Dermatophagoides extract-treated confluent type II epithelial cells (cA549) and human lung mesenchymal cell growth

Dermatophagoides extract–treated confluent type II epithelial cells (cA549) and human lung mesenchymal cell growth Anthony Capetandes, PhD; Nathanael S. Horne, MD; and Marianne Frieri, MD, PhD

Background: Chronic severe persistent asthma is associated with damaged epithelial cells with discontinuous tight junctions that contribute to dysregulated fibroblast and endothelial cell (mesenchymal) growth. Dermatophagoides species– derived proteases have been shown to cause damage to epithelial cell tight junctions. Objective: To determine whether Dermatophagoides species can stimulate confluent A549 (cA549), a cell type with discontinuous tight junctions that approximate differentiated type II cells, to undergo altered growth and secrete putative soluble factors that affect the growth of human lung fibroblasts and microvascular endothelial cells. Methods: Dialyzed Dermatophagoides pteronyssinus or Dermatophagoides farinae extracts (0, 300, 600, and 1,000 AU/mL) were cultured with and without cA549 in serum-free media for 24 hours. After changes in cA549 growth were recorded, conditioned media from extracts with cA549 (CM) and without cA549 (control media [CTLM]) were transferred to fibroblasts and endothelial cells for 24 hours. Fibroblast and endothelial cell growth responses to CM and CTLM were observed and measured. Results: All conditions showed greater than 95% cell viability. Confluent A549 showed dose-dependent growth changes characterized by increased aggregation when incubated with 300, 600, and 1,000 AU/mL of D pteronyssinus in serum-free media relative to control. The CM, but not the CTLM, induced dose-dependent aggregation by fibroblasts and endothelial cells. Fibroblasts also showed decreased adhesion when incubated with CM. Dermatophagoides farinae–treated cA549 showed similar but weaker results. The use of serum, boiled CM, or boiled extract inhibited these findings. Conclusions: Dialyzed Dermatophagoides species extracts altered cA549 growth and stimulated the secretion of factors that dysregulate mesenchymal cell growth in vitro. Ann Allergy Asthma Immunol. 2005;95:381–388.

INTRODUCTION A new concept of asthma pathogenesis has recently been reviewed1 that involves the epithelial-mesenchymal trophic unit (EMTU).2 The EMTU is usually quiescent, characterized by pulmonary epithelial maintenance of an attenuated fibroblast sheath3 and a well-defined microcirculation.4,5 Reactivation of the EMTU by TH2-mediated inflammation results in epithelial cell damage and the release of active transforming growth factor ␤ (TGF-␤) that contributes to dysregulated fibroblast growth.2 Electron microscopy of the histoarchitecture of the alveoli showed that type II epithelial cells display cytoplasmic extensions through apertures in the endothelial cell basement membrane that contact fibroblasts.6 This physical interaction may be disrupted by activated eosinophils and macrophages in juxtaposition to alveolar epithelial cells.7–9 This evidence suggests that alveolar epithelial cell damage causes reactivation of the EMTU, with subsequent dysregulation of mesenchymal cell growth.2 Division of Allergy Immunology and Clinical Immunopathology, Departments of Medicine and Pathology, Nassau University Medical Center, East Meadow, New York. Dr Horne was the first place recipient of the Von Pirquet Award in November 2004 for part of this research. Received for publication January 27, 2005. Accepted for publication in revised form April 19, 2005.

