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Cancer Research 68, 8429, October 15, 2008. doi: 10.1158/0008-5472.CAN-08-1758
© 2008 American Association for Cancer Research
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Immunology

Cell Motility in Chronic Lymphocytic Leukemia: Defective Rap1 and {alpha}Lβ2 Activation by Chemokine

Kathleen J. Till1, Robert J. Harris1, Andrea Linford1, David G. Spiller2, Mirko Zuzel1 and John C. Cawley1

1 Division of Hematology, School of Cancer Studies and 2 Center for Cell Imaging, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom

Requests for reprints: Kathleen J. Till, University of Liverpool, Daulby Street, Liverpool, L12 4XZ, United Kingdom. Phone: 44-151-706-4282; Fax: 44-151-706-4311; E-mail: k.j.till{at}liv.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Chemokine-induced activation of {alpha}4β1 and {alpha}Lβ2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of {alpha}Lβ2; engagement of {alpha}4β1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in {alpha}Lβ2 activation and determine how it is corrected. We show here that the {alpha}Lβ2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both {alpha}4β1 and {alpha}Lβ2 is defective, autocrine VEGF and chemokine are necessary to activate {alpha}4β1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for {alpha}Lβ2 activation for motility and TEM. The present study not only clarifies the nature of the {alpha}Lβ2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL. [Cancer Res 2008;68(20):8429–36]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
It has been known for many years that extensive accumulation of malignant lymphocytes in the lymph nodes, spleen, and bone marrow of chronic lymphocytic leukemia (CLL) patients is associated with progressive disease and an adverse prognosis. This tissue infiltration provides a microenvironment favoring the survival and proliferation of the malignant cells and also leads to suppression of hematopoiesis and immune function. Indeed, tissue invasion by malignant cells is such an important pathogenetic process in CLL that it forms the basis of current clinical staging systems (1, 2). For all these reasons, understanding the mechanisms governing tissue invasion in CLL is a subject of major clinical importance.

Tissue invasion in CLL requires the motility and transendothelial migration (TEM) of the malignant cells. These processes involve chemokines, activation of both {alpha}4β1 (VLA-4; CD49d/29) and {alpha}Lβ2 (LFA-1; CD11a/18) integrins, and ligand binding]to VCAM-1 and intercellular adhesion molecule-1 (ICAM-1), respectively]. We have therefore focused on the role of chemokines, and these two integrin heterodimers, in CLL cell motility on and through endothelium (3, 4). Our most recent work (4) showed that the chemokine-induced clustering of {alpha}Lβ2 is defective in CLL cells and that combined stimulation by {alpha}4β1 engagement and autocrine vascular endothelial growth factor (VEGF) overcomes this defect, allowing {alpha}Lβ2-dependent motility and TEM. We also showed that CLL cells, which express little or no {alpha}4β1 at their surface, display a markedly reduced ability to undergo chemokine-stimulated TEM (3). These observations have substantial clinical relevance because we have shown a strong correlation between {alpha}4β1 expression and clinical lymphadenopathy (3). The importance of {alpha}4β1 for tissue invasion by malignant cells is further underscored by the recent demonstration that expression of this integrin is a major prognostic indicator in CLL (5).

Regarding {alpha}Lβ2, its activation involves both conformational changes and lateral mobility (clustering) at the cell surface (6–10). In our previous work, we showed that there is a failure of chemokine-induced polar clustering of this integrin in {alpha}4β1+ CLL clones (4) but did not attempt to further characterize the nature of the defect in activation of {alpha}Lβ2.

The aims of the present study, therefore, were to define the nature of the defect in activation of CLL cell {alpha}Lβ2. In addition, we sought to establish the mechanisms by which stimulation involving chemokine, {alpha}4β1 engagement, and autocrine VEGF overcomes this defect. These studies are of obvious clinical relevance, because it has long been known that malignant lymphocyte recirculation is abnormal in CLL (11) and there is currently much interest in the therapeutic potential of reagents that block {alpha}4β1, {alpha}Lβ2, and VEGF (12–14).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Patients and Donors
The study involved 19 patients with CLL, all of whom had typical CLL with respect to morphology and surface marker expression (CD5-positive and CD23-positive with dim light chain-restricted immunoglobulin). All patients had a total WBC count of >50 x 109/L, and mononuclear cell preparations therefore always had ≥90% CLL cells. A given CLL clone was regarded as {alpha}4+ when surface fluorescence-activated cell sorting (FACS) analysis showed a clear peak shift with an MFI of >10. For {alpha}4– clones, the stained peak virtually overlapped that of the class-specific immunoglobulin control. Further patient details are given in Supplementary Table S1.

