- Email: [email protected]
S10.3. Cystic fibrosis, antimicrobial peptides and gene therapy
S10
Supplement / The Netherlands Journal of Medicine 54 (1999) S3 –S84
toms in CF. Frank J. Kelly. King’ s College London, St. Thomas’ Hospital, London, UK. Neurological sequelae are the only well-defined clinical signs of vitamin E deficiency. Although low blood levels of vitamin E are common in unsupplemented CF patients, overt neurological disease is relatively rare. Indeed it is usually only apparent in the presence of severe liver disease. When neurological symptoms are seen in CF they are usually similar to those associated with abetalipoproteinaemia or Friedreich’s ataxia. Patients show abnormalities of the cerebellum, posterior columns and peripheral nerves. Exactly how vitamin E deficiency leads to these neurological problems is not clearly delineated. Nervous tissue has a high lipid content and therefore requires vitamin E to prevent oxidation. Indeed, nervous tissue avidly retains vitamin E, loosing it very slowly compared to other tissues. In the absence of sufficient vitamin E, nervous tissue is subject to increased oxidative stress and this likely leads to neuronal demyelation. An incomplete understanding of vitamin E metabolism has, however, left many questions unanswered. Recent developments in stable isotope technology, including the availability of deuterium-labelled tocopherol, have led to progress in this field. Studies with control subjects have shown that even non-CF patients have variable abilities to absorb vitamin E. Importantly however, an individual’s ability to absorb vitamin E is relatively stable over time. Clearly the factors that influence absorption are not fully understood. For example, smoking appears to have a negative influence. A number of specific metabolites of vitamin E, several of which appear in urine, have now also been identified. One, or more of these, may represent a new, non-invasive means of examining vitamin E requirements in humans. If this proves to be the case it will be of benefit in the management of CF.
S10.2. Pulmonary antimicrobial peptides: expression and activity. P.B. McCray Jr., H.P. Jia, P.K. Singh, S.M. Travis, M.J. Welsh, B.F. Tack. Departments of Pediatrics, Microbiology and Internal Medicine, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, IA, USA. Innate immune factors present in airway surface liquid (ASL) contribute to host defenses against inhaled microbes. One layer of these defenses consists of antimicrobial peptides, including the defensins and cathelicidins. Such peptides are produced several cells in the lung, including airway epithelia, macrophages and neutrophils. Loss of peptide activity in ASL may contribute to the chronic airway infection that characterizes CF. We studied the expression and activity of the human b-defensins (HBD) in the lung. The HBD-1 and HBD-2 mRNAs were expressed in surface and submucosal gland epithelia from non-CF and CF patients. In cultured epithelia, interleukin-1b stimulated the expression of HBD-2, but not HBD-1 mRNA and peptide. HBD-1 was found in BAL from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL from patients with CF or inflammatory lung disease but not in normal volunteers. Both HBD-1 and HBD-2 showed salt-sensitive bactericidal activity. HBD-2 may be important in the response to inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation. One cathelicidin peptide, LL37, has
been identified in humans and is a product of neutrophils and some epithelia. We studied the antimicrobial activities of cathelicidin peptides from several species including human, rabbit, mouse and rat. A wide range of microbicidal activity was noted against CF-associated pathogens including Pseudomonas, Staph, Alcaligenes, and Xanthomonas species. Of note, some peptides retained full activity in 175 mM NaCI. Another feature of these peptides was very rapid kill kinetics, occurring within minutes. Topical application of peptide antibiotics, including the cathelicidins, may have applications for the treatment or prevention of CF lung disease.