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It has been shown that proteases derived from inhaled aerosolized Dermatophagoides species contribute to the damaged epithelial cell tight junctions induced by eosinophilmediated airway inflammation.10 –14 The epithelial cell damage may be manifested in cell culture by changes in growth (defined herein as the presence of aggregation, morphologic changes, altered adhesion, and mitogenesis) relative to the control. We, therefore, tested the effects of dialyzed standardized extracts of Dermatophagoides pteronyssinus and Dermatophagoides farinae on the growth of confluent A549 (cA549), a transformed type II alveolar epithelial cell that approximates differentiation on attaining confluence15 and that possesses discontinuous tight junctions.16 The decreased barrier function of compromised tight junctions of epithelial cells permits diffusion of inhaled Dermatophagoides species protease-rich particles to the mesenchymal cells associated with alveoli.6,13,17 Hence, the growth of human pulmonary fibroblasts and microvascular endothelial cells was tested in the presence of D pteronyssinus and D farinae extracts. Furthermore, reactivation of the EMTU has been hypothesized as a mechanism of dysregulation of mesenchymal cell growth2 that may be mediated by soluble factors released by epithelial cells in the presence of dust mite proteases.18 –24 We, therefore, tested the growth of human pulmonary fibroblasts and microvascular endothelial cells in response to putative

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factors secreted into the culture media conditioned by cA549 treated with D pteronyssinus or D farinae extract (CM). MATERIALS AND METHODS Dialysis of Standardized Dust Mite Allergen Extract Standardized extracts of D pteronyssinus and D farinae (10,000 AU/mL) stock in 50% glycerin were a gift from ALK-Abello Inc, Port Washington, NY. All extracts contained the major allergens of Der p1, Der p2, Der f1, and Der f2 (50 – 80 ␮g/mL). The extracts were dialyzed using dialysis bags (Spectra/Por Biotech RC Irradiated Membrane; Spectrum Laboratories Inc, Rancho Dominguez, CA) with a molecular weight cutoff value of 8 kDa and several changes of sterile endotoxin-screened phosphate-buffered saline (PBS) (Invitrogen Gibco, Carlsbad, CA) at 1°C to 6°C in 48 hours. In a few experiments, the D pteronyssinus extracts used were from another manufacturer (Nelco Laboratories, Deer Park, NY). The manufacturers claim less than 1% culture medium material in the extract used to grow the dust mite. Cell Culture Standard cell culture conditions included a humidified 37°C cell culture incubator with 5% carbon dioxide. The A549 (passage 85 to 110; American Type Culture Collection CCL185) were grown to confluence (cA549) in Dulbecco Modified Eagle Medium supplemented with 5% to 10% fetal calf serum and penicillin G, 100 U/mL, plus streptomycin, 100 ␮g/mL. Stock cultures of normal human lung fibroblasts (NHLFs) and human microvascular endothelial cells from the lung (HMVECLs) were grown and maintained in proprietary growth factor– defined, serum-reduced media FGM2 and EGM2, respectively (Cambrex Bio-Science, Walkersville, MD). For preliminary experiments, confluent normal human bronchial epithelial (cNHBE) cells were grown in proprietary growth media (Cambrex Bio-Science). All experiments that used dialyzed dust mite extracts were performed with serumfree media (SFM) supplemented with 1⫻ insulin-transferrinselenium (supplied as 500⫻) (Cambrex Bio-Science) and penicillin G, 100 U/mL, plus streptomycin, 100 ␮g/mL. Generation of CM and Control Media After attaining confluence, the culture media were removed from the cA549, followed by gentle washing with endotoxinscreened PBS (Invitrogen Gibco) to remove traces of residual growth factors typically found in serum. After PBS removal, SFM without (0) or with 300, 600, or 1,000 AU/mL of dialyzed standardized D pteronyssinus or D farinae extract was immediately added to the cA549. Growth changes by cA549 were photographed after 24 to 48 hours of incubation with D pteronyssinus extract or 48 to 72 hours of incubation with D farinae extract. After this incubation period, the SFM was labeled “conditioned media” (CM) with corresponding concentrations of dust mite extract (0 CM, 300 CM, 600 CM, and 1,000 CM) from cA549 or cNHBE cells. Control media (CTLM) were generated by incubating the SFM for the same amount of time without (0) or with 300, 600, or 1,000 AU/mL

