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BACKGROUND: Lymphedema is histologically characterized by inflammation, and recent studies have shown that this inflammatory reaction contributes to the pathology of this disorder. Although it is clear that lymphedematous tissues are “inflamed,” the precise nature of this inflammatory response remains unknown. This knowledge is important since understanding how tissues respond to lymphatic stasis is a necessary step for developing targeted treatments for lymphedema. The aim of this study was therefore to characterize the cellular inflammatory response to lymphatic stasis.
METHODS: Axillary lymph node dissection (ALND) or skin incision without lymph node removal were performed in adult female mice to simulate changes that occur clinically after lymph node dissection. To study the effects of more severe lymphatic stasis, we excised both the superficial and deep lymphatics of the tail skin (EX) and compared these tissues with controls that underwent skin incision without lymphatic ablation. Animals were sacrificed 3 or 6 weeks after surgery and upper extremity or tail tissues distal to the zone of lymphatic ablation of experimental and control animals were harvested, digested, and single-cell suspensions were prepared. Multi-color flow cytometry on CD45+ (leukocytes) enriched and non-enriched samples was performed to identify inflammatory cell subtypes.
RESULTS: The number of leukocytes (CD45+) was significantly increased in tissues of ALND limbs and EX-treated animals compared to controls at 6 weeks post-operatively (p<0.05). The percentage of mature CD4+ T cells (CD4+TCRB+) within the CD45+ population was similarly increased following ALND (1.3-fold) and EX (2.1-fold) compared to controls (p<0.01); while, no significant differences in CD8+ T cell (CD8+TCRB+) infiltrate were present. In the tail model, the percentage of alternatively-activated (M2) macrophages (CD11b+CD206^hi) was increased compared to controls (1.4-fold, p<0.05) and compared to classically-activated (M1) macrophages (CD11b+CD206^lo, 4.3-fold, p<0.001). Following ALND, the percentage of M1 macrophages was significantly increased compared to controls (p<0.01), and although there was no difference in M2 macrophages, the percentage of infiltrating M2 macrophages increased by 6 weeks post-operatively while the M1 population decreased. Finally, the percentage of dendritic cells (CD11c+MHCII+) was increased in ALND and EX groups relative to controls (p<0.01) while the percentage of neutrophils (CD11bLy6g^hi) and monocytes (CD11bLy6c^hi) did not differ significantly among groups at 6 weeks post-operatively.
CONCLUSIONS: These results suggest that chronic lymphatic fluid stasis following lymph node dissection and lymphatic disruption promotes CD4+ T cell infiltration and alternatively-activated (M2) macrophage differentiation. These findings are important since they demonstrate that the inflammatory response to lymphatic stasis is highly differentiated and not simply a global increase in inflammation. The highly specific nature of this inflammatory response may enable targeted treatments designed to inhibit pathologic responses in lymphedema (e.g. fibrosis, adipose deposition) while preserving beneficial or homeostatic effects of inflammation (i.e. lymphangiogenesis, angiogenesis, tissue turnover).