Critical Role of Protein Kinase C ( PKC ) in the Onset of Airway Hypersensitivity in Ova-Sensitized Guinea Pig Model of Asthma

Background and Objectives: Protein kinase C (PKC) activation plays an important role in activation of T-lymphocytes in asthma. Airway hypersensitivity is one of the main characteristic features of asthma, the mechanism of onset of which is not clearly understood. Therefore, the objective was to elucidate the role of PKC in etiopathogenesis of airway hypersensitivity in asthma. Methods: Male guinea pigs (n = 30) were sensitized with ovalbumin and day of initial allergen-specific immune response determined by intradermal test, airway hypersensitivity, BALF cytology and lung histopathology. Total PKC activity, PKC isoenzymes and phosphoinositides were assessed in airway smooth muscles (ASM) and peripheral blood lymphocytes. Results: Intradermal test revealed that day 9 was the earliest time of allergen-specific response and onset of airway hypersensitivity to ovalbumin. It was associated with significant increase in total and differential (lymphocytes and eosinophils) BALF counts and grade I peribronchiolar chronic lymphocytic inflammation in lung. On day 14, grade II infiltration of lymphocytes and eosinophils with onset ofstructural remodelingofproximal and distal airways was seen. Total PKC activity, expression of PKCα, PKCε and phosphoinositides increased significantly in ASM and lymphocytes on day 9 and were maximum on day 14. There was no change in PKC-τ expression. Conclusions: Activation of PKC, particularly PKCα and PKCε, mediated signal transduction pathway plays a critical role in lymphocyte infiltration and onset of airway hypersensitivity, airway remodeling and asthma pathophysiology. The present study is the first one on the mechanism of the etiopathogenesis of the disease, which shows a direct evidence of the role of PKC mediated pathway in the initiation and onset of airway hypersensitivity in ovalbumin sensitized guinea pig model.


Introduction
Asthma affects approximately 10% of the population in the United States and its prevalence has almost doubled in the past 20 years [1,2].In India, its prevalence varies between 2.4% to 31.14% from different parts of the country [3,4].Airway inflammation, persistent airway hyper-responsiveness (AHR) and airway obstruction are the main characteristics of asthma [5].Onset of thisdisease starts with the sensitization to an allergen, followed by IgE-mediated response, mast celldegranulation, bronchoconstriction and recruitment of inflammatorycells.In addition, during progression of the disease, structural changes in the airways like; goblet cell hyperplasia, smooth muscle thickening and subepithelial and airway wall fibrosis are known to occur [6][7][8][9][10].
Asthma is triggered by various stimuli including virus, environmental pollutants, tree and weed pollens, cold air, exercise etc. [11,12].The response of airway cells to these stimuli is mediated through activation of distinct transmembrane signaling intermediates [13].The activation of protein kinase C (PKC) signaling pathway is one of the key players in asthma pathogenesis [14,15].Previously, we and others have shown that inhibition of PKC reduces the activation of lymphocytes, inhibits the expression of Th2 cytokines by T lymphocytes from asthmatic patients [16,17], abolishes airway smooth muscles constriction [18], and inhibits the proliferation and structural changes in airway smooth muscle cells from asthmatic rats [19].In another study, inhibition of PKC by calphostin-C, prevented proliferation of bovine tracheal smooth muscle cells following activation of mannose receptors by β-hexosaminidase and also abolished Ca 2+ -dependent and -independent PKC activity [20] which suggests involvement of different isoenzymes of PKC.
Thus, it is evident that PKC plays a pivotal role in the pathophysiology of asthma.In most of the studies, investigations were carried out after the disease was established and there is no report available on the role of PKC in the etiopathogenesis of the disease.We, therefore, hypothesized that PKC mediated signal transduction pathway might be playing an important role in the onset of the diseases.Since airway inflammation, hypersensitivity and tissue remodeling are the characteristic features of asthma, therefore, first of all we investigated the day of onset of these features followed by the process of PKC activation and expression of its isoenzymes at the onset of the disease using guinea pig model of asthma developed by ovalbumin induced IgE response.The studies were conducted on the airway smooth muscles being the targetsd in lung and the lymphocytes as the inflammatory cweslls (obtained from peripheral blood) and brocn-choalveolar lavage fluid (BALF) for cytological changes and lungs for histopathological changes to assess the pulmonary inflammation.Our results demonstrate that the first ova-specific immune response i.e. onset of the hypersensitivity, can be observed on day 9 after ova-sensitization in the guinea pigs, which coincided with the onset of airway hypersensitivity, increased total PKC activity, and increased expression of PKCα and PKCε isoenzymes.These changes were associated with mild peribronchiolar lymphocytic infiltration and progressive eosinophilic infiltration, suggesting role of PKC in the pathogenesis of the onset of airway inflammation and the disease.

