Cutaneous Late Phase Response Research Articles

A randomised placebo-controlled trial investigating efficacy and mechanisms of low-dose intradermal allergen immunotherapy in treatment of seasonal allergic rhinitis

BackgroundWe previously reported that repeated low-dose grass pollen intradermal allergen injection suppresses allergen-induced cutaneous late-phase responses, comparable with conventional high-dose subcutaneous and sublingual immunotherapy.ObjectiveTo evaluate the efficacy and mechanism of grass pollen intradermal immunotherapy for treatment of allergic rhinitis.DesignA Phase II, double-blind, randomised controlled parallel-group trial.SettingSingle-centre UK study.ParticipantsAdults aged 18–65 years, with grass pollen-induced allergic rhinoconjunctivitis.InterventionsSeven 2-weekly intradermal injections were given into the forearm, containing eitherPhleum pratensesoluble grass pollen extract (7 ng of the major allergen Phl p 5) or histamine control.Main outcome measuresThe primary outcome was a combined symptom and medication score (CSMS) during the 2013 grass pollen season. Secondary clinical outcomes were overall symptom scores; individual symptoms scores for nose, mouth, eyes and lungs; overall medication scores; CSMSs during the peak season; visual analogue scale (VAS) scores for nose and eye symptoms; Mini Rhinitis Quality of Life Questionnaire scores; health-related quality-of-life scores (European Quality of Life-5 Dimensions, 5-levels); a global evaluation of symptoms, number of symptom-free and medication-free days; number of days when prednisolone was used; and adverse events. Mechanistic studies included measurement of late-phase skin response sizes, allergen-specific antibody titres, analysis of skin biopsies and basophil activation tests.ResultsThere was no significant difference in CSMSs between treatment arms [difference in median area under curve (AUC) 14, 95% confidence interval (CI) –172.5 to 215.1;p = 0.80]. Paradoxically, among the secondary outcomes, nasal symptoms measured with daily scores were higher in the active arm (difference in median AUC 35, 95% CI 4.0 to 67.5;p = 0.03), with a trend for higher nasal symptoms measured by VASs (difference in median AUC 53, 95% CI –11.6 to 125.2;p = 0.05). No differences were seen in other clinical outcomes in the main intention-to-treat analysis. In mechanistic studies, active treatment increasedP. pratense-, Phl p 1- and Phl p 5-specific immunoglobulin E (allp = 0.001) compared with the control. T cells cultured from skin biopsies of active intradermal immunotherapy subjects showed higher T helper type 2 cell (Th2) marker CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) expression (p < 0.05) and lower T helper type 1 cell marker CXCR3 [chemokine (C-X-C Motif) receptor 3] expression (p < 0.05), respectively. Interleukin 5 messenger ribonucleic acid, measured by microarray, was more highly expressed by cultured skin T cells in the active arm (p < 0.05). Late-phase skin responses to grass pollen were still inhibited up to 7 months after intradermal immunotherapy (p = 0.03), but not at 10–13 months’ time points.LimitationsGrass pollen doses were not increased during the course, as our proof-of-concept trial showed that repeating the same doses was sufficient to achieve almost complete late-response suppression. Injections were not continued throughout the season, as previous subcutaneous grass pollen immunotherapy trials have demonstrated preseasonal regimen efficacy.ConclusionsIntradermal immunotherapy suppressed late-phase skin responses to allergen, but was not clinically effective. The intervention appeared to have an immunological priming effect and exacerbated certain seasonal symptoms, notably in the nose.Future workFurther studies on low-dose intradermal grass pollen immunotherapy are not recommended because of our demonstrated worsening of allergic rhinitis symptoms and immunological priming. The findings are of great significance for other novel immunotherapies targeting the skin, such as epicutaneous techniques.Trial registrationCurrent Controlled Trials ISRCTN78413121.FundingThis project was funded by the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research partnership.

