Published ahead of print on September 18, 2003, doi:10.1164/rccm.200305-690OC
© 2004 American Thoracic Society Late Asthmatic Reactions Induced by Inhalation of Allergen-derived T Cell PeptidesDepartment of Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College London, London, United Kingdom; and Clinical Research Center, National Sagamihara Hospital, Sagamihara, Kanagawa, Japan Correspondence and requests for reprints should be addressed to Prof. A. Barry Kay, Department of Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK. E-mail: a.b.kay{at}imperial.ac.uk
In individuals with atopy and asthma, allergen-derived T cell peptides injected intradermally induce isolated late asthmatic reactions (LARs) followed by bronchial hyporesponsiveness to peptide, inhibition of the allergen-induced cutaneous late-phase reaction, and altered T cell function in vitro. Laboratory animal data indicate that "activation" and "tolerance" also occur if peptides are inhaled. In this study, we show that inhalation of Fel d 1–derived peptides induced isolated LAR in individuals with asthma sensitive to cat allergen comparable with that previously demonstrated using intradermal injection. LARs were accompanied by eosinophilia and nonsignificant elevations of total cysteinyl leukotrienes in the sputum. Unlike the intradermal route, repeated inhalation of peptides was not associated with abrogation of the LAR and produced a sputum eosinophilia comparable with the first exposure. In addition, there was no inhibition of the cutaneous late-phase reaction to whole cat dander. Thus, isolated LAR induced by inhaled, allergen-derived peptides represent a novel model of provoked asthma and are not associated with the induction of hyporesponsiveness ("tolerance") in the skin or lung.
Key Words: allergy • T lymphocytes • epitopes • inflammation • tolerance We previously showed that intradermal administration of short, overlapping T cell peptide epitopes, derived from the major cat allergen Fel d 1, induced isolated, major histocompatability complex (MHC) Class II–restricted, late asthmatic reactions (LARs) in sensitized individuals without the requirement for an early IgE/mast cell–dependent response (1). Thus, direct activation of allergen-specific T cells represented a novel challenge system to investigate the role of the T cell in initiating airway narrowing, independent of the early reaction. Surprisingly, LARs induced by intradermally injected Fel d 1 peptides were neither associated with infiltration of eosinophils or other inflammatory cells nor elevated concentrations of histamine and leukotrienes (LTs) in bronchial biopsies or bronchoalveolar lavage fluid (2). This suggested that part of the asthma process might involve pathways to airway narrowing that do not require infiltrating inflammatory cells. However, as the challenge was systemic (intradermal), an alternative explanation is that T cell activation may have occurred in perivascular tissue distal to bronchoscopic sampling. For these reasons, we hypothesized that the mucosal (inhalational) route of challenge with T cell peptide epitopes would also induce isolated LARs and that luminal airway inflammation would be detectable as shown by sputum induction. In the present study we show, for the first time, that peptides can induce isolated LARs by the inhaled route and that peptide-induced airway narrowing is associated with sputum eosinophilia. Furthermore, unlike LARs provoked by intradermal challenge (1, 3, 4) these were not associated with subsequent "tolerance" even to peptides in the target organ or to whole allergen in the skin. Some of the results of these studies have been previously reported in the form of abstracts (5, 6).
Subjects and Study Design Volunteers who had asthma and were sensitive to cat allergen were recruited and characterized clinically as defined previously (1). The study was approved by the Royal Brompton and Harefield NHS Trust Ethics Committee. All volunteers gave written informed consent. All subjects demonstrated provocative concentration resulting in a 20% decrease in FEV1 (PC20) to histamine of 8 mg/ml or less. ß2-agonists were withheld on the study day, and inhaled corticosteroids were discontinued for a minimum of 1 week before each visit. Further details are provided in the online supplement.
