Negative feedback between secretory and cytosolic phospholipase A2 and their opposing roles in ovalbumin-induced bronchoconstriction in rats
Sarit Offer,1
Saul Yedgar,2
Ouri Schwob,2
Miron Krimsky,2
Haim Bibi,3
Abraham Eliraz,4
Zecharia Madar,1 and
David Shoseyov5
1Institute of Biochemistry, Faculty of Agriculture, The Hebrew University, and 4Pulmonary Unit, Kaplan Hospital, Rehovot; 2Department of Biochemistry, Hebrew University-Hadassah Medical School, and 5Department of Pediatrics, Hadassah University Hospital, Mount Scopus, Jerusalem; and 3Department of Pediatrics, Barzilai Medical Center, Ashkelon, Israel
Submitted 28 May 2004
; accepted in final form 14 November 2004
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ABSTRACT
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Phospholipase A2 (PLA2) hydrolyzes cell membrane phospholipids (PL) to produce arachidonic acid and lyso-PL. The PLA2 enzymes include the secretory (sPLA2) and cytosolic (cPLA2) isoforms, which are assumed to act synergistically in production of eicosanoids that are involved in inflammatory processes. However, growing evidence raises the possibility that in airways and asthma-related inflammatory cells (eosinophils, basophils), the production of the bronchoconstrictor cysteinyl leukotrienes (CysLT) is linked exclusively to sPLA2, whereas the bronchodilator prostaglandin PGE2 is produced by cPLA2. It has been further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production. This seems to suggest that sPLA2 and cPLA2 play opposing roles in asthma pathophysiology and the possibility of a negative feedback between the two isoenzymes. To test this hypothesis, we examined the effect of a cell-impermeable extracellular sPLA2 inhibitor on bronchoconstriction and PLA2 expression in rats with ovalbumin (OVA)-induced asthma. It was found that OVA-induced bronchoconstriction was associated with elevation of lung sPLA2 expression and CysLT production, concomitantly with suppression of cPLA2 expression and PGE2 production. These were reversed by treatment with the sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and reduced CysLT production and sPLA2 expression, concomitantly with enhanced PGE2 production and cPLA2 expression. This study demonstrates, for the first time in vivo, a negative feedback between sPLA2 and cPLA2 and assigns opposing roles for these enzymes in asthma pathophysiology: sPLA2 activation induces production of the bronchoconstrictor CysLT and suppresses cPLA2 expression and the subsequent production of the bronchodilator PGE2.
asthma; cysteinyl leukotrienes; prostaglandin E2; secretory phospholipase A2 inhibitors
PHOSPHOLIPASE A2 (PLA2) is a family of enzymes that hydrolyzes cell membrane phospholipids (PL) to produce lysophospholipids (LysoPL) and free fatty acids. The PLA2 family consists of secreted and intracellular enzymes. The intracellular PLA2 include the cytosolic (cPLA2), which is specific for arachidonic acid (AA)-carrying PL, and the calcium-independent PLA2 (iPLA2), which has no fatty acid preference. The secretory PLA2 (sPLA2) enzymes are secreted by activated inflammatory and other mammalian cells in inflammatory conditions, such as pancreatitis, respiratory distress, sepsis, asthma, and more (7, 25).
By hydrolyzing cell membrane PL, PLA2 enzymes initiate the production of numerous lipid mediators of diverse pathological states. When the released fatty acid is AA, it is metabolized into the eicosanoid families, mainly via the lipoxygenase pathways, producing the leukotrienes (LT), and the cyclooxygenase pathways, producing prostaglandins (PG) and thromboxanes. Eicosanoids of the different families are involved in the development of almost any pathological condition, especially those related to inflammatory/allergic processes (6, 41, 42).
