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The chemokine stromal cell-derived factor-1 (SDF-1) that is released by bone marrow (BM) stromal cells and contributes to stem cell homing may also play a role in the trafficking of leukaemic cells. We analysed SDF-1-induced intracellular calcium fluxes in leukaemic blasts from the peripheral blood of patients with newly diagnosed acute myeloid leukaemia (AML) and lymphoblastic leukaemia (B-lineage ALL), determined the effect of BM stromal cell-conditioned medium on in vitro transendothelial migration (TM) and measured expression of the SDF-1 receptor, CXCR4, by flow cytometry. AML FAB M1/2 blasts did not show calcium fluxes and TM was not stimulated. In myelomonocytic AML (M4/5), however, SDF-1 induced significant calcium fluxes and TM was increased twofold by the conditioned medium. M3 and M4 blasts with eosinophilia (M4eo) showed intermediate activity and M6 blasts showed no functional activity. In ALL, strong calcium fluxes and increased TM (2.5-fold) were observed. Accordingly, expression of CXCR4 was low in undifferentiated (M0) AML, myeloid (M1/2) AML and erythroid (M6) AML, but high [mean fluorescence (MF) > 50] in promyelocytic (M3) AML, myelomonocytic (M4/5) AML and B-lineage ALL. We conclude that, in AML, SDF-1 is preferentially active in myelomonocytic blasts as a result of differentiation-related expression of CXCR4. Functional activity of SDF-1 and high expression of CXCR4 in B-lineage ALL is in accordance with the previously described activity of SDF-1 in early B cells. SDF-1 may contribute to leukaemic marrow infiltration, as suggested by increased CXCR4 expression and migratory response in BM-derived blasts compared with circulating cells.
Conditioned medium from bone marrow stromal cells has been shown to contain chemotactic factors that act not only on mature leucocytes, but also on immature haematopoietic progenitor cells ( Aiuti et al, 1997 ; Möhle et al, 1998) . Recently, stromal cell-derived factor-1 (SDF-1) was identified as the predominant chemotactic factor produced by bone marrow stromal cells ( Bleul et al, 1996a ; Aiuti et al, 1997 ). SDF-1 mediates its effects through the GTP-binding protein (G-protein)-coupled 7-transmembrane chemokine receptor CXCR4 ( Loetscher et al, 1994; Nagasawa et al, 1994; Bleul et al, 1996b ; Oberlin et al, 1996 ). In contrast to other chemokines, the interaction of SDF-1 and CXCR4 appears to be specific without cross-reactivity with other chemokines or chemokine receptors, resulting in a similar phenotype of SDF-1- and CXCR4-deficient mice ( Nagasawa et al, 1996; Ma et al, 1998 ; Zou et al, 1998 ). These animals show dramatically reduced bone marrow haematopoiesis, while fetal liver haematopoiesis is less affected, suggesting an important role for SDF-1 and CXCR4 in haematopoietic stem cell homing, particularly to the bone marrow. In addition, SDF-1 and CXCR4 are also involved in embryogenesis, including heart development, neuronal cell migration and vascular development ( Nagasawa et al, 1996 ; Ma et al, 1998 ; Tachibana et al, 1998; Zou et al, 1998 ).
Stromal cell-derived factor-1 belongs to the CXC chemokine family that is characterized by an intervening residue separating the first two cysteine residues within a conserved motif ( Wells et al, 1996 ). Other members of the CXC chemokine family primarily act on T lymphoctes (e.g. ligands of the chemokine receptor CXCR3 such as IP-10, Mig and others; Loetscher et al, 1996 ), B lymphocytes (e.g. BCA-1, the ligand of CXCR5; Legler et al, 1998 ) or are potent chemoattractants for granulocytes [ligands of CXCR1 and CXCR2 such as interleukin (IL)-8 and others; Smith et al, 1991 ]. In contrast to other members of the CXC chemokine family that are produced upon cytokine stimulation (e.g. increased IL-8 expression during inflammation), SDF-1 is constitutively produced by stromal cells ( Bleul et al, 1996a ). Moreover, SDF-1 is not only released in the bone marrow, but also in other tissues ( Tashiro et al, 1993 ). This suggests that the biological function of SDF-1 is not limited to haematopoietic stem cell homing. SDF-1 most probably contributes to extravasation of leucocytes in the absence of inflammation, which is important for lymphocyte trafficking ( Bleul et al, 1997 ). In addition, the chemokine receptor CXCR4 acts as a co-receptor, together with CD4, for the entry of the human immunodeficiency virus into T lymphocytes, which is blocked by SDF-1 or antibodies to CXCR4 ( Feng et al, 1996; Oberlin et al, 1996 ).
