IntroductionB-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of a monoclonal population of CD5 ϩ neoplastic B cells in blood, secondary lymphoid tissues, and the marrow. 1 Chemokines are essential for lymphocyte trafficking and homing during immune surveillance. 2 However, the mechanisms that regulate the dissemination of CLL cells to different tissue compartments are largely unknown. Earlier, we reported that the chemokine receptor CXCR4 induces spontaneous migration of CLL cells beneath marrow stromal cells. 3 More recently, the importance of CXCR4 for homing of neoplastic B cells to the marrow was confirmed in vivo. 4 We also reported that a small proportion of monocyte-derived cells from the blood of CLL patients differentiates into large, adherent nurselike cells (NLCs) that attract CLL cells and protect them from undergoing spontaneous or druginduced cell death. [5][6][7] NLCs express high levels of CD68 and can be detected in secondary lymphoid tissues from patients with CLL. 6 NLCs therefore appear to be an integral part of the CLL microenvironment within secondary lymphoid tissues, and may be comparable with lymphoma-associated macrophages (LAMs) in follicular lymphoma. 8 The molecules involved in the cross talk between CLL cells and NLCs are only partially understood. NLCs secrete CXCL12 and thereby attract CLL cells and support their survival. 5,9 Moreover, NLCs express B-cell-activating factor of the tumor necrosis factor (TNF) family (BAFF), a proliferation-inducing ligand (APRIL), 9 CD31, and plexin-B1 10 that also protect CLL cells from apoptosis, indicating that NLCs use multiple distinct pathways to support CLL cell survival.In contrast to CXCR4, which is broadly expressed by normal and malignant hematopoietic and nonhematopoietic cells, 11 the chemokine receptor CXCR5 is expressed only by mature recirculating B cells, a small subset of CD4 ϩ and CD8 ϩ T cells, and skin-derived migratory dendritic cells (reviewed in Muller et al 12 ). CXCR5 initially was isolated from Burkitt lymphoma and designated Burkitt lymphoma receptor 1 (BLR1). 13 CXCR5 gene-deleted mice display defective formation of primary follicles and germinal centers in the spleen and Peyer patches, and lack inguinal lymph nodes. 14 Subsequently, the ligand for CXCR5 was identified and termed B-cell-attracting chemokine 1 (BCA-1). 15,16 According to our current chemokine classification, BCA-1 now is designated CXCL13. 17 CXCL13 is a homeostatic chemokine that is constitutively secreted by stromal cells in B-cell areas of secondary lymphoid tissues (follicles), where B cells encounter antigen and differentiate. Generally, CXCR5 induces recruitment of circulating naive B cells to follicles. 15,[18][19][20] Regarding the microanatomic positioning within the germinal center (GC), dark and light zones of the GC can be distinguished, and centroblasts localize to the dark zones via CXCR4. There, centroblasts rapidly divide and undergo somatic hypermutation of the antibody variable region genes. Subsequently, t...
IntroductionChronic lymphocytic leukemia (CLL), the most prevalent form of adult leukemia in Western countries, is characterized by the progressive accumulation of phenotypically mature, monoclonal B lymphocytes in the peripheral blood, lymph nodes, and bone marrow. These long-lived CLL B cells are mostly arrested in the G 0 /G 1 phase of the cell cycle and display features consistent with a defect in programmed cell death (apoptosis), such as overexpression of Bcl-2-family proteins. 1,2 Despite their apparent longevity in vivo, CLL cells undergo spontaneous apoptosis in vitro, once removed from their in vivo microenvironment and placed into suspension culture without supportive stromal cells. 3,4 Spontaneous apoptosis can be prevented by coculture with various stromal cells, such as marrow stromal cells (MSCs), follicular dendritic cells, or nurse-like cells. [4][5][6][7][8] This prosurvival effect of stromal cells is largely dependent on direct cell contact between CLL and stromal cells. 4,5,9 Chemokine secretion by stromal cells and expression of corresponding chemokine receptors on leukemia cells play a critical role in directional migration (chemotaxis) and adhesion of leukemia cells to MSCs, both in vitro 10 and in vivo. 11 CXCL12, previously called stromal cell-derived factor-1, is a chemokine constitutively secreted by MSCs that attracts and confines CLL cells to stromal cells via its cognate receptor CXCR4 expressed at high levels on CLL cells. 10,12 This mechanism is shared with normal hematopoietic stem cells that require this receptor for homing to stromal niches in the marrow. 13,14 Besides its activity on adhesion and migration of CLL cells, 10 which is partially dependent on PI3K activation, 15 CXCL12 also has a direct prosurvival effect on CLL cells. 8,16 Once they engage in adhesion to stromal cells, CLL cells become resistant to the cytotoxic effects of drugs commonly used to treat CLL patients, such as fludarabine 17 or corticosteroids. 4 This primary drug resistance mechanism, also called cell adhesion-mediated drug resistance, 18 may account for minimal residual disease in tissue compartments such as the marrow and relapses commonly seen in treatment of CLL patients. [19][20][21] We previously demonstrated that CXCR4 antagonists can partially resensitize CLL cells to cytotoxic drugs in cocultures with MSCs, 17 a finding that is currently pursued in clinical trials in leukemia patients, 22 using the small molecule CXCR4 antagonist AMD3100 (now called Plerixafor). However, from our previous work 17 and other studies, 23,24 it is also apparent that targeting of CXCR4 only partially overcomes stromal cell-mediated drug resistance; therefore, other CLL-microenvironment interactions may represent alternative therapeutic targets.Phosphoinositide 3-kinases (PI3Ks) are among the most commonly activated signaling pathways in human cancers. [25][26][27] In freshly isolated CLL cells, PI3Ks are constitutive activated, 28 and CLL patients with unmutated immunoglobulin variable heavy chain genes, which ...
