The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level

BACKGROUND: The proteolytic activity of the house dust mite allergen Der p 1 has recently been shown to bias Th cell subset development in favour of Th2. Apart from its direct effect on T cells, it is conceivable that the proteolytic activity of Der p 1 may induce the generation of dendritic cells (DCs) that favour a Th2 response. OBJECTIVE: To study the effect of the proteolytic activity of Der p 1 on DC functions; namely cell surface phenotype, IL-12 production and ability to favour a Th2 response. METHODS: We have generated immature DCs from peripheral blood monocytes, matured them with LPS in the presence of either proteolytically active or inactive Der p 1 and compared their functions using flow cytometric analysis. RESULTS: Here we demonstrate for the first time that DCs that have been matured in the presence of proteolytically active Der p 1 produce significantly less IL-12, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. The suppression of IL-12 production was due to the cleavage of CD40 by the proteolytic activity of Der p 1, hence rendering the DCs less responsive to stimulation through the CD40L-CD40 pathway. Furthermore, we demonstrate that DCs that have been matured in the presence of proteolytically active Der p 1 induce the production of significantly less IFN-gamma and more IL-4 by CD4 T cells, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. CONCLUSIONS: Collectively, our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in directing DCs to induce Th2 subset development.     

Heterogeneous proteolytic specificity and activity of the house dust mite proteinase allergen Der p I

Background Exposure of the skin or respiratory tract to proteinases is frequently associated with allergic sensitization. This is of particular significance in the domestic indoor environment where the proteolytic activity of Der p I, the group I allergen of the house dust mite Dermatophagoides pteronyssinus, may influence the allergenicity of mites. Using class-specific proteinase inhibitors and active-site affinity chromatography, we have previously shown that Der p I exhibits a mixed cysteine-serine proteinase activity. Measurement of the amount of cleavage, however, did not determine whether the inhibitors used were targeting exactly the same proteolytic mechanism. Objective To resolve this issue, we have examined whether the cleavage specificity of the cysteine and serine proteinase activities of Der p I was the same. Methods HPLC and mass spectrometry were used to analyse and identify the products of a Der p I-digested peptide substrate and thus identify the peptide bonds cleaved. Results Der p I cleaves different peptide bonds, depending upon the class of proteolytic mechanism used. In the model peptide substrate insulin B chain, the cysteine and serine proteinase activities of Der p I showed preference for glutamic acid and arginine respectively in the P1 position. Conclusion These data suggest the existence of more than one mechanistic form of the allergen immunologically identified as Der p I. If proteolytic activity is indeed a function of allergenicity, this information may have important implications for the pathogenicity of Der p I and the ability of innate antiproteinase defences in the respiratory tract to prevent immune sensitization.     

Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen,by human dendritic cells

BACKGROUND: Immature dendritic cells (DCs) take up antigens in peripheral tissues and, after antigen processing, mature to efficiently stimulate T cells in secondary lymph nodes. In allergic airway diseases DCs have been shown to be involved in the induction and maintenance of a T(H)2-type profile. OBJECTIVE: The present study was undertaken to determine pathways of Der p 1 (a house dust mite allergen) uptake by human DCs and to compare Der p 1 uptake between DCs from patients with house dust mite allergy and DCs from healthy donors. METHODS: Monocyte-derived DCs (MD-DCs) were obtained from patients with house dust mite allergy (n = 13) and healthy donors (n = 11). Der p 1 was labeled with rhodamine. Der p 1 uptake by MD-DCs was analyzed by means of flow cytometry and confocal microscopy. RESULTS: Rhodamine- labeled Der p 1 was demonstrated to be taken up by MD-DCs in a dose-, time-, and temperature- dependent manner. The involvement of the mannose receptor (MR) in the Der p 1 uptake was demonstrated by using (1) inhibitors of the MR- mediated endocytosis (mannan and blocking anti-MR mAb), which inhibited the Der p 1 uptake from 40 % to 50 %, and (2) confocal microscopy showing the colocalization of rhodamine-labeled Der p 1 with FITC-dextran. Interestingly, compared with DCs from healthy donors, DCs from allergic patients expressed more MR and were more efficient in Der p 1 uptake. CONCLUSION: These results suggest that the MR could play a key role in the Der p 1 allergen uptake by DCs and in the pathogenesis of allergic diseases in dust mite -sensitive patients.     

