Vet Pathol 41:666–672 (2004)

Expression of Nitric Oxide Synthase 2 and Cyclooxygenase-2 in Swine Experimentally Infected with Actinobacillus pleuropneumoniae W.-S. CHO

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C. CHAE

Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Kwanak-Gu, Seoul, Republic of Korea Abstract. The expression of inflammatory mediators was examined in pigs experimentally infected with Actinobacillus pleuropneumoniae. The activity of nitric oxide synthase 2 (NOS2) and cyclooxygenase-2 (COX2) was determined by measuring nitric oxide (NO) and prostaglandin E2 (PGE2) in bronchoalveolar lavage fluid in response to A. pleuropneumoniae in vivo. By in situ hybridization and immunohistochemistry, both NOS2 and COX-2 enzymes were detected in neutrophils and macrophages that had infiltrated into alveolar spaces. The sharp increase in PGE2 concentration preceded the increase in the concentrations of NO. NO levels were highly correlated with PGE2 level (rs ⫽ 0.7218, P ⬍ 0.05). The NO levels were positively correlated with lung lesion scores (rs ⫽ 0.9087, P ⬍ 0.05) until 24 hours postinoculation (hpi) as were the lung lesion scores and PGE2 levels (rs ⫽ 0.925, P ⬍ 0.01). High levels of PGE2 produced by COX-2 are generated in early infection (6 hpi). However, in later stages of infection (12–36 hpi), there is participation of NO and PGE2 accompanied by coinduction of both NOS2 and COX-2. Key words: Actinobacillus pleuropneumoniae; cyclooxygenase; inflammation; nitric oxide; nitric oxide synthase; prostaglandin.

Nitric oxide (NO) is one of the smallest endogenous biologic mediators, and it is synthesized by three isoforms of reduced nicotinamide adenine dinucleotide phosphate–dependent enzymes, the nitric oxide synthases (NOS).21 Isoforms of the enzyme are constitutively expressed in neuronal cells (NOS1) and endothelial cells (NOS3), respectively. The third isoform, NOS2, is induced after stimulations with various proinflammatory cytokines and lipopolysaccharide (LPS).23 NO reacts rapidly with superoxide to produce peroxynitrite, which in turn reacts with proteins to produce nitrotyrosine.18 Two isoforms of cyclooxygenase (COX) have been recognized.27 COX-1 is a constitutively expressed enzyme found in many tissues, whereas COX-2 is an inducible enzyme predominantly expressed at the sites of inflammation.15 The generation of prostaglandin (PG) and thromboxane results from the conversion of arachidonic acid to PG H2 through COX.6 Actinobacillus pleuropneumoniae, an encapsulated gram-negative bacterium, causes severe, often fatal fibrinohemorrhagic necrotizing pleuropneumonia in pigs. Pulmonary lesions caused by A. pleuropneumoniae are similar to those seen in endotoxic shock, and the LPS of A. pleuropneumoniae alone can cause lesions similar to those seen in naturally occurring porcine pleuropneumonia.14,29 Macrophages are induced to

release cytokines such as tumor necrosis factor–␣ (TNF-␣) and interleukin-1 (IL-1) early in the course of porcine pleuropneumonia,1,7,13 and they are believed to be primary mediators of the LPS effects in porcine pleuropneumonia. The objective of this study was to investigate the expression and localization of NOS2 and COX-2 in lungs from pigs experimentally infected with A. pleuropneumoniae. The activity of NOS2 and COX-2 was also confirmed by measuring NO and PGE2, respectively. Materials and Methods Experimental design Forty 7-week-old colostrum-deprived pigs were randomly allocated to infected (n ⫽ 24) or control groups (n ⫽ 16). In the infected group, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 as described previously.1 Three infected and two uninfected pigs were euthanatized at 3, 6, 9, 12, 24, 36, 48, 60 hours postinoculation (hpi). Bronchoalveolar lavage (BAL) fluid was collected from the lung of each as described previously.1 After centrifugation of BAL fluid at 5,000 ⫻ g for 10 minutes, 4 C, the supernatant was used to determine NO2⫺/NO3⫺ and PGE2 levels. Microscopic pulmonary lesion assessment Each lung lobe was sectioned, randomly coded, and subjectively scored by two observers for lesion severity (scale 0–5) in each lesion for one or more of the following chang-