Human umbilical vein endothelial cells (HUVEC) were prepared as previously described (3). Normal peripheral blood mononuclear cells (PBM) were either from the blood of normal volunteers or from buffy coats prepared by National Blood Transfusion Service.

All samples were obtained with informed consent and with the approval of the Liverpool Research and Ethics Committee, Royal Liverpool and Broadgreen University Hospitals Trust and the Research and Development Committee, Liverpool Women's Hospital.

Cell Preparation and Culture
CLL cells and PBM were isolated from peripheral blood and buffy coats by Ficoll-Hypaque density gradient centrifugation. Normal B cells were purified from PBM, either by positive selection using CD19-conjugated magnetic beads or by negative selection using a B-cell isolation kit (Miltenyi Biotech; >98% and >95% CD20+, respectively). Lymphocytes were cultured (5% CO2 in air) at 37°C in RPMI containing 1% bovine serum albumin (BSA; Sigma), 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin (Invitrogen).

In certain experiments, cells were cultured on polyHEMA-coated plates (Sigma) in serum-free medium (Invitrogen) containing 0.1% BSA. Cells cultured under these conditions are deprived of the exogenous stimuli received in vivo from serum and adhesion; however, endogenous stimuli persist and the cells remain viable (15).

Live Cell Imaging and TEM
CLL cells (5 x 105) were added to 3.5 mm2 Petri dishes (IWAKI) coated with HUVEC or with VCAM-1 or ICAM-1 (both at 10 µg/mL; R&D Systems), and motility was measured as previously described (4). TEM of CLL cells was measured after 6 h (3).

Inhibition Studies
Cells were incubated at 4°C for 30 min with blocking monoclonal antibodies (mAb) to either {alpha}4 (5 µg/mL) or {alpha}L (10 µg/mL) or with isotypic control mAbs at the relevant concentrations (all mAbs from R&D). In addition, cells were incubated with an inhibitor of VEGF receptor kinase activity, SU5416 (10 µmol/L; Merck Biosciences), or with 0.25% DMSO (the diluent) alone for 2 h at 37°C.

Staining
Activation antigens. Lymphocytes ± chemokine [CXCL12 (100 ng/mL), CXCL13 (10 ng/mL), CCL21 (1 µg/mL)] or ± Mg2+ + EGTA were stained with conformation-dependent anti-{alpha}Lβ2 mAbs (all at 10 µg/mL) according to the method of Cabanas and Hogg (16); staining was analyzed by FACS or confocal microscopy. The chemokines (all from R & D) were used at concentrations that induced maximal CLL cell TEM (ref. 3; data not shown). The conformation-dependent mAbs used were mAb24 (16), NKI-L16 (17), and 327A and 327C (ref. 9; kindly donated by Nancy Hogg, LRF London; Carl Figdor, University Medical Center Nijmegen; and ICOS Corporation, respectively). As a measure of total {alpha}L, cells were also stained with a conformation-independent anti-{alpha}L mAb (10 µg/mL; Santa Cruz Biotechnologies).

Rap1. CLL cells were either untreated, incubated with CXCL12 (100 ng/mL) for 5 min or with an exchange protein directly activated by cAMP (EPAC) agonist (8-Br-2'-0-Me-cAMP, 10 µg/mL; Biolog) for 1 min and then cytospun before fixation in methanol. Slides were blocked in 10% BSA before being stained with an antibody to Rap1 (4 µg/mL) or with control rabbit immunoglobulin (both from Santa Cruz), followed by goat anti-rabbit immunoglobulin-AlexaFluor568.

Adhesion Assay
A bead assay was used to measure adhesion (18). Briefly, slides were coated with BSA (10%), ICAM-1 (10 µg/mL), or VCAM-1 (10 µg/mL) ± CXCL12 (100 ng/mL). Latex beads (Sigma) were also coated with ICAM-1, VCAM-1, or BSA (all 10 µg/mL). CLL cells were then added to the coated slides and incubated with the labeled beads for 4 h, fixed in 1% formaldehyde, and stained with hematoxylin. The number of beads that had bound to 100 cells was then counted.

Rap1 Assay
1 x 107 cells were lysed, and an aliquot was removed to examine total protein levels. Rap1-GTP was then pulled down from the remaining lysate using Ral-GDS-RBD beads (Upstate Biotechnology; ref. 19). Levels of the GTPase were then measured in the lysate and pull-down by Western blotting using a Rap1 antibody (Santa Cruz).

