American Journal of Respiratory Cell and Molecular Biology

House dust mites (HDM) are the most common source of aeroallergens and in genetic susceptible individuals can cause symptoms ranging from atopic dermatitis to bronchial asthma. Der p 1, a major target of the human immune responses to HDM, through its enzymatic properties can modulate the adaptive immune system by the cleavage of CD23 and CD25. The consequences of this would be to promote allergic inflammatory responses. Furthermore, by disrupting epithelial tight junctions Der p 1 facilitates the transport of allergen across the epithelium. Here, we report that Der p 1 has additional effects on the innate defense mechanisms of the lung, by inactivating in vitro and ex vivo the elastase inhibitors human (h) α1-proteinase inhibitor (h-A1-Pi), mouse (m-), (but not human [h])-SLPI and h-elafin. We confirm that Der p 1 contain both cysteine and serine proteinases, and extend this finding to demonstrate for the first time that h-elafin is particularly sensitive to the biological activity of the latter. Because these elastase inhibitors have antimicrobial, as well as antielastase activity, our results suggest that inactivation of these innate components of the lung defense system by Der p 1 may increase the susceptibility of patients with allergic inflammation to infection.

Asthma is a chronic obstructive disease of the lower airways characterized by intermittent exacerbations of reversible airways obstruction, airways inflammation, and bronchial hyperreactivity (1). The chronic immune response in asthma and other atopic diseases is characterized by a predominately Th2 T cell response, associated with interleukin (IL)-4, IL-5, IL-13, and IgE production (1). Although a third of all cases of asthma can be characterized as intrinsic nonallergic, where the eliciting agent is unknown (2), it is clear that in most cases of asthma (extrinsic) the eliciting factor is of allergen origin, with a strong atopic component (3).

Prevalence of asthma varies in studies between 5 and 25% (1), and has doubled in the industrialized world over the past 20 yr (4). Factors implicated in this increase in prevalence include altered indoor environment such as warmer housing, increased use of broad spectrum antibiotics with altered bacterial infection profiles, dietary changes, and increased aeroallergen exposure.

Exposure to a number of aeroallergens has been shown to contribute to both immediate hypersensitivity and chronic asthma, among these the common indoor allergens produced by the house dust mite (HDM). In human atopic disease, inflammatory responses to the group I antigens of Dermatophagoides pteronyssinus and Dermatophagoides farinae (Der p 1 and Der f 1) are well documented (56). The Der p 1 cysteine protease is a 25-kD glycoprotein present in significant quantities in HDM fecal pellets, and is suggested to have a digestive role in the gut of the mite. A number of recent studies have demonstrated that Der p 1 is capable of cleaving human proteins with potentially immunomodulatory effects including α1-proteinase inhibitor (A1-Pi), (7), CD23 (the human low-affinity IgE receptor) (8), CD25 (the α subunit of the human IL-2 receptor) (9), and tight junctions of bronchial epithelium, leading to increased bronchial epithelial permeability (10). In addition to A1-Pi (1112), the lung also secretes the mucosal/alarm antiproteases secretory leukocyte proteinase inhibitor (SLPI) and elafin, which all contribute significantly to not only antielastase activity but also to activation of innate immunity (1316). For example, using an adenovirus-overexpression strategy, we have recently reported that elafin has chemotactic activities for neutrophils and protects the lung in a Pseudomonas aeruginosa model of acute inflammation (1718).

Accordingly, we sought to investigate in the present study whether or not Der p 1 was able to inactivate these important antiproteases and consequently could shift the “elastase–antielastase balance” in favor of a proinflammatory environment to facilitate both the initiation and the maintenance of the asthmatic response.

We have demonstrated here that all three antiproteases studied can potentially be degraded by Der p 1 in vitro and ex vivo. Furthermore, we have investigated in detail the enzymologic properties of Der p 1 against synthetic substrates and selective inhibitors to clarify the nature of the proteases that inactivate innate antiprotease defences. As a consequence, we have uncovered that in addition to the well-characterized cysteine proteinase present in Der p 1, a co-purifying serine protease is extremely active against human elafin.

Materials

Human (h) A1-Pi was purchased from Sigma (Poole, Dorset, UK) and human (h) synthetic elafin was manufactured by Albachem (Edinburgh, UK) as described previously (19). Recombinant human (h) SLPI was purchased from R&D Systems (Minneapolis, MN) and recombinant murine (m)-SLPI was a gift from Dr. C. Wright (Amgen, Thousand Oaks, CA). Human neutrophil elastase (HNE) was obtained from Elastin Products (Owensville, MO). E-64 (L-trans-epoxysuccinyl-leucyl-amido[4-guanidino]butane; Sigma) is an active site directed irreversible inhibitor of cysteine proteases which inhibits Der p 1 (20).

Human bronchoalveolar lavage fluid (BALF) from six patients with established acute respiratory distress syndrome (21) was a generous gift from Dr. S. Donnelly. The samples were pooled and incubated with Der p 1, as described below.

