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Bronchial Mast Cells Are the Dominating LTC₄S-Expressing Cells in Aspirin-Tolerant Asthma

Institute of Oral Biology, University of Oslo, Oslo; Department of Circulation and Medical Imaging, Medical Faculty NTNU, Trondheim; and Department of Lung Medicine, University Hospital of Trondheim, Trondheim, Norway

Address correspondence to: Dr. Trond S. Halstensen, Institute of Oral Biology, University of Oslo, PB 1052, Blindern, 0316 Oslo, Norway. E-mail: thalsten{at}odont.uio.no

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increased bronchial production of leukotriene C₄ (LTC₄) in asthma is assumed to derive from infiltrating eosinophils expressing LTC₄-synthase (LTC₄S). Multicolor immunohistofluorescence examination of bronchial cryosections from 30 treated, untreated, or bronchial antigen–provoked aspirin-tolerant individuals with asthma and nine control subjects revealed that the dominating LTC₄S-expressing cells were mast cells (> 80%), and not eosinophils. Whereas 95% of the mast cells expressed high levels of LTC₄S, only 8–27% of the eosinophils expressed low levels. Image analysis revealed a significantly higher LTC₄S expression levels in mast cells than in eosinophils. The bronchial mRNA levels for LTC₄S did not correlate with the densities of LTC₄S-positive eosinophils or mast cells. Treated individuals with asthma with more than 12% reversibility had significantly higher density of LTC₄S-positive mast cells than those with less reversibility, and it correlated significantly with reduction in lung function (FEV₁-predicted), both before and after salbutamol inhalation. Thus, mucosal mast cells, and not eosinophils, were the dominating LTC₄S-containing cells in both untreated and treated aspirin-tolerant asthma. The density of LTC₄S-positive mast cells correlated, moreover, with both the reduction in lung function and the degree of reversibility in treated asthma.

Abbreviations: diffraction interface contrast, DIC • 5-lipo-oxygenase activating protein, FLAP • glycol methacrylate, GMA • horseradish peroxidase, HRP • inhaled corticosteroid, ICS • interleukin, IL • leukotriene, LT • leukotriene C₄-synthase, LTC₄S • microsomal glutation S-transferase, mGST-II • phosphate-buffered saline, PBS • prostaglandin, PG • periodate-lysine-paraformaldehyd, PLP • reverse transcriptase–polymerase chain reaction, RT-PCR

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The eicosanoids are lipid mediators derived from arachadonic acid and consist of the cysteinyl leukotrienes (cys-LTs) and prostaglandins (PG). The cys-LTs LTC₄, LTD₄, and LTE₄ induce potent bronchoconstriction, mucosal edema, increased mucus secretion, and eosinophilic recruitment and were previously known as the "slow reacting substance of anaphylaxis" (1, 2). During cell activation, cytosolic phospholipase A₂ releases arachadonic acid that is relocated to the 5-lipo-oxygenase activating protein (FLAP) and converted in two steps to cys-LTA₄ by 5-lipoxygenase (5-LO). LTA₄ is either converted to the dihydroxy leukotriene (LT) B₄ by cells expressing LTA₄-hydrolase or to cys-LTC₄ by cells expressing LTC₄-synthase (LTC₄S), which conjugates LTA₄ to reduced glutathione. The metabolites LTD₄ and LTE₄ are produced by sequential cleavage. The neutrophil granulocyte activator LTB₄ is the main product in the absence of LTC₄S (for review see Ref. 3). Patients with asthma have increased production of cys-LT both during asthmatic attack (4) and during the late asthmatic reactions (4, 5). In particular, patients with aspirin-induced asthma have increased urinary cys-LTC₄ metabolites (LTE₄) after aspirin challenge and asthmatic reactions (for review see Ref. 6). Single-color immunohistochemical examination of acetone-fixed glycol methacrylate (GMA)-embedded bronchial biopsies identified the infiltrating eosinophilic granulocytes to be the main LTC₄S containing cells both in aspirin-intolerant and conventional, aspirin-tolerant asthma (7). The concentration of LTC₄ in induced sputum obtained at baseline and 24 h after antigen challenge in patients with aspirin-tolerant asthma has also been shown to correlate with the numbers of eosinophils in the sputum (5). Thus, eosinophils have been considered to be the main cellular source for LTC₄S in the asthmatic bronchial mucosa.

Although inhaled corticosteroid (ICS) generally is considered the most efficient treatment for asthma (8), additional treatment with LT receptor antagonists induces further symptom relief in a proportion of the patients (2, 9, 10). LT antagonists are therefore predominantly recommended as add-on drugs taken in addition to ICS treatment (2, 10). The bronchial density of eosinophils is often reduced to almost none after ICS treatment (11). If mucosal eosinophils are the main cell type containing LTC₄S as reported previously (7), what mucosal cell may then contain the LTC₄S required for LTC₄ production in treated patients?

The aim of this study was to examine the density and phenotype of the LTC₄S-expressing cells in asthma with particular emphasis on treated patients. By applying three different LTC₄S-peptide antisera in multicolor immunhistofluorescence microscopy on bronchial cryosections, we surprisingly identified the mast cell, and not the eosinophils, to be the main LTC₄S expressing cells both in untreated, bronchial antigen–provoked and in treated patients with asthma. The bronchial density of LTC₄S-positive mast cells correlated, moreover, with reversibility and reduction in lung function (FEV₁-predicted) in treated patients.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study Subjects
Information about the 30 patients with asthma is listed in Table 1. The control subjects (n = 9, median age 27 yr, range 20–75 yr) were skin prick test–negative, nonatopic, never-smoking healthy students (n = 6), hospital staff members (n = 2), and a patient without asthma. Additional bronchial (n = 1) and nasal polyp cryo-biopsies (n = 3) from four patients with aspirin-induced asthma, and GMA-embedded bronchial biopsies from patients with treated aspirin-tolerant asthma (n = 5), were obtained for immunohistochemical control purposes. The regional ethical committee approved the study and all subjects gave informed consents.

