Colchicine prevents tumor necrosis factor-induced toxicity in vivo.

Tumor necrosis factor (TNF) toxicity was induced in vivo by intravenous administration of 15 micrograms of recombinant murine TNF-alpha per kg to galactosamine-sensitized mice. Within 8 h, the animals developed a fulminant hepatitis. Intravenous administration of 0.5 mg of colchicine per kg at 19 and 4 h prior to TNF challenge protected the animals against hepatitis. Lipopolysaccharide (LPS)-stimulated, bone marrow-derived macrophages from C3H/HeN mice released significant amounts of TNF in vitro. When such macrophages were intravenously given to LPS-resistant galactosamine-sensitized C3H/HeJ mice, these animals died within 24 h. Preincubation of these transferred macrophages with colchicine did not suppress the LPS-inducible TNF release from these cells. Concordantly, administration of macrophages exposed to colchicine in vitro resulted in full lethality. However, in vivo pretreatment of C3H/HeJ mice with colchicine 19 and 4 h prior to the transfer of LPS-stimulated macrophages prevented lethality. In LPS-responsive NMRI mice which had been protected against galactosamine-LPS-induced hepatitis by pretreatment with colchicine, TNF was still released into the blood. We conclude from our findings that the in vivo protection by colchicine is mediated by blocking TNF action on target cells while the effector cells of LPS toxicity, i.e., the macrophages, remain responsive.     

Axial and appendicular bone mineral density in patients with long-term deficiency or excess of calcitonin

Whether calcitonin deficiency causes and calcitonin excess prevents bone loss is controversial. We therefore measured plasma calcitonin levels and bone mineral density at the radius (by single photon absorptiometry) and lumbar spine (dual photon absorptiometry) in patients with an excess or deficiency of calcitonin. We studied 21 patients who had undergone subtotal thyroidectomy 6.8 to 29 years previously and had no calcitonin secretory reserve, and 11 patients who had received a diagnosis of medullary thyroid carcinoma 6.8 to 23 years previously and had chronic hypercalcitoninemia. Bone-density values, expressed as Z-scores (i.e., as the number of standard deviations above or below the normal means adjusted for age and sex), were indistinguishable from normal in the patients who had undergone thyroidectomy (means +/- SE: radius, 0.36 +/- 0.15; spine, 0.27 +/- 0.17). In the patients with medullary thyroid cancer, radial bone-density values were normal (-0.26 +/- 0.39), but spinal density was significantly reduced (-0.75 +/- 0.17, P less than 0.01). There were no significant correlations between the duration of calcitonin excess or deficiency and the bone density at either site. Bone mineral density was not affected by whether or not thyroxine replacement therapy was given. We conclude that skeletal mass is not affected by endogenous plasma calcitonin in adults.     

In vivo regulation of inducible no synthase in immune-mediated liver injury in mice

Inducible nitric oxide synthase (iNOS) has been shown to play an important role in the development of liver injury. iNOS deficiency protects mice from hemorrhage/resuscitation as well as from cytokine-mediated liver injury, for example, after administration of concanavalin A (con A). Here we investigated the in vivo effects of tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma, two mediators of con A-induced liver injury, the TNF receptor (TNFR) usage leading to iNOS expression, and its connection with nuclear factor kappaB (NF-kappaB) activation. In conclusion, iNOS expression in vivo is dependent on both TNF-alpha and IFN-gamma. Although con A-induced liver injury depends on both TNFR1 and TNFR2, TNF-dependent iNOS expression is mediated exclusively by TNFR1 and requires NF-kappaB activation.     

Antigen presentation,autoantibody production,and therapeutic targets in autoimmune liver disease

The liver is an important immunological organ that controls systemic tolerance. The liver harbors professional and unconventional antigen-presenting cells that are crucial for tolerance induction and maintenance. Orchestrating the immune response in homeostasis depends on a healthy and well-toned immunological liver microenvironment, which is maintained by the crosstalk of liver-resident antigen-presenting cells and intrahepatic and liver-infiltrating leukocytes. In response to pathogens or autoantigens, tolerance is disrupted by unknown mechanisms. Intrahepatic parenchymal and nonparenchymal cells exhibit unique antigen-presenting properties. The presentation of microbial and endogenous lipid-, metabolite- and peptide-derived antigens from the gut via conventional and nonconventional mechanisms can educate intrahepatic immune cells and elicit effector responses or tolerance. Perturbation of this balance results in autoimmune liver diseases, such as autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis. Although the exact etiologies of these autoimmune liver diseases are unknown, it is thought that the disruption of tolerance towards self-antigens and microbial metabolites and lipids, as well as alterations in bile acid composition, may result in changes in effector cell activation and polarization and may reduce or impair protective anti-inflammatory regulatory T and B cell responses. Additionally, the canonical and noncanonical transmission of antigens and antigen:MHC complexes via trogocytosis or extracellular vesicles between different (non) immune cells in the liver may play a role in the induction of hepatic inflammation and tolerance. Here, we summarize emerging aspects of antigen presentation, autoantibody production, and the application of novel therapeutic approaches in the characterization and treatment of autoimmune liver diseases.     

