Hyper-responsiveness of IPF/UIP fibroblasts: Interplay between TGFβ1, IL-13 and CCL2
Introduction
Idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is a chronic debilitating interstitial lung disease that is characterized by the presence of honeycombing within the lung (Dacic & Yousem, 2003). The incidence of IPF/UIP has been estimated to be 27–29 cases/100,000, however, it may even be as high as 150 cases/100,000 in individuals over the age of 75 (Coultas, Zumwalt, Black, & Sobonya, 1994). Moreover, there is no current effective treatment for this chronic lung disease and the 2 to 3-year-median survival is 50%. The honeycombing may arise due to increased collagen deposition, thus resulting in reduced lung elasticity and causing retraction and ultimate collapse of normal alveolar structure. Furthermore, the extent of honeycombing and fibrosis is very heterogeneous within the lung, with dense areas of excess collagen often adjoining normal lung parenchyma or interstitial tissue rich in monocytic infiltrates (Flaherty et al., 2001; Travis, Matsui, Moss, & Ferrans, 2000; Katzenstein & Myers, 1998).
There is a large pool of fibroblasts within the lung, which control the homeostatic balance of collagen generation and breakdown, thereby serving to remodel the lung following injury. Fibroblasts generate collagen in response to various growth factors such as TGFβ1 (Fine & Goldstein, 1987). Furthermore, TGFβ1 induces fibroblast differentiation into myofibroblasts, and this is a common feature in the IPF/UIP lung tissue (Phan, 2002). These differentiated myofibroblast cells are capable of generating collagen. Further, the presence of alpha-smooth muscle actin (αSMA) in these cells confers a contractile phenotype (Kuhn & McDonald, 1991; Ohta, Mortenson, Clark, Hirose, & King, 1995). In normal wound healing, fibroblasts generate collagen and growth factors to direct the reparative processes. Once tissue architecture is restored, fibroblast collagen generation decreases and the cells go through apoptosis, thus preventing excess scar formation (Desmouliere, Redard, Darby, & Gabbiani, 1995). In contrast, IPF/UIP fibroblasts are more resistant to apoptosis compared to fibroblasts isolated from non-fibrotic tissue (Moodley et al., 2003, Tanaka et al., 2002). In addition, there is also a pronounced extent of fibroblast proliferation found in the lungs of IPF/UIP patients (Travis et al., 2000; Katzenstein & Myers, 1998). Therefore, IPF/UIP fibroblasts persist at sites of fibrosis, continuously adding to an environment of aberrant excessive collagen deposition.
As well as being more resistant to apoptosis, previous work has shown other phenotypic differences in fibroblasts isolated from sites of fibrosis. For example, fibroblasts isolated from the lungs of IPF/UIP patients have increased expression of IL-13 receptor subunits (Jakubzick, Choi, Carpenter, et al., 2004). IL-13 is a Th2-type cytokine that is found at elevated levels in the lungs of IPF/UIP patients (Hancock, Armstrong, Gama, & Millar, 1998) and in patients with other fibrotic diseases including systemic sclerosis (Hasegawa, Fujimoto, Kikuchi, & Takehara, 1997; Hasegawa, Sato, Nagaoka, Fujimoto, & Takehara, 2003). IL-13 is pro-fibrotic in vitro and in in vivo models of fibrosis and directly promotes collagen generation and proliferation of fibroblasts (Saito, Okazaki, Sugawara, Yamamoto, & Takizawa, 2003; Ingram, Rice, Geisenhoffer, Madtes, & Bonner, 2004; Oriente et al., 2000). Signaling of IL-13 through IL13Rα2 has been recently demonstrated to be pro-fibrotic through induction of TGFβ1 (Fichtner-Feigl, Strober, Kawakami, Puri, & Kitani, 2006). Another pathway where phenotypic differences in receptor expression have been reported includes the CCL2: CCR2 pathway. This chemoattractant cytokine CCL2 (CC-chemokine ligand 2, monocyte chemoattractant protein 1 or MCP1) binds to CCR2. Increased levels of CCL2 have been described in patients with IPF/UIP (Iyonaga et al., 1994, Suga et al., 1999). Fibroblasts isolated from sites of scleroderma have increased CCR2 expression (Carulli et al., 2005). Mice have two functional orthologs of human MCP1/CCL2, JE/CCL2 and MCP5/CCL12, which have been shown to signal with similar high affinity through murine CCR2. Interestingly, inhibition of CCR2 signaling in vivo by utilization of either CCR2 receptor knockout mice or JE/MCP5 ligand neutralization results in attenuated collagen deposition in multiple animal models of fibrosis (Moore et al., 2001, Moore et al., 2005, Moore et al., 2006).
An interplay between IL-13 and CCR2 has been previously described using an animal model of pulmonary specific over-expression of IL-13. Breeding the transgenic IL-13 mice onto a CCR2 deficient background inhibited several of the pathologies including subepithelial fibrosis (Zhu et al., 2002). Therefore, the rationale of this study was to determine if the pathways described in the murine system apply to clinical setting of IPF/UIP. In this study we evaluated the role of CCL2 in both human IPF/UIP and non-fibrotic fibroblasts to determine whether this chemokine differentially activated these cells. Fibroblasts isolated from lung tissue of IPF/UIP patients displayed higher levels of fibrotic hallmarks at baseline and these phenotypes were augmented following stimulation. Furthermore, cells isolated from non-fibrotic lung tissue were relatively non-responsive to CCL2, however, CCL2 behaved as a pro-fibrotic mediator on IPF/UIP fibroblasts. In addition, this study highlighted a significant interplay between TGFβ1, IL-13 and CCL2 in human diseased cells, in that all of these mediators induced an upregulation of TGFβ and IL-13 receptors. We then evaluated the effect of CCL2 neutralization in vivo and in vitro. JE/MCP5 neutralization during bleomycin-induced pulmonary fibrosis inhibited collagen deposition and a number of genes including JE and CTGF. In contrast, inhibition of CCL2 in vitro showed only a modest attenuation of TGFβ1 induced upregulation of specific pro-fibrotic genes in IPF/UIP fibroblasts.