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es: 1) necrosis, 2) hemorrhage, 3) inflammatory cell infiltration into alveolar spaces, 4) thrombosis, and 5) pleuritis. Each lung lobe was assigned a score to reflect the approximate percentage of the entire lung represented by that lobe. Ten points each were assigned to the right cranial lobe, right middle lobe, cranial part of the left cranial lobe, and caudal part of the left cranial lobe. The accessory lobe was assigned 5 points. The right and left caudal lobes were each assigned 27.5 points to reach a total of 100 points representing the entire (100%) lung as described previously.16 Measurement of NO2⫺/NO3⫺ production To determine the amount of NO production by A. pleuropneumoniae–infected lung, BAL fluids were assayed for the stable end products of NO oxidation, NO2⫺/NO3⫺, in a colorimetric assay kit (Alexis Bioch Corp., San Diego, CA) based on Griess reaction according to manufacturer’s instruction.4 Measurement of PGE2 production PGE2 production was measured using a commercially available enzyme-linked immunosorbent assay kit (Oxford Biomedical Research, Oxford, MI) according to manufacturer’s instruction. Reverse transcription polymerase chain reaction BAL cells were used for extraction of NOS2 and COX-2 RNA, respectively. RNA was extracted from these cells with Trizol LS reagent (GIBCO BRL, Grand Island, NY) according to the manufacturer’s instructions. RNA extracts were treated with deoxyribonuclease (DNase) I (GIBCO BRL) to eliminate genomic DNA contamination. The LPS-stimulated alveolar macrophages were prepared as described previously and used as positive control for NOS2 and COX-2 induction.7,10 The reverse transcription polymerase chain reaction (RT-PCR) was performed as described previously.7,9 In situ hybridization of NOS2 and COX-2 nucleic acid RT-PCR products of NOS2 and COX-2 were purified using a 30-kDa cutoff membrane ultrafiltration filter. The nucleotide sequences of the purified PCR products were determined by use of BigDye chemistry with the ABI Prism Sequencer (Applied Biosystems, Foster City, CA). The purified RT-PCR products were sequenced before labeling by random priming with digoxigenin-deoxyuridine triphosphate (dUTP) (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. In situ hybridization was performed as described previously.7,10 Lung tissues from pigs naturally infected with A. pleuropneumoniae were used as positive control of NOS2 and COX-2 as described previously.7,10 In situ hybridization of A. pleuropneumoniae DNA The probe for A. pleuropneumoniae was generated from a genomic library of A. pleuropneumoniae by PCR and labeled by random priming with digoxigenin-dUTP (Boehringer Mannheim) according to the manufacturer’s instructions.22 In situ hybridization for A. pleuropneumoniae was carried out as described previously.22

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Immunohistochemistry Immunohistochemical examination for NOS2, COX-2, and nitrotyrosine was carried out as described previously.8,9,11 Three antibodies were used: 1) polyclonal rabbit anti-NOS2 antibody (Biotechnology Inc., Santa Cruz, CA), 2) polyclonal rabbit anti-mouse COX-2 antibody (Cayman Chemical, Ann Arbor, MI), and 3) monoclonal mouse anti-nitrotyrosine antibody (Zymed Laboratories, Inc., San Francisco, CA). Lung tissues from pigs naturally infected with A. pleuropneumoniae were used as positive control of NOS2, COX-2, and nitrotyrosine.8,9,11 Statistical analysis One-way analysis of variance was used to assess statistical differences in the increase in NO2⫺/NO3⫺ and PGE2 production and lung lesion score among different time-point groups. Pearson correlation analysis was used to assess relationship between NO2⫺/NO3⫺ level and lung lesion score and between PGE2 level and lung lesion score. P ⬍ 0.05 was considered to be statistically significant.

Results Histopathology

Microscopically, the distribution of lesion was generalized in all lobes examined. In infected pigs euthanatized at 3 hpi, the alveolar septa were thickened with congested blood vessels, and the alveolar spaces filled with erythrocytes or fibrin (or both), proteinaceous fluid, and small numbers of streaming degenerate leukocytes. Interlobular septa were congested and thickened by proteinaceous fluid. Lesions at 6 hpi were similar but more severe, and interlobular septa were markedly distended by fibrin and proteinaceous fluid. By 9, 12, and 24 hpi, alveolar spaces contained large numbers of neutrophils and macrophages, proteinaceous fluid, and fibrin. Interstitial tissue was infiltrated by neutrophils and macrophages, and alveolar septa were thickened by proteinaceous fluid. Interlobular septa were markedly thickened by neutrophils, macrophages, and fibrin. Septal lymphatics were dilated. Pigs necropsied at 36 and 48 hpi had variously sized foci of coagulation necrosis often centered around bronchioles. Alveolar spaces were filled with fibrin and neutrophils. Lesions were not seen in uninfected negative control pigs. Lung lesion score

