Fibrocytes are associated with vascular and parenchymal remodelling in patients with obliterative bronchiolitis

Lung transplantation (LTP) represents a treatment option in many end-stage lung diseases such as chronic obstructive lung disease, cystic fibrosis, and idiopathic pulmonary fibrosis (IPF). Unfortunately, as many as 60% of patients who have been transplanted develop obliterative bronchiolitis (OB) which is a form of chronic rejection, within 5 years [1]. It is also known that bone marrow transplantation (BMT) after, for example, acute and chronic leukaemia, anaemia, and rare immunodeficiency disorders, can also lead to OB at a frequency of 2-11% [2‐4], with similar pathological characteristics as after LTP. The reason that some patients develop OB is still unclear, but one of the most frequent risk factors is repeated acute rejection, followed by lymphocytic bronchitis or bronchiolitis [5]. The histological findings are described as epithelial injury, with mononuclear inflammation and fibrotic lesions that lead to intraluminal polypoid plugs of granulation tissue within the terminal and respiratory bronchioles. The lesions consist of fibroblasts and/or myofibroblasts and extracellular matrix (ECM). The main producers of ECM are lung fibroblasts and phenotypes derived from fibroblasts. There are three current hypotheses concerning the origin of these cells: 1.) proliferation and/or differentiation of resident fibroblasts [6]; 2.) epithelial mesenchymal transition (EMT) [7‐9]; and 3.) recruitment of circulating progenitors such as fibrocytes to the lung, where they differentiate further into specific fibroblast phenotypes. Fibrocytes are identified by specific combinations of mesenchymal markers such as prolyl 4-hydroxylase and α-smooth muscle actin (αSMA), together with haematopoietic markers such as CD34, leukocyte markers such as CD45 [10, 11], and chemokine receptors such as CXCR4 [12].

In a previous study, our group showed that there is accumulation of sub-epithelial fibrocytes in patients with mild asthma [13]. There was a positive correlation between the number of fibrocytes and the thickness of the lamina reticularis layer of the basement membrane, which is also a characteristic feature of asthma. Furthermore, fibrocytes have been observed near fibroblastic foci in patients with IPF [14]. Bröcker et a. also identified recipient-derived αSMA-positive cells in lung tissue in a study of two patients with OB after bone marrow transplantation [15]. When fibrocytes move from the circulation to the injured lung tissue, there is a gradual loss of haematopoietic markers while the expression of mesenchymal markers increases [16].

It has been shown that LTP patients have a reduced number of blood vessels in the small airways before developing obliteration of the airway [17], and that LTP patients with BOS (bronchiolitis obliterans syndrome) have higher microvascular density in endobronchial biopsies than controls. The changed composition of the vessels has been hypothesised to be a tissue reaction to relative hypoxia and hypercarbia [18].

Together, the previous findings have led to the hypothesis that fibrocytes may be an additional source of mesenchymal cells in fibrotic lesions. If so, fibrocytes either circulating or in tissue, could be a possible biomarker for remodelling in OB, and the vascular remodelling in these patients might facilitate an increased influx of fibrocytes into the tissue. These events may play an important role in the development of fibrosis in OB. Hereby we show that the number of fibrocytes identified in tissue was correlated to structural remodelling of both the pulmonary vessels and the alveolar parenchyma.

This study shows a correlation between structural remodelling of several compartments of the lung and the number of fibrocytes identified in tissue from patients with OB after transplantation. The fibrocytes were identified in both the patient and the control groups by combining detection of different markers (CXCR4/CD34/CD45R0) with detection of prolyl 4-hydroxylase. However, more fibrocytes were identified in the patient group - and especially in the LTP patients - than in the controls. The combination CXCR4 together with prolyl 4-hydroxylase identified the highest number of fibrocytes relative to the other combinations and the patient group also had a larger proportion of endothelial layer combined with a larger amount of vessel lumen compared to the controls. The number of fibrocytes in lung tissue was also correlated to remodeling changes of the pulmonary vessels. Moreover, there was a gradual increase in endothelial layer seen over time from transplantation to autopsy.

There is currently a debate as to whether fibrocytes are good or bad in airway remodelling. Recently, however, various studies have indicated that there is an association between the occurrence of fibrocytes and pathological structural changes of the airways, paralleled by a decline in lung function. In idiopathic pulmonary fibrosis, it has been demonstrated that there is a correlation between the number of fibrocytes lung tissue and the number of fibroblastic foci [14]. Also, Moeller et al. have shown that the number of circulating fibrocytes is elevated during an exacerbation and that the number of these cells may be useful as a predictor of early mortality in patients with IPF [20]. Furthermore, Nihlberg et al. have demonstrated that in asthma patients basement membrane thickness may be correlated to the number of fibrocytes in lung tissue [13]. Also other studies have shown who the number of circulated fibrocytes are increased by severity of the disease [21, 22]. In accordance with this, the present study has shown a correlation between the number of fibrocytes and degree of structural remodeling of the alveolar parenchyma as well as degree of remodeling of the vessels. These findings indicate that the fibrocytes have a negative effect, at least at the end-stage of OB. Fibrocytes appear to indicate an ongoing chronic reparative process, at least in the pulmonary diseases described.

