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Abstract 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.

Abstract Background Increased 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. Methods Cells 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. Results At 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. Conclusion These 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.

In elderly acute myeloid leukemia (AML) patients post-remission treatment options are associated with high comorbidity rates and poor survival. Dendritic cell (DC)-based immunotherapy is a promising alternative treatment strategy. A novel allogeneic DC vaccine, DCP-001, was developed from an AML-derived cell line that uniquely combines the positive features of allogeneic DC vaccines and expression of multi-leukemia-associated antigens. Here, we present data from a phase I study conducted with DCP-001 in 12 advanced-stage elderly AML patients. Patients enrolled were in complete remission (CR1/CR2) (n = 5) or had smoldering disease (n = 7). All patients were at high risk of relapse and ineligible for post-remission intensification therapies. A standard 3 + 3 dose escalation design with extension to six patients in the highest dose was performed. Patients received four biweekly intradermal DCP-001 injections at different dose levels (10, 25, and 50 million cells DCP-001) and were monitored for clinical and immunological responses. Primary objectives of the study (feasibility and safety) were achieved with 10/12 patients completing the vaccination program. Treatment was well tolerated. A clear-cut distinction between patients with and without detectable circulating leukemic blasts during the vaccination period was noted. Patients with no circulating blasts showed an unusually prolonged survival [median overall survival 36 months (range 7-63) from the start of vaccination] whereas patients with circulating blasts, died within 6 months. Long-term survival was correlated with maintained T cell levels and induction of multi-functional immune responses. It is concluded that DCP-001 in elderly AML patients is safe, feasible and generates both cellular and humoral immune responses.

Multiple myeloma (MM) is characterized by progressive immune dysregulation, loss of myeloma-specific immunity, and an immunosuppressive milieu that fosters disease growth and immune escape. Accordingly, cancer vaccines that reverse tumor-associated immune suppression represent a promising therapeutic avenue of investigation. We examined the potential of an allogeneic cellular vaccine to generate immune responses against MM tumor cells. The DCOne vaccine is comprised of a human myeloid leukemia cell line differentiated into a fully functional dendritic cell, expressing a range of tumor-associated antigens that are also known targets in MM. We found that the myeloma-specific antigens expressed by the DCOne vaccine can traffic via extracellular vesicles to surrounding antigen-presenting cells, thus stimulating autologous T-cell responses. Indeed, coculture of peripheral blood mononuclear cells from patients with MM with the DCOne vaccine resulted in the expansion of activated CD8 T cells expressing interferon-γ and perforin, with no significant change in the percentage of CD4 T cells producing interleukin-10. Further, coculture of patient's tumor cells with peripheral blood mononuclear cells and DCOne induced cytotoxic T-lymphocyte-mediated killing of autologous MM cells. These findings demonstrate that the allogeneic DCOne vaccine can induce T-cell activation and myeloma-specific immunity via cross presentation of antigens by native antigen-presenting cells.

Aims: Vaccination with acute myeloid leukemia (AML)-derived dendritic cells (DCs) is a promising immunotherapeutic approach to prevent relapse of AML. However, in clinical practice AML-derived DC culture is unfeasible in 40% of cases. Here, we demonstrate that AML cells can be expanded in vitro prior to differentiation with cocktails of cytokines with known myeloid growth-promoting effects., Results: Nine out of 13 initially CD14(-) samples gain de novo CD14 (>10%) expression (69% increment; p = 0.01) after in vitro expansion. These expanded CD14(+) leukemic cells displayed a high probability (six out of six initially CD14(-) samples tested) to differentiate into DCs upon culture with GM-CSF, TNF-α and IL-4., Conclusion: Induction of CD14 on initially CD14(-) AML cells potentially increases the number of patients eligible for DC-based immunotherapy.