Purification of Der p 1 from HDM Fecal Pellet Extract

Sixty grams of house dust mite powder (gift from Dr. T. Wayne, University of Perth, Perth, Australia) was dissolved into 1 liter of Dulbecco's phosphate-buffered saline (PBS) (calcium and magnesium-free) containing 0.5M NaCl, 0.01% (wt/wt) sodium azide, pH 7.4. Mouse anti–Der p1 monoclonal antibody 4C1 (Indoor Biotechnologies, Clwyd, UK) was coupled to 5 g of Cyanogen-activated Sepharose 4B (Amersham Biosciences UK Limited, Pollard's Wood, Buck, UK), suspended, and washed in an affinity column according to manufacturer's instructions. One hundred milliliters of Der p 1 solution was applied to the column at a flow rate of 20 ml/h. The column was washed with 400 ml PBS 0.5 M NaCl and elution performed at 20 ml/h using 5 mM glycine in 50% ethylene glycol pH 10.0. Fractions were assayed for absorbance at 280 nm, and were extensively dialyzed for 18 h against 10 liters PBS. These fractions will be denoted CS-Der p 1 for the rest of the study. Further purification was performed on half of the dialysate by applying it to an affinity column containing 100 mg of soybean trypsin inhibitor (SBTI) coupled to 5 g of Cyanogen-activated Sepharose 4B (Pharmacia Biotech), according to the manufacturer's instructions. Fractions were collected and reassayed for absorbance at 280 nm to collect serine-protease depleted Der p 1 and re-dialysed extensively against PBS. Der p 1, which had been serine protease depleted by this process, was denoted C-Der p 1 for the rest of the study. The protein concentration of both C/CS-Der p 1 was assessed using a bicinchoninic acid plate assay (Pierce and Warriner Ltd., Chester, Cheshire, UK). Purity of the preparations was assessed using migration on 12% SDS-PAGE analysis (gels stained with using Biorad Silver Stain). Samples were stored in aliquots at –20°C.

Enzymatic Analysis of Der p 1 Fractions
7-amino-4 methylcoumarin (AMC) substrate hydrolysis.

A range of synthetic fluorescent substrates (Novabiochem, Nottingham, UK) were used to analyze Der p 1 substrate selectivity, and included Cbz-Arg-Arg-AMC, H-Arg-AMC, Cbz-Phe-arg-AMC, Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-Pro-Arg- AMC, H-Pro-Phe-Arg-AMC, Suc-Ala-Ala-Pro-Phe-AMC (22) and Suc-Gly-Pro-Leu-Gly-Pro-AMC (23). They were dissolved in dimethyl sulfoxide (DMSO) to generate a 10-mM stock and hydrolysis of each substrate (10 μM final) by 6 μg of CS-Derp1 was performed in triplicate in 0.5 M Tris-HCl (pH 8.0) containing 5 mM cysteine and 2% DMSO. Hydrolysis was measured by monitoring the release of AMC every 10 s for 2 min, using a Hoeffer TK 100 mini-fluorimeter (excitation wavelength of 365 nm and emission detection at 465 nm, San Francisco, CA). Substrate concentrations were kept constant by allowing no more than 5% hydrolysis. The rate of hydrolysis was determined by linear regression analysis using the software program SIGMA PLOT, version 4.00. The concentration of AMC was determined from the regression line: [AMC] = (y − c)/m, where y is the fluorescent units released from hydrolysis, c is the intercept, and m is the slope of the regression line.

pH assays.

The pH profile of purified CS-Derp1 against the synthetic substrates Boc-Gln-Ala-Arg-AMC and Tosyl-Gly-Pro-Arg-AMC was determined in the following buffers:

0.1 M citric acid/sodium citrate buffer (pH 3–6), 0.1M phosphate buffer (pH 6–8) and 0.5 M Tris-HCl (pH 8–9.5). All reactions were performed in the presence of 5 mM cysteine.

Km determination.

The rate constant (Km) of CS-Der p 1 for synthetic substrates was determined by monitoring the initial rates of hydrolysis at the optimum pH for each substrate. The range of substrate concentrations used for Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-pro-Arg-AMC, and Cbz-Phe-Arg-AMC were 0–600 μM, 0–10 μM, and 0–100 μM, respectively. The initial rates of hydrolysis (Vo) were plotted against the differing substrate concentrations [S] to ensure that curvature was seen. The data were also represented as a straight line using the Hanes-Woolf plot; it was then applied to the Michaelis-Menten rate equation as described below using a nonlinear regression analysis program, in SIGMA PLOT version 4.00, to obtain the Km and maximum initial velocity (Vmax).

Differential inhibition of Der p 1.

CS-Der p 1 and C-Der p 1 enzymatic activities were further characterized by differential inhibition by using E64 and APMSF, inhibitors of cysteinyl and serine proteinases, respectively. CS-Der p 1 and C-Der p 1 were preincubated with 10 μM E64 or 100 μM APMSF before assaying residual activity as described above using either Boc-Gln-Ala-Arg-AMC (10 μM final in 0.1 M phosphate buffer pH 6.0) or Tosyl-Gly-Pro-Arg-AMC (10 μM final in 0.5 M Tris-HCl pH 8.0). All buffers contained 5 mM cysteine.

