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Keywords:

  • AML;
  • ALL;
  • SDF-1;
  • CXCR4;
  • transendothelial migration

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

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.

PATIENTS and METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Isolation of AML blasts from the peripheral blood Fifty patients with newly diagnosed AML and ALL were included in this analysis. The patient characteristics are shown in Table I. Peripheral blood was drawn into heparinized syringes. Mononuclear cells (MNCs) were isolated by Ficoll (Seromed-Biochrom, Berlin, Germany) density centrifugation. The diagnosis of AML and ALL was made by standard morphology and cytochemistry of peripheral blood and bone marrow films according to the French–American–British (FAB) criteria ( Bennett et al, 1985 ) and by immunophenotyping using a comprehensive panel of monoclonal antibodies (mAbs) against myeloid and lymphoid-associated antigens, as proposed by the EGIL group ( Bene et al, 1995 ). The percentage of blasts in the MNCs used for the experiments ( Table I) was assessed by flow cytometry using the individual, characteristic immunophenotype, as described below (flow cytometry). Cases without a distinct blast population in the immunofluorescence analysis as well as cases with a diagnosis of blast transformation of chronic myeloid leukaemia or other myeloproliferative disorders and secondary acute leukaemias (preceding myelodysplastic syndrome) were excluded. In some cases, bone marrow samples were processed in parallel with peripheral blood samples.

Table I.  Patient characteristics and case numbers. Data for gender and age refers to the total number of patients in each acute leukaemia subtype. In addition, the case numbers in different experiments (measurement of calcium mobilization, transendothelial migration, expression of CXCR4 by flow cytometry, flow cytometry and transmigration of peripheral blood and bone marrow-derived blasts in parallel) are shown. The mean percentage of blasts in the samples used for the experiments is also given.
Type N (total) Sex (male/ female) Age (median, range) N (Ca2+ fluxes) N (TM) N (FACS) N (FACS) (PB vs. BM) N (TM) (PB vs. BM) Blasts (%, PB, mean)
  1.  * All patients common/pre-B-ALL (five c-ALL patients, one cytoplasmic IgM + pre-B-ALL patient).

  2.  TM, transmigration; FACS, flow cytometry analysis; PB, peripheral blood; BM, bone marrow.

AML
 M021/157·5 (55–60)265
 M132/156 (33–78)3331147
 M2146/863 (18–75)1312133361
 M331/243 (37–62)22329
 M4eo52/353 (40–59)53449
 M464/239 (29–79)6663355
 M583/549 (40–72)8773375
 M631/264 (55–74)21264
ALL *63/345 (16–70)66681

Measurement of intracellular free calcium mobilization Chemokine-mediated receptor stimulation results in the mobilization of intracellular free calcium ( Murphy, 1994). Therefore, intracellular Ca2+ was measured using the fluorescent calcium indicator, Fluo-3 (Molecular Probes, Leiden, Netherlands), which was loaded into the cells by a method described by Vandenberg & Ceuppens (1990), based on the loading procedure for quin2 ( Tsien et al, 1982 ). The cells (107/ml) were incubated in Hanks' balanced salt solution (HBSS) (Sigma-Aldrich, Deisenhofen, Germany) containing 10 μmol/l Fluo-3 for 30 min at 37°C. After a 1:5 dilution in HBSS/1% fetal calf serum (FCS) and a further incubation for 40 min at 37°C, the cells were washed three times, resuspended at a final concentration of 106 cells/ml in HEPES-buffered saline (137 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l Na2HPO4, 5 mmol/l glucose, 1 mmol/l CaCl2, 0·5 mmol/l MgCl2, 1 g/l bovine serum albumin (BSA), 10 mmol/l HEPES, pH 7·4) and incubated for 10 min at 37°C. After stimulation with SDF-1 (rhSDF-1β, final concentration 100 ng/ml; R & D Systems GmbH, Wiesbaden, Germany), the fluorescence [fluorescein isothiocyanate (FITC) channel] was continuously analysed using a FACScalibur flow cytometer (Becton-Dickinson) in 5 s acquisition intervals. Leukaemic blasts were gated in a forward scatter/side scatter (FSC/SSC) dot plot. The characteristics of this gate were chosen from the immunofluorescence analysis (as described below) to ensure that the majority of events corresponded with leukaemic blasts. As a control, fluorescence changes were measured without chemokine stimulation.