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of dialyzed D pteronyssinus or D farinae extract without cA549 (0 CTLM, 300 CTLM, 600 CTLM, and 1,000 CTLM). In preliminary experiments, cNHBE cells were also used to generate CM. The CM and CTLM from either cA549 or bronchial epithelial cells were removed at the same time and stored at ⫺40°C until ready for addition to mesenchymal cells. Mesenchymal Cell Culture With CM or CTLM Normal human lung fibroblasts were subcultured at 40% to 60% of confluence, and HMVECLs were grown to 70% to 80% of confluence in proprietary culture media. Before adding the CM or CTLM from the cA549 or cNHBE cells, the culture media were removed from the mesenchymal cells, which were then washed gently with PBS. The CM or CTLM were then immediately added to the mesenchymal cells and incubated for 24 to 48 hours. At the end of incubation, the mesenchymal cells were observed and photographed for changes in growth. Immediately after CM or CTLM removal, the mesenchymal cells were assayed for viability, cell density, and adhesion using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenol tetrazolium bromide (MTT) incorporation assay (see the following subsection). MTT Incorporation Assay for Viability and Cell Number At the end of the experimental time points with Dermatophagoides species extract with cA549 or cNHBEs, or CM and CTLM with mesenchymal cells, MTT (Sigma-Aldrich Co, St Louis, MO), 250 ␮g/mL, in respective growth medium was added immediately, followed by incubation for 3 hours at 37°C. During this incubation period, viable cells incorporated the MTT and enzymatically converted it to a blue formazan end product. The MTT/growth media was then removed, and the cells with (viable) and without (not viable) blue formazan color were counted. The percentage of viability was then calculated ([number of formazan-positive cells per field / (number of formazanpositive cells ⫹ formazan-negative cells per field)] ⫻ 100). To quantify the relative cell numbers between dust mite extract treatments vs controls with cA549, or CM vs CTLM with mesenchymal cells, the blue formazan color was dissolved using 70% isopropanol in 0.04N hydrochloride for 1 hour at 21°C, and optical density was measured using a 96-well plate reader at an absorbance of 550 nm (A550) with the background subtracted at A650. A sufficient volume of isopropanol hydrochloride with plate sealing was used to minimize differences in well-to-well evaporation. The intensity and intracellular distribution of the blue formazan end product was uniform with all cells tested in all conditions. MTT Incorporation Assay for Mesenchymal Cell Adhesion At the end of the 24-hour incubation period in a 24-well plate, CM or CTLM were removed from NHLFs or HMVECLs; MTT, 250 ␮g/mL, in proprietary growth media for either NHLFs or HMVECLs, was immediately added and incubated for 3 hours at 37°C. In experiments with NHLFs, this incubation period with MTT resulted in adherent and floating aggregated cells that showed blue formazan end product. The solution containing the floating aggregated cells was removed

