Introduction

The prevalence of allergic diseases has increased dramatically over the past few decades, with population prevalence rates reaching 30% in the industrialized nations. The defining feature of allergic disorders is their association with aberrant levels, and targets, of immunoglobulin E (IgE) production. Allergy is thought to result from maladaptive immune responses to ubiquitous, otherwise innocuous environmental proteins, referred to as allergens.1 Allergens, by definition, are proteins that have the ability to elicit powerful T helper lymphocyte type 2 (Th2) responses, culminating in IgE antibody production (atopy). Although allergens represent a minute fraction of the proteins that humans are routinely exposed to, allergenicity is a very public phenomenon, with the identical proteins functioning as allergens in allergic patients. Why specific proteins cause such aberrant T- and B-cell responses is a basic mechanistic question that has remained largely unanswered.

Allergens derive from a variety of environmental sources, such as plants (trees, grasses), fungi (Alternaria alternata), arthropods (mites, cockroaches), and other mammals (cats, dogs, cows). Allergens constitute a diverse range of molecules, varying in size from small to large multidomain proteins. As they are derived from complex living organisms, they serve a broad range of functions in their respective hosts, from structural to enzymatic. For example, the common house dust mite allergens include several cysteine proteases (Der p 1, Der p 3), serine proteases (Der p 3, Der p 6, Der p 9), chitinases (Der p 15, Der p 18), lipid-binding molecules (Der p 2), and structural molecules such as tropomyosin (Der p 10). Some are species specific; others are molecules with broad biochemical homology that are found in many species. Much work has centered on the study of the allergen epitopes recognized by T and B cells. However, as there is no compelling evidence for common structural characteristics among the diverse T- and B-cell epitopes recognized in allergic responses,2, 3, 4 it appears doubtful that the presence of such B- and T-cell epitopes are sufficient to endow a protein with allergenic potential. Other factors, such as the size, glycosylation status, resistance to proteolysis, and enzymatic activity, have been suggested to be important in allergenicity. However, none of these factors has been consistently linked with allergenic potential. For example, glycosylation appears not to be a common critical determinant of allergenicity as both glycosylated and nonglycosylated proteins function as food allergens. It may be that there are many structural paths to allergenicity, but the absence of any common structural motif or conformational sequence pattern leaves open the possibility that proteins with allergic potential exhibit a necessary commonality of biological function. Indeed, it has been recently proposed that allergens are linked by their ability to activate the innate immune system of mucosal surfaces, triggering an initial influx of innate immune cells that subsequently promote Th2-polarized adaptive immune responses. It should be noted that, reductive experimental systems aside, natural exposure is not to single, purified proteins, but to complex mixtures of molecules. It may well be that the innate immune-activating molecules are not identical to the proteins recognized by allergic responses, although this would still beg the question as to why those particular proteins are so recognized among the many present during exposure. In this review, we address recent advances in our understanding of the diverse innate immune-activating properties of allergens that appear to endow them with a propensity for promoting Th2 immune responses.

Protease Activity and Allergic Sensitization

Several allergens have cysteine or serine protease activity, including diverse allergens from arthopods (e.g., house dust mites,5, 6, 7 German cockroaches,8 fungi (A. alternata),9 and Cladosporium herbarum,10 mammals (e.g., Felis domesticus),11 plants (e.g., pollens from ragweed12). In addition, many forms of occupational allergy are associated with encounters with proteolytic enzymes such as those used in the manufacture of detergents (alkaline detergents),13 or in the food industry (papain).14

