AJP - Lung Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 292: L62-L67, 2007. First published September 1, 2006; doi:10.1152/ajplung.00181.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/L62    most recent
00181.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okamoto, K.
Right arrow Articles by Rubin, B. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okamoto, K.
Right arrow Articles by Rubin, B. K.

Secretory phospholipases A2 stimulate mucus secretion, induce airway inflammation, and produce secretory hyperresponsiveness to neutrophil elastase in ferret trachea

Kosuke Okamoto ,* Jung-Soo Kim,* and Bruce K. Rubin

Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Submitted 18 May 2006 ; accepted in final form 8 August 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Secretory phospholipases A2 (sPLA2) are increased in the bronchoalveolar lavage fluid of patients with asthma and acute respiratory distress syndrome. Intratracheal sPLA2 instillation induces acute lung injury in the rat and guinea pig. We hypothesized that sPLA2 would stimulate mucus secretion in vitro and that intratracheal sPLA2 exposure would induce mucus hypersecretion and airway inflammation in the ferret trachea in vivo. In vitro, porcine pancreatic sPLA2 at a concentration of 0.5 or 5 U/ml significantly increased mucous glycoconjugate (MG) secretion from the excised ferret trachea. P-bromophenacylbromide (a sPLA2 inhibitor), quercetin (a lipoxygenase inhibitor), or MK-886 (a 5-lipoxygenase inhibitor), each at 10–4 M, significantly reduced sPLA2-induced MG secretion. sPLA2-stimulated MG secretion was decreased in Ca2+-free medium. In vivo, ferrets were intubated for 30 min once per day for 3 days using an ETT coated with 20 units of porcine pancreatic sPLA2 mixed in water-soluble jelly. Constitutive MG secretion increased 1 day after sPLA2 exposure and returned to control 5 days later. Human neutrophil elastase (HNE) at 10–8 M increased MG secretion in the sPLA2-exposed trachea compared with that in the control trachea, but methacholine at 10–7 M did not. sPLA2-induced secretory hyperresponsiveness continued for at least 5 days after sPLA2 exposure ended. sPLA2 increased tracheal inflammation, MG secretion, and secretory hyperresponsiveness to HNE probably through enzymatic action rather than by activation of its receptor.

mucous glycoconjugate; lipoxygenase; mucin; asthma

SECRETORY PHOSPHOLIPASES A2 (sPLA2) are a family of lipolytic enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids generating free fatty acids, including arachidonic acid (AA), and lysophospholipids leading to the generation of eicosanoids, including leukotrienes (LT). There are more than 19 different isoforms (groups) of PLA2 in humans, denoted by Roman numerals. PLA2s acting on membrane phospholipids have been implicated in intracellular membrane trafficking, differentiation, proliferation, and apoptosis.

sPLA2 activity is increased in the serum and bronchoalveolar lavage fluid (BALF) of patients with asthma (1) or the acute respiratory distress syndrome (ARDS) (14, 37) and in animals with acute lung injury (7–9).

Allergen challenge induces vasodilation, airway smooth muscle constriction, and increased mucus secretion, all characteristic of asthma (27). The intratracheal administration of LTs C4 and D4 can cause this same constellation of symptoms (5), and 5-lipoxygenase (5-LO) inhibitors decrease LT-stimulated mucous glycoconjugate (MG) secretion in cultured human lung tissue (5, 23).

We hypothesized that sPLA2 would stimulate mucus secretion in vitro and that intratracheal sPLA2 exposure would induce inflammation and mucus hypersecretion in vivo and make the airway more responsive to inflammatory mediators. We also evaluated potential mechanisms for sPLA2-stimulated MG secretion.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All reagents and supplies were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. This study was approved by the Animal Care and Use Committee of Wake Forest University.

Animals and assay of tracheal secretion. Male adult ferrets (Marshall Farms, North Rose, NY) weighing ~2 kg were used in this study. The ferret tracheal airway is rich in submucosal glands and is a robust model for evaluating the regulation of MG secretion (12, 16–18, 21). For ex vivo studies, ferrets were killed by intraperitoneal injection of pentobarbital (120 mg/kg). Immediately after death, the trachea from larynx to carina was removed and divided into eight roughly equal segments, weighed, and immersed into Krebs-Henseleit solution (KHS; 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 3.4 mM CaCl2, 2 g/l D-glucose, pH 7.4) at ferret body temperature, 38°C. After resting for 1 h in KHS, the segments were incubated for 30 min with KHS to measure constitutive secretion (period 1) and then incubated for 30 min with porcine pancreatic sPLA2 at a concentration of 0.005, 0.05, 0.5, and 5 U/ml or with KHS alone to measure stimulated secretion (period 2). After incubation, the buffer was collected, and secreted MG was analyzed as described below. We randomized the administration of sPLA2 and KHS to different segments in each experiment.

