The pro-inflammatory cytokine TNF-α inhibits lymphatic pumping via activation of the NF-κB-iNOS signaling pathway

Abstract

Objective: Mesenteric lymphatic vessel pumping, important to propel lymph and immune cells from the intestinal interstitium to the mesenteric lymph nodes, is compromised during intestinal inflammation. The objective of this study was to test the hypothesis that the pro-inflammatory cytokine TNF-α, is a significant contributor to the inflammation-induced lymphatic contractile dysfunction, and to determine its mode of action.

Methods: Contractile parameters were obtained from isolated rat mesenteric lymphatic vessels mounted on a pressure myograph after 24-hours incubation with or without TNF-α. Various inhibitors were administered, and quantitative real-time PCR, Western blotting, and immunofluorescence confocal imaging were applied to characterize the mechanisms involved in TNF-α actions.

Results: Vessel contraction frequency was significantly decreased after TNF-α treatment and could be restored by selective inhibition of NF-кB, iNOS, guanylate cyclase, and ATP-sensitive K⁺ channels. We further demonstrated that NF-кB inhibition also suppressed the significant increase in iNOS mRNA observed in TNF-α-treated lymphatic vessels and that TNF-α treatment favored the nuclear translocation of the p65 NF-κB subunit.

Conclusions: These findings suggest that TNF-α decreases mesenteric lymphatic contractility by activating the NF-κB-iNOS signaling pathway. This mechanism could contribute to the alteration of lymphatic pumping reported in intestinal inflammation.

Keywords: cytokine; inflammation; lymphatic vessel contraction; nitric oxide synthase; nuclear factor-kappa B.

Figure 1. Effects of TNF-α on the contractile activity of pressurized rat mesenteric lymphatic vessels

A. Original recordings of the contractile activity of lymphatic vessels pressurized at 5 cmH₂O after a 24-h incubation without (sham) and with increasing concentrations (0.1, 1.0 and 10 ng/ml) of TNF-α. Time scale bar applies to all traces. B. TNF-α caused a concentration-dependent decrease in contraction frequency (top bar graph), but did not affect the amplitude of contractions or the tone index (n=4; *P<0.05, **P<0.01, repeated measures of ANOVA with Dunnett’s post-hoc test).

Figure 2. Effect of PDTC on the TNF-α-induced decrease in lymphatic contraction frequency

A. Original recordings comparing the contractile activity of lymphatic vessels pressurized at 5 cmH₂O after a 24-h incubation in DMEM/F12 media (sham), in media plus 3 ng/ml TNF-α and in media/TNF-α plus 10 μM PDTC. Time scale bar applies to all traces. B. Summary bar graph of the effect of PDTC on TNF-α-induced decreased response relative to the contraction frequency measured in the respective sham vessels (n=4–5; *P<0.05 vs own sham, paired Student t-test and ^##P<0.05, repeated measures of ANOVA with Bonferroni’s post-hoc test).

Figure 3. Effect of TNF-α on NF-κB activation in lymphatic vessels

A. Representative western blots of unphosphorylated (left panel) and phosphorylated (right panel) IκB protein following 4-h incubation without (sham) and with 3ng/ml and 10 ng/ml TNF-α. B. Summary densitometry bar graph of the level of IκB and p-IκB in the three vessel groups normalized to the level of SM22 (n=4; *P<0.05, **P<0.01, repeated measures of ANOVA with Dunnett’s post-hoc test). C. Level of nuclear translocation of the NF-κB subunit, p65 in lymphatic cells, illustrated by confocal immunofluorescence. Representative images depicting nucleus (blue, DAPI) and p65 (red) in a sham (top left panel) and a TNF-α-treated lymphatic vessel (middle panel, 10 ng/ml, 3-h incubation). Maximal projection of a stack of 3 images taken at the level of the muscles, as denoted by the vertical (perpendicular to the vessel length) orientation of most of the nucleus (note immune cells identified by their round nucleus, arrowheads). Nuclear translocation (pink nuclear staining) is apparent in most cells of the TNF-α-treated vessel as confirmed by the summary bar graph representing the level of nuclear p65 fluorescence (right panel; n=4; *P<0.05 vs own sham, paired Student t-test). Nuclear translocation of the p65 subunit is also evident in cultures of lymphatic endothelial (bottom left panels) and muscle cells (bottom right panels) isolated from the same rat mesenteric vessels when treated with TNF-α. Images are representative of 3–5 similar experiments.

