Sandra van Wetering

Defensins: Key players or bystanders in infection, injury, and repair in the lung? ☆ ☆☆

Antimicrobial peptides have been identified as key elements in the innate host defense against infection. Recent studies have indicated that the activity of antimicrobial peptides may be decreased in cystic fibrosis, suggesting a major role for these peptides in host defense against infection. One of the most intensively studied classes of antimicrobial peptides are defensins. Defensins comprise a family of cationic peptides that in human subjects can be divided into the alpha- and beta-defensin subfamilies. The alpha-defensins are produced by neutrophils and intestinal Paneths cells, whereas beta-defensins are mainly produced by epithelial cells. Although studies on beta-defensins have so far focused on their antimicrobial activity, studies on alpha-defensins have suggested a role of these peptides in inflammation, wound repair, and specific immune responses. alpha-Defensins, which accumulate in airway secretions of patients with various chronic inflammatory lung disorders, were shown to be cytotoxic toward airway epithelial cells and to induce chemokine secretion in several cell types. Furthermore, the capacity of alpha-defensins to promote bacterial adherence to epithelial cells in vitro further supports a role for these peptides in the pathogenesis of chronic obstructive pulmonary disease and cystic fibrosis. Increased numbers of neutrophils are also present in the airways of patients with asthma, suggesting that neutrophils are involved in the pathogenesis of this disease. Because defensins are able to induce histamine release by mast cells and increase the airway hyperresponsiveness to histamine, it is tempting to speculate that defensins may also contribute to the inflammatory processes in asthma. Besides these proinflammatory effects, alpha-defensins may also display anti-inflammatory activities, including regulation of complement activation and proteinase inhibitor secretion. Finally, defensins may be involved in wound repair because defensins increase epithelial cell proliferation. Thus recent defensin research has revealed potential links between the innate and acquired immune system.

Human neutrophil defensins induce lung epithelial cell proliferation in vitro

Repair of injured airway epithelium is often accompanied by an influx of leukocytes, and these cells have been suggested to contribute to the repair process. The aim of the present study was to investigate the effect of neutrophil defensins—antimicrobial peptides present in large amounts in the neutrophil— on proliferation of cultured lung epithelial cells. Neutrophil defensins at 4–10 μg/ml enhanced proliferation of the A549 lung epithelial cell line as assessed using cell counting, BrdU incorporation, and the tetrazolium salt MTT assay. Higher, cytotoxic concentrations of defensins decreased cell proliferation. Whereas defensin‐induced cell proliferation was not inhibited by the EGF receptor tyrosine kinase inhibitor AG1478, it was completely inhibited by the mitogen‐activated protein (MAP) kinase kinase (MEK) inhibitor U0126, suggesting that defensins mediate cell proliferation via an EGF receptor‐independent, MAP kinase signaling pathway. Although the cytotoxic effect of defensins was inhibited by α1‐proteinase inhibitor, the defensin‐induced cell proliferation was not affected. These data suggest that neutrophil defensins may possibly be involved in epithelial repair in the airways by inducing lung epithelial cell proliferation.

Effects of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione

BackgroundIncreased airway epithelial proliferation is frequently observed in smokers. To elucidate the molecular mechanisms leading to these epithelial changes, we studied the effect of cigarette smoke condensate (CSC) on cell proliferation, wound closure and mitogen activated protein kinase (MAPK) activation. We also studied whether modulation of intracellular glutathione/thiol levels could attenuate CSC-induced cell proliferation.MethodsCells of the bronchial epithelial cell line NCI-H292 and subcultures of primary bronchial epithelial cells were used for the present study. The effect of CSC on epithelial proliferation was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Modulation of epithelial wound repair was studied by analysis of closure of 3 mm circular scrape wounds during 72 hours of culture. Wound closure was calculated from digital images obtained at 24 h intervals. Activation of mitogen-activated protein kinases was assessed by Western blotting using phospho-specific antibodies.ResultsAt low concentrations CSC increased proliferation of NCI-H292 cells, whereas high concentrations were inhibitory as a result of cytotoxicity. Low concentrations of CSC also increased epithelial wound closure of both NCI-H292 and PBEC, whereas at high concentrations closure was inhibited. At low, mitogenic concentrations, CSC caused persistent activation of ERK1/2, a MAPK involved in cell proliferation. Inhibition of cell proliferation by high concentrations of CSC was associated with activation of the pro-apoptotic MAP kinases p38 and JNK. Modulation of intracellular glutathione (GSH)/thiol levels using N-acetyl-L-cysteine, GSH or buthionine sulphoximine (BSO), demonstrated that both the stimulatory and the inhibitory effects of CSC were regulated in part by intracellular GSH levels.ConclusionThese results indicate that CSC may increase cell proliferation and wound closure dependent on the local concentration of cigarette smoke and the anti-oxidant status. These findings are consistent with increased epithelial proliferation in smokers, and may provide further insight in the development of lung cancer.

