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1Department of Microbiology, Hanyang University College of Medicine, Korea.
2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Korea.
3Department of Parasitology, Yonsei University College of Medicine, Korea.
Received August 21, 1995; Accepted September 22, 1995.
Abstract
The protozoan parasite, Entamoeba histolytica, is one of major causative agents of intestinal disease all over the world. In acute experimental infection, the early host response to E. histolytica is characterized by an infiltration of neutrophils. However, the chemotactic signal for this response is not well known. Based on the finding that human epithelial cells produce the potent neutrophil chemoattractant and activator, interleukin-8 (IL-8), IL-8 gene expression was examined thoroughly in human colon epithelial cells exposed to E. histolytica trophozoites. Cellular RNAs were extracted from HT-29 or Caco-2 human colon epithelial cells exposed to E. histolytica trophozoites for 30 minutes, 1 and 3 hours. IL-8 mRNA transcripts were measured by reverse transcriptional polymerase chain reaction (RT-PCR) using synthetic standard RNA. The number of IL-8 mRNA molecules increased from 30 minutes to 3 hours of exposure period, reaching 3.1 × 107 molecules/µg of total RNA. Expression pattern of IL-8 mRNA transcripts was parallel to the amounts of IL-8 protein measured by enzyme-linked immunosorbent assay (ELISA). Lysates of E. histolytica also induced expression of mRNA for IL-8 in colon epithelial cells. These results suggest that acute inflammatory reaction by E. histolytica may be initially triggered by proinflammatory cytokines such as IL-8 secreted from epithelial cells of the colon.
Figures
Fig. 1 Quantitationof IL-8 mRNA using RT-PCR. Varying copy numbers of synthetic standard RNA were co-reverse transcribed with 1 µg of cellular RNA extracted from colon epithelial cells exposed to E. histolytica trophozoites. After reverse transcription, the cDNA reaction mixture was amplified using IL-8 specific primers. Electrophoresis of PCR products were done and the intensities of their band were quantified using imaging densitometor. At the equimolar point, the number of mRNA molecules for IL-8 could be calculated.
Fig. 2 IL-8 mRNA expression for numbers of HT-29 cel vs. numbers of exposed-E. histolytica. After HT-29 cells were exposed to E. histolytica for 3 hours, cellular RNAs were extracted by guanidinium thiocyanate-phenol-chloroform method. And IL-8 mRNA was observed by RT-PCR using specific primers. The (+) represents positive control for IL-8 and β-actin. The (-) represents negative control which was performed by omitting the RNA during cDNA synthesis and specific PCR amplification. The 107 shows the intensity for RT-PCR using 107 molecules of synthetic standard RNA instead of cellular RNA. The "ctrl" represents the band of RT-PCR products for edllular RNA extracted from HT-29 cells which did not expose to E. histolytica trophozoites. β-actin in included as a control since it is a constitutively expressed "housekeeping" gene.
Fig. 3 IL-8 mRNA profiles in HT-29 cells exposed to E. histolytica. After the colon epithelial cells, HT-29, were exposed to E. histolytica during 30 minutes, 1 and 3 hours, cellular RNAs were extracted by guanidinium thiocyanate-phenol-chloroform method. The ratio of HT-29 cells/E. histolytica was 20:1. IL-8 mRNA was observed by RT-PCR using specific primers. The (+) represents positive control for IL-8 and β-actin. The (-) represents negative control which was performed by omitting the RNA during cDNA synthesis and specific PCR amplification. The 107 shows the intensity for RT-PCR using 107 molecules of synthetic standard RNA instead of cellular RNA. The ctrl represents the band of RT-PCR products for cellular RNA extracted from HT-29 cells which did not expose to E. histolytica trophozoites. β-actin in included as a control since it is a constitutively expressed "housekeeping" gene.
Fig. 4 IL-8 mRNA expression in Caco-2 cells treated with lysates of E. histolytica. After Caco-2 cells were treated with lysates of E. histolytica trophozoites for 3 hours, cellular RNAs were extracted by guanidinium thiocyanate-phenol-chloroform method. And IL-8 mRNA was observed by RT-PCR using specific primers. The (+) represents positive control for IL-8 and β-actin. The (-) represents negative control which was performed by omitting the RNA during cDNA synthesis and specific PCR amplification. The 107 shows the intensity for RT-PCR using 107 molecules of synthetic standard RNA instead of cellular RNA. The "ctrl" represents the band of RT-PCR products for cellular RNA extracted from Caco-2 cells which did not treated with lysates of E. histolytica trophozoites. β-actin included as a control since it is a constitutively expressed "housekeeping" gene.
Fig. 5 Amounts of IL-8 proteins induced by HT-29 cells exposed to E. histolytica. After the HT-29 cells were exposed to (■) and not to (□) E. histolytica trophozoites for 30 minutes, 1 and 3 hours, culture supernatants were obtained. The ratio of HT-29 cells/E. histolytica was 20:1. The IL-8 levels were determined by ELISA. Results are expressed as mean and standard error. Each panel represents the mean of separate three experiments.
Tables
Table 1 Quantitation of IL-8 mRNA expressed in HT-29 cells exposed to E. histolytica
Table 2 Quantitation of IL-8 mRNA expression for numbers of HT-29 cells vs. numbers of exposed E. histolytica
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