Cytokine fingerprinting: characterization of chemical allergens.

Chemical allergy is a common and important occupational health issue. Allergic sensitization induced by chemicals may take a variety of forms, including allergic contact dermatitis (skin sensitization) and allergic asthma and rhinitis (sensitization of the respiratory tract). There is a need to identify and characterize chemicals that have the potential to cause such sensitization reactions. Although a number of methods are available for the prospective analysis of skin sensitizing activity, there are currently no widely accepted tests for the identification of chemical respiratory allergens. We here describe a novel approach, cytokine fingerprinting, that has the potential to distinguish between chemical contact and respiratory allergens. The pattern of cytokine production by draining lymph node cells (LNCs) is evaluated following repeated topical exposure of mice to test chemicals. Experience to date reveals that contact allergens stimulate the selective development of type 1 immune responses associated with the secretion by draining LNCs of interferon gamma (IFN-gamma), but little interleukin-4 (IL-4) or interleukin-10 (IL-10). In contrast, chemical respiratory allergens are found to induce the appearance of preferential type 2 immune responses characterized by IL-4 and IL-10 production, but comparatively low levels of IFN-gamma. It is proposed that cytokine fingerprinting may permit the simultaneous identification and characterization of those chemicals that have the potential to cause allergic sensitization.     

Early immune events in the induction of allergic contact dermatitis

The skin is a barrier site that is exposed to a wide variety of potential pathogens. As in other organs, pathogens that invade the skin are recognized by pattern-recognition receptors (PRRs). Recently, it has been recognized that PRRs are also engaged by chemical contact allergens and, in susceptible individuals, this elicits an inappropriate immune response that results in allergic contact dermatitis. In this Review, we focus on how contact allergens promote inflammation by activating the innate immune system. We also examine how innate immune cells in the skin, including mast cells and dendritic cells, cooperate with each other and with T cells and keratinocytes to initiate and drive early responses to contact allergens.     

Recombinant allergens for analysing T-cell responses

T-cell responses constitute a central element of allergic disease and a model for studying Th1 and Th2 cytokine pathways. Most studies to date have used extracts of allergens which contain variable quantities of different allergens and non-allergenic antigens. Recombinant allergens provide the tools for studying the responses to allergens in a reproducible and dose-dependent manner and the different T-cell responses of allergic and non-allergic subjects provide a method for verifying the responses and their relationship to allergic sensitisation. Most allergies show dominant responses to one or a few major allergens. These allergens have been described for the common allergies and have been produced as recombinant allergens. A particular problem for allergens is that many are mixtures of proteins from multi-gene families or are highly polymorphic. Information now exists so the sequence variation can be represented. Purified recombinant allergens produced by standard expression systems stimulate the expected T-cell responses from the peripheral blood of allergic and non-allergics to allergen extracts. Although stimulation with recombinant allergens which are not produced with a natural IgE binding activity can provide a measure of allergenicity, the altered tertiary structure can reduce Th2 responses. The sequence information now available provides the means to use PCR to produce cDNA for the production of recombinant allergens from readily available sources. The production of the highly reactive recombinant Der p 2 allergen of house dust mite from natural sources is described.     

Molecular determinants for antibody binding on group 1 house dust mite allergens

House dust mites produce potent allergens, Der p 1 and Der f 1, that cause allergic sensitization and asthma. Der p 1 and Der f 1 are cysteine proteases that elicit IgE responses in 80% of mite-allergic subjects and have proinflammatory properties. Their antigenic structure is unknown. Here, we present crystal structures of natural Der p 1 and Der f 1 in complex with a monoclonal antibody, 4C1, which binds to a unique cross-reactive epitope on both allergens associated with IgE recognition. The 4C1 epitope is formed by almost identical amino acid sequences and contact residues. Mutations of the contact residues abrogate mAb 4C1 binding and reduce IgE antibody binding. These surface-exposed residues are molecular targets that can be exploited for development of recombinant allergen vaccines.     

Domestic arthropods and their allergens

Allergy prevalence has increased worldwide over the last 25 years along with industrialization and westernized lifestyles. Indoor allergens are primarily responsible for the sensitization and development of atopic diseases. The main indoor allergens are known to be derived from various arthropods which account for up to 80% of the kingdom Animalia. The two classes of arthropods are Insecta, which includes cockroaches, flies, mosquitoes, ants and silverfishes, and Arachnida, which includes mites, spiders, ticks, and scorpions and are the main sources of the allergens. Excreted materials, cast-overs from skin-molting, and dead debris are sources of allergens that can sensitize genetically predisposed individuals and elicit allergic disorders. The use of molecular biology techniques has contributed to the identification and characterization of an ever-increasing number of allergens. However, key determinants and allergen properties that drive allergic responses are poorly understood. The biological characterization of allergens will provide an understanding of the pathogenesis of allergic diseases and contribute to the development of new diagnostic and therapeutic approaches.     