The effects of various treatments on Rap1-GTP levels were examined. Cells were treated with chemokines or the EPAC agonist (both for 1 min) or with VCAM-1 ± CXCL12 for 5 min. The B-cell receptor (BCR) was stimulated with either F(Ab)2 goat anti-human IgM (10 µg/mL) cross-linked with F(Ab)2 rabbit anti-goat immunoglobulin (both from Jackson Immunoresearch Laboratories) or with antihuman IgM coated beads (Sigma) for 5 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Our previous studies of CLL cell motility, which showed that chemokines fail to induce polar clustering of {alpha}Lβ2, involved only clones expressing {alpha}4β1 (4). These investigations were restricted in this way because clones expressing {alpha}4β1 had a markedly greater ability to undergo TEM in response to chemokine, and because such TEM correlated with clinical lymphadenopathy (3, 4). Therefore, it seemed important to investigate {alpha}Lβ2 function in clones lacking {alpha}4β1 to establish whether or not the defect in {alpha}Lβ2 function is a general feature of CLL cells.

The {alpha}Lβ2 of {alpha}4β1– CLL cells does not cluster or mediate motility in response to chemokine. Using live cell imaging, we first examined the distribution of {alpha}Lβ2 on {alpha}4β1– CLL cells in contact with HUVEC (which express ICAM-1 and produce chemokines) or with purified ICAM-1 + chemokines (CXCL12 or CCL21). The {alpha}Lβ2 of the CLL cells, in contrast to that of normal B cells, did not become clustered in a polar fashion on either HUVEC (n = 4; 3 ± 3% clustered versus 47 ± 5% for normal B cells) or ICAM-1 + chemokine (n = 4; 4 ± 6% clustered versus 31 ± 1% for normal B cells). Furthermore, the cells did not undergo {alpha}Lβ2-dependent motility.

We concluded at this stage that CLL clones, regardless of their expression of {alpha}4, have a defect in the polar clustering of {alpha}Lβ2 induced by chemokines and that this is associated with defective movement dependent on this integrin heterodimer.

Clustering of integrins during cell activation is often accompanied by changes in their conformation (6, 7, 20). Therefore, we next examined the conformation of {alpha}Lβ2 on untreated and chemokine-stimulated CLL cells and compared it with that of normal B cells.

The {alpha}Lβ2 of CLL cells is variably activated and not altered by chemokine stimulation. In unstimulated cells, {alpha}Lβ2 has a bent conformation, which has low affinity for ligand (8). Upon cell stimulation, the molecule extends to an intermediate affinity form on which an epitope detected by the antibody NKI-L16 is exposed (8, 21). Further conformational changes in the ligand-binding I domain are responsible for high-affinity ligand binding (21–23); these changes are detected by mAb24 and/or mAbs 327A and 327C.

The immobilization of chemokine and engagement of {alpha}Lβ2 affect the expression of the activation epitopes in a different way than do soluble chemokines (21). Therefore, using the above mAbs, we first examined unstimulated cells and then determined the effects of both soluble and immobilized chemokine(s) + ligand. In these experiments, Mg2+ treatment was used as a positive control because this divalent cation forces the exposure of the epitopes detected by mAb24 and 327A and 327C while decreasing that of NKI-L16 (21). Normal B cells were examined for comparison, and because their expression of {alpha}Lβ2 activation epitopes and the effect of chemokines thereon had not been clearly defined in the literature.

{alpha}4β1+ CLL cells resembled normal B cells in their expression of the {alpha}Lβ2 activation epitopes, except that the conformation detected by NKI-L16 was more strongly expressed (P = 0.032; Fig. 1 ). For both {alpha}4β1+ CLL and normal B cells, Mg2+ markedly increased the expression of the mAb24 epitope (P < 0.03) and had a less marked, but significant, effect (P < 0.03) on the expression of the 327A and 327C epitopes. In contrast, unstimulated {alpha}4β1– CLL cells, in addition to expressing the NKI-L16 epitope, consistently displayed higher levels of the activation epitopes detected by mAbs 327A, 327C, and mAb24 (P < 0.05). Moreover the expression of these epitopes was not enhanced by Mg2+, suggesting that the {alpha}Lβ2 of these cells is already fully activated (Fig. 1).