Intradermal grass pollen immunotherapy increases TH2 and IgE responses and worsens respiratory allergic symptoms

BackgroundRepeated low-dose grass pollen intradermal allergen injection suppresses allergen-induced cutaneous late-phase responses comparably with conventional subcutaneous and sublingual immunotherapy.ObjectiveWe sought to evaluate the efficacy and safety of grass pollen intradermal immunotherapy in the treatment of allergic rhinitis.MethodsWe randomly assigned 93 adults with grass pollen–induced allergic rhinitis to receive 7 preseasonal intradermal allergen injections (containing 7 ng of Phl p 5 major allergen) or a histamine control. The primary end point was daily combined symptom-medication scores during the 2013 pollen season (area under the curve). Analysis was by intention to treat. Skin biopsy specimens were collected after intradermal allergen challenges, and late-phase responses were measured 4 and 7, 10, or 13 months after treatment.ResultsThere was no significant difference in the primary end point between treatment arms (active, n = 46; control, n = 47; median difference, 14; 95% CI, −172.5 to 215.1; P = .80). Among secondary end points, nasal symptoms were worse in the intradermal treatment group, as measured based on daily (median difference, 35; 95% CI, 4.0-67.5; P = .03) and visual analog scale (median difference, 53; 95% CI, −11.6 to 125.2; P = .05) scores. In a per-protocol analysis intradermal immunotherapy was further associated with worse asthma symptoms and fewer symptom-free days. Intradermal immunotherapy increased serum Phleum pratense–specific IgE levels (P = .001) compared with those in the control arm. T cells cultured from biopsy specimens of subjects undergoing intradermal immunotherapy had higher expression of the TH2 surface marker CRTH2 (P = .04) and lower expression of the TH1 marker CXCR3 (P = .01), respectively. Late-phase responses remained inhibited 7 months after treatment (P = .03).ConclusionIntradermal allergen immunotherapy suppressed skin late-phase responses but was not clinically effective and resulted in worsening of respiratory allergic symptoms.

A Randomized Placebo-Controlled Trial of Intradermal Grass Pollen Immunotherapy for Seasonal Allergic Rhinitis

Rationale: We previously reported that repeated intradermal injection of grass pollen (nanograms of allergen) almost completely suppresses allergen-induced cutaneous late phase responses (JACI 2012 130(4):918-24). Similar suppression of late responses is seen after high dose subcutaneous and sublingual immunotherapy. We therefore evaluated the efficacy of grass pollen intradermal immunotherapy (IDIT) for treatment of allergic rhinitis. Methods: 93 adults with grass pollen allergic rhinitis were randomized to receive 7 pre-seasonal IDIT injections (7 ng of Phl p 5 major allergen) or histamine control at 2-weekly intervals. The primary end point was combined daily symptom/medication scores (CSMS) during 2013 pollen season. Skin biopsies were taken after the pollen season following intradermal allergen challenges. Cutaneous late responses were measured 4 and either 7, 10 or 13 months post-treatment. Results: No difference in CSMS was observed between treatment arms. Paradoxically, amongst secondary endpoints, nasal symptoms measured with daily scores (P=0.03) and visual analog scales (P=0.05) were higher in the IDIT group. IDIT also increased serum Phl p-specific IgE (P=0.001) compared to the control arm. T cells cultured from skin biopsies of IDIT subjects showed higher and lower expression of surface markers for Th2 (P=0.04) and Th1 (P=0.01) cells, respectively, together with higher expression of IL-5 mRNA measured by microarray. Late responses to grass pollen were still inhibited 7 months after IDIT (P=0.03). Conclusions: Although grass pollen IDIT suppressed skin late responses, it was not clinically effective but resulted in immunological priming and worsening of allergic rhinitis symptoms.

The Inhibitory Effects of Low-Dose Ionizing Radiation in IgE-Mediated Allergic Responses.

Ionizing radiation has different biological effects according to dose and dose rate. In particular, the biological effect of low-dose radiation is unclear. Low-dose whole-body gamma irradiation activates immune responses in several ways. However, the effects and mechanism of low-dose radiation on allergic responses remain poorly understood. Previously, we reported that low-dose ionizing radiation inhibits mediator release in IgE-mediated RBL-2H3 mast cell activation. In this study, to have any physiological relevance, we investigated whether low-dose radiation inhibits allergic responses in activated human mast cells (HMC-1(5C6) and LAD2 cells), mouse models of passive cutaneous anaphylaxis and the late-phase cutaneous response. High-dose radiation induced cell death, but low-dose ionizing radiation of <0.5 Gy did not induce mast cell death. Low-dose ionizing radiation that did not induce cell death significantly suppressed mediator release from human mast cells (HMC-1(5C6) and LAD2 cells) that were activated by antigen-antibody reaction. To determine the inhibitory mechanism of mediator released by low-dose ionizing radiation, we examined the phosphorylation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, and protein kinase C, as well as the intracellular free Ca2+ concentration ([Ca2+]i). The phosphorylation of signaling molecules and [Ca2+]i following stimulation of FcεRI receptors was inhibited by low dose ionizing radiation. In agreement with its in vitro effect, ionizing radiation also significantly inhibited inflammatory cells infiltration, cytokine mRNA expression (TNF-α, IL-4, IL-13), and symptoms of passive cutaneous anaphylaxis reaction and the late-phase cutaneous response in anti-dinitrophenyl IgE-sensitized mice. These results indicate that ionizing radiation inhibits both mast cell-mediated immediate- and delayed-type allergic reactions in vivo and in vitro.

