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Inflammation in Cystic Fibrosis

Mediators of Inflammation 5, 121-143 (1996) Inflammation in Cystic Fibrosis International Symposium


Introduction
Chronic airway inflammation is a consistent finding in patients with cystic fibrosis (CF). Early bacterial colonization and infection of the airways with persisting pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa are generally thought to provoke the activation of cells of the specific and nonspecific immune system and the migration of blood neutrophils to the lung. The consequences of chronic inflammation are decreasing lung function and premature death of CF patients. The mechanisms leading to inflammation in CF patients and the complex cellular interactions in inflammation mediated by a number of mediators including proteinases, reactive oxygen species and cytokines as well as their inhibitors are complex. Progress in understanding this important aspect of CF may be accelerated by discussions and collaborations between researchers with different skills. It is this aim which led to an international symposium on inflammation in CF where a number of relevant topics were discussed and the present knowledge summarized by experts in their fields. Such knowledge will hopefully result in more potent and selective anti-inflammatory therapeutic strategies, and thus increase life expectancy in CF.
Clinical and pathophysiological aspects of lung inflammation were discussed in detail. Michel Aubier, Paris, France focuses on compartimentalized lung inflammation in patients with pulmonary infections. Then, .udck Cl/ment, Paris, France reports on recent studies showing that inflammation in CF may precede lung infection in contrast to the paradigm that infection precedes inflammation. If this finding can be further substantiated, the mechanisms are to be elucidated and new ap.proaches to monitor pulmonary inflammation in very young infants are needed. Interestingly, the expression and/or regulation of inflammatory mediators from macrophages and epithelial cells may be different in normal individuals and CF patients. Melvin Berger, Cleveland, LISA reports that CF bronchial epithelial cells contain much less IL-10 than those from normal healthy individuals, hypothesizing whether defective CFTR function could alter epithelial cell cytokine production. Jean- Michel Dayer, Geneva, Switzerland presents the complex network of pro-inflammatory cytokines and their inhibitors stressing on the concept of a possible dissociation between immunosuppressive and anti-inflammatory actions of cytokine inhibitors and its potential impact on therapeutic strategies. Damage and remodelling of CF airway epithelium caused by inflammation may also facilitate adhesion of P. aeruginosa as described by Sophie Girod de Bentzmann, Reims, France.
The mechanisms of neutrophil recruitment to the CF airway lumen, the release of lysosomal enzymes and the preventive strategies have been of major interest in this symposium. Based on experimental models of lung inflammation, Peter Ward, Ann Arbor, USA discusses the mediators for neutrophil recruitment and lung disease. Jay" Nadel, San Francisco, USA describes a complex signalling process involving compounds from bacteria, the airway epithelium and the neutrophil causing chemotaxis and lysosomal enzyme release. Elaine Tuomanen, New York, USA reports on trials in which antibodies to CD18 or molecules which bind to receptors of CD18 blocked neutrophil migration. Robert Stockley, Birmingham, UK summarizes the current knowledge about the genes of the neutrophil serine proteinases, elastase, cathepsin G and proteinase-3 and discusses modulatory processes for elastase expression during cell differentiation. Gerd D6ring, Tiibingen, Getanany reports on trials using aerosolized proteinase inhibitor to suppress neutrophil elastase activity in CF airways.
Oxidative stress, as a consequence of oxidant/ anti-oxidant imbalances, is another important topic of this symposium. V&ronique Witko-Sarsat, Paris, France reviews the current knowledge on the mechanisms of neutrophil formation of reactive oxygen species and long-lived oxidants and presents recent data demonstrating that homozygote and heterozygote CF individuals overexpress neutrophil oxidative activity. Frank J. Kelly, London, UK demonstrates that biological markers of oxidative stress and antioxidant scavengers are present in plasma, urine and sputum of CF patients, suggesting that antioxidant therapy could reduce lung injury in CF. In contrast, Dieter Worlitzsch, Ttbingen, Getanany explains the absence of detectable hydrogen peroxide in the exhaled breath condensates of CF patients with the presence of high sputum concentrations of myeloperoxidase and catalase.
Based on the clinical experience, conventional anti-inflammatory drugs, presented by Michael Konstan, Cleveland, USA may only reduce the inflammatory manifestations of the airways; therefore gene therapy is discussed as an alternative approach to prevent lung inflammation. Catherine Figarella, Marseille, France reports on the expression and function of CFTR in CF and normal airways. Patricia Lemarchand, Paris, France discusses the hurdles of gene replacement therapy for defective CFTR and for oxidative stress. In vitro studies by Meksantier Edehnan, Paris, France reveal that cytokines and aspirin may downregulate CFTR gene expression suggesting that inflammation may impair gene therapy. Gabriel Bellon, Lyon, France reports on the results of a CFTR gene replacement trial in six CF patients and Claire Danel, Paris, France focuses on the problem of lung inflammation consecutive to gene transfer.
In conclusion, Inflammation in Cystic Fibrosis closely reflects the hostile fce of Janus chosen by the Editor-in-Chief, Ivan Bont, as 'the symbol par excellenc for Mediators of Inflammation.
We hope that the basic and novel aspects on the role of inflammation in CF and therapeutic strategies discussed in this symposium will have an important impact on the management of chronic inflammatory lung disease in CF patients.
Normally, bacteria are prevented from reaching the alveoli by several defense mechanisms located along the normal upper airways. Furthermore, bacteria reaching the alveoli are usually phagocytized and killed by resident macrophages. When normal clearance mechanisms are overwhelming, a complex response develops. At a peripheral tissue site, invasion by a bacterial pathogen produces an inflammatory response that is accompanied by the activation of local macrophages by bacterial products such as endotoxin and cell-wall components. The early stage of infection may be characterized by the generation of multiple signals, including a cascade of endogenous host mediators triggered by bacterial products. Among these mediators, cytokines and particularly tumour necrosis factor-cz (TNF-cz), interleukin-1]3 (IL-113), interleukin-6 (It-6) and It- 8 are thought to mediatemany host responses to bacterial infection. These cytokines are involved in the response to infection, including the activation of immune cells leading to the production of specific antibodies. They are also involved in the recruitment and activation of monocytes and neutrophils to areas of infection following activation of endothelial cells.
Although excessive cytokine production during severe infection has many deleterious effects that may lead to death, 2 various animal models support the beneficial role of cytokines in natural host resistance to local infection. This suggests that locally produced cytokines ma contribute to eradicating the invading pathogen. Indeed, some cytokines appear to have more local effects within their tissue of origin than systemic effects. [4][5][6] In this regard, several human and animal studies have demonstrated significant differences between the inflammatory response in intravascular models of infection and in localized bacterial infection such as meningitis. 4'5 However, little is known about the in situ inflammatory response developing in the human lung during localized bacterial infection.
In two recent studies, 7'8 the in situ inflammatory response developing in the human lung during a localized bacterial infection was studied in 15 patients with unilateral community-acquired pneumonia (CAP). The local response in the involved lung was compared with that in the controlateral, non-involved lung as well as with the systemic blood response. Eight healthy volunteers served as control subjects. Concentrations of tumour necrosis factor-(TNF-a), interleukin-1]3 (IL-113), and interleukin-6 (IL6) and It-8 were measured by ELISA in bronchoalveolar lavage (BAL) fluids (n 15), serum (n 15), and alveolar macrophage and monocyte culture supernatant (n 8). The concentrations of TNF-cz, IL-]3, IL6 and IL-8 in BAL fluid were significantly higher in the involved lung than in the paired non-involved lung (p < 0.01) or in healthy subjects (p < 0.02, p < 0.01, p < 0.001, p < 0.0001, respectively). Serum IL-6 concentrations were higher in patients than in control subjects, whereas IL-1 ]3, TNF-cz and IL-8 concentrations did not differ in the two groups. Alveolar macrophages from the involved lung spontaneously released higher concentrations of IL-1]3, IL-6, and TNF-cz (p < 0.05) than did macrophages from the non-involved lung, which served as control. However, macrophages were hyporesponsive in terms of cytokine production to further stimulation by lipopolysaccharide (LPS) in the noninvolved and involved lung compared with controls, whereas peripheral blood monocytes were not. These data in humans provide strong evidence that during unilateral CAP, the inflammatory response is compartmentalized within the human lung and is limited to the site of infection.
Mthough TNF-cz and IL-1]3 are believed to have synergistic .effects that induce lung damage and Mediators of Inflammation Vol 5 1996 123 leakage through the capillary wall, 9 a beneficial role has also been ascribed to cytokines in limiting bacterial spread. The localized and compartmentalized production of cytokines within the lung in unilateral pneumonia confirms that these substances play an important role in the local lung response to infection. Concomitantly, the observed hyporeactivity in LPS-induced cytokine synthesis might be the result of a homeostatic process that would limit the local inflammatory response and thus protect the lung from the deleterious effects of excessive cytokine production, which might otherwise contribute to tissue damage and particularly of alveolar epithelial cells.
