Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice.

Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-α and interferon-γ were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.

Sonic extracts from a bacterium related to periapical disease activate gelatinase A and inactivate tissue inhibitor of metalloproteinases TIMP-1 and TIMP-2.

AIM: To examine the effects of sonicated bacterial extracts (SBEs) from three related to periapical disease bacteria (Porphyromonas gingivalis, P. endodontalis and F. nucleatum) on the activation of matrix metalloproteinase (MMP-2) and the inactivation of tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2). METHODOLOGY: Each SBE was added to cultures of human periodontal ligament (PL) cells or HT1080 cells and their supernatants were analysed by zymography for MMP-2. Each SBE was added to PL cell cultures, and the amount of TIMP-1 was determined by ELISA. P. gingivalis SBE was incubated with HT1080 cell culture supernatants, and the amounts of TIMP-1 and TIMP-2 were determined by ELISA. Statistical analysis was performed with the paired Student's t-test. RESULTS: In extracts of PL cells that had been incubated in the presence of P. gingivalis SBE, one representing pro-MMP-2 (72 kDa) and a band corresponding to the active MMP-2 (66 kDa) were observed; but in the other extracts it was not detected. When HT1080 cells were treated with P. gingivalis SBE, the pro-MMPs was processed into 86- and 66-kDa fragments, but in the other extracts, the processing did not occur when the other SBEs were used. When PL cells were incubated with the same SBEs, the amount of TIMP-1 was markedly decreased (P < 0.01), but in the other extracts, it was not. The amounts of both TIMP-1 and TIMP-2 were decreased in a dose-dependent manner when HT1080 cell culture supernatant was incubated with P. gingivalis SBE. CONCLUSIONS: These findings suggest that P. gingivalis SBE may cause connective tissue to be destroyed, contributing to the process of periapical disease, by activating pro-MMP-2 as well as by inactivating TIMP-1 and TIMP-2.

Cloning and sequencing of the cDNA encoding a mouse tissue inhibitor of metalloproteinase-2.

A mouse cDNA encoding a tissue inhibitor of metalloproteinases (mTIMP-2) was cloned and the 1695-bp sequence was determined. While high homology was observed with the sequences encoding the human (hTIMP-2) and bovine (bTIMP-2) genes, mTIMP-2 contained a long 3'-nontranslated region not observed in the hTIMP-2 or bTIMP-2 genes.

Tissue inhibitor of metalloproteinases from human bone marrow stromal cell line KM 102 has erythroid-potentiating activity, suggesting its possibly bifunctional role in the hematopoietic microenvironment.

In this study, we demonstrated that tissue inhibitor of metalloproteinases (TIMP) produced by human bone marrow stromal cell line KM-102 had erythroid-potentiating activity (EPA) which stimulates the proliferation of erythroid progenitor cells. We, then, propose a scheme for the bifunctional role of TIMP/EPA in hematopoietic microenvironment, that is, the maintenance of the integrity of bone marrow matrix and the proliferation of erythroid progenitor cells proceeding on the matrix.

Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases.

Tissue inhibitor of metalloproteinases (TIMP) from cultured bovine dental pulp inhibits human rheumatoid synovial matrix metalloproteinase 3 (MMP-3) with a stoichiometry of 1:1 on a molar basis. Among the serine proteinases examined, human neutrophil elastase, trypsin and alpha-chymotrypsin destroyed the inhibitory activity of TIMP against MMP-3 by degrading the inhibitor molecule into small fragments. In contrast, the inhibitory activity of TIMP was not significantly reduced by the actions of cathepsin G, pancreatic elastase and plasmin. These data indicate that neutrophils which infiltrate tissues in various inflammatory conditions may play an important role in regulating TIMP activity in vivo through the action of neutrophil elastase.

A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells.

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.

Stimulation of TIMP-1 and metalloproteinase production in co-cultures of human tumor cells and human fibroblasts.

The co-cultures of five different human tumor cell lines with human normal fibroblasts significantly stimulated the production of tissue inhibitors of metalloproteinases-1 (TIMP-1) when compared to cultures of individual cells. In the co-culture of T24 human urinary bladder carcinoma cells and CCD18 human fibroblasts, production of both TIMP-1 and metalloproteinases was stimulated, and the stimulatory effects were dependent on the cellular ratio between the fibroblasts and carcinoma cells. On day 6 of culture, collagenase and stromelysin were stimulated at a ratio of CCD18 fibroblasts to T24 cells of 1:0.1, while the maximum TIMP-1 production occurred at a ratio of 1:1. Thus, the cellular ratio in the interaction of carcinoma cells with host fibroblasts affects the production of TIMP-1 and metalloproteinases and hence modulates the balance between them.

