TNF-A Antibody Therapy in Combination with the T-Cell–Specific Antibody Anti-TCR Reverses the Diabetic Metabolic State in the LEW.1AR1-Iddm Rat
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2880 Diabetes Volume 64, August 2015 Anne Jörns,1,2 Ümüs Gül Ertekin,1 Tanja Arndt,1 Taivankhuu Terbish,1 Dirk Wedekind,3 and Sigurd Lenzen1 TNF-a Antibody Therapy in Combination With the T-Cell–Specific Antibody Anti-TCR Reverses the Diabetic Metabolic State in the LEW.1AR1-iddm Rat Diabetes 2015;64:2880–2891 | DOI: 10.2337/db14-1866 Anti–tumor necrosis factor-a (TNF-a) therapy (5 mg/kg b-cell death (1–3). The infiltrate in the pancreatic islets is body weight), alone or combined with the T-cell–specific composed of different immune cell types, especially macro- antibody anti–T-cell receptor (TCR) (0.5 mg/kg body phages and T cells (2–4). Upon activation, the immune cells weight), was performed over 5 days immediately after release proinflammatory cytokines and other cytotoxic disease manifestation to reverse the diabetic metabolic mediators, causing b-cell apoptosis (3,5). state in the LEW.1AR1-iddm rat, an animal model of hu- Effective prevention strategies require a combination man type 1 diabetes. Only combination therapy starting therapy (6–8) to target the proinflammatory cytokines pro- at blood glucose concentrations below 15 mmol/L re- duced in the different immune cell types. Of crucial impor- stored normoglycemia and normalized C-peptide. In- tance is in this context the proinflammatory cytokine tumor b creased -cell proliferation and reduced apoptosis led necrosis factor-a (TNF-a). It is expressed in all immune cell to a restoration of b-cell mass along with an immune types infiltrating the pancreatic islets in patients with T1D ISLET STUDIES cell infiltration–free pancreas 60 days after the end as well as in the spontaneous mouse (NOD mouse) and rat of therapy. This combination of two antibodies, anti- (BB, Komeda, and LEW.1AR1-iddm rats) models (3,9). TCR/CD3, as a cornerstone compound in anti–T-cell Cumulative evidence from studies in a variety of other therapy, and anti–TNF-a, as the most prominent and a autoimmune diseases supports the contention that the effective therapeutic antibody in suppressing TNF- fl action in many autoimmune diseases, was able to re- in ammatory process in the affected organs always goes – – verse the diabetic metabolic state. With increasing along with a high TNF-a expression (9 13). Anti TNF-a blood glucose concentrations during the disease pro- therapy is therefore an established therapeutic principle – fl gression, however, the proapoptotic pressure on the (14 16) in rheumatoid arthritis, in ammatory bowel dis- residual b-cell mass increased, ultimately reaching eases, lupus erythematosus, Sjögren syndrome, psoriasis, a point where the reservoir of the surviving b-cells was and Hashimoto thyroiditis (10,15,17). insufficient to allow a restoration of normal b-cell mass Anti–TNF-a monotherapy in T1D is ineffective (18–21) through regeneration. The present results may open and may even aggravate the disease (22,23). It is probably a therapeutic window for reversal of diabetic hyper- not surprising in an autoimmune disease such as T1D, in glycemia in patients, worthwhile of being tested in which the pancreatic b-cells are so extremely vulnerable clinical trials. (24), that these cells not only become dysfunctional but are also quickly destroyed. As has been documented in diabetic LEW.1AR1-iddm Type 1 diabetes (T1D) is an immune cell–mediated disease (IDDM) rats, as well as in pancreases from patients with that causes insulin dependence due to selective pancreatic T1D (3), the islet-infiltrating immune cells produce and 1Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany This article contains Supplementary Data online at http://diabetes 2Centre for Anatomy, Hannover Medical School, Hannover, Germany .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1866/-/DC1. 3 Institute for Laboratory Animal Science, Hannover Medical School, Hannover, A.J. and Ü.G.E. contributed equally to this work. Germany © 2015 by the American Diabetes Association. Readers may use this article as Corresponding author: Sigurd Lenzen, [email protected]. long as the work is properly cited, the use is educational and not for profit, and Received 9 December 2014 and accepted 11 March 2015. the work is not altered. diabetes.diabetesjournals.org Jörns and Associates 2881 release, along with interleukin (IL)-1b, TNF-a as the dom- previously described (36). To identify changes in the inating proinflammatory cytokine (3,5,25). pancreatic islets, one biopsy was collected on the day Combination therapies, at variance from monothera- of T1D manifestation, immediately before the start of pies, have shown promising results in T1D (25–29) therapy, and another at the end of therapy; the remaining and other autoimmune diseases (11,14,15). We there- pancreas, comprising head and tail, pancreas-draining fore investigated