Among the latter, type II alveolar epithelial cells (ATII cells) have been shown to play an active role in the regulation of the inflammatory reaction within alveolar space. Indeed, ATII cells are ideally located to have a role in modulating immunologic activity in the alveolar space and both in vitro and in vivo data suggest that ATII cells could participate in the intra-alveolar cytokine network by secreting interleukin-8 (IL-8), 11 interferon, 2 monocTte chemoattractant protein-1, transforming growth factor beta 4 plateletderived growth factor 5' and granulocyte monocyte colony-stimulating factor v and more recently IL-6 under appropriate stimulation. 8 The latter cytokine has been shown to exert some anti-inflammatory functions. In vitro, at pathologic concentrations, 11-6 inhibits T cell responses via the activation of macrophages that secrete TGF-[3, a cytokine that impairs both T 18 cell and thymocyte proliferative responses.
Moreover, IL-6 downregulates IL-113 and TNFcz gene expression in human activated monocytes, 9 modulates the synthesis of czl-anti-trypsin in vitro in human mononuclear phagocytes 2 and induces a dysfunction of natural killer functions of lymphocytes. 21 In vivo, in rats, intra-tracheal IL-6 partially inhibited the neutrophil alveolitis induced by intra-tracheal tPS. 22 Thus, it appears that cellular communication between immune and non-immune cells is an essential process in the initiation maintenance, and resolution of the intra-alveolar inflammatory response. A better understanding of the processes which regulate the inflammatory processes within the alveolar space may lead to a more rational and new therapeutic approach in patients with infectious or non-infectious alveolitis. Correlations and interactions in the production of interleukin 6 (IL-6), ILl, and tumor necrosis factor (TNF) in human blood mononuclear cells:  Vol 5 1996 infection leading to respiratory insufficiency by obstructive lung disease. Colonization of the lower respiratory tract by bacteria such as Pseudomonas aeruginosa has been thought initially to be a critical event in the initiation and development of inflammatory processes. However, it is likely that the cascade associated with disease progression involves first pulmonary inflammation and subsequently infection of the lower respiratory tract which may therefore result from the inflammatory insult.
The current view of the pathogenesis of lung disease in CF with initial inflammation is supported by recent studies. Controlled analyses of bronchoalveolar lavage fluid from infants with CF have documented the presence of increased inflammatory parameters including neutrophils counts, activity of free neutrophil elastase and IL-8 levels. Interestingly, these markers of pulmonary inflammation were found even in the absence of common bacterial CF-related pathogens, of respiratory viruses and of fungi.
These recent observations raise two issues. First, understanding of the mechanisms associated with initiation of the inflammatory response in infants with CF appears to be critical for the development of new therapeutic strategies. Several hypotheses can be discussed. The cells most involved in the onset of inflammatory response are the local macrophages. From recent studies, it is believed that airway and alveolar macrophages are an important source of IL-8 in CF. The reason for an increased expression of IL-8 by macrophages is still unclear. It may be induced by external factors such as the abnormal mucus which accumulates in the lower airways in CF. It may also reflect an altered function of the macrophages. Indeed, expression of the cystic fibrosis transmembrane conductance regulator gene has been documented in macrophages and this gene seems to play a role in the control of several cellular functions including cytokine production and secretion. Another element that may be involved in the amplification of the inflammatory response is the respiratory epithelium. The cells that compose the epithelium of the airways and of the alveoli are known to have the ability of producing a wide range of inflammatory mediators. They serve as part of the local immune system, providing structures and functions crucial for the maintenance of normal pulmonary function. It is now well established that alteration of the pulmonary epithelium plays an important role in the pathogenesis of many respiratory diseases. In CF, epithelial cells can participate in the progressive destruction of airways and lung tissue through interactions with bacteria and their products, as well as through interactions with cells present in the airways. These cell-cell interactions are certainly involved in the up-regulation of inflammatory mediators released by the respiratory cells. However, the mechanisms leading to altered expression of the molecules of the inflammatory response as well as the role of cystic fibrosis transmembrane conductance regulator gene in this control remain poorly defined.
The second issue relates to the need of early markers of inflammatory response in infants. Indeed, if inflammation is an initial event in disease progression, efforts should be made to define tools of interest for evaluation of local inflammation in very young children. In this view, bronchoalveolar lavage is certainly of great interest; however, its relative invasiveness precludes its repeated utilization during the follow-up of the patients. Clearly, new approaches of pulmonary inflammation evaluation in infants are now required in order to optimize the therapeutic strategies which could be proposed. These approaches should include studies of sputum samples or of aspirates from the upper airways.
Experimental models of lung inflammation Peter/t Ward Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan, USA Animal models of inflammation have provided important information regarding mechanisms by which inflammatory cellular responses occur and the manner by which tissue injury develops. The most comprehensive understanding of acute lung inflammatory injury in rats has come from the use of the IgG immune complex model in which alveolar deposition of immune complexes leads to in situ complement activation. These complexes, together with complement activation products, activate lung macrophages to generate the early response cytokine, TNF-cz and IL-1. Concurrently, these cells generate the C-C chemokine MIP-lcz, which seems to have autocrine positive feed-back effects, enhancing macrophage production of TNF-cz and IL-1. The primary role of these two cytokines is to cause upregulation of lung vascular ICAM-1 and E-selectin. Through a sequence of adhesion promoting-interactions, these adhesion molecules facilitate the influx of neutrophils into the alveolar compartment.
Neutrophil recruitment requires E-selectin, 1CAM-1 and CDlla/CD18 within the vascular compartment and CDllb/CD18 and ICAM-1 within the airway compartment. The chemotactic Mediators of Inflammation Vol 5 1996 125 influx of neutrophils into lung is mediated at can lead to increased amounts of serum proleast in part, by a C-X-C chemokine,  teases in the extracellular fluid able to induce Injury to the alveolar and vascular walls occurs epithelial remodelling. It has been clearly shown via production of nitric oxide (NO) generated by that human leucocyte elastase can induce a wide inducible NO synthase (iNOS), which has two variety of epithelial remodelling including sources in lung: alveolar Type II epithelial cells mucous to serous cell conversion, 5 mucous cell and macrophages. Induction of iNOS appears to hyperplasia and epithelial shedding] In CF, be regulated by three cytokines: TNF-a IL-1 and chronic airway infection associated with chronic interferon-gamma. Lung matrix damage occurs airway inflammation represent sources of epithefollowing release from macrophages and neutro-lial injury. Baltimore et al. have described in an phils of serine proteinases and metalloproteinextensive histopathologic study, the intense ases.
remodelling of airway epithelium, in CF patients. These lung inflammatory reactions are self-Lesion of airway epithelium, which represents the limited, apparently because of concomitant ultimate degree of epithelial damage is followed appearance of IL-10 which suppresses macro-by repair. The repair process of airway epithephage production of TNF-: and IL-10. Endogenlium has been described by Zahm et al. 9 in an in ous production of IL-10 has been shown by the vitro model of wound repair of airway epitheappearance of mRNA and IL-10 protein in lung lium. Depending on the size of the wound, following deposition of immune complexes, airway epithelial cells are able to spread, migrate Blocking of endogenous IL-10 leads to a signifi-and proliferate in order to restore the integrity cant increase in lung injury. These data demonof the epithelial barrier. Denuded basement strate that lung inflammatory reactions are membrane and in particular laminin 2 represent mediated by a variety of pro-inflammatory cytosites of adherence for Pseudomonas aeruginosa, kines and regulated by at least one anti-inflammaone of the most important pathogens recovered tory cytokine (IL-10).
