Th2 differentiation is necessary for soft tissue fibrosis and lymphatic dysfunction resulting from lymphedema

Abstract

Lymphedema is a dreaded complication of cancer treatment. However, despite the fact that >5 million Americans are affected by this disorder, the development of effective treatments is limited by the fact that the pathology of lymphedema remains unknown. The purpose of these studies was to determine the role of inflammatory responses in lymphedema pathology. Using mouse models of lymphedema, as well as clinical lymphedema specimens, we show that lymphatic stasis results in a CD4 T-cell inflammation and T-helper 2 (Th2) differentiation. Using mice deficient in T cells or CD4 cells, we show that this inflammatory response is necessary for the pathological changes of lymphedema, including fibrosis, adipose deposition, and lymphatic dysfunction. Further, we show that inhibition of Th2 differentiation using interleukin-4 (IL-4) or IL-13 blockade prevents initiation and progression of lymphedema by decreasing tissue fibrosis and significantly improving lymphatic function, independent of lymphangiogenic growth factors. We show that CD4 inflammation is a critical regulator of tissue fibrosis and lymphatic dysfunction in lymphedema and that inhibition of Th2 differentiation markedly improves lymphatic function independent of lymphangiogenic cytokine expression. Notably, preventing and/or reversing the development of pathological tissue changes that occur in lymphedema may be a viable treatment strategy for this disorder.

Figure 1.

Sustained lymphatic stasis results in CD4⁺ cell inflammation. A) Representative photograph demonstrating persistent tail lymphedema in an experimental mouse 6 wk postoperatively as compared with resolving edema in a control mouse. B) CD4⁺ cell counts proximal and distal to the wound in control and lymphedematous animals. C) Quantification (top panel) and representative flow cytometry analysis (bottom panel) demonstrating effects of gradients of lymph stasis on mature T-helper cell inflammation in mice with tail lymphedema 3 and 6 wk after surgery. Note accumulation of mature T-helper cells in the distal (lymphedematous) regions. D, E) Number of Th1 (D) and Th2 (E) cells in proximal and distal regions of control and lymphedematous mouse tails 6 wk after surgery. F) Representative Western blot and relative fold changes comparing protein expression in the distal regions of control and lymphedematous tails 6 wk after surgery.

Figure 2.

Lymph node dissection results in CD4⁺ cell inflammation. A) Quantification (left panel) and representative flow cytometry analysis (right panel) demonstrating accumulation of mature T-helper cells in upper extremity soft tissues after ALND 3 and 6 wk after surgery. B) Representative Western blot of protein harvested from the upper extremity 6 wk postoperatively with relative fold-increase comparing ALND to sham. C) Number of CD4⁺ cells in normal and lymphedematous upper extremities of lymphedema patients. D) Correlation of the number of CD4⁺ cells in the lymphedematous limb with severity of lymphedema. E, F) Number of IL-4⁺ cells/mm² (E) and IL-13⁺ cells/mm² (F) in normal and lymphedematous upper extremities of patients with lymphedema.

Figure 3.

CD4⁺ cells are necessary for fibrosis and lymphatic dysfunction. A) Representative photographs of nude, CD4KO, and wild-type mouse-tails 6 wk after tail skin/lymphatic excision. Note fixed contracture (J shape) of wild-type mouse-tail. B) Tail volume changes (% change from preoperative) 6 wk after surgery. C) Subcutaneous tissue thickness and representative cross-sectional histology 6 wk after surgery. Gross photograph and site of tissue harvest (dashed line) are shown for orientation. Note markedly decreased subcutaneous adipose deposition (brackets) in nude and CD4KO mice. D) CD45⁺ cells/HPF in CD4KO and WT mouse tails 6 wk after tail skin and lymphatic excision. E, F) Scar index (E) and collagen deposition (F) of distal tail tissues 6 wk postoperatively. G) Peak nodal uptake of ^99mTc injected in the distal tail 6 wk after surgery. H) Proximal tail fluorescence (left) and representative photographs (right) 6 wk after surgery, comparing CD4KO and wild-type mice.

Figure 4.

