In situ localization and regulation of thromboxane A2 synthase in normal and LPS-primed lungs

L. Ermert1, M. Ermert2, H.-R. Duncker2, F. Grimminger3, and W. Seeger3

1 Department of Pathology, 2 Institute of Anatomy and Cell Biology, and 3 Department of Internal Medicine, Justus-Liebig-University Giessen, 35385 Giessen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thromboxane (Tx) A2 synthase catalyzes the conversion of prostaglandin H2 to the unstable metabolite TxA2, which is a potent mediator of vasoconstriction and bronchoconstriction. The cellular localization of TxA2 synthase was examined by immunohistochemistry and in situ hybridization in human and rat lung tissues. Bronchial epithelial cells, bronchial smooth muscle cells, peribronchial nerve fibers, single cells of bronchus-associated lymphoid tissue, single cells located in the alveolar septum, and alveolar macrophages exhibited positive immunostaining for TxA2 synthase protein in lung tissue of both species. In addition, vascular smooth muscle cells of muscular and partially muscular vessels displayed strong (rat) and moderate (human) immunostaining for TxA2 synthase. In situ hybridization performed in the rat lungs demonstrated TxA2 synthase mRNA localization in accordance with the immunostaining pattern. Perfusing isolated rat lungs with endotoxin for 1 and 2 h resulted in a marked increase in TxA2 synthase protein staining intensity in most cell types as measured by quantitative image analysis, whereas the in situ hybridization signal was unchanged. We conclude that the pulmonary distribution of TxA2 synthase displays close similarity between rat and human lung tissues and matches well with the previously described immunolocalization of cyclooxygenase-1 and cyclooxygenase-2 in this tissue. Endotoxin challenge is suggested to cause a rapid upregulation of TxA2 synthase at the posttranscriptional level. These data provide a morphological basis for the understanding of the role of TxA2 in the regulation of lung bronchial and vascular tone and in immunologic events.

lipopolysaccharide; immunohistochemistry; in situ hybridization; arachidonic acid metabolism; prostanoids; cyclooxygenase; endotoxin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EICOSANOIDS ARE ENZYMATIC METABOLITES of arachidonic acid with important biological functions modulating coagulation (11, 27), vasoreactivity, and bronchomotor activity (28, 34). In addition, they have been implicated in immunologic defense mechanisms (13, 20, 39). Thromboxane (Tx) A2 synthase catalyzes the conversion of the unstable intermediate prostaglandin H2 to TxA2, which is a potent constrictor of vascular and bronchial smooth muscle (13, 27). TxA2 has been associated with the pathogenesis of lung diseases such as pulmonary hypertension, asthma, and acute respiratory distress syndrome (27, 29). In experimental endotoxemia, e.g., the early pulmonary hypertension is largely mediated via TxA2 synthesis in many animal models (28, 35). Similarly, pulmonary vasoconstriction and ventilation-perfusion mismatch in response to bacterial exotoxins were shown to be attributable to Tx synthesis (8, 34, 36, 38).

To date, the cellular origin of Tx generation in the lung parenchyma and bronchial tissue is largely unknown. Much effort has been made to identify and exclude circulating cells as possible sources, but neither in thrombopenic sheep (30) nor in neutropenic rats (2) was endotoxin-induced TxA2 formation found to be reduced. Originally isolated from platelets (23), TxA2 synthase was detected in several different organs (21). In particular, lung, kidney, and spleen have been found to express the mRNA of TxA2 synthase (20, 39). Western blot analysis and immunoassays of human organ homogenates displayed the highest enzyme abundance in the lung, liver, and colon (25). Up to now, however, the cellular localization within the lung is largely unknown. Only ubiquitously occurring cell types like polymorphonuclear leukocytes, monocytes, and alveolar macrophages distributed within the lung tissue are unambiguously known to express TxA2 synthase (21, 24-26).

