Li, Winslow, Davis, Mellins, Utz, Crabtree, Parnes. Manuscript preparation. Jewish Medical and Research Center, Denver, CO). Detection of ANAs and kidney immune complex deposition For analyses of glomerular immune complexes, frozen kidney sections were stained with fluorescein isothiocyanate (FITC)Cconjugated anti-mouse IgG antibodies (Jackson ImmunoResearch, West Grove, PA). More than a dozen glomeruli on each kidney section were evaluated. For detection of ANAs, HEp-2 cells were incubated with mouse serum, followed by staining with FITC-conjugated anti-mouse IgG antibodies. Sera from lupus-prone MRL-lpr/lpr mice were used as a positive control. To determine in vitro production of anti-HEL IgM antibodies, purified naive mature (CD43?) B cells were incubated with or without HEL antigen (0.5 sections in paraffin, and stained with hematoxylin and eosin. All slides were examined by a board-certified veterinary pathologist (CRD), in a blinded manner, using light microscopy. Organs were evaluated for the extent of changes in specific histopathologic features of inflammation, where 0 = no change, 1 = minimal to mild EMD638683 change, 2 = moderate change, and 3 = severe change. Genotype analysis Genotyping was carried out by polymerase chain reaction (PCR) analyses of genomic DNA, using microsatellite markers of simple sequence-length polymorphisms distributed among the 19 autosomal chromosomes. Control DNA for PCR analysis of each marker was obtained from a C57BL/6ByJ mouse and from E14.1 Rabbit Polyclonal to BMX embryonic stem cells. Statistical analysis Student’s values less than or equal to 0.05 were considered significant. Results Phenotypic characterization of CD72-deficient anergic B cells An HEL anergic B cell model in mice (26) was utilized to investigate whether CD72 plays a role in B cell anergy. C57BL/6 mice carrying transgenes (BCR specific to HEL) and transgenes were bred with CD72?/? mice to assess the EMD638683 function EMD638683 of CD72 in the presence and absence of a defined self antigen (HEL). Control and CD72?/? mice expressing alone (and transgenes (double-transgenic [dTg]) were used to study the role of CD72 in regulating self tolerance. B cells from 0.04) (Figure 1A). Open in a separate window Figure 1 Analyses of B cell proliferation and B cell death in vitro in 0.04; ** = 0.001. C, Anti-HEL autoantibody production in mature splenic dTg B cells after stimulation with HEL antigen with or without anti-CD40. Bars show the mean and SEM of duplicate cultures. D, Assessment of B cell proliferation, using 3H-thymidine incorporation, after stimulation with HEL (0.5 0.001) (Figure 1B). Thus, it appears that, given an equivalent number of B cells in vivo, dTg mice with CD72 deficiency will have a 6-fold increase in anti-HEL autoantibody production as compared with dTg control mice. Antibody production was also examined in vitro. 0.01. E, ERK activity after stimulation with HEL. Results were obtained by in vitro kinase assay using Elk as the substrate. Total Elk was used to show equal loading. F, Akt phosphorylation and Akt kinase activity. Results were obtained by in vitro kinase assay using glycogen synthase kinase 3 (GSK3) as the substrate. EMD638683 Total GSK3 was used to show equal loading. Results in E and F are representative of at least 2 independent experiments. See Figure 1 for other definitions. Mitogenic signaling requires the activation of MAPK signaling pathways. Both the basal ERK activity and the BCR-induced ERK activity were increased in dTg CD72?/? B cells (Figure 2E). The EMD638683 basal activity of JNK was higher in dTg anergic B cells from CD72?/? mice than in those from control mice. Moreover, after stimulation with HEL antigen, JNK activity was also induced to a greater extent in dTg CD72?/? B cells (results not shown). Similarly, p38 kinase activity was induced to a higher level in the absence of CD72 (results not shown). These results indicate that MAPK signaling appears to be generally increased in dTg anergic B cells that lack CD72-mediated negative regulation. Akt signaling contributes to the survival of many primary and transformed cell types. B cells from the dTg CD72?/? mice, which had increased survival after stimulation, also had increased Akt phosphorylation and higher Akt kinase activity (Figure 2F). Taken together, these results indicate that CD72 likely influences BCR-induced proliferation and survival of dTg anergic B cells at the early stage of BCR signaling as downstream effectors are increased in the absence of CD72 (8). Interaction of CD72 with Cbl-b and regulation of Cbl-b phosphorylation We next examined.