Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Heterogeneity in the Bone Marrow B Cell Compartment: Implications for Pathogenesis of Human RA.

Palanichamy,  Arumugam, Cistrone,  Chris, Hossler,  Jennifer, Wei,  Chungwen, Kobie,  James, Sanz,  Inaki, Anolik,  Jennifer

Purpose:

Recent studies suggest that the bone marrow (BM) may be an active compartment in the development of autoimmune responses in RA. However the precise phenotype, function and regulation of mature B cells in BM remain largely unexplored in humans. Investigation of BM compartment may provide critical insights into the role of B cells in the pathogenesis of RA

Methods:

B cells in BM and matched peripheral blood (PB) samples from 10 subjects (5 RA and 5 normal controls (NC)) were analyzed by 11 color flow cytometry. For analysis DAPI+ dead, CD3+ T and CD24hi/CD38hi early B cells were negatively gated and CD19+ mature B cells were divided into memory subsets (switched (SM): IgD-, CD27+; unswitched (USM): IgD+, CD27+; double negative (DN): IgD-, CD27-) and true naïve (N: IgD+, CD27-) population and expression of markers associated with activation and differentiation including FcRH4 and B220 were examined. In 2 NC, single B cells from memory subsets were sorted, reverse transcribed, Ig-VH3 genes amplified by nested PCR, sequenced and mutational imprints compared between BM (n=58) and PB (n=57).

Results:

Frequency of CD27+ memory B cells in RA BM remained lower compared to matched PB or normal BM (18.24±2.9 v 29.04±13.2 or 25.16±13.0; RA BM v RA PB or normal BM) which reflected in a reduction of SM B cells (7.87±3.0 v 16.86±13.6; RA BM V NC BM). Examination of expanded markers, such as B220 (which is previously shown to be expressed on a majority of CD27- and a subset of CD27+ B cells) revealed an increased number of B220 expressing BM mature naïve and USM cells in RA as opposed to NC (P<0.05). Furthermore elevated levels of B220 expressing cells were noticed in naïve and memory subsets in RA PB compared to BM suggesting tissue specific differential expression. Analysis of the FcRH4 (an inhibitory FcR homolog,) showed a slightly higher number of USM and naïve B cells from RA PBL that was FcRH4+ compared to the BM. On the molecular level, Ig gene analysis of memory subsets between BM and PB of normal controls showed no differences in the overall mutational frequencies between BM and PB. However significant changes were noted in the pattern of mutational targeting in Ig-VH framework (FR1–3) and hypervariable regions (CDR1 and 2). Of note, SM B cells from BM exhibited a 2 fold higher mutations in FR2 compared to PB, whereas in USM B cells, a 2 fold increased mutations were seen in CDR1 compared to PB. Both SM and USM had higher numbers of G mutations in hotspot motifs in PB. Mutational analyses of RA BM and PB memory subsets are underway.

Conclusion:

Molecular analysis of BM and matched PB hints increased AICDA enzymatic activity in PB revealed by G mutations and tissue specific generation/selection of distinctly targeted B cell subsets. Distinct mature B cells with consistent heterogeneity in RA BM suggest that these cells may play a role in the mounting autoimmune responses. Further analyses are needed to elucidate the functionality of these cells.

To cite this abstract, please use the following information:
Palanichamy, Arumugam, Cistrone, Chris, Hossler, Jennifer, Wei, Chungwen, Kobie, James, Sanz, Inaki, et al; Heterogeneity in the Bone Marrow B Cell Compartment: Implications for Pathogenesis of Human RA. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1085
DOI: 10.1002/art.28852

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