Transendothelial migrationIn vitro analysis of migration across microvascular endothelium was performed, as described previously ( Möhle et al, 1997 ), using the human endothelial cell line ECV304 ( Takahashi & Sawaski, 1992; Möhle et al, 1995 ) which was cultivated in Medium 199 (Seromed-Biochrom, Berlin, Germany), supplemented with 20% FCS. For the transmigration experiments, ECV304 cells were seeded on 3 μm transwell microporous membranes, which separated the upper and lower chambers in six-well tissue culture plates (Transwell, Corning-Costar, Bodenheim, Germany), and grown to confluency. The medium was then replaced with Medium 199/5% FCS. SDF-1-containing conditioned medium from the bone marrow stromal cell line MS-5 was added to the lower chamber underneath the membrane, as described previously ( Möhle et al, 1998 ), and 5 × 105 cells were placed into the upper chamber. After 14 h, the transmigrated cells were enumerated. To assess migration of the blasts only rather than migration of the remaining non-malignant leucocytes in cases with a blast percentage < 95%, the cell counts of the input cells and transmigrated cells were corrected by multiplying with the proportion of blasts (as assessed by flow cytometry using individual characteristic surface markers as decribed below). Partially blocking CXCR4 mAb (clone 12G5, Pharmingen) was added to the cells at a final concentration of 10 μg/ml.

Flow cytometry 1–2 × 105 cells were incubated for 30 min at 4°C with the phycoerythrin (PE)-conjugated mAb to CXCR4 (Clone 12G5; Bleul et al, 1997 ; Pharmingen, San Diego, California). An isotype-identical mAb served as a control. The cells were analysed using a FACScalibur flow cytometer (Becton-Dickinson). To calculate the percentage of positive cells, a proportion of ≤ 1% false positive events was accepted in the negative control sample. If the percentage of blasts in the analysis of the immunophenotype was less than 95% (after gating in a FSC/SSC dot plot), co-expression analysis of the PE-labelled CXCR4 antibody and FITC-labelled antibodies (Becton-Dickinson) against individually characteristic markers such as CD34, CD13, CD15, CD117, CD10, CD19 or low expression of CD45 (according to the immunophenotype) was used to selectively analyse CXCR4 expression in the malignant blast population. The CXCR4-PE mean fluorescence (MF) intensity of the blasts was then calculated and expressed in arbitrary units.

Statistical analysis Data from independent experiments were expressed as mean ± SEM (standard error of the mean, for n ≥ 3). For the assessment of statistical significance, the parameter-free Mann–Whitney U-test (differences between leukaemic subtypes) or Wilcoxon matched-pairs test (differences between peripheral blood and bone marrow-derived blasts) were applied.

Results

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

SDF-1-induced intracellular free calcium mobilization

Intracellular free calcium was measured semiquantitatively as relative Fluo-3 mean fluorescence. After application of SDF-1 (100 ng/ml), a rapid, transient flux of intracellular Ca2+ was observed in AML samples, dependent on the FAB subtype, and in ALL samples, as shown in Fig 1. As a negative control, fluorescence changes without chemokine stimulation were also measured. Myeloid (FAB M1 and M2) blasts did not show intracellular calcium mobilization, similar to erythroid (FAB M6) blasts. However, in promyelocytic (FAB M3) and particularly in myelomonocytic (FAB M4 and M5) blasts, rapid calcium fluxes were observed in response to SDF-1. AML FAB M4 with eosinophilia (M4eo) blasts showed intermediate reactivity, similar to AML M3 blasts. In B-lineage ALL, application of SDF-1 consistently induced calcium fluxes in all samples analysed. The difference in the SDF-1-induced relative Fluo-3 fluorescence at 5 s was statistically significant when myeloid (FAB M1/2) and myelomonocytic (FAB M4/5) AML blasts were compared (P < 0·01) or when myeloid (FAB M1/2) AML and ALL blasts were compared (P < 0·05). The CXC chemokine IL-8 (100 ng/ml, equimolar to 100 ng/ml SDF-1, owing to a nearly equal molecular weight), which is highly reactive in granulocytes, did not induce significant intracellular calcium fluxes in AML blasts of all subtypes (data not shown).