Evaluation of secretory pathways. Inhibitors were used to evaluate potential mechanisms for sPLA2-induced MG secretion. Atropine (a muscarinic agonist) at 10–4 M, p-bromophenacylbromide (p-BPB; a sPLA2 inhibitor) at 10–5 and 10–4 M, 5,8,11,14-eicosatetraynoic acid (a lipoxygenase inhibitor) at 10–4 M, quercetin (a lipoxygenase inhibitor, Sigma) at 10–4 M, MK-886 (a specific 5-LO inhibitor; Calbiochem, San Diego, CA) at 5 x 10–5 and 10–4 M, and indomethacin (a cyclooxygenase inhibitor, Sigma) at 10–4 M were used. Ferret tracheal segments were rested in KHS. After resting for 1 h, the segments were incubated for 30 min in KHS with an inhibitor or KHS alone (period 1) and then incubated for another 30 min in KHS containing 0.5 or 5 U/ml sPLA2 with or without an inhibitor or KHS alone (period 2).

Tracheal secretion in calcium-free medium. MG secretion in Ca2+-free medium was tested because sPLA2 requires millimolar concentrated Ca2+ for enzymatic activity, whereas receptor-mediated sPLA2 activity has been reported to be calcium independent (13, 19). Ferret tracheal segments were first rested in KHS (1.3 mM Ca2+, pH 7.4). After resting for 1 h, the segments were incubated for 30 min with KHS (period 1) and then incubated for another 30 min in KHS containing porcine pancreatic sPLA2 at 5 U/ml or KHS alone in the presence (+) or absence (–) of 2 mM EGTA (period 2). Secreted MG was analyzed as described below.

Intratracheal sPLA2 exposure. Ferrets were anesthetized with 40 mg/kg ketamine and 5 mg/kg xylazine and intubated for 30 min once per day for 3 days using an endotracheal tube (ETT) coated with 20 units of porcine pancreatic sPLA2 mixed in 300 µl of water-soluble K-Y jelly (Johnson & Johnson, New Brunswick, NJ). The position of the ETT was checked with a neonatal bronchoscope. Tracheae from larynx to carina were removed 1, 5, or 10 days after the last sPLA2 exposure (sPLA2 day 1, sPLA2 day 5, sPLA2 day 10). Control animals were intubated with an ETT coated with 300 µl of K-Y jelly alone for 30 min per day for 3 days, and tracheae were removed 1 day after the last intubation. Each trachea was divided into eight roughly equal segments, and the segments were rested in KHS. A middle segment was fixed in 10% formalin and processed for histological evaluation. After resting for 1 h, ferret tracheal segments were incubated for 30 min with KHS to measure constitutive MG secretion (period 1) and then incubated for 30 min in KHS with a secretagogue or KHS alone to measure stimulated MG secretion (period 2). To evaluate MG secretion in response to cholinergic or serine protease stimulation, methacholine (MCh) at 10–7 M or human neutrophil elastase (HNE; Elastin Products, Owensville, MO) at 10–8 M was used based on our previous studies (16).

MG secretion. Ferret tracheal mucins have high blood group titers, reflecting an abundance of galactose-N-acetyl-a1–3 (fucose-a1–2) galactose-R (21). These blood group antigens can be detected by Dolichos biflorus agglutinin (DBA) lectin. DBA immunohistochemistry has shown specific binding to goblet cells and submucosal glands in ferret trachea (16).

A sandwich enzyme-linked lectin assay was used to measure MG secretion as previously described (16–18). A 96-well microtiter plate was coated with 60 µl of DBA (6 µg/ml in PBS) and incubated at 4°C overnight. After rinsing five times with PBS with 0.5% Tween 20 (PBS-Tween), the plate was exposed to sample buffer and incubated at room temperature for 2 h. It was then incubated for 1.5 h with 50 µl of DBA conjugated with horseradish peroxidase (0.25 µg/ml) in PBS containing 1% bovine serum albumin. Before and after this step, the plate was washed five times with PBS-Tween. One hundred and fifty microliters of tetramethylbenzidine (0.42 mM) in citrate-acetate buffer (pH 6.0) was added to each well and incubated for 10 min. The reaction was stopped by adding 50 µl of 4.7 N H2SO4. Color development was read as the difference in absorbance at 450 and 650 nm. The concentration of MG was calculated by comparison with asialo ovine submaxillary mucin at 20–200 ng/ml. The amount of MG was expressed as mucin weight in the incubated buffer per trachea tissue weight (ng/gm tissue).

