Idiopathic pulmonary fibrosis (IPF) is the most common and severe idiopathic interstitial pneumonia 12. In affected portions of the lung irreversible remodelling of tissue architecture takes place, which is histopathologically described as "usual interstitial pneumonia". In recent years evolving opinions regarding the pathogenesis of this specific chronic fibrosing disease have arose and different models have been proposed 3456. Recently, the "inflammatory theory" of IPF/UIP has been challenged on the assumption that abnormal epithelial-mesenchymal interactions and aberrant wound healing are in fact crucial pathogenetic events 34. Although this new scheme is appealing, many points remain unresolved, including the nature of "fibroblast-foci" (FF), as well as the molecular mechanisms responsible for alveolar loss, honeycomb modifications, abnormal fibrosis and severe tissue remodelling. In previous studies we provided evidence that the wnt/β-catenin signalling pathway is abnormally activated in IPF/UIP, acting on both the alveolar and bronchiolar components 789. The central role played by the wnt/β-catenin pathway in lung embryogenesis and pathology is further demonstrated by the complex functions exerted by this pathway in regulating a variety of crucial mechanisms, including cell proliferation, apoptosis, cell migration, and angiogenesis 10. Accordingly, the wnt-signalling pathway regulates branching morphogenesis in the lung, and can produce severe modifications in the developmental potential of embryonic lung differentiation when aberrantly expressed 11. Interestingly, the wnt/β-catenin pathway is a central trigger of epithelial-mesenchymal transition (EMT), an important process occurring during critical phases of embryonic development, tumour progression, and fibrotic tissue repair in different organs including the lung 121314. This possibility is relevant since such a new scheme could completely change the pathogenic scenario for IPF/UIP, a devastating disease where new therapeutic options are necessary 15.

We hypothesise that uncontrolled activation of wnt-β-catenin pathway can profoundly influence tissue remodelling in IPF/UIP by triggering pronounced cell migration and proliferation at sites of aberrant expression, thus interfering with the physiologic molecular program determining correct tissue repair. A variety of molecules involved in cell migration and invasion are in fact targets of β-catenin transcriptional activation and/or regulation, including matrylisin/MMP7 (a metalloproteinase with multifunctional roles including the induction of epithelial cell migration, apoptosis and metaplastic conversion) 91617, laminin-5 gamma-2 chain (LAM5γ2; a potent migration-inducing factor expressed by epithelial cells in healing tissues) 18, tenascin-C (a component of the extracellular matrix expressed during development, neoplastic invasion and wound-healing) 19, and fascin (an actin-binding protein involved in cell motility of epithelial cells) 2021. Abnormal expression of matrilysin/MMP7 has been demonstrated in UIP samples by both analysis of gene expression and immunohistochemistry 922, and tenascin has been found to be expressed at high levels in fibroblast foci. However, only limited information is available in UIP regarding the expression of other molecules involved in cell migration, such as LAM5γ2, fascin, and heat-shock protein-27 (HSP27), a multifunctional stress-inducible molecule involved in the modulation of actin microfilament dynamics and cell migration 232425.

In this study we have investigated the immunohistochemical expression of LAM5γ2, fascin, and HSP27 in 30 cases of UIP, and in a large variety of biopsies of other pulmonary diseases used as controls.

All studies were carried out in compliance to the Helsinki declaration and in accordance with Italian law, following the ethical recommendations of the Institutions where they were performed.

All the 30 samples of IPF were morphologically described as usual interstitial pneumonia by the presence of typical modifications. These included patchy interstitial fibrosis alternating with normal or minimally affected parenchymal tissue, and honeycombing. FF, morphologically defined as circumscribed collections of loose organizing connective tissue formed by spindle-shaped myofibroblasts, was present in all samples, and quantified on serial sections by immunostaining for αSMA and tenascin. The number of FF was highly variable in different samples, ranging from 1 to >10 per cm². The epithelial cells overlying FF were heterogeneous and appeared as either flat, cuboidal, or ciliated.

The pathogenesis of idiopathic interstitial pneumonia is poorly defined and remains the subject of intense debate and research. Although high-throughput molecular analysis has been applied to UIP samples with some success 2227, the precise definition of molecular events occurring at sites of disease activity will require direct in situ analysis of lung biopsies.

In this paper we provide in situ evidence that the epithelial component overlying fibroblast foci (FF) expresses a set of molecules involved in inducing cell motility and invasiveness, including LAM5γ2, fascin and HSP27. The relevance of our findings is related to the specific functions of the investigated molecules, as well as the characteristic tissue localisation of their abnormal expression. The morphology and location of this cellular component is in fact particularly intriguing, since positive cells appeared as linear clusters of bronchiolar basal cells within FF, wedged between luminal epithelial cells and myofibroblasts. The recognition of these negative-positive-negative three-layered lesions (that we termed "sandwich" fibroblast-foci or SW-FF) was particularly evident using LAM5γ2 and HSP27 as markers (Figs. 1 and 2).