Allergen-induced expression of IL-25 and IL-25 receptor in atopic asthmatic airways and late-phase cutaneous responses

IL-25 is thought to participate in allergic inflammation by propagating T(h)2-type responses. To address the hypothesis that allergen provocation increases expression of IL-25 and its receptor IL-25R in the asthmatic bronchial mucosa and skin dermis of atopic subjects. Sequential single and double immunostaining was used to evaluate the numbers and phenotypes of IL-25 and IL-25R immunoreactive cells in bronchial biopsies from mild atopic subjects with asthma (n= 10) before and 24 hours after allergen inhalation challenge and skin biopsies from atopic subjects (n= 10) up to 72 hours after allergen subepidermal injection. IL-25 immunoreactivity was expressed by a majority of epidermal cells in both organs at baseline and was not further augmented by challenge. IL-25R immunoreactive cells were rare in the epidermis before or after challenge. Allergen challenge was associated with significantly (P< .01) increased expression of IL-25 and IL-25R immunoreactivity in the submucosa of both organs. IL-25 immunoreactivity colocalized with eosinophils, mast cells, and endothelial cells, whereas IL-25R immunoreactivity colocalized with eosinophils, mast cells, endothelial cells, and T lymphocytes. In both organs, correlations were observed between increases in IL-25 expression and the magnitudes of the late-phase allergen-induced clinical responses. Allergen provocation induces functionally relevant, increased expression of IL-25 and its receptor in the asthmatic bronchial mucosa and dermis of sensitized atopic subjects. In addition to T cells, eosinophils, mast cells, and endothelial cellsare potential sources and targets of IL-25 in the course ofallergic inflammation.

Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs

All available therapies for human allergic disease target IgE mediated pathologic responses after IgE has been produced. We are developing tetracyclines as anti-allergy drugs to prevent IgE production, based on our findings that minocycline or doxycycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses (ELISA, mast cell mediated cutaneous late phase responses); the tetracyclines also strongly suppress peak IgE responses of BPO-KLH sensitized mice (ELISPOT assay, ELISA, skin tests). The antibiotic activity of the tetracyclines is not required for suppression of IgE responses; inclusion of minocycline or doxycycline in sterile culture prevents anti-CD40/IL-4 mediated induction of memory IgE responses by PBMC of allergic asthmatic patients (ELISA), and induction of specific memory IgE responses by spleen cells of BPO-KLH sensitized mice (ELISPOT assay, ELISA). The tetracyclines affect an epsilon specific pathway because IgM, IgG and IgA responses did not decrease. Further, in humans, DTH responses to recall antigens did not decrease. In related studies, we found that two distinct T cell subsets: CD4+CD60 negative and CD8+CD60+ (CD60 is a ganglioside) (humans) and CD4+ Asialo GM1 ganglioside negative and CD8+Asialo GM1 ganglioside+ (mice), both are required for induction of memory IgE responses. Phosphorylated (phos) p38 MAP kinase, but not phos ERK or phos JNK expression by CD4+ and CD8+, including CD8+CD60+, T cells is increased in allergic asthmatic humans, as is IL-4 and IL-10 production. The tetracyclines appear to target T cell pathways to induce suppression of IgE responses because they suppress phos p38 MAP kinase expression by both CD4+ and CD8+, including CD8+CD60+, T cell subsets, and IL-4 and IL-10, while upregulating IL-2 and IFN gamma, and suppressing IgE responses. Our finding that tetracyclines do not require antibiotic activity to suppress IgE responses opens the door to development of new tetracycline-based and other therapeutics for human allergic disease.