A relationship between time and increased severity of lung lesion score was demonstrated. The lung lesion score in A. pleuropneumoniae–inoculated pigs was significantly different (P ⬍ 0.001) at 3, 6, and 12 hpi (P ⬍ 0.001) compared with previous hpi and was maintained through 60 hpi (Fig. 1). Lung lesion score was zero in the uninfected negative control pigs. The lung lesion scores were not significantly different between the two observers.

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Fig. 1. Pulmonary lesion score from pigs experimentally infected with Actinobacillus pleuropneumoniae. * indicates significantly different at each time point compared with previous time (P ⬍ 0.05). Fig. 2. Concentration of NO2⫺/NO3⫺ and PGE2 in BAL fluid from the lung of pigs experimentally infected with Actinobacillus pleuropneumoniae. * indicates significantly different at each time point compared with previous time (P ⬍ 0.05). NO ⫽ nitric oxide; PG ⫽ prostaglandin.

NO2⫺/NO3⫺ and PGE2 concentrations

RT-PCR analyses of NOS2 and COX-2 mRNAs

A strong relationship between time and increased NO2⫺/NO3⫺ concentration was demonstrated. NO2⫺/ NO3⫺ concentration in A. pleuropneumoniae–inoculated pigs was significantly different (P ⬍ 0.001) at 12, 24, and 36 hpi compared with previous hpi, and maximum concentrations were seen at 24 hpi. The concentration of NO2⫺/NO3⫺ declined markedly by 36 hpi (P ⬍ 0.001) (Fig. 2). Concentrations of NO2⫺/NO3⫺ were not detected at any time in the uninfected negative control pigs. The concentration of PGE2 in BAL fluid was significantly different at 6 hpi (P ⬍ 0.001) from that at 3 hpi, and a maximum concentration was seen at 24 hpi. Concentrations of PGE2 were sustained between 6 and 36 hpi but declined markedly by 48 hpi (P ⬍ 0.001) (Fig. 2). PGE2 was not detected in BAL fluid collected at any time from uninfected negative control pigs. NO2⫺/NO3⫺ level was correlated to PGE2 level (rs ⫽ 0.7218, P ⬍ 0.05). Statistically, correlation between NO2⫺/NO3⫺ levels and mean lung lesion score (rs ⫽ 0.9087, P ⬍ 0.05) was high for intervals up to 24 hpi as was the correlation between lung lesion score with PGE2 level (rs ⫽ 0.925, P ⬍ 0.01). However, there was no correlation between NO2⫺/NO3⫺ level and mean lung lesion score and between PGE2 level and mean lung lesion score thereafter.

RT-PCR analyses of lung tissue from pigs experimentally inoculated with A. pleuropneumoniae demonstrated expression of NOS2 and COX-2 messenger RNA (mRNA). Amplification of template complementary DNA with primers of NOS2 or COX-2, respectively, resulted in amplified products corresponding to those of the predicted size, namely, 491 bp (NOS2) and 437 bp (COX-2). Sequenced PCR products were confirmed as NOS2 and COX-2 (data not shown). Expression of NOS2 and COX-2 genes was not detected in lung tissue from the uninfected negative control pigs. In situ hybridization

In lung tissue from pigs at 12, 18, and 24 hpi, a strong hybridization signal for NOS2 was seen in degenerate leukocytes bordering zones of coagulative necrosis and in alveolar spaces. The hybridization signal was consistently detected in neutrophils and macrophages within alveolar spaces but was not detected in these same cells in intravascular locations. Identification of cell types expressing NOS2 genes was occasionally difficult, but examination of serial sections stained with hematoxylin and eosin confirmed that the NOS2 genes were present in neutrophils and macrophages. COX-2 expression was particularly intense in clustered leukocytes that had streaming nuclear chromatin,