Ethics Statement
The study was approved by the Animal Ethics Commit-tee of Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India.

Study Design
The experiments were conducted on guinea pigs.The animals were sensitized by ovalbumin using standards protocol.The control group received the vehicle alone.The sensitization of the animals was checked every day by intradermal (ID) test using ovalbumin.The day, the sensitized animals showed a positive reaction for the first time, was recorded.The airway responsiveness to ovalbumin was measured by non-invasive body-plethysmography technique daily starting from on day 0 (baseline) till the day when the sensitization was observed by intradermal test for the first time (onset) and finally the day of maximum positivity.Besides it, the cytology BALF and histopathology of lung tissue was also performed to assess the airway changes in the lungs.These parameters established day 9 to be the day of onset of hypersensitivity and day 14 to be the day of maximum hyper-responsiveness.The animals were divided in two main groups, the control an experimental.Each group had 15 animals.Five animals from each group were taken for experiments on day 0, day 9 and day 14.
Blood was collected from each animal directly from the heart for preparing the peripheral blood lymphocytes.The animals were then sacrificed by cervical dislocation.For assessment of inflammatory changes, bronchoalveolar lavage fluid (BALF) was collected for cytology and lung removed and preserved for histopathological examination.For biochemical studies, the trachea and bronchi were removed and airway smooth muscles (ASM) dissected out.The ASM homogenate and lymphocyte lysate were prepared, followed by identification, characterization and quantification of phosphoinositides by thin layer chromatography (TLC), total PKC activity assay by radio-binding and histone phosphorylation methods, PKC isoenzymes by Western blot and identification of target proteins of PKC by protein phosphorylation.The data were analyzed statistically.P < 0.05 was considered significant.The study was approved by the Animal Ethics Committee of the Institute.

Animals
Male Dunkin-Hartley guinea pigs (30 nos.) weighing 350 -450 g were obtained from the V.P. Chest Institutemaintained animal colony.The animals were kept under the condition of husbandry on standard pellet diet and water ad libitum and maintained as per the guidelines for care and use of animals in scientific research issued by Indian National Science Academy, India.The project was approved by the Animal Ethics Committee of the Institute.The animals were divided in two main groups, Group I: control and group II: experimental.Each group had 15 animals.Five animals from each group were taken for experiments on day 0, day 9 and day 14.

Sensitization of Guinea Pigs
The animals were sensitized with 100 µg ovalbumin and 100 mg aluminum hydroxide per ml of normal saline [21].Control group animals received the vehicle alone.The antigen-adjuvant solution was agitated vigorously on a vortex mixer for 90 minutes and 0.5 ml injected intraperitoneally and 0.5 ml subcutaneously by dividing the amount equally among 5 different sites near the lymph nodes, viz.cervical, axillary, lumbar regions, neck and inguinal.This protocol results in sensitization of guinea pigs.

Intradermal Test (i.d.)
For intradermal test, left pinna of the ear was cleaned with alcohol and dorsal surface was injected with 25 µl of normal saline or same volume of 0.2 mg/ml ovalbumin.Histamine solution (5 mg/ml) and physiological saline were used as positive and negative controls, respectively.After 12 to 15 minutes the skin was checked for red, raised, itchy areas (wheal), formation of which indicates an allergen-specific positive immune reaction.Sensitization was considered complete by the doubling of wheal size.The thickness of the ear lobes of the animals was measured.

Specific Airway Conductance (SGaw)
SGaw was determined by dividing airway conductance by the lung volume (SGaw = Gaw/TGV).It was measured by non-invasive whole body plethysmography technique [20].Briefly, the box pressure and airflow was measured (Validyne PM 18 differential transducers) and the signal amplified (CD 12 carrier demodulators).The box pressure signal was fed on the X channel and airflows on the Y channel of oscilloscope (Hewlett-Packard, USA) to obtain an X-Y plot [22].

