Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

et al. (2012) Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients. PLoS ONE 7(12): e51867. doi:10.1371/journal.pone.0051867 Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients Refik Pul 0 Franco Morbiducci 0 Jelena S kuljec 0 Thomas Skripuletz 0 Vikramjeet Singh 0 Ute Diederichs 0 Niklas Garde 0 Elke Verena Voss 0 Corinna Trebst 0 Martin Stangel 0 Martin Sebastian Weber, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Germany 0 1 Department of Neurology, Hannover Medical School , Hannover, Germany, 2 Center for Systems Neuroscience, Hannover , Germany Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 mg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14++CD16+ cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/ 61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14++CD16+ monocyte subset. - Funding: This study was partially supported by TEVA Pharmaceutical Industries. The open access publication has been supported by the German Research Foundation (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: RP declares that he has received travel funds from Baxter, Bayer HealthCare, Biogen, Novartis, Sanofi-Aventis, and Merck Serono in the last six years. TS declares that he has received travel funds from Novartis, Merck, and Bayer Healthcare. NG, FM and JS declare that they have no competing interests. CT has received honoraria for consultation and expert testimony as well as travel grants from Bayer Health Care, Biogen Idec, Diamed Medizintechnik GmbH, Fresenius Medical Care, Merck Serono, Novartis, Sanofi-Avnetis, Talecris Biotherapeutics GmbH, and Teva Pharmaceutical Industries. MS has received honoraria for scientific lectures and/or travel funds from Baxter, Bayer Healthcare, Biogen Idec, CSL Behring, Merck-Serono, Novartis, Sanofi-Aventis, Talecris, and Teva. EVV declares that she received travel funds from Novartis and speakers honoraria from Biogen idec in the last six years. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. . These authors contributed equally to this work. Circulating blood monocytes are critical effectors of the innate immune response regulating the adaptive immunity [1]. During inflammatory disorders, such as multiple sclerosis (MS), monocytes are repeatedly recruited from the periphery, thereby reinforcing the local inflammatory reaction within the central nervous system (CNS) [2,3]. Inflammatory lesions in the CNS of MS patients and animals with experimental autoimmune encephalomyelitis reveal an abundant presence of mononuclear phagocytic macrophages which originate from resident microglia and infiltrating monocytes [4,5]. Equipped with a large array of scavenger receptors these cells are involved in repair mechanisms by removing myelin breakdown products since it has been identified that myelin debris is a substantial inhibitor of remyelination [6]. Depletion of blood monocytes resulted in impaired remyelination underlining that this replenishing pool of myeloid cells is required to meet the demands for efficient repair [7,8]. Among the approved drugs for MS treatment, GA has been shown to exert immunomodulatory effects not only on T cells but also on cells of the myelo-monocytic lineage [9,10,11,12,13,14]. Accordingly, treatment of dendritic cells (DCs) with GA increased the secretion of the anti-inflammatory cytokine interleukin (IL)-10 and decreased that of the major Th1 polarizing factor IL-12 as well as tumor necrosis factor (TNF)-a [9,11]. Moreover, GA broadly inhibited the activation of monocytes and promoted development of type II monocytes which secreted more IL-10 and transforming growth factor-b, and less IL-12 and TNF-a [14]. Independently of antigen specificity, these monocytes directed differentiation of Th2 cells and T regulatory cells (Tregs) [15]. Adoptive transfer of type II monocytes reversed EAE, suppressed Th17 cell development, and promoted both Th2 differentiation and expansion of Tregs in recipient mice [10,15]. Recently, we demonstrated that GA drives microglia to adopt a type II APC similar to monocytes and DCs suggesting a general effect on myelo-monocytic cells [16]. We additionally discovered that GA directly and distinctly increases phagocytic activity of primary rat microglia. In the present study, we focused on human blood monocytes and examined whether this effect can be observed in monocytes of MS patients treated with GA. Since GA has been shown to bind strongly to the integrin macrophage-1 antigen (CD11b/CD18) and to increase the level of the T cell immunoglobulin mucin-3 (TIM-3) mRNA in peripheral blood mononuclear cells (PBMC), we performed further in vitro experiments to evaluate possible mechanisms explaining the increased phagocytic activity [17,18]. Materials and Methods Patients A total of 13 GA treated (20 mg per day subcutaneously) and 20 non-treated subjects who met the criteria for relapsing-remitting MS according to revised McDonald criteria (2005) were enrolled [19]. A (...truncated)