SDS-PAGE and Western Blot Analysis

Protein samples were submitted to SDS-PAGE and/or analyzed by Western Blot analysis as described (24). Briefly, after electrophoretic transfer onto Hybond nitrocellulose membranes (Amersham, Bucks, UK), the membranes were probed sequentially with anti–h-elafin rabbit IgG (1:1,000 dilution, 1 h incubation at room temperature) and horseradish peroxidase–conjugated goat anti-rabbit IgG (1:2,500 dilution, 20 min at room temperature; Dako, Ely, Cambridgeshire, UK). Western blots were developed by enhanced chemiluminescence (ECL kit; Amersham).

Elastase Activity Assay

50 mM Tris 0.5 M NaCl 0.1% Triton pH 8 buffer was added to defined quantities of HNE on a 96-well ELISA plate (Linbro; Flow Laboratories, McLean, VA) to a volume of 200 μl. Fifty microliters of synthetic elastase substrate N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide (Sigma) was added to each well, and cleavage of substrate was monitored through change in absorbance (at 405 nm), measured specrophotometrically, at defined time points, using a Dynex MRX II plate reader (Dynatech, Billinghurst, UK).

Anti-HNE activity of test solutions (either purified proteins or BALF) was assessed by addition of the solution to the HNE/buffer mix, maintaining a total volume of 200 μl. The plate was then incubated for 15 min at 37°C before addition of substrate as described above. It was demonstrated that Der p 1 fractions, dithiotreitol (DTT) and L-cysteine, alone or in combination, did not influence the rate of HNE cleavage of substrate (data not shown).

Enzymatic Activity of Der p 1

We first sought to establish the enzymatic specificities of our preparation of CS-Der p 1, after purification from the 4C1 affinity chromatography column. Several synthetic substrates were used as listed in Table 1

TABLE 1 Substrate specificity of CS-Der p 1


Substrate

Class

Specific Activity
 (nM of AMC released/s/mg ± SD)
Cbz-Arg-Arg-AMCCysteinyl3.7 ± 0.61
H-Arg-AMCCysteinylNot detected
Cbz-Phe-Arg-AMCCysteinyl and serine10.7 ± 1.75
Boc-Gln-Ala-Arg-AMCCysteinyl and serine480 ± 8.8
Tosyl-Gly-Pro-Arg-AMC“Trypsin-like” serine100 ± 0.58
H-Pro-Phe-Arg-AMCSerine8.165 ± 0.88
Suc-Ala-Ala-Pro-Phe-AMCSerineNot detected
Suc-Gly-Pro-Leu-Gly-Pro-AMC
Metalloprotease
Not detected

Substrate specificity of CS-Der p 1 was analyzed using a number of synthetic fluorogenic substrates (10 μM final concentration). All assays were carried out in 0.5 M Tris-HCl pH 8.0 containing 5 mM cysteine. Results are expressed as nM of 7-amino-4-methylcoumarin (AMC) released/s/mg ± SD.

. Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-Pro-Arg-AMC, and Cbz-Phe-Arg-AMC were the best substrates for the CS-Der p 1 preparation, suggesting that a serine protease and a cysteine protease were the active molecules responsible for enzymatic activity. In addition, by assessing the optimal pH of enzymatic activity, we showed that CS-Der p 1 maximal enzymatic activity was observed at pH 6.0 and 8.0, against Boc-Gln-Ala-Arg-AMC and Tosyl-Gly-Pro-Arg-AMC, providing further evidence for the involvement of cysteine and serine proteases, respectively.

The affinity of these proteases was further studied in Figure 1

. The mean Km values obtained from three separate experiments for Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-Pro-Arg-AMC, and CBz-Phe-Arg-AMC were 254, 2.3, and 13 μM, respectively, suggesting that the affinity for serine selective substrates (last two) was significantly higher than for the cysteine protease substrate (first one). The specificity of these proteases was further confirmed using the synthetic inhibitors APMSF and E-64, of serine and cysteine proteases, respectively (20). Figure 2A shows that E-64, but not APMSF, inhibited the enzymatic activity of CS-Der p 1 against the cysteine protease substrate Boc-Gln-Ala-Arg-AMC. The cysteine protease presence was further shown by the abrogation of its activity when the cysteine-activating agent L-Cysteine (L-Cys) was omitted. Conversely, APMSF, but not E64, inhibited the activity of CS-Der p 1 against the serine protease substrate Tosyl-Gly-Pro-Arg-AMC (Figure 2B).

Having established that CS-Der p 1 contained both serine protease and cysteine protease components, we segregated both activities by submitting the CS-Der p 1 fraction to SBTI-affinity chromatography.

The resulting purified C-Der p 1 was then tested on both Tosyl-Gly-Pro-Arg-AMC and Boc-Gln-Ala-Arg-AMC. We found that C-Der p 1 was active against Boc-Gln-Ala-Arg-AMC and only E-64 abolished that activity (Figure 2C). There was no recordable activity against Tosyl-Gly-Pro-Arg-AMC (data not shown), establishing that indeed, C-Der p 1 only contained the cysteine proteinase activity.