Cyclooxygenase (COX), the key enzyme of the prostanoid pathway, exists in two isoforms. The inducible isoform, COX-2, was initially assumed to be expressed only under inflammatory conditions, whereas COX-1 was known to occur constitutively (10, 18). Ermert et al. (7) recently investigated the cellular localization of COX-1 and COX-2 in normal rat lung tissue by immunohistochemistry, noting a distinct pattern for each isoenzyme form: whereas COX-1 was predominantly found in bronchial epithelial cells and alveolar macrophages, COX-2 was localized mainly to vascular smooth muscle cells of partially muscular vessels and to macrophage-like cells in the perivascular and peribronchial connective tissue.

The biological significance of this finding is, however, largely dependent on the distribution of the downstream enzymes TxA2 synthase and prostaglandin I2 synthase because the arising metabolites possess largely opposite effects. We investigated the cellular distribution of TxA2 synthase in rat and human lung tissues to broaden the morphological basis for understanding the pulmonary effects of Tx generation. Good correspondence of the immunostaining pattern between rat and human tissue was noted, and the distribution of TxA2 synthase closely matched the previously described distribution of both COX-1 and COX-2.

During inflammation, e.g., in response to endotoxin [lipopolysaccharide (LPS)], marked changes in Tx synthesis may occur, which are commonly attributed to a regulation of COX-2 synthesis (17, 22, 26). This does not, however, exclude additional regulatory events on the TxA2 synthase level. Previous studies (15, 37) in freshly isolated and cultured cells suggested a posttranscriptional mechanism for TxA2 synthase regulation rather than regulation on the transcriptional level. In addition to addressing the pulmonary distribution of TxA2 synthase under baseline conditions, we investigated changes in the cellular expression of this enzyme in perfused rat lungs undergoing LPS provocation as a model of acute pulmonary inflammation.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Animals and Reagents

The CD rats (Sprague-Dawley) were obtained from Charles River (Sulzfeld, Germany). Animal housing and all experimental procedures were performed in conformity with local and American guidelines for the care and use of laboratory animals. A polyclonal rabbit anti-TxA2 synthase antibody and secondary antibodies were obtained from Biotrend (Cologne, Germany). The anti-TxA2 synthase antibody is directed against the purified enzyme from porcine lung. A second polyclonal anti-TxA2 synthase antibody was obtained from Cayman (Ann Arbor, MI). FITC-labeled triple oligonucleotides against rat TxA2 synthase; triple random control oligonucleotides; oligonucleotides against alpha -tubulin, beta -actin, and poly(T); and in situ hybridization (ISH) buffer were provided by Biognostik (Göttingen, Germany). Tyramide signal amplification and horseradish peroxidase-labeled anti-FITC were obtained from NEN Life Science (Boston, MA). The Vector red and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate kits were acquired from Vector Laboratories (Burlingame, CA). All other biochemicals were obtained from Merck (Darmstadt, Germany) and Sigma (Deisenhofen, Germany).

Lung Isolation and Perfusion

The rats (male, body weight 350-400 g) were deeply anesthetized with pentobarbital sodium (100 mg/kg body weight ip). After local anesthesia with 2% xylocaine and a median incision, the trachea was dissected, and a tracheal cannula was immediately inserted. Subsequently, mechanical ventilation was started with 4% CO2, 17% O2 and 79% N2 (tidal volume 4 ml, frequency 65 breaths/min, and end-expiratory pressure 3 cmH2O) with a small-animal respirator (KTR-4, Hugo Sachs Elektronic). A median laparatomy was performed, and, subsequently, the rats were anticoagulated with 1,000 U of heparin. After a midsternal thoracotomy, the right ventricle was incised, a cannula was fixed in the pulmonary artery, and the apex of the heart was cut off to allow pulmonary venous outflow. Simultaneously, pulsatile perfusion with buffer solution was started. The buffer contained 2.4 mM CaCl2, 1.3 mM MgCl2, 4.3 mM KCl, 1.1 mM KH2PO4, 125.0 mM NaCl, and 25 mM NaHCO3 as well as 13.32 mM glucose (pH ranged between 7.35 and 7.40).

