Severity of lung injury in cyclooxygenase-2-deficient mice is dependent on reduced prostaglandin E(2) production

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P < 0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P < 0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.     

C-C chemokine receptor 2 (CCR2) deficiency improves bleomycin-induced pulmonary fibrosis by attenuation of both macrophage infiltration and production of macrophage-derived matrix metalloproteinases

Macrophage infiltration is implicated in various types of pulmonary fibrosis. One important pathogenetic process associated with pulmonary fibrosis is injury to basement membranes by matrix metalloproteinases (MMPs) that are produced mainly by macrophages. In this study, C-C chemokine receptor 2-deficient (CCR2-/-) mice were used to explore the relationship between macrophage infiltration and MMP activity in the pathogenesis of pulmonary fibrosis, using the bleomycin-induced model of this disease process. CCR2 is the main (if not only) receptor for monocyte chemoattractant protein-1/C-C chemokine ligand 2 (MCP-1/CCL2), which is a critical mediator of macrophage trafficking, and CCR2 -/- mice demonstrate defective macrophage migration. Pulmonary fibrosis was induced in CCR2-/- and wild-type (CCR2+/+) mice by intratracheal instillation of bleomycin. No significant differences in the total protein concentration in bronchoalveolar lavage (BAL) fluid, or in the degree of histological lung inflammation, were observed in the two groups until day 7. Between days 3 and 21, however, BAL fluid from CCR2-/- mice contained fewer macrophages than BAL fluid from CCR2+/+ mice. Gelatin zymography of BAL fluid and in situ zymography revealed reduced gelatinolytic activity in CCR2-/- mice. Immunocytochemical staining showed weaker expression of MMP-2 and MMP-9 in macrophages in BAL fluid from CCR2-/- mice at day 3. Gelatin zymography of protein extracted from alveolar macrophages showed reduced gelatinolytic activity of MMP-2 and MMP-9 in CCR2-/- mice. At days 14 and 21, lung remodelling and the hydroxyproline content of lung tissues were significantly reduced in CCR2-/- mice. These results suggest that the CCL2/CCR2 functional pathway is involved in the pathogenesis of bleomycin-induced pulmonary fibrosis and that CCR2 deficiency may improve the outcome of this disease by regulating macrophage infiltration and macrophage-derived MMP-2 and MMP-9 production.     

Prostaglandin E2 synthesis and suppression of fibroblast proliferation by alveolar epithelial cells is cyclooxygenase-2-dependent

Alveolar epithelial cells (AECs) may influence neighboring fibroblasts by the elaboration of prostaglandin E(2) (PGE(2)). This prostanoid can be synthesized via "constitutive" cyclooxygenase (COX)-1 and "inducible" COX-2 enzyme isoforms. We compared AECs isolated from wild-type (WT), COX-1 knockout (KO), and COX-2 KO mice to determine the contribution of COX isoforms to AEC PGE(2) synthesis and capacity for suppression of fibroblast proliferation in co-cultures. WT AECs constitutively expressed both COX-1 and COX-2 isoforms by immunoblot analysis. COX-1 KO cells and WT cells comparably augmented PGE(2) synthesis following incubation with lipopolysaccharide or interleukin-1, whereas COX-2 KO cells were unable to do so. Surprisingly, however, constitutive generation of PGE(2) was also dramatically reduced only in COX-2 KO cells. When co-cultured with WT murine lung fibroblasts, AECs from WT and COX-1 KO animals suppressed serum-induced fibroblast proliferation, whereas COX-2-deficient AECs caused a modest enhancement in fibroblast proliferation. These results indicate that PGE(2) synthetic capacity in AECs is predominantly COX-2-dependent under both basal and stimulated conditions. They also demonstrate conclusively that AECs can modulate fibroblast function by the elaboration of suppressive prostanoids. These alterations in AEC phenotype likely contribute to the propensity for pulmonary fibrosis observed in COX-2-deficient mice.     