Having demonstrated enzymatically that CS-Der p 1 contained both cysteine and serine protease activities (Table 1, and Figures 1, 2A, and 2B), whereas C-Der p 1 only contained the former (Figure 2C), the activity of both fractions on the lung elastase inhibitors h-A1-Pi, h-elafin, h-SLPI, and m-SLPI was investigated.

Interaction between Der p 1 and Human A1-Pi

The interaction of Der p 1 (CS and C) with h-A1-Pi was analyzed by SDS-PAGE: Figure 3A

shows that incubation (molar ratio 2:1) for 2 h at 37°C cleaved native h-A1-Pi and generated a proteolytic fragment of 49 kD, in accordance with the literature (7). This cleavage was produced by both CS-Der p 1, containing both cysteine and serine proteases (lane 3), and C-Der p 1, containing the cysteine protease only (lane 5). In addition, the cleavage was dependent upon the activation of Der p 1 with L-Cys (not shown). Also, E-64 prevented h-A1-Pi degradation from both Der p 1 preparations (lanes 4 and 6). Incubation of h-A1-Pi alone, or with 20 mM L-cysteine, did not produce detectable degradation (lanes 1 and 2, respectively).

The same Der p 1/A1-Pi ratio was used for the functional assay where the residual anti-HNE activity of h-A1-Pi was measured (Figure 3B).

At all time points (0, 1, 2, 4 h), the results are expressed as percentage of residual HNE activity, as compared with HNE incubated with buffer alone. Mirroring the results from the SDS-PAGE analysis, we observed that both CS-Der p 1 and C-Der p 1 functionally inactivated h-A1-Pi (as determined by increased HNE activity over time). Incubation of Der p 1 with E-64 prevented h-A1-Pi inactivation because HNE activity remained minimal throughout the experiment, again demonstrating that indeed, the cysteine proteinase of Der p 1 is responsible for the activity. It was also demonstrated that Der p 1 and L-cys, alone or in combination, did not influence HNE activity (not shown).

Interaction between Der p 1 and Human Elafin

The interaction between Der p 1 and h-elafin was analyzed as described above, using SDS-PAGE and Western blot analysis (Figure 4A)

.

Cleavage of h-elafin was obtained at a CS-Der p 1/h-elafin ratio of 0.5:1 (Figure 4A, bottom, lane 4). Decreasing the ratio resulted in the gradual preservation of h-elafin integrity (Figure 4A, bottom, lanes 4–8). L-Cys alone did not influence h-elafin migration (lane 2). Very significantly, the interaction between CS-Der p 1 and h-elafin in the absence of L-Cys still induced h-elafin degradation (Figure 4A, bottom, lane 3), suggesting that the h-elafin degrading activity is at least partly due to the serine protease contained in CS-Der p 1.

Expectedly, C-Der p 1, which only has cysteine proteinase activity, needed activation with L-Cys to be able to degrade h-elafin (Figure 4A, top, lanes 4–8, compared with lane 3, without L-Cys). Taken together, the data presented here suggest that the serine and cysteine activities of Der p 1 are both able to degrade h-elafin.

For the functional assay (Figure 4B), an “intermediate” molar ratio Der p 1/h-elafin of 0.3:1 was chosen on the basis that a ratio of 0.5:1 cleaved most of the native elafin molecule after 2 h (Figure 4A, lane 4), whereas a ratio of 0.1:1 was less efficient (Figure 4A, lane 5).

The results reveal that both CS-Der p 1 and C-Der p 1 functionally inactivated h-elafin. Incubation of CS-Der p 1 with E-64 failed to prevent h-elafin inactivation, supporting the Western blot analysis results that indeed the serine protease contributes very significantly to the h-elafin–degrading activity in the CS fraction. Nevertheless, C-Der p 1, which only has cysteine proteinase activity, lost its h-elafin–inactivating activity upon incubation with E-64, suggesting that both serine and cysteine proteases have the capacity to inactivate h-elafin.

Interaction between Der p 1 and Human SLPI

h-SLPI SDS-PAGE analysis (Figure 5)

was performed on a 15% gel as described for A1-Pi and h-elafin. h-SLPI was incubated for 2 h at 37°C with C-Der p 1 and CS-Der p 1 (molar ratio of C/CS-Der p 1/h-SLPI varying from 0.25:1 to 1:1) in the presence or absence of L-Cys. The resistance of the h-SLPI protein to cleavage by C/CS-Der p 1 is demonstrated in Figures 5A and 5B. Because h-SLPI is a small protein containing eight disulfide bonds (13), we hypothesized that its resistance to Der p 1 could be due to the high compactness of the molecule. We therefore investigated whether reduction of the disulfide bonds of h-SLPI with DTT would favor the subsequent proteolytic activity of Der p 1 (Figure 5C).