LysoPL activate white cells and increase their vascular permeation (31), act as growth factors (especially lysophosphatidic acid), enhance airway smooth muscle (ASM) contractility and cell proliferation (37), and induce eosinophil activation and infiltration (27). Lysophosphatidyl serine, in particular, activates histamine secretion by mast cells (22). Lysophosphatidylcholine is the precursor of platelet-activating factor, a strong proinflammatory mediator (8, 10). Together, PLA2 enzymes are directly and indirectly involved in diverse pathological processes, mainly those involved in inflammation and allergy.
iPLA2 is considered a housekeeping enzyme involved in the maintenance of membrane PL composition, although a recent study demonstrated that in some inflammatory states, specifically adjuvant-induced arthritis in rats, this enzyme is the first to produce inflammatory eicosanoids (12). On the other hand, activation of cPLA2 and sPLA2 has unequivocally been shown to initiate the production of proinflammatory lipid mediators, eicosanoids in particular (6). The interrelationship between cPLA2 and sPLA2 in the induction of inflammatory processes is not unequivocally clear, as disparate results have been reported. For example, cPLA2 is required for the induction of sPLA2 expression in fibroblasts and macrophages (21), and cPLA2 inhibitors block sPLA2-dependent AA release (36). Conversely, in other cells (e.g., murine mesangial cells, human neutrophils, bone marrow mast cells, and others), sPLA2 activates cPLA2 and eicosanoid production (1113, 1719, 36). Together, these studies suggest that the relative contribution of the two PLA2 types to inflammatory conditions is tissue dependent and might differ between tissues and cell types. Yet, it is generally accepted that both cPLA2 and sPLA2 take part and often act synergistically, in positive feedback mechanism, in inducing inflammatory/allergic processes. However, this generalization seems to be too inclusive, particularly in regard to airway function, in which different eicosanoids appear to play opposing roles: cysteinyl LT (CysLT) are strong bronchoconstrictors and are involved in airway inflammation and remodeling occurring in asthma (17), whereas PGE2, which is generally considered a strong proinflammatory agent, is a potent bronchodilator and can inhibit ASM proliferation (28, 40). Moreover, it has been shown that intratracheal administration of PGE2 to rats reduced ovalbumin (OVA)-induced elevation of CysLT level in the bronchoalveolar lavage (BAL) (23). On these grounds, it has been postulated that the lung is a privileged site for the beneficial actions of PGE2, since in the lung, as opposed to other parts of the body, PGE2 has a role in limiting the immune-inflammatory responses as well as in tissue repair processes (40). In accordance with that, it was further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production (17). It thus seems that although both eicosanoids are derived from AA subsequent to PLA2 activation, the production of CysLT and PGE2 in challenged airways follows different routes. A possible explanation for these disparate routes may be found in reports that in inflammatory cells (basophils, eosinophils), LT are produced from an AA pool linked to sPLA2, whereas PG are produced from an AA pool provided by cPLA2 (7, 24). Together, this seems to suggest an inverse relationship between sPLA2 and cPLA2 and disparate, possibly opposing, roles in asthma pathophysiology.
To test this hypothesis, in the present study we have examined the effect of a cell-impermeable extracellular inhibitor of sPLA2 (ExPLI) on bronchoconstriction, concomitantly with PLA2 expression and eicosanoid production, in rats with OVA-induced asthma. The ExPLIs, designed and synthesized in the laboratory of S. Yedgar (42), have been previously found effective in amelioration of inflammatory processes in cell cultures and in animal models. These include inhibition of lipid membrane hydrolysis by diverse types of sPLA2 (9), inhibition of group V sPLA2 activation in LPS-stimulated P388D macrophages (3), endotoxin-induced sepsis in rats, as expressed by reduced mortality rate, blood cytokine level (TNF-
, IL-6), and expression of type IIA sPLA2 and inducible nitric oxide synthase in liver and kidney of septic rats (4), suppression of sPLA2 and PGE2 production by LPS-stimulated cultured rat brain glial cells and experimental allergic encephalomyelitis in rats and mice (29), and trinitrobenzensulfonic acid-induced colitis in rats, expressed by reduced mortality rate, histology, and blood sPLA2 activity (20). Using the ExPLIs, in the present study we have found that OVA-induced asthma in rats is associated with elevation of sPLA2 and suppression of cPLA2 activity and expression in the lung.
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MATERIALS AND METHODS
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Animals.