in CF. Airway epithelial cells engaged in the repair process represent also important sites of adherence for P. aeruginosa. We have recently described the significantly increased adherence Process of lesion and repair of airway of P. aeruginosa to airway epithelial cells which epithelium spread at the edge of the wound from CF and non-CF origins. Phenotypic alterations of repair- of the wound, are responsible for the overexpression of epithelial receptors for P. aeruginosa In the normal human respiratory tract, the pseu-adherence. Among epithelial receptors, asialylated dostratified airway epithelium is protected from membrane glycolipids such as asialo GM1 were aerocontaminants including viruses, bacteria and recovered at the surface of repairing airway pollutants, by the combination of a mucus gel epithelial cells acting as receptors for P. aerugilayer and of a mucociliary clearance mechanism nosa adherence. Although asialo GM1 represents by means of the ciliary activity, a site of adherence at the surface of CF repairing In pathological conditions, such as in cystic airway epithelial cells, we identified asialo GM1 at fibrosis (CF), the mucociliary clearance is the surface of non-CF repairing airway epithelial impaired due, in particular, to hypersecretion cells, acting as receptors for P. aeruginosa. and hyperviscosity of respiratory mucus. The Moreover, the apical asialo GM1 expression on defective mucociliary clearance in CF, associated repairing airway epithelial cells decreases along with marked chronic inflammation creates local the repair process. This suggests that the primary propitious conditions for epithelial injury. Epithe-defect of CFTR in CF cannot represent the lial injury can vary from epithelial remodelling, unique explanation for P. aeruginosa adherence including loss of ciliated cells to epithelial shedto airway epithelial cells. Moreover, airway epitheding with focal basal cells still attached to the lial cells actively synthesize and secrete fibronec-14 basement membrane, tin during repair which also represents a site of Various molecules, bacteria or viruses have adherence for P. aeruginosa. 15 We are currently been extensively described as being able to identifying the FN receptor on repairing airway induce such epithelial damages. These include epithelial cells. We hypothesize that dramatic ciliostastic effects, mucous hyperplasia, 2 and phenotypic alterations of airway epithelial cells epithelial shedding. '4 during lesion and repair widely contribute to Apart from bacterial or viral products, local chronic airway infection with P. aeruginosa in immune response to bacterial or viral infections CF. Moreover, the susceptibility of repairing airway epithelium to P. aeruginosa adherence decreases along the repair process in parallel to asialo GM1 expression, suggesting that immediately after wounding, the onset of repair of the airway epithelium represents a critical phase during which it would be useful to protect the regenerating airway epithelium. Cystic fibrosis (CF) lung disease is characterized by inadequate clearance of airway secretion and increased mucus production causing obstructive lung disease and chronic bronchial infections leading to progressive respiratory failure and death. The running, hypothesis, by which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to a defect in mucociliary clearance, is one where the defects in ion transport (decrease of chloride conductance) found in epithelia of conducting airways lead to dehydration of mucus, insipissation of secretions and defective clearance of inhaled pathogens. This in turn might explain why CF airways are liable to infection but does not explain the persistant colonization with Pseudomonas aeruginosa and the excessive mucous production. For instance it is known that patients with 'immotile cilia syndrome' have a more profound slowing of airway mucus transport than that seen in CF, but they suffer only from mild airway infections. Critical to an understanding of the basic molecular pathology of the CF airway, is a definition of the normal expression of CFTR in the human lung. In a pioneer study, Engelhardt et al., using in situ hybridization and immunochemistry, found a very low expression of CFTR in superficial epithelia of the proximal airways but a predominant site of CFTR expression in submucosal glands. In these glands, high levels of CFTR-RNA and protein are found in all cells of serous tubules of the distal gland, and in 1-3% of cells in the more proximal collecting ducts. Using immunogold electron microscopy, Jacquot et al. demonstrated further that CFTR protein was more specifically associated with the secretory granules of glandular serous cells. 2 Later, CFTR was also detected in a subpopulation of epithelial cells at every level of the distal lung, including proximal, terminal, and respiratory bronchioles and the alveoli but with a substantial variation in the level of CFTR expression between samples. The fact that the highest level of CFTR expression was found in submucosal glands, is not surprising if we consider that the major problem in CF is an alteration of the rheology of mucus, and that these glands have long been recognized as being the main secretory structures in the mucosa of the bronchotracheal tree. In humans, the glandular volume is estimated to be about 4 ml, the volume of mucus cells present in the airways surface epithelium being 1/40 of this value. The role of submucosal glands in the CF pulmonary disease does not seem to be in doubt and the fact that the lungs of knockout mice, which lack CFTR entirely and normally have ve few submucosal glands, are minimally affected supports this hypothesis.
We recently characterized a primary cell culture model of human tracheal gland serous cells (HTGS), which, studied at the third passage, respond to cholinergic and adrenergic agonists and synthesize serous secretory markers such as the secretory leukocyte proteinase inhibitor as well as high molecular-weight glycoproteins. 5' These cells do express CFTR mRNA and contain low conductance CFTR-like chloride selective channels] We could demonstrate that cultured CF-HTGS cells are subjected to an important defect in the regulation of the secretory process, since we observed in these cells a constitutive hypersecretion of proteins and a hyporesponsiveness to the two physiological neurotransmitters: acetylcholine and norepinephrine. 8 These defects are specific of CF-HTGS cells and were not found in cultured HTG cells derived from patients with other inflammatory diseases like sarcoidose or chronic obstructive pneumopathological disease (COPD). This observation indicates that in cystic fibrosis, besides ionic defects, there is a defect in protein secretion leading us to suspect a more general defect in the secretory process. In addition, our preliminary results show that normal HTGS cells cultured in the presence of the LPS of P. aeruginosa, behave in the same way as CF-HTGS cells, strongly suggesting that CF cells could be constitutively in an inflammatory state.
To conclude, many arguments, be they genetic, molecular or electrophysiologic, are now in favour of the theory that glandular tracheal cells are a privileged target of cystic fibrosis. The fact that their cellular mechanisms are not completely understood and that these cells are profoundly lodged in the mucosa will most likely influence the approaches of somatic gene therapy for the CF lung.  The airway submucosal glands and surface goblet cells normally provide protective mechanisms against inhaled irritants. In chronic diseases of the airways, mucus hypersecretion occurs and is an important cause of airway obstruction in cystic fibrosis (CF). Large numbers of neutrophils and high concentrations of catalytically active neutrophil proteases exist in CF sputum, and neutrophil elastase is the most potent secretagogue ever examined. Thus, sputum from patients with CF is capable of stimulating secretion from gland cells in culture even when the sputum is diluted 30000-fold! These findings implicate neutrophil elastase as a potentially important cause of airway hypersecretion. If neutrophil elastase is a cause of the hypersecretion, how is the elastase released from neutrophils? In the case of the goblet cells in the lumen, dead neutrophils are, at least in part, the probable cause of secretion, because CF sputum contains high concentrations of active neutrophil elastase. In the case of gland cells, elastase in the lumen does not cause secretion and intact neutrophils do not normally (or easily) release significant elastase into the medium. I suggest that a complex signalling process involving the surface of the secretory cells activates second messengers in the neutrophil, causing exocytosis of elastase-containing granules in close contact with the secretory cell surface. What is the mechanism of neutrophil recruitment in CF airways? CF sputum contains high concentrations of interleukin-8 (IL-8) and a blocking antibody to IL-8 inhibits substantially the chemotactic activity of CF sputum, suggesting that IL-8 plays a significant role in neutrophil recruitment in CF.
In CF, Pseudomonas aeruginosa (PA) is an important infecting organism, and the supernatant of PA organisms causes time-dependent production of IL-8 in airway epithelium. Incubation of PA supernatant with normal airways obtained from lung transplant donors resulted in tL-8 expression selectively in the airway epithelium. To examine whether feedback mechanisms perpetuate or exaggerate IL-8 expression and neutrophil recruitment, we performed animal studies in vivo. We cloned and expressed IL-8 in dogs and studied IL-8 expression in a bypassed segment of trachea. PA supernatant caused early selective expression of IL-8 in airway epithelium, and this was due to a novel, small molecular weight, water soluble product from PA. Several hours later, IL-8 was strongly expressed in the recruited neutrophils, and this was due to PA lipopolysaccharide (LPS). Thus, neutrophil recruitment is potentiated. In natural infection, this would continue until the neutrophils killed all PA bacteria, and then the mechanism would be rapidly turned off.
In summary, neutrophil recruitment and release of products is a complicated process. Epithelial and neutrophil products form a positive feed-back system (which is an advantage to the normal host but apparently disadvantageous to CF patients). This inflammatory cascade provides multiple opportunities for potential therapeutic intervention.
Prevention of leukocyte diapedesis bacterial pneumonia Elaine Tuomanen Rockefeller University, New York, USA While it is well accepted that endotoxin is the major noxious component of gram negative bacteria, it is less well appreciated that cell walls are equally inflammatory in the context of gram positive disease. The signs and symptoms of pneumonia induced by cell walls mimic that of living bacteria in animal models. Clinical strains and their isogenic lab derivatives which have defects in release of cell wall fragments induce an attenuated pattern of disease. 2 These cell wall fragments are potently bioactive with some bein8 specifically toxic to pulmonary epithelial cells, while others induce the influx of leukocytes or increased p,ermeability of the pulmonary 4 endothelium. Given that antibiotic therapy induces a rapid and extremely potent release of either endotoxin or cell wall fragments, it is recognized that decreasing injury during infection necessarily involves an effort to decrease the inflammation generated by antibiotic therapy itself. In this context, the ability to modulate leukocyte diapedesis, especially during antibiotic therapy of pulmonary infection, is currently of intense interest.
Recently, it has become clear that endotoxin and cell walls activate inflammatory mediators in mechanistically different ways. 5 This complicates the development of strategies aimed at downmodulating inflammation as a mechanism of reducing tissue injury. However, a common end-result of mediator release in both gram positive and gram negative infection is leukocyte diapedesis, a process which in turn is a key contributor to pulmonary damage. Inhibition of leukocyte trafficking is therefore not only a rational target for adiunct therapy to serious pneumonia, it is also one of the very few targets shared by gram positive and gram negative,diseases.
