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- Christerson, Ulrika, 1970-, et al.
(författare)
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Cytosolic phospholipase A2 and activity upon stimulation with A23187, trypsin and TNF-2 (cPLA2) and calcium-independent2 (iPLA2) in the human mast cell line HMC-1
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background : Mast cells (MC) are considered to be involved in the pathogenesis of Crohn´s disease (CD). A key regulatory event in the production of eicosanoids in MC is the mobilization of arachidonic acid (AA) by the action of phospholipases A2 (PLA2s). The expression of protease activated receptor-2 (PAR-2) is increased on intestinal mast cells in CD, but it is not known if this receptor may influence the PLA2 activity in MC. Although extensively studied in rodent mast cells, not much is known about the PLA2s in human mast cells. The principle aims of the present study were to investigate if the cell line HMC-1 could serve as a model for studies on cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) in human mast cells, and if stimulation of PAR-2 may influence the activity of the two enzymes. Methods: A human mast cell line, HMC-1, was used for the studies. PLA2 activity was analyzed by quantification of radiolabeled fatty acids. PLA2 expression was investigated by RT-PCR and immunocytochemistry. Results: A23187 and the PAR-2-activator trypsin induced a fatty acid release that was not restricted to AA. Only the A23187-induced AArelease was reduced by a combined cPLA2 and iPLA2 inhibitor and by a specific iPLA2 inhibitor. The protein expression of cPLA2, as determined by immunocytochemistry, was very low compared to the expression of iPLA2. TNF-α specifically increased the cPLA2 mRNA expression but had no effect on the protein expression of the PLA2s, as determined by immunocytochemistry. Conclusions: Stimulation with a calcium ionophore may activate iPLA2-dependent AA release, but neither cPLA2 nor iPLA2 seems to be involved in PAR-2-stimulated AA release. TNF-α stimulates cPLA2 mRNA expression, but does not influence the immunocytochemically detected protein level. The expression of cPLA2 and iPLA2 in HMC-1 cells, together with its ability to release radiolabeled fatty acids upon stimulation, makes this cell line a promising model for further studies on cPLA2 and iPLA2 in human MCs.
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| 2. |
- Christerson, Ulrika, 1970-, et al.
(författare)
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Group IIA secretory phospholipase A2 (sPLA2- IIA) and group V secretoryphospholipase A2 (sPLA2-V) in the human mast cell line HMC-1
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Mast cells (MC) are considered to be major effector cells in the pathophysiology of Crohn´s disease (CD). The variety of agents released from activated MC includes secretory phospholipase A2s (sPLA2s), small enzymes known to have both autocrine and paracrine actions of potential importance in inflammatory conditions. The expression of protease-activated receptor-2 (PAR-2) is increased on ileal mucosal MC in CD, however it is not known if this receptor influences the release of sPLA2s from MC. Despite extensive studies in rodent MC, not much is known about sPLA2s in human MC. The principle aims of this study were to investigate if the human mast cell line HMC-1 could serve as a model for studies on two sPLA2s implicated in inflammation, namely sPLA2-IIA and sPLA2-V, and if stimulation of PAR-2 influences the release of the two enzymes.Methods: HMC-1 cells were used for studies on sPLA2 expression by RT-PCR and immunocytochemistry. Degranulation and sPLA2 release was investigated by β-hexosaminidase assay and ELISA, respectively. sPLA2 expression in ileal CD specimens was investigated by immunohistochemistry. Results: In HMC-1 cells the basal expression of sPLA2-IIA seemed to be more pronounced than of sPLA2-V but only sPLA2-V was induced by TNF-α. A23187, but not the PAR-2 stimulator trypsin, caused release of the two enzymes. Both sPLA2-IIA and -V were detected in ileal MC of the CD specimens, but sPLA2-IIA seemed to be more abundant. Conclusions: HMC-1 cells express both sPLA2-IIA and sPLA2-V but the expression may bedifferently regulated by inflammatory cytokines. HMC-1 cells release sPLA2-IIA and sPLA2-V upon appropriate stimulation, although not by PAR-2 stimulation. These results make HMC-1 promising as a model for studies on these two enzymes in human MC. Indeed, the finding that both sPLA2-IIA and sPLA2-V are present in ileal MC in CD strengthens the relevance of such a model.
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| 3. |
- Christerson, Ulrika, 1970-, et al.
(författare)
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Possible Involvement of Intracellular Calcium-Independent Phospholipase A(2) in the Release of Secretory Phospholipases from Mast Cells-Increased Expression in Ileal Mast Cells of Crohn's Disease
- 2019
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Ingår i: Cells. - : MDPI. - 2073-4409. ; 8:7, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Increased activity of secretory phospholipases A(2) (sPLA(2)) type-II was previously observed in ileum of Crohn's disease (CD). Our aims were to explore the involvement of calcium-independent (i)PLA(2 beta) in the release of sPLA(2)s from the human mast cell (MC) line (HMC-1) and investigate expressions of cytosolic (c)PLA(2) alpha, iPLA(2)beta, sPLA(2)-IIA and sPLA(2)-V in MCs of CD ileum. The release of sPLA(2) was investigated in HMC-1 by immunocytochemistry and ELISA. The expression intensities of PLA(2)s in mucosal MCs, and the proportion of PLA(2)-positive MCs, were investigated in normal ileum and in ileum from patients with CD by immunohistochemistry. The calcium ionophore-stimulated release of sPLA(2)-IIA and sPLA(2)-V from HMC-1 was reduced by the iPLA(2)-inhibitor bromoenol lactone. All four PLA(2)s were detectable in mucosal MCs, both in normal ileum and in CD, but the proportion of iPLA(2)beta-containing mucosal MCs and the expression intensity of sPLA(2)-IIA was increased in CD. Results indicate that iPLA(2)beta is involved in the secretion of sPLA(2)s from HMC-1, and suggest that iPLA(2)beta-mediated release of sPLA(2) 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 PLA(2)s in the inflammatory processes of CD.
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