Fig 1.
FcγR-Ig blocks the macrophage mediated antibody-coated endothelial cell death.
(A) RAW 264.7 cells were co-cultured with HUVECs coated with anti-human fibronectin Ab or HUVECs alone in the presence or absence of CD16A-Ig and CD32A-Ig at 37°C. (A) The cells were analyzed after 2hr for binding of RAW264.7 cells with HUVECs using plate inversion method. (B) After 8hr, the HUVECs viability was analyzed using CellTiter-Blue Assay method. (C) After 12hr caspase-3 assay was carried out by calorimetric method. Blocking mAbs (2.4G2), CD16A-Ig, and CD32A-Ig were pre-incubated for 1 hr at 4°C and then continuously present during their incubation at 37°C. In all the experiments RAW 264.7 cells pretreated with 2.4G2 mAb, antigen alone, antibody alone, HUVEC treated with medium alone served as controls. Data shown are the average of three individual experiments; each experiment was carried in triplicate. P value < 0.05 considered as significant (*) and P< .005 as highly significant (**), NS: non-significant.
Fig 2.
Immune-complex induced nitric oxide secretion is prevented by dimeric FcγR-Ig molecules.
(A) RAW 264.7 cells were cultured with different concentrations (0–300μg/ml) of soluble ICs in serum free RPMI1640 medium for 12 and 24 hrs. Untreated RAW264.7 cells and cells treated with antigen, antibody and 2.4G2 mAb served as specificity controls. The culture medium was collected at specified time points, and the nitrite concentration was determined using the Griess reagent. (B) RAW 267.4 cells were cultured with 100 and 200μg/ml of soluble ICs for different time points (0–24 hr). The culture medium was collected at specified time points, and the nitrite concentration was determined using the Griess reagent. (C) CD16A-Ig and CD32A-Ig blocked the binding of ICs to RAW 264.7 cells thereby inhibited the NO production. In all the experiments RAW 264.7 cells pretreated with 2.4G2 mAb, antigen alone, antibody alone, cells treated with medium alone served as specificity controls. Data shown are the average of three individual experiments; each experiment was carried in triplicate. P < 0.05 considered as significant (*) and P< 0.005 as highly significant (**), NS: non-significant.
Fig 3.
Immune-complex induced iNOS upregulation inhibited by decoy FcγR-Igs.
(A) RAW 264.7 cells (2×106 cells/ml) were treated with the indicated concentrations of immune complex (25–300μg/ml) in serum free RPMI 1640 medium for 24 hr. (B) RAW264.7 cells were treated with 200μg/ml of ICs for different time points (0.5- 24hr). (C) CD16A-Ig and CD32A-Ig prevented iNOS upregulation in RAW264.7 cells. RAW264.7 cells (2×106/ml) in serum free medium were blocked from binding to ICs (200μg/ml) by incubating in the presence or absence of CD16A-Ig and CD32A-Ig (100μg/ml) for 24hr at 37°C. Untreated RAW264.7 cells, LPS treated, antigen alone, antibody alone and 2.4G2 mAb treated cells served as specificity controls. Cell lysate was used to detect the iNOS upregulation by Western blot analysis GAPDH was used as internal control. The blots were developed and scanned as described in detail in Materials and Methods. Protein band intensities were analyzed using ImageJ software (NIH, Bethesda, MA) and the relative band intensities were presented. Western blot pictures are representative of three individual experiments. Bar graphs are average of three individual experiments. P< 0.05 considered as significant (*) and < 0.005 as highly significant (**), NS: non-significant.
Fig 4.
Inhibition of iNOS in RAW264.7 cell prevents NO release and HUVEC death.
The RAW264.7 cells were pre-treated with 1400W (100ng/ml) 1hr at 37°C. The HUVECs and pre-treated RAW264.7 cells were co-cultured for 4 (A) 8 (B) and 12hrs (C). The viability of the HUVECs was determined using CellTitre Blue kit method. RAW264.7 cells pre-treated with 2.4G2 were over layered on antibody-coated HUVECs served as specificity control. Uncoated HUVECs, antibody-coated HUVECs and uncoated HUVECs over layered with 2.4G2 pre-treated RAW264.7 cells were served as additional controls. Data shown are the average of three individual experiments; each experiment was carried in triplicate. P<0.05 considered as significant (*) and P<0.005 as highly significant (**), NS: non-significant.
Fig 5.
Exogenous nitric oxide initiates intrinsic apoptotic pathway in HUVECs, which was unaltered by decoy FcγR-Igs.
Concentration-dependent apoptosis was induced by nitric oxide in cultured HUVECs (1x 106 cells) stimulated with different concentrations of S-nitroso-N-acetyl-penicillamine (SNAP) (0–0.5mM) for 24 hr with or without CD16A-Ig was analyzed at mRNA level. Total RNA extracted from the SNAP treated cells was used to make cDNA. QRTPCR analysis of apoptosis related genes (A) Bak (B) Bax (C) Cytochrome-C and (D) Caspase-3 were carried out. Data shown are the average of six individual experiments; each experiment was carried in triplicate. P<0.01 were considered as significant (*) and P<0.005 were as highly significant (**), NS: non-significant.
Fig 6.
Exogenous nitric oxide induces caspase-3 activation and upregulation in HUVECs.
(A) Cells treated with different concentrations of S-nitroso-N-acetyl-penicillamine (SNAP) (0- 2mM) for 24hr. Untreated HUVECs served as specificity control. Cells were lysed and total proteins extracted. Western blot was carried out using an antibodies specific for caspase-3 and β-actin (internal control). (B) Caspase-3 enzymatic activity was performed using total protein extracts. Western blot pictures are representative of three individual experiments. Caspase-3 enzyme activity graph s is average of three individual experiments. P<0.05 considered as significant (*) and P<0.005 as highly significant (**), NS: non-significant.
Fig 7.
Hypothetical model of inhibition of IC-mediated, NO-induced apoptotic pathway in HUVECs by decoy FcγR-Igs.
Circulating ICs deposits in the blood vessels during IC-mediated inflammatory disorders. Effector cells upon binding to the IC get activated. These cells result in the secretion of pro-inflammatory factors like cytokines and toxic superoxide radicals such as nitric oxide in copious amounts. Nitric oxide ability to interact with cellular components in the endothelium results in triggering apoptotic signaling pathway and subsequently cell death and tissue damage. Decoy FcγR-Igs competes with cell surface FcγRs expressed on effector cells and block the access to the ICs deposits in various organs including blood vessels. This will eventually inhibit effector cell mediated endothelial inflammation and damage observed in autoimmune vasculitis.