Migration of leukocytes from blood into the alveolar space is a three-step process. The first step involves margination of the leukocyte by the interaction of its surface carbohydrates with selectins on the surfaces of the endothelial cells. Thus, circulating carbohydrates or selectin analogues, competitively inhibit the ability of leukocytes to find inflamed endothelia. The second step is the activation of the rolling leukocyte by chemokines, particularly platelet activating factor (PAF). PAF plays a major role in pulmonary inflammation and PAF receptor antagonists are specifically useful in this context. 4 The third step is the actual transmigration event from. blood to alveoles. The key molecules for this event are the CD18 family of leukocyte integrins. Antibodies to CD18 block leukocyte accumulation in lung when given intravenously. Similarly, molecules that bind receptors for CD18, such as ICAMs on the endothelium, also block leukocyte movement. However, there is one instance in which this is not the case. There appears to be a unique CD18-independent migration that accounts for 50% of the leukoctyes entering the lung during pneumococcal pneumonia. 4' Given the availability of three steps in leukocyte migration as potential anti-inflammatory targets, there exists a number of options for leukocyte-directed anti-inflammatory therapy. This approach is likely to be fruitful for the development of useful partner drugs for the amelioration of pulmonary damage during pneumonia as well as acute systemic states such as ARDS. Leukocytes contain several serine proteinases, enhanced. This suggests that in a heightened including neutrophil elastase, proteinase 3 inflammatory state associated with infection, too (Wegeners antigen) and Cathepsin G. These much neutrophil elastase may be detrimental and three enzymes have been implicated in the would be consistent with our concepts of pathogenesis of chronic lung disease since both chronic infection in man. 7 neutrophil elastase and Cathepsin G have also The human equivalent of the beige mouse is been shown to cause chronic bronchial disease the Chediak Higashi syndrome. Mature neutroand for this reason have been implicated in the phils show abnormal empty granule formation. pathogenesis of several chronic progressive lung The cells have a chemotactic defect and do not diseases, contain neutrophil elastase. At present little is known about these protei-Studies have shown that Cathepsin G is also nases and their regulation. The genes are distrib-absent in the mature neutrophils and confirmauted at different sites. Neutrophil elastase and tion has been obtained recently that proteinase 3 proteinase 3 are found on chromosome 19, is also deficient (unpublished observations in D13.3 whereas Cathepsin G is found on chrocollaboration with P. Hiemstra, Leiden). mosome 14 Q 11.2. 2 These genes are expressed However, the studies of bone marrow have during differentiation of neutrophil in the bone shown that immature neutrophils express both marrow and currently no evidence exists to indi-the elastase and Cathepsin G gene and that these cate that they are expressed in the fully differen-enzymes are present prior to full maturation. 8 tiated cell. The best characterized gene, at Since these serine proteinases are coded for on present, is neutrophil elastase which is expressed different parts of the human genome and since at the promyelocyte stage during the differentiaat least two of them are expressed in early differtion of the neutrophil and its expression, thereentiation in the bone marrow, it seems likely that fore, is both tissue specific and time specific the defect in Chediak Higashi syndrome is a during the differentiation process, common one that is involved in post-translational The 5' flanking region of the neutrophil elasmodification of the products. Studies have shown tase gene contains a CAAT box, TATA box and that all three enzymes are produced as a prepro-GC rich elements that are consistent with a funcprotein. The amino terminal sequence is cleaved tional promoter. Nucleotide deletion studies have to provide the pro-enzyme and a final dipeptide suggested that other regulatory sequences are is cleaved at a common site as indicated in the present within 200 base pairs upstream from the Fig. 1. Recent studies have suggested that the initiation codon. Han and colleagues recognized defective enzyme may be a cysteine dipeptidase both positive and negative regulatory sequences but confirmation is awaited.
(-149 to -102 and -196 to -153 respectively) Studies in lung disease have shown that the whereas Srikanth and Rado 4 identified a further amount of neutrophil elastase in leukocytes from positive regulatory sequence (-106 to-76). The patients with bronchiectasis is increased comsequences are active in promonocytic cells and pared to age matched healthy controls and subnot in other cell lines including the hep G2 or jects with emphysema. 9 This increase in HELA cells, indicating that they may be tissue expression of enzyme is not unique since specific, patients with rheumatoid arthritis and 'diabetes Studies with promonocytic and promyelocytic have also been shown to have inceased neutrocell lines have shown that expression of the elasphil elastase content of the PMN. The mechantase gene can be decreased by factors that stimu-isms involved in his excess enzyme production late cell differentiation. In addition the use of anti-sense oligonucleotide probes, to proteinase gene transcripts in the HL60 cell line has actually been shown to induce differentiation towards a monocyte. 5 The implications of this latter finding is currently unknown.
The importance of the neutrophil elastase gene is indicated in several genetic defects. In animals, the beige mouse is a genetic variant in t. . are unknown, but may clearly influence the subsequent tissue damage that occurs. The results would suggest that mechanisms exist for modulating the amount of neutrophil elastase within the PMN and this presumably occurs during differentiation. The reasons for increased neutrophil elastase content may relate to increased transcription, more efficient translation of the messenger RNA, modifications in intracellular processing and storage of transport to the cell membrane. Further studies of these processes may determine the exact mechanism but also provide potential therapeutic targets for the modulation of proteinase expression. 6. Tanaka   There is a growing body of evidence that the consequences of neutrophil activation in the infected airways of patients with cystic fibrosis (CF) are at least partly responsible for respiratory failure and death in CF. Neutrophils release lysosomal metalloproteinases and serine proteinases upon activation and besides active collagenase and cathepsin G, high levels of elastase have been detected in bronchial lavage or sputum samples of CF patients. '2 Apparently, an elastase/anti-elastase imbalance is present in the inflamed CF airways and high amounts of the major endogenous elastase inhibitor, czl-protein-ase inhibitor (czl-PI), are proteolytically cleaved and inactivated. The other serine proteinase inhibitors, secretory leukocyte protease inhibitor (SLPI) and cz2-macroglobulin seem to play a minor role in protecting the lower respiratory tract from neutrophil elastase. Neutrophil elastase plays a major role in the pathophysiology of chronic inflammation in CF as well as in other disorders. Besides inactivation of czl-PI, it cleaves lung elastin and fibronectin, stimulates airway gland secretion, reduces the ciliary beat frequency and impairs opsonophagocytosis at the levels of immunoglobulins, complement and the complement receptor CRI on neutrophils. These events may start very early in the life of a CF patient, since elevated levels of elastase have been found in CF patients younger than 6 months. 3'4 In older patients, they may be present not only in severely diseased patients but also in CF patients with stable, clinically mild lung disease.
The proven elastase/anti-elastase imbalance suggests a strategy of increasing the antiproteinase levels in the lung by supplementation with suitable inhibitors. A wide variety of inhibitors has been developed for neutrophil elastase, but only a few may be applied in humans, czl-PI is one of them. A trial with aerosolized czl-PI in a small number of CF patients for several weeks gave promising results: when 12 patients received 1.5-3.0 mg/kg every 12  In order to achieve its principal function which is the host defence against pathogens, neutrophil can use two different microbicidal systems.
The non-oxygen dependent pathway consists of proteinases and antibiotic proteins stored in azurophil granules. Neutrophil elastase, which is a serine proteinase released upon neutrophil activation, has led to extensive studies in the context of cystic fibrosis (CF). The high levels of neutrophil elastase in CF airways has evidenced the importance of neutrophil-derived mediators in the inflammatory process in CF. The second microbicidal system is the oxygendependent pathway leading to oxidant genera-2 tion. Unlike proteinases, no extensive studies on the oxidant-generating systems have been carried out in CF. However, a growing body of evidence has shown an imbalance between oxidants and antioxidants in CF, thus pointing out an oxidative stress.
Oxidative metabolism activation, known as the respiratory burst, first involves NADPH oxidase. Activation requires cellular components from both the cytosol and the cell membrane. This enzymatic complex is an electron transport chain driven by an NADPH oxidase. The membrane proteins are composed of a cytochrome b558 which consists of a highly glycosylated large 91 kD subunit (gp91phox, glycoprotein 91 kD phagocyte oxidase) and which contains binding sites for NADPH and FAD and a small 22 kD protein subunit (p22 phox). The  are p47phox (which binds to gp91phox after phosphorylation by protein kinase C), p67phox and a GTP-binding protein, Racl or Rac2, which hydrolyzes GTP in GDP and is involved in the dissociation of the active complex. After assembly, the NADPH complex is functional, resulting in the univalent reduction of molecular oxygen to superoxide anion. This superoxide is converted to hydrogen peroxide (H202) spontaneously or by the means of superoxide dismutase. In the presence of iron salts, superoxide anion and hydrogen peroxide may interact further to produce hydroxyl radicals (OH.) (see Fig. 1).