Fiberoptic bronchoscope was performed in accordance with international guidelines (12). Bronchial biopsies (n = 115) were taken from the second- and third-generation carina and processed for cryosectioning.

Patients did not inhale ß-agonist 8 h before the bronchial provocation tests (BPT). The two patients were skin prick–positive for dog (10,000 SQ produced 4+), and Dermatophagoides pteronyssinus (33,000 SQ produced 3+), respectively. Allergen extracts (ALK-Abelló A/S, Hørsholm, Denmark) were administered by a controlled tidal volume breathing technique (13). The dog-allergic patients had a PD20 of 100 SQ allergens, whereas the mite-allergic patient had a PD20 of 6,400 SQ. The patients received salbutamol inhalation just after the provocation and 40 mg metylpredisolon intravenously 2 h thereafter. Bronchial biopsies were performed 24 h after provocation, and only the dog-allergic patient had a late phase reaction (mild).

Tissue Processing
All specimens were immediately fixed in ice-chilled periodate-lysine containing 1% paraformaldehyde (1% PLP) for 4 h at 4°C (14). The specimens were infiltrated in Histocon (Histolab, Götebotg, Sweden) for 1 h. Specimens were oriented on a thin slice of carrot, embedded in OCT (Tissue-Tek, Miles Laboratories, IN), snap-frozen in liquid nitrogen, and stored at -20°C. Cryosections cut serially at 4 µm were dried overnight at room temperature, enwrapped in aluminum foil, and stored at –20°C until used.

The additional bronchial biopsies (n = 5) from the patients with treated asthma were placed in acetone containing 2 nM phenyl-methyl-sulfonyl-fluoride (P-7626) and iodoacetamide (I-6125; both from Sigma-Aldrich, St. Louis, MO) at –20°C, fixed overnight, and embedded in GMA resin similar to that used by Cowburn and coworkers (7) and by Seymour and colleagues (15).

Baculovirus Expressed Microsomal Glutation S-trasferase II
A Baculovirus expression vector for microsomal glutation S-transferase II (mGST-II) and LTC₄S (kindly provided by Joseph A. Mancini, Merck Frost Centre for Therapeutic Research, Kirkland, PQ, Canada; 16) was used to infect 3 x 10⁷ Sf9 cells cultured in Grace's insect cell media or SF900-II (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (PAA Laboriatories, Linz, Austria). Infected and noninfected cells were harvested after 96 h, washed, re-suspended in phosphate-buffered saline (PBS; pH 7.4), sonicated on ice at full power (3 x 10 s) and centrifuged for 10 min at 500 x g. The supernatants was collected and centrifuged at 100 000 x g for 1 h at 4°C. The pellet was resuspended in 1 ml PBS supplemented with 0.05% Tween-20 and 1 mM EDTA, sonicated as above and frozen at -70 before use.

Western Blots
The microsomal fraction (200 µg) from mGST-II infected, LTC₄S-infected, and noninfected Sf9 cell lines was precipitated in acetone overnight at -20°C, centrifuged (15,000 rpm, 15 min., 4°C), dried, and dissolved in loading buffer. Additional cell pellets from the LTC₄S-infected cells, uninfected Sf9 control cells, and the human mast cell leukemia cells (HMC-1; kindly provided by J. H. Butterfield, Division of Allergic Diseases and Internal Medicine, Mayo Clinic, Rochester, MN) and 5 µl prestained proteins standard (SeeBlue Pluss2; Invitrogen) were electrophoresed in 15% SDS-PAGE, blotted, and incubated with the following antisera: anti–LTC₄S-peptide_37–51 antiserum (1:10,000); a C-terminal LTC₄S-peptide_136–150 antiserum (1:666); antiserum to whole LTC₄S, (1:1,000; both kindly provided by Frank K. Austen and Bing K. Lam at the Department of Medicine, Harvard Medical School, Boston, MA); an anti–mGST-II antiserum (1:1,000; kindly provided by Joseph A. Mancini, Merck Frost Centre for Therapeutic Research) and nonimmune rabbit antiserum (1:500) for 90 min. The immune reactions were revealed with alkaline phosphatase–conjugated anti-rabbit IgG (1:5,000 in 2% goat serum; Sigma-Alrich, A3812) for 30 min, washed, and developed in BCIP/NBT for 10–20 min.

Immunohistochemistry
We used a multicolor immunohistofluorescence staining technique basically as published elsewhere (17). Briefly, 4 µm cryosections were blocked by 5% dry milk powder in 20% horse serum for 20 min, then incubated for 1 h at ambient temperature with rabbit anti-serum raised to peptide aa 37–51 from human LTC₄-synthase (1:1,000; kindly provided by Drs. Donald Nicholson and John Vaillancourt at Merck Research Laboratories, Rahway, NJ; 18) mixed with an mAb to mast cells (1:500, clone AA1; Dako, Glostrup, Denmark) or eosinophils (1:500, mAb EG2; Pharmacia, Uppsala, Sweden). The primary incubation was followed by biotinylated-horse anti-mouse IgG (2 µg/ml; Vector Laboratories, Burlingame, CA) for 1.5 h followed by 30 min incubation with ALEXA-488–conjugated goat anti-rabbit IgG (10 µg/ml), mixed with ALEXA-594 streptavidin (0.25 µg/ml; both Molecular Probes, Eugene, OR) in 12.5% bovine serum albumin, 2% normal heat-inactivated horse serum, and 0.8% human globulin (Pharmacia and Upjohn, Uppsala, Sweden). 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (300 nmol; Molecular Probes) was added to visualize the nucleus when appropriate. The eosinophils were identified with diffraction interface contrast microscopy (DIC) (19, 20) and/or by both mAb EG2 positivity and DIC on optimally fixed sections from every patient with asthma and every control subject. T cells (CD3+) were examined for LTC₄S expression by combining the LTC₄S antiserum with mAb RIV9 to CD3 (mouse IgG3, 1:20; Monosan, am Unden, the Netherlands) on 25 sections from 10 patients with asthma and 8 control subjects.

