PAF mediates cigarette smoke-induced goblet cell metaplasia in guinea pig airways

Goblet cell metaplasia is an important morphological feature in the airways of patients with chronic airway diseases; however, the precise mechanisms that cause this feature are unknown. We investigated the role of endogenous platelet-activating factor (PAF) in airway goblet cell metaplasia induced by cigarette smoke in vivo. Guinea pigs were exposed repeatedly to cigarette smoke for 14 consecutive days. The number of goblet cells in each trachea was determined with Alcian blue-periodic acid-Schiff staining. Differential cell counts and PAF levels in bronchoalveolar lavage fluid were also evaluated. Cigarette smoke exposure significantly increased the number of goblet cells. Eosinophils, neutrophils, and PAF levels in bronchoalveolar lavage fluid were also significantly increased after cigarette smoke. Treatment with a specific PAF receptor antagonist, E-6123, significantly attenuated the increases in the number of airway goblet cells, eosinophils, and neutrophils observed after cigarette smoke exposure. These results suggest that endogenous PAF may play a key role in goblet cell metaplasia induced by cigarette smoke and that potential roles exist for inhibitors of PAF receptor in the treatment of hypersecretory airway diseases.     

Periostin regulates goblet cell metaplasia in a model of allergic airway inflammation

Periostin is a 90-kDa member of the fasciclin-containing family and functions as part of the extracellular matrix. Periostin is expressed in a variety of tissues and expression is increased in airway epithelial cells from asthmatic patients. Recent studies have implicated a role for periostin in allergic eosinophilic esophagitis. To further define a role for periostin in Th2-mediated inflammatory diseases such as asthma, we studied the development of allergic pulmonary inflammation in periostin-deficient mice. Sensitization and challenge of periostin-deficient mice with OVA resulted in increased peripheral Th2 responses compared with control mice. In the lungs, periostin deficiency resulted in increased airway resistance and significantly enhanced mucus production by goblet cells concomitant with increased expression of Gob5 and Muc5ac compared with wild type littermates. Periostin also inhibited the expression of Gob5, a putative calcium-activated chloride channel involved in the regulation of mucus production, in primary murine airway epithelial cells. Our studies suggest that periostin may be part of a negative-feedback loop regulating allergic inflammation that could be therapeutic in the treatment of atopic disease.     

Resolution of LPS-induced airway inflammation and goblet cell hyperplasia is independent of IL-18

Background

The resolution of inflammatory responses in the lung has not been described in detail and the role of specific cytokines influencing the resolution process is largely unknown.

Methods

The present study was designed to describe the resolution of inflammation from 3 h through 90 d following an acute injury by a single intratracheal instillation of F344/N rats with LPS. We documented the inflammatory cell types and cytokines found in the bronchoalveolar lavage fluid (BALF), and epithelial changes in the axial airway and investigated whether IL-18 may play a role in the resolution process by reducing its levels with anti-IL-18 antibodies.

Results

Three major stages of inflammation and resolution were observed in the BALF during the resolution. The first stage was characterized by PMNs that increased over 3 h to 1 d and decreased to background levels by d 6–8. The second stage of inflammation was characterized by macrophage influx reaching maximum numbers at d 6 and decreasing to background levels by d 40. A third stage of inflammation was observed for lymphocytes which were elevated over d 3–6. Interestingly, IL-18 and IL-9 levels in the BALF showed a cyclic pattern with peak levels at d 4, 8, and 16 while decreasing to background levels at d 1–2, 6, and 12. Depletion of IL-18 caused decreased PMN numbers at d 2, but no changes in inflammatory cell number or type at later time points.

Conclusion

These data suggest that IL-18 plays a role in enhancing the LPS-induced neutrophilic inflammation of the lung, but does not affect the resolution of inflammation.     

