Retagging identifies dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN) protein as a novel receptor for a major allergen from house dust mite

Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.     

Monoclonal antibodies to recombinant Der p 2, a major house dust mite allergen: specificity, epitope analysis and development of two-site capture ELISA.

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides. A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 micrograms/ml with the D. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts.     

Acid-induced polymerization of the group 5 mite allergen from Dermatophagoides pteronyssinus.

House dust mites are the most important source of indoor allergens and cause allergic diseases. Our studies here suggest that the group 5 allergen from Dermatophagoides pteronyssinus (Der p 5) is monomeric at neutral pH, but forms filaments at low pH. Circular dichroism measurements show Der p 5 is a helical protein, and the protein sequence reveals Der p 5 contains coiled-coil helices. The acid-induced filament assembly could be explained in part by the high content of charged residues (40%) in the coiled-coil structure. Interestingly, some of the known Dermatophagoides allergens also contain a heptad repeat, which could potentially form coiled coils. Therefore, coiled-coil helices may be one of the common structural motifs of mite allergens that contribute to their allergenicity.     

Sequence polymorphisms of Der f 1, Der p 1, Der f 2 and Der p 2 from Korean house dust mite isolates

Amino acid sequence variations have possible influences on the allergenicity of allergens and may be important factors in allergen standardization. This study was undertaken to investigate the sequence polymorphisms of group 1 and 2 allergens from Korean isolates of the house dust mites Dermatophagoides farinae and D. pteronyssinus. cDNA sequences encoding group 1 and 2 allergens were amplified by RT-PCR and compared the deduced amino acid sequences. Der f 1.0101, which appeared in 64.0 % of the 50 sequences analyzed, was found to be predominant. Among the Der p 1 sequences, Der p 1.0102 and 1.0105 were predominant (58 %). Among the Der f 2 sequences, Der f 2.0102 (40.7 %) and a new variant with Gly at position 42 (27.8 %) were predominant. The deduced amino acid sequences of 60 Der p 2 clones were examined, and 28 variants with 1-5 amino acid substitutions were found. Interestingly, all of the Der p 2 sequences had Thr instead of Lys at position 49. Two variants (Leu40, Thr49, and Asn114 (26.6 %); Val40, Thr49, and Asn114 (20.0 %)) were found to be the most predominant forms of Der p 2. Der p 1 has a high rate of sporadic substitutions and the group 2 allergens show a more regular pattern with orderly associations of amino acid substitutions. Der f 1 and Der p 2 from Korean mite isolates have unique amino acid sequence polymorphisms. These findings provide important data for house dust mite allergen standardization.     

Functional Interaction of Common Allergens and a C-type Lectin Receptor, Dendritic Cell-specific ICAM3-grabbing Non-integrin (DC-SIGN), on Human Dendritic Cells

Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the α1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le^x). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-α expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-α expression in MDDCs via, in part, Raf-1 signaling pathways.     

House dust mite allergens in domestic homes in Cheonan, Korea

House dust mites produce inhalant allergens of importance to allergic patients. We measured the major group 1 allergens, Der p 1 and Der f 1, from the house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farina, respectively in 100 randomly selected domestic homes from Cheonan, Korea. Dust samples were collected by vacuuming from the living room floor and 1 mattress in each home. Der p 1 and Der f 1 were measured by double monoclonal ELISA. Der p 1 levels were very low, with geometric mean levels for floors and mattresses being 0.11 microgram/g (range: 0.01-4.05) and 0.14 microgram/g (range: 0.01-30.0), respectively. Corresponding levels of Der f 1 were higher, 7.46 microgram/g (range: 0.01-262.9) and 10.2 microgram/g (range: 0.01-230.9) for floors and mattresses, respectively. D. farinae appears to be the dominant house dust mite in Cheonan.     

