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. 1999 Dec;155(6):1977-84.
doi: 10.1016/S0002-9440(10)65516-2.

Basic Fibroblast Growth Factor Synthesis by Human Peritoneal Mesothelial Cells: Induction by interleukin-1

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Free PMC article

Basic Fibroblast Growth Factor Synthesis by Human Peritoneal Mesothelial Cells: Induction by interleukin-1

M V Cronauer et al. Am J Pathol. .
Free PMC article

Abstract

Peritoneal mesothelial cells are uniquely located to regulate cellular events in the peritoneal cavity and are an important source for various cytokines and growth factors. This study was conducted to analyze the capacity of human peritoneal mesothelial cells (HPMCs) to synthesize and release basic fibroblast growth factor (bFGF) and to characterize its regulation by inflammatory cytokines. HPMCs constitutively synthesized and released considerable amounts of bFGF as detected by a specific immunoassay. Almost 80% of bFGF (1547 +/- 173 pg/10(5) cells) was localized intracellularly. Approximately 20% of the bFGF (357 +/- 27 pg/10(5) cells) was associated with extracellular matrix components on the HPMC surface. Small amounts of bFGF (<1%) were detectable in tissue culture supernatants (8.4 +/- 1.4 pg/10(5) cells). Treatment of HPMCs with interleukin-1beta (IL-1beta; 1 ng/ml) resulted in a significant increase in bFGF production. The intracellular bFGF content showed a rapid but only transient increase, which was significant above background levels after 24 hours (41% increase; P < 0.05). This increase in intracellular bFGF concentration was associated with an induction of the release of bFGF. Within 96 hours, the release of bFGF to the cell surface and into the supernatant increased by 58% (564 +/- 52.4 pg/10(5) cells; P < 0.01) and by 214% (26.4 +/- 3.2 pg/10(5) cells; P < 0.001), respectively. Neither tumor necrosis factor-alpha nor interferon-gamma affected bFGF synthesis by HPMCs. Stimulation of HPMCs with IL-1beta increased steady-state levels of bFGF-specific mRNA. Immunohistochemical analyses of peritoneal tissue revealed constitutive expression of bFGF by HPMCs. This in situ expression proved to be most pronounced in areas of serosal inflammation in activated HPMCs. Our study demonstrates that HPMCs synthesize and release significant amounts of bFGF and that the expression of this growth factor is significantly up-regulated by the proinflammatory cytokine IL-1beta. The data support the view that HPMCs are key regulators of abdominal disease processes such as peritonitis, peritoneal fibrosis, or peritoneal tumor metastasis.

Figures

Figure 1.
Figure 1.
Cellular content and release of bFGF by HPMCs. Cells were treated for 96 hours with the proinflammatory cytokines IL-1β, TNF-α, or IFN-γ (1 ng/ml each). bFGF was measured in duplicate by ELISA in the supernatants (Supernatant), the trypsin-releasable fraction (Pericellular), and cellular extracts (Intracellular). Results are the mean of bFGF expressed as pg/10 cells, from HPMCs cultured from five separate donors. *** P < 0.001, ** P < 0.01
Figure 2.
Figure 2.
Detection of bFGF-specific transcripts by RT-PCR. Total RNA was isolated from HPMCs from three separate donors, reverse transcribed to cDNA, and specific cDNA fragments of the bFGF and the β2-microglobulin cDNAs were amplified in a duplex PCR, using fluorescence-labeled primers as described in Material and Methods. The products were separated on a sequencing gel by means of the ABI 370A sequencer and quantified by using ABI GeneScan software. The figure presents a black and white picture of the virtual gel picture obtained with ABI GeneScan software, showing the bFGF and the β2-microglobulin fragments amplified from three samples. All PCR reactions were performed in duplicate.
Figure 3.
Figure 3.
Quantification of bFGF mRNA expression in HPMC treated with IL-1β. HPMCs were treated with IL-1β for 3, 12, and 24 hours, respectively. bFGF-specific mRNA levels were determined quantitatively as described in Material and Methods in relation to the housekeeping gene β2-microglobulin. Results are expressed as bFGF/β2-microglobulin ratios and represent the mean values of mesothelial cultures derived from three separate donors.
Figure 4.
Figure 4.
Immunohistochemical detection of bFGF in peritoneal tissue. A-C: Normal peritoneal tissue covered by a flat layer of mesothelial cells. A: The cells are identified by their expression of the cytokeratin subtypes 8 and 18. B: Section stained with H&E. C: Serial section next to section in B stained with a polyclonal antibody against bFGF. HPMC showed a weak expression of bFGF. Weak positivity was also noticed in capillary endothelial cells of the submesothelial tissue. D-F: Acute peritoneal inflammation. D: HPMCs are infiltrated by polymorphonuclear cells and covered by an exudate of fibrin. E: The cells appear activated with enlarged cytoplasm and are highlighted by staining for cytokeratin subtypes 8 and 18. F: Serial section next to section in E revealed increased expression of bFGF in activated HPMCs. Granulocytes were negative. Only weak positivity was noticed in the submesothelial tissue. Original magnification, ×400.

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