in the IDDM rat, an animal model of lymph nodes, and serum, were collected 60 days after humanT1D,whichmirrorsthe human T1D situation the end of therapy (25). Tissue specimens were fixed for most closely (3,5,30,31), whether a combination therapy microscopic analyses (25,36). Blood glucose concentrations of anti–TNF-a, which suppresses TNF-a release from all were determined daily (Glucometer Elite; Bayer, Leverkusen, immune cells, with anti–T-cell receptor (TCR), which Germany). Serum C-peptide was analyzed with a rat-specific suppresses proinflammatory cytokine release from ac- ELISA (Mercodia, Uppsala, Sweden) and serum cytokine pro- tivated T cells, can be successful. Anti-TCR is an anti- tein concentrations with a multiplex immunoassay kit (Bio- body against T cells that binds to an epitope of the Rad, Munich, Germany) (25,36). a/b-chains of the TCR (32). It is comparable in action Morphological Analyses to anti-CD3, which binds to the ´-chain of the CD3 Serial sections were stained with the avidin-biotin com- molecule (33). Both antibodies bind noncovalently to plex method or the double-immunofluorescence tech- the TCR/CD3 complex and modulate T cells by affect- nique with primary antibodies for b-cells and immune ing the trans-signaling pathway (32,33). Anti-TCR is cells (3,36). The antibodies against TNF-a and T cells used in rats because anti-CD3 is available for mice recognized epitopes other than those targeted by the (33) and humans (33,34) but not for rats (25,35). We antibodies used for therapy. b-Cell apoptosis was quan- show in this study that this combination therapy tified by TUNEL (Roche, Mannheim, Germany), as well scheme is capable of successfully reversing the diabetic as proliferation by a double staining with Ki67 antibody metabolic state. (COOH-terminal; Acris, Herford, Germany) and insulin – RESEARCH DESIGN AND METHODS (D3E7; AbD Serotec). At each time point, 25 40 pan- creatic islets were studied (25,36). A minimum of 1,000 Animals b-cells were counted in the proliferation and apoptosis Congenic rats (for details see http://www.mh-hannover analyses (25,36). Islet infiltration was scored as de- .de/3642.html) were bred and maintained as described scribed (25). The b-cell mass, identified by insulin and (25,30). Experimental procedures were approved by the GLUT2 staining on sequential sections, was calculated District Government of Hannover (LAVES, nos. 33-42502- by the ratio of the b-cell area (mm2)tothewholepan- 05/958 and 509.6-42502-03/684). creatic area, determined four times in distances of 100 Experimental Groups mm each and multiplied by the pancreas weight. Anal- Four experimental groups were studied. Group 1 (n =5) yses were performed using an Olympus BX61 micro- comprised healthy, normoglycemic IDDM rats without scope (25,36). therapy; group 2 (n = 12) comprised acutely diabetic In Situ RT-PCR IDDM rats treated for 5 consecutive days with rat- Pancreatic sections from all experimental groups were specificanti–TNF-a (5 mg/kg body weight [bw] i.v.; pro- placed on three-chamber slides. The in situ RT-PCR vided by Janssen Research & Development, Spring analysis was performed on a special thermal cycler (MJ House,PA).Group3(n = 12) comprised diabetic rats Research, Waltham, MA) (25,36). Primer sequences are treated for 5 consecutive days with anti–TNF-a and in provided in Supplementary Table 2. addition with anti-TCR (0.5 mg/kg bw i.v.) (R73; AbD Serotec, Munich, Germany). Group 4 (n =5)comprised Real-time RT-PCR untreated diabetic IDDM rats. All therapies were started The quality of the mRNA isolated from pancreas- within 1 day after T1D onset at blood glucose concen- draining lymph nodes was controlled with the Experion trations .10mmol/L.Animalsingroups1and4re- electrophoresis system (Bio-Rad). mRNA was quanti- ceived 0.9% NaCl solution. Anti-TCR causes slight fied in real-time RT-PCR reactions performed with the lymphocyte reduction in the periphery (,20%), which DNA Engine Opticon fluorescence detection system disappears within 1 week after cessation of therapy (MJ Research) (36). Primer sequences are provided in (25). Anti–TNF-a was without effect and also did not Supplementary Table 3. further reduce the lymphocyte count in combination Statistics therapy with anti-TCR (data not shown). The CD4-to- Results are presented as mean values 6 SEM. Compari- CD8 T-cell ratios were not affected by any of the treat- sons among the different therapy groups and the normo- ments (Supplementary Table 1). glycemic or diabetic control rats were analyzed with the Pancreatic Biopsies and Tissue Processing Mann-Whitney U test and ANOVA, and correlation coef- Biopsies of tissue (30 mg) were obtained from the pan- ficients were calculated according to Pearson with the creas tail of each animal under isoflurane anesthesia as Prism 5 program (GraphPad Inc., San Diego, CA). 2882 TNF-a/Anti-TCR Therapy Reverses Diabetic State Diabetes Volume 64, August 2015 RESULTS both parameters in the diabetic rats.