Chapter Four Anti-Tnfα Antibodies Induce Regulatory Macrophages in an Fc Region Dependent Manner

Towards therapeutic disease control in inflammatory bowel diseases Vos, A.C.W. Citation Vos, A. C. W. (2011, September 8). Towards therapeutic disease control in inflammatory bowel diseases. Retrieved from https://hdl.handle.net/1887/17819 Version: Corrected Publisher’s Version Licence agreement concerning inclusion of doctoral License: thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/17819 Note: To cite this publication please use the final published version (if applicable). Chapter four 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 Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands Gastroenterology 2011;140:221-230 74 Chapter 4 Abstract Anti-TNFα antibodies are effective in Crohn’s disease whereas soluble TNFα receptors have failed to show clinical efficacy. The molecular mechanism that underlies the differences between these compounds has not been elucidated. Here we aimed to examine the mechanism of action of the immunosuppressive effect of anti-TNFα antibodies on activated T cells. We studied the effect of anti-TNFα antibodies infliximab and adalimumab, the soluble TNFα receptor etanercept, pegylated F(ab’) fragment certolizumab and certolizumab-IgG on primary activated T cells. T cells were grown in isolation or in a mixed lymphocyte reaction (MLR). Proliferation was measured by 3H thymidine incorporation and apoptosis was examined using Annexin V labeling and a colorimetric assay for activated caspase-3. Mac- rophage phenotype was assayed by flow cytometry and cytokine secretion. Infliximab and adalimumab reduced proliferation in an MLR, whereas etanercept and certolizumab did not. This effect was completely abolished after blocking Fc receptors. Inflixi- mab F(ab’)2 fragment failed to inhibit proliferation whereas certolizumab-IgG gained the ability to inhibit proliferation. In the MLR anti-TNFs induced a new population of macrophages in an Fc region dependent manner. These macrophages were found to have an immunosuppressive phenotype, in terms of their capacity to inhibit proliferation of activated T cells, production of anti-inflammatory cytokines and the expression of the regulatory macrophage marker CD206. Regulatory macrophages have immunosuppressive properties and play an important role in wound healing. Our data show that anti-TNFs induce regulatory macrophages in an Fc region dependent manner. This mechanism of action of anti-TNFs may contribute to the resolution of inflammation. Anti-TNF antibodies induce regulatory macrophages in an Fc region dependent manner 75 Introduction Crohn’s disease (CD) is a chronic inflammatory bowel disease that results from a dysregu- lated immune response of unknown aetiology. 1-3 Tumour necrosis factor alpha (TNFα), a cytokine produced by activated macrophages, monocytes and T cells, 4 is a key mediator in immune responses and is increased in serum and intestine in CD and synovium in rheu- matoid arthritis (RA). 5-7 Since their introduction in the ’90s, anti-TNFα antibodies are commonly used for the treatment of Crohn’s disease. Various classes of anti-TNFs have been introduced, and although all classes efficiently neutralize TNFα, they show different efficacy profiles. The antibodies infliximab 8, 9 and adalimumab 10, 11 were shown to be effective in both inducing and main- taining remission in CD patients. On the other hand, two soluble receptors; etanercept and onercept were ineffective in inducing remission in CD patients. 12, 13 Also, the humanized anti-TNFα antibody CDP571 which was designed as an IgG4 to reduce interaction with Fc receptors in the hope to reduce side effects also failed to show effectiveness in CD. 14 These clinical data strongly suggest that neutralizing TNFα may not be the sole mechanism of action of anti-TNFα treatment in Crohn’s disease. Several effector mechanisms of anti-TNFα treatment have been proposed that are independ- ent of TNFα neutralizing activity. Examples are the induction of apoptosis in T cells and monocytes via binding of membrane bound TNFα (mTNFα), 15-17 antibody-dependent-cell- mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity. 18, 19 Furthermore, a role for Fc receptors has been proposed based on the association between a polymorphism in the Fc gamma receptor IIIa and the biological response to infliximab.20 Fc receptors bind the Fc region of an antibody or antibody-complex, resulting in myeloid cell activation, phagocytosis and cytokine secretion. The importance of Fc receptors in the mechanism of action of other antibody therapies such as anti-CD20 and anti-Human Epidermal growth factor Receptor 2 (Her2) has been described before. 