ALBERT

Beschreibung: CD74 is an integral membrane protein that was thought to function mainly as an MHC class II chaperone. However, CD74 was recently shown to have a role as an accessory-signaling molecule. Our studies demonstrated that CD74 regulates B-cell differentiation by inducing a pathway leading to the activation of transcription mediated by the NF-κB p65/RelA homodimer and its coactivator, TAFII105. Here, we show that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-κB activation, entry of the stimulated cells into the S phase, elevation of DNA synthesis, cell division, and augmented expression of BCL-XL. These studies therefore demonstrate that surface CD74 functions as a survival receptor.

Beschreibung: Most mature follicular B cells circulate within the periphery in a quiescent state, without actively contributing to an acute immune response. Lasting B-cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. We recently demonstrated that cell surface CD74 controls mature B-cell survival. Stimulation of cell surface CD74 leads to NF-κB activation, which enables entry of the stimulated B cells into the S phase, induction of DNA synthesis, and cell division, and augments the expression of survival genes. In the present study, we investigated CD74 target genes to determine the identities of the molecules whose expression is modulated by CD74, thereby regulating B-cell survival. We report that CD74 activates the p65 member of the NF-κB family, which in turn up-regulates the expression of p53-related TAp63 proteins. TAp63 then binds and transactivates the Bcl-2gene and induces the production of Bcl-2 protein, thereby providing the cells with increased survival capacity. Thus, the CD74/NF-κB/TAp63 axis defines a novel antiapoptotic pathway in mature B cells, resulting in the shaping of both the B-cell repertoire and the immune response.

Beschreibung: Scientific Background: Previous studies have shown that chronic lymphocytic leukemia (CLL) lymphocytes express relatively large amounts of CD74 mRNA compared to normal B cells. We have recently demonstrated in a murine model that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-κ B activation, entry of the stimulated cells into the S phase, elevation of DNA synthesis, cell division, and augmented expression of BCL-XL. These findings therefore demonstrated that surface CD74 functions as a survival receptor. In the current study we aimed to determine whether activation of cell surface CD74 in B-CLL cells leads to induction of a signaling cascade resulting in cell survival. Meterials and methods: B cells were purified from the peripheral blood of CLL patients of different stages. CD74 stimulation was achieved using anti-CD74 or MIF (a natural occurring ligand of CD74). IL-8 expression and function was determined by RT-PCR, western blot, ELISA and Annexin V staining. Results: In all CLL patients there was a significantly increased expression of cell surface CD74. Activation of cell surface CD74 initiates a signaling cascade that results in secretion of Interleukin 8 (see Fig1), which in turn regulates cell survival(see Fig 1,2). Conclussion: Our data show that over-expression of CD74 in CLL is an important survival mechanism, operational from the very early stages of the disease, and inherent in all further stages. This survival mechanism thus appears to be an early and significant event in the pathogenesis of the disease. Our findings open prospects for novel therapeutic strategies aimed at this survival pathway. Legends: Fig 1: CD74 induces IL-8 and Bcl-2 expression in CLL B cells. (A,C) Cells were incubated in the presence or absence of anti-CD74 antibody, Id2, a control antibody or MIF for 18 h. (A,C) RNA was purified and levels of IL-8, Bcl-2 and actin mRNA were analyzed. The results presented are representative of 7 early-stage and 5 advanced-stage B-CLL patients. (B) ) Cells’ conditioned medium was collected and their IL-8 levels were analyzed by ELISA. The results presented are representative of 3 independent experiments. Fig 1:. CD74 induces IL-8 and Bcl-2 expression in CLL B cells. (A,C) Cells were incubated in the presence or absence of anti-CD74 antibody, Id2, a control antibody or MIF for 18 h. (A,C) RNA was purified and levels of IL-8, Bcl-2 and actin mRNA were analyzed. The results presented are representative of 7 early-stage and 5 advanced-stage B-CLL patients. (B) ) Cells’ conditioned medium was collected and their IL-8 levels were analyzed by ELISA. The results presented are representative of 3 independent experiments. Fig 2: IL-8 secreted following CD74 stimulation regulates B-CLL cell survival.
 (A, B) B-CLL cells were incubated in the presence or absence of anti-CD74 (B), anti-IL-8 (A, B) or a control antibody (c-jun; A) for 48 h. Cells were stained with annexin V and analyzed by FACS. The results presented are representative of 3 early-stage and 4 advanced-stage B-CLL patients. (C) B-CLL cells were incubated in the presence or absence of a control antibody (c-jun), anti-CD74, anti-IL-8 or IL-8 for 48 h. Cell death was analyzed by ELISA. The graph shows the average of one experiment, representative of 4. Fig 2:. IL-8 secreted following CD74 stimulation regulates B-CLL cell survival.
 (A, B) B-CLL cells were incubated in the presence or absence of anti-CD74 (B), anti-IL-8 (A, B) or a control antibody (c-jun; A) for 48 h. Cells were stained with annexin V and analyzed by FACS. The results presented are representative of 3 early-stage and 4 advanced-stage B-CLL patients. (C) B-CLL cells were incubated in the presence or absence of a control antibody (c-jun), anti-CD74, anti-IL-8 or IL-8 for 48 h. Cell death was analyzed by ELISA. The graph shows the average of one experiment, representative of 4.