[N acetyl-aspartyl glutamic acid (NAAGA) inhibits the adhesion of leukocytes to activated endothelial cells and down-modulates the cytokine-induced expression of adhesion molecules].
BACKGROUND
Cell adhesion plays a pivotal role in most ocular surface inflammatory diseases. Adhesion molecules mediate cell-to-cell and cell-to-matrix adhesion. Their expression is up-regulated by pro-inflammatory stimuli such as cytokines, histamine or complement-derived anaphylatoxins. The dipeptide N acetyl-aspartyl glutamic acid (NAAGA) is used as unpreserved topical eyedrops in the treatment of allergic conjunctivitis. NAAGA is known to inhibit leukotriene synthesis, histamine release by mast cells, and complement-derived anaphylatoxin production.


OBJECTIVE
To investigate the potential capability of NAAGA to interfere with leukocyte adhesion to endothelial cells and modulate cytokine-induced expression of adhesion molecules.


METHODS
Human blood-derived leukocytes were co-cultured with human umbilical vein endothelial cells (HUVECs) in the absence or the presence of 1000 UI/mL human recombinant TNFalpha, 10(-4) M histamine di-hydrochloride or 5x10(-6) M human recombinant C5a, and in the absence or presence of NAAGA (final concentration 2.45%). Adhesion of leukocytes to HUVECs was calculated by subtracting the number of nonadherent leukocytes from the total number of leukocytes. Expression of adhesion molecules was assessed by flow cytometry using anti-CD11b, anti-CD49d, anti-ICAM-1 (CD54), anti-ICAM-2 (CD102), anti-VCAM-1 (CD106) and anti-ELAM-1 (CD62E) monoclonal antibodies.


RESULTS
NAAGA was found to totally inhibit adhesion of unstimulated leukocytes, or leukocytes activated with C5a, TNFalpha, or histamine, to TNFalpha-stimulated HUVECs (P=0.0001). Adhesion of leukocytes to unstimulated HUVECs was not modified by NAAGA. Similar results were obtained with endothelial cells stimulated by histamine or C5a. Taken together, these data indicate that NAAGA totally abrogates cell adhesion under inflammatory conditions, without interfering with the physiological adhesion of leukocytes to normal endothelium. At the molecular level, NAAGA inhibited histamine-induced expression of CD11b (P=0.0004) and CD49d (P=0.0045) on granulocytes. On TNFalpha-activated HUVECs, NAAGA induced a significant decrease in the VCAM-1 expression level (P<0.0001) and totally reversed TNFalpha-induced overexpression of ICAM-1 (P=0.0069), ICAM-2 and ELAM-1 (P<0.0001), without interfering with baseline expression of these molecules.


CONCLUSIONS
These results show that the antiallergic compound NAAGA directly inhibits leukocyte adhesion to endothelial cells induced by pro-inflammatory stimuli, and abrogates TNFalpha-induced expression of adhesion molecules on granulocytes and endothelial cells. This capacity to block overexpression of selectins and integrins induced by pro-inflammatory stimuli confers to NAAGA a potential as an anti-inflammatory drug, since interfering with adhesion molecule expression is probably one of the most efficient ways to curb leukocyte recruitment to inflammatory sites.
Bouhlal
H
H
Hôpital Broussais, INSERM U430, 96, rue Didot, 75014 Paris.
Blondin
C
C
Haeffner-Cavaillon
N
N
Goldschmidt
P
P
D015703:Antigens, CD / Q000096:biosynthesis; D039481:CD11b Antigen / Q000096:biosynthesis; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology*; D019040:E-Selectin / Q000096:biosynthesis; D004727:Endothelium / Q000166:cytology* / Q000502:physiology*; D006801:Humans; D039441:Integrin alpha4 / Q000096:biosynthesis; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D007962:Leukocytes / Q000187:drug effects* / Q000502:physiology*; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis
D015703:Antigens, CD; D039481:CD11b Antigen; D015815:Cell Adhesion Molecules; D004151:Dipeptides; D019040:E-Selectin; C065289:ICAM2 protein, human; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D039441:Integrin alpha4; C027172:isospaglumic acid