Lung Defense through IL-8 Carries a Cost of Chronic Lung Remodeling and Impaired Function.

IL-8-dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases, including chronic obstructive pulmonary disease, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible, pathological changes associated with elevated levels of IL-8 in the lung. To better understand the duality of IL-8-dependent host immunity to bacterial infection and lung pathology, we expressed human IL-8 transgenically in murine bronchial epithelium, and investigated the impact of overexpression on lung bacterial clearance, host immunity, and lung pathology and function. Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation and activation, and chemotaxis. There was enhanced protection against challenge with Pseudomonas aeruginosa, and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL-2, and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen OprF, indicating a regulatory T-cell phenotype. However, this enhanced bacterial immunity came at a high price of progressive lung remodeling, with increased inflammation, mucus hypersecretion, and fibrosis. There was increased expression of Ccl3 and reduced expression of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all of which resulted in impaired lung function with reduced compliance, increased resistance, and bronchial hyperreactivity as measured by whole-body plethysmography. These results show that IL-8 overexpression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodeling, and damaged tight junctions, leading to impaired lung function.

The pneumonectomy model of compensatory lung growth: insights into lung regeneration.

Pneumonectomy (PNX) in experimental animals leads to a species- and age-dependent compensatory growth of the remaining lung lobes. PNX mimics the loss of functional gas exchange units observed in a number of chronic destructive lung diseases. However, unlike in disease models, this tissue loss is well defined, reproducible and lacks accompanying inflammation. Furthermore, compensatory responses to the tissue loss can be easily quantified. This makes PNX a potentially useful model for the study of the cellular and molecular events which occur during realveolarisation. It may therefore help to get a better understanding of how to manipulate these pathways, in order to promote the generation of new alveolar tissue as therapies for destructive lung diseases. This review will explore the insights that experimental PNX has provided into the physiological factors which promote compensatory lung growth as well as the importance of age and species in the rate and extent of compensation. In addition, more recent studies which are beginning to uncover the key cellular and molecular pathways involved in realveolarisation will be discussed. The potential relevance of experimental pneumonectomy to novel therapeutic strategies which aim to promote lung regeneration will also be highlighted.

Innate responsiveness of CD8 memory T-cell populations nonspecifically inhibits allergic sensitization.

BACKGROUND: Infection or stimulation of the innate immune system by nonspecific microbial antigens is thought to educate the immune system to respond appropriately to allergens, preventing allergy. OBJECTIVE: To determine the immunologic pathways that might explain how infection/microbial exposure inhibits allergic sensitization. METHODS: Immunologic studies of non-antigen-specific functions of CD8 memory cells, their maturation in vivo, and their effects in a mouse asthma model, to test the hypothesis that CD8 memory is shaped by innate immunity in a way that can inhibit allergic disease. RESULTS: We found that CD8 memory T-cell (CD8 Tm) populations bridge innate and adaptive immunity by responding to either antigen or cytokines alone. CD8 Tm populations partially subvert the clonal selection process by activating their neighbors through induction of dendritic cell IL-12. Stimulation of innate or acquired immunity in the lung or gut causes expansion/maturation of CD8 Tm populations, which provide an early source of cytokines, enhance T(H)1 immunity, and inhibit allergic sensitization and airway inflammation/hyperresponsiveness in a non-antigen-specific fashion. CONCLUSION: CD8 T-cell-mediated immune memory is long-lived and can retain its capacity for rapid cytokine release in a nonantigen-specific fashion. This novel type of memory enhances T(H)1 over T(H)2 immunity and prevents allergic sensitization after exposure to environmental antigens or infection.

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation.

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.

Probucol [4,4'-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1,1-dimethylethyl)phenol]] inhibits compensatory remodeling and promotes lumen loss associated with atherosclerosis in apolipoprotein E-deficient mice.