The lungs were carefully excised, avoiding any damage, while being perfused with buffer solution and suspended in an upright position. Next, a cannula was inserted through the left ventricle and fixed in the left atrium to obtain a closed perfusion circuit without leakage. Subsequently, the lungs, freely suspended from a force transducer, were placed in a temperature-equilibrated housing chamber (37°C).

After extensive rinsing of the vascular bed, the lungs were recirculatively perfused with a pulsatile flow of 13 ml/min. The alternate use of two separate perfusion circuits, each containing 100 ml, allowed the repetitive exchange of perfusion fluid. Perfusion pressure, ventilation pressure, and weight of the isolated organ were registered continuously. The left atrial pressure was set at 2 mmHg under baseline conditions (0 referenced at the hilum) to guarantee zone III conditions at end expiration throughout the lung.

The lungs selected for the study were those that 1) had a homogeneous white appearance without signs of hemostasis or edema formation, 2) had pulmonary arterial and ventilation pressures in the normal range, and 3) were isogravimetric during a steady-state period of 30 min.

Experimental Protocol

Five rat lungs were perfused for ~5 min for washout of blood. In control experiments, the rat lungs (n = 5) were perfused for 2 h solely with buffer fluid. In additional experiments, LPS was admixed in concentrations of 1,000 or 10,000 ng/ml to the recirculating buffer fluid. After administration of 1,000 or 10,000 ng/ml of LPS, both 1- and 2-h perfusion periods were employed (n = 5 lungs/group).

After termination of perfusion, the rat lungs were instilled with TissueTek OCT compound (Sekura Firetek, Zoeterwoude, The Netherlands) and frozen in liquid N2. The rat lungs were dissected into tissue blocks from all lobes and stored at -80°C. Sections 10 µm in thickness were cut from frozen tissue blocks of both left and right lungs.

Human lung specimens were obtained from 15 patients who underwent lung surgery for lung cancer. In addition, three specimens were taken from excised lung tissue in which the tumor was diagnosed as being benign (chondroid hematoma) or in which there was metastasis of nonlung origin (renal cell carcinoma and rectal adenocarcinoma). Normal lung tissue remote from the lung cancer tissue was freshly obtained and embedded in TissueTek OCT compound. Cryostat sections of 10 µm were made from three to four tissue blocks per case.

Immunohistochemistry

The sections were fixed for 5 min with a 3% paraformaldehyde solution and washed in 0.01 M PBS containing 150 mM NaCl (pH 7.6) three times for 5 min each. They were treated for 15 min with a 1% Triton solution. The sections were preincubated in PBS containing 5% goat serum, 1% BSA, and 0.05% Tween 20 to block nonspecific binding. Overnight incubation with the polyclonal rabbit anti-TxA2 synthase primary antibody (Biotrend) diluted 1:100 in PBS containing 1% BSA and 0.05% Tween 20 was carried out at 4°C. The second polyclonal antibody obtained from Cayman was used accordingly in a dilution of 1:50. The sections were then washed in PBS and incubated with a goat anti-rabbit F(ab')2-alkaline phosphatase (AP) conjugate (Biotrend) diluted 1: 2,000 in the same dilution buffer overnight at 4°C. Afterward, three washes of the sections for 5 min each in PBS were performed. Subsequently, the sections were developed with a Vector red substrate kit (Vector). Levamisol (2.5 mM) was added to inhibit endogenous AP activity. Counterstaining of the sections was performed with methyl green. Control staining was performed by omission of the primary antibody and substitution with nonspecific serum at the same dilution.