Characterization of an exosite binding inhibitor of matrix metalloproteinase 13

Matrix metalloproteinase 13 (MMP13) is a key enzyme implicated in the degradation of the extracellular matrix in osteoarthritis. Clinical administration of broad spectrum MMP inhibitors such as marimastat has been implicated in severe musculo-skeletal side effects. Consequently, research has been focused on designing inhibitors that selectively inhibit MMP13, thereby circumventing musculo-skeletal toxicities. A series of pyrimidine dicarboxamides were recently shown to be highly selective inhibitors of MMP13 with a novel binding mode. We have applied a molecular ruler to this exosite by dual inhibition studies involving a potent dicarboxamide in the presence of two metal chelators of different sizes. A larger hydroxamate mimic overlaps and antagonizes binding of the dicarboxamide to the exosite whereas the much smaller acetohydroxamate synergizes with the dicarboxamide. These studies elucidate the steric requirement for compounds that fit exclusively into the active site, a mandate for generating highly selective MMP13 inhibitors.     

Potential clinical implications of recent matrix metalloproteinase inhibitor design strategies

Analysis of matrix metalloproteinases (MMPs) expression profiles in various pathologies correlated with their presence in promoting disease progression. Drugs were designed to inhibit MMPs in an extreme manner by chelating the active site zinc ion. This approach did not distinguish between the 24 members of the MMP family and had devastating consequences during clinical trials. Subsequent knockout mouse studies showed that some MMPs are beneficial in regulating tumor growth, metastasis and indirectly stimulating the immune system. The broad-spectrum inhibitor approach was rethought and modified in order to increase specificity by taking into account the non-conserved secondary binding sites or differences in structures within MMPs and also generating antibodies. These showed interesting results in vitro and in vivo. The recent technological advances that allow us to better understand the function and structure of MMPs are aiding in the development of selective inhibitors.     

Design and synthesis of an orally active matrix metalloproteinase inhibitor

A series of 4-(4-phenoxy)benzoylamino-4-methoxymethyloxymethyl butyric acid hydroxamates, which were derived from l-glutamic acid, were synthesized and evaluated as matrix metalloproteinase inhibitors. Most of the compounds listed in exhibited strong inhibitory activity against MMP-2 and MMP-9, as well as even stronger inhibitory activity against MMP-3, but showed relatively weak inhibition of MMP-1. Structure-activity relationships are discussed.     

Engineered sarafotoxins as tissue inhibitor of metalloproteinases-like matrix metalloproteinase inhibitors

The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.     

Agarose plug instillation causes goblet cell metaplasia by activating EGF receptors in rat airways

We hypothesized that foreign bodies in airways cause inflammation leading to goblet cell metaplasia. Instilled agarose plugs lodged in the bronchi of pathogen-free rats caused a time-dependent increase in Alcian blue-periodic acid-Schiff staining that was detected within 24 h and markedly increased at 72 h. Control bronchi contained no pregoblet or goblet cells, but plugged bronchi contained many pregoblet and goblet cells and a decrease in nongranulated secretory cells. In situ hybridization showed no expression of MUC5AC in control airways, but plugged airways showed a marked expression. Control bronchi showed sparse staining for epidermal growth factor receptor (EGFR) protein, but plugged bronchi showed intense EGFR staining in the epithelium. Pretreatment with an EGFR tyrosine kinase inhibitor (BIBX1522) prevented Alcian blue-periodic acid-Schiff staining and MUC5AC gene expression in plugged bronchi. Pretreatment with tumor necrosis factor-alpha neutralizing antibody or pretreatment with cyclophosphamide abolished plug-induced EGFR protein expression and goblet cell metaplasia. Thus instillation of agarose plugs induces profound goblet cell metaplasia by causing EGFR expression and activation.     

Effect of a matrix metalloproteinase inhibitor on host resistance against Listeria monocytogenes infection

