A sharp gradient of S1P is maintained between circulation and tissues, with much higher levels in lymph (high nanomolar range) and blood (micromolar range) than within tissues (53
). Current technologies have not permitted interstitial S1P concentrations within lymphoid tissue to be directly measured. However, S1P causes internalization of S1PR1, and surface levels of this receptor have been used as a proxy for the relative amounts of S1P within a tissue to show that they are likely in the low or sub-nanomolar range (34
). Regulation of both S1P production and degradation are critical for proper S1P distribution in vivo
and for immune cell exit from lymphoid organs (55
). S1P is produced by sphingosine kinases intracellularly in all cell types, but the cell types important for the generation of secreted S1P in the extracellular space seem to be more specific. Red blood cells are an important source of plasma S1P, which contributes to thymic and splenic egress (53
), and non-hematopoietic sources contribute to plasma S1P as well (53
). Lymphatic endothelial cells produce S1P necessary for exit of lymphocytes from lymph nodes into lymph (57
), suggesting that different cell types are important for S1P production in different compartments.
S1P lyase is critical for the maintenance of the S1P gradient between circulation and tissue, as it contributes to S1P degradation and low S1P levels within tissues (55
). However, treatment of mice with an inhibitor of S1P lyase did not disrupt S1PR2 function in the GC, suggesting S1P lyase may not be critical for determining the S1P distribution pattern needed to promote GC organization (JAG and JGC, unpublished data). In addition to S1P lyase, sphingosine phosphate phosphatase 1 (Sgpp1) and Sgpp2 and three lipid phosphate phosphatases (LPP1-3) possess S1P-degradative ability. Recent evidence shows that LPP3 expression on endothelial and epithelial cells in the thymus contributes to the maintenance of low S1P levels that permit T-cell exit from the thymus into circulation (58
How S1P is distributed within B-cell follicles in such a way to exert effects on GC B-cell centering and clustering is not yet fully understood. Staining of S1PR1 in tissue sections has been used to show internalization of the receptor on B cells close to or within S1P-containing lymphatic vessels (59
), suggesting that this method could be used as an indirect test of the S1P levels to which B cells are exposed within follicles. In several attempts with this technique, we were not able to detect differences in the ratio of surface to intracellular S1PR1 on B cells in inner and outer regions of the follicle (JAG and JGC, unpublished data). This may in part have been due to background staining with the polyclonal rabbit antibody (Santa Cruz Biotech), as we observed B-cell staining in follicles of mice conditionally lacking S1PR1 from B cells (S1PR1f/- Mb1Cre mice; JAG and JGC, unpublished data). Even with adequate sensitivity, one reason that this technique might fail to detect an S1P gradient in the follicle is that B cells traverse distances of several microns per minute, possibly moving between regions of differing S1P concentration in time frames that are faster than the rate of S1PR1 internalization and recycling. However, several findings imply that S1P is present in a decaying gradient in the follicle (). The first is that S1PR2 overexpression in B cells favors their movement to the center of the follicle (31
). S1PR2 engagement by S1P inhibits migration to chemoattractants, so by extension if cells are present in a uniform field of attractant (such as CXCL13) S1PR2-expressing cells are most likely to move in the direction of lowest S1P. Second, the influence of S1PR2 on GC cell distribution in mixed chimeras, to be discussed further below, was lessened when chimera hosts lacked the ability to produce S1P, but not when S1P production was lacking specifically in FDCs (32
). Thus, the important source(s) of S1P are stromal cells that are not the FDCs in the center of the follicle, implying that much of the relevant S1P is being produced outside the follicle center.
S1P’s half-life in plasma is on the order of 15 min, suggesting tight temporal and spatial control of S1P distribution within tissues would be possible (56
). Red blood cells in the plasma do not express S1P-degrading enzymes (53
), while B cells in the follicle do and can efficiently degrade S1P in vitro
), suggesting that the S1P half-life in the follicle is likely very short and that B cells could actively maintain low S1P levels. In particular, B cells express higher levels of the ectoenzyme LPP3 than T cells, suggesting they may have a specialized ability to degrade extracellular S1P (32
). Further, there is not extensive entry of naive B cells or other cell types into the GC throughout the GC response, suggesting that circulatory S1P would not be carried into the GC and would likely be degraded by B cells in the follicle before it reached the GC. As such, it seems reasonable to speculate that the center of the follicle and the GC in particular is a region of low S1P, allowing S1PR2 to contribute to maintaining GC confinement. It will be useful to determine if B cell-specific expression of enzymes with S1P-degrading potential is important for maintaining low levels of S1P in the follicle, and to narrow the source of relevant S1P to a specific subset of stromal cells.