Basophils Infiltrate Skin Lesions of Eosinophilic Pustular Folliculitis (Ofuji’s Disease)

© 2011 The Authors. doi: 10.2340/00015555-1052 Journal Compilation © 2011 Acta Dermato-Venereologica. ISSN 0001-5555 Basophils are blood leukocytes that constitute less than 0.5% of total leukocytes. Basophils have been shown to infiltrate in cutaneous hypersensitivity responses in guinea pigs (cutaneous basophil hypersensitivity) (1, 2). Basophil infiltration is also observed in allergen-induced late-phase cutaneous responses in human atopic subjects (3) and has been implicated in allergic human diseases (4). Recent findings in mice have demonstrated clearly that basophils are essential initiator cells of immunoglobulin (Ig)E-mediated chronic allergic inflammation (5), and they are capable of functioning as a source of interleukin (IL)-4 and contributing to Th2-type immunity (6). Eosinophilic pustular folliculitis (EPF) is an inflammatory skin disease characterized by pruritic follicular papulopustules of unknown aetiology. Histologically, a number of eosinophils infiltrate around and into hair follicles in the dermis, whereby it is suggested that the Th2-type immune response is involved in this pathological mechanism (7). However, basophils in skin lesions have not been studied in detail because these cells, unlike eosinophils, cannot be detected by routine histological staining techniques. To address this, we performed immunohistochemical staining to detect tissue basophils in EPF lesions. MATErIALS AND METHoDS

Early production of thymic stromal lymphopoietin precedes infiltration of dendritic cells expressing its receptor in allergen‐induced late phase cutaneous responses in atopic subjects

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine that triggers dendritic cell-mediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL-7 receptor alpha (IL-7Ralpha) chain and the TSLP receptor (TSLPR), which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD4(+) T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen-induced late-phase reaction (LPR) in atopic subjects. Skin biopsies were obtained from atopic subjects (n = 9) at various times after cutaneous allergen challenge. In situ hybridization and immunohistochemistry were used to determine TSLP mRNA expression and to measure infiltration of TSLPR(+) DC in skin LPR. RT-PCR and flow cytometry were employed to analyse TSLPR expression on isolated blood DC. Allergen-induced skin TSLP expression occurred as early as 1 h after allergen challenge, whereas TSLPR(+) and CD11c(+) cells infiltrated relatively late (24-48 h). The majority of TSLPR(+) cells were DC co-expressing blood DC antigen-1 (BDCA-1) or BDCA-2. Freshly isolated blood DC expressed both TSLPR and IL-7Ralpha chains. Maturation and stimulation with TSLP or polyriboinosinic-polyribocytidylic acid in vitro upregulated the expression of both TSLPR and IL-7Ralpha chains in DC but not in chemoattractant receptor-homologous molecule expressed on Th2 cells(+) CD4(+) T cells. The data suggest that TSLP plays a role in augmenting, through DC recruitment and activation, the development of Th2-type T cells in allergic inflammation.

Minocycline Therapy Inhibits Development of Mast Cell Mediated Cutaneous Late Phase Responses

RATIONALE: We have previously reported minocycline treatment of allergic asthmatic patients has oral steroid-sparing effects, improves symptoms and spirometry (Daoud, 2008), suppresses ongoing human and murine IgE responses in vivo (Joks, 2008) and in vitro induction of memory IgE responses (Smith-Norowitz, 2002) in isotype specific manner. Effect of minocycline on induced cutaneous allergic inflammation has not been studied.

