Brief Report: Treatment of Tumor Necrosis Factor-Transgenic Mice With Anti-Tumor Necrosis Factor Restores Lymphatic Contractions, Repairs Lymphatic Vessels, and May Increase Monocyte/Macrophage Egress

Abstract

Objective: Recent studies have demonstrated that there is an inverse relationship between lymphatic egress and inflammatory arthritis in affected joints. As a model, tumor necrosis factor (TNF)-transgenic mice develop advanced arthritis following draining lymph node (LN) collapse, and loss of lymphatic contractions downstream of inflamed joints. It is unknown if these lymphatic deficits are reversible. This study was undertaken to test the hypothesis that anti-TNF therapy reduces advanced erosive inflammatory arthritis, associated with restoration of lymphatic contractions, repair of damaged lymphatic vessels, and evidence of increased monocyte egress.

Methods: TNF-transgenic mice with advanced arthritis and collapsed popliteal LNs were treated with anti-TNF monoclonal antibody (10 mg/kg weekly) or placebo for 6 weeks, and effects on knee synovitis, lymphatic vessel ultrastructure and function, and popliteal LN cellularity were assessed by ultrasound, histology, transmission electron microscopy (TEM), near-infrared indocyanine green imaging, and flow cytometry.

Results: Anti-TNF therapy significantly decreased synovitis (∼5-fold; P < 0.05 versus placebo), restored lymphatic contractions, and significantly increased the number of popliteal LN monocyte/macrophages (∼2-fold; P < 0.05 versus placebo). TEM demonstrated large activated macrophages attached to damaged lymphatic endothelium in mice with early arthritis, extensively damaged lymphatic vessels in placebo-treated mice with advanced arthritis, and rolling leukocytes in repaired lymphatic vessels in mice responsive to anti-TNF therapy.

Conclusion: These findings support the concept that anti-TNF therapy ameliorates erosive inflammatory arthritis, in part via restoration of lymphatic vessel contractions and potential enhancement of inflammatory cell egress.

Figure 1. Anti-TNF therapy restores lymphatic contraction in TNF-Tg mice with advanced arthritis

Male TNF-Tg mice (n=6) were randomized to anti-TNF or IgG placebo treatment following PD-US confirmation of PLN collapse and baseline NIR-ICG imaging to confirm loss of lymphatic contractions. Following commencement of therapy, the mice underwent NIR-ICG imaging every 2-weeks for 6-weeks to assess recovery of lymphatic contractions. Representative NIR images of the lower limb obtained 1 hour after ICG injection (A, B), and of the injected foot 24 hours later (D, E), after 6-weeks of therapy are shown. Note that ICG-filled lymphatic vessels were most apparent in the anti-TNF treated mice (arrow in B). Representative examples of the raw signal intensity data used to quantify lymphatic contraction are shown to illustrate the recovery observed at 6-weeks in anti-TNF treated mice versus the absence of contractions in placebo (C). Note that residual ICG was greater in the feet of placebo treated mice (D vs. E). The quantified lymphatic contraction frequency (F, two-way ANOVA with multiple comparisons corrected for by controlling the False Discovery Rate showed a treatment effect with p=0.01), and % ICG clearance at 6-weeks (G) are presented as the mean ± SEM (*p<0.05 via Mann-Whitney test).

Figure 2. Ultrastructural evidence of lymphatic vessel repair and rolling leukocytes in anti-TNF treated TNF-Tg mice that recovered lymphatic vessel contractions

WT and TNF-Tg mice (n=3) underwent TEM imaging as described in the Materials and Methods. Low magnification (2500×) imaging was used to identify lymphatic vessels, as shown in the representative WT (A). After identifying the lymphatic vessel, vessel walls were imaged at 10,000× magnification. Representative images from vessel walls are shown from: WT (B), untreated TNF-Tg with expanding PLN (C), untreated TNF-Tg with collapsed PLN (D), placebo (IgG) treated TNF-Tg with collapsed PLN (E), anti-TNF treated TNF-Tg with collapsed PLN (F). Highlighted in the images are the lumen (L), endothelial cells (EC) and smooth muscle cells (SMC). Note the absence of cells in the lumen in WT (B), the activated macrophage (Mϕ) with initial pseudopod attachment to EC afferent to expanded PLN (C), complete Mϕ attachment to EC afferent to collapsed PLN (D), exacerbated destruction of the lymphatic vessel in placebo treated mice as evidenced by vacuole formation in EC (red arrows in (E), mononucleated leukocytes that appear to be rolling along the endothelium in the anti-TNF treated TNF-Tg mice (red asterisks in F) indicative of restored cellular egress.