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from the plate and transferred to microcentrifuge tubes and centrifuged at 14,000 rpm for 4 minutes at 21°C. The supernatant was removed with minimal dislodging of the blue formazan–stained pellet. The blue formazan–stained cell pellet was solubilized using 250 ␮L of 70% isopropanol in 0.04N hydrochloride for 1 hour at 21°C and vortexed, and then 150 ␮L was aliquoted immediately to a 96-well plate at volume with plate sealing to minimize the effects of evaporation. Cells that remained adhered to the plastic were solubilized using 70% isopropanol in 0.04N hydrochloride for 1 hour at 21°C and then were aliquoted (150 ␮L) immediately to a 96-well plate and sealed to minimize the effects of evaporation. The optical density of the blue formazan end product from the pellets and adherent cells was measured at A550 with the background subtracted at A650. The number of detached cells represented by the pellets was compared with the number of adhered cells. Statistical Analysis All data conformed to a Gaussian distribution as tested using the D’Agostino-Pearson omnibus test. These parametric data were analyzed for significance at ␣ ⫽ .05 using the 2-tailed unpaired t test for comparisons between 2 groups or analysis of variance (ANOVA) for comparisons among 3 or more groups. The ANOVA at P ⬍ .05 was followed by selected post hoc tests to determine which groups were statistically significantly different, as indicated in the text or figures. Graphed data showed power in the range of 0.6 to 0.8 at a significance level of ␣ ⫽ .05. If the power analysis showed power less than 0.6, then the sufficient number of additional samples required to achieve significance at ␣ ⫽ .05 with an ideal power of 0.8 was calculated, and the experiment was repeated. Graphed data are presented as mean ⫾ 2 SDs. Statistical software used included Prism (GraphPad Software Inc, San Diego, CA) and SigmaStat (Systat Software Inc, Point Richmond, CA). RESULTS D pteronyssinus and D farinae Extracts Cause Reversible Changes in Growth by Viable cA549 The cA549 cultured for 24 to 48 hours with SFM without D pteronyssinus extract showed a monolayer of flat contiguous cells that formed a cobblestone appearance (Fig 1A). In contrast, cA549 grown with SFM plus 300, 600, or 1,000 AU/mL of D pteronyssinus extract showed disrupted monolayers characterized by increased numbers of aggregate formation and cell clumping accompanied by expanded zones of acellularity between 24 and 48 hours in a dose-dependent manner (Fig 1B-D; only the 24 hour point is shown). At 1,000 AU/mL of D pteronyssinus extract, the clumped cA549 were characterized by elongated and raised cells that appeared to fold over each other (Fig 1D). The cA549 cultured with serum-reduced media plus D pteronyssinus extract at any concentration showed a confluent monolayer without aggregation (Fig 1A). The D farinae induced qualitatively weaker aggregation in a dose-dependent manner at more than 48 to 72 hours (data not shown). Similar observations were made with cNHBEs with higher concentrations of D pteronyssinus

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(n ⫽ 3) (data not shown). These findings (Fig 1) were observed in all experiments performed with 2 different lot numbers of Dermatophagoides species (Alk-Abello Inc) and with 2 experiments from a second vendor (Nelco Laboratories). The MTT incorporation assay showed greater than 95% viability by cA549 in all conditions (data not shown). The increased aggregation seen in Figure 1B-D resulted in decreased cell numbers throughout the range of concentrations of D pteronyssinus and D farinae in all experiments (n ⫽ 6) (data not shown). The cA549 aggregates induced with 600 or 1,000 AU/mL of D pteronyssinus demonstrated decreased adhesion at 24 hours of incubation as evidenced by the use of mechanical detachment without trypsin for subculturing. In contrast, trypsin was required for the detachment of aggregated cA549 treated for 12 and 24 hours with 300 AU/mL of D pteronyssinus and 600 and 1,000 AU/mL of D pteronyssinus at 12 hours. When the cell aggregates from mechanical detachment were subcultured in serum-reduced media, the aggregates of cA549 attached to the cell culture plastic and grew to confluence, with a contiguous monolayer without areas of acellularity (Fig 1A). Subcultured cells composing the monolayer showed greater than 95% viability, identical to the viability in SFM without D pteronyssinus and D farinae or serum-reduced media. Control dialysate with PBS did not induce acellular areas in the monolayer, cell aggregation, or adhesion loss, and boiling the dust mite extracts for 10 minutes destroyed the proaggregatory activity (data not shown). Finally, 4 of 6 experiments showed a small but significant 10% to 15% increase in the secretion of TGF-␤1 by cA549 stimulated with 1,000 AU/mL but not 300 or 600 AU/mL of D pteronyssinus (n ⫽ 4; P ⫽ .02 by ANOVA, Student-Newman-Keuls post hoc test) (data not shown). CM, But Not CTLM, From cA549 Induce Aggregation With Adhesion Loss of Viable NHLFs To determine whether putative mediators of growth were secreted by D pteronyssinus–induced aggregated cA549, NHLFs grown to 40% to 60% confluence were cultured with CM for 24 hours and evaluated for changes in growth relative to CTLM. Normal human lung fibroblasts showed normal morphologic features, with greater than 95% viability by MTT incorporation when cultured with 0 CM or 0, 300, 600, and 1,000 CTLM (Fig 2A). Normal human lung fibroblasts also showed a dose-dependent increase in aggregation when cultured with 300, 600, and 1,000 AU/mL of D pteronyssinus extract in SFM (data not shown) that was similar to the dust mite extract effect on cA549 (Fig 1B-D). In contrast, 600 and 1,000 CM induced viable NHLFs to detach with floating aggregates in a dose-dependent manner (Fig 2C-D). Adhered, but not floating, aggregates of NHLFs were observed with 300 CM (Fig 2B). In all experiments, the number of cell aggregates in 600 CM was greater than that in 1,000 CM, with a corresponding decrease in aggregate size in 600 CM relative to 1,000 CM. At 1,000 CM, a few large floating aggregates were observed, with minimal adherent cells. Adherent, aggregated, and floating aggregates of NHLFs showed greater than 95% viability. Aggregated NHLFs in 600 or 1,000 CM