For the inhalation challenges 28 subjects, in five groups, attended for nebulized diluent and peptide 1 week apart (Figure 1A)
. Group 1 (n = 4) received 0.01 µg peptides, Group 2 (n = 6) 0.1 µg, Group 3 (n = 6) 1.0 µg, Group 4 (n = 10) 5 µg, and Group 5 (n = 9) 10 µg. FEV1 was recorded at 0, 5, 10, 20, 30, 40, 50, and 60 minutes and hourly thereafter for 5 hours, followed by sputum induction. Five subjects entered more than one group, attending on consecutive days for increasing peptide dose, after failing to react to lower doses (previous studies [3] have demonstrated that significant hyporesponsiveness develops
To assess cutaneous hyporesponsiveness to whole allergen after inhalation of peptides, nine more subjects received an intradermal injection of whole cat dander extract (30 BU; ALK-Abello, Horsholm, Denmark) (Figure 1B). The late-phase reaction was measured at 360 minutes (7). Subjects then inhaled the diluent and underwent spirometry, followed 1 week later by peptide challenge (5 µg). Fourteen days after peptide, subjects developing LAR received repeat intradermal cat dander challenge. A separate cohort of 24 subjects, recruited in 3 groups of 8 as part of a previous study (3), received intradermal peptides—Group 1: 1 µg (each peptide), Group 2: 2.5 µg, and Group 3: 5 µg (Figure 1C). Spirometry was recorded at baseline and for 6 hours. To assess the development of bronchial hyporesponsiveness, six responders (one from Group 1, two from Group 2, and three from Group 3) underwent repeat peptide injection at the same dose after 2 to 14 weeks. To investigate the induction of whole allergen hyporesponsiveness in these six subjects, intradermal cat dander challenge was performed at baseline and 14 days after initial peptide injection.
Peptide Synthesis and Validation Chain 1: EICPAVKRDVDLFLTGT LFLTGTPDEYVEQVAQY EQVAQYKALPVVLENA KALPVVLENARILKNCV RILKNCVDAKMTEEDKE KMTEEDKENALSLLDK KENALSVLDKIYTSPL Chain 2: LTKVNATEPERTAMKK TAMKKIQDCYVENGLI SRVLDGLVMTTISSSK ISSSKDCMGEAVQNTV AVQNTVEDLKLNTLGR
Inhalational Challenge
Sputum Induction and Processing
Statistical Analyses
Isolated LARs Induced by Inhalation of T Cell Peptide Epitopes Derived from Fel d 1 A total of 35 dose administrations were performed via the inhaled route. Eight subjects (responders) experienced isolated LARs with varying symptoms of chest tightness, wheezing, and nasal congestion (Figure 2) . The frequency of reactions at individual doses was: one at 0.1 µg (16.7%), one at 1.0 µg (16.7%), four at 5.0 µg (40%), and two at 10 µg (22.2%) (Figure 3A) . The maximal decrease in FEV1 ranged from 20.2 to 36.3% of baseline, the mean maximal decrease being 26.3%. No early asthmatic reactions were observed. The remaining subjects (nonresponders) did not develop immediate or late bronchial reactions to the peptides.
An area under the curve analysis comparing the percentage change in FEV1 on inhaled diluent control versus peptide days, in the eight responders, was statistically significant (p = 0.0052). The response of the eight reactors to the inhaled bronchodilator given at 6 hours (the end of the observation time) is shown in Figure 4 . Reversal of the FEV1 was observed in all subjects.
After intradermal injection of peptides, one subject experienced a LAR at 1 µg (12.5%), two at 2.5 µg (25%), and four at 5 µg (50%) (Figure 3B). The reactions in the 5-µg group were significantly greater than in either the 2.5-µg (p = 0.031) or the 1-µg group (p = 0.015). Thus, both the frequency and magnitude of reactions were dose dependent.
