Influences on effector functions of monocyte-derived macrophages in corneal allograft rejection

Abstract

Purpose: To examine inhibition of recruitment of monocyte and related macrophages and factors derived from these cells in the context of corneal graft rejection.

Methods: Flow cytometry was used to examine monocyte recruitment at the site of corneal graft rejection, and multiplex protein assays to quantify pro-inflammatory mediators in this context. An inflammatory milieu was modelled by stimulation of human macrophages with lipopolysaccharide (LPS) or interferon (IFN)γ. Transwell apparatus were used to model monocyte migration towards inflammatory foci across an endothelial barrier and the effect of targeting chemokine pathways was tested by depleting specific chemokines or using neutralising antibodies to selected chemokine receptors. The cytotoxic effect on human corneal endothelium was tested by confocal microscopy enumeration of nuclei after ex vivo exposure to inflammatory mediators and inflammatory monocytes.

Results: Aqueous humour samples showed a selective enrichment for classical (CD14++/CD16-) monocytes during corneal graft rejection. We established the repertoire of monocyte chemokines upregulated in inflammatory responses and tested the effect of targeting these pathways on monocyte recruitment. We show that depletion of individual chemokines only partially attenuates monocyte recruitment, and that targeting of chemokine receptors may be more effective. Confocal microscopy showed that macrophage derived inflammatory milieus in this model had significant cytopathic effect on human corneal endothelial cells.

Conclusions: We have built on the evidence from human and rodent studies supporting a role for monocyte recruitment in the immunopathogenesis of corneal rejection. We have developed human experimental models to identify candidates for therapeutic targeting to inhibit monocyte recruitment and cytopathic mechanisms for corneal endothelial injury during corneal allograft rejection. These models may lead to novel immunotherapeutic strategies to modulate the pathogenesis of rejection and prolong the survival of corneal allografts.

Keywords: 481 cornea: endothelium • 490 cytokines/chemokines • 741 transplantation