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Figure 1. Growth response of cA549 at 24 hours in culture with serum-free media with and without dialyzed Dermatophagoides pteronyssinus extract at the following concentrations: 0 AU/mL of extract showing a monolayer of contiguous cells (A), 300 AU/mL of extract showing the initial aggregate formations (dashed ovals) with minimal acellular zones (dotted circles) (B), 600 AU/mL of extract showing smaller aggregates and larger acellular zones (C), and 1,000 AU/mL of extract showing additional diminishment of aggregates with cell clumping and expanded acellular zones (D). Similar results with weaker aggregation were observed with Dermatophagoides farinae extract (not shown). Monolayers of contiguous confluent A549 were aggregation free, as observed in (A), when cultured for 24 hours with serum-reduced media or boiled D pteronyssinus extract in serum-free media. Viability of confluent A549 was greater than 95% in all conditions as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide incorporation assay. The photomicrographs are representative of 30 experiments (n ⫽ 3 per experiment).

detached by gentle mechanical disruption and transferred to FGM2 adhered to the cell culture plastic within 1 hour and formed typical fibroblast morphologic features within 24 hours, suggesting that the NHLF cells that formed the aggregates were not apoptotic (data not shown). These observations were duplicated with D farinae, but the degree of aggregation was qualitatively weaker than that of D pteronyssinus. In addition, preliminary experiments using CM from cNHBEs treated with 300, 600, and 1,000 AU/mL of D pteronyssinus, but not 0 CM or CTLM, also caused a dose-dependent increase in NHLF aggregation (n ⫽ 4) (data not shown). The MTT incorporation showed a dose-dependent increase in the numbers of cells in the floating aggregates of NHLFs

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when cultured in 300, 600, and 1,000 CM relative to 0 CM (Fig 3). This increase was accompanied by increased numbers of floating aggregates with decreased adhered cells (Fig 2B-D). In contrast, NHLFs in 0, 300, 600, and 1,000 CTLM showed no floating aggregates (Fig 3A). This was accompanied by normal morphologic features without aggregation and no loss of adhesion (Fig 2A). When the MTT incorporation of the floating NHLF aggregates in 300, 600 and 1,000 CM was plotted again and compared with the adhered NHLFs (Fig 3B), a nonproportional dose-dependent inverse relationship was found. This suggested that one proportion of the detached NHLFs contributed to the size and number of floating aggregates, whereas the remaining proportion most likely

