Kinetics of Collagenase and Neutral Protease Release by Neutrophils Exposed to Microcrystalline Sodium Urate

Abstract

The rates of release of the various enzymes from PMN leukocytes exposed to MSU crystals were measured. Lysozyme and neutral protease appeared to be released simultaneously and release appeared to be essentially complete by 60 minutes. In contrast, collagenase was detected only after 30 minutes incubation, reached peak concentration at 90 minutes and dropped noticeably by 180 minutes. The presence of these enzymes was not due to cell lysis since only 10% of the total cellular LDH was present in the supernates. The levels of total and active collagenase in the supernatants were measured. In contrast to latent collagenase, active collagenase levels increased continually throughout the incubation period. The gradual increase in level of active collagenase may explain the corresponding drop of latent collagenase in the longer incubation (90 minutes or more) as the latter apparently is converted to active form. The effects of collagenase on Type I collagen were examined by SDS gel electrophoresis.

Purification to homogeneity of latent and active 58-kilodalton forms of human neutrophil collagenase

“…Since the only white cells that contain collagenase are granulocytes (Christner et al, 1982), the buffy coats were extracted directly without further fractionation. The alternative of isolating viable neutrophils from fresh blood and stimulating secretion of HNC with phorbol myristic acetate (Hasty et al, 1986) or other agents (Oronsky et al, 1973;Cheung et al, 1983;Hibbs et al, 1984;Weiss et al, 1985) produces a starting material of higher initial purity but gives a lower yield and is harder to scale up. The purification procedure described below gives the same results whether buffy coats from fresh or outdated blood are used as the starting material.…”

“…In contrast to HFC, human neutrophil collagenase (HNC) is stored within the cell in specific granules (Lazarus et al, 1968;Murphy et al, 1977) and can be released by a variety of stimuli (Oronsky et al, 1973;Cheung et al, 1983;Hibbs et al, 1984; Weiss et al, 1985;Hasty et al" 1986). HNC is immunologically distinct from HFC (Hasty et al, 1984(Hasty et al, , 1987a and has been reported to have a different molecular weight (Murphy et al, 1980;Macartney & Tschesche, 1983; Sorsa et al, 1985;Hasty et al, 1986) and different collagen specificity (Horwitz et al, 1977;Hasty et al, 1987b).…”

Tophus-derived monosodium urate monohydrate crystals are biologically much more active than synthetic counterpart

“…During phagocytosis of particulate materials, including MSUM crystals, phagocytic cells generate and release increased amounts of proteolytic enzymes [17], free radicals [7][8][9][10][11] and other molecular species that may damage normal cell and tissue constituents [12]. Luminol-enhanced CL production by PMN cells is largely due, during phagocytosis of particulate materials, to a release of myeloperoxidase from azurophilic granules and to a formation of H202 and hypochlorus acid [18].…”

Collagenolytic metalloenzymes of the human neutrophil

“…Regardless of the molecular basis underlying the latency of neutrophil collagenase, it is clear that the enzyme must be activated in an intact cell system before it can catalyze collagen degradation. Although a variety of processes have been postulated to play potential roles in the activation of neutrophil collagenase [6,7], only a handful of studies have actually examined the regulation of the enzyme in an intact cell system [12,27,28]. Until recently, the accumulated data suggested that neutrophils could release latent collagenase extracellularly, but that the cells have almost no intrinsic ability to activate * It should be noted that activation fragments released from zymogens can exert inhibitory effects on the active enzyme.…”

“…the enzyme [12,27,28]. (Indeed, it seems that the apparent inability of the neutrophil to activate its collagenase stifled further interest in the enzyme.)…”