The relative increase of MG in each tracheal segment was calculated as the ratio of the actual amount of secreted MG in period 2 incubation divided into that of period 1. A secretory index (SI) for stimulated MG secretion was calculated using the ratio of stimulated MG secretion divided by that of unstimulated secretion (both period 1 and 2: KHS) in each animal. A percent (%) change of SI was calculated using the SI of stimulated MG secretion divided by that of unstimulated (KHS) secretion in each animal.

Histological evaluation. A middle segment of trachea was fixed in 10% formalin, embedded in paraffin, and processed for histological analysis using light microscopy. The tissue was cut in 4-µm sections, and the slides were stained with hematoxylin and eosin to measure the accumulation of inflammatory cells and overall severity of inflammation. The accumulation of inflammatory cells was variable, possibly related to the degree of tissue exposure to the sPLA2-coated tubes. Thus we could not accurately quantify the amount of inflammation by cell counts so we determined the type of cellular infiltrate and how long the inflammatory cells and loss of epithelial integrity persisted after sPLA2 exposure ceased.

Statistical analysis. Statistical analysis of data was performed using the StatView 5.0 statistics package (SAS, Cary, NC). After confirming that data were normally distributed, data were analyzed using ANOVA Scheffé's F test or two-tailed unpaired t-test. P values of ≤ 0.05 were considered statistically significant. Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
sPLA2 stimulates MG secretion in the ferret trachea in vitro. sPLA2 rapidly stimulated MG secretion in the excised ferret trachea in a dose- and a time-dependent manner (Fig. 1). sPLA2 at a concentration of 0.5 (P < 0.05) and 5 (P < 0.0001) U/ml significantly increased MG secretion compared with KHS control.


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 1. Secretory PLA2 (sPLA2) stimulates mucous glycoconjugate (MG) secretion in the ferret trachea. Top: tracheal segments were immersed in Krebs-Henseleit solution (KHS). After resting for 1 h, segments were incubated in KHS for 30 min (period 1) and then for another 30 min in KHS with sPLA2 at a concentration of 0.005 (n = 7), 0.05 (n = 7), 0.5 (n = 31), or 5 (n = 14) U/ml or KHS alone (n = 35) (period 2). Secreted MG was measured by enzyme-linked lectin assay (ELLA). The relative increase of MG in each tracheal segment was calculated as the ratio of the amount of secreted MG in period 2 incubation divided into that of period 1. A secretory index (SI) for stimulated MG secretion was calculated as the ratio of stimulated MG secretion divided by that of unstimulated secretion. All data are expressed as means ± SE. *P < 0.05 or **P < 0.0001 compared with KHS. Bottom: kinetics of sPLA2-induced MG secretion. Tracheal segments were incubated in KHS with porcine pancreatic sPLA2 at a concentration of 0.5 or 5 U/ml sPLA2 or KHS alone (each: n = 6), and the buffers were collected at 5, 10, 15, and 30 min after incubation. All data are shown as concentration of secreted MG and expressed as means ± SE. *P < 0.0001 or **P < 0.0005 compared with each KHS group; +P < 0.01 compared with KHS group.

 
sPLA2 and LO inhibitors decrease sPLA2-induced MG secretion. Atropine at 10–4 M had no effect on sPLA2-induced MG secretion. sPLA2-induced MG secretion was significantly decreased by p-BPB (an sPLA2 inhibitor) at 10–4 M (P < 0.01), quercetin (a LO inhibitor) at 10–4 M (P < 0.01), or MK-886 (a specific 5-LO inhibitor) at 5 x 10–5 M (P < 0.01) (Fig. 2). MK-886 at 10–4 M also inhibited sPLA2-induced MG secretion (P < 0.005) as shown in Fig. 3, top. Indomethacin (a cyclooxygenase inhibitor) at 10–4 M did not affect sPLA2-induced MG secretion (Fig. 2).


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 2. Inhibition of sPLA2-induced MG secretion. Tracheal segments were incubated for 30 min in KHS with an inhibitor or with KHS alone (period 1) and then for another 30 min in KHS alone or KHS containing sPLA2 at 0.5 U/ml with or without an inhibitor (period 2). Shown are sPLA2 at 0.5 U/ml alone (n = 23), atropine at 10–4 M (n = 3), p-bromophenacylbromide (p-BPB) at 10–5 (n = 6) and 10–4 M (n = 6), quercetin at 10–4 M (n = 4), eicosatetraenoic acid (ETYA) at 10–4 M (n = 4), MK-886 at 5 x 10–5 M (n = 5), and indomethacin at 10–4 M (n = 6). Data are shown as percent change of SI compared with KHS group and expressed as means ± SE. MG secretion stimulated by sPLA2 at 0.5 U/ml was significantly decreased by p-BPB at 10–4 M, quercetin at 10–4 M, and MK-886 at 5 x 10–5 M, compared with sPLA2 at 0.5 U/ml alone (* P < 0.01).

