Targeting lymphangiogenesis after islet transplantation prolongs islet allograft survival

Abstract

Background: Lymphatics are important for their conduit functions of transporting antigen, immune cells, and inflammatory mediators to draining lymph nodes and to the general circulation. Lymphangiogenesis is involved in many pathologic processes; however, the roles for lymphatic responses in transplantation have not been thoroughly investigated.

Methods: Mice were made diabetic by a single high dose of streptozotocin and then received islet allografts. Animals were treated with three different lymphatic inhibitors. FTY720, an analog of sphingosine 1-phosphate, inhibited lymphocyte migration into afferent and efferent lymphatics. Sunitinib, a kinase inhibitor, blocked several receptors, including vascular endothelial growth factor receptor 3 (VEGFR3), the major growth factor receptor for lymphatic endothelial cells. Anti-VEGFR3 monoclonal antibody specifically inhibited VEGFR3. Diabetes was determined by daily monitoring of blood glucose levels. Inflammation within islet grafts was assessed by immunohistochemistry for insulin, T cells (CD3), and lymphatics (LYVE-1).

Results: After transplantation, lymphangiogenesis occurred in islet allografts and in draining lymph nodes. FTY720, sunitinib, and anti-VEGFR3 each inhibited lymphangiogenesis in the islets and significantly prolonged allograft survival. Immunofluorescent staining demonstrated that administration of each of the lymphatic inhibitors resulted in preservation of islets and β-cells along with a markedly reduced infiltration of T cells into the grafts.

Conclusion: Lymphangiogenesis occurs in islet allografts in response to inflammation and plays a key role in the islet inflammation in alloimmunity. Interfering with lymphatic function leads to inhibition of lymphangiogenesis and prolonged or indefinite allograft survival. These observations suggest new therapeutic targets for rejection and tolerance.

FIGURE 1

Lymphangiogenesis occurs in rejecting islet allografts. C57BL/6 mice were rendered diabetic by a single high-dose injection of streptozotocin. Islets from BALB/c donors were implanted under the renal capsule. Rejecting islet grafts (14 days posttransplantation) were stained for β-cells (insulin) and lymphatic vessels (LYVE-1). 100× magnification.

FIGURE 2

Inhibition of lymphangiogenesis delays rejection of islet allografts. Diabetic C57BL/6 recipients transplanted with BALB/c islets were treated with FTY720, sunitinib, or anti-vascular endothelial growth factor receptor 3 (VEGFR3) monoclonal antibody (mAb), for 2 weeks starting on the day of transplantation, and groups of recipients were treated with FTY720 (1 mg/kg daily), sunitinib (40 mg/kg daily), or anti-VEGFR3 mAb (32 mg/kg three times/week) for 2 weeks starting on the day of transplantation. Graft survival (n=5 per group). P value versus controls: FTY720 less than 0.001, sunitinib, and anti-VEGFR3 less than 0.005. PBS, phosphate-buffered saline.

FIGURE 3

Inhibition of lymphatic function prevents T-cell infiltration and preserves islets. (A) and (C) Immunofluores-cent analysis of β-cells (insulin), T cells (CD3), and lymphatic vessels (LYVE-1) in islet grafts 7 days (A) and 14 days (C) after transplantation. Dashed lines show boundary between kidney cortex (a) and graft (b). 100× magnification. Scale bars: 100 μm. (B) and (D) Quantitative analysis of insulin, CD3, and LYVE-1 staining of islet grafts 7 days (B) and 14 days (D) after transplantation. Two to three slides/mouse, two to three mice/group. Data are presented as mean±standard deviation. ns, not significant. **P less than or equal to 0.01; ***P less than or equal to 0.001.

FIGURE 4

Inhibition of lymphatic function prevents draining lymph node (LN) lymphangiogenesis. (A) Immunofluorescent analysis of T cells (CD3), lymphatic vessels (LYVE-1), and blood vessels (peripheral LN addressin [PNAd]) of draining renal LNs 7 days after transplantation. Normal, normal mesenteric LN. 100× magnification. Scale bars: 100 μm. (B) Quantitative analysis of LYVE-1 and PNAd in draining renal LNs 7 days after transplantation. Two to three sections/LN, two to three mice/group. Data are presented as mean±standard deviation. *P less than or equal to 0.05, **P less than or equal to 0.01, ***P less than or equal to 0.001 versus phosphate-buffered saline (PBS) treatment.