Nontumoral accessory cells such as marrow stromal cells (MSC) or nurselike cells (NLC), which constitute the leukemia microenvironment, constitutively secrete the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). CXCL12 transduces signals via its receptor CXCR4, which is expressed at high levels by Chronic Lymphocytic Leukemia (CLL) B cells. Via the CXCL12-CXCR4 axis, CLL cells migrate and adhere to stromal cells. Adhesion to stromal cells protects CLL cells from spontaneous and drug-induced apoptosis in a contact-dependent fashion. Signaling pathways regulating these processes in CLL B cells are largely unknown. Here, we examined the importance of phosphatidyl-inositide 3-kinases (PI3-K) for migration and viability of CLL B cells using non-specific and isoform-specific PI3-K inhibitors. The importance of PI3-K for migration of CLL cells to CXCL12 was determined by transwell chemotaxis and pseudoemperipolesis (PEP) assays. Inhibition of PI3-K resulted in a significant reduction of CLL cell migration in chemotaxis and PEP assays. In comparison to untreated CLL cells, Ly 294002 inhibited chemotaxis to 65 ± 4.6% of untreated controls. Using a panel of isoform-specific PI 3-K inhibitors (PI-103, PIK-90, IC87114, TGX-115, ZK-75), we observed inhibition of chemotaxis by the multi-targeted compounds PI-103 (51.4 ± 0.2%) and PIK-90 (57.5 ± 8.9%), whereas p110beta and delta inhibition had no effect. We conclude from this part of the study that PI3-kinases play an important role for CXCR4 signaling in CLL B cells, mediating migratory responses and protection from apoptosis. Experiments with inhibitors of PI3-K with higher target selectivity suggest a dominant role for the class I PI3-K p110alpha for migration in response to CXCL12. Because adhesion to stroma mediates protection from chemotherapeutic drugs, we tested PI3-K inhibitors alone and in combination with fludarabine in CLL-stroma co-cultures. Pre-treatment of CLL cells with the PI3-K inhibitors Ly 294002, PI-103, and PIK-90 resulted in a significant decrease in viability of CLL cells co-cultured with and without stroma. Moreover, PI3-K isoform specific inhibitors enhance the cytotoxicity of Fludarabine and partially reverse the protective effect of stromal cells on fludarabine-induced apoptosis. Collectively, this study establishes that PI3-Ks play an important role in CXCR4 signaling for CLL cell migration and adhesion to stromal cells. New, isoform-specific PI3-K inhibitors enhance the cytotoxicity of fludarabine in suspension cultures and in co-cultures with stromal cells. Therefore, the therapeutic potential of PI3-K inhibitors alone or in combination with fludarabine should further be investigated. Figure. Figure.
