Deletion Of The Von Hippel-Lindau Gene Interferes With The Development Of Peripheral B-Cell Subsets

The development of hematopoietic cells occurs in highly specialized microenvironments within bone marrow, thymus and spleen. Hematopoietic stem and progenitor cells are positioned at the lowest end of a bone marrow oxygen gradient, which implies a role for the hypoxic-response pathway in regulating hematopoiesis. In this pathway the von Hippel-Lindau protein (pVhl) is the central negative regulator and continuously mediates the proteasomal destruction of the hypoxia-inducible factor-1α (Hif-1α). Under hypoxic conditions Hif-1α destruction is inhibited and results in the expression of hypoxia-inducible genes.

In order to study the role of pVhl in hematopoiesis we crossed Vhl conditional mice with vav-iCre mice to induce a constitutive hematopoiesis-specific Vhl deletion (VhlKO^vav). As expected, we observed a dramatic expansion of spleen erythropoiesis, however bone marrow lin^-Sca-1⁺c-kit⁺CD48^-CD150⁺ hematopoietic stem cell numbers of VhlKO^vav mice did not significantly differ from control mice. The most striking observation in VhlKO^vav mice was that B-cell numbers in bone marrow and spleen were decreased by 53% and 78%, respectively. In order to exclude that the loss of B-cells in VhlKO^vav mice was due to a B-cell extrinsic effect we crossed Vhl conditional mice to the B-cell specific deleter lines CD19-Cre and Mb1-Cre (VhlKO^CD19 and VhlKO^Mb1 mice). Flow cytometric analysis also revealed decreased peripheral B-cell numbers in VhlKO^CD19 mice and an even more pronounced B-cell loss in VhlKO^Mb1 mice (B-cells spleen, mean±SEM; control (n=9), 77±2.4x10⁶; VhlKO^Mb1 (n=12), 1.3±0.2x10⁶; p<0.001). This demonstrated that pVhl is cell-autonomously required for the normal development of the B-cell compartment. To more accurately define the Vhl-null B-cell developmental defect we analyzed bone marrow B-cell subsets of VhlKO^Mb1 and control mice. Early B-cell progenitor numbers defined by the surface markers B220, CD43, CD24, BP-1 (Hardy’s classification, fractions A-D) of VhlKO^Mb1 mice were not altered. In contrast, we observed a significant 41% reduction of the immature B220⁺CD43^-IgM⁺IgD^- and a 46% reduction of the mature B220⁺CD43^-IgM⁺IgD⁺ bone marrow VhlKO^Mb1 B-cell numbers compared to controls. In peripheral blood VhlKO^Mb1 immature and mature B-cell numbers were even more decreased (by 81% and 86%, respectively). We hypothesized that increased CXCR4 expression, which is negatively regulated by pVhl, could be responsible for the decreased bone marrow egress of immature bone marrow B-cells. Indeed, we observed a more than twofold increase of CXCR4 expression of VhlKO^Mb1 compared to control bone marrow immature B-cells (MFI, mean±SEM; control (n=3), 72±15; VhlKO^Mb1 (n=3), 162±23; p<0.05). Strikingly, VhlKO^Mb1 spleens were almost devoid of follicular B220⁺CD21/35^intmCD23⁺IgD⁺IgM⁺ and marginal zone B220⁺CD21/35^highCD23^-IgM^high B-cells and accordingly follicular structures could not be observed in histological sections. In VhlKO^Mb1 lymph nodes the follicular B-cell numbers were also dramatically decreased. Next, we flow sorted residual splenic VhlKO^Mb1 B-cells and were able to confirm deletion of the Vhl-gene by PCR. Target genes of the hypoxic-response pathway such as Pgk1, Vegf and Bnip3 were 10- to more than 100-fold higher expressed in sorted VhlKO^Mb1 compared to control B-cells. As a possible reason for the low VhlKO^Mb1 peripheral B-cell numbers we identified a more than twofold reduction in CD62L expression by immature blood B-cells (MFI, mean±SEM; control (n=3), 3127±250; VhlKO^Mb1 (n=3), 1528±66; p<0.05) which presumably impaired their homing ability to peripheral lymphoid organs. Additionally, we detected an increased B-cell apoptosis rate of VhlKO^Mb1 B-cells in the spleen. Finally, we were able to show that decreased follicular splenic B-cell numbers of VhlKO^CD19 mice could be completely rescued by additionally breeding Hif-1α conditional alleles into the system (Hif-1αVhlKO^CD19mice).

In summary, we identified pVhl as a key regulator of peripheral B-cell maturation. We show that pVhl-mediated negative regulation of the hypoxic-response pathway is required for normal peripheral B-cell differentiation. Our data suggest that B-cell pVHL loss-of-function leads to decreased bone marrow egress and decreased lymphoid organ homing of immature B-cells mediated by the dysregulation of CXCR4 and CD62L.