Collection of Broncho-Alveolar Lavage Fluid and Cytology
The bronchoalveolar lavage fluid (BALF) was collected by exposing the trachea as per the method described earlier [23], and physiological saline (8 ml, 37˚C) was injected in the lung and lavaged twice.The lavage fluid obtained was centrifuged at 500 × g for 10 min at 4˚C.The cell pellet was given osmotic shock to eliminate the erythrocyte contamination as described by Bansal [24].
The cell suspension was centrifuged as before, pellet washed twice with saline and resuspended in 100 µl saline.The total cell count was performed using a Neubaur's Counting Chamber.Smears were stained with Giemsa stain and differential count performed on at least 300 cells in each case.

Histopathology of Lung
A histopathological study of lungs was carried out as described by Underwood et al. [25].Briefly, the lungs were removed, half the portion fixed by slowly inflating with buffered formaline and subsequently embedding in paraffin.5 µm thick sections were stained with haematoxylin and eosin.Lung parenchyma was assessed for changes in bronchi and bronchioles.Peribronchiolarinflammation was assessed and graded on the scale of 0 -3 [26].Gomori's stain for reticular fibers to demonstrate thickening of the sub-epithelial lamina reticularis and Masson Trichrome stain to demonstrate sub-epithelial collagenization were also used.

Preparation of Airway Smooth Muscles (ASM) Extract
The trachea was harvested after sacrificing the animal, and adhering extraneous material to it was carefully removed, smooth muscle portion dissected out, washed in chilled saline and soaked over a clean filter paper.The tissue was then weighed and suspended in 9 volumes of homogenizing buffer (25 mM tris HCl pH 7.5, 2 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 250 mM sucrose) and homogenized in a glass homogenizer in an ice-water bath.Homogenate was then centrifuged at 700 × g for 5 min as above and supernatant used for subsequent analysis.

Preparation of Peripheral Blood Lymphocytes (PBL) Lysate
The peripheral blood lymphocytes were isolated by the method of Boyum [27].Briefly, blood was diluted 1:1 with saline and carefully layered over histopaque (specific gravity 1.077) and centrifuged at 2000 rpm (Rotor no. 9, Rota 4R-V/FM Plasto Crafts, Mumbai, India) for 10 minutes.The opaque ring at the interface containing lymphocytes was collected, diluted with saline and centrifuged at 1500 rpm for 10 minutes.The pellet was washed twice with saline.Contaminating erythrocytes were lysed by incubating the pellet in 0.85% ammonium chloride solution.The PBL lysate was prepared by sonicating the cell suspension in the homogenizing buffer in an Ultrasonicator (Misonix Ultrasonic Processor XL-2020) by giving five bursts of 30 sec each at an interval of one min at 0˚C in an ice-water bath.The cell lysate was centrifuged at 700 × g for 5 minutes and supernatant saved for subsequent analysis.

Protein Estimation
Protein contents of the ASM extracts and PBL lysate were quantified by the method of Lowry et al. [28].
Equal amount of proteins from different experimenttal groups was used for further analyses and determinations.

Phosphoinositide Assay
Phosphoinositides were extracted and resolved by TLC as described by Bansal et al. [29].Briefly, the ASM homogenate or lymphocytes lysate (0.5 ml) in 10 mM LiCl solution containing one mg total protein was thoroughly mixed with 1.8 ml of acidified chloroformmethanol mixture (CHCl 3 :CH 3 OH:HCl; 100:200:2; v/v/v), followed by addition of 0.6 ml of chloroform and 0.6 ml of 2 M KCl.After thorough mixing, the contents were centrifuged at 500 g for 5 min at room temperature, aqueous and organic phases carefully separated and saved in separate tubes.The pellet was washed again with 2 volumes of acidified chloroformmethanol mixture and aqueous and organic phases carefully separated and pooled.Organic phase containing phospholipids was dried under the stream of N 2 and stored at 4˚C till analyzed.Phospholipids were resolved by TLC on silica gel G plates impregnated with 1% potassium oxalate and 1 mM EDTA [30].
The spots corresponding to the standards were scraped.The bands of each phosphoinositide were processed for estimation of total phosphorus by the direct gel digestion method of Misra [31].

