K. Fehr

Interleukin 1 Activity in the Synovial Fluid of Patients with Rheumatoid Arthritis

SummaryThe synovial fluids (SF) of patients with rheumatoid arthritis (RA) were investigated for their effects on thymocytes of C3H/HeJ mice. Of the 20 SF tested, 17 (85%) showed an augmentation of the phytohaemagglutinin (PHA) induced thymocyte stimulation. Out of 16 SF of patients with osteoarthrosis, such an activity was detected in only one (6.25%). Further characterisation of the amplification factor revealed that (1) the SF of RA patients augmented both the PHA and the Concanavalin A response of the thymocytes (2) in the absence of mitogens, SF-treated thymocytes showed an increased uptake of 3H-thymidine, (3) the SF did not propagate the growth of an interleukin 2 dependent ovalbumin specific T cell clone, but (4) the SF were found to be required for optimal interleukin 2 release by spleen cells stimulated with suboptimal doses of lectin. Based on these biological effects the factor in the SF of RA patients is suggested to represent an interleukin 1 (IL-1). IL-1 produced in cultures by activated macrophages has been shown to stimulate T and B cell functions and to induce the production of collagenase and prostaglandins by cultured synovial cells. Both properties of IL-1 could be relevant in the pathogenesis of RA.

Inhibition of human elastase from polymorphonuclear leucocytes by a glycosaminoglycan polysulfate (Arteparon

Abstract Human lysosomal elastase from polymorphonuclear leucocytes is inhibited by the glycosaminoglycan polysulfate Arteparon. The inhibition is of a mixed type: hyperbolic uncompetitive. The interaction between inhibitor and enzyme occurs via electrostatic forces, and the binding is very tight ( K i ranges from 10 −8 to 10 −7 M). Depending on the chain length of the polysulfated glycosaminoglycan, two, three or five enzyme molecules can be tightly bound by a single inhibitor molecule. These findings suggest a possible therapeutic role of Arteparon as an inhibitor of elastase, a potent mediator of connective tissue breakdown, since the enzyme is inhibited by a drug concentration as small as 1 μg/ml or less.

Analysis of glycosaminoglycans in human serum after oral administration of chondroitin sulfate

SummaryChondroitin sulfate was administered orally to six healthy volunteers, six patients with rheumatoid arthritis and six patients with osteoarthritis. Blood was collected at intervals before and after treatment and the glycosaminoglycan concentration was analyzed in serum using a sensitive assay based on the metachromatic reaction with 1,9-dimethylmethylene blue. The glycosaminoglycan concentration in serum before and after ingestion of chondroitin sulfate was statistically unchanged in all of the subjects studied. We suggest that chondroprotection by orally administered chondroitin sulfate is a biologically and pharmacologically unfounded theory. Any possible benefit to osteoarthritic patients after ingestion of chondroitin sulfate should be sought at the gastrointestinal rather than at the plasmatic or articular cartilage level.

Degradation in vivo of articular cartilage in rheumatoid arthritis and juvenile chronic arthritis by cathepsin G and elastase from polymorphonuclear leukocytes

SummaryPeroxidase-anti-peroxidase (PAP) staining and specific antibodies against cathepsin G and elastase from polymorphonuclear leukocytes (PMN) were applied to pannus-free and microscopically intact superficial articular cartilage. Restricted local deposits containing cathepsin G and elastase were found in three of ten patients with seropositive rheumatoid arthritis (RA), in one of three patients with seronegative RA and in one patient with juvenile chronic arthritis (JCA). Similarly, localized deposits of IgG and C3 were found in the patients with seropositive RA and JCA, but not in the patient with seronegative RA. Adjacent sections exhibited esterase activity in and around the PMN. In proteinase-positive areas from patients with seropositive RA the inhibitors α1-proteinase inhibitor (α1-PI) and α2-macroglobulin (α2-MG) were present in two of three and one of three patients, respectively. In JCA only α1-proteinase inhibitor was present, and in seronegative RA no inhibitors were found. No staining of articular cartilage was observed in a patient with psoriatic arthritis. One of three cases with osteoarthritis exhibited patchy superficial staining for IgG only. In articular cartilage covered by pannus, in three patients with seropositive RA, in one with seronegative RA and in the patient with JCA a few regions with variably dense PMN infiltrates were observed. Cathepsin G, elastase and esterase activity were found in and around the PMN. In one of the three patients with seropositive RA the adjacent cartilage-pannus junction exhibited distinct staining for cathepsin G and elastase, but not for IgG/C3 and proteinase inhibitors. The findings suggest that cathepsin G and elastase from PMN are involved in the breakdown of rheumatoid articular cartilage in the presence, as well as in the absence, of entrapped immune-complex-like material, and that proteinase inhibitors are often lacking in such regions.

Inhibition of human elastase from polymorphonuclear leucocytes by gold sodium thiomalate and pentosan polysulfate (SP-54®)

Abstract Human lysosoma) elastase, the serine proteinase from the azurophil granules of polymorphonuclear leucocytes, is inhibited by gold thiomalate and pentosan polysulfate (SP-54®). The kinetic mechanism of the inhibition was studied using succinyl-alanyl-alanyl-prolyl-valyl-4-methyl-7-coumarylamide and (t-butyloxycarbonyl-alanyl-p-nitrophenylester as subsrates. The degree of inhibition was also tested using insoluble elastin as substrate. Independent of the substrate, the maximal inhibition of elastase by gold thiomalate and pentosan polysulfate was 40% and 60%, respectively. Pentosan polysulfate behaved as a simple intersecting, hyperbolic, non-competitive inhibitor and k′i. the dissociation constant of the E-S-I complex, was 1.8 × 10−7M. The interaction between inhibitor and enzyme is driven by electrostatic forces. Gold thiomalate showed a hyperbolic mixed type inhibition (intersecting, slope-hyperbolic, intercept-hyperbolic, non-competitive inhibition) with k′i = 5.4 × 10−5M. The overall kinetic mechanism of lysosomal elastase conforms to that of other serine proteinases. With both the ester and peptide substrates the rate limiting step of the reaction has been identified with the formation of the acyl-enzyme.

