Systemic lupus erythematosus (SLE), also known as Lupus disease, is an autoimmune disease in which the immune cells of the body attack body’s own tissues.
Authors: Ansa Naeem, Eman Azhar
In this disease, the long-lived plasma cells are are responsible for the production of anti- cardiolipin and anti-RNA antibodies, whereas the short-lived plasma cells, which are more susceptible to anti-proliferation therapies, are believed to be the producers of anti- DNA antibodies.
In this research article, Microarray analysis of gene expression is used to characterize circulating plasma cells in subjects with people suffering from SLE. Flow cytometry is used to sort plasma cells and comparator B cell populations from active SLE blood, normal blood and normal tonsil. The gene expression profiles of the sorted B cell populations are then compared.
1. Peripheral Blood and Tonsil Populations:
Tonsil B cell populations were obtained from young patients (age 2–10). The tonsils of these patients were disaggregated and separated by Ficoll gradient centrifugation.
The mononuclear cell layer was harvested, washed in phosphate-buffered saline (PBS), and resuspended in ACK lysing buffer to remove small numbers of red blood cells. After washing and resuspension in 10 mL PBS with 10% bovine serum albumin, cells were counted and prepared for staining for flow cytometry
Peripheral blood mononuclear cells, PBMCs, were stained with mouse anti-human IgD FITC (Pharmingen) or goat anti-human IgD FITC and mouse anti-human CD19 PE. Each of the SLE subjects had increased numbers of circulating plasma cells (PC) phenotypically expressing CD19dimIgD−CD38++.
The tonsillar mononuclear cells were incubated with mouse anti-human IgD FITC or goat anti-human IgD FITC, anti-CD38 APC and mouse anti-human CD19 PE. Stained cells were analyzed with the use of the FACS Calibur or CyAN or sorted with a MoFlo Cell Sorter. Paint-a-Gate, CellQuest and Summit were used to analyze data generated by flow cytometry.
3. Microarray analysis of gene expression
Sorted B cell subpopulations were placed in TRIZOL for RNA extraction. Isolated RNA was further purified with the RNeasy mini Kit and processed for microarray analysis using the standard Affymetrix protocols. Briefly, 1–10 µg RNA was reversed transcribed into cDNA. The template cDNA was purified for amplification and in vitro transcription to cRNA. cRNA was biotin labeled, purified and hybridized.
Data and Statistical analysis
The Ig secreting signatures of tonsil PC and PB were defined by comparing the tonsil PC to the tonsil naïve, pre-germinal center/germinal center founder (GCF), germinal center (GC) or dark zone, and post germinal center memory B cells to determine the differential gene expression. These populations are defined based on cell surface markers referred to in the methods.
To visualize the individual gene expression levels within each sample, each gene is represented as a color with bright red representing the greatest expression level, bright green the least and black as equivocal.
The genes that were significant in each comparison were tabulated and the median of the gene expression value was computed for each gene across the each column or sample in the heatmap.
To visualize the individual gene expression levels within each sample, each gene is represented as a color with bright red representing the greatest expression level, bright green the least and black as equivocal. The genes that were significant in each comparison were tabulated and the median of the gene expression value was computed for each gene across the each column or sample in the heatmap.