Background: Smoking in asthma occurs frequently and is associated with increased symptom severity, an impaired response to corticosteroids, and accelerated lung function decline. Airway pathology in smoking asthmatics is characterized by neutrophilia and epithelial changes such as goblet cell hyperplasia and increased proliferation. Bronchial CD8(+) T cells are implicated in lung function decline in asthma and COPD. We hypothesized that smoking modifies airway inflammation in asthma by increasing the number of CD8(+) T cells at an early stage., Objectives & Methods: To study effects of smoking on airway pathology in bronchial biopsies from atopic patients with controlled intermittent or mild persistent asthma (12 smokers, 9.7 py and 11 never-smokers, 0.0 py; 20-50 yrs; FEV1 > 70% predicted; PC20MCh < 8 mg/mL, no ICS) using immunohistochemistry., Results: Smoking asthmatics showed higher numbers of bronchial CD8(+) T cells (55.8 vs 23.9 cells/0.1 mm(2); p = 0.001) and CD68(+) macrophages (7.5 vs 4.6 cells/0.1 mm(2), p = 0.012), and a lower CD4(+)/CD8(+) cell ratio (0.16 vs 0.40; p = 0.007) compared with non-smoking asthmatics, but no difference in neutrophils. Furthermore, the % intact epithelium was higher in smoking asthmatics (49.3 vs 23.3, p = 0.001)., Conclusion: Smoking asthmatics with a limited smoking history show a distinct pattern of airway pathology characterized by a bronchial infiltrate of CD8(+) T cells and CD68(+) macrophages, and epithelial remodelling resembling COPD-like features. This raises the hypothesis that early presence of CD8(+) T cells contributes to disease progression in smoking asthmatics., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

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.

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.

The composition of the airway epithelium is dynamic and epithelial differentiation is regulated by endogenous mediators as well as inhaled substances. In atopic asthma the differentiation of the epithelium is altered. Various studies have addressed the ability of cultured airway epithelial cells to release the eosinophil-attractant chemokines eotaxin, eotaxin-2 and eotaxin-3 using epithelial cell lines or poorly differentiated primary cells. Since little is known about the role of the epithelial differentiation state in the response of epithelial cells to stimuli that increase production of mediators such as the eotaxins, we analyzed the effect of differentiation state on the production of the eotaxins. In particular, we investigated the effects of the Th2 cytokines IL-4 and IL-13 on eotaxin-2 and -3 production by primary human bronchial epithelial cells and examined whether their production is affected by epithelial cell differentiation using both submerged and air-liquid interface (ALI) cultures. The results show that both IL-4 and IL-13 increase eotaxin-2 and -3 mRNA expression and protein release in submerged- and ALI-cultures. Moreover, epithelial differentiation in ALI-cultures appeared an important determinant in the regulation of eotaxin-2 and -3. Mucociliary differentiation of the epithelial cells was induced by culture in the presence of a high concentration of retinoic acid (RA), whereas low concentrations of RA resulted in a flattened squamous epithelial phenotype. Mucociliary differentiated ALI-cultures expressed and released more eotaxin-3 upon stimulation with IL-4/IL-13, whereas eotaxin-2 production was predominantly found in squamous differentiated ALI-cultures. TNFalpha reduced IL-4-induced eotaxin-2 release in submerged cultures but not in ALI-cultures; no effects on eotaxin-3 synthesis were observed. The results indicate that epithelial differentiation is an important determinant in Th2 cytokine-induced eotaxin-2 and -3 release by airway epithelial cells. These findings may provide new insights into the role of airway epithelial differentiation and Th2 cytokines in the pathogenesis of inflammatory lung disorders such as asthma.

Background: Increased 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., Methods: Cells 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., Results: At 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., Conclusion: These 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.

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.

Human airways are frequently exposed to potentially harmful agents that cause tissue injury. Upon such injury, a repair process is initiated that comprises cell migration, proliferation, and differentiation. We have previously shown that human neutrophil defensins (human neutrophil peptides 1-3 [HNP1-3]) induce airway epithelial cell proliferation. Because of the role of cell proliferation in epithelial wound repair, we investigated the effect of HNP1-3 on airway epithelial wound closure and mucin gene expression in vitro. Using NCI-H292 airway epithelial cell cultures, we demonstrated that HNP1-3 cause a dose- and time-dependent increase of wound closure as well as increased cell migration. Furthermore, HNP1-3 caused a biphasic activation of the mitogen-activated protein kinase extracellular-regulated kinase 1 and 2 (ERK1/2). Both the effects of HNP1-3 on wound closure and ERK1/2 activation were blocked by specific inhibitors of the mitogen-activated protein kinase kinase MEK, whereas inhibitors of epidermal growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase, and Src did block defensin-enhanced wound closure but not ERK1/2 activation. Finally, HNP1-3 increased mRNA encoding the mucins MUC5B and MUC5AC, suggesting a role for defensins in mucous cell differentiation. These results indicate that neutrophil defensins increase epithelial wound repair in vitro, which involves migration and proliferation, and mucin production. Neutrophil defensin-enhanced wound repair appears to require epidermal growth factor receptor activation and downstream signaling pathways.