Hydroxy acid-based matrix metalloproteinase (MMP) inhibitors have been shown to inhibit tumor infiltration and growth, endotoxin shock, and acute graft-versus-host disease. Blockade of the release of soluble tumor necrosis factor-alpha (TNF-alpha) and CD95 ligand (CD95L; FasL) from cell-associated forms is reportedly involved in the mechanism of the drug effect. We investigated the effect of a MMP inhibitor, KB-R7785, on host resistance against Listeria monocytogenes infection, in which TNF-alpha is essentially required for the defense, in mice. The administration of KB-R7785 exacerbated listeriosis, while the drug prevented lethal shock induced by lipopolysaccharide and D-galactosamine. KB-R7785 inhibited soluble TNF-alpha production in spleen cell cultures stimulated by heat-killed L. monocytogenes and the drug treatment reduced serum TNF-alpha levels in infected mice, whereas the compound was ineffective on the modulation of interferon-gamma and interleukin-10 production. The effect of KB-R7785 was considered to be dependent on TNF-alpha because the drug failed to affect L. monocytogenes infection in anti-TNF-alpha monoclonal antibody-treated mice and TNF-alpha knockout mice. Anti-CD95L monoclonal antibody was also ineffective on the infection. These results suggest that induction of infectious diseases, to which TNF-alpha is critical in host resistance, should be considered in MMP inhibitor-treated hosts.     

Membrane type 1 matrix metalloproteinase-associated degradation of tissue inhibitor of metalloproteinase 2 in human tumor cell lines

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for the membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent activation of pro-MMP-2 on the cell surface. MT1-MMP-bound TIMP-2 has been shown to function as a receptor for secreted pro-MMP-2, resulting in the formation of a trimolecular complex. In the presence of uncomplexed active MT1-MMP, the prodomain of cell surface-associated MMP-2 is cleaved, and activated MMP-2 is released. However, the behavior of MT1-MMP-bound TIMP-2 during MMP-2 activation is currently unknown. In this study, (125)I-labeled recombinant TIMP-2 ((125)I-rTIMP-2) was used to investigate the fate of TIMP-2 during pro-MMP-2 activation by HT1080 and transfected A2058 cells. HT1080 and A2058 cells transfected with MT1-MMP cDNA (but not vector-transfected A2058 cells) were able to bind (125)I-rTIMP-2, to activate pro-MMP-2, and to process MT1-MMP into an inactive 43-kDa form. Under these conditions, (125)I-rTIMP-2 bound to the cell surface was rapidly internalized and degraded in intracellular organelles through a bafilomycin A(1)-sensitive mechanism, and (125)I-bearing low molecular mass fragment(s) were released in the culture medium. These different processes were inhibited by hydroxamic acid-based synthetic MMP inhibitors and rTIMP-2, but not by rTIMP-1 or cysteine, serine, or aspartic proteinase inhibitors. These results support the concept that the MT1-MMP-dependent internalization and degradation of TIMP-2 by some tumor cells might be involved in the regulation of pericellular proteolysis.     

X-ray structure of a novel matrix metalloproteinase inhibitor complexed to stromelysin

A new class of matrix metalloproteinase (MMP) inhibitors has been identified by screening a collection of compounds against stromelysin. The inhibitors, 2,4,6-pyrimidine triones, have proven to be potent inhibitors of gelatinases A and B. An X-ray crystal structure of one representative compound bound to the catalytic domain of stromelysin shows that the compounds bind at the active site and ligand the active-site zinc. The pyrimidine triones mimic substrates in forming hydrogen bonds to key residues in the active site, and provide opportunities for placing appropriately chosen groups into the S1' specificity pocket of MMPS: A number of compounds have been synthesized and assayed against stromelysin, and the variations in potency are explained in terms of the binding mode revealed in the X-ray crystal structure.     

Discovery of a new chemical lead for a matrix metalloproteinase inhibitor

A series of N-benzoyl gamma-aminobutyric hydroxamic acids were synthesized and evaluated as matrix metalloproteinase inhibitors. First, we focused on chemical modification of the N-benzoyl residue. Introduction of electron-rich para-substituents was effective to increase the inhibitory activity. Especially, some of the analogs with relatively more planar N-acyl residues, such as 10 and 11, demonstrated more potent activity. Second, chemical modification of the gamma-aminobutyric hydroxamic acid moiety was carried out to optimize the three-dimensional arrangement of the two pharmacophores (hydroxamic acid and N-acyl residues). Among the tested, the gamma-aminobutyric hydroxamic acid moiety was found to be the best spacer for connecting the above-mentioned two pharmacophores. Synthesis and structure-activity relationships are discussed.     

