α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction

Abstract

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic β-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in β-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

Figure 1

AAT treatment improves islet survival and function in a syngeneic mouse intraportal islet transplantation model. A: Kaplan-Meier analysis shows significantly higher percentages of PT normoglycemia in diabetic mice that received AAT (2 mg/kg) Q2D or Q3D compared with control (Ctrl), which received vehicle. B: AAT-treated mice reached normoglycemia faster than control mice. C: IVGTT shows that AAT treatment (Q2D) enhances blood glucose disposal 30 days after transplantation. Inset, AUC of IVGTT. D: Serum concentrations of human AAT before the last dose in each group (n = 8–9 in each group). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 by Student t test.

Figure 2

AAT reduces immediate release of C-peptide and inhibits islet cell death after transplantation. A: Serum C-peptide levels in AAT-treated and control (Ctrl) recipients 3 and 6 h PT as measured by ELISA. B: Islets in liver sections from AAT-treated mice showed fewer TUNEL⁺ dead cells compared with islets from control mice 24 h after islet transplantation. Nuclei are stained with DAPI (blue) and β-cells with anti-insulin antibody (red). Arrowheads point to TUNEL⁺ cells (green) within an islet. C: Percentage of TUNEL⁺ cells per islet graft as determined after microscopic evaluation of intrahepatic islets randomly selected from three different recipients in Ctrl (25 islets with sizes of 10,900.5 ± 3,700.7 µm²) and AAT (20 islets with sizes of 9,480.4 ± 4,840.2 µm²) groups. Results are expressed as means ± SD. *P < 0.05, **P < 0.01 by Student t test.

Figure 3

AAT suppresses neutrophil and macrophage infiltration and serum TNF-α production. A: Accumulation of neutrophils identified by naphthol AS-D chloroacetate esterase staining in AAT (n = 3) or control (Ctrl; n = 3) grafts 6 h PT. Serial paraffin sections of an islet graft were stained with anti-insulin (left, brown staining) and naphthol AS-D chloroacetate esterase staining (right, purple staining). Arrowheads point to neutrophils. B: Numbers of neutrophils within the islet were lower in the AAT group than in the control group 6 h after islet transplantation. A total of 25 Ctrl islets (average size: 8,500.9 ± 6,816.6 µm²) and 27 AAT islets (average size: 10,549.2 ± 6,596.9 µm²) were counted. C: Representative photomicrographs show fewer infiltrated macrophages surrounding transplanted islets identified by the anti-F4/80 antibody in AAT compared with Ctrl mice 6 h after islet transplantation. Red staining identifies macrophages, green staining identifies β-cells, and blue represents nuclear material from all cells. D: Serum TNF-α levels measured by ELISA are higher in serum of Ctrl (n = 6) than AAT-treated (n = 4) recipients 6 h after islet transplantation. Scale bars are 100 μm for panels A and C and 20 μm for insets in panel A. *P < 0.05 AAT vs. Ctrl by Student t test.

Figure 4

AAT inhibits activation of coagulation after intraportal transplantation. A: Reduced TF expression in AAT-treated grafts compared with control (Ctrl) grafts. Immunohistochemistry staining of TF (green) in transplanted islets (red for insulin) from AAT or Ctrl mice. Arrows point to TF⁺ cells. B: Relative fluorescence intensity of TF divided by intensity of insulin in grafts from AAT or Ctrl mice. C: Change of plasma TAT levels in AAT and Ctrl mice at 3 and 6 h PT. D: Fibrinogen deposition in transplanted islets 6 h after intraportal transplantation in Ctrl and AAT mice. Serial paraffin sections of an islet graft were stained with anti-insulin (left) and anti-fibrinogen (right). The dashed line outlines islet grafts based on the insulin staining. The arrow indicates fibrin deposition on islet graft. Scale bars = 100 μm. Data are expressed as mean ± SD. *P < 0.05 AAT vs. Ctrl by Student t test.

