Lymphatic endothelial cells efferent to inflamed joints produce iNOS and inhibit lymphatic vessel contraction and drainage in TNF-induced arthritis in mice

Abstract

BackgroundIn this study, we sought to determine the cellular source of inducible nitric oxide synthase (iNOS) induced in lymphatic endothelial cells (LECs) in response to tumor necrosis factor (TNF), the effects of iNOS on lymphatic smooth muscle cell (LSMC) function and on the development of arthritis in TNF-transgenic (TNF-Tg) mice, and whether iNOS inhibitors improve lymphatic function and reduce joint destruction in inflammatory erosive arthritis.MethodsWe used quantitative polymerase chain reactions, immunohistochemistry, histology, and near-infrared imaging to examine (1) iNOS expression in podoplanin + LECs and lymphatic vessels from wild-type (WT) and TNF-Tg mice, (2) iNOS induction by TNF in WT LECs, (3) the effects of iNOS inhibitors on expression of functional muscle genes in LSMCs, and (4) the effects of iNOS inhibitors on lymphatic vessel contraction and drainage, as well as the severity of arthritis, in TNF-Tg mice.ResultsLECs from TNF-Tg mice had eight fold higher iNOS messenger RNA levels than WT cells, and iNOS expression was confirmed immunohistochemically in podoplanin + LECs in lymphatic vessels from inflamed joints. TNF (0.1 ng/ml) increased iNOS levels 40-fold in LECs. LSMCs cocultured with LECs pretreated with TNF had reduced expression of functional muscle genes. This reduction was prevented by ferulic acid, which blocked nitric oxide production. Local injection of L-N6-(1-iminoethyl)lysine 5-tetrazole-amide into inflamed paws of TNF-Tg mice resulted in recovery of lymphatic vessel contractions and drainage. Treatment of TNF-Tg mice with ferulic acid reduced synovial inflammation as well as cartilage and bone erosion, and it also restored lymphatic contraction and drainage.ConclusionsiNOS is produced primarily by LECs in lymphatic vessel efferent from inflamed joints of TNF-Tg mice in response to TNF and inhibits LSMC contraction and lymph drainage. Ferulic acid represents a potential new therapy to restore lymphatic function and thus improve inflammatory arthritis by inhibiting local production of nitric oxide by LSMCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0963-8) contains supplementary material, which is available to authorized users.

The pro‐inflammatory cytokineTNF‐α inhibits lymphatic pumping via activation of theNF‐κB‐iNOSsignaling pathway

“…Although the PCR experiments on intact lymphatics do not permit us to determine in which cell type the iNOS upregulation occurred, we observed that both lymphatic endothelial and muscle cells in culture exhibited upregulated iNOS mRNA expression following TNF-α treatment (Rehal, Roizes & von der Weid, unpublished data) and we can reasonably infer that both cell types are sensitive to TNF-α and contribute to lymphatic contractile dysfunction by upregulating iNOS expression and NO production. This proposal is further supported by other studies demonstrating iNOS upregulation in cultured lymphatic endothelial cells isolated from sheep, mouse and rat mesenteric lymphatic vessels upon stimulation with LPS, TNF-α, and other cytokines [17,42,45] and in mesenteric lymphatic vessels from old rats, which display an inflamed phenotype[51] Alternatively, NO could also be generated by immune cells reminiscent of macrophages that have recently been reported to populate the wall of mesenteric lymphatic vessels [12,16,40]. Indeed, some of these cells display NF-κB activation (see arrowheads in Fig.…”

“…[38][39][40] While in physiological states this action essentially relies on eNOS, 41,42 several studies demonstrated that NO produced under the catalytic action of iNOS plays a major role in lymphatic dysfunction observed during inflammation. 1,2,43,44 upon stimulation with LPS, TNFα, and other cytokines 26,50,51 and in mesenteric lymphatic vessels from old rats, which display an inflamed phenotype 52 Alternatively, NO could also be generated by immune cells reminiscent of macrophages that have recently been reported to populate the wall of mesenteric lymphatic vessels. 30,53,54 Indeed, some of these cells display NF-κB activation (see arrowheads in Figure 3C).…”

(5R)-5-hydroxytriptolide (LLDT-8) prevents collagen-induced arthritis through OPG/RANK/RANKL signaling in a rat model of rheumatoid arthritis

“…Nitric oxide synthase (NOS) is a key enzyme in the generation of NO (29). During the development of OA, cartilage cells are impaired, caused by a release of inflammatory factors, which leads to the generation of NO (30). Released NO inhibits cartilage cell proliferation and …”

Attenuated Joint Tissue Damage Associated With Improved Synovial Lymphatic Function Following Treatment With Bortezomib in a Mouse Model of Experimental Posttraumatic Osteoarthritis

“…This may be related to the high levels of inducible nitric oxide synthase (iNOS) produced by macrophages and LECs. Inducible NOS directly affects lymphatic muscle cells via excess nitric oxide . We observed that in mice carrying the tumor necrosis factor transgene (TNF‐Tg), a model of rheumatoid arthritis (RA), macrophages were attached to LECs in the collecting lymphatic vessel that drains the inflamed joints .…”