This study aimed to investigate phenotype of RP105(−) B cell subsets in patients with systemic lupus erythematosus (SLE). Flow cytometry was used for phenotyping RP105-negaive B cell subsets. Based on CD19, RP105, and CD138 expression, RP105(−) B cells consist of at least 5 subsets of late B cells, including CD19(+)RP105(int), CD19(+) RP105(−), CD19(low) RP105(−) CD138(−), CD19(low) RP105(−)CD138(int), and CD19(low) RP105(−) CD138(++) B cells. Especially, CD19(+)RP105(int) and CD19(low) RP105(−)CD138(int) B cells are significantly larger than other RP105(−) B cell subsets in SLE. By comparison of RP105(−) B cell subsets between patients with SLE and normal subjects, these subsets were detectable even in normal subjects, but the percentages of RP105(−) B cell subsets were significantly larger in SLE. The phenotypic analysis of RP105(−) B cell subsets suggests dysregulation of later B cell subsets in SLE and may provide new insights into understanding regulation of B cells in human SLE.
Systemic lupus erythematosus (SLE) is a typical systemic autoimmune disease characterized by production of various autoantibodies including anti-double-strand (ds) DNA antibodies from B cells [
RP105 (CD180) is one of the homologues of Toll-like receptors (TLRs). RP105 expresses on mature B cells, macrophages, and dendritic cells (DCs) [
We have previously reported that enlarged population of RP105-lacking [RP105(−)] B cells in peripheral blood (PB) is an outstanding feature in patients with active SLE [
Late B cells, including plasmablasts and plasma cells, play critical roles in humoral immune response and autoimmune diseases [
Patients with active SLE (
The following monoclonal antibodies (mAbs) were used in our studies fluorescein isothiocyanate-(FITC-) conjugated, phycoerythrin- (PE-) conjugated, or allophycocyanin- (APC-) conjugated antihuman CD19, FITC-conjugated or PE-conjugated antihuman RP105, FITC- or PE-conjugated anti-CD19, anti-CD20, anti-CD22, anti-CD24, anti-CD27, anti-CD28, anti-CD30, anti-CD31, anti-CD38, anti-CD40, anti-CD62L, anti-CD70, anti-CD72, anti-CD77, anti-CD79b, anti-CD80, anti-CD86, anti-CD95, anti-CD97, anti-CD126, anti-CD138, anti-CD147, anti-CD164, anti-CD200, anti-CD209, anti-CD267, anti-CD275, anti-CD279, anti-CCR7, anti-CXCR5 (CD185), anti-HLA-DR, anti-IgG, anti-IgM, anti-IgD, anto-TLR5, anti-TLR6, PE-conjugated anti-CD10, anti-CD21, anti-CD23, anti-CD25, anti-CD27, anti-CD28, anti-CD45RO, anti-CD69, anti-CD77, anti-CD122, anti-CD125, anti-CD132, anti-CD150, anti-CD152, anti-CD184 (CXCR4), anti-CCR2, anti-CCR10, anti-CX40, and anti-TLR2 were purchased from BD Bioscience (San Jose, CA, USA). The mAbs to human BCMA (B cell maturation antigen) (Vicky-1, rat IgG1), BAFF-R (B cell activating factor receptor) (11C1, mouse IgG1), and TACI (transmembrane activator and calcium modulator ligand interactor; CD267) (1A1, rat IgG2a) were obtained from ALEXIS Biochemical (Piscataway, NJ, USA). FITC- or PE-conjugated isotype-matched control mAbs were purchased from BD Bioscience. PerCP- (Peridinin chlorophyll protein-) conjugated CD138 was also obtained from BD Bioscience.
Heparinized peripheral venous blood was obtained from patients with SLE. PB mononuclear cells (PBMCs) were separated immediately by centrifugation over Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). PBMCs were washed twice and resuspended at 1 × 106 cells/mL in staining buffer.
Direct immunofluorescence was carried out with PE- or FITC-conjugated antibodies against surface antigens and stained with FITC- or PE-conjugated anti-RP105, PerCP-conjugated anti-CD138, and APC-conjugated anti-CD19 mAbs. Irrelevant isotype-matched control antibodies were used to determine background fluorescence. These samples were analyzed with the saved setting of gate. More than 500 000 viable, antibody-labeled cells were identified according to their forward and side scattering, electronically gated, and analyzed on a FACScalibur flow cytometer (Becton Dickinson). Results were expressed as percent of positive cells or mean fluorescence intensity (MFI) using WINMDI software (
Statistical analysis was performed with the Mann-Whitney
In SLE patients, there is a large population of RP105(−) B cells [
Subsets of RP105(−) B cells. (a) Representative flow cytometric profiles of RP105 expression on CD19(+) B cells from an active SLE patient and a normal subject. The population was subdivide (1) subset 1; CD19(+)RP105(int), (2)subset 2; CD19(+)RP105(−), and (3) presubset; CD19(low)RP105(−). (b) CD138 levels after gating CD19(low)RP105(−) presubset. The presubset cells were further subdivided into 3 subpopulations, subset 3; CD138(−), subset 4; CD138(int) and subset 5; CD138(++). (c) The percentages of subsets in PB of each patient and normal subject. *
We identified 4 populations of B cells in the panel of CD19 and RP105. The populations were CD19(+)RP105(+) B cells (subset 0) and three RP105(−) B cell subpopulations, CD19(+)RP105(int; intermediate) (subset 1), CD19(+)RP105(−) (subset 2), and CD19(low)RP105(−) B cells (presubset). The population of CD19(low)RP105(−) B cells (presubset) was further subdivided into 3 subpopulations, CD19(low)RP105(−)CD138(−) (subset 3), CD19(low)RP105(−)CD138(int) (subset 4), and CD19(low)RP105(−)CD138(++) (subset 5) B cells according to CD138 expression levels after gating presubset (Figure
The percentages of subsets in PB of each patient and normal subject were shown in Figure
In order to characterize these subsets, we analyzed various antigens on B cells (Figures
Flow cytometric analysis of various antigens on and in B cell subsets from a patient with SLE. Positive cell ratio or MFI (mean fluorescence intensity) was shown in flow cytometric profiles.
