After 24 h the rats were challenged with 10 mg of spent mite medium in 1 ml of 0.05% Evans Blue (ICN) in saline. The reciprocal of the highest dilution producing a blue spot was taken as the titer and expressed in log2 units (7). For the ELISA Maxisorb plates (Nunc) were coated with 100 µl of rat anti-mouse IgE antibody (2 µg/ml, cat. no. 553413; PharMingen) diluted in 0.1 M Na2HPO4 (pH 9.0) and incubated overnight at 4°C. The plates were washed 5 times with 50 mM TrisHCl, 0.9% NaCl and 0.05% Tween 20, and blocked by adding 250 µl/well of 2% BSA in 0.05 M TrisHCl, 0.9% NaCl for 1 h at room temperature. The plates were then washed 5 times, and 100 µl of either sample or standard diluted in DELFIA Assay buffer (Wallac, Turku, Finland) was added and incubated for 4 h with gentle shaking at room temperature. Dilutions of a pool of hyperimmune sera were used to construct a standard curve and the titers were calculated from the standard which was given a concentration of 100 arbitrary units. The plates were washed 5 times and 100 µl of Der p 1 (5 µg/ml) diluted in DELFIA assay buffer added for 2 h at room temperature. After 5 washes, 100 µl of the biotinylated mouse anti-Der p 1 mAb 4C1 diluted in DELFIA Assay buffer was incubated for 1 h at room temperature. After 5 washes, samples were incubated with 100 µl of DELFIA europium-labeled streptavidin (Wallac) 30 min with a final 8 washes. Then 200 µl of DELFIA enhancement solution (Wallac) was added at room temperature. The plates were read 15 min later with a Victor2 1420 Multilabel counter (Wallac) and the titers calculated against the standards.
Delayed hypersensitivity
Mice sensitized with Der p 1 in CFA were challenged on day 7 with an intradermal injection of 10 µl of Der p 1 in PBS in the ventral side of the pinnae of the ear. After 24 h the thickness of both ears was measured with an engineer's micrometer and the difference taken as a measure of delayed hypersensitivity. The swelling was expressed in units of 102 mm and compared to the swelling produced by challenging non-sensitized mice.
Bronchoalveolar lavage (BAL)
The lungs of freshly euthanized mice were perfused by injecting PBS through the left atrium of the heart. The airway to the right lobes of the lung was clamped and the right lung was removed and placed in 10% saline-buffered formaldehyde for histological staining. The trachea was exposed and an incision made between the cartilaginous rings immediately below the mandible. A cannula consisting of 2 cm of 1 mm polyethylene tubing (Dural Plastics and Engineering, Dural, NSW, Australia) connected to a 23G needle was inserted, and the left lung was lavaged with 3x300 µl of PBS/0.2% BSA. For differential cell counts, cytospin preparations of 5x104 cells in 100 µl were made using a cytocentrifuge (Elliot, Shandon, Pittsburg, PA). Cells were dried for 30 min and then stained with Diff Quik (Lab Aids, Narrabeen, Australia) as per the manufacturer's instructions. Paraffin tissue sections were stained with hematoxylin & eosin by standard procedures.
Statistical methods
Groups of five mice were set up for each experiment, which were performed at least in duplicate. The significance was determined by Student's t-test or MannWhitney tests.
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Results
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Bystander responses
The intranasal administration of peptides containing the major epitope of Der p 1 has been shown to inhibit the responses of mice sensitized s.c. with Der p 1 in CFA as measured by IL-2 release in in vitro recall responses of lymph node cells. The results of previous experiments designed to examine whether or not responses to a bystander antigen injected with the Der p 1 were also inhibited, produced equivocal results with a trend to inhibition at the P < 0.1 level (7). To re-examine this, mice were given three intranasal doses of 10 µg of p114128 and after 10 days immunized with 100 µg Der p 1 in CFA containing 100 µg of OVA. Using this protocol the responses to Der p 1 and OVA could be consistently and significantly inhibited to the same degree as the inhibition of Der p 1 responses (Fig. 1
).

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Fig. 1. Bystander suppression. Mice were treated with three daily doses of p114128 or PBS, and then immunized with a mixture of Der p 1 and OVA in CFA. The results show the mean SD of IL-2 release for groups of five mice. The responses of the peptide-treated group to Der p 1 (A) and OVA (B) were significantly reduced, P < 0.05 Student's t-test.
