Study On The Mechanism Of Keratocyte Collagen Degradation Induced By Pseudomonas Aeruginosa

The opportunistic Gram-negative pathogen Pseudomonasaeruginosa is a causative agent of severe keratitis. Corneal infection inhumans with P. aeruginosa is commonly characterized by rapidinfiltration of polymorphonuclear leukocytes (PMNs), corneal ulceration,and various degrees of stromal destruction.That is mediated by bothpseudomonal and host factors. P. aeruginosa can produce pathogenicfactors,such as elastase,lipopolysaccharide,and exotoxin A ,etc. Thekeratocytes are the only cells in the corneal matrix. Corneal infectionresults in the activation of keratocytes at the wound edge.They cansynthesize ECM proteins and MMPs,and express mRNA and proteins ofproinflammatory cytokines, such as IL-1,IL-1Ra, IL-6, and TNF-α asreaction to the bacterial. We think that the elastase from P. aeruginosaand the MMPs from the cells of host has stimulatory effect on collagindegradation. IL-1β is a potent proinflammatory cytokine and involved inthe effector phase of inflammatory and immune responses. We devisethis experiment to investigation the ralationship of them.We detected the effect of pseudomonal plathogenic factors ,keratocytes, and IL-1Ra on the corneal matrix collagin degradation.Wewant to investigation the problems followed: (1) The mechanism ofkeratocytes collagin degradation induced by P. aeruginosa. (2) Theeffect of IL-1Ra on collagen degradation by cultured keratocytes. Method: Type Ⅰ collagen gels with or without suspendedkeratocytes were incubated and treated with different concentrations ofpseudomonal culture medium for 24 hours .Then the amount ofhydroxyproline was measured spectrophotometrically.The effect ofIL-1Ra on collagen degradation was also examined.Results: 1.Pseudomonal culture medium stimulated collagendegradation in dose-dependent manner,especially when keratocytesare existed. 2.IL-1Ra inhibites collagen degradation mediated bykeratocytes significantly but cannot effect collagen degradation inducedby P. aeruginosa without keratocyte.Discussion:The culture supernatant of P. aeruginosa bothdegrades collagen directly and promotes collagen degradationmediated by keratocytes,the latter effect being likely attributable,at leastin part, to the activation of proMMPs. P. aeruginosa can produceelastase and alkaline protease.Those play an important role oncorneal ulceration and stromal destruction.Pseudomonal elastaseactivates proMMPs produced by keratocytes and PMNs by limitedproteolysis. Such protease activation and consequent collagendegradqtion may persest even after the bacteria have beeneliminated.Thus,the administration of antibiotics at this stage is unlikelyto affect corneal keratitis that persists as a result of proteases alreadyreleased from bacteria and the consequent activation of proMMPs anddegradation of the ECM.Our result demonstrates that IL-1Ra can inhibites keratocytecollagen degradation mediated by keratocytes but cannot effectcollagen degradation caused by P. aeruginosa without keratocyte. Thishints us that IL-1 play a important role on coneal stromaldestruction,and that can be inhibite by IL-1Ra partly.Other studydemonstrates that IL-1 can regulate the expression of MMPs.Somestudy demonstrated that IL-1? had a stimulatory effect on expression ofMMP-1,MMP-3 and MMP-9 in cultured corneal epithelial cells. It hasshown that IL-1βcan cause the MMPs expression at the cell culturesystem. It have been confirmed that IL-1Ra can obviously restrain thefunction of IL-1α and IL-1β.Reasonable application of IL-1Ra canreduce the damage of corneal structure.IL-1Ra has become a new wayof treatment .MMPs are zinc-binding, proteolytic enzymes that act collectively todegrade most components of the extracellular matrix (ECM). MMPactivity is regulated at multiple levels, including gene transcription,proenzyme activation, and inhibition by tissue inhibitors of matrixmetalloproteinases (TIMPs). Every kind of elastase can activateMMPs.Some study have shown that plasminogen promotes collagendegradation by keratocytes in culture,suggesting that a plas inogenactivator-plasmin system contributes to this process.In our experimentPseudomonal elastase activates proMMPs produced by keratocytes.Conclusion: 1.Pseudomonal culture medium stimulated collagendegradation in dose-dependent manner,especially when keratocytesare existed.This suggest that pseudomonal culture medium bothdegrades type Ⅰcollagen directly and promotes collagen degradationmediated by keratocytes,the latter effect being likely attributable,at leastin part ,to the activation of MMPs produced by keratocytes.2.IL-1Rainhibites collagen degradation mediated by keratocytes significantly butcannot effect collagen degradation without keratocyte. This hints us thatIL-1 play a important role on coneal stromal destruction,that mayattribute to the stimulatory effect on the expression and activation ofMMPs.