Prevalence of arthropod antibodies in Korean patients with allergic rhinitis.

Arthropod antigens are main causative agents which induce allergic responses in humans. However, little information is known about the prevalence of specific arthropod allergens in Koreans with allergic diseases. The current study was designed to determine the positive rates of arthropod antibodies by the Korean inhalant panel of MAST-CLA. One hundred sixty patients, who were diagnosed with allergic rhinitis from an out-patient center at the Soonchunhyang University Chunan Hospital, were studied between August 1998 to July 2000. The overall positive rate, at least more than one specific antibody of arthropods such as Dermatophagoides farinae (Df), Dermatophagoides pteronyssinus (Dp), and cockroach mix (Cm), was 46.9%. Each positive rate of Df, Dp, and Cm was 45.0%, 43.1%, and 8.8%, respectively. A significant agreement among arthropod allergens was observed (Df and Dp: 95.6%, Kappa = 0.911, P < 0.001). Our data supported the fact that arthropods were the most common allergens in Korean patients with allergic rhinitis; however, the MAST-CLA should be modified to increase specificity of arthropod allergens.     

Impact of cutaneous IL-10 on resident epidermal Langerhans' cells and the development of polarized immune responses

Prolonged topical exposure of BALB/c mice to chemical contact and respiratory allergens stimulates, respectively, preferential Th1- and Th2-type responses with respect to serum Ab isotype and cytokine secretion phenotypes displayed by draining lymph node cells. We now report that differential cytokine secretion patterns are induced rapidly in the skin following first exposure to the contact allergen 2,4-dinitrochlorobenzene (DNCB) and the respiratory sensitizer trimellitic anhydride (TMA). TMA induced early expression of IL-10, a cytokine implicated in the negative regulation of Langerhans cell (LC) migration, whereas exposure to DNCB resulted in production of the proinflammatory cytokine IL-1beta. Associated with this, TMA provoked LC migration with delayed kinetics compared with DNCB, and local neutralization of IL-10 caused enhanced LC mobilization in response to TMA with concomitant up-regulation of cutaneous IL-1beta. We hypothesize that these differential epidermal cytokine profiles contribute to the polarization of immune responses to chemical allergens via effects on the phenotype of activated dendritic cells arriving in the draining lymph node. Thus, TMA-exposed dendritic cells that have been conditioned in vivo with IL-10 (a potent inhibitor of the type 1-polarizing cytokine IL-12) are effective APCs for the development of a Th2-type response.     

Allergenic tropomyosins and their cross-reactivities

The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock. A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen. Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates. Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.     

Contact allergy caused by fragrance mix and Myroxylon pereirae (balsam of Peru)--a retrospective study

Because of their widespread use, fragrances are among the most common causes of contact allergic dermatitis, second only to nickel. During a five-year period 3,065 patients with contact dermatitis were patch tested using a specific mix of fragrances. 509 (16.6%) patients were allergic to the fragrance mix, while 258 (8.4%) patients exhibited an allergic reaction to Myroxylon pereirae (balsam of Peru). Between those 509 patients, 157 were patch tested with eight individual substances contained in the fragrance mix: cinnamal, cinnamyl alcohol, eugenol, isoeugenol, geraniol, hydroxycitronellal, alpha-amyl cinnamal and Evernia prunastri (oak moss). The most frequent allergens were isoeugenol 57.9% (91/157), eugenol 55.4% (87/157), cinnamyl alcohol 34.4% (54/157) and Evernia prunastri (oak moss) 24.2% (38/157). There were 62 patients (39.5%) who exhibited an allergic reaction to both the fragrance mix and Myroxylon pereirae (balsam of Peru). The results prove the importance of avoiding allergens in daily life, especially in industrial and cosmetic products. In order to prevent ACD, better cooperation between industry and dermatologists is needed.     

