IVIG regulates the survival of human but not mouse neutrophils.

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.

Heme-Exposed Pooled Therapeutic IgG Improves Endotoxemia Survival.

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.

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes.

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.

Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

The protective effect of modified intravenous immunoglobulin in LPS sepsis model is associated with an increased IRA B cells response.

Intravenous immunoglobulin preparations (IVIg) that have undergone a mild oxidizing treatment with ferrous ions have an increased polyspecificity, which is not associated with a higher propensity to form aggregates. Among other biological properties of the modified IVIg, a protective effect in LPS sepsis model stands out as the native preparation is totally devoid of it or even exacerbates sepsis. A recent finding identified an LPS induced subset of B1 lymphocytes that migrate from the peritoneal cavity to the spleen acquiring the expression of CD93, GM-CSF as well as the capacity to control sepsis. This report demonstrates that modified IVIg, but not the native preparation, causes a further increase in this population during LPS sepsis. Partial targeted suppression of the peritoneal B cell proliferation by an intracellular dye abrogates this effect and the clinical benefit of modified IVIg.

Exposure of IgG to an acidic environment results in molecular modifications and in enhanced protective activity in sepsis.

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.

Protein destabilizing agents induce polyreactivity and enhanced immunomodulatory activity in IVIg preparations.

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.

Intravenous immunoglobulin up-regulates the expression of the inhibitory FcgammaIIB receptor on B cells.

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 FcgammaIIB receptors. Further, this exposure enhanced the ability of a chimeric antibody, cross-linking FcgammaRIIB 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.