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Antibody responses to Der p 1 immunization in alum and DTH to immunization in CFA
Mice were given one, three or five daily intranasal doses of 10 µg of p114128 or one dose of 50 µg. At 10 days after the last administration the mice were injected i.p. with 10 µg of Der p 1 in alum and the ensuing serum antibody responses measured at different time intervals. The IgE titers in mice which received either five intranasal doses or the one 50 µg dose were 4 log2 units lower than controls (i.e. an average of 1/16th of controls) and the other groups were the same as the controls (Fig. 2
). To confirm the inhibition of IgE, sera were assayed by capture IgE ELISA for anti-Der p 1. There was a close correlation with the PCA. The week 4 titers for the PBS treated group were 725.7 ± 303.9 (mean ± SD, ELISA units) and for the mice treated with 5x100 µg peptide 21.58 ± 9.741 units. For comparison, experiments were conducted to determine the effect of different 10 µg doses of intranasal peptide on delayed hypersensitivity produced after the injection of Der p 1 in CFA. It was also found that five but not three intranasal doses were markedly inhibitory (Table 1
). The sera of the same groups of alum immunized mice which were used to show the inhibition of IgE titers were assayed for IgG1 and IgG2a antibody (Figs 3a and 3b
). No differences were found between the controls and the intranasal peptide groups.

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Fig. 2. IgE responses of mice treated with intranasal peptide and immunized with Der p 1 in alum. Mice were treated with one, three or five daily doses of 10 µg of p114128 (1, 3 or 5) or 1 dose of 50 µg p114128 (50) or five doses of PBS (0) and injected i.p. with Der p 1 in alum. The IgE titers of groups of five mice (mean ± SD log2 titer) were significantly decreased in groups treated with five daily doses of 10 µg peptide or one 50 µg dose, P < 0.05 Student's t-test.
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Fig. 3. IgG responses of mice treated with intranasal peptide and immunized with Der p 1 in alum. Mice were treated with one, three or five daily doses of 10 µg of p114128 or one dose of 50 µg p114128 or five doses of PBS and injected i.p. with 10 µg Der p 1 in alum. The responses of groups of five mice (mean ± SD arbitrary ELISA units) show no differences between the groups for IgG1 (A) or IgG2a (B).
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Respiratory immunization
Respiratory immunization was performed by the intranasal administration of 100 µg of Der p 1 with 10 µg of mutated E. coli enterotoxin as adjuvant. The titers induced were similar to those induced by the injection of Der p 1 in alum and persisted for at least 8 weeks. Pilot experiments had shown that IgE responses to intranasal Der p 1 alone were transient and variable, and that lower doses of the Der p 1 or the mutated toxin induced lower responses. To examine the effect of intranasal peptides on IgE responses, experiments first were conducted with the same doses and regime of intranasal peptide or Der p 1 administration which had produced the inhibition of IgE and DTH to parenteral immunization. No inhibition was found (data not shown). The dose of intranasal peptide was then increased to five consecutive intranasal doses of 100 µg. No effect on IgE titer was found (Fig. 4
) after either a primary or boosted response. The IgG1 antibody titers were not affected by the intranasal peptide but prior intranasal administration of the whole intranasal Der p 1 enhanced the IgG1 titers (Fig. 5a
). The IgG2a titers were not affected by intranasal peptide or Der p 1 (Fig. 5b
). To test for a possible direct inhibitory effect of the mutated toxin on the induction or maintenance of intranasal tolerance, mice were given five daily doses of intranasal p114128, and after 10 days treated with intranasal mutated toxin and injected i.p. with Der p 1 in alum. The mutated toxin did not affect the IgE titers induced by the injected Der p 1 and the inhibition induced by the intranasal peptide was unaffected (Fig. 6
).

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Fig. 4. IgE responses of mice treated with intranasal peptide or antigen and immunized by respiratory sensitization. Mice were given five daily doses of 100 µg of p114128 or whole Der p 1 or PBS and then sensitized with the intranasal installation of 100 µg of Der p 1 with mutated E. coli enterotoxin. The IgE titers of all of groups of five mice (mean ± SD log2 titer) showed no significant differences in primary or boosted responses (week 9).
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Fig. 5. IgG responses of mice treated with intranasal peptide or antigen and immunized by respiratory sensitization. Mice were given five daily doses of 100 µg of p114128, whole Der p 1 or PBS and then sensitized with the intranasal installation of 100 µg of Der p 1 with mutated E. coli enterotoxin. The responses of groups of five mice (mean ± SD arbitrary ELISA units) show that (A) intranasal pretreatment with Der p 1 enhanced the IgG1 responses when boosted (week 9) while peptide had no effect and (B) no differences were found for IgG2a responses.