S10.3. Cystic fibrosis, antimicrobial peptides and gene therapy. R. Bals, J.M. Wilson. Institute for Human Gene Therapy, University of Pennsylvania, and The Wistar Institute, PA, USA. A breach of the innate host defense mechanisms of the airways contributes to lung disease in cystic fibrosis. Antimicrobial peptides are part of this defense system. Several epithelial antimicrobial peptides are produced in airway epithelial cells and secreted into the airway surface fluid (ASF). Human b-defensin 1 and 2 (hBD-1, hBD-2) belong to the b-defensin family that is characterized by the presence of three disulfide found in a specific pattern. LL-37 / hCAP-18 is the only member of the cathelicidins found in human. These antimicrobial peptides contribute the host defense of the ASF showing a broad activity against bacteria and fungi. They work synergistically with other secretory antimicrobial substances, such as lysozyme and lactoferrin. To reveal the role of these substances as part of the innate immune system animal models will have to be used. Mouse b-defensins 1 and 2 and murine cathelin like protein (MCLP) are homologs of hBD-1, hBD-2 and LL-37 / hCAP-18 and provide possibilities to study the host defense system. To answer the question whether decreased concentrations of antimicrobial peptides might contribute to the primary defect in CF lung disease, we measured the concentrations of hBD-1, hBD-2 and LL-37 / hCAP-18 in bronchoalveolar lavage fluid from normal and CF infants and washings from cells cultured in a human bronchial xenograft model. All these peptides were found in equivalent concentrations in CF and normal. The question remains what alteration of the ASF causes the defect in antimicrobial activity. To answer the question whether the macromolecular composition of CF ASF is different from normal, we isolated ASF from xenografts. The desalted material was used in quantitative antimicrobial dilution assays using a low salt buffer system. We found that ASF from CF xenografts lost its antimicrobial significantly earlier as compared to normal material. These results indicate the absence of an antimicrobial factor that contributes to host defense under normal conditions in this culture system. Finally we tested whether overexpression of endogenous antimicrobial substances by gene transfer results in correction of the killing defect in CF. Gene transfer of LL-37 / hCAP-18 cDNA into CF xenografts resulted in restoration of the antimicrobial activity of the ASF. This strategy represents a possible treatment approach for infectious diseases. A primary defect of the host defense system of the airways
Supplement / The Netherlands Journal of Medicine 54 (1999) S3 –S84 contributes to the development of CF lung disease. Secondary defects caused by tissue destruction determine the further progression of the disease. Better understanding of the host defense system will provide new therapeutical options in the future.
S10.4. Role of neutrophil defensins in inflammatory lung disease. P.S. Hiemstra. Department of Pulmonology, Leiden University Medical Center, The Netherlands. Neutrophils are thought to play an important role in a variety of inflammatory lung diseases, including cystic fibrosis and chronic obstructive pulmonary disease (COPD). Neutrophils may cause tissue injury through the release and production of a variety of mediators, including products released from the azurophilic granules of these cells. These granules contain products that may cause lung injury, and include serine proteinases such as elastase, and neutrophil defensins. Neutrophil defensins are cationic, small, antimicrobial and cytotoxic peptides that are members of the a-defensin subfamily of defensins. Large amounts of neutrophil defensins have been shown to be present in purulent secretions of patients with cystic fibrosis and COPD. It is now known that neutrophil defensins not only kill micro-organisms and host cells, but may also affect inflammatory and repair processes in the lung due to a variety of actions on various target cells. In our studies, we have focused on the in vitro effect of neutrophil defensins on airway epithelial cells, a main target for neutrophilmediated injury. These studies were performed using both airway epithelial cell lines and subcultures of primary bronchial epithelial cells. Our results demonstrate that neutrophil defensins induce synthesis of the neutrophil chemotactic chemokines IL-8 and ENA-78 in epithelial cells, and that this effect is inhibited by dexamethasone and a1-proteinase inhibitor. Furthermore, at the same concentration (100 mg / ml) defensins cause an increased release of the elastase inhibitor secretory leukocyte proteinase inhibitor (SLPI) from these cells, indicating that defensins may also stimulate anti-inflammatory mechanisms. At lower concentrations (20 mg / ml), defensins increase the adherence of Haemophilus influenzae to airway epithelial cells in vitro, indicating that in certain conditions defensins may facilitate H. influenzae infections in the lower airways despite their antimicrobial activity. Finally, at even lower concentrations of defensins (5–10 mg / ml) these peptides were found to increase proliferation of airway epithelial cells, which indicates that they may play a role in epithelial repair processes. In summary, these results show that neutrophil defensins may affect inflammatory and repair processes in the lung, and that the nature of the effect of defensins is dependent in part on its local concentration. Furthermore, our results show that part of the extracelluar activities of defensins are restricted by a1proteinase inhibitor.
