Plasma preparation BALB/c normal mice and lupus-prone MRL/MpJ-or BALB/c mice were diluted in 500 l of PBS

Plasma preparation BALB/c normal mice and lupus-prone MRL/MpJ-or BALB/c mice were diluted in 500 l of PBS. showed that antibodies from MRL-mice bound better to MPs from apoptotic cells than those from NZB/W mice. Together, these studies indicate important differences in the serological features of the two strains as reflected by the capacity of antibodies to bind to MPs. and studies, cell death can lead to extracellular DNA that varies in molecular size and properties. In addition to apoptosis and necrosis, NETosis, which is characterized by the extrusion of high molecular DNA to form an anti-bacterial mesh, produces extracellular DNA either locally or systemically [5, 6]. These considerations suggest that elucidating the antigenic components of complexes is important for understanding the generation of the complexes and devising strategies to block their formation and activity. As shown in studies on cell free DNA and RNA in the blood, circulating nuclear molecules can exist in either a soluble (or free) or particulate forms. The most abundant particles in blood are called microparticles (MPs) [7]. MPs are small membrane-bound vesicles that are usually 0.1 to 1 1 Clec1b micron in diameter and differ from exosomes which are much smaller and originate from the cell interior. While platelets can release MPs during activation, MPs from nucleated cells most likely derive from blebs during apoptosis; blebs are bubble like structures that form on the cell surface and detach by a budding process. The function of blebs is not known, although these structures can contain nuclear as well as cytoplasmic molecules which undergo translocation during apoptosis. MPs have important pro-inflammatory and pro-thrombotic activities and can mediate intercellular communication via their molecular contents [8, 9]. Importantly, blebs are a major source of nuclear autoantigens that are targeted in SLE, with their presence in these structures potentially enhancing immunogenicity [10, 11]. In a previous study, we explored the antigenicity of MPs generated by cell lines undergoing apoptosis [12]. Using flow cytometry (FACS), we showed that murine monoclonal autoantibodies as well as IgG from the plasma of lupus patients can bind particles. These studies showed further that the plasma of lupus patients have dramatically increased numbers of particles expressing IgG, indicative of IC formation, with levels of IgG-positive particles correlating with levels of anti-DNA. Other investigators have reported similar results [13, 14]. Together, these studies raise the possibility that MPs may be an important source Amuvatinib hydrochloride of ICs in lupus, differing in dimensions, molecular composition and immunological activity compared to ICs formed from circulating nuclear molecules. In the current study, we have extended this analysis to murine autoimmunity and investigated the role of MPs in generating circulating ICs in the NZB/W and MRL-lupus models. For this purpose, we used FACS analysis to measure IgG-positive MPs in the plasma from mice collected over time and further investigated the binding of plasma IgG to purified MPs. As results of these studies show, the two strains differ markedly in the number of IgG-positive particles in plasma as well as the ability of plasma IgG to bind to particles of or origin. Whereas MRL-mice, like patients with SLE, consistently have circulating IgG-positive Amuvatinib hydrochloride MPs, NZB/W mice have a much lower number of such particles that occur sporadically among individual animals. The plasmas of these strains also differ in their ability to bind to MPs generated and NZB/W mice differ in the specificity of autoantibodies as well as the structure of immune complexes. 2. Materials and Methods 2.1. Plasma preparation BALB/c normal mice and lupus-prone MRL/MpJ-or BALB/c mice were diluted in 500 l of PBS. The pool was first centrifuged at 1,000 x g for 10 min and then recentrifuged at 16,000 x g for 30 min to sediment the MPs. The MP pellet was washed in PBS by centrifuging again at 16,000 x g for 30 min. The resulting MP pellet was resuspended in 500 l of PBS for use in assays. Jurkat, THP-1 and HL-60 cells were obtained from the Duke University Comprehensive Cancer Center Cell Culture Facility and were cultured at 37C and 5% CO2 in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 20 g/ml gentamicin (Invitrogen) and 10% fetal bovine serum (Hyclone, Logan, UT). Cells were adjusted to a concentration of 2.5 106 cells/ml and treated with 10 M etoposide for 20 hr to induce apoptosis. Microparticles were obtained by differential centrifugation as described above 2.4. Determination of microparticle numbers MPs from mouse plasma and Amuvatinib hydrochloride MPs from human cell lines were analyzed.