Regulation of inflammation and fibrosis by macrophages in lymphedema

Abstract

Lymphedema, a common complication of cancer treatment, is characterized by inflammation, fibrosis, and adipose deposition. We have previously shown that macrophage infiltration is increased in mouse models of lymphedema. Because macrophages are regulators of lymphangiogenesis and fibrosis, this study aimed to determine the role of these cells in lymphedema using depletion experiments. Matched biopsy specimens of normal and lymphedema tissues were obtained from patients with unilateral upper extremity breast cancer-related lymphedema, and macrophage accumulation was assessed using immunohistochemistry. In addition, we used a mouse tail model of lymphedema to quantify macrophage accumulation and analyze outcomes of conditional macrophage depletion. Histological analysis of clinical lymphedema biopsies revealed significantly increased macrophage infiltration. Similarly, in the mouse tail model, lymphatic injury increased the number of macrophages and favored M2 differentiation. Chronic macrophage depletion using lethally irradiated wild-type mice reconstituted with CD11b-diphtheria toxin receptor mouse bone marrow did not decrease swelling, adipose deposition, or overall inflammation. Macrophage depletion after lymphedema had become established significantly increased fibrosis and accumulation of CD4(+) cells and promoted Th2 differentiation while decreasing lymphatic transport capacity and VEGF-C expression. Our findings suggest that macrophages home to lymphedematous tissues and differentiate into the M2 phenotype. In addition, our findings suggest that macrophages have an antifibrotic role in lymphedema and either directly or indirectly regulate CD4(+) cell accumulation and Th2 differentiation. Finally, our findings suggest that lymphedema-associated macrophages are a major source of VEGF-C and that impaired macrophage responses after lymphatic injury result in decreased lymphatic function.

Keywords: diphtheria toxin; fibrosis; inflammation; lymphatic function; lymphedema; macrophages.

Fig. 1.

Lymphedema results in increased macrophage infiltration. A: representative high-powered field (HPF) (×100) histological sections of matched control (normal limb) and lymphedematous tissues obtained from a patient with unilateral upper-extremity breast cancer-related lymphedema. Red arrows show EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1) positively stained macrophages. B: cell counts of EMR⁺ macrophages in control and lymphedematous limbs of patients with breast cancer-related upper extremity lymphedema (n = 8; *P < 0.01). C: representative HPF (×80) histological images of tissues obtained from mice that underwent tail incision without lymphatic ligation (control) and lymphedematous tails 2 and 6 wk after surgery stained for F4/80⁺ macrophages. D: cell counts of F4/80⁺ cells in control and lymphedematous tail tissues 2 and 6 wk after tail surgery (n = 10; *P < 0.01). E: representative flow diagrams of tail tissues obtained from control and lymphedematous tail tissues 6 wk after surgery identifying cells that express CD206 and CD11b. F: quantification of M1 (CD11b⁺CD206^lo) and M2 (CD11b⁺CD206^hi) macrophages in control and lymphedematous tail tissues 6 wk after surgery (n = 8; *P < 0.01).

Fig. 2.

Chimeric CD11b/diphtheria toxin receptor (DTR) have sustained macrophage depletion without systemic toxicity. A: diagrammatic representation of bone marrow chimeras created from wild-type (WT) mice reconstituted with bone marrow from CD11b-DTR mice. B: immunofluorescent staining of chimeric bone marrow showing costaining of CD11b and DTR. C: representative flow diagram of macrophages (B220⁺/CD11b⁺) harvested from the peripheral lymph nodes of animals treated with vehicle control or DT (3 wk after 3 times weekly treatment). D: quantification of macrophages in peripheral lymph nodes of mice treated with or without DT for 3 wk (*P < 0.01). E: representative immunohistochemical staining of lymphedematous tail tissues harvested 3 wk after 3 times weekly treatment with or without DT. Arrows show positively stained macrophages. F: quantification of F4/80⁺ cells per HPF (×80 magnification) in lymphedematous tissues of mice treated with or without DT for 3 wk (n = 8; *P < 0.01).

Fig. 3.

Depletion of macrophages does not alter tail volumes or adipose deposition. A: tail volumes of mice treated with vehicle control, DT beginning immediately after surgery (immediate DT), or DT beginning 3 wk after surgery (3-wk DT). B: representative cross-sectional histological sections of control, immediate DT, and 3-wk DT-treated mice (×2.5 magnification) harvested 6 wk after surgery. Sections were harvested 1.5 cm distal to the zone of lymphatic injury. C: quantification of adipose area in control, immediate DT, and 3-wk DT groups from cross-sectional histological sections harvested 6 wk after surgery. D: dermal thickness of various groups analyzed in cross-sectional histological sections (n = 8; *P < 0.05). E: representative HPF photomicrograph (×80) of tail tissues stained for CD45. F: quantification of CD45⁺ cells in tail sections of mice treated with vehicle control, immediate DT, or DT 3 wk after surgery.