S11.1. Interaction of Aspergilus fumigatus with the defense system of the airways. H.F. Kauffman. Univ. Hospital, Clin. Internal Med, Lab. Allergology and Pulmonology, Groningen, The Netherlands. Aspergillus fumigatus, innate defense, epithelial cells, proteases. Healthy airways have an effective innate defense system that
S11
generally will prevent intense contact with the systemic immune response. Therefore, interactions of inhaled fungi are generally not destructive for the airways except for pathogenic fungi e.g. Aspergillus fumigatus (Af ). As defense strategies can be recognised, 1. the rate of clearance by transport of impacted materials of the mucus layer by epithelial beating frequency; 2. the killing capacity by epithelial derived antifungal proteins e.g. lysozyme, SLPI and other proteins; 3. the opsonization of fungal materials (spores) by different proteins e.g. sIgA, C-type lectins, and surfactant proteins that will prevent binding to airway wall cells and give help to 4. the fungal killing capacity by phagocytic cells (alveolar macrophages and neutrophils). Af has the capacity to impair this defense system at different levels. Inactivation of cilia beating frequency, degradation of antifungal proteins and impairment of the phagocytic killing capacity, have been shown. Patients with CF are exceptionally prone to infection with Af. The development of ABPA in these patients has been shown to induce an increased decline in lung function parameters. This increased decline can possibly be ascribed to the damaging role of toxic and proteolytic proteins that can be released by the infiltrating eosinophils, which is characteristic for ABPA. Recently we have shown that proteolytic activities excreted by Af if grown on a collagen medium (EColCF), induces in-vitro epithelial detachment and induction of cytokines. The decline in cytokine productions at increasing quantities of EcolCF extracts, appears to be due to degradation of the cytokines by proteolytic activities present in EcolCF. Epithelial detachment and degradation of cytokines will be discussed in relation to the pathogenic properties of Af.
S11.3. Clinical manifestations of Aspergillus fumigatus in children and adults with CF. G.P.M. Mannes. Department of Pulmonary Diseases, Leyenburg Hospital, The Hague, The Netherlands. In humans Aspergillus species have been associated with a wide variety of diseases in different organs. In cystic fibrosis (CF) patients almost all Aspergillus manifestations are caused by Aspergillus fumigatus (Af ) and are localized in the lungs. The exact contribution of Af to the disease process in Cf patients is poorly understood however. Pulmonary aspergillosis is classified into four groups: 1. 2. 3. 4.
Non-pathogenic saprophytic colonization. Hypersensitivity-induced aspergillosis. Pulmonary aspergilloma. Invasive aspergillosis.
Sometimes patients may exhibit characteristics of two different types of aspergillosis. Ad 1.: Af may cause saprophytic infestation of the respiratory tract without causing manifest tissue damage. The presence of the fungus is either harmless or leads only to a subclinical response. In CF patients the reported prevalence of Af-positive sputum cultures is ranging from 10–60%. Ad 2.: Sensitization to Af as detected by skin prick test for Af and / or by determination of specific serum IgE antibodies to Af is
Supplement / The Netherlands Journal of Medicine 54 (1999) S3 –S84
toms in CF. Frank J. Kelly. King’ s College London, St. Thomas’ Hospital, London, UK. Neurological sequelae are the only well-defined clinical signs of vitamin E deficiency. Although low blood levels of vitamin E are common in unsupplemented CF patients, overt neurological disease is relatively rare. Indeed it is usually only apparent in the presence of severe liver disease. When neurological symptoms are seen in CF they are usually similar to those associated with abetalipoproteinaemia or Friedreich’s ataxia. Patients show abnormalities of the cerebellum, posterior columns and peripheral nerves. Exactly how vitamin E deficiency leads to these neurological problems is not clearly delineated. Nervous tissue has a high lipid content and therefore requires vitamin E to prevent oxidation. Indeed, nervous tissue avidly retains vitamin E, loosing it very slowly compared to other tissues. In the absence of sufficient vitamin E, nervous tissue is subject to increased oxidative stress and this likely leads to neuronal demyelation. An incomplete understanding of vitamin E metabolism has, however, left many questions unanswered. Recent developments in stable isotope technology, including the availability of deuterium-labelled tocopherol, have led to progress in this field. Studies with control subjects have shown that even non-CF patients have variable abilities to absorb vitamin E. Importantly however, an individual’s ability to absorb vitamin E is relatively stable over time. Clearly the factors that influence absorption are not fully understood. For example, smoking appears to have a negative influence. A number of specific metabolites of vitamin E, several of which appear in urine, have now also been identified. One, or more of these, may represent a new, non-invasive means of examining vitamin E requirements in humans. If this proves to be the case it will be of benefit in the management of CF.
