Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells

Abstract

Although a number of sPLA2 (secretory phospholipase A2) enzymes have been identified in mammals, the localization and functions of individual enzymes in human pathologic tissues still remain obscure. In the present study, we have examined the expression and function of sPLA2s in human lung-derived cells and in human lungs with pneumonia. Group IID, V and X sPLA2s were expressed in cultured human bronchial epithelial cells (BEAS-2B) and normal human pulmonary fibroblasts with distinct requirement for cytokines (interleukin-1b, tumour necrosis factor a and interferon-g). Lentivirus- or adenovirus-mediated transfection of various sPLA2s into BEAS-2B or normal human pulmonary fibroblast cells revealed that group V and X sPLA2s increased arachidonate release and prostaglandin production in both cell types, whereas group IIA and IID sPLA2s failed to do so. Immunohistochemistry of human lungs with pneumonia demonstrated that group V and X sPLA2s were widely expressed in the airway epithelium, interstitium and alveolar macrophages, in which group IID sPLA2 was also positive, whereas group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and group IIE and IIF sPLA2s were minimally detected. These results suggest that group V and X sPLA2s affect lung pathogenesis by facilitating arachidonate metabolism or possibly through other functions.

Figure 1. Expression of endogenous sPLA₂s and other PGE₂-biosynthetic enzymes in cultured human lung-derived cells

(A) Expression of sPLA₂s in BEAS-2B and NHPF with or without stimulation for 24 h with cytokines, as assessed by RT–PCR (30 cycles). (B) Expression of various PGE₂-biosynthetic enzymes in these cells with or without stimulation for the indicated periods with cytokines, as assessed by immunoblotting. (C) Production of PGE₂ in these cells with or without stimulation for the indicated periods with cytokines. Representative results of 3–4 independent experiments are shown.

Figure 2. AA metabolism in human bronchial epithelial BEAS-2B cells transfected with various PLA₂s by the lentivirus system

(A, B) Individual PLA₂s stably expressed in BEAS-2B cells by the lentivirus system were detected by Northern blotting (A) and immunoblotting (B). Dark arrows indicate the bands for individual sPLA₂s, and grey arrows may correspond to their unprocessed forms. Molecular masses are indicated in kDa. (C) Immunocytostaining of sPLA₂s expressed in BEAS-2B cells. Fluorescent signals for individual sPLA₂s were visualized with specific antibodies (×200 magnification) (panel a) or signals for GM130, a Golgi marker (×400 magnification) (panel b). In panel c, the sPLA₂-IIA signal was unaffected by treatment of the transfectants with 1 mg/ml heparin (×100 magnification). Parental cells exhibited minimal staining for sPLA₂s. Other sPLA₂s displayed similar staining patterns (results not shown). (D) sPLA₂ activities in the supernatant and membrane-associated fractions of the transfectants. In the latter fraction, sPLA₂s were solubilized by treatment of the cells with a medium containing 1 M NaCl. Values show the amounts of sPLA₂s estimated by comparison of the activities between the samples and the authentic sPLA₂ standards. (E) [³H]AA release by BEAS-2B cells stably transfected with individual PLA₂s. The cells were incubated for 4 h with or without TNFα in culture medium. (F) PGE₂ production by BEAS-2B cells stably transfected with PLA₂s in combination with either of the two COX isoenzymes. The cells were incubated for 4 h with TNFα in a medium containing 10% FCS. Inset: expression of COX-1 and -2 proteins in the transfectants, as assessed by immunoblotting. Representative results of 3–5 independent experiments are shown in (A–D), and values are means±S.E.M. for four independent experiments in (E, F).

Figure 3. AA metabolism in BEAS-2B cells transfected with various PLA₂s by the adenovirus system

(A) BEAS-2B cells stably expressing COX-2 were infected with adenoviruses for various PLA₂s or control (LacZ) [multiplicity of infection (MOI)=10] for 36 h and stimulated for 12 h with TNFα in culture medium, and the supernatants were then taken for PGE₂ assay. Values are means±S.E.M. for three independent experiments. *P<0.05 compared with the control. (B) Time-course study of adenovirus infection. BEAS-2B cells stably expressing COX-2 were stimulated for the indicated periods with TNFα in the presence of adenoviruses bearing various sPLA₂s. The supernatants and cells were harvested at each time point to quantify the levels of PGE₂ and sPLA₂ expression respectively. A representative result of three independent infection experiments is shown. Inset: expression of individual PLA₂s was assessed by Northern blotting.

