Inflammation and Lymphedema Are Exacerbated and Prolonged by Neuropilin 2 Deficiency

Abstract

The vasculature influences the progression and resolution of tissue inflammation. Capillaries express vascular endothelial growth factor (VEGF) receptors, including neuropilins (NRPs), which regulate interstitial fluid flow. NRP2, a receptor of VEGFA and semaphorin (SEMA) 3F ligands, is expressed in the vascular and lymphatic endothelia. Previous studies have demonstrated that blocking VEGF receptor 2 attenuates VEGFA-induced vascular permeability. The inhibition of NRP2 was hypothesized to decrease vascular permeability as well. Unexpectedly, massive tissue swelling and edema were observed in Nrp2^-/- mice compared with wild-type littermates after delayed-type hypersensitivity reactions. Vascular permeability was twofold greater in inflamed blood vessels in Nrp2-deficient mice compared to those in Nrp2-intact littermates. The addition of exogenous SEMA3F protein inhibited vascular permeability in Balb/cJ mice, suggesting that the loss of endogenous Sema3F activity in the Nrp2-deficient mice was responsible for the enhanced vessel leakage. Functional lymphatic capillaries are necessary for draining excess fluid after inflammation; however, Nrp2-mutant mice lacked superficial lymphatic capillaries, leading to 2.5-fold greater fluid retention and severe lymphedema after inflammation. In conclusion, Nrp2 deficiency increased blood vessel permeability and decreased lymphatic vessel drainage during inflammation, highlighting the importance of the NRP2/SEMA3F pathway in the modulation of tissue swelling and resolution of postinflammatory edema.

Supplemental Figure S1

The cutaneous phenotypes +/− inflammation and +/− neuropilin (Nrp)-2 deficiency. A: Balb/cJ mouse 1 day after delayed-type hypersensitivity (DTH). B: Lymphangiography in Balb/cJ mice 3 days after DTH. Arrows point to areas of pooled dye from lymphatic vessels. C: Total cell lysate proteins from human fibroblasts (HF), human microvascular endothelial cells (EC), human dermal lymphatic endothelial cells (LEC), porcine aortic endothelial cells (PAE; negative control), PAEs transfected with NRP1 (N1), and PAEs transfected with NRP2 (N2; positive control) were immunoblotted for NRP2 and vascular endothelial growth factor receptor (VEGFR)-3 expression. β-Actin served as a loading control. D: Immunoblot of pure semaphorin (SEMA)-3F protein and wild-type (WT) ear lysate for Sema3F expression. E: Paraffin mouse skin section stained with SEMA3F (brown) and hematoxylin (blue). F: SEMA3F-transfected A375SM tumors served as a positive control for SEMA3F immunostaining. The section was counterstained with hematoxylin (blue color). G and H: Adult WT (G) and Nrp2 knockout mice (H) stained for lymphatic vessel endothelial hyaluronic acid receptor (LYVE)-1, 1 day after DTH. I and J: Embryonic day 18 skin sections were stained for LYVE1. Scale bars: 2 mm (B); 100 μm [E–J (scale bar in G applies to G and H; scale bar in I applies to I and J)].

Figure 1

Inflammation up-regulates neuropilin (Nrp)-2 in the endothelium. A: Ear swelling versus time after delayed-type hypersensitivity (DTH) reaction in C57BL/6 mice. B: Hematoxylin and eosin (H&E) staining of ear sections before (day 0; top panel) and after (day 3; bottom panel) DTH shows epidermal and dermal hyperplasia; asterisks denote dilated vessels. C: Immunoblot of ear lysates from C57BL/6 mice after DTH. Nrp2 receptor and soluble Nrp2 (top panel) increases by day 1 and is greatest on day 5 after DTH. Integrin α5, a loading control, is shown in the bottom panel. D: X-gal (blue) and eosin (pink) staining in Nrp2^+/LacZ mice ears before (day 0; top panel) and after (day 2; bottom panel) DTH. Nrp2-expressing vessels (arrows) are only present in inflamed ears. E–G: Nrp2 is up-regulated in endothelial cells (ECs) and lymphatic ECs. Immunostaining for Nrp2 (E and G; brown) or podoplanin (Pdpn) (F; blue). Tissue in E and G was counterstained with hematoxylin (blue nuclei). In serial sections of wild-type Nrp2^+/+ ears on day 3 after DTH, arrows point to lymphatic vessels stained with Nrp2 and Pdpn; asterisks denote Nrp2-positive blood vessels negative for Pdpn staining. As a control, Nrp2^−/− ears on day 3 after DTH were negative for Nrp2 immunostaining (G, lack of brown); melanocytes in the skin are brown due to endogenous melanin. Data are expressed as means ± SD. n = 5 (A) Scale bars = 100 μm [B, D, and E–G (scale bar in E applies to F and G)].