Linked suppression in peripheral T cell tolerance to the house dust mite derived allergen Der p 1

BACKGROUND: Peripheral tolerance is required to maintain balance within the immune system. A feature of peripheral tolerance is linked suppression, in which tolerance induced to a single T cell epitope inhibits the response to all epitopes in the same protein. It is suggested that this phenomenon is mediated by regulatory T cells through either the activity of immunopressive cytokines or direct cell contact. In previous experiments we failed to detect inhibitory cytokines when T cells from mice rendered tolerant by intranasal delivery of the immunodominant peptide of Der p 1 (p 1, 110-131) were restimulated with peptide in vitro. Therefore, the aim of this study was to determine if cognate interactions between T cells mediated by Notch/Delta signaling induce and maintain peripheral T cell tolerance. METHODS: Using in situ hybridization and viral mediated gene transfer, the expression and function of Delta1 were investigated in a murine model of T cell tolerance to Der p 1 in vivo. RESULTS: Delta1 expression is increased on peripheral T cells during the induction of tolerance with high-dose peptide delivered intranasally and when tolerant animals are rechallenged under immunogenic conditions. Peptide p 1, 110-131-specific CD4+ T cells transfected with Delta1 inhibited the response of antigen-primed T cells and induced linked suppression. CONCLUSIONS: High-dose peptide delivered intranasally induces transient expression of Delta 1 on inhibitory CD4+ T cells. Ligation of the Notch1 receptor on neighbouring T cells by Delta1+ regulatory T cells inhibits clonal expansion of the former and mediates linked suppression.     

House-dust-mite allergen (Der p 1) levels in university colleges

Background In coastal Australia, mean house-dust-mite allergen concentration is 20-40 times higher in homes than in public buildings. Allergen concentrations in university colleges, which share some eharacteristics of both homes and public buildings, are not known. The study aimed to compare bed mite-allergen concentration in colleges with local homes.
Methods Mattress dust was collected from three colleges (n = 60 m each) and local homes (N=68) during summer, Der p 1 was measured by ELISA. Information was collected on the floor plan of the colleges, cleaning practices, age of building, and orientation of room.
Results Most college mattresses (94%) had Der p 1 concentrations less than the mean of homes in the same climate. The geometric means of Der p 1 m the mattresses of the colleges were as follows: A, 8.9 pg Der p 1/g fme dust (95% CI 6.9,11.5); B, 1.9 (1.5,2.3); and C, 1.5 (1.2,2.0), compared to homes, 22,5 (17.6, 28.7). The percentages of college mattresses with less than 2 jig/ g were 7%, 48%, and 58%, respectively, compared to 4% for homes. Higher Der p 1 concentrations were weakly associated with age of building in college A, and orientation in college B, Der p 1 concentrations were independent of floor level and age of mattress.
Conclusions These findings indicate that low allergen concentrations are achievable without extreme hygiene and cleaning measures in a climate which supports mite proliferation in homes.     

House-dust mite allergen Der p 1: amount or concentration?

There is a lack of consensus as to the best way to quantify settled house-dust mite Der p 1 allergen from planar and nonplanar surfaces. To investigate the most appropriate measure of settled house-dust mite Der p 1 allergen, we determined the relationship between Der p 1 allergen content and collected dust weight from planar-surface (mattress) and nonplanar-surface (living-room) samples in 58 homes. There was a direct relationship between Der p 1 content and dust weight for both planar-surface (mattress, n =217) samples ( r =0.50, P <0.0001) and nonplanar-surface (living-room, n =48) samples ( r =0.61, P <0.0001). Correction for the surface area of the planar surface (mattress) vacuum-cleaned resulted in no additional benefit. We conclude that expression of Der p 1 level per weight of collected dust appears to be the best method for both planar and nonplanar surfaces.     

