| Rheumatoid arthritis (RA) is a chronic inflammatory joint disease in which patients often suffer from arthritic flare. Using longitudinal contrast-enhanced (CE)-MRI to study knee arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice, we observed that the popliteal lymph nodes (PLN) firstly "expand" in size and contrast enhancement, and then suddenly "collapse" during arthritic flare. Since CE-MRI is too costly for phenotyping and longitudinal analyses of PLN, our aim was to develop ultrasound (US) methods that could replace MRI. In our initial study, we demonstrated a significant correlation between PLN volumes determined by US vs. MRI. However, since PLN collapse is more closely associated with lymphatic draining function than volume, we evaluated CE-US methods to distinguish changes in lymphatic transport, which was shown as a biomarker of arthritic flare. Unfortunately, delivery of the contrast agent prior to US significantly impairs lymphatic function, making it unsuitable for phenotyping PLNs. Thus, we went on to develop power Doppler (PD) US methods to phenotype PLN with greater accuracy and cost effectiveness vs. CE-MRI.;Another important prior observation we made is that arthritic flare is associated with the loss of lymphatic pulse. From other models of inflammation, lymphatic pulse is known to be controlled by endothelial nitric oxide synthase (eNOS), and inhibited by inducible NOS (iNOS) expressed in Gr-1+ cells. To test the hypothesis that eNOS/iNOS dysregulation is responsible for the loss of lymphatic pulse during arthritic flare in TNF-Tg mice, we performed IHC and in vivo pharmacological intervention studies with selective and non-selective iNOS inhibitors. The IHC results demonstrated that large numbers of iNOS expressing Gr-1+ cells exist in collapsed PLN. By evaluating the lymphatics with NIR-ICG imaging, we observed that the specific iNOS inhibitor L-NIL increased lymphatic pulse and afferent lymphatic drainage in TNF-Tg mice. Additionally, the micro-CT results showed that bone erosions were ameliorated in L-NIL treated TNF-Tg mice compared with placebo. Collectively, these results suggest a model that the accumulation of iNOS-expressing Gr-1+ cells accelerates the onset of flare in the setting of inflammatory arthritis via inhibition of lymphatic drainage, and identifies this pathway as a potential target for RA therapy. |
