30 To determine the CD74/MIF downstream signalling cascade in B cells from SLE-afflicted mice, we tested the production of the anti-apoptotic molecules https://www.selleckchem.com/products/jq1.html Bcl-2 and Bcl-xL, and of the pro-apoptotic molecule Caspase-8 and assessed the effect of treatment with hCDR1 on their expression. Figure 4(a)

presents the mean levels of the anti-apoptotic Bcl-2 and Bcl-xL gene expression relative to the expression in the vehicle-treated group. As shown, the expression of Bcl-2 and Bcl-xL was significantly reduced in B cells of hCDR1-treated mice, compared with B cells of vehicle-treated (P = 0·03 and P = 0·01) or control peptide-treated (P = 0·0001 and P = 0·05) mice, respectively. The down-regulating effect of hCDR1 on the latter genes was also confirmed at the protein level by Western blot analysis. Figure 4(b)

shows that the expression of the pro-apoptotic Caspase-8 was significantly up-regulated in B cells of hCDR1-treated mice (P = 0·001 and 0·02, respectively), compared with B cells of vehicle-treated or control-peptide-treated mice. The up-regulating effect of hCDR1 on Caspase-8 was also confirmed at the protein level using Western blot analysis. Hence, hCDR1 down-regulates the anti-apoptotic molecules Bcl-2 and Bcl-xL, which were elevated, and up-regulates the pro-apoptotic molecule Caspase-8, GDC-0068 purchase which was diminished, in B cells of SLE-afflicted mice. We further investigated the association Phosphatidylethanolamine N-methyltransferase between the expression levels of the anti-apoptotic and pro-apoptotic molecules and the rates of apoptosis in B cells from the experimental mice. Figure 5(a) shows the B220+ cells

that were stained for Annexin-V out of the propidium iodide-negative cells. An increase in the percentage of Annexin-V-positive cells was found in B cells of hCDR1-treated mice compared with the vehicle-treated and control-peptide-treated mice. To directly demonstrate whether the up-regulation of B-cell apoptosis by hCDR1 was mediated through the down-regulation of MIF (Fig. 2), we incubated spleen cells isolated from vehicle-treated or hCDR1-treated mice in the presence or absence of MIF and analysed them by Annexin-V staining. It can be seen that the addition of MIF to the vehicle-treated B cells induced almost no change in the low levels of apoptosis, as determined by the Annexin-V staining. The figure shows that the number of Annexin-V-positive cells was increased in the hCDR1-treated cells but the addition of MIF to the latter cells resulted in a significant reduction of B-cell apoptosis. Hence, MIF, CD74 and CD44 regulate B-cell survival in SLE-afflicted mice and following hCDR1 treatment the expression of these molecules and their downstream cascade are diminished. Kidney and central nervous system (CNS) involvement are common in SLE. We demonstrated previously that treatment with hCDR1 ameliorated kidney damage,4,6,7,31–33 CNS pathology and cognitive behaviour5 in the SLE-afflicted mice.