S10.2. Pulmonary antimicrobial peptides: expression and activity. P.B. McCray Jr., H.P. Jia, P.K. Singh, S.M. Travis, M.J. Welsh, B.F. Tack. Departments of Pediatrics, Microbiology and Internal Medicine, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, IA, USA. Innate immune factors present in airway surface liquid (ASL) contribute to host defenses against inhaled microbes. One layer of these defenses consists of antimicrobial peptides, including the defensins and cathelicidins. Such peptides are produced several cells in the lung, including airway epithelia, macrophages and neutrophils. Loss of peptide activity in ASL may contribute to the chronic airway infection that characterizes CF. We studied the expression and activity of the human b-defensins (HBD) in the lung. The HBD-1 and HBD-2 mRNAs were expressed in surface and submucosal gland epithelia from non-CF and CF patients. In cultured epithelia, interleukin-1b stimulated the expression of HBD-2, but not HBD-1 mRNA and peptide. HBD-1 was found in BAL from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL from patients with CF or inflammatory lung disease but not in normal volunteers. Both HBD-1 and HBD-2 showed salt-sensitive bactericidal activity. HBD-2 may be important in the response to inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation. One cathelicidin peptide, LL37, has
been identified in humans and is a product of neutrophils and some epithelia. We studied the antimicrobial activities of cathelicidin peptides from several species including human, rabbit, mouse and rat. A wide range of microbicidal activity was noted against CF-associated pathogens including Pseudomonas, Staph, Alcaligenes, and Xanthomonas species. Of note, some peptides retained full activity in 175 mM NaCI. Another feature of these peptides was very rapid kill kinetics, occurring within minutes. Topical application of peptide antibiotics, including the cathelicidins, may have applications for the treatment or prevention of CF lung disease.
S10.3. Cystic fibrosis, antimicrobial peptides and gene therapy. R. Bals, J.M. Wilson. Institute for Human Gene Therapy, University of Pennsylvania, and The Wistar Institute, PA, USA. A breach of the innate host defense mechanisms of the airways contributes to lung disease in cystic fibrosis. Antimicrobial peptides are part of this defense system. Several epithelial antimicrobial peptides are produced in airway epithelial cells and secreted into the airway surface fluid (ASF). Human b-defensin 1 and 2 (hBD-1, hBD-2) belong to the b-defensin family that is characterized by the presence of three disulfide found in a specific pattern. LL-37 / hCAP-18 is the only member of the cathelicidins found in human. These antimicrobial peptides contribute the host defense of the ASF showing a broad activity against bacteria and fungi. They work synergistically with other secretory antimicrobial substances, such as lysozyme and lactoferrin. To reveal the role of these substances as part of the innate immune system animal models will have to be used. Mouse b-defensins 1 and 2 and murine cathelin like protein (MCLP) are homologs of hBD-1, hBD-2 and LL-37 / hCAP-18 and provide possibilities to study the host defense system. To answer the question whether decreased concentrations of antimicrobial peptides might contribute to the primary defect in CF lung disease, we measured the concentrations of hBD-1, hBD-2 and LL-37 / hCAP-18 in bronchoalveolar lavage fluid from normal and CF infants and washings from cells cultured in a human bronchial xenograft model. All these peptides were found in equivalent concentrations in CF and normal. The question remains what alteration of the ASF causes the defect in antimicrobial activity. To answer the question whether the macromolecular composition of CF ASF is different from normal, we isolated ASF from xenografts. The desalted material was used in quantitative antimicrobial dilution assays using a low salt buffer system. We found that ASF from CF xenografts lost its antimicrobial significantly earlier as compared to normal material. These results indicate the absence of an antimicrobial factor that contributes to host defense under normal conditions in this culture system. Finally we tested whether overexpression of endogenous antimicrobial substances by gene transfer results in correction of the killing defect in CF. Gene transfer of LL-37 / hCAP-18 cDNA into CF xenografts resulted in restoration of the antimicrobial activity of the ASF. This strategy represents a possible treatment approach for infectious diseases. A primary defect of the host defense system of the airways
Supplement / The Netherlands Journal of Medicine 54 (1999) S3 –S84 contributes to the development of CF lung disease. Secondary defects caused by tissue destruction determine the further progression of the disease. Better understanding of the host defense system will provide new therapeutical options in the future.