Expressions of important B cell markers (MFI) on RP105(−) B cells (subset 1–5) and RP105(+) B cells (subset 0). The levels of CD19, CD20, and CD24 were significantly lower in subset 3, 4, and 5 B cells compared to subset 0, 1, and 2 B cells (
On the other hand, CD38 and CD27 expressions were the lowest on subset 0 B cells. The levels of CD38 and CD27 gradually increased from subset 1 to subset 4 and 5 B cells. While subset 0, 1, and 2 B cells expressed surface IgD and IgM, subset 3, 4, and 5 B cells had lower levels of surface Igs. All the subsets of RP105(−) B cells expressed HLA-DR. However, the levels of HLA-DR gradually decreased. Low expression of CD25 was detectable in subset 0, 1, 2, and 3 B cells.
CXCR5 (CD185), a homing receptor, presented in subset 0, 1, and 2 B cells, but it disappeared in the subset 3, 4, and 5 B cells. The expression of CXCR4 (CD184) was the highest in subset 0 B cells but was lost in subset 4 and 5 B cells.
More interestingly, of BAFF receptors, BAFF-R expression was higher in subset 0, 1, and 2 B cells, but BCMA expression was conversely higher in subset 3, 4, and 5 B cells.
From subset 0 to subset 2 B cells, CD72, CD79b, and CD200 expressions were found, but those were lower on subset 3, 4, and 5 B cells. CD86, CD95, CD97, and CD126 were positive on subset 3, 4, and 5 cells. CD1a, CD1b, CD10, CD40, CD77, and CD80 were low or negative in all subsets. CD31, CD49d, and CD45RA were constantly positive on all subsets (data not shown).
We investigated and summarized the phenotype of RP105(−) B cell subsets in patients with active SLE. RP105(−) B cells consist of at least 5 subsets of late B cells, including CD19(+)RP105(int), CD19(+)RP105(−), CD19(low)RP105(−)CD138(−), CD19(low)RP105(−)CD138(int), and CD19(low)RP105-(−)CD138(++) B cells. The phenotypic analysis of RP105(−) B cells suggests mature phenotype of these RP105(−) B cells, CD20(low or lost) CD22(low or lost) CD27(high) CXCR5(low or lost).
We analyzed whether our finding of subsets of RP105(−) B cells is valid in healthy subjects. Although circulating RP105(−) B cells are very rare in healthy subjects, subsets of RP105(−) B cells were identified. Therefore, comparison of the identified B cell subsets in healthy subjects with SLE patients could lead to relevant observations.
Virtually, patterns of phenotype of RP105(−) B cells from normal subjects seemed similar to those from SLE patients. However, the expression levels of several antigens were significantly different between SLE patients and normal subjects (Figure
Comparison of surface expression of various antigens with significant difference of mean of MFI on RP105(−) B cell subsets between SLE patients and normal subjects.
RP105 (CD180) is mainly expressed on mature B cells and regulates B cell function in humans and mice [
We identified 5 subpopulations of RP105(−) B cells with different phenotype that are categorized as follows: subset (1) CD19(+)RP105(int), (2) CD19(+)RP105(−), (3) CD19(low)RP105(−)CD138(−), (4) CD19(low)RP105(−)CD138(int), and (5) CD19(low)RP105(−)CD138(++) B cells. Each subset of RP105(−) B cells showed different phenotype of B cells.
IgD and CD38 were classically used to subdivide PB B cells into four quadrants, IgD(+)CD38(−) naïve B cells (Bm1 and Bm2), IgD(+)CD38(+) Pre-GC B cells (Bm2’a and Bm2’b), IgD(−)CD38(+) GC B cells (Bm3 and Bm4), and IgD(−)CD38(−) memory B cells (Bm5) [
CD27 expression is commonly used as an exclusive marker for human memory B cells [
It is reported that in vitro human activated B cells and plasmablasts/plasma cells express CD19, whose levels, however, are lower on plasmablasts and plasma cells compared to activated B cells [
Regarding homing receptors, differentiated plasma cells are characterized by a disappearance of CXCR5, a progressive reduction in CXCR4 [
RP105(−) B cells have abundant intracellular Igs [
Collectively, each subset represents the step of B cell differentiation towards plasma cells. However, to confirm each steps comparison between PB and bone marrow plasma cells is required. Further analysis of in vitro culture of B cells that develop and differentiate into plasma cells with morphological change, functional studies, and expression of transcription factors is also important. In our results, we present the existence of increased various late B cell subsets in SLE patients.
We performed phenotype analysis in healthy subjects. The results were shown in Figures
Recently, it has been reported that increased circulating CD138(int) B cells, producing autoantibodies, are related to autoimmunity in MRL/lpr lupus mice [
Further studies will be required to determine mechanisms of appearing, differentiation, and proliferation of RP105(−) B cell in human SLE. It is possible that inappropriate enlarged population of various subsets of RP105(−) B cells in PB is greatly related to pathophysiology in SLE.
No conflict of interests has been declared by the authors.
The authors thank M. Fujisaki and K. Eguchi for their assistance with the research. S. Koarada is supported by Grant-Aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan (no. 22591077).