Acute pancreatitis with organ dysfunction associates with abnormal blood lymphocyte signaling: controlled laboratory study

Abstract

Introduction: Severe acute pancreatitis is associated with systemic inflammation, compensatory immune suppression, secondary infections, vital organ dysfunction, and death.Our study purpose was to delineate signaling profiles of circulating lymphocytes in acute pancreatitis complicated by organ dysfunction.

Methods: Sixteen patients with acute pancreatitis, dysfunction of vital organ(s), and immune suppression (proportion of HLA-DR Human Leukocyte Antigen - DR - positive monocytes < 80%) participated. Healthy volunteers served as reference subjects. Using phospho-specific whole blood flow cytometry we studied lymphocyte phosphorylation of nuclear factor-κB (NFκB), mitogen-activated protein kinases p38 and extracellular signal-regulated kinases (ERK)1/2, and signal transducers and activators of transcription (STATs) 1, 3, and 6. Statistical comparisons were performed with the Wilcoxon-Mann-Whitney test.

Results: In blood samples supplemented with tumor necrosis factor, E. coli or S. aureus, phosphorylation levels of NFκB were lower and levels of p38 were higher in patients with acute pancreatitis than healthy subjects. Low NFκB activation involved CD3+CD4+ and CD3+CD8+ lymphocytes. ERK1/2 phosphorylation induced by co-stimulation with phorbol 12-myristate 13-acetate and calcium ionophore A23187 was depressed in patients. STAT3 was constitutively activated in patients' CD3+CD4+ and CD3+CD8+ lymphocytes. Also, IL-6-induced STAT1 phosphorylation was impaired while IL-4-induced STAT6 phosphorylation was enhanced.

Conclusions: Lymphocytes of patients with acute pancreatitis, organ dysfunction and immune suppression show impaired NFκB activation, which increases infection risk and enhanced p38 activation, which sustains inflammation. Secondly, they indicate constitutive STAT3 activation, which may favor Th17 lineage of CD4+ lymphocyte differentiation. Thirdly, they reveal impaired STAT1 activation and enhanced STAT6 activation, denoting a shift from Th1 towards Th2 differentiation.

Figure 1

NF-κB signaling. Levels of phosphorylated NF-κB p65 (pNF-κB) in lymphocytes (Ly) of healthy subjects (circles) and patients (quadrangles) with acute pancreatitis were measured in whole blood samples left without supplement (open symbols) or supplemented (closed symbols) with TNF (10 ng/ml, five minutes), E. coli, S. aureus, S. epidermidis, LPS, or MDP. Responses to TNF, determined as: A. Fluorescence intensity (FI) of all Ly and subsets of Ly (N = 9 to 10); B. As a proportion of pNFκB-positive Ly and their FI. C. The sample histograms of TNF-stimulated (white) and non-stimulated (gray) Ly. The M1 (marker) denotes proportion of pNF-κB-positive Ly. D. Responses to bacteria, LPS, and MDP, determined as the FI of all Ly (N = 8 to 10). E. As proportion of pNFκB-positive cells among all Ly and their FI. RFU, relative fluorescence units. In A and D, horizontal lines denote median, and in B and E, data are given as mean ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001.

Figure 2

The effects of infliximab and anakinra on bacteria-induced NF-κB phosphorylation in lymphocytes. Whole blood samples of healthy subjects were left without cytokine inhibitor or mixed with infliximab, an anti-TNF mAb, anakinra, an IL-1 receptor antagonist, or both, and then left without further supplement or supplemented with E. coli, S. aureus, or S. epidermidis. *P < 0.05 (N = 4 to 6). ⁺ Significantly different (P < 0.05) from respective E. coli, S. aureus, and S. epidermidis groups with the exception of S. epidermidis with infliximab only (P = 0.055), or infliximab + anakinra (P = 0.054).

Figure 3

ERK1/2 signaling. Levels of ERK1/2 phosphorylation in lymphocytes of healthy subjects (circles) and patients (quadrangles) were measured in whole blood samples without supplement (open circles) or supplemented (closed symbols) with combination of PMA (1 μM) and Ca-ionophore (1 μM). RFU, relative fluorescence units. *P < 0.05 (N = 8 to 10).

Figure 4

STAT3 signaling. Levels of phosphorylated STAT3 (pSTAT3) in lymphocytes of healthy subjects (circles) and patients (quadrangles) in whole blood samples without supplement (open symbols) or supplemented (closed symbols) with IL-6 (100 ng/ml, five minutes). A. Fluorescence intensity (RFU, relative fluorescence units); B. sample histograms; C. proportion of pSTAT3-positive cells among all lymphocytes (N = 12 to 13); D. Fluorescence intensity; E. proportion of pSTAT3-positive lymphocytes among; F. fluorescence intensity of pSTAT3-positive lymphocytes among subsets of non-stimulated lymphocytes. Horizontal lines in A and C-F denote median and M1 (marker) in B denotes proportion of pSTAT3-positive lymphocytes. *P < 0.05, **P < 0.01, *** P < 0.001.

Figure 5

STAT1 and STAT6 signaling. Levels of A. pSTAT1 fluorescence intensity (FI); B. pSTAT6 FI in lymphocytes (Ly) of healthy subjects (circles) and patients (quadrangles) in whole blood samples left without supplement (open symbols) or supplemented (closed symbols) with IL-6 (100 ng/ml, five minures) in A and IL-4 (100 ng/ml, five minutes) in B. RFU, relative fluorescence units. *P < 0.05.