Figure 1
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  		Figure 1. Effect of soluble chemokine stimulation on the activation of {alpha}Lβ2 as measured by FACS analysis of cells stained with mAbs detecting activation epitopes. The values shown are the MFIs ± SEM of the test mAb, whereas the MFIs of the IgG1 class-specific control ± SEM indicate background staining. The percentage of positive cells is not shown, because for all the mAbs, >95% of cells were stained. For normal B cells, lymphocytes from three donors were examined, whereas for CLL, five {alpha}4– and five {alpha}4+ clones were examined. Where the error bars for the {alpha}4– clones were relatively large, this was the result of variations in expression of epitopes from clone to clone, but for each individual clone, stimulation by chemokine did not significantly increase reactivity (in a paired t test, P > 0.05; see also Supplementary Fig. S1); in particular, the apparent increase in NKI-L16 (*) after chemokine treatment was not significant.

 
Although the baseline levels of {alpha}Lβ2 expression were different in {alpha}4β1+ and {alpha}4β1– CLL clones, soluble chemokines (CXCL12, CXCL13, and CCL21; all relevant to the pathogenesis of CLL; refs. 3, 24, 25) had no effect on the expression of the activation epitopes. As expected (9, 16, 21), soluble chemokines increased the expression of the 327A and 327C epitopes on normal B cells, but had no effect on mAb24 epitope expression. However, in contrast to T cells (21), the unstimulated normal B cells expressed little or no NKI-L16 epitope and levels were increased after exposure to soluble chemokines (Fig. 1 and Supplementary Fig. S1). The observed changes were not attributable to altered expression of total {alpha}Lβ2 because, as shown using a conformation-independent mAb, levels of the integrin were not affected by chemokine or Mg2+ (data not shown).

To test the effect of immobilized chemokine and ligand binding (9, 16, 26), we examined cells on either HUVEC or ICAM-1 + chemokine. On both surfaces, activation epitopes were not increased on either {alpha}4β1+ and {alpha}4β1– CLL cells (Fig. 2A and Supplementary Fig. S2). In contrast, on normal B cells, which become motile on both surfaces, the expression of mAb24 (Fig. 2A) and NKI-L16 epitopes (Supplementary Fig. S2) was enhanced and became redistributed in a polar fashion.


Figure 2
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  		Figure 2. A, effect of immobilized chemokine and ligand engagement on the expression of the mAb24 activation epitope of {alpha}Lβ2 on normal B and CLL cells. B, adhesion of CLL cells to ICAM-1–coated beads. A, representative confocal images of CLL clones (n = 5 each for {alpha}4+ and {alpha}4–) and highly purified normal B cells (n = 3) stained for mAb24; reactivity with the other MAbs is shown in Supplementary Fig. S2. The images are composites of more than one field; at least five cells are present in each panel. However, in certain panels, the cells are only faintly seen because of their low or absent expression of the epitope detected by mAb24. For reasons that are unclear, incubation of {alpha}4– cells on ICAM-1 + CXCL12 resulted in reduced expression of the epitopes detected by all the conformation-dependent antibodies. B, CLL cells were incubated on either BSA-coated or VCAM-1–coated plates. The number of ICAM-1–coated beads that adhered to these cells was then counted. The numbers assigned to a particular clone in this and other figures (3 and 5) correspond to the patient no. in Supplementary Table S1.

 
Taken together, these results indicate that, in CLL, the induction of {alpha}Lβ2 activation epitopes by chemokines is defective and that the {alpha}Lβ2 of {alpha}4β1– clones is in a fully activated conformation.

It next seemed important to see whether these different activation states of {alpha}Lβ2 in {alpha}4β1+ versus {alpha}4β1– CLL clones result in functional differences. Because both cell types are not motile on ICAM-1 ± chemokine, we examined their ability to bind ligand-coated beads.

{alpha}4+ and {alpha}4– CLL clones differ in their {alpha}Lβ2-mediated adhesion. {alpha}4β1+ CLL clones incubated on BSA were unable to bind the ICAM-1–coated beads. However, when the slide was coated with VCAM-1 to engage their {alpha}4β1, the cells bound the beads. In contrast, CLL clones lacking {alpha}4β1 bound the ICAM-1–coated beads when plated on either BSA or VCAM-1 (Fig. 2B).

These results indicate that the fully extended {alpha}Lβ2 on {alpha}4β1– cells is able to bind ICAM-1 without stimulation, whereas the form of {alpha}Lβ2 on {alpha}4β1+ cells requires stimulation via {alpha}4β1–VCAM-1 interaction for adhesion to become demonstrable.