 

Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 3. Top: MK-886, an (5-lipoxygenase) inhibitor, inhibited sPLA2-induced MG secretion. Tracheal segments (each group: n = 8) were incubated for 30 min in KHS with MK-886 at 10–4 M or KHS alone (period 1) and then for another 30 min in KHS alone or KHS containing sPLA2 at 5 U/ml with or without MK-886 at 10–4 M (period 2). Data are shown as SI and expressed as means ± SE. *P < 0.0001 and **P < 0.005. Bottom: MG secretion in calcium-free medium. Tracheal segments were incubated for 30 min in KHS (1.3 mM Ca2+) (period 1) and then for another 30 min in KHS alone, KHS with EGTA at 2 mM, or KHS containing sPLA2 at 5 U/ml with or without EGTA at 2 mM (each group: n = 4) (period 2). Data are shown as SI and expressed as means ± SE. *P < 0.0001, **P < 0.01, and ***P < 0.0005.

 
Mucin secretion in calcium-free KHS. sPLA2 at 5 U/ml significantly increased MG secretion in the ferret trachea (KHS in 1.3 mM Ca2+), and secretion was significantly decreased in Ca2+-free medium (KHS in 1.3 mM Ca2+ + 2.0 mM EGTA) as shown in Fig. 3. Constitutive MG secretion was also decreased in Ca2+-free medium compared with KHS control (P < 0.01).

Intratracheal sPLA2 exposure induced MG hypersecretion. Ferrets were intubated once per day for 3 days with an ETT coated with 20 units of sPLA2 mixed in water-soluble K-Y jelly. Constitutive MG secretion significantly increased 1 day after the last sPLA2 exposure compared with control (P < 0.0005) and returned to control levels at 5 days (sPLA2 day 5), as shown in Fig. 4. HNE at 10–8 M markedly and synergistically stimulated MG secretion in the sPLA2-exposed trachea (P < 0.01), and this hyperresponsiveness to HNE continued for 5 days (P < 0.01) and returned to control by day 10 (Fig. 5). MCh at 10–7 M increased MG secretion in the sPLA2-exposed trachea no more than from the nonexposed trachea.


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Constitutive MG secretion in the sPLA2-exposed trachea. Ferrets were intubated with an endotracheal tube (ETT) coated with 20 units of sPLA2 mixed in water-soluble K-Y jelly for 30 min once daily for 3 days. One (sPLA2 day 1), 5 (sPLA2 day 5), or 10 (sPLA2 day 10) days after the last sPLA2 exposure, tracheae were removed. Control animals were intubated with an ETT coated with jelly alone, and tracheae were removed 1 day after the third and last intubation (control intubated). After resting for 1 h in KHS, segments were placed in fresh KHS for 30 min. Secreted MG was measured by ELLA. Data are shown as secreted MG. Shown are control unintubated: no treatment (n = 36), control intubated (n = 40), sPLA2 day 1 (n = 40), sPLA2 day 5 (n = 32), and sPLA2 day 10 (n = 32). Data are shown as absolute amount of secreted MG and expressed as means ± SE. *P < 0.0005 compared with control unintubated.

 

Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 5. Human neutrophil elastase (HNE) and methacholine (MCh) stimulate MG secretion in the sPLA2-exposed trachea. Ferret tracheal segments were placed in KHS for 30 min to measure constitutive MG secretion (period 1) and then were incubated for another 30 min in KHS with HNE at 10–8 M, MCh at 10–7 M, or KHS alone (period 2). Shown are control unintubated (period 2: KHS and HNE at 10–8 M, MCh at 10–7 M: n = 15, 11, 10), control intubated (n = 14, 13, 13), sPLA2 day 1 (n = 14, 13, 13), sPLA2 day 5 (n = 10, 11, 11), and sPLA2 day 10 (n = 9, 12, 11). All data are shown as SI for MG secretion and expressed as means ± SE. *P < 0.01 compared with control unintubated.