Myeloperoxidase is the second important enzyme in the oxidative metabolism of neutrophils. Myeloperoxidase is stored in azurophil granules and represents 5% of the neutrophil dry weight. In the presence of H202 and chlorine, it catalyses the formation of hypochlorous acid (HOC1). 4 The formation of singlet oxygen, a potent microbicidal agent, has also been described. 5 HOCl can react with endogenous amines (R-NH2) to generate chloramines (RNH-Cl). 6 Taurine, a beta-amino acid considered as a HOC1 scavenger and very abundant in neutrophils is the most common substrate used to form chloramines. The oxidants such as superoxide anion, H202 and even HOC1 are very reactive and labile species whereas chloramines are stable enough to accumulate in the extracellular milieu; they have thus been called 'long-living oxidants'. The presence of long-living oxidants have been recently reported in sputum from CF patients. 7 It becomes evident from several studies that superoxide anion or H202 may not be the microbicidal species and it is usually admitted that HOCl, terhaps via the formation of singlet oxygen, 5 can be the most toxic substance. 8 The exploration of neutrophil oxidative metabolism in CF has led to divergent results, because of the wide heterogeneity in the clinical state of the CF patients and because of the difference in the methodology.
It seems that defects in intracellular transduction found in CF epithelial cells were not present at the level of the neutrophil. In fact, increase in cellular cyclic AMP triggered by several pharmacological agents (adrenaline, theophyllin and forskolin) had the same inhibitory effect on neutrophil cellular functions such as superoxide anion production, degranulation, membrane depolarization in CF subject as in controls. 9 It has been shown that the luminol enhanced chemiluminescence which measured the intracellular MPO-dependent oxidant formation of neutrophils stimulated by opsonized zymosan had the same intensity in CF patients and in controls. However, the kinetics of the reaction were different: the time to reach the maximum was decreased in CF. Moreover, this parameter appears to be correlated with the clinical state of the patients. These authors thus concluded that neutrophils from CF patients were 'primed'. Other studies on monocytes and neutrophils have shown an increase of luminol enhanced chemioluminescence following stimulation by f-MLP, PMA and A23187.11 Investigations on neutrophil oxidative metabolism are hampered by the fact that it is difficult to differentiate between a possible activation state due to recurrent infections of CF patients and a possible 'constitutive' defect which could be specific of CF. Investigation of neutrophils from CF heterozygote subjects, who are obligate carriers of the CFTR mutation and clinically asymptomatic, could provide a mean to discriminate between the two possibilities. It has been reported that monocytes from CF homozygotes and heterozygotes showed increased adherence on glass. 12 In .a recent study carried out in our laboratory, we have shown a disturbance in MPO-dependent oxidant generation in neutrophils from CF homozygotes and heterozygotes as compared to controls. The mechanism underlying this hyperactivity seems to involve the sodium/proton exchange system as pharmacological blockers of this exchange system such as amiloride or EIPA or a sodium-deprived buffer corrected this hyperactivity (Witko-Sarsat et al., submitted for publication).
In conclusion, the neutrophil plays a pivotal role in CF inflammation and remains the more potent in cellular oxidant-generating systems. It thus seems of special relevance to explore their mechanisms of formation, to study the consequence of their massive release as well as the clinical consequences.
Although the genetic defect responsible for cystic fibrosis (CF) is well characterized, the events leading to the gradual destruction of lung tissue and the consequent decrease in lung function remain unclear. In an attempt to improve this situation our Group has over recent years, addressed the specific role of oxidative stress and antioxidant defences in the pathogenesis of CF1-5. Two overriding features have characterized the findings from this series of studies. First, gross deficiencies of fat-soluble antioxidants such as cz-tocopherol (vitamin E) have not been found. This presumably reflects the good clinical and nutritional management now provided in CF clinics.
Second and more disturbing, despite relatively normal antioxidant concentrations, many CF patients exhibit evidence of oxidative stress. Blood plasma obtained from CF patients has a decreased oxygen radical trapping capacity of plasma in CF patients, indicating an increased susceptibility to oxidative damage compared to control subjects. Confirming this finding, we have demonstrated the presence of oxidized lipids (hydroperoxides) and proteins (carbonyls) in the plasma of CF patients, implying free radical-mediated tissue damage. 2 In addition, we have shown that the DNA oxidation product, 8-hydroxydeoxyguanosine is present in the urine of CF patients. As already mentioned the interesting, and yet disturbing aspect of all these findings is that oxidative stress was detected in CF children whose circulating antioxidant defences (vitamin E, ascorbic acid, uric acid and total plasma sulphydryls) were within the normal agerelated range. These findings, therefore, not only provide confirmation that increased radical production is a component of CF aetiology, but that normal concentrations of circulating antioxidants are not sufficient in many CF patients to offset the consequences of their increased oxidative burden.
Oxidative stress and the decline in lung function in CF Establishing the presence of markers of oxidative injury in fluids such as plasma and urine has provided a biochemical basis for free radical damage in CF, but determining the clinical relevance of these findings is clearly more important. In this respect we recently found that the age-related decrease in lung function in CF children is associated with lower plasma cz-tocopherol, ascorbic acid and sulphydryl concentrations. Moreover, the age-related reduction in pulmonary function correlated with elevated plasma malondialdehyde in CF children. 4 In addition, patients with severe lung dysfunction (FEV1 < 50% predicted) had higher plasma concentrations of lipid hydroperoxides than those with mild to moderate lung dysfunction (FEV1 > 50% predicted). We believe that these findings warrant a closer examination of the association between lung function and oxidative stress in CF. To this end, studies are required which focus directly on the lung. Antioxidant defences and markers of oxidative stress in sputum Sputum arises in the lung and as such may provide a more suitable medium than plasma to examine for markers of oxidative stress and antioxidants. In a pilot study, conducted in 11 CF patients we have demonstrated that it is possible, with minor modifications to existing methods, to measure cz-tocopherol, uric acid, ascorbic acid and total sulphydryls in sputum. Indeed, the concentrations and composition of these antioxidants indicate that sputum is a reasonable surrogate for the lining fluids of the upper respiratory tract. Despite this wide array of extracellular antioxidants, evidence was obtained for both protein and lipid oxidation in sputum from CF patients. We have not yet had the opportunity to determine whether the markers of oxidative stress in sputum correlate with those present in plasma of the same patients. Neither have we addressed the association between antioxidant defences, markers of oxidative stress in sputum and lung function in CF patients. Antioxidant therapy for CF Two findings have led us to believe that antioxidant supplementation, to above control levels, may prove to be a useful therapeutic benefit in CF. First, markers of oxidative stress, which am not present in control age-matched children, are present in many CF patients, whose circulating antioxidant defences fall within the normal agematched range. Second, CF patients with the highest plasma antioxidant concentrations exhibit the best pulmonary function. We hypothesize that CF patients endure cyclic periods of oxidative stress associated with chronic respiratory infection and that when this oxidative stress exceeds the patients pulmonary antioxidant defences oxidative-lung injury ensues. 5 This injury, in combination with that arising from the protease/antiprotease imbalance, contributes to the pulmonary pathology seen in CF patients and in time results in compromised lung function. Combating this oxidative stress with additional antioxidant therapy may help to reduce lung injury, and hence decrease the rate of loss of lung function. In cystic fibrosis (CF), the most common autosomal recessive disorder of whim populations, abnormal exocrine gland secretions lead to chronic endobronchial infections with opportu-nistic bacterial pathogens such as Pseudomonas aeruginosa. As a consequence, large numbers of neutrophils are recruited to the infected airways where cell activation and lysosomal enzyme release occur. Neutrophils respond to stimulation with a burst of oxygen consumption and the production of reactive oxygen species such as superoxide anion, hydrogen peroxide, hydroxyl radical and possibly singlet oxygen. As with lysosomal proteinases, these species are not only released into the phagolysosome but also outside of the phagocyte where they may be toxic to the host.