Airway inflammation and responsiveness in prostaglandin H synthase-deficient mice exposed to bacterial lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) is a risk factor for exacerbation of asthma and causes airway inflammation. The aim of this study was to examine the effects of disruption of prostaglandin (PG) H synthase (PGHS)-1 and PGHS-2 genes on pulmonary responses to inhaled LPS. PGHS-1(-/-), PGHS-2(-/-), and wild-type (WT) mice were exposed to 4 to 6 microg/m(3) LPS via aerosol. Enhanced pause (PenH), a measure of bronchoconstriction, was assessed using a whole-body plethysmograph before and immediately after a 4-h LPS exposure. Bronchoalveolar lavage (BAL) was performed after LPS exposure to assess inflammatory cells, cytokines/chemokines (tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2), and PGE(2). The degree of lung inflammation was scored on hematoxylin-and-eosin-stained sections. PGHS-1 and PGHS-2 protein levels were determined by immunoblotting. All mice exhibited increased PenH and methacholine responsiveness after LPS exposure; however, these changes were much more pronounced in PGHS-1(-/-) and PGHS-2(-/-) mice relative to WT mice (P < 0.05). There were no significant differences in inflammation as assessed by BAL fluid (BALF) cells or lung histology between the genotypes despite reduced BALF cytokines/chemokines and PGE(2) in PGHS-1(-/-) and PGHS-2(-/-) mice relative to WT mice (P < 0.05). PGHS-2 was upregulated more in PGHS-1(-/-) mice compared with WT mice after LPS exposure. We conclude that: (1) airway inflammation and hyperresponsiveness are dissociated in PGHS-1(-/-) and PGHS-2(-/-) mice exposed to LPS; (2) the balance of PGHS-1 and PGHS-2 is important in regulating the functional respiratory responses to inhaled LPS; and (3) neither PGHS-1 nor PGHS-2 is important in regulating basal lung function or the inflammatory responses of the lung to inhaled LPS.     

Cyclooxygenase-2 deficiency exacerbates bleomycin-induced lung dysfunction but not fibrosis

Cyclooxygenase (COX)-derived eicosanoids have been implicated in the pathogenesis of pulmonary fibrosis. Uncertainty regarding the influence of COX-2 on experimental pulmonary fibrosis prompted us to clarify the fibrotic and functional effects of intratracheal bleomycin administration in mice genetically deficient in COX-2. Further, the effects of airway-specific COX-1 overexpression on fibrotic and functional outcomes in wild-type and COX-2 knockout mice were assessed. Equivalent increases in airway cell influx, lung collagen content, and histopathologic evidence of fibrosis were observed in wild-type and COX-2 knockout mice 21 d after bleomycin treatment, suggesting that COX-2 deficiency did not alter the extent or severity of fibrosis in this model. However, bleomycin-induced alterations in respiratory mechanics were more severe in COX-2 knockout mice than in wild-type mice, as illustrated by a greater decrease in static compliance compared with genotype-matched, saline-treated control mice (26 +/- 3% versus 11 +/- 4% decreases for COX-2 knockout and wild-type mice, respectively; P < 0.05). The influence of COX-1 overexpression in airway Clara cells was also examined. Whereas the fibrotic effects of bleomycin were not altered in wild-type or COX-2 knockout mice overexpressing COX-1, the exaggerated lung function decrement in bleomycin-treated COX-2 knockout mice was prevented by COX-1 overexpression and coincided with decreased airway cysteinyl leukotriene levels. Collectively, these data suggest an important regulatory role for COX-2 in the maintenance of lung function in the setting of lung fibrosis, but not in the progression of the fibrotic process per se.     