Fig. 4.

Depletion of macrophages increases tissue fibrosis and impairs lymphatic function. A: representative HPF photomicrographs (×40) of tail sections stained for type I collagen. B: quantification of type I collagen staining in tail sections of mice treated with vehicle control, immediate DT, or DT beginning 3 wk after surgery (n = 8; *P < 0.01). C: representative photomicrographs of tail sections stained with Sirius red and imaged using polarized light microscopy (×40 magnification). D: scar index of various groups quantified from Sirius red-stained tissues (n = 8; *P < 0.01). E: representative Tc⁹⁹ heat maps of tails from control, immediate DT, and 3 wk postsurgery DT-treated animals. Hot spot at the bottom of the photograph is the injection site. Small areas of uptake at the top of the picture are the sacral lymph nodes (white arrow). F–H: graphs depicting decay adjusted uptake (F), peak nodal uptake (G) (n = 8; *P < 0.01), and rate of uptake (H) (n = 8; *P < 0.05) in the sacral lymph nodes of animals in various groups.

Fig. 5.

Macrophage depletion increases CD4⁺ cell infiltration and Th2 differentiation. A: representative HPF (×80) photomicrographs of tail sections stained for CD4⁺ cells. B: quantification of CD4⁺ cells per HPF in tissue sections harvested from animals in various groups (n = 8; *P < 0.01). C: representative HPF (×80) photomicrographs of tail sections stained for Gata-3⁺ cells. D: quantification of Gata-3⁺ cells per HPF in various experimental groups (n = 8; *P < 0.01). E: IL-13 gene expression relative to control animals in experimental animals treated with DT either immediately after surgery or beginning 3 wk postoperatively. Data are presented as fold change vs. control corrected for GAPDH expression (n = 8; *P < 0.05).

Fig. 6.

Macrophage depletion decreases VEGF-C expression. A: representative photomicrographs (×40) of tissue sections stained with LYVE-1 in various experimental groups. B: quantification of the number of LYVE-1⁺ vessels in tail tissue sections (n = 8; *P < 0.01). C: quantification of LYVE-1⁺ vessel area in tail tissue sections. D: VEGF-C protein expression in total cellular protein harvested from tail tissue sections of various experimental groups (n = 8; *P < 0.01). E and F: expression of Prox-1 (E) and LYVE-1 (F) in tail tissues in various experimental groups. Values are expressed as mRNA level fold change relative to control animals that were not treated with DT (n = 8; *P < 0.05).

Fig. 7.

Depletion of macrophages after lymphedema is established decreases adipose deposition. A: tail volumes of mice treated with or without DT beginning 6 wk after surgery when lymphedema had become established. B: representative cross-sectional histology of tail sections stained with hematoxylin and eosin (×2.5 magnification). C: quantification of adipose area in control and depleted animals (n = 8; *P < 0.01). D: quantification of dermal thickness in control and depleted animals (n = 8; *P < 0.05). E: representative HPF photomicrographs (×80) of tail tissues harvested from control and DT-depleted animals stained for CD45. F: quantification of CD45⁺ cells in tail tissues of control and macrophage-depleted animals. G: representative HPF photomicrographs (×80) of tail tissues harvested from control and DT-depleted animals stained for CD4. H: quantification of CD4⁺ cells per HPF in tail tissues of control and macrophage-depleted animals (n = 8; *P < 0.01). I: representative photomicrograph of tail tissues from control and macrophage-depleted animals stained with LYVE-1. J: quantification of LYVE-1⁺ vessels/HPF in control and experimental animals. K: quantification of LYVE-1⁺ vessel areas in control and experimental animals. L: VEGF-C protein expression in total cellular protein harvested from tail tissue sections of experimental and control animals (n = 8; *P < 0.01).

Fig. 8.

Late macrophage depletion increases fibrosis and impairs lymphatic function. A: representative photomicrographs (×40) of tail tissues from control and macrophage-depleted animals stained for type I collagen. B: quantification of type I collagen staining area in experimental and control animals (n = 8; *P < 0.01). C: representative photomicrographs (×40) of tail sections stained with Sirius red and imaged using polarized light microscopy. D: scar index of control and experimental animals quantified from Sirius red-stained tissues (n = 8; *P < 0.05). E: representative Tc⁹⁹ heat maps from control and macrophage-depleted animals. F–H: graphs depicting decay adjusted uptake (F), peak nodal uptake (G), and rate of uptake (H) in the sacral lymph nodes of animals in control and macrophage depleted animals (n = 8; *P < 0.05). I: representative photomicrographs (×40) of tissue sections stained with LYVE-1 in various experimental groups. J: quantification of the number of LYVE-1⁺ vessels in tail tissue sections. K: quantification of LYVE-1⁺ vessel area in tail tissue sections. L: VEGF-C protein expression in total cellular protein harvested from tail tissue sections of control and macrophage-depleted animals (n = 8; *P < 0.01).