While strides have been made in the understanding of how intravenous immunoglobulin (IVIg) ameliorates autoimmune diseases by using mouse models of immune thrombocytopenia (ITP) and inflammatory arthritis, the precise cellular target of IVIg has remained elusive. To determine which cell populations interact with IVIg upstream of the inhibition of phagocytosis, we have developed a method for ‘priming’ specific leukocyte populations with IVIg to study their potential role in ameliorating ITP upon passive transfer, which we describe herein. Using this technique, we found that splenic CD11c+ dendritic cells reacted with IVIg, or several IVIg mimetic regimes, were primed to ameliorate ITP upon transfer to thrombocytopenic mice. We anticipate that this novel methodology could also be used to determine the mechanism of action of IVIg in other autoimmune diseases in addition to ITP.
The precise mechanism of action of intravenous immunoglobulin (IVIg) in the amelioration of autoimmune diseases such as immune thrombocytopenia (ITP) has, until now, been difficult to ascertain (1,2). Studies using gene knockout mice have demonstrated that in the treatment of murine ITP, IVIg requires the expression of the inhibitory receptor FcγRIIB (3,4) and that IVIg requires CSF-1-dependent macrophages to increase FcγRIIB expression on CSF-1-independent macrophages (5). IVIg has also been demonstrated to affect the function and maturation of dendritic cells in vitro (6,7). However, the precise cellular target(s) of IVIg has remained, until recently, an enigma. In an attempt to answer these questions, we have developed a method for isolating and ‘priming’ splenic leukocytes or specific splenic cell populations with i) IVIg, ii) soluble immune complexes (sIC) or iii) antigen-specific antibodies plus crosslinking antibodies. Splenic white cells are used for priming as is (whole leukocytes) or purified into CD11b negative and positive or CD11c negative and positive populations using magnetic bead-coupled monoclonal antibodies. Cells are then incubated with IVIg, sIC or antigen-specific antibodies (as above) at 37oC for 30 min, washed and injected into mice one day prior to injection of anti-platelet antibody to induce ITP (8). This method avoids the actual administration of IVIg and allows the researcher to isolate specific cell populations, prime them and passively transfer them to thrombocytopenic mice in order to determine their potential role in IVIg function. Recently, using this technique we found activating Fcγ receptors on CD11c+ dendritic cells to be the target of IVIg and IVIg mimetics in the amelioration of ITP in our mouse model (9). We anticipate that this new technique could also be used to determine the mechanism of action of IVIg in other models of autoimmune disease.