Pieter S. Hiemstra

An angiogenic role for the human peptide antibiotic LL-37/hCAP-18

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

Innate immunity in the lung: how epithelial cells fight against respiratory pathogens

The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 litres of air. The observation that respiratory infections are nevertheless rare is testimony to the presence of an efficient host defence system at the mucosal surface of the lung. The airway epithelium is strategically positioned at the interface with the environment, and thus plays a key role in this host defence system. Recognition systems employed by airway epithelial cells to respond to microbial exposure include the action of the toll-like receptors. The airway epithelium responds to such exposure by increasing its production of mediators such as cytokines, chemokines and antimicrobial peptides. Recent findings indicate the importance of these peptides as effector molecules of innate immunity by killing microorganisms, but also as regulators of inflammation, immunity and wound repair. Finally, the clinical relevance of the functions of the airway epithelium in innate immunity is discussed.

In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation

The reported requirement of functional Toll-like receptor (TLR)4 for resistance to Gram-negative pyelonephritis prompted us to localize the expression of TLR2 and TLR4 mRNA in the kidney at the cellular level by in situ hybridization. The majority of the constitutive TLR2 and TLR4 mRNA expression was found to be strategically located in the renal epithelial cells. Assuming that the TLR mRNA expression is representative of apical protein expression, this suggests that these cells are able to detect and react with bacteria present in the lumen of the tubules. To gain insight in the regulation of TLR expression during inflammation, we used a model for renal inflammation. Renal inflammation evoked by ischemia markedly enhanced synthesis of TLR2 and TLR4 mRNA in the distal tubular epithelium, the thin limb of Henle’s loop, and collecting ducts. The increased renal TLR4 mRNA expression was associated with significant elevation of renal TLR4 protein expression as evaluated by Western blotting. Using RT-PCR, the enhanced TLR2 and TLR4 mRNA expression was shown to be completely dependent on the action of IFN-γ and TNF-α. These results indicate a potential mechanism of increased immunosurveillance during inflammation at the site in which ascending bacteria enter the kidney tissue, i.e., the collecting ducts and the distal part of the nephron.

The Antimicrobial Peptide LL-37 Activates Innate Immunity at the Airway Epithelial Surface by Transactivation of the Epidermal Growth Factor Receptor

Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.

Monocyte chemoattractant protein 1, interleukin 8, and chronic airways inflammation in COPD

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death, with cigarette smoking among the main risk factors. Hallmarks of COPD include chronic airflow obstruction and chronic inflammation in the airway walls or alveolar septa. An earlier study reported elevated numbers of macrophages and mast cells within the bronchiolar epithelium in smokers with COPD, compared with smokers without. Since specific chemokines may be involved in this influx, the in situ protein and mRNA expression of monocyte chemoattractant protein 1 (MCP‐1) and of interleukin 8 (IL‐8) were studied in tumour‐free peripheral lung tissue resected for lung cancer of current or ex‐smokers with COPD (FEV1<75%; n=14) and without COPD (FEV1>84; n=14). MCP‐1 was expressed by macrophages, T cells, and endothelial and epithelial cells. Its receptor, CCR2, is expressed by macrophages, mast cells, and epithelial cells. IL‐8 was found in neutrophils, epithelial cells, and macrophages. In subjects with COPD, semi‐quantitative analysis revealed 1.5‐fold higher levels of MCP‐1 mRNA and IL‐8 mRNA and protein in bronchiolar epithelium (p<0.01) and 1.4‐fold higher levels of CCR2 in macrophages (p=0.014) than in subjects without COPD. The bronchiolar epithelial MCP‐1 mRNA expression correlated with both CCR2 expression on macrophages and mast cells (p<0.05) and the numbers of intra‐epithelial macrophages and mast cells (p<0.04). The epithelial IL‐8 expression did not correlate with the numbers of neutrophils, macrophages, CD45RO+, CD8+, or mast cells. These data suggest that MCP‐1 and CCR2 are involved in the recruitment of macrophages and mast cells into the airway epithelium in COPD. Copyright

Reduction in sputum neutrophil and eosinophil numbers by the PDE4 inhibitor roflumilast in patients with COPD

Background: Roflumilast is a targeted oral once-daily administered phosphodiesterase 4 (PDE4) inhibitor with clinical efficacy in chronic obstructive pulmonary disease (COPD). Results from in vitro studies with roflumilast indicate that it has anti-inflammatory properties that may be applicable for the treatment of COPD. Methods: In a crossover study, 38 patients with COPD (mean (SD) age 63.1 (7.0) years, post-bronchodilator forced expiratory volume in 1 s (FEV1) 61.0 (12.6)% predicted) received 500 μg roflumilast or placebo once daily for 4 weeks. Induced sputum samples were collected before and after 2 and 4 weeks of treatment. Differential and absolute cell counts were determined in whole sputum samples. Markers of inflammation were determined in sputum supernatants and blood. Spirometry was performed weekly. Results: Roflumilast significantly reduced the absolute number of neutrophils and eosinophils/g sputum compared with placebo by 35.5% (95% CI 15.6% to 50.7%; p = 0.002) and 50.0% (95% CI 26.8% to 65.8%; p<0.001), respectively. The relative proportion of sputum neutrophils and eosinophils was not affected by treatment (p>0.05). Levels of soluble interleukin-8, neutrophil elastase, eosinophil cationic protein and α2-macroglobulin in sputum and the release of tumour necrosis factor α from blood cells were significantly reduced by roflumilast compared with placebo treatment (p<0.05 for all). Post-bronchodilator FEV1 improved significantly during roflumilast compared with placebo treatment with a mean difference between treatments of 68.7 ml (95% CI 12.9 to 124.5; p = 0.018). Conclusion: PDE4 inhibition by roflumilast treatment for 4 weeks reduced the number of neutrophils and eosinophils, as well as soluble markers of neutrophilic and eosinophilic inflammatory activity in induced sputum samples of patients with COPD. This anti-inflammatory effect may in part explain the concomitant improvement in post-bronchodilator FEV1.

