Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall

Abstract

The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity?

The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter).

MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC.

We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

Introduction

The human respiratory tract consists of a vast surface area, which provides a very thin air–blood tissue barrier of a few hundred nanometres in the gas exchange region. Because this huge area is continuously exposed to a broad array of environmental antigen ranging from harmful to innocuous the respiratory system is equipped with a series of structural and functional barriers for protection (Nicod, 2005, Rothen-Rutishauser et al., 2005). One of the most important barrier components is the lung epithelial cellular layer sealed with tight junctions (TJ) at the apical side of the cells (Schneeberger and Lynch, 1984, Godfrey, 1997). This tight structure consisting of cells and their cell–cell adhesion proteins serves as a structural and functional barrier between the air-exposed lumen of the lung with airway- or alveolar macrophages located at its surface and the tissue below, containing amongst other components blood capillaries, lymph vessels and important cells of the immune system like e.g. dendritic cells (DC) (Holt et al., 2008). The epithelial tissue barrier principally prevents deposited particulate antigen from the translocation into the tissue below the epithelium.

A number of studies demonstrate the sampling of particulate and soluble antigen by macrophages and by DC at the apical side of the epithelium (Jakubzick et al., 1993, Vermaelen et al., 2001). Furthermore, in vivo studies report DC to transport the sampled antigen across the epithelial barrier to the draining lymph nodes (Vermaelen et al., 2001, Havenith et al., 1993, Jahnsen et al., 2006, Wikstrom and Stumbles, 2007). These findings are consistent with our in vitro results using a triple cell co-culture model of the airway wall which consists of the bronchial epithelial cell (EC) line 16HBE14o- or the human alveolar-like EC line A549 together with human blood monocytes derived macrophages (MDM) located at the apical side of the epithelium and human blood monocytes derived dendritic cells (MDDC) located at its base (Rothen-Rutishauser et al., 2005, Rothen-Rutishauser et al., 2008, Blank et al., 2007). We found MDDC extending cellular processes between the EC from the base to the apical side to take up 1 μm polystyrene particles and transport them to the base (Blank et al., 2007). Furthermore, MDDC were found to move completely from the base to the apical side of the epithelial monolayer to sample particles (Blank et al., 2007). Interestingly, particle bearing MDM were also found to extend their cytoplasmic processes from the apical side to the base of the epithelium (Blank et al., 2007). However, the epithelial integrity (i.e. the integrity of the TJ), which was verified by measuring trans-epithelial electrical resistance (TEER) or the paracellular transport of mannitol during particle exposure was not affected by trans-epithelial processes extended by MDDC and MDM (Rothen-Rutishauser et al., 2005, Blank et al., 2007).

How is it possible for MDDC and MDM to build such a network across and even transmigrate through the epithelium without disrupting the epithelial TJ integrity? From studies with an in vitro model of the human gut epithelium (Rescigno et al., 2001a, Rescigno et al., 2001b) and from the characterization of a population of DC residing in the mucosa of mouse airways (Sung et al., 2006) it is known, that dendritic cells express TJ proteins which may build TJ like complexes with EC in order to preserve the epithelial integrity.

In the present study we demonstrate in live cell experiments that MDM and MDDC actively collaborate since MDDC take up particles from MDM. Furthermore, we show that not only MDDC but also MDM express the TJ proteins occludin, claudin-1, zonula occludens-3(ZO-3), junctional adhesion molecule-A (JAM-A) and the AJ protein E-cadherin. These findings support our postulate of MDM and MDDC interacting with each other by a trans-epithelial network of slender cytoplasmic processes. The expression of TJ and AJ proteins in those cells might help to preserve the epithelial integrity during trans-epithelial interactions. We interpret this trans-epithelial cellular network as a pathway for particle translocation through the epithelial barrier by cells of the immune defence system.