Formation Of Insoluble Immune Complexes Research Articles

Failure to detect anti-idiotypic antibodies in the autoimmune response to IA-2 in Type 1 diabetes

The concept that immune responses to self antigens are regulated by anti-idiotypic networks has attracted renewed interest following reports of circulating factors within IgG fractions of serum that impair detection of autoantibodies with autoantigen. Thus, preclearance of sera with bead-immobilised monoclonal autoantibodies to the Type 1 diabetes autoantigen GAD65, or prebinding of serum antibodies to protein A Sepharose prior to addition of antigen, increases immunoreactivity detected in serum samples consistent with the trapping on the beads of anti-idiotypic antibodies that block antibody binding to the autoantigen. The aim of this study was to investigate the presence of anti-idiotypic antibodies to another major target of autoantibodies in Type 1 diabetes, IA-2. As previously observed for GAD65, preadsorption of serum samples with immobilised monoclonal IA-2 autoantibody, or prebinding to protein A Sepharose, resulted in substantial increases in subsequent immunoprecipitation of radiolabeled IA-2 in a proportion of samples. However, control experiments indicated that the increases seen on pre-incubation with immobilized autoantibodies were caused by displacement of the antibody by serum IgG, whereas impaired detection of immunoreactivity in liquid-phase radiobinding assays was the result of formation of insoluble complexes that bind poorly to protein A. The results emphasise the importance of direct demonstration of specific binding of antibodies to the idiotype in the study of idiotypic networks in autoimmunity. Variability between patients in formation of insoluble immune complexes has implications for the design and standardization of autoantibody assays for diabetes prediction.

Interactions of histidine-rich glycoprotein with immunoglobulins and proteins of the complement system

This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.

S. aureus IgG-binding proteins SpA and Sbi: Host specificity and mechanisms of immune complex formation

The evasion of the host immune response is central to the pathogenicity of Staphylococcus aureus, and is facilitated by the ability of the cell wall-associated protein A (SpA) to bind immunoglobulin G Fc fragments, thereby impeding phacocytosis and classical pathway complement fixation. SpA also acts as a B-cell superantigen through interactions with the heavy-chain variable part of Fab fragments, and sequesters immunoglobulins by forming large insoluble immune complexes with human IgG. Here we show that the formation of insoluble immune complexes is mediated by the binding of (V H3 +) Fab fragments in addition to Fc, and that SpA forms soluble complexes with IgG Fc fragments. We compared these results with those for Sbi, a second staphylococcal immunoglobulin-binding protein, and note that this protein requires only the Fc fragment for precipitation with human IgG. Homology models of immunoglobulin-binding domains of SpA and Sbi in complex with Fc reveal the molecular basis of the Fab-independent formation of insoluble complexes by Sbi. Finally, we compared the sequences of the spa and sbi genes from human strains to those infecting a range of animal hosts to determine whether Sbi and SpA have acquired specificity for host IgG. We note remarkable sequence conservation within the IgG-binding domains of these genes, consistent with a lack of host specificity. The Fab-independent binding of IgG by Sbi could have significant clinical implications. The use of SpA in immunoadsorption therapy can cause severe side-effects, thought to be mediated by FcγR recognition and complement fixation. The formation of insoluble immune complexes with Sbi occurs only via Fc binding and free Fc regions are unlikely to be available for FcγR recognition and complement fixation.

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

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.

Histidine-rich glycoprotein regulates the binding of monomeric IgG and immune complexes to monocytes.

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein which we have shown previously inhibits the formation of insoluble immune complexes (IC). In this study we examined the ability of HRG to regulate the binding of monomeric IgG and IC to monocytes. Initial studies demonstrated that HRG interacts with FcgammaRI on the monocytic cell line THP1 and blocks the binding of monomeric IgG to these cells. However, despite totally blocking the binding of monomeric IgG to FcgammaRI, pre-incubation of THP1 cells with HRG had no effect on the binding of IC to these cells. In contrast, depending on the HRG:IgG molar ratio, pre-incubation of monomeric IgG with HRG resulted in either enhanced or reduced IgG binding to FcgammaRI. Similarly, under certain highly defined conditions, incorporation of HRG in IgG-containing IC potentiated the binding of IC to THP1 cells. The key conditions involved incorporating approximately equimolar concentrations of HRG and IgG in the IC, the IC being formed at a near equivalence antigen:antibody ratio and usually physiological concentration (20 microM) of Zn(2+) being present. Collectively these observations indicate that HRG is an important regulator of IC uptake by monocytes. Thus HRG can interact with FcgammaRI on monocytes and block monomeric IgG binding, whereas when incorporated in IgG containing IC, HRG can enhance the uptake of IC by monocytes, probably via its heparan sulfate binding domain.