It is not yet certain whether toxic oxygen metabolites produced by stimulated PMN also contribute to lung injury in CF, as has been suggested in other airway diseases. 2 However, indirect evidence may support this hypothesis; high sputum concentrations of extracellular myeloperoxidase (MPO), a PMN-derived enzyme which transforms hydrogen peroxide (H202) into highly reactive oxygen metabolites, have been detected in CF patients,and lung function has been inversely correlated with MPO levels. 4-In addition, deficiency of the antioxidant glutathione and increased lipid peroxidation 8 have been reported to occur in CF patients. The presence of endogenous scavengers such as uperoxide dismutase (SOD) which dismutates superoxide anion to hydrogen peroxide, or catalase which changes hydrogen peroxide to water and oxygen, has not been investigated .in CF sputum or bronchoalveolar lavage samples until now. The human catalase gene in airway epithelial cells is a 'house keeping gene' which cannot be augmented by hyperoxia. 9 Nevertheless, the amount of tissue damage and degree of protein inactivation by oxidative attack in CF and other disorders has not been clearly defined. Of considerable interest is whether alproteinase inhibitor (al-PI) undergoes oxidative inactivation. A number of studies have shown that Met5, an important residue of the serine proteinase binding site, is particularly susceptible to oxidative attack, the result being a substantial reduction in the ability of the inhibitor to bind neutrophil elastase. 1 Provided that further proof of the hypothesized oxidant/antioxidant imbalance in CF can be obtained, which possibilities do we have to protect the lung epithelium and biological important proteins, fatty acids, carbohydrate moieties and DNA from oxygen radical-induced damage? Several strategies have been developed including the application of rhSOD 11 or selenium, 12 aerosolization of N-acetylcysteine as a precursor of 13 glutathione and the augmentation of the antioxidant screen of the CF epithelial surface using replication-deficient recombinant adenoviral vectors 2 or liposomes 4 containing human catalase cDNA. 2
Inhibition can also be contrived by interfering at the following levels: (1) the stimulus inducing the production of proinflammatory molecules (e.g. anti-CD14) in relation to LPS stimulation; anti-CD11 and anti-CD69 in relation to cell-cell interaction resulting in the production of IL-1, TNFcz, PGE2 and metalloprotease; (2) the transduction, transcription and translational processes; (3) the maturation and secretion of cytokines; (4) the functional structure of cytokines (muteins). Factors to be considered when examining the concept of cytokine/cytokine inhibitors are: (1) the chronology of appearance of the molecules; (2) their relative levels are more important than absolute levels; (3) the potentially beneficial effect, for a short period, of a proinflammatory molecule in host defense; (4) the complexity of some cytokine systems such as the IL-1 system which prossesses at least two types of natural antagonists (II-lRa, II-ISR) leading to the paradox of enhanced pro-inflammatory activity; (5) the formation of unstable cytokine/cytokine inhibitor complexes. Therefore, the concept of cytokine inhibitors may not be considered an 'on/off' but a 'buffer' system. Another important aspect of the potential impact of cytokine inhibitors is the dissociation between their immunosuppressive and antiinflammatory action. TNF-R: Fc fusion protein, for instance, is hundred times more potent, in inhibiting metalloproteases and PGE2 production by fibroblasts than in inhibiting T cell proliferation and cytokine production on T cells. It is therefore important in devising therapeutical approaches in the future to pinpoint inhibitors of cytokines that are relevant primarily in immune-inflammatory or destructive events. on measuring cytokine concentrations and determining their sites of production within the lung itself, using bronchoalveolar lavage (BAL) and airway brushing specimens. Concentrations of the pro-inflammatory cytokines IL-1, IL-6 and TNF-cz are all dramatically elevated in BAL fluids from CF patients as compared to healthy controis. While no IL-8 is found in the BAL fluid of healthy controls, CF patients' fluids contained greater than 30 ng/ml of this potent neutrophil attractant, even when they were stable and remote from exacerbation. CF patients' BAL fluids contained modest elevations of the regulatory factors IL-1 receptor antagonist and TNF-cz soluble receptor fragments but the ratios of antagonist to cytokine were greatly reduced in the CF patients. IL-10, a regulatory cytokine, which suppresses production of the pro-inflammatory cytokines, was absent or markedly reduced in the CF specimens but present at 3.5 ng/ml in specimens from healthy controlsl All of these differences were more pronounced in CF patients infected with Pseudomonas as compared with patients infected with Haemophilus influenzae and/or Staphyloccocus aureus, but similar changes were present in the latter patients as well. In one case of an infant in which intensive antibiotic treatment was able to eradicate detectable infection, the concentrations of IL-6 and IL-8 fell after treatment but were still markedly elevated as compared with controls. No IL-10 was detectable before or after antibiotic treatment.
To understand this imbalance towards proinflammatory cytokines, we evaluated the possible cellular sources of these cytokines in the CF lung. Fresh macrophages in CF BAL specimens, fixed and stained immediately after they were obtained from the lung, showed positive immunofluorescenc for IL-1, IL-6, IL-8 and TNF-0t and resembled healthy controls' macrophages which had been incubated with lipopolysaccharide in vitro before staining. In contrast, fresh macrophages from the controls which were not incubated in vitro were predominately negative. Interestingly, fresh macrophages from the CF patients stained positively for IL-10. This was also observed in LPS-treated macrophages from healthy controls but not in untreated controls' cells. Further studies, aimed at determining the source of IL-10 in the healthy controls' lavage fluid, showed that fresh bronchial epithelial cells obtained by brushing had positive immunofluorescence for IL-10, contained IL-10 mRNA, and secreted functionally active IL-10 when placed in primary culture in vitro. CF patients' bronchial epithelial cells contained much less IL-10 by immunofluorescence. These findings suggest that in the normal lung, the bronchial epithelial cells produce the anti-inflammatory cytokine IL-10 to down-regulate local inflammatory responses and prevent epithelial damage, whereas this suppressive mechanism is lost in the infected lung in CF. In contrast, in CF the bronchial epithelial cells are actively producing IL-6 and IL-8, which further exacerbate the inflammatory process.
Taken together, these observations suggest that the bronchial epithelial cells may play a major role in regulating local immunologic and inflammatory processes in the lung. This hypothesis, in turn, raises the questions of whether defective CFTR function could alter epithelial cell cytokine production, even in the absence of infection; and/or whether the responses of CF epithelial cells to stimuli that could upregulate IL-8 production and/or downregulate IL-10 production differ from the responses of normal cells.
Cystic fibrosis results from mutations in a 250 kb gene encoding the CFTR protein, a cyclic AMP-regulated Cl-channel. CFTR protein is located in the apical membranes of secretory epithelial cells. 1'2 Mutations in the gene result in the altered synthesis, processing or function of the protein. The dysfunction of this channel is, at least partly, responsible for defective fluid secretion in CF patients. The disease has pleiotropic manifestations dominated by abnormalities of the airway epithelium surface which hypersecretes a thick mucus favouring inflammation and chronic infections.
Inflammation triggers the formation of a large number of both intracellular and tissular mediators able to affect the cell functions. For example, interferons (IFN), a family of multifunctional proteins produced in response to viral and bacterial infections, are major mediators of the host defence against the infectious agents. 4 IFN0t and IFN[3 are mainly synthesized by lymphocytes and fibroblasts, respectively. IFN,, a key cytokine of the immune network, is synthesized by T lymphocytes and natural killer cells after antigen stimulation, and its crucial role in the defence against parasitic and viral infections was recently highlighted. 5 Another mediator of inflammation, the tumour necrosis factor 0t, TNFcz, is synthesized and released by macrophages/monocytes as well as by B and T cells, Kupffer cells, glial cells and adipocytes, 6 and provokes pleitropic intracellular effects. IFNT and TNF0t also alter transepithelial ion transport but the mechanisms responsible for this action are not well defined.
Both the bacterial colonization and the tissular mediators of inflammation may affect the cell functions. However, whether they influence directly or indirectly CFTR function was not clearly established. This prompted the investigation of the effects of IFNs and of TNFcz on CFTR gene expression.
Because inflammation and infection constitute the practical problem in cystic fibrosis, antibiotics and anti-inflammatory drugs are systematically administrated to treat CF patients. The present state of the art about these molecules suggests that they might affect the epithelial cell functions. Thus, we investigated the actions exerted by two different anti-inflammatory drugs (aspirin and dexamethasone) on CFTR gene expression.
Two adenocarcinoma cell lines (HT29 and T84 cells) known to express large amounts of CFTR protein served as experimental models. We found that IFN,, at low concentrations (from 0.1 up to 100 IU/ml), down-regulated CFTR gene expression at mRNA and protein levels through a dose-dependent post-transcriptional mechanism. The effect was time-dependent: the IFN,-induced mRNA decrease was significant at 3 hours and maximal after a 12 hour-incubation. INFz and J3 were inactive on CFTR gene expression in T84 and HT29 cells, suggesting that the effect of IFN, was specific. The IFN,-induced inhibition of CFTR gene expression evidenced at mRNA and protein levels was also found at the functional level: treatment with IFN, reduced the cAMPdependent Cl-fluxes. In HT29 cells, TNFcz, which is concomitantly produced with IFN, during the inflammatory and immune reactions, also reduced CFTR gene expression, and acted synergistically with IFN,. v These results suggest that the inflammatory processes may enhance the CF symptomatology and impair the efficacity of a gene therapy. Thus, to prevent the negative action of the two cytokines, we are investigating the effects of two molecules, representative of the distinct families of anti-inflammatory drugs, i.e., dexamethasone as an example of steroid-like molecules and aspirin as a non-steroid molecule, on CFTR gene expression. Under experimental conditions used, dexamethasone (ltmol) does not exert any effect, either at mRNA or at a functional level. On the contrary, 48 hour-treatment of the T84 cells either with aspirin or with salicylic acid (0.5 to I mM) decreased the amount of CFTR transcripts and the cAMP-dependent Cl fluxes. The aspirin effect does not require de novo protein synthesis, since it subsists after treatment of the cells with cycloheximide. This effect is not due to a primer effect on adenylate cyclase since the forskolin-induced cAMP production is not modified by the treatment of the cells with aspirin (or salicylic acid).