In Situ Hybridization

The sections were fixed for 5 min with a 3% paraformaldehyde solution. They were incubated in a methanol-glacial acetic acid solution for 10 min at -20°C before they were treated with 0.2% glycine in 0.01 M PBS for 5 min and with a 1% Triton solution in 0.1× saline-sodium citrate for 15 min. The sections were preincubated in ISH buffer for 30 min to block nonspecific binding. Overnight incubation with ISH-buffer containing triple FITC-labeled oligonucleotides (TxA2 synthase and random control) at a concentration of 50 pmol/ml each and positive control oligonucleotides [alpha -tubulin, beta -actin, and poly(T)] at 50 pmol/ml was carried out at 30°C. A stringent wash was carried out two times for 5 min each at 40°C in 0.1× saline-sodium citrate buffer. Afterward, the sections were blocked with 1.5% H2O2 for 30 min and incubated with an anti-FITC-horseradish peroxidase conjugate at a concentration of 1:1,000 overnight at 4°C. Signal amplification was performed with tyramide signal amplification-FITC (1:50) for 30 min at 20°C. The sections were incubated overnight at 4°C with an anti-FITC-F(Ab')2-AP-conjugated antibody diluted 1:2,000. The sections were then washed once in PBS for 5 min and in Tris buffer (pH 9.5) two times for 5 min each. Subsequently, the sections were developed with a BCIP/NBT substrate kit for 35 min. Counterstaining was performed with nuclear fast red. Control staining was performed by omission of the oligonucleotides and incubation of triple random control oligonucleotides.

Image Analysis

An image-analysis system consisting of a 12-bit cooled charge-coupled device camera (Sensys KAF 1400, Photometrics, Tucson, AZ) mounted on a fully automated Leica (Wetzlar, Germany) DM RXA microscope was used to digitize gray-scale images to a dual-pentium 200-MHz host computer. Microscope settings were kept constant throughout all measurements (×40 oil objective, Leica PL Fluotar 40x/0.75). A stabilized 12-V tungsten-halogen lamp (100 W) was used for illumination. Microdensitometry was performed with a custom-designed filter manufactured by Omega Optical (Brattleboro, VT) for absorbance measurement of the Vector red substrate (central wavelength 525 nm, half bandwidth 10 ± 2 nm). The optimal central wavelength was determined by measurement of the substrate in a Leica MPV SP microscope photometer system (courtesy of Leica). See Chieco et al. (3) for a review of the absorbance measurement technique.

Adjustment of all microscope settings was stored and recalled before measurement. Calibration of the measurement system with a reference slide was done before measurement. Gray-scale images were digitized to 12-bit accuracy, resulting in an intensity scale ranging from 0 to 4,095. Image analysis was performed by means of the image-analysis program ImagePro 3.0 (Mediacybernetics). For direct visualization of staining intensity, a pseudocolor scale with 11 colors, each representing an equal sector of the intensity scale, was chosen and applied to the images. Background measurement was performed to evaluate the influence of nonspecific staining and/or absorption of unstained tissue.

From each section, five images per stained structure were digitized, and the area of interest was manually defined. The mean gray values were automatically measured and subsequently transferred into the spreadsheet program EXCEL (Microsoft, Redmond, WA).

Anatomic segments of the vascular tree were defined according to the classification described by Hislop and Reid (12). In brief, elastic arteries and muscular and partially muscular vessels were distinguished by the structure of the vessel wall. Large arteries at the lung hilum were defined by the thickness of their muscle layer and the occurrence of elastic fibers, whereas hilum veins were identified by cardiac muscle cells, which accompany the pulmonary veins of rats into the lung parenchyma (12). A vessel was termed muscular or partially muscular when smooth muscle cells were observed in the subendothelial layer forming a muscular layer that surrounded the vessel either fully or partly, respectively. The vessels located in the preacinar regions were predominantly of a fully muscular type (associated with bronchioli and terminal bronchioli), whereas within the acinar region (associated with respiratory bronchioli and alveolar ducts), mainly partially muscular and nonmuscular vessels were found in accordance with previous data (12, 16, 31).

Statistical Analysis

Analysis of variance was used to evaluate differences among the groups. A value of P < 0.05 was considered significant. Data are given as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rat Lung Tissue

Immunostaining. Both polyclonal antibodies used showed identical immunolocalization in rat as well as in human lung tissue. Nonspecific staining and background staining were low for lung tissue of both species. Staining was absent from sections where the primary antibody was omitted or nonspecific serum was applied (Fig. 1D).


