Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling

Abstract

Peritoneal B cells represent a heterogeneous mixture of mature peripheral B lineage subsets with distinct developmental and functional characteristics. Here, we report that a single intraperitoneal injection of intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) results in the stable fluorescent labeling of resident lymphocytes, without dissipation of the tracer compound into other peripheral lymphoid organs. Using this in situ labeling procedure followed by multicolor flow cytometry or tissue fluorescence at various periods for up to 4 weeks post-labeling, we demonstrate that the distinct peritoneal leukocyte sub-populations and, within the B lineage, B-1 and B-2 B-cell subsets have different exchange kinetics with extraperitoneal sites under steady-state conditions. The B cells labeled with CFSE showed only minimal localization to other peripheral lymphoid tissues. On the other hand, a substantial fraction of both B-1 and B-2 subsets labeled with CFSE accumulated within the pleural cavity, although at a lower frequency than in the peritoneum. We also show that exposure to LPS induces a rapid re-distribution of peritoneal B lymphocytes and an enhanced entry of B-1 cells in the pleural cavity, in addition to augmenting the egress and the division-linked reduction of CFSE fluorescence of both B-1 and B-2 cells. These data indicate that following their in situ labeling, peritoneal lymphocytes show preferential accumulation in serosal cavities, although with a differential efficiencies for T, B-1 and B-2 lymphocyte subsets.