The phenotype of the non-eosinophil (DIC-negative, mAb EG2-negative), non–mast cell (tryptase-negative), but LTC₄S-positive cells were examined in multicolor immunohistofluoresence staining by using rabbit antiserum to LTC₄S and/or IgE (1:2,000; Dako) in various combinations with mAbs to: c-kit (1:5,000, IgG1; Dako), Fc-RI (clone CAR-1, 1:2,000, IgG2b; Kyokuto Pharmaceutical, Tokyo, Japan), CD68 (KP1; DAKO), and HLA-DR (mAb L123, 1:200, IgG2a; Becton and Dickinson, Franklin Lakes, NJ) on sections (n = 54) containing the highest fraction of such cells. A solution of 0.06% hydrogen peroxide was applied for 30 min to block endogenous peroxidase. Secondary reagents were various combinations of ALEXA-488–conjugated (1:1,000), ALEXA-549–conjugated (1:4,000; both from Molecular Probes), biotin-conjugated, or horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG-subclass–specific antisera (1:200; both from Southern Biotechnology, Birmingham, AL). Binding of HRP-conjugated and biotinylated reagents were visualized by various combinations of TSA-direct 7-diethylaminocoumarin-3-carboxylic acid (AMCA) or fluorescein isothicyanate (both from NEN, Life Science, Boston, MA) and/or ALEXA-549 streptavidin (1:4,000; Molecular Probes). The rabbit anti-LTC₄S was then visualized by either ALEXA-488–conjugated goat anti-rabbit IgG (1:200; Molecular Probes) or PAP-complexes (DAKO) and TSA-AMCA. DAPI was added to visualize the nucleus, when appropriate. These combinations made it possible to examine LTC₄S, in combination with c-kit (CD117) or tryptase and FcR-I^+ve on nuclear stained (DAPI) cells, identifying eosinophils by DIC-microscopy. Similar examination was also performed mixing antiserum to LTC₄S with mAb to CD68 (KP1, IgG1; DAKO) and mAb to HLA-DR (IgG2a, BD).

LTC₄S in macrophages were further examined in a sequential four-color immunohistofluoresence staining procedure where mAb AA1 to tryptase mixed with antiserum to LTC₄S were applied first, followed by biotinylated goat anti-mouse IgG (Vector). A solution of 0.06% hydrogen peroxide was applied for 30 min to block endogenous peroxidase. The sections were thereafter incubated with mAb KP1 to CD68 (1:20; Dako) to identify macrophages, followed by HRP-conjugated goat anti-mouse IgG1 (1:200; Southern Biotechnology) that was visualized by tyramide-AMCA (NEN, Life Science) and finally incubated with ALEXA-549 streptavidin (1:4,000) and ALEXA-488–conjugated goat anti-rabbit IgG (1:200; both Molecular probes) for 30 min. The sections were rinsed in PBS (pH 7.4) at room temperature for 10 min between each incubation steps. The latter staining procedure made it possible to identify LTC₄S-expressing cells (green) in either CD68+ macrophages (pure AMCA blue) or mast cells (mAb AA1 red and CD68 blue) in the same sections by using appropriate fluorescence filters. Nuclear DAPI was selectively visible with 365-nm excitation filters. A further examination for LTC₄S in macrophages was performed with antiserum to the C-terminal part of LTC₄S on bronchial sections (n = 19) from 4 control subjects and 12 aspirin-tolerant patients with asthma. This anti-LTC₄S antiserum produced the best signal/noise ratio. The anti-peptide antiserum was mixed with various combinations of mAbs to CD68 (KP1; Dako), HLA-DQ (BD), CD1c (BDCA-1; Miltenyi Biotec) and c-Kit.

To test for nonspecific immunoreactivity, all sections were incubated with nonimmune heat-inactivated rabbit serum at same concentration as the antiserum (1:1,000), mixed with normal mouse serum (1:500) or 12.5% bovine serum albumin instead of the primary antibodies. No specific immunohistochemical staining was observed in these sections.

To control the specificity of the LTC₄S-peptide_37–51 antiserum used in the present study, we performed parallel experiments where bronchial cryosections (n = 12) from untreated patients and control subjects were immunostained with optimal concentrations of the anti–LTC₄S-peptid_43–56 antiserum (directed to the active site); anti–LTC₄S-peptide_136–150 antiserum to the C-terminal part of LTC₄S known not to cross react with mGST-II; and an IgG fraction (2 mg/ml) of an antiserum from the same LTC₄S-immunized animals that were used to generate the affinity-purified antiserum that Cowburn and associates (7) and Seymour and coworkers (15) used in their studies (provided by Frank K. Austen, and Bing K. Lam). Optimal concentrations of these antisera were used in combination with mAb EG2, or mAbs to c-kit and mast cell tryptase (mAb AA1), and processed as the other sections. The results were compared with the staining pattern produced by the LTC₄S-peptide_37–51 antiserum.

The immunohistochemical specificity of the anti-LTC₄S peptide_37–51 and the anti-LTC₄S peptide_136–150 (to the C-terminal part) was tested twice by adding the microsomal fraction of the LTC₄S-, and mGST-II baculovirus–infected Sf9 cell line to the anti–LTC₄S-peptide antisera. The anti–LTC₄S-peptide_37–51 antiserum (1:3,000, 1:5,000) and the C-terminal LTC₄S-peptide_136–150 antiserum (1:1,000, 1:15,000) was mixed with the microsomal protein fraction of LTC₄S-infected (0.23 and 0.45 mg/ml), mGST-II baculovirus–infected (0.23 and 0.46 mg/ml), and uninfected Sf9 cell line (0.33 and 0.66 mg/ml) and left on a shaker for 20 h at 4°C. The mixture was centrifuged, and the supernatant was used for immunostaining in parallel with antiserum not mixed with microsomal fractions. Bronchial cryosections were incubated for 1 h and subsequently stained as described above.