肺结核并发纤维化患者BALF和血清MMP及TIMP-1检测的临床意义

目的:探讨肺结核并发纤维化患者支气管肺泡灌洗液(BALF)及血清中基质金属蛋白9(MMP-9)及基质金属蛋白酶组织抑制剂l(TIMP-1)含量与肺纤维化及肺功能的关系.方法:酶联免疫(ELISA)法检测我院2009年3月至2009年9月68例肺结核并发纤维化患者(纤维化组)及30例无肺部疾病患者(对照组)的支气管肺泡灌洗液及血清中MMP-9及TIMP-1的含量,同时进行肺功能检查.结果:纤维化组患者的BALF及血清MMP-9的含量与对照组怠者相比无统计学意义,但纤维化组患者的BALF及血清TIMP-1的含量显著高于对照组(P＜0.05),且纤雏化组患者的BALF及血清MMP-9/TIMP-1比值低于对照组(P＜0.05).患者的动脉血氧分压、肺活量等肺功能指标与TIMP-1呈正相关,而与MMP-9/TIMP-1比值呈负相关(P＜0.05).结论:肺结核患者肺纤维化的发生可能与TIMP-1水平升高及MMP-9/TIMP-1比值降低相关,降低TIMP-9的水平可能是防止肺结核惠者并发肺纤维化的一种新途径.     

IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.     

Loss of pro-apoptotic Bim promotes accumulation of pulmonary T lymphocytes and enhances allergen-induced goblet cell metaplasia

Immunological tolerance during prolonged exposure to allergen is accompanied by a shift in the lymphocyte content and a reduction of goblet cell metaplasia (GCM). Bim initiates negative selection of autoreactive T and B cells and shut down of T cell immune responses in vivo. The present study investigated whether Bim plays a role in the resolution of GCM during prolonged exposure to allergen. Loss of Bim increased T lymphocyte numbers in the bronchoalveolar lavage at 4 and 15 days of allergen exposure. The numbers of pulmonary CD4(+)8(-), CD4(-)8(+), and gammadelta T cells were significantly higher in naive and allergen-challenged bim(-/-) mice compared with wild-type (WT) littermates. When activated, pulmonary bim(-/-) T cells produced increased levels of IFNgamma compared with bim(+/+) T cells. No differences were noted in the total numbers of epithelial cells per millimeter of basal lamina between bim(+/+) and bim(-/-) mice, and the rate of resolution over 15 days of exposure was similar in both groups of mice. However, GCM was significantly enhanced and expression of IL-13Ralpha2 was reduced in bim(-/-) mice compared with WT mice at 4 days. Furthermore, treatment of bronchiolar explant cultures with increasing IFNgamma levels reduced immunostaining for IL-13Ralpha2. Collectively, these studies suggest that, during prolonged exposure to allergen, Bim plays no role in the resolution of GCM, but increased IFNgamma levels in bim(-/-) mice may be responsible for reduced expression of IL-13Ralpha2 and enhanced GCM despite similar levels of IL-13 in bim(+/+) and bim(-/-) mice.     

A matrix metalloproteinase mediates long-distance attenuation of stem cell proliferation

Ligand-based signaling can potentiate communication between neighboring cells and between cells separated by large distances. In the Drosophila melanogaster ovary, Wingless (Wg) promotes proliferation of follicle stem cells located ∼50 µm or five cell diameters away from the Wg source. How Wg traverses this distance is unclear. We find that this long-range signaling requires Division abnormally delayed (Dally)-like (Dlp), a glypican known to extend the range of Wg ligand in the wing disc by binding Wg. Dlp-mediated spreading of Wg to follicle stem cells is opposed by the extracellular protease Mmp2, which cleaved Dlp in cell culture, triggering its relocalization such that Dlp no longer contacted Wg protein. Mmp2-deficient ovaries displayed increased Wg distribution, activity, and stem cell proliferation. Mmp2 protein is expressed in the same cells that produce Wg; thus, niche cells produce both a long-range stem cell proliferation factor and a negative regulator of its spreading. This system could allow for spatial control of Wg signaling to targets at different distances from the source.     

