Low-dose pulse methotrexate inhibits articular destruction of adjuvant arthritis in rats.

The purpose of this study was to determine whether low-dose methotrexate pulse therapy, which had recently become important in the treatment of rheumatoid arthritis, was effective for controlling the progression of articular destruction in rats with adjuvant arthritis. Intraperitoneal methotrexate at a dose of 0.05 or 0.1 mg kg-1 twice weekly inhibited inflammation in rats with adjuvant arthritis, as shown by reduction of the hind-paw volume. Methotrexate also inhibited articular destruction, as shown by X-ray findings. Although the mechanisms by which low-dose pulse methotrexate acts on rat adjuvant arthritis are still unclear, our results imply that it might effectively slow the progression of articular destruction in rheumatoid arthritis in man. In addition, assessment of articular destruction in this animal model might be useful when evaluating new treatments for rheumatoid arthritis.

The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells.

Thrombomodulin (TM) is an anticoagulant endothelial cell surface glycoprotein containing six tandem epidermal growth factor (EGF)-like structures. We prepared a recombinant TM peptide (rTME1-6, from R214GHWA to DSGK466 of native TM) composed of these six EGF-like structures and investigated the effect of rTME1-6 peptide on the growth of the Swiss 3T3 fibroblast cell line. It was found that rTME1-6 induced proliferation of Swiss 3T3 cells and accelerated [3H]thymidine uptake into their DNA. [3H]Thymidine uptake increased in a dose-dependent manner, plateauing at 50 ng/mL rTME1-6, which was 1.8 times the control level. rTME1-6 peptide (50 ng/mL) also accelerated the DNA synthesis of human dermal fibroblasts (HDFs), A549 (a human lung cancer cell line), HepG2 (a human hepatocarcinoma cell line), and U937 cells (a human monocytic cell line) to 1.5, 1.6, 1.4, and 1.2 times the control level, respectively. The magnitude of the acceleration of DNA synthesis in Swiss 3T3 induced by rTME1-6 was approximately 20% of that of EGF on a molar basis. The uptake of [3H]thymidine was accelerated synergistically by coculture of the cells with rTME1-6 and insulin, similar to the coculture with EGF and insulin. The effects of rTME1-6 were abolished by addition of polyclonal antihuman TM IgG, whereas the actions of insulin and EGF were not influenced. Glucose uptake in Swiss 3T3 cells also increased 1.6 times over control levels by culture with 50 ng/mL rTME1-6 (1.25 nmol/L), compared with 2.7 times by 10 ng/mL EGF (1.66 nmol/L). Binding of [125I]EGF (0.5 ng/mL, 0.083 nmol/L) by the cells was inhibited by about 60% by addition of an eight-fold molar excess of nonlabeled EGF (0.664 nmol/L), whereas no inhibition of [125I]EGF binding was observed, even in the presence of a 1,000-fold molar excess (83 nmol/L) of rTME1-6. Specific binding of [125I]rTME1-6 on the cells showed a saturation curve, and the apparent concentration of rTME1-6 required for half maximum binding of the peptide on the cells was calculated to be 31.5 ng/mL. Thus, the overall results indicated that the rTME1-6 peptide had mitogenic activity for Swiss 3T3 cells, accelerated DNA synthesis and glucose uptake, and that the mitogenic activity might be mediated by binding of the peptide to a specific site different from the EGF receptor.

The role of leukocyte factors on uterine cervical ripening and dilation.

To clarify the role of leukocytes effused into uterine cervix at term pregnancy, the effect of conditioned medium (MCM) of rabbit peritoneal macrophages on the production of specific collagenase by cervical cells was investigated, in vitro. MCM stimulated uterine cervical cells to induce a 10-fold increase in collagenase production as compared with the control. Similarly, production of gelatinolytic metalloproteinase (an endogenous procollagenase activator) increased to about 4-fold of the control cultures, whereas MCM did not affect [3H]thymidine incorporation into DNA. The enhancement of collagenase production was depressed by the treatment of cells with 10(-6) M cycloheximide. The MCM also contained lymphocyte-activating activity (interleukin-1). These data suggest that rabbit uterine cervical cells are able to produce both specific collagenase and gelatinolytic metalloproteinase in response to interleukin-1, and that leukocytes effused into the cervix may participate in the ripening and dilation of uterine cervix at term pregnancy.

Dehydroepiandrosterone sulfate stimulates collagenase synthesis without affecting the rates of collagen and noncollagen protein syntheses by rabbit uterine cervical fibroblasts.

The effects of dehydroepiandrosterone 3-sulfate (DHAS) and 17 beta-estradiol (E2) on collagen and noncollagen protein syntheses by rabbit uterine cervical cells were studied, and their effects on latent collagenase synthesis were compared. DHAS (1 X 10(-6) M) stimulated the synthesis of latent collagenase and did not affect the cell number and [3H]thymidine incorporation into DNA, whereas E2 had no effect on collagenase synthesis. On the other hand, neither DHAS (1 X 10(-6) M) nor E2 (1 X 10(-10)-1 X 10(-6) M) showed effects on collagen and noncollagen protein syntheses. These results suggest that the stimulative effect of DHAS on cervical ripening is mediated mainly by the stimulation of collagen catabolism, and that E2 does not concern the changes in the concentration of collagen and noncollagen protein in uterine cervix of the rabbit during pregnancy at term.

Procollagenase activator produced by rabbit uterine cervical fibroblasts.