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Fig. 6. Effect of mutant toxin adjuvant on the peptide-induced inhibition of parenteral IgE responses. Mice were given five daily intranasal doses of 100 µg of p114128 or PBS and then injected i.p. with 10 µg Der p 1 in alum. At the time of the i.p. injection groups were given either intranasal PBS (+PBS) or the mutant enterotoxin (mT). The IgE titers of all of groups of five mice (mean ± SD log2 titer) showed that IgE was inhibited by the peptide treatment P < 0.05 Student's t-test. The treatment with mutated enterotoxin did not affect the IgE titers in the peptide or PBS pretreated groups.
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To examine the effects on recall responses of T cells, mice were given five daily doses of 100 µg of p114128 and then immunized with 100 µg intranasal Der p 1 and mutated toxin. After 10 days the superficial cervical lymph nodes were removed and responses to Der p 1 were measured by the cytokine released after in vitro stimulation. No effect was found on the release of IL-2, IL-5, IL-10 and IFN-
, but the amount of IL-4 and IL-13 released was reduced to 50% in the group treated with intranasal peptides (Table 2
). Replicate experiments produced the same degree of inhibition. To examine lung inflammation, mice were immunized with intranasal Der p 1 with mutant toxin adjuvant and after 4 weeks were challenged with three intranasal doses of 100 µg of Der p 1 given on 3 consecutive days. Two days after the last instillation the cellular infiltrate was examined by BAL and by histological sections. The infiltrate was primarily lymphocytic. Mice immunized with peptide pretreatment (5 daily doses of 100 µg ) had significantly higher lymphocytic infiltrate (12,002 ± 5620 compared to 988 ± 473, P < 0.01, MannWhitney test). The increase was also clearly apparent on examination of histological sections stained with hematoxylin & eosin.
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Discussion
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The intranasal administration of peptides containing CD4 T cell epitopes can inhibit Th1-type immunity as shown by the ability of peptides from myelin basic protein (MBP) to inhibit experimental autoimmune encephalomyelitis (EAE) (1). This peptide pretreatment inhibited both IFN-
and IL-4 responses and increased IL-10 production, which if blocked by neutralizing antibody prevented the inhibition of disease (11). Liu et al. (12) found MBP peptides decreased IFN-
but without affecting IL-4 and transforming growth factor (TGF)-ß production, and other reports found increased IL-4 and TGF-ß responses (2,13). Intranasal peptides from the acetylcholine receptor (AChR) have been shown to prevent the induction of myasthenia gravis (14) and IL-2 release from sensitized T cells, and in a study using whole AChR, intranasal administration decreased IFN-
and IL-4, while increasing TGF-ß (3). In the cases of collagen-induced arthritis (4) and anti-GAD65 responses in spontaneous diabetes (5), intranasal peptides have inhibited disease and converted Th1 to Th2 responses by inhibiting IFN-
and increasing IL-4.
A similar increase in Th2 responses was indicated by the work of Wolvers with an exogenous antigen, OVA (15). Here intranasal administration of OVA decreased DTH and increased IgE production following immunization with CFA. Previous studies with Der p 1 showed that peptides blocked the induction of DTH in mice immunized with Der p 1 with CFA, but not IgE (7). This type of result and a similar literature on oral tolerance has led to speculation that Th2 cytokines may be involved in the tolerance or may be more resistant to the toleration. The results here are the first reports of the effect of intranasal synthetic peptide on a Th2 immunization protocol and show IgE can be readily inhibited with a low-dose regimen or by a single high-dose application, by at least 80%.
The initial studies on Der p 1 peptides examined responses to the allergen injected in CFA because this regimen readily induced responses to individual T cell epitopes which could be measured with different peptides. It was shown that peptides containing only one T cell epitope could markedly inhibit sensitization to all epitopes on Der p 1 as measured by in vitro IL-2 release or by in vivo DTH (6,7). This intramolecular suppression, where regulatory responses to one epitope blocked responses to the whole molecule, as well as the ability of the peptides to block responses of primed mice (6), has suggested some utility in modifying hypersensitivity reactions to complex antigens. It is, however, important to study IgE responses to a Th2-type immunization. The results here show that the daily installation of five quite low doses (10 µg) of peptide can inhibit IgE titers, induced by the i.p. injection of Der p 1 in alum. The intranasal doses required were the same as those required for the inhibition of DTH in animals sensitized with allergen in CFA. A possible reason why many studies have shown that Th2 cytokine responses can be increased in responses induced to antigen in CFA is that there is that is a release from regulation by the dominant Th1 responses. It is also possible that mechanisms of intranasal and oral tolerance require the injection of antigen to become activated, and this, depending on whether it was a Th1 or Th2 stimulus, could influence the inhibitory activity.