Cigarette smoke-induced pulmonary inflammation, but not airway remodelling, is attenuated in chemokine receptor 5-deficient mice

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory response of the airways and lungs to noxious particles and gases, mostly cigarette smoke (CS). Pathological changes characteristic of COPD include airway wall thickening, peribronchial fibrosis, peribronchial lymphoid follicles and destruction of lung parenchyma (emphysema). The recruitment of inflammatory cells into the lung in response to CS is thought to play an important role in the development of COPD. OBJECTIVE: Our aim was to study the contribution of chemokine receptor 5 (CCR5) to the pathogenesis of COPD and specifically whether the development of airway remodelling is a direct result of airway inflammation or rather occurs through an independent mechanism. METHODS: In this study, C57BL/6 wild-type mice and CCR5-deficient mice were subjected to sub-acute (4 weeks) and chronic (24 weeks) CS exposure. RESULTS: Both sub-acute and chronic CS exposure significantly increased CCR5 mRNA expression and protein levels of CCR5 ligands [macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and regulated upon activation, normal T expressed and secreted (RANTES)], and induced the recruitment of neutrophils, macrophages, dendritic cells, and lymphocytes to the bronchoalveolar lavage (BAL) of wild-type mice. Chronic CS exposure also increased the number and extent of peribronchial lymphoid follicles. In CCR5 knockout (KO) mice, these CS-induced increases in CCR5 ligands, inflammatory cells in BAL and peribronchial lymphoid follicles were all significantly attenuated compared with wild-type animals. Importantly, chronic CS exposure induced airspace enlargement in wild-type mice, while CCR5 KO mice were partially protected against the development of emphysema. However, CCR5 deficiency did not affect CS-induced airway wall remodelling, because chronic CS exposure induced a similar increase in airway wall thickness, smooth muscle mass and peribronchial deposition of collagen and fibronectin in both wild-type and CCR5 KO mice. CONCLUSION: Our data suggest that CCR5 contributes to pulmonary inflammation and to the development of emphysema in response to CS. CCR5 is, however, not implicated in CS-induced airway wall remodelling, suggesting that the mechanisms that lead to airway inflammation are distinct to those responsible for airway remodelling.     

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.     

COX-2 is widely expressed in metaplastic epithelium in pulmonary fibrous disorders

Idiopathic usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF) and asbestosis represent progressive and often fatal pulmonary fibrous disorders, whereas cryptogenic organizing pneumonia (COP), desquamative interstitial pneumonia (DIP), and respiratory bronchiolitis-interstitial lung disease (RB-ILD) usually are reversible or nonprogressive conditions. Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and myofibroblast transition, its production depending on cyclooxygenase-2 (COX-2). In patients with UIP/IPF, levels of PGE2 and COX-2 are reduced in fibroblasts, and levels of PGE2 in bronchioalveolar lavage fluid may be lowered. We analyzed the immunohistochemical expression of COX-2 in UIP/IPF, asbestosis, COP, DIP, and RB-ILD. Our results show that the metaplastic epithelium in UIP/IPF, asbestosis, and COP is widely COX-2+, whereas COX-2 positivity is scant in DIP and RB-ILD. The mesenchymal cells remained negative. Our results suggest that irrespective of the underlying disease, lung injury that causes extensive fibrosis induces wide expression of COX-2 in the regenerating metaplastic epithelium.     

Interleukin-9 reduces lung fibrosis and type 2 immune polarization induced by silica particles in a murine model