Antigen Der f I from the dust mite Dermatophagoides farinae: structural comparison with Der p I from Dermatophagoides pteronyssinus and epitope specificity of murine IgG and human IgE antibodies

The physicochemical and antigenic properties of an allergen purified from Dermatophagoides farinae, Der f I, were compared with Der p I from Dermatophagoides pteronyssinus. On SDS-PAGE, Der f I migrated as a single polypeptide chain with the same m.w. as Der p I (24,000). Two isoallergenic peaks of Der f I were identified on preparative isoelectric focusing (pI 5.7 to 6.3 and pI 6.6 to 6.95). Fractions from each peak were shown to have an identical amino acid composition (which was similar but not identical to Der p I) and the same N-terminal amino acid sequence. There was a good correlation between quantitative intradermal skin tests to both purified allergens and to D. farinae extract in mite-allergic patients, with positive results when using as little as 10(-5) micrograms/ml of Der f I. The majority of sera with detectable IgE antibody to D. farinae also had IgE antibody to Der f I both among children (29/42 = 69%) and adults (55/63 = 87%). By RAST, there was an excellent correlation between IgE antibody to Der f I and Der p I in sera from 42 mite-allergic children (n = 0.94, p less than 0.001). Polyclonal IgG antibodies from six mice immunized with Der f I showed preferential binding to that allergen, and most monoclonal antibodies (16 of 18) raised against Der f I did not bind Der p I. However, two monoclonal antibodies from this fusion showed cross-reactive binding to both allergens. Immunoabsorption experiments, using D. pteronyssinus and D. farinae extracts coupled to Sepharose, showed that a large proportion of murine antibodies (74% to Der p I and 60 to 93% to Der f I) could not be absorbed by the heterologous extract on the immunosorbent. In contrast, in sera from seven mite-allergic patients, most of the specific IgE and IgG antibody (i.e., greater than or equal to 82%) was removed by either immunosorbent. Thus, Der f I and Der p I represent a homologous pair of major allergens which possess both cross-reacting and species-specific epitopes. The antibody response in mice immunized with either allergen in complete Freund's adjuvant was largely directed against species-specific epitopes, whereas in allergic humans, IgE- and IgG-specific antibodies bound predominately to cross-reacting epitopes.     

Cooperative effects of Th2 cytokines and allergen on normal and asthmatic bronchial epithelial cells

In sensitized individuals, exposure to allergens such as Dermatophagoides pteronyssinus (Der p) causes Th2 polarization and release of cytokines, including IL-4 and IL-13. Because Der p extracts also have direct effects on epithelial cells, we hypothesized that allergen augments the effects of Th2 cytokines by promoting mediator release from the bronchial epithelium in allergic asthma. To test our hypothesis, primary bronchial epithelial cultures were grown from bronchial brushings of normal and atopic asthmatic subjects. RT-PCR showed that each culture expressed IL-4R(alpha), common gamma-chain, and IL-13R(alpha)(1), as well as IL-13R(alpha)(2), which negatively regulates IL-13 signaling; FACS analysis confirmed IL-13R(alpha)(2) protein expression. Exposure of epithelial cultures to either Der p extracts, TNF-alpha, IL-4, or IL-13 enhanced GM-CSF and IL-8 release, and this was partially suppressible by corticosteroids. Simultaneous exposure of the epithelial cultures to IL-4 or IL-13 together with Der p resulted in a further increase in cytokine release, which was at least additive. Release of TGF-alpha was also increased by TNF-alpha and combinations of IL-4, IL-13, and Der p; however, this stimulation was only significant in the asthma-derived cultures. These data suggest that, in an allergic environment, Th2 cytokines and allergen have the potential to sustain airway inflammation through a cooperative effect on cytokine release by the bronchial epithelium. Our novel finding that IL-4, IL-13, and allergen enhance release of TGF-alpha, a ligand for the epidermal growth factor receptor that stimulates fibroblast proliferation and goblet cell differentiation, provides a potential link between allergen exposure, Th2 cytokines, and airway remodelling in asthma.     

In planta expression of a mature Der p 1 allergen isolated from an Italian strain of Dermatophagoides pteronyssinus

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.     