The trimeric protein laminin-5 (α3, β3, γ2-chain) is an integral part of the basal lamina of stratified epithelia where it plays a crucial role in the organization of the basal stem-cell niche by providing epithelial-mesenchymal connections by interacting with integrin α6β4 28. These interactions are critical for regulating cell migration, an event required in different processes, such as wound healing, embryogenesis and metastatic dissemination 29. The γ2 chain of laminin-5 (LAM5γ2) acts as a soluble cell motility factor in a variety of conditions after its cleavage by metalloproteinases, and enhanced expression of LAM5γ2 is considered one of the best marker of invasiveness in different carcinomas 30313233. At the invasive front of colorectal carcinoma the cytoplasmic accumulation of LAM5γ2 in neoplastic cells is the result of synergistic activation of the LAMC2 gene by β-catenin, TGFβ1, and hepatocyte-growth factor (HGF), molecules that all have been variably involved in the pathogenesis of IPF/UIP 918343536. Our findings regarding LAM5γ2 expression are partially at variance with those recently described by Lappi-Bianco et al 37, who observed LAM5γ2 expression in regenerating epithelial cells in both COP and IPF/UIP, but did not note the characteristic immunoreactivity in basal cells at FF. In our study, the bronchiolar nature of epithelial cells overlying FF was assessed by sensitive and specific immunophenotyping using recently-introduced robust markers such as ΔN-p63, that were not used in the aforementioned study, thus possibly explaining this apparent discrepancy.

Fascin is a 55 kD protein that binds actin, organising it into well ordered bundles thus contributing to the formation of the various cell protrusions (filopodia, spikes, lamellipodial ribs and dendrites) necessary for cell adhesion and motility 2138. Fascin is not normally expressed in pulmonary epithelial cells, but is up-regulated in a number of carcinomas 3940. Interestingly, fascin can also associate with β-catenin, utilising the same binding sites used by E-cadherin and co-localising at cell-cell borders and leading edges 20.

Heat shock protein-27 is a small molecule rapidly induced and phosphorylated by heat shock and other stressing agents 41. HSP27 behaves as an actin-capping protein interfering with its polymerisation, thus regulating cell adhesion and motility under the control of p38 MAPK (p38 mitogen-activated protein kinase) 242542. In addition, HSP27 can mediate resistance against cell death induced by stress and differentiation 4344. The mechanisms accounting for the cytoprotective functions of HSP27 are complex, since HSP27 directly interacts with several apoptotic effectors. Using a specific antibody, we demonstrated that the HSP27 protein expressed at FF is phosphorylated, arguing in favour of its biological functionality.

Recent pathogenic models of IPF/UIP have been proposed, suggesting that disturbed re-epithelialisation occurs at sites of abnormal tissue damage and repair 347. The demonstration of increased cell migration at sites of ongoing remodelling is in line with these models, and also with the abnormal wnt-pathway activation occurring at the same sites as previously suggested by us (9).

Finally, according to our data, the sandwich-foci observed in the large majority of UIP samples using LAM5γ2 and HSP27 antibodies could represent a useful new marker for characterisation of IPF/UIP. The UIP pattern, although well defined in its morphological features, is not completely specific for IPF, and both mimicking and difficult cases arise. Accordingly, full diagnostic agreement regarding IPF/UIP evaluation on lung biopsy is not reached even among expert lung pathologists 45. The sandwich-pattern is easily recognisable on routine tissue samples and high quality antibodies for both LAM5γ2 and HSP27 are available. Further studies are in progress to validate the utility of this promising marker in the differential diagnosis of interstitial pneumonias on a larger series of cases.

The molecular abnormalities demonstrated in this study suggest that abnormal proliferation and migration of epithelial basal cells overlying myofibroblasts in FF have a major role in the pathological remodelling characterising IPF/UIP, leading to bronchiolar colonisation with substitution of the alveolated parenchyma and eventual progression towards lung fibrosis and functional loss. Activation of the wnt-pathway and increased expression of proteins involved in cell migration and invasiveness are involved in this process.

FF: fibroblast foci

IPF/UIP: idiopathic pulmonary fibrosis/usual interstitial pneumonia

EMT: epithelial-mesenchymal transition

SW-FF: sandwich-fibroblast foci

UIP: usual interstitial pneumonia

IPF: idiopathic pulmonary fibrosis

MMP7: matrix metalloproteinase 7

LAM5γ2: laminin-5 gamma-2 chain

HSP27: heath-shock protein 27

NSIP: non-specific interstitial pneumonia

AIP/DAD: acute interstitial pneumonia with diffuse alveolar damage

DIP: desquamative interstitial pneumonia

EAA: extrinsic allergic alveolitis

LCH: Langerhans cell histiocytosis

AEP: acute eosinophilic pneumonia

ACIF: airway-centred interstitial fibrosis

DPB: diffuse panbronchiolitis

CK5: cytokeratin 5

SMA: smooth muscle actin

SPA: surfactant apoprotein A

OP/COP: organising pneumonia/cryptogenic organising pneumonia

MAPK: mitogen activated protein kinase

The author(s) declare that they have no competing interests.

MC designed the study, evaluated slides microscopically, drafted and edited the manuscript. AZ participated in study design, manuscript drafting and revision. DR collected study specimens and helped in manuscript revision. ML evaluated slides microscopically and revised the manuscript. LM and SP carried out the immunoassays. MGE evaluated slides microscopically and revised the manuscript. AC selected patients for inclusion in the study and revised the manuscript. BM evaluated slides microscopically and revised the manuscript. VP participated in study design, selected patients for inclusion in the study and revised the manuscript.