Pulmonary inflammation and edema induced by phospholipase A2: global gene analysis and effects on aquaporins and Na+/K+-ATPase.

Victims of snakebite quickly succumb to severe respiratory failure, which can be fatal if left untreated. One of the most toxic components of snake venom is phospholipase A2 (PLA2; EC 3.1.1.4). PLA2 isolated from the elapid, Naja sputatrix, induced pulmonary inflammation and edema when administered intravenously and intratracheally to rats. Analysis of pulmonary gene expression profiles using oligonucleotide microarrays revealed 60 genes whose expression was altered by at least 3-fold in response to intratracheal instillation of PLA2 for 3 h as compared with controls. In addition to genes encoding cytokines and chemokines responsible for inflammatory processes, the Na+/K+-ATPase gene has been found to be involved in edema formation. Real-time PCR, Western blot, and immunohistochemical analyses confirmed that the expression of AQP1 and AQP5 mRNAs and proteins was decreased. Besides providing an experimental model for studies on the pathophysiology of the lung, this investigation yields a clue to the mechanisms by which endogenous PLA2s could mediate inflammation in conditions such as allergy and rheumatoid arthritis.

Snake venoms are chemically complex mixtures of pharmacologically active proteins, many of which also have enzymatic properties (1). Because venoms serve not only as a source of digestive enzymes but also as a defense mechanism, these proteins can target multiple tissues, causing a simultaneous poisoning of multiple physiological systems. From these venoms, hundreds of toxins have been purified and characterized. One of the components that contributes significantly to the lethality of snake venoms is phospholipase A 2 (PLA 2 ) 1 (EC 3.1.1.4) (2). Venom PLA 2 s are low-molecular weight (13,000 -14,000 Da), secretory phospholipases containing seven disulfide bonds (3). Like all PLA 2 s, venom PLA 2 catalyzes the hy-drolysis of the sn-2 fatty acyl bond of phospholipids to release free fatty acids and lysophospholipids in the presence of Ca 2ϩ (4). However, unlike secretory pancreatic PLA 2 , which serves a mainly digestive function, venom PLA 2 exhibits a host of additional pharmacological properties that include antiplatelet, anticoagulant, hemolytic, neurotoxic (presynaptic), myotoxic, hemorrhagic, inflammatory, and cardiotoxic activities (5)(6)(7).
Neutral PLA 2 purified from the venom of the elapid, Naja sputatrix (nsPLA 2 ; Malayan spitting cobra) was used in this study. This is a Group IA PLA 2 that has a disulfide bridge between the half-cysteines at positions 11 and 69. PLA 2 found in mammalian pancreatic juice belongs to Group IB as it contains a pancreatic loop. However, both groups are secretory PLA 2 s. Another group of secretory PLA 2 s, Group II PLA 2 s are found in crotalid venom and mammalian cells (3,8). In our laboratory, we have found that when nsPLA 2 is injected intravenously into rats, it brought about death characterized by paralysis following muscle spasms and respiratory failure. This was accompanied by inflammation and edematous swelling of lung tissue. Respiratory compromise is a sign of moderate to severe envenomation and is observed in many victims of snake bites (9 -11). In fact, envenomation by elapids has been shown to produce respiratory distress followed by death within 10 min (12). Being a presynaptically acting PLA 2 , nsPLA 2 blocks acetylcholine release at the neuromuscular junction, leading to muscle paralysis (13) and airway paralysis as well. The mechanism by which it causes pulmonary inflammation and edema, however, is not understood. Maintenance of clear airspaces and regulation of fluid movement between the vascular, interstitial, and airway compartments are vital for normal lung function. Disruption of this homeostasis will severely affect gas exchange and lung mechanics, leading eventually to respiratory failure. Previous studies of venom toxin-induced lung inflammation provide clues to pathophysiology (14 -16) but expression studies were at best limited to a few selected genes.
In this report, we investigate the mechanisms of nsPLA 2induced inflammation and fluid accumulation by studying the gene expression profiles of lungs from rats treated intravenously and intratracheally with nsPLA 2 for 10 and 1 and 3 h, respectively, using oligonucleotide microarrays. This technology allows the simultaneous quantitation and comparison of the expression of ϳ8000 genes and expressed sequence tags found in rat genome arrays. Here, we show that the expression of inflammatory cytokines and related genes (e.g. cyclooxygenase-2) was significantly up-regulated while interestingly, the expression of water-transporting membrane channels such as aquaporins and the Na ϩ /K ϩ -ATPase pump was decreased. These observations shed light on the pathways activated by nsPLA 2 during pulmonary inflammation and consequently, on the causes of respiratory failure upon snake envenomation.