Using a C/CS-Der p 1/SLPI molar ratio of 0.5:1, the preincubation of h-SLPI with 2 mM DTT rendered it susceptible to further cleavage by both C-Der p 1 (compare Figure 5C, left and right, lane 2) and CS-Der p 1 (compare Figure 5C, left and right, lane 4). At the dilutions of DTT used in the experiment, we checked that DTT itself was not affecting the activity of Der p 1 (not shown). Interestingly, both C-Der p 1 (Figure 5C, right, lane 3) and CS-Der p 1 activities (Figure 5C, right, lane 5) were abolished to a similar degree with E-64, suggesting that it is the cysteine protease of Der p 1, which plays a role in the degradation of DTT-treated h-SLPI.

Interaction between Der p 1 and Murine SLPI

Contrary to h-SLPI (Figure 5), m-SLPI was very sensitive to CS-Der p 1 inactivation (Figure 6)

, even in the absence of denaturing agent. Significantly, in the absence of L-Cys–activating agent (lane 2), there was no inactivation of m-SLPI, suggesting that the cysteine proteinase in CS-Der p 1 plays the most important role in that process.

Interaction between Der p 1 and Human BALF

Because the three human elastase inhibitors A1-Pi, elafin, and SLPI studied above are the only three major elastase inhibitors so far identified in the lung, we were prompted to test if Der p 1 was able to inactivate the elastase-inhibitory capacity in human BALF. Because patients with established acute respiratory distress syndrome (ARDS) have a very high level of active antiproteases in their BALF (21), we chose to study a pool of six BAL samples.

As demonstrated in our previous study (21), we show here that untreated BALF had a marked anti-HNE activity over the time course of the experiment, with HNE inhibition ranging between 85 and 98%. Incubation of BALF with L-Cys alone did not change this activity. By contrast, both C-Der p 1 and CS-Der p 1, when incubated with L-Cys, degraded the anti-HNE activity of this pool of BALFs (Figure 7)

. CS-Der p 1 in the presence of L-cysteine resulted in the highest inactivation, suggesting that the cysteine proteinase is the most active molecule in that setting. In support of this finding, incubation with E-64 prevented the BALF anti-HNE activity from being depleted.

Der p 1 is one of the commonest aeroallergens associated with atopic asthma (2526). A number of recent studies have implicated the cysteine protease activity of Der p 1 in the pathogenesis of asthma (710). We have in the present article explored further issues relating to the enzymatic properties of Der p 1 in relation to its activity on the lung defense molecules A1-Pi, SLPI, and elafin.

The main protein present in conventionally affinity-purified Der p 1, which we have named CS-Der p 1 here, is undoubtedly a cysteine proteinase, as evidenced by silver-stained gel purity and N-terminal sequencing (7, 27). However, previous work suggested that there is a minor serine protease contaminant in that preparation, which was removed with an additional SBTI-affinity chromatography step (2830). Our results confirm and extend these findings by showing, using a wide variety of synthetic substrates, that indeed cysteine and protease activities coexist in CS-Der p 1, and that the SBTI additional step removed the serine protease contaminant, because the remaining protein (C-Der p 1) was only able to degrade the cysteine protease substrate (Figure 2). However, we have been unable to purify and identify the serine protease component, probably because of its very high affinity for SBTI, which makes it very difficult to elute from the column (A. Brown, unpublished data).

Because of the importance of the elastase inhibitors A1-Pi, elafin, and SLPI as key molecules in the defense of the lung (13), we were prompted to determine whether or not CS-Der p 1 and C-Der p 1 were able to biochemically and functionally inactivate these molecules.

In accordance with our previous study (7), we found that CS-Der p 1 could inactivate h-A1-Pi, and further established here that it is the cysteine proteinase of Der p 1 that is responsible for the h-A1-Pi–degrading activity, because CS- and C-Der p 1 have similar activities. In contrast, for h-elafin, we found that the serine protease had the most potent inactivating activity, as illustrated by the fact that even in the presence of an inactive cysteine protease (without L-Cys) h-elafin was almost completely degraded. Furthermore, when the cysteine protease was inhibited by E-64, the activity of h-elafin was still degraded to the same extent as when no E-64 was added. These findings suggest that the serine protease, albeit probably present as a minor contaminant in the CS-Der p 1 fraction, has a much greater affinity for h-elafin than the cysteine protease.

Interestingly, h-SLPI and m-SLPI were not equally sensitive to Der p 1. Indeed, m-SLPI was much more sensitive than h-SLPI (compare Figures 5 and 6), with the latter only becoming susceptible to cleavage by Der p 1 after pretreatment with DTT, which disrupts the molecule by reduction of its internal disulfide bonds. This difference in sensitivity is notable in view of the significant homologies between m-SLPI and h-SLPI (58% overall) (31) and between human elafin (which is sensitive to Der p 1) and human SLPI (which is not, 47% homology) (13). It is interesting that the three molecules found in the present study as being sensitive to Der p 1 (h-elafin, h-A1-Pi, and m-SLPI) have an alanine at or near the antiprotease-reactive site (at positions P1, P4, and P4, respectively) whereas there is no alanine in h-SLPI. Indeed, when Der p 1 cleavage sites on h-A1-Pi were studied (7), the enzymes were shown to cleave on either side of the alanine residue. In addition, m-SLPI has an alanine-arginine motif in the P3-P4 position, also present in the cysteine proteinase synthetic substrate Boc-Gln-Ala-Arg-AMC used in this study.