We examined the effect of interleukin (IL)-9, a cytokine active on B and T lymphocytes and associated with bronchial asthma, on the development of lung fibrosis induced by crystalline silica particles. Therefore, we compared the response to silica (1 and 5 mg/animal, intratracheally) in transgenic mice that constitutively express high levels of IL-9 (Tg5) and their wild-type counterparts (FVB). At 2 and 4 mo after treatment with silica, histologic examination and measurement of lung hydroxyproline content showed that the severity of fibrosis was significantly less important in Tg5 mice than in their wild-type counterparts. Intraperitoneal injection of IL-9 in C57BL/6 mice also reduced the amplitude of silica-induced lung fibrosis. The reduction of lung fibrosis by IL-9 was associated with a significant expansion of the B-lymphocyte population, both in bronchoalveolar lavage (BAL) and in the pulmonary parenchyma. In wild-type animals, silica-induced fibrosis correlated with markers of a T helper 2-like response such as upregulation of IL-4 levels in lung tissue and an increased immunoglobulin (Ig) G1/IgG2a ratio in BAL. Immunohistochemical studies demonstrated that the upregulation of IL-4 associated with the development of fibrosis was mainly localized in inflammatory alveolar macrophages. In transgenic mice, the level of IL-4 in lung homogenates was not significantly affected by silica treatment, and a reduced IgG1/IgG2a ratio was observed upon treatment with silica. The levels of interferon-gamma were significantly decreased after silica treatment in both strains. Together, these observations point to an antifibrotic effect of IL-9 in pulmonary fibrosis associated with a limitation of the type 2 polarization which accompanies lung fibrosis.     

Heat-killed BCG induces biphasic cyclooxygenase 2+ splenic macrophage formation--role of IL-10 and bone marrow precursors

Previous studies have shown that prostaglandin E(2) (PGE(2)) release by splenic F4/80(+) cyclooxygenase (COX)-2(+) macrophages (M?) isolated from mice, treated with mycobacterial components, plays a major role in the regulation of immune responses. However, splenic M?, isolated from untreated mice and treated in vitro with lipopolysaccharide and interferon-gamma, express COX-1 and COX-2 within 1 day but release only minimal amounts of PGE(2) following elicitation with calcium ionophore A23187. For further characterization of in vivo requirements for development of PGE(2)-releasing M? (PGE(2)-M?), C57Bl/6 [wild-type (WT)], and interleukin (IL)-10-deficient (IL-10(-/-)) mice were treated intraperitoneally with heat-killed Mycobacterium bovis bacillus Calmette-Guerin (HK-BCG). One day following injection, COX-2 was induced in splenic M? of both mouse strains. However, PGE(2) biosynthesis by these M? was not increased. Thus, expression of COX-2 is not sufficient to induce PGE(2) production in vivo or in vitro. In sharp contrast, 14 days after HK-BCG treatment, PGE(2) release by COX-2(+) splenic M? increased as much as sevenfold, and a greater increase was seen in IL-10(-/-) cells than in WT cells. To further determine whether the 14-day splenic PGE(2)-M? could be derived from bone marrow precursors, we established a chimera in which bone marrow cells were transfused from green fluorescent protein (GFP)-transgenic donors to WT mice. Donors and recipients were treated with HK-BCG simultaneously, and marrow transfusion was performed on Days 1 and 2. On Day 14 after BCG treatment, a significant number of spleen cells coexpressed COX-2 and GFP, indicating that bone marrow-derived COX-2(+) M? may be responsible for the increased PGE(2) production.     

Murine TLR4 is implicated in cigarette smoke-induced pulmonary inflammation

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with an abnormal inflammatory response of the lungs to noxious particles or gases. We investigated whether Toll-like receptor 4 (TLR4) is implicated in cigarette smoke (CS)-induced pulmonary inflammation in a murine model of COPD. METHODS: C3H/HeOuJ (Tlr4(WT)) and C3H/HeJ (Tlr4(defective)) mice were exposed to air or CS for 5 weeks (subacute) and 26 weeks (chronic), and pulmonary inflammation was evaluated. RESULTS: In Tlr4(WT) mice, subacute and chronic CS exposure induced a substantial pulmonary infiltration of macrophages, neutrophils, lymphocytes and dendritic cells (DCs), that was absent in air-exposed mice. CS exposure increased the costimulatory marker expression on DCs, the levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluid and induced the pulmonary expression of matrix metalloproteinase-12 (MMP-12), TLR4 and TLR2. In contrast, after subacute CS exposure, Tlr4(defective) mice showed a limited (5-fold lower) increase of DCs and lymphocytes in BAL fluid, lower costimulatory marker expression on DCs and lower MCP-1 and TNF-alpha levels in BAL fluid compared to Tlr4(WT) animals. After chronic CS exposure, however, the difference in pulmonary inflammation between Tlr4(WT) and Tlr4(defective) mice was less pronounced and both strains showed similar MCP-1 and TNF-alpha levels in BAL and similar pulmonary MMP-12, TLR4 and TLR2 expression. CONCLUSIONS: We demonstrated that the TLR4 mutation in C3H/HeJ mice is protective against CS-induced pulmonary influx of neutrophils, DCs and lymphocytes upon subacute CS exposure. However, TLR4 is only of minor importance in chronic CS-induced inflammation in mice.     