Acute myelogenous leukaemia (AML) and acute lymphoblastic (ALL) leukaemia represent malignant counterparts of haematopoietic progenitor and precursor cells characterized by a variable degree of maturation, as assessed by morphological analysis or expression of differentiation-related antigens such as CD34 ( Foon et al, 1982 ; Vaughan et al, 1988 ). Previous studies have shown that AML blasts from most patients constitutively produce the CXC chemokine IL-8, but only rarely express the functionally active IL-8 receptor ( Tobler et al, 1993 ), which is in accordance with the predominant activity of IL-8 in mature, post-mitotic granulocytes ( Smith et al, 1991 ). On the other hand, we have demonstrated that even the most primitive CD34+/CD38− haematopoietic progenitor cells express CXCR4 and respond to SDF-1 with increased transendothelial migration (TM) ( Möhle et al, 1998 ). In these preliminary results, we have also shown invariable expression of CXCR4 in myeloid cell lines and AML blasts, but high levels in B lymphoma cell lines. To further analyse functional activity of SDF-1 in acute leukaemia, SDF-1-induced intracellular calcium mobilization and transendothelial migration in response to conditioned medium from bone marrow stromal cells (MS-5) were investigated in circulating, leukaemic blasts from patients with AML and B-lineage ALL. In addition, expression of CXCR4 was determined by flow cytometry and bone marrow-derived blasts were compared with circulating cells. The results show that reactivity to SDF-1 is related to myelomonocytic differentiation in AML, is consistently found in B-lineage ALL, and may contribute to bone marrow and tissue infiltration of the leukaemic blasts.
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In this study, we demonstrate that the chemokine SDF-1, which plays an important role in the homing of haematopoietic progenitor cells to the bone marrow stroma, is functionally active in acute myeloid leukaemia with myelomonocytic differentiation (AML FAB M4 and M5) and in acute lymphoblastic leukaemia of the B lymphocyte lineage. In these subtypes, SDF-1 was capable of inducing a rapid flux of intracellular free calcium and significantly enhanced transendothelial migration in vitro, as a result of high expression of the chemokine receptor CXCR4. The influence of the non-malignant cells (which were variably contained in the samples) on the measurements was minimized by simultaneous flow cytometric analysis of characteristic markers of the leukaemic phenotype, which allowed selective assessment of CXCR4 expression and migration of the blast population. We have previously used a similar approach to selectively measure adhesion of mobilized haematopoietic progenitor cells to endothelium without the need to separate progenitors from the peripheral blood mononuclear cells ( Möhle et al, 1995 ).
Only measurement of the calcium fluxes might be variably influenced by the non-malignant cells because expression of cell surface antigens could not be assessed simultaneously. However, the qualitative results (presence or absence of significant SDF-1-induced calcium fluxes) were consistent with the CXCR4 expression analysis and SDF-1-induced transendothelial migration. In contrast, the quantitative results of calcium mobilization (maximal relative Fluo-3 fluorescence) did not correlate with CXCR4 fluorescence intensity or SDF-1-induced migration. This may reflect the fact that, in addition to CXCR4 expression, SDF-1-induced calcium fluxes (when measured as a relative shift of the Fluo-3 fluorescence) also depend on other factors such as cell size, efficacy of intracellular accumulation of the calcium indicator Fluo-3, repertoire of GTP-binding proteins and amount of intracellularly stored calcium. Indeed, SDF-1 induced a stronger shift of the relative Fluo-3 fluorescence in mature granulocytes, which expressed only moderate levels of CXCR4, when compared with B-lineage chronic lymphocytic leukaemia (B-CLL) cells, which even overexpressed CXCR4 ( Bautz et al, 1997; Möhle et al, 1999b ).
Given the fact that SDF-1 is constitutively produced by stromal cells from the bone marrow and other tissues ( Tashiro et al, 1993 ; Bleul et al, 1996a ), one might speculate that it contributes to marrow and tissue infiltration of leukaemic blasts. Indeed, infiltration of non-haematopoietic tissues such as gum or skin is most often observed in AML with monocytic differentiation (AML FAB M4 and M5) ( Cuttner et al, 1980 ), which expresses the greatest level of CXCR4 among all AML subtypes. However, quantitative data on SDF-1 production in different tissues are missing and bone marrow infiltration is also observed in AML subtypes with low or absent expression of CXCR4. It is unknown whether typical sites of leukaemic infiltration are characterized by a greater expression and production of SDF-1 compared with other tissues that are not invaded by the blasts. However, expression of SDF-1 has been detected in endothelial cells and pericytes of the skin ( Pablos et al, 1999 ). As presentation of SDF-1 by endothelial cells stimulates the integrin-mediated arrest of circulating cells on the vascular endothelium ( Peled et al, 1999 ), expression of CXCR4 by leukaemic blasts might facilitate leukaemic skin infiltration. Similarly, SDF-1 has been detected in lymph nodes and could therefore contribute to infiltration by ALL blasts ( Bleul et al, 1998 ). Particularly during embryonic development, however, SDF-1 is widely expressed and plays an important role in the developmant of various tissues and organs, including the central nervous system, vascular system, gastrointestinal tract and liver ( Ma et al, 1998 ; Tachibana et al, 1998 ; Zou et al, 1998 ; Coulomb-L'Hermin et al, 1999 ).