Additionally, the data may provide evidence for a physiological role played by water and ion channels in the maintenance of lung fluid balance.

MATERIALS AND METHODS
Purification of nsPLA 2 -N. sputatrix crude venom was obtained from Sigma. The nsPLA 2 was purified using Sephadex G-100 gel filtration chromatography (Amersham Biosciences) followed by reverse phase high perofrmance liquid chromatography and then characterized as described (16). The purified fractions were quantitated using the Bradford assay (Bio-Rad).
Experimental Groups-All animals were handled according to the guidelines (58) given by the Council for International Organization of Medical Sciences (CIOMS) on animal experimentation (WHO, Geneva, Switzerland). Male Sprague-Dawley rats (200 -220 g) were maintained on an ad libitum intake of standard laboratory chow and drinking water prior to and after treatments. Various doses of nsPLA 2 , which has an LD 50 value of 125 g/200 g body weight in Sprague-Dawley rats, were administered either intravenously or intratracheally. For intravenous administration, the rats were restrained and nsPLA 2 was delivered via tail vein injection. For intratracheal administration, the rats were anaesthetized intraperitoneally with a mixture (100 l/100 g body weight) consisting of an equal volume of hypnorm (0.315 mg of fentanyl and 10 mg of fluanosome; Jansen Pharmaceutica, Beerse, Belgium) and midazolam (5 mg of dormicum and 2 ml of water; Roche Diagnostics). An anterior midline incision was made and nsPLA 2 was instilled into the trachea using a 27 [1,2]-gauge needle and a 1-ml syringe (BD Biosciences). The tracheal site was closed using 3/0 silk sutures (Aesculap AG & Co. KG, Tuttlingen) and the rats were allowed to recover. Consequently, there were a total of 3 treatment groups. (a) Intravenous administration of 240 g/200 g body weight of nsPLA 2 for 10 h (PLA2 IV 10 h; n ϭ 6); (b) intratracheal administration of 48 g/200 g body weight of nsPLA 2 for 1 h (PLA2 IT1hr, n ϭ 6); and (c) 3 h (PLA2 IT3hr, n ϭ 9). Control rats (n ϭ 15) were treated similarly but with 100 l of vehicle (0.9% NaCl) instead. All rats were sacrificed at the various time points by cervical dislocation and whole lungs were rapidly removed, pooled, and snap-frozen in liquid nitrogen. Frozen lungs were stored at Ϫ70°C until use.
RNA Isolation-Total RNA was isolated from nsPLA 2 -treated and control lungs by a single-step method using Trizol reagent (Invitrogen) from lung tissues. The RNA samples were subsequently treated with RNase-free DNase I at 37°C for 20 min and stored at Ϫ70°C until use.
Protein Isolation-Lung tissue from nsPLA 2 -treated and control rats were isolated and placed in chilled isolation buffer containing 0.9% NaCl and 50 mM EDTA. Whole tissues were homogenized in 3 ml of ice-cold homogenization buffer containing 50 mM Tris-HCl adjusted to pH 7.4, 300 mM mannitol, and 50 mM EDTA. Homogenates were centrifuged at 100 ϫ g for 20 min at 4°C. The supernatant was removed and centrifuged at 14,000 ϫ g for 30 min. The resulting supernatant was collected and the total protein concentration in each sample was measured using the Bradford assay (Bio-Rad).
Western Blot Analysis-Total membrane proteins (70 g/sample) were separated by Tris-Tricine SDS-PAGE as described (18). Electrotransfer was carried out for 1 h on ice onto nitrocellulose membranes (Protran TM , Schleicher & Schuell). Membranes were blocked for 1 h in 1% blocking solution (skim milk in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 3 mM KCl, and 0.1% Tween 20)) followed by incubation overnight at 4°C with affinity purified, polyclonal anti-AQP1 (a kind gift from Dr. Mark Knepper, National Institutes of Health) at a dilution of 1 g/ml and anti-AQP5 at a dilution of 2 g/ml (Alpha Diagnostics International, San Antonio, TX) in 0.5% blocking solution. After three washes of 10 min each in TBST, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat antirabbit (AQPs 1 and 5; Bio-Rad) secondary antibody at a dilution of 1:50,000 in 0.5% blocking solution, washed twice for 10 min each in TBST, and visualized via enhanced chemiluminescence (SuperSignal; Pierce) with variable exposures (Kodak-MS film). To confirm equivalent loading of samples, separate sets of gels were run simultaneously and stained with Coomassie Brilliant Blue. Films of Western blots were scanned (Acer SWZ3300U) and labeling intensities of the bands were quantitated using AIS software (Synoptics Imaging Systems).