Regardless of the mechanism of Der p 1 differential inhibition of lung antiproteases, our in vitro experiments performed on the isolated human proteins SLPI, A1-Pi, and elafin revealed that the latter two molecules were mostly susceptible to the cysteine and serine proteinases present in Der p 1, respectively, whereas the in vitro resistance of h-SLPI to Der p 1 would suggest that the former may not be a significant Der p 1 substrate in vivo.

To determine the importance of these findings ex vivo in human lung samples, we obtained BALF from patients with established ARDS, with the knowledge that these patients would have high levels of antiproteases (21) and would provide a good marker for lung sensitivity to Der p 1. Indeed, incubation of BALF fluid with Der p 1 of either preparation (C/CS-Der p 1) reveals that both proteases lead to a marked loss of antielastase activity in the fluid. This finding is in keeping with our data, which show the high susceptibility of human antielastases A1-Pi and elafin to cleavage by the mite proteases. Because concentrations in excess of 3 ng/ml of Der p 1 have been detected in the BAL of allergic individuals (32) and the antiproteases studied here are also present in ng/ml in BAL (21), similar molar ratios of Der p 1:antiproteases as the ones used in vitro are likely to be present in vivo, making our findings pathophysiologically relevant. The studies presented here add to our knowledge on the biological activities of Der p 1 and the role of its enzymatic functions on the development and persistence of asthma by compromising the antiprotease defences of the lung. In addition, Der p 1 has a direct effect on some of the components of the adaptive immune system, including CD23 on B cells and CD25 on T cells, which would increase IgE synthesis by disrupting a negative feedback signal and by favoring a Th1 to Th2 shift by affecting interferon-γ responses, respectively (8, 9, 20, 27). The consequence of this would be to promote allergic inflammatory responses. Furthermore, Der p 1 has destructive activities on structural cells in that it disrupts epithelial cells junctions (10), which facilitates transport of the allergen across the epithelium.

Overall, these findings have potential implications at at least three levels. First, they suggest that following exposure of the bronchial mucosa to HDM fecal pellet, the resultant solubilized mite proteases can access the lung interstitium, promote Th2 responses, and skew the elastase/antielastase balance toward elastase and thus a proinflammatory state. Indeed, elastase, in addition of cleaving a variety of substrates within the lung (33), can upregulate the potent neutrophil chemokine IL-8 (3435), with ensuing neutrophilic inflammation, which in extreme instances could trigger the onset of fatal asthma (3640).

Second, the HNE inhibitor h-A1-Pi can inhibit protease-mediated airway hyperresponsiveness (AHR) in an allergic model (41). It therefore follows that inactivation of A1-Pi by Der p 1 may be deleterious by facilitating AHR.

Third, A1-Pi, SLPI, and elafin possess properties in addition to their antielastase activity. They can function as antimicrobials, either directly or indirectly (1516, 1819, 42) and, at least for elafin and SLPI, can “prime” the innate immune system (17, 43). Thus, inactivation of these additional innate immunity functions may contribute to the infectious exacerbations found in patients with asthma (4445).

In addition, our report suggests for the first time that the serine protease of Der p 1 may also be an important therapeutic target and identifies elafin, a key molecule in lung defense as an important substrate for this protease (1314, 46).

Further work will be needed to establish in vivo whether a dual blockade of the cysteine and serine proteases of Der p 1 will be beneficial to patients with HDM-induced allergic inflammation, by modulating type 2 responses and/or by restoring lung antimicrobial functions.

The authors thank the UK National Asthma Campaign for support Grant Reference 99/009. They also acknowledge the MRC for the support of this work and for providing a Clinical Training fellowship grant to K.F. They also thank Pr. K. Brocklehurst of Queen Mary's and Westfield College, University of London, for his advice on the enzyme kinetic studies, and Dr. P. T. Reid, Western General Hospital, Edinburgh, for stimulating discussions.