Inbred Brown Norway male rats (4 wk old) obtained from Harlan were used in this study. The Hebrew University Animal Welfare Committee approved all protocols.
Induction of asthma.
Asthma was induced in rats by sensitization with OVA (Sigma, Rehovot, Israel) according to a previously described protocol (33): on day 0, rats received a single subcutaneous injection of 1 mg of OVA plus aluminum hydroxide (200 mg/ml in 0.9% NaCl; Sigma) and an intraperitoneal injection of 1 ml containing 6 x 109 heat-killed Bordetella pertussis bacteria (Pasteur Marieux). Repeated bronchial allergen challenge was performed from day 14 every other day for 1 mo by inhalation of OVA (1 mg/ml in 0.9% normal saline) for 5 min each time in a 20-l box connected to an ultrasonic nebulizer (LS 230 System Villeneuve Sur Lot).
Treatments.
Rats were divided into three treatment groups (n = 10): 1) no sensitization and no treatment (/), used as naïve control; 2) sensitization plus challenge with OVA and no treatment (OVA/OVA), used as positive control; and 3) sensitization plus challenge with OVA and treatment with ExPLI (OVA/ExPLI). The OVA/OVA group received a subcutaneous injection of 1 ml of saline before each challenge.
Treatment with ExPLI.
In this study, we employed HyPE, designed and synthesized in the laboratory of S. Yedgar, by linking phosphatidylethanolamine to truncated hyaluronic acid (mol wt
50 kDa) (4, 15). Each rat received a subcutaneous injection of 1 ml of saline containing 15 mg of HyPE (to obtain a concentration of
1 mg/ml of body fluid = 20 µM) before each challenge.
Assessment of bronchoconstriction.
Unrestrained conscious rats were placed in a whole body plethysmograph (Buxco Electronics, Troy, NY) connected to a pneumotach (Type 0000, EMKA Technologies) at one end and to a 10-ml bottle at the other end. The pneumotach was connected to a preamplifier (model MAX2270, Buxco Electronics). Analog signals from the amplifier were converted to a digital signal by an analog-to-digital card (LPM-16; National Instruments, Austin, TX). Bronchoconstriction measures were expressed as the enhanced pause (Penh). Penh = (PEF/PIF) x [(Te Tr)/Tr], where PEF is peak expiratory flow, PIF is peak inspiratory flow, Te is expiratory time, and Tr is relaxation time = time of the pressure decay to 36% of total box pressure during expiration, according to Ref. 14.
BAL.
BAL was performed on day 30. The rats were tracheotomized and incannulated. BAL was performed with 5 x 10 ml of PBS. BAL fluid was collected into 50-ml sterile tubes, centrifuged (to collect cell-free supernatant), and stored at 80°C.
Lung histological preparation.
Lungs were fixed by inflation with paraformaldehyde at a pressure of 20 cmH2O and embedded in paraffin. Tissue slices were stained with eosin-hematoxylin and periodic acid-Schiff (PAS) for cell identification.
Immunohistochemistry of cPLA2 in rat lung tissue.
Five-micrometer sections were subjected to staining using Histostain-SP kit (Zymed, San Francisco, CA). Polyclonal anti-rabbit cPLA2 antibody, generously provided by Dr. Lisa Marshall [Glaxo-Smith-Kline (GSK), King of Prussia, PA], was used as first antibody. The antibody complex was visualized by applying aminoethylcarbazol for 310 min.
Computerized staining intensity measurement.
Staining intensity was quantified by measuring density lamination using the Image Pro Plus V3 computer program (Media Cybernetics, Baltimore, MD). In this method, one measures the light transmittance, which is inverse (reciprocal) to the density of the staining color (i.e., the more cPLA2 in the tissue, the lesser the light transmittance). Accordingly, cPLA2 staining was determined by the difference between the maximal light transmittance and that of the measured sample and expressed by total density lumination in lung tissues and point counting in specific cell types.
Protein expression of cPLA2 and sPLA2 in lung tissue.
