Paired immunoglobin-like receptors A and B are new targets for inducing dendritic cells tolerance in mice.
The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with the recombinant murine interleukin-10 (IL-10) and recombinant human transforming growth factor beta1 (TGF-beta1) respectively to develop the T-DC and stimulated with lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small interfering RNAs (siRNA) molecule for PIR-B was chemically synthesized and transfected into DC2.4 cells (Si-DC) by lip2000. The expression of PIRs on DC2.4 cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and Western blot. Realtime reverse transcriptase-polymerase chain reaction (Realtime-PCR) was applied for measurement of PIR-A and CD80, CD86, MHC-II mRNA expression. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR) using (3)H-thymidine incorporation test. The concentration of IFN-gamma in supernatants of MLR from distinct groups was analyzed by ELISA. The results showed that PIR positive rate was (28.65+/-8.12)% examined by FCM on DC2.4 cells. PIR positive rate was increased dramatically to (54.21+/-6.34)%, (58.78+/-4.70)%, (48.24+/-6.75)% respectively for IL-10, TGF-beta1 and LPS induction (P<0.01), but there was no significantly different among the three groups (P>0.01). The semi-quantitative RT-PCR and Western blot revealed that IL-10 and TGF-beta1 induced the higher PIR-B level and lower PIR-A level. On the contrary, the LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. Realtime PCR examination demonstrated that PIR-A and co-stimulating molecules such as CD80, CD86 and MHC-II were increased significantly after stimulation with LPS. Compared with the DC2.4 cells and the LPS-DC, the T-DCs inhibited alloactivated T cell proliferation and down-regulated the IFN-gamma secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P<0.01) and enhanced the IFN-gamma secretion (P<0.01). It was concluded that up-regulating the PIR-B and down-regulating the PIR-A expression were the general feature of phenotype and constructed the new targets for dendritic cells to acquire immune tolerance in mice. Overexpression of PIR-B can inhibit the up-regulation of the PIR-A, CD80, CD86 and MHC-II expression, which might be the molecular mechanism for the T-DC.
Liu
Zhengrong
Z
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. liuzhengrong618@yahoo.com.cn
Li
Weiming
W
Zhang
Min
M
Zhou
Hao
H
Han
Hong
H
Zou
Ping
P
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000494:pharmacology
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1; D016753:Interleukin-10