 
Histopathology of the sPLA2-exposed trachea. One day after the last sPLA2 exposure (sPLA2 day 1), there was an influx of inflammatory cells in both the epithelium and the lamina propria. The inflammatory cells comprised a mix of polymorphonuclear and mononuclear cells. Five days after the last sPLA2 exposure (sPLA2 day 5), fewer inflammatory cells were in the epithelium, but there was increased airway mucus secretion. The lamina propria was relatively free of inflammation. Ten days after the last sPLA2 exposure (sPLA2 day 10), inflammatory cell numbers were similar to the airway not exposed to sPLA2. The lamina propria resembled control tissues, and the basal cell layer of the epithelium was intact. There was no evidence of increased inflammatory cells in the control intubated airways.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
sPLA2 activity is increased in the serum or BALF of patients with asthma (2) or ARDS (14, 37). We show here that sPLA2 is a rapid and potent MG secretagogue, this effect is dose dependent, and it can be blocked by Ca2+ depletion or by LT inhibitors but not by cholinergic inhibitors. In vivo, sPLA2 also induces a state of "secretory hyperresponsiveness" to HNE but not to cholinergic stimulation as we further discuss below.

In the guinea pig, intratracheal instillation of 10 or 30 units of sPLA2 rapidly increases the thickness of interalveolar septa by edema and epithelial cell swelling (7, 8). We applied sPLA2 directly to the tracheal epithelium by intubating ferrets with an ETT coated with sPLA2 mixed in water-soluble jelly for 30 min once per day for 3 days. This technique was described previously by our group for exposure of the airway to bacterial endotoxin (18). Our results in vivo suggest that sPLA2 induces local airway tissue injury and stimulates mucus hypersecretion.

sPLA2 type IB is abundant in pancreatic fluid of many species and is sometimes referred to as pancreatic type sPLA2. sPLA2 type IIA is sometimes called inflammatory type, because it is highly expressed in the plasma or synovial fluids of patients with inflammatory diseases such as rheumatoid arthritis (30) and septic shock (38). sPLA2 type IB is produced as an inactive proenzyme and activated by proteolytic enzymes (25, 26). sPLA2 type IB was used in this study because it has similar enzymatic characteristics to sPLA2 type IIA, it has a widespread tissue distribution including in the lung (31, 33, 36), and it has potent pathophysiological activity (1, 11, 15). Both sPLA2 type IB and IIA are 14-kDa proteins (35) and require millimolar amounts of Ca2+ for enzymatic activity. Activated sPLA2 type IB is detected in the serum and urine of subjects with acute lung injury but not in healthy controls (28).

sPLA2 type IB has also been shown to regulate cellular function in vitro. It stimulates cell proliferation in Swiss 3T3 cells (1) and pancreatic cancer cells (11) and prostaglandin production and sPLA2 type IIA expression in rat mesangial cells (15). These actions of sPLA2 type IB are thought to be receptor mediated. A sPLA2 type IB specific receptor has been identified (20). The receptor-mediated actions of this enzyme appear to be independent of Ca2+ (13, 19), whereas the enzymatic activity is Ca2+ dependent. p-BPB can inhibit sPLA2 enzymatic activity but not its receptor-mediated action (4).

Consistent with this, we demonstrate that p-BPB decreased sPLA2 type IB-stimulated MG secretion. These results suggest that sPLA2 type IB may stimulate MG secretion in the ferret trachea via its enzyme activity and indirectly through LT synthesis rather than by a receptor-mediated action. The results of MG secretion in Ca2+-free KHS support this because Ca2+-free KHS decreased sPLA2-induced MG secretion, and Ca2+-free medium also decreased constitutive MG secretion.

AA is a substrate for LO in the AA cascade generating eicosanoids, including LTs that are potent secretagogues (5, 23). Indomethacin, a cyclooxygenase inhibitor, did not affect sPLA2-induced MG secretion, supporting the hypothesis that cyclooxygenase pathway products (e.g., prostaglandins) do not play an important role in sPLA2-induced MG secretion. Quercetin, an LO inhibitor, decreased sPLA2-induced MG secretion, suggesting that sPLA2 may stimulate MG secretion by activating the LO pathway. MK-886, a specific 5-LO inhibitor, decreased sPLA2-induced MG secretion in the ferret. MK-886 binds a membrane-bound 5-LO-activating protein, thereby inhibiting the translocation and activation of 5-LO (6, 29). MK-886 at 5 x 10–5 M also significantly deceased sPLA2-induced MG secretion. Thus sPLA2 probably stimulate tracheal MG secretion through the activation of LO and the 5-LO pathway.