We next examined the effect of chemokine. Treatment of {alpha}4+ or {alpha}4– clones with CXCL12 had no effect on the binding of ICAM-1–coated beads to cells incubated on BSA and did not enhance such binding when the cells were plated on VCAM-1 (data not shown). These findings are in accord with our observations that chemokine does not affect either the conformation (data above) or clustering (4) of the {alpha}Lβ2 of CLL cells.

We then turned to the mechanism(s) involved in the defect in chemokine-induced activation of CLL cell {alpha}Lβ2. Because signaling via Rap1 is central to chemokine-induced activation of {alpha}Lβ2 (27–29), we next examined the activation of this GTPase in CLL cells.

The chemokine-stimulated GTP loading of Rap1 is defective in most CLL clones. We first used Western blotting to examine Rap1 protein in CLL cells and found that the protein is variably expressed (Fig. 3A ; n = 12, {alpha}4+ n = 6, {alpha}4– n = 6). We then used a pull-down assay to determine the GTP loading of Rap1 in the same CLL clones. GTP-loaded Rap1 was readily detected in the cells of all cases studied, but levels varied from clone to clone (Fig. 3A). In all 12 clones, levels of GTP-Rap1 correlated with the amount of Rap1 protein expressed (R2 = 0.91; Supplementary Fig. S3A). However, there was no significant difference between {alpha}4+ and {alpha}4– clones with regard to total Rap1 protein, Rap1-GTP, or Rap1-GTP/Rap1 ratios (P > 0.3; Supplementary Fig. S3B).


Figure 3
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  		Figure 3. Rap1 and Rap1-GTP in CLL cells: effects of stimulation. A, Western blots of Rap1 and Rap1-GTP. The analysis was carried out using lysates from equal numbers of cells from each case of CLL (eight representative clones); Rap1-GTP was pulled down from aliquots of these lysates. B, the effect of chemokine and an agonist of EPAC (EPAC-A) on CLL-cell Rap1-GTP levels. The nonresponsive CLL is an example of the majority of clones in which Rap1-GTP was not increased on chemokine/EPAC-A stimulation. In contrast, the responsive CLL is an example of the minority of clones in which stimulation increased the GTP loading of Rap1. Normal B cells (n = 3) are shown for comparison, whereas WEHI 231 cells constituted a positive control. C, CLL cells were treated with either CXCL12 or an agonist of EPAC, stained for Rap1, and examined by confocal microscopy. Representative cell sections of clones, which did or did not GTP load their Rap1 in response to chemokine, are shown [n = 2 responders (1 {alpha}4β1+) and 2 nonresponders (1 {alpha}4β1+)]. Note that, in the responsive clone, Rap1 has become redistributed to patches at the cell surface.

 
We next examined the effect of CXCL12 on GTP loading of Rap1 in the same and additional cases. In 11 of the 16 clones studied (9 {alpha}4+ and 7 {alpha}4–), chemokine did not enhance the GTP loading of Rap1 (Fig. 3B). As expected, CXCL12 enhanced Rap1-GTP loading in highly purified normal B cells (n = 3; Fig. 3B). When CLL clones were stimulated with two other chemokines (CCL21 and CXCL13), identical results were obtained (Supplementary Fig. S3C). Taken together, these results indicate that chemokine-induced activation of Rap1 is defective in ~70% of CLL clones.

We next sought to confirm this conclusion using a different method. On activation, Rap1 is translocated to the cell membrane where it regulates integrin activation (30, 31). We therefore used confocal microscopy to examine the location of Rap1 in CLL cells before and after chemokine stimulation. In CLL clones, which failed to GTP load their Rap1 in response to chemokine, the distribution of the GTPase was unchanged (Fig. 3C). In contrast, in the minority of CLL clones in which chemokine did induce Rap1 activation, the GTPase was translocated to the membrane in a clustered fashion (Fig. 3C).

Having established that the chemokine-induced activation of Rap1 is frequently defective in CLL, we next examined possible mechanisms involved.

The defect in chemokine-induced Rap1-GTP loading in CLL cells is at the level of the GTPase. The GTP loading of Rap1 in response to chemokine is mediated by CalDAG-GEFI (32). However, Rap1 can be activated by a number of other guanine nucleotide exchange factors (GEF), including EPAC, which is highly expressed in CLL cells (33). Therefore, we next measured Rap1-GTP in CLL cells treated with an agonist of EPAC, arguing that if the defect is upstream of Rap1, the EPAC agonist would stimulate Rap1-GTP loading. In fact, in cases in which CXCL12 did not increase GTP loading, the EPAC agonist also did not increase Rap1-GTP levels (n = 11; Fig. 3B). In the minority of clones where chemokine did enhance the GTP loading of Rap1 (n = 5) and in normal B cells (n = 3), the EPAC agonist increased GTP-Rap1 to the same extent as chemokine (Fig. 3B).