Protein Kinase C (PKC) Assay
PKC in ASM extracts and PBL lysate was assayed by two different methods viz.radio-ligand binding assay and histone IIIS phosphorylation as described earlier [32].
For radio-ligand binding assay, briefly, to 25 µl of reaction mixture (50 mM tris HCl, pH 7.5, 1 mM CaCl 2 , 0.1% BSA, 20 mM MgCl 2 •6H 2 O, 2 mM DTT, 5 mg/ml phosphatidyl serine (PS) and 1 µCi/ml [ 3 H]-PDBu) in triplicate, 25 µl sample (30 -50 µg protein) was added to each tube to start the reaction and contents incubated at 30˚C for 25 minutes.In one of the test tubes, phorbol 12-myristate 13-acetate (PMA) was added (final conc 5 µM) which served as control for the non-specific binding.The reaction was terminated by adding 2 ml chilled 10 mM tris-hydrochloride (pH 7.5).The contents were filtered on what man glass fibre filter (25 mm GF/C), washed twice with chilled 10 mM tris HCI (pH 7.5) and processed for radioactivity counting in a liquid scintillation counter (Beckman LS 6500).PKC activity was expressed as femtomoles [ 3 H]-PDBu bound per mg protein under the experimental conditions.
For histone IIIS phosphorylation assay, the standard reaction mixture (50 μl) containing 50 mM tris HCl pH 7.5, 50 µM ATP, 10 mM MgCl 2 •6H 2 O, 100 µM CaCl 2 •2H 2 O, 50 µg histone IIIs, 5 µg PS and 32 P-γ-ATP (0.1 µCi, sp.activity 4000 Ci/mM) was kept on ice.The reaction was started by addition of 50 µl of sample containing 50 µg proteins.The tubes were vortexed and incubated for 3 minutes at 30˚C in a metabolic shaker.The reaction was stopped by the addition of one ml chilled 20% trichloroacetic acid (TCA).Acid precipitable material was collected on a Millipore membrane filter HA type (25 mm, 0.45 µm) under vacuum using a manifold filtration device (Millipore).The filters were washed with one ml of TCA (10%) followed by washing with 2 ml ethanol, transferring of the filters into scintillation vials containing 5 ml dioxane-based scintillation fluid and counting of radioactivity using Beckman LS 6500 liquid scintillation counter.PKC activity was expressed as femtomoles of 32 P-transferred to histone IIIs per mg protein under experimental conditions.

Identification of PKC Isoenzymes by Western Blot Analysis
Equal amounts ofthe ASM extract and PBL lysate proteins (50 µg) were separately mixed with sample buffer, boiled for 5 minutes, resolved by SDS-PAGE on 10% polyacrylamide gel electrophoresis and transferred to PVDF membrane.Positive control for each isoenzyme was run simultaneously.The membrane was incubated with blocking buffer (2% BSA in 10 mM Tris HCl; pH 7.5, 100 mM NaCl, 0.1% tween-20) at 37˚C for 45 minutes and washed thrice with wash buffer (10 mM Tris HCl; pH 7.5, 100 mM NaCl and 0.1% tween-20) for 10 minutes each.The membrane was incubated with primary antibodies (monoclonal, diluted as per the instructions of the manufacturer, M/S Transduction Laboratories, Lexington, Kentucky, USA) for overnight at 4˚C and then washed thrice in wash buffer as before and incubated with second antibody (HRP-conjugate) for 30 min at 37˚C.Bands were visualized using a horseradish peroxidase-conjugated anti-rabbit IgG Ab (Cell Signaling Technology) and chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA).Signals on X-ray film were scanned using a Bio-Rad GS 800 densitometer.For stripping, membranes were incubated with stripping buffer (Thermo Scientific, Rockford, IL, USA) at 37˚C for 30 min, then washed, blocked and reprobed overnight at 4˚C with monoclonal mouse anti-β-actin antibody (1:10.000;Sigma).

Statistical Analysis
Statistical analysis was done by using "Prism" software.Students "t" test was applied to compare the groups.P < 0.05 was considered significant.

Determination of Onset of Immune
Response, Airway Hypersensitivity and Inflammatory Changes in OVA-Sensitized Guinea Pigs

Determination of Day of Onset of Immune Response
Intradermal test (i.d.) is the most common screening method for allergy evaluation.A positive i.d. is an indirect measurement of allergen-specific IgE level.First of all, we determined the ovalbumin-specific immune response in the sensitized animals by i.d.On day 0, there was no reaction by treatment of normal saline or ovalbumin, while histamine, used as positive control, showed a 6 mm diameter wheal.On day 9, in sensitized group, the ovalbumin-specific wheal size in experimental group was 3.2 ± 0.28 mm (P < 0.0001 as compared to the control group).This was the first day when animals in experimental group showed positive response to i.d.post-sensitization, which indicated the onset of immune reaction to ovalbumin.Further, on day 14, the sensitized animals showed maximum intradermal test positivity (6.22 ± 0.02 mm diameter, P < 0.0001 as compared to the control group) against ovalbumin, indicating complete sensitization.