Despite their apparent longevity in vivo, isolated Chronic Lymphocytic Leukemia (CLL) B cells generally undergo spontaneous apoptosis in vitro when cultured under conditions that support the growth of human B cell lines. This suggests that interactions between CLL cells and a distinct tissue microenvironment in the marrow and the lymphatic tissues, where CLL cells are in close contact with accessory cells (mesenchymal stromal cells, and CD68+ “nurselike cells”/NLC), are critical for the progression of the disease. NLC can be detected in secondary lymphoid tissues from CLL patients and appear to be an integral part of the CLL microenvironment, comparable to lymphoma-associated macrophages in follicular lymphoma. The molecules involved in CLL-NLC cross talk are only partially understood. Therefore, we examined the gene expression profile of purified CLL B cells using Affymetrix U133 Plus 2.0 Arrays to define distinct expression profiles induced in purified CLL B cells by co-culture with NLC. When compared to freshly isolated blood CLL B cells, we found that CLL B cells co-cultured for 14 days with NLC displayed high level expression of two T cell chemoattractants, macrophage inflammatory protein-1a and b (MIP-1 a, MIP-1b, also called CCL3 and CCL4). CCL3 and CCL4 expression levels correlated with ZAP-70 expression by the CLL cells, suggesting that B cell receptor signaling is involved in inducing the expression of these chemokines. Supernatants from CLL-NLC co-cultures harvested 7 and 14 days after initiation of the cultures revealed high CCL3 and CCL4 protein levels (up to >30 ng/ml) by ELISA, predominantly in the same CLL cases that displayed high CCL3, CCL4 and ZAP-70 expression by expression profiling. Moreover, serum samples from CLL patients were tested for CCL3 and CCL4 protein expression by ELISA. These studies demonstrated higher CCL3 and CCL4 serum levels in CLL patients when compared to healthy volunteers. CCL3 and CCL4 serum levels were found to be higher in CLL patients that were CD38+, displayed non-mutated IgVH genes, and b2 microglobulin levels that are > 4 mg/L. In vitro, B cell receptor (BCR) triggering of CLL cells, using anti-IgM antibodies, induced a rapid and robust induction of CCL3 and CCL4 protein production by CLL B cells. In contrast, CD40 triggering did not induce expression of these chemokines. These studies reveal a novel mechanism of cross-talk between CLL B cells and their microenvironment, namely the induction of two T cell chemokines, CCL3 and CCL4, by CLL-NLC interaction. Thus, we provide the first evidence that neoplastic B cells are an important source of T cell chemokines, that can induce recruitment of T cells of helper/effector phenotype to sites of cognate T cell-CLL interactions. Besides inducing the outgrowth of NLC, this is another mechanism how CLL cells actively create a microenvironment that favors their growth and survival. Figure Figure
Trafficking and homing of lymphocytes between the blood, bone marrow, and lymphoid tissues is a multistep process that involves selectins, integrins, and chemokine receptors. We previously demonstrated the importance of CXCR4 chemokine receptors for CLL migration to stromal cells, such as mesenchymal stromal cells and monocyte-derived nurselike cells (NLC). However, the importance of other molecules for CLL cell trafficking is less defined. Expression of CD38 and ZAP-70 by the leukemia cells recently has been described to enhance CLL cell migration in response to the CXCR4 ligand, CXCL12 which may explain the poor prognosis of CLL patients expressing these molecules. In stroma co-cultures, a fraction of input CLL cells spontaneously migrates beneath and underneath the stromal cells, where they are protected form spontaneous and drug-induced apoptosis. This migration phenomenon is termed “pseudoemperipolesis” (PEP) and could explain the evasion of a subpopulation of CLL cells from cytotoxic therapy, leading to minimal residual diseases commonly seen after conventional CLL therapy. To determine which molecules are involved in CLL cell migration beneath stromal cells, we evaluated the expression of migration-related markers on CLL cells in pseudoemperipolesis assays. CLL PBMC were isolated by Ficoll and plated onto marrow stromal cell monolayers in 12-well plates. After 5 hours incubation, supernatant cells were washed off. The stromal layer containing the migrated cells was documented photographically, and then detached with trypsin. Supernatant and migrated CLL cells were stained with saturating concentrations of the following antibodies: anti-CD5, anti-CD38, anti-CD40, anti-CXCR5, anti-CD44, anti-CXCR4, anti-CD62L, anti-CD49d, anti-CD80, anti-CD54, anti-CD86, anti-CXCR3, anti-CCR7, and anti-CD19. Aliquots of supernatant and migrated cells also were subject to cell counting by FACS to determine the relative proportion of migrated CLL cells. Comparing mean fluorescence intensities (MFI) of migrated versus non-migrated cells, we noticed a significant down-regulation of CD62L expression on the migrated cells (10 ± 2 vs. 34 ± 19, MFI mean ± standard deviation, n = 15, P < 0.05), down-regulation of CXCR4 (570 ± 188 vs. 819 ± 400, n = 15, P < 0.05), higher expression of CD49d (116 ± 81 vs. 62 ± 42, n = 15, P < 0.05), and higher expression of CD38 (19 ± 11, vs. 12 ± 7, n = 15, P < 0.05). No significant differences where observed for the expression of the other markers. In conclusions, this study demonstrates that spontaneous migration of CLL cells beneath marrow stromal cells involves selectins, CXCR4, and VLA4 integrins. CD62L is an adhesion molecule that recognizes carbohydrate groups on endothelial cells as well as high endothelial venules of lymphoid tissues. Down-regulation of CD62L after migration indicates that L-selectin is important for this process which also involves down-modulation of CXCR4 in response to CXCL12 secreted by stromal cells. The higher expression of CD49d on migrated CLL cells confirms the role of this integrin in the interaction with VCAM-1 or fibronectin on marrow stromal cells. On the other hand the higher expression of CD38 on migrated CLL cells suggests that CD38 pathway may be involved in this migratory response to stromal cell. In conclusion, our findings suggest that CD62L, CXCR4, CD49d, and CD38 are involved in cognate interactions between CLL cells and stromal cells in the multistep process of CLL migration beneath marrow stromal cells.
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