Reduction of excitotoxicity and associated leukocyte recruitment by a broad-spectrum matrix metalloproteinase inhibitor

An important step in the cascade leading to neuronal cell death is degradation of laminin and other components of the brain extracellular matrix by microglia-derived proteases. Excitotoxic cell death of murine hippocampal neurones in vivo can be prevented by inhibitors of tissue plasminogen activator (tPA) or by inhibitors of plasmin. Plasmin is a potent activator of the matrix metalloproteinases (MMPs), which are made by resident and recruited leukocytes following CNS injury. In this study, we show, using Taqman RT-PCR, that MMP mRNAs, but not other calcium-dependent proteases such as calpain mRNAs, are acutely up-regulated after an excitotoxic challenge in vivo. alpha(2)-antiplasmin or BB-3103, a broad-spectrum inhibitor of the MMPs, co-injected with kainic acid into the striatum, inhibits excitotoxic cell death in the rat striatum, and reduces both the number of recruited macrophages and the size of the lesion. We also show that leukocyte populations differentially express MMPs, which may account, in part, for the expression profile we observe in the challenged brain. Our results show that inhibition of the MMPs in the rat will prevent kainic acid-induced cell death in the brain. These studies suggest that MMP inhibitors have therapeutic potential for use in stroke, and support the increasing evidence that microglial activation may contribute to neuronal cell death.     

Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines.

Activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP is believed to play a critical role during tumor progression and metastasis. In this study we present evidence that MT1-MMP plays a key role for the in vitro invasiveness of malignant melanoma. Melanoma cell lines secreted latent MMP-2 when cultured on plastic. However, when cells were grown in floating type I collagen lattices, only high invasive melanoma cells activated proMMP-2. Activation could be inhibited by antibodies against MT1-MMP, by addition of recombinant tissue inhibitor of metalloproteinases (TIMP)-2 and by inhibition of MT1-MMP cleavage. MT1-MMP protein was detected as an inactive protein in all cell lines cultured as monolayers, whereas in collagen gels, active MT1-MMP protein was detected in the membranes of both high and low invasive melanoma cells. Production of TIMP-2 was about 10-fold higher in low invasive cells as compared with high invasive melanoma cells and was further increased in the low invasive cells upon contact to collagen. Thus, in melanoma cells TIMP-2 expression levels might regulate MT1-MMP-mediated activation of proMMP-2. High invasive melanoma cells displayed increased in vitro invasiveness, which was inhibited by TIMP-2. These data indicate the importance of these enzymes for the invasion processes and support a role for MT1-MMP as an activator of proMMP-2 in malignant melanoma.     

Successful ovulation in plasminogen-deficient mice treated with the broad-spectrum matrix metalloproteinase inhibitor galardin

Many studies have suggested the hypothesis that the plasminogen activator (PA) system and the matrix metalloproteinase (MMP) system, either separately or in combination, may provide the proteolytic activity that is required for rupture of the follicular wall at the time of ovulation. Our recent studies on ovulation in plasminogen (plg)-deficient mice have, however, shown that plasmin is not required for normal ovulation, leading us to the hypothesis that MMPs may be a more important source of proteolysis for this process. To investigate the role of MMPs and also the possibility of a functional overlap or synergy between the MMP and PA systems during ovulation, we have studied ovulation efficiency in wild-type and plg-deficient mice treated with the broad-spectrum MMP inhibitor galardin. We found that in both wild-type mice and heterozygous plg-deficient (plg+/-) mice that had been treated with galardin prior to ovulation, there was a mild (18-20%) reduction in ovulation efficiency. Surprisingly, galardin treatment of plg-deficient (plg-/-) mice only caused an additional 14% reduction in ovulation efficiency as compared to vehicle-treated plg-/- mice. Our data therefore suggest that although MMPs may play a role in degradation of the follicular wall, they may not be obligatory for ovulation. In contrast to previous studies on tissue remodeling during wound healing and placental development, we have demonstrated that there is no obvious functional overlap or synergy between the PA and MMP systems, which has previously been thought to be essential for the ovulatory process.