CD4(+) cells regulate fibrosis and lymphangiogenesis in response to lymphatic fluid stasis

Abstract

Introduction: Lymphedema 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.

Methods: We 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.

Results: Lymphedema 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.

Conclusions: Lymphedema 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Chronic lymphedema results in a mixed inflammatory cell response.

A. Photograph of mouse-tails 6 weeks after skin/lymphatic excision (lymphedema; left) or skin incision (control; right). B. Representative cross sectional histology of mouse tails comparing lymphedema (left) and control (right) mice 6 weeks after surgery. Cross sections were obtained 2 centimeters distal to the tail wound (arrow in figure A). Note subcutaneous fat deposition (brackets), dilated lymphatics, and inflammation in lymphedema section. C. Flow cytometry analysis for CD45+ cells in single cell suspensions prepared from tail tissue 2 cm distal to the wound of lymphedema or control mice (n = 5–7/group) 6 weeks after surgery. The percentage of CD45+ cells as a function of total cell population is shown. A representative histogram is shown to the right. D., E. Flow cytometry analysis of T-helper, T-cytotoxic, natural killer T cells (NKT), B cell (Figure D) and neutrophils, monocytes, macrophage, and dendritic cells (Figure E) in single cell suspensions of lymphedematous or control mice (n = 5–7/group) 6 weeks after surgery. Representative dot plots are shown to the right. Oval gates indicate double positive cell populations.

Figure 2. Axillary lymph node dissection results in a T cell inflammatory reaction. A.

Flow cytometry analysis for CD45+ cells in single cell suspensions prepared from upper extremity soft tissues 1.5 cm distal to the axillary wound in animals treated with axillary lymph node dissection (ALND) or axillary incision without lymphadenectomy (sham; n = 5–7/group) 3 or 6 weeks after surgery. A representative histogram is shown to the right. B. Flow cytometry analysis of T-helper, T-cytotoxic, natural killer T cells (NKT), B cell (top panel) and neutrophils, monocytes, macrophage, and dendritic cells in single cell suspensions of upper extremity soft tissues harvested 3 weeks after ALND or sham incision (n = 5–7/group). Representative dot plots are shown below. C. Flow cytometry analysis of T-helper, T-cytotoxic, natural killer T cells (NKT), B cell (top panel) and neutrophils, monocytes, macrophage, and dendritic cells in single cell suspensions of upper extremity soft tissues harvested 6 weeks after ALND or sham incision (n = 5–7/group). Representative dot plots are shown below.

Figure 3. CD4 cell depletion reduces lymphedema. A.

Flow cytometry analysis of splenic single cell suspensions from mice treated with isotype control antibodies or depleted of CD4+ cells (upper panel) or CD8+ cells (lower panel) using neutralizing antibodies (n = 5–7/group). Representative dot plots are shown to the right. B. Representative photograph of control, CD8+ cell depleted, or CD4+ cell depleted mice 6 weeks after tail superficial and deep lymphatic excision. Note near complete resolution of edema in CD4+ treated animals and loss of fixed tail contracture (“J” shape seen in control or CD8+ treated animals). C. Representative cross sectional histology and quantification of subcutaneous tissue thickness (brackets) in control, CD8+, and CD4+ depleted animals. D. Tail volumes in control, CD8+, or CD4+ depleted animals over the course of the experiment. CD4+ or CD8+ cell depletion was begun 2 weeks after surgery (arrow). E. Analysis of lymphatic vessel diameter (podoplanin+ vessels) in control, CD8+, or CD4+ depleted animals (left) and representative photomicrographs (right). Lymphatic vessel diameter is shown in brackets.

Figure 4. CD4+ cell depletion reduces lymphedema induced chronic inflammation. A, B, C.

Representative photomicrographs of CD45 (figure A), F4/80 (figure B), and CD4 (figure C) immunohistochemical staining in tail tissues of control, CD8+, or CD4+ cell depleted animals 6 weeks after tail superficial and deep lymphatic excision. Quantification of cell numbers per high-powered field (hpf) are shown below for each cell type. D. Representative (of triplicate experiments) western blots from tail tissues for Th1 (IFN-y, Tbet), T-reg (FoxP3), and Th2 (Gata-3) markers in control, CD8+, and CD4+ depleted animals 6 weeks after tail superficial and deep lymphatic excision. Quantification of band density relative to controls (arbitrarily set at 1 and represented by dotted line) is shown to the right.

Figure 5. CD4+ cell depletion decreases fibrosis and improves lymphatic function. A.

Scar index analysis (below) and representative photomicrographs of polarized light microscopic views (above) in control, CD8+, and CD4+ depleted animals (n = 5–7 per group) 6 weeks after surgery. B. Representative photomicrographs of type I collagen immunohistochemistry (above) and calculation of type I collagen staining in the dermis (positive pixels/mm2; below) in control, CD8+, and CD4+ depleted animals 6 weeks after surgery. C. Calculation of type I:type III collagen staining ratio in tail tissue sections from control, CD8+, and CD4+ depleted mice 6 weeks after surgery. D. Representative (of triplicate experiments) western blot analysis of a-sma, E-cadherin, type III collagen, pSMAD, and TGF-B1 in protein lysates obtained from tail tissues of control, CD8+, and CD4+ cell depleted animals 6 weeks after surgery. Quantification of band density relative to controls (arbitrarily set at 1 and represented by dotted line) is shown to the right. E. Representative microlymphangiography (upper) and quantification of tissue florescence proximal to the tail wound (ratio of proximal to distal florescence) in control, CD8+, and CD4+ depleted mice 6 weeks after surgery. Note crossing of the tail wound by florescent marker only in CD4+ depleted mice. F. Lymphoscintigraphy and sacral lymph node uptake in control, CD8+, and CD4+ depleted mice 6 weeks after surgery. Representative heat map is shown to the right (white arrow = injection site; red circle = sacral lymph nodes).