Kinetics of the Different Susceptibilities of the Four Human Immunoglobulin G Subclasses to Proteolysis by Human Lysosomal Elastase

Human lysosomal clastase cleaves human monoclonal IgG into components that closely resemble the fragments produced by papain digestion IgG1 produced Fab, Fe and Fab‐Fc fragments; cleavage of IgG2 produced Fab‐Fc, Fab and Fc fragments: IgG3 gave rise to almost pure Fab und Fch (Fc covalently Joined to the extended hinge region polypetide of IgG3). and from IgG4, F(ab)2, Fab and Fc fragments were recovered. The relative susceptibilities of the four human IgG subclasses to proteolytic attack by elastase were studied kinetically and showed the following decrease order of susceptibility: IgG3 >IgG1 >IgG2 >IgG4. The Fab fragment from papain digestion of IgG1 and the corresponding fragment from clastase digestion showed indistinguishable molecular weights and immunochemical identity

Degradation in vivo of articular cartilage in rheumatoid arthritis by leucocyte elastase from polymorphonuclear leucocytes

SummaryUsing a specific substrate, no leucocyte elastase activity could be detected in 55 synovial fluids, including 29 from patients with rheumatoid arthritis (RA). However, a high percentage of samples contained phagocytic inclusions of elastase, α1-proteinase inhibitor (α1-PI) and α2-macroglobulin (α2-MG) in both the polymorphonuclear (PMN) and mononuclear phagocytes. Immunofluorescence and indirect peroxidase-antiperoxidase staining of articular cartilage (ACA) from 52% of 21 patients with RA and one with juvenile RA (JRA) showed presence of elastase in the superficial layer of microscopically intact but proteoglycan depleted pannus-free ACA. In histologically altered pannus-free RA-ACA superficial elastase deposits were found in 24% of the cases. Adjacent ACA sections contained IgG, C 3, α1-PI and rarely α2-MG. RA-ACA below or surrounded by pannus showed close contact with intact and decaying PMN in 62% and 48% of the cases, respectively. ACA specimens from patients with degenerative disease and systemic lupus were negative. These findings strongly suggest that PMN leucocyte elastase is operative in the degradation of RA-ACA and JRA-ACA, and that this activity is largely dependent upon the presence of entrapped immune complexes in such cartilage.

Action of collagenase and elastase from human polymorphonuclear leukocytes on human articular cartilage

SummaryCollagenase from human polymorphonuclear leukocytes (neutrophil collagenase) attacks collagen type II in solution at a rate intermediate to those of type I and III collagens. This enzyme alone is not able to initiate degradation of native human articular cartilage. If the cartilage is first treated with leukocyte elastase, collagenase slowly degrades collagen.Confirming earlier findings by other investigators, elastase has a dual action on cartilage: The enzyme removes proteoglycans, thus demasking collagen fibers and giving collagenase access to them, and solubilizes collagen at a sizable rate.Although neutrophil collagenase cleaves collagen type II in solution at a high rate, the native, cross-linked status of collagen in cartilage makes it a relatively poor substrate for this enzyme. On a weight by weight scale, elastase and collagenase display about the same collagenolytic potential on human articular cartilage.The elastase/collagenase system from human polymorphonuclear leukocytes could represent a cooperative proteolytic complex in the destruction of cartilage in rheumatoid arthritis.

A handy assay for collagenase using reconstituted fluorescein-labeled collagen fibrils

Abstract This report describes an assay for measuring the activity of collagenases using reconstituted fibrils of fluorescein-labeled soluble collagen as substrate. The labeling of commercially available material is easy and not expensive. The assay is very sensitive, reproducible, and is linear with collagenase concentration up to 100% consumption of the reconstituted fibrils. Readings of collagenolytic activity can be determined directly by measuring the fluorescence of the released labeled peptides without further processing of the data.

The ultrastructural localization of fibronectin in the lining layer of rheumatoid arthritis synovium: the synthesis of fibronectin by type B lining cells.

SummaryThe distribution of fibronectin in the lining layer of inflamed rheumatoid arthritis (RA) synovium has been ultrastructurally investigated using an anti-human plasma fibronectin antibody.The hyperplasia of lining cells was prominent in the lining layer of the inflamed RA synovium. A high level of fibronectin was localized in this region. Ultrastructurally the fibronectin was observed on the surface of both type A (type M) and type B (type F) cells, and in the extracellular fibrin-like material. This glycoprotein was detectable in rough endoplasmic reticulum (RER), some Golgi apparatus, and peripheral vesicles of type B cells. On the other hand, RER and Golgi apparatus of type A cells failed to be immunostained with the antibody. Type A cells occasionally contacted each other with interdigitation of cytoplasmic processes, and a high amount of fibronectin was localized in this region.These findings indicate that fibronectin is synthesized along the classic secretory pathway through RER and Golgi apparatus of type B cells. On the contrary, type A cells seem not to be associated with the local synthesis of the glycoprotein. Fibronectin may play a structural role in organizing these proliferated lining cells by promoting cell adhesion. The synthesis of fibronectin by proliferated type B cells may be responsible in part for the local increase of this glycoprotein in the lining layer of RA synovium.