High circulating levels of biologically inactive IL-6/SIL-6 receptor complexes in systemic juvenile idiopathic arthritis: evidence for serum factors interfering with the binding to gp130

Abstract

We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.

Fig. 1

Assay for the IL-6/gp130 binding activity. (a) Efficacy of two different mAbs to the sIL-6R (22·1 and R1/9) for capture in the evaluation of the IL-6/gp130 binding activity. Wells were incubated at 37°C for 1 hour with buffer alone (j) or with soluble gp130FLAG (250 ng/ml), was incubated at 37°C for 1 h in the absence () or in the presence of rhsIL-6R alone (100 ng/ml, ;), or of rhsIL-6R (100 ng/ml) and rhIL-6 (3 ng/m)(h) and then added to wells coated with the indicated mAb. j None. The IL-6/sIL-6R complex bound to gp130FLAG was revealed with a mAb to the FLAG epitope. Results are shown as total O.D., as means + SE, represented by the horizontal bars, of triplicate determinations. (b) Dose–response curves of the assay for the IL-6/gp130 binding activity obtained by adding increasing concentrations of rhIL-6 to rhsIL-6R (100 ng/ml) () or to a normal human serum (s) in the presence of gp130FLAG (250 ng/ml). Reaction mixtures were incubated at 37°C for 1 h and then tested, as described in the methods section, in the assay for IL-6/gp130 activity using the mAb 22·1 to the sIL-6R for capture. Results are shown as specific O.D., as means + SE, represented by the vertical bars, of triplicate determinations. (c) Dose–response curves of the assay for the IL-6/gp130 binding activity obtained by reading the A405 after a 30 minute (j), a 2-h (▴) and a 6-h (d) incubation with the substrate p-NPP. Results are shown as specific O.D., as means and SE, represented by the vertical bars, of triplicate determinations.

Fig. 2

Comparison of the levels of IL-6 estimated by the B9 cell assay, the IL-6/gp130 binding activity assay and the immunoassay for the IL-6/sIL-6R complex in s-JIA sera.

Fig. 3

Comparison of the IL-6 levels estimated (a) by the human myeloma XG-1 cells (h) or (b)by the human hepatoma Hep 3b cells (h) with those estimated by the murine hybridoma B9 cells (), by the IL-6/gp130 binding activity assay () and the immunoassay for the IL-6/sIL-6R complex (j) in representative sera from patients with s-JIA (4 out of 15 tested in the XG-1 assay and 4 out of 8 tested in the Hep 3b assay). Results are shown as means + SE, represented by the vertical bars, of triplicate determinations.

Fig. 4

s-JIA sera interfere with the binding of the IL-6/sIL-6R complex to gp130. (a) Amount of IL-6/gp130 binding activity generated in controls and s-JIA sera following exogenous addition of IL-6. Sera were preincubated with 4 ng/ml of recombinant human IL-6 and then tested in the IL-6/gp130 binding activity assay. Results are shown as means + SE, represented by the vertical bars, of triplicate determinations. The horizontal dotted line indicates the 4 ng/ml amount of IL-6 that was exogenously added. (b) Dose dependent inhibition of the IL-6/gp130 binding activity obtained adding increasing percentages of sera of patients with active s-JIA (s-JIA Pool) to a normal control serum incubated with 1 ng/ml of rhIL-6.

Fig. 5

Serum levels of soluble gp130 (a) and autoantibodies to IL-6 (b) in patients with systemic JIA (s-JIA) (open bar in panel a) and in healthy controls (CTRL) (hatched bar in panel a). Sgp130 levels were measured by ELISA as described in the method section. For the determination of antibodies to IL-6, a reference Ig preparation with high levels of autoantibodies to hIL-6 (IgG anti-IL-6), or sera from patients with s-JIA (n = 11) and from controls (n = 8) were incubated with ¹²⁵I-rIL-6 and then subjected to chromatography on Sephadex G-75 as described in the method section. Results are expressed as the ratio between bound and total added ¹²⁵I-rIL-6. Results are shown as means + SD, represented by the vertical bars.

Fig. 6

CRP binds IL-6 without interfering with its biological activity. (a) IL-6 is coimmunoprecipitated with CRP. Recombinant human IL-6 (500 ng) was incubated with an anti-CRP polyclonal antibody conjugated to Protein A-Sepharose in the absence (lane 2) or in the presence of 5 µg of human purified CRP (lane 3). Lane 1 shows recombinant human IL-6. The immunoprecipitates were subjected to SDS-PAGE and Western Blotting, and the presence of IL-6 was revealed with a mAb to hIL-6. MWM, molecular weight markers. (b) CRP does not affect the biological activity of IL-6 on XG-1 cells. A control human serum was preincubated with rhIL-6 (100 pg/ml) for 1 h at 37°C in the absence or in the presence of human purified CRP (100 µg/ml) and then tested on the XG-1 cells. Results are expressed as mean + SE, represent by the vertical bars, of triplicate determinations. (c) CRP does not affect the binding of the IL-6/sIL-6R complex to gp130. rhIL-6 (5 ng/ml) was added to a normal control serum in the absence or in the presence of exogenous CRP (100 µg/ml) and then tested in the IL-6/gp130 binding activity assay. Results are expressed as mean + SE, represent by the vertical bars, of triplicate determinations.