Determination of Airway Hyper-Responsiveness
during Onset of Immune Response After determining the allergen specific immune response, we measured the specific airway conductance (SGaw) on days 1 -14 in control and sensitized guinea pigs.On day 0, the SGaw of control group was 0.95 ± 0.02 sec −1 cm H 2 O −1 (Mean ± SEM) and that of experimental group (immediately after sensitization) was 0.89 ± 0.03.There was no significant change in SGaw between day 1 and 8 (data not shown) in sensitized group as compared to the control group.However, on day 9, there was >35% (36.88% ± 0.55%) fall in SGaw after ovalbumin inhalation in experimental group while in control group the fall was only 21.80 ± 1.250 and the changes were statistically significant (P < 0.0001), indicating that day 9 was the accurate time point of onset of the airway hypersensitivity after sensitization.On day 14, there was a further decrease (43.91% ± 2.131%) in SGAw (P < 0.0001) as compared to control (21.14 ± 1.60).Since there was no significant change in SGawon day 7 and 8, we concluded day 9 to be the day of onset of airway hypersensitivity.

Determination of Cytological, Pathological
and Biochemical Changes during the Onset of Airway Hypersensitivity

Histopathological Changes in Airways
The histopathological changes in the proximal and distal airways were determined to assess the initiation of airway hypersensitivity.In the proximal airways of sensitized animals, an increase in thickness of airway smooth muscle and multiple areas of epithelial denudation was observed on day 14 when compared to control (Figures 2(a) and (b).These changes were not observed on day 9.The distal airways revealed grade 1, peribronchiolar chronic lymphocytic inflammation when compared to control on day 9 (Figures 3(a) and (b)).On day 14, pathological changes were of grade 2 which corresponded to infiltration of lymphocytes and eosinophils in the lung (Figure 3(c)).Further, the onset ofstructural remodelingofproximal and distal airwayswas observed in experimental group on day 14 in which ASM hypertrophy was seen in proximal airways (Figure 4(a)) while distal airways showed a mild increase in thickness of the subepithelial lamina reticularis (Figures 4(b)-(d)) as compared to control group.

PKC Activity and Expression of Its Isoenzymesin Airway Smooth Muscles (ASM) and Lymphocytes
To test our hypothesis that PKC could be a critical player during the initiation of airway hypersensitivity after sensitization, we examined the total PKC activity in ASM (Figures 5(a) and (b)) and lymphocytes (Figures 5(c) and (d)) isolated from control and ovasensitized group on days 0, 9 and 14.On day 0, there was no significant change in the PKC activity in ASM and lymphocyte as compared to control group.However, on day 9, PKC activity, measured by two different methods, increased  significantly in ASM (P = 0.0023 and 0.0025 by radioligand and histone III phosphorylation methods, respectively) and lymphocytes (P = 0.0120 and 0.0001 respectively).On day 14, the PKC activity further increased significantly in ASM (P = 0.0004 and 0.0011 respectively) as well as lymphocytes (P = 0.008 and 0.0027 respectively).The increase in PKC activity on day 9 and further was >45%, suggesting physiological activation of the enzyme at the onset of the airway hypersensitivity.
Since increase in the activity as well as expression of PKC coincide with the days of Ova-induced airway hypersensitivity, these results suggest that PKC could be a key mediator of initiation of airway hypersensitivity in guinea pigs.

Metabolism of Phosphoinositides in Airway Smooth Muscles and Lymphocytes
Since PKC activity is regulated by the metabolic pool of phosphoinositides and their turnover, we next examined the changes in the total phosphoinositides in ASM and lymphocytes during the initiation of airway hypersensitivity.The phosphoinositides extracted and estimated were phosphatidyl inositol (PI), phosphatidyl inositol 4-monophosphate (PIP) and phosphatidyl inositol 4,5bisphosphate (PIP 2 ).In experimental group on day 0, there was no change inPI, PIP and PIP 2 levels in ASM and lymphocytes as compared to control group (Table 1).On day 9, the level of PI increased significantly in ASM (P = 0.0181) and lymphocytes (P = 0.0087) as compared to control group.Similarly, the levels of PIP increased significantly in ASM (P = 0.0285) and lymphocytes (P = 0.0076) as compared to control group.The levels of PIP 2 also increased significantly in ASM and lymphocytes (P = 0.0258 and 0.0020) as compared to control group on day 9. On day 14, the levels of these phosphoinositide intermediates increased further in both ASM and lymphocytes as summarized in Table 1.These results suggest that increased activity of PKC isoenzymes during initiation of airway hypersensitivity could be associated with increased levels of phosphoinositides in ova-sensitized and challenged animal.