Proteins were identified in homogenized lung tissue (100 µg of protein) using standard Western blot. A specific polyclonal antibody against cPLA2 (used as first antibody) was provided by Dr. Lisa Marshall and was diluted [Tris buffer saline Tween 20 (TBST) buffer + 0.1% BSA] 1:1,000. Anti-sPLA2 antibody (Santa Cruz) was diluted 1:500 (vol/vol) in TBST buffer plus 0.1% BSA. The immune reaction was detected by enhanced chemiluminescence.
Determination of cPLA2 activity.
cPLA2 activity was determined by the hydrolysis of radioactively labeled lipid membranes (liposomes) containing AA-carrying PL, as previously described (20). Lung tissues were homogenized in a polytron in ice-cold buffer (1:5 wt/vol) containing Tris·HCl (pH 7.6), EDTA (1 mM), EGTA (1 mM), and DTT (5 mM) to abolish sPLA2 activity. The phospholipid substrate (liposomes) was composed of 1-stearoyl-2-[14C]-arachidonyl phosphatidylcholine (Amersham), lysophosphatidylcholine, and dioleoyl-phosphatidylcholine (Sigma), suspended in Tris buffer, and supplemented with 1 mg/ml of BSA. The reaction was initiated by the addition of 100 µl of homogenate and 2 mM CaCl2 and incubated at 37°C in a shaking water bath for 1 h. Naja Mocambique PLA2 type 1 (Sigma) was used as positive control. The reaction was stopped by the addition of 1.25 ml of Doles reagent (isopropyl alcohol/heptane/0.5 M H2SO4, 20:5:1, by vol), 1 ml of heptane, and 0.75 ml of water. The mixture was vortexed and centrifuged (1,000 g for 10 min). The organic phase (0.8 ml) was added to 20 mg of silica with 1 ml of heptane and was then centrifuged to remove remnants of PL. The supernatant was mixed with scintillation fluid, and 14C radioactivity was determined in a scintillation counter. PLA2 activity was defined as the percentage of 14C-labeled fatty acid in the total radioactivity.
CysLT.
CysLT levels were measured in BAL using a kit for direct enzyme immunoassay according to the manufacturers instructions (Amersham Pharmacia Biotech). The specificity of the kit was 100% for leukotriene C4, 100% for leukotriene D4, and 70% for leukotriene E4. The result range was between 0 and 48 pg.
Determination of PGE2.
PGE2 levels were determined in BAL fluid by RIA using specific antibody and a radioligand as previously described by Pinto et al. (29). Anti-PGE2 and standard PGE2 were purchased from Sigma (St. Louis, MO). 3[H]PGE2 (150200 Ci/mmol) was purchased from Amersham.
Statistical analysis.
All data are expressed as means ± SE. One-way ANOVA was used to compare treatment groups. Pairwise comparisons were performed by the Tukey-Kramer honestly significant difference test (P = 0.05). Where necessary, data were log transformed before analysis to stabilized variances. In all analyses, P < 0.05 was considered statistically significant.
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RESULTS
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Effect of ExPLI on OVA-induced bronchoconstriction.
To examine the relationship of respiratory function to the expression and activity of the PLA2 isoenzymes, we first validated the applicability of the ExPLI (HyPE) as an sPLA2 inhibitor on respiratory functions of sensitized rats with OVA-induced asthma. As shown in Fig. 1, HyPE effected a dramatic improvement in respiratory functions. Figure 1 shows that the administration of HyPE at a dose corresponding to
10 µM in body fluid induced an
10-fold reduction of the OVA-induced bronchoconstriction.

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Fig. 1. Effect of phosphatidylethanolamine linked to hyaluronic acid (HyPE), administered subcutaneously, on early asthmatic reaction induced by ovalbumin (OVA) inhalation. Bronchoconstriction was induced in OVA-sensitized rats by inhalation of OVA and expressed by the difference in enhanced pause (Penh) measured before and 5 min after allergen challenge (see MATERIALS AND METHODS for details). Data are means ± SE for 10 rats. aP < 0.01; b, cP < 0.05.
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CysLT and PGE2 in BAL of asthmatic rat.