Effect of Dexamethasone on the Cutaneous Late Phase Response

The cutaneous late phase response (LPR) demonstrates characteristics of the inflammatory response associated with acute onset or exacerbations of atopic dermatitis (AD). The current study describes the characterization of the cutaneous LPR in monkeys. Ascaris suum (Asc) sensitive, male cynomolgus monkeys (6-10 kg, n = 4-6) were anesthetized and intradermal injections (40 μL) of PBS or Asc were administered over the thorax/abdominal area. The early phase response (EPR), wheal and flare, was measured 15 minutes post Asc. The LPR was evaluated by analyzing the cellular infiltrate from punch biopsies obtained 20 h after allergen exposure. During treatment studies, monkeys were administered dexamethasone (1 mg/kg; i.m.) 1 h prior to allergen. Asc exposure resulted in a wheal and flare that was significantly (p ≤ 0.04) larger than the paired PBS response. At 20 h post Asc injection a moderate inflammatory response in the upper and deep dermis (p = 0.03) was observed. The LPR was characterized by dermal eosinophilia, and an increase in CD4+ and CD25+ cells, indicative of infiltration and activation of T cells. Pretreatment with dexamethasone had no effect on the Asc-induced EPR, but inhibited the LPR. Dexamethasone significantly inhibited upper dermal inflammation associated with the LPR (p = 0.05) and eosinophilia in both the upper (p = 0.001) and deep (p = 0.05) dermis. With Asc intradermal injection, both an EPR and LPR cutaneous response is observed in the cynomolgus monkey. Acute administration of dexamethasone significantly inhibited the cutaneous LPR. These data elucidate the potential utility of this model for the evaluation of novel therapeutics for AD.

Immune response to allergens in sheep sensitized to house dust mite

BackgroundHouse dust mite (HDM) allergens are a major cause of allergic asthma. Most studies using animal models of allergic asthma have used rodents sensitized with the 'un-natural' allergen ovalbumin. It has only recently been recognized that the use of animal models based on HDM provide a more relevant insight into the allergen-induced mechanisms that underpin human allergic disease. We have previously described a sheep model of human allergic asthma that uses Dermatophagoides pteronyssinus HDM. The present study extends our understanding of the immune effects of HDM and the allergens Der p 1 and Der p 2 in the sheep model of asthma.MethodsPeripheral blood sera from non-sensitized (control) sheep and sheep sensitized to HDM was collected to determine immunoglobulin (Ig) reactivities to HDM, Der p 1 and Der p 2 by ELISA. Bronchoalveolar lavage (BAL) fluid collected following allergen challenge was also assessed for the presence of HDM-specific antibodies. To examine the cellular immune response to HDM allergens, T cell proliferation and cutaneous responses were assessed in sensitized and control sheep.ResultsStrong HDM- and Der p 1-specific IgE, IgG1, IgG2 and IgA serum responses were observed in sensitized sheep, while detectable levels of HDM-specific IgG1 and IgA were seen in BAL fluid of allergen-challenged lungs. In contrast, minimal antibody reactivity was observed to Der p 2. Marked T cell proliferation and late phase cutaneous responses, accompanied by the recruitment of eosinophils, indicates the induction of a cellular and delayed-type hypersensitivity (DTH) type II response by HDM and Der p 1 allergen, but not Der p 2.ConclusionThis work characterizes the humoral and cellular immune effects of HDM extract and its major constituent allergens in sheep sensitized to HDM. The effects of allergen in HDM-sensitized sheep were detectable both locally and systemically, and probably mediated via enzymatic and immune actions of the major HDM allergen Der p 1. This study extends our understanding of the actions of this important allergen relevant to human allergic asthma and its effects in sheep experimentally sensitized to HDM allergens.

Thymic Stromal Lymphopoietin (TSLP) and Receptor (TSLPR) and CD11c+ Dendritic Cells (DC) in Allergen-induced Cutaneous Late Phase Responses (LPR) in Atopic Subjects

RATIONALE: TSLP is an IL-7-like cytokine that triggers dendritic cell-mediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL-7 receptor alpha (IL-7Rα) chain and the TSLP receptor (TSLPR) which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD4+ T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen-induced LPR in atopic subjects.

Changes in Morphology and Key Cytokine Gene Expression after Intradermal Injection of Louse (Bovicola ovis) Antigen in Sheep with Naturally Occurring Atopic Dermatitis

