Midkine Is Part of the Antibacterial Activity Released at the Surface of Differentiated Bronchial Epithelial Cells

Abstract

To resist infections, robust defense mechanisms of the airways are essential. Retinoic acid promotes differentiation and maintains the phenotypic characteristics of bronchial epithelium. In addition, it induces the expression of the antibacterial growth factor midkine (MK). In the present study, we explored the expression and antibacterial activity of MK in an airway context. MK was detected in bronchial epithelial cells of large airways and type 2 pneumocytes of normal lungs by immunohistochemistry. Immunoelectron microscopy revealed a surface-associated distribution, both on the ciliated apical and basolateral sides, and MK was detected in sputum obtained from healthy individuals by ELISA. In vitro, MK killed the common respiratory pathogen Streptococcus pneumoniae at below micromolar concentrations, an activity retained in the presence of sodium chloride at physiological concentrations. The MK molecule consists of two domains with three anti-parallel β-sheets and a COOH-terminal tail. Although both the NH2- and COOH-terminal domains alone showed antibacterial activity, the COOH-terminal domain including the tail region possessed higher bactericidal activity, i.e. in the order of the holoprotein. Retinoic acid-induced differentiation of primary bronchial epithelial cells, using an air-liquid interface system, revealed bactericidal activity in the apical airway surface liquid, an activity that was reduced after immunoprecipitation of MK. This study shows that airway epithelial cells of large airways and alveoli have a constitutive production of MK that is part of the bactericidal activity present in the air surface liquid, at least in vitro, and may thus be an important part of this arm of airway host defense.

Staphylococcus aureus-induced clotting of plasma is an immune evasion mechanism for persistence within the fibrin network

“…Specimens were fixed in 2.5 % glutaraldehyde in 0.15 M cacodylate buffer (pH 7.2), prepared for electron microscopic analysis and analysed with a Zeiss DSM 960A electron microscope as described previously (Nordin et al, 2013).…”

Expression of MIG/CXCL9 in Cystic Fibrosis and Modulation of Its Activities by Elastase of <b><i>Pseudomonas aeruginosa</i></b>

“…Tissue samples from CF lung explants obtained during transplantation were fixed in PBS containing 4% paraformaldehyde with 0.1% glutaraldehyde and prepared for immunostaining and transmission electron microscopy as described [21]. In short, ultrathin sections were incubated with primary antibodies against MIG/CXCL9, followed by detection with a secondary goat anti rabbit antibody (conjugated with 10 nm colloidal gold; Electron Microscopy Sciences, Fort Washington, Pa., USA; diluted 1:10-1:20).…”

“…The bacteria were further diluted to a concentration of (2 × 10 3 cfu/ml) and 40 µl of the bacteria suspension was incubated with protein of interest at different concentrations or in Tris-glucose buffer alone (control) at 37°C, 5% CO 2 for 1 h. Thereafter, aliquots of the incubation were plated on agar in a series of dilutions to promote growth. If no colonies are formed, the bacteria have been killed [21]. …”

Structure and function of midkine as the basis of its pharmacological effects

“…Inflammation, which is enhanced by MK, is part of the immune system. MK also has anti‐microbial activities and is regarded as a component of the innate immune system (Svensson et al ., ; Nordin et al ., ). Furthermore, MK has been found to enhance the survival of B cells (Cohen et al ., ).…”

“…Thus, MK is strongly expressed during embryogenesis, especially in the mid‐gestation period (Kadomatsu et al ., ; Mitsiadis et al ., 1995b). In adults, significant MK expression is observed only in restricted sites such as the kidney (Kadomatsu et al ., ), gut (Aridome et al ., ), epidermis (Inazumi et al ., ), bronchial epithelium (Nordin et al ., ), lymphocytes (Cohen et al ., ) and macrophages (Inoh et al ., ), while MK expression is induced in many tissues after injury (Yoshida et al ., ; Obama et al ., ; Horiba et al ., ; Sato et al ., ).…”