Figure 2

A neuropilin (Nrp)-2 ligand, semaphorin (Sema)-3F, inhibits vascular permeability. A and B: Vascular permeability was compared between Nrp2^+/+ [wild type (WT)], Nrp2^+/− [heterozygous (HET)], and Nrp2^−/− [knockout (KO)] mice in modified Miles assays after delayed-type hypersensitivity (DTH). A: The mean leakage values of Evans Blue dye [optical density, 620 nm] extracted from unchallenged (−) and challenged (+) ears in the same mice from each group on day 2 after inflammation were plotted and compared to that of WT ears injected with exogenous vascular endothelial growth factor (VEGF) A (positive control) (representative data). B: Relative vascular permeability in each group compared with that in WT (combined data from 3 experiments). Baseline (unchallenged) vascular leakage was not significantly different among the three genotypes. Vascular permeability was doubled in Nrp2^−/− KO mice compared to that in Nrp2^+/+ WT mice. C and D: Vascular permeability in Miles assays in Balb/cJ mice. Permeability was induced with phosphate-buffered saline (PBS; control), VEGF, or a mixture of VEGF and SEMA3F (S3F; at 20× or 200× of VEGF) or VEGF and anti-VEGF (at 200× of VEGF). C: Representative mouse skin image. D: Mean relative permeability values in the VEGF, VEGF + SEMA3F, VEGF + anti-VEGF groups compared to that in the PBS control group (from three independent experiments). VEGF SEMA3F and VEGF αVEGF were compared and found to be statistically similar. Data are expressed as means ± SEM. n = 3 mice per group (A and B); n = 11 mice total (D). ^∗P < 0.05.

Figure 3

Prolonged inflammation and lymphedema in mice lacking neuropilin (Nrp)-2. A: Ear swelling versus time after delayed-type hypersensitivity (DTH) reactions in Nrp2^+/+ [wild type (WT); circle], Nrp2^+/− [heterozygous (HET); triangle], and Nrp2^−/− [knockout (KO); square] mice (representative of three independent experiments). B: Hematoxylin and eosin (H&E) staining of ear sections from WT (bottom panel) or Nrp2 KO (top panel) mice after DTH (day 4). Nrp2^−/− ears are thicker than WT littermate ears; swelling is more pronounced in the outer ear. C: Cryosections stained for lymphatic vessel endothelial hyaluronic acid receptor (LYVE)-1 (red) 1 day after challenge reveal lymphatic capillaries. Ear sections from WT mice (left panel) show dilated superficial lymphatic capillaries near the epidermis (arrows) and in the deeper dermis near the cartilage. Nrp2 KO mice (right panel) show only deeper lymphatic vessels and lack superficial capillaries. D and E: The distance from the epidermis to the nearest lymphatic capillary was measured in inflamed ears in adult mice (D) and in normal embryonic day 18 (E18) skin (E). Lymphatic capillaries were 5-fold deeper in the inflamed ears and 2.5-fold deeper in normal embryonic skin. F: The retention of Evans Blue dye was quantified after 16 hours in WT and Nrp2 KO ears on day 4 after DTH. Lymphatic drainage was 2.5-fold less in the Nrp2 KO mice compared to that in the WT mice. Data are expressed as means ± SEM. n = 5 mice per group (A and F); n = 4 mice per group (D and E). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bars = 100 μm.