Fundamental roles of the innate-like repertoire of natural antibodies in immune homeostasis

The composition of the early immune repertoire is biased with prominent expression of spontaneously arising B cell clones that produce IgM with recurrent and often autoreactive binding specificities. Amongst these naturally arising antibodies (NAbs) are IgM antibodies that specifically recognized amaged and senescent cells, often via oxidation-associated neo-determinants. These NAbs are present from birth and can be further boosted by apoptotic cell challenge. Recent studies have shown that IgM NAb to apoptotic cells can enhance phagocytic clearance, as well as suppress proinflammatory responses induced via Toll-like receptors, and block pathogenic IgG-immune complex (IC)-mediated inflammatory responses. Specific antibody effector functions appear to be involved, as these anti-inflammatory properties are dependent on IgM-mediated recruitment of the early recognition factors of complement. Clinical surveys have suggested that anti-apoptotic cell (AC) IgM NAbs may modulate disease activity in some patients with autoimmune disease. In mechanistic studies, anti-AC NAbs were shown to act in dendritic cells by inhibition of the mitogen-activated protein kinase (MAPK) pathway, a primary signal transduction pathway that controls inflammatory responses. This immunomodulatory pathway has an absolute requirement for the induction of MAPK phosphatase-1. Taken together, recent studies have elucidated the novel properties of a class of protective NAbs, which may directly blunt inflammatory responses through a primitive pathway for regulation of the innate immune system

Protective Roles of Natural IgM Antibodies

Antibodies are a vital part of the armamentarium of the adaptive immune system for the fine-tuning of the recognition and response to foreign threats. However, in health there are some types of antibodies that instead recognize self-antigens and these contribute to the enhancement of primitive innate functions. This repertoire of natural IgM antibodies is postulated to have been selected during immune evolution for their contributions to critical immunoregulatory and housekeeping properties. The clearance of dying cells is one of the most essential responsibilities of the immune system, which is required to prevent uncontrolled inflammation and autoimmunity. In the murine immune system, natural IgM antibodies that recognize apoptotic cells have been shown to enhance the phagocytic clearance of dead and dying cells and to suppress innate immune signaling pathways. In the mouse, natural IgM are often the products of B-1 cell clones that arise during immune development without an absolute requirement for exogenous antigenic stimulation. In patients with systemic lupus erythematosus, IgM autoantibodies, which bind to neo-epitopes on apoptotic cells, have been demonstrated to be present at significantly higher levels in patients with lower disease activity and with less severe organ damage. While certain specificities of IgM autoantibodies correlate with protection from lupus renal disease, others may convey protective properties from lupus-associated atherosclerotic cardiovascular disease. New and unexpected insights into the functional roles of IgM antibodies are still emerging, especially regarding the functions of natural antibodies. Herein, we review recent progress in our understanding of the potential roles of natural IgM autoantibodies in the regulation of immune homeostasis and for protection from autoimmune and inflammatory diseases

LAG-3-deficient mice produce higher amounts of cytokine following mercury treatment.

<p>Wild-type and <i>Lag-3^−/−</i> mice were either given a challenge dose of HgCl₂ (30 µg/mouse s.c. on days 0, 2 and 4) or were left untreated. Splenocytes were harvested on day 8 and then stimulated in vitro with plate bound anti-CD3 and anti-CD28 antibodies. Supernatant were collected after 72 hrs to determine the levels of cytokine by ELISA (n = 3 or 4). Two way ANOVA was used to determine the statistical significance between the groups where *indicates <i>p</i><0.05 and ***indicates <i>p</i><0.0001.</p

Anti-LAG-3 monoclonal antibody breaks established tolerance against mercury-induced autoimmunity.

<p>A.SW mice were tolerized to Hg and 7 days later they received a challenge dose of Hg in conjunction with anti-LAG-3 or control antibody as depicted in the Figure. Serum IgG1 and IgE levels were measured by ELISA and autoantibody titers were determined by IF assay (n = 5). The IgG and IgE concentrations depicted at wk -1 represent the baseline levels of antibodies in serum of untreated mice and no autoantibodies were detectable in untreated mice. To determine statistical significance between the groups two way ANOVA was used where *indicates <i>p</i><0.05.</p

Exposure to Hg results in higher expression of LAG-3 on CD4⁺T cells and in an increased amount of soluble LAG-3 in serum.

<p>(A) B6.SJL mice were left untreated or given 3 injections of HgCl₂ s.c. on days 0, 2 and 4. Splenocytes were harvested on day 8 and analyzed by flow cytometry. Figure depicts a representative dot plot of total splenocytes gated for LAG-3⁺ CD4⁺ T cells. (B) Results are expressed as frequency of LAG-3⁺ CD4⁺ T cells in each group (n = 3 or 4). (C) A.SW mice were challenged with 3 injections of HgCl₂ (30 µg/mouse) on days 0, 2 and 4. Concentration of soluble LAG-3 (sLAG-3) in serum was measured by ELISA (n = 4 or 5). Unpaired t test was used for statistical analysis where *indicates <i>p</i><0.05.</p

A short course of HgCl₂ injections is sufficient to induce mercury-induced autoimmunity in LAG-3-deficient B6.SJL mice.

<p>(A) Wild-type or <i>Lag-3^−/−</i> B6.SJL mice were challenged with HgCl₂ (30 µg/mouse s.c.) on days 0, 2 and 4. Serum was analyzed by ELISA to determine IgG1 and IgE polyclonal antibodies and ANoA titers were measured by IF assay (n = 5 or 6). (B) Kidneys of mice from this experiment were harvested after 4 weeks, sectioned and stained with goat anti-mouse IgG-FITC to detect IgG deposit. The extent of kidney disease was scored depending on the amount and intensity of IgG staining as described in details in Material and Method section. To determine statistical significance between the groups of fig. 2A and 2B two way ANOVA was used where *indicates <i>p</i><0.05, **indicates <i>p</i><0.005 and ***indicates <i>p</i><0.0001.</p

LAG-3-deficient mice cannot be tolerized to mercury.

<p>Wild-type or LAG-3-deficient mice were given a tolerogenic dose of HgCl₂ (3 µg/mouse given i.p.) on day -7. Mice were then exposed to 3 injections of HgCl₂ (30 µg/mouse given s.c) on days 0, 2 and 4. Serum Ig were measured by ELISA and ANoA titers were determined by IF assay (n = 5 or 6). To determine statistical significance between the groups two way ANOVA was used where *indicates <i>p</i><0.05.</p