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Fig. 1.   Immunohistochemical localization of thromboxane (Tx) A2 synthase in normal rat (A-C) and human (E and F) lung tissue. Control staining with nonspecific immune serum showed no staining reaction (rat lung; D). BALT, bronchus-associated lymphoid tissue. Within rat lung tissue, vascular smooth muscle cells (VSMC) of small arteries (A) and partially muscular vessels (B) displayed strong immunostaining. Most prominent immunostaining was noted in single cells within alveolar septum (A and B, arrows). Bronchial epithelial cells (BE) were heavily stained, whereas bronchial smooth muscle cells (BSMC) showed moderate staining for TxA2 synthase (C). In human lung tissue, BE and alveolar macrophages (*) within alveolar spaces exhibited strong TxA2 synthase-positive immunostaining (E and F, respectively).

In normal rat lung tissue, smooth muscle cells of the pulmonary vessels exhibited strong staining, which could be detected in all muscular and partially muscular vessels (Fig. 1, A and B). Extrapulmonary elastic arteries were not stained, in contrast to the pulmonary veins at the lung hilum that exhibited a strong staining reaction (Table 1). In the airways of normal rat lung tissue, a marked staining was observed in the bronchial epithelial cells (Fig. 1C) and in the pseudostratified ciliated columnar epithelium of the trachea. Bronchial smooth muscle cells were also found to be stained but mainly in the first- and second-generation bronchi where the muscle layer is prominent (Fig. 1C). In addition, peribronchial and perivascular nerve fibers and cells within the bronchus-associated lymphoid tissue exhibited positive staining for TxA2 synthase (Table 1). In addition, epithelial cells of the tracheal glands were determined to be positive for TxA2 synthase. Within the lung parenchyma, single cells located in the alveolar septum and especially around small vessels showed extensive staining (Fig. 1, A and B, respectively). Alveolar macrophages were found to be stained throughout all lung sections (Table 1).

                              
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Table 1.   Image analysis of TxA2 synthase immunostaining intensity in normal and LPS-primed rat lung tissues

Two-hour buffer-perfused lungs were not different in TxA2 synthase expression from that in freshly excised blood-free rinsed lungs.

In response to LPS, a significant increase in staining intensity was detected within 1 h. The increase in staining intensity was visualized by pseudocolor depiction, e.g., in bronchial epithelial cells of large bronchi and bronchial smooth muscle cells (Fig. 2, A and B), vascular smooth muscle cells of small muscular vessels (Fig. 2, C and D), and alveolar macrophages and single cells within the alveolar septum (Fig. 2, C-F). The increase in staining intensity occurred within 1 h and did not significantly change up to 2 h of LPS incubation (Table 1, Fig. 3). In normal rat lung tissue, there was no marked difference in immunostaining of muscular and partially muscular vessels; however, the increase in staining intensity in response to LPS was slightly more marked in the muscular vessels (up to ~150% of baseline) compared with that in the partially muscular vessels (up to ~130%).


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Fig. 2.   Image analysis with pseudocolor depiction of immunohistochemical TxA2 synthase staining in control (A, C, and E) and lipopolysaccharide (LPS)-primed (B, D, and F) rat lung tissue. TxA2 synthase staining intensity was increased in BE and BSMC in response to LPS (10,000 ng/ml, 1 h; B). Increased staining intensity was also noted in VSMC of small muscular vessels (C, arrows) and in single cells within alveolar septum (10,000 ng/ml of LPS, 2 h; D, arrows). In addition to single cells within septum (E and F, arrows), alveolar macrophages (*) displayed a stronger staining reaction after LPS application (1,000 ng/ml of LPS, 2 h; F).



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Fig. 3.   Quantitative evaluation of immunohistochemical TxA2 synthase staining in response to LPS. A: 1st- and 2nd-generation BE. B: BSMC. C: alveolar macrophages. D: VSMC of partially muscular vessels. Intensity of TxA2 synthase staining was measured in rat lungs after 1-h (striped bars) and 2-h (solid bars) perfusion time after LPS application. Staining intensity is presented as percent of gray-scale values measured in control rat lungs. Values are means ± SE from 5 independent experiments. Significant difference compared with respective control group: * P < 0.05; ** P < 0.01; *** P < 0.001.

