TRAIL and DcR1 expressions are differentially regulated in the pancreatic islets of STZ- versus CY-applied NOD mice

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is an important component of the immune system. Although it is well acknowledged that it also has an important role in Type 1 Diabetes (T1D) development, this presumed role has not yet been clearly revealed. Streptozotocin (STZ) and Cyclophosphamide (CY) are frequently used agents for establishment or acceleration of T1D disease in experimental models, including the non-obese diabetic (NOD) mice. Although such disease models are very suitable for diabetes research, different expression patterns for various T1D-related molecules may be expected, depending on the action mechanism of the applied agent. We accelerated diabetes in female NOD mice using STZ or CY and analyzed the expression profiles of TRAIL ligand and receptors throughout disease development. TRAIL ligand expression followed a completely different pattern in STZ- versus CY-accelerated disease, displaying a prominent increase in the former, while appearing at reduced levels in the latter. Decoy receptor 1 (DcR1) expression also increased significantly in the pancreatic islets in STZ-induced disease. Specific increases observed in TRAIL ligand and DcR1 expressions may be part of a defensive strategy of the beta islets against the infiltrating leukocytes, while the immune-suppressive agent CY may partly hold down this defense, contributing further to diabetes development.

Figure 1

Mean blood glucose levels following STZ- or CY-inductions. NOD mice received single intraperitoneal injections of 150 mg/kg STZ or 200 mg/kg CY or vehicle (physiological saline or citrate buffer) at 10 weeks of age. Fifteen mice were used for initial inductions in both STZ and CY groups. Three mice were sacrificed at each point for immunohistochemical pancreas analysis starting from day 1 for the CY group and day 2 for the STZ group. The given values represent the mean blood glucose values for the live subjects, including those to be sacrificed, at each point.

Figure 2

Lymph node staining of the TRAIL ligand and its DcR1 decoy receptor. Specificities of the primary antibodies were confirmed on lymph node sections. Stainings for TRAIL and DcR1 are shown. C stands for the negative control staining, where only the secondary antibody was used in the immunohistochemical procedure.

Figure 3

Histologic examination of the pancreatic islets of STZ- or CY-treated NOD mice for TRAIL ligand expression. NOD mice received single intraperitoneal injections of 150 mg/kg STZ or 200 mg/kg CY. Pancreata were isolated at days 0, 2, 4, 7, 11, and 14 in the STZ group, and at days 0, 1, 4, 7, 14, and 21 in the CY group. Three mice were sacrificed at each point. Representative images are shown for negative, weak, moderate, and strong stainings for the TRAIL ligand expression. (a) Sample stainings for STZ-treated mice are shown through (A)–(D), and for CY-treated mice through (E)–(H): (A) and (E) show negative staining for TRAIL in the pancreatic islet in the STZ- and CY-treated groups, respectively; the surrounding acinar cells display weak IC staining (arrow) in (A). Weak IC (B), moderate IC (C), and strong nuclear (D) positivity in the pancreatic islets. Acinar cells display strong nuclear positivity in (D); (F) presents weak (thin arrow) and moderate (thick arrow) IC stainings within the islet, (G) moderate (thin arrow) and strong (thick arrow) IC positivity in the islet, (H) moderate IC and strong nuclear staining in majority of the islet, and moderate nuclear positivity in acinar cells. Control staining (secondary antibody only) is shown in (I). Smaller islets are depicted at 400X magnification for clarity, while other images were taken at 200x. (b) Immunohistochemical staining results for TRAIL were evaluated as described in Section 2. Changes at days 2, 4, and 14 observed in the STZ group, and the alteration observed at day 14 in the CY group were statistically significant (P < 0.05). Error bars represent ±SEM. IC: intracytoplasmic.

Figure 4

DcR1 expression analysis in STZ- or CY-treated NOD mice. Pancreata were isolated at days 0, 2, 4, 7, 11, and 14 in the 150 mg/kg STZ-injected group and at days 0, 1, 4, 7, 14, and 21 in the 200 mg/kg CY-injected group. Representative images are shown for negative, weak, moderate, and strong stainings for DcR1 expression. (a) Sample stainings for STZ-treated mice are displayed through (A)–(D), and for CY-treated mice through (E)–(H): negative staining for DcR1 is shown in (A) and (E); moderate IC staining is evident in some acinar cells in (E), depicted by arrow. (B) Weak IC positivity in the islet, moderate IC staining in the acinar cells, (C) weak IC, moderate nuclear (thin arrow) and strong nuclear (thick arrow) staining in the islet, moderate nuclear staining in acinar cells, (D) weak IC and strong nuclear positivity in the islet, along with strong nuclear positivity in acinar cells, (F) weak IC staining in the islet, moderate IC positivity in some acinar cells, (G) moderate (thin arrow) and strong (thick arrow) positivity in the islet, no staining in acinar cells, (H) moderate IC, as well as strong nuclear stainings in the islet, moderate nuclear staining in the acinar cells, (I) control staining with secondary antibody alone. Smaller islets are depicted at 400x magnification for clarity. Other images were taken at 200x. (b) Immunohistochemical staining evaluations for DcR1 were performed as described in Section 2. Each bar represents mean data from three mice. Increase at day 14 in the STZ group is significant (P < 0.05). Error bars represent ±SEM. IC: intracytoplasmic.

Figure 5

Expression profiles for DcR2 and DR5 in STZ- or CY-applied, NOD mice. Sample collection days for immunohistochemical analyses are given under bars. The collection days shown correspond to the common pancreata collection points in both groups. The mean values for 3 different subjects are presented in each bar. Error bars represent ±SEM. Statistical significance (*) was determined by Mann-Whitney U test applied following the Shapiro-Wilk normality test. A P value of 0.05 was considered significant.

Figure 6

TRAIL ligand and marker expressions in pancreatic acinar cells of STZ- or CY-applied NOD mice. Expression values before agent application (day 0) and at the last pancreas collection points (Day 14 for STZ application and day 21 for CY application) are given. The mean values of 3 different subjects are presented in each bar. Error bars represent ±SEM. Statistical significance (*) was determined by Mann-Whitney U test applied following the Shapiro-Wilk normality test. A P value of 0.05 was considered significant.

Figure 7

Survival patterns and mean blood glucose level profiles for NOR mice. (a) Mortality rates for NOR mice. Female NOR mice received the same agent doses as the NOD mice, that is, single intraperitoneal injections of 150 mg/kg STZ (n = 10), or 200 mg/kg CY (n = 10) or vehicle (physiological saline or citrate buffer) at 10 weeks of age. Eighty percent of the STZ-injected mice died at day 7. (b) Changes in blood glucose levels in NOR mice. Nonfasting blood sugar levels of the STZ-, CY-, or vehicle-injected NOR mice groups were measured by a portable glucose meter. Data are given as mean ± SEM.