Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
A Novel CR3 Agonist Attenuates Pro-Inflammatory Signaling in Subjects with Common and Variant ITGAM Polymorphism, rs1143679.
Jain1, Manish, Amato1, Michael, Buyon1, Jill P., Gupta2, Vineet, Clancy1, Robert M.
The mechanism underlying initiation and perpetuation of inflammationwith eventual end organ injuryin Systemic Lupus Erythematosus (SLE) is not yet defined. Equally unclear are mechanisms to attenuate such inflammation. An important candidate for initiation and perpetuation of the inflammatory response may be the chronic stimulation of resident leukocytes through toll like receptors (TLR). Complement Receptor 3 (CR3), a heterodimeric receptor on the surface of various types of leucocytes, is known to decrease pro-inflammatory signals by dendritic cells when ligated to iC3b. The goal of this study was to evaluate whether a TLR-mediated pro-inflammatory stimulus is attenuated by a novel iC3b mimetic specific for CR3known as LA1on other cells expressing CR3: macrophages and neutrophils. ITGAM polymorphism rs1143679, encoding for a non-conserved R77H substitution in the CD11b alpha chain of CR3, is known to be associated with SLE across various ethnic groups. While the polymorphism is theorized to affect ligand binding to CR3, its functional significance is unknown. A sub-goal was to evaluate if rs1143679 carrier status attenuated the effect of LA1.
The effect of LA1 on basal and stimulated responses by macrophages and neutrophils of human subjects (twenty healthy donors) was evaluated. Rs1143679 carrier status of subjects was determined by allelic discrimination. Macrophages derived from CD14+ monocytes of healthy human donors were treated with R848 (a specific TLR7 ligand, 1 uM), with and without LA1 (a recently described CR3 agonist, 15 uM). Quantification of TNF-a secretion, the readout of TLR7 activation, was assessed by ELISA. Neutrophils derived from healthy subjects were evaluated for superoxide anion production after stimulation with FMLP (.1 uM) in the absence and presence of LA1 (15 uM). Neutrophils were also evaluated for adhesion to a fibrinogen-coated surface in the presence of LA1.
Treatment of macrophages with R848 significantly stimulated TNF-a release compared with macrophages alone (1265 ± 297 pg/ml versus 26 ± 30 pg/ml, respectively, p = 0.006, n = 7). Pre-exposure to LA1 followed by treatment with R848 impaired TNF-a secretion from macrophages (R848 + LA1: 700 ± 249 pg/ml, p=0.015). Exposure of macrophages to LA1 in absence of R848 had no effect on cellular morphology, and did not induce TNF-a over 24 hrs. The % inhibition by LA1 of TNF-a in rs1143679 non-carriers was 63% (n=5) vs. 17% (n=2) in rs1143679 carriers (heterozygous). The addition of LA1 to neutrophils 15 min before FMLP resulted in the inhibition of O° production (13 nmol O*/5 min vs 7 nmol O*/5 min). As expected, pretreatment with LA1 alone stimulated the adherence of neutrophils to fibrinogen-coated surface (8±10 PMNs baseline, 5,930 ± 250 PMNs, LAI, 15 uM).
As previously shown in dendritic cells, inflammatory responses in macrophages and neutrophils are able to be attenuated in a CR3-specific fashionin this case, by novel agonist LA1. These results also suggest significant cross-talk between CR3 and other pro-inflammatory pathways, such as TLR7. A novel functional difference in immune response to iC3b mimetic LA1, based on carrier-status of the rs1143679 polymorphism, is suggested.
To cite this abstract, please use the following information:
Jain, Manish, Amato, Michael, Buyon, Jill P., Gupta, Vineet, Clancy, Robert M.; A Novel CR3 Agonist Attenuates Pro-Inflammatory Signaling in Subjects with Common and Variant ITGAM Polymorphism, rs1143679. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :1675