Suppressive activity of alveolar macrophages and blood monocytes from interstitial lung diseases: role of released soluble factors

Two groups of patients suffering from interstitial lung diseases (ILD) namely sarcoidosis (SA) and idiopathic pulmonary fibrosis (IPF) were investigated for alveolar macrophages (AM), secretion of prostaglandin E2 (PGE2) and interleukin 1 (IL-1), together with superoxide anion (O2-) production. Peripheral blood monocytes (PBMO) of the same patients were examined concomitantly for suppressive activity. Consistent with previous results, AM obtained by bronchoalveolar lavage (BAL) from ILD patients markedly suppressed the effects of PHA stimulation of autologous peripheral blood lymphocytes (APL): 61.8 +/- 9.7% suppressive activity compared to 15.5 +/- 15.4% in the control group (CO) P less than 0.001. The AM suppressive activity was correlated with an increase in PGE2 secretion: 3.861 +/- 2.194 ng/10(5) cells/ml in the IPF group, but not in the sarcoid group: 0.217 +/- 0.116 ng/10(5) cells/ml (P less than 0.001 between them). On the other hand, IL-1 secretion by AM was greatly increased in sarcoid patients (308 +/- 196 U/ml) but was within the normal limits in IPF (27.3 +/- 28.8 U/ml, P less than 0.01 between them). Therefore, an inverse correlation was found between degree of PGE2 secretion and IL-1 release by AM in ILD. O2-production by AM was markedly increased in all ILD patients but this mechanism is apparently not involved in suppressive activity. PBMO originating from ILD patients were less suppressive than the corresponding AM.     

Correlation between PGE2 production and suppressor activity of alveolar macrophages from patients with interstitial lung diseases

The suppressive activity of alveolar macrophages (AM) obtained from bronchoalveolar lavage (BAL), on PHA stimulation of autologous peripheral blood lymphocytes (APL) was evaluated. The effect on lymphocyte stimulation was evaluated by coculturing the AM and APL cells at a ratio of 1:1. PGE2 released by AM during the culture period was measured by a radioimmune assay. The patients included in the study were 11 cases with interstitial lung disease (ILD), 8 cases of lung cancer (CA), and 5 controls (CO). Addition of AM of patients from the CA group resulted in slight suppression of lymphocyte stimulation in 4 cases, slight enhancement in 3 cases and no effect in one case. AM from the CO group induced slight suppression in 4 out of 5 cases. AM from all 11 ILD cases exerted a significant high suppressive activity (65.6% +/- 18.2 - P less than 0.001 by comparison with the CO and CA groups). In ILD cases, a dichotomous pattern was found in regard to relation between high suppressive activity of AM and release of PGE2: in idiopathic pulmonary fibrosis (IPF) patients, high suppressive activity of AM (70.4% +/- 15.4) correlated well with elevated secretion of PGE2: 3.58 +/- 0.26 ng/ml/10(5) cells (P less than 0.02 compared to CO). AM from sarcoidosis patients suppressed PHA stimulation by 61.6% +/- 19.3 but secreted only 0.357 +/- 0.26 ng/ml/10(5) cells of PGE2 (P less than 0.02 compared with the idiopathic pulmonary fibrosis group). Therefore, it seems that other factors, in addition to PGE2, might be involved in the suppressive activity of AM from interstitial lung diseases.     