Human T cells that have been conditioned by the proteolytic activity of the major dust mite allergen Der p 1 trigger enhanced immunoglobulin E synthesis by B cells

BACKGROUND: We have previously demonstrated that the proteolytic activity of Der p 1 selectively cleaves human CD25, the 55 kDa alpha subunit of the IL-2 receptor. As a result of cleavage of surface CD25, peripheral blood T cells produce less IFN-gamma and more IL-4, thereby leading to progressive polarization of the T cells towards a Th2 cytokine profile. Therefore, these observations underline the potential role of the proteolytic activity of Der p 1 in creating a microenvironment conducive for IgE synthesis. OBJECTIVE: To study the effect of T cells that have been conditioned by the proteolytic activity of Der p 1 on IgE synthesis by B cells. METHODS: We have examined this concept in experiments whereby T cells that have been exposed to either proteolytically active or inactive Der p 1 were cocultured with autologous B cells and IgE antibody synthesis was monitored. RESULTS: Here we demonstrate for the first time that coculturing T cells that have been in contact with proteolytically active Der p 1 with autologous B cells leads to augmentation of IgE antibody responses. CONCLUSIONS: The proteolytic activity of Der p 1 conditions human T cells, which then become empowered to trigger enhanced IgE synthesis by B cells.     

IgE and monoclonal antibody binding by the mite allergen Der p 7

Background The recently characterized group 7 house dust mite allergens give positive skin-test reactions in 53% of allergie patients. Objective This study was performed to compare the IgE bindinig activity of natural and recombinant Der p 7, to measure the binding in allergic sera in comparison to major allergen Der p 2 and characterize the response by competitive inhibition with monoclonal antibodies. Methods IgE anti Der p 2 and Der p 7 antibodies against the recombinant allergens and monoclonal binding activities were measured by a solid phase radioimmune assay. Reslts A competitive binding assay showed that rDer p 7 inhibited 91% of IgE-binding to natural Der p 7 in 2 sera and 73% in a further two. The IgE binding of rDer p 2 and Der p 7 from 41 sera was then compared. Of the sera 88% and 46% respectively showed positive binding. All of the 19 sera which bound Der p 7 also bound Der p 2 but 11 (58%) had bound IgE to Der p 7 as high or higher than the binding to Der p 2. These sera were mostly high responders to both allergens. A panel of six monoclonal antibodies produced against either rDer p 7 or rDer f 7 was used for epitope analysis. All of these reaeted with eaeh allergen by enzyme linked immunosorbent assay (ELISA) and immunoblotting. Two patterns of cross inhibition of monoclonal antibody binding were observed and of five monoclonal antibodies tested, lour could inhibit the binding of IgE (WH9, WH22. WPS and HD19) while one (WH14) could not. Conclusions Although Der p 7 only reacts with 50% of allergic sera it often has a high IgE binding activity and may be more important than the major Der p 2 allergen in a high percentage of subjects. The combined competitive inhibition experiments show the IgE response is directed at several specilieities.     

Sensitisation to the lipid-binding apolipophorin allergen Der p 14 and the peptide Mag-1

BACKGROUND: The IgE-binding peptides Mag 1 and Mag 3 and the high-molecular-weight protein M-177 have been identified as parts of the apolipophorin-like group 14 house dust mite allergen. By analogy with the homologous insect proteins, apolipophorins are hydrophobic proteins found in lipid bodies and lipid transport particles. This explains why they degrade and are poorly represented in extracts. METHODS: We have examined the T cell stimulation induced by a 341-residue recombinant Der p 14 peptide equivalent to the Mag 1 polypeptide examined by others. RESULTS: Peripheral blood mononuclear cells from both allergic and non-allergic donors responded strongly in in vitro proliferation assays to the Der p 14 peptide to induce markedly more (3)H-thymidine incorporation than Der p 2 and the release of Th2 cytokines. CONCLUSIONS: The apolipophorin-like group 14 allergens, despite their predicted hydrophobicity and lipid-binding activity, can induce high IgE responses and T cell stimulation. They appear to be important mite allergens unsuited to representation by aqueous extracts of mites.     