Adverse reactions to foods

Allergic reactions to foods represent a prominent, actual and increasing problem in clinical medicine. Symptoms of food allergy comprise skin reactions (urticaria, angioedema, eczema) respiratory (bronchoconstriction, rhinitis), gastrointestinal (cramping, diarrhea) and cardiovascular symptoms with the maximal manifestation of anaphylactic shock. They can be elicited by minute amounts of allergens. The diagnosis of food allergy is done by history, skin test, in vitro allergy diagnosis and — if necessary — oral provocation tests, if possible placebo-controlled. Avoidance of respective allergens for the allergic patient, however, is often complicated or impossible due to deficits in declaration regulations in many countries. Increasing numbers of cases including fatalities, due to inadvertent intake of food allergens are reported. It is therefore necessary to improve declaration laws and develop methods for allergen detection in foods. Allergens can be detected by serological methods (enzyme immunoassays, in vitro basophil histamine release or in vivo skin test procedures in sensitized individuals). The problem of diagnosis of food allergy is further complicated by cross-reactivity between allergens in foods and aeroallergens (pollen, animal epithelia, latex etc.). Elicitors of pseudo-allergic reactions with similar clinical symptomatology comprise low-molecular-mass chemicals (preservatives, colorings, flavor substances etc.). For some of them (e.g. sulfites) detection assays are available. In some patients classic allergic contact eczema can be elicited systemically after oral intake of low-molecular-mass contact allergens such as nickel sulfate or flavorings such as vanillin in foods. The role of xenobiotic components in foods (e.g. pesticides) is not known at the moment. In order to improve the situation of the food allergic patient, research programs to elucidate the pathophysiology and improve allergen detection strategies have to be implemented together with reinforced declaration regulations on a quantitative basis.     

Endogenous attenuation of allergic lung inflammation by syndecan-1

The airway plays a vital role in allergic lung diseases by responding to inhaled allergens and initiating allergic inflammation. Various proinflammatory functions of the airway epithelium have been identified, but, equally important, anti-inflammatory mechanisms must also exist. We show in this study that syndecan-1, the major heparan sulfate proteoglycan of epithelial cells, attenuates allergic lung inflammation. Our results show that syndecan-1-null mice instilled with allergens exhibit exaggerated airway hyperresponsiveness, glycoprotein hypersecretion, eosinophilia, and lung IL-4 responses. However, administration of purified syndecan-1 ectodomains, but not ectodomain core proteins devoid of heparan sulfate, significantly inhibits these inflammatory responses. Furthermore, syndecan-1 ectodomains are shed into the airway when wild-type mice are intranasally instilled with several biochemically distinct inducers of allergic lung inflammation. Our results also show that syndecan-1 ectodomains bind to the CC chemokines (CCL7, CCL11, and CCL17) implicated in allergic diseases, inhibit CC chemokine-mediated T cell migration, and suppress allergen-induced accumulation of Th2 cells in the lung through their heparan sulfate chains. Together, these findings uncover an endogenous anti-inflammatory mechanism of the airway epithelium where syndecan-1 ectodomains attenuate allergic lung inflammation via suppression of CC chemokine-mediated Th2 cell recruitment to the lung.     

The role of innate immunity activation in house dust mite allergy

House dust mite (HDM) allergy is a frequent inflammatory disease found worldwide. Although allergen-specific CD4(+) Th2 cells orchestrate the HDM allergic response, notably through induction of IgE directed towards mite allergens, recent studies have demonstrated that innate immunity activation also plays a critical role in HDM-induced allergy pathogenesis. HDM allergens can not only be considered proteins that induce adaptive Th2-biased responses in susceptible subjects but also as strong activators of innate immune cells, including skin keratinocytes and airway epithelial cells. The contribution of microbial adjuvant factors, derived from HDM carriers or the environment, is also essential in such cell stimulation. This review highlights how HDM allergens, together with microbial compounds, promote allergic responses through pattern recognition receptor-dependent pathways.     

A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease

The respiratory allergens that induce experimental Th cell type 2-dependent allergic lung inflammation may be grouped into two functional classes. One class of allergens, in this study termed type I, requires priming with adjuvants remote from the lung to overcome airway tolerogenic mechanisms that ordinarily preclude allergic responses to inhaled Ags. In contrast, the other, or type II, allergen class requires neither remote priming nor additional adjuvants to overcome airway tolerance and elicit robust allergic lung disease. In this study, we show in an experimental model that diverse type II allergens share in common proteolytic activity that is both necessary and sufficient for overcoming airway tolerance and induction of pulmonary allergic disease. Inactivated protease and protease-free Ag fragments showed no allergenic potency, demonstrating that only active protease acting on endogenous substrates was essential. Furthermore, induction of airway tolerance could be aborted and allergic lung disease established by simply adding purified protease to a type I allergen. Thus, exogenous proteases are common to type II allergens and may be generally required to overcome the innate resistance of the airway to Th cell type 2 activation and allergic inflammation, raising concern for their potential contribution to diseases such as asthma.     

