Autoactivation profiles of calcium-dependent matrix metalloproteinase-2 and -9 in inflammatory synovial fluid: effect of pyrophosphate and bisphosphonates.

BACKGROUND: The presence of matrix metalloproteinase-2 and -9 (MMP-2, MMP-9), gelatinase A and B, in synovial fluid is typical in inflammatory connective tissue diseases especially rheumatoid arthritis (RA). Because MMPs are synthesized as latent proforms, a pathophysiologic understanding of MMP regulation has focused on mechanisms of activation that remain to date largely unresolved. METHODS: Synovial fluid was collected by aseptic aspiration from RA patients and incubated with and without physiologic levels of calcium and other modifiers (pyrophosphate, bisphosphonates, and the tissue inhibitors of MMPs (TIMPs), under conditions that activate MMPs. MMP-2 and -9 were then characterized by substrate gel electrophoresis (gelatin zymography) to resolve both latent and activated 'partially proteolyzed' forms. RESULTS: Gelatin zymography revealed that RA synovial fluid contained latent neutrophil MMP-9 (92, 130, 225 kDa) and fibroblast MMP-2 (72 kDa). A small amount of activated MMP-2 (64 kDa) was also noted. Incubation of synovial fluid without calcium resulted in MMP-9 activation to 87, 116, and 209 kDa forms. MMP-9 activation was, however, substantially delayed in the presence of physiologic calcium (2.5 mmol/l). MMP-2 did not demonstrate any appreciable activation with or without physiologic calcium. MMP-9 activation likely occurred via an autoactivation mechanism since it was susceptible to inhibition by the tissue inhibitor of MMP-9 (TIMP-1). Pyrophosphate and bisphosphonates (alendronate and risedronate) were ineffective in blocking synovial fluid MMP-9 autoactivation. Some early MMP-9 activation was noted with alendronate despite the presence of physiologic calcium. DISCUSSION: Although RA synovial fluid contained abundant MMP-2 and MMP-9, only MMP-9 underwent autoactivation to lower molecular weight forms. MMP-9 was transiently stable in the presence of physiologic calcium concentration, whereas autoactivation was more pronounced without exogenous calcium. The apparent lack of MMP-2 autoactivation with or without calcium, likely resulted from the coexistence of its bound endogenous inhibitor, TIMP-2. The role of differential autoactivation of MMPs activity in inflammatory arthritic disease is discussed.

An indium:calcium phosphate colloid that specifically targets fibrin.

The ability of indium to target fibrin in vitro was evaluated. The radionuclide (114m)Indium (114mIn) was prepared as a soluble and colloidal (In:In) form, as well as, a mixed indium:calcium phosphate (In:CaP) colloid. Soluble 114mIn was prepared by maintaining acid pH (50 mM HCl). Colloidal 114mIn (In:In) was prepared under slightly basic conditions (50 mM Tris-Cl, pH 7.6). The mixed In:CaP colloid was prepared by incubation of 114mIn with calcium (10 mM) and phosphate (250 microM) under slightly basic conditions (50 mM Tris-Cl, pH 7.6). To assess fibrin binding, the three 114mIn preparations were mixed with diluted human plasma (source of fibrinogen). Fibrin polymerization was initiated by addition of calcium (5 mM) and thrombin (0.5 U/ml). Following incubation (15 min, 37 degrees C), the fibrin matrix was condensed, removed from the reaction mixture, and washed briefly. Fibrin uptake of 114mIn (soluble, colloidal, or In:CaP) was determined by gamma counting. Results demonstrated that soluble 114mIn exclusively bound a plasma protein electrophoretically and immunologically identified as transferrin. Although both colloidal 114mIn and 114mIn:CaP bound fibrin, the mixed 114mIn:CaP colloid demonstrated substantially higher fibrin binding activity (about 2-fold). The target of indium binding was confirmed as fibrin due to the presence of characteristic cross-linked gamma-gamma dimers (100 kDa) and beta-monomers (58 kDa) by SDS-PAGE. 114mIn colloid and the mixed 114mIn:CaP colloid demonstrated no ability to bind fibrin's precursor, fibrinogen. 114mIn:CaP fibrin binding was associated with formation of CaP, as evidenced by its dependence on phosphate concentration. The biocompatibility of CaP including its ability to bind 114mIn and specifically target fibrin may be of potential value for diagnostic imaging studies to identify regions of occult vascular stenosis (i.e., atherosclerotic plaques, deep vein thrombosis, pulmonary embolus).

Gelatinolytic and fibrinolytic activity in fresh-frozen plasma.

