Protease inhibitors in rheumatoid synovial fluid

Summary Paired plasma and synovial fluids from 17 patients with seropositive rheumatoid arthritis were examined for electrophoretic homogeneity/heterogeneity and enzymic inhibitory capacity of the protease inhibitors. The high degree of saturation (90%) of the polyvalent protease inhibitor ₂-macroglobulin in the rheumatoid synovial fluid contrasts sharply with the low saturation of ₁-antitrypsin. The inhibitory reactivity of the non-complexed fraction of both of these dominating antiproteases was retained (85–90%). Thus, a selective inactivation of synovial ₁-antitrypsin could not be demonstrated. ₁-Anti-chymotrypsin revealed electrophoretic homogeneity in all synovial fluids. Electrophoretic heterogeneity of the plasmin inhibitor antiplasmin was detected in the majority of synovial fluids indicating plasmin activation. The existence of a protease-antiprotease imbalance in the rheumatoid joint was indicated by the high degree of saturation of ₂-macroglobulin and a cleavage of C3 in rheumatoid synovial fluids.     

Protease activities in gastric and colon cancer tissues

Activities of cathepsin B, cathepsin L, and plasminogen activators (urinary type plasminogen activator and tissue type plasminogen activator) were assayed in homogenates of cancer tissue, normal tissue closely surrounding the cancer tissue, and normal tissue distant from the cancer tissue from 30 patients undergoing surgery for gastric cancers and 10 patients undergoing surgery for colon cancers. Activities of those proteases were also assayed in homogenates of adenoma tissue from 10 patients undergoing polypectomy for colon polyps. In the gastric cancer tissue homogenates, the activities of cathepsin B, cathepsin L and tissue type plasminogen activator were significantly higher than in normal tissues. By contrast, the activities of urinary type plasminogen activator of gastric cancer tissues were significantly lower than normal tissues. In the colon cancer tissue homogenates, the activities of cathepsin, B, cathepsin L, and urinary type plasminogen activator were significantly higher than in normal tissues. On the other hand, the activities of tissue type plasminogen activator of cancer tissues were significantly lower than normal tissues. But there were no significant differences in the activities of plasminogen activators between the cancer tissues and adenoma tissues. These results suggest that cathepsin B and cathepsin L play an important role in gastric and colon cancer proliferation and evolution, although the roles of plasminogen activators in gastric and colon cancer proliferation and evolution and in the colon adenoma-carcinoma sequence are still unknown.     

Protease inhibitors in rheumatoid synovial fluid: a quantitative analysis

The concentrations of the main endogenous inhibitors of granulocyte proteases (anti-leukoprotease, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and alpha 2-macroglobulin) were estimated in paired samples of synovial fluid and serum/plasma from seropositive rheumatoid arthritics and controls. Rheumatoid synovial fluid contained significantly higher levels of all inhibitors except antileukoprotease. The influence of the synovial membrane on these concentrations was taken into account by comparing the ratio between the observed concentration and that predicted from a certain regression curve fitted to a set of non-inhibitory reference proteins of extra-articular origin (orosomucoid, albumin, and ceruloplasmin). Divergences were interpreted as the net result of intra-articular production or consumption of the inhibitor in question. The results suggested a consumption of antileukoprotease and alpha 1-antitrypsin in the rheumatoid joint, while the increased levels of alpha 1-antichymotrypsin and alpha 2-macroglobulin probably reflected the altered trans-synovial membrane protein flux with some reservation for alpha 2-macroglobulin.     

Mast cell numbers in rheumatoid synovial tissues

Synovial biopsy specimens from 20 patients with rheumatoid arthritis were subjected to quantitative analysis for several parameters of inflammation and for enumeration of synovial tissue mast cells. Strong positive correlations were found between numbers of mast cells per cubic millimeter of synovial tissue and the following synovial tissue parameters: inflammatory index (a quantification of lymphocytic infiltration), Leu-3a grade (T helper/inducer lymphocytes), Leu-1 grade (T lymphocyte), and plasma cell grade. A strong negative correlation was found between the synovial mast cell count and the extent of sublining layer fibrin deposition. Correlations between synovial mast cell count and Leu-2a grade, ratio of Leu-3a grade:Leu-2a grade, OKM1 grade, HLA–DR grade, and lining layer thickness grade did not reach statistical significance. In addition, we obtained synovial specimens from 6 of the patients both before and after long-term therapy with oral methotrexate and from 3 of the patients before, and 1 week after, an intraarticular injection of steroid. The 3 patients who had an intraarticular steroid injection showed a 67–96% decrease in the number of synovial tissue mast cells; there was no significant change in the number of synovial mast cells in the tissues of the 6 patients who received oral methotrexate. These observations are the first documentation of a quantitative relationship between the number of mast cells and the number and phenotypic profile of infiltrating lymphocytes in an inflamed tissue, which in this case, is human synovium. Our findings suggest that mast cells are involved in the pathologic interactions in rheumatoid arthritis and might play a role in the early phases of exacerbations of disease activity.     

