Markus Biburger

IL‐10, regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A–induced liver injury in mice

The liver appears to play an important role in immunological tolerance, for example, during allo‐transplantation. We investigated tolerance mechanisms in the model of concanavalin A (ConA)‐induced immune‐mediated liver injury in mice. We found that a single injection of a sublethal ConA dose to C57BL/6 mice induced tolerance toward ConA‐induced liver damage within 8 days. This tolerogenic state was characterized by suppression of the typical Th1 response in this model and increased IL‐10 production. Tolerance induction was fully reversible in IL‐10−/− mice and after blockade of IL‐10 responses by anti‐IL10R antibody. Co‐cultures of CD4+CD25+ regulatory T cells (Tregs) and CD4+CD25− responder cells revealed Treg from ConA‐tolerant mice being more effective in suppressing polyclonal T cell responses than Treg from control mice. Moreover, Treg from tolerant but not from control mice were able to augment in vitro IL‐10 expression. Depletion by anti‐CD25 monoclonal antibody (MAb) indicated a functional role of Tregs in ConA tolerance in vivo. Cell depletion studies revealed Tregs and Kupffer cells (KC) to be crucial for IL‐10 expression in ConA tolerance. Studies with CD1d−/− mice lacking natural killer T (NKT) cells disclosed these cells as irrelevant for the tolerogenic effect. Finally, cellular immune therapy with CD4+CD25+ cells prevented ConA‐induced liver injury, with higher protection by Treg from ConA‐tolerized mice. Conclusion: The immunosuppressive cytokine IL‐10 is crucial for tolerance induction in ConA hepatitis and is mainly expressed by CD4+CD25+ Treg and KC. Moreover, Tregs exhibit therapeutic potential against immune‐mediated liver injury. (HEPATOLOGY 2007;45:475–485.)

α-Galactosylceramide-Induced Liver Injury in Mice Is Mediated by TNF-α but Independent of Kupffer Cells

NKT cells expressing phenotypic markers of both T and NK cells seem to be pivotal in murine models of immune-mediated liver injury, e.g., in Con A-induced hepatitis. Also α-galactosylceramide (α-GalCer), a specific ligand for invariant Vα14 NKT cells, induces hepatic injury. To improve the comprehension of NKT-cell mediated liver injury, we investigated concomitants and prerequisites of α-GalCer-induced hepatitis in mice. Liver injury induced by α-GalCer injection into C57BL/6 mice was accompanied by intrahepatic caspase-3 activity but appeared independent thereof. α-GalCer injection also induces pronounced cytokine responses, including TNF-α, IFN-γ, IL-2, IL-4, and IL-6. We provide a detailed time course for the expression of these cytokines, both in liver and plasma. Cytokine neutralization revealed that, unlike Con A-induced hepatitis, IFN-γ is not only dispensable for α-GalCer-induced hepatotoxicity but even appears to exert protective effects. In contrast, TNF-α was clearly identified as an important mediator for hepatic injury in this model that increased Fas ligand expression on NKT cells. Whereas intrahepatic Kupffer cells are known as a pivotal source for TNF-α in Con A-induced hepatitis, they were nonessential for α-GalCer-mediated hepatotoxicity. In α-GalCer-treated mice, TNF-α was produced by intrahepatic lymphocytes, in particular NKT cells. BALB/c mice were significantly less susceptible to α-GalCer-induced liver injury than C57BL/6 mice, in particular upon pretreatment with d-galactosamine, a hepatocyte-specific sensitizer to TNF-α-mediated injury. Finally, we demonstrate resemblance of murine α-GalCer-induced hepatitis to human autoimmune-like liver disorders. The particular features of this model compared with other immune-mediated hepatitis models may enhance comprehension of basic mechanisms in the etiopathogenesis of NKT cell-comprising liver disorders.