Expression of β-defensin 1 and 2 mRNA by human monocytes, macrophages and dendritic cells

Human β‐defensins are broad‐spectrum antimicrobial peptides known to be produced by epithelial cells. It was recently shown that β‐defensins also display chemotactic activity for dendritic cells (DC) and T cells, and thus may serve to link innate and adaptive immunity. The aim of the present study was to explore expression of mRNA for these peptides in mononuclear phagocytes and DC. The results revealed that monocytes, monocyte‐derived‐macrophages (MDM), and monocyte‐derived‐dendritic cells (DC) all express human‐β‐defensin‐1 (hBD‐1) mRNA. hBD‐1 mRNA expression by monocytes and MDM was increased after activation with interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS) in a dose‐ and time‐dependent fashion. Alveolar macrophages showed an intense hBD‐1 expression, which could not be further increased. Expression of hBD‐1 mRNA by immature DC was low, and increased considerably after maturation. Monocytes, MDM, alveolar macrophages and DC showed a limited expression of human β‐defensin‐2 (hBD‐2) mRNA, which could only be increased in monocytes and alveolar macrophages by IFN‐γ and/or LPS in a dose‐ and time‐dependent fashion. Immunocytochemical stainings demonstrated the expression of hBD‐2 peptide by freshly isolated blood monocytes and alveolar macrophages in cytospin preparations.

Effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin‐8 in nasal lavage in asthmatic subjects in vivo

Background Asthma exacerbations are closely associated with respiratory virus infections. However, the pathophysiological consequences of such infections in asthma are largely unclear.

Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma

Sputum induced by inhalation of nebulized hypertonic saline is increasingly used to monitor airways inflammation in asthma. The aim of this study was to assess the repeatability of measuring cellular and soluble markers of inflammation in whole sputum samples as obtained by sputum induction in patients both with mild and moderate-to-severe asthma. Twelve patients with mild, atopic asthma without inhaled steroid treatment and nine patients with moderate-to-severe, atopic asthma treated with inhaled steroids were studied on two separate days at least 2 days apart. Whole sputum samples, induced by inhalation of hypertonic (4.5%) saline, were homogenized, and analysed for differential cell counts and for concentrations of albumin, fibrinogen, interleukin-8 (IL-8), and eosinophil cationic protein (ECP). Repeatability was expressed as intraclass correlation coefficient (Ri), and as coefficient of repeatability (CR) in percentage cells or in doubling concentration. Samples from two patients with mild asthma contained more than 80% squamous cells and were excluded from analysis. The repeatability for cell differential counts in both groups combined was: for neutrophils, Ri = 0.57 and CR = 31.0; for eosinophils, Ri = 0.85 and CR = 12.4; and for lymphocytes, Ri = 0.76 and CR = 6.9. The repeatability of the fluid phase measurements was: for albumin, Ri = 0.71 and CR = 3.2; for fibrinogen, Ri = 0.88 and CR = 2.8; for IL-8, Ri = 0.66 and CR = 2.2; and for ECP, Ri = 0.82 and CR = 1.1. We conclude that the repeatability of cellular and soluble markers of inflammation in induced sputum from patients with mild and moderate-to-severe asthma is satisfactory. Hence, induced sputum, processed by using the whole expectorated sample, seems to be a valuable method to monitor airway inflammation in asthma.

Antileukoprotease: An Endogenous Protein in the Innate Mucosal Defense against Fungi

Previous studies have suggested that endogenous protease inhibitors may participate in the mucosal host defense. Antileukoprotease (ALP) is an important protease inhibitor found on various mucosal surfaces, including those of the respiratory and genital tracts. This study reports on the antimicrobial activity of recombinant (r) ALP toward the human fungal pathogens Aspergillus fumigatus and Candida albicans. rALP expressed pronounced fungicidal activity toward metabolically active A. fumigatus conidia and C. albicans yeast cells; however, metabolically quiescent A. fumigatus conidia were totally resistant. In contrast with the protease inhibitory activity of rALP, the fungicidal activity was localized primarily in the NH2-terminal domain. On a molar base, the fungicidal activity of rALP was comparable with that of human defensins and lysozyme. In addition, rALP caused inhibition of C. albicans yeast cell growth. By exhibiting antifungal activity, ALP may play an important role in the innate mucosal defense against human pathogenic fungi.