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Suppression of nuclear factor kappa B and CD18-mediated leukocyte adhesion to the corneal endothelium by
M Shimoyama1, S Shimmura, K Tsubota
1Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
Insights
Leukocyte adhesion to corneal endothelium involves CD18 and is reduced by dexamethasone. Dexamethasone may work by inhibiting nuclear factor kappa B (NFkappaB) activation.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Leukocyte adhesion to the corneal endothelium plays a role in ocular inflammation.
- Understanding the molecular mechanisms of this adhesion is crucial for developing targeted therapies.
Purpose of the Study:
- To confirm CD18 antigen mediates leukocyte adhesion to corneal endothelial cells.
- To investigate if dexamethasone suppresses this adhesion by inhibiting nuclear factor kappa B (NFkappaB) activation.
Main Methods:
- Cultured bovine corneal endothelium was stimulated with tumor necrosis factor alpha (TNFalpha).
- Leukocyte adhesion was assessed with and without anti-CD18 antibody or dexamethasone pretreatment.
- NFkappaB localization was determined using confocal immunocytochemistry.
Main Results:
- TNFalpha significantly increased leukocyte adhesion to corneal endothelial cells.
- Anti-CD18 antibody and dexamethasone pretreatment significantly reduced this adhesion.
- Dexamethasone treatment inhibited TNFalpha-induced nuclear translocation of NFkappaB.
Conclusions:
- CD18 on activated leukocytes mediates adhesion to corneal endothelium.
- Dexamethasone inhibits leukocyte adhesion, potentially by suppressing NFkappaB nuclear translocation.
Purpose:
To demonstrate that leukocyte adhesion to cultured corneal endothelial cells is mediated by the CD18 antigen, and to determine whether dexamethasone directly suppresses adhesion by inhibiting activation of nuclear factor kappa B (NFkappaB).
Methods:
Cultured bovine corneal endothelium was stimulated for 6 hours by 40 micron/ml tumor necrosis factor alpha (TNFalpha). Dexamethasone was added 1 hour before TNFalpha stimulation in the dexamethasone group. After stimulation, neutrophils separated from a healthy human volunteer were added with or without anti-CD18 antibody. The culture plate was settled for 15 minutes at 37 degrees C, and then neutrophils were activated by N-formyl-methionyl-leucyl-phenylalanine for 5 minutes. Nonadherent neutrophils were removed by sealing and inverting the culture well. The intracellular localization of NFkappaB after TNFalpha simulation was determined by confocal immunocytochemistry using an anti-p65 antibody.
Results:
Neutrophil adhesion to cultured corneal endothelial cells increased significantly on exposure to TNFalpha (451.4+/-45.4 cells/mm2, n = 16) compared to control (156.7+/-27.3 cells/mm2, n = 16, P < 0.01). This increased adhesion was suppressed by the addition of anti-CD18 antibody (157.6+/-25.1 cells/mm2, n = 8, P < 0.01) and by pretreatment with 10(-7) M dexamethasone (207.9+/-31.5 cells/mm2, n = 10, P < 0.01). Immunocytochemistry 60 minutes after stimulation revealed that NFkappaB was located in the cytoplasm in unstimulated cells; however, the addition of TNFalpha caused NFkappaB to translocate into the nucleus. Pretreatment with dexamethasone tapered NFkappaB translocation into the nucleus.
Conclusions:
Leukocyte adhesion to the corneal endothelium was shown to be mediated by CD18 expressed on activated leukocytes. Pretreatment of the endothelium with dexamethasone inhibited leukocyte adhesion; this may be due in part to the suppression of NFkappaB entry into the nucleus.