Fresh-frozen plasma (FFP) was evaluated for gelatinolytic and fibrinolytic activity. Gelatin zymography revealed that gelatinase A (MMP-2) was constitutively present in FFP whereas gelatinase B (MMP-9) was present at variable levels. The presence of MMP-9 likely represents differential release from neutrophils during FFP collection or processing. Although fibrin matrices generated from FFP or freshly prepared plasma contained characteristic crosslinked gamma-gamma dimers and beta-monomers, matrices generated from FFP were resistant to spontaneous plasmin-dependent fibrinolysis. This observation likely stems from the plasminogen activator instability and could potentially lead to a hypofibrinolytic state. The impact of these in vitro findings to protease balance in patients receiving multiple FFP doses remains to be determined.

Selective detection of autoimmune antibodies to single- and double-stranded DNA by enzyme immunoassay.

Presence in serum of anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) is one of the diagnostic criteria for systemic lupus erythematosus. Anti-single stranded DNA antibodies (anti-ssDNA) also occur, but their clinical significance is unclear. Use of enzyme immunoassay (EIA) kits that are specific for anti-dsDNA is desirable but problematic, since preparation of dsDNA of high integrity is difficult (ie, regions of ssDNA may persist). This study evaluated an EIA kit that uses low Mw plasmid/bacteriophage DNA as a nucleic acid source. Anti-dsDNA results were compared to results obtained by an anti-ssDNA EIA and an immunofluorescent assay (IFA) that uses Crithidia luciliae (specific for anti-dsDNA). Consecutive serum samples (n=139, 88% female) submitted to the clinical laboratory for anti-dsDNA analysis were evaluated. EIA precision was determined at three levels. Intra-assay precision [mean +/- SD (CV)] for anti-dsDNA: 36 +/- 3.5 IU/ml (9.8%); 98 +/- 4.4 (4.5%); and 245 +/- 5.8 (2.4%); and anti-ssDNA. 40 +/- 1.4 U/ml (3.5%); 190 +/- 4.8 (2.5%); and 283 +/- 10.3 (3.7%) (n=8). Inter-assay precision for anti-dsDNA: 36 +/- 6.3 IU/ml (17.5%); 90 +/- 5.9 (6.6%); and 207 +/- 20.9 (10.1%); and anti-ssDNA: 48 +/- 8.2 U/ml (17.0%); 193 +/- 12.7 (6.6%); and 263 +/- 21.5 (8.2%) (n=8). Linearity was assessed with (a) high dsDNA/high ssDNA and (b) low dsDNA/high ssDNA samples (no high dsDNA/low ssDNA samples were identified). Linearity (>200 IU/ml) was found for both sample types with a correlation coefficient (r) of 0.995-0.999. Anti-dsDNA immunoreactivity was not apparent with the low dsDNA/high ssDNA sample. More patients were positive for anti-ssDNA (54%), compared to anti-dsDNA (17%). IFA confirmation (Crithidia) indicated a relative sensitivity and specificity of 94.1% and 93.4% for the anti-dsDNA EIA. IFA positivity correlated with increased anti-dsDNA level: 20% (30-60 IU/ml); 70% (61-200 IU/ml); and 89% (>200 IU/ml). Of specimens that were anti-ssDNA positive and anti-dsDNA negative (n=51), only one was IFA positive. When IFA was compared to an anti-dsDNA EIA kit that used high Mw calf thymus DNA, lower relative specificity (88.2%) and sensitivity (72.6%) was obtained. Anti-ssDNA was found in many false positive specimens (87%).

Zymographic analysis of latent and activated forms of matrix metalloproteinase-2 and -9 in synovial fluid: correlation to polymorphonuclear leukocyte infiltration and in response to infection.

BACKGROUND: Matrix metalloproteinase-2 and-9 (MMP-2, MMP-9), and gelatinase A and B participate in the degradation of the extracellular matrix proteins in a variety of inflammatory connective tissue diseases including arthritis. METHODS: Synovial fluid was collected by aseptic aspiration from patients with rheumatoid arthritis (RA), osteoarthritis (OA), gout, infected joint, septic arthritis, and systemic lupus erythematosus (SLE). Synovial fluid was subjected to cell count with polymorphonuclear leukocyte (PMN) differential, Gram staining and culture as necessary. MMP-2 and -9 were characterized by substrate gel electrophoresis (gelatin zymography) to resolve latent and activated 'partially proteolyzed' forms. RESULTS: Gelatin zymography revealed that MMP-9 (92, 130, 225 kDa) in synovial fluid was associated with extent of white blood cell infiltration specifically PMNs. In contrast, fibroblast MMP-2 (72 kDa) was present in all synovial fluids irrespective of PMN count. No MMP-9 was detected in the osteoarthritic specimen with low PMN count. Higher PMN count was associated with the presence of activated MMPs, especially in specimens that were confirmed culture positive. Activated synovial fluid MMPs persisted despite resolution of infection. DISCUSSION: Latent and activated MMP-2 and MMP-9 in synovial fluids fluctuate in proportion to PMN infiltration and specifically in response to infection. The presence of activated MMPs post-therapy would suggest that use of specific MMP inhibitors be indicated to eliminate activated MMPs that apparently persist post-infection.