Purinergic receptors are expressed throughout the respiratory system in diverse cell types. The efficiency of mucus clearance in the airways, the cascade leading to tissue injury, and inflammation are modulated by autocrine/paracrine release of nucleotides and signaling by purinergic receptors. We assessed the role of purinergic receptors in innate host defense of the lung in vivo by infecting mice deficient in P2Y1, P2Y2, or both receptors with intratracheal instillation of Pseudomonas aeruginosa. After P. aeruginosa challenge, all double knockout (P2Y1/P2Y2−/−) mice succumbed within 30 h of challenge, whereas 85% of the wild-type mice survived. Thirty-three percent of wild-type mice survived beyond 96 h. Single knockout mice, P2Y1−/−, or P2Y2−/−, exhibited intermediate survivals. Twenty-four hours following intratracheal instillation of a sublethal dose of P. aeruginosa, the level of total protein in bronchoalveolar lavage fluid was 1.8-fold higher in double knockout than in wild-type mice (P < 0.04). Total cell count in bronchoalveolar lavage fluids at 4 h and levels of IL-6 and macrophage inflammatory protein-2 in lung homogenates at 24 h postchallenge were significantly reduced in P2Y1/P2Y2−/− mice relative to wild-type mice. These findings suggest that purinergic receptors exert a protective role against infection of the lungs by P. aeruginosa by decreasing protein leak and enhancing proinflammatory cytokine response.
- bacterial clearance
- cystic fibrosis
the lung is protected against infection and inflammation from inhaled microbes by a host defense system that includes a mucociliary escalator in the airways, antimicrobial proteins in airway surface liquid, and macrophages in the alveoli. Purinergic receptors are expressed in the respiratory system by diverse cell types where they regulate the rate of mucus clearance (18, 20, 29), surfactant secretion (30), pulmonary vasodilatation (2, 16, 21), and several alveolar macrophage functions (3, 14, 15).
Purinergic receptors are important in the modulation of inflammatory cascades (9, 10) including release of TNF-α, IL-8, and IL-1β (5, 17, 38), in the activation of inducible nitric oxide synthase (8, 15, 38), in enhancing chemotaxis of macrophages and neutrophils (9, 15, 41), in adhesion of neutrophils to endothelial cells (7), and in macrophage apoptosis (23). Superoxide production in neutrophils is modulated by nucleotides (36). Both ATP and granulocyte/macrophage colony-stimulating factor (GM-CSF) retard neutrophil apoptosis via the MAPK family (12, 13, 32). ADP stimulates respiratory burst in alveolar macrophages (14).
Purinergic agonists may play a role in the pulmonary response to P. aeruginosa infection. Interaction of flagellin, an abundant protein in P. aeruginosa, with the asialglycoprotein (ASGM1) on airway epithelial surfaces induces release of ATP, which then acts in an autocrine/paracrine fashion through P2Y receptors to activate phospholipase C, increase intracellular calcium, activate MAPK, and increase mucin gene transcription (28). The flagellin:ASGM1 interaction also induces release of IL-8, GM-CSF, and granulocyte colony-stimulating factor, through a calcium-mediated pathway (19, 25, 31). Together, purinergic receptor and their agonists appear to be important in host defense of the lungs.
Infection of the lower respiratory tract with P. aeruginosa is prevalent in immunocompromised patients, in preterm neonates, and among patients with cystic fibrosis. P. aeruginosa is the most common cause of intensive care unit-associated pneumonia (4). In the current study, we assessed the role of the two predominant subtypes of purinoceptors expressed in the lungs in the regulation of inflammation and infection in vivo. Mice lacking purinoceptors (P2Y1 and/or P2Y2) as a result of gene targeting were infected intratracheally with P. aeruginosa. Survival, bacterial clearance, inflammatory response, and epithelial integrity were assessed.