We next used confocal microscopy to confirm these results. As expected, where EPAC did not induce GTP loading of Rap1, the GTPase was not translocated to the cell membrane and vice versa (Fig. 3C).

We also stimulated the Rap1 of CLL cells by BCR cross-linking, which activates the GTPase via a third GEF (34). BCR stimulation using both immobilized and soluble anti-IgM failed to induce further GTP loading of Rap1 in the clones that did not respond to chemokines or to the EPAC agonist and vice versa (n = 4 clones; all responded to BCR cross-linking by increasing [Ca2+]i; Supplementary Fig. S3C).

The fact that stimulation by pathways involving three different GEFs did not induce GTP loading of Rap1 indicates that the defect in chemokine-induced activation of Rap1 is not upstream of the GTPase.

The defect in chemokine-induced Rap1-GTP loading is the result of in vivo stimulation. There is now considerable evidence that CLL cells have been activated in vivo (35, 36), and the results presented, thus far, support this view. We consequently hypothesized that the absence of Rap1-GTP loading in response to chemokine is the result of such stimulation. We therefore cultured CLL cells in serum-free medium on polyHEMA-coated plates (to prevent exogenous stimulation; ref. 15) and examined Rap1-GTP levels ± chemokine stimulation. In clones that did not increase their Rap1-GTP loading in response to chemokine, Rap1-GTP levels dropped after 24 to 48 h of culture and loading in response to chemokine became demonstrable (Fig. 4A ). Furthermore, after such culture, {alpha}4– clones displayed a markedly increased ability to undergo {alpha}Lβ2-dependent TEM in response to chemokine (Fig. 4B). This confirms the notion that in vivo stimulation leads to GTP loading of Rap1 and chemokine unresponsiveness.


Figure 4
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  		Figure 4. Effect of culture on Rap1-GTP loading and TEM of CLL cells. CLL clones, which failed to increase Rap1-GTP in response to chemokine, were used. A, Rap1-GTP and its response to CXCL12 were examined. The results were similar regardless of {alpha}4 expression, and a representative example of each is shown (n = 4; two {alpha}4+ and two {alpha}4–). For each time point, the Rap1 protein levels were very similar in control and stimulated cells. B, TEMs of {alpha}4– cells before and after culture are shown (representative of n = 2).

 
We next turned to the minority of CLL clones, which were able to GTP load their Rap1 in response to chemokine. We hypothesized that such CLL clones would differ from the majority of cases (4) in being able to cluster their {alpha}Lβ2 and undergo TEM in response to chemokine in a manner independent of autocrine VEGF and {alpha}4. In fact, the chemokine did indeed induce polar clustering of {alpha}Lβ2 in these cells and they underwent {alpha}Lβ2-dependent TEM even when {alpha}4 engagement and autocrine VEGF were blocked (n = 3, two {alpha}4+ and one {alpha}4–; data not shown).

Having shown that the defect in {alpha}Lβ2 function observed in most CLL clones is the result of in vivo stimulation of Rap1, we next examined the mechanism(s) by which {alpha}4 engagement and autocrine VEGF overcome this defect.

Autocrine VEGF and chemokine activate {alpha}4β1 for ligand binding, and together, these stimuli induce {alpha}Lβ2 clustering in a Rap1-independent manner. It is well established that cross-talk from {alpha}4β1 can induce activation of {alpha}Lβ2 by an unknown mechanism (37, 38). We hypothesized that this cross-talk occurs independently of Rap1. We therefore incubated {alpha}4+ CLL cells (which did not GTP load their Rap1 in response to chemokine) on VCAM-1, in the presence or absence of CXCL12; no increase in Rap1-GTP was seen (data not shown; n = 3) despite the induction of {alpha}Lβ2 clustering in the presence of chemokine. This indicates that {alpha}4β1 engagement in the presence of chemokine induces clustering independently of Rap1.

