Fibrin induction of interleukin-8 expression in corneal endothelial cells in vitro.
OBJECTIVE
Classically, acute and chronic inflammations are characterized by fibrin deposition and a dynamic influx of leukocytes. This leukocyte recruitment, and associated activation, is thought to be dependent on the generation of leukocyte chemotactic factors (LCF). Although the functional existence of LCF in ocular tissue has been demonstrated, the identity, source(s), and mechanisms of induction of these LCF are unclear. The authors investigate the hypothesis that in vitro corneal endothelial cells produce LCF in response to fibrin-induced activation. They further hypothesize that in vivo this fibrin-induced expression of LCF contributes to the leukocyte accumulation associated with ocular injury.


METHODS
Bovine corneal endothelial cells (BCEC) were co-cultured for 3 to 72 hours with physiologic concentrations of highly purified fibrin (0.125 to 2.0 mg/ml) polymerized in situ. At harvest, the conditioned medium was separated from the fibrin matrix by centrifugation and characterized for the presence and nature of polymorphonuclear leukocyte chemotactic activity. This fibrin-induced LCF was compared to known LCF, such as interleukin-8 (IL-8), using standard physical, chemical, and immunologic parameters.


RESULTS
Conditioned medium from fibrin-treated BCEC exhibited a dose- and time-dependent induction of LCF activity, as verified by checkerboard analysis. This LCF activity was not immunoprecipitated by a polyclonal antibody to bovine fibrinogen, and it was heat stable (60 degrees C, 90 minutes) and protease labile. Isoelectric focusing and gel filtration analysis revealed a major peak of chemotactic activity at pH 8.5 to 9.0 and a molecular weight of 10 kd, respectively. Radioimmunoassay of conditioned medium from fibrin-treated BCEC for IL-8 demonstrated an 11-fold increase in IL-8 antigen for fibrin-treated BCEC compared to control BCEC.


CONCLUSIONS
In vitro, fibrin induces BCEC expression of LCF activity that includes IL-8. In vivo, this fibrin induction of LCF from corneal endothelial cells probably serves to control both leukocyte recruitment and activation within the anterior chamber in general and to corneal endothelium in particular. These studies provide a foundation for understanding the nature, sources, and mechanisms of the LCF generation that contributes to endocular inflammation.
Department of Pathology, University of Connecticut School of Medicine, Farmington.
M L
ML
Ramsby
D L
DL
Kreutzer
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D005337:Fibrin / Q000494:pharmacology*; D016209:Interleukin-8 / Q000096:biosynthesis*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009504:Neutrophils / Q000276:immunology
D016209:Interleukin-8; D005337:Fibrin