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Figure 2. Subconfluent normal human lung fibroblasts (NHLFs) cultured for 24 hours in the following conditions as shown by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenol tetrazolium bromide incorporation assay for cell adhesion and viability: serum-free media, 0 CM, or 300, 600, or 1,000 CTLM showing adhered nonaggregated cells (A); 300 CM with adhered aggregated cells (B); 600 CM with adhered and floating aggregated cells (C); and 1,000 CM with floating aggregated cells without adhered cells (D). In all conditions shown, greater than 95% of adhered and aggregated NHLFs were viable. The photomicrographs are representative of 20 experiments (n ⫽ 3 per experiment).

were not viable. Furthermore, the number of adherent cells decreased in a dose-dependent manner with increasing concentrations of D pteronyssinus in either CM or CTLM (Fig 3B). This suggested that 2 contemporaneous effects were present: (1) a direct effect of D pteronyssinus that decreased the cell density of NHLFs without causing floating aggregates in the presence of CTLM and (2) the effect of putative soluble factors released into the CM by the cA549 when stimulated with D pteronyssinus.

than 95% viable. The HMVECLs grown in 300, 600, and 1,000 CM and detached with gentle mechanical disruption grew to form confluent monolayers when subcultured with EGM2 on cell culture plastic. In addition, preliminary experiments with CM from cNHBEs treated with 600 and 1,000 AU/mL of D pteronyssinus, but not 0 or 300 CM or CTLM, also caused a dose-dependent increase in HMVECL aggregation (n ⫽ 2) (data not shown).

CM, But Not CTLM, From cA549 Induce Aggregation With Diminished Adhesion by Viable HMVECLs Subconfluent HMVECLs cultured with 0 CM or 0, 300, 600, and 1,000 CM or CTLM showed dose-dependent changes in growth (Fig 4). The HMVECLs in all conditions were greater

DISCUSSION The results of this study show that dialyzed standardized D pteronyssinus and D farinae extracts cause viable cA549 to undergo changes in growth (defined in this article as the presence of aggregation, morphologic changes, diminished

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Figure 3. Effect of conditioned media (CM) or control media (CTLM) on normal human lung fibroblast (NHLF) adhesion and floating aggregates as measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT) incorporation at 24 hours. A, Absorbance of MTT of floating aggregates of NHLF in CM (F) relative to CTLM (f) at each concentration of Dermatophagoides pteronyssinus extract. OD indicates optical density; asterisk, P ⬍ .003, by 2-tailed unpaired t test; plus, P ⬍ .001, by analysis of variance, post hoc test for linear trend (n ⫽ 6). Error bar represents SD. B, Absorbance of MTT of NHLF floating aggregates in response to CM (F) and adhered NHLFs in response to CM (E). OD indicates optical density; asterisk, P ⬍ .005, by 2-tailed unpaired t test; plus, P ⬍ .001, by analysis of variance, post hoc test for linear trend (n ⫽ 6). Error bar represents SD. All adherent and aggregated NHLFs showed greater than 95% viability. Similar but weaker results were observed with Dermatophagoides farinae. Data are representative of 20 experiments.

adhesion, or decreased cell numbers) and to secrete soluble mediators into CM that cause aggregation of NHLFs and HMVECLs in a dose-dependent manner. These observations were not seen with dialysis buffer or sham dialysis with PBS and were inhibited by serum and by boiled dust mite extract or CM for 10 minutes. The CTLM, generated by incubating