Protease activity in female Ornithodoros tholozani ticks.

The protease activity in guts of Ornithodoros tholozani females was studied in vitro. The intracellular protease in Ornithodoros tholozani guts has a pH optimum of about 3.0. Hemoglobin is the preferred substrate, and bovine serum albumin is digested very slowly. In this respect the protease resembles cathepsin D. Unfed ticks contain a small amount of protease in the gut. After feeding the level of protease increases gradually for several days until peak protease activity is attained. The level of gut protease activity depends on the size of the blood meal taken and on the interval after feeding. After a period of peak protease activity, the level of protease declines. The level of gut protease in unmated females (kept at 27 degrees C) did not reach prefeeding levels within 100 days. The level of gut proteolytic activity, as determined by in vitro protease assays, does not reflect the degree of blood digestion which takes place in vivo. After a period of rapid digestion, lasting for about two weeks, the undigested part of the blood meal remains unchanged in the lumen of the gut. At that time the gut tissue contains considerable levels of protease, which can be demonstrated by in vitro assays. Presumably, the protease remains active inside the gut cells, although the uptake of hemoglobin from the gut lumen has ceased. The results are compared to those obtained in other tick species.     

Immunohistochemical localization of prostaglandin e in rheumatoid synovial tissues

Synovial tissues removed at synovectomy from patients with active rheumatoid arthritis (RA) or osteoarthritis (OA) were examined for prostaglandin E (PGE) by immunohistochemical techniques using rabbit antisera specific for PGE. Marked increments of PGE were noted in RA synovia in comparison to OA, with staining concentrated in synovial lining cells, interstitial inflammatory cells, and endothelial cells of blood vessels. A correlation was noted between the degree of synovial lymphocytic infiltrate and the intensity of PGE staining. These studies provide initial cellular localization of PGE in such tissues.     

Marked elevation of serum N-acetyl-beta-D-hexosaminidase activity in rheumatoid rheumatoid arthritis

OBJECTIVE: To study N-acetyl-beta-D-hexosaminidase (NAHase) activity in the sera of rheumatoid arthritis (RA) patients and to determine its source. METHODS: NAHase activity in the serum and synovial fluid of RA patients was measured with p-nitrophenyl beta-N-acetylglucosaminide as substrate. The p-nitrophenol released was measured spectrophotometrically in an ELISA reader. Rabbit articular chondrocytes in primary culture were stimulated with interleukin-1 beta (IL-1 beta). RESULTS: Serum NAHase activity was higher in 35% of the RA patients than in healthy patients. The median activity was about twice that of the serum of healthy volunteers. RA patients with high serum NAHase activity also had more joint destruction (85%) than those with normal NAHase activity (57%, p < 0.05), but their inflammatory status was similar. The source of NAHase in RA was investigated by assaying it in RA synovial fluids (SF) and measuring its release from articular chondrocytes in primary culture. NAHase activity was detected in all 23 RA SF, at a median concentration that was 2 times that of the serum. NAHase activity in the medium of articular chondrocytes was stimulated by IL-1 beta (p < 0.005 compared to unstimulated cells), suggesting that cartilage is a source of serum and SF NAHase activity. CONCLUSION: The serum concentration of the matrix hydrolase, NAHase, is higher in destructive RA than in inflammatory RA.     

Production of agglutinators and rheumatoid factors in plasma cells of rheumatoid and nonrheumatoid synovial tissues

Immunohistochemical studies were performed in synovial tissues from 40 patients with rheumatoid arthritis (RA), 9 with juvenile rheumatoid arthritis (JRA), 7 with psoriatic arthritis, and 4 with various rheumatic diseases. Overall synthesis of IgG– and/or IgM–rheumatoid factor (RF) was found in all patients with seropositive RA and JRA, in 75% of patients with seronegative RA, and in 1 patient with psoriatic arthritis. Agglutinator producing cells were found in 77% of the samples from seropositive RA and in 44% and 56% from seronegative RA and JRA patients, respectively. The percentage of IgG plasma cells synthesizing one or more of the 5 types of agglutinators studied was approximately 10% of plasma cells synthesizing IgG–RF. Intercellular and intracellular immune complex deposits were also found in patients with seropositive and seronegative RA and JRA. These findings suggest that synthesis of agglutinators by synovial tissue plasma cells of RA and JRA patients is a distinct—but definitely less prominent—function than that of RF synthesis.     