Monocyte Subsets Responsible for Immunoglobulin G-Dependent Effector Functions In Vivo

Immunoglobulin G (IgG) antibodies confer protection against pathogenic microorganisms, serve as therapeutics in tumor therapy, and are involved in destruction of healthy tissues during autoimmune diseases. Understanding the molecular pathways and effector cell types involved in antibody-mediated effector functions is a prerequisite to modulate these activities. In this study we used two independent model systems to identify innate immune effector cells required for IgG activity in vivo. We first defined the precise repertoire of receptors for the IgG Fc fragment (FcγR) on innate immune effector cells in the blood and on tissue-resident macrophage populations. Despite expression of relevant activating FcγRs on various phagocyte populations, our data indicate that the majority of these cell types are dispensable for IgG activity in vivo. In contrast, IgG-dependent effector functions were selectively impaired in animals lacking the CX(3)CR1(hi)Ly6C(lo)CD11c(int) monocyte subset, which expressed the full set of FcγRs required for IgG activity.

IVIg-mediated amelioration of ITP in mice is dependent on sialic acid and SIGNR1.

Intravenous immunoglobulin G (IVIg) therapy is widely used to treat autoimmune and inflammatory diseases. Recent evidence suggests that in mice, splenic resident cells might be important for the anti‐inflammatory activity of IVIg in a model of serum transfer arthritis. Splenectomized human immunothrombocytopenia (ITP) patients, however, still respond to IVIg therapy. To investigate whether the requirement of the spleen is essential for mouse ITP, we used a passive model of induced ITP and demonstrated that IVIg activity was functional in splenectomized animals. Further analysis showed that the IVIg‐mediated amelioration of platelet phagocytosis was fully dependent on terminal sialic acid residues in the IVIg preparation and could be blocked with a specific ICAM3 grabbing nonintegrin‐related 1 (SIGNR1) specific antibody. These results suggest that, similar to the human system, a spleen‐independent but sialic acid‐ and SIGNR1‐dependent pathway is responsible for IVIg‐mediated suppression of autoantibody‐dependent platelet depletion in mice.

The other side of immunoglobulin G: suppressor of inflammation

Immunoglobulin G (IgG) molecules can have two completely opposite functions. On one hand, they induce proinflammatory responses and recruit innate immune effector cells during infection with pathogenic microorganisms or autoimmune disease. On the other hand, intravenous infusion of high doses of pooled IgG molecules from thousands of donors [intravenous IG (IVIG) therapy] represents an efficient anti‐inflammatory treatment for many autoimmune diseases. Whereas our understanding of the mechanism of the proinflammatory activity of IgG is quite advanced, we are only at the very beginning to comprehend how the anti‐inflammatory activity comes about and what cellular and molecular players are involved in this activity. This review will summarize our current knowledge and focus upon the two major models of either IVIG‐mediated competition for IgG‐triggered effector functions or IVIG‐mediated adjustment of cellular activation thresholds used to explain the mechanism of the anti‐inflammatory activity.

Mechanisms of action of intravenous immunoglobulins

Intravenous immunoglobulin (IVIg) has been used for nearly three decades as an efficient anti-inflammatory therapeutic regimen in a growing number of autoimmune diseases. Despite this their success in clinical application, the mechanism of action of IVIg therapy remains elusive. During the last few years, several mechanisms dependent on either the IgG variable or constant fragment have been proposed to explain the potent immunomodulatory activity of IVIg. This review will discuss which molecular and cellular pathways might be involved in the anti-inflammatory activity of IVIg and for which types of autoimmune diseases they might be relevant.

The neuropeptide calcitonin gene-related peptide (CGRP) prevents inflammatory liver injury in mice.