Culture medium from rabbit uterine cervical fibroblasts contained a procollagenase and a neutral proproteinase which acts as a procollagenase activator. These two proenzymes have been purified by a combination of ion-exchange, affinity and gel chromatographies. The purified neutral proproteinase showed Mr 60,000 with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This neutral proproteinase was activated by trypsin, 4-aminophenylmercuric acetate (APMA) and plasmin, and the active species of the proteinase had Mr 53,000 when activated by APMA; kallikrein and urokinase did not activate this proproteinase. The purified neutral proteinase was inhibited by EDTA, 1,10-phenanthroline and rabbit plasma, but not by serine proteinase inhibitors, suggesting that this proteinase is a metal-dependent proteinase. The purified enzyme could also degrade gelatin, casein, proteoglycan and type IV procollagen. The purified procollagenase had Mr 55,000 and was activated by trypsin, APMA and the active neutral proteinase. These activations were accompanied by decrease in Mr, and the activated species had an Mr which was approx. 10,000 less than that of the procollagenase. In particular, procollagenase activation with neutral proteinase depended on incubation time and proteolytic activity of proteinase. These results indicate that activation of procollagenase by the rabbit uterine neutral proteinase is related to limited proteolysis in the procollagenase molecule.

Hormonal control of collagenase inhibitor production in rabbit uterine cervical fibroblast-like cells.

Rabbit uterine cervical fibroblast-like cells maintained in fetal calf serum-free medium were found to biosynthesize and secrete a collagenase inhibitor into the culture medium. All the properties of this inhibitor were similar to those that have been described so far for the tissue inhibitor of metalloproteinases. Both progesterone and 17 beta-estradiol significantly increased the level of collagenase inhibitor without the proliferation of cells. These data suggest that both progesterone and estradiol regulate collagenolysis in the uterus bifunctionally by acceleration of the inhibitor production in addition to their known inhibitory actions towards collagenase biosynthesis.

Effects of dehydroepiandrosterone sulphate on the production of collagenase and gelatinolytic metalloproteinase by rabbit uterine cervical cells in primary cultures.

Cells from pregnant rabbit uterine cervix in primary cultures produced specific collagenase and gelatinolytic metalloproteinase, both in latent forms. The two enzymes were separated by CM-52 cellulose chromatography. Dehydroepiandrosterone 3-sulphate (DHAS) stimulated the production of the latent collagenase by the cells in a dose-dependent manner and the maximal effect, which was about 2-fold of control cultures, was observed when 1 X 10(-6) M DHAS was added to the medium containing 10% (v/v) foetal calf serum (FCS). DHAS also stimulated the production of the gelatinolytic metalloproteinase. The significant stimulative effects were also confirmed when the cells were incubated with 1 X 10(-7) M DHAS in the medium containing 9% (v/v) dextran-coated charcoal treated FCS and 1% (v/v) FCS. The uptake of [3H]DHAS by the cells from pregnant rabbits was 5-10-fold greater than that from nonpregnant rabbits. When confluent cultures were incubated with 6.5 X 10(-8) M [3H]DHAS for 2 d, 3.4% and 0.03% of total radioactivity added were accumulated as [3H]oestradiol-17 beta ([3H]E2) in the medium and cell layer, respectively. Nevertheless, the addition of 1 X 10(-9) M and 1 X 10(-11) M E2 or dehydroepiandrosterone (DHA) to the confluent cultures caused no significant effect on the collagenase production. These results strongly suggest that the stimulative effect of DHAS on the production of collagenase and gelatinolytic metalloproteinase by the rabbit uterine cervical cells was due to unchanged DHAS and not due to E2 or DHA converted from DHAS.

Effect of dehydroepiandrosterone sulfate on collagenase production in rabbit uterine cervix culture.

Rabbit uterine cervical explants were found to produce a typical collagenase, latent form, in tissue culture, 4-Aminophenylmercuric acetate and trypsin were potent activators of the enzyme. The enzyme was purified simply in one step of CM-52 cellulose ion-exchange chromatography, and then further characterized. Addition of dehydroepiandrosterone sulfate (DHAS) to culture medium significantly stimulated collagenase production, but DHAS did not directly activate the enzyme. In addition, dehydroepiandrosterone and 17 beta-estradiol, the main metabolites of DHAS in vivo, depressed enzyme production. Our previous result, that increases in cytoplasmic DHAS-binding protein in rabbit uterine cervices parallel the progress of pregnancy, and these results suggest that DHAS might have direct actions toward cervical ripening.

Partial purification and characterization of gelatinase and metal dependent peptidase from rabbit uterus and their synergistic action on gelatin in vitro.

Two metal dependent proteases were investigated in rabbit uterus using a synthetic substrate, 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gin-D-Arg (Dnp-peptide). One was extracted by homogenization in 50 mM Tris-HCl/0.25% Triton X-100/100 mM CaCl2, pH 7.4, from rabbit uterus, and the other from the insoluble fraction by heating at 60 degrees C for 4 min in 50 mM Tris-HCl/100 mM CaCl2, pH 7.4. Both enzymes were partially purified by gel filtration, ion-exchange chromatography and chromatofocusing, and further characterized. The soluble enzyme was a metal dependent peptidase, and hydrolysed 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg as well as Dnp-peptide. Its molecular weight was about 7.0 X 10(4), and the cleavage sites for Dnp-peptide were Gln-Gly and possibly Gly-Ile in the ratio of 3:1. On the other hand, the enzyme extracted from the insoluble fraction was present as a latent form, and was found to be activated by 4-aminophenylmercuric acetate but not by trypsin. The activated enzyme hydrolysed gelatin, in addition to Dnp-peptide, indicating that the enzyme is a gelatinase. The molecular weight was about 7.4 X 10(4) for the active form, and the cleavage site for Dnp-peptide was only the Gly-Ile bond. The rabbit uterine metal dependent peptidase obtained here had negligible activity on gelatin, but once it had been cleaved by the above gelatinase, the presence of metal dependent peptidase accelerated the action of gelatinase. Thus, the actions of both enzymes on gelatin were found to be synergistic.