Paired immunoglobin-like receptors A and B are new targets for inducing dendritic cells tolerance in mice

Abstract

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 β1 (TGF-β1) 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 ³H-thymidine incorporation test. The concentration of IFN-γ 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-β1 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-β1 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 alloactived T cell proliferation and down-regulated the IFN-γ secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P<0.01) and enhanced the IFN-γ 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.

Keywords

dendritic cell / tolerance / receptor / paired immunoglobin-like / siRNA

References

[1] GadM., ClaessonM. H., PersenA. E.. Dendritic cells in peripheral tolerance and immunity. APMIS, 2003, 111(7–8): 766-775

[2] SatoK., YamashitaN., BabaM., et al.. Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells. Blood, 2003, 101(9): 3581-3589

[3] SatoK., YamashitaN., YamashitaN., et al.. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse. Immunity, 2003, 18(3): 367-379

[4] HiranoA., LukeP. P., SpechtS. M., et al.. Graft hyporeactivity induced by immature donor-derived dendritic cells. Transpl Immunol, 2000, 8(3): 161-168

[5] BellinghausenI., BrandU., SteinbrinkK., et al.. Inhibition of human allergic T-cell responses by IL-10-treated dendritic cells: differences from hydrocortisone-treated dendritic cells. J Allergy Clin Immuno, 2001, 108: 242-249

[6] KubagawaH., BurrowsP. D., CooperM. D.. A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells. Proc Natl Acad Sci U S A, 1997, 94(10): 5261-5266

[7] ManavalanJ. S., RossiP. C., VladG., et al.. High expression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells. Transpl Immunol, 2003, 11(3–4): 245-258

[8] BanchereauJ., BriereF., CauxC., et al.. Immunobiology of dendritic cells. Annu Rev Immunol, 2000, 18: 767-811

[9] RutellaS., DaneseS., LeoneG.. Tolerogenic dendritic cells: Cytokine modulation comes of age. Blood, 2006, 108(5): 1435-1440

[10] ChangC. C., CiubotariuR., ManavalanJ. S., et al.. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat Immunol, 2002, 3(3): 237-243

[11] Suciu-FocaC. N., PiazzaF., HoE., et al.. Distinct mRNA microarray profiles of tolerogenic dendritic cells. Hum Immunol, 2001, 62(10): 1065-1072

[12] MaedaA., KurosakiM., KurosakiT.. Paired immunoglobulin-like receptor (PIR)-A is involved in activating mast cells through its association with Fc receptor gamma chain. J Exp Med, 1998, 188(5): 991-995

[13] YamashitaY., OnoM., TakaiT.. Inhibitory and stimulatory functions of paired Ig-like receptor (PIR) family in RBL-2H3 cells. J Immunol, 1998, 161(8): 4042-4047

[14] TakaiT.. A novel recognition system for MHC class I molecules constituted by PIR. Adv Immunol, 2005, 88: 161-192

[15] NakamuraA., KobayashiE., TakaiT.. Exacerbated graft-versus-host disease in Pirb-/-mice. Nat Immunol, 2004, 5(6): 623-629

[16] PereiraS., ZhangH., TakaiT., et al.. The inhibitory receptor PIR-B negatively regulates neutrophil and macrophage integrin signaling. J Immunol, 2004, 173(9): 5757-5765

[17] TakaiT.. Paired immunoglobulin-like receptors and their MHC class I recognition. Immunology, 2005, 115(4): 433-440

[18] OkadaN., SaitoT., MasunagaY., et al.. Efficient antigen gene transduction using Arg2Gly2Asp fiber-mutant adenovirus vectors can potentiate antitumor vaccine efficacy and maturation of murine dendritic cells. Cancer Res, 2001, 61(21): 7913-7919

[19] NiH. T., SpellmanS. R., JeanW. C., et al.. Immunization with dendritic cells pulsed with tumor extract increases survival of mice bearing intracranial gliomas. J Neurooncol, 2001, 51(1): 1-9