S10.4. Role of neutrophil defensins in inflammatory lung disease. P.S. Hiemstra. Department of Pulmonology, Leiden University Medical Center, The Netherlands. Neutrophils are thought to play an important role in a variety of inflammatory lung diseases, including cystic fibrosis and chronic obstructive pulmonary disease (COPD). Neutrophils may cause tissue injury through the release and production of a variety of mediators, including products released from the azurophilic granules of these cells. These granules contain products that may cause lung injury, and include serine proteinases such as elastase, and neutrophil defensins. Neutrophil defensins are cationic, small, antimicrobial and cytotoxic peptides that are members of the a-defensin subfamily of defensins. Large amounts of neutrophil defensins have been shown to be present in purulent secretions of patients with cystic fibrosis and COPD. It is now known that neutrophil defensins not only kill micro-organisms and host cells, but may also affect inflammatory and repair processes in the lung due to a variety of actions on various target cells. In our studies, we have focused on the in vitro effect of neutrophil defensins on airway epithelial cells, a main target for neutrophilmediated injury. These studies were performed using both airway epithelial cell lines and subcultures of primary bronchial epithelial cells. Our results demonstrate that neutrophil defensins induce synthesis of the neutrophil chemotactic chemokines IL-8 and ENA-78 in epithelial cells, and that this effect is inhibited by dexamethasone and a1-proteinase inhibitor. Furthermore, at the same concentration (100 mg / ml) defensins cause an increased release of the elastase inhibitor secretory leukocyte proteinase inhibitor (SLPI) from these cells, indicating that defensins may also stimulate anti-inflammatory mechanisms. At lower concentrations (20 mg / ml), defensins increase the adherence of Haemophilus influenzae to airway epithelial cells in vitro, indicating that in certain conditions defensins may facilitate H. influenzae infections in the lower airways despite their antimicrobial activity. Finally, at even lower concentrations of defensins (5–10 mg / ml) these peptides were found to increase proliferation of airway epithelial cells, which indicates that they may play a role in epithelial repair processes. In summary, these results show that neutrophil defensins may affect inflammatory and repair processes in the lung, and that the nature of the effect of defensins is dependent in part on its local concentration. Furthermore, our results show that part of the extracelluar activities of defensins are restricted by a1proteinase inhibitor.
S11.1. Interaction of Aspergilus fumigatus with the defense system of the airways. H.F. Kauffman. Univ. Hospital, Clin. Internal Med, Lab. Allergology and Pulmonology, Groningen, The Netherlands. Aspergillus fumigatus, innate defense, epithelial cells, proteases. Healthy airways have an effective innate defense system that
S11
generally will prevent intense contact with the systemic immune response. Therefore, interactions of inhaled fungi are generally not destructive for the airways except for pathogenic fungi e.g. Aspergillus fumigatus (Af ). As defense strategies can be recognised, 1. the rate of clearance by transport of impacted materials of the mucus layer by epithelial beating frequency; 2. the killing capacity by epithelial derived antifungal proteins e.g. lysozyme, SLPI and other proteins; 3. the opsonization of fungal materials (spores) by different proteins e.g. sIgA, C-type lectins, and surfactant proteins that will prevent binding to airway wall cells and give help to 4. the fungal killing capacity by phagocytic cells (alveolar macrophages and neutrophils). Af has the capacity to impair this defense system at different levels. Inactivation of cilia beating frequency, degradation of antifungal proteins and impairment of the phagocytic killing capacity, have been shown. Patients with CF are exceptionally prone to infection with Af. The development of ABPA in these patients has been shown to induce an increased decline in lung function parameters. This increased decline can possibly be ascribed to the damaging role of toxic and proteolytic proteins that can be released by the infiltrating eosinophils, which is characteristic for ABPA. Recently we have shown that proteolytic activities excreted by Af if grown on a collagen medium (EColCF), induces in-vitro epithelial detachment and induction of cytokines. The decline in cytokine productions at increasing quantities of EcolCF extracts, appears to be due to degradation of the cytokines by proteolytic activities present in EcolCF. Epithelial detachment and degradation of cytokines will be discussed in relation to the pathogenic properties of Af.
S11.3. Clinical manifestations of Aspergillus fumigatus in children and adults with CF. G.P.M. Mannes. Department of Pulmonary Diseases, Leyenburg Hospital, The Hague, The Netherlands. In humans Aspergillus species have been associated with a wide variety of diseases in different organs. In cystic fibrosis (CF) patients almost all Aspergillus manifestations are caused by Aspergillus fumigatus (Af ) and are localized in the lungs. The exact contribution of Af to the disease process in Cf patients is poorly understood however. Pulmonary aspergillosis is classified into four groups: 1. 2. 3. 4.
Non-pathogenic saprophytic colonization. Hypersensitivity-induced aspergillosis. Pulmonary aspergilloma. Invasive aspergillosis.
Sometimes patients may exhibit characteristics of two different types of aspergillosis. Ad 1.: Af may cause saprophytic infestation of the respiratory tract without causing manifest tissue damage. The presence of the fungus is either harmless or leads only to a subclinical response. In CF patients the reported prevalence of Af-positive sputum cultures is ranging from 10–60%. Ad 2.: Sensitization to Af as detected by skin prick test for Af and / or by determination of specific serum IgE antibodies to Af is