In persons with asthma, bronchial hyperresponsiveness is defined by the decrease in airflow, generally FEV1, to a variety of stimuli including MCh, histamine, hyperosmolar challenge, exercise, or cold air. In this study, we show that local tracheal exposure to sPLA2 exposure induced tracheal inflammation, producing a long-lasting enhanced secretory response to HNE but not to MCh stimulation (Fig. 5). We have termed this enhanced and synergistic secretory response secretory hyperresponsiveness. This suggests that sPLA2 causes hypertrophy or hyperplasia of secretory cells or glands (analogous to asthma increasing the amount of smooth muscle in the airway) and/or that the secretory apparatus is sensitized after sPLA2 exposure to produce a greater secretory response to HNE, analogous to the concept of "twitchy airways." Because secretory hyperresponsiveness to NHE returned to control levels 10 days after the last sPLA2 exposure, we hypothesize that in this model, the airway has been primed to respond to NHE in an exaggerated manner.

Bowton and colleagues (3) studied the effect of LY-333013, a potent inhibitor of group II sPLA2, on allergen-induced bronchoconstriction following inhaled allergen challenge in atopic asthmatics. Compared with placebo, LY-333013 had no impact on bronchial hyperresponsiveness defined by pulmonary function changes following inhaled allergen challenge (3). However, when Masuda and colleagues (24) examined the expression and function of sPLA2 in human lung-derived cells and in human lungs with pneumonia, they showed that whereas groups V and X sPLA2 were widely expressed in the airway epithelium, interstitium, and alveolar macrophages, group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and groups IIE and IIF sPLA2 were minimally detected. These results suggest that groups V and X sPLA2 are involved in disease pathogenesis in the human lung to a greater extent than the group II sPLA2 enzymes.

Inflammatory cells consisting of polymorphonuclear and mononuclear cells rapidly accumulated in the sPLA2-exposed trachea by day 1, but by day 10 after exposure, the sPLA2-exposed tracheae appeared the same as control tracheae. Neutrophils release chemical mediators that are potent airway secretagogues (5, 34). The number of neutrophils and the amount of neutrophil elastase is increased in sputum of patients with cystic fibrosis or severe asthma (10, 32). HNE enhanced MG hypersecretion in the sPLA2-exposed trachea compared with control trachea, suggesting that secretory hyperresponsiveness may depend both on the existence of underlying inflation and on continued stimulation by inflammatory mediators such as HNE.

The experimental procedures in vivo were based on an endotoxin-inflamed tracheal model that we developed (16). Intratracheal sPLA2 exposure produced similar changes in MG secretion and inflammatory cells in the ferret trachea as the endotoxin model. Constitutive MG secretion increases after endotoxin or sPLA2 exposure, and MG secretion markedly increases in response to HNE, but not to MCh, compared with control trachea. The influx of inflammatory cells is common in both models. It has been reported that bacterial endotoxin stimulates sPLA2 mRNA expression and enzymatic activity in the rat lung (22). Although endotoxin is known to trigger inflammation both through CD14 and CD137 and Toll-like receptors 4 and 2 signaling, it is possible that some of this airway hypersecretory response to endotoxin occurs through a sPLA2-dependent pathway.


   ACKNOWLEDGMENTS
 
We thank Lauren Vannoy for technical assistance and Dr. David Bass for first suggesting these experiments.

Present address of K. Okamoto: Mie University School of Medicine, Department of Otolaryngology, Tsu, Japan; present address of J.-S. Kim: Department of Otolaryngology, College of Medicine, Kyungpook National University, Daegu, Korea.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. K. Rubin, Dept. of Pediatrics, Wake Forest Univ. School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1081 (e-mail: brubin{at}wfubmc.edu)

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. Section 1734 solely to indicate this fact.