image

Figure 1. SDF-1-induced mobilization of intracellular free calcium in leukaemic blasts from peripheral blood. After addition of SDF-1, the release of intracellular free calcium in Fluo-3-loaded leukaemic blasts was measured by continuous flow cytometry (FITC-channel) in 5 s aquisition intervals. Data were expressed as relative mean fluorescence compared with the baseline level before chemokine stimulation (time = 0 s). A rapid, transient flux of intracellular calcium was observed in AML FAB M4, M5 and ALL blasts in response to SDF-1 (100 ng/ml). In contrast, AML FAB M1, M2 and M6 blasts did not respond to SDF-1. In AML FAB M3 and M4eo blasts, intermediate calcium fluxes were observed. As a negative control, fluorescence changes were measured without addition of the chemokine.

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In vitro transendothelial migration

To mimic the bone marrow microenvironment, conditioned medium from bone marrow stromal cells (cell line MS-5) was added to the lower chamber of the transmigration system, which enhanced transendothelial migration of AML and ALL blasts in vitro, as shown in Fig 2. In accordance with the absence of significant calcium mobilization in response to SDF-1, stromal cell-derived conditioned medium did not stimulate migration of AML FAB M1, M2 and M6 blasts. In contrast, increased migration of M3, M4, M5 and B-lineage ALL blasts was observed. The difference in the SDF-1-induced migration was statistically significant when myeloid (FAB M1/2) and myelomonocytic (FAB M4/5) AML blasts were compared, or when myeloid (FAB M1/2) AML and ALL blasts were compared.

image

Figure 2. In vitro transendothelial migration of circulating, leukaemic blasts in response to stromal cell-conditioned medium containing SDF-1. 5 × 105 cells were added to the upper chamber of the transmigration system. After 14 h, transmigrated cells were recovered from the lower chamber and counted. Relative transmigration was calculated by dividing the number of transmigrated cells in response to chemokines by the number of spontaneously migrating cells (control = fresh medium added to the lower chamber), which was measured in parallel. In cases with a percentage of blasts below 95%, all cell numbers were corrected after flow cytometric analysis using characteristic surface antigens according to the immunophenotype of the malignant blasts. Transendothelial migration of AML FAB M3, M4eo, M4, M5 and ALL blasts was enhanced, when SDF-1-containing conditioned medium was added to the lower chamber. In contrast, migration of AML M1, M2, and M6 blasts was not influenced by the conditioned medium. The difference in SDF-1-induced transmigration between myeloid (M1/2) and myelomonocytic AML (M4/5) or ALL was statistically significant.

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Expression of CXCR4 on the cell surface of leukaemic blasts from the peripheral blood

Representative examples of the flow cytometric analysis are shown in Fig 3. Leukaemic blasts were gated in a FSC/SSC dot plot. For further discrimination of the malignant blasts from the residual non-malignant leucocytes, co-expression analysis of characteristic surface markers (FITC-labelled antibodies) and CXCR4 (PE-labelled) was performed (in the examples shown: CD34 for AML M2 and CD10 for c-ALL). On average ( Fig 4), high expression (mean fluorescence MF > 50) of CXCR4 was found in AML M3, M4 and M5 blasts, which was in accordance with the observed functional effects of SDF-1 in these subtypes. Only in AML FAB M4 with eosinophilia (M4eo) blasts, which also showed intermediate functional response to SDF-1, the average CXCR4 mean fluorescence was below 50. Flow cytometric data were also available for AML M0 blasts, which were negative for CXCR4 (functional data not available). In B-lineage ALL, CXCR4 was even overexpressed, resulting in a mean fluorescence of between 100 and more than 1000. For example, in Fig 3, a brighter CXCR4 fluorescence of the leukaemic blasts (CD10+) could be observed compared with the residual non-malignant mononuclear cells (CD10). The difference in the CXCR4 expression was statistically significant when myeloid (FAB M1/2) and myelomonocytic (FAB M4/5) AML blasts were compared or when myeloid (FAB M1/2) AML and ALL blasts were compared.

image

Figure 3. Analysis of CXCR4 expression on AML and ALL blasts by flow cytometry. The results of two representative flow cytometric analyses are shown. Leukaemic blasts from peripheral blood were gated in a FSC/SCC dot plot. Co-expression analysis of specific surface markers (CD34 and CD10) and CXCR4 was used to further discriminate leukaemic blasts (indicated by a bold rectangular gate) from remaining non-malignant, mononuclear cells. CXCR4 expression was low in AML FAB M2 blasts from a representative patient and brightly positive in common ALL (cALL) blasts. Isotype identical IgG antibodies served as negative controls.

