Possible Involvement of Intracellular Calcium-Independent Phospholipase A2 in the Release of Secretory Phospholipases from Mast Cells-Increased Expression in Ileal Mast Cells of Crohn's Disease

Abstract

Increased activity of secretory phospholipases A₂ (sPLA₂) type-II was previously observed in ileum of Crohn's disease (CD). Our aims were to explore the involvement of calcium-independent (i)PLA₂β in the release of sPLA₂s from the human mast cell (MC) line (HMC-1) and investigate expressions of cytosolic (c)PLA₂α, iPLA₂β, sPLA₂-IIA and sPLA₂-V in MCs of CD ileum. The release of sPLA₂ was investigated in HMC-1 by immunocytochemistry and ELISA. The expression intensities of PLA₂s in mucosal MCs, and the proportion of PLA₂-positive MCs, were investigated in normal ileum and in ileum from patients with CD by immunohistochemistry. The calcium ionophore-stimulated release of sPLA₂-IIA and sPLA₂-V from HMC-1 was reduced by the iPLA₂-inhibitor bromoenol lactone. All four PLA₂s were detectable in mucosal MCs, both in normal ileum and in CD, but the proportion of iPLA₂β-containing mucosal MCs and the expression intensity of sPLA₂-IIA was increased in CD. Results indicate that iPLA₂β is involved in the secretion of sPLA₂s from HMC-1, and suggest that iPLA₂β-mediated release of sPLA₂ from intestinal MCs may contribute to CD pathophysiology. Ex vivo studies on isolated mucosal mast cells are however needed to clarify the precise role of MC PLA₂s in the inflammatory processes of CD.

Keywords: Crohn’s disease; inflammation; mast cells; phospholipases A2.

Figure 1

Expression of iPLA₂β and cPLA₂α in HMC-1. Cells were stimulated with TNFα (25 ng/mL) or culture medium (control) for 48 h. (A) Reverse Transcriptase-PCR analysis; PCR products were identified as iPLA₂α (184 bp), cPLA₂β (737 bp) or 18S rRNA (531 bp). Results are presented as duplicate samples representative of three independent experiments. (B) Immunocytochemical analysis; green staining is for either iPLA₂β or cPLA₂α. Cell nuclei were visualized with propidium iodide staining (red) (magnification × 600). Results are representative for three independent experiments.

Figure 2

Expression of sPLA₂-IIA and sPLA₂-V in HMC-1. Cells were either stimulated with TNFα (25 ng/mL) or culture medium (control) for 48 h. (A) Reverse Transcriptase-PCR analysis; the PCR products were identified as sPLA₂-IIA (238 bp), sPLA₂-V (559 bp) or 18S rRNA (531 bp). Note that the cDNA for sPLA₂-IIA was diluted ten times compared to the cDNA for sPLA₂-V. Samples are two representatives out of seven independent runs. (B) Immunocytochemical analysis. Green staining is for sPLA₂-IIA or sPLA₂-V and red staining is for visualization of cell nuclei (magnification × 600). Results are representative for three independent experiments.

Figure 3

Release of radiolabeled fatty acids from A23187-stimulated HMC-1 cells. Control cells were incubated with culture medium only. (A) Time-dependent release of arachidonic acid (AA). (B) Time-dependent release of oleic acid (OA). (C) Effect of combined stimulation with calcium ionophore A23187 and phorbol myristate acetate (PMA) on the release of AA. PMA and/or A23187 were added for 4 h. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control cells. Data from three independent experiments.

Figure 4

Effect of PLA₂ inhibitors on the calcium ionophore A23187-stimulated release of radiolabeled arachidonic acid (AA) from HMC-1. Cells were pre-treated for 30 min with various concentrations of PLA₂ inhibitors, prior to incubation with A23187 (2 µM) for an additional 4 h. (A) Effect of the combined cPLA₂ and iPLA₂ inhibitor methyl arachidonyl fluoro-phosphonate (MAFP). (B) Effect of the specific iPLA₂ inhibitor bromoenol lactone (BEL). *p < 0.05, ** p < 0.01, *** p < 0.001 vs. A23187-stimulated cells. Data from three independent experiments.

Figure 5

Degranulation and release of sPLA₂-IIA and sPLA₂-V in A23187-stimulated HMC-1. Cells were stimulated for 4 h with various concentrations of calcium ionophore A23187. Control cells were incubated with culture medium only. (A) Release of β-hexosaminidase. (B) ELISA analysis. Release of sPLA₂-IIA. (C) Immunocytochemical analysis, visualizing the effect of A23187 on the release of sPLA₂-IIA and sPLA₂-V. Green staining is for sPLA₂-IIA or sPLA₂-V and red staining is for visualization of cell nuclei (magnification × 600). Note that the expression of sPLA₂-V had to be upregulated by TNFα, as described in Figure 2A and B. **p < 0.01, ***p < 0.001 vs. controls. Data from three independent experiments.

Figure 6

Effect of iPLA₂ inhibition on A23187-induced degranulation and release of sPLA₂-IIA and sPLA₂-V in HMC-1. (A) Effect of the specific iPLA₂ inhibitor bromoenol lactone (BEL) on the release of β-hexosaminidase. (B) Effect of the specific iPLA₂ inhibitor BEL on the release of sPLA₂-IIA measured by ELISA. (C) Immunocytochemical analysis, visualizing the effect on the release of sPLA₂-IIA and sPLA₂-V. Green staining is for sPLA₂-IIA or sPLA₂-V and red staining is for visualization of cell nuclei (magnification × 600). Note that the expression of sPLA₂-V had to be upregulated by TNFα, as described in Figure 2A and B, to be illustrated. ** p < 0.01, *** p < 0.001 vs. controls. Data from three independent experiments.

Figure 7

Expression of iPLA₂β, cPLA₂α, sPLA₂-IIA and sPLA₂-V on mast cells (MCs) in the intestinal mucosa of 5 patients with Crohn’s disease (CD) and 5 controls. (A) Percentage of MCs expressing iPLA₂β. Arrows indicate MCs co-localizing with iPLA₂β in a patient with CD. (B) Percentage of MCs expressing cPLA₂α. Arrows indicate MCs co-localizing with cPLA₂α in a control patient. Arrow-head indicates cPLA₂α expression in a cell not positive for MC tryptase. (C) Expression intensity of sPLA₂-IIA on MCs. Arrows indicate MCs co-localizing with sPLA₂-IIA in a patient with CD. Arrow-head indicates sPLA₂-IIA expression in a cell not positive for MC tryptase. (D) Expression intensity of sPLA₂-V on MCs. Arrows indicate MCs co-localizing with sPLA₂-V in a control patient.