Although this is the first report of the effect of the intranasal administration of synthetic peptides on IgE responses, the effect of intranasal antigen or aerosols has been previously studied. Holt et al. (16) originally found that chronic intranasal administration of OVA inhibited IgE responses of mice injected with antigen in alum and that chronic intranasal Der p 1 could inhibit IgE responses of low responder rats (17), although responses of high responders and mice were unaffected. Like the results described here for high-responder mice, IgG was not inhibited. Although some contribution from ingested allergen could not be excluded, as is the case here, the dose of allergen ingested was shown to be far lower than that required in standard oral tolerance regimens (16). Later studies showed that OVA aerosols produced a similar effect which was associated with the development of IFN-
-producing cells (18). Recent studies using a short intranasal exposure protocol similar to that used here have also found selective IgE tolerance antibody (19,20) but IFN-
was not increased (20). In contrast, the study of Wiedermann (21) with whole intranasal Bet v 1 allergen found that IgE and all IgG isotypes were markedly reduced, as well as T cell proliferation and the release of IL-4, IL-5, IL-10 and IFN-
in in vitro stimulation assays. The ability to inhibit responses with intranasal peptides differs very significantly from the reported effect of s.c. injection of peptide on Th2 responses to OVA. The injection of peptide containing the dominant OVA epitope stimulated instead of inhibited IgE responses (22). Inhibition could only be achieved by injecting the whole protein. In the experiments here, the intranasal administration of the whole protein, but not peptide, significantly stimulated IgG1 produced by respiratory immunization. The use of peptide may therefore be preferable for aeroallergen immunotherapy.
The demonstration of bystander inhibition here confirms previous experiments which showed a strong trend to inhibition (7). The responses of both the bystander protein and Der p 1 were similarly inhibited. The mechanism may be the same or different to the intramolecular suppression described previously. One mechanism is that the inhibition can be mediated by contact inhibition signals mediated by the Notch pathway at the time of antigen presentation (23). Clearly this could also exist if the co-administered antigens were processed and presented by the same antigen-presenting cells. The production of immunosuppressive TGF-ß has not been detected in the Der p 1 model, but regulation by IL-10 as described by Burkhart et al. (11) in the EAE model could be another mechanism.
All previous studies on intranasal inhibition have only examined responses induced by parenteral sensitization. Although the experiments of Wiedermann et al. (21) with whole Bet v 1 allergen used a respiratory challenge, the response was critically dependent on i.p. sensitization in alum. Immunoregulation of responses induced via the respiratory mucosa can be different to other sites. Wolvers et al. (24) have directly shown by lymph node transplantation that, for producing intranasal tolerance, the respiratory nodes cannot be substituted by nodes draining other tissues. Constant et al. (25) have reported that C57BL6J mice develop Th2 responses to the respiratory inoculation of Leishmania instead of the strong protective Th1 responses induced by injection. The circulation of cells may also be different. The nodes draining the upper respiratory tract (15,26) do produce some MadCAM integrin, the ligand for
4ß7 integrin, found in the gut, and T cells in the epithelium have the
Eß7 integrin also found in the gut (27). Most T cells in lung inflammation have the
4ß1 integrin which is found on memory cells which circulate to most tissues (28,29) and the
Eß7 does not therefore appear to be of paramount importance (30,31). T cells with the
Eß7-integrin, however, do predominate in the bronchial epithelium along with the epithelial ligand E-selectin (27). While the function of these cells has not been ascertained they can persist in the epithelium where they can be regulated by TGF-ß (32). The gut intraepithelial T cells have been shown to respond to down-regulatory signals from the epithelium which do not affect spleen T cells so it is possible the
Eß7-bearing intraepithelial T cells may have a role in dampening mucosal responses (33). Other studies have indicated that while the homing of memory cells, such as those with
4ß1 integrin, may not be tissue specific there is a mechanism for the preferential survival of cells in the tissue of priming (46). In a novel mechanism found for stromal-derived factor-1, low concentrations of chemokines can attract while high concentrations can repulse the migration of T cells (34). This has not been studied in the lung, but the eosinophil influx into lung inflammation can be blocked by anti-CXC4R, the only known ligand for SDF-1 (35). Another factor to consider is that cytokine delivered to or produced in the mucosa can prevent the induction of mucosal tolerance and lead to immune responses (20,36).