Several lines of evidence suggest that proteases may facilitate allergen sensitization. First, intrinsic protease activity appears to be linked with sensitization ability in several allergens. Removal of proteases from Aspergillus fumigatus,15 German cockroach frass,16 American cockroach Per a 10 antigen,17 Epi p1 antigen from the fungus Epicoccum purpurascens,18 or Cur 11 antigen from the mold Curvularia lunata19 was reported to decrease airway inflammation and airway hyperresponsiveness in mouse models of allergic asthma. Second, direct exposure of mice to proteolytic enzymes such as papain can induce allergic sensitization.20 Moreover, co-administration of active proteases from A. fumigatus with the tolerogenic antigen, ovalbumin (OVA), resulted in allergic sensitization.15 As co-exposure to a tolergenic protein with a protease can induce allergic sensitization, proteases found in ambient air derived from bacterial and viral species may have accessory roles. Finally, subcutaneous injection of a serine protease inhibitor, nafamostat mesilate, during sensitization to house dust mite extracts blunted the development of allergic inflammation and airway hyperresponsiveness.21

The exact mechanisms by which proteases can promote allergic sensitization are not well understood. Several mechanisms have been postulated. First, protease activity may increase transepithelial access of allergens to critical cells of the innate immune response, such as dendritic cells (DCs). For example, the cysteine protease Der p 1 can alter epithelial permeability through disruption of epithelial tight junctions and a reduction in ZO-1 and occludin content.6 Consistent with this, proteolytic enzymes from a number of tree and grass pollens have also been shown to degrade ZO-1 and disrupt tight junctions.22 Moreover, Der p 1 has been shown to cleave α-1-anti-trypsin, inhibiting its ability to protect the respiratory tract against serine proteases such as Der p 3 and Der p 9. This may disrupt the protease–antiprotease balance in mucosal tissues, enhancing the activity of both endogenous and exogenous proteases and leading to enhanced tissue damage and immune activation.

Second, the cysteine protease activity of several mite allergens (Der p 1, Der f 1) may directly impair innate defense mechanisms in the lung by degrading and inactivating lung surfactant proteins (SPs)-A and -D.23 SP-A and SP-D are calcium-dependent carbohydrate-binding proteins with multiple innate immune functions, including bacterial agglutination and modulation of leukocyte functions. Importantly, SP-D and SP-A have been shown to protect against allergic inflammation induced by A. fumigatus in mice,24, 25 likely through binding to glucan moieties of inhaled allergens and facilitation of their clearance.

Proteases may have more direct immunomodulatory actions as well. Proteases from mites, cockroaches, and fungi can increase the expression of cytokines, including interleukin (IL)-6, IL-8, and granulocyte monocyte colony-stimulating factor,5, 6, 8, 9 which may lead to the recruitment, activation, and/or enhanced survival of DCs at the mucosal surface. In addition, Der p 1 has been shown to influence the expression of co-stimulatory molecules such as CD40 on DCs. Der p 1 can cleave CD40 on human monocyte-derived DCs, resulting in inhibition of the production of the pivotal Th1-differentiating cytokine, IL-12.26 The suppression of CD40 signaling and IL-12 production may induce a shift toward Th2 responses. A similar effect has been observed with the mold Aspergillus (Asp). Exposure of healthy human monocyte-derived DCs to Asp induced their maturation and enhanced their ability to prime Th2 immune responses in allogeneic naive T cells as compared with naive T cells primed with lipopolysaccharide (LPS)-activated DCs.27 When the proteolytic activity of Asp was neutralized by chemical inactivation, Asp failed to upregulate co-stimulatory molecules on DCs, and these DCs did not prime a Th2 response in naive T cells. The skewed Th2 response was thought to occur as a result of suppressed IL-12 production by Asp-primed DCs. Interestingly, although the exact mechanisms by which allergen-derived proteases influence the decision-making capability of DCs are not well understood, recent reports suggest that protease containing allergens such as Der p 1 can target two C-type lectins, DC-SIGN and DC-SIGNR.28 Loss of DC-SIGN expression after Der p 1 treatment led to a reduction in its binding to its ligand, ICAM-3, on naive T cells, which is thought to be important in Th1 signaling.28 Thus, the combined proteolytic activities of Der p 1 on surface expression of molecules, such as CD40 and DC-SIGN, could have profound effects on the decision-making capability of DCs, biasing adaptive immunes response toward a Th2 pattern of response.