Histology and Immunohistochemistry-Histopathologic changes and immunohistochemical staining for AQP1 and AQP5 were evaluated in nsPLA 2 -treated and control lungs fixed in 4% paraformaldehyde. Fixed lung tissues were embedded in paraffin, sectioned at 7-m, and stained with hematoxylin and eosin for morphologic analysis. Paraffin-embedded tissues were sectioned at 5 m and processed as described (19) for immunohistochemical staining using the same AQP1 and AQP5 antibodies as those for Western blot analysis.
Microarray GeneChip TM Analysis-Total RNA isolated from rat lungs was processed and hybridized to each array of the RG_U34A GeneChip TM Array Set according to the protocols described in the GeneChip TM expression analysis technical manual (Affymetrix, Santa Clara, CA). Two chips were used for each experimental group. Data from each treatment were scaled to an average intensity of 800 and a comparison of signal intensities using Microsoft Excel showed results from duplicate chips highly correlated with R 2 values ϭ 0.98. Relative mRNA expression levels were expressed as plus or minus fold changes compared with saline-treated controls using Microarray Suite software 5.0 (Affymetrix). All genes showing a change of 3-fold or more in at least one experimental condition were included in subsequent analyses. Selforganizing map clustering was performed using GeneSpring 5.1 (Silicon Genetics, Redwood City, CA). Values below 0 were set to 0 and per chip normalization was carried out by dividing each measurement by the 50th percentile of all measurements in that sample. Per gene normalization was performed by dividing each gene by the median of its measurements in all samples. Normalized profiles were grouped on the basis of similarity in their temporal expression patterns using the self-organizing map. Cluster members were classified according to their biological functions as described in the NetAffx data base (Affymetrix).
Wet-Dry Lung Weight Ratios-Whole lungs were excised from ns-PLA 2 -treated and control rats and weighed to obtain lung wet weights (n ϭ 3 per group). Lungs were dried in a 62°C oven with desiccant and weighed again after 7 days to obtain dry weights.
Statistical Analysis-Statistical analyses were performed using unpaired Student's t test and results were expressed as mean Ϯ S.E. A p value of Ͻ0.05 was considered statistically significant.

RESULTS
Intravenous injection of nsPLA 2 (240 g/200 g body weight; 2ϫ LD 50 ) into rats caused death characterized by paralysis and severe respiratory distress after ϳ10 h. Morphologic observation of the lungs indicated gross swelling and inflammation. To quantitatively assess pulmonary edema in animals injected with nsPLA 2 intravenously, whole lung wet-to-dry weight ratios were determined. The wet-to-dry weight ratio of whole lungs from rats 10 h after introduction of nsPLA 2 intravenously proved to be significantly increased (170 Ϯ 1% of salinetreated controls, p Ͻ 0.05; Fig. 1B). To study the effects of nsPLA 2 on the lungs alone, the toxin was administered locally via intratracheal instillation (48 g/200g body weight, 0.4ϫ LD 50 ) for 1 and 3 h. Because nsPLA 2 was applied directly to the lungs of rats by intratracheal administration, a much lower dose of nsPLA 2 (0.4ϫ LD 50 ) was required to induce inflammation and edema as compared with that for intravenous injection (2ϫ LD 50 ), which would have caused nsPLA 2 to enter the systemic circulation. Lung histology was used to assess inflammation in rats after intratracheal instillation of nsPLA 2 for 3 h (Fig. 1A). Pulmonary infiltrate and edema fluid were observed in the alveolar airspaces 3 h after nsPLA 2 administration (Fig.  1A, center and right panels) as compared with the salinetreated control (Fig. 1A, left panel), which showed clear airspaces. The infiltrate was not detected in bronchial or pulmo-nary vascular regions. Alveolar epithelia remained intact and there were no indications of cell membrane disruption or lysis. Lung sections obtained from rats treated intravenously with nsPLA 2 for 10 h showed the same extent of cellular infiltration and fluid accumulation as those for PLA2 IT3hr lungs whereas lung sections from rats treated intratracheally with nsPLA 2 for 1 h showed a lesser extent of inflammation (results not shown). Quantitative measurement of edema showed that the wet-todry weight ratios of PLA2 IT1hr and PLA2 IT3hr lungs were significantly increased (138 Ϯ 2 and 185 Ϯ 1% of saline-treated controls, respectively, p Ͻ 0.05; Fig. 1B). To understand the molecular mechanisms underlying the nsPLA 2 -induced pulmonary inflammation and