Figure 2

Nitric oxide secreted by myelin phagocytosing macrophages inhibits lymphocyte proliferation in vitro. (a) Comparison of lymphocyte proliferation following coculture with untreated, myelin-, zymosan- (Zym) or latex bead-treated macrophages. The relative proliferation is defined as the percentage of proliferating cells in experimental cultures divided by values in control lymphocyte cultures without macrophages (dotted line). Data represent the mean of two independent experiments. (b) To determine the capacity of mye-macrophages to present myelin antigens, MBP-reactive lymphocytes were cocultured with macrophages or mye-macrophages (ratio 1/10) in the absence of thymocytes and MBP. Data represent the mean of two independent experiments. (c) The role of respectively IDO, arginase and NO in the observed inhibition of proliferation, macrophage coculture (black bars) and mye-macrophage coculture (white bars), was determined by addition of an IDO inhibitor (1-MT), an arginase inhibitor (Nor-NOHA) and an iNOS inhibitor (L-NMMA). Dotted line represents the proliferation of control lymphocyte cultures without macrophages. Data represent four independent experiments. (d) Relative NO concentration in supernatants following a 4d coculture with MBP-reactive lymphocytes. Dotted line represents the NO production of control lymphocyte cultures without macrophages. Data represent the mean of 4 independent experiments. (e) Comparison of lymphocyte proliferation following coculture with untreated and myelin-treated macrophages using transwell inserts. Dotted line represents the proliferation of control lymphocyte cultures without macrophages. Data represent the mean of two independent experiments. (f) Ex vivo proliferation (black bar) and viability (white bar) of lymph node cells following a coculture with MBP and myelin. Proliferation was compared to cultures in which no myelin was added. Lymph nodes were isolated 9d-post MBP-immunization. Data represent three independent experiments. Ma: Macrophages, LN: Lymph node, TW: transwell inserts.