Leukocyte adhesion is mediated totally and transendothelial migration partially by heterotypic interactions between the beta1- and beta2-integrins on the leukocytes and their ligands, Ig-like cell adhesion molecules (Ig-CAM), VCAM-1, and ICAM-1, on the endothelium. Both integrins and Ig-CAMs are known to have signaling capacities. In this study we analyzed the role of VCAM-1-mediated signaling in the control of endothelial cell-cell adhesion and leukocyte transendothelial migration. Antibody-mediated cross-linking of VCAM-1 on IL-1beta-activated primary human umbilical vein endothelial cells (pHUVEC) induced actin stress fiber formation, contractility, and intercellular gaps. The effects induced by VCAM-1 cross-linking were inhibited by C3 toxin, indicating that the small GTPase p21Rho is involved. In addition, the effects of VCAM-1 were accompanied by activation of Rac, which we recently showed induce intercellular gaps in pHUVEC in a Rho-dependent fashion. With the use of a cell-permeable peptide inhibitor, it was shown that Rac signaling is required for VCAM-1-mediated loss of cell-cell adhesion. Furthermore, VCAM-1-mediated signaling toward cell-cell junctions was accompanied by, and dependent on, Rac-mediated production of reactive oxygen species and activation of p38 MAPK. In addition, it was found that inhibition of Rac-mediated signaling blocks transendothelial migration of monocytic U937 cells. Together, these data indicate that VCAM-1-induced, Rac-dependent signaling plays a key role in the modulation of vascular-endothelial cadherin-mediated endothelial cell-cell adhesion and leukocyte extravasation.

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 microg/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 alpha1-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.

The integrity of the endothelium is dependent on cell-cell adhesion, which is mediated by vascular-endothelial (VE)-cadherin. Proper VE-cadherin-mediated homotypic adhesion is, in turn, dependent on the connection between VE-cadherin and the cortical actin cytoskeleton. Rho-like small GTPases are key molecular switches that control cytoskeletal dynamics and cadherin function in epithelial as well as endothelial cells. We show here that a cell-penetrating, constitutively active form of Rac (Tat-RacV12) induces a rapid loss of VE-cadherin-mediated cell-cell adhesion in endothelial cells from primary human umbilical veins (pHUVEC). This effect is accompanied by the formation of actin stress fibers and is dependent on Rho activity. However, transduction of pHUVEC with Tat-RhoV14, which induces pronounced stress fiber and focal adhesion formation, did not result in a redistribution of VE-cadherin or an overall loss of cell-cell adhesion. In line with this observation, endothelial permeability was more efficiently increased by Tat-RacV12 than by Tat-RhoV14. The loss of cell-cell adhesion, which is induced by Tat-RacV12, occurred in parallel to and was dependent upon the intracellular production of reactive oxygen species (ROS). Moreover, Tat-RacV12 induced an increase in tyrosine phosphorylation of a component the VE-cadherin-catenin complex, which was identified as alpha-catenin. The functional relevance of this signaling pathway was further underscored by the observation that endothelial cell migration, which requires a transient reduction of cell-cell adhesion, was blocked when signaling through ROS was inhibited. In conclusion, Rac-mediated production of ROS represents a previously unrecognized means of regulating VE-cadherin function and may play an important role in the (patho)physiology associated with inflammation and endothelial damage as well as with endothelial cell migration and angiogenesis.

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-1alpha. Stromal cell-derived factor-1alpha-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.