CTLA-4 signaling regulates the intensity of hypersensitivity responses to food antigens, but is not decisive in the induction of sensitization

Although food allergy has emerged as a major health problem, the mechanisms that are decisive in the development of sensitization to dietary Ag remain largely unknown. CTLA-4 signaling negatively regulates immune activation, and may play a crucial role in preventing induction and/or progression of sensitization to food Ag. To elucidate the role of CTLA-4 signaling in responses to food allergens, a murine model of peanut allergy was used. During oral exposure to peanut protein extract (PPE) together with the mucosal adjuvant cholera toxin (CT), which induces peanut allergy, CTLA-4 ligation was prevented using a CTLA-4 mAb. Additionally, the effect of inhibition of the CTLA-4 pathway on oral exposure to PPE in the absence of CT, which leads to unresponsiveness to peanut Ag, was explored. During sensitization, anti-CTLA-4 treatment considerably enhanced IgE responses to PPE and the peanut allergens, Ara h 1, Ara h 3, and Ara h 6, resulting in elevated mast cell degranulation upon an oral challenge. Remarkably, antagonizing CTLA-4 during exposure to PPE in the absence of CT resulted in significant induction of Th2 cytokines and an elevation in total serum IgE levels, but failed to induce allergen-specific IgE responses and mast cell degranulation upon a PPE challenge. These results indicate that CTLA-4 signaling is not the crucial factor in preventing sensitization to food allergens, but plays a pivotal role in regulating the intensity of a food allergic sensitization response. Furthermore, these data indicate that a profoundly Th2-biased cytokine environment is insufficient to induce allergic responses against dietary Ag.     

Contact allergy to special and standard allergens in patients with venous ulcers

The aim of the study was to determine the prevalence of contact sensitivity in patients with leg ulcers, and possible difference in the rate of contact hypersensitivity to standard series of allergens used in patch testing, and to particular topical agents used in local therapy of leg ulcers in special series, patients with and without atopy. The study included 60 patients, 45 female and 15 male, aged 37-85 (mean 68.37 female and 51.13 male), 30 of them with and 30 without allergic contact dermatitis (ACD) of the leg (control group). The mean duration of leg ulceration was 5.62 years. The two groups of patients underwent testing to standard series allergens and target series allergens including mupirocin, bepanthene, silver sulfadiazine, chloramphenicol + clostridiopeptidase, betamethasone dipropionate, hydrocortisone + oxytetracycline, momethasone, alginate, hydrocolloid, lanolin, pyrogallol, Vaseline, permanganate, Rivanol, povidone-iodine, gentamicin, i.e. local agents most frequently used by the patients. Contact allergic hypersensitivity to standard series allergens was demonstrated in 25 patients with a total of 49 positive reactions and a mean of 1.6 reactions per patient. Positive reactions were most commonly recorded to balsam of Peru, fragrance mix and neomycin sulfate. There were 12 positive reactions to target series allergens, mean 0.4 reactions per patient. Forty-five positive reactions, mean 0.1 reactions per patient, were recorded in the control group. Positive reactions were most commonly demonstrated to corticosteroid ointments, lanolin and bepanthene. Study results did not confirm a statistically significantly higher rate of sensitization to particular topical agents frequently used in the treatment of patients with venous ulcers. Patch testing to standard and special series allergens should be performed in case of prolonged leg ulcer epithelization.     

Effect of Mouse Strain in a Model of Chemical-induced Respiratory Allergy

The inhalation of many types of chemicals is a leading cause of allergic respiratory diseases, and effective protocols are needed for the detection of environmental chemical–related respiratory allergies. In our previous studies, we developed a method for detecting environmental chemical–related respiratory allergens by using a long-term sensitization–challenge protocol involving BALB/c mice. In the current study, we sought to improve our model by characterizing strain-associated differences in respiratory allergic reactions to the well-known chemical respiratory allergen glutaraldehyde (GA). According to our protocol, BALB/c, NC/Nga, C3H/HeN, C57BL/6N, and CBA/J mice were sensitized dermally with GA for 3 weeks and then challenged with intratracheal or inhaled GA at 2 weeks after the last sensitization. The day after the final challenge, all mice were euthanized, and total serum IgE levels were assayed. In addition, immunocyte counts, cytokine production, and chemokine levels in the hilar lymph nodes (LNs) and bronchoalveolar lavage fluids (BALF) were also assessed. In conclusion, BALB/c and NC/Nga mice demonstrated markedly increased IgE reactions. Inflammatory cell counts in BALF were increased in the treated groups of all strains, especially BALB/c, NC/Nga, and CBA/J strains. Cytokine levels in LNs were increased in all treated groups except for C3H/HeN and were particularly high in BALB/c and NC/Nga mice. According to our results, we suggest that BALB/c and NC/Nga are highly susceptible to respiratory allergic responses and therefore are good candidates for use in our model for detecting environmental chemical respiratory allergens.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments and second structure information of Fag s 1: Fagales allergen from <Emphasis Type="Italic">Fagus sylvatica</Emphasis>