Histidine-rich glycoprotein binds to human IgG and C1q and inhibits the formation of insoluble immune complexes.

Purification of the complement component C1q from human serum using an established method resulted in the copurification of two 30 kDa proteins with an N-terminal sequence identical to human histidine-rich glycoprotein (HRG). Therefore, to explore the possibility that HRG can interact with C1q, we examined the ability of 81 kDa (native) and the 30 kDa proteins (presumably proteolytic N-terminal fragments of HRG) to bind to C1q, using both ELISA and optical biosensor techniques. Both forms of HRG were found to bind to the human complement component C1q and also to purified human and rabbit IgG by ELISA. Kinetic analyses of the HRG-C1q and HRG-IgG interactions using the IAsys biosensor indicate two distinct binding sites with affinities Kd1 0.78 x 10(-8) M and Kd2 3.73 x 10(-8) M for C1q, and one binding site with affinity Kd 8.5 x 10(-8) M for IgG. Moreover, the fact that both native and 30 kDa HRG bind to C1q and to IgG suggests that the IgG and C1q binding regions on HRG are located in the 30 kDa N-terminal region of the HRG molecule. The Fab region of IgG is likely to be involved in the HRG-IgG interaction since HRG also bound to F(ab')2 fragments with an affinity similar to that seen with the complete IgG molecule. Interestingly, the binding between HRG and IgG was significantly potentiated (Kd reduced from 85.0 to 18.9 nM) by the presence of physiological concentrations of Zn2+ (20 microM). Conversely, the presence of Zn2+ weakened the binding of HRG to C1q (Kd increased from 7.80 to 29.3 nM). Modulation of these interactions by other divalent metal cations was less effective with relative potencies being Zn2+ > Ni2+ > Cu2+. An examination of the effect of native and 30 kDa HRG on the formation of insoluble immune complexes (IIC) between ovalbumin and polyclonal rabbit anti-ovalbumin IgG revealed that physiological concentrations of HRG can markedly inhibit IIC formation in vitro. The results show that human HRG binds to C1q and to IgG in a Zn2+-modulated fashion, and that HRG can regulate the formation of IIC in vitro, thus indicating a new functional role for HRG in vivo.

The formation of insoluble immune complexes between ovalbumin and anti-ovalbumin IgG occurs in at least two distinct phases dependent on reactant concentration and ionic strength

The mechanism regulating the formation of insoluble immune complexes (IIC) in serum in certain disease states is not well understood. Ovalbumin and rabbit anti-ovalbumin IgG was used to study the formation of IIC in vitro in a stirred reaction vessel; and the radii of IIC that formed was determined by light scattering techniques. Using an initial IgG concentration of 1 mg/ml at equivalence antigen: antibody ratio IIC formation was detected within 5 s, and the complexes increased in radii to approx. 100 nm after 20–30 s (phase 1). This was followed by a phase (phase 2) in which the complexes rapidly increased in radii to the point where Mie scattering was reached ( ∼ 200 nm). The time of onset of the second phase decreased with increasing initial IgG concentrations at a fixed antigen: antibody ratio; and was at a minimum at equivalence antigen: antibody ratio, but increased at both antigen and antibody excess ratios. Immune complexes formed using F(ab′) 2 fragment showed a similar pattern to those formed using IgG. A similar pattern was seen in the presence of the complement component CIq which potentiated IIC formation in phase 2, and human serum (1:10 dilution) which attenuated IIC formation in both phases. For complex formation using IgG and ovalbumin the presence of NaCI at concentrations up to 0.6 M led to a progressive increase in the time of onset of phase 2; potencies of inhibition by other sodium halides followed the lyotropic series NaF < NaCl < Nal. The results suggest that formation of IIC occurs in at least two distinct phases, and that the second phase leading to the generation of very large insoluble complexes is associated with a rapid polymerisation of the complexes by a mechanism that is not dependent on Fc:Fc interactions.

