Stimulatory effect of pseudomonal elastase on collagen degradation by cultured keratocytes.
OBJECTIVE
The pathobiology of corneal ulceration induced by Pseudomonas aeruginosa was investigated by characterization of the pseudomonal pathogenic factors responsible for degradation of the collagen matrix.


METHODS
Three-dimensional gels of type I collagen containing (or not) rabbit keratocytes were incubated in the presence of either culture supernatant of P. aeruginosa strain PAO1 or pseudomonal pathogenic factors (elastase, lipopolysaccharide, or exotoxin A), and the extent of collagen degradation was assessed after 24 hours by measurement of released hydroxyproline. Activation of matrix metalloproteinases (MMPs) produced by keratocytes was also examined by gelatin zymography and immunoblot analysis.


RESULTS
In the absence of keratocytes, the PAO1-conditioned medium increased the extent of collagen degradation. The conditioned medium also promoted keratocyte-mediated collagen degradation. Of the pseudomonal pathogenic factors examined, only elastase degraded collagen directly as well as stimulated keratocyte-mediated collagen degradation. Culture supernatant of elastase-deficient P. aeruginosa (lasR or lasB) mutants had no effect on collagen degradation in the absence or presence of keratocytes. Elastase also induced the conversion of the inactive precursors of MMP-1, -2, -3, and -9 produced by keratocytes to the active forms of the enzymes.


CONCLUSIONS
These results suggest that pseudomonal elastase both degrades type I collagen directly and promotes collagen degradation mediated by keratocytes, the latter effect being likely attributable, at least in part, to the activation of proMMPS:
Nagano
T
T
Department of Ophthalmology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan.
Hao
J L
JL
Nakamura
M
M
Kumagai
N
N
Abe
M
M
Nakazawa
T
T
Nishida
T
T
D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005098:Exotoxins / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism; D010196:Pancreatic Elastase / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003094:Collagen; D003470:Culture Media; D005098:Exotoxins; D008070:Lipopolysaccharides; D020782:Matrix Metalloproteinases; D010196:Pancreatic Elastase; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A