Figure 4. Effect of TNF-α on the gene expression of NOSs and COXs in lymphatic vessels

Bar graphs representing time-course mRNA expression as measured by qPCR of genes for iNOS (A), eNOS (B) COX-2 (C) and COX-1 (D) in fresh lymphatic vessels (control), and in vessels incubated for 6, 12 and 24 h in normal media (sham) or in the presence of TNF-α (3 ng/ml). Gene expressions were normalized to the expression of the housekeeping gene β-actin (n=3–7; *P<0.05, **P<0.01, two-way ANOVA with Bonferroni’s post-hoc test).

Figure 5. Effect of 1400W on the TNF-α-induced decrease in lymphatic contraction frequency

A. Original recordings comparing the contractile activity of lymphatic vessels pressurized at 5 cmH₂O after a 24-h incubation in DMEM/F12 media (sham), in media plus 3 ng/ml TNF-α and in media/TNF-α plus 10 μM 1400W. Time scale bar applies to all traces. B. Summary bar graph of the effect of 1400W on TNF-α-induced decreased response relative to the contraction frequency measured in the respective sham vessels (n=5; *P<0.05 vs own sham, paired Student t-test and ^##P<0.01, repeated measures of ANOVA with Bonferroni’s post-hoc test. C. Summary bar graph of the absence of effect of a 24-h incubation with 10 μM indomethacin on TNF-α-induced decreased response relative to the contraction frequency measured in the respective sham vessels (n=4).

Figure 6. Effect of PDTC on the TNF-α-induced upregulation of iNOS in lymphatic vessels

Bar graphs representing iNOS mRNA expression as measured by qPCR in lymphatic vessels without (control), and after 6 h of incubation in media alone (sham), 10 μM PDTC, TNF-α (3 ng/ml) and TNF-α plus PDTC. Gene expression was normalized to the expression of β-actin (n=4; **P<0.01 vs sham, one-way ANOVA with Bonferroni’s post-hoc test).

Figure 7. Effect of ODQ on the TNF-α-induced decrease in lymphatic contraction frequency

A. Original recordings comparing the contractile activity of lymphatic vessels pressurized at 5 cmH₂O after a 24-h incubation in DMEM/F12 media (sham), in media plus 3 ng/ml TNF-α and in media/TNF-α plus 10 μM ODQ. Time scale bar applies to all traces. B. Summary bar graph of the effect of ODQ on TNF-α-induced decreased response relative to the contraction frequency measured in the respective sham vessels (n=4; **P<0.01 vs own sham, paired Student t-test; ^#P<0.05, repeated measures of ANOVA with Bonferroni’s post-hoc test).

Figure 8. Effect of glibenclamide on the TNF-α-induced decrease in lymphatic contraction frequency

A. Original recordings of the contractile activity of lymphatic vessels pressurized at 5 cmH₂O after a 24-h incubation in media plus 3 ng/ml TNF-α before (left trace) and during incubation with 10 μM glibenclamide (right trace). Time scale bar applies to both traces. B. Summary bar graph of the effect of glibenclamide administration on TNF-α-induced decreased response relative to the contraction frequency measured in sham vessels (n=4; *P<0.05, paired Student t-test; ^#P<0.05 vs sham, repeated measures of ANOVA with Dunnett’s post-hoc test).

Figure 9. Expression of TNF-α and its receptor in ileum and mesenteric lymphatic vessels of TNBS-treated rats

Bar graphs representing mRNA expression as measured by qPCR of genes for TNF-α (A) and TNFR1 (B) in lymphatic vessels and ileum collected 3 days after surgical injection of saline (sham) and TNBS in rat ileum. Gene expressions were normalized to the expression of the housekeeping gene β-actin (n=4; *P<0.05, **P<0.01, ***P<0.001, unpaired Student t-test).