* K. Okamoto and J.-S. Kim contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Arita H, Hanasaki K, Nakano T, Oka S, Teraoka H, Matsumoto K. Novel proliferative effect of phospholipase A2 in Swiss 3T3 cells via specific binding site. J Biol Chem 266: 19139–19141, 1991.[Abstract/Free Full Text]
  2. Bowton DL, Seeds MC, Fasano MB, Goldsmith B, Bass DA. Phospholipase A2 and arachidonate increase in bronchoalveolar lavage fluid after inhaled antigen challenge in asthmatics. Am J Respir Crit Care Med 155: 421–425, 1997.[Abstract]
  3. Bowton DL, Dmitrienko AA, Israel E, Zeiher BG, Sides GD. Impact of a soluble phospholipase A2 inhibitor on inhaled allergen challenge in subjects with asthma. J Asthma 42: 65–71, 2005.[CrossRef][Web of Science][Medline]
  4. Chang T, Chang CH, Wagner DR, Chey WY. Porcine pancreatic phospholipase A2 stimulates secretine release from secretin-producing cells. J Biol Chem 274: 10758–10764, 1999.[Abstract/Free Full Text]
  5. Coles SJ, Neill KH, Reid LM, Austen KF, Nii Y, Corey EJ, Lewis RA. Effects of leukotrienes C4 and D4 on glycoprotein and lysozyme secretion by human bronchial mucosa. Prostaglandins 25: 155–170, 1983.[CrossRef][Web of Science][Medline]
  6. Dixon RAF, Diehl RE, Opas E, Rands E, Vickers PJ, Evans JF, Gillard JW, Miller DK. Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis. Nature 343: 282–284, 1989.
  7. Durham SK, Selig WM. Phospholipase A2-induced pathophysiologic changes in the guinea pig lung. Am J Pathol 136: 1283–1291, 1990.[Abstract]
  8. Durham S, Selig WM. Pathophysiologic changes induced by phospholipase A2 in the isolated, perfused guinea pig lung. Exp Lung Res 16: 323–338, 1990.[Web of Science][Medline]
  9. Edelson JD, Vadas P, Villar J, Mullen JBM, Pruzanski W. Acute lung injury induced by phospholipase A2. Am Rev Respir Dis 143: 1102–1109, 1991.[Web of Science][Medline]
  10. Fahy JV, Kim KW, Liu J, Boushey HA. Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation. J Allergy Clin Immunol 95: 843–852, 1995.[CrossRef][Web of Science][Medline]
  11. Hanada K, Kinoshita E, Itoh M, Hirata M, Kajiyama G, Sugiyama M. Human pancreatic phospholipase A2 stimulates the growth of human pancreatic cancer cell line. FEBS Lett 373: 85–87, 1995.[CrossRef][Web of Science][Medline]
  12. Hyde DM, Samuelson DA, Blakeney WH, Kosch PC. A correlative light microscopy, transmission microscopy and scanning electron microscopy study of the ferret lung. Scan Electr Microsc 3: 891–898, 1979.
  13. Ishizaki J, Kishino J, Teraoka H, Ohara O, Arita H. Receptor-binding capability of pancreatic phospholipase A2 is separable from its enzymatic activity. FEBS Lett 324: 349–352, 1993.[CrossRef][Web of Science][Medline]
  14. Kim DK, Fukuda T, Thompson BT, Cockrill B, Hales C, Bonventre JV. Bronchoalveolar lavage fluid phospholipase A2 activities are increased in human adult respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol 269: L109–L118, 1995.[Abstract/Free Full Text]
  15. Kishino J, Ohara O, Nomura K, Kramer RM, Arita H. Pancreatic-type phospholipase A2 induces group II phospholipase A2 expression and prostaglandin biosynthesis in rat mesangial cells. J Biol Chem 269: 5092–5098, 1994.[Abstract/Free Full Text]
  16. Kishioka C, Okamoto K, Kim JS, Rubin BK. Regulation of secretion from mucous and serous cells in the excised ferret trachea. Respir Physiol 126: 163–171, 2001.[CrossRef][Web of Science][Medline]
  17. Kishioka C, Okamoto K, Hassett DJ, Mello DD, Rubin BK. Pseudomonas aeruginosa alginate is a potent secretagogue in the isolated ferret trachea. Pediatr Pulmonol 27: 174–179, 1999.[CrossRef][Web of Science][Medline]
  18. Kishioka C, Okamoto K, Kim JS, Rubin BK. Hyperosmolar solutions stimulate mucus secretion in the ferret trachea. Chest 124: 306–313, 2003.
  19. Lambeau G, Ancian P, Nicolas JP, Beiboer SHW, Moinier D, Verheij H, Lazdunski M. Structural elements of secretory phospholipases A2 involved in the binding to M-type receptors. J Biol Chem 270: 5534–5540, 1995.[Abstract/Free Full Text]
  20. Lambeau G, Ancian P, Barhanin J, Lazdunski M. Cloning and expression of a membrane receptor for secretory phospholipases A2. J Biol Chem 269: 1575–1578, 1994.[Abstract/Free Full Text]
  21. Leigh MW, Cheng PW, Boat T. Developmental changes of ferret tracheal mucin composition and biosynthesis. Biochem 28: 9440–9446, 1989.[CrossRef][Medline]
  22. Ljungman AG, Tagesson C, Lindahl M. Endotoxin stimulates the expression of group II PLA2 in rat lung in vivo and in isolated perfused lungs. Am J Physiol Lung Cell Mol Physiol 270: L752–L760, 1996.[Abstract/Free Full Text]