Recently, study of the occupational allergen, papain (commonly used in the food industry), has led to the novel hypothesis that proteases can directly prime Th2 immune responses through actions on basophils.20 Data suggest that papain can cleave a yet-to-be identified host sensor which, in turn, activates basophils to produce IL-4, and thymic stromal lymphopoietin-promoting Th2 differentiation. This intriguing work supports the concept that host detection of protease activity associated with allergens may provide a unique pathway of innate immune activation. The generality of this pathway, as well as its molecular identification, remains to be defined.

Allergen-derived proteases can have direct effects on adaptive immune responses as well, through cleavage of molecules such as CD25 and CD23. Specifically, Der p 1 has been shown to be able to cleave the α-chain of the IL-2 receptor (CD25) on human T cells29 (Figure 1). As a result, T cells exposed directly to Der p 1 show markedly reduced Th1 cytokine production and enhanced Th2 cytokine production, something dependent on the protease activity or Der p 1. Cleavage of CD25 might also, of course, alter regulatory function, as IL-2 stimulation is required for the maintenance of regulatory T cells in the periphery. The overall effect may be to shift the balance of immune responses from a tolerogenic response to one favoring a Th2 pattern of response. Allergenic proteases such as Der p 1 have also been shown to be able to cleave the low-affinity receptor for IgE, CD23, from the surface of human B cells, releasing the soluble form of the receptor30 (Figure 1). As the membrane-bound form of the IgE receptor is thought to exert its effect as a negative regulator of IgE synthesis, Der p 1 cleavage of CD23 could potentially disrupt the negative feedback signal and enhance IgE synthesis, thereby amplifying the allergic response. Antitrypsin can inhibit this effect of Der p 1 on CD23 cleavage, suggesting that disruption of the balance between proteases and protease inhibitors might have a role in allergic sensitization. It should be noted that whether intact proteases such as Der p 1 actually gain functional access to lymphocytes in vivo remains an open question.

Figure 1
figure 1

Schematic of innate immune mechanisms activated by allergens.

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The biological effects of some allergenic proteases may also be mediated through activation of the protease-activated receptor 2 (PAR2). PARs (1, 2, 3, 4) are a family of proteolytically activated G-protein-coupled receptors. Proteases cleave within the N terminus of the receptors and expose a tethered ligand domain that binds and activates the cleaved receptor. Several lines of evidence suggest that PAR2, in particular, may be important in allergic sensitization. It is expressed by many cells in the lung, including airway epithelial cells,31 fibroblasts,32 macrophages33 and mast cells,34 and, importantly, patients with asthma have been shown to exhibit increased expression of PAR2 on respiratory epithelial cells.35 Several house dust mite allergens (Der p1, Der p 3, Der p 9), along with German cockroach extract,36 have been shown to be able to cleave and activate PAR2 (Figure 1). Several reports have shown that activation of PAR2 by house dust mite extract,7 German cockroach,37 or the mold allergen Pen c 1338 leads to increased cytokine production by airway epithelia. Specifically, airway epithelial cells were shown to increase the expression of thymic stromal lymphopoietin through the activation of PAR2 when treated with papain, trypsin, or the fungus Alterneria.9 As thymic stromal lymphopoietin promotes DC polarization of naive T cells to a Th2 phenotype, these results suggest that PAR2 activation may serve as a link between innate and adaptive immune responses.

