IgG molecules are exposed on a regular basis to acidic conditions during immunoaffinity purification procedures, as well as during the production of some therapeutic immunoglobulin preparations. This exposure is known to induce in them an antigen‐binding polyreactivity. The molecular mechanisms and the possible biological significance of this phenomenon remain, however, poorly understood. In addition to the previously reported ability of these modified IgG antibodies to interact with a large panel of self‐antigens, enhanced binding to non‐self‐antigens (bacterial), an increased ability to engage in F(ab′)2/F(ab′)2 (idiotype/anti‐idiotype) interactions and an increased functional antigen‐binding affinity are reported here. The newly acquired ‘induced polyreactivity’ of low‐pH buffer‐exposed IgG is related to structural changes in the immunoglobulin molecules, and is at least partly attributable to the enhanced role of the hydrophobic effect in their interactions with antigen. Our results suggest that data from many previous studies on monoclonal and polyclonal IgG antibodies purified by low‐pH buffer elution from protein A or protein G immunoaffinity columns should be reconsidered, as the procedure itself may have dramatically affected their antigen‐binding behavior and biological activity. Low‐pH buffer‐treated pooled therapeutic immunoglobulins acquire novel beneficial properties, as passive immunotherapy with the pH 4.0 buffer‐exposed, but not with the native therapeutic intravenous immunoglobulin preparation, improves the survival of mice with bacterial lipopolysaccharide‐induced septic shock.

Intravenous immunoglobulin (IVIG) are purified IgG preparations made from the pooled plasma from thousands of healthy donors and are being tested in preclinical mouse models. Inherent challenges, however, are the pluripotency of IVIG and its xenogeneicity in animals. IVIG can alter the viability of human neutrophils via agonistic antibodies to Fas and Siglec-9. In this study, we compared the effects of IVIG on human and mouse neutrophils using different death assays. Different commercial IVIG preparations similarly induced cytokine-dependent death in human neutrophils, whereas they had no effects on the survival of either peripheral blood or bone marrow neutrophils from C57BL/6 or BALB/c mice. F(ab’)2 but not Fc fragments of IVIG induced death of human neutrophils, whereas neither of these IVIG fragments, nor agonistic monoclonal antibodies to human Fas or Siglec-9 affected the viability of mouse neutrophils. Pooled mouse IgG, which exhibited a different immunoprofile compared to IVIG, also had no effect on mouse cells. Together, these observations demonstrate that effects of IVIG on neutrophil survival are not adequately reflected in current mouse models, despite the key role of these cells in human inflammatory and autoimmune diseases.

Natural polyreactive IgG antibodies are found in the sera of all healthy individuals. The in vitro exposure of pooled human IgG to protein-destabilizing chemical or physical factors has been previously shown to result in the exposure of their "hidden" polyspecificity. We hypothesize that such an enhancement of their pre-existing immunoreactivity may occur in vivo in the aggressive microenvironment of inflammation sites. An increase in the antigen binding intensity as well as of the number of recognized antigens was observed in the sera of IgG-infused immunodeficient SCID mice with induced local inflammation. The expansion of the IgG pathogen-binding repertoire may have important biological consequences.

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as "genomic storm" in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous "danger" signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes. online address: http://www.molmed.org

Intravenous immunoglobulin (IVIg) preparations are known to modulate autoimmune/inflammatory diseases through several F(ab') 2 -and Fc-dependent mechanisms. In this study, we show that the in vitro and the in vivo exposure of B lymphocytes from lupus-prone and from healthy mice to IVIg results in an increased expression of their surface inhibitory FccIIB receptors. Further, this exposure enhanced the ability of a chimeric antibody, cross-linking FccRIIB and immunoglobulin receptors on DNA-specific B lymphocytes, to suppress IgG anti-DNA antibody production. F(ab') 2 fragments of IVIg had a similar activity as the intact preparation, whereas Fc fragments had no effect. This study describes a novel approach with clinical relevance for modulating B lymphocyte activity.

Normal pooled human IVIg are produced using various blood protein fractionation technologies and as a result they may well differ in their biological properties. We have demonstrated that exposure of IVIg, for a period as short as 15 min, to protein-destabilizing agents like acidic pH, ROS or pro-oxidative ferrous ions dramatically increases the panel of recognized Ag including pro-inflammatory cytokines. We now show that exposure of IVIg to ferrous ions modifies some IgG molecules without denaturating them and enhances the protective activity of the preparation in experimental septic shock.

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Antibody repertoires of healthy humans and animals contain a fraction of antibodies able to acquire additional polyspecificity following exposure to several biologically relevant redox molecules (free heme, reactive oxygen species, ferrous ions, HOCl, etc.). The physiological role of these "hidden" polyspecific antibodies is poorly understood. Similar to inherently polyspecific antibodies, those with induced polyspecificicty may also have immunoregulatory properties. We have previously shown that a pooled human IgG preparation, modified by the exposure to ferrous ions, acquires the ability to significantly improve survival of animals with polymicrobial sepsis or aseptic systemic inflammation induced by bacterial lipopolysaccharide or zymosan administration. In the present study, we have analyzed the effects of administration of heme-exposed pooled human IgG in the same models of sepsis and aseptic systemic inflammation. The administration of a single dose of heme-exposed pooled IgG has resulted in a significant increase in the survival of mice with endotoxinemia, but not in those with polymicrobial sepsis and zymosan-induced severe generalized inflammation. Finally, we have provided evidence that the anti-inflammatory effect of heme-exposed IgG can be explained by scavenging of pro-inflammatory mediators.