MATERIALS AND METHODS
The four groups of mice used in the current study included mice deficient in the purinoceptor P2Y1 (P2Y1−/−), mice deficient in P2Y2 (P2Y2−/−), mice deficient in both P2Y1−/− and P2Y2−/− (P2Y1/P2Y2−/−), and wild-type (WT) 129P3/J mice. Generation and characterization of P2Y1−/−, P2Y2−/−, and P2Y1/P2Y2−/− mice were previously described (6, 11). Mice were comparable with respect to weight (15–25 g), lung structure and function, longevity, and fecundity. Animals were housed in barrier containment and studied under Institutional Animal Care and Use Committee-approved protocols in the animal facilities of the Children's Hospital Research Foundation, Cincinnati, Ohio. Animals were studied between 5 and 6 wk or at 12–15 mo of age. Routine viral serologies on sentinel mice were negative.
A stock culture of P. aeruginosa, PAO1 strain, was used in the current study. To minimize variability in virulence, all bacteria were selected from aliquots of the same passage that had been frozen at −70°C in 2X yeast tryptone (2XYT) media with 15% glycerol. For each experiment, an aliquot was thawed and plated on trypticase soy agar/sheep blood plate. A single colony was inoculated into 6 ml of 2XYT broth and incubated at 37°C for 16–20 h with continuous shaking. The broth was centrifuged at 500 g, and the bacterial pellet was washed in 10 ml of sterile PBS and resuspended in 5 ml of PBS. The bacterial concentration was enumerated as colony-forming units (CFU) by quantitative culture on blood agar plates. Bacterial suspension was stored at 4°C until injection within 24 h of preparation. The dose of P. aeruginosa required to kill 50% of WT 129P3/J mice (LD50) was determined in preliminary studies. Subsequent dosing in the current study was based on these data. For each experiment, the inoculum was confirmed by plating dilutions of the aliquot used for intratracheal injection. For assessment of concentrations of inflammatory mediators and total proteins in bronchoalveolar lavage fluid (BALF), control mice received sterile PBS intratracheally.
Five- to six-week-old P2Y1−/−, P2Y2−/−, P2Y1/P2Y2−/−, or WT mice (n = 20 for each group) were challenged with P. aeruginosa (1 × 108 CFU administered intratracheally as described previously) (1). Animals were evaluated every 6 h. Animals had access to water and food ad libitum. To determine whether the difference in survival was mitigated by age, survival study was repeated on 12- to 15-mo-old WT and P2Y1/P2Y2−/− mice as described above.
Five- to six-week-old P2Y1−/−, P2Y2−/−, P2Y1/P2Y2−/−, or WT mice were challenged with P. aeruginosa (1 × 107 CFU administered intratracheally). At 4 or 24 h following bacterial challenge, lungs were harvested and bacterial burden was assessed on lung homogenates as described previously (1). The remaining lung homogenates from WT and P2Y1/P2Y2−/− mice were centrifuged for 15 min at 1,000 g, and the supernatant was stored at −20°C until assayed for cytokine levels. For each time point, five or six mice were infected for each genotype. Data from two experiments were pooled.
BALF cell count and differential.
At 4 or 24 h following challenge with P. aeruginosa, WT and P2Y1/P2Y2−/− mice were anesthetized and exsanguinated as described above. Lungs were lavaged three times with 1-ml aliquots of cold normal saline. An aliquot of BALF was used for protein determination. The remaining BALF was centrifuged for 10 min at 500 g. The supernatant was stored at −20°C until processed for nitrate/nitrite measurements. The pellet was resuspended in 500-μl volume of PBS for total cell count and differential. Cell differentials were determined after cytospin and staining with May-Grünwald Giemsa stain (Diff-Kwik; Thermo Shandon, Pittsburgh, PA).
Protein levels in BALF.
Protein levels in the BALF from P. aeruginosa-infected or PBS-treated WT and P2Y1/P2Y2−/− mice were determined with bicinchoninic acid assay (TPRO-562; Sigma, St. Louis, MO) 24 h following intratracheal challenge. All samples were assayed in duplicate. A standard curve was generated with bovine serum albumin (33).
Levels of inflammatory mediators.