Staining intensity was not found to be increased after LPS application in the epithelial cells of small bronchioli and nerve fibers. In addition, immunostaining was always negative in the endothelial cells of all types of vessels (Table 1). For background staining, a mean gray value of 734 ± 17.3 was determined.

In situ hybridization. A strong TxA2 synthase mRNA signal was detected in the vascular smooth muscle cells of small muscular and partially muscular vessels in rat lung tissue (Fig. 4, A and B, respectively). The vascular smooth muscle cells of extrapulmonary arteries did not exhibit positive staining for TxA2 synthase mRNA in accordance with negative TxA2 synthase immunostaining. In addition, alveolar macrophages (Fig. 4D) and single cells within the alveolar septum (Fig. 4B) exhibited a strong mRNA signal for TxA2 synthase. Bronchial epithelial cells displayed a moderate TxA2 synthase mRNA signal and bronchial smooth muscle cells displayed a weak TxA2 synthase mRNA signal (Fig. 4C). In response to LPS, TxA2 synthase mRNA staining did not change within the 2-h period for all cell types investigated.


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Fig. 4.   In situ hybridization of TxA2 synthase in rat lung tissue. A strong TxA2 synthase mRNA signal was detected in VSMC of small muscular [A; note elastica interna with unstained endothelial cells (arrowhead)] and partially muscular (B) vessels. BE and BSMC also displayed positive mRNA signal (C). Alveolar macrophages (*) exhibited a marked TxA2 synthase mRNA signal (D). Poly(T) mRNA detection as an example of a positive control (E) showed staining in all cell types. Negative controls treated with a triple random control oligonucleotide (F) showed no staining reaction of lung tissue.

Control sections incubated with random control oligonucleotides did not show nonspecific staining (Fig. 4F). Positive control sections incubated with oligonucleotides for detection of alpha -tubulin, beta -actin and poly(T) mRNAs exhibited strong mRNA signals (Fig. 4E).

Human Lung Tissue

Immunostaining. TxA2 synthase was uniformly expressed in all human lung specimens. Bronchial epithelial cells were the most prominently stained structures in human lung tissue (Fig. 1E). Due to diagnostic procedures, a complete sampling of the bronchial tree could not be achieved. Therefore, a possible difference between bronchi of different sizes, as shown in the rat lung, could not be investigated. Strong staining was noted in alveolar macrophages throughout the specimens (Fig. 1F). Within the alveolar septum, single cells displayed positive staining for TxA2 synthase. The vascular smooth muscle cells of the small muscular and partially muscular vessels were weakly stained. Larger vessels such as pulmonary arteries accompanying the bronchi were not stained.

Bronchial smooth muscle cells exhibited weak but detectable staining. Nerve fibers and especially cells of nerve ganglia were positive for TxA2 synthase according to rat lung tissue.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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In previous investigations addressing cellular sources of TxA2 in lung tissue (24, 25), immunohistochemistry employing a monoclonal antibody detected TxA2 synthase in alveolar and interstitial macrophages. A far more broad distribution was, however, expected from the fact that functional studies suggested a role for Tx in vasoconstrictor events (6, 28, 35) and that many more cell types displayed COX-1 and/or COX-2 immunostaining in a preceding study in rat lungs (7). The isoenzyme COX-1, which was anticipated to be constitutively expressed, was prominent in bronchial epithelial cells, alveolar macrophages, muscle cells resembling cardiac muscle surrounding the pulmonary vein at the hilum, endothelial cells of large arteries, and bronchial smooth muscle cells. The isoform COX-2 was similarly found to be constitutively expressed in the lung tissue and was localized predominantly to vascular smooth muscle cells, macrophage-like cells in the perivascular and peribronchial connective tissues, and bronchial epithelial cells (7). In addition, COX-2 immunostaining was identified in bronchial smooth muscle cells, single cells scattered in the alveolar septum, alveolar macrophages, and cells of the bronchus-associated lymphoid tissue. Virtually all vascular smooth muscle cells of a distinct part of the vascular tree, the partially muscular vessels, displayed an intense staining for COX-2 (7).