Discussion
Asthma is a multifactorial airway inflammatory disease, characterized by airway inflammation, structural changes in the airway wall associated with progressive decline in lung function, and airway hyperresponsiveness (AHR) [5].Most of the studies on bronchial asthma on the mechanism of stimulus-induced changes have been conducted after the manifestation of the disease, which do not represent its etiopathogenesis.We, therefore, investigated the biochemical changes in airway hyper reactive guinea pig model of asthma and have presented evidence that PKC isoenzymes could be key mediators in the initiation of airway hypersensitivity during the onset of asthma.Previously, several investigators have suggested that PKC plays an active role in the progression and ex-acerbation of asthma [16,17].We have also demonstrated acerbation of asthma [16,17].We have also demonstrated that PKC antagonist sphingosine reduced the T lymphocytes activity isolated from asthmatic patients [16].Further, PKC inhibitor, Ro31-8220, has been shown to inhibit the expression of Th2 cytokines from asthmatic T lymphocytes [17], abolish airway smooth muscles constriction [18] and inhibit the proliferation and structural changes in airway smooth cells of asthmatic rats [19].In another study, Lew et al. [20] showed that calphostin-C, a PKC inhibitor, prevented proliferation of bovine tracheal smooth muscle cells following activation of mannose receptors by β-hexosaminidase and also abolished Ca 2+ -dependent and -independent PKC activity.These evidences clearly demonstrate the role of PKC in asthma, but its role during the onset of the disease is not clearly understood.We therefore investigated the biochemical and molecular changes in airway hyper reactive guinea pig model of asthma and have presented evidence that PKC isoenzymes could be key mediators in the initiation of airway hypersensitivity during the onset of asthma.Immunoglobulin E (IgE) plays a central role in the pathogenesis of allergic diseases, including asthma [33,34].Allergic sensitization results from the formation of antigen-specific IgE in response to common inhalant allergens [35,36].In our present study, on day 9 after sensitization the animals in the experimental group showed a significant positive response in i.d. which indicate that allergen-specific IgE were synthesized in enough amounts which could respond to the antigen as early as day 9.When these animals inhaled ovalbumin, they showed a significant airway hypersensitivity on day 9, suggesting that day 9 was the accurate time point for the onset of the disease.The changes were further confirmed by histopathology of the lungs, e.g.there was a significant increase in the total BALF cell counts on day 9, the first day of initiation of hypersensitivity in experimental group, which further increased (3.5 fold) on day 14, similar to the findings of Sakai et al. [37].PKC is one of the key enzymes that mediate inflammatory signals [38].It represents a structurally homologous group of 12 isoforms which are divided into three sub-groups that differ in their cofactor requirements.These are conventional(c) PKC isoforms (α, β I , β II and γ) that require Ca 2+ and diacylglycerol (DAG) for activation, novel (n) PKC isoforms (δ, ε, ζ, θ and μ) that are Ca 2+ independent but require DAG and the atypical (a) PKC isoforms, namely ζ, ι and λ (the mouse homologue of human PKCι), that do not require Ca 2+ or DAG [14,39].
One of the major signaling pathways that involves PKC is the hydrolysis of membrane PIP 2 by phospholipase C (PLC) that generates IP 3 and DAG [14,40], which then activate PKC [40].Activated PKC then phosphorylates downstream signaling protein kinases leading to activation of transcription factors such as NF-kB which in turn transcribe many inflammatory genes resulting in inflammatory response and pathophysiological changes in affected cells.In views of this, the role of PKC isoenzymes is crucial in the activation of inflammatory signals (Figure 6).PKC activity is influenced by the metabolic pool of phosphoinositides and their turnover [40].The increase in phosphoinositides pool may be due to their increased biosynthesis or decreased breakdown by the phosphomonoesterases.The phosphoinositides are metabolized by two different pathways.In one pathway activation of phosphoinositide-specific phospholipase C (PLC) leads to the generation of diacylglycerol (DAG) and inositol triphosphate (IP 3 ) which act as second messengers and activate PKC [42].In the other pathway, phosphatidyl inositol −3 kinase (PI-3K) phosphorylates and converts the inositol of various phosphoinositides into respective phosphoinositide 3 phosphate species [41,42], the levels of which are regulated by specific phosphatases.We observed that the total contents of the phosphoinositides (PI, PIP, and PIP 2 ) were significantly increased on day 9 after Ova-sensitization, which coincided with significantly increased antigen-specific immune response and airway hypersensitivity.The increase in the levels of PIs was associated with corresponding increase in the total PKC enzyme activity in airway smooth muscles and blood lymphocytes.These changes were further confirmed by the histopathological changes of the distal airways which showed lymphocytic infiltration (pathological grade 1), suggesting that increased activity and expression of PKC could lead to the activation of T cells and play a pivotal role in the pathophysiology, specifically during initiation of asthma.The increased PKC α activity on day 9 suggests the activation of conventional PKCs which require calcium and DAG for their activation [39].It is known that increase in intracellular calcium is transient and tightly regulated.After being released from intracellular pools, the level of calcium returns to normal rather quickly [40], suggesting that initiation of airway hypersensitivity and inflammation on day 9 could involve activation of PKC α, which probably trigger the onset of the disease.However, for long term sustenance of the pathology as in chronic asthma, calcium-independent mechanism will be required [40].Ono et al. have suggested that novel PKCs do not require calcium for their activation and have been implicated in the progression of asthma [43].The significantly increased expression of PKCε by day 14, when the immune response was fully established, as evident by maximum i.d.positivity and airway hypersensitivity, indicates that sustained activation of novel PKCs in ASM and lymphocytes could be responsible for the progression of the disease.Since in our study there was no change in the expression of PKC-τ on day 9, it could be stipulated that atypical PKCs, whose activation is independent of calcium and/or DAG, do not play any significant role in the onset of the airway inflammation [39].These findings suggest that the onset of airway inflammation requires Ca 2+ and DAG initially and activation of DAG-dependent mechanism in the later stage of asthma pathogenesis.
In summary, the results of this study suggest that PKC signal transduction pathway participates in onset of airway hypersensitivity and inflammation by regulating activation of lymphocytes and ASM.Therefore, the use of PKC antagonist may be a promising approach towards prevention of asthma.