Concentrations of IL-6, IL-1β, macrophage inflammatory protein (MIP)-2, keratinocyte-derived chemokine (KC), IFN-γ, TNF-α, and GM-CSF in total lung homogenates from WT or P2Y1/P2Y2−/− mice were assessed 24 h following intratracheal challenge with P. aeruginosa by ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Nitrate and nitrite levels in BALF were measured by the Greiss reaction using a standardized assay kit (R&D Systems). All plates were read on a microplate reader (Quant, BIO-TEK Instruments). The correlation coefficients of all assays were >0.97.
Analysis of lung water.
Twenty-four hours after intratracheal infection with 1 × 107 CFU of P. aeruginosa, mice were anesthetized and exsanguinated via abdominal aorta transection. Lungs were excised en bloc and weighed (wet wt) and then desiccated for 72 h in an 80°C oven and reweighed (dry wt). Ratios of wet-to-dry lung weights were calculated.
Twenty-four hours after lung infection with 1 × 107 CFU of P. aeruginosa, mice were killed with a lethal injection of pentobarbital sodium. Lungs were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin as described previously (1). Representative sections from each lung lobe were evaluated by two investigators, masked to the genotype, and scored for edema, inflammation, and proteinaceous debris on a scale of 1 to 4 as described previously (24).
Twenty hours after intratracheal infection with 1 × 107 CFU of P. aeruginosa, mice were injected with 0.2 ml of bromodeoxyuridine (BrdU) labeling reagent (Zymed, San Francisco, CA) or injected with PBS (control) intraperitoneally. Lungs were inflation-fixed and processed as described above. Lung sections were stained for BrdU, and BrdU-positive cells were counted from representative sections from each of the five lung lobes. A grid was superimposed on images, and labeled cells were enumerated using Metamorph image analysis software (Universal-Imaging, Downingtown, PA). Data from two high-power fields per lung section were pooled.
Data are expressed as means ± SE. Mean values were compared by Student's t-test, using a Bonferroni adjustment for multiple comparisons when appropriate. Findings were considered statistically significant at levels of P < 0.05. Kaplan-Meir survival curves were generated, and differences in survival were analyzed with log rank test.
Purinergic receptors and survival after acute lung infection with P. aeruginosa.
To assess the role of purinoceptors in innate host defense of the lungs, 6-wk-old WT, P2Y1−/−, P2Y2−/−, or P2Y1/P2Y2−/− mice were injected intratracheally with 1 × 108 CFU of P. aeruginosa. Survival of P2Y1/P2Y2−/− mice and P2Y1−/− mice was significantly decreased compared with WT mice (P < 0.04). All P2Y1/P2Y2−/− mice succumbed by 30 h. In contrast, there was 55 and 85% survival in P2Y2−/− and WT mice, respectively (Fig. 1A). To determine whether survival in P2Y1/P2Y2−/− mice was mitigated by age, survival studies were repeated on P2Y1/P2Y2−/− and WT mice at 12–15 mo of age. At 72 h postinfection, 70% of P2Y1/P2Y2−/− mice had succumbed compared with 30% mortality in WT mice (P < 0.02; Fig. 1B).
Role of purinoceptors in inflammatory response to P. aeruginosa infection.
