The scavenger receptor, cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester

Abstract

Gp-340 is a glycoprotein belonging to the scavenger receptor cysteine rich (SRCR) group B family. It binds to host immune components such as lung surfactant protein D (SP-D). Recent studies found that gp-340 interacts directly with pathogenic microorganisms and induces their aggregation, suggesting its involvement in innate immunity. In order to investigate further its potential immune functions in the appropriate cell lines, the expression of gp-340 in four conventional immune cell lines (U937, HL60, Jurkat, Raji), and two innate immune-related epithelial cell lines (A549 derived from lung and AGS from stomach), was examined by RT-PCR and immunohistochemistry. The resting immune cell lines showed weak or no gp-340 mRNA expression; while the two epithelial cell lines expressed gp-340 at much higher level, which was differentially regulated by phorbol myristate acetate (PMA) treatment. In the A549 cells, gp-340 was up-regulated along with the PMA-induced proinflammatory expression of both IL-6 and IL-8. In AGS cells, PMA down-regulation of gp-340 was seen in parallel with an up-regulation of the two mature gastric epithelial specific proteins TFF1 (trefoil factor 1) and TFF2, which are implicated as markers of terminal differentiation. Analysis of the distribution of gp-340, together with the TFFs and SP-D in normal lung and gastric mucosa, supported further our in vitro data. We conclude that the differential regulation of gp-340 in the two epithelial cell lines by PMA indicates that gp-340 s involvement in mucosal defence and growth of epithelial cells may vary at different body locations and during different stages of epithelial differentiation.

Fig. 1

RT-PCR analysis of gp-340 mRNA expression in different cell lines (a) and the levels of the gp-340 transcripts in the gastric epithelial cell line AGS and pulmonary epithelial cell line A549 (b). A transferrin receptor (TfR) specific product from the cDNAs of all the cells was used as an internal control. The results shown are representatives of three independent experiments.

Fig. 2

Analysis of protein synthesis for gp-340, TFF1, TFF2 and SP-D using immunohistochemistry (a, b and c) and flow cytometry (d), in unstimulated and PMA stimulated (100 ng/ml) AGS (a) and A549 cells (b): i and v, gp-340; ii and vi, TFF1 (a) or SP-D (b); iii and vii, TFF2 (a) or TFF1 (b); iv and viii, control staining using isotype monoclonal antibody Hyb99–1 (IgG1) to substitute the specific antibodies at the same concentration. (c) Intracellular distribution of gp-340 in AGS cells (i and ii) and A549 cells (iii and iv). Original magnifications: (a) and (b) ×200; (c) ×1000. The results displayed represent two independent experiments. (d) FACS analysis demonstrates the PMA effect on the percentage of cells gated positive (%Gp) for SP-D in A549 cells and for TFF1 in AGS cells. The histograms in red represent background staining using control antibodies, as explained in Materials and methods. Data are representative of three independent experiments.

Fig. 3

RT-PCR analysis of mRNA expression of the molecules gp-340, SP-D, TFF1, TFF2, IL-6 and IL-8 in epithelial cell lines A549 (a) and/or AGS (b). RNA templates were extracted from unstimulated and PMA stimulated A549 and AGS cells, converted to cDNAs using oligo (dT)₁₅ primers. Molecules of interest were amplified using specific primer pairs, as described in Table 1. TfR-specific product was used as an internal control.

Fig. 4

Immunohistochemical localization of gp-340, TFF1, TFF2 and SP-D in normal human lung and/or gastric mucosa. Tissue sections were processed and stained as described in Materials and Methods. Images from high magnification (×400) show the specific expression of gp-340 (a) and SP-D (b) in type II cells but absent in type I cells in epithelial cells lining around alveolar sac. Expression of TFF1 was missing in normal human distal lung (c). Images from low magnification (×50) demonstrate the complete thickness of the epithelial layer of the gastric mucosa, and strong granular signals of gp-340 (d) dominantly in the neck region, but barely detectable toward luminal and bottom side; TFF1 (e) and TFF2 (f) were found primarily in the superficial mucin-producing epithelial cells. The profiles represent staining results from at least three different normal tissue samples.

Fig. 5

Phase contrast microscopy showing the effects of PMA on the morphology of AGS and A549 cells. AGS cells (a) and A549 cells (d) before PMA addition. Morphological changes were observed in most AGS cells after PMA stimulation for 6 h, with asymmetric shape and elongated cytoplasm extending toward one end of the cells (b). The same type of change was not observed in A549 cells, which maintained an even distribution of cytoplasm with stretching edges toward all directions (e). 24 h after PMA addition, the morphology of AGS cells returned to normal (c); A549 cells obtained well-defined borders along cellular junctions in confluence (f). Original magnification, × 200. The images were identical in five independent experiments.

Fig. 6

Immunohistochemical analysis of the localizations for gp-340, and a proliferation marker Ki-67 illustrated epithelial proliferation zone, in the gastric mucosa. Serial sections of normal human gastric mucosa were stained with monoclonal antibodies Hyb213–6 (a and c), and monoclonal antibody Ki-67 (b and d). Original magnifications: (a) and (b) ×100; (c) and (d) × 200.