Conclusion
The study established the day 9 th to be the day of onset of airway inflammation and hypersensitivity in the guinea model of ovalbumin and day 14 th to be the day when the hypersensitivity is fully developed.During the onset of the disease, the activation of PKC, particularly PKCα and PKCε, mediated signal transduction pathway plays a critical role in lymphocyte infiltration and onset of airway hypersensitivity, airway remodeling and asthma pathophysiology.This study is the first one which shows a direct evidence of the role of PKC mediated pathway in the onset of airway hypersensitivity and the mechanism of the etiopathogenesis of the disease in ovalbumin sensitized guinea pig model.

Figure 1 .
Figure 1.BALF differential cell count (a) Control group i.e. on day 0, comprising predominantly of AM, (b) On day 9, Significant increase in N and E (P = 0.002 and P = 0.016 respectively) as compared to control and (c) on day 14, AM showed significant decrease to 53.20% ± 4.23% (P < 0.0001) and a significant increase in E to 10.80% ± 2.48%, (P = 0.0001) as compared to control.AM = Alveolar Macrophages, N = Neutrophils, E = Eosinophils and L = Lymphocytes.

Figure 2 .Figure 3 .
Figure 2. Pathological changes in proximal airways on day 14.H&E stain ×40.(a) Control group; showing normal histopathology and(b) Experimental group on day 14 showed epithelial denudation and airway smooth muscle hypertrophy.

Figure 5 .
Figure 5. Assessment of PKC activity in Airway smooth muscle (ASM) and blood derived-lymphocytes.The PKC activity was measured by radio-ligand binding assay (a and c), expressed as mean ± SEM femtomoles of [ 3 H] DBu bound/mg protein in ASM or per 10 6 lymphocytes and Histone phosphorylation (b, d), expressed as mean ± SEM femtomoles of 32 P transferred to IIIs histone per mg proteins in ASM or per 10 6 lymphocytes.The activity is shown in control and experimental groups on day 0, day 9 and day 14 representing baseline, the onset and full sensitization respectively (n = 5 in each group).