To determine whether the loss of purinergic receptors resulted in perturbed inflammatory response to acute infection, 6-wk-old WT, P2Y1−/−, P2Y2−/−, or P2Y1/P2Y2−/− mice were injected intratracheally with a sublethal dose of P. aeruginosa. Bacterial burden and levels of inflammatory mediators in lung homogenates, total protein, nitrite/nitrates, and cell counts in BALF were assessed at 4 and/or 24 h following intratracheal instillation. There were no significant differences in the killing and systemic dissemination of P. aeruginosa between WT and P2Y1/P2Y2−/− mice at this dose as assessed from quantitative culture of lung and splenic homogenates (see Table 2). Levels of inflammatory mediators involved in signaling by purinergic receptors were significantly increased following challenge with P. aeruginosa. Concentrations of MIP-2 and IL-6 were 2-fold higher in WT mice than in P2Y1/P2Y2−/− mice (P < 0.04), whereas the concentration of GM-CSF in P2Y1/P2Y2−/− mice was >12-fold lower than in lung homogenates from WT mice (P = 0.08; Fig. 2). Although the concentrations of KC, IL-1β, TNF-α, and IFN-γ were all increased following infection with P. aeruginosa, the levels were not significantly different between infected WT and P2Y1/P2Y2−/− mice. Four hours following challenge with P. aeruginosa, total cell counts were 1.7-fold higher inBALF from WT compared with P2Y1/P2Y2−/− mice (P < 0.04). At 24 h, there were no significant differences in total and differential cell counts and nitrite and nitrate levels in BALF from WT and P2Y1/P2Y2−/− mice (Table 1). Levels of total protein in BALF were increased 1.8-fold in the P2Y1/P2Y2−/− mice relative to the WT mice (P < 0.04; Fig. 3).
Analysis of lung water.
Twenty-four hours after intratracheal instillation of 1 × 107 CFU P. aeruginosa in WT and P2Y1/P2Y2−/− mice, wet-to-dry weight ratio following 72 h of desiccation was 1.9-fold higher in P2Y1/P2Y2−/− mice (4.6 ± 0.13 vs. 2.4 ± 0.16 in WT, P < 0.06; Fig. 4).
Lung injury and repair.
Hematoxylin/eosin-stained and BrdU-labeled lung sections from four P. aeruginosa-infected WT or P2Y1/P2Y2−/− mice were scored on a scale of 1 to 4 by two investigators (T. Korfhagen and H. Akinbi) who were masked to the genotype of the mice for alveolar edema, inflammation, and proteinaceous debris. Lungs from both WT and P2Y1/P2Y2−/− mice showed severe lobar pneumonia on histopathology. There were no significant differences in histological scores between the WT and P2Y1/P2Y2−/− mice (Table 2). Similarly, the numbers of BrdU-positive cells/lobe were not significantly different between WT and P2Y1/P2Y2−/− mice (Table 2).
Several in vitro studies on the functions of P2Y purinoceptors suggest an important role in host defense. However, the role of P2Y purinoceptors in innate host defense of the lungs in vivo has never been tested directly. After acute challenge of the lungs with P. aeruginosa, survival was significantly decreased in 6-wk-old mice that were deficient in both P2Y receptors and to a lesser degree in mice deficient in P2Y1 compared with WT mice. Survival was less affected in P2Y2−/− mice, suggesting that P2Y1 may play a more important role in P. aeruginosa infection. At 1 yr of age, more WT and purinergic receptor-deficient mice survived challenge with P. aeruginosa as a result of lower bacterial inoculum per gram of body weight or due to age-related differences. However, the difference in mortality between the two groups of mice persisted.
Mortality following infection with P. aeruginosa appears to be due mainly to pneumonia, as all infected mice developed severe respiratory distress consistent with the diffuse alveolar consolidation on histopathology in all four groups of mice. Differences in mortality are unlikely to be secondary to extrapulmonary effects of deficiency of purinoceptors as there were no significant differences between P2Y1/P2Y2−/− and WT mice in systemic dissemination of P. aeruginosa as reflected in the numbers of P. aeruginosa CFU cultured from splenic homogenates 24 h postinfection. Other causes of mortality were not excluded in the current study. Increased susceptibility of purinergic receptor-deficient mice is similar to what has been reported for several cystic fibrosis mice (G551D, S489X) (27, 40). Similar to G551D CF mice, concentration of MIP-2 was lower in P2Y1/P2Y2−/− mice following challenge with intratracheal P. aeruginosa (27). However, unlike S489X CF mice challenged with intratracheal P. aeruginosa-laden agarose beads (39), levels of TNF-α were not significantly different between P2Y1/P2Y2−/− and WT mice.
