I Nagaoka1, S Hirota. 1. Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan. nagaokai@med.juntendo.ac.jp
Abstract
OBJECTIVE: Matrix metalloproteinase (MMP)-9 plays an important role in neutrophil extravasation and migration by its ability to degrade the major components of basement membrane. To evaluate the expression of neutrophil MMP-9 under inflammatory conditions, we examined the levels of MMP-9 and its mRNA in neutrophils of glycogen-induced peritoneal inflammation. MATERIALS AND METHODS: Male Hartley guinea pigs weighing 250-300 g were intraperitoneally injected with 0.17% glycogen solution, and 13-15 h after the injection, blood and peritoneal neutrophils were isolated. The levels of MMP-9 and its mRNA were analyzed by gelatin zymography and Northern blotting, respectively. Furthermore, MMP-9 activities in the peritoneal supernatants were measured. RESULTS: MMP-9 level in peritoneal neutrophils was essentially the same as that in blood neutrophils, although peritoneal neutrophils were assumed to have extracellularly released MMP-9 from the granules during infiltration into the peritoneal cavity. Interestingly, MMP-9 mRNA was expressed more abundantly in peritoneal neutrophils than in blood neutrophils (p<0.01). Moreover, MMP-9 levels in blood and peritoneal neutrophils were reduced to 30-45% of non-treated controls by actinomycin D (500 microg/kg) or cycloheximide (10 mg/kg)-treatment (p<0.05). In contrast, MMP-9 activity increased in the peritoneal supernatants of glycogen-injected animals was not significantly affected by actinomycin D- or cycloheximide-treatment. In addition, when blood neutrophils of non-injected animals were stimulated with 10(-7) M N-formyl-Met-Leu-Phe, 10 microg/ml lipopolysaccharide, 10 ng/ml phorbol 12-myristate 13-acetate, 10(-8) M IL-8 or 100 U/ml tumor necrosis factor-a, expression of MMP-9 mRNA was markedly increased (p<0.05). CONCLUSIONS: The present observations indicate that MMP-9 gene is transcribed, and MMP-9 protein is synthesized in neutrophils during glycogen-injected peritoneal inflammation. Moreover, it is likely that MMP-9 protein in the peritoneal supernatants is mostly derived from preformed MMP-9 which is stored in the neutrophil granules and extracellularly released during infiltration of neutrophils into the peritoneal cavity. Finally, the transcription of MMP-9 gene can be upregulated in neutrophils by stimulation with inflammatory mediators, even after neutrophils have been matured.
OBJECTIVE:Matrix metalloproteinase (MMP)-9 plays an important role in neutrophil extravasation and migration by its ability to degrade the major components of basement membrane. To evaluate the expression of neutrophil MMP-9 under inflammatory conditions, we examined the levels of MMP-9 and its mRNA in neutrophils of glycogen-induced peritoneal inflammation. MATERIALS AND METHODS: Male Hartley guinea pigs weighing 250-300 g were intraperitoneally injected with 0.17% glycogen solution, and 13-15 h after the injection, blood and peritoneal neutrophils were isolated. The levels of MMP-9 and its mRNA were analyzed by gelatin zymography and Northern blotting, respectively. Furthermore, MMP-9 activities in the peritoneal supernatants were measured. RESULTS:MMP-9 level in peritoneal neutrophils was essentially the same as that in blood neutrophils, although peritoneal neutrophils were assumed to have extracellularly released MMP-9 from the granules during infiltration into the peritoneal cavity. Interestingly, MMP-9 mRNA was expressed more abundantly in peritoneal neutrophils than in blood neutrophils (p<0.01). Moreover, MMP-9 levels in blood and peritoneal neutrophils were reduced to 30-45% of non-treated controls by actinomycin D (500 microg/kg) or cycloheximide (10 mg/kg)-treatment (p<0.05). In contrast, MMP-9 activity increased in the peritoneal supernatants of glycogen-injected animals was not significantly affected by actinomycin D- or cycloheximide-treatment. In addition, when blood neutrophils of non-injected animals were stimulated with 10(-7) M N-formyl-Met-Leu-Phe, 10 microg/ml lipopolysaccharide, 10 ng/ml phorbol 12-myristate 13-acetate, 10(-8) M IL-8 or 100 U/ml tumor necrosis factor-a, expression of MMP-9 mRNA was markedly increased (p<0.05). CONCLUSIONS: The present observations indicate that MMP-9 gene is transcribed, and MMP-9 protein is synthesized in neutrophils during glycogen-injected peritoneal inflammation. Moreover, it is likely that MMP-9 protein in the peritoneal supernatants is mostly derived from preformed MMP-9 which is stored in the neutrophil granules and extracellularly released during infiltration of neutrophils into the peritoneal cavity. Finally, the transcription of MMP-9 gene can be upregulated in neutrophils by stimulation with inflammatory mediators, even after neutrophils have been matured.