Groups of louse-infested and louse-naïve lambs (n=6 or 7) were used in two experiments to determine the sequential tissue response (macroscopical, microscopical and key cytokine mRNA) to intradermal injection of crude louse (Bovicola ovis) antigen over a period of 72 or 96 h. Histamine diphosphate and phosphate-buffered saline/glycerol (antigen vehicle control) solutions were also injected intradermally in each lamb for comparison. In both experiments, louse-infested lambs showed immediate and late-phase responses (LPRs) to louse antigen that differed significantly from the responses in the louse-naïve lambs. In experiment 1, biopsy samples taken at 7, 24, 48 and 96 h after injections showed more extensive dermal inflammation and leucocyte infiltration in response to louse antigen in louse-infested than in louse-naïve lambs. Eosinophils were significantly more numerous in the dermis of louse-infested lambs after all treatments and increased substantially in these lambs after antigen injection. Additionally, the louse-infested lambs differed from the naïve lambs in showing significantly higher mononuclear leucocyte and basophil infiltration and significantly lower neutrophil infiltration after antigen injection. In experiment 2, biopsy samples taken 4, 24, 48 and 72 h after injections showed trends in eosinophil infiltration of the dermis similar to those observed in experiment 1. Peak IL-4 mRNA expression was detected 4 h after antigen injection in the louse-infested lambs and remained significantly elevated at 24 h as compared with the results in the louse-naïve lambs. No significant difference in IFN-gamma mRNA expression between the louse-infested and the louse-naïve lambs was observed. These results indicated that louse-infested lambs show a cutaneous LPR analogous to that observed in atopic human beings and dogs. However, some differences were observed, including the longer duration of the LPR, the profuse eosinophil infiltration, and an absence of increased IFN-gamma mRNA expression.

Inhalation Tolerance Is Induced Selectively in Thoracic Lymph Nodes but Executed Pervasively at Distant Mucosal and Nonmucosal Tissues

Under immunogenic conditions, both the site of initial Ag exposure and consequent T cell priming in specific draining lymph nodes (LNs) imprint the ensuing immune response with lasting tissue-selective tropism. With respect to immune tolerance, whether the site of tolerance induction leads to compartmentalized or, alternatively, pervasive tolerance has not been formally investigated. Using a murine model of inhalation tolerance, we investigated whether the induction of respiratory mucosal tolerance precludes the development of de novo Th2 sensitization upon subsequent exposure to the same Ag at distant mucosal (gut) and nonmucosal (cutaneous) sites. By tracking the proliferation of CFSE-labeled OVA-TCR transgenic CD4(+) T cells upon OVA inhalation in vivo, we defined the site of tolerance induction to be restricted to the thoracic LNs. Expectedly, inhalation tolerance prevented de novo Th2 sensitization upon subsequent exposure to the same Ag at the same site. Importantly, although gut- and skin-draining LNs were not used during tolerance induction, de novo Ag-specific proliferation and Th2 differentiation in these LNs, as well as memory/effector Th2 responses in the gut (allergic diarrhea) and skin (late-phase cutaneous responses) were inhibited upon immunogenic challenge to the same Ag. Interestingly, this pervasive tolerogenic phenotype was not associated with the presence of suppressive activity throughout the lymphatics; indeed, potent suppressive activity was detected solely in the spleen. These data indicate that while inhalation tolerance is selectively induced in local thoracic LNs, its tolerogenic activity resides systemically and leads to pervasive immune tolerance in distant mucosal and nonmucosal sites.

Cutaneous Antigen Priming via Gene Gun Leads to Skin-Selective Th2 Immune-Inflammatory Responses

It is becoming increasingly evident that the compartmentalization of immune responses is governed, in part, by tissue-selective homing instructions imprinted during T cell differentiation. In the context of allergic diseases, the fact that "disease" primarily manifests in particular tissue sites, despite pervasive allergen exposure, supports this notion. However, whether the original site of Ag exposure distinctly privileges memory Th2 immune-inflammatory responses to the same site, while sparing remote tissue compartments, remains to be fully investigated. We examined whether skin-targeted delivery of plasmid DNA encoding OVA via gene-gun technology in mice could generate allergic sensitization and give rise to Th2 effector responses in the skin as well as in the lung upon subsequent Ag encounter. Our data show that cutaneous Ag priming induced OVA-specific serum IgE and IgG1, robust Th2-cytokine production, and late-phase cutaneous responses and systemic anaphylactic shock upon skin and systemic Ag recall, respectively. However, repeated respiratory exposure to aerosolized OVA failed to instigate airway inflammatory responses in cutaneous Ag-primed mice, but not in mice initially sensitized to OVA via the respiratory mucosa. Importantly, these contrasting airway memory responses correlated with the occurrence of Th2 differentiation events at anatomically separate sites: indeed cutaneous Ag priming resulted in Ag-specific proliferative responses and Th2 differentiation in skin-, but not thoracic-, draining lymph nodes. These data indicate that Ag exposure to the skin leads to Th2 differentiation within skin-draining lymph nodes and subsequent Th2 immunity that is selectively manifested in the skin.