Probucol [4,4'-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1,1-dimethylethyl)phenol]] was withdrawn from the United States market because it failed to inhibit atherosclerosis in human femoral arteries, yet the drug was shown subsequently to inhibit atherosclerosis in human carotid arteries, and probucol monosuccinate ester is presently being tested in a phase III clinical trial as an antiatherosclerotic compound based on its anti-inflammatory properties. Inflammatory macrophages are implicated in arterial remodeling associated with atherosclerosis, and probucol inhibits experimental atherosclerosis in part by decreasing macrophages in lesions. However, the impact of probucol on remodeling is unknown, although such knowledge could help explain why the drug's benefit on human atherosclerosis is controversial. We therefore examined the effect of probucol on remodeling of the common carotid artery in apolipoprotein E-deficient mice. We observed that during de novo atherosclerosis, plaque growth was fully compensated by expansive remodeling, such that lumen area was unaffected. Early lesions were composed almost entirely of macrophages, and their contribution to lesion area progressively decreased thereafter. Probucol significantly decreased plaque area, expression of vascular cell adhesion molecule-1, and proliferation of intimal cells, resulting in delayed macrophage accumulation in the vessel. Probucol also decreased the production and activity of matrix metalloproteinases-2 and -9, independent of the plasmin protease system, and this was associated with an inhibition of expansive remodeling, resulting in lumen loss. These studies show that probucol attenuates compensatory remodeling associated with de novo atherosclerosis, probably via its anti-inflammatory properties. Our findings suggest that lumen volume is not a suitable surrogate to assess the antiatherosclerotic activity of probucol and related drugs.

Protective effect of vitamin E supplements on experimental atherosclerosis is modest and depends on preexisting vitamin E deficiency.

Vitamin E has failed to protect humans from cardiovascular disease outcome, yet its role in experimental atherosclerosis remains less clear. A previous study (Proc. Natl. Acad. Sci. USA 97:13830-13834; 2000) showed that vitamin E deficiency caused by disruption of the alpha-tocopherol transfer protein gene (Ttpa) is associated with a modest increase in atherosclerosis in apolipoprotein E gene deficient (Apoe(-/-)) mice. Here we confirm this finding and report that in Apoe(-/-)Ttpa(-/-) mice dietary alpha-tocopherol (alphaT) supplements restored circulating and aortic levels of alphaT, and decreased atherosclerosis in the aortic root to a level comparable to that seen in Apoe(-/-) mice. However, such dietary supplements did not decrease disease in Apoe(-/-) mice, whereas dietary supplements with a synthetic vitamin E analog (BO-653), either alone or in combination with alphaT, decreased atherosclerosis in Apoe(-/-) and in Apoe(-/-)Ttpa(-/-) mice. Differences in atherosclerosis were not associated with changes in the arterial concentrations of F(2)-isoprostanes and cholesterylester hydro(pero)xides, nor were they reflected in the resistance of plasma lipids to ex vivo oxidation. These results show that vitamin E at best has a modest effect on experimental atherosclerosis in hyperlipidemic mice, and only in situations of severe vitamin E deficiency and independent of lipid oxidation in the vessel wall.

Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging.

Oxidative stress is implicated in atherogenesis, yet most clinical trials with antioxidants, particularly vitamin E, have failed to protect against atherosclerotic diseases. A striking exception is probucol, which retards atherosclerosis in carotid arteries and restenosis of coronary arteries after angioplasty. Because probucol has in vitro cellular-protective effects independent of inhibiting lipid oxidation, we investigated the mode of action of probucol in vivo. We used three models of vascular disease: apolipoprotein E-deficient mice, a model of atherosclerosis; rabbit aortic balloon injury, a model of restenosis; and carotid injury in obese Zucker rats, a model of type 2 diabetes. Unexpectedly, we observed that the phenol moieties of probucol were insufficient, whereas its sulphur atoms were required for protection. Probucol and its sulphur-containing metabolite, but not a sulphur-free phenolic analogue, protected via cell-specific effects on inhibiting macrophage accumulation, stimulating reendothelialization, and inhibiting vascular smooth muscle cell proliferation. These processes were mediated via induction of heme oxygenase-1 (HO-1), an activity not shared by vitamin E. Our findings identify HO-1 as the molecular target of probucol. They indicate 2-electron rather than radical (1-electron) oxidants as important contributors to atherogenesis, and point to novel lead compounds for therapeutic intervention against atherosclerotic diseases.

Probucol inhibits in-stent thrombosis and neointimal hyperplasia by promoting re-endothelialization.

