CD4+ Cells Regulate Fibrosis and Lymphangiogenesis in Response to Lymphatic Fluid Stasis

Abstract

IntroductionLymphedema is a chronic disorder that occurs commonly after lymph node removal for cancer treatment and is characterized by swelling, fibrosis, inflammation, and adipose deposition. Although previous histological studies have investigated inflammatory changes that occur in lymphedema, the precise cellular make up of the inflammatory infiltrate remains unknown. It is also unclear if this inflammatory response plays a causal role in the pathology of lymphedema. The purpose of this study was therefore to characterize the inflammatory response to lymphatic stasis and determine if these responses are necessary for the pathological changes that occur in lymphedema.MethodsWe used mouse-tail lymphedema and axillary lymph node dissection (ANLD) models in order to study tissue inflammatory changes. Single cell suspensions were created and analyzed using multi-color flow cytometry to identify individual cell types. We utilized antibody depletion techniques to analyze the causal role of CD4+, CD8+, and CD25+ cells in the regulation of inflammation, fibrosis, adipose deposition, and lymphangiogenesis.ResultsLymphedema in the mouse-tail resulted in a mixed inflammatory cell response with significant increases in T-helper, T-regulatory, neutrophils, macrophages, and dendritic cell populations. Interestingly, we found that ALND resulted in significant increases in T-helper cells suggesting that these adaptive immune responses precede changes in macrophage and dendritic cell infiltration. In support of this we found that depletion of CD4+, but not CD8 or CD25+ cells, significantly decreased tail lymphedema, inflammation, fibrosis, and adipose deposition. In addition, depletion of CD4+ cells significantly increased lymphangiogenesis both in our tail model and also in an inflammatory lymphangiogenesis model.ConclusionsLymphedema and lymphatic stasis result in CD4+ cell inflammation and infiltration of mature T-helper cells. Loss of CD4+ but not CD8+ or CD25+ cell inflammation markedly decreases the pathological changes associated with lymphedema. In addition, CD4+ cells regulate lymphangiogenesis during wound repair and inflammatory lymphangiogenesis.

IL-6 regulates adipose deposition and homeostasis in lymphedema

“…By comparing tissues harvested from mice that had undergone axillary lymph node dissection with a more severe model of lymphatic injury in the mouse tail, we have also shown that the degree of adipose deposition is related to the severity of lymphatic obstruction and tissue inflammation (6). The association between inflammation and adipose deposition is important and supported by our previous studies demonstrating that depletion of T cells or blockade of T-helper type 2 (Th2) cell inflammation potently inhibits adipose tissue deposition in the mouse-tail model (6,40). However, although it is evident that inflammatory changes are necessary for adipose deposition in lymphedema, it remains unclear which inflammatory pathways regulate adipose homeostasis in lymphedematous tissues.…”

“…Matched tissue biopsies were obtained from the normal and lymphedematous upper extremity of patients with post-surgical lymphedema (grades I-III; see below) by Professor Waldemar Olszewski at the Polish Academy of Science. Full-thickness (5 mm) skin biopsies were fixed, paraffinembedded, and sectioned at a thickness of 5 m using our previously described methods (40). The Institutional Review Boards (IRB) of the Polish Academy of Science and Memorial Sloan-Kettering Cancer Center (MSKCC) approved all studies.…”

“…We used our previously described axillary lymph node dissection (ALND) model to study the effects of lymphatic injury without significant adipose deposition (40). In this model, the axillary lymph nodes are completely excised through a 1-cm skin incision.…”

“…Control animals are treated with an axillary skin incision without lymph node excision. To study the effects of more significant lymphatic injury and adipose deposition, we used a well-described mouse tail model of lymphedema (3,4,6,40). In this model, the superficial and deep lymphatic system of the midportion of the tail are disrupted by excising a 2-mm portion of the skin and microsurgically ligating the deep lymphatic channels that run along the lateral tail veins.…”

“…In this model, the superficial and deep lymphatic system of the midportion of the tail are disrupted by excising a 2-mm portion of the skin and microsurgically ligating the deep lymphatic channels that run along the lateral tail veins. We have previously shown that this intervention results in sustained lymphatic fluid stasis lasting at least 6 wk in the distal region of the tail with resultant inflammation, adipose deposition, and fibrosis that is histologically similar to clinical lymphedema (3,4,6,40). Control animals were treated with an incision in the same region of the tail without deep lymphatic ligation.…”