The Role of Macrophage Elastase (MMP-12) in the Inflammatory Response in the Airways
· Claude Bertrand, AstraZeneca, United Kingdom
· Dr Soazig Nenan, France
· Vincent Lagente, INSERM U620, Université de Rennes 1, France
Macrophage elastase (MMP-12) is a metalloproteinase able to degrade extracellular matrix components such as elastin. As other MMPs, MMP-12 may be involved in acute and chronic lung injury. However, its precise role in the inflammatory cascade of the airways during obstructive diseases is not clearly understood. We demonstrated that rhMMP-12 instilled in mice airways is able to induce severe inflammatory cell recruitment with an early accumulation of neutrophils associated with an increase in pro-inflammatory cytokines and in gelatinases followed by a relatively stable recruitment of macrophages in the lungs over a period of about ten days. Recent studies suggest that resident alveolar macrophages and recruited neutrophils are not essential to induce the delayed macrophage recruitment. However, epithelial cells could be the target for MMP-12 in our model. We also reported that a corticosteroid, dexamethasone, a PDE4 inhibitor, rolipram and a non selective MMP inhibitor, marimastat were able to reverse some of these inflammatory events. These data indicate that our MMP-12 model could highlight some of the inflammatory response seen in COPD and could be used for the pharmacological evaluation of new anti-inflammatory mechanism of action. Because of its ability to induce an inflammatory response and tissue remodeling, it may be possible to consider MMP-12 as an essential component of the process leading to the development of the disease.
MMP / TIMP Imbalance in the Development of Fibrosis
· Annie Pardo, Universidad Nacional Autónoma de México, Mexico
Lung fibrotic remodeling is characterized by fibroblast/myofibroblast activation, and excessive extracellular matrix (ECM) accumulation leading to progressive organ dysfunction and usually terminal outcome. Studies in humans and experimental models support a critical role for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the development of pulmonary fibrosis. By oligonucleotide microarrays, an overexpression of several MMPs, primarily, MMP-7, MMP-1, MMP-2 and MMP-9 have been found in idiopathic pulmonary fibrosis (IPF). One of the most upregulated genes is MMP-7 whose immunoreactive protein is mainly localized in reactive epithelial cells and bound to ECM. This enzyme is able to degrade several matrix and substrates, such as proteoglycans, laminin, fibrin/fibrinogen, and others. Furthermore, matrilysin knockout mice are dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin, suggesting that MMP-7 plays an important role in lung fibrogenesis and that it can be a potential therapeutic target. MMP-1 is found primarily in reactive alveolar epithelial cells as well as in bronchiolar epithelial cells lining honeycomb cystic spaces. Moreover, MMP-1 is virtually absent in fibroblasts of the interstitial fibrotic areas where collagens are being deposited. The role of epithelial MMP-1 is presently unknown but it might be related with cell migration. Considering that one of the main substrates of collagenases are fibrillar collagens, the lack of the expression of this enzyme in interstitial fibroblasts might explain in a simplistic way the presence of scars that do not undergo resorption. By contrast, TIMPs are usually found in the interstitial compartment. TIMP-1 is expressed by interstitial cells associated to fibrous tissue and TIMP-2 is specifically expressed by myofibroblasts within fibroblast foci. TIMP-3 binds to the ECM and is primarily localized to the elastic lamina of vessels and in fibroblasts. Therefore, despite the high expression of some MMPs in IPF, the strong interstitial localization of the TIMPs as compared to interstitial collagenases, supports the notion that a non-degrading fibrillar collagen microenvironment is present in this disease.
