Interleukin‐1β stimulates collagenase production by cultured human periodontal ligament fibroblasts

Abstract

To examine the effects of interleukin-1 beta (IL-1 beta) on collagenase production by human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) in culture, collagenase activity in conditioned media was determined using a novel procedure that circumvented interference by enzyme inhibitors. Fibroblasts obtained from five paired periodontal ligament and gingival tissues were cultured for two weeks, and then incubated for a further 72 h in alpha-MEM supplemented with various concentrations of IL-1 beta (0 to 1250 pg/ml). The conditioned media from individual cultures were harvested and treated with dithiothreitol to inactivate TIMPs, and then with APMA, to activate the latent collagenase. Collagenase activity was measured fluorometrically using FITC-collagen as a substrate. IL-1 beta induced a approximately 2.4 to 5.2-fold increase in collagenase activity in PLF compared to a approximately 1.4 to 2.2-fold increase in GF. These results are in contrast to previous studies in which collagenase activity was measured in the presence of TIMPs, and indicate that PLF are more sensitive to IL-1 beta than GF. Since both PLF and GF are present in periodontal lesions, it is possible that collagenase secretion stimulated by exposure to inflammatory cell products such as IL-1 beta may participate in the destruction of collagen fibers involved in periodontal attachment.

Factors Affecting IL-1-Mediated Collagen Metabolism By Fibroblasts and the Pathogenesis of Periodontal Disease: A Review of the Literature

“…Several studies have shown that IL-1 gives rise to elevated levels of immunoreactive procollagenase as well as steady-state transcripts in both gingival fibroblasts (GF) and periodontal ligament fibroblasts (PLF) (Meikle et al, 1989;Richards and Rutherford, 1990;Lark et al, 1990;Tewari et al, 1994). PLF appear to be more sensitive to IL-1I3 than GF and exhibit a 2.4-5.2-fold increase in collagenase activity as compared with a 1.4-2.2-fold increase for GF (Ohshima et al, 1994). No significant change in inhibitor synthesis was observed after treatment of PLF and GF with IL-I13 (Meikle et al, 1989) as well as TIMP-1 mRNA level in PLF (Alvares et al, 1995).…”

Jatropha curcas latex inhibits the release of collagenase by gingival fibroblast

“…13 Several methods are known to detect collagenase products among others is collagenase activity assay with collagen as substrate which products are then separated by sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE). 8,10 The purpose of this study was to evaluate the effect of Jatropha curcas latex on the release of collagenase by fibroblast cells. Materials for collagenase assay: APMA buffer: 7 mg APMA (4-aminophenyl mercuric acetate) was weighed in a glass test tube.…”

“…9 Enzyme secretion can be induced by several agents as endotoxin, immune complex, and IL-1. 8 In periodontal disease, collagenase increase with the severity of the disease. 13 Several methods are known to detect collagenase products among others is collagenase activity assay with collagen as substrate which products are then separated by sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE).…”

“…To make APMA buffer, 1 ml APMA solution was added to 9 ml collagenase buffer (according to Ohshima). 8 Collagenase assay buffer: Tris 50 mM pH 7.5, CaCl 2 10 mM, NaCl 150 mM. Collagen (ICN).…”

“…7 Collagenase is a protease secreted by macrophage, neutrophyl, and also by activated fibroblast. [8][9][10][11] It has the unique ability to degrade collagen generating ¾ A fragment, which is characteristic to collagen degraded by collagenase. 12 This enzyme is important in connective tissue remodeling occurring in normal processes or wound healing, for degradation and resolution of inflammation, however, in certain disease it may result in articular destruction.…”