Recently, high expression and functional activity of CXCR4 has been described in B-lineage chronic lymphocytic leukaemia ( Burger et al, 1999 ; Möhle et al, 1999b ). However, malignant infiltration of tissues other than bone marrow and lymphatic organs is not usually observed in this disease. One might therefore assume that other factors such as adhesion molecules also play an important role in tissue infiltration of leukaemic blasts. As far as the bone marrow microenvironment is concerned, bone marrow endothelial cells, as well as stromal cells, constitutively express integrin ligands (e.g. VCAM), while the corresponding integrins (e.g. VLA-4) are found on leukaemic blasts ( Bendall et al, 1993 ; Yanai et al, 1994 ; Jacobsen et al, 1996 ). Adhesion molecule-mediated tropism for the bone marrow might also account for marrow infiltration of AML subtypes with weak response to SDF-1 and low CXCR4 expression.
On the other hand, the greater level of CXCR4 expression and increased SDF-1-induced migration in bone marrow-derived leukaemic blasts compared with the peripheral blood support the idea that interaction between SDF-1 and CXCR4 at least partially contributes to bone marrow infiltration of leukaemic blasts expressing CXCR4. We have previously shown that functional responsiveness to SDF-1 correlates with the expression level of CXCR4 ( Möhle et al, 1998 ). Similarly, a lower expression of CXCR4 in mobilized, circulating CD34+ haematopoietic progenitor cells has been reported compared with bone marrow-derived progenitors that was associated with a decreased responsiveness to SDF-1 ( Aiuti et al, 1997 ; Möhle et al, 1999a ).
SDF-1 is the major chemoattractant released by bone marrow stromal cells that acts on haematopoietic cells such as lymphocytes and progenitors ( Bleul et al, 1996a ; Aiuti et al, 1997 ). Although production of SDF-1 is not confined to the bone marrow, and CXCR4 and SDF-1 are involved in embryonic and adult trafficking of non-haematopoietic cells, recent findings suggest a particular role of CXCR4 and its ligand SDF-1 in the bone marrow microenvironment ( Nagasawa et al, 1996 ; Tanabe et al, 1997 ; Tachibana et al, 1998 ; Zou et al, 1998 ). For example, transition of haematopoiesis from the fetal liver to the bone marrow critically depends on the presence of SDF-1 ( Nagasawa et al, 1996 ). It is therefore conceivable that expression of CXCR4 in a functionally active form on malignant haematopoietic cells contributes to the tropism for the bone marrow microenvironment.
In our in vitro experiments, the bone marrow microenvironment was mimicked by the bone marrow stromal cell-conditioned medium added underneath an endothelial cell layer. The results demonstrate that the amount of SDF-1 produced by stromal cells is sufficient to build up a transendothelial gradient that supports migration of the leukaemic blasts. Therefore, SDF-1 is likely to influence trafficking of acute leukaemic blasts in vivo also.
Compared with monocytes and lymphocytes, expression of CXCR4 is lower in haematopoietic progenitor cells (particularly mobilized, circulating progenitors) and mature myeloid cells (e.g. granulocytes) ( Bleul et al, 1996b ; Bautz et al, 1997; Möhle et al, 1999a). Thus, the low level of CXCR4 and absent functional response to SDF-1 in undifferentiated AML, AML FAB M1, M2 and erythroid AML (FAB M6) compared with myelomonocytic AML, could reflect differentiation-related differences in CXCR4 expression.
The strongest expression of CXCR4 in acute leukaemia was observed in ALL (B-lineage). SDF-1/CXCR4 may play a particular role in malignancies of the early B lymphocyte lineage. Indeed, SDF-1 was initially identified as a ‘Pre B cell growth stimulating factor’ (PBCSF) ( Nagasawa et al, 1994 ), which corresponds with the finding that, during B lymphocyte differentiation, CXCR4 is expressed on B-cell precursors and may play a role in the trafficking of normal B lymphocytes and their precursors ( D'Apuzzo et al, 1997 ). Moreover, impaired B lymphopoiesis in SDF-1- and CXCR4-deficient mice suggests a central role for SDF-1/CXCR4 in B-cell development ( Nagasawa et al, 1996 ). In addition to its effect on migration, SDF-1 may also play a role as a growth factor in malignancies of the B lymphocyte lineage. We have recently shown that overexpression of CXCR4 may contribute to bone marrow infiltration of low-grade B lymphoproliferative disorders ( Möhle et al, 1999b ), which may also be the case in acute lymphoblastic leukaemia.
In conclusion, functional responsiveness to SDF-1 is observed in myelomonocytic AML and B-lineage ALL, as a result of high expression of the chemokine receptor CXCR4 in these subtypes. As high expression of CXCR4 occurs early during B and T lymphocyte differentiation and, for example, precedes expression of terminal deoxynucleotidyl transferase ( Ishii et al, 1999 ), measurement of CXCR4 might be useful in acute leukaemia phenotyping in order to discriminate between undifferentiated AML or AML with aberrant expression of lymphatic antigens and progenitor/precursor ALL. In addition, the chemotactic effect of SDF-1 secreted by stromal cells may contribute to the marrow and tissue infiltration frequently observed in monocytic AML and ALL.