Figure 4. AA metabolism by various PLA₂s in NHPF

(A, B) Individual sPLA₂s stably expressed in NHPF cells by the lentivirus system were detected by Northern blotting (A) and immunoblotting under non-reducing conditions (B). Cells infected with adenovirus for sPLA₂-IIF were also subjected to immunoblotting under both reducing [2-mercaptoethanol (+)] and non-reducing [2-mercaptoethanol (−)] conditions (B) MOI, multiplicity of infection. (C) Confocal laser microscopic analyses of sPLA₂s expressed in NHPF. Fluorescent signals for individual sPLA₂s stably transfected into NHPF by the lentivirus method were visualized with specific antibodies (×100 magnification) (panel a). Parental cells showed no appreciable staining (panel a). Magnified views (×400) of the staining of sPLA₂-IIA and -X are shown in panel b. sPLA₂-IIA was mainly located in the Golgi apparatus (arrows), which was verified by double immunostaining with the Golgi marker GM130 (results not shown), whereas sPLA₂-X was also distributed in the ER (light blue arrow). Signal for sPLA₂-IIA was detected equally in cells before and after treatment with 1 mg/ml heparin (×100) (panel c). Signal for sPLA₂-IIF, which was transfected into NHPF by the adenovirus method, was found in the Golgi as well as in the cytosolic aggregates (arrowheads, panel d). (D) sPLA₂ activities in the supernatant (S) and membrane-associated (C) fractions of the transfectants obtained by the lentivirus (for sPLA₂-IIA, -V and -X) or adenovirus (for sPLA₂-IIF) method. In the latter fraction, sPLA₂s were solubilized from cell surfaces by treatment with medium containing 1 M NaCl. (E) [³H]AA release from NHPF stably transfected with individual PLA₂s after incubation for 4 h in culture medium. Addition of cytokines did not augment [³H]AA release by sPLA₂s (results not shown). (F) PGE₂ production by NHPF stably transfected with PLA₂s after incubation for 4 h with or without various cytokines. (G) PGE₂ production in NHPF transfected with various PLA₂s by means of the adenovirus system. The cells were infected for 36 h with various doses of adenoviruses bearing PLA₂s or control (LacZ) and then stimulated for 12 h with IL-1β. Inset: expression of individual PLA₂s was assessed by Northern blotting. (H) Adenoviruses for WT (wild-type) sPLA₂-X, its catalytically inactive mutant (Gly³⁰→Ser) and control were infected (MOI=10) into NHPF, and PLA₂ activity in the supernatants (upper panel) and PGE₂ production after stimulation for 12 h with IL-1β (lower panel) were assessed. Inset: expressions of WT and mutant sPLA₂-X were verified by Northern blotting. Representative results of 3–5 independent experiments are shown in (A–D) and (H) and values are means±S.E.M. for four independent experiments in (E–G).

Figure 5. Immunohistochemical localization of sPLA₂s in human lungs affected by pneumonia

(A) Staining of sPLA₂-IIA. Bronchial chondrocytes (panels a and b, green arrow) and arterial VSMC (panels a and d, pink arrows) were positively stained, whereas alveolar macrophages (panel c, yellow arrows) and bronchial epithelial cells (panel a) were negative. Areas with no or mild inflammation showed no sPLA₂-IIA staining (panel e). (B) Staining of sPLA₂-V. Bronchial epithelial cells (panels a and b, red arrowheads), interstitial fibroblasts (panel b, light blue arrowhead) and alveolar macrophages (panel c, yellow arrowheads) were intensely positive, whereas arterial VSMC were not stained (panel a). Although sPLA₂-V staining was weak in areas with no or mild inflammation (panel d), scattered expression was found in bronchial epithelial cells (panel e). (C) Staining of sPLA₂-X. Alveolar epithelial cells, macrophages (panels a and b) and bronchial epithelial cells (panel c) were positively stained for sPLA₂-X. sPLA₂-X was also detected in the interstitium beneath the bronchial epithelium, but not in VSMC, in the control lungs (panel d). Scattered staining of sPLA₂-X was also evident in the alveolar epithelium of the control lungs (panel e). (D) Staining of sPLA₂-IID. Bronchial, but not alveolar, epithelial cells (panels a and b) as well as alveolar macrophages (panels b and c), were positively stained. Mildly inflamed lesions were not stained (panel d). No appreciable staining for sPLA₂-IIE and -IIF was observed (results not shown).