Method: Mouse (WT (C57/Bl6) or α7nAchR-/-) or human neutrophils (PMNs) were incubated with C5a (100nM) in a presence or absence of cholinergic agonists, GTS21 (specific α7nAChR agonist) (1μM) or control medium. TNFα concentrations were analyzed by ELISA. IkB phosphorylation was detected by western blot. Peritoneal reverse passive Arthus reaction was initiated by injecting OVA i.v. and anti-OVA IgG i.p. After 1.5hrs PMN recruitment in peritoneal lavage fluid was assessed by FACS.
Result: Compared to WT mice, naïve α7nAchR-/- mice had a greater number of neutrophils, macrophages and mast cells in peritoneal cavity (0.58±0.2x104 vs. 7.4±4.1x104 p<0.05; 96±5 x104 vs 144±13 x104 p<0.05; 35±5x104 vs 58±8x104) with later two expressing higher levels of C5aR and FcγR on their surface, although bone marrow granulopoesis was comparable in both strains. Bone marrow derived α7-/- macrophages spontaneously produced higher TNFα in culture medium than WT macrophages (33±6 vs 82±23 pg/ml) and TNFα levels were higher in the peritoneal cavity of naïve α7nAchR-/- animals (120±20 vs 197±43 pg/ml). When α7nAchR-/- mice were challenged with the reverse passive Arthus reaction, early neutrophil accumulation was enhanced to approximately 245% of wild type levels (51±12x106 vs. 124±27x106cell/ml; p=0.024) and TNFα in peritoneal lavage fluid was also markedly increased (243±60 vs. 439±96 pg/ml; p=0.023). To examine the basis of the suppressive effect of ligation nAchR, we stimulated PMNs with C5a in the presence and absence of specific α7nAChR agonist, GTS21. C5a mediated NFkB activation was markedly decreased by GTS21 in association with blockade of phosphorylation of its inhibitor IKB; GST21 did not alter phosphorylation of IKB in unstimulated cells.
Conclusion: These results indicate that α7nAchR expressed on innate immune cells play critical role in suppressing inflammation at sites of immune complex deposition and suggest that tonic stimulation of α7nAchR attenuates the FcγR- and C5aR-mediated activation of the NFkB-proinflammatory cytokine axis, a pathway that activates resident and newly recruited leukocytes. Our findings have implications for tissue injury in SLE and RA and suggest new strategies to reduce damage at the sites of immune complex deposition.
Disclosure: M. Vukelic, None; G. Koo, None; P. M. Redecha, None; J. E. Salmon, None.