As discussed above, among the lipid mediators involved in asthma pathophysiology, CysLT and PGE2 are said to play opposing roles. CysLT are considered key mediators of bronchoconstriction, whereas PGE2 is a bronchodilator. The results of the present study support this notion: Fig. 2 shows that in parallel to the OVA-induced bronchoconstriction, the level of CysLT in BAL was markedly elevated, and treatment with the ExPLI reduced it to that of naïve rats. Contrary to CysLT, the PGE2 level in the BAL of asthmatic rats was markedly reduced, and this was strongly reversed by treatment with HyPE, bringing it to the level of the normal rats, as shown in Fig. 3.

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Fig. 2. Effect of HyPE on cysteinyl leukotriene (CysLT; LTC4, LTD4, and LTE4) level in the bronchoalveolar lavage (BAL) of OVA-induced asthmatic rats. BAL was collected upon death, and CysLT levels were determined by enzyme immunoassay, as described in MATERIALS AND METHODS. Data are means ± SE for 10 rats. a, bP < 0.01. There was no significant difference between HyPE-treated and naïve rats.
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Fig. 3. Effect of HyPE on PGE2 levels in BAL of rats with OVA-induced asthma. PGE2 levels were determined in BAL fluid by RIA (see MATERIALS AND METHODS). Data are means ± SE for 10 rats. Bars that have a common letter are significantly different at P < 0.01.
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cPLA2 and sPLA2 in lungs of asthmatic rats.
As noted in the Introduction, it has been previously suggested that LT are produced from an AA pool provided by sPLA2, whereas PG are produced from an AA pool provided by cPLA2 (1, 25). Along this line, we have determined the levels of the PLA2 isoforms in rat lungs following the different treatments, first by immunohistochemistry.
cPLA2 immunoreactivity was measured by the density of the immunochemical staining. As illustrated in Fig. 4, in general, cPLA2 was observed in alveolar macrophages, lymphocytes at the site of inflammatory infiltrate (mainly in the untreated asthmatic rats), in ASM cells, and in airway-ciliated epithelial cells. Staining with PAS reagent for mucus-secreting (goblet) cells was negative, implying that goblet cells were not formed, as expected in acute allergic inflammation. It is well known that mucus metaplasia is not significant in acute inflammation and generally occurs in later stages of chronic asthma. In the control and asthmatic rats, the staining in the epithelial cells was mainly in the apical membrane, whereas in the HyPE-treated group, in which the staining was enhanced, cPLA2 was localized primarily in the cytoplasm of the ASM and epithelial cells.

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Fig. 4. Immunohistochemistry of cytosolic phospholipase A2 (cPLA2) in lung of rat with OVA-induced asthma. A: naïve, control rats. B: OVA-challenged rat. C: OVA-challenged rats treated with HyPE (x40 magnification). D: total staining density. Data are means ± SE for 50 arbitrarily chosen fields. a, bP < 0.05; cP < 0.01. E: point counting of cPLA2 staining in epithelial cells (expressed as percentage of stained/total epithelial cells). Data are the average of points counted in 3 separate sections for each treatment. a, bP < 0.01.
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The corresponding quantitation of cPLA2 is depicted in Fig. 4D, presenting total staining density, and Fig. 4E, presenting point counting of cPLA2 in the epithelial cells. The point-counting procedure could not be applied to ASM layer, since it exhibited multiple nuclei, which could not be sufficiently distinguished from the cytoplasm. Figure 4, D and E, shows that cPLA2 expression was reduced in the asthmatic, untreated rats, but treatment with HyPE reversed this process and enhanced the cPLA2 level even beyond that of naïve rats. Comparison of Fig. 3 with Fig. 4 shows that the changes in cPLA2 paralleled those in PGE2 production. In the same experiment, sPLA2 could not be detected by immunochemical staining of lung tissues, and this could be either due to inaccessibility of sPLA2 (which is membrane bound) to its antibody and/or due to the sPLA2 secretion to the extracellular medium.
