The acute-phase reactant rabbit serum amyloid A 3 (SAA3) was identified as the major difference product in Ag-induced arthritis in the rabbit, a model resembling in many aspects the clinical characteristics of rheumatoid arthritis (RA) in humans. In Ag-induced arthritis, up-regulated SAA3 transcription in vivo was detected in cells infiltrating into the inflamed joint, in the area where pannus formation starts and, most notably, also in chondrocytes. The proinflammatory cytokine IL-1β induced SAA3 transcription in primary rabbit chondrocytes in vitro. Furthermore, rSAA3 protein induced transcription of matrix metalloproteinases in rabbit chondrocytes in vitro. In the human experimental system, IL-1β induced transcription of acute-phase SAA (A-SSA; encoded by SAA1/SAA2) in primary chondrocytes. Similar to the rabbit system, recombinant human A-SAA protein was able to induce matrix metalloproteinases’ transcription in chondrocytes. Further, immunohistochemistry demonstrated that A-SAA was highly expressed in human RA synovium. A new finding of our study is that A-SSA expression was also detected in cartilage in osteoarthritis. Our data, together with previous findings of SAA expression in RA synovium, suggest that A-SAA may play a role in cartilage destruction in arthritis.

In recent years, numerous studies have documented that macrophages have important secretory activities. Activated macrophages have been shown to secrete plasminogen activator (1), collagenase (2), and an elastase-like enzyme (3), in addition to lysozyme (4), which is also secreted by nonactivated macrophages. Macrophages have also been shown selectively to release B-glucuronidase and other acid hydrolases when they are stimulated by undigestible particles (5), lymphokines (6), or complement products (7). In this paper, we show that macrophages synthesize and secrete considerable amounts of lysosoma] enzymes over long periods of culture, independently of external stimuli. Materials and MethodsMacrophage Cultures. Macrophages from male OF1 mice (Sandoz Ltd., Basel, Switzerland) weighing 20-24 g were collected by peritoneal lavage with ice-cold complete medium (see below) containing 20 U of heparin per ml, according to a standard method (8). The mice were either untreated, or treated 4 days before cell harvesting by an intraperitoneal injection of 0.75 ml of one of the following agents: Brewer's thioglycollate (TA) 1 medium, a 10% proteose-peptone (PP) medium (beth from Difco Laboratories, Detroit, Mich.), or a suspension of streptococcus A cell wall material (SA) in phosphate-buffered saline (PBS). Washed type A~ streptococci were disrupted by a modification of the method of Colman and Williams (9). The cells were shaken for 20-30 min in a Braun cell disintegrator (Apparateben Braun, Melsungen, W. Germany) with 0.1-mm glass beads at -60°C. The contents of the disintegrator were suspended in water, filtered through glass wool to remove the beads, and the bacterial fragments were pelleted by centrifugation at ~ 50,000 g. rain. The pellets were washed with water and lyophilized. This material was suspended in PBS (0.8 mg/ml) before use.The medium, a minor modification of that used by Cohn and Bensen (8), was made up as follows. To 89 ml of a solution of 140 mg of sodium bicarbonate in double-distilled water was added 10 ml of 10-fold concentrated medium 199 (Hanks' salts; Grand Island Biological Co., Grand Island, N. Y.), and then I ml of I M Hepes. Penicillin (100 IU/ml), streptomycin (0.1 mg/ml), and mycostatin (30 U/ml) were added, and the mixture was sterilized by filtration through Millipere filters, type GSWP, with a pore diameter of 0.22 ~m (Millipore AG, Kloten, Switzerland). This medium was stored at 4°C for up to 2 wk. Before use, 1 ml of a sterile 200 mM solution of Lglutamine and serum, usually 5 ml of sterile acid-treated (1) fetal bovine serum (FBS; Flow Laboratories, Rockville, Md.) per 100 ml was added.The peritoneal cell suspensions were centrifuged at 120 g for 5 rain at room temperature, the cells were resuspendod in culture medium and plated on 60-ram plastic culture dishes (Falcon Plastics, Div. Becten, Dickinson & Co., Cockeysville, Md.) at a density of 4-6 × 10 ~ cells per dish in 3 ml of medium. The cultures were kept for 2-3 h at 37°C, and nonadherent cells were then ' Abbreviations used in t...