To test for tissue processing–induced differences we used GMA-embedded bronchial biopsies from treated patients with asthma (n = 5) similar to those used by Cowburn and colleagues (7) and by Seymour and coworkers (15) that were cut at 2 µm, dried for 2 h, enwrapped in aluminum foil, and stored at –20°C until used. The sections were incubated overnight at 4°C with the same primary antibody combinations, followed by the same secondary regents as for cryosections for 2 h in each step.

Microscopy, Evaluation, and Scoring
The samples were observed in a blind manner in a Zeiss Axioplan 2 microscope equipped with a plan-neofluar x40/1.3 oil lens and appropriate fluorochrome filters including single-, double-, and triple-color filter blocks that allow simultaneous examinations of green, red, and blue emissions (Carl Zeiss, Göttingen, Germany). The numbers of positive stained cells were counted in a 230-µm-deep subepithelial zone using an ocular photo-grid and the density per mm² was estimated. Cryosections from two to four different located bronchial biopsies from both patients with asthma and from control subjects were examined. A median area of 1.2 mm² (range 0.6–4 mm²) submucosa per patient was evaluated. The densities of the various cell types within each patient was estimated by summarizing the actual number of evaluated cells in each section and divided by the total area examined.

Computer-Assisted Digital Image Analysis
Single-color images were captured with a MicroMax CCD digital camera system (Princeton Instruments, Roper Scientific, Inc., Princeton, NJ) and the imaging software package MetaMorph 3.0 (Universal Imaging Corporation, Downingtown, PA). The LTC₄S images were all exposed for 750 ms to ensure equal exposure on all sections and to prevent overexposure of the most intensely stained cells. The 12-bit images were combined into 24-bit multicolor images. Electronically 800% zoomed images were used to analyze LTC₄S positivity by measuring the average nuclear staining intensity within a manually drawn area. The eosinophils were immunohistochemically identified with mAb EG2 and its typical granularity in DIC microscopy. The DAPI-identified nucleus within the mAb EG2–positive cells were identified on single-stained images and used as template. A circle was carefully drawn around the DAPI-positive nucleus to include the perinuclear location of the LTC₄S immunoreactivity without prior knowledge of the cells' LTC₄S positivity. This circle was then automatically copied onto the LTC₄S-stained image in a site-specific manner and the average LTC₄S immunoreactivity within the area was digitally measured. The LTC₄S positivity in the mAb EG2–negative but LTC₄S-positive cells were measured and taken to reflect LTC₄S immunoreactivity in mast cells, as all DIC-negative but LTC₄S-positive cells had been identified as tryptase-positive and/or c-kit–positive mast cells on neighboring sections. LTC₄S-negative nuclei adjacent to the positive ones were used to estimate the background. The average immunofluorescence from the LTC₄S-negative nuclei were used as threshold levels for the specific staining within each image. Median threshold levels were subtracted from the LTC₄S immunoreactive level. Some LTC₄S-negative eosinophils got a negative value because the non-eosinophilic, non–LTC₄S-positive control cells produced stronger nonspecific immunofluorescence signal than the LTC₄S-negative eosinophils.

mRNA Isolation, Competitive RT-PCR, Gel Electrophoresis, and Scanning Densitometry
Bronchial biopsies (n = 10) from six untreated and two bronchial antigen–provoked aspirin-tolerant patients with asthma, one treated patient, and one control subject were available for mRNA extraction. The biopsies were immediately placed in 0.5 ml RNAlater (Ambion, Austin, TX) for 24 h at 4°C and then placed at -20°C until processed. Total RNA was extracted using TRI-reagents (Molecular Research Center Inc., Cincinnati, OH) supplemented with GenElute LPA (Sigma-Aldrich cat# 5–6575) as RNA carrier, according to suppliers' instructions. mRNA (0.5 µg) were reverse transcribed with 2.5 µM poly₁₈-dT and 400U M-MLV (Rnase H minus) reverse transcriptase (Promega, Madison, WI) in 50 µl reaction buffer at 42°C for 1 h, followed by 81°C for 10 min, and finally diluted 50 times with ddH₂O. The cDNA levels in the samples were examined with a competitive reverse transcriptase (RT)-polymerase chain reaction (PCR) assay using 5 µl of the diluted cDNA in serial dilution with increasing concentrations (200, 1,000, 5,000, 2,500 copies per tube) of the pQB2 multistandard plasmid (21) with 100 ng of the primers for ß-actin (sense5'-gggtcagaaggattcctatg-3'; antisense 5'-ggtctcaaacatgatctggg-3') in 25 µl PCR reactions under standard condition using 20" at 94°C, 30" at 57°C, and 45" at 72°C in 40 cycles. Ten microliters of the PCR products (10 µl) were electrophoresed in 2% agarose gels (Sigma-Aldrich) containing 0.5 µg/ml ethidium bromide (Sigma-Aldrich). The PCR products were photographed with a Polaroid DS-34 instant camera, using 667 films. The ß-actin cDNA levels were used to adjust the amount of cDNA used for LTC₄S PCR. Ten microliters of diluted cDNA was used with 0.6 µM of the LTC₄S primers (5'cgaggaacagcgggaagtac-3'; 5'-gagtcctgctgcaagcctacttc-3') that jumped an exon–intron junction in a PCR reaction using 64°C for 30" and 72°C for 45" for 40 cycles. Negative controls included water and genomic DNA. Ten microliters of the PCR product were electrophoresed in 3% agarose gel containing SYBR Green I nucleic acid gel stain (1:10,000; Molecular Probes) and photographed. A dilution series of mRNA extracted from human small intestine were used to calibrate the LTC₄S RT-PCR. Both the ß-actin and LTC₄S PCR-product images were scanned on an Agfa photoscanner at 8 bits gray scale. The unmanipulated TIFF image was imported to the imaging software package TotalLab (Nonlinear Dynamics Ltd., New Castle upon Tyne, UK), where the intensity of the bands was recorded as pixel volumes. The logarithmic value of the ratio of PCR band intensity (pixel volume) for ß-actin to standard plasmid was plotted against the logarithmic value of the plasmid concentration in a log-log scatter diagram where the straight line crossed the y axis at the logarithmic value of the ß-actin concentration in the samples. The pixel value of the LTC₄S PCR intensity was linear to the concentration of the template as estimated with the serial dilution of extracted mRNA from small intestine. The pixel value of the LTC₄S PCR was then divided by the estimated ß-actin concentration to generate the correlated LTC₄S pixel value/ß-actin. This value was then compared with the bronchial density of eosinophils, LTC₄S-positive eosinophils or mast cells in the patients.