Several mouse models of allergic inflammation have also underscored a potential role for PAR2 in allergic sensitization. For example, one recent study showed that tolerance to inhaled OVA could be overcome by co-administration of PAR2-activating peptides to the airways, promoting allergic sensitization.39 Other studies have shown that overexpression of PAR2 in mice renders them susceptible to allergic airway inflammation when sensitized and challenged locally with OVA, as compared with wild-type controls.40 In contrast, PAR2-deficient mice were protected against allergic inflammation. Although these data strongly support the concept that protease activity may lead to airway allergic sensitization through PAR2 activation, there are a few reports suggesting that PAR2 activation may also reduce airway inflammation. For example, despite the fact that TLR4 (vide infra) and PAR2 signaling have been shown to exhibit cooperativity,41 PAR2-activating peptides have been reported to inhibit LPS-induced neutrophil influx into mouse airways.42 In a rabbit model of experimental asthma, sensitization to the pollen Parietaria judaica, followed by allergen challenge in the presence or absence of a PAR2-activating peptide, led to PAR2-mediated attenuation of the development of airway hyperresponsiveness and airway eosinophilia.43 It is possible that the discrepancies in these results are due to the timing of PAR2 activation, with activation by exogenous proteases during generation of immune responses having different effects than that occurring in the midst of an ongoing inflammatory response.

Toll-Like Receptors, Lipid-Binding Activity, and Allergic Sensitization

Recent recognition of the critical roles of innate pattern recognition receptors such as Toll-like receptors, NOD-like receptors, RIG-I-like receptors, and C-type lectin receptors, in activation and instruction of antigen-presenting cells44 has led to the exploration of the role of these pathways in allergic responses. It will be noted that, although TLR-promoted activation of Th1 responses by DCs is well studied and well understood, the receptors and pathways promoting Th2 immune responses have been considerably less tractable to experimental investigation.

Numerous studies have probed the role of TLR4 signaling in allergic inflammation. Epidemiological studies have reported an inverse correlation between high levels of bacterial products, such as LPS in the ambient environment during very early life and the subsequent development of atopy and allergic disease.45, 46, 47 It has been postulated, pace the hygiene hypothesis, that such exposures mediate robust counter-regulatory tone in the developing immune system.48 LPS exposure can also exacerbate established asthma, however, probably by direct stimulation of airway pro-inflammatory responses.49 Experimental mouse models have provided mechanistic insight into the ability of LPS exposure to regulate the development of allergic asthma. As predicted by the hygiene hypothesis, LPS dose appears to be a critical variable. Although airway sensitization with OVA along with “very low dose” (<1 ng) LPS was reported to induce tolerance, sensitization in the presence of “low-dose” (100 ng) LPS promoted TLR-dependent, Th2 inflammation, and sensitization in the presence of “high-dose” (100 μg) LPS led to a Th1 (and likely a regulatory) response.50, 51 Recent studies using bone marrow chimeras indicate that TLR4 signaling in radioresistant cells, not radiosensitive hematopoietic cells, is necessary and sufficient for DC activation and priming of allergic effector T helper responses in the lung in response to dust mite extracts.52 As for other Toll-like receptors, TLR2 ligands have been shown to be able to promote53 or inhibit54 Th2 differentiation and allergic inflammation in the lung.

A recent study has reported a more direct link between TLR signaling and allergic sensitization. Among defined dust mite antigens, Der p 2 and Der f 2 have the highest rates of skin test positivity in atopic patients.55 Notably, sequence homology places these allergens in the MD-2-related lipid-recognition domain family of proteins56, 57—MD-2 being a secreted protein that is the LPS-binding member of TLR4 signaling complex. As the crystal structures of Der p 2 and MD-2 exhibit structural homology, Trompette et al.58 examined whether Der p 2 exhibited functional homology as well. Indeed, they reported that Der p 2 can facilitate TLR4 signaling through direct interactions with the TLR4 complex, reconstituting LPS-promoted TLR4 signaling in the absence of MD-2 and facilitating such signaling in the presence of MD-2 (Figure 1). They further found that Der p 2 could facilitate LPS signaling in primary antigen-presenting cells, with or without MD-2 being present. Finally, they reported that the in vitro functional and biochemical activities of Der p 2 show its in vivo allergenicity—Der p 2 promotes experimental allergic asthma in a TLR4-dependent manner, retaining this property in mice with a genetic deletion of MD-2. These data suggest that Der p 2's propensity to be targeted by the adaptive immune response is a function of its autoadjuvant properties. In the light of this study, it should be noted that efficient generation of effector T-cell responses has been shown to depend on the presence of TLR ligands in the specific DC phagosome that contains the antigen.59 In the case of Der p 2, antigen and TLR ligand are, perforce, colocalized. These data also suggest the possibility that Der p 2-mediated facilitation of TLR4 signaling under conditions of bacterial product exposure—those associated with increasing rates of aeroallergy in the urban, Westernized world—may shift the LPS–response curve from the tolerizing into the Th2-inducing range. Der p 2 may also promote exacerbation of established asthma by facilitating TLR4 signaling by airway epithelial cells—cells reported to express TLR4, but little or no MD-2, in the basal state.60