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dust mite extract in SFM without cA549, did not cause aggregation of mesenchymal cells but did cause a dosedependent decrease in cell numbers, most likely due to residual protease activity. A small, but statistically significant, increase in TGF-␤1 secretion by cA549 was also found, as predicted by the current model of EMTU reactivation in airway remodeling.2 The direct addition of D pteronyssinus and D farinae extract to mesenchymal cells in SFM decreased cell numbers, suggesting the presence of cell death, but did not induce adhesion loss, as seen with CM. In fewer experiments, similar results were found using CM generated with dust mite extract–treated cNHBEs. Other studies showed that purified Der p1 stimulated subconfluent A549 cells in SFM to secrete proinflammatory cytokines in a protease-activated receptor 2– dependent manner18 and increased the permeability of bronchial epithelial cells,25 although no associated aggregation or changes in cell number were described. We suspect that this might be due to differences in experimental conditions and the use of nanomolar concentrations of Der p1,25 which may be below the threshold for stimulating aggregation but sufficient for stimulating cytokine secretion. In addition, our data are consistent with the reports that subconfluent A549 in SFM showed morphologic changes, cell detachment, and cytokine secretion when treated with extracts of Alternaria alternata,26 Alternaria fermata,26 D pteronyssinus,27 and Lepidoglyphus destructor,27 although the changes in mesenchymal cell biology seen in Figures 2, 3, and 4 were not reported. It is possible that the media used to grow the D pteronyssinus or D farinae commercially may have contributed to the changes in cA549 and mesenchymal cell growth. The manufacturers claim that the dust mite extract contains less than 1% yeast and pork medium used to grow the dust mite. Testing the effect of the proprietary medium or recombinant Der p1 on cell growth, however, was not possible at this time. We reasoned that cell growth changes mediated by D pteronyssinus– or D farinae– derived proteases are concentration dependent and, therefore, might be mimicked by serially diluted cell culture– grade trypsin. Serial dilution of trypsin incubated for 24 to 48 hours at 37°C (identical conditions as the CM) identified a concentration 102 to 103 lower than stock (0.5 g/L) that caused aggregation in a manner similar to that seen with the dust mite extracts with viable cA549. In addition, boiling the dialyzed dust mite extract for 10 minutes destroyed its ability to induce cA549 aggregation and adhesion loss. Because the aggregation and diminished adhesion was inhibited by serum, destroyed by boiling, and mimicked by diluted trypsin, we suspect that mite-derived protease(s) mediated this observation, not the media components used to commercially grow D pteronyssinus or D farinae, although the dust mite protease may not be trypsin per se. This study provides in vitro support for the recent finding that dust mite proteases may contribute to reactivation of the EMTU28 and supports the role of proteases and protease-activated receptors in allergic inflammation of the lung.18,29 Mesenchymal cell growth is important in early life when infants are exposed to dust mite allergens

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Figure 4. Effect of conditioned media (CM) or control media (CTLM) on subconfluent human microvascular endothelial cells from the lung (HMVECLs) growth relative to controls at 24 hours. A, Serum-free media, 0 CM, or 300, 600, or 1,000 CTLM showing adhered nonaggregated cells. Also shown are cells with increasing extent of aggregation with decreased cell numbers with 300 CM (B), 600 CM (C), and 1,000 AU/mL of Dermatophagoides pteronyssinus (D). The HMVECLs show greater than 95% viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide incorporation assay. Similar results were observed with Dermatophagoides farinae with 3 experiments. Data are representative of 3 experiments (n ⫽ 3 per experiment).

and other triggers that can play a role in the progression of remodeling from childhood asthma to adult asthma.1 Although this study is an in vitro model of mesenchymal cell aggregation, our findings lead us to hypothesize that a contemporaneous double-hit from dust mite– derived proteases in the presence of eosinophil-derived factors causes the rate of pulmonary epithelial cell damage to be greater than repair in individuals with chronic severe persistent asthma. With further development (ie, the addition of known TH2-related inflammatory mediators to co-cultures

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of epithelial and mesenchymal cells), this culture setting may provide the opportunity to mechanistically explore this double-hit hypothesis. Identification of the dust mite– and cA549-derived mediators that alter the growth of epithelial and mesenchymal cells may provide targets for novel pharmacologic interventions.30 ACKNOWLEDGMENTS We thank Nisar Alvi, MD, Y.-C. Huang, MS, and E. Sequerra, MD, for their technical assistance.

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Requests for reprints should be addressed to: Marianne Frieri, MD, PhD Division of Allergy Immunology Nassau University Medical Center Bldg Q, Room 105 East Meadow, NY 11554 E-mail: [email protected]

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