edema, microarray analysis was performed on intratracheally treated lungs. Lung tissue was pooled from 3 rats per treatment group to minimize interindividual variations. Each GeneChip in the RG_U34A Array Set represents ϳ8800 genes and expressed sequence tags. All probe sets designated "absent" by the analysis software were discarded and only genes whose expression changed by 3-fold or greater in at least one pairwise comparison were deemed significant. These probe sets were subjected to self-organizing map clustering where genes were grouped according to their expression patterns in response to nsPLA 2 administration over two time points. Two clusters were selected and they contained genes whose expression was changed after nsPLA 2 treatment for 1 h followed by a dramatic increase or decrease in expression after 3 h (Fig. 2A). The changes at both time points occurred in the same direction in all cases and hence, indicate that a longer exposure to nsPLA 2 only augmented its effects at the molecular level. Cluster 1 represents genes whose expression was induced after intratracheal instillation of nsPLA 2 and which may play important roles in the development of inflammation and edema. Cluster 2 represents genes that were downregulated by nsPLA 2 . Despite the apparent similarity in expression profiles within each cluster, the representation of each gene by its normalized signal intensity shows the varying extent to which nsPLA 2 alters gene expression. These genes were further assigned to categories according to their known functions in physiological pathways and the results are shown in Table I, together with the corresponding -fold changes in expression. Taken together, Cluster 1 consists of genes primarily involved in causing and perpetuating inflammation (cytokines, chemokines and acute phase proteins), signal transduction (growth factors, receptors, kinases, phosphatases, and transcription factors), and oxidative damage (generators of free radicals and products of oxidative stress), whereas Cluster 2 contain gene groups that are less well defined, including those encoding signaling proteins (e.g. cyclase-associated protein homologue and protein kinase, MUK), proteins involved in lipid metabolism (e.g. pancreatic phospholipase A 2 and fatty acid synthase), a protease (cathepsin D), an antioxidant (glutathione S-transferase), and a heat shock protein (HSP-90). Expectedly, inflammatory genes such as macrophage inflammatory protein-1 (ϩ42.2-and ϩ104.0-fold increases 1 and 3 h, respectively) and interleukin-6 (ϩ7.0-and ϩ362.0-fold after 1 and 3 h, respectively) were most highly up-regulated in response to nsPLA 2 treatment as compared with the saline-treated control. Interestingly, the expression of cyclooxygenase-2 (COX-2) was increased 27.9-fold whereas lipoxygenase-5 expression was unchanged. Both these enzymes generate distinct eicasanoids from arachidonic acid. Induction of COX-2 alone indicates that this pathway is activated by nsPLA 2 rather than the lipoxygenase-5 pathway and its downstream products mediate inflammation.
A gene that may provide a clue to the development of pulmonary edema is Na ϩ /K ϩ -ATPase-␣. Its expression was significantly decreased (Ϫ3.7-fold) 3 h after intratracheal instillation. Real-time PCR analysis was carried out to validate the results obtained by microarray (Fig. 3). It was shown that the expression of Na ϩ /K ϩ -ATPase-␣ was indeed down-regulated (ϳ3.3-fold, p Ͻ 0.05) by nsPLA 2 after 3 h while its expression was not significantly increased after 1 h (ϳ0.7-fold). The catalytic ␣ subunit of the ATPase exchanges intracellular Na ϩ for extracellular K ϩ , generating an electrochemical gradient that allows coupling of the transport of other molecules into the cells, including the osmotic flow of water (20). Active Na ϩ transport across the alveolar epithelium has been shown to be necessary for the resolution of pulmonary edema (21-23) as Na ϩ flux produces a transepithelial osmotic gradient that causes water to move passively from the airspaces to the alveolar interstitium. Hence, a decrease in Na ϩ /K ϩ -ATPase expression would cause fluid to accumulate in the lungs.  , ϫ310; right panel, ϫ490). Determination of wet-to-dry weight ratios from whole lungs of rats (n ϭ 3 per group) treated intratracheally for 1 (PLA2 IT 1hr) and 3 h (PLA2 IT 3hr) and intravenously for 10 h (PLA2 IT 10hr) with ns-PLA 2 revealed an increase in lung fluid after nsPLA 2 administration as compared with the saline-treated controls (B). Values obtained as grams of wet weight/ grams of dry weight are expressed as percentage of control Ϯ S.E. *, p Ͻ 0.05.