Fagales allergens belonging to the Bet v 1 family account responsible for the majority of spring pollinosis in the temperate climate zones in the Northern hemisphere. Among them, Fag s 1 from beech pollen is an important trigger of Fagales pollen associated allergic reactions. The protein shares high similarity with birch pollen Bet v 1, the best-characterized member of this allergen family. Of note, recent work on Bet v 1 and its homologues found in Fagales pollen demonstrated that not all allergenic members of this family have the capacity to induce allergic sensitization. Fag s 1 was shown to bind pre-existing IgE antibodies most likely primarily directed against other members of this multi-allergen family. Therefore, it is especially interesting to compare the structures of Bet v 1-like pollen allergens, which have the potential to induce allergic sensitization with allergens that are mainly cross-reactive. This in the end will help to identify allergy eliciting molecular pattern on Bet v 1-like allergens. In this work, we report the ¹H, ¹⁵N and ¹³C NMR assignment of beech pollen Fag s 1 as well as the secondary structure information based on backbone chemical shifts.     

Laboratory animal allergy: an update

Allergic reactions are among the most common conditions affecting the health of workers involved in the care and use of research animals. Between 11 and 44% of the individuals working with laboratory animals report work-related allergic symptoms. Of those who become symptomatic, 4 to 22% may eventually develop occupational asthma that can persist even after exposure ceases. Allergic symptoms consist of rashes where animals are in contact with the skin, nasal congestion and sneezing, itchy eyes, and asthma (cough, wheezing, and chest tightness). The generation of immunoglobulin E (IgE) antibodies is a prerequisite for the production of allergic symptoms. The mechanism by which IgE antibodies develop is becoming clearer. The propensity to produce IgE is genetically determined, and pre-existing allergy may be a risk factor for the development of laboratory animal allergy (LAA). However, exposure to animal allergens is the major risk factor for the development of LAA. Techniques to measure the airborne concentration of laboratory animal allergens have been developed. Research on animal allergens themselves indicates that many of the mouse and rat urinary proteins belong to a family of proteins called lipocalins, which share sequence homology with antigens of the parasitic agent that causes schistosomiasis. The fact that parasite infections also trigger IgE antibody responses may account for the development of LAA in persons who have never had any previous allergy. The prevention of LAA should be a major goal of an effective health and safety program in the animal research facility, and it can be accomplished by education and training of employees, reduction of exposure (including the use of personal protective gear), and changes in facility design. Medical surveillance programs can also play a role in improving health of individuals working with laboratory research animals. Early recognition of symptoms and evidence of sensitization can lead to interventions to reduce exposure and thereby avoid the long-term health consequences of LAA.     

No Adjuvant Effect of Bacillus thuringiensis-Maize on Allergic Responses in Mice

Genetically modified (GM) foods are evaluated carefully for their ability to induce allergic disease. However, few studies have tested the capacity of a GM food to act as an adjuvant, i.e. influencing allergic responses to other unrelated allergens at acute onset and in individuals with pre-existing allergy. We sought to evaluate the effect of short-term feeding of GM Bacillus thuringiensis (Bt)-maize (MON810) on the initiation and relapse of allergic asthma in mice. BALB/c mice were provided a diet containing 33% GM or non-GM maize for up to 34 days either before ovalbumin (OVA)-induced experimental allergic asthma or disease relapse in mice with pre-existing allergy. We observed that GM-maize feeding did not affect OVA-induced eosinophilic airway and lung inflammation, mucus hypersecretion or OVA-specific antibody production at initiation or relapse of allergic asthma. There was no adjuvant effect upon GM-maize consumption on the onset or severity of allergic responses in a mouse model of allergic asthma.     

Human Langerhans Cells Control Th Cells via Programmed Death-Ligand 1 in Response to Bacterial Stimuli and Nickel-Induced Contact Allergy

Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4⁺T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6⁺/CCR4⁺ T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6⁺ cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6⁺/CCR4⁺ cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.