Figure 6. T-regulatory cell inflammation is potently increased by lymphatic fluid stasis and lymphedema. A.

Flow cytometry analysis for T-regulatory (T-reg) cells in single cell suspensions prepared from upper extremity soft tissues 1.5 cm distal to the axillary wound in animals treated with axillary lymph node dissection (ALND) or axillary incision without lymphadenectomy (sham; n = 5–7/group) 3 or 6 weeks after surgery. B. Flow cytometry analysis for T-regulatory (T-reg) cells in single cell suspensions prepared from tail tissue 2 cm distal to the wound of lymphedema or control mice (n = 5–7/group) 6 weeks after surgery. C. Flow cytometry of splenic single cell suspensions for CD4+/CD25- cells after treatment with control or CD25 neutralizing antibodies. Note no significant decrease in the overall number of CD4+ cells. Representative dot plot is shown to the right. D. Flow cytometry of splenic single cell suspensions for CD4+/CD25+/Foxp3+ (T-regs) cells after treatment with control or CD25 neutralizing antibodies. Note no significant decrease in the overall number of CD4+ cells. Representative dot plot is shown to the right.

Figure 7. CD25+ cell depletion does not improve lymphedema, decrease fibrosis, or augment lymphatic function. A.

Representative photograph of control or CD25+ depleted mice 6 weeks after tail superficial and deep lymphatic excision. B. Representative cross sectional histology and quantification of subcutaneous tissue thickness (brackets) in control and CD25+ cell depleted animals. C. Tail volumes in control and CD25+ cell depleted animals over the course of the experiment. CD25+ cell depletion was begun 2 weeks after surgery (arrow). D. Representative (of triplicate experiments) western blots from tail tissues for Th1 (IFN-y, Tbet), and Th2 (Gata-3, IL4) markers in control and CD25+ cell depleted animals 6 weeks after tail superficial and deep lymphatic excision. Quantification of band density relative to controls (fold change) is shown to the right. E. Lymphoscintigraphy and sacral lymph node uptake in control and CD25+ cell depleted mice 6 weeks after surgery. Representative heat map is shown to the right (white arrow = injection site; red circle = sacral lymph nodes). F. Scar index analysis (below) and representative photomicrographs of polarized light microscopic views (above) in control and CD25+ cell depleted animals (n = 5–7 per group) 6 weeks after surgery. G. Representative photomicrographs of type I collagen immunohistochemistry (above) and calculation of type I collagen staining in the dermis (positive pixels/mm2; below) in control and CD25+ cell depleted animals 6 weeks after surgery. H. Calculation of type I:type III collagen staining ratio in tail tissue sections from control and CD25+ cell depleted mice 6 weeks after surgery. I. Representative (of triplicate experiments) western blot analysis of a-sma, E-cadherin, type III collagen, pSMAD, and TGF-B1 in protein lysates obtained from tail tissues of control and CD25+ cell depleted animals 6 weeks after surgery. Quantification of band density relative to controls (fold change) is shown to the right.

Figure 8. Loss of CD4+ but not CD8+ or CD25+ cells increases lymphangiogenesis. A.

LYVE-1+ vessel counts (left) and representative figures (right) of tail sections from control, CD8+, CD4+, or CD25+ cell depleted mice 6 weeks after surgery. B. Podoplanin+ vessel counts (left) and representative figures (right) of tail sections from control, CD8+, CD4+, or CD25+ cell depleted mice 6 weeks after surgery. C. VEGF-C+ cells/high powered field (hpf) counts (left) and representative figures of VEGF-C immunohistochemistry in tail tissues from control, CD8+, CD4+, or CD25+ cell depleted animals (right) 6 weeks after surgery. D. Representative (of triplicate experiments) western blot analysis of VEGF-A, and VEGF-C expression in protein lysates obtained from tail tissues from control, CD8+, CD4+, or CD25+ cell depleted animals 6 weeks after surgery. Quantification of band density relative to controls (arbitrarily set at 1 and represented by dotted line) is shown to the right.

Figure 9. CD4+ cells regulate inflammatory lymphangiogenesis.

A. LYVE-1+ vessel density in popliteal lymph nodes 7 days after CFA/OVA induced lymph node lymphangiogenesis in control, CD4+ cell depleted, or CD4 knockout (CD4KO) mice. Representative cross sectional histology of the lymph node (blue DAPI stain, red LYVE-1 stain) are shown to the right. B., C. Expression of VEGF-A (A) and VEGF-C (B) protein by ELISA in popliteal lymph nodes harvested 7 days after CFA/OVA induced lymph node lymphangiogenesis in control or CD4 depleted mice.