The immunohistochemical localization of TxA2 synthase in the present study showed close similarity of the cellular distribution of TxA2 synthase in rat and human lung tissues. In rat lungs, TxA2 synthase was demonstrated to be present in epithelial cells lining the trachea as well as in cells lining the entire bronchial airway. In situ hybridization showed a lower expression of mRNA in bronchial epithelial cells than anticipated by the strong staining intensity for TxA2 synthase protein in immunohistochemistry. This could be due to a low turnover rate of mRNA and/or protein in bronchial epithelial cells compared with that in alveolar macrophages or vascular smooth muscle cells.

Bronchial epithelial cells have been shown to express both COX isoenzymes constitutively in both rat (7) and human (4) lung tissues. Cell culture studies gave evidence for some baseline release of different prostanoids from bronchial epithelial cells (1, 19). The present demonstration of strong TxA2 synthase immunostaining in the epithelial cell layer of the entire bronchial tree strongly suggests an important role of Tx in this compartment. Such a role may obviously be related to the regulation of bronchomotor tone but is not necessarily restricted to this aspect. Findings of mRNA expression of TxA2 synthase in organs like bone marrow, spleen, and thymus have enforced the suggestion that TxA2 may have an important functional role in the immune system (33, 39). The bronchial epithelium represents a barrier with a large external surface protecting against airborne noxious agents and invading organisms. It has been suggested that prostanoid release from the bronchial epithelium might be part of a nonspecific immune response, contributing to a natural defense mechanism (4, 13, 14, 19). Our finding that bronchial epithelial cells together with alveolar macrophages display intensive immunostaining for TxA2 synthase may support this view.

There has been evidence that TxA2 release may be responsible for bronchoconstriction and may participate in the development of bronchial asthma (27, 29). Expression of mRNA in the bronchial smooth muscle of rat lungs was low, in accordance with a moderate expression of TxA2 synthase protein under baseline conditions. This might well be different under inflammatory conditions such as asthma, with the possible upregulation of TxA2 synthase. Regardless, the immunohistochemical detection of TxA2 synthase in bronchial smooth muscle cells already in noninflamed lungs suggests that these cells may synthesize low amounts of TxA2 under baseline conditions, which might be implicated in the physiological regulation of bronchial tone.

Next to bronchial epithelial cells, single cells located in the alveolar septum exhibited intense staining for TxA2 synthase enzyme and mRNA. We assume that a major percentage of these cells represent intracapillary polymorphonuclear leukocytes (5, 9), which have been shown to produce TxA2 in vitro (21) and, like alveolar macrophages, at sites of inflammation (13). Some of the cells in the alveolar septum could as well be lung fibroblasts (21) or interstitial macrophages (25), which were also previously described to contain TxA2 synthase.

In addition, vascular smooth muscle cells were found to strongly express TxA2 synthase enzyme and mRNA signal in normal rat lungs. The most intensive staining among vessels was noted in small muscular and partially muscular vessels, which similarly display strong COX-2 expression in normal rat lungs (7). This constitutive expression of COX-2 has been implicated in physiological vasotone regulation via prostanoid synthesis (7). Recent hemodynamic measurements in isolated perfused rat lungs strongly supported this view: selective COX-2 inhibitors entirely suppressed an arachidonic acid-induced pulmonary arterial pressure increase (9) known to be largely mediated via rapid synthesis of TxA2 (6, 28, 32). In addition, COX-2 inhibition blocked Tx release into the vascular space in a preceding study (9). Immunolocalization of TxA2 synthase in smooth muscle cells of partially muscular vessels now suggests that these cells may be largely responsible for the arachidonic acid-induced vasoconstrictor response in the lung vasculature, being operative via a COX-2-TxA2 synthase axis. Moreover, a strong TxA2 synthase mRNA signal, indicating high mRNA turnover, suggests that this axis might be involved in vasomotor regulation of the pulmonary circulation, also under physiological conditions.