Section snippets
Reagents
Cell culture grade human recombinant TGFβ1, IL-13, PDGF-AB and CCL2 were obtained from R&D Systems Inc. (R&D Systems Inc., Minneapolis, MN). All TaqMan reagents were purchased from Applied Biosystems (Foster City, CA).
Antibodies
To neutralize JE and MCP5, rat anti-murine JE and human anti-murine MCP5 monoclonal antibodies were administered concomitantly on days 2, 6 and 11 (0.5 mg/mAb/dose/i.p.; reagents generated in-house at Centocor). Rat IgG was administered to control mice at the same concentration (1
Enhanced fibrotic phenotype at baseline in IPF/UIP fibroblasts
To determine if phenotypic differences occur between fibroblasts isolated from the lungs of IPF/UIP patients fibroblasts isolated from non-fibrotic lung tissue, unstimulated cell lines were analyzed for fibrosis-associated gene expression. Fibroblasts isolated from IPF/UIP lung biopsies had an increase in fibrogenic phenotype at baseline in comparison to non-fibrotic fibroblasts (Fig. 1). All of the fibroblast cell lines evaluated were >99% fibroblast-like (see Section 2), however, any minor
Discussion
Our findings have highlighted a significant connection between TGFβ1, CCL2 and IL-13 during lung fibrosis. Previous work has indicated an interplay between TGFβ1, IL-13 and CCL2 in murine fibrosis models. CCR2 knockout mice are protected from bleomycin-induced lung fibrosis (Gharaee-Kermani, McCullumsmith, Charo, Kunkel, & Phan, 2003). Furthermore, pulmonary fibroblasts isolated from the lungs CCR2 knockout mice have decreased TGFβ1-induced αSMA expression and decreased TGFβRII, highlighting
References (44)
- et al.
Increased expression of integrin alphavbeta5 induces the myofibroblastic differentiation of dermal fibroblasts
Am J Pathol
(2006) - et al.
Stat6-deficient mice develop airway hyperresponsiveness and peribronchial fibrosis during chronic fungal asthma
Am J Pathol
(2002) - et al.
Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching
Am J Pathol
(2003) - et al.
The effect of transforming growth factor-beta on cell proliferation and collagen formation by lung fibroblasts
J Biol Chem
(1987) - et al.
CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis
Cytokine
(2003) - et al.
Monocyte chemoattractant protein-1 in idiopathic pulmonary fibrosis and other interstitial lung diseases
Hum Pathol
(1994) - et al.
Human pulmonary fibroblasts exhibit altered interleukin-4 and interleukin-13 receptor subunit expression in idiopathic interstitial pneumonia
Am J Pathol
(2004) - et al.
CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury
Am J Pathol
(2005) The myofibroblast in pulmonary fibrosis
Chest
(2002)- et al.
Platelet-derived growth factor in idiopathic pulmonary fibrosis
J Clin Invest
(1990)
Increased expression of integrin alpha(v)beta3 contributes to the establishment of autocrine TGF-beta signaling in scleroderma fibroblasts
J Immunol
Proteoglycan deposition in pulmonary fibrosis
Am J Respir Crit Care Med
CXCL11 attenuates bleomycin-induced pulmonary fibrosis via inhibition of vascular remodeling
Am J Respir Crit Care Med
Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts: Evidence for autocrine regulation of myofibroblast differentiation
Arthritis Rheum
The epidemiology of interstitial lung diseases
Am J Respir Crit Care Med
Histologic classification of idiopathic chronic interstitial pneumonias
Am J Respir Cell Mol Biol
Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar
Am J Pathol
Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: Down-regulation by camp
FASEB J
IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis
Nat Med
Histopathologic variability in usual and nonspecific interstitial pneumonias
Am J Respir Crit Care Med
Production of interleukin 13 by alveolar macrophages from normal and fibrotic lung
Am J Respir Cell Mol Biol
Elevated serum levels of interleukin 4 (IL-4), IL-10, and IL-13 in patients with systemic sclerosis
J Rheumatol
Cited by (127)
Comprehensive review of potential drugs with anti-pulmonary fibrosis properties
2024, Biomedicine and PharmacotherapyResearch on the mechanism of berberine in the treatment of COVID-19 pneumonia pulmonary fibrosis using network pharmacology and molecular docking
2022, Phytomedicine PlusCitation Excerpt :Therefore, fibroblasts are tended from the peripheral circulation to the alveolar interstitium, which increases the risk of pulmonary fibrosis (Ekert et al., 2011; Wolters et al., 2014b; Murray et al., 2010). Studies have also shown that by inhibiting or attenuating CCL2, pulmonary fibrosis can be inhibited (Moore et al., 2005; Moore et al., 2006; Murray et al., 2008). In addition, CCR2 can induce fibroblasts to proliferate and differentiate into myofibroblasts, and the survival of fibroblasts also depends on the expression of IL-6 induced by CCL2,on the other handIL-6 enhances the inflammatory response and further aggravates pulmonary fibrosis (Wolters et al., 2014a; Liu et al., 2007).
A novel phosphodiesterase 4 inhibitor, AA6216, reduces macrophage activity and fibrosis in the lung
2020, European Journal of PharmacologyIdiopathic pulmonary fibrosis is associated with tight junction protein alterations
2020, Biochimica et Biophysica Acta - Biomembranes