BACKGROUND/AIMS Calcitonin gene-related peptide (CGRP) is a potent vasodilator and supposed to be responsible for neurogenic inflammation involved in migraine. Its role in inflammatory diseases of other organs is controversial and poorly investigated regarding liver inflammation, although the organ is innervated by CGRP containing primary sensory nerve fibers. METHODS Male Balb/c and IL-10(-/-) mice were pretreated with either alphaCGRP or the CGRP receptor antagonists CGRP(8-37) or BIBN4096BS. Immune-mediated liver injury was induced by administration of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNFalpha) to galactosamine (GalN)-sensitized mice and evaluated by serum transaminase activities and cytokine levels. Furthermore, intrahepatic CGRP receptor expression and hepatic CGRP concentrations were examined. RESULTS CGRP receptor 1 was expressed by immune cells and hepatocytes in human and murine liver. During liver injury CGRP receptor expression was increased whereas hepatic CGRP concentrations concomitantly decreased. While CGRP receptor antagonists failed to affect liver damage, pretreatment with alphaCGRP protected mice from GalN/LPS-induced liver injury by suppression of the pro-inflammatory cytokine response independently from IL-10 but related to the induction of the transcriptional repressor inducible cAMP early repressor (ICER). In contrast, alphaCGRP failed to protect against GalN/TNFalpha-induced liver failure. CONCLUSION In the liver, CGRP exerts anti-inflammatory properties, which are characterized by a reduced production of pro-inflammatory cytokines.

B cells and CD22 are dispensable for the immediate antiinflammatory activity of intravenous immunoglobulins in vivo

Intravenous immunoglobulins (IVIgs) efficiently suppress a variety of autoimmune diseases. Over the past few years several potential mechanisms underlying this antiinflammatory activity have become apparent. Among these, terminal sialic acid residues in the sugar moiety of the immunoglobulin G constant fragment have been shown to be critical for the antiinflammatory activity of IVIgs in models of rheumatoid arthritis and immunothrombocytopenia (ITP). More recently, B cells and the sialic acid‐binding protein CD22 were suggested to be involved in this IVIg‐dependent immunomodulatory pathway. To study whether B cells are directly involved in IVIg‐mediated suppression of acute autoimmune diseases, we tested the activity of IVIgs in mice deficient in B cells or CD22. We show that neither B cells nor CD22 are critical for the immediate antiinflammatory activity of IVIgs in mouse models of rheumatoid arthritis and ITP.

Kupffer Cell-Expressed Membrane-Bound TNF Mediates Melphalan Hepatotoxicity via Activation of Both TNF Receptors

Isolated hepatic perfusion of nonresectable liver cancer using the combination of TNF and melphalan can be associated with a treatment-related hepatotoxicity. We investigated whether, apart from TNF, also melphalan is cytotoxic in primary murine liver cells in vitro and investigated mediators, mode of cell death, and cell types involved. Melphalan induced a caspase-dependent apoptosis in hepatocytes, which was not seen in liver cell preparations depleted of Kupffer cells. Neutralization of TNF prevented melphalan-induced apoptosis and liver cells derived from mice genetically deficient in either TNFR 1 or 2, but not from lpr mice lacking a functional CD95 receptor, were completely resistant. Cell-cell contact between hepatocytes and Kupffer cells was required for apoptosis to occur. Melphalan increased membrane-bound but not secreted TNF in Kupffer cells and inhibited recombinant TNF-α converting enzyme in vitro. Melphalan induced also severe hepatotoxicity in the isolated recirculating perfused mouse liver from wild-type mice but not from TNFR 1 or 2 knockout mice. In conclusion, this study shows that melphalan elicits membrane TNF on Kupffer cells due to inhibition of TNF processing and thereby initiates apoptosis of hepatocytes via obligatory activation of both TNFRs. The identification of this novel mechanism allows a causal understanding of melphalan-induced hepatotoxicity.

FcγRIIB: a modulator of cell activation and humoral tolerance

An immune response needs to be tightly regulated to prevent excessive inflammation, which may result in the destruction of healthy tissues. At the molecular level, the strength of an immune response is determined by the integration of a multitude of positive and negative signals. This review will focus on IgG-dependent immune responses and discuss how the inhibitory receptor FcγRIIB may be involved in regulating both the afferent and efferent phases of such a response. Furthermore, we will discuss recent evidence suggesting that FcγRIIB may have important functions beyond the negative regulation of signals transduced by the B-cell receptor or activating FcγRs and could be responsible for the activity of agonistic antibodies in vivo.