Inhaled Peptide–induced LARs and Sputum Eosinophilia
Sputum Cys LTs after Inhaled Peptide–induced LARs Inhaled peptide LARs were associated with a nonsignificant increase in the sputum concentration of cys LTs from a median of 357.4 pg/ml (range, 177.9–1,512) after diluent challenge to 745.1 pg/ml (range, 462.8–1,841) after peptide challenge (p = 0.06). Nonresponders failed to demonstrate a similar increase after inhalation of peptides (diluent vs. peptide challenge, p = 0.30) (Figure 6) . A significant correlation was observed between concentration of cys LTs and eosinophil percentage in sputum collected during the inhaled peptide LARs (r = 0.89, p = 0.03). However, there was no relationship between the magnitude of the decrease in FEV1 and the change in either eosinophils (r = -0.32, p = 0.56) or LTs (r = -0.03, p = 1.00) in responders.
Effects of Repeat Challenge Six subjects who experienced a LAR after intradermal challenge of Fel d 1 peptides underwent a second intradermal challenge between 2 and 14 weeks later. The results were compared with the first challenge and also with a control day (Figure 7A) . The first challenge led to a significant decrease in FEV1 compared with that of the control day (p = 0.039), whereas the subsequent challenge did not provoke a LAR (second peptide challenge vs. control day, p = 0.84). This difference between the first and second peptide challenges was statistically significant (p = 0.008).
The eight responders who had experienced LARs provoked by inhalation also had repeat inhalational challenges at between 2 and 38 weeks. A progressive and marked decline in FEV1 was observed on both occasions (second challenge vs. control day, p = 0.0006), and there was no significant difference between the two peptide challenges in the area under the curve analysis (p = 0.079) (Figure 7B). The individual results giving the time interval between the first and second inhalational challenge and the percentage decreases in FEV1 are shown in Table 1 .
Analysis of the differential cell count on induced sputum from six repeat responders showed a significant eosinophil increase at second peptide challenge (median increase 17.5%; range, 9.2–26.5; p = 0.0312) (Figure 8) . There was no significant difference between the changes in eosinophil count observed when Challenge 1 was compared with Challenge 2.
Effect of Repeat Challenge on the Cutaneous Late-Phase Allergic Reaction to Whole Allergen Two weeks after administration of intradermal peptides there was a significant (p = 0.036) decrease in the whole allergen–induced cutaneous late-phase reaction (Figure 9A) . In contrast, no difference was observed in the magnitude of the whole allergen–induced late-phase reaction 2 weeks after inhalation of peptides (p = 0.56) (Figure 9B).
We have previously described IgE-independent, MHC-restricted isolated LARs after intradermal administration of allergen-derived T cell peptide epitopes (1–4). Here, we demonstrate that comparable reactions, that is to say a LAR without a preceding early asthmatic reaction, can be elicited by peptides given via the inhaled route. This is a novel finding and, in combination with the findings of lack of IgE-mediated histamine release in vitro (1, 3) and human leukocyte antigen restriction of T cell responses to Fel d 1 peptides (1), further supports the view that direct activation of T cells is an initiating event in the LAR. In a previous intradermal study, 9/40 individuals who were sensitive to cat allergen responded to intradermal administration of three short overlapping peptides derived from chain 1 of Fel d 1 (1). In the current investigation, 12 short overlapping peptides (each 16–17 residues long) spanning most of Chain 1 and Chain 2 of Fel d 1 were employed. These were designed to elicit reactions in a greater proportion of individuals challenged, as more T cell epitopes were likely to be present and capable of interacting with a larger number of MHC Class II restriction elements. Responsiveness was also a reflection of the dose administered, with the threshold for developing a LAR varying among individuals. Out of eight responders via the inhaled route, two had attended on successive days for peptide challenges (0.1 µg/1.0 µg/5.0 µg and 1.0 µg/5.0 µg, respectively) with a LAR occurring only at the highest dose. In the intradermal group we observed that there was a dose-dependent increase in both the frequency and magnitude of reactions, with 50% of subjects responding at a dose of 5 µg. As this was the highest dose employed, a larger dose may have resulted in more frequent responses. Because a dilutional effect occurs when a drug is administered systemically, due to the volume