IL-5 production by allergen-stimulated T cells following grass pollen immunotherapy for seasonal allergic rhinitis

SUMMARY Grass pollen immunotherapy for the treatment of seasonal allergic rhinitis (‘summer hayfever’) results in improvement in symptoms, a reduction in the early and late phase responses to allergen provocation and decreased tissue eosinophilia. Immunotherapy may act by altering the pattern of cytokine production by allergen-specific T cells from a ‘Th2-type’ (IL-4 and IL-5) profile to a ‘Th1-type’ (interferon-gamma (IFN-γ)) profile. We set out to determine whether clinical improvement following specific allergen immunotherapy is accompanied by reduced production of the pro-eosinophilic and archetypal ‘Th2-type’ cytokine, IL-5. Peripheral blood mononuclear cells (PBMC) were isolated from (i) 13 patients who had received 6 or 7 years' continuous conventional immunotherapy with timothy grass pollen (Phleum pratense); (ii) 14 patients who had received 3 or 4 years of conventional immunotherapy followed by 3 years of placebo treatment; (iii) 12 matched seasonal rhinitic patients who had never received immunotherapy; and (iv) 17 non-atopic normal controls. PBMC were stimulated with 20 μg/ml and 200 μg/ml P. pratense extract, or 10 μg/ml of Mycobacterium tuberculosis purified protein derivative (PPD), at 2 × 106 cells/ml and 5 × 106 cells/ml. IL-5 concentrations in culture supernatants collected after 6 days' culture were measured by ELISA. IL-5 production in response to stimulation with P. pratense extract was highly reproducible and was elevated in both of the immunotherapy treated groups and the untreated rhinitics relative to non-atopic controls (P &amp;lt; 0.005 for each group relative to non-atopic controls, under each of the four conditions tested). However, no significant reduction was observed in IL-5 production when immunotherapy treated patients were compared with untreated rhinitic controls. Moreover, abrogation of the cutaneous late-phase responses to allergen following treatment was not associated with reduced IL-5 production by allergen-stimulated peripheral blood T cells. Reduced IL-5 production by peripheral blood T cells may not be necessary for immunotherapy to be effective. Local immunodulation of T cell responses may play a role in this form of treatment.

The effect of montelukast on allergen‐induced cutaneous responses in house dust mite allergic children

The benefits of a leukotriene modifier on allergen-induced cutaneous responses have not been studied in children. We hypothesized that a leukotriene-receptor antagonist would provide protection against allergen-induced early and/or late phase cutaneous responses in susceptible children. In a randomized, double-blind, placebo-controlled, cross-over study in 6- to 15-year-old house dust mite allergic children, we compared the benefit of either montelukast or a matching placebo for 2 days on cutaneous responses induced by intradermal injection of house dust mite extract. Responses were measured as the mean of the longest diameter and its longest perpendicular of the induration response after 10, 20 min, 4 h and 6 h of the challenge. A total of 30 children were randomized to placebo or montelukast. The mean decline from baseline was significantly greater (p = 0.04) in the montelukast-treated children [-5.20 mm (-12.03-1.63)][median (95% CI)] than in the placebo-treated children [0.13 mm (-6.46-6.73] at 6th hour of the challenge, as was the mean diameter (p = 0.04), but did not differ in the remaining time points. We concluded that in house dust mite allergic children, montelukast 5 mg provided significant improvement in allergen-induced cutaneous late-phase responses, suggesting its contribution to allergic skin responses and its potential therapeutic value.

The Editors’ Choice

The Editors’ Choice

Inhibition of human T-cell responses by allergen peptides.

