Immune cells control skin lymphatic electrolyte homeostasis and blood pressure

Abstract

The skin interstitium sequesters excess Na+ and Cl- in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl- accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl-, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.

Figure 1. TONEBP in MPS cells is essential for VEGFC-driven lymphatic capillary hyperplasia and clearance of Cl^– in the skin and buffers systemic blood pressure.

(A) Representative whole-mount staining of lymphatic capillary density (anti–Lyve-1 antibody, green) in ears of FVB mice (WT control group) fed LSD and HSD and of LysM^WTTonEBP^fl/fl mice (without MPS-specific TONEBP deletion) and LysM^creTonEBP^fl/fl mice (with MPS-specific TONEBP deletion), both after HSD. Scale bar: 500 μm. (B) Representative VEGFC protein (85 kDa) expression in FVB mice and LysM^WTTonEBP^fl/fl controls compared with that in LysM^creTonEBP^fl/fl mice. β-Actin (42 kDa) expression was used as a loading control. The mice were fed HSD. (C) TonEBP and Vegfc mRNA expression and VEGFC and CD68 protein expression in skin as well as cutaneous lymphatic capillary density (LCD; arbitrary units) and MAP (mmHg) in LysM^WTTonEBP^fl/fl mice (n = 16) and in LysM^creTonEBP^fl/fl mice (n = 12) fed a HSD. (D) Na⁺ and Cl^– content and concentrations in skin compared with plasma concentrations in the same mice. (E) GAG charge densities in the same mice. *P (genotype) < 0.05.

Figure 2. Blocking the VEGFC/VEGFR3 interaction with mF4-31c1 eliminates the MPS/VEGFC–driven lymphatic capillary hyperplasia after HSD and leads to salt-sensitive blood pressure increase despite unaltered VEGFC/VEGFR2–mediated increased eNOS expression.

(A) Quantification of lymphatic capillary density by whole-mount staining (anti–Lyve-1 antibody) in whole ears of FVB mice fed LSD or HSD, with and without mF4-31c1 treatment. Red squares are the computerized quantitated area; numerical values are lymphatic capillary density (arbitrary units). (B) Representative Western blots for CD68 (100 kDa), VEGFC (85 kDa), eNOS (132 kDa), and p-eNOS (140 kDa) in mouse skin. β-Actin (42 kDa) expression was used as a loading control. (C) Lymphatic capillary density in ear and MAP in the mice. In mice fed HSD, blockade of lymphatic capillary hyperplasia was paralleled by a 17-mmHg increase in MAP with mF4-31c1. (D–F) Relationship among lymphatic capillary density and MAP, skin Cl^– content, and skin Cl^– concentration in the same mice. SKW, skin water content; SKCl^–, skin Cl^– content; rSKCl^–, skin Cl^– content relative to DW. *P < 0.05 versus LSD WT; ^†P < 0.05 versus LSD plus mF4-31c1; ^‡P < 0.05 versus HSD WT; ^#P (diet*mF4-31c1) < 0.05.

Figure 3. Relationship among Na⁺ and Cl^– accumulation, water retention, MAP, and unmeasured anions in mice without and with mF4-31c1 treatment.

Relationship between (A) Cl^– accumulation and (B) Na⁺ accumulation in the skin and MAP in control and in mF4-31c1–treated mice fed a HSD. Elevated blood pressure with anti-VEGFR3 treatment was paralleled by increased skin Cl^– content but not with increased skin Na⁺ content. (C) Skin Na⁺ content, skin Cl^– content, and Cl^–-to-Na⁺ ratio in the mice. With HSD, blockade of cutaneous lymphatic capillary density by mF4-31c1 treatment selectively increased skin Cl^– content. (D) Relationship between skin Na⁺ (orange) and Cl^– (blue) accumulation and skin water content in control mice and in mF4-31c1–treated mice fed HSD. Increasing skin Na⁺ or Cl^– content increased skin water. However, the skin Cl^–-to-water ratio was shifted to the right with mF4-31c1 treatment (0.035 ± 0.006 mmol/ml [control HSD] versus 0.050 ± 0.010 mmol/ml [HSD plus mF4-31c1]; P < 0.05), indicating a reduction in the gap between skin Na⁺ and Cl^– content, which represents unmeasured anionic osmolytes. rSKNa⁺, skin Na⁺ content relative to DW; rSKW, skin water content relative to DW. *P < 0.05 versus LSD WT; ^†P < 0.05 versus LSD plus mF4-31c1; ^‡P < 0.05 versus HSD WT.

Figure 4. A skin-specific VEGFC trap in mice with overexpression of sVEGFR3 in keratinocytes (K14-FLT4 mice) leads to cutaneous lymphatic capillary hypoplasia, skin electrolyte retention, and salt-sensitive blood pressure increase.

(A) Representative staining of MPS cells in ears (anti-CD68 staining; red) of WT and K14-FLT4 mice given LSD or HSD. Scale bar: 50 μm. (B) Representative whole-mount staining of lymphatic capillaries (anti–Lyve-1 staining; green) in the same group of mice. Scale bar: 200 μm. (C) Representative Western blots for CD68 (100 kDa) and VEGFC (85 kDa) expression in mouse skin from the same groups. β-Actin (42 kDa) expression was used as a loading control. (D) Representative Western blots for eNOS (132 kDa) and p-eNOS (140 kDa), with β-actin (42 kDa) as a loading control. (E) Lymphatic capillary density and MAP in WT and in K14-FLT4 mice fed LSD or HSD. *P < 0.05 versus LSD WT; ^†P < 0.05 versus LSD K14-FLT4; ^‡P < 0.05 versus HSD WT; ^#P (diet*K14-FLT4) < 0.05.

Figure 5. HSD leads to skin Na⁺ and Cl^– storage and osmotic stress that is not reflected in plasma.

(A) Na⁺, Cl^–, and osmolality in plasma in rats remained unchanged with HSD, while Na⁺ and Cl^– content and concentrations increased in skin tissue. (B) HSD increased TonEBP and Vegfc mRNA expression and MPS cell count as well as MAP and interstitial fluid pressure (IFP) in the same rats. (C) Electron-dispersion x-ray scanning electron microprobe analysis of Na⁺ and Cl^– concentrations in skin lymph capillaries in DOCA-HSD rats. The arrow denotes lymphatic capillary site from which the spectra were obtained. Scale bar: 20 mm. Lymphatic capillary Na⁺ was higher than that in plasma. The Cl^– values were not significantly different. (D) Na⁺ concentration and osmolality in skin microdialysate and in plasma in rats. (E) Direct plasma and skin-tissue vapor pressure osmolality measurements in rats after 2 weeks of LSD and HSD. DOCA, deoxycorticosterone acetate. *P (diet) < 0.05; ^†P (fluid composition) < 0.05.