Background/Purpose:

B cells can have a negative regulatory role, mainly mediated by interleukin 10 (IL-10) and we recently showed that IL-10 producing B cells (B10 cells) are deficient in patients with rheumatoid arthritis (RA). However, B10 cells still remain to be better characterized and finding new markers of B10 cells is an important challenge. Annexin V (AnV) is a protein binding to phosphatidylserine (PS), a phospholipid usually located on the inner leaflet of the plasma membrane that could be externalized during some process such as apoptosis. Previous works have suggested that AnV staining, a known marker for apoptosis, could also be expressed on viable and stimulated B cells. We therefore aimed to explore the relationship between PS exposure (assessed by AnV staining) and B10 cells.

Methods:

We used PBMCs from 6 healthy controls and 7 RA patients. AnV staining was compared between human B10 cells and IL-10 non-producing B cells. Levels of CpG induced B10 cells from flow cytometry sorted AnV^pos and AnV^neg B cells were compared. Apoptosis was studied in AnV^pos and AnV^neg B cells, using different methods: DAPI staining, cellular cycle study with propidium iodide (PI) and DiOC6 staining. PS was then blocked using biotynilated AnV and glyburide to evaluate the impact on B cell IL-10 production.

Results: B10 cells externalized more PS than IL-10 non-producing B cells with respectively 27.75 [17.03-44.38]% and 12.35 [9.49-14.20]% of AnV^pos B cells (p= 0.03; n= 6 healthy controls). In RA patients, the proportion of AnV^pos B cells was also significantly higher among B10 cells than among IL-10 non-producing B cells (p= 0.03; n= 6). After CpG stimulation, the differentiation into B10 cells was more important for AnV^pos B cells than for AnV^neg B cells (5.50 [2.21-8.80]% vs 2.50 [0.75-6.09]% respectively, p= 0.03; n= 6 healthy controls). This exposure of PS does not seem to be related to apoptosis since proportions of dead cells (DAPI+) were similar between AnV^pos and AnV^neg B cells (p= 0.3; n= 6 healthy controls). Moreover, the proportions of B cells in an early apoptotic stage (DiOC6-) were similar between AnV^pos and AnV^neg B cells (p= 0.4; n=4 healthy controls). Lastly, cellular cycle study showed that, compared to AnV^neg B cells, AnV^pos B cells have a higher proliferation index (p= 0.03; n= 6 healthy controls). PS blockade decreased the levels of B10 cells with 6.59 [3.71-8.12]% of B10 cells for culture medium alone; 2.99 [1.15-5.61]% with biotynilated AnV and 3.20 [1.26-4.80]% with glyburide (p= 0.03 and p= 0.06 for biotynilated AnV vs media and glyburide vs media respectively, n= 6 healthy controls).

Conclusion: This study showed that a positive AnV staining is observed on viable B cells suggesting that AnV is not only a marker for apoptosis. In healthy controls and RA patients, B10 cells have an increased AnV staining. These AnV^pos B cells differentiate more into B10 cells and phosphatidylserine (PS) blockade inhibits B10 cells generation. These results strongly suggest a link between PS exposure and B10 cells. This could be useful to generate B10 cells in future therapeutic issues, for example for RA patients.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/phosphatidylserine-outer-layer-translocation-is-implicated-in-il-10-secretion-by-human-regulatory-b-cells/