In recent years, the discovery of specific water channels known as aquaporins has deepened our understanding of water transport and homeostasis in cells. There are now 11 known AQPs of which AQPs 1 and 5 are found in the distal lung in rats (24). Although noticeably absent from Fig. 2 (expression of AQPs 1 and 5 was altered less than 3-fold by nsPLA 2 ; data not shown), we decided to investigate for any significant changes in the expression of AQPs 1 and 5 in nsPLA 2 -treated rat lungs. Quantitative real-time PCR analysis was carried out for the same pooled RNA samples using a trio of specific primers and probe for each gene (Fig. 4A). The relative expression of AQPs 1 and 5 in PLA2 IV10hr, PLA2 IT1hr, and PLA2 IT3hr lungs was obtained after normalizing against an internal control (18 S ribosomal RNA) and a calibrator, in this case, their salinetreated controls (relative gene expression ϭ 1). Both AQPs 1 and 5 were significantly down-regulated after intratracheal instillation of nsPLA 2 for 3 h (AQP1, ϳ20-fold and AQP5, ϳ10-fold, respectively, p Ͻ 0.01) while only AQP1 expression was significantly decreased after 1 h (ϳ1.7-fold, p Ͻ 0.05). Intravenous injection of nsPLA 2 also dramatically reduced the expression of AQPs 1 and 5 after 10 h (ϳ100 fold, p Ͻ 0.01).
To determine whether the decrease in mRNA levels of AQPs 1 and 5 resulted in a corresponding decrease in their protein levels, Western blot analysis was carried out on PLA2 IV10hr, PLA2 IT1hr, and PLA2 IT3hr lungs. Using an affinity purified polyclonal antibody directed against the AQP1 carboxyl-terminal synthetic peptide, Western blot analysis revealed bands at 28 and ϳ35 kDa, representing the native and glycosylated forms, respectively (Fig. 4B). Densitometry of the 28-kDa AQP1 band showed a significant decrease in protein levels 1 and 3 h after intratracheal instillation of nsPLA 2 (9 Ϯ 0.5 and 3 Ϯ 0.2% of the saline-treated controls, respectively, p Ͻ 0.01) and 10 h after intravenous injection of nsPLA 2 (13 Ϯ 1% of the salinetreated control, p Ͻ 0.01; Fig. 4C). AQP5 protein expression was analyzed by Western blot using an affinity purified polyclonal antibody directed against the AQP5 carboxyl-terminal synthetic peptide and a band at 29 kDa was revealed (Fig. 4B). Protein levels were significantly decreased 3 h after intratracheal instillation of nsPLA 2 (29 Ϯ 3% of the saline-treated control, p Ͻ 0.01) and 10 h after intravenous injection of ns-PLA 2 (4 Ϯ 0.5% of the saline-treated control, p Ͻ 0.01; Fig. 4C). AQP5 protein levels of PLA2 IT1hr lungs were not statistically distinct from that of the control (98 Ϯ 2%, p Ͻ 0.01; Fig. 4C). Clearly, there is considerable overlap in the results obtained for both mRNA and protein determinations of changes in AQPs 1 and 5 expression in rats administered nsPLA 2 .