21, 22 In Fcγ-/- mice which are unable to bind Fc regions, both anti-CD20 and anti-Her2 lost their efficacy in reducing tumor size, demonstrating a key role for the Fc receptor in the mechanism of action of these antibodies. Although a number of mechanisms have been suggested, it is still unclear why certain anti-TNFs are effective in Crohn’s disease and other anti-TNFs are not. Many studies on the mechanism of action of anti-TNFα focus on binding to mTNFα and reverse signaling in T cells, 18, 23 whereas the effect and importance of binding to Fc receptors has not been established thus far. In this study, we found that in order for an anti-TNFα to inhibit T cell proliferation in vitro, the compound needs to bind to mTNFα on activated T cells and posses an Fc region to interact with the Fc receptor on antigen presenting cells. Upon this binding, a distinct macrophage subset is induced with immunosuppressive capacities, including the production of anti-inflammatory cytokines and inhibition of T cell proliferation. 76 Chapter 4 Material and methods Antibodies and Reagents Infliximab, certolizumab, adalimumab and etanercept were prepared according to manufacturers’ recommendations. Certolizumab-IgG was obtained from UCB (UCB, Belgium), and IgG1k was obtained from Sigma. Cell isolation and culture Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated by Ficoll Paque density-gradient centrifugation. After washing, monocytes were isolated by Percoll density-gradient centrifugation. CD3 positive T cells were isolated from PBMCs using nega- tive magnetic bead separation (Invitrogen). For T cell activation, cells were activated with αCD3/αCD28 antibodies (Sanquin) at the indicated concentration or αCD3/αCD28 beads (Invitrogen) (1 bead/5 cells). PBMCs and T cells were cultured in RPMI 1640 containing 10% heat-inactivated FCS. Dendritic cells (DCs) were obtained by culturing monocytes with GM-CSF (50 ng/mL, R&D), IL-4 (50 ng/mL, R&D) for 7 days in AIM-V medium. To generate macrophages, monocytes were cultured in 6 wells plates for 5 days. Infliximab induced macrophages (Mφind) were isolated from MLR cultures using CD14 microbeads according to manufacturer’s protocol (Miltenyi). Next, cells were cultured in RPMI 1640, containing 10% heat-inactivated FCS. For light microscopy, cells were adhered to poly-L-lysine coated coverslips and stained using DiffQuick. Binding to membrane TNFα For mTNFα binding assays, infliximab, certolizumab, adalimumab, etanercept and control IgG were labeled with a fluorescent dye using a commercially available kit according to the manufacturers’ instructions (Alexa Fluor 647 protein labeling kit, Pierce). CD3 positive T cells were activated with αCD3/CD28 antibodies for 48 hours. Cells were collected, washed three times in FACS buffer (PBS containing 1% BSA) and incubated with various amounts of labeled anti-TNFα compound or IgG control for 30 minutes on ice. Binding of infliximab F(ab’)2 fragment was assessed in a competition assay; activated T cells were incubated with labeled infliximab (10μ g/mL) and increasing concentrations of unlabeled infliximab F(ab’)2 fragment at the same time. After washing, binding to mTNFα was analyzed by flow cytometry. Allogeneic mixed lymphocyte reaction (MLR) PBMCs from two healthy donors were cultured in a 1:1 ratio in RPMI 1640 culture medium. After 48 hours of activation, cells were treated with the indicated compound (infliximab, adalimumab, certolizumab, etanercept, Certolizumab-IgG or IgG control, all at 10 μg/mL) for up to 7 days where indicated. When appropriate, Fc receptors were saturated by treating MLRs with IgG (10 μg/mL, Sigma) for 6 – 16 hours and next treated with anti-TNF compound or IgG control for 2 days. Finally, proliferation was measured using a 3H-thymidine incorporation assay. Anti-TNF antibodies induce regulatory macrophages in an Fc region dependent manner 77 Assays for apoptosis PBMCs or isolated T cells were activated in an MLR or with CD3/CD28 antibodies for 48 hr, and treated with anti-TNF compound or control (10 μg/mL). Cells were collected, washed three times, and stained with Annexin V and Pi and analyzed by flowcytometry. For measurement of caspase-3 enzymatic activity, a colorimetric assay was used as described before. 24 Cell lysates were generated from MLR cultures and protein concentration was determined by BCA analysis (Pierce). Lysates were incubated with a saturating concentration of 25μM specific enzyme substrate Ac-Aps-Glu-Val-Asp-AMC (Ac-DEVD-AMC, Bachem, Germany) in 100mM HEPES buffer with 10% sucrose, 10mM dithiothreitol and 0.1% Nonidet-P40. Samples were incubated at 37°C and fluorescent AMC release was moni- tored (Fluostar Optima plate reader). FACS analysis Human monocytes and DCs were plated in 6 well plates (2 x 106 cells/well) and cultured with or without LPS (Sigma, 100 ng/mL) for 16 hours, and treated with anti-TNFα compound or IgG control (Sigma, 10 μg/mL) for 25 – 48 hours. Cells were harvested, washed, and stained for αCD14-FITC, αCD40-FITC, αCD80-Pe, αCD83-APC, αCD86-APC, αHLA- DR-FITC and appropriate controls (all BD) for 30 minutes on ice.

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