E. Coli Heat-labile Enterotoxin-induced Cytokine Production in Mice
|Shanghai Ocean University,
|Clinical Veterinary Medicine
|Escherichia coli heat-labile enterotoxin (LT) cytokines real-timefluorescence quantitative PCR macrophages Luminex flow cytometry
Escherichia coli heat-labile enterotoxin (LT) of enterotoxigenic E. coli (ETEC) is atoxic factor, which can cause diarrhea to human and animals. Furthermore it is one ofthe most powerful mucosal immune adjuvant and a strong immunomodulatory effects,in stimulating a variety of cytokines. However because of its strong intestinal toxicity, itcan not be used as adjuvant. So many nontoxic or attenuated mutants protein werestudied in order to make this adjuvant available in use. Among the mutant proteins,LTRG has much lower toxicity than the wild type LT and showed good mucosaladjuvant. In this study, the recombinant protein LTRG was used to immunize the mice,or to stimulate murine macrophages. The mRNA levels of cytokines, including IL-1β,IL-12, IFN-γ, TNF-α, IL-4, IL-6were detected by using quantitative PCR. Furthermorecytokines production in the supernant of cells were detected using Luminex xMAP technology and T lymphocytes changes in immunized mice were analyzed using flowcytometry.Different doses of LTRG protein were selected in stimulating the mousemacrophages in vitro and1ug/ml LTRG protein have the best stimulation and induce thehigh level IL-1β and IL-4mRNA transcription. Detection of macrophage cytokinemRNA showed IL-1β, TNF-α and IL-6mRNA of LT stimulation reached the highestlevel and show significant difference (p<0.01) with the control group at2h,4h and12hpost stimulation. IL-12, IFN-γ, IL-4mRNA of LT immunization reached the highestlevel and show significant difference (p<0.01) with the control24h post stimulation.LTRG stimulated macrophage cytokine mRNA reached highest level a bit delaycomparing with LT treatment cells, but showed higher mRNA level of six cytokines.In order to know the adjuvant activity of LTRG protein, LTRG was co-stimulatedto mice macrophages with BSA and the cytokine mRNA were also detected. The resultsshowed TNF-α and IL-6mRNA were the highest and showed significant difference(p<0.01) at8h and24h. While other cytokine mRNAs in LTRG+BSA treated cellsreached the highest level and show significant difference (p<0.01) at12h. Cytokines production in macrophages were detected by Luminex xMAP technology. TheTNF-α and IL-6concentration of LTRG treated reached the highest level comparingwith the PBS control and showed significant difference (p<0.01) at36h and48h; TNF-αand IL-6concentration of LTRG+BSA reached the highest level and showed significantdifference (p<0.01) at24h comparing with the BSA control. These results showed thatLTRG can activate murine macrophage inflammatory response, which means LTRG canenhance the antigen-presenting cell function and induce both humoral and cellularimmune responses.The mRNA of cytokines produced in mice spleen immunized with LT or LTRGprotein were also detected in different times. IL-1β, IL-12, IFN-γ, TNF-α, IL-4, IL-6mRNA were in the highest level at48h or60h p.i. and show significant difference(p<0.01) with PBS control. The results showed that the LT and LTRG protein caninduce transcription of cytokines mRNA. Furthermore CD4+and CD8+positive Tlymphocytes in spleens of immunized mice were detected using flow cytometrytechnique. And LT and LTRG protein can both greatly enhance the percentage of spleenCD4+and CD8+T lymphocyte. This indicated that LT and LTRG can enhance the Tlymphocyte immune activity.In summary, mRNA and cytokine secretion in the cell and mice stimulated with LTor LTRG indicated that LT and LTRG can enhance transcription and synthesis ofcytokines IL-1β, IL-12, IFN-γ, TNF-α, IL-4, IL-6, which were driven by Th1and Th2cells. Furthermore, CD4+and CD8+were also increased which means LT and LTRG canincrease T lymphocyte nonspecific immune activity. However LTRG can enhance theimmune reaction similar with that with LT protein, but the peak of cytokinetranscription was delayed comparing with that of LT protein.