Abstract

Anti-TNFα Antibodies Induce Regulatory Macrophages in an Fc Region-dependent Manner

Anne Christine W. Vos, Manon E. Wildenberg, Marjolijn Duijvestein,
Auke P. Verhaar, Gijs R. van den Brink and Daniel W. Hommes

The introduction of anti-tumor necrosis factor alpha (anti-TNFα) agents has been an important breakthrough in the treatment of Crohn's Disease (CD). However, not all anti-TNFα agents are effective in CD, suggesting that neutralizing soluble TNFα is not the most important mechanism of action in CD. The aim of this study was to examine the mechanism of action of anti-TNFα in vitro. Induction of apoptosis in T cells and in a mixed lymphocyte reaction (MLR) was determined by annexin V staining or a caspase 3 activity assay. Inhibition of proliferation was measured with a ³H-thymidine incorporation assay. To evaluate the phenotype of macrophages, cytokine profiles were determined by CBA, proliferation in a secondary MLR was measured by ³H-thymidine incorporation, and expression of markers was measured with flow cytometry. None of the anti-TNF agents showed effects on proliferation or apoptosis in activated T cells grown in isolation. Infliximab and adalimumab reduced proliferation in an MLR, whereas etanercept and certolizumab did not. This effect was completely abolished after blocking Fc receptors. Also, the infliximab F(ab')2 fragment, lacking the Fc region, did not inhibit proliferation in an MLR, whereas certolizumab Ig (with an Fc region) gained the ability to inhibit proliferation, indicating a role for the Fc receptor in this assay. Anti-TNF agents induced a new population of macrophages in an Fc region-dependent manner. These macrophages appeared to have a strong immunosuppressive phenotype, as indicated by their capacity to inhibit proliferation of activated T cells, produce anti-inflammatory cytokines and express the regulatory macrophage marker CD206. We showed that anti-TNF agents can induce regulatory macrophages in an Fc-region dependent manner. Regulatory macrophages have immunosuppressive properties and play an important role in wound healing. This mechanism of action of anti-TNF agents may play a role in mucosal healing in patients with IBD.

Epigenetics and Autoimmunity at a Glance

Pierre Youinou

Epigenetics is defined as stable and heritable changes in gene expression that are not accompanied by alterations in the DNA sequence. Epigenetic modifications involve either methylation of cytosine in CpG dinucleotides or covalent post-translational modifications of the histones. The first piece of evidence of epigenetic involvement in the development of autoimmunity comes from in vivo studies showing that prolonged treatment with DNA methylation inhibitors induces a lupus-like disease. Interestingly, such an effect has been reproduced after adoptive transfer of DNA-hypomethylated CD4+ T cells and -hypomethylated B cells. The second piece of evidence comes from the analysis of twins, showing that changes in DNA methylation reflect twin discordances in systemic lupus erythematosus (SLE). The third piece of evidence comes from analysis of the different blood cell populations in SLE, synoviocytes in rheumatoid arthritis, or neural cells in multiple sclerosis. In SLE, we and others have established that T and B cells are characterized by a profound DNA methylation defect associated with a reduction of DNA methyltransferases and histone acetylation. As a consequence, promoter DNA demethylation permits transcriptional activation of normally repressed genes such as those for cytokines, activated cell surface receptors, and human endogenous retrovirus. Such effects have been related to a blockage in the protein kinase C delta/Erk pathway and/or a growth arrest at the G0/G1 interface. One of the most important aspects of epigenetic regulation is the possibility of reversion; for example, blocking the IL-6 autocrine loop in SLE B cells restores DNA methylation, thus creating possibilities for the development of new therapeutics and diagnostic biomarkers.

Guided Growth of the Spine

Eric Wall and Donita Bylski-Austrow

The success of guided growth methods to correct limb angular deformity has generated interest in applying growth modification methods to correct angular deformity of the spine. The history of growth modulation dates back almost 100 years. Recent animal studies on spine growth guidance have identified the reasons for prior failure of staples and tethers to modify spine growth, and have prompted new device designs to solve these problems. Preclinical animal studies also have identified the mechanism of spine growth modification at the microscopic level. Recent attempts to correct spine growth in children with scoliosis have shown some success amongst the failures. Several technical challenges remain, given that a growth modulation device in the human spine usually crosses the intravertebral joint and disc in addition to the growth plate. Recent advances in both genetic and skeletal maturity identification have improved the prediction of those children who have the highest risk of scoliosis progression and brace failure. If the technical difficulties of spine growth modification are solved, it may become the primary surgical treatment for scoliosis in a growing child or adolescent.

Neuropeptide Effects in the Trigeminal System
- Relevance to the Pathophysiology of Migraine

Karl Messlinger, Jochen K. Lennerz, Mirjam Eberhardt
and Michael J. M. Fischer

The neuropeptides substance P, calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) have been considered important mediators in migraine and other primary headaches. CGRP and VIP have been found at increased concentrations in jugular venous plasma during attacks of migraine or cluster headache, and CGRP receptor antagonists have recently been shown to be effective in migraine therapy. Substance P and CGRP are produced from a subset of trigeminal afferents and VIP from parasympathetic efferents. Release of these neuropeptides in the meninges causes arterial vasodilatation or mast cell degranulation and plasma extravasation. CGRP released from central terminals of trigeminal afferents in the spinal and medullary dorsal horn seems to facilitate nociceptive transmission via a presynaptic mechanism. The central effect of CGRP is substantiated by the suppression of nociceptive c-fos activation and neuronal activity in the spinal trigeminal nucleus following CGRP receptor inhibition. The distribution of immunoreactivity of CGRP receptor components supports these proposed functions and additionally indicates possible CGRP effects mediated by glial CGRP receptors. Infusion of nitric oxide (NO) donors is known to induce delayed headache attacks in migraineurs. The same treatment in rats increased the stimulated CGRP release from isolated trigeminal ganglion and increased the number of CGRP- and neuronal NO synthase-immunoreactive trigeminal ganglion neurons. The increase in spinal trigeminal activity evoked by NO donors in rats was reversed by CGRP receptor inhibition. The currently available data point to multiple sites of CGRP action in trigeminal nociception and the pathogenesis of migraine.

Targeted Therapy for Cholangiocarcinoma

Gregory J. Gores, Boris Blechacz and Rory Smoot

Cholangiocarcinoma is a frequently lethal neoplasm originating from the intra- and extra-hepatic biliary tree. It often arises in the context of chronic biliary tract inflammation and fibrosis. Given this context for biliary tract carcinogenesis, we have examined the role of inflammatory cytokines in biliary tract carcinogenesis and progression. The inflammatory cytokine IL6 is a potent survival factor for cholangiocarcinoma cells. IL6, via a STAT3 signaling cascade, induces expression of the anti-apoptotic Bcl-2 protein Mcl-1. Mcl-1 is the most potent survival factor of the Bcl-2 family of proteins against TRAIL-mediated cytotoxicity. Mechanisms to inhibit STAT3 signaling by sorafenib down-regulate Mcl-1 expression and sensitize cholangiocarcinoma cells to TRAIL-mediated cytotoxicity. Indeed, sorafenib sensitizes cholangiocarcinoma cells to TRAIL cytotoxicity both in vitro and in vivo in a syngenic/orthotopic rodent model. These data suggest that mechanisms to inhibit IL6 signaling and STAT3 signaling in combination with TRAIL therapy make a viable therapeutic strategy for the treatment of human cholangiocarcinoma.