Histidine-rich glycoprotein prevents the formation of insoluble immune complexes by rheumatoid factor

Abstract

In previous studies we have shown that histidine-rich glycoprotein (HRG), a relatively abundant plasma protein, can bind to immunoglobulin G (IgG) and inhibit the insolubilization of IgG-containing immune complexes (IC). It was of interest, therefore, to determine whether HRG can inhibit the formation of insoluble IC (IIC) resulting from the interaction of rheumatoid factor (RF) with human IgG-containing IC. Light scattering techniques were used to examine the effect of HRG on the formation of IIC between RF and IC containing human IgG according to three different models. In all three models physiological concentrations of HRG could block the formation of IIC induced by RF. Optical biosensor studies of the RF-IgG interaction also revealed that HRG can mask the epitopes on IgG recognized by RF. Additional studies examined whether HRG can solubilize already formed IIC and demonstrated that HRG can, in fact, partially solubilized IIC. These data indicate that HRG can regulate the formation of IIC induced by RF at three levels: namely by inhibiting the initial recognition of IgG containing IC by RF, by inhibiting the subsequent insolubilization of IgG containing IC by RF and by solubilizing already formed IIC. Collectively, these findings suggest that HRG may be an important inhibitor of the formation of pathogenic IC in diseases such as systemic lupus erythematosus and rheumatoid arthritis.

Figure 1

Formation of IIC between human IgG and either rabbit anti‐human IgG antibodies or RF as measured by light scattering at 350 nm. The results in (a) show the increase in absorbance as a function of time caused by formation of IIC containing rabbit anti‐human IgG (69 µg/ml) and human IgG at the antigen : antibody ratios of 0·056 (▪), 0·1 (□). 0·17 (•) and 0·22 (○). The results in (b) show the increase in absorbance as a function of time caused by formation of IIC containing human RF (8 IU/ml) and aggregated human IgG at concentrations of 20 (▪), 40 (□), 60 (•), 80 (○) and 120 (▵) µg/ml. The results in (c) show the increase in absorbance as a function of time caused by formation of IIC containing human RF (8 IU/ml) and monomeric human IgG at concentrations of 60 (▪), 120 (□) and 240 (•) µg/ml. Data are representative of three separate experiments.

Figure 2

Effect of HRG on the formation of IIC between human IgG and either rabbit anti‐human IgG antibodies or human RF as measured by light scattering at 350 nm. The results in (a) show the increase in absorbance as a function of time due to formation of IIC containing human IgG and rabbit anti‐human IgG (69 µg/ml) at equivalence antigen : antibody ratio for a control experiment (▪ no additions), and for experiments carried out in the presence of 22·5 (□), 45 (•), 60 (○), 75 (▴) and 150 (▵) µg/ml of human HRG. The results in (b) show the increase in absorbance as a function of time due to formation of IIC containing RF (8 IU/ml) and aggregated human IgG (120 µg/ml) for a control experiment (▪ no additions), and for experiments carried out in the presence of 37·5 (□), 75 (•), 150 (○) and 300 (▵) µg/ml of human HRG. Data are representative of three separate experiments.

Figure 3

Effect of HRG on the formation of IIC between RF and STP aggregated biotinylated human IgG (b‐IgG). In (a) b‐IgG (30 µg/ml) was incubated with STP at 6 (▪), 12 (□), 18 (•), 24 (○) and 60 (▴) µg/ml for 20 min, then RF (8 IU/ml) was added to the STP–b‐IgG complexes and IIC formation was monitored by light scattering. In (b) b‐IgG was preincubated without (▪) or with human HRG at 15 (□), 30 (•), 75 (○), and 150 (▴) µg/ml for 20 min before aggregation by streptavidin (STP) (18 µg/ml) for 20 min and the addition of RF (8 IU) to form IIC. Data are representative of three separate experiments.

Figure 4

Interaction of RF with immobilized human IgG1κ. (a) Human IgG1κ immobilized onto the sensing surface of a biosensor cuvette was reacted with different concentrations (2–16 IU/ml) of RF, the overlay plots representing the binding of different RF concentrations to the immobilized IgG1κ. (b) The value of k_obs for the binding curve for each RF titre was determined using the linearization method (Fast Fit program) and each value plotted against the concentration of RF. The plot of k_obs against RF concentration (IU/ml) approximates a straight line (•); the slope represents k_on and the y‐intercept represents k_off for this interaction. (c) In some experiments before monitoring the binding of RF (8 IU/ml) to IgG1κ the cuvette was either untreated (no HRG) or pretreated (+HRG) with HRG (15 µg/ml) for 5 min. Each data point in (b) represents the mean ± SEM obtained from three separate experiments. The data in (a) and (c) are representative of three separate experiments.

Figure 5

Effect of HRG on solubilization of already formed IIC. In (a) IIC were formed between ovalbumin and anti‐ovalbumin IgG (900 µg/ml) at different antigen : antibody ratios (0·005–0·05) and then HRG (150 µg/ml) was examined for its ability to solubilize the IIC overnight at 37° (▪) and compared with IIC incubated in the absence of HRG (•). Data expressed as percent precipitation of anti‐ovalbumin IgG (for details see Materials and methods). (b) Shows the ability of different concentrations of HRG, following incubation overnight at 37°, to solubilize ovalbumin antiovalbumin IgG (900 µg/ml) IIC formed at an equivalence (0·03) antigen: antibody ratio. Each data point in (b) represents mean ± SEM of three experiments. Asterisks indicate the ability of different concentrations of HRG to significantly solubilize the preformed IIC when compared to IIC formation in the absence of HRG, i.e. *P = 0·02, **P = 0·006.