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image

Figure 4. CXCR4 mean fluorescence intensities in AML subtypes and ALL. CXCR4-PE mean fluorescence intensities were calculated from dot plots (examples are shown in Fig 3). Mean fluorescence intensities > 50 (on average) were found in AML M4 and M5, and ALL, which corresponded with the functional effects of SDF-1 preferentially in these subtypes. Also in AML M3, which showed only intermediate response to SDF-1, a mean fluorescence > 50 (on average) was observed. The difference in CXCR4 expression between myeloid (M1/2) and myelomonocytic AML (M4/5) or ALL was statistically significant.

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Comparison of CXCR4 expression in circulating and bone marrow-derived blasts

In ten cases of AML, both peripheral blood and bone marrow samples were available and analysed in parallel. As expected, the percentage of blasts tended to be greater in the cells derived from bone marrow compared with the peripheral blood (66% vs. 59%). Particularly in cases with AML M4 or M5, bone marrow-derived blasts expressed a greater level of CXCR4 ( Fig 5A). On average, the CXCR4 mean fluorescence was approximately twofold higher in bone marrow-derived blasts compared with circulating cells ( Fig 5B). For AML M4/5 blasts, the difference between peripheral blood and bone marrow was statistically significant.

image

Figure 5. Comparison of CXCR4 expression on peripheral blood and bone marrow-derived blasts. Peripheral blood and bone marrow samples were analysed in parallel. In CXCR4-positive AML, bone marrow-derived blasts consistently showed a greater expression level compared with circulating blasts. In A, two representative examples (fluorescence histograms) are shown. To analyse the CXCR4-PE mean fluorescence intensities of PB and BM-derived blasts (B), the FAB subtypes were divided into two groups: low CXCR4 expression (FAB M1 and M2, n = 4) and high CXCR4 expression (FAB M4 and M5, n = 6). In both groups, the mean fluorescence intensities were on average approximately twofold greater in BM compared with PB-derived blasts. The difference in CXCR4 expression between peripheral blood and bone marrow-derived myelomonocytic AML (M4/5) blasts was statistically significant.

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In vitro transendothelial migration of circulating and bone marrow-derived blasts, effect of blocking CXCR4 mAb

According to the higher expression of CXCR4, bone marrow-derived blasts migrated more avidly in response to SDF-1-containing, conditioned medium compared with circulating blasts ( Fig 6). Particularly, bone marrow-derived AML M4/5 blasts efficiently migrated in response to the conditioned medium. For AML M4/5 blasts, the difference between peripheral blood and bone marrow was statistically significant. Migration of both circulating and bone marrow-derived blasts was inhibited by the partial blocking of CXCR4 mAb 12G5 (10 μg/ml), indicating that SDF-1 is the predominant chemotactic activity contained in the stromal cell conditioned medium, which is in accordance with previous studies ( Bleul et al, 1996a ; Aiuti et al, 1997 ; Möhle et al, 1998 ).

image

Figure 6. Comparison of in vitro transendothelial migration of peripheral blood and bone marrow-derived blasts. Particularly in myelomonocytic AML (FAB M4 and M5), bone marrow (BM)-derived blasts migrated more efficiently in response to SDF-1-containing conditioned medium compared with peripheral blood (PB)-derived blasts (statistically significant for AML M4/5 blasts). Addition of a blocking CXCR4 mAb inhibited migration, indicating that SDF-1 is the predominant chemotactic activity for leukaemic blasts contained in the stromal cell-conditioned medium.

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Discussion

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank Christine Zimmermann and Petra Mayer for excellent technical assistance. This study was supported in part by grants from Deutsche Forschungsgemeinschaft (SFB 510).

References

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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