Nitric oxide secretion in human conjunctival fibroblasts is inhibited by alpha linolenic acid.
OBJECTIVE
It is known that both human conjunctival fibroblasts (HCF) and corneal epithelial (HCE) cells contribute to the inflammatory process in the ocular surface by releasing inflammatory cytokines. In addition, nitric oxide (NO) has an important role in inflammatory responses in the ocular surface. In the present study, we aimed to characterize the capacity of these cells to release nitric oxide in response to cytokines and Lipopolysaccharide (LPS), and show that Alpha-linoleic acid (ALA) inhibits these responses.


METHODS
HCF, HCE cells, peripheral blood mononuclear cells (PBMCs) and co-culture of HCF and PBMC were treated with different combinations of inflammatory inducers, including interleukin)IL- (6, tumor necrosis factors (TNF)-α, interferon (IFN)- γ and IL-1β and LPS. Nitrite levels were measured in cell supernatants with and without ALA by the Griess reaction test at 24, 48 and 72 h respectively. Expression of nitric oxide synthase 2 (NOS-2) was evaluated by real-time PCR.


RESULTS
All cytokine combinations had an inducible effect on nitrite secretion in HCF, PBMC and co-cultured PBMC and HCF, but not in HCE cells. Treatment with a combination of IL-6, LPS, TNF-α, IFN- γ and IL-1β induced the highest nitrite secretion (2.91 fold, P < 0.01) as compared to cells incubated in medium alone. nitrite secretion was reduced by 38.9 % (P < 0.05) after treatment with ALA alone. Co-culturing PBMC with HCF with and without ALA treatment demonstrated similar results in nitrite level as,compared to PBMC alone. In addition, ALA significantly decreased NOS-2 expression in HCF by 48.9 % (P < 0. 001) after 72 h.


CONCLUSIONS
The decrease in nitrite release and inhibition of NOS-2 expression indicate that ALA may have an anti-inflammatory effect both on HCF and on peripheral immune cells. This indicates that ALA may serve as a potent anti-inflammatory agent in ocular surface inflammation.
Departments of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Nir
N
Erdinest
Departments of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Noam
N
Shohat
Clinical Immunology and Allergy Unit, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Eli
E
Moallem
Departments of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Claudia
C
Yahalom
Departments of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Hadas
H
Mechoulam
Departments of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Irene
I
Anteby
Department of Neurology at The Agnes Ginges Center for Human Neurogenetics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Haim
H
Ovadia
Departments of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.|Cornea and Refractive Surgery Service, Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, 91120 Israel.
Abraham
A
Solomon
Alpha linolenic acid; Conjunctival fibroblasts; Corneal epithelium; Nitric oxide