Hemin-activated macrophages home to the pancreas and protect from acute pancreatitis via heme oxygenase-1 induction

Abstract

Hemin upregulates heme oxygenase-1 (HO-1), a stress-induced enzyme implicated in protection from a variety of injuries while its related isoform HO-2 is constitutively expressed. The role of hemin or HO-1 in the pancreas and their potential modulation of pancreatic injury are unknown. We show that HO-1 is induced in pancreatitis caused by caerulein and more prominently in severe pancreatitis caused by feeding a choline-deficient diet (CDD). Intraperitoneal hemin administration dramatically increases peritoneal and pancreas macrophages that overexpress HO-1 in association with pancreatic induction of the chemoattractants monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha but not RANTES or macrophage inflammatory protein-2. Hemin administration before CDD feeding protected 8 of 8 mice from lethality while 7 of 16 controls died. Protection is mediated by HO-1-overexpressing macrophages since hemin-primed macrophages home to the pancreas after transfer to naive mice and protect from CDD-induced pancreatitis. Suppression of hemin-primed peritoneal cell HO-1 using HO-1-specific small interfering RNA prior to cell transfer abolishes protection from CDD-induced pancreatitis. Similarly, hemin pretreatment in caerulein-induced pancreatitis reduces serum amylase and lipase, decreases pancreatic trypsin generation, and protects from lung injury. Therefore, hemin-like compounds or hemin-activated macrophages may offer novel therapeutic approaches for preventing acute pancreatitis and its pulmonary complication via upregulation of HO-1.

Figure 1

Effect of hemin or pancreatic injury on HO-1 induction and inhibition of pancreatic injury by hemin. (A and B) Total tissue homogenates were obtained from pancreata of caerulein-injected (cae-injected) or CDD-fed mice. Two age- and sex-matched mice were used for each time point. Homogenates were tested by blotting, using antibodies to HO-1 or HO-2. (C) Mice (3 mice/condition) were given hemin (H) by i.p. injection (4 times during 1 week or 8 times during 2 weeks). Two control (Co) mice were injected with vehicle alone. Pancreatic homogenates were then obtained and blotted with anti–HO-1 and anti–HO-2. (D–G) Mice were injected with saline (S), hemin, or vehicle (V) (8 mice/group) 3 times (arrows) followed by feeding with CDD for 3 days, then harvesting of the pancreata. HO-1 and HO-2 were analyzed by blotting of pancreatic homogenates (3 mice/group). The number of mice that died in each cohort of 8 mice is shown, and the survival difference was significant (P < 0.03) when comparing controls (saline and vehicle) with hemin-injected mice. Representative H&E stainings of pancreata from mice that survived CDD feeding are shown. Scale bar: 50 μm. Note the marked pancreas edema (ED) and necrosis (N) in the saline- (not shown) and vehicle-injected mice as compared with the hemin group.

Figure 2

Effect of hemin on caerulein-induced pancreatitis and its pulmonary complications. (A and B) Mice were injected i.p. with hemin or vehicle (7 mice/group) every other day (3 times; arrows) followed by injection with either saline or caerulein hourly (7 times; arrows) and then harvesting of the pancreata and lungs at 12 hours after first saline or caerulein injection. Sera were collected for amylase/lipase measurement. (C–E) Pancreatic and lung tissues (from A and B) were used to determine trypsin (pmol/mg protein) and MPO (ng/mg wet tissue) enzyme activities and to assess extent of tissue injury. Data are presented as mean ± SD. A representative H&E staining of lung is shown. Scale bar: 50 μm. Note the marked hemorrhage and alveolar collapse in vehicle- (E) compared with hemin- (F) pretreated mice receiving caerulein.

Figure 3

Hemin increases HO-1⁺ pancreatic macrophages in association with MCP-1 and MIP-1α induction. (A–D) Pancreas from CDD-fed mice was triple stained using antibodies to HO-1 (A), the macrophage marker F4/80 (B), and the granulocyte marker Gr-1 (C). (D) A merged image of the triple stain. Scale bar: 20 μm. (E) Mice were treated i.p. with hemin or vehicle 1 or 3 times during a 1-week period followed by feeding with CDD or regular chow for 3 days. Pancreata were collected followed by staining and counting F4/80⁺ macrophages in 10 randomly selected high-power fields (HPFs). The mean ± SD (3 mice/group) and P values for the indicated comparisons are shown. (F) Hemin or vehicle was injected i.p. once followed by harvesting of pancreata after 24 hours. A noninjected control group was also included. mRNA levels were then estimated from pancreata of 3 mice per group as a ratio of the indicated chemokine to β-actin (mean ± SD). Changes in RANTES/MIP-2 mRNA levels were not significant after vehicle or hemin administration.

Figure 4

Effect of hemin on in vivo macrophage homing to the pancreas using bioluminescence imaging. (A) Experimental scheme of cell transfer. Hemin was injected 3 times i.p. (arrows) into luciferase-overexpressing (luciferase^+/+) mice, then peritoneal cells were harvested and macrophages were selected using anti–Mac-1 magnetic beads. Luciferase/Mac-1 double-positive cells were transferred i.p. to wild-type (luciferase^–/–) mice that were preinjected with 1 dose of hemin or vehicle 24 hours prior to the transfer. This hemin injection was necessary to induce pancreatic chemokines (Figure 3F). (B–D) Live images of recipient intact anesthesized mice were taken 5 minutes (B) or 24 hours (C) after cell transfer. Livers and pancreata were removed 24 hours after the transfer, followed by imaging (D). The signal intensity scale bar is shown below each image. (E–G) A duplicate of the pancreata shown in part D (from recipients receiving donor cells from hemin-injected animals) was double stained with antibodies to luciferase (E) and F4/80 (F). Similar double staining of pancreata from recipient animals receiving donor cells from V-injected mice showed background staining (e.g., E [inset] for the anti-luciferase staining). (G) A merged image of the double stain. Scale bar: 50 μm.

Figure 5

Protective effect of hemin-primed peritoneal cells on CDD-induced pancreatitis. (A) Peritoneal cells are isolated from hemin- or vehicle-pretreated mice (3 injections indicated by arrows), then transferred i.p. into naive recipient littermates followed by immediate initiation of CDD feeding. (B–E) After 2.5 days of CDD, sera were collected to measure amylase and BUN (mean ± SD, 6 mice/group). Pancreata were also collected for gross tissue assessment and for histological staining and scoring. Scale bar: 50 μm. Vac, vacuoles; ED, edema; Hem, hemorrhage; m1 and m2, pancreatic tissues from 2 separate mice per hemin- or vehicle-pretreated group.

Figure 6

Inhibition of the protective effect of hemin-primed peritoneal cells using HO-1 knock-down in vivo. (A and B) Peritoneal cells were isolated from hemin-treated mice that were also injected i.p. (arrows) on alternate days with saline, HO-1 siRNA (constructs 1 and 2), or scramble control (Sc) siRNA. The peritoneal cells were then used to examine HO-1 and HO-2 levels by immunoblotting. (C and D) Peritoneal cells were harvested from hemin/construct 2 HO-1 siRNA or hemin/scrambled siRNA–injected mice, then transferred into naive recipient littermates followed by immediate initiation of CDD feeding. At 2.5 days of CDD feeding, pancreata were collected for histologic scoring. Data for histology score are presented as mean ± SD (n = 5–6/group), and are 6.4 ± 1.7 (scrambled) and 12.7 ± 4.5 (HO-1 siRNA). (E–J) Pancreata from C and D were double stained using antibodies to F4/80 and HO-1. Scale bar, 20 μm.

Figure 7

Schematic of the effect of hemin on mouse HO-1 induction and homing of peritoneal macrophages to the pancreas. Hemin induced the expression of several pancreas chemokines and increased peritoneal macrophages and their HO-1 expression. Peritoneal macrophages that overexpress HO-1 homed to the pancreas and were able to provide significant protection from pancreatic injury.