In our previous work, we showed that the {alpha}Lβ2-dependent motility/TEM of CLL cells requires autocrine VEGF in addition to {alpha}4β1 engagement and chemokine (4). We therefore next examined the relative contributions of {alpha}4β1 engagement, autocrine VEGF, and chemokine to the polar clustering of {alpha}Lβ2 required for motility and TEM. We first showed that activation of {alpha}4β1 for adhesion to ligand requires combined stimulation by both VEGF and chemokine. Thus, unstimulated CLL cells were unable to bind VCAM-1–coated beads. However, chemokine markedly enhanced the binding of such beads, but this was largely abrogated by a VEGF receptor kinase inhibitor, SU5416 (Fig. 5A ). We next showed that {alpha}4β1 engagement alone does not generate the stimulus for the polar clustering of {alpha}Lβ2. To do this, we incubated CLL cells on an immobilized anti-{alpha}4 mAb and showed that the {alpha}Lβ2 did not become clustered (Fig. 5B), and the cells did not become motile (data not shown). However, addition of chemokine-induced marked clustering and motility, which were inhibited by SU5416 (Fig. 5B; not shown).


Figure 5
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  		Figure 5. Contributions of {alpha}4 engagement, chemokine, and autocrine VEGF to the activation of {alpha}4 and {alpha}L. A, CLL cells, which were either untreated or preexposed to SU5416, were incubated on plates coated with BSA ± CXCL12. The number of VCAM-1–coated beads that adhered to these cells was then counted. B, {alpha}4+ CLL cells, which were again either untreated or had been pre-exposed to SU5416, were stained with an FITC-conjugated anti-{alpha}L mAb. They were then incubated on Petri dishes, which were either uncoated or coated with an anti-{alpha}4 mAb ± CXCL12. Polar clustering of {alpha}L was then examined by live cell imaging.

 
Therefore, autocrine VEGF and chemokine act in concert to stimulate {alpha}4 for adhesion to ligand, and this, together with autocrine VEGF and chemokine, is required for the polar clustering of {alpha}Lβ2, which is necessary for CLL cell TEM (these interactions are shown schematically in Fig. 6 ).


Figure 6
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  		Figure 6. Cartoon summarizing the nature of the defect in activation of CLL-cell {alpha}Lβ2 and its correction by engagement of {alpha}4β1, chemokine, and autocrine VEGF. A, the sequence of events leading to chemokine-induced {alpha}Lβ2 and {alpha}4β1 activation in normal B cells. B, in CLL cells, {alpha}Lβ2 and Rap1 are already activated as a result of in vivo stimulation, and chemokine does not induce additional loading of Rap1. As a result, neither {alpha}Lβ2 or {alpha}4β1 are further activated by chemokine alone. VEGF and chemokine activate {alpha}4β1–VCAM-1 adhesion, and the three stimuli together then induce Rap1-independent polar clustering of {alpha}Lβ2 and TEM in response to chemokine.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The aim of the present study was to characterize the defect in chemokine-induced {alpha}Lβ2 activation that we have previously identified in {alpha}4β1+ CLL cells (3, 4).

Because many CLL clones express little or no {alpha}4β1 (3, 5), we started the present studies by examining chemokine-induced {alpha}Lβ2 clustering on these cells. The {alpha}L of {alpha}4– CLL cells also failed to undergo polar clustering or {alpha}Lβ2-dependent motility when incubated on ligand in the presence of chemokine. Because the {alpha}Lβ2 of normal B cells becomes clustered under these conditions (4), this indicates that defective {alpha}L clustering in response to chemokine is a feature of CLL cells, regardless of their expression of {alpha}4β1.

We next found that the {alpha}Lβ2 of CLL cells tested directly ex vivo is in an activated conformation, but the degree of activation varied among cases, with {alpha}4– clones expressing the high-affinity, fully activated form of {alpha}Lβ2; furthermore, these cells were able to bind ICAM-1. In contrast, {alpha}4β1+ cells expressed the intermediate-affinity form of {alpha}Lβ2 and were unable to bind ligand. Because the intermediate affinity form of {alpha}Lβ2 is important in mediating motility (39), it is likely that the in vivo activation of {alpha}Lβ2 in {alpha}4– cells contributes to the defect in motility/TEM of such CLL clones.