C1q is a nucleotide binding protein and is responsible for the ability of clusterin preparations to promote immune complex formation

Clusterin prepared from human serum by monoclonal antibody affinity chromatography was devoid of the ability to increase the rates of formation of insoluble immune complexes associated with clusterin preparations obtained by polyclonal IgG affinity chromatography. Clusterin did not bind to AMP-Sepharose but the protein responsible for increasing the rates of formation of insoluble immune complexes did bind to this affinity matrix. This protein was identified as complement protein C1q on the basis of its behaviour on SDS/PAGE and reactivity in sandwich ELISA with monoclonal antibodies specific for C1q. C1q (identified from its behaviour on SDS/PAGE, immunoreactivity with C1q-specific monoclonal antibodies and N-terminal sequencing data) was purified from serum by AMP-Sepharose chromatography. The binding of C1q to AMP-Sepharose was inhibited by adenine nucleotides.

The Effects of Histidine Residue Modification on the Immune Precipitating Ability of Rabbit IgG

Treatment of anti-ovalbumin rabbit IgG with diethylpyrocarbonate (DEPC) at concentrations up to 100 μM led to a progressive decrease in the rates of formation of insoluble immune complexes, without affecting the final extent of immune complex formation. DEPC concentrations approximately 10-fold higher were needed to give comparable decreases in the rates of immune complex formation by F(ab′)2. Treatment of DEPC-treated IgG with hydroxylamine led to substantial restoration of the rates of formation of insoluble immune complexes. Carbethoxylation of two histidine residues per IgG molecule had little effect on rates of formation of insoluble immune complexes, but these rates were markedly decreased in samples of IgG with four to five histidines per molecule modified. There were parallel decreases in the protein A-binding activity and in the rates of formation of insoluble immune complexes in IgG treated with increasing concentrations of DEPC. The presence of complement protein C1q restored the rates of formation of insoluble immune complexes of DEPC-treated IgG.

Clusterin enhances the formation of insoluble immune complexes

Clusterin was purified from human serum by sequential affinity chromatography over IgG-, protein A- and Con A-Sepharose. The protein was ∼ 70 kDa by SDS/PAGE under non-reducing conditions and was resolved into ∼ 35 kDa bands under reducing conditions. The protein reacted with clusterin-specific Mabs in ELISA and in Western blots. Its N-terminal sequences agreed with those published for clusterin. An antiserum specific for clusterin made by the above method detected it in complement membrane attack complexes on rabbit erythrocyte membranes. The interaction of clusterin with IgG was physiologically relevant because it was found to increase the rate of formation of insoluble immune complexes.

The role of Fc:Fc interactions in insoluble immune complex formation and complement activation

The initial rate of formation of insoluble immune complexes from rabbit IgG and ovalbumin was approximately 12 times that for formation of F(ab') 2-ovalbumin complexes. At low IgG concns, in the range 0.7–2.7 n M, the formation of insoluble immune complexes was characterised by a lag phase, especially for complexes formed in low antigen excess, compared to antibody excess. Guanidine HC1 (at concns up to 0.5 M) and urea (at concns up to 1 M) decreased the initial rates of formation of IgG immune complexes more than F(ab') 2 immune complexes. Pre-formed IgG immune complexes were solubilised at lower guanidine HC1 concns than were F(ab') 2 immune complexes. Clq enhanced the initial rate of formation of IgG immune complexes at Clq:IgG ratios up to 1:1. Higher Clq concns decreased the initial rate of formation of the complexes. Urea (1 M) blocked the Clq mediated enhancement of immune complex formation.

Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

To examine whether the ability of complement to form soluble immune complexes plays a role in preventing immune complex-mediated diseases, we analyzed the capacity of complement to inhibit immune precipitation (IIP) and to solubilize preformed immune aggregates (SOL) in 23 sera of patients with various hypocomplementemic states, and we correlated the results of these studies with the clinical syndromes found in the various patients. In sera with deficiency or depletion of early classical pathway components, IIP was profoundly altered, whereas SOL was delayed but in the normal range. In contrast, in sera with C3 depletion but intact classical pathway and in properdin-deficient serum, IIP was initially preserved, whereas SOL was abolished. Since the incidence of immune complex diseases in various hypocomplementemic states correlates with the severity of IIP defects, but not with reduced SOL, it is suggested that IIP is an essential biological function of complement that prevents the rapid formation of insoluble immune complexes in vivo.