Efficient presentation of house dust mite allergen Der p 2 by monocyte-derived dendritic cells and the role of beta 2 integrins

Monocyte-derived dendritic cells (MDDC) are potent antigen-presenting cells (APC). The APC functions of MDDC in allergy were examined. MDDC presentation of the major house dust mite allergen Der p 2 resulted in 4-12-fold higher T-cell proliferation and markedly higher IFN-gamma and IL-5 production than PBMC cultures. Comparable T-cell proliferation was obtained with 10-fold fewer MDDC than purified monocytes. MDDC cultured from adherent cells, or CD14+, CD11b+ or Percoll-purified monocytes were comparable in presenting soluble Ag, and in stimulating allogeneic MLR. Importantly, MDDC presentation of Der p 2 resulted in both Th1 and Th2 stimulation, although MDDC are known to produce high levels of IL-12 and stimulate biased Th1 responses. The basis for the potent APC function of MDDC was further examined. MDDC were found to be highly phagocytic. Immunoprecipitation studies showed markedly elevated ICAM-1 expression but > 10-fold reduction in LFA-1 expression on MDDC compared with monocytes. Monoclonal antibody (MoAb)-blocking experiments showed that ICAM-LFA-1 interaction was essential for MDDC stimulation of Der p 2-specific T-cell proliferative responses. These results show that the use of MDDC as APC provides a simple, sensitive and versatile method for detecting T-cell responses to allergens and that the strong phagocytic capability and the increased ICAM-1 expression of MDDC contribute significantly to their Ag presentation potency.     

Characterization of the house dust mite allergen Der p 7 by monoclonal antibodies

The house dust mite allergen Der p 7, which was defined by cDNA cloning, has been shown to react with about 50% of allergic sera and corresponds to or is antigenically related to at least three different sized components in mite extracts. To characterize these entities, monoclonal antibodies (MoAbs) were generated by immunizing BALB/c mice affinity-purified Der p 7-GST (glutathione S-transferase) fusion protein. MoAbs WH9 and WH22 showed positive reactivity to recombinant Der p 7 negative reactivity to GST and the Der p 5-GST fusion protein in ELISA and immunoblotting. The specificity of both MoAbs was confirmed by inhibition of the ELISA activity by recombinant Der p 7 but not by the recombinant Der p 5. Immunoblot analysis demonstrated that both MoAbs showed reactivities to components with molecular weights (mol. wt.) of 31, 30 and 26kDa reactive to both MoAbs. At least six major forms with different pI or size were indicated by 2-D gel analysis. In addition to characterization of Der p 7, both MoAbs may also be considered for use in the standardization of Der p 7 in mite extracts.     

Comparative enzymology of native and recombinant house dust mite allergen Der p 1

Background:  The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool.
Methods:  Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined.
Results:  Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism.
Conclusion:  The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris . This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens.     

Human T cell subset commitment determined by the intrinsic property of antigen: the proteolytic activity of the major mite allergen Der p 1 conditions T cells to produce more IL-4 and less IFN-gamma

The house dust mite Dermatophagoides pteronyssinus allergen Der p 1 elicits IgE antibody responses in a significant proportion of patients suffering from dust mite allergy. We have recently shown that Der p 1 proteolytically cleaves a cell surface molecule involved in the homeostatic control of human IgE synthesis, namely the IL-2 receptor (CD25) on T cells. As a result, these T cells show markedly diminished proliferation and IFN-gamma secretion in response to stimulation by anti-CD3 antibody. However, these observations still leave open the important issue of whether CD25 cleavage, and the consequent suppression of IFN-gamma secretion, leads to enhanced IL-4 secretion, and whether such cytokine changes would be exhibited by both CD4 and CD8 T cells. Here we demonstrate for the first time that the proteolytic activity of Der p 1 biases human CD4 and CD8 T cells towards a type 2 cytokine profile. Our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in creating a microenvironment conducive for IgE synthesis.     