Figure 5

AAT protects islets from cytokine-induced cell death by suppression of JNK phosphorylation and NF-κB activation. AAT treatment reduces p-JNK after cytokine treatment as measured by Western blot. A: Representative immunoblot of p-JNK and total JNK and histone H3 by Western blot. B: Ratio of p-JNK to total JNK from immunoblots quantified by densitometry. Data are presented as mean ± SD for five individual experiments. C: Cell death measured in βTC3 cells pretreated with AAT at 0.1 mg/mL or 0.5 mg/mL and/or 20 nmol/L SP600125, the JNK phosphorylation blocker (JNK inh), for 1 h and stimulated with cytokines for 48 h using the Cell Death ELISA kit. Experiments were repeated four times. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 using one-way ANOVA, followed by Tukey post hoc analysis. D: NF-κB activity (percentage to control [Ctrl]) as measured by p65 level in the nuclear fraction by ELISA in livers bearing islet grafts. Experiments were repeated three times. E: Representative immunoblot of inhibitor of κB-α (IκBα) and β-actin in βTC3 cells pretreated with vehicle or 0.5 mg/mL AAT, and stimulated with cytokine for 0–30 min as analyzed by Western blot. *P < 0.05 AAT vs. Ctrl by Student t test.

Figure 6

AAT reduces serum C-peptide and TAT levels and inhibits inflammatory cells infiltration to human islet grafts. A: Serum C-peptide levels in AAT-treated and control (Ctrl) recipients 1, 3, and 6 h PT as measured by ELISA. B: Change of plasma TAT levels in AAT and Ctrl mice. *P < 0.05, **P < 0.01 vs. Ctrl by two-way repeated-measures ANOVA, followed by Bonferroni correction. C: Less neutrophil infiltration was identified in AAT grafts compared with control grafts 6 h PT. Serial paraffin sections of an islet graft were stained with anti-insulin (left) and naphthol AS-D chloroacetate esterase staining (right). The dashed line illustrates islets with positive insulin staining, and arrowheads point to neutrophils. Scale bars = 100 μm. D: Number of neutrophils per islets in AAT and control mice (n = 20 per group) 6 h after islet transplant. E: Representative photomicrographs show more infiltrated macrophages surrounding Ctrl vs. AAT islet grafts as identified by staining with the anti-F4/80 antibody 6 h after islet transplantation. Green staining identifies macrophages, red staining identifies insulin⁺ cells, and blue represents nuclei. *P < 0.05 AAT vs. Ctrl by Student t test.

Figure 7

AAT reduces TF expression and inhibits neutrophil infiltration to human islet in miniature tube assay. A: Reduced TF expression was observed in human islets (Hi) transplanted to AAT-treated recipients compared with controls 15 min after exposure to NOD-SCID blood. GM, growth medium. B: Relative fluorescence intensity of TF divided by intensity of insulin from the AAT or control (Ctrl) group. ImageJ software was used to measure three to nine islets in each group. More polymorphonuclear cell infiltrations were observed in Ctrl islets compared with AAT-treated islets as determined by naphthol AS-D chloroacetate esterase staining 15 min after exposure to NOD-SCID blood as measured by immunohistochemistry staining (C) and cell counting (D). Scale bars = 50 μm. More than 30 islets were counted in each group. *P < 0.05 AAT vs. Ctrl, ***P < 0.001 by Student t test.

Figure 8

AAT suppressed JNK phosphorylation in human islets in vitro. A: Immunohistochemistry staining of TF in human islets 15 min after exposure to NOD-SCID blood. B: Relative fluorescence intensity of p-JNK divided by intensity of insulin from AAT or control (Ctrl) group. In each group, 10 islets were measured using ImageJ software. C: Human islets were treated with cytokines with or without preincubation with AAT for the indicated times. Representative immunoblot of p-JNK is shown. Ratio of p-JNK to total JNK from immunoblots was quantified by densitometry. Scale bars = 50 μm. *P < 0.05 AAT vs. Ctrl by Student t test.