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Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Previous studies have shown that CLL B lymphocytes express relatively large amounts of CD74 mRNA relative to normal B cells. In the present study, we analyzed the molecular mechanism regulated by CD74 in B-CLL cells. The results presented here show that activation of cell-surface CD74, expressed at high levels from an early stage of the disease by its natural ligand, macrophage migration-inhibition factor (MIF), initiates a signaling cascade that contributes to tumor progression. This pathway induces NF-B activation, resulting in the secretion of IL-8 which, in turn, promotes cell survival. Inhibition of this pathway leads to decreased cell survival. These findings could form the basis of unique therapeutic strategies aimed at blocking the CD74-induced, IL-8-dependent survival pathway.apoptosis ͉ invariant chain B cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of CD5 ϩ B lymphocytes in peripheral blood, lymphoid organs, and bone marrow (1). The hallmark of the disease is decreased apoptosis, resulting in accumulation of these malignant cells. Previous studies have shown that chronic lymphocytic leukemia (CLL) lymphocytes express relatively large amounts of mRNA for CD74, which is the cell-surface form of invariant chain (Ii), as compared with normal B cells (2, 3). CD74 is a nonpolymorphic type II integral membrane protein, which was originally thought to function mainly as an MHC class II chaperone (4). However, CD74 recently was found to play an additional role as an accessory-signaling molecule. In macrophages, CD74 demonstrates high-affinity binding to the proinflammatory cytokine, macrophage migration-inhibitory factor (MIF). MIF binds to the extracellular domain of CD74; this complex is required for MIF-mediated MAPK activation and cell proliferation (5). Moreover, the bacterium Helicobacter pylori was shown to bind to CD74 on gastric epithelial cells and to stimulate IL-8 production (6).In a previous study, we showed that CD74 is involved directly in shaping the B cell repertoire (7, 8) by a pathway leading to the activation of transcription mediated by the NF-B p65/RelA homodimer and its coactivator, TAFII105 (9). NF-B activation is mediated by the cytosolic region of CD74 (CD74-ICD), which translocates to the cell nucleus (10). This signal is terminated by degradation of the active CD74-ICD fragment (11,12). Moreover, we demonstrated recently that CD74 stimulation with an agonistic CD74 antibody leads to NF-B activation, enabling entry of the stimulated B cells into the S phase, an increase in DNA synthesis, cell division, and augmented expression of members of the Bcl-2 protein family. Thus, these findings indicate that surface CD74 functions as a survival receptor (13).In the present study, we sought to determine whether CD74 functions as a survival receptor in B-CLL cells. Our results show that MIF-induced CD74 activation initiates a signaling cascade that results in secr...

Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-1 due to low B2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL12. CLL cells failed to transmigrate across VCAM-1-expressing, ICAM-1-expressing, and CXCL12-expressing endothelium, whereas when LFA-1 expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1-dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1-independent and VLA-4-independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen.

Lasting B-cell persistence depends on survival signals that are transduced by cell surface receptors. Here, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia (CLL) cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase zeta (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival, by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population is reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74 induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and CLL cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way towards understanding the mechanisms shaping B cell survival, and suggest novel therapeutic strategies based on the blockade of the midkine/RPTPζ-dependent survival pathway.