Epitope mapping of the house-dust-mite allergen Der p 2 by means of site-directed mutagenesis

Recombinant Der p 2, expressed in the baker's yeast Saccharomyces cerevisiae. was used as a tool to determitie IgE- and monoclonal antibody (mAb)-binding sites on this allergen. For this purpose, mutant molecules were produced by application of site-directed mutagenesis. The amino-acid residues spanning cys21-cys27 and cys73-cys78 were deleted, thus preventing loop formation through disulfide bonds. Charged residues in three predicted antigenic sites (residues 45–48, 67-1-69. and 88–90) were replaced by alanine residues. IgE- and mAb reactivity to these mutants was compared to that to “wild type” Der p 2. Residues spanning cys73-cys78 were involved in the antigenic binding site for mAb aDpX. Mutations in the areas adjacent to this loop (i.e. 67–69 and 88–90) had similar effects on this mAb (10- to 20-fold decreases in reactivity were observed), supporting the suggestion that these areas are involved in this antigenic structure. The area of residues 45–48 was shown to be involved in an epitope for mAb 2B12. The reactivity of mAb 7A1 was influenced by substitutions of residues 45–48 as well as 88–90. Deletion of the residues spanning cys21-cys27 resulted in decreased reactivity to three mAbs (lOEll, αDpX. and 7A1). From these observations, it may be concluded that binding of different mAbs is influenced by the same mutations and that the binding of single mAbs is influenced by two or more mutations scattered over the allergen molecule. These findings can point in two directions: minor aminoacid changes result in disruption of the overall conformation of the allergen, or distant sites are close together in the three-dimensional structure of the allergen. Decreased IgE reactivity was observed with all mutant molecules, varying between patients. The observed effects ranged from 5- to 1000-fold. Deletion of the amino-acid residues spanning cys21-cys27 and cys73-cys78 had the strongest effect on IgE reactivity, where decreases up to 1000-fold were observed. Such mutants might be useful tools to improve the safety of allergen-specific immunotherapy.     

Murine allergic respiratory responses to the major house dust mite allergen Der p 1.

BACKGROUND: Although many studies have examined chronic asthma, limited data exist on acute immunopathogenic events induced by allergens. The aim of the study was to investigate the acute cellular, serologic and histopathologic events in airway inflammation produced by intranasal challenge of mice sensitised to the major house dust mite allergen Der p 1. METHODS: C57BL/6 mice were immunised subcutaneously with Der p 1 in alum. Mice were bled and challenged intranasally with Der p 1 on day 14 and killed on day 17. Lungs were fixed in situ, processed and stained with haematoxylin and eosin. The degree of inflammation and eosinophil infiltration was quantified by image analysis. Specific IgE was determined by passive cutaneous anaphylaxis. Cells from spleen and draining lymph nodes were cultured for 24 h with Der p 1, and IL-3/GM-CSF released into supernatants was measured by bioassay. RESULTS: Intranasal challenge of sensitised mice induced eosinophilic influx into the large and small airways and the alveolar regions of the lung, mucus plugging and in severe cases numerous Charcot-Leyden crystals. The quantitation of the inflammation induced by different sensitisation and challenge doses showed that optimal inflammation could be produced using only 1 microg of allergen for both sensitisation and challenge. The degree of inflammation was not related to the titre of IgE antibody and was indeed produced in its absence. T cell reactivity of spleen cells to the allergen was decreased suggesting cell migration or inactivation. CONCLUSIONS: Mice sensitised and challenged intranasally with as little as 1 microg of Der p 1 produced an extensive pulmonary eosinophilic inflammation which shared many of the features of the inflammation found in asthma. The small amount of allergens required and the use of intranasal challenge should provide a useful model.     

Sequence polymorphisms of the Der p 3 house dust mite allergen

Background The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectricfocusing studies with the natural Der p 3 protein have indicated that several isoforms exist. Objective To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen. Methods Five cDNA clones of Der p 3 have been isolated from a λ gt 10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco RI digested genomic DNA was performed. Results Southern blot analysis of Eco RI digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones ditfered only in their 3′untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen. Conclusions These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group I and group 2 house dust mite allergens.