In human lung tissue, however, vascular and bronchial smooth muscle cells were less intensely stained compared with bronchial epithelial cells or alveolar macrophages. This difference from rat lung tissue might be due to species variations regarding the ability of the lung to produce lipid mediators (14, 38). A previous suggestion (14) that differences in the expression of enzymes downstream from COX may underlie such species variations is supported by the immunohistochemical results of the present study.

Overall, compared with the recent localization of the COX isoenzymes, the cellular staining pattern of TxA2 synthase closely matches that of COX-1 and COX-2 together, with the exception of macrophage-like cells in perivascular and peribronchial connective tissues, which were strongly positive for COX-2 and did not express TxA2 synthase, and endothelial cells, which were positive for COX-1 but did not express TxA2 synthase. This finding is well in line with the present view that endothelial cells predominantly generate vasodilatory prostanoids from the unstable endoperoxide intermediate. All other structures containing one or both COX isoenzymes were also found to express TxA2 synthase in the present study.

TxA2 is supposed to play an important role in host defense during sepsis and has been implicated in functions of the immune system (33). In response to LPS, Tx synthesis has been shown to be largely increased (17, 22, 32); however, the mechanisms regulating Tx synthesis are not clear. Previous studies (17, 22, 26), which mainly investigated prostanoid synthesis on a transcriptional level, suggested that increased LPS-induced prostanoid generation depends predominantly on rapid induction of COX-2 mRNA. The present study provides evidence that, in addition, TxA2 synthase itself is rapidly increased in many cell types within a 1-h period after LPS application. Measurement of staining intensity by quantitative image analysis showed a significant increase in TxA2 synthase protein in both groups undergoing LPS challenge (1,000 or 10,000 ng/ml). In accordance with previous in vitro studies (17, 22, 26), there was, however, no evidence for an increased expression of TxA2 synthase mRNA in LPS-primed lung tissue. These findings thus support the view of a posttranscriptional regulation of Tx synthesis, which has been previously discussed (15, 37). In these preceding studies, posttranscriptional regulation mechanisms involving alternate splicing of TxA2 synthase mRNA (37) and substrate inactivation of the enzyme (15), both of which may limit the biosynthesis of TxA2, were addressed, but additional mechanisms may well be operative. Overall, both upregulation of COX-2, the preceding enzymatic step in TxA2 formation, at the transcriptional level and enhanced expression of TxA2 synthase due to changes at the posttranscriptional level may contribute to the markedly amplified Tx synthesis in LPS-primed lungs.

The influence of TxA2 on the development of lung disease is still ambiguous. Eicosanoids have been implicated in the pathogenesis of chronic obstructive pulmonary disease, cystic fibrosis, persistent pulmonary hypertension of the newborn, primary pulmonary hypertension, asthma, and acute respiratory distress syndrome (13, 29). Nonphysiological Tx synthesis occurring in these diseases may contribute to excessive bronchoconstrictor and/or vasoconstrictor events. The present study offers a morphological basis for a better understanding of these events. In addition, the presence of intensive immunostaining of TxA2 synthase in various cell types in the noninflamed lungs and the increase in staining intensity after LPS challenge strongly suggest a regulatory role under both physiological and pathophysiological conditions. Adaptation of vascular tone to the regional demand of flow, bronchomotor regulation below the threshold of overt bronchospasm, and immunologic surveillance at the large bronchial and alveolar surface of the lung are examples of regulatory mechanisms in this context. Additional studies are required for further elucidating the role of coexpression of COX-1 and/or COX-2 and their downstream enzymes in different compartments of the lung.


    ACKNOWLEDGEMENTS

We thank G. Müller for excellent technical assistance. We are grateful to Dr. R. L. Snipes (Department of Anatomy, Justus-Liebig-Giessen University, Giessen, Germany) for linguistically reviewing the manuscript.


    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 547 (Kardiopulmonales Gefässsystem).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Ermert, Institut fuer Anatomie und Zellbiologie, Aulweg 123, 35385 Giessen, Germany (E-mail: leander.ermert{at}anatomie.med.uni-giessen.de).

Received 19 March 1999; accepted in final form 11 October 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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