Immunohistochemistry was carried out to determine the localization and expression of AQPs 1 and 5 in the lungs of rats FIG. 2. Self-organizing map and functional clustering of genes that exhibited a 3-fold or greater change in expression after intratracheal instillation of nsPLA 2 for 1 and 3 h. Two selected self-organizing map clusters are shown and they contain genes whose expression profiles were altered in a similar pattern after nsPLA 2 administration. The normalized average signal intensity for each gene was plotted against time. treated intratracheally with nsPLA 2 for 1 and 3 h. AQP1 had previously been shown to be present in bronchial and alveolar vascular endothelium and fibroblasts in the distal rat lung whereas AQP5 was localized in the apical epithelial of Type I pnuemocytes (23). The localization of AQP1 remained unchanged in nsPLA 2 -treated and saline-treated control lungs. By light microscopy, staining for AQP1 was decreased in PLA2 IT1hr and PLA2 IT3hr lungs (Figs. 5, B and C, respectively) as compared with that for the control (Fig. 5A). AQP1 staining was decreased more prominently in PLA2 IT3hr than in PLA2 IT1hr lungs. Similarly for AQP5, staining was reduced in ns-PLA 2 -treated lungs in comparison to the saline-treated control, with PLA2 IT3hr lungs showing a more marked decrease in AQP5 staining (Fig. 5, D-F). Although staining for both AQPs 1 and 5 was clearly observed in the alveolar regions, the exact cell types that were stained could not be differentiated. DISCUSSION The biochemical, immunological, and pharmacological properties of snake venoms have been subjects of keen research for a long time. These venoms contain many proteins and polypeptides that possess enzymatic activity as well as exhibit patho- physiological properties. Not only have the studies of these toxins aided in the treatment and management of snake envenomation, they have also been a source of research tools crucial for the elucidation of molecular mechanisms underlying physiological processes. A component of all snake venoms, phospholipase A 2 (25), is of particular interest. PLA 2 enzymes are ubiquitous in nature and can be classified into two groups: intracellular or extracellular/secretory. Intracellular PLA 2 is cytosolic and can be Ca 2ϩ -dependent or independent. Extracellular PLA 2 is divided into four groups and all require millimolar concentrations of Ca 2ϩ for enzymatic activity. Venom PLA 2 belongs to Group I of the secretory PLA 2 family, as do pancreatic and mammalian PLA 2 , and is characterized by its low molecular mass and high disulfide bond content (26,27). To date, venom PLA 2 has been used to evaluate the structural function of phospholipids and their roles as mediators of biological processes like the phospholipids methylation system (28,29), to determine asymmetric organization of phospholipids in biomembranes (30 -32), as models of mammalian intracellular phospholipases that are difficult to obtain in sufficient quantities for meaningful studies and to aid in the understanding of presynaptic control mechanisms of neurotransmitter release (33)(34)(35).
The main cause of death upon cobra envenomation is peripheral respiratory paralysis because of both presynaptic and postsynaptic neurotoxins present in the venom (36). However, intravenous injection of nsPLA 2 , a presynaptically acting neurotoxin, into rats induced marked inflammation and edema in the lungs. Given the prolonged period of envenomation prior to death (Ͼ10 h) and the massive accumulation of pulmonary fluid during this time, inflammation must play an important role in venom-induced respiratory failure, an aspect often overlooked. This observation led us to investigate the proinflammatory and edema-inducing properties of nsPLA 2 . Whereas these effects are known (14,37,38), little had been done to decipher the molecular determinants involved in venom PLA 2 -induced inflammation and edema, especially in the lungs.
The results of this study had identified 60 pulmonary genes whose expression was changed by nsPLA 2 (Fig. 6). A majority of the genes induced by nsPLA 2 are mediators of inflammation (Fig. 2). The expression of cytokines, interleukin-1␤ (IL-1␤), interleukin-6 (IL-6), tumor necrosis factor-␣ (TNF-␣; Fig. 2A, Cluster 1), and small inducible cytokine subfamily A20, was all increased. IL-6, in particular, was induced 362-fold above the control. IL-6 is secreted by macrophages, T cells, and mast cells and serves to enhance the maturation of activated T and B cells. IL-1␤ and TNF-␣ are known to stimulate the release of chemokines, specifically monocyte chemotactic protein-1 and IL-8 (39), which recruit neutrophils to sites of inflammation. IL-1␤ is also able to stimulate the secretion of macrophage inflammatory protein-1 by monocytes (40). Macrophage inflammatory protein-1 is a chemokine whose expression was increased 104-fold 3 h after intratracheal instillation of nsPLA 2 . A, real-time PCR analysis of total RNA isolated from lungs of rats (n ϭ 3 per group with each measurement performed in triplicate) treated intratracheally for 1 and 3 h and intravenously for 10 h with 48 and 240 g/200 g body weight nsPLA 2 , respectively, showed that AQPs 1 and 5 mRNA expression was significantly decreased relative to the saline-treated controls. B, Western blot analysis was performed to determine the changes in AQPs 1 and 5 protein levels in lung homogenates from PLA2 IT1hr, PLA2 IT3hr, and PLA2 IV10hr rats (n ϭ 3). Equal amounts of protein were loaded as determined by the Bradford assay. Probing with anti-AQP1 revealed 28-and ϳ35-kDa bands, corresponding to the native and glycosylated forms of the protein, respectively. Anti-AQP5 detected a 29-kDa band, corresponding to the AQP5 protein. C, changes in protein expression were quantitated by densitometry which showed that ns-PLA2 administration reduced AQPs 1 and 5 protein levels significantly except in PLA2 IT1hr lungs. Values are expressed as ϮS.E. **, p Ͻ 0.01.