Matrix metalloproteinase 2 and 9 in esophageal cancer.

To search for the biochemical properties of esophageal carcinoma relevant to its aggressive behavior, we studied metalloproteinases released from surgical specimens of the carcinoma. In an assay with [H-3]-labeled gelatin, esophageal carcinoma tissues showed gelatinolytic activities clearly higher than those of paired normal mucosae. EDTA and TIMP-1 could strongly suppress these activities, suggesting that the activities belong to metalloproteinases. In addition, levels of TIMP-1 expression did not show good correlation with these activities, suggesting that tumor-specific elevation of gelatinolytic activity depended on metalloproteinase per se rather than the suppression of TIMP-1-secretion. By zymographic analysis, two gelatinase bands of 82- and 62-kDa were found specifically in carcinoma tissues, in addition to the other 6 bands detected both in normal and carcinoma tissues. Immunoprecipitation and immunoblotting of gelatinases with anti-MMP-9 or anti-MMP-2 monoclonal antibody, and treatment of the enzymes with APMA showed that these 82- and 62-kDa gelatinases were cleaved products of MMP-9 and MMP-2, respectively. These results imply that enhanced secretion and proteolytic activation of MMP-2 and MMP-9 take place specifically in the esophageal carcinoma tissues. Moreover, the levels of gelatinolytic activity expressed good correlation with the organ metastasis rate of the carcinoma, suggesting that MMPs play an important role in tumor metastasis.

Purification and characterization of bovine dental pulp collagenase inhibitor.

Root pulps from bovine unerupted wisdom teeth produce a potent collagenase inhibitor together with latent collagenase when cultured in Eagle's minimal essential medium (Biochem. Int. 5, 763, 1982). The inhibitor was purified more than 700-fold from the explant medium using Con A-Sepharose, Ultrogel AcA 44 and DE-52 cellulose columns. It showed a single band (MW = 36,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but showed multiple bands on basic (pH 8.3) polyacrylamide gel electrophoresis and electrofocusing. The inhibitor is a sialo-glycoprotein containing approx. 20% carbohydrate by weight and its composition suggests that it contains complex-type oligosaccharides. The electrophoretic heterogeneity of the inhibitor was proved to be due to the attachment of different numbers of sialic acid residues. All the SH groups were demonstrated to exist as six disulfide linkages which might be involved in the inhibitory activity. The bovine pulp inhibitor does not combine with collagen. The addition of the inhibitor to activated collagenase resulted in dose-dependent inhibition of the enzyme activity, but the interaction between the inhibitor and activated collagenase is not tight enough for the complex to remain intact during gel filtration column chromatography. A rabbit antiserum was prepared against the inhibitor, and immunoglobulin purified from the antiserum can completely abolish the inhibitory activity of the inhibitor.

Purification and characterization of bovine dental sac collagenase.

1. Active type collagenase was purified as much as 140-fold from the explant medium of bovine dental sacs and showed a single band on disc gel electrophoresis. Purified collagenase cleaved native collagen at only one locus under physiological conditions, but hydrolyzed neither gelatin nor alpha-casein. The optimal pH was about 7.8. 2. The molecular weight of active type enzyme was 35,000 by gel filtration and 34,000 by gel electrophoresis. The activation of latent type of collagenase resulted in the reduction of molecular weight from 45,000 to 38,000 by gel filtration. 3. A small but detectable amount of collagenase was directly extracted from frozen and thawed bovine dental sacs. In explant media of frozen and thawed tissue and fresh tissue with actinomycin D, some activity was detected for the first 2 days, but essentially no collagenase activity was detected in the explant medium after day 3. 4. The latent type collagenase was activated by trypsin, 4-aminophenylmercuric acetate (4-APMA), thiocyanate and deoxycholate (DOC). DOC showed irreversible dissociation of latent type enzyme in similar fashion to that exerted by 4-APMA. 5. The purified collagenase was inhibited by bovine serum, EDTA, o-phenanthroline, cysteine and dithiothreitol.

Phagocytosis of titanium particles and necrosis in TNF-alpha-resistant mouse sarcoma L929 cells.