Statistical Analysis
Data on cell density counts are presented as median values with range and the Mann-Whitney nonparametric test was analyzed with the SPSS software package version 11.0 for Windows (Chicago, IL). Regression analysis of the percentage predicted FEV₁ and the density of LTC₄S-positive cells and the RT-PCR results for LTC₄S against the density of eosinophils, LTC₄S-positive eosinophils, or mast cells was performed by Kendals nonparametric test. A value of P < 0.05 was considered statistically significant.

Methodologic Results and Considerations
Western blots. The production of recombinant proteins in the Baculovirus-infected Sf9 cells was examined in Western blotting (not shown). Antiserum to mGST-II reacted with a 16-kD band in mGST-II–infected cell lines only. The C-terminal anti-LTC₄S peptide_136–150 antiserum reacted with a 16-kD band both in the cell pellet and in the microsomal fraction from the LTC₄S-infected Sf9 cells, and in the cell pellet from the HMC-1 cell lines, but not with mGST-II–infected or uninfected Sf9 cells lines (not shown). The anti–mGST-II antiserum detected the 16-kD band in mGST-II–transfected cell lines only. The anti–LTC₄S-peptide_37–51 antiserum detected similar bands in the mGST-II and LTC₄S infected cell lines, at 25 kD. Thus, both cell pellets and the microsomal fractions from the infected Sf9 cells contained the relevant proteins.

Specificity of the antisera. Identification of LTC₄S-expressing cells is critically dependent on the specificity of the antisera. We compared the staining patterns produced by the LTC₄S-peptide_37–51 antiserum (predominantly used in the study), with the staining pattern produced by the LTC₄S-peptide_43–56 antiserum (directed against the active site), and the LTC₄S-peptide_136–150 antiserum (directed to the C-terminal part of LTC₄S; Figure 1) . Whereas both the LTC₄S-peptide_37–51 antiserum and the active-site peptide_43–56 antiserum is directed to regions in the LTC₄S that show amino acid homology with mGST-II (73% and 78% homology, respectively), the C-terminal peptide_136–150 antiserum is directed to a region with no amino acid sequence similarity to either mGST-II (0.06% homology) or the family member 5-lipoxygenase-activating protein (FLAP, 0.1% homology). Blast search of the DNA and amino acid sequence in the National Center for Biotechnology Information (www.ncbi.nih.gov) blast search service did not identify any human protein with sequence similarities to the C-terminal region. Western blotting with the C-terminal anti-peptide_136–150 antiserum identified the 16 kD LTC₄S- band in both the cell pellet and in the microsomal fraction from the LTC₄S-transfected Sf9 cells and in cell pellets from the mast cell line HMC-1, but not from mGST-II–infected Sf9 cells (not shown). Moreover, mixing the C-terminal anti-LTC₄S antiserum with the microsomal fraction from LTC₄S-infected Sf9 cells (but not if mixed with mGST-II–infected cells) completely blocked the anti-LTC₄S immunoreactivity in bronchial cryosections (not shown). Thus the C-terminal–peptide_136–150 antiserum did not crossreact with mGST-II.

View larger version (146K): [in this window] [in a new window]   Figure 1. (A–D) The predominant LTC4S-containing cells (green in A and B, arrows) were not eosinophils (red in A and C, arrowheads), although LTC4S positivity was observed on some eosinophils (cells labeled c and d in A). Digital image analysis with three-color line scan (D) of two non-eosinophils (mast cells labeled a and b in A) and two eosinophils (labeled c and d in A) illustrated the intensity and location of the perinuclear LTC4S immunoreactivity (green line in D) in these cells. Note the lack of reactivity in one eosinophil (unlabeled red cell in A). (E–G) These strong LTC4S-immunoreacting cells (green in E, arrows) were mast cells as they co-expressed c-kit (red in F and G, arrows). Note the LTC4S-negative eosinophils, which are visualized by their prominent granules in DIC microscopy (G, arrowheads). (H–J) Similar results were observed in GMA-embedded acetone-fixed specimens, where the dominating LTC4S-expressing cells (green in H and J, arrows) were not eosinophils as they were mAb EG2–negative (red in F and G, arrowheads). (K–P) Although the anti–LTC4S-peptide37–51 antiserum predominantly identified mast cells (green arrowed cell in K and M) and not eosinophils (mAb EG2+ red cells in L and M) in untreated aspirin-tolerant patients with asthma, it detected such immunoreactivity (green cells, arrowheads in N) in bronchial eosinophils (mAb EG2+ red cells, arrowheads in N, O, and P) in the aspirin-intolerant patients. Note the strong green LTC4S-immunoreactivity in non-eosinophils (one is arrowed in N and P) which represented LTC4S-expressing mast cells. Digital multi overlay images from three-color immunohistofluorescence labeling for LTC4S (anti–LTC4S-peptide31–51 antiserum, green); eosinophils (red mAb EG2+ cells in A, C, I, J, L, M, O, and P); or mast cells (c-kit, red in F and G), and nuclei (DAPI, blue), superimposed on diffraction interface contrasted (DIC) images in A, G, J, M, and P. Bronchial cryosection from treated (H–J) and untreated aspirin-tolerant patients with asthma (A–C; E–G; K–M) and one aspirin-intolerant patient (N–P).