In the course of granulocyte-dominated lung inflammation, granulocytes migrate across the endothelium and epithelium of the lung and cause severe tissue damage. To study this process in more detail, we developed a bilayer transmigration model composed of primary human endothelial and lung epithelial cells, simultaneously cultured on opposite sides of Transwell filters. Electron microscopical analysis showed that the morphology of the cells and the expression of junctional proteins remained unaltered and that matrix components were deposited onto the filter. Intriguingly, neutrophil migration was more efficient across the bilayers than across single epithelial monolayers and did not differ from migration across single endothelial monolayers. Coculture experiments showed that endothelial cells stimulated epithelial cells to release IL-6 and that epithelial cells enhanced release of IL-8 from endothelial cells. Together these data reveal bidirectional signaling and enhanced neutrophil migration in a transmigration model of primary human epithelial and endothelial cells.

Background: To protect against the extracellular activity of serine proteinases, the lung is equipped with serine proteinase inhibitors including secretory leukocyte proteinase inhibitor (SLPI) and elafin. Both SLPI and elafin are locally produced by airway epithelial cells, but the mechanisms that regulate the expression of these proteinase inhibitors are relatively unknown. Previous studies using airway epithelial cell lines indicated that neutrophil elastase (NE) increases SLPI mRNA transcripts while decreasing SLPI protein release. Similar results were observed for elafin. The aim of the present study was to investigate the effect of NE on SLPI and elafin synthesis in cultures of human primary bronchial epithelial cells (PBEC)., Methods: Subcultures of human PBEC were incubated with NE, followed by preparation of cell-free supernatants and cellular lysates and determination of SLPI and elafin protein levels by enzyme-linked immunoadsorbent assay. The effect of NE on SLPI mRNA transcripts was determined by Northern blot analysis., Results: The results showed that NE increased SLPI mRNA expression while decreasing SLPI protein release. This NE-induced decrease was associated with an increase in cell-associated SLPI, providing an explanation for the apparent paradox of increased SLPI mRNA transcripts and decreased SLPI protein levels present in supernatants. In addition, NE had a stimulatory effect on the release of elafin by airway epithelial cells, whereas no increase in cell-associated elafin was observed., Conclusions: The results from the present study indicate that NE may play a role in the regulation of the antiproteinase screen in the lung and the formation of a protective surface at the epithelial site.

Adherence of Haemophilus influenzae to bronchial epithelial cells is enhanced by neutrophil defensins, which are released from activated neutrophils during inflammation [Gorter et al. (1998) J. Infect. Dis. 178, 1067-1078]. In this study, we showed that the adherence of H. influenzae to various epithelial, fibroblast-like and endothelial cell types was significantly enhanced by defensins (20 microg ml(-1)). Defensins stimulated also the adherence of Moraxella catarrhalis, Neisseria meningitidis and nonencapsulated Streptococcus pneumoniae to the NCI-H292 cell line. In contrast, defensins did not affect the adherence of Pseudomonas aeruginosa, encapsulated S. pneumoniae, Escherichia coli and Staphylococcus epidermidis. These results suggest that the defensin-enhanced adherence might support the adherence and possibly persistence of the selected bacterial species using the respiratory tract as port of entry.

Secretory leukocyte proteinase inhibitor (SLPI) is a serine proteinase inhibitor that is produced locally in the lung by cells of the submucosal bronchial glands and by nonciliated epithelial cells. Its main function appears to be the inhibition of neutrophil elastase (NE). Recently, NE was found to enhance SLPI mRNA levels while decreasing SLPI protein release in airway epithelial cells. Furthermore, glucocorticoids were shown to increase both constitutive and NE-induced SLPI mRNA levels. In addition to NE, stimulated neutrophils also release alpha-defensins. Defensins are small, antimicrobial polypeptides that are found in high concentrations in purulent secretions of patients with chronic airway inflammation. Like NE, defensins induce interleukin-8 production in airway epithelial cells. This induction is sensitive to inhibition by the glucocorticoid dexamethasone and is prevented in the presence of alpha(1)-proteinase inhibitor. The aim of the present study was to investigate the effect of defensins on the production of SLPI and the related NE inhibitor elafin/SKALP in primary bronchial epithelial cells (PBECs). Defensins significantly increase SLPI protein release by PBECs in a time- and dose-dependent fashion without affecting SLPI mRNA synthesis. In the presence of alpha(1)-proteinase inhibitor, the defensin-induced SLPI protein release is further enhanced, but no effect was observed on SLPI mRNA levels. Dexamethasone did not affect SLPI protein release from control or defensin-treated PBECs. In addition, we observed a constitutive release of elafin/SKALP by PBECs, but this was not affected by defensins. The present results suggest a role for defensins in the dynamic regulation of the antiproteinase screen in the lung at sites of inflammation.