屋尘螨Ⅰ类变应原Der p1的体内定位

对过敏性疾病主要过敏原屋尘螨Dermatophagoides pteronyssinusⅠ类变应原（Der p1）进行了抗原定位研究。选用经表达和纯化的Der p1抗原蛋白,按常规方法免疫小鼠,分离血清获抗重组Der p1的抗体（Ⅰ抗）,用抗鼠IgG荧光抗体为Ⅱ抗,屋尘螨经石蜡切片,在荧光显微镜下对其特异性变应原的定位进行观察。HE染色显示屋尘螨消化系统占据大部分体腔。组织免疫荧光发现屋尘螨Ⅰ类变应原主要定位于螨的中肠组织及肠内容物,而螨的生殖系统、排泄系统等脏器以及几丁质甲壳均呈阴性反应。据此认为屋尘螨Ⅰ类变应原存在于螨的肠内容物和中肠组织中。     

Thymic stromal lymphopoietin is produced by dendritic cells

Thymic stromal lymphopoietin (TSLP) is a type 1 cytokine that contributes to lymphopoiesis and the development of asthma and atopic dermatitis. TSLP acts on multiple lineages, including dendritic cells (DCs), T cells, NKT cells, eosinophils, and mast cells, mediating proliferation and survival and linking innate and adaptive immune responses. TSLP is produced by a range of cells, including epithelial cells, fibroblasts, stromal cells, and keratinocytes. DCs are important primary targets of TSLP, and we unexpectedly demonstrated that DCs also produce TSLP in response to TLR stimulation and that this is augmented by IL-4. Moreover, we demonstrated that when mice were challenged with house dust mite extract, lung CD11c(+) DCs expressed TSLP mRNA at an even higher level than did epithelial cells. These data suggested that DCs not only respond to TSLP but also are a source of TSLP during pathogen and/or allergen encounter.     

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Modelling and Bioinformatics Analysis of the Dimeric Structure of House Dust Mite Allergens from Families 5 and 21: Der f 5 Could Dimerize as Der p 5

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.     

Activation mechanism of recombinant Der p 3 allergen zymogen: contribution of cysteine protease Der p 1 and effect of propeptide glycosylation

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T(P1)R.     

The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.     

The Dermatophagoides pteronyssinus group 2 allergen contains a universally immunogenic T cell epitope

The use of T cell epitope-containing peptides for the induction of anergy in allergen sensitization is limited by genetic restriction that could be circumvented by using universally immunogenic epitopes. We attempted to identify such epitopes on Dermatophagoides pteronyssinus group 2 allergen (Der p 2), a major allergen of D. pteronyssinus T cells from BALB/c (H-2(d)), C57BL/6 (H-2(b)), C3H (H-2(k)), and SJL (H-2(s)) mice that were immunized with rDer p 2, recognized an immunodominant region encompassing residues 21-35. A synthetic 21-35 peptide (p21-35) induced strong dose-dependent in vitro T cell proliferation with cells of the four mouse strains and required processing for MHC class II presentation. Substitution of Ile(28) with Ala resulted in reduction of T cell proliferation in each strain. Ile(28) could represent an important MHC class II anchoring residue for T cell response to p21-35. An immunodominant T cell epitope of Der p 2 therefore behaves as a universal epitope and could be a suitable candidate for T cell anergy induction.     