Tissue processing and immunohistochemical controls. Immunohistochemistry on the GMA-embedded bronchial biopsies revealed similar staining pattern as in cryosections (Figure 1). All mast cells displayed strong perinuclear LTC₄S immunoreactivity, whereas the eosinophils were predominantly negative.

The low staining intensity for LTC₄S in eosinophils was apparently not due to the anti-LTC₄S peptide_37–51 antiserum's (or the other anti-peptide antisera's) inability to detect LTC₄S in eosinophils because similar staining of nasal polyps and bronchial mucosa from patients with aspirin-intolerant asthma revealed strong perinuclear LTC₄S staining in the majority of the eosinophils (Figure 1).

Thus, all LTC₄S-peptide antisera reacted with mast cells, but only the strongest ones (the anti–C-terminal and the anti-peptide_37–51 antisera) detected LTC₄S in a fraction of the eosinophils in aspirin-tolerant patients with asthma.

Identification of eosinophils. The eosinophils were mainly identified by DIC microscopy (19), but additional immunohistofluorescence identification of eosinophils (mAb EG2) was performed on sections from all patients and control subjects. The mAb EG2 was previously considered to identify activated eosinophils, but detailed immunohistofluorescence analysis revealed that mAb EG2 binds to all eosinophils if sections are appropriately fixed (20). We used biopsies that had been fixed for 4 h in 1% paraformaldehyde containing PLP. Optimal immunoreactivity for mAb EG2 was achieved if the cryosections were additional prefixed with 1% paraformaldehyde containing PLP for 10 h at 4°C. When not appropriately fixed, mAb EG2 reacted with fewer cells that also included non-eosinophils (no granularity in DIC microscopy) close to mAb EG2-negative, but DIC-positive cells. The latter phenomenon suggested that mAb EG2–reactive material had leaked out of the eosinophils and bound to adjacent cell membranes during immunohistofluorescence staining, as also shown previously (20).

The LTC₄S immunoreactivity was localized to the nuclear membrane in both eosinophils and mast cells. Cytoplasmic staining was observed in some mast cells as a localized dot resembling immunoreactivity in the Golgi apparatus.

	Results

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Increased Bronchial Density of Lamina Propria LTC₄S-Positive Cells in Untreated Asthma
LTC₄S was strongly expressed in scattered submucosal, and occasional intraepithelial, mononuclear cells. Nuclear staining revealed that the LTC₄S was predominantly located in the nucleus and perinuclear membrane, with occasional Golgi-associated dotting in the cytoplasma (Figure 1). The submucosal density of LTC₄S-containing cells in the untreated patients with asthma (median 68 cells/mm²; range 35–114 cells/mm², n = 12), was significantly (P < 0.05) higher than in treated asthma (median 40 cells/mm², range 6–121 cells/mm², n = 16), but not significantly different from that in control subjects (median 47 cells/mm², range 40–64 cells/mm², n = 9; Figure 2) . The density of LTC₄S-containing cells was also high in the two patients with bronchial antigen–provoked asthma (median 70 cells/mm², range 55–85 cells/mm²).

View larger version (20K): [in this window] [in a new window]   Figure 2. The mucosal density of LTC4S-positive cells was significantly higher in untreated than in treated asthma, but not different from the control subjects. The bars indicate median values. There was no association between type of medication and bronchial LTC4S-positive cell density. The controls were all non-atopics without medications. Atopics are squares and non-atopics are triangles. The medication is indicated as: long acting ß2-agonist only (symbols with the bottom half filled); inhaled corticosteroids only (symbols with the right half filled); inhaled corticosteroids and long acting ß2-agonistic inhalation (filled symbols), and untreated patients (open symbols). Provocated: bronchial antigen–provoked patients with asthma.

The density of LTC₄S-positive mast cells (mAb AA1–positive) in treated patients with asthma (median 37 cells/mm², range 6–98 cells/mm², n = 16) was slightly lower than in untreated patients with asthma (median 44 cells/mm², range 33–96 cells/mm², n = 12) and the control subjects (median 45 cells/mm², range 32–62 cells/mm², n = 9), but the difference did not reach statistical significance (P > 0.05).

The percentage of LTC₄S-positive tryptase-negative non-eosinophils (EG2-negative, nongranular in DIC microscopy) was increased (> 5%) in some of the sections in most of the untreated (n = 10), half of the treated (n = 8), and one of the challenged patients with asthma, as well as in two of the control subjects. Multicolor immunohistofluoresence staining showed that practically all such cells expressed c-kit and FcR-I (Figure 1). Thus, the tryptase-negative LTC₄S-positive cell were c-kit–positive, FcR-I–positive mast cells, with only occasional (0–12 cells per section) unidentified LTC₄S-positive cells. These cells were presumably macrophages because separate tracing revealed scattered c-kit–negative, HLA-DQ–positive, CD1c-negative cells with weak perinuclear LTC₄S positivity in both controls (n = 4, median 0, range 0–1 cell/section) and in untreated patients with aspirin-tolerant asthma (n = 12; median 3, range 0–12 cells/section). These cells also co-expressed CD68 on adjacent sections (not shown). No LTC₄S was observed in CD3-positive T cells.

