Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms.

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.

Author contributions: RB-J, AD, SM, ASN, ZMT, PB and DC performed the experimental work.AD, MCU, DC and STM performed the Affymetrix analysis. IDS performed statistical analysis, SRJ conceived the study, obtained the funding, saw the patients and is guarantor for the study.

Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14 þ and CD14À mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor kB ligand (RANKL) þ /À macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51 þ , CD14À, cathepsin K þ , TRAP þ ) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL ± M-CSF, CD14 þ , but not CD14À, mononuclear cells differentiated into TRAP þ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14 þ cell cultures. CD14À cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14 þ macrophage precursors. CD14À mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14 þ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.Laboratory Investigation (2012) 92, 600-605; doi:10.1038/labinvest.2012.5; published online 13 February 2012 KEYWORDS: aneurysmal bone cyst (ABC); bisophosphonate; bone resorption; giant cell; osteoclast Benign and malignant tumours that contain numerous osteoclast-like giant cells frequently arise in bone and are categorised on the basis of tissue location and histomorphology. 1,2 The mechanism whereby giant cells accumulate and contribute to the osteolysis that accompanies growth of these tumours in bone is not certain. Primary aneurysmal bone cyst (ABC) is a benign cystic giant cell-rich tumour that arises most commonly in the metaphyseal region of a long bone; 1,2 the cysts are filled with blood and the fibrous cyst wall contains fibroblast-like stromal cells, scattered macrophages and osteoclast-like giant cells. The lesion most commonly arises in the first two decades of life and is a clonal proliferation associated with a characteristic rearrangement of the short arm of chromosome 17. [3][4][5] Osteoclasts are formed from marrow-derived circulating precursors, which express a monocyte/macrophage phenotype. 6,7 These mononuclear phagocyte precursors express the receptor activator of nuclear factor kB (RANK) and, in the presence of macrophage-colony stimulating...