Therapeutic Targeting of Classical and Lectin Pathways of Complement Protects from Ischemia-Reperfusion-Induced Renal Damage

Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163(+), SWC3a(+), CD4a(+), and CD8a(+) cells. C1-inhibitor administration led to significant inhibition of tubular damage and tubular epithelial cells apoptosis. Interestingly, we report that focal C4d-deposition colocalizes with C1q and MBL at the peritubular and glomerular capillary levels also in patients with delayed graft function. In conclusion, we demonstrated the activation and a pathogenic role of classical and lectin pathways of complement in a swine model of ischemia-reperfusion-induced renal damage. Therefore, inhibition of these two pathways might represent a novel therapeutic approach in the prevention of delayed graft function in kidney transplant recipients.

Migration of Human Hematopoietic Progenitor Cells Across Bone Marrow Endothelium Is Regulated by Vascular Endothelial Cadherin

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34+ cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34+ cells in response to stromal cell-derived factor-1α. Stromal cell-derived factor-1α-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34+ cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.

Interactions between neutrophil-derived antimicrobial peptides and airway epithelial cells

Most antimicrobial peptides have been discovered based on activity‐guided purification procedures, which used assays to determine their antimicrobial activity. Nevertheless, recent studies have shown that antimicrobial peptides also exert a range of other functions. Based on these observations, antimicrobial peptides are now not only implicated in host defense against infection but also in other immune reactions, inflammation, and wound‐repair processes. The activities of neutrophil defensins and the cathelicidin hCAP‐18/LL‐37, antimicrobial peptides that are abundantly expressed in the human neutrophil, are the subject of an increasing number of studies. Exposure to neutrophil defensins and hCAP‐18/LL‐37 results in increases in mediator expression and release, chemotaxis, and proliferation of inflammatory and epithelial cells and fibroblasts, and the mechanisms underlying these effects have been partly elucidated. This review is focused on the effects of neutrophil defensins and hCAP‐18/LL‐37 on airway epithelial cells.

IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

Allogeneic dendritic cell (DC) vaccination as an “off the shelf” treatment to prevent or delay relapse in elderly acute myeloid leukemia patients: results of Phase I/IIa safety and feasibility study

A novel immunotherapy platform is represented by the DCOne cell line, which originates from a human myeloid leukemia cell line, endogenously expresses a range of leukemia associated antigens, including PRAME and WT-1, and can be differentiated into mature dendritic cells (DC). This cell line is being developed to replace patient-derived DC vaccines. The first indication in which mature DC derived from DCOne have been tested clinically is Acute Myeloid Leukemia (AML). A Phase I/IIa study enrolled 12 AML patients (age range 58-71) who were either in CR1/CR2 (n=5) or had smouldering disease (n=7). Patients had received all available standard care, were at high risk of relapse and ineligible for available post-remission therapies. A typical 3+3 design was used, starting with 4 bi-weekly intradermal DCOne DC vaccinations of 10E6 (n=3), 25E6 (n=3) or 50E6 cells(n=6). Patients were monitored for clinical and immunological responses for 126 days. Primary endpoints were safety and feasibility, and secondary endpoints were clinical and immunological responses. Treatment was well tolerated in all patients, with only injection site reactions (< grade 2). During the study 3 patients died: 2 from infections and 1 from leukemia. Clear evidence for induction of multi-functional immune responses was obtained, including increased post vaccination delayed type hypersensitivity reactions, increases in CD4+ and CD8+ T cell proliferative responses and/or seroconversion to DCOne DC and/or AML blasts in 6 out of 9 patients. Also, 3 out of 7 patients that were evaluable by IFNγ ELIPOTs showed vaccination-induced specific T cell responses to WT-1 and/or PRAME. Patients who survived more than 6 months post-vaccination showed remarkable survival (4 patients are still alive 27, 21, 12 and 9 months after study entry). One of these patients, with smouldering AML at entry, achieved CR2 after vaccination; one patient who was in CR2 at entry relapsed 8 months after vaccination, entered CR3 following a single low dose of 5-AZA and survived until 23 months. The two most recently enrolled patients recruited are still in remission at 12 and 9 months. We conclude that vaccination with DCOne derived DC is feasible in AML, and generates both cellular and humoral immune responses. The hypothesis that DCOne-derive DC induce immune responses against patient leukemia cells correlate with clinical benefit, will now be investigated in a multi-center randomized Phase II trial in AML patients in first remission.