These data suggest that production of IFN, and TNFcz in response to infections might worsen lung dysfunction in CF patients, and create a localized CF phenotype in non-CF patients with chronic bacterial or inflammatory lung disorders. Furthermore, our results also point out new action of aspirin commonly used as an anti-inflammatory drug in CF. High doses of aspirin, when applied chronically, might also diminish CFTR-related C1secretion and worsen status of the patient.
Anti-inflammatory therapy for fibrosis: rational and prospects cystic Michael W. Konstan  Many lines of evidence suggest that the accumulation of neutrophils into the airways is excessive and plays a major role in the lung destruction that continues to be the primary cause of morbidity and mortality in CF. In addition to the destructive effects, there are many ways in which neutrophil products cause dysfunction of the lung and its defence mechanisms. These have been amply reviewed by other speakers at this symposium. Several recent studies 2' including our own work on patients with relatively mild lung disease, 4 make it clear that this excessive inflammatory process begins very early during the course of CF and continues unabatedly, although its intensity does vary from time to time. 5 Chronic infection in the lung undoubtedly is a major stimulus for this inflammatory response, and exacerbations of infection clearly lead to increased numbers of neutrophils and their damaging products. Some recent observations su,gest that inflammation may precede infection and/or that regulation of inflammation in the CF lung may be inherently abnormal, but the role of infectious exacerbations in increasing the inflammatory damage cannot be denied.
Clearly, treatment designed to decrease the inflammatory damage must include efforts to prevent and/or decrease the amount of infection. However, once Pseudomonas becomes firmly established, infection is rarely eradicated despite continuing evolution of new antibiotics. Bronchodilators, airway clearance techniques and DNase also contribute to the goal of preseeeing lung function, but despite all these efforts, the inflammatory process continues and inexorably worsens. For these reasons, additional strategies, such as anti-inflammatory agents that attempt to compensate for the basic defect (i.e., by the use of amiloride and/or UTP) and gene therapy are all being developed.
In contemplating anti-inflammatory therapy, it is obvious that the main target must be the neutrophil itself. The initial step in migration of neutrophils into the lung involves activation and attraction of the cells by soluble mediators. Many studies have documented that CF lung secretions contain high concentrations of all of the major classes of neutrophil chemo-attractants, most notably, LTB4, C5a and IL-8 (reviewed in 1). It is therefore unlikely that antagonists of the production or receptor for any single one of these would have a major effect. Pharmacologic agents which might interfere with the adhesion molecules employed by the neutrophil to crawl out of the small blood vessels and into the epithelium have not yet reached clinical trials. Thus, the most practical approach is to interfere directly with neutrophil migration and function.
Because of the likely side effects of corticosteroids, which subsequently proved problematic in a large clinical trial in the US, 6 we investigated the use of non-steroidal anti-inflammatory drugs (NSAIDs), in particular, ibuprofen. Ibuprofen has direct effects in decreasing neutrophil migration and degranulation and at high but achievable concentrations, blocks the release of LTB 4 as well as inhibiting cyclo-oxygenase (reviewed in 7). Using the delivery of neutrophils to the alveolar crevices of the gingiva in the mouth as a model for the intrabronchial infection in the lung, we showed that ibuprofen interfered with movement of neutrophils in vivo in normal volunteers as well as in CF patients. 8 Most importantly, we found that administration of ibuprofen to animals with the agar bead model of chronic Pseudomonas infection reduced the area of inflammation in the lung without increasing the burden of infectious organisms] After these preliminary studies, and once we had defined the pharmacokinetics of ibuprofen in patients with CF, we initiated a four-year clinical trial of this drug in patients with mild lung disease. Compared with placebo, ibuprofen-treated patients had significantly less decline in pulmonary function and Brasfield chest X-ray scores, preserved their percentage of ideal body weight, and tended to have fewer hospital admissions. 9 The effect was most pronounced in the youngest age group (5-13 years), the annual rate of decline in the percent predicted FEV1 was 88% slower for ibuprofentreated patients compared to placebo. There were no significant adverse effects. Clinical use of ibuprofen is now being implemented throughout the US and other parts of the world. Establishing the appropriate dose by pharmacokinetic studies in each patient is important in individualizing therapy. Other NSAID trials have also been initiated and preliminary results are promising as well (reviewed in 10). Reports showing that inflammation is found even in infants with CF and our results which showed that the most dramatic effects of ibuprofen occurred in the youngest patients, suggest that anti-inflammatory therapy should be initiated very early in life. Until the potential of gene therapy is realized, anti-inflammatory agents are likely to be an increasingly important part of the therapeutic armamentarium in CF.
Gene therapy for cystic fibrosis (CF) has been first developed to replace in vivo the CF defective gene. Rapid progress towards gene transfer has been made since the identification of the CF gene, and the initial complementation experiments provided encouragement to investigators in search of gene transfer vectors to deliver the CF gene to airways. The ideal vector would efficiently deliver the gene to the appropriate target cells without causing toxicity or inflammation. Although none of the available vectors meets these criteria, studies in a number of animal models demonstrated gene transfer to the lung, and human trials have been initiated using recombinant adenoviruses and liposomes as vectors for gene transfer to airway epithelium. However, the underlying pathophysiology of CF remains unclear, and several issues have come up for successful CF gene therapy. First, at the cellular level, there is abundant evidence that CFTR is important for several cellular functions. Although the clinically relevant respiratory manifestations of CF are in the airways, it is not possible to evaluate CFTR function reproducibly in the human bronchial epithelium in vivo, and thus it is difficult to demonstrate at the site of the major effective therapy for end-stage pulmonary failure clinical manifestations of the disease that a gene caused by CF. However, lung transplantation is therapy strategy in humans actually corrects the associated with complications such as reperfufunctional deficiencies (including Clconducsion injury and graft rejection. Pulmonary tance and hyperabsorption of Na +) associated oedema with acute respiratory failure occurs in with mutations in the CFTR gene. Second, at the about 20% of patients after lung transplantation organ level, CFTR has been localized to both the and is associated with increased early mortality. surface epithelium and submucosal glands in the This acute respiratory failure results from a lungs. None of the vectors being used in human complex lung injury with both ischaemic and trials efficiently achieves gene transfer to submureperfusion components. Gene therapy targeted cosal glands. Thus, if CFTR function in submucoto the graft offers a promising approach to the sal glands is necessary for normal airway prevention of these complications, by the transclearance, current approaches are not likely to fer of antioxidant genes such as catalase and/or impact on disease pathogenesis in the cartilagen-superoxide dismutase genes. As adenovirus ous airways. Finally, the relative sparing of pulvectors can transfer genes in vivo to the lung monary disease in CF patients who carry an vasculature, we evaluated the feasibility of gene allele encoding a partially defective CFTR mole-transfer to the lung graft in a porcine model of cule suggests that less than complete genetic left lung allotransplantation, using an adenovirus reconstitution will be necessary for therapeutic vector containing a reporter gene. Adenovirusefficiency. However, in vivo adenovirus-mediated mediated gene transfer to the lung graft was feagene transfer to the airway epithelia of nonhusible ex vivo, but several parameters limited gene man primates tends to be of low efficiency and transfer efficiency and need to be improved patchy, before clinical application is attempted. In summary, several key issues will need to be addressed for successful CF  Anti-oxidative gene therapy for CF A major part of the airway damage in CF is mediated by oxidants released by inflammatory cells on the ai.rway epithelial surface, particularly Germ tlerapy ir 'tR: fibroi neutrophils. Because the number of neutrophils Gabriel Bellon and Andrea Paviran* in the epithelial lining fluid (ELF) of individuals Hospices Civils de Lyon, Unite "Pneumologie with CF are 100to 1000-fold greater than in Pediatrique-Mucoviscidose", Centre Hospitalier Lyon normal subjects, the potential burden for oxi-Sud, 69310 Pierre Benite and *Transgene SA, 11 rue dants on the airway epithelium is enormous. The de Molsheim, 67082 Strasbourg, France oxidant-antioxidant imbalance is placed further in favour of the oxidants in that respiratory ELF At present it is conceivable to think that gene levels of glutathione are markedly reduced in CF therapy represents a way to treat or even prevent compared with that in normal individuals. A strat-the respiratory manifestations of cystic fibrosis. egy to protect the epithelium is to augment the Consistent with such a concept, there is sufficient anti-oxidant screen of the epithelial surface, thus evidence that Ad-CFTR, a recombinant replicaallowing the inflammatory cells on the epithelial tion-deficient adenovirus expressing the human surface to phagocytize and kill bacteria, yet procystic fibrosis transmembrane conductance regutecting the epithelium from damage by oxidants lator cDNA, can vectorize the expression of a released by the phagocytes during this process, functional CFTR (cystic fibrosis transmembrane Preliminary in vitro studies have demonstrated conductance regulator) to the nasal and airway that adenovirus mediated gene transfer of the epithelia. human catalase gene could protect human The clinical protocol was designed to assess epithelial cells against oxidant stress, the safety of single escalating doses of a replication defective adenovirus expressing the cystic Improvement of lung graft survival by gene fibrosis transmembrane conductance regulator therapy gene (Ad-CFTR) when administered to the tra-Lung transplantation has rapidly evolved into an cheobronchial portion of the airways and whether biological efficacy of CFTR delivery lowing the use of recombinant adenoviruses, could be demonstrated, including the induction of cellular immunity, Six cystic fibrosis patients received nasal instilrecombination to produce a replication compelation and subsequent aerosol (Optineb(R), Air tent virus, production of neutralizing antibodies Liquide, Paris, France) administration of Ad-CFTR reducing the efficacy of further application and the following day. Doses (pfu) applied to the inflammation at the site of therapy, with potential nose were 105 (patients SG and PB), 10 systemic consequences. Animal studies have (patients FP and EP) and 4 x 10 (patients DS addressed some of these important safety issues, and FG), while aerosolized doses were 10 especially in rodents and non-human primates.