Defensins are small, cationic antimicrobial peptides that are present in the azurophilic granules of neutrophils. Earlier studies have shown that defensins may influence complement activation by specific interaction with activated C1, C1q, and C1-inhibitor. In the present study, we show that the defensin human neutrophil peptide-1 (HNP-1) is able to inhibit activation of the classical complement pathway by inhibition of C1q hemolytic activity. The binding site for HNP-1 on C1q is most likely located on the collagen-like stalks, as a clear, dose-dependent binding of HNP-1 to either intact C1q or to the collagen-like stalks of C1q was demonstrated using enzyme-linked immunosorbent assay (ELISA). Besides binding of HNP-1 to C1q, also a limited binding to C1 and to a mixture of C1r and C1s was observed, whereas no binding to C1-inhibitor was found. Because binding of HNP-1 to C1-inhibitor has been suggested in earlier studies, we also assessed the binding of HNP-1 to mixtures of C1-inhibitor with either C1r/ C1s or C1. No binding was found. Using a competition ELISA, it was found that HNP-1, but not protamine, inhibited binding of biotin-labeled HNP-1 to C1q in a dose-dependent fashion. In the fluid phase, preincubation of HNP-1 with C1q resulted in complex formation of HNP-1 and C1q and generation of stable complexes. In conclusion, HNP-1 is able to bind to C1q in the fluid phase and inhibits the classical complement pathway. This mechanism may be involved in the control of an inflammatory response in vivo.

Patients with chronic obstructive pulmonary disease (COPD) frequently have recurrent lower respiratory tract infections with nonencapsulated Haemophilus influenzae. The infected mucosa of these patients is infiltrated with neutrophils, which upon activation may release antimicrobial peptides, including defensins. It was shown that defensins isolated from neutrophils or from sputum samples of COPD patients did not kill H. influenzae from these patients, but they did stimulate its adherence to human bronchial epithelial cells in a time- and dose-dependent manner. Maximal stimulation was observed after 3 h in the presence of > or = 10 micrograms/mL defensins, resulting in 65 +/- 36 cfu/cell (61-fold increase). The enhanced adherence was not solely due to charge effects and was specifically blocked by alpha 1-proteinase inhibitor. Because adherence is the first step in the onset of respiratory tract infections, our findings indicate that neutrophil defensins likely contribute to the pathogenesis of H. influenzae infection in the lower respiratory tract.

Neutrophil accumulation in the lung may contribute to tissue injury as observed in inflammatory diseases. Both oxidative and non-oxidative mechanisms are involved in neutrophil-mediated tissue injury. Non-oxidative mechanisms include the release of neutrophil granule proteins such as the serine proteinases elastase and cathepsin G, and the non-enzymatic defensins. Because stimulated neutrophils are thought to release their products simultaneously, we investigated possible interactions between purified defensins and serine proteinases with respect to induction of cellular injury and their ability to induce interleukin-8 (IL-8) synthesis in cells of the lung epithelial cell line A549. Whereas defensins induced cell lysis, elastase and cathepsin G induced detachment of A549 cells. Co-incubation of elastase and cathepsin G revealed an additive effect on detachment, whereas defensins inhibited serine proteinase-induced detachment. Vice versa, both serine proteinases reduced defensin-induced cell lysis. Furthermore, elastase and cathepsin G prevented defensin-induced IL-8 synthesis. In contrast, no inhibitory interaction between cathepsin G and defensins was observed with respect to their antibacterial activity. The results from this study indicate that, at sites of inflammation, neutrophil-mediated injury might be regulated by interactions between released defensins and serine proteinases.