In the oral cavity, titanium is an excellent biocompatible material. However, it is reported that high ratios of intracellular titanium particles can cause cell apoptosis or necrosis by as-yet unknown mechanisms. The purpose of this study was to investigate the response of tumor necrosis factor alpha (TNF-alpha)-resistant L929 fibroblasts to titanium particles. Cells were cultured in Eagle's medium supplemented with fetal bovine serum and L-glutamine. Titanium particle sizes were less than 9 micro. Cytotoxicity was assayed by a cell counting kit, trypan blue dye exclusion test and lactate dehydrogenase (LDH) leakage. The production of reactive oxygen species (ROS) was detected by a confocal laser scanning microscope (CLSM) using dichlorofluorescein diacetate as a fluorescent probe. Morphology was viewed by a CLSM and with an X-ray microanalyser (XMA). When titanium particles were added to cells, the viability decreased to around 50% at a particle concentration of 2.0%. The number of dead cells and LDH activity in the culture media increased significantly between 1 and 2 days. However, formation of active oxygen species did not occur, since no dichlorofluorescein fluorescence was observed. A scanning electron photomicrograph (SEM) revealed a large number of particles covering or adhering to cellular components in lysed cells compared with flattened control cells attached to the substrate. The XMA showed that the titanium accumulation was coincident with the deformed cell shape. The CLSM also confirmed that particles were within the cells. From these results it was concluded that titanium particles ingested in large quantities into the cell induced necrosis by a pathway other than by producing ROS.

Immunohistochemical localization of collagenase inhibitor in bovine dental pulp, pulp cells in monolayer culture, and in some oral connective tissues.

The development of an antiserum, monospecific to the collagenase inhibitor, from bovine dental pulps permitted localization of immunoreactive inhibitor protein, by means of both immunofluorescence and immunoperoxidase-staining techniques in sections of bovine dental pulps. The immunoreactive inhibitor protein in bovine dental pulps is present both in cells and extracellular matrices. When cultured in Eagle minimal essential medium, coronal pulps from bovine-unerupted teeth were shown, by assay of the medium, to produce only about 1/10 of the amount of inhibitor produced by the root pulps. When compared by immunohistochemical observation, however, essentially no differences in fluorescent activity was found between coronal and root pulps. Specific cytoplasmic staining was seen both in explanted root-pulp tissues and in immature fibroblast-like pulp cells from monolayer cell cultures of bovine root pulps, which indicate that the pulp cells are responsible for inhibitor production. Sections of dental follicle and gingiva from the same animal, showed a distribution of immunoreactive inhibitor protein similar to that in dental pulps.

Characteristics of collagenase in experimental periapical granulomas from the teeth of dogs.

Periapical granulomas were induced, with a success rate of about 60% (66 granulomas produced out of 109 roots treated) in mandibular premolars. The average wet weight of the granulomas, 46.1 +/- 34.5 mg (mean +/- SD, n = 22), was sufficient to allow individual specimens to be used for most of the biochemical analyses. High collagenase activity was extracted directly from the granulomas with 4 M urea solution. The enzyme was a typical animal collagenase (EC 3.4.24.7) which clove native collagen molecules into three-quarter (alpha A) and one-quarter (alpha B) length fragments. The collagenase was activated by 1 mM p-aminophenylmercuric acetate. This activated enzyme broke down collagen I rather than collagen III preferentially, which is similar to the activity of human polymorphonuclear leucocyte collagenase. The molecular weights of the latent and activated collagenases were 67 and 49 K, respectively.

CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages.

Interferon-inducible protein-10 (IP-10)/CXCL10, which is a ligand for CXC chemokine receptor 3 (CXCR3), is known to be involved in the pathogenesis of pulmonary sarcoidosis. However, the roles of monokine induced by interferon gamma (Mig)/CXCL9 and interferon-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, which are also CXCR3 ligands, remain unclear. Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in both bronchoalveolar lavage fluid (BALF) and serum in patients with pulmonary sarcoidosis were measured by enzyme-linked immunosorbent assay (ELISA). The expression of these chemokines in alveolar macrophages was examined using ELISA, quantitative real-time polymerase chain reaction and immunostaining. In BALF, Mig/CXCL9 and IP-10/CXCL10 were significantly elevated in stage II sarcoidosis as compared with the levels in healthy volunteers. In serum, Mig/CXCL9 and I-TAC/CXCL11 were increased in stage II of the disease. The levels of all CXCR3 ligands in BALF were correlated with the numbers of both total and CD4(+) lymphocytes. Alveolar macrophages were stained positive for all CXCR3 ligands and produced increased amounts of these chemokines. Positive staining of the three chemokines was also observed in the epithelioid and giant cells in the sarcoid lungs. These findings suggest that Mig/CXCL9 and I-TAC/CXCL11 as well as IP-10/CXCL10 play important roles in the accumulation of Th1 lymphocytes in sarcoid lungs.