Several other members of the MD-2-like lipid-binding family are major allergens,4 suggesting generality for these findings. More broadly, however, more than 50% of defined major allergens are thought to be lipid-binding proteins,4 something that suggests that intrinsic adjuvant activity by such proteins and their lipid cargo is likely to have wide generality as a mechanism underlying the phenomenon of allergenicity. Further studies defining the lipids normally bound by these allergens, the receptors thereby activated, and the pathways of innate and adaptive immune response promoted by such activation are awaited. It should be noted that, in addition to activation of Toll-like receptors, lipid ligands are known to be important promoters (and targets) of innate lymphocyte responses.

Carbohydrate Structures and Allergic Sensitization

Recent data also suggest an importance of complex carbohydrates in promoting Th2 immune responses. Helminth-derived carbohydrates such as lacto-N-fucopentaose III have been shown to promote Th2 responses through their ability to activate DCs in vivo.61 In addition, lacto-N-fucopentaose III has been shown to be able to promote Th2 responses to a co-administered, unrelated antigen such as human serum albumin.62 Though the mechanism underlying DC activation by lacto-N-fucopentaose III glycoconjugates has not been fully elucidated, it involves ligation of C-type lectins on the DC, leading to subsequent antagonism of TLR signaling. Support for a broad role for complex carbohydrates, in particular glucans, in allergen-associated Th2 immune responses is emerging. Glucans are a diverse class of naturally occurring glucose polymers, which can be short or long, branched or unbranched, exist as α- or β-isomers, and be soluble or particulate. For the purpose of this discussion, we are mostly concerned with the β-glucans, which contain a polyglucose, (1 → 3)-β-D-glucan, and are commonly found in the cell walls of fungi, pollens, and certain bacteria. In plants, polymers of β-glucans are thought to protect the developing pollen during meiosis, and are later destroyed by the enzyme (1 → 3)-β-D-glucanase to liberate the microspores. Although β-glucans are widely expressed, they are not found in mammalian cells. As such, they can exert their effect as pathogen-associated molecular patterns, triggering immune responses through activation of specific innate pattern recognition receptors.

The immunostimulatory properties of β-glucans have been recognized for decades, since their identification as the immunoactive component of mushrooms.63 Recently, β-glucans have been shown to be able to promote Th2 responses. For example, it has been reported that β-glucan structures present in the peanut glycoallergen Ara h 1 have Th2-inducing characteristics;64 native, but not deglycosylated, Ara h 1 has been shown to activate human monocyte-derived DC and induce IL-4- and IL-13-secreting Th2 cells.

