Alarmin expression is induced by acute lymphedema and necessary for early lymphangiogenesis.
Jamie C. Zampell, Tomer Avraham, MD, Alan Yan, MD, Babak Mehrara, MD.
Sloan Kettering Institute, New York, NY, USA.
BACKGROUND: : Lymphedema is a devastating complication of lymph node dissection associated with progressive swelling and soft tissue fibrosis. While the pathologic development of lymphedema is poorly understood, it is known that acute lymphatic fluid stasis potently induces genes regulating inflammation and fibrosis, including those encoding alarmins. These molecules serve to activate the immune system and restore homeostasis by promoting reconstruction of damaged tissue. The purpose of this study was to evaluate the mechanisms by which alarmins and acute lymphatic fluid stasis act to initiate the pathologic events leading to chronic lymphedema.
METHODS: We used a mouse tail model of lymphedema in which superficial lymphatics are ablated by a 2 mm full thickness skin excision followed by microsurgical ligation of deep collecting lymphatics. Control animals underwent tail incisions without lymphatic disruption. Alarmin (HMGB1, HSP70, S100A8) expression was evaluated temporally (1, 2, 3, and 6 weeks post-operatively) and spatially (at intervals along the tail with respect to tail edema) using immunohistochemistry and western blot analysis. Mice deficient in toll-like receptor (TLR) 2, 4, or 9 (receptors required for intracellular signaling events mediated by alarmins) additionally underwent tail operations, and lymphatic regeneration was analyzed by tail volume measurement, microlymphangiography, and immunohistochemistry.
RESULTS: Animals with lymphedema had a 3-fold increase in alarmin expression as compared to controls (incisions without lymphedema; p<0.01). Peak alarmin expression corresponded with regions of severe lymphedema and correlated histologically with inflammation, lymphatic dysfunction, and tissue fibrosis. NF-kB activation, a downstream effector of alarmin signaling, additionally correlated with the gradient of alarmin expression. Lymphedematous tissues analyzed 6 weeks post-operatively demonstrated persistence of alarmins. Interestingly, TLR deficient animals developed severe, persistent lymphedema (2-fold increase in tail volume), fibrosis (p<0.05), and reduced lymphatic regeneration after tail surgery, indicating that early alarmin expression is necessary for the events modulating lymphatic regeneration.
CONCLUSIONS: Our findings demonstrate that acute lymphatic fluid stasis induced alarmin expression in the early post-operative phase and led to sustained expression of these signals at later post-operative periods. Our results additionally suggest that alarmin expression was necessary for TLR activation and promotion of early lymphatic regeneration. Further investigation will be necessary to determine whether sustained alarmin expression by lymphatic fluid stasis is associated with chronic lymphatic dysfunction and fibrosis.
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