of distribution, the dose of peptides required to achieve an equivalent effect when applied directly to the site of action was expected to be lower (although this takes no account of relative stabilities with each route). Indeed, we observed that the minimum inhaled dose required to produce a LAR was one log-fold less than the lowest intradermal dose. Unexpectedly, the dose–response relationship in the inhalational group appeared to give high-dose inhibition. This may due to the small numbers in the study and will require further investigation. The kinetics of inhaled peptide–induced LARs was similar in both intradermally provoked reactions and LARs after inhaled whole allergen challenge. A pilot bronchoscopy study after intradermal challenge had shown that bronchoalveolar lavage and bronchial biopsies obtained during LARs had neither increased numbers of inflammatory cells (including eosinophils) nor elevated concentrations of LTs, histamine, histamine-releasing factors, or prostanoids (2). Inhalationally provoked reactions, however, were associated with luminal airway eosinophilia. Th2 cytokines, together with cysteine cysteine chemokines, especially eotaxin, lead to selective eosinophil migration, accumulation, and activation. These events may initiate airway smooth muscle contraction and edema through the production of eosinophil membrane–derived cys LTs (11). Previous work has shown that concentrations of cys LTs in induced sputum are significantly higher in individuals with asthma than in normal control subjects and increase further after acute exacerbations (12). Furthermore, the concentration of sputum cys LTs increases 24 hours after allergen inhalation in individuals with asthma and atopy, and the increase correlates with an elevation in sputum eosinophil numbers (13). We have shown here that the LAR after peptide inhalation was associated with a nonsignificant increase in induced sputum cys LTs. Furthermore, the concentration of cys LTs correlated with the eosinophil percentage at 6 hours after challenge. The significant correlation between cys LTs and eosinophil numbers suggests that eosinophils may be a major source of cys LTs within the airway and that these lipid mediators may play a role in the production of the LAR. However, neither cys LTs nor eosinophils alone correlated with the magnitude of LAR, suggesting that additional factors might influence the intensity of the asthmatic response. Thus, our current findings suggest that peptide- and whole allergen–induced LARs share a common mechanism and that the mucosal route of challenge produces inflammatory changes detectable by superficial sampling.
A number of studies have shown that administration of allergen-derived peptides can induce the development of T cell tolerance in naive and antigen-primed mice (14–17). This is preceded by transient T cell activation. For example, intranasally administered peptides derived from the house dust mite allergen Der p 1, inhibited T cell responses in previously sensitized mice and in addition prevented sensitization in naive mice (14). Initially, transient activation of MHC Class II–restricted CD4+ T cells from lymph node and splenic tissue was observed (15). In a model of bee venom allergy, an admixture of three long peptides covering the whole sequence of phospholipase A2 was employed. Again, prophylactic inhalation led to suppression of a subsequent specific IgE response in naive animals, whereas a therapeutic approach in sensitized mice was associated with a greater than 60% reduction in phospholipase A2–specific IgE, together with a decline in the interleukin-4:IFN-
We observed that a second intradermal injection of Fel d 1–derived peptides was associated with a marked reduction or absence of the LAR (clinical correlate of T cell activation), with up to 40 weeks required for return to baseline values (3). Thus, rechallenge with the same dose of peptide within approximately 40 weeks revealed partial or complete attenuation of the response. Furthermore, it was demonstrated that the cutaneous late-phase reaction to whole cat dander was also inhibited, even in subjects who did not experience an initial LAR. These observations were associated with a significant decrease in peptide- and whole allergen–induced proliferation of peripheral blood mononuclear cells and the production of interleukin-4, interleukin-13, and IFN- In the present study, we examined whether this same phenomenon occurs after inhaled peptide–induced LARs. As maximal attenuation of bronchial responsiveness did not occur until 2 weeks after the first intradermal challenge, we chose to rechallenge responders at 2 weeks or more after their first