Platelet-activating factor receptor mRNA is localized in eosinophils and epithelial cells in rat small intestine: regulation by dexamethasone and gut flora

Abstract

Platelet-activating factor (PAF) is a potent mediator involved in bowel injury. We investigated PAF receptor transcription and its mRNA localization in the small intestine of normal (conventionally fed) and germ-free rats, by competitive polymerase chain reaction (PCR) and in situ hybridization. A dose of PAF (1.5 microg/kg, i.v.) insufficient to cause gross bowel injury was injected into rats. Some rats were pretreated with dexamethasone (1 mg/kg). We found: (1) PAF receptor (PAF-R) mRNA localized predominantly in lamina propria eosinophils and in epithelial cells; (2) PAF increased PAF-receptor signals in the epithelial cells; (3) Dexamethasone depleted eosinophils in the intestine and markedly decreased PAF-receptor transcripts; the response to PAF was also weaker than control rats; (4) Germ-free rats had less PAF-R mRNA than normal rats, and showed a weaker response to PAF than conventionally fed rats. Thus, we conclude: (1) PAF receptor mRNA is constitutively expressed in the epithelium and in lamina propria eosinophils in the intestine. (2) PAF-R transcription is up-regulated by PAF and gut flora, mostly in the epithelium. (3) PAF-R transcription is down-regulated by glucocorticoids, mainly as a result of eosinophil depletion. These results suggest a functional role for PAF receptors both in host defence and the inflammatory response in the small intestine.

Figure 2

PAF up-regulates PAF-R transcripts in intestinal epithelial cells. (a) Total RNA (0·5 μg) extracted from isolated small intestinal epithelial cells was coamplified by RT-PCR in the presence of 4·16 × 10⁷ (lane 1), 2·08 × 10⁷ (lane 2), 1·04 × 10⁷ (lane 3) and 0·52 × 10⁷ (lane 4) molecules of competitor RNA. After EcoRI digestion, the competitor PCR products were cut into two fragments (380 bp and 205 bp), whereas the target PCR products remain to be 585 bp. Both competitor and target DNA were analyzed by 1·2% agarose-gel electrophoresis followed by staining with SYBR Green I. Lane M, marker of 100 bp ladder. Normal: normal conventionally fed rats, no treatment. PAF: 90 min after PAF (1·5 μg/kg, i.v.) treatment; Sham: sham-operated, 90 min. (b) Standard curve plotting log of (product of target RNA/product of cRNA) ratio against log of known cRNA standard (added to the reaction mixture). The concentration of PAF-R mRNA in each sample was calculated by extrapolating from the intersection of its own standard curve (where the amounts of the target and the competitor are equal, or log₁₀ = 0) to the x-axis (log₁₀cRNA(molecules).). (▴) normal; (○) sham 90 min; (•) PAF 90 min. (c) PAF-R mRNA in isolated epithelial cells. Results are mean±SEM (n = 3).

Figure 3

Up-regulation of PAF-R transcripts in rat small intestine following PAF injection. (a) animals killed at time 0. (b) animals killed 90 min after PAF (1·5 μg/kg, i.v.). Lane M, marker of 100 bp ladder. S, sham-operated; P, PAF only; D, rats pretreated with dexamethasone (1 mg/kg, i.p., 24 hr before the experiment); D+P, dexamethasone-pretreated, injected with PAF; G, sham-operated, germ-free rats; G+P, germ-free rats injected with PAF. See Fig. 2(a) legend for methods and symbols. (c) PAF (1·5 μg/kg, i.v.) was injected at time 0. Animals were killed at different time points for PAF-R mRNA quantification. (•) PAF only. (▵) Dexamethasone (1 mg/kg, i.p., at −24 hr) followed by PAF injection. (▪) Germ-free rats injected with PAF. (□) Sham-operated, germ-free rats. (○) Sham-operated, conventionally fed rats. n = 4 or 5 for each point.

Figure 1

Expression of PAF receptor transcripts in eosinophils of lamina propria and in epithelial cells in rat small intestine. In situ hybridization was performed with [³⁵S]-labelled PAF-R antisense RNA probe (a–g) and sense RNA probe control (h). Rats were killed at 90 min (c–f) or 30 min (g, h) after PAF (1·5 μg/kg, i.v.) injection: (a) (light field, H & E) and (b) (dark field) are from normal, conventionally fed rats; (c) (dark field) and (d) (dark field) from PAF-injected rats; (e) (light field) and (f) (dark field) from PAF-injected, dexamethasone (1 mg/kg, i.p.) pretreated (24 hr before PAF) rats; (g) (light field, antisense) and (h) (light field, sense control) from PAF-injected, germ-free rats. Magnification: × 210 (a, b, c, g and h) and × 110 (d, e and f). H & E stain (a, b) or eosin stain (c–h). The H & E stain in (a) highlights the eosinophils (stained red, arrows) within the lamina propria. The same slide is used for both light and dark field microscopy. Note that (a) and (b) show the same area, and the location of the positive cells in (b) matches exactly the eosinophils in (a). The amount of PAF-R transcripts in epithelial cells in control animals is low (a, b). In contrast, rats injected with PAF showed a more intense positivity of PAF-R mRNA, brought out especially by dark field examination (c, d,and f). (e, f) (Depicting the same area on the slide) show that dexamethasone treatment resulted in the disappearance of both eosinophils (e) and PAF-R transcript-containing cells (f) in the lamina propria. However, the PAF-R transcripts are still abundant in the epithelial cells located at the periphery of villi (f). (h) (Sense probe) shows that the background (black grains) in eosinophils (arrows) is very low.

Figure 4

Change of mean arterial pressure (top panel), peripheral WBC count (middle panel) and haematocrit (lower panel) after administration of PAF. PAF (1·5 μg/kg, i.v.) was injected at time 0. See Fig. 3(c) legend for symbols. Results are mean±SEM (n = 4–6 for each point). Post hoc tests of analysis of variance results is performed between sham and PAF groups and between the conventionally fed group receiving PAF and the groups of germ-free and dexamethasone-pretreated rats receiving PAF. Significant differences (P < 0·05) are found between the following groups. Upper panel (blood pressure): between sham and PAF groups at 5, 15, and 30 min; between conventionally fed rats receiving PAF and germ-free rats receiving PAF at 15 and 30 min; and between conventionally fed rats receiving PAF and dexamethasone-pretreated groups at 15 and 30 min. Middle panel (WBC count): between germ-free control and germ-free receiving PAF groups at 30 min. Lower panel (Hct): between control and PAF groups at 30 and 90 min; between conventionally fed rats receiving PAF and germ-free rats receiving PAF at 30 and 90 min.