Density of LTC₄S-Positive Mast Cells Correlated with Lung Function and Reversibility
A linear regression analysis between the submucosal density of LTC₄S-positive mast cells and the percentage of FEV₁ expected showed a significantly inverted correlation in patients with asthma treated for more than 1 yr, both before (Kendalls = -0.562, P = 0.004) and after Salbutamol inhalation (Kendalls = -0.543, P = 0.005; Figure 3) .

View larger version (11K): [in this window] [in a new window]   Figure 3. The density of bronchial LTC4S-positive mast cells correlated significantly with the reduction in percentage FEV1 predicted in patients with treated asthma, both before (A, Kendalls = -0.562; P < 0.001) and after Salbutamol inhalation (B, Kendalls = -0.543; P = 0.005). Atopic patients are indicated with squares and non-atopics with triangles. Filled symbols are patients with more than 12% reversibility.

Density of Eosinophils and their LTC₄S Positivity
The previous identification of eosinophils as the dominating LTC₄S-positive cells in both aspirin-intolerant and aspirin-tolerant asthma (7) made it important to ensure correct identification of the eosinophils. Immunohistofluorescence with mAb EG2 and/or the characteristic granularity observed in DIC microscopy (17) were used to identify the eosinophils (Figure 1). Although the submucosal density of eosinophils was significantly higher in untreated (median 13, range 2–248 eos/mm²) than in treated asthma (median 2, range 0–91 eos/mm², P < 0.01) and control subjects (median 4, range 0–97 eos/mm², P < 0.05), the increase was mainly observed in atopic patients (median 54, range 9–248 eos/mm², n = 6). Nonatopic patients had significantly lower density (median 6, range 2–19 eos/mm², P < 0.05). One of the bronchial antigen–provoked patients had particularly high density of eosinophils (126 eos/mm²), whereas the other had 21 eos/mm².

There were too few eosinophils in the bronchial mucosa from treated patients with asthma to perform a reliable estimation of eosinophilic LTC₄S positivity in each patient. Grouped data from all sections examined in the 16 treated patients with asthma identified 205 eosinophils, of which 18% expressed LTC₄S. All immunoreactivity was located to the nuclear membrane (Figure 1). Grouped data from the untreated nonatopic patients revealed similar percentage (18% of 74 eosinophils), whereas 30% of 553 eosinophils expressed LTC₄S in untreated atopic patients. However, the percentage of LTC₄S-positive eosinophils in all patients with asthma as a group was not significantly different from the control subjects, in which 22% of 73 eosinophils expressed LTC₄S (grouped data). Bronchial antigen provocation did apparently not induce de novo LTC₄S expression in eosinophilis, because biopsies obtained 24 h after such provocation in two atopic patients showed that only 11% of 257 eosinophils (11 sections from 9 biopsies), contained LTC₄S.

The bronchial density of LTC₄S-positive eosinophils was considerably lower than the density of LTC₄S-positive mast cells in almost all patients and control subjects. Only one of the untreated atopic patients had a higher density of LTC₄S-positive eosinophils than LTC₄S-positive mast cells (Figure 4) .

View larger version (23K): [in this window] [in a new window]   Figure 4. The bronchial densities of LTC4S-positive mast cells (filled symbols) were significantly higher than the density of LTC4S-positive eosinophils (open symbols) in all patient groups. The mast cells were the dominating LTC4S-expressing cell in all patients except in one untreated atopic patient. Atopic patients are represented with squares and non-atopic patients with triangles. The LTC4S-positive mast cell density and the density of LTC4S-positive eosinophils within each patient is linked by lines. Medians are indicated by horizontal bars.

View larger version (21K): [in this window] [in a new window]   Figure 5. The mast cells (MC) contained significantly more LTC4S-positive immunoreactivity than the eosinophils (Eos). Computer-assisted digital image analyzes on average perinuclear/nuclear LTC4S-positive immunoreactivity in mast cells and eosinophils.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increased density of LTC₄S containing cells observed in the untreated aspirin-tolerant asthma was similar to the findings in aspirin-intolerant asthma (7). However, in contrast to previous reports (7, 15) the dominating LTC₄S-expressing cells were mast cells and not eosinophils. Whereas LTC₄S has been observed in only 1–5% of the bronchial mast cells in seasonal allergic asthma (15) and aspirin-tolerant asthma (7), respectively, the present study observed strong perinuclear LTC₄S immunoreactivity in 95% of the mast cells in all patient groups and control subjects, compared with weak immunoreactivity in 20% of the eosinophils. This may explain why the density of LTC₄S-expressing cells in the present study was considerably higher ( 50–60 cells/mm²) than previously reported (3–5 cell/mm²; 7, 15).

The different tissue-processing techniques could apparently not explain these discrepancies because multicolor immunohistofluorescence staining for mast cells, eosinophils, and LTC₄S in similar GMA-embedded acetone-fixed specimens as used previously (7, 15) confirmed that all mast cells expressed high levels of LTC₄S. It is therefore more likely that differences in antibodies and/or tissue examination techniques have influenced the results. Cowburn and colleagues (7) and Seymour and associates (15) used an affinity-purified antiserum raised against isolated LTC₄S, single-color immunohistochemical staining on neighboring semithin sections, and a camera lucida to co-localize the LTC₄S to different cell types. The present study used an antipeptide antiserum (18) and multicolor immunohistofluorescence staining allowing simultaneous examination of LTC₄S immunoreactivity on DAPI-identified nuclei in cells that simultaneously were identified as mast cells or eosinophils.

Although the LTC₄S-peptide_37–51 antiserum used in this study was generated to an amino acid sequence with high amino acid homology to mGST-II (16, 22), it crossreacted only weakly with this molecule in immunohistochemistry (see METHODOLOGIC CONSIDERATIONS). Adding recombinant mGST-II to the anti-LTC₄S peptide_37–51 antiserum did not change its reactivity toward mast cells or eosinophils. Moreover, parallel experiments with the three different anti-LTC₄S peptide antisera revealed that they all detected LTC₄S in its typically perinuclear location (23) in mast cells.