inhalation of peptides. The two subjects who had received peptides on successive days were rechallenged with the dose they received on the day of the LAR, i.e., 5.0 µg in both cases. Responders attended for repeat inhalation of peptides at varying intervals ranging from 2 to 38 weeks. Repeat LARs were induced in all cases, and no significant difference was observed between the two challenges. Furthermore, a significant sputum eosinophilia was found again. A second group of responders was used to examine the effects of a single inhaled dose of peptides on peripheral tolerance. We found no effect of peptides on the cutaneous late-phase reaction to whole allergen. Taken together, these results confirm the lack of peripheral T cell tolerance conferred by inhaled peptides. This is in marked contrast to murine studies where use of the nasal route has been successfully exploited to generate mucosal tolerance in both allergic and autoimmune disease models, albeit with much higher milligram/kilogram doses (14–16, 18, 19). One possible reason is species differences in the degree of development of nasal-associated lymphoid tissue, a constituent of the immune system with the capacity to induce tolerance. The dependence of rodents on their nose may have resulted in an evolutionary suppression of inflammatory responses to substances received intranasally. Alternatively, the dose of peptide entering the bloodstream in these murine models was likely to be much greater than that in the current study in humans. The ability of peptides to activate T cells and induce LARs appears to be unrelated to the ensuing immunomodulation observed after intradermal administration. Thus, activation of memory T cells in the airways may result in bronchoconstriction, whereas other mechanisms may be involved in the induction of hyporesponsiveness. The fact that the latter has been observed in the absence of the former in certain subjects in earlier studies (3) supports this notion. Perhaps the most likely explanation for the differential ability to induce antigen-specific hyporesponsiveness is the distribution of the peptide after challenge. In conclusion, we have demonstrated that inhalation of allergen-derived T cell peptide epitopes can elicit an isolated late-phase response in sensitized subjects by an IgE-independent mechanism. The luminal airway inflammation observed suggests that peptide- and whole allergen–induced LARs share a common mechanism associated with increases in LTs and the infiltration of inflammatory cells. However, inhaled peptide–induced LARs do not precede peptide- or whole allergen–specific hyporesponsiveness, suggesting that inhalation may be an ineffective route for peptide immmunotherapy in the treatment of allergic diseases.
The authors thank Justine Arbery (research nurse) who assisted in clinical observations, Jackie Turner for statistical analyses, ALK (UK) for whole cat dander extract for in vivo administration, CBF LETI S.A. (Madrid, Spain) for whole cat dander extract for in vitro experiments, and Dr. Haruhisa Mita for measurement of cys LTs.
Supported by the Medical Research Council and the National Asthma Campaign (UK). This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Conflict of Interest Statement: F.R.A. has no declared conflict of interest; W.L.G.O. has no declared conflict of interest; N.H. has no declared conflict of interest; M.L. has acted as a consultant to Powderject Pharmaceuticals PLC in the field of peptide vaccine development for allergic diseases, has received $80,000 in consultancy fees from June 2002–present, and is a coinvestigator on a grant from Powderject Pharmaceuticals PLC with a value of approximately $800,000, and has received approximately $160,000 following the sale of Powderject Pharmaceuticals PLC to Chiron Vaccines; A.B.K. has acted as a consultant to Powderject Pharmaceuticals PLC in the field of peptide vaccine development for allergic diseases, has received $106,000 in consultancy fees from June 2002–present, and is a coinvestigator on a grant from Powderject Pharmaceuticals PLC with a value of approximately $800,000, and has received approximately $300,000 following the sale of Powderject Pharmaceuticals PLC to Chiron Vaccines. Received in original form May 23, 2003; accepted in final form September 11, 2003
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ABSTRACT
METHODS
1 week after peptide challenge). In these individuals, sputum induction was performed after the highest dose. A LAR was defined as a decrease in FEV1 more than or equal to 20% from baseline. Eight subjects who developed a LAR after peptides (responders) repeated the study after 2 to 38 weeks. 
ratio (