Using a sublethal dose of P. aeruginosa that still induced severe respiratory distress and alveolar consolidation, we investigated other possible mechanisms of impaired survival after P. aeruginosa challenge in the P2Y1/P2Y2−/− mice. Protein levels in the BALF from P2Y1/P2Y2−/− mice were increased 1.8-fold relative to WT mice 24 h postchallenge with P. aeruginosa. This suggests that purinoceptors may ameliorate protein leak after P. aeruginosa infection. This is similar to the intestine in which epithelial barrier permeability is perturbed following infection partly due to hypoxia-induced increased activity of CD73, the ectoenzyme responsible for degrading AMP into adenosine (33, 37). Alternatively, increased protein leak into the alveolar space could result from increased capillary leak secondary to endothelial injury.
Identifying the role of an individual cytokine in the complex milieu of the infected lung is complicated by the array of cell types capable of secreting cytokines (macrophages, neutrophils, endothelial and epithelial cells) and the complex interacting networks of cytokines and growth factors produced following infection. However, impaired survival in the receptor-deficient mice may be related to perturbation of specific components of the inflammatory cascade in P2Y1/P2Y2−/− mice. IL-1-induced cytokine cascade is impaired in macrophages in P2X7-deficient mice, resulting in delayed apoptosis of these cells (35). In the current study, levels of both IL-6 and MIP-2 were significantly lower in P2Y1/P2Y2−/− mice following intratracheal instillation of P. aeruginosa. GM-CSF response to P. aeruginosa infection was also attenuated in P2Y1/P2Y2−/− mice.
In vitro, interaction of the flagellin protein of P. aeruginosa and ASGM1 induces ATP release and subsequent autocrine activation of epithelial purinoceptors resulting in increased intracellular calcium, activation of MAP and ERK kinases, and induction of mucin gene transcription (28). Interestingly, the binding of flagellin to ASGM1 also induces secretion of GM-CSF and IL-8 by epithelial cells (19, 25, 31). Flagella are highly immunogenic (26). The role of epithelial purinoceptors as an intermediate step between flagellin binding to ASGM1 may be pivotal for cytokine secretion and may explain, in part, the decreased levels of proinflammatory cytokines in P2Y1/P2Y2−/− following P. aeruginosa infection. In future experiments, the role of flagellin in the pathogenesis of P. aeruginosa infection in the lungs would be assessed by comparing the levels of inflammatory mediators and lung histopathological changes following challenge of purinergic receptor-deficient mice challenged with either WT P. aeruginosa or fliC, a flagellin-mutant strain of P. aeruginosa.
The role of P2Y purinoceptors in infection of the lung and the impact on survival have not been demonstrated previously. The impact on survival appears delayed, with no difference in mortality noted during the first 12 h after infection of P2Y1/P2Y2−/− and WT mice. Bacterial clearance and neutrophil influx, which occur early in infection (13, 22, 42), were not significantly different at 24 h postinfection with a sublethal dose of P. aeruginosa, consistent with the delayed impact on survival. The impact of deficiency of purinoceptors on other inflammatory markers, such as lysozyme, lactoferrin, surfactant protein (SP)-A, and SP-D may also help clarify the complex inflammatory network impacting survival of these mice.
In the current study, survival in P2Y1/P2Y2−/− mice and to a lesser extent in mice lacking P2Y1 purinoceptor was impaired following lung challenge with P. aeruginosa. The mechanism for the impaired survival may be related to an attenuation of the inflammatory cascade and impaired alveolar wall integrity. These findings suggest that the P2Y1 and possibly P2Y2 purinoceptors may play a protective role in pulmonary infections with P. aeruginosa. Although the current study did not assess the benefit of exogenously administered nucleotides, our data indicate that deficiency of purinoceptors is associated with increased susceptibility to infection with P. aeruginosa and suggest that inhaled nucleotide therapy, currently in clinical trials, may be a useful adjunct to therapies aimed at decreasing colonization and infection with P. aeruginosa in patients with cystic fibrosis.
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- Copyright © 2005 the American Physiological Society