Our data demonstrate that while NF-κB p50-/- mice are clearly deficient in MZ B cells for the first 2 months of life, MZ B cells appear with age, such that by 6 months a normal splenic MZ B cell compartment has been reconstituted. There are at least four distinct but not mutually exclusive mechanisms that could account for the late appearance of cells within the MZ of p50-/- mice. First, these B cells could be memory B cells, which have been reported to accumulate in the MZ . While we cannot formally rule out this possibility, B cells from p50-/- mice respond poorly to antigenic challenge, especially T-dependent antigens . Consequently, it is unlikely that memory B cells would be generated in these mice, irrespective of their age. Alternatively, the emergence of MZ B cells in p50-/- mice could represent the accumulation of these cells through homeostatic proliferation, in which cells proliferate to fill niches in response to deficits in specific subsets of cells . While splenic B cells from p50-/- mice will undergo homeostatic proliferation as a consequence of their transfer into SCID recipients , the ability of the few MZ B cells in the p50-/- donor spleen to expand following transfer was not examined . Nevertheless, we view it as unlikely that homeostatic proliferation accounts for the accumulation of MZ B cells in the spleens of older p50-/- mice, since the accumulation of cells brought on by homeostatic proliferation is generally observed over a much shorter period of time, days to a couple of weeks, rather than the months required to reconstitute MZ B cells in p50-/- mice. A third possibility is that a NF-κB p50-dependent signal transduction pathway that activates a developmentally required transcription program in MZ B cells or their precursors is overcome by compensatory mechanisms with aging. NF-κB p50 is involved in a number of pathways crucial for B cell development and maturation, including B cell receptor (BCR) signalling and responses to members of the TNF family [33–35] Signal strength through the BCR has been proposed to be an important determinant of B cell lineage fate decisions, including the MZ B cell pool [36, 37] BAFF signalling is also important for the selection of B cells into the MZ B cell pool , while an intact LT-β R signalling pathway is required for the proper positioning of these cells . The accumulation of MZ B cells in older p50-/- mice could reflect compensatory mechanisms in one or more of these signal transduction pathways important for the emergence or maintenance of MZ B cells. A fourth possibility is that the small numbers of MZ B cells that successfully emerge in p50-/- mice survive for extended periods of time, resulting in the slow accumulation of this subset of cells with age. Hao and Rajewsky  have demonstrated that the half-life of MZ B cells is greater than that of FO B cells. Thus, a simple explanation for the accumulation of MZ B cells in p50-/- mice with age is that these cells preferentially survive, and thus their frequency relative to the FO B cell pool and other B cells in the spleen increases slowly with age.
B cells from p50-/- mice are compromised in their ability to respond to specific antigen challenge, and are completely unresponsive to mitogenic substances such as LPS . However, our data suggests that MZ B cells from p50-/- mice display some functionality, including the binding, transport and deposition of IgM-IC onto FDCs, a function unique to MZ B cells . Like IgM-containing T-dependent immune complexes, MZ B cells also bind T-independent type 2 (TI-2) antigens in a complement and CR1/2-dependent manner . Despite the fact that p50-/- mice are widely considered to be devoid of MZ B cells, the presence of small numbers of these B cells in p50-/- mice were in fact noted in the initial studies of Pillai and colleagues, and these appeared to bind low levels of TI-2 antigens . However, further analysis of the consequences of this binding, such as the transfer of immune complexes to FDCs, was not evaluated. The results of the current study not only extend these previous studies, but demonstrate that one consequence of the small amount of IgM-IC binding manifests itself by the limited, but none-the-less detectable, deposition of these complexes onto FDCs. This suggests that this function of MZ B cells is completely independent of the activation of NF-κB, at least those complexes containing p50. These results extend other evidence suggesting that the activation of MZ B cells through engagement of the BCR may not be required for transport and deposition of IgM-IC, since MZ B cells from CD19-deficient mice, which also have deficits in MZ B cells, are also functional in this assay .
Together, these results raise the question of whether MZ B cells require activating signals to migrate into B cell follicles following IgM-IC binding to CR1/2. MZ B cell migration can be detected in 1 h or less after the binding of IgM-IC . The binding of IgM-IC to these cells may interfere with one or more of the mechanisms proposed to play a role in MZ B cell positioning, including retention by macrophages within the MZ , integrin-mediated retention , or responsiveness to lysophospholipids in the blood . Once released, the MZ B cells would be expected to migrate into follicles in response to chemoattractants such as BLC . These considerations suggest that follicular migration of MZ B cells may be the "default" pathway, whereas BCR engagement, which is known to direct MZ B cells not into the follicles but to the T-B interface [45–47], actively alters the fate of antigen specific MZ cells away from follicular migration.
Despite the increase in frequency (and numbers) of MZ B cells in p50-/- mice, the deposition of IgM-IC within the follicles does not improve with age, but declines severely. This appears to be due to a decrease in function of FDCs, which are fewer in number and are less pronounced, as evidenced by CD21 staining (Figure 5). Thus, the decreased ability of FDCs to retain complement containing immune complexes observed in old mice , appear to emerge with accelerated kinetics in p50-/- mice.
NF-κB p50 is only one of a growing number of genes, including a number of transcription factors, whose expression influences the development of MZ B cells (reviewed in ), highlighting the complex interactions that must converge to generate this B cell compartment. The fact that the requirements for individual proteins, such as p50, could change with age suggests that compensatory mechanisms exist which can overcome cellular defects, and/or that more than one pathway to generate these cells may exist.