Although mast cells were the dominating LTC₄S-containing cell, their density did not correlate with the bronchial LTC₄S mRNA level. The relationship between protein content and mRNA level may be more complicated as cellular LTC₄S protein appears to accumulate over several days as shown for interleukin (IL)-4–stimulated umbilical cord–derived mast cells (24). The bronchial biopsies from patients with highest bronchial density of LTC₄S-positive eosinophils contained, nevertheless, not significantly more LTC₄S-positive mRNA than those with few eosinophils, supporting the concept that eosinophils were not the dominating LTC₄S-expressing cells.

Clinically, response to asthma treatment is often associated with a distinct reduction in mucosal eosinophils (11), implying that activated eosinophils participate in generating the asthmatic symptoms. However, relatively few bronchial eosinophils contained any LTC₄S immunoreactivity even in untreated and antigen-provoked aspirin-tolerant patients with asthma. This was apparently not due to the inability of the antipeptide antisera to detect LTC₄S in eosinophils, because the majority of eosinophils in bronchial and nasal specimens from the aspirin-intolerant patients with asthma included for methodologic evaluations, contained strong LTC₄S immunoreactivity. The inhibition studies showed, moreover, that recombinant LTC₄S, but not mGST-II, completely removed or significantly reduced the mast cell and eosinophilic immunoreactivity for the C-terminal and the LTC₄S-peptide_37–51 antiserum, respectively. Thus, all antipeptide antisera detected LTC₄S predominantly in mast cells and only weakly in a small fraction of the eosinophils. All the patients were, however, stable at the time of bronchoscopy, and the situation might have been different during an acute asthmatic attack.

The density of LTC₄S-positive mast cells was reduced from high levels in untreated asthma to lower than normal levels in treated patients with asthma with good lung function and no reversibility. This suggested that a reduction in mucosal LTC₄S-positive mast cells may be associated with clinical response to treatment. This may also explain why the treated patients with asthma with poor lung function (FEV₁ expected) and > 12% reversibility contained significantly higher densities of LTC₄S-positive mast cells than those with good lung functions.

Activated mast cells secrete several pro-inflammatory cytokines (e.g., IL-4, IL-13, tumor necrosis factor-) and proteolytic enzymes like tryptase, metalloproteinases, and the fibroblast activating transforming growth factor-ß (for review see Ref. 25). Thus, chronic mast cell activation may produce several factors involved in airway remodeling (24). This may explain why the treated patients with asthma with high density of LTC₄S-positive mast cells were still obstructive after salbutamol inhalation. The latter suggested that activated mucosal mast cells participate in airway remodeling, but not necessarily through their LTC₄ production, because approximately all mast cells expressed LTC₄S.

Whether the production capacity for LTC₄ is proportional to the cellular content of LTC₄S is unknown, but it would suggest that bronchial LTC₄ primarily derive from activated mast cells. IgE-mediated mast cell activation induces both LTC₄ and PGD₂ production (27). Induced sputum obtained after bronchial antigen provocation in atopic patients contained, however, increased amount of LTC₄ only, and not PGD₂ (5). The level of LTC₄ was related to the number of eosinophils in the sputum (5), which suggested that the LTC₄ predominantly derived from bronchial eosinophils and not mast cells. However, the concentration of LTC₄ and PGD₂ in induced sputum may not reflect the production within the mucosa. It rather reflects the concentration in the fluid lining the bronchoalveolar epithelium, which may be dependent on activated (28) LTC₄S-positive eosinophils that have transversed the epithelium during the antigen-induced asthmatic reaction. Examination of LTC₄ and prostaglandin metabolites in the urine of patients with asthma before, during, and after bronchial allergen–provoked asthma have identified increased production of the mast cell–specific products 9,11ß-PGF₂, and the LTC₄ degradation product, LTE₄, shortly after an asthmatic attack and during the late asthmatic reaction (27–30). This was observed without any increased concentration of the eosinophil-specific secretory product EPX (for reference see Ref. 30), suggesting that these substances were produced by mast cells only. Thus, mast cells, rather than eosinophils, may be the major LTC₄-producing cell in the bronchial mucosa. Other cells like the vascular endothelium may, in addition, produce LTC₄ via mGST-II (31).

To what extent the infiltrating eosinophils influence the asthmatic reaction is still undetermined, and its importance has been severely questioned by the negative effect of an anti–IL-5 study (32). Eosinophilic infiltration and activation depend on local production of IL-5. Treating patients with asthma with antibodies to IL-5 reduced bronchial eosinophils significantly, but did not influence asthmatic symptoms.

In conclusion, mucosal mast cells were the dominating LTC₄S-expressing cell in untreated, antigen-challenged, and treated aspirin-tolerant patients with asthma as well as in control subjects. This may explain why steroid treatment does not reduce antigen-induced leukotriene production (33, 34) and why ICS-treated patients with asthma, presumably with few bronchial eosinophils, may improve on additional leukotriene receptor antagonist therapy (35).

	Acknowledgments

 
The authors thank Drs. Frank K. Austen, Bing K. Lam (Department of Medicine, Harvard Medical School, Boston, MA), Donald Nicholson, and John Vaillancourt (Merck Research Laboratories, Rahway, NJ) for the generous gift of antisera to LTC₄S, and Joseph A. Mancini (Merck Frosst Centre for Therapeutic Research, Kirkland, PQ, Canada) for the recombinant mGST-II– and LTC₄S-expressing baculovira and for recombinant mGST-II. Solveig Stig is acknowledged for excellent technical assistance.

Received in original form August 29, 2002

Received in final form June 16, 2003

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