In both {alpha}4+ and {alpha}4– CLL clones, chemokines and ligand binding did not induce further conformational activation of {alpha}Lβ2. In contrast, the {alpha}Lβ2 of normal B cells, like that of unstimulated T lymphocytes (9, 16, 21), was in a nonactive conformation and became activated after chemokine stimulation. Thus, the {alpha}Lβ2 of CLL cells differs from that of normal B cells, not only with regard to its activated conformation and inability to undergo clustering, but also in its failure to undergo chemokine/ligand-induced conformational changes. Because the {alpha}Lβ2 of {alpha}4+ clones became clustered on HUVEC (4) but did not change its conformation (present study), our results indicate that {alpha}4 engagement overcomes the defect in {alpha}Lβ2 by inducing clustering without changing the conformation of the integrin heterodimer. In contrast, in the absence of the possibility of {alpha}4 engagement, the {alpha}Lβ2 of {alpha}4– CLL clones does not cluster and also cannot undergo conformation changes in response to chemokine, because the integrin heterodimer is already in its fully extended high-affinity state. Because polar clustering is necessary for {alpha}Lβ2-mediated motility (7, 20, 29) and this integrin is essential for lymphocyte TEM (40, 41), the present results explain why {alpha}4– CLL cells display markedly reduced TEM in response to chemokine.

We next examined Rap1 activation, because this GTPase is central to integrin activation by chemokines (27, 30). We found that Rap1-GTP is readily detected in the cells of all CLL clones. Rap1-GTP has been shown to induce expression of the intermediate affinity form {alpha}Lβ2, whereas induction of the high-affinity form is Rap1-independent (42). This is fully in accord with our results, which show that both the GTPase and the NKI-L16 epitope are detected in all CLL clones and that the high-affinity conformation observed in the {alpha}4– cells is not linked to levels of Rap1-GTP.

Importantly, the GTP loading of Rap1 was not increased by chemokine in the majority of CLL clones examined. In these clones, chemokine did not induce polar clustering of {alpha}Lβ2 and TEM. In contrast, in a minority of CLL clones, chemokine did activate the GTPase and induce polar clustering of {alpha}Lβ2 and subsequent TEM. Taken together, these data suggest that the inability of chemokine to induce Rap1 activation is central to the defective function of {alpha}Lβ2 observed in CLL.

We next returned to the majority of CLL clones in which chemokine failed to induce GTP loading of Rap1 and attempted to define the cause of this defect. The fact that stimuli activating Rap1 via different GEFs all failed to increase Rap1-GTP levels in these cells indicates that the defect is at the level of Rap1 loading. Furthermore, the defect is clearly the result of in vivo stimulation because cell culture in the absence of exogenous stimulation led to a reduction of Rap1-GTP levels and to a restoration of chemokine responsiveness as measured by Rap1 activation and TEM. We have not identified the nature of this in vivo stimulus, but it is tempting to speculate that the BCR is involved because stimulation of this receptor by (auto)antigen is central to the pathogenesis of CLL (43, 44). It is possible that such stimulation of CLL cells causes their Rap1 to be fully loaded and that these cells have been more stimulated in vivo than have the cells which responded to chemokine. Also, because deficiency of the Rap1–GTPase-activating protein, SPA-1, has been linked to a CLL-like disorder in knockout mice (45), it is possible that SPA-1 levels/function are reduced in human CLL, thereby reducing the ability of Rap1 to convert GTP to GDP. These possibilities will be the subject of a future study.

Finally, we examined the mechanism by which {alpha}4β1 engagement overcomes the defect in chemokine-induced activation of {alpha}Lβ2. We found that such engagement does not activate Rap1 and then, using combinations of stimuli and a VEGF kinase inhibitor, we showed that autocrine VEGF and chemokine together activate {alpha}4β1 for adhesion. Such {alpha}4β1 ligand binding alone was not sufficient to induce polar clustering of {alpha}Lβ2. Rather, ligand binding, autocrine VEGF, and chemokine were all needed for polar clustering of {alpha}Lβ2 and subsequent motility/TEM. These novel interactions are summarized in Fig. 6.

In conclusion, the present studies are the first to show that the in vivo stimulation thought to be important in the pathogenesis of CLL (35, 36, 43, 44) results in conformational activation of {alpha}Lβ2 and failure of chemokine-induced Rap1 activation. Together, these changes lead to a defect in the function of both this integrin heterodimer and {alpha}4β1. We have already shown that most CLL clones differ from normal B cells in requiring {alpha}4β1 engagement and autocrine VEGF to undergo TEM and entry to the proliferation centers of lymph nodes (4). Therefore, the present study, by defining the individual roles of chemokine, autocrine VEGF, and {alpha}4β1–VCAM-1 binding in correcting the defect in chemokine-induced activation of {alpha}Lβ2, emphasizes the therapeutic potential of inhibitors of {alpha}4β1 and/or VEGF.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Leukemia Research Fund (UK).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 5/ 9/08. Revised 7/18/08. Accepted 8/ 8/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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