Exposure to Polyhexamethylene Guanidine Exacerbates Bronchial Hyperresponsiveness and Lung Inflammation in a Mouse Model of Ovalbumin-Induced Asthma

Humidifier disinfectants (HDs) exposure has now been associated with acute lung injury and pulmonary fibrosis; polyhexamethylene guanidine (PHMG) has been confirmed to cause severe lung inflammation and fibrosis in mice. Recent evidence also indicates that HDs exposure increases the asthma risk in children, but the underlying mechanisms remain unclear. We aimed to investigate the effects of PHMG exposure on asthma in mice and the potential underlying mechanisms. BALB/c mice were intranasally administered PHMG (0.1 mg/kg/day; 5 days per week) during 2 episodes of ovalbumin (OVA) sensitization and were then challenged with 1% OVA by inhalation. Bronchial hyperresponsiveness (BHR), inflammatory cell influx into bronchoalveolar lavage (BAL) fluid, serum total and OVA-specific immunoglobulin (Ig) E levels, and histopathological changes in the lung were analyzed. The levels of asthma-related cytokines and chemokines were assayed in the lung tissues to evaluate possible mechanisms. Exposure to PHMG following OVA sensitization and challenge significantly enhanced BHR, inflammatory cell counts in BAL fluid, airway inflammation, and total serum IgE levels in the asthma mouse model. In addition, the levels of chemokine ligand (CCL) 11 and serpine F1/pigment epithelium-derived factor (SERPINF1) were significantly elevated in the lungs of these mice compared to those in the control and OVA-treated only groups. Our findings suggest that PHMG can enhance the development of allergic responses and lung inflammation via CCL11- and SERPINF1-induced signaling in a mouse model of asthma.     

机动车尾气诱发大鼠慢性阻塞性肺疾病并上调气道上皮细胞COX-2/PGE2/EP受体信号通路

目的：探究机动车尾气(MVE)长期暴露引起大鼠慢性阻塞性肺疾病(COPD)发生时,气道上皮细胞中环加氧酶2(COX-2)/前列腺素E₂(PGE₂)/E-前列腺素类激素(EP)受体信号通路成员的表达变化。方法：(1)动物实验：健康雄性SD纯系大鼠(SPF级)16只,随机分为2组：MVE暴露组(n=8)和空白对照(CTL)组(n=8)。采用MVE暴露6个月的方法建立COPD大鼠模型。造模结束后,使用Buxco小动物有创肺功能仪检测大鼠肺功能;肺组织切片行HE染色并评估肺组织病理变化;ELISA法检测大鼠支气管肺泡灌洗液(BALF)中炎症因子白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和PGE₂的水平,评估大鼠肺部炎症情况;采用免疫荧光及Western blot法检测肺组织COX-2及EP受体蛋白水平;提取肺组织核蛋白,Western blot检测MVE对肺组织NF-κB核转位的影响。(2)细胞实验：采用MVE细颗粒物(PM2.5)标准品刺激人正常支气管上皮细胞BEAS-2B。ELISA法检测细胞培养液中PGE     

Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes in the lung.