Effect of Neutrophil Depletion on MMPs/TIMP-1 Imbalance in Bleomycin-Induced Pulmonary Fibrosis in Mice
· Boris Manoury, INSERM U620, Université de Rennes 1, Rennes, France, France
· Soazig Nénan, INSERM U620, Université de Rennes 1, Rennes, France, France
· Isabelle Guénon, INSERM U620, Université de Rennes 1, Rennes, France, France
· Vincent Lagente, INSERM U620, Université de Rennes 1, Rennes, France, France
· Elisabeth Boichot, INSERM U620, Université de Rennes 1, Rennes, France, France
Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix resulting in respiratory failure. The turn over of extracellular matrix is partially regulated by proteases such as metalloproteinases (MMPs) and their inhibitors (TIMPs). We investigated the impact of polymorphonuclear neutrophils (PMN) depletion on the MMP/TIMP-1 imbalance and the development of bleomycin-induced fibrosis in mice. C57BL/6 mice were injected i.p. with 200µL of rabbit anti-mouse PMN antibody. Control mice received vehicle solution. One day and 14 days after bleomycin administration (0.3 mg/mouse), bronchoalveolar lavages (BAL) were performed and lungs were removed for analysis. Administration of anti-PMN antibodies blunted the neutrophil influx detected in BAL and in whole blood one day after bleomycin administration (89 % decreased; P < 0.05). At day 14, hydroxyproline content reflected the collagen deposition in the lung tissue was increased both in anti-PMN treated and control mice, without difference between groups. At day one, bleomycin elicited a raise in pro-MMP-9 level in BAL that was significantly attenuated in anti-PMN depleted mice, whereas bleomycin increased TIMP-1 release in both groups in a similar manner. Pro-MMP-9 activity was significantly correlated with the neutrophil count in the BAL fluids (P = 0.0003). Anti-PMN administration elicits stronger IL-6 level in BAL of bleomycin-treated mice. Anti-PMN administration had no effect on MMP-2 that was induced in BAL by bleomycin, both at day 1 and day 14. Higher RNA levels were observed in PMN-treated mice at day one for MMP-9 and MMP-2 and at day 14 for MMP-2 only. At day 14, bleomycin elicited a raise of TIMP-1 protein and RNA levels regardless to anti-PMN treatment, whereas MMP-9 returned to basal level. PMN-depletion and the associated modifications in pro-MMP-9 / TIMP-1 imbalance in lung during the early inflammatory phase do not alter susceptibility to bleomycin-induced pulmonary fibrosis.
Induction Of ADAMTS-4 (Aggrecanase-1) Gene Expression By Interleukin-1 In Articular Chondrocytes Is Mediated By Mitogen-Activated Protein Kinases
· Rasheed Ahmad, Department of Medicine and Research Centre of CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada
· Mohammed El Mabrouk, Department of Medicine and Research Centre of CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada
· Judith Sylvester, Department of Medicine and Research Centre of CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada
· Ataf Chaudry, Department of Medicine and Research Centre of CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada
· Francois Huynh, Department of Medicine and Research Centre of CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada
· Muhammad Zafarullah, Department of Medicine and Research Centre of CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada
PURPOSE:
Interleukin-1 (IL-1β)
is the main proinflammatory stimulant of cartilage catabolism in arthritis.
Aggrecanases (ADAMTS are enzymes implicated in tissue remodeling and cleavage of
aggrecan, a major protein of cartilage matrix responsible for its compressive
stiffness. IL-1 also inhibits matrix synthesis. We investigated the molecular
mechanisms of IL-1 signal transduction leading to ADAMTS-4 RNA induction in
human articular chondrocytes.
METHODS: Confluent normal human knee articular chondrocytes maintained in
serum-free medium were pretreated either with different pharmacological
inhibitors or transiently transfected with antisense oligonucleotides or small
interfering RNAs (siRNAs) and stimulated further for 24 h with IL-1β
(10 ng/ml). ADAMTS-4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA
levels were analyzed by RT-PCR.
RESULTS: IL-1β
induced ADAMTS-4 RNA in high-density human chondrocyte monolayer cultures.
Pretreatment with specific inhibitor of ERK-MAPK, U0126 and transfection with
antisense ERK or its siRNA partially inhibited IL-1-induced expression of
ADAMTS-4 RNA. Protein 38 and JNK inhibitors, SB203580 and SP600125 also
down-regulated such induction. A leukotreine and c-Fos (component of activating
protein or AP-1 transcription factor) inhibitor, nordihydroguiaretic acid (NDGA)
suppressed the ADAMTS-4 RNA induction. Further, AP-1 and nuclear factor kappa B
(NF-κB)
transcription factors inhibitors, pyrrolidine dithiocarbamate (PDTC) and
curcumin partially or completely inhibited aggrecanase-1 induction. The levels
of internal control, GAPDH RNA remained constant.
CONCLUSIONS: These results suggest the involvement of ERK-, p38- and
JNK-MAPKs as well as AP-1 and NF-κB
transcription factors in the IL-1 induction of ADAMTS-4 in chondrocytes.
Inhibition of these targets by the specific pharmacological or genetic agents
could be useful for reducing ADAMTS-4 driven cartilage resorption in arthritis.