(patients SG and PB), 108 (patients FP and EP) These studies are critical for assessing the potenand 5.4 x 10 a (patients DS and FG), respectively, tial toxicity associated with direct instillation of No acute toxic effects, no increase in the titer of recombinant adenoviruses, such as the adenoanti-adenovirus antibodies and no spreading or virus containing human CFTR (AdCFTR), on shedding of Ad-CFTR were detected. In one airways and lung parenchyma. patient Ad-CFTR DNA was found in the urine and blood two days after aerosolization. Ad-Pulmonary inflammation in animal species CFTR DNA was detected in nasal and bronchial The cotton rat provides a good model for safety brush samples, in BAL, in saliva and tonsils 21, 8, evaluations of human adenovirus-derived 14 and 4 days post virus administrat?on, respecvectors. 6 In this animal model, with regard to the tively. Ad-CFTR mRNA (RT-PCR on bronchial expression of the transfected gene in targeted cells) and CFTR protein (immunochemistry on cells, all cell types in the respiratory airway nasal and bronchial cells) were detected up to epithelium expressed the gene, with a good dis-14 days following Ad-CFTR administration, tribution of the vector throughout the respiratory These results show that the nebulization of tract as far as the terminal bronchioles but with Ad-CFTR is a possible approach for treating the relatively little gene expression in alveolar epitherespiratory manifestation of cystic fibrosis, lium. 5 To accomplish gene therapy successfully, genetic observed." Furthermore, in animals evaluated information must be transferred and expressed histologically for pulmonary fibrosis 28 days after in the target cells. Various vectors such as retro-vector administration, no increase in collagen viruses, adenoviruses, adeno-associated viruses, fibres or architectural disorders were observed in liposomes and molecular conjugates have been the peribronchiolar, perivascular, alveolar or developed to enhance this process. Adenovirus-pleural areas.
based vectors have a number of properties that In non-human primates, baboons or rhesus make them attractive vehicles for human gene monkeys, the major adverse effect associated therapy. 5'7 Their ability to transfer genetic mate-with intrabronchial instillation of recombinant rial efficiently into lung epithelial cells has led adenoviruses was pulmonary alveolar inflammathem to be chosen for the first trials of human tion. The severity and location of the inflammagene therapy for cystic fibrosis (CF). 2'3'5'7'8'10'11 tory infiltrate depended upon the concentration The success of this approach will depend not of the virus to which the lung tissue was only on the level and duration of transgene exposed, and the inflammation was not restricted expression but also on its safety. Important ques-solely to the area where the virus was directly tions remain regarding the clinical toxicity of infused. Occurrence of inflammation outside the administration of adenoviral vectors to the CF targeted regions is likely due to spillover during lung. In theory, several problems may occur folvirus infusions. 9 Post-mortem microscopic analy-sis of the lung parenchyma demonstrated a dose Clinical trials dependent increase in inflammatory cells, primar-Clinical trials have been initiated in CF patients, ily lymphocytes in the area where AdCFTR was based on nasal and/or lung directed AdCFTR in delivered and which persisted for at least 2 order to evaluate the safety and efficiency of months in some animals. This inflammatory gene transfer. One of these studies was perreaction appeared to develop over 2-3 weeks formed on four CF patients in whom varying following gene administration. It started with doses of CFTR recombinant adenovirus (up to mild to moderate perivascular accumulation of 2 X 10 9 pfu) were administered in the nose and lymphocytes, and extended to the lung intersti-two days later in the lungs. 2' Protein and mRNA tium in animals receiving the highest dose (10 9 expression were documented in bronchial and and 10 m pfu). Clinical tests did not accurately nasal epithelium. Nevertheless, within 12 hours reflect the presence of lung inflammation, with of administration of the virus, one of the subjects the exception of alveolar infiltrates on chest developed a clinical syndrome with fever, hyporadiographs and an increase in lymphocytes in tension, multilobar infiltrates and a marked bronchoalveolar lavage (BAL). 1'9 Abnormalities increase in circulating levels of the inflammatory that were seen on chest radiographs resolved mediator interleukin-6. No evidence of recombicompletely on follow-up examinations. The nation or of serum neutralizing antibodies was animals' physical appearances and behaviour found. This probably represented an inflammaappeared normal throughout the studies. In all tory reaction within the lung, centred around the these species, cotton rats or non human pri-site of the application of the adenovirus. The mates, examination of non-respiratory organs symptoms resolved over a period of one month failed to show any abnormalities attributed to the with no apparent long-term effects. No evidence transfection, of any contaminating agent was found. This subject received the highest dose of adenovirus Mechanisms that contribute to the inflammatory (2 x 109 pfu) in this study, thus it is likely that responses this syndrome was a dose-related phenomenon, The precise mechanism of the lung inflammaoffsetting the transfection efficiency advantage of tion observed in animals after instillation of adenoviruses. recombinant adenovirus is unknown. Similar his-Animal models, although showing inflammatopathologic appearances after administration of tory responses, did not predict the intensity and different types of replication-deficient adenovirus rapidity of the onset of this clinical syndrome, such as AdRSVBgal (containing the Escherichia underlining the importance of such human coli lac Z) or Ad CFTR (containing the normal studies. This could be explained by species difhuman CFTR cDNA) indicate that the inflamma-ferences, or by significant pre-existing lung tory response is probably related to adenovirus disease observed in CF subjects. Lack of animal components rather than to the transfected gene. models of CF have limited the evaluation of gene It is unlikely that cell lysis from viral replication therapy, because extrapolation to human therapy initiated the injury. No viral inclusions were seen is difficult. In particular, of the animal models and no recombinant or wild type viruses were available, none mimics the human CF lung with detected by culture. The more likely cause to significant pre-existing disease. the pulmonary inflammation is a response to For some authors, human nasal studies could viral antigens, possibly related to the antigenicity help predict dose related difficulties observed in of viral coat protein and cytotoxic T-lymphocyte lung parenchyma. 12 In fact, more extensive responses. Time course and perivascular lymtesting of gene transfer agents in the nasal phocytic infiltrates are compatible with a epithelium prior to the lung do not seem helpful response to foreign antigens. The early appear-because it has been shown that the nasal epitheance of neutrophils is consistent with a non-spe-lium does not mimic the responses observed in cific host response. Observations have shown the lower airways, particularly in CF subjects. that bronchial epithelial cells release interleukin-Even though the CF transmembrane conductance 8 (IL-8) after exposure to various mediators regulator mutations are expressed similarly in the such as mmour necrosis factor (TNF), and that nasal and bronchial epithelium in CF, the inflam-IL-8 acts as a neutrophil chemo-attractant matory consequences appear to be different, thought to mediate the non-specific chronic pulwith little inflammation and no change in the monary inflammation in CF. Replication-deficient proportion of epithelial cells in the nasal epitheadenoviral vectors have also been shown to lium, compared with marked neutrophil inflamincrease IL-8 and ICAM-1 mRNA and protein marion on the epithelial surface and significant expression in A549, a lung epithelial human cell changes, in epithelial populations in the large line. 13 airways. 4 Regarding the toxicity associated with administration of El-deleted adenovirus into the airways of animals, investigations performed in a number of species, including rodents and non-human primates, showed that there is a dose dependent inflammatory response at the site of gene transfer that diminishes without permanent sequelae. Experience with intrapulmonary instillation of virus in patients is limited, but is usually unremarkable at the low doses administered to patients, except in the case of one patient. This syndrome was not predicted by the animal experiments, thus showing the importance of human studies, especially in CF. In this disorder, the lack of an animal lung model and the intense pulmonary inflammation observed complicate the evaluation of potential adenovirus vector-related toxicity.