iNOS dependent and independent phases of lymph node expansion in mice with TNF-induced inflammatory-erosive arthritis

Abstract

IntroductionA pivotal effect of lymphatic vessel (LV) function in joint homeostasis was identified in the tumor necrosis factor-transgenic (TNF-Tg) mouse model of rheumatoid arthritis (RA). Specifically, loss of LV contractions is associated with progressive synovitis and erosions. Furthermore, draining lymph node expansion is a biomarker of arthritic progression, and both macrophages and lymphatic endothelial cells express inducible nitric oxide synthase (iNOS), which disrupts LV contraction and transport of immune cells to the draining lymph nodes. Therefore, to directly assess these relationships, we tested the hypothesis that TNF-Tg mice with global genetic ablation of iNOS (iNOS−/−) will show delayed draining lymph node expansion, maintained LV contractions, and decreased synovitis and erosions.MethodiNOS−/−× TNF-Tg female and male mice, and control littermates (iNOS−/−, TNF-Tg, and WT), were examined with (1) ultrasound to determine popliteal lymph node (PLN) volume and (2) near-infrared imaging (NIR) to assess popliteal LV contraction frequency, and differences between genotypes were assessed at 3, 4, 5, and 6 months of age. Knees and PLN were harvested at 4 months in females and 6 months in males, to assess synovitis, bone erosions, and cellular accumulation in PLN sinuses via histology.ResultsInitially, an increase in PLN volume was observed for both female and male iNOS−/−× TNF-Tg and TNF-Tg compared to their WT and iNOS−/− counterparts at 2 and 3 months, respectively. Subsequently, TNF-Tg PLNs continue to increase in volume, while iNOS−/−× TNF-Tg did not increase in volume from the initial timepoints. WT and iNOS−/− PLN volume was unchanged throughout the experiment. LV contraction frequency was increased at 4 months in females and 5 months in males, in the iNOS−/−× TNF-Tg mice compared to the TNF-Tg. Synovitis and erosions were moderately reduced in iNOS−/−× TNF-Tg versus TNF-Tg knees in females, while no differences in knee pathology were observed in males.ConclusionsGenetic iNOS ablation maintains draining lymph node volume and LV function during TNF-induced inflammatory arthritis and is associated with moderately decreased joint inflammation and damage.

Altered Lymphatic Vessel Anatomy and Markedly Diminished Lymph Clearance in Affected Hands of Patients With Active Rheumatoid Arthritis

“…After 28 days of therapy, the NO inhibitor did not significantly decrease DAS28 scores or synovitis scoring via ultrasound compared to placebo, while prednisone significantly reduced both outcome measures (43). Interestingly, the contraction frequency defect in murine TNF-transgenic end-stage arthritis can be recovered with both chemical iNOS inhibition and global genetic iNOS ablation, but has no effect on arthritis severity (15,16). The limited effectiveness of NO inhibitors in the treatment of RA may be due to the various roles NO plays in the bone and lymphatic microenvironments (16,44), in addition to our finding that RA patients do not have LV contraction deficits (Figure 3).…”

“…Interestingly, the contraction frequency defect in murine TNF-transgenic end-stage arthritis can be recovered with both chemical iNOS inhibition and global genetic iNOS ablation, but has no effect on arthritis severity (15,16). The limited effectiveness of NO inhibitors in the treatment of RA may be due to the various roles NO plays in the bone and lymphatic microenvironments (16,44), in addition to our finding that RA patients do not have LV contraction deficits (Figure 3). Our findings suggest that LV contraction frequency may not significantly contribute to lymphatic clearance of the web spaces, and thus therapeutic intervention targeting LV contraction frequency may not be efficacious in treating RA.…”

“…In the TNFtransgenic murine model of arthritis, no changes in contraction frequency were observed during the onset of disease, but during end-stage disease there was a complete cessation of contractions, with significantly diminished clearance, lymph velocity, and lymph pumping pressure (7)(8)(9)15). Interestingly, this contraction frequency defect can be recovered with both chemical inducible nitric oxide synthase (iNOS) inhibition and genetic iNOS ablation, but has no effect on arthritis severity (15,16). One potential explanation for our clinical finding is that there were fewer total functional vessels to carry the lymph fluid despite maintenance of contraction frequency, resulting in diminished clearance.…”

“…Lymphatic anatomy of the hands and forearm have been characterized previously, and we used these data as references (28)(29)(30)(31). ROIs were placed over the LVs, NIR/ICG signal intensity was measured over time, and boluses of dye flowing through the LVs, marked by peaks in signal intensity, were counted (by RDB and HMK) in order to generate contractions per minute, similar to previously described murine NIR methods (16). Contractions were quantified as both the sum and the average of all contractions on the basilic or cephalic bundle, as described in Supplementary Figures 1A-D (http://onlin e libr ary.wiley.com/doi/10.1002/art.41311/ abstract).…”

“…The lymphatic system provides homeostatic turnover of interstitial fluid and trafficking of immune cells to the closest lymph node (4,5). Multiple studies on murine RA models have demonstrated dramatic changes in both lymphatic vessel (LV) contractions and the joint-draining lymph node volume with disease progression (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). During the early and middle phases of musculoskeletal inflammation, the joint-draining lymph node undergoes a rapid increase in volume to several fold greater than unaffected lymph nodes.…”