Population growth and allergen accumulation of <Emphasis Type="Italic">Dermatophagoides pteronyssinus</Emphasis> cultured at 20 and 25°C

The house dust mites, Dermatophagoides pteronyssinus and D. farinae are cultured commercially and in research laboratories and material is harvested from these cultures to make extracts that are used for diagnosis, immunotherapy and research. Temperature and other climatic conditions can influence population growth rates, dynamics of allergen production, and the associated endotoxin, enzyme and protein levels of the mite material harvested from these cultures. Here we determined how temperature affected these parameters. Dermatophagoides pteronyssinus was cultured at 20 and 25°C at 75% relative humidity, and at 2-week intervals the concentrations of mites, Der p 1 and Der p 2 allergens, endotoxin, and selected enzymes were determined. Mite density increased exponentially but growth rate and final population density were greater at 25°C compared to 20°C. The combined allergen (Der p 1 + Der p 2) concentrations accumulated in the cultures at about the same rate at both temperatures. However, individual Der p 1 and Der p 2 accumulation rates varied independently at the two temperatures. Der p 1 accumulated faster at 20°C whereas Der p 2 accumulated faster at 25°C. The amount of Der p 1 in whole cultures was greater than the amount of Der p 2. The concentration of allergen for washed mites harvested from the cultures was much less than for the whole cultures. Our study demonstrated that temperature is an important factor in population growth and the dynamics of allergen production in cultured mites.     

Crystal Structures of Mite Allergens Der f 1 and Der p 1 Reveal Differences in Surface-Exposed Residues that May Influence Antibody Binding

The group 1 mite allergens Der f 1 and Der p 1 are potent allergens excreted by Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. The human immunoglobulin E antibody responses to the group 1 allergens show more cross-reactivity than the murine immunoglobulin G antibody responses, which are largely species specific. Here, we report the crystal structure of the mature form of Der f 1, which was isolated from its natural source, and a new high-resolution structure of mature recombinant Der p 1. Unlike Der p 1, Der f 1 is monomeric both in the crystalline state and in solution. Moreover, no metal binding is observed in the structure of Der f 1 despite the fact that all amino acids involved in Ca²⁺ binding in Der p 1 are completely conserved in Der f 1. Although Der p 1 and Der f 1 share an extensive sequence identity, comparison of the crystal structures of both allergens revealed structural features that could explain the differences in murine IgG and human IgE antibody responses to these allergens. There are structural differences between Der f 1 and Der p 1 that are unevenly distributed on the allergens' surfaces. This uneven spatial arrangement of conserved versus altered residues could explain both the specificity and cross-reactivity of antibodies against Der f 1 and Der p 1.     

Monocyte-derived dendritic cells exposed to Der p 1 allergen enhance the recruitment of Th2 cells: major involvement of the chemokines TARC/CCL17 and MDC/CCL22

Dendritic cells (DC) are potent antigen - presenting cells that can orientate the immune response towards a Th1 or a Th2 type. DC produce chemokines that are involved in the recruitment of either Th1 cells, such as IP10 (CXCL10), Th2 cells such as TARC (CCL17) and MDC (CCL22), or non-polarized T cells such as RANTES (CCL5) and MIP-lalpha (CCL3). We investigated whether monocyte-derived DC (MD-DC) generated from healthy donors or from patients sensitive to Dermatophagoides pteronyssinus (Dpt) and exposed to the cysteine-protease Der p 1(allergen of Dpt), could upregulate the expression of chemokines involved in type 1 or type 2 T cell recruitment. MD-DC were pulsed with either Der p 1 or with LPS as the control and the chemokines produced were evaluated using ELISA and chemotaxis assays. Der p 1-pulsed DC from allergic patients showed increased TARC (CCL17) and MDC (CCL22) production without modifying IP-10 (CXCL10) release. Der p 1-pulsed DC from healthy donors showed only increased IP-10 (CXCL10) secretion. RANTES (CCL5) and MIP-lalpha (CCL3) production were similarly increased when DC were from healthy or allergic donors. The selective Th2 clone recruitment activity of supernatants from Der p 1-pulsed DC of allergic patients was inhibited by anti-TARC (CCL17) and anti-MDC (CCL22) neutralizing Abs. By using anti-IP10 (CXCL10) blocking Abs, supernatants of Der p 1-pulsed DC from healthy donors were shown to be involved in the recruitment of Th1 cells. These results suggest that in allergic patients exposed to house dust mites, DC may favour the exacerbation of the Th2 response via the increase in type 2 chemokine production.