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Fig. 3. Lung tissues for pig experimentally infected with Actinobacillus pleuropneumoniae at 36 hpi. Fig. 3a. Actinobacillus pleuropneumoniae DNA was detected in the streaming alveolar leukocytes. Fig. 3b. Serial section showing that contiguous cells positive for NOS2 nucleic acid. In situ hybridization; cDNA probe; nitroblue tetrazolium/5-bromocresyl3-indolylphosphate, methyl green counterstain. Fig. 4. Lung tissues for pig experimentally infected with Actinobacillus pleuropneumoniae at 48 hpi. Fig. 4a. NOS2 antigen (red reaction) was detected in inflammatory cells. Fig. 4b. Serial section showing that contiguous cells positive for nitrotyrosine antigen (red reaction). Immunohistochemistry; alkaline phosphatase, red substrate, hematoxylin counterstain.

and neutrophils and macrophages within alveolar spaces frequently had strong signals in pigs necropsied at 12, 24, and 36 hpi. Expression of NOS2 and COX-2 was always detected within inflammatory lesions but was minimal in nonaffected portions of the lung. The close cell-to-cell correlation between NOS2 and COX2 expression in serial sections from lungs was confirmed by in situ hybridization. Pretreatment with ribonuclease A eliminated hybridization signal for NOS2 and COX-2 from pigs experimentally infected with A. pleuropneumoniae. Sections from positive control showed hybridization signal for NOS2 and COX-2. No hybridization signal was detected in lung sections from an uninfected negative control pigs. A. pleuropneumoniae organisms were also detected

within inflammatory lesions, but they were minimal in nonaffected portions of the lung from experimentally infected pigs. In situ hybridization of serial sections of lung indicated that areas containing numerous A. pleuropneumoniae DNA-positive cells also have numerous NOS2- and COX-2–positive cells (Fig. 3a, b). Pretreatment with DNase I eliminated hybridization signal for A. pleuropneumoniae. Hybridization signal was not detected in lung sections from uninfected negative control pigs. Immunohistochemistry of NOS2 and COX-2 protein

A strong immunohistochemical signal for NOS2 and COX-2 was detected in lung tissue from all pigs experimentally infected with A. pleuropneumoniae. Pos-

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itive cells typically exhibited a red cytoplasmic reaction product without background staining. Labeling of NOS2 and COX-2 proteins were particularly intense in clustered leukocytes with streaming nuclear chromatin. Less-intense immunostaining was seen in the dense zone of degenerate cells in granulation tissue surrounding chronic foci of necrosis. Positive cells for NOS2 and COX-2 proteins generally had large oval nuclei and abundant cytoplasm typical of macrophages. A less-intense signal was seen in the neutrophils within alveolar spaces. NOS2 and COX-2 protein was always associated with macrophages and neutrophils in lesions but was minimal in unaffected portions of lung. Sections from positive control showed immunohistochemical staining for NOS2 and COX-2, and immunohistochemical staining was not detected in lung sections from uninfected negative control pigs. Immunohistochemistry of nitrotyrosine

Intense immunostaining for nitrotyrosine residue was seen within the lung lesions from A. pleuropneumoniae–infected pigs. Staining was especially strong in neutrophils and macrophages in the periphery of the lesions and within the alveolar spaces. There was close cell-to-cell correlation when serial sections were examined by immunohistochemistry for nitrotyrosine and NOS2 in lung samples from A. pleuropneumoniae–infected pigs (Fig. 4a, b). Nitrotyrosine antigen was always associated with macrophages and neutrophils in the pneumonic lung lesions, but it was minimal in unaffected portions of lung of A. pleuropneumoniae–infected pigs. Sections from uninfected negative control pigs did not exhibit an immunohistochemical staining for nitrotyrosine. Discussion The results of this study demonstrated that induction of NOS2 and COX-2 is a direct response to A. pleuropneumoniae because the expression of NOS2 and COX-2 was greater in lesions of pneumonia than in unaffected portions of lung from infected pigs, Furthermore, in situ hybridization of serial sections of lung indicated that the majority of areas containing numerous NOS2- and COX-2–positive cells also have numerous A. pleuropneumoniae DNA-positive cells. Expression of both genes is markedly upregulated both at the mRNA and protein levels in the lungs of swine. Statistical correlations of NO and PGE2 levels and lung lesion score suggest that NOS2 and COX-2 play a role in the pathogenesis of acute pleuropneumonia in swine. In situ hybridization of serial sections of lung indicated that areas containing numerous NOS2- and COX-2–positive cells also have numerous A. pleuropneumoniae DNA-positive cells in this study. Al-