An acute (2 h) exposure of humans to 0.4 ppm ozone initiates biochemical changes in the lung that result in the production of components mediating inflammation and acute lung damage as well as components having the potential to lead to long-term effects such as fibrosis. However, many people are exposed to lower levels of ozone than this, but for periods of several hours. Therefore, it is important to determine if a prolonged exposure to low levels of ozone is also capable of causing cellular and biochemical changes in the lung. Nonsmoking males were randomly exposed to filtered air and either 0.10 ppm ozone or 0.08 ppm ozone for 6.6 h with moderate exercise (40 liters/min). Bronchoalveolar lavage (BAL) was performed 18 h after each exposure, and cells and fluid were analyzed. The BAL fluid of volunteers exposed to 0.10 ppm ozone had significant increases in neutrophils (PMNs), protein, prostaglandin E2 (PGE2), fibronectin, interleukin-6 (IL-6), and lactate dehydrogenase (LDH) compared with BAL fluid from the same volunteers exposed to filtered air. In addition, there was a decrease in the ability of alveolar macrophages to phagocytize yeast via the complement receptor. Exposure to 0.08 ppm ozone resulted in significant increases in PMNs, PGE2, LDH, IL-6, alpha 1-antitrypsin, and decreased phagocytosis via the complement receptor. However, BAL fluid protein and fibronectin were no longer significantly elevated. We conclude that exposure of humans to as low a level as 0.08 ppm for 6.6 h is sufficient to initiate an inflammatory reaction in the lung.     

Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice

Didecyldimethylammonium chloride (DDAC) is used worldwide as a germicide, in antiseptics, and as a wood preservative, and can cause adverse pulmonary disease in humans. However, the pulmonary toxicity of DDAC has not yet been thoroughly investigated. Mice were intratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500 μg/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150 μg/kg of DDAC caused weakly induced inflammation, and exposure to 15 μg/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150 μg/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C–C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health.     

Requirement for chemokine receptor 5 in the development of allergen-induced airway hyperresponsiveness and inflammation

Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2-induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5(-/-)) and wild-type (CCR5(+/+)) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5(-/-) mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5(+/+) mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5(-/-) mice were lower than in CCR5(+/+) mice. Decreased numbers of lung T cells were also detected in CCR5(-/-) mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5(+/+), but not transfer of CCR5(-/-) cells, into CCR5(-/-) mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5(+/+) T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.     

Type I interleukin-1 receptor is required for pulmonary responses to subacute ozone exposure in mice

Interleukin (IL)-1, a proinflammatory cytokine, is expressed in the lung after ozone (O(3)) exposure. IL-1 mediates its effects through the type I IL-1 receptor (IL-1RI), the only signaling receptor for both IL-1alpha and IL-1beta. The purpose of this study was to determine the role of IL-1RI in pulmonary responses to O(3.) To that end, wild-type, C57BL/6 (IL-1RI(+/+)) mice and IL-1RI-deficient (IL-1RI(-/-)) mice were exposed to O(3) either subacutely (0.3 ppm for 72 h) or acutely (2 ppm for 3 h). Subacute O(3) exposure increased bronchoalveolar lavage fluid (BALF) protein, interferon-gamma-inducible protein (IP)-10, soluble tumor necrosis factor receptor 1 (sTNFR1), and neutrophils in IL-1RI(+/+) and IL-1RI(-/-) mice. With the exception of IP-10, all outcome indicators were reduced in IL-1RI(-/-) mice. Furthermore, subacute O(3) exposure increased IL-6 mRNA expression in IL-1RI(+/+), but not IL-1RI(-/-) mice. Acute (2 ppm) O(3) exposure increased BALF protein, IL-6, eotaxin, KC, macrophage inflammatory protein (MIP)-2, IP-10, monocyte chemotactic protein-1, sTNFR1, neutrophils, and epithelial cells in IL-1RI(+/+) and IL-1RI(-/-) mice. For IL-6, eotaxin, MIP-2, and sTNFR1, there were small but significant reductions of these outcome indicators in IL-1RI(-/-) versus IL-1RI(+/+) mice at 6 hours after exposure, but not at other time points, whereas other outcome indicators were unaffected by IL-1RI deficiency. These results suggest that IL-1RI is required for O(3)-induced pulmonary inflammation during subacute O(3) exposure, but plays a more minor role during acute O(3) exposure. In addition, these results suggest that the induction of IL-6 via IL-1RI may be important in mediating the effects of O(3) during subacute exposure.