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Macrophages are found in large numbers at sites of chronic inflammation . They enter the lesions as monocytes (1) and develop in situ into large cells which synthesize and release an impressive variety of enzymes such as lysosomal hydrolases (2, 3), plasminogen activator (4), elastase (5), and collagenase (6) . These enzymes are most likely involved in the extensive destruction of cellular and extracellular tissue elements which accompanies chronic inflammation .Since macrophages are highly phagocytic, we have investigated the role of phagocytosis in their activation . We have studied the activation process in cultures of mouse peritoneal macrophages which were exposed to different types of particles . We have followed macrophage activation by assaying, in the cells as well as in the culture media, a number of enzyme activities which are known to change in the course of this process . In this paper, we present experiments which show that macrophages obtained from untreated mice become activated in culture after phagocytosis of zymosan or formaldehyde-treated sheep erythrocytes but do not become activated after phagocytosis of latex beads . Materials and MethodsMacrophage Cultures. Macrophages were obtained from male OF1 mice (Sandoz Ltd., Basel, Switzerland) weighing 20-24 g . The mice were either untreated, or treated 4 days before cell harvesting with Brewer's thioglycollate broth or a suspension of streptococcus A cell wall fragments (3) . The cell culture techniques and the biochemical assay methods used were described in an earlier paper (3) .Phagocytosis . Particles were prepared as follows . Zymosan (Koch-Light Laboratories Ltd ., Colnbrook, Buckinghamshire, England) was boiled in phosphate-buffered saline (PBS) l for 1 h, washed three times by centrifugation, resuspended in PBS (50 mg/ml), and autoclaved at 120°C (7) . Latex beads, 1 .01 ftm in diameter (Serva GmbH & Co., Heidelberg, W . Germany), were suspended in PBS containing 0 .01% Tween 80, washed three times by centrifugation, resuspended in PBS/Tween 80 and irradiated with ultraviolet light before use (8) . Sheep erythrocytes were washed five times in PBS by centrifugation, resuspended in PBS containing 3% formaldehyde for 2 days at 4°C, and then washed five more times in PBS (9) .Phagocytosis experiments were performed on cells which were allowed to adhere to the culture dishes at 37°C during 3 h after harvest . Particles were added in a small volume of PBS to the culture dishes (60 mm) containing 4-6 X 106 adherent cells and 3 ml of medium (3) . The particle-to-cell ratios were between 7 and 30 :1 for zymosan (see figures for details), 5 :1 for formaldehyde-treated sheep erythrocytes, and 40 :1 for the latex beads . Phagocytosis was stopped after 1 h by draining the medium and eliminating noningested particles by washing three times with PBS . Culturing in particle-free medium was then continued for up to 13 days under standard conditions (3) . The medium was changed at different intervals but at least every 3rd day .' Abbreviations used in thi...

Synthetic calcium pyrophosphate dihydrate crystals and, to a lesser extent, synthetic hydroxyapatite crystals increased the amount of interleukin-l/mononuclear cell factor released by human blood monocytes, as measured by collagenase and prostaglandin Ez production by rabbit chondrocytes, human dermal fibroblasts, and adherent rheumatoid synovial cells. The same crystals also directly induced collagenase and prostaglandin Ez secretion by rabbit chondrocytes, and potentiated the action of interleukin-llmononuclear cell factor on chondrocytes. These mechanisms may be important in the pathogenesis of the destructive arthropathies associated with these crystals.The processes that lead to irreversible articular cartilage destruction in inflammatory and degenerative