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though NOS2 is not induced by LPS in porcine alveolar macrophages,24 in vitro study clearly showed that A. pleuropneumoniae have detectable effects on the activities in NOS2 and COX-2 in porcine alveolar macrophages.12 Therefore, altered gene and protein expression of NOS2 and COX-2 may be a direct effect of A. pleuropneumoniae organisms. Alternatively, expression may also be the result of cytokines produced in response to A. pleuropneumoniae LPS. Expression of TNF-␣ and IL-1 has been detected in A. pleuropneumoniae–infected lungs by in situ hybridization,1,13 but how these molecules interact is not completely understood. It is possible that TNF-␣ and IL-1 act synergistically with A. pleuropneumoniae LPS to induce both NOS2 and COX-2 expression by macrophages and other inflammatory cells in infected lungs. Both in situ hybridization and immunohistochemistry confirmed increased expression of NOS2 and COX-2 by inflammatory cells in A. pleuropneumoniae–induced pleuropneumonia. Simultaneous detection of nucleic acid and protein by in situ hybridization and immunohistochemistry, respectively, provides direct molecular evidence that these inflammatory cells produced NO and PGE2 and play a major role in the expression of NOS2 and COX-2 in pigs infected with A. pleuropneumoniae. This concept is also supported by other studies in which increased NOS2 and COX-2 expression was detected in isolated alveolar macrophages or neutrophils after stimulation by LPS or inflammatory cytokines.17,20 Histologic lesions in peracute pneumonia caused by A. pleuropneumoniae are characterized by hemorrhage, edema, and fibrin deposition within alveolar spaces.3,19,20 In this study, congestion, hemorrhage, and edema were striking histologic features of disease. PGE2 synthesized by COX-2 contributes to these lesions of acute inflammation by causing vasodilation and increased vascular permeability.6,25 Although NO produced by NOS2 is also a potent vasodilator and contributes to the formation of edema during acute inflammation,5 NO2⫺/NO3⫺ levels in this study did not increase markedly in early infection. In this regard, the early phase (3–6 hours) of pneumonia caused by A. pleuropneumoniae may be mediated by eicosanoids, and NO may not play a role. Our data also show that the participation of NO and PGE2 occurs in the later phase (12–24 hours) of infection, with maximal NO and PGE2 levels expressed in BAL fluid by 24 hpi. Our data agree with previous reports in other species where the sharp increase in PGE2 concentration preceded the sharp increase in the concentration of NO2⫺/ NO3⫺. Data from this study also indicate a correlation exists between the expression of NOS2 and COX-2 pathways and the migration of inflammatory cells into alveolar spaces. A regulatory role for NO in the in

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vivo production of prostanoids has been observed in rats treated with LPS,27 in the carrageenan rat air pouch model,25 and in a model of renal inflammation in rabbit.26 In those studies, NO released from NOS2 activated the induced COX, resulting in increased production of PGs. Thus, NO and PGs may play mutually modulatory and synergistic roles in the inflammatory response caused by A. pleuropneumoniae infection. Inflammatory cells infiltrating the alveolar spaces express NOS2 and colocalize in cells with nitrotyrosine immunoreactivity in this study. Because NOS2 and nitrotyrosine colocalize in inflammatory cells in alveolar spaces, it is possible that NO and superoxide produced by inflammatory cells react to form peroxynitrite, which, in turn, reacts with proteins to produce nitrotyrosine. Nitrotyrosine formation was initially proposed as a specific marker for the detection of the endogenous peroxynitrite formation.2 However, other reactions can induce tyrosine nitration irrespective of the presence of peroxynitrite. The reaction of nitrite with hypochlorous acid and of myeloperoxidase with hydrogen peroxide can catalyze and form nitrotyrosine.28 Increased staining for nitrotyrosine is therefore considered to be an indication of increased nitrative stress rather than a specific marker of the generation of peroxynitrite. Porcine pleuropneumonia is an acute complex disease process involving numerous mediators of cellular and plasma origin with elaborate, interrelated biologic effects. This study offers further insight into the association with NOS2 and COX-2 pathways in the inflammatory response in pleuropneumonia and strongly suggest that NOS2 and COX-2 are involved in pleuropneumonia because one or more cell populations responded to A. pleuropneumoniae by a rapid and intense increase/induction of NOS2 and COX-2 expression.

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Acknowledgement This study was supported by a grant of the Korea Health 21 R&D Project (02-PJ1-PG3-20799-0001), the Ministry of Health and Welfare, Republic of Korea.

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Request reprints from Dr. C. Chae, Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu 151–742, Seoul (Republic of Korea). E-mail: [email protected].