It can be argued that the correlation studies are of less value as there is already a significant difference between the control- and patient group in number of fibrocytes and structural related changes of the tissue. Nevertheless, the patient material consists of a mixed population of various disease states (Table 1) with several interlaced values between lowest and highest points of the correlation line, indicating true correlations.

Fibrocytes could contribute this chronic reparative process in several ways. When the fibrocytes are leaving the circulation and passing through the vascular barrier into tissue, they are able to secrete a cascade of factors: pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and haematopoietic growth factors that can change the surrounding environment and the vascular bed by promoting endothelial cell migration, proliferation, and/or tube formation [23]. Fibrocytes are also able to produce ECM and to differentiate into fibroblasts or myofibroblasts [24], which are the main producers of both collagen and proteoglycans such as decorin, versican, and biglycan [25], and thus could contribute to the development of the fibrotic lesions seen in OB. This differentiation is possible when the fibrocyte has entered the tissue after rolling. Vessel changes in the bronchial circulation occur in lung diseases such as asthma [26], but also in patients with OB [18]. The remodelling of the pulmonary circulation has not been fully investigated. For example, in IPF the changes depend both on the progress of the fibrosis and on the distance from fibroblastic foci, with more vessels early in the disease and outside fibroblastic foci [27]. In the present study, all the patients were at the end-stage of the disease and the remodelling process had continued over time.

The group of patients who had undergone BMT seemed to be more homogenous than the LTP group, both regarding the number of fibrocytes and the degree of remodelling of vessels. This may be explained by the age of the patients. All the patients had a close age range - between 10 and 15 years of age at the time of BMT and between 12 and 30 years old when they were lung transplanted. There was a positive correlation between the number of fibrocytes identified by CD34 and prolyl 4-hydroxylase and age at the time of autopsy in LTP patients. Our findings are further supported by a study by Mora et al. that showed that the number of circulating fibrocytes in mice varied with the age of the mouse, with a higher number being found in the older animals [28].

It has been reported that patients with OB after LTP have elevated levels of transforming growth factor beta (TGF-β) in respiratory epithelial lining fluid and that cells from bronchoalveolar lavage show increased expression of TGF-β [29]. Even though the level of TGF-β in tissue has only been evaluated in animal models of chronic allograft rejection [30], it has been shown that TGF-β is a central cytokine in the development of fibrosis [31]. Furthermore, the expression of VEGF and haematopoietic growth factors can regulate the TGF-β-dependent development of fibrotic lesions [32].

A potentially important agent of both fibrocyte differentiation and of the remodeling of vessels is therefore TGF-β. This cytokine has been shown to differentiate blood-derived fibrocytes to αSMA-expressing myofibroblasts [22]; in addition, it has also been shown to increase the expression of certain ECM molecules that could function in stabilising vessels.

It is hypothesised that hypoxia is a major contributor to the induction of remodelling of vessels and in the balance between deposition and degradation of ECM. This could occur due to an increased level of nitric oxide which would result in vasodilation, increased vasopermeability, and initiation of angiogenesis while the hypoxia itself would increase expression of VEGFR. Hypoxia is a well-characterized condition, which elicits expression of hypoxia-induced factor (HIF) [33], SDF-1 [34], and CXCR4 [35] and may give rise to recruitment of progenitor cells such as fibrocytes. HIF can upregulate expression of prolyl 4-hydroxylase [36], a key enzyme in the biosynthesis of collagen, and thus more collagen and ECM would be synthesised and accumulated. It has also been shown that SDF-1 can bind to ECM molecules [37], where the ECM can function as a reservoir and also help to sustain the fibrotic reaction. SDF-1 can also be released from the ECM under specific conditions such as injury or stress.

In summary, these novel findings have provided us with some insight into the complexity of OB. Both the remodelling of vessels and the thickening of the alveolar parenchyma are related to the number of fibrocytes, which can be assumed to have an important role in OB. Future studies of these cells will help us to understand the mechanisms behind this very serious complication of transplantation. To further increase the understanding about the fibrogenesis in OB, complementary studies on additive effects of EMT and resident fibroblast on this process are warranted.