Granulocyte macrophage colony-stimulating factor expression in human herpetic stromal keratitis: implications for the role of neutrophils in HSK.
OBJECTIVE
Granulocyte macrophage colony-stimulating factor (GM-CSF) is thought to play a key role in chronic inflammatory diseases by governing the survival and function of infiltrating neutrophils. The objective of this study was to determine the putative role of GM-CSF in the pathogenesis of human herpetic stromal keratitis (HSK).


METHODS
Primary human corneal fibroblast (HCF) cultures and a telomerase-immortalized human corneal epithelial (HCE) cell line representative of native HCE were stimulated with the known HSK-inducing cytokines interferon (IFN)-gamma, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha. Alternatively, the T-cell cytokine IL-17 was added solely or simultaneously. Human neutrophils were incubated with conditioned medium (CM) of the HCF and HCE stimulated with the aforementioned cytokines, or recombinant GM-CSF, and their viability or activation status was determined by flow cytometry. GM-CSF and IL-8 secretion levels in the CM were determined by ELISA. The antibody-dependent cellular cytotoxicity (ADCC) of neutrophils toward herpes simplex virus (HSV)-infected HCFs was determined by flow cytometry. The expression of GM-CSF was determined in HSK and control corneal buttons by real-time RT-PCR and immunohistology.


RESULTS
Compared with IFN-gamma, CM of either cell type stimulated with IL-1beta, or in the case of HCE cells, stimulated with TNF-alpha or IL-17, delayed neutrophil apoptosis significantly. Only in HCFs did IL-17 exhibit a synergistic effect with TNF-alpha. The antiapoptotic activity was attributable in part to the GM-CSF secreted by the activated HCFs and HCE cells. GM-CSF stimulation of neutrophils induced their activation and the secretion of IL-8. GM-CSF did not increase significantly the ADCC reaction of neutrophils toward HSV-infected HCFs. Finally, GM-CSF was expressed in corneas of the patients with HSK but not in control subjects.


CONCLUSIONS
The data suggest that GM-CSF, expressed by cornea-resident cells such as HCFs and HCE cells, may play a role in the immunopathogenesis of HSK by prolonging the survival and modulating the effector function of corneal infiltrating neutrophils.
Duan
Rui
R
Institute of Virology, Erasmus Medical Center, Rotterdam, The Netherlands.
Remeijer
Lies
L
van Dun
Jessica M
JM
Osterhaus
Albert D M E
AD
Verjans
Georges M G M
GM
D000920:Antibody-Dependent Cell Cytotoxicity; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000502:physiology; D006801:Humans; D007124:Immunoenzyme Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D016209:Interleukin-8 / Q000378:metabolism; D016849:Keratitis, Herpetic / Q000378:metabolism*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000502:physiology*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D053583:Interleukin-1beta; D016209:Interleukin-8; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor