Differential Adhesion Pattern of B Cell Chronic Lymphocytic Leukemia Cells

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Differential adhesion pattern of B cell chronic lymphocytic leukemia cells SI Behr1,2,3, D Korinth1 and F Schriever1 ¨ 1Department of Hematology and Oncology, Virchow University Hospital, Humboldt Universitat Berlin; and 2Institute of Anthropology and ¨ Human Biology, Freie Universitat Berlin, Germany

Binding of B cell chronic lymphocytic leukemia (B-CLL) cells Engaging CD19 on B-CLL cells specifically induced homo- to other cells and to extracellular matrices influences the typic aggregation through the ligand pair integrin ␣ pathophysiology and the clinical presentation of the B-CLL dis- L ease. It is still unknown which adhesion pathways regulate the (leukocyte function antigen-1 (LFA-1),CD11a)/intercellular traffic of B-CLL cells within distinct histologic compartments adhesion molecule-1 (ICAM-1) and through CD21. We specu- of lymphoid organs. In addition, it is not yet clarified which late that these adhesion mechanisms influence the migration mechanisms mediate the intercellular adhesion of B-CLL cells. and metastasis of B-CLL cells in vivo. The present study sought to identify the mechanisms that are involved in the binding of B-CLL cells to secondary lymphoid organs in situ and in the homotypic aggregation of these cells. B-CLL cells specifically bound to germinal centers of normal Materials and methods human tonsils via the adhesion pair integrin ␣4␤1/vascular cell adhesion molecule-1 (VCAM-1). Among a large panel of anti- B-CLL samples and cell culture bodies tested only mAbs against CD19 induced homotypic ␣ adhesion of B-CLL cells via the adhesion molecules integrin L B-CLL cells were isolated by Ficoll gradient centrifugation (leukocyte function antigen-1 (LFA-1)), intercellular adhesion from the peripheral blood of B-CLL patients (n = 33) after molecule-1 (ICAM-1) and CD21. Anti-CD19-induced aggregation required protein synthesis. We hypothesize that the observed obtaining informed consent. All studies were approved by the heterotypic and homotypic adhesion of B-CLL cells reflects the Ethics Committee of the Virchow Hospital of the Humboldt ability of these leukemic cells to migrate in vivo. University. Samples were only used from patients who had not Keywords: chronic lymphocytic leukemia cells; adhesion received chemotherapy within the previous 3 weeks. Disease stages were defined according to Rai et al.8 Purity and phenotype pattern of the B-CLL samples were characterized by flow Introduction cytometry (FacScan; Becton Dickinson, Heidelberg, Germany). Diagnosis of the CLL cases was confirmed by Intercellular adhesion of B cell chronic lymphocytic leukemia examining co-expression of CD5, CD19, CD23 and surface (B-CLL) cells is relevant for the pathophysiology of the B-CLL immunoglobulin (slg). B cells from the peripheral blood of disease. Migration of B-CLL cells through the peripheral normal healthy donors were prepared by Ficoll gradient cen- blood, selective adhesion to endothelial cells and traffic trifugation and elimination of T lymphocytes by rosetting with through primary and secondary lymphoid organs is determ- sheep red blood cells. For in vitro culture, cells were main- ined by a set of functionally relevant adhesion molecules. Dif- tained at 5% CO2 in RPMI-1640 medium supplemented with ferential usage of these adhesion pathways by B-CLL cells also 10% FCS, 1% glutamine and 1% penicillin/streptomycin. seems to have an important impact on the clinical presen- Effect of mAbs on proliferation and on apoptosis of CLL tation of B-CLL. Members of the integrin family of adhesion cells was investigated by culturing cells (1 × 106/ml) for 72 molecules are differentially expressed in lymphoid leuke- and 48 h, respectively, with various antibodies (5 ␮g/ml) and mias.1–5 Expression of ␤1, ␤2 and ␤3 integrins on B-CLL cells with tetradecanoyl phorbol acetate (TPA; 0.005 ␮g/ml) and correlates with worsening of the prognosis of the disease.2 In dexamethasone (100 ng/ml) as controls. Stimulation of cells addition, sera of patients with acute leukemia contain elev- was quantified using a nonradioactive detection assay accord- ated concentrations of secreted vascular cell adhesion mol- ing to the instructions of the manufacturer (EZ4U; Biomedica, ecule-1 (VCAM-1)6 and levels of secreted ICAM-1 correlate Vienna, Austria) and an ELISA reader. This assay quantifies with progression of B-CLL.7 It is currently unknown, however, cell proliferation by determining the capability of the mito- how these different adhesion pathways guide the traffic of B- chondria to reduce uncolored tetrazolium salts (MTT) into CLL in lymphoid organs and how they determine the inter- intensely coloured formazan derivatives.9 Quantification of cellular adhesion of the leukemic cells. apoptosis and necrosis was performed by flow cytometric The current study aims at defining the adhesion molecules analysis of CLL cells labeled with Annexin-V-FITC10 (Bender that are responsible for the binding of B-CLL cells to distinct MedSystems, Vienna, Austria) and with propidium iodide, histologic compartments of normal lymphoid tissues in situ. respectively. Furthermore, we sought to identify the structures on B-CLL cells that regulate the homotypic aggregation of these cells. B-CLL cells were found to bind via the ligand pair integrin Reagents and monoclonal antibodies (mAbs) ␣4␤1/vascular cell adhesion molecule-1 (VCAM-1) selectively to the germinal centers of secondary lymphoid follicles. mAbs against the following antigens were obtained from Dianova-Immunotech (Hamburg, Germany): CD3 (clones X35 ␣ and UCHT-1), CD5 (BL1a), CD11a (integrin L (25.3.1)) , Correspondence: F Schriever, Virchow University Hospital, Humboldt CD18 (7E4), CD19 (J4.119), CD20 (B9E9/HRC20), CD21 University, Biomedical Research Building, Augustenburger Platz 1, (BL13), CD26 (BA5), CD29 (K20), CD49b (integrin ␣2 (Gi9)), 13353 Berlin, Germany; Fax: 011 49 30 450 59488 ␣ ␣ 3Current address: Max-Planck-Institute of Infectious Biology, Berlin, CD49c (integrin 3 (M-KID 2)), CD49d (integrin 4 (HP2/1)), Germany CD54 (ICAM-1(84H10)), CD58 (LFA-3 (AICD58)), CD102 Received 6 March 1997; accepted 18 September 1997 (ICAM-2 (B-T1)), CD77 (38-13), CD81 (JS64) and CD106 Differential adhesion of B-CLL cells SI Behr et al 72 (VCAM-1 (1G11)). All mAbs were used at a concentration of homotypic aggregation of B-CLL cells was determined using 10 ␮g/ml unless otherwise indicated. Fluorescein isothiocyan- different inhibitors as described.12 B-CLL cells (2 × 106/ml) ° ate (FITC-conjugated mouse IgM (Dako, Glostrup, Denmark) were incubated for 30 min at 37 C with 5% CO2 in media served as a negative control for flow cytometry. Cyclohexim- containing 102 nM staurosporine, 50 ␮M H7, 50 mg/ml ide, herbimycin and propidium iodide were obtained from genistein, 10 ␮g/ml cycloheximide, 1 ␮g/ml herbimycin, Sigma (St Louis, MO, USA). Cytochalasin B, genistein and H7 0.1 mM cytochalasin B or were treated with control media. were purchased from Calbiochem (La Jolla, CA, USA). Stauro- Control media had the same concentrations of DMSO solvent sporine was obtained from Boehringer Ingelheim as were used in media-containing inhibitors. Cells were sub- (Heidelberg, Germany). sequently washed, were treated as described above with mAbs and were examined for reduced homotypic aggregation.

Frozen section binding assay Statistical analyses To study adhesion of B-CLL cells to tonsils in situ,anin vitro frozen section assay was performed with modifications.11–16 Significance of differences of the expression of cell surface Normal human tonsils, obtained from routine tonsillectomies, molecules was determined using the Mann–Whitney U test. were snap frozen, cut (8 ␮m), placed on microscope slides Significance of difference between binding pattern of CLL and dried overnight. B-CLL cells were applied at 2.4 × 107 cells of patients with low (0–II) and high (III and IV) Rai stages cells/ml in 300 ␮l adhesion medium (containing 2.4 mM was determined using the Fisher exact test.

MgCl2) on tissue sections within a 2-cm diameter circle marked with a water-repellent ‘Pap-pen’ (Dako, Carpinteria, CA, USA) and were incubated under rotation (70 revolutions Results per min) for 20 min at room temperature. Rotation was used since under static conditions no specific binding to histologic Adhesion of B-CLL cells to germinal centers of human compartments was noted (not shown). Thereafter, slides were tonsils via integrin ␣4␤1/VCAM-1 decanted, were fixed for a minimum of 5 h in fresh, cold 3%- glutaraldehyde/PBS and were counterstained with hemalaun. To determine the in situ binding pattern of B-CLLs on second- Bound cells were identified by light microscopy (×250). To ary lymphoid tissues, an adhesion assay on human tonsils was determine adhesion molecules involved in the tissue binding, performed. Of 14 different cases examined, CLL cells of seven B-CLL cells were incubated with a set of mAbs, washed and patients bound selectively to the germinal centers of tonsils examined for reduced tissue adherence. To block correspond- (Figure 1). Binding B-CLL samples and cells that failed to ing adhesion molecules on the tissue, tonsils were incubated adhere differed in the patients’ stages of the disease (Table 1). with mAbs and washed prior to applying the cells. Decrease In contrast to the cases with non-binding B-CLL cells, most of binding was assessed in at least three separate experiments patients whose CLL cells showed adhesion had advanced by comparison with the adhesion of cells treated with isotype- stages of their disease (Rai III and IV; P = 0.029). In addition, matched control mAbs. Cell binding was morphometrically binding B-CLL cells expressed integrin ␣4, whereas cells that quantified with digitalized, video-supported light microscopy did not adhere were almost negative for this adhesion mol- using a Zeiss Axioplan microscope (Jena, Germany) and the ecule (P = 0.0017; Figure 2). In contrast, differential ␣ ISIS 3 software (MetaSystems, Belmont, MA, USA). Inhibition expression of integrin L, an antigen involved in germinal of binding was scored as positive (complete inhibition of center binding of T lymphocytes,16 did not correlate with the binding) and negative (no reduction of cell binding). binding pattern of the CLL cells. To define the binding structures responsible for the in situ adhesion, mAbs were incubated with the B-CLLs and were tested for their ability to Homotypic adhesion assay inhibit tissue adherence. Among a large panel of mAbs against adhesion molecules (CD11a, CD18, CD29, CD49b, CD49c, To determine induction of homotypic aggregation of B-CLL CD49d, CD54, CD58, CD102, CD106) only antibodies cells and normal B cells by mAbs, we used a modified method against integrin ␣4 (CD49d) and against the corresponding ␤1 described by Rothlein et al.17 Cells (2 × 106 cells/ml) were chain (CD29) were able to block binding of B-CLL cells. ° cultured at 37 C and 5% CO2 with a series of different mAbs When tonsils were incubated with a mAb against a ligand of (5 ␮g/ml) in duplicates in flat-bottom 96-well microtiter plates. integrin ␣4␤1, VCAM-1, binding was inhibited, whereas a Isotype-matched mAbs were used as negative controls. Aggre- negative control mAb did not impair germinal center binding. gate formation was determined by inverse light microