Heme oxygenase-1 is induced in peripheral blood mononuclear cells of patients with acute pancreatitis: a potential therapeutic target

Abstract

Heme oxygenase-1 (HO-1) induction by hemin or Panhematin protects against experimental pancreatitis. As a preclinical first step toward determining whether HO-1 upregulation is a viable target in acute pancreatitis (AP) patients, we tested the hypothesis that HO-1 expression in peripheral blood mononuclear cell (PBMC) subsets of hospitalized patients with mild AP is upregulated then normalizes upon recovery and that cells from AP patients have the potential to upregulate their HO-1 ex vivo if exposed to Panhematin. PBMCs were isolated on days 1 and 3 of hospitalization from the blood of 18 AP patients, and PMBC HO-1 levels were compared with PMBCs of 15 hospitalized controls (HC) and 7 volunteer healthy controls (VC). On day 1 of hospitalization, AP patients compared with VCs had higher HO-1 expression in monocytes and neutrophils. Notably, AP monocyte HO-1 levels decreased significantly upon recovery. Panhematin induced HO-1 in ex vivo cultured AP PBMCs more readily than in HC or VC PBMCs. Furthermore, PBMCs from acutely ill AP patients on day 1 were more responsive to HO-1 induction compared with day 3 upon recovery. Similarly, mouse splenocytes had enhanced HO-1 inducibility as their pancreatitis progressed from mild to severe. In conclusion, AP leads to reversible PBMC HO-1 upregulation that is associated with clinical improvement and involves primarily monocytes. Leukocytes from AP patients or mice with AP are primed for HO-1 induction by Panhematin, which suggests that Panhematin could offer a therapeutic benefit.

Fig. 1.

Leukocyte subset expression of heme oxygenase-1 (HO-1) using fluorescence-activated cell sorting (FACS) analysis. Peripheral blood mononuclear cells (PBMCs; obtained from patients on the first day of admission) were stained for leukocyte-specific [monocytes (A), neutrophils (B), B cells (C), T cells (D)] surface markers as described in materials and methods and then stained for intracellular HO-1. The cells were analyzed by FACS and corrected for isotype control antibody and normalized by using Spherotech beads to allow comparison between patients. Statistical analysis was done via the Mann-Whitney U-test. VC, volunteer controls; HC, hospitalized controls; AP, acute pancreatitis; MFI, mean fluorescence intensity.

Fig. 2.

Leukocyte subset expression of HO-1 over time. PBMCs were obtained from VC and AP patients on day 1 (d1) and day 3 (d3) and stained for leukocyte surface markers and intracellular HO-1. The cells were then analyzed by FACS and corrected for isotype control antibody and normalized by use of Spherotech beads to allow comparison between individuals and over time. Samples from VC are shown in broken lines with mean values shown in bold broken bold line; samples from AP are shown in solid lines with mean values shown in bold solid line. Statistical analysis (day 1 vs. day 3) was based on the Wilcoxon's signed-rank test. A: monocytes. B: neutrophils. C: B cells. D: T cells.

Fig. 3.

Panhematin (PH)-mediated HO-1 induction in AP patients, HC patients, and VC PBMCs. A: PBMCs from AP patient blood samples obtained on hospital admission day 1 and day 3 were isolated by density centrifugation. PBMCs were cultured for 16, 24, or 48 h with PH or vehicle (VE) alone. B: PBMCs were isolated from VC healthy individuals and HC patients and cultured for 24 h as in A. For both A and B, cultured PBMCs were lysed in a homogenization buffer as described in materials and methods, and 10 μg total protein from each sample was separated by SDS-PAGE for Western blotting to compare HO-1 levels. Duplicate gels were also stained with Coomassie blue to confirm equal protein loading. Bar graphs show HO-1 density quantification normalized to protein loaded. *P < 0.05 compared with VE.

Fig. 4.

PH-mediated HO-1 induction in leukocyte subsets from AP patients and healthy VC. PBMCs from AP patients at the time of admission and from VC donors were isolated by density centrifugation. PBMC were cultured for 24 h with either PH or VE alone. The cells were subsequently stained for leukocyte surface markers followed by staining for intracellular HO-1. The immune-stained cells were then analyzed by FACS. Histograms with light and dark lines are from VE- and PH-treated cells, respectively. Bar graph shows % HO-1 induction as determined from the FACS histograms. Solid and open bars represent results from AP and VC donors, respectively. *P < 0.05.

Fig. 5.

PH-mediated HO-1 induction in mouse splenocytes during experimental pancreatitis. A: FVB/n mice were fed regular chow (a) or CDE for 24 (b), 48 (c), or 60 h (d) to induce mild, moderate, and severe pancreatitis, respectively. Pancreata were isolated and fixed in 10% formalin, and sections were stained with hematoxylin and eosin and scored. Serum was used for determination of lipase levels. Scale bar, 50 μm. B: leukocytes from mouse spleens of the FVB/n mice fed CDE for 24, 48, or 60 h were isolated and cultured for 24 h with either PH or VE alone. Cultured leukocytes were lysed in a homogenization buffer as described in materials and methods, and 10 μg total protein from each sample was separated by SDS-PAGE followed by Western blot analysis to compare HO-1 protein expression levels. Equal protein loading was confirmed by assessing levels of the constitutively-expressed HO-2 isoform. Bar graphs show the density of HO-1 relative to HO-2. *P < 0.05 compared with VE.

Fig. 5.

PH-mediated HO-1 induction in mouse splenocytes during experimental pancreatitis. A: FVB/n mice were fed regular chow (a) or CDE for 24 (b), 48 (c), or 60 h (d) to induce mild, moderate, and severe pancreatitis, respectively. Pancreata were isolated and fixed in 10% formalin, and sections were stained with hematoxylin and eosin and scored. Serum was used for determination of lipase levels. Scale bar, 50 μm. B: leukocytes from mouse spleens of the FVB/n mice fed CDE for 24, 48, or 60 h were isolated and cultured for 24 h with either PH or VE alone. Cultured leukocytes were lysed in a homogenization buffer as described in materials and methods, and 10 μg total protein from each sample was separated by SDS-PAGE followed by Western blot analysis to compare HO-1 protein expression levels. Equal protein loading was confirmed by assessing levels of the constitutively-expressed HO-2 isoform. Bar graphs show the density of HO-1 relative to HO-2. *P < 0.05 compared with VE.