TNF-alpha induced endothelial MAdCAM-1 expression is regulated by exogenous, not endogenous nitric oxide

Abstract

Background: MAdCAM-1 is an adhesion molecule expressed in Peyer's patches and lymphoid tissues which is mobilized by cytokines like TNF-alpha and is a major determinant of lymphocyte trafficking to the gut in human inflammatory bowel disease (IBD). It has been suggested that both reactive oxygen and nitrogen metabolites participate in regulating adhesion molecule expression in response to TNF-alpha.

Methods: To examine how exogenous and endogenous sources of NO modulate MAdCAM-1 induction by TNF-alpha, we pre-treated mouse lymphatic endothelial cells with either long or short acting NO donors prior to TNF-alpha-stimulation, and measured MAdCAM-1 induction at 24 h.

Results and discussion: DETA-NO, a long-acting NO donor, and SperNO, a rapid releasing NO donor both inhibited TNF-alpha-stimulated MAdCAM-1 expression in a concentration dependent manner. Both NO donors also reduced a4b7-dependent lymphocyte endothelial adhesion. Inhibition of endogenous NO production by either L-NAME, a non-selective NOS inhibitor, or by 1400 w, a selective iNOS inhibitor failed to induce, or potentiate TNF-alpha regulated MAdCAM-1 expression.

Conclusions: Exogenous NO donors may be beneficial in the treatment of IBD, while endogenous nitric oxide synthases may be less effective in controlling adhesion molecule expression in response to cytokines.

Figure 1

RT-PCR analysis of MAdCAM-1 and GAPDH in SVEC exposed to TNF-α with or without DETA-NONOate. In controls, only a faint MAdCAM-1 transcript is detected (474 bp). MAdCAM-1 band is easily detected after TNF-α stimulation (20 ng/ml). DETA-NONOate (100 μM) blocked TNF-α-induced MAdCAM-1 transcript. In each panel, 307 bp bands indicate GAPDH transcripts, used to evaluate amount and quality of RNA. Similar results were obtained in 3 separate experiments.

Figure 2

Effect of NO on MAdCAM-1 in SVEC. Cells were pretreated with (A) DETA-NONOate or (B) Spermine-NONOate (SNO) with indicated concentrations and then exposed to TNF-α (20 ng/ml) stimulation in the continued presence of NO donors. MAdCAM-1 expression is significantly increased after TNF-α. DETA-NONOate or Spermine-NONOate (SNO) blocked the TNF-α induced MAdCAM-1 expression dose dependently. Values represent mean ± SE; each group (n = 3). ^* p < 0.001 vs. untreated controls, ^# p < 0.001 vs. TNF-α treatment. (C) Effect of NO on p65 NF-κB translocation. Immunoblots of NF-κB p65 in nuclear extracts from SVEC. Nuclear extracts (40 μg) were loaded in each well. (B) Inhibition of TNF-α activation of NF-κB in SVEC. After 1 h of TNF-α (20 ng/ml) treatment nuclear p65 of NF-κB is induced. Pretreatment of the cells with DETA-NONOate (100 μM) or Spermine-NONOate (100 μM) reduced the TNF-α induced nuclear translocation of the NF-κB p65 subunit.

Figure 3

Effect of NOS inhibitors on MAdCAM-1 in SVEC. Cells were pretreated with vehicle, L-NAME or 1400ω with indicated concentrations and then exposed to TNF-α (20 ng/ml) stimulation in the continued presence of the NOS inhibitor. NOS inhibitors themselves did not induce MAdCAM-1 expression nor did they increase MAdCAM-1 in TNF-α treated cells. Similar results were obtained in 3 separate experiments.

Figure 4

Adhesion of TK-1 cells on SVEC. TNF-α induced significant increase of TK-1 cells adhesion. This increased adhesion was significantly inhibited by pretreatment with MAdCAM-1 Ab. Pretreatment of DETA-NONOate (100 μM) also significantly inhibited TNF-α (20 ng/ml, 24 h) -induced TK-1 adhesion on SVEC. Each value represents the mean ± SE; each group (n = 4). ^* p < 0.001, ^** p < 0.05 vs. untreated control, ^# p < 0.001 vs. TNF-α treatment.