96 Biochimica et Biophysica Acta, 1033(1990) 96-102 Elsevier BBAGEN23224 Synovial fluids from infected joints contain metalloproteinase - tissue inhibitor of metaUoproteinase (TIMP) complexes Tim Cawston, Penny McLaughlan, Robert Coughlan, Valerie Kyle and Brian Hazleman Rheumatology Research Unit, Addenbrooke's Hospital, Cambridge (U.K.) (Received16 June 1989) Keywords: CoUagenase; Tissue inhibitor; Synovial fluid; az-Macroglobulin; Metalloproteinase; Proteinase inhibitorcomplex Samples of synovial fluids aspirated from patients with septic arthritis prior to the commencement of any treatment contained active metailoproteinases but no proteinase inhibitory activity. We therefore assayed these samples for proteinase-inhibitor complexes. Although no biologically active az-macroglobuiin or tissue inhibitor of metal- loproteinase (TIMP) was present in the fluids, immunoassay of the samples clearly showed that high molecular weight proteinase-TIMP complexes were present. It is proposed that high levels of active metaUoproteinaSes are released from neutrophils into septic synovial fluids and that these proteinases complex all the available TIMP, forming metallopro- teinase-TIMP complexes. Introduction Joint infection is a serious disease [1-6]. Delay in diagnosis and institution of appropriate therapy is a factor that contributes to the mortality and morbidity in this disease. Destruction of cartilage collagen and pro- teoglycan begins early in the course of infection and a very rapid loss of cartilage often occurs. Since Curtiss and Klein [7] examined the effect of staphylococcal pus and various proteolytic enzymes on the structural integrity of articular cartilage in vitro, a number of other workers [8,9] have used animal models to determine the mechanisms of joint destruction in septic arthritis. All these studies suggested that proteo- lytic enzymes were responsible for the removal of both cartilage collagen and proteoglycan, but the source of these enzymes was not discovered. Harris et al. [10] considered the various enzymes that might be responsi- ble for collagen degradation and described a case of septic arthritis. Synovial fluid analysis revealed active collagenase to be present and they suggested that this enzyme was derived from the leukocytes. Barrett [11] has shown that active leukocyte proteinases can degrade cartilage in vitro. In a previous study [12], we examined septic synovial fluids and found that in the early course of the disease high levels of active neutrophil metalloproteinases capa- ble of digesting all the components of the extracellular matrix were present. In addition, these fluids contained no proteinase inhibitors such as a2-macroglobulin, the serum inhibitor which can block the action of all four classes of proteinase [13], and the tissue inhibitor of metalloproteinase (TIMP), a specific metalloproteinase inhibitor [14]. These inhibitors are usually present in excess in rheumatoid synovial fluid [15]. The levels of TIMP in rheumatoid synovial fluid are higher than corresponding serum values, suggesting local synthesis within the joint, whilst levels of synovial fluid a 2- macroglobulin are lower, suggesting it is still partially excluded from the joint by its relatively large size [16]. In septic fluids, if levels of these inhibitors are unde- tectable using bioassays, whilst active metalloprotein- ases are present it is likely that the inhibitory capacity of the fluid has been exceeded and all the available inhibitors are complexed to proteinases. In this study we have analysed synovial fluids from patients with bacteriologically confirmed septic arthritis to determine whether the fluid contains metalloproteinase-TIMP complexes. Abbreviations: APMA, 4-aminophenyl mercuricacetate;TIMP,tissue inhibitor of metaUoproteinases; HAF, human amnioticfluid; HFL, human fetallung fibroblast. Correspondence: T.E. Cawston, Rheumatology Research Unit, Ad- denbrooke's Hospital,HillsRoad, Cambridge,CB2 2QQ, U.K. Materials and Methods Hyaluromdase (bovine testes) type IV-S was from Sigma (London), Poole, Dorset, U.K. 4-Aminophenyl mercuric acetate (APMA) was from Aldrich, Gil- 0304-4165/90/$03.50 © 1990 ElservierSciencePublishers B.V.(Biomedical Division)