Inhibition of Th2 differentiation prevents initiation of fibrosis and improves lymphatic function. A) Representative photographs of tails from animals treated with IL-4mAb, IL-13mAb, or isotype control antibodies (control) for 6 wk beginning immediately after tail skin/lymphatic excision. Note lack of fibrotic contracture in the tails of IL-4/IL-13mAb-treated mice, as compared to fixed contracture of controls. B) Tail volume changes (% change from preoperative) 6 wk after surgery. C) Subcutaneous tissue thickness and representative cross-sectional histology 6 wk after surgery. Note marked decreased adipose deposition in IL-4mAb/IL-13mAb-treated mice (brackets). D) Scar index and representative histology (×40) of distal tail tissues 6 wk postoperatively. E) Collagen deposition and representative histology (×20) of distal tail tissues 6 wk postoperatively. F) Western blot analysis of distal tail tissues (arrow indicates wound; dotted line indicates harvest site) comparing IL-4mAb and control animals 6 wk after surgery. Fold decrease in expression in IL-4mAb animals relative to controls is shown. G) Serum IL-4 and IL-13 levels in animals treated with IL-4mAb or control antibody 6 wk after surgery. H) Number of Th1 (IFN-γ⁺/CD4⁺) and Th2 (IL-13⁺/CD4⁺) cells in distal tail tissues of IL-4mAb and control antibody-treated animals 6 wk postoperatively. I) Representative heat maps (top panels) and peak nodal uptake of ^99mTc in sacral lymph nodes (white arrows) following distal tail injection 6 wk after surgery. J) Representative microlymphangiography and proximal tail fluorescence 6 wk after surgery.

Figure 5.

Inhibition of Th2 differentiation decreases established lymphedema. A–C) Representative photographs (A), tail volumes (B), and subcutaneous tissue thickness (C) of tails from animals 6 wk after skin excision and lymphatic disruption (pretx), following 3 wk treatment with IL-4/control antibodies (post-tx) beginning 6 wk after surgery, and following an additional 3-wk treatment withdrawal (post-tx withdrawal). D) Western blot of distal tail tissues from animals following 3-wk treatment with IL-4/control antibodies beginning 6 wk postoperatively (fold-decrease=normalized expression from IL-4mAb-treated animals relative to controls). E) Serum IL-4 and IL-13 levels in animals post-treatment with IL-4/control antibodies for 3 wk. F) Number of Th1/Th2 cells in distal tail tissues posttreatment with IL-4/control antibodies.

Figure 6.

Inhibition of Th2 differentiation decreases established fibrosis and improves lymphatic function. Scar index (A; representative ×40 views) and collagen staining (B; representative ×20 views) of distal tail tissues posttreatment with IL-4/control antibodies. C) Representative heat maps (top panels) and peak nodal uptake of ^99mTc in sacral lymph nodes following distal tail injection posttreatment (Ab Tx) and following treatment withdrawal (Tx withdrawal). D) Representative microlymphangiography and proximal tail fluorescence posttreatment (Ab Tx) and following treatment withdrawal (Tx withdrawal) with IL-4/control antibodies. E) Number of α-sma⁺ capillary lymphatic vessels/hpf.

Figure 7.

Inhibition of JAK1/2 does not prevent fibrosis or preserve lymphatic function and fibrosis independently inhibits lymphatic function. A) Representative photographs of JAK1/2 inhibitor or control-treated mouse tails 6 wk postoperatively. B, C) Change in tail volume (B) and subcutaneous tissue thickness (C) in JAK1/2 inhibitor or control treated mice 6 wk postoperatively. D) Peak nodal uptake of ^99mTc in JAK1/2 inhibitor or control-treated animals 6 wk postoperatively. E) Subcutaneous tissue thickness of animals treated with bleomycin or PBS. F, G) Peak nodal uptake of ^99mTc by sacral lymph nodes (F) and microlymphangiography/proximal fluorescence (G) in animals treated with bleomycin, bleomycin and IL-4mAb, or PBS control. H) Quantification of lectin⁺ capillary lymphatics as a percentage of total lymphatics (left panel) and representative whole mount lectin stain (right panel) in bleomycin/IL-4mAb and bleomycin/control antibody-treated animals. White arrows indicate capillary lymphatics; dotted circles represent pooled areas of lectin in interstitial space.

Figure 8.

IL-4 blockade does not increase VEGF-A or VEGF-C expression. A) LYVE-1⁺ vessel counts in IL-4/control antibody-treated animals beginning immediately after surgery and continued for 6 wk (initiation) or beginning 6 wk after surgery and continued for 3 wk (progression). B) Representative Western blots (of triplicate experiments) for LYVE-1 and PROX1 expression in distal tail protein of animals treated with IL-4mAb/control antibody for 6 wk beginning immediately after surgery (left) or after treatment with IL-4mAb/control antibodies for 3 wk beginning 6 wk postoperatively (right). Normalized fold-increase in expression relative to controls is shown. C, D) Representative Western blots (of triplicate experiments) from distal tail protein isolated from animals treated with IL-4mAb/control antibody for 2 or 6 wk beginning immediately after surgery (C; initiation of fibrosis) or after treatment with IL-4/control antibodies for 3 wk beginning 6 wk after surgery (D; progression of fibrosis). Normalized fold-increase in expression relative to controls is shown. E) LYVE-1⁺ lymphatic vessel density of popliteal lymph nodes in animals treated with IL-4mAb/control antibodies 2 wk post-CFA/OVA injection. F) Expression of VEGF-A/C (by ELISA) in popliteal lymph nodes 2 wk post-CFA/OVA. G) Schema depicting proposed mechanism of lymphedema development.