Basophils activated via TLR signaling may contribute to pathophysiology of type 1 autoimmune pancreatitis

Abstract

Background: Pathophysiology of type 1 autoimmune pancreatitis (AIP) is still unclear. We previously reported that M2 macrophages might play an important role in type 1 AIP. Recently, it has been reported that basophils regulate differentiation to M2 macrophages. In this study, we investigated basophils from the pancreatic tissue and peripheral blood of individuals with type 1 AIP.

Methods: By using immunohistochemistry, we investigated basophils in pancreatic tissue from 13 patients with type 1 AIP and examined expression of toll-like receptors (TLRs) by these cells. Additionally, we obtained peripheral blood samples from 27 healthy subjects, 40 patients with type 1 AIP, 8 patients with alcoholic chronic pancreatitis, 10 patients with bronchial asthma, and 10 patients with atopic dermatitis, and analyzed activation of basophils by stimulating them with ligands of TLR1-9. We also compared TLR expression in basophils from the tissue and blood samples.

Results: Basophils were detected in pancreatic tissues from 10 of 13 patients with type 1 AIP. Flow cytometric analysis revealed that the ratios of basophils activated by TLR4 stimulation in type 1 AIP (9.875 ± 1.148%) and atopic dermatitis (11.768 ± 1.899%) were significantly higher than those in healthy subjects (5.051 ± 0.730%; P < 0.05). Levels of basophils activated by TLR2 stimulation were higher in seven type 1 AIP cases. Furthermore, stimulation of TLR2 and/or TLR4, which were expressed by basophils in pancreas, activated basophils in peripheral blood.

Conclusions: Basophils activated via TLR signaling may play an important role in the pathophysiology of type 1 AIP.

Keywords: Autoimmune pancreatitis; Basophil; M2 macrophage; TLR.

Fig. 1

Identification of resting and activated basophils from peripheral blood by flow cytometry. All leukocytes were stained with CD3, CD294, and CD203c. All leukocytes formed discrete FSC/SSC populations after fixation and lysis (a). CD3-positive events (R1) were removed due to the isolation of lymphocytes and monocytes (b). Basophils were defined as CD294 (CRTH2)-positive events (R2) (c). CD203c is a specific marker of activated basophils. Line A was drawn so that 2–5% of basophils were included above it, and we defined them as activated basophils. We compared the percentage of basophils above line A before and after the stimulation (d). These experiments were performed using the Allergenicity Kit (Beckman Coulter Company, Brea, CA, USA)

Fig. 2

H&E staining (a, e), immunohistochemical findings for basophils [low magnification view, ×100] (b, f), [high magnification view, ×400] (c, g), and negative controls [high magnification view, ×400] (d, h) in pancreatic tissue samples of two cases with type 1 autoimmune pancreatitis (AIP). Infiltration of basophils was confirmed by using mouse anti-basophil antibodies (2D7)

Fig. 3

H&E staining (a), immunohistochemical findings for basophils [low magnification view, ×100] (b), [high magnification view, ×400] (c), and negative controls [high magnification view, ×400] (d) in a pancreatic tissue sample with alcoholic chronic pancreatitis (ACP)

Fig. 4

Comparison of the total number of basophils in three different 1-mm² fields of view in pancreatic tissue samples from patients with type 1 AIP or alcoholic chronic pancreatitis (ACP). The average number of basophils in samples from patients with type 1 AIP was 8.615 ± 2.528 (n = 13). However, basophils were not detected in the pancreatic tissue of patients with ACP (n = 10)

Fig. 5

Double-immunofluorescence staining for basophils, TLR2, and TLR4 in pancreatic tissue of patients with type 1 autoimmune pancreatitis. Images show staining for basophils (2D7; red, a, e), TLR2 (green, b), TLR4 (green, f), and 4′,6-diamidino-2-phenylindole (DAPI, blue, c, g). Merged image of basophils and TLR2 (d)/TLR4 (h)

Fig. 6

The ratio of basophils activated by the stimulation of TLR1–TLR9. We analyzed peripheral basophils in healthy subjects (n = 27), patients with type 1 autoimmune pancreatitis (AIP, n = 40), patients with alcoholic chronic pancreatitis (ACP, n = 8), patients with bronchial asthma (n = 10), and patients with atopic dermatitis (n = 10). Basophils were stimulated with the TLR1–9 ligands, Pam₃CSK4 (400 ng/mL, InvivoGen, San Diego, CA, USA), HKLM (10⁸ cells/mL, InvivoGen), poly:IC (50 μg/mL, Sigma, St. Louis, MO, USA), LPS (1 μg/mL, Sigma), FLA-ST (10 μg/mL, InvivoGen), FSL-1 (5 μg/mL, InvivoGen), imiquimod (5 μg/mL, InvivoGen), ssRNA40/LyoVec (5 μg/mL, InvivoGen), or ODN2006 (10 μg/mL, AdipoGen, San Diego, CA, USA), respectively. Data are presented as the mean ± standard error of the mean. Statistical comparisons of quantitative data were carried out by the Tukey–Kramer test. Differences were considered to be significant when the value of P was less than 0.05 (P < 0.05)