Exposure to β-glucans has also been shown to induce airway hyperresponsiveness in allergic humans.65 Studies in guinea pigs have shown that direct delivery of (1 → 3)-β-D-glucan to the airways can induce the recruitment of lung eosinophils and lymphocytes.66 In mice, exposure to soluble β-glucan isolated from Candida albicans67 markedly exacerbated OVA-induced eosinophilic airway inflammation, concomitant with enhanced lung expression of Th2 cytokines and IL-17A. Exposure to β-glucans and OVA increased the number of cells bearing major histocompatibility complex class II and the expression of antigen-presenting cell-related molecules such as CD80, CD86, and DEC205 on bone marrow-derived DCs. In support of a role of β-glucans in the recruitment and activation of DCs at mucosal surfaces, recent studies have shown that β-glucans contained in house dust mite extracts and in molds may initiate immune responses at the mucosal surface. House dust mite extract-mediated induction of the release of the chemokine, CCL20, which recruits immature DCs, by human airway epithelial cells in culture, was shown to occur through β-glucan and Syk-dependent signaling pathways68 (Figure 1). Although the exact lectin receptor mediating these effects was not identified in these studies, the results suggested that β-glucan moieties contained in house dust mite extracts might mediate early processes leading to immature DC recruitment to the airways. This concept is supported by another recent study that showed that dectin-2 receptor signaling pathways (Dectin-2/FcRγ/Syk) mediated the production of cysteinyl leukotrienes in bone marrow-derived DCs following stimulation with house dust mite extracts or Aspergillus69 (Figure 1). Taken together, these findings identify the dectin-2/FcRγ/Syk axis as a novel receptor-mediated pathway by which several potent allergens are recognized by innate immune cells at the airway surface, linking them with the development of Th2-skewed adaptive immune responses.

Consistent with a role for lectins in promoting Th2 immune responses, blockade of the mannose receptor, an endocytic C-type lectin receptor, significantly reduced Der p 1 uptake by DCs.70 These findings are consistent with previous findings suggesting that engagement of the mannose receptor by selected ligands on human DCs leads to the induction of a DC phenotype favoring Th2 polarization.71 Although the study of the role of glucans as Th2-inducing pathogen-associated molecular patterns is only in its infancy, data to date suggest that carbohydrate moieties contained in common allergens function as strong Th2 inducers through activation of variety of C-type lectin receptors on DCs.

Oxidative Activity and Allergen Sensitization

It has been recently shown that common allergenic pollen grains contain nicotinamide adenine dinucleotide phosphate (reduced) oxidase activity as well as allergens.72 Such pollen grains have been shown to increase the levels of reactive oxygen species significantly in cultured cells, and to be able to induce allergic airway inflammation in experimental animals73 (Figure 1). Pretreatment of these pollen grains with nicotinamide adenine dinucleotide phosphate oxidase inhibitors attenuated their capacity to increase reactive oxygen species levels in airway epithelial cells and subsequent airway inflammation. Similarly, pretreatment of mice with antioxidants has been shown to prevent the development of pollen-mediated asthma in mice. Interestingly, delaying antioxidant treatment until after pollen challenge was ineffective, suggesting that the oxidase activity is of critical importance during the period of innate immune activation. Although the mechanisms remain to be defined, it has been speculated that nicotinamide adenine dinucleotide phosphate oxidase activity initiates immune activation through its ability to recruit inflammatory cells, possibly through the induction of IL-8 by p38 mitogen-activated protein kinase.74 Of interest, genetic polymorphisms in genes regulating oxidative stress have been shown to be associated with susceptibility to asthma in several populations.75

Conclusions

Although allergens are a diverse group of molecules, it is becoming increasingly clear that their allergenicity likely resides in their ability to activate various innate immune pathways at mucosal surfaces, rather than in any structural similarities. Complex allergens contain multiple innate immune-activating components, which trigger the initial mucosal influx of innate immune cells that subsequently promote Th2-polarized adaptive immune responses. Although the study of innate activating properties of allergens is in its infancy, it is clear that a better molecular understanding of the fundamental origins of allergenicity may well lead to the development of new therapeutic strategies to block allergen recognition and the ensuing inflammatory cascade effectively.

Disclosure

The authors declared no conflict of interest.