To further elaborate on the changes in cPLA2 and sPLA2, we determined the protein expression of these isoforms in the lung tissue homogenates following the different treatments. As shown in Fig. 5, using this method, sPLA2 could also be detected in the lung tissue. This figure shows that, similar to the immunohistochemical staining (Fig. 4), cPLA2 protein expression was lower in the asthmatic rats and was recovered by treatment with ExPLI. At the same time, sPLA2 protein expression was elevated in the lung of asthmatic rats and was reduced by treatment with ExPLI.

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Fig. 5. Effect of HyPE on cPLA2 and secretory phospholipase A2 (sPLA2) expression in lung of rats with OVA-induced asthma. Shown are Western blot (A) and corresponding densitometry (B) of cPLA2 and sPLA2 in lung homogenates of rats with OVA-induced asthma, treated as indicated. In B, for each enzyme, the density values were normalized to corresponding naïve rats.
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PLA2 activity in lung tissues.
Subsequent to the changes in PLA2 protein levels in lung, the related activities in lung tissues were measured by the hydrolysis of lipid membrane composed of AA-containing PL, which is a substrate for both isoforms. To differentiate the cPLA2 activity from that of sPLA2, the hydrolysis was determined in the absence of the reducing agent DTT (to obtain total activity) and in the presence of DTT, which abolishes sPLA2 activity (14), thus enabling the determination of cPLA2 activity alone (see MATERIALS AND METHODS). As shown in Fig. 6, cPLA2 activity was sharply suppressed in lung tissues of the asthmatic rats and was augmented to that of the control healthy rats by treatment with HyPE. The corresponding changes in sPLA2 enzymatic activity in lung tissues (calculated by deduction of the cPLA2 from total activity) were in the opposite direction to that of cPLA2, as was expected, i.e., elevated in the lungs of asthmatic rats and reduced by treatment with HyPE (not shown). It should be noted that attempts to determine sPLA2 activity in BAL were not successful, probably due to excessive dilution during the process of BAL collection.

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Fig. 6. Effect of HyPE on cPLA2 activity in homogenates of lung tissue of rats with OVA-induced asthma. cPLA2 activity was determined by the capacity of homogenates of lung tissue of rats, treated as indicated, to hydrolyze 2-arachidonic acid (AA)-phosphatidylcholine containing liposomes in the presence of DTT, as described in MATERIALS AND METHODS. Data are means ± SE for 8 rats. aP < 0.05; bP < 0.01.
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DISCUSSION
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The contributions of cPLA2 and sPLA2 to the development of inflammatory/allergic diseases have been the subject of disparate reports and sometimes of disagreement. As discussed above, since cPLA2 is specific to AA-carrying PL, and its activation has been linked to production of PG, it has been suggested that this isoform is the important one in the production of proinflammatory eicosanoids. On the other hand, sPLA2 activation and participation in pathophysiology have been observed in many inflammatory conditions (7, 25). In addition, it has been suggested that in cells that express sPLA2, the bulk of AA is produced by sPLA2 of different types, particularly under inflammatory conditions (2, 25, 34), and sPLA2 enzymes have thus been considered the inflammatory enzymes (13, 18). As discussed in the Introduction, many studies suggested appositive feedback and a synergistic action between sPLA2 and cPLA2, but the relative contribution of the two isoenzymes to inflammatory conditions and the interrelation between them might differ between tissues and cell types.
With regard to asthma, previous studies suggested that cPLA2 plays a role in its pathophysiology, and it has been reported that cPLA2-deficient mice had decreased sensitivity to induction of asthma by OVA (35, 38). On the other hand, it has been reported that in ASM, cPLA2 is responsible for the release of PGE2 (30), which acts as an antagonist to CysLT by inducing bronchodilation and inhibiting smooth muscle cell mitogenesis (17, 40), and that "the capacity of airway epithelium to CysLT synthesis is inversely related to its ability to make PGE2" (17). In accordance with that, it has been reported that in cells that play a key role in asthma, namely human eosinophils and basophils, PGE2 is produced from a cPLA2-linked AA pool, whereas CysLT are produced from a different, sPLA2-linked AA pool (7, 24). Indeed, sPLA2 is found at high levels in BAL and bronchotracheal smooth muscle cells (32, 39). These reports thus indicate opposing roles for these enzymes in asthma pathophysiology. This hypothesis is clearly supported by the findings of the present study, summarized in Table 1. This table shows that in asthmatic rats, OVA-induced bronchoconstriction is accompanied by elevation of airway sPLA2 protein expression and CysLT production, concomitantly with suppression of cPLA2 protein expression and activity, and PGE2 production. These processes are reversed when the rats are treated with cell-impermeable sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and decrease in sPLA2 expression and CysLT production, concomitantly with enhancement of PGE2 production and cPLA2 expression. Hence, this study shows, for the first time in vivo, inverse modulation of sPLA2 and cPLA2 in asthma pathophysiology and further suggests that their activities are interdependent.
