1B). As shown in the figure, we co-precipitated pro-IL-16 and MHC class II molecules and confirmed the association between pro-IL-16 and MHC class II molecules. More importantly, the level of pro-IL-16

was increased by LPS treatment of resting B cells for 15 min, and increased JQ1 nmr expression of pro-IL-16 protein was inhibited by anti-I-Ad MHC class II antibody treatment. This inhibitory effect was haplotype-specific and was not detected when we used a monoclonal antibody (10-3.6.2) specific to an unrelated haplotype (I-Ak) (data not shown). To characterize the form of IL-16 present in 38B9 resting B cells, we performed Western blot analysis using a commercial antibody specific to the C-terminal part of mouse IL-16, which can recognize both precursor and mature forms of IL-16 (Fig. 1C). Extracts prepared from 38B9 cells showed a single band at 80 kDa, representing pro-IL-16, but there was no band at 20 kDa (C-terminal mature form of IL-16) or at 60 kDa (remaining N-terminal part of pro-IL-16). In contrast, control EL4 cells, which are mouse CD8+ T cells known to express IL-16, showed only a single band at 20 kDa, indicating the presence of the mature form of IL-16. These results suggest that the precursor form of IL-16, rather than the mature form, is predominantly BIBW2992 mouse expressed in 38B9 resting B cells. We assumed that

cleaved mature IL-16 was rapidly secreted rather than stored in the cytoplasm of B cells because we detected the expression of caspase-3, which is involved in pro-IL-16 cleavage, in 38B9 resting B cell lysates through Western blot analysis (data not shown). Collectively, we confirmed that pro-IL-16 is associated with MHC class II molecules

and that it is involved in MHC class II-mediated inhibitory signalling in resting B cells. It is known that cleavage of the C-terminal portion of pro-IL-16 Selleckchem Gefitinib by caspase-3 yields the mature form of IL-16 [23, 24]. Mature IL-16 is secreted, and the N-terminal fragment of pro-IL-16 or full-length pro-IL-16 translocates into the nucleus where pro-IL-16 or full-length pro-IL-16 induces G0/G1 cell-cycle arrest [18, 19]. Cytoplasmic pro-IL-16 can therefore be considered as a precursor of secreted IL-16, while pro-IL-16 in the nuclear compartment acts as cell-cycle regulator. Those previous reports and our observation of an association between pro-IL-16 and MHC class II-mediated negative signalling in resting B cells prompted us to determine whether pro-IL-16 has an inhibitory effect on B cell proliferation, as shown in T cells. Consequently, we initially examined the intracellular location of pro-IL-16 in resting B cells (Fig. 2). Western blot analysis of nuclear and cytoplasmic fractions prepared from resting B cells demonstrated that pro-IL-16 was present in both the cytoplasmic and nuclear compartments (Fig. 2A).