It was one of the most highly up-regulated genes, revealing the critical role that macrophage inflammatory protein-1 plays in chemotaxis and the release of inflammatory mediators. The increase in TNF-␣ gene expression correlated with the decreased expression of the tissue inhibitor of metalloproteinase-1. Metalloproteinases cause TNF-␣ release by facilitating the processing of TNF-␣ by TNF-␣-converting enzyme, TACE (39). Besides being an inflammatory cytokine, TNF-␣ has been hypothesized to be involved in the systemic manifestations of snake envenomations, especially hypotension. How TNF-␣ mediates the lowering blood pressure is not clear. It was also suggested that TNF-␣ might be responsible for the development of local edema (41), a point that is discussed further in the text. Interestingly, IL-18 precursor transcript was down-regulated while there was an increase in IL-1 receptor II transcript. During airway inflammation underlying asthma, immunoregulatory cytokines like IL-12 and IL-18 are down-regulated, presumably to strengthen the type-2 helper cell-driven inflammatory process (42). This may explain the reduced expression of IL-18 precursor in nsPLA 2 -induced inflammation here. IL-1 receptor II serves as a decoy receptor for IL-1␤ (43). Concomitant up-regulation of IL-1 ␤ and its decoy receptor likely represents a physiological mechanism to tightly regulate the inflammatory process.
Recent studies (44,45) have implicated IL-1␤ in yet another role during the inflammatory response. Freund et al. (43) observed that IL-1␤ induced an increase in both the mRNA and protein levels of nerve growth factor in human airway smooth muscle cells. It was also shown that nerve growth factor produced by macrophages and lung fibroblasts and epithelial cells augmented allergic early phase reaction (45). Analysis of gene expression in lungs of rats treated with nsPLA 2 revealed that nerve growth factor-induced gene and -factor A transcripts were both substantially up-regulated (Table I). Nerve growth factor is likely to mediate interactions between immune and nerve cells during inflammation.
A gene of particular interest whose expression had been induced by nsPLA 2 is cyclooxygenase-2. COX-2 and lipoxygenase-5 metabolize arachidonic acid, a product of phospholipid hydrolysis by PLA 2 , via distinct pathways to produce eicosanoids, namely prostaglandins and leukotrienes, respectively. Both these pathways appear to operate cooperatively as inhibition of any one pathway will simply divert the arachidonic acid pool to the other. In addition, prostaglandins and leukotrienes induce the same processes and are both agonists and antagonists of the same pathological processes (27). Hence, it is intriguing to note that nsPLA 2 only induced the COX-2 pathway, implying that the inflammation is mediated primarily through prostaglandins. A possible explanation is that COX-2 synthesis can be up-regulated by TNF-␣ and IL-1␤ as a means of regulating the release of several inflammatory mediators (46). Prostaglandins produce vasodilatation and increase vascular permeability. They also contribute to hyperalgesia, fever, platelet aggregation, and bronchoconstriction. In addition, prostaglandin E 2 has been shown to function as a secondary messenger in the pathways regulating the expression of cytokine receptors (47). Notably, pancreatic PLA 2 expression was reduced in response to nsPLA 2 . Pancreatic PLA 2 , although serves a mainly digestive function, is known to stimulate the expression of cytosolic PLA 2 and COX-2 (48). The down-regulation of its expression during nsPLA 2 -induced inflammation is possibly another example of how finely tuned and closely regulated the inflammatory response is.
