IFN-Regulated Chemokines Are Associated with SLE Disease Activity

<p>Shown are 20 serum analytes that exhibited significant positive (highlighted in yellow) or negative (highlighted in blue) correlation coefficients with clinical measures of SLE. The IFN gene score was calculated from 82 IFN-inducible transcripts measured by concurrent whole blood gene expression microarrays. The chemokine protein “score” was calculated using the seven IFN-regulated CC and CXC chemokines highlighted in red. * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001; **** <i>p</i> < 0.0001. <i>p</i>-Values were obtained by permutation testing.</p

Coordinate Dysregulation of Serum Protein Levels in SLE

<p>Hierarchical clustering was applied to protein levels of the identified analytes (“Serum Protein Levels”). Individual data points represent the log₂ ratio of the analyte concentration to the mean of control concentrations (scale shows linear fold-differences). “In Vitro Control PBMC Gene Expression” columns present gene expression microarray data obtained by incubating normal control PBMCs in vitro with medium alone or with type I IFN for 6 and 24 h. Data are normalized to control medium-alone conditions (scale depicts linear fold-differences). Grey boxes indicate missing data. Analytes induced by type I IFN in vitro (>2-fold and >500 expression unit mean difference) are highlighted in red font.</p

Genes Differentially Expressed between Ctrl-M^hi and Cre-M^lo Cell Populations

<p>Shown are representative genes that were generally either (A) up-regulated or (B) down-regulated in Cre-M^lo cells compared with Ctrl-M^hi cells. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030082#pbio-0030082-g002" target="_blank">Figure 2</a> legend for details.</p

Immature B Cells That Lose Basal Signaling Show Induction of LC Rearrangements

<div><p>(A) PCR analysis of endogenous Ig light chain rearrangements (V-Jλ3, V-Jλ1, RS, and V-Jκ1) in genomic DNA of FACS-sorted HEL-Ig/Rag2-GFP BM cells incubated with medium alone or with 300 ng/ml herbimycin A for 24 h. IgM^a+GFP⁺ cells were sorted from herbimycin A-treated cultures, and IgM^a+GFP⁻ were sorted from control cultures. Data are from three independent experiments. CD14 is a loading control. −, negative control (C57Bl/6J tail DNA); +, positive control (C57Bl/6J spleen DNA).</p> <p>(B) Genomic DNA from the same cell populations described in (A) was subjected to ligation-mediated PCR to detect double-strand signal end DNA breaks at Jκ1. Controls in right three lanes of blot: H₂O, control; −, negative control (S17 stroma); +, positive control (C57Bl/6 BM).</p> <p>(C) Genomic DNA was extracted from B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre immature B cells 3 d following incubation with IFNαβ. Quantitative PCR analysis was used to determine the fold-induction of LC rearrangements in B1-8f/3-83κ/Mx-Cre immature B cells treated with IFN compared to medium alone. Data represent the mean ± standard deviation of two (V-Jλ1), three (V-Jλ3), or four experiments (V-Jκ1, RS).</p></div

Microarray Gene Expression Analysis Demonstrates Co-Clustering of Cre-Deleted IgM^lo Cells with IgM⁻ Cell Populations

<div><p>(A) FACS sorting strategy for Ctrl-M^hi, Cre-M^hi, and Cre-M^lo immature B cells incubated with IFNαβ (1,000 units/ml) for 2 d. The numbers shown indicate the percentage of gated cells.</p> <p>(B) Affymetrix microarrays were used to identify genes differentially expressed between IFN-treated Ctrl-M^hi and Cre-M^lo cells. Analysis identified 327 transcripts that met the following criteria: 2-fold or greater change in mean expression level, a more than 200-unit difference in mean expression values, and Student's t-test <i>p</i> < 0.01. Individual expression values for each gene were divided by the mean of expression levels for three control IgM⁺ cell populations: IFN-treated Ctrl-M^hi; IgM^hi cells sorted from 5-d IL-7 HEL-lg BM cultures (HEL-M^hi); and sorted from normal Balb/c BM (FxE). Other populations included Cre-M^hi, Hardy Fraction D pre-B cells (FxD) sorted from normal Balb/c BM, and lgM⁻ cells sorted from 5-d IL-7 cultures of control B6 BM (B6-M⁻). Data were transformed into log₂ space, and represent fold-differences relative to the IgM⁺ cell populations (see scale bar). Data from 293 transcripts (duplicates and all but four representative Ig HC and LC transcripts removed) were clustered and visualized using CLUSTER and TREEVIEW [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030082#pbio-0030082-b51" target="_blank">51</a>]. Red represents genes expressed at higher levels, while green represents genes expressed at lower levels, than the mean of IgM⁺ cells. Each column represents an individual sorted cell population.</p></div

Inducible Cre-Mediated Deletion of the B1-8f HC Allele Leads to Loss of Surface Ig from Immature B Cells

<div><p>(A) Flow cytometry showing surface phenotype of B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre BM after 5-d IL-7 culture.</p> <p>(B) Flow cytometric analysis of B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre BM culture cells incubated with IFNαβ (1,000 or 5,000 units/ml), in the absence of IL-7, for 1, 2, or 3 d. The cell populations shown are gated on lymphocytes by forward and side scatter, and then for B220. At the end of the 5-d IL-7 culture, more than 90% of the cells were viable and B220⁺. The numbers shown indicate the percentage of gated cells.</p></div

Protein Expression Confirms Reversal of Development in Immature B Cells Losing Surface IgM

<p>B1-8f/3-83κ control and B1-8f/3-83κ/Mx-Cre cell populations were harvested at the end of a 3- or 4-d culture with IFNαβ (3,000 units/ml), stained with mAbs for cell surface proteins, and analyzed by flow cytometry. Shown are the expression levels of B220-gated Ctrl-M^hi (thick line) and Cre-M^lo (thin line) cells at the end of the culture period. Essentially identical results were observed when Cre-M^hi and Cre-M^lo cells were compared (data not shown).</p