1. Global initiative for asthma. National Institutes of Health, National Heart, Lung, and Blood Institute. 2002. Available at: www.ginasthma.com
2. Peat, J. K., B. G. Toelle, E. J. Gray, M. M. Haby, E. Belousova, C. M. Mellis, and A. J. Woolcock. 1995. Prevalence and severity of childhood asthma and allergic sensitisation in seven climatic regions of New South Wales. Med. J. Aust. 163:22–26.
3. Burrows, M., F. D. Martinez, M. Halonen, R. A. Barbee, and M. G. Cline. 1989. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N. Engl. J. Med. 320:271–277.
4. Seaton, A., D. J. Godden, and K. Brown. 1994. Increase in asthma: a more toxic environment or a more susceptible population? Thorax 49:1–4.
5. Sporik, R., S. T. Holgate, T. A. E. Platts-Mills, and J. J. Coswell. 1990. Exposure to house-dust mite allergen (Der p 1) and the development of asthma in childhood. N. Engl. J. Med. 323:502–507.
6. Custovic, A., S. Taggart, and H. Francis. 1996. Exposure to house dust mite allergens and the clinical activity of asthma. J. Allergy Clin. Immunol. 98:64–72.
7. Kalsheker, N. A., S. Deam, L. Chambers, S. Sreedharan, K. Brocklehurst, and D. A. Lomas. 1996. The house dust mite allergen Der p1 catalytically inactivates alpha 1-antitrypsin by specific reactive centre loop cleavage: a mechanism that promotes airway inflammation and asthma. Biochem. Biophys. Res. Commun. 221:59–61.
8. Hewitt, C. R. A., A. P. Brown, B. J. Hart, and D. I. Pritchard. 1995. A major house dust mite allergen disrupts the immunoglobulin E network by selectively cleaving CD23: innate protection by antiproteases. J. Exp. Med. 182:1537–1544.
9. Schulz, O., H. F. Sewell, and F. Shakib. 1998. Proteolytic cleavage of CD25, the alpha subunit of the human T cell interleukin 2 receptor, by Der p 1, a major mite allergen with cysteine protease activity. J Exp. Med. 187:271–275.
10. Wan, H., H. L. Winton, C. Soeller, E. R. Tovey, D. C. Gruenert, P. J. Thompson, G. A. Stewart, G. W. Taylor., D. R. Garrod, M. B. Cannell, and C. Robinson. 1999. Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions. J. Clin. Invest. 104:123–133.
11. Sallenave, J.-M., G. M. Tremblay, J. Gauldie, and C. D. Richards. 1997. Oncostatin M, but not interleukin-6 or leukemia inhibitory factor, stimulates expression of alpha1-proteinase inhibitor in A549 human alveolar epithelial cells. J. Interferon Cytokine Res. 17:337–346.
12. Boutten, A., P. Venembre, N. Seta, J. Hamelin, M. Aubier, G. Durand, and M. S. Dehoux. 1998. Oncostatin M is a potent stimulator of α1-antitrypsin secretion in lung epithelial cells: modulation by transforming growth factor-β and interferon-γ. Am. J. Respir. Cell Mol. Biol. 18:511–520.
13. Sallenave, J. M. 2000. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease. Respir. Res. 1:87–92.
14. Sallenave, J. M. 2002. Antimicrobial activity of antiproteinases. 2002. Biochem. Soc. Trans. 30:111–115.
15. Hiemstra, P. S., R. J. Maassen, J. Stolk, R. Heinzel-Wieland, G. J. Steffens, and J. H. Dijkman. 1996. Antibacterial activity of antileukoprotease. Infect. Immun. 64:4520–4524
16. Wiedow, O., J. Harder, J. Bartels, V. Streit, and E. Christophers. 1998. Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes. Biochem. Biophys. Res. Commun. 248:904–909.
17. Simpson, A. J., G. A. Cunningham, D. J. Porteous, C. Haslett, and J. M. Sallenave. 2001. Regulation of adenovirus-mediated elafin transgene expression by bacterial lipopolysaccharide. Hum. Gene Ther. 12:1395–1406.
18. Simpson, A. J., W. A. Wallace, M. E. Marsden, J. R. Govan, D. J. Porteous, C. Haslett, and J. M. Sallenave. 2001. Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection. J. Immunol. 167:1778–1786.
19. Simpson, A. J., A. I. Maxwell, J. R. Govan, C. Haslett, and J. M. Sallenave. 1999. Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens. FEBS Lett. 452:309–313.
20. Schulz, O., B. J. Sutton, R. L. Beavil, J. Shi, H. F. Sewell, H. J. Gould, P. Laing, and F. Shakib. 1997. Cleavage of the low-affinity receptor for human IgE (CD23) by a mite cysteine protease: nature of the cleaved fragment in relation to the structure and function of CD23. Eur. J. Immunol. 27:584–588.
21. Sallenave, J. M., S. C. Donnelly, I. S. Grant, C. Robertson, J. Gauldie, and C. Haslett. 1999. Secretory leukocyte proteinase inhibitor is preferentially increased in patients with acute respiratory distress syndrome. Eur. Respir. J. 13:1029–1036.
22. Graf, L., C. S. Cralk, A. Patthy, S. Roczniak, R. J. Fletterick, and W. J. Rutter. 1987. Selective alteration of substrate specificity by replacement of aspartic acid with lysine in the binding pocket of trypsin. Biochemistry 26:2616–2623.
23. Kojima, K., H. Kinoshita, T. Kato, T. Nagatsu, K. Takada, and S. Sakakibara. 1979. A new and highly sensitive fluorescence assay for collagenase-like peptidase activity. Anal Biochem 100:43–50.