Immunohistochemical localization of prostaglandin E in rheumatoid synovial tissues.

Synovial tissues removed at synovectomy from patients with active rheumatoid arthritis (RA) or osteoarthritis (OA) were examined for prostaglandin E (PGE) by immunohistochemical techniques using rabbit antisera specific for PGE. Marked increments of PGE were noted in RA synovia in comparison to OA, with staining concentrated in synovial lining cells, interstitial inflammatory cells, and endothelial cells of blood vessels. A correlation was noted between the degree of synovial lymphocytic infiltrate and the intensity of PGE staining. These studies provide initial cellular localization of PGE in such tissues.     

Analysis of cell populations in rheumatoid arthritis synovial tissues.

Knee synovium, taken from patients with rheumatoid arthritis at the time of arthroplasty, was studied immunohistologically. Focal perivascular lymphoid infiltrates of different sizes were examined in detail to evaluate changes in cell populations as the infiltrate size increased. T cells formed the largest component of mononuclear cells of all aggregates. The large grade 3 aggregates contained substantial numbers of B cells arranged around a central venule and cells bearing the CD45RA+ phenotype. In contrast, the small grade 1 aggregates contained few B cells and the T-cell population contained relatively greater numbers of CD8+ cells. Cells bearing the CD45RO+ phenotype exceeded CD45RA+ cells in grade 1 aggregates. Detailed analysis of mononuclear cell aggregates of different sizes in the rheumatoid synovium suggests that the composition of each aggregate depends on the total number of mononuclear cells it contains.     

Identification of immunoglobulins and complement in rheumatoid articular collagenous tissues

Ninety-three patients with a variety of joint diseases were studied for evidence of immune complexes in articular collagenous tissues. Frozen sections of freshly obtained biopsies of hyaline articular cartilage and menisci were stained with fluoresceinated monospecific antisera for evidence of human immunoglobulins (IgG, IgM, IgA) and the β_1c component of complement. The criterion for the presence of complexes was the staining of two or more immunoglobulins and β_1c in an identical location of sequentially cut sections. Of the 42 patients with rheumatoid arthritis (RA) 83% were positive by this criterion. In those with classic RA the incidence was 92%. Sixteen patients with fresh joint trauma or nonarthritic disease had negative findings. Among 26 patients with noninflammatory disease, 4 of 8 with polyarthritis whose features suggested primary degeneration, 1 of 11 patients with secondary degenerative arthritis, and a single case of synovial osteochondromatosis had positive findings. Among 9 patients with miscellaneous inflammatory arthritides, all of 3 with psoriatic arthritis were negative; however 2 of 6 with other inflammatory arthritides were positive. The findings in classic RA suggest that immune complexes are deposited in the articular collagenous tissues. The persistence of these complexes may play a significant role in the chronicity of the synovitis.     

Protease content and fibrinolytic activity of human leukocytes

The fibrinolytic activity of human leukocytes was studied by the fibrin platemethod and by the histochemical fibrin slide method, using plasminogen-richand plasminogen-free fibrin substrates. Lysis is caused by a protease. Plasminogen activator is absent. The slide method showed the effect of leukocytes tobe weak in comparison to that produced by the plasminogen activator incapillary endothelial cells invading fibrin deposits in the body.

Submitted on April 26, 1966 Accepted on June 26, 1966     

Increased collagenase activity in human rheumatoid meniscus

Collagenase activity of the knee joint menisci of patients suffering from rheumatoid arthritis was approximately 3-fold higher than that found in menisci of control patients. The mean collagenase activity in the macroscopically more diseased parts of the rheumatoid menisci was significantly higher than that in the less damaged areas. The specific degradation products resulting from the cleavage of human meniscoid type II collagen by rheumatoid meniscoid collagenase were demonstrated by SDS-polyacrylamide gel electrophoresis. Addition of N-ethylmaleimide, which activates latent mammalian collagenases, did not further increase collagenase activity in rheumatoid menisci. Thus in rheumatoid meniscus, collagenase may be synthesized and then activated, probably by proteolytic enzymes involved in the inflammatory reaction.