Synthesis of latent collagenase and collagenase inhibitor by bovine aortic medial explants and cultured medial smooth muscle cells.

Bovine aortic medial tissue and medial smooth muscle cells were demonstrated for the first time to synthesize a latent collagenase together with collagenase inhibitor in culture. Molecular weights of the latent collagenase and its inhibitor derived from aortic medial tissue explant were estimated to be about 52 K by gel filtration and 26.5 K by electrophoresis, respectively. Activated aortic collagenases cleaved type I collagen in solution into 3/4 (alpha A) and 1/4 (alpha B) length cleavage fragments and were inhibited by EDTA, o-phenanthroline, dithiothreitol, bovine serum, and highly purified dental pulp and aortic collagenase inhibitors. The aortic inhibitors showed inhibitory activity against all the animal collagenases tested, except for bacterial collagenase. Double-immunodiffusion analysis using a monospecific antiserum prepared against dental pulp inhibitor showed that the aortic inhibitors are immunologically identical to the pulp inhibitor. Using the same antiserum, we found immunoreactive collagenase inhibitor protein to be localized along the collagen fibers between elastic membranes in aortic medial tissue.

Extraction of latent-type collagenase from cultured bovine dental pulps with NaCl or urea or by collagen degradation.

All of the collagenase activity extracted from cultured bovine dental pulp tissue with NaCl or urea solutions was due to enzyme in a latent form and identified as a typical animal collagenase. Isotonic sucrose solution solubilized no detectable collagenase activity from cultured dental pulps. Also, no collagenase activity was extracted from either fresh bovine dental pulps or those cultured in Tyrode's solution containing 50 micrograms/ml cycloheximide. A two-day difference was observed between the appearance of collagenase activity in the cultured pulps and in the culture medium. The activity profiles in the culture media showed essentially no difference with or without the addition of cyclohexamide on and after the 10th day of culture, indicating that the biosynthesis of collagenase in the cultured pulp might have terminated around that time. About 80% of the total pulp collagenase activity was extracted by a bacterial collagenase procedure. Nearly half (43.5%) of the collagenase activity extracted from the cultured pulps with NaCl or urea solution was precipitated with collagen molecules in the presence NaCl, and most of the precipitated activity retained in the precipitate even after washing it with NaCl-buffer solution. These facts suggest a close association of collagenase with the collagen in cultured dental pulp tissue.

Salivary gland morphogenesis: possible involvement of collagenase.

When 12-day rudiments of mouse submandibular glands were cultured, they formed an average two clefts within 24 h. The average number of the clefts, however, increased up to five in the presence of bovine tissue inhibitor of metalloproteinases (TIMP). Contrary to this, either bacterial or bovine interstitial collagenase in the medium completely inhibited the cleft formation of the glands. Electron microscopic observations revealed that the amounts of collagen bundles at the cleft points significantly increased with TIMP, but decreased with bacterial collagenase. These results strongly support the idea that endogenous collagenase regulates the cleft formation through the modulation of fibrillar architectures containing collagen in the extracellular matrix. Recently, our immunohistochemical studies clarified that collagen III, known to be rich in embryonic tissue, accumulated preferentially at the cleft points of the epithelium, and may play a crucial role in submandibular gland morphogenesis.

Dual regulation of collagenase activity in bovine dental pulps.

A monospecific rabbit anti-inhibitor serum did not cross-react with either intact latent collagenase or with the one pretreated by p-aminophenylmercuric acetate. Furthermore, immunoglobulin G purified from the antiserum quantitatively inhibited the anti-collagenolytic activity of the inhibitor in either the presence or absence of p-aminophenylmercuric acetate. But the immunoglobulin G did not affect latent collagenase at all. These facts, along with other lines of evidence, strongly support the possibility that the inhibitor may not be responsible for the latency of collagenase and allow us to propose a dual regulatory mechanism of collagenase activity; that is, an inactive form, per se, may thus be additionally kept in the inactive state by the existence of an inhibitor which is synthesized in the same pulp tissues.