Other genes that were induced by nsPLA 2 included inducible nitric-oxide synthase, DNA damage-inducible transcript (Gadd 45), and oxidative stress-inducible protein-tyrosine phosphatase. Arachidonic acid plays an essential role in the activation of NADPH oxidase, which generates the superoxide anion by neutrophils (49). The production of reactive oxygen species is a natural consequence of inflammation and is responsible for tissue damage that occurs in the later stages of the inflammatory process. In addition, nitric oxide is a vasodilator that increases endothelium permeability during inflammation. The expression of methallothein, an antioxidant that has the ability to scavenge superoxide anion, was reassuringly increased al- FIG. 5. Immunohistochemical analysis of AQPs 1 and 5 localization and expression in lung sections from ns-PLA 2 -treated rats. Paraffin sections (5 m) were stained with anti-AQP1 and -AQP5 and then counterstained with hematoxylin. AQPs 1 and 5 staining was decreased in lungs treated with 48 g/200 g body weight nsPLA 2 for 1 (B and E, respectively) and 3 h (C and F, respectively) as compared with the salinetreated controls (A and D, respectively). though it is unclear as to why glutathione S-transferase transcript was reduced. Various transcription factors (e.g. junB and krox24/krox20), signaling molecules (e.g. cyclase-associated protein homolog, SSeCKs, phospholipase-C␤, kinases, and receptors), and inflammation-related genes (e.g. adrenomedullin precursor, gro, plasminogen activator inhibitor-1, and atrial natriuretic peptide clearance receptor-3) were also differentially expressed in response to nsPLA 2 and are important regulators of the inflammatory response.
The development of edema after intratracheal instillation of nsPLA 2 may partly be attributed to the decreased mRNA transcripts encoding the Na ϩ /K ϩ -ATPase. There is clear evidence for the role of Na ϩ /K ϩ -ATPase in edema fluid clearance (50 -52). It is now known that active Na ϩ transport rather than hydrostatic or protein osmotic gradients regulates in vivo alveolar fluid clearance (51). Activation of the channel by ␤-agonists or increasing channel expression by adenoviral transfer of either subunit (␣1 or ␤1) enhanced edema clearance significantly (51). Consequently, a reduced expression would result in pulmonary fluid accumulation. The recent discovery of Na ϩ / K ϩ -ATPase in alveolar epithelial type I cells (52) may implicate aquaporins in edema formation. The physiological significance of AQPs 1 and 5 has been vigorously debated. Studies using knockout mice had concluded that aquaporins played only minor roles in airway humidification, airway surface liquid hydration, and isosmolar fluid reabsorption (53,54). However, the high water permeability of alveolar epithelia (AQP5) and vascular endothelia (AQP1) and the presence of both Na ϩ /K ϩ -ATPase and AQP5 in the type I cells may suggest a cooperative role in maintaining lung fluid balance. Studies demonstrating decreased pulmonary vascular permeability in AQP1-null humans (55) and reduced expression of AQPs 1 and 5 in inflamed lung after acute viral infection (56) also strengthen this view. Indeed, results from our study have shown that AQPs 1 and 5 mRNA and protein levels were significantly decreased in the edematous lungs of rats treated with nsPLA 2 . Active Na ϩ transport across the alveolar epithelium via apical sodium channels and basolateral Na ϩ /K ϩ -ATPase generates a vectorial Na ϩ flux that produces a transepithelial osmotic gradient that causes water to move passively from airspaces to the alveolar interstitium (51) via AQP5. A decrease in Na ϩ /K ϩ -ATPase expression reduces this Na ϩ flux and clearance is impaired. It is unclear if reduced expression of the sodium pump is directly regulated by nsPLA 2 or by an inflammatory mediator. The corresponding decrease in AQPs 1 and 5 expression may be an adaptive response to the change in osmotic pressure or a consequence of active signal transduction pathways. TNF-␣ had been shown to inhibit AQP5 expression via a molecular pathway that involves binding to the p55 TNF-␣ receptor and activating NF-B transcription factor (57). Hence, the induction of TNF-␣ expression by nsPLA 2 led to the downregulation of AQP5 expression. The discrepancy between the differential gene expression for AQPs 1 and 5 as determined by microarray analysis and real-time PCR indicates that conditions and detection sensitivities vary between techniques, despite the apparent similarity in the principles of hybridization chemistry underlying both methods. This highlights the necessity to employ additional techniques to support significant data.
The results of our study initially provide evidence that pulmonary inflammation and edema are likely causes of respiratory failure and death in victims of snake envenomation, in addition to possible airway paralysis. Subsequent analysis of pulmonary gene expression profiles in the lungs of rats intratracheally administered nsPLA 2 revealed an intricate web of cross-talk among signaling pathways that mediated pulmonary inflammation and provided evidence for the physiological significance of water and ion channels in lung fluid homeostasis. Additionally, venom PLA 2 -induced inflammation may serve as an experimental model for studying the mechanisms of action of endogenous secretory PLA 2 s, which are implicated in conditions such as allergic airway inflammation, sepsis, acute pancreatitis, asthma, rheumatoid arthritis and eventually, adult respiratory distress syndrome.