Alcohol affects the late differentiation of progenitor B cells

Abstract

Aims: Previous studies show that alcohol exposure can affect the differentiation of progenitor B cells. Before final commitment to a B lineage, progenitor B cells usually undergo several important stages. However, it is still unclear whether alcohol alters B cell differentiation at which stages. The aim of this study was to determine which stage(s) of progenitor cell differentiation are affected by alcohol and to elucidate the mechanism(s) responsible for the effect of alcohol on B cell differentiation.

Methods: Oligoclonal-neonatal-progenitor (ONP) cells from bone marrow cells of 2-week-old mice were cultured under different conditions in vitro with or without the exposure of 100 mM alcohol. Phenotype analysis was performed at different time points and expression levels of transcription factors (TFs) and cytokine receptors were measured quantitatively and kinetically.

Results: After 3 days in vitro culture, ONP cells differentiated into two populations: B220(-)CD11b(-) and B220(-)CD11b(+) cells. B220(-)CD11b(-) cells can further differentiate into B lineage cells only with the support of B220(-)CD11b(+) cells. Cells exposed to 100 mM of alcohol during the first 3 days of culture showed no statistically significant difference in B cell formation after 12 days compared with the control group. However, cells exposed to alcohol from Day 4 till the end of culture yield very few B cells. Expression levels of TFs and cytokine receptors were down-regulated kinetically among ONP cells co-cultured with the addition of 100 mM alcohol.

Conclusions: Alcohol affects the ONP cell differentiation into B lineage at a late stage. Alcohol also down-regulates the expression level of TFs and cytokine receptors resulting in the impairment of B cell differentiation.

Fig. 1.

ONP cells can differentiate into different lineages in vitro. ONP cells can differentiate into both myeloid and lymphoid lineage. (A) showed ONP cells yielded CD19⁺ cells after 12 days bulk culture in liquid medium containing SCF, FL, IL-3 and IL-7. However, when cultured in above medium with SCF, FL, IL-3 and GM-CSF, ONP cells yielded only CD11b⁺ cells (B). (C) and (D) showed the different morphologies of clones derived from a single ONP cell after cultured for 17 days with different culture condition.

Fig. 2.

B220⁻CD11b⁻ cells can differentiate into B cells with the support of OP9 stromal cells. ONP cells were sorted and cultured with SCF, FL and IL-3 for 3 days, phenotype analysis showed <5% cells differentiated into B220⁻CD11b⁻ cells (A). When sorted B220⁻CD11b⁻ cells separately and cultured with OP9 stromal cells for another 9 days, over 90% of these cells differentiated into B cells (B).

Fig. 3.

Alcohol affects the late stage of the ONP cell differentiation into a B lineage. ONP cells were sorted and first cultured with SCF, FL and IL-3 for 3 days. After 3 days in vitro culture, cells were differentiated into two populations: B220⁻CD11b⁻ and B220⁻CD11b⁺ cells (A). These B220⁻CD11b⁻ cells started to lose their surface expression of c-Kit (F). B220⁻CD11b⁻ cells were sorted and ‘splint’ into two wells, cells in one well were cultured with OP9 stromal cells without alcohol for another 9 days and yielded B220⁺ cells (B). These B220⁺ cells were further stained with anti-CD19 (B-cell marker) showed over 90% of CD19 positive expression (C). Cells in the other well were cultured with OP9 stromal cells with the addition of 100 mM of alcohol for another 9 days. Very few cells differentiated into a B lineage (D) with minimal expression of CD19 (E).

Fig. 4.

Alcohol does not affect the early stage of the ONP cell differentiation. ONP cells were sorted and cultured with SCF, FL, IL-3 and 100 mM of alcohol for the first 3 days. Cells were analyzed after 3 days and showed two cell populations: B220⁻CD11b⁻ and B220⁻CD11b⁺ cells (A). There is no statistically significant difference between early alcohol-treated and normal control groups (P > 0.05, A and Fig. 3A). These B220⁻CD11b⁻ cells also lost their surface expression of c-Kit (F). After 3 days of culture, B220⁻CD11b⁻ cells were also sorted and ‘split’ into two wells, cells were all co-cultured with the support of OP9 stromal cells. Cells in one well without the exposure of alcohol were differentiated into B220⁺ cells (B) with strong CD19 expression (C) and reached no statistically significant difference (B, C versus Fig. 3B and 3C P > 0.05). Cells in the other well with the addition of 100 mM of alcohol yielded no B cells (D and E).

Fig. 5.

Alcohol down-regulated the transcription factor expression during the ONP cell differentiation. ONP cells were harvested at day 0, 3, 6, 9 and 12 during in vitro culture in both normal control and alcohol groups. The expression level of EBF, Pax-5 and IL-7Rα were analyzed using real-time PCR. Results showed the relative expression levels of TFs EBF (A), Pax5 (B) and cytokine receptor IL-7Rα (C) were increased kinetically in the control group but not in the alcohol group.