Although the concomitant elevation of sPLA2 and suppression of cPLA2 suggests a linkage between the enzymes, simultaneous appearance of two phenomena does not necessarily suggest a cause-and-effect relationship between them, and the two effects might be independent results of a common effector. In accordance with that is the aforementioned report that in human eosinophils, CysLT production results from sPLA2 activity independent of cPLA2 (24). However, the present study shows that treatment of the asthmatic rats with the sPLA2 inhibitor not only suppressed this sPLA2 expression (and corresponding CysLT production) but also upregulated cPLA2 expression (and corresponding PGE2 production). This strongly suggests a negative feedback between sPLA2 and cPLA2 types.
A possible explanation for this phenomenon can be that a product of PL hydrolysis by sPLA2 downregulates cPLA2 expression and activity. For example, sPLA2, which is enhanced in airways of allergen-stimulated asthmatic patients, produces excessive amounts of LysoPL (5, 8), which in turn induces cPLA2 phosphorylation (18, 19, 26). Although cPLA2 phosphorylation is generally assumed to induce the enzymes activation, the opposite was found (in vascular smooth muscle cells) when cPLA2 was phosphorylated by cGMP-dependent kinases, which inhibited the enzymes activity as expressed by AA production (26). It is likely that a similar process is responsible for the sPLA2-associated suppression of cPLA2 activity and PGE2 production and its reversal by the sPLA2 inhibitor. Yet, in the present study, we have found that sPLA2 activation is associated not only with suppression of cPLA2 activity (Fig. 6) but also with its protein expression (Figs. 4 and 5), and this is reversed by the ExPLI. sPLA2 is able to affect cPLA2 expression and activity by induction of cell signaling via a receptor-mediated process, independently of its lipolytic activity (1). Although this usually leads to increased cPLA2 synthesis, on the grounds of the present study we cannot rule out an opposite effect in OVA-induced bronchoconstriction, as suggested by the negative feedback modulation of sPLA2 and cPLA2. It is therefore possible that sPLA2 modulates cPLA2 in more than one way, and the exact mechanism(s) of this interrelationship in OVA-induced bronchoconstriction has yet to be explored.
All in all, the present study, which shows for the first time in vivo that OVA-induced bronchoconstriction is controlled by inverse modulation of sPLA2 and cPLA2, supports the hypothesis that these enzymes have opposing roles in the pathophysiology of asthma. These findings also support the previous reports that in airway epithelial cells, CysLT, the major bronchoconstricting eicosanoids, are produced subsequent to sPLA2 action, whereas the bronchodilator PGE2 is produced from a separate, cPLA2-linked AA pool. It is plausible to conclude that in OVA-induced asthma in rats, sPLA2 activity contributes to the disease development while cPLA2 activity contributes to its amelioration, and the control of the negative feedback between these isoenzymes by inhibition of sPLA2 might introduce a potentially novel approach in the treatment of asthma.
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GRANTS
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This study was supported by a grant to S. Yedgar from the Walter and Greta Stiel Chair and an Israel Ministry of Health grant (to D. Shoseyov and S. Yedgar).
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FOOTNOTES
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Address for reprint requests and other correspondence: S. Yedgar, Dept. of Biochemistry, Hebrew Univ.-Hadassah Medical School, Jerusalem, Israel 91120 (E-mail: yedgar{at}md.huji.ac.il)
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.
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