24. Sallenave, J. M., A. Silva, M. E. Marsden, and A. P. Ryle. 1993. Secretion of mucus proteinase inhibitor and elafin by Clara cell and type II pneumocyte cell lines. Am. J. Respir. Cell Mol. Biol. 8:126–133.
25. Platts-Mills, T. A. E., and M. D. Chapman. 1987. Dust mites: immunology, allergic disease and environmental control. J. Allergy Clin. Immunol. 80:755–775.
26. Platts-Mills, T. A. E., D. Vervloet, W. R. Thomas, R. C. Aalberse, and M. D. Chapman. 1997. Indoor allergens and asthma: report of the third international workshop. J. Allergy Clin. Immunol. 100:S2–S24.
27. Schulz, O., P. Laing, H. F. Sewell, and F. Shakib. 1995. Der p I, a major allergen of the house dust mite, proteolytically cleaves the low-affinity receptor for human IgE (CD23). Eur. J. Immunol. 25:3191–3194.
28. Hewitt, C. R. A., H. Horton, R. M. Jones, and D. I. Pritchard. 1997. Heterogeneous proteolytic specificity and activity of the house dust mite proteinase allergen Der p 1. Clin. Exp. Allergy 27:201–207.
29. Hewitt, C. R. A., S. Forster, C. Phillips, H. Horton, R. M. Jones, A. P. Brown, B. J. Hart, and D. I. Pritchard. 1998. Mite allergens: significance of enzyme activity. Allergy 53:60–63.
30. Schulz, O., H. F. Sewell, and F. Shakib. 1998. A sensitive fluorescent assay for measuring the cysteine protease activity of Der p 1, a major allergen from the dust mite Dermatophagoides pteronyssinus. Mol. Pathol. 51:222–224.
31. Zitnik, R. J., J. Zhang, M. A. Kashem, T. Kohno, D. E. Lyons, C. D. Wright, E. Rosen, I. Goldberg, and A. C. Hayday. 1997. The cloning and characterization of a murine secretory leukocyte protease inhibitor cDNA. Biochem. Biophys. Res. Commun. 232:687–697.
32. Ferguson, P., and D. H. Broide. 1995. Environmental and bronchoalveolar lavage Dermatophagoides pteronyssinus antigen levels in atopic asthmatics. Am. J. Respir. Crit. Care Med. 151:71–74.
33. Bieth, J. G. 1986. Elastases: catalytic and biological properties. In Regulation of Matrix Accumulation. R. Mecham, editor. New York: Academic Press. 217–230.
34. Sallenave, J. M., J. Shulmann, J. Crossley, M. Jordana, and J. Gauldie. 1994. Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes. Am. J. Respir. Cell Mol. Biol. 11:733–741.
35. Nakamura, H., K. Yoshimura, N. G. McElvaney, and R. G. Crystal. 1992. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J. Clin. Invest. 89:1478–1484.
36. Little, S. A., K. J. MacLeod, G. W. Chalmers, J. G. Love, C. McSharry, and N. C. Thomson. 2002. Association of forced expiratory volume with disease duration and sputum neutrophils in chronic asthma. Am. J. Med. 112:446–452.
37. Sur, S., T. B. Crotty, and G. M. Kephart. 1993. Sudden onset fatal asthma: a distinct entity with few eosinophils and relatively more neutrophils in the airway submucosal. Am. Rev. Respir. Dis. 148:713–719.
38. Fahy, J. V., K. W. Kim, J. Liu, and H. A. Boushey. 1995. Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbations. J. Allergy Clin. Immunol. 95:843–852.
39. Ordonez, C. L., T. E. Shaughnessy, M. A. Matthay, and J. V. Fahy. 2000. Increased neutrophil numbers and IL-8 levels in airway secretions in acute severe asthma—clinical and biological significance. Am. J. Respir. Crit. Care Med. 161:1185–1190.
40. Jakanon, A., C. Uasuf, and W. Maziak. 1999. Neutrophilic inflammation in severe persistent asthma. Am. J. Respir. Crit. Care Med. 160:1001–1008.
41. Forteza, R., Y. Botvinnikova, A. Ahmed, A. Cortes, R. H. Gundel, A. Wanner, and W. M. Abraham. 1996. The interaction of alpha 1-proteinase inhibitor and tissue kallikrein in controlling allergic ovine airway hyperresponsiveness. Am. J. Respir. Crit. Care Med. 154:36–42.
42. Cantin, A. M., and D. E. Woods. 1999. Aerosolized prolastin suppresses bacterial proliferation in a model of chronic Pseudomonas aeruginosa lung infection. Am. J. Respir. Crit. Care Med. 160:1130–1135.
43. Ding, A., N. Thieblemont, J. Zhu, F. Jin, J. Zhang, and S. Wright. 1999. Secretory leukocyte protease inhibitor interferes with uptake of lipopolysaccharide by macrophages. Infect. Immun. 67:4485–4489.
44. Johnston, S. L. 1998. Viruses and asthma. Allergy 53:922–932.
45. Gern, J. E. 2000. Viral and bacterial infections in the development and progression of asthma. J. Allergy Clin. Immunol. 105:S497–S502.
46. Schalkwijk, J., O. Wiedow, and S. Hirose. 1999. The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core. Biochem. J. 340:569–577.

*Both authors contributed equally to this study.

Address correspondence to: J.-M. Sallenave, Rayne Laboratory, MRC Centre for Inflammation Research, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, UK. E-mail:

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