CCR2 deficiency disrupts the peripheral differentiation of B
cells
To investigate whether the lack of CCR2 influences B cell development
and differentiation, we first examined B cell subsets using FCM. The
CCR2 KO B cells extracted from BM were incubated with anti-BP-1 and
-CD24 Abs to distinguish B cell precursors (Fig. 1A-B ). Our
results suggest that CCR2 deficiency caused little to no alterations to
the development of B cell in BM since the proportion and absolute cell
number of precursor B cell subsets in both WT and CCR2 KO mice were
similar (Fig. 1C ). However, this was not the case for the
peripheral B cell differentiation process. By testing peripheral B cell
subsets (Fig. 1D-F ), we discovered that the proportion and cell
number of follicular (FO) B cells
(B220+IgMlowIgDhigh)
as well as the cell number of transitional type-2 (T2)
(B220+IgMhighIgDhigh)
B cells from the CCR2 KO mice were significantly
diminished compared to those
observed in WT mice
(Fig.
1G-H ). Nevertheless, both the ratio and cell number of transitional
type-1 (T1)
(B220+IgMlowIgDlow),
marginal zone (MZ)
(B220+CD23lowCD21high),
and germinal center (GC)
(B220+GL-7highCD95high)
B cells in WT and CCR2 KO mice were comparable (Fig. 1I-K ).
Furthermore, no differences were
observed for B1a
(CD19+IgD-IgM+CD5-CD11b+)
and B1b
(CD19+IgD-IgM+CD5+CD11b+)
B cells (Fig. 1L ) when comparing WT and KO mice (Fig.
1M-N ). To rule out the potential effect of other immune cells on the
abnormally differentiated B cells observed in CCR2 KO mice, BM chimeras
were generated using irradiated C57BL/6J (CD45.1+)
recipients through tail vein injection of BM cells extracted from WT and
CCR2 KO (CD45.2+) mice. Thus, we examine the inherent
reduction of FO B cells in CCR2 deficient mice (Fig. S1A-H ).
Since the kidneys are most susceptible to develop systemic autoimmune
disease, we analyzed the glomerulus of CCR2 KO mice and observed a
significant increase in IgG immune complex deposition. Additionally,
CCR2 KO mice showed increased anti-dsDNA Ab titers (Fig. 1O-P ).
While examining other organs, we observed that CCR2 KO mice exhibited
more severe lung lymphocyte infiltrations compared to that of WT mice
(Fig. 1Q, Fig. S2A ). The transcription factor T-bet expressed
by B cell has been previously associated with
autoimmunity,25 and as
expected, CCR2 KO B cells exhibited higher expression levels of T-bet
than those of the WT B cells (Fig. 1R ). Collectively, these
findings suggest that CCR2 plays an important role in the integrity
maintenance of peripheral B cell differentiation and the regulation the
peripheral autoimmunity.