Abstract
Background Pentraxin3 (PTX3) is overexpressed in kidneys of patients developing lupus nephritis (LN). Active LN is associated with reduced anti-PTX3 antibodies. However, abnormalities of B cell differentiation against PTX3 have not been characterized in systemic lupus erythematosus (SLE).
The aim of our study is to characterize PTX3-specific (PTX3+) B cells in peripheral blood of SLE patients with or without LN and healthy donors (HD).
Methods SLE patients without LN, biopsy-proven LN and matched HD were analyzed. Active LN was defined as proteinuria >0.5 g/day or CrCl <60 ml/min/1.73 m2 with active urinary sediment. Peripheral B cells were analyzed for direct PTX3 binding by flow cytometry using PTX3 labeled with cyanine 5 (Cy5) and phycoerythrin (PE).
Identification of PTX3+ B cells among CD19+CD20+ B cells. (A) Three representative dot plots of the PTX3-specific B cells before and after blocking with unlabeled PTX3. Only B cells staining positive for both PTX3-Cy5 and PTX3-PE were considered (light gray square). (B) Quantification of PTX3 biding among B cells before and after blocking of PTX3. Footnotes: Cy5, cyanin 5; PE, phycoerythrin
PTX3+ B cells are decreased in patients with lupus nephritis and are mainly confined to CD27-IgD+ B cells. (A) Absolute numbers of PTX3+ B cells (cell/mL) within left) total; middle) naïve or right) memory B cells in HD (n=22) and SLE (n=26) and LN (n=12) patients. (B) Frequencies of PTX3+ B cells (left), naïve (middle) and memory (right) are decreased in LN (n=12) in comparison with HD (n=22) and SLE (n=26). (C) Distribution of CD27 and IgD expression by PTX3+ B cell subsets are shown. Enrichment in the naïve pool with decreases in the other subsets was found in HD (n=22) and SLE (n=26), but not in LN (n=12). (D) Pie charts of percentages of PTX3+ CD27IgD subsets within the PTX3+ B cell pool are consistent with distribution of absolute numbers. Mann-Whitney U test. (*<0.05, **<0.01, ***<0.001). Footnotes: SLE, systemic lupus erythematosus; HD, healthy donors; LN, lupus nephritis; PTX3 pentraxin3
Results Initially, a flow cytometry based assay to identify PTX3 +B cells was developed by demonstrating simultaneous binding of PTX3-Cy5 and PTX3-PE. Specificity of B cells was validated by blocking experiments using unlabeled PTX3 (figure 1). We could identify circulating PTX3 +B cells in HD and patients. Notably, LN patients showed a significantly diminished number of PTX3 +B cells (SLE vs. LN p=0.033; HD vs. LN p=0.008). This decrease was identified in naïve and memory B cell compartments (naïve: SLE vs. LN p=0.028; HD vs. LN p=0.0001; memory: SLE vs. LN p=0.038, HD vs. LN p=0.011) (figure 2).
Conclusions Decreased PTX3 +B cells in LN within the naïve and memory compartment suggest their negative selection at early stages of B cell development potentially related to a decreased regulatory function. PTX3 +B cells could candidate for autoantigen-defined regulatory B cells as a striking abnormality of LN patients