Of the many significant advances in the field of immunology that have occurred in the last 30 years arguably the most important, at least from a T-cell biologists point of view, has been the definition of major histocompatibility complex (MHC) restriction1 and the crystal structure of human leucocyte antigen (HLA) molecules.2–4 These demonstrations have enabled detailed study of the way in which T cells recognize antigen and also how this process differs from that in B cells. With a few notable exceptions, production of specific immunoglobulin by B cells is T-cell dependent. Furthermore, recruitment of non-lymphoid effector cells into areas of inflammation is a process largely marshalled by the T lymphocyte. It follows therefore that inhibition of T-cell function may have beneficial outcomes in situations in which detrimental immune responses occur and give rise to pathology. Suppression of T-cell function has been achieved with the use of pharmacological agents, such as glucocorticosteroids and cyclosporin, and more recently with biological agents, such as monoclonal antibodies directed against CD3 and CD4. Whilst achieving the desired aim of immunosuppression, these approaches suffer from serious weaknesses, the most obvious of which is a lack of specificity for the antigen(s) which drive the inflammatory process. The same may be said to be true of other therapeutic approaches currently being developed which aim to neutralize the effects of individual mediators in the inflammatory cascade. Here, a notable success has been the use of monoclonal antibodies to tumour necrosis factor (TNF) to treat patients with rheumatoid arthritis.5 However, the long-term consequences of removing individual mediators remain to be seen. Of concern is the recent report of subjects receiving anti-TNF therapy developing a lupus-like syndrome thought to be a direct result of the therapy itself.6 The challenge of introducing specificity into strategies aimed at modulating T-cell responses is one that has been met with considerable success in animal models by the use of peptide epitopes from the antigen in question. Peptide-induced tolerance has been demonstrated in models of experimental autoimmune encephalomyelitis (EAE),7,8 collagen-induced arthritis9 and, more recently, in models of allergic disease. Briner and colleagues sensitized mice to the cat allergen Fel d I and subsequently demonstrated the ability of allergen-derived peptides to inhibit T-cell cytokine and antibody production.10 Hoyne and colleagues demonstrated the ability of peptides from the house dust mite allergen Der p II to down-regulate T-cell and antibody responses to challenge with intact protein and also to prevent sensitization by prior administration.11,12 Bauer administered a dominant T-cell epitope of Bet v I to CBA/J mice either prior to or following sensitization with the whole allergen. No change in antibody isotype was observed but T-cell proliferative responses were inhibited by both treatments.13 More recently, mice sensitized to bee venom allergen phospholipase A2 (PLA2), were treated by intranasal administration of a mixture of three long peptides spanning the entire molecule. A marked reduction in specific immunoglobulin E (IgE) was observed coupled with a decreased interleukin-4 (IL-4) to interferon-γ (IFN-γ) ratio. Pre-administration of peptide resulted in a failure to develop IgE sensitization to subsequent immunization with the whole molecule.14 In addition to animal models, the ability of high-dose peptides to modulate human T-cell responses has also been demonstrated. In vitro studies with human T cells were performed by Lamb and colleagues, who demonstrated the induction of antigen-specific non-responsiveness following the incubation of T-cell clones with supraoptimal doses of specific ligand in the absence of professional antigen-presenting cells.15 Collectively, these studies provided a logical basis for the translation of a peptide-based therapeutic approach in man.

Interleukin-10 levels increase in cutaneous biopsies of patients undergoing wasp venom immunotherapy.

We have studied the influence of wasp venom immunotherapy (VIT) on cellular recruitment and cytokine mRNA expression during allergen-induced cutaneous late-phase responses (LPR). Nine subjects with a history of wasp sting anaphylaxis, and specific IgE in their sera underwent wasp VIT. Skin biopsies were taken 24 h after intradermal diluent and allergen before and after 3 months VIT. Pre-immunotherapy, there were significant allergen-induced increases in EG2(+) eosinophils, elastase(+) neutrophils, CD68(+) macrophages and IL-10 protein(+) cells, and increased expression of mRNA for IL-4, IL-13, IFN-gamma, IL-12, IL-10, TGF-beta, RANTES and eotaxin. When these allergen-induced changes in cytokine mRNA and cellular profiles were compared with those obtained after 3 months VIT there was a significant reduction in IL-4 mRNA (p=0.012) and increase in IL-10 protein(+) cells (p=0.004) with a trend to an increase in IL-10 mRNA (p=0.054). There were also significant reductions in eosinophils (p<0.004) and the size of the cutaneous LPR (p<0.01) but no change in mRNA to IFN-gamma, IL-13 or IL-12. Therefore, VIT is associated with a significant increase in cells positive for IL-10 protein but not IL-12 or IFN-gamma. These results suggest that induction of IL-10 may be important in VIT and occur independently of the switch to a Th1 phenotype. IL-10 generation may down-regulate IL-4 expression and eosinophil recruitment.