The Responses Of Monocytes/Macrophages To LPS And CpG Oligodeoxynucleotides And Their Related Mechanisms

Part I: Identification of a Novel RGS Molecule and Its TuningEffects on Cytokine Production by Lipopolysaccharide TreatedTHP-1 MonocytesRegulator of G-protein signaling (RGS) proteins are GTPase activating proteins thatinhibit signaling in various cellular responses controlled by heterotrimeric G proteins. Here wereport a novel RGS molecule cloned from human dendritic cells (DCs), designated DC-RGS,and its role in regulating the responsiveness of monocytes to lipopolysaccharide (LPS) in vitro.We show that DC-RGS is widely distributed and mainly expressed in myeloid cells. Inhibitionof DC-RGS expression in THP-l cells by antisense technique differentially sensitizedLPS-induced production of tumor necrosis factor (TNF)-ct, interleukin (IL)-1 j3, IL-6, IL-8 and11-12. Moreover, inhibition of DC-RGS expression partially restored secretion of TNF-ct and11-6by LPS desensitized TFW-1 cells. Overexpression of DC-RGS in THP-1 cells did notsignificantly affect cytokine production compared with mock control. Thus, DC-RGS, a novelRGS cloned from DCs, plays a fine-timing role in LPS responses and participates in regulatingresponsiveness of monocytes to LPS. We proposed that RGS proteins together with G proteinsmight participate in innate immunity and subsequently affect the adaptive immune responses.Part II: Evidence for Activation-independent RepressionMechanism of PPARy in LPS and CpG ODN Responses: Relevanceto DesensitizationLipopolysaccharide (LPS) desensitization or endotoxin tolerance, a state of hypo-responsiveness to LPS induced by pretreatment of low dose of LPS, is characterized bydecreased proinflammatory factors production in response to secondary LPS challenge even in10high dose. Here we showedoligodeoxynucleotides containing unmethylated CpG motif (CpGODN), similar to LPS, could also resulted in desensitization as evidenced from reduced nitricoxide (NO) and cytokine IL-12p40 production by murine macrophage Raw264.7 cells. LPSand CpG ODN could cross-desensitize each other to induce NO and IL-12p40 production butCpG ODN was more potential. Interestingly, 1-8 production by THIP-l human monocytescouldn't be desensitized by LPS or/and CpG ODN. We then tested the hypothesis thatperoxisome proliferator-activated receptor (PPAR) a and PPARy, ligand-dependent nuclearreceptors that have been implicated in negative-modulating macrophage cell activation, mightbe involved in desensitization state induction. We showed that pretreatment with ligands ofPPARcC (agonist Wy-14643) and PPARy (agonist 15-d-PGJ2 and antagonist BADGE), but notwith PPARy agonist pioglitazone, reduced LPS or CpG ODN-induced NO and IL-12p4Oproduction. Further, enhanced expression of PPARQ or PPARy (including a dominant-negativePPARy mutant) by transient transfection, mimics desensitization induction, attenuated LPS orCpG ODN-induced NO and ]IL-l2p4O production, but did not affect IL-S production. Westernblot analysis showed that LPS or CpG ODN treatment resulted in altered kinetics of PPAR7and NF-icB p50 subunit expression while no alteration of PPARcL expression in THP-1 cells.LPS or CpG ODN pretreatment significantly increased expression of PPART. Thus we suggestthat PPARy might participate in signaling and desensitization induced by LPS and CpG ODNin activation-independent manner and constitutively.Part Ill: Modulating Effects of B21-2, a Monoclonal AntibodyAgainst MHC class II molecules, on Danger Signals-Induced NOProduction by Antigen-Presenting CellsMajor histocompatibilty complex class II molecules (M1HC II) are efficiently expressedby antigen-presenting cells (APCs), including macrophages, dendritic cells (DCs) and B cells.Macrophages and DCs could be induced to produce nitric oxide (NO) by danger signals, suchas bacterial endotoxin lipopolysaccharide (LPS)...