Regulatory T Cell–Derived IL-10 Ameliorates Crescentic GN

Regulatory T cells (Tregs) exert their immunosuppressive activity through several immunoregulatory mechanisms, including the production of anti-inflammatory cytokines such as IL-10. Although several studies suggest a role for Tregs in modulating crescentic GN, the underlying mechanisms are not well understood. Here, using IL-10 reporter mice, we detected IL-10–producing Foxp3⁺ T cells in the kidney, blood, and secondary lymphoid tissue in a mouse model of crescentic GN. Specific inactivation of Il10 in Foxp3⁺ Tregs eliminated the ability of these cells to suppress renal and systemic production of IFNγ and IL-17; these IL-10–deficient Tregs lost their capacity to attenuate renal tissue injury. These data highlight the suppressive functions of Tregs in crescentic GN and suggest the importance of Treg-derived IL-10 in ameliorating disease severity and in modulating both the Th1 and most notably Th17 immune response.The discovery of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in the 1990s and their indispensable role in (self) tolerance and autoimmunity marked the beginning of a new era in immunology.¹ Since then, different suppressive mechanisms mediated by various Treg cell subsets were identified,^2,3 particularly in well studied models of autoimmune diseases such as Crohn''s disease,⁴ multiple sclerosis,⁵ or rheumatoid arthritis.^6,7 Until now, only limited numbers of studies have assessed the function of regulatory T cells in crescentic GN. Adoptive cell transfer experiments in mice showed the beneficial role of exogenous wild-type (wt) CD4⁺CD25⁺ Tregs in attenuation of crescentic GN,⁸ whereas CCR6- and CCR7-deficient CD4⁺CD25⁺ Tregs failed to protect mice against GN.^9,10 Recently, our own published data revealed the importance of endogenous Foxp3⁺ Tregs in suppressing the Th1 immune response and consequently ameliorating the disease severity in the T cell–dependent GN model of nephrotoxic nephritis (NTN).¹¹ Concurrently, Ooi and coworkers confirmed the relevance of endogenous Foxp3⁺ Tregs in an accelerated model of experimental crescentic GN.¹² However, the mechanisms of Treg cell-mediated suppression in crescentic GN are still unclear. One important player might be the anti-inflammatory cytokine IL-10, which is known to be released by Tregs in order to suppress immune responses and therefore might protect against autoimmunity.¹³ Indeed, endogenous IL-10 regulates the Th1 immune response in an accelerated model of experimental crescentic GN, as kidney damage is aggravated in IL-10–deficient mice.¹⁴ However, the source of protective IL-10 still needs to be clarified. Because IL-10 detection and tracking in vivo is difficult, most findings are based on studies with IL-10^−/− mice.Therefore, to study the cell-specific function of IL-10, we used a double-knockin reporter mouse model (Foxp3-IRES-mRFP (FIR) x IL-10 ires gfp-enhanced reporter [tiger]), which enables detection of the well-defined and simultaneous expression of IL-10 (green fluorescent protein [GFP]) and Foxp3 (monomeric red fluorescent protein [mRFP]). Indeed, we detected a distinct population of renal mRFP⁺(Foxp3⁺) Tregs expressing GFP (IL-10) upon induction of NTN. Thus, to investigate the role of Treg cell-derived IL-10 in NTN, we first adoptively transferred CD4⁺CD25⁺ Tregs from wt or IL-10^−/− mice into wt mice subsequently challenged with nephrotoxic sheep serum. Adoptively transferred wt Tregs attenuated the course of NTN, whereas IL-10^−/− Tregs did not. Furthermore, to analyze the role of endogenous IL-10 produced by Tregs, we generated Foxp3^YFP-Cre x Il10^flox/flox mice, in which IL-10 is selectively inactivated in Foxp3⁺ Tregs.¹⁵ Indeed, lack of Treg-derived IL-10 resulted in an aggravated course of NTN. In summary, we demonstrated a crucial role of Treg cell-derived IL-10 in regulating the Th1 and most notably the Th17 immune response in NTN. Hence, this study contributes to the understanding of the suppressive mechanisms of Tregs in crescentic GN and will have biologic implications for designing therapeutic approaches.     

Cytokine modulation and suppression of liver injury by a novel analogue of thalidomide

Thalidomide has been shown to reduce the production of tumor necrosis factor-alpha (TNF-alpha), a cytokine with deleterious pathophysiologic effects in various diseases. In search of thalidomide analogues with improved TNF-alpha inhibiting properties, 5-ethyl-1-phenyl-5-(3,4,5,6-tetrafluorophthalimido)barbituric acid (TFBA) was found to be superior to thalidomide. Besides TNF-alpha, TFBA also suppressed interleukin-6 and interleukin-10 production of isolated monocytes. The possibility that TFBA exerts its action by increasing levels of cAMP via inhibition of phosphodiesterase-4 activity was excluded. TFBA had no influence on T cell proliferation; neither did it inhibit TNF-alpha production in peripheral blood mononuclear cells stimulated by anti-CD3 monoclonal antibody. When applied to mice treated with D-galactosamine and lipopolysaccharide, TFBA prevented a rise in serum TNF-alpha, had no effect on interleukin-6 levels and led to an increase in interleukin-10 production. The changes in cytokine production observed in vitro and in vivo were reflected by similar changes in the mRNA expression. Moreover, TFBA significantly reduced liver transaminase levels in D-galactosamine/lipopolysaccharide-treated mice and thus efficiently protected the animals from liver injury. Thus, according to its properties, TFBA has the potential of modulating an immune response by acting as an anti-inflammatory agent.