Previous experiments had shown that the intranasal administration of Der p 1 led to transient but substantial immune responses in the draining lymph nodes and spleen. The T cells from these tissues could release IL-2, IFN-
and granulocyte macrophage colony stimulating factor (GM-CSF), but no IL-4 or TGF-ß was detected. A further intranasal exposure to peptide after 21 days induced a similar immune response where, in contrast, parenteral challenge at this time with Der p 1 or peptide in CFA showed the characteristic reduction in responses. The current experiments were designed to examine the effect of the peptides on an immunogenic mucosal stimulation capable of inducing persistent responses, similar to injection in adjuvant. The responses induced by a single intranasal administration of Der p 1 with mutated enterotoxin elicited IgE titers similar to those induced by the injection of Der p 1 in alum. The administration of peptide did not alter the IgE titer in response to this sensitization or the production of IgG1 and IgG2a antibody isotypes. Thus, while 5x10 µg or 1x50 µg could inhibit the IgE production to immunization with Der p 1 in alum or DTH to Der p 1 in CFA, neither these doses or a very high dose regimen of 5x100 µg of peptide affected antibody production. The administration of the mutated enterotoxin adjuvant itself did not abrogate the mucosal regulation because administration of the mutated toxin when the immunization was by injection with alum did not affect the inhibition.
While the intranasal peptide did not inhibit the IgE responses to respiratory immunization it did have some effect on Th2 cytokines. When the cells from the draining lymph nodes were tested for their ability to produce cytokines in vitro there was no difference in the release of IL-2, IL-5, IL-10 or IFN-
, but there was a consistent reduction in their ability to release IL-4 and IL-13, to about half the level of control responses. This clearly was not sufficient to reduce IgE antibody but IL-4 and IL-13 have many direct effects on mucosal inflammation such as the induction of chemokines by epithelial cells including eotaxin (37), the induction of adhesion molecules VCAM-1 (38,39) and ICAM-1 (40) on epithelial and vascular cells, the induction of 15-lipoxygenase and ecosanoid inflammatory mediators (41), the stimulation of mucus secretion (42,43), the induction of GM-CSF (39) and effects on tissue matrix (44). Indeed, aerosols of soluble IL-4R has been shown to produce rapid improvements in asthmatic lung function indicating an effect beyond the regulation of lymphocyte differentiation (45). The knowledge that these cytokine responses can be decreased by intranasal peptides also indicates that it is possible that a more efficient protocol may be devised. The peptides unexpectedly did not inhibit IL-2 and IFN-
and thus a more chronic treatment may skew responses away from the Th2-type 1 hypersensitivity. The lungs of mice pretreated with peptide also had an increased lymphocyte infiltrate after challenge with Der p 1. This is a potentially important result showing the need to examine appropriate models of sensitization. More detailed studies on the activity of these cells and their effect on respiratory function are, however, required. The split cytokine regulation has been previously evident in that despite the marked inhibition of IL-2 in responses to immunization with CFA, the intranasal peptide administration has consistently had little effect on GM-CSF/IL-3 release (6). The lack of any increase in IL-10 production is of interest because of the contrast with the marked increase found in mice treated with intranasal peptide and immunized with MPB in CFA (11). The regulatory effects manifested by intranasal peptide appear to depend on the type of sensitization.
In summary, therefore, the intranasal administration of peptide containing the major T cell epitope of the allergen Der p 1 can readily inhibit IgE production to a parenteral injection of Der p 1 in alum as well the inhibition of DTH responses induced by the injection of antigen in CFA. The peptide administration could inhibit some responses induced by sensitization via the respiratory mucosa, as show for IL-4 and IL-13 but even high doses of intranasal peptide did not affect IgE via this route. Although the route and method of antigen challenge has important quantitative and qualitative effects on the outcome, immunomodulation was observed for both type 1 and cell-mediated hypersensitivity responses.
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Acknowledgments
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This work was supported by the National Health and Medical Research Council of Australia.
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Abbreviations
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AChR acetylcholine receptor |
BAL bronchoalveolar lavage |
CFA complete Freund's adjuvant |
DTH delayed-type hypersensitivity |
EAE experimental autoimmune encephalomyelitis |
GM-CSF granulocyte macrophage colony stimulating factor |
MBP myelin basic protein |
OVA ovalbumin |
PCA passive cutaneous anaphylaxis |
TGF transforming growth factor |
 |
Notes
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Transmitting editor: A. Radbruch
Received 18 December 2000,
accepted 25 June 2001.
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References
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