  23. Marom Z, Shelhamer JH, Bach MK, Kaliner M. Effects of arachidonic acid, monohydroxyeicosanotetraenoic acid, and prostaglandin on the release of mucous glycoproteins from human airways in vitro. J Clin Invest 67: 1695–1702, 1981.[Web of Science][Medline]
  24. Masuda S, Murakami M, Mitsuishi M, Komiyama K, Ishikawa Y, Ishii T, Kudo I. Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells. Biochem J 387: 27–38, 2005.[CrossRef][Web of Science][Medline]
  25. Mukherjee AB, Miele L, Pattabiraman N. Phospholipase A2 enzymes: regulation and physiological role. Biochem Biophys 48: 1–10, 1994.[CrossRef]
  26. Nakano T, Fujita H, Kikuchi N, Arita H. Plasmin converts pro-form of group I phospholipase A2 into receptor binding, active form. Biochem Biophys Res Commun 198: 10–15, 1994.[CrossRef][Web of Science][Medline]
  27. Piacentini GL, Kaliner MA. The potential roles of leukotrienes in bronchial asthma. Am Rev Respir Dis 143: S96–S99, 1991.[Web of Science][Medline]
  28. Rae D, Porter J, Newman NB, Sumar N, Bennett D, Taylor JH. Type I phospholipase A2 propeptide in acute lung injury. Lancet 344: 1472–1473, 1994.[CrossRef][Web of Science][Medline]
  29. Rouzer CA, Ford-Hutchinson AW, Morten HE, Gillard JW. MK-886, a potent and specific leukotriene biosynthesis inhibitor, blocks and reverses the membrane association of 5-lipoxygenase in ionophore challenged leukocytes. J Biol Chem 265: 1436–1442, 1990.[Abstract/Free Full Text]
  30. Sakamoto K, Arakawa H, Yamashita S, Mizuta H, Takaki K, Ogawa M. Membrane-associated phospholipase A2 detected by a radioimmunoassay is a sensitive marker of inflammation in rheumatoid arthritis. Res Commun Chem Pathol Pharmacol 76: 279–285, 1992.[Web of Science][Medline]
  31. Sakata T, Nakamura E, Tsuruta Y, Tamaki M, Teraoka H, Tojo H, Ono T, Okamoto M. Presence of pancreatic-type phospholipase A2 mRNA in rat gastric mucosa and lung. Biochem Biophys Acta 1007: 124–126, 1989.[Medline]
  32. Salva PS, Doyle NA, Graham L, Eigen H, and Doerschuk CM. TNF-alpha, IL-8, soluble ICAM-1, and neutrophils in sputum of cystic fibrosis patients. Pediatr Pulmonol 21: 11–19, 1996.[CrossRef][Web of Science][Medline]
  33. Seilhamer JJ, Randall TL, Yamanaka M, Johnson LK. Pancreatic phospholipase A2: isolation of the human gene and cDNAs from porcine pancreas and human lung. DNA 5: 519–527, 1986.[Web of Science][Medline]
  34. Sommerhoff CP, Nadel JA, Basbaum CB, Caughey GH. Neutrophil elastase and cathepsin G stimulate secretions from cultured bovine airway gland serous cells. J Clin Invest 85: 682–689, 1990.[Web of Science][Medline]
  35. Tischfield JA. A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J Biol Chem 272: 17247–17250, 1997.[Free Full Text]
  36. Tojo H, Ono T, Kuramatsu S, Kagamiyama H, Okamoto MA. Phospholipase A2 in the supernatant fraction of rat spleen. J Biol Chem 263: 5724–5731, 1988.[Abstract/Free Full Text]
  37. Vadas P. Elevated plasma phospholipase A2 levels: correlation with the hemodynamic and pulmonary changes in gram-negative septic shock. J Lab Clin Med 104: 873–881, 1984.[Web of Science][Medline]
  38. Vadas P, Scott K, Smith G, Rajkovic I, Stefanski E, Schouten BD, Singh R, Pruzanski W. Serum phospholipase A2 enzyme activity and immunoreactivity in a prospective analysis of patients with septic shock. Life Sci 50: 807–811, 1992.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. M. Munoz, A. Y. Meliton, L. N. Meliton, S. M. Dudek, and A. R. Leff
Secretory group V phospholipase A2 regulates acute lung injury and neutrophilic inflammation caused by LPS in mice
Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L879 - L887.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
J. C. Abanses, S. Arima, and B. K. Rubin
Vicks VapoRub Induces Mucin Secretion, Decreases Ciliary Beat Frequency, and Increases Tracheal Mucus Transport in the Ferret Trachea
Chest, January 1, 2009; 135(1): 143 - 148.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
B. P. Hurley and B. A. McCormick
Multiple Roles of Phospholipase A2 during Lung Infection and Inflammation
Infect. Immun., June 1, 2008; 76(6): 2259 - 2272.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/L62    most recent
00181.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okamoto, K.
Right arrow Articles by Rubin, B. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okamoto, K.
Right arrow Articles by Rubin, B. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.