BACKGROUND: Evidence suggests that delayed re-endothelialization is responsible for in-stent thrombosis. Probucol inhibits neointimal thickening in animals via enhanced re-endothelialization and is the only oral drug that consistently inhibits restenosis after coronary angioplasty in humans. Here, we examined the effects of probucol on re-endothelialization and neointimal formation in a stent model. METHODS AND RESULTS: New Zealand White rabbits were fed a hypercholesterolemic diet with probucol (1%) or without (control) (n=11 each) for 6 weeks. At 2 weeks, endothelial denudation and stenting of the iliac artery was performed. Iliac arteries were harvested at week 6, and stented segments sectioned and analyzed. Compared with control, probucol increased in-stent re-endothelialization (74+/-6% in controls versus 93+/-3% in probucol-treated; P=0.008), and decreased average luminal stenosis (58+/-27 versus 31+/-16%; P=0.01) and stent depth (619+/-310 versus 314+/-158 microm; P=0.009). Compared with control, probucol also decreased accumulation of macrophages in the neointima. Furthermore, none of the probucol-treated rabbits had in-stent thrombosis, whereas four of eleven control rabbits showed thrombosis (P=0.04). CONCLUSIONS: Probucol demonstrates anti-restenotic and appears to have anti-thrombotic properties that are likely related to its ability to promote in-stent re-endothelialization.

Processes involved in the site-specific effect of probucol on atherosclerosis in apolipoprotein E gene knockout mice.

OBJECTIVE: To elucidate processes by which the antioxidant probucol increases lesion size at the aortic sinus and decreases atherosclerosis at more distal sites in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: Male apoE(-/-) mice were fed high-fat chow with 1% (w/w) probucol or without (controls) for 6 months, before aortic sinus, arch, and descending aorta were analyzed separately for lesion size and composition. Compared with control, probucol significantly increased lesion size by 33% at the sinus, but it inhibited atherosclerosis at the descending aorta by 94%. Sites where atherosclerosis was inhibited contained substantially fewer macrophages, less lipids (cholesterol and cholesteryl esters), and endogenous antioxidant (alpha-tocopherol), but not oxidized lipids, and the extent to which probucol metabolism occurred was increased. Compared with control, aortic sinus lesions of probucol mice contained a substantially increased content of extracellular matrix, but decreased total cell and macrophage density, comparable levels of lipids and alpha-tocopherol, and decreased concentrations of oxidized lipids (cholesteryl ester hydroperoxides, F2-isoprostanes, and 7-ketocholesterol). CONCLUSIONS: Probucol affects atherosclerosis in apoE(-/-) mice independent of the accumulation of arterial lipid oxidation products, thereby dissociating the 2 processes. Rather, probucol exerts antiinflammatory activity by decreasing accumulation of macrophages in lesions, and it promotes a more stable lesion composition at the aortic sinus.

Coenzyme Q(10) supplementation inhibits aortic lipid oxidation but fails to attenuate intimal thickening in balloon-injured New Zealand white rabbits.

Oxidized lipoproteins are implicated in atherosclerosis, and some antioxidants attenuate the disease in animals. Coenzyme Q(10) (CoQ(10)) in its reduced form, ubiquinol-10, effectively inhibits lipoprotein oxidation in vitro and in vivo; CoQ(10) supplements also inhibit atherosclerosis in apolipoprotein E gene knockout (apoE-/-) mice. Here we tested the effect of dietary CoQ(10) supplements on intimal proliferation and lipoprotein lipid oxidation in balloon-injured, hypercholesterolemic rabbits. Compared to nonsupplemented chow, CoQ(10) supplementation (0.5% and 1.0%, w/w) significantly increased the plasma concentration of CoQ(10) and the resistance of plasma lipids to ex vivo oxidation. CoQ(10) supplements also increased the content of CoQ(10) in the aorta and liver, but not in the brain, skeletal muscle, kidney, and heart. Surprisingly, CoQ(10) supplementation at 1% increased the aortic concentrations of all lipids, particularly triacylglycerols, although it significantly inhibited the proportion of triacylglycerols present as hydroperoxides by > 80%. The observed increase in vessel wall lipid content was reflected in elevated plasma concentrations of cholesterol, cholesteryl esters and triacylglycerols, and hepatic levels of mRNA for 3-hydroxy-3-methylglutaryl-coenzyme A reductase. CoQ(10) supplements did not attenuate lesion formation, assessed by the intima-to-media ratio of injured aortic vessels. Thus, like in apoE-/- mice, a high dose of supplemented CoQ(10) inhibits lipid oxidation in the artery wall of balloon-injured, hypercholesterolemic rabbits. However, unlike its antiatherosclerosis activity in the mice, CoQ(10) does not inhibit intimal hyperplasia in rabbits, thereby dissociating this disease process from lipid oxidation in the vessel wall.