Fig 1.
Identification of recombinant human SSB protein.
Recombinant human Sjögren's syndrome antigen B (rSSB) preparations were analyzed in 12% SDS-PAGE followed by Coomassie blue staining (A), and Western blotting reaction (B) with purified human polyclonal anti-SSB antibodies, mouse monoclonal anti-histidine tag antibodies (anti-HisTag) or mouse monoclonal antibodies against human SSB. A distinct band with a molecular weight around 50 kDa was identified as recombinant human SSB protein.
Fig 2.
IL-8 production from normal human PMNs and retinoid acid-induced granulocytic differentiated HL-60 (RA) cells.
Cells (1x106 cells/ml) were treated with different stimuli and IL-8 levels in culture supernatants were measured. A, Dose-responsive (left panel) and time-dependent (right panel) effects of rSSB on IL-8 production from normal human PMNs. Incubation with rSSB (10 μg/ml) for 2 hours were chosen in the ongoing experiments. B, Comparison of IL-8 production from normal PMNs after incubation with rSSB (10 μg/ml) and LPS (100 ng/ml) for 2 hours. Medium effect was subtracted in both groups. C, The effect of polymyxin-B on rSSB- or LPS-stimulated IL-8 production from normal PMNs. D, The effect of different modifications of rSSB molecules on IL-8 production from normal PMNs, including: C: controls (intact rSSB molecule as 100%), R: RNase A-digested rSSB (n = 2), P: proteinase-K digested rSSB (n = 3) and H: heat-denatured rSSB (n = 4). E, An additive effect of TNF-α pretreatment on rSSB-induced IL-8 production from normal PMNs. F, Effect of rSSB, anti-SSB antibody and rSSB-anti-SSB immune complex on IL-8 production from normal PMNs. G, The dose-responsive (left channel) and time-dependent (right channel) effects of rSSB (10 μg/ml) on IL-8 production from HL-60 (RA) cells. *p<0.05; **p<0.001
Fig 3.
Uptake of FITC-rSSB by normal human PMNs and HL-60 cells.
A, Kinetic uptake of FITC-rSSB (10 μg/ml) by normal human PMNs after incubation for 5–60 minutes. The percentage of FITC-rSSB uptake was analyzed by flow cytometry after quenching the extracellular FITC fluorescence by adding 0.4% trypan blue. B, Comparisons of FITC-BSA (10 μg/ml) and FITC-rSSB (10 μg/ml) uptake by HL-60 (RA) cells after incubation for 15 minutes at 4°C or 37°C. The percentage of cell death was also compared at 4°C and 37°C for 15 minutes by propidium iodide (PI) staining. C, Comparisons of the percentage of FITC-rSSB (40 μg/ml) uptake by HL-60 (RA) cells in the presence of medium (Med), LPS (100 ng/ml), non-specific IgG (10 μg/ml) or purified human polyclonal anti-SSB antibody (10 μg/ml) for 15 minutes. D, Uptake of FITC-rSSB by HL-60 cells was observed with fluorescence microscopy. A representative case of 2–3 independent experiments is shown in A-D.
Fig 4.
Effect of chloroquine (CQ) and NH4Cl on IL-8 gene expression in HL-60 (RA) cells and human PMNs.
A, The effect of CQ (5–50 μg/ml) and NH4Cl (30 mM) pre-incubation on rSSB-induced IL-8 gene expression in HL-60 (RA) cells was determined by ELISA (after 2 hours of stimulation) and Q-PCR (after 1 hour of stimulation). Relative ratios were calculated compared with the medium pretreatment group (set at 1.0). B, Effect of CQ (10 μg/ml) pre-incubation on rSSB-stimulated IL-8 mRNA expression (after 1 hour of stimulation) and IL-8 protein secretion (after 2 hours of stimulation) by normal PMNs. Relative ratios were calculated compared to medium control. C, Effect of CQ (5 μg/ml) pre-incubation on IL-8 production from rSSB-incubated PMNs from patients with SLE.
Fig 5.
Signaling pathways for rSSB-induced IL-8 production in normal human PMNs and HL-60 (RA) cells.
A, Activation and phosphorylation of p38, ERK1/2 and JNK MAPK pathways by rSSB (10 μg/ml) in normal human PMNs. B, Activation and phosphorylation of p38 and ERK1/2 MAPK pathways by rSSB (10 μg/ml) in HL-60 (RA) cells. A representative blot of 3 independent experiments is shown in (A) and (B). C, The effects of specific inhibitors for p38 (SB203580, SB 10 μM), MEK-1 (PD98059, PD, 10 μM), p38 and MEK-1 (SB + PD, 10 μM each), and Gαi-protein-coupled receptors (pertussis toxin, PTX, 100 ng/ml) on rSSB (10 μg/ml)- or LPS (100 ng/ml) induced IL-8 production were compared. The relative ratio of IL-8 production in the medium pretreatment group was set at 1, and multiple comparisons were adjusted by Bonferroni correction. D, Nuclear translocation of NF-κB subunits p65 and p50 induced by rSSB (10 μg/ml) at 1 hour compared to medium control in HL-60 (RA) cells. *p<0.05. E, Nuclear translocation of NF-κB subunits p65 and p50 induced by rSSB (10 μg/ml) at 1 hour compared to medium control in normal human PMNs. A representative result of 2 independent experiments is shown in E.
Fig 6.
Schematic diagram illustrating the interactions of SSB with PMNs and the signaling pathways.
During infections or autoimmune responses, SSB molecules are released from damaged tissues and dead cells. The free SSB molecules activate PMN via MAPK pathways and NF-κB nuclear translocation to transcribe the IL-8 gene. TNF-α and SSB-anti-SSB immune complexes, which may utilize TNF-α receptors (TNFR) and Fc-gamma receptors (FcγR), respectively, augment IL-8 production probably through a final common pathway.