Lymphocyte-derived microparticles induce bronchial epithelial cells’ pro-inflammatory cytokine production and apoptosis

Abstract

Objective

The aim of this study was to determine if human CEM (human lymphoblastoma) T cell-derived microparticles (LMPs) could directly induce human bronchial epithelial cells (BECs) apoptosis and cytokine production. We also tested if LMPs phagocytosis by BECs played a role in mediating these effects.

Methods

We generated LMPs from CEM (human lymphoblastoma) T cells to investigate their effects on a human BEC cell line (16HBE) in vitro.

Results

BECs (16HBE cells) incubation with LMPs resulted in significant production of inflammation-associated cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8, in a dose- and time-dependent manner. LMPs also induced increased activities of caspase-3, caspase-8, and caspase-9 in BECS, which resulted in increased BECs apoptosis as assessed by flow cytometry (Annexin V and propidium iodide staining) and transmission electronic microscopy (TEM). Interestingly, LMPs effects on BECS were inhibited by the phagocytosis inhibitors cytochalasin D and chloroquine.

Conclusions

These results suggest that phagocytosis plays an important role in mediating the effects of LMPs on BECs. Thus, increased LMP concentrations may contribute to increased respiratory inflammatory responses and innate immune response maintenance in airway epithelium after LMPs engulfment by endothelial cells.

Introduction

Microparticles (MPs) are submicron-sized membranous vesicles (diameters between 0.1 and 1 μm) that contain cell surface proteins and cytoplasmic components and are shed by cells under stress, such as activated and apoptotic cells (Burnier et al., 2009, Hugel et al., 2005). The levels of circulating microparticles, such as leukocyte-derived MPs (LMPs), are significantly increased in patients with pulmonary hypertension (Amabile et al., 2008). Increased levels of pro-coagulant alveolar microparticles are found in alveolar edema samples or bronchoalveolar lavage fluids (BALF) from patients with acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and COPD (Bastarache et al., 2009, Mutschler et al., 2002). Thus, MPs appear to be involved in the pathogenesis of numerous respiratory diseases, particularly those characterized by chronic inflammation.

Damage to bronchial epithelial cells (BECs) that causes ciliary dysfunction is a key property of the pathogenesis of airway inflammation and is thought to be an important contributor to the development of chronic inflammatory pathological changes and airway hyper-responsiveness. A variety of cells types, such as lymphocytes, other leukocytes, platelets, and endothelial cells, can access the bronchi and alveolar spaces. Thus, these cells could be the sources of intra-bronchial and intra-alveolar MPs that accumulate on the surfaces of BECs and function as activating molecules to induce functional changes (Buesing et al., 2011).

There is considerable evidence that substantially increased numbers of circulating LMPs are associated with several pathological conditions of immune and inflammatory disorders, such as rheumatoid arthritis, malaria, septicemia, HIV infection, and preeclampsia (Aupeix et al., 1997, Berckmans et al., 2005, Couper et al., 2010, Meziani et al., 2006, Mostefai et al., 2008). A proteomics analysis of LMPs from malignant lymphocytes identified 413 proteins, including 117 membrane proteins, many of which were associated with various pathologies (Miguet et al., 2006). Thus, LMPs are biomarkers and vectors that carry biological information between cells (Morel et al., 2011).

LMPs derived from human Jurkat T-lymphoma cells are rapidly engulfed by RAW 264.7 mouse macrophages and consequently induce macrophage apoptosis (Distler et al., 2005a). We also observed that LMPs derived from human CEM (human lymphoblastoma) T cells strongly reduced human umbilical vein endothelial proliferation without inducing endothelial cell death (Yang et al., 2008). LMPs also caused a dose-dependent inhibition of Lewis lung carcinoma cells growth, although these effects were associated with increased cellular apoptosis (Yang et al., 2010). Interestingly, resting and cytokine-stimulated human BECs can selectively recognize and engulf apoptotic eosinophils (Sexton et al., 2004, Walsh et al., 1999). However, no studies have investigated the effects of LMPs on BECs with regard to these cells’ phagocytic activities.

Previous studies have also shown that BECS played key roles in immune regulation during the development of bronchial inflammation that was linked to either contact with inflammatory cells or activation by inflammatory mediators in response to pathogen-associated molecular pattern molecules (Bals and Hiemstra, 2004). Monocyte/macrophage-derived microparticles induced increased expression of IL-8 in human bronchial epithelial and alveolar cells (Cerri et al., 2006, Neri et al., 2011). Distler et al. (2005b) confirmed that LMPs induced the expressions of various inflammatory mediators, including IL-6, IL-8, MCP-1, MCP-2, and MMPs, in synovial fibroblasts in rheumatoid arthritis.

Thus, in this study we investigated if human CEM (human lymphoblastoma) T cell-derived microparticles (LMPs) could directly induce apoptosis and cytokine production by human BECs. We also examined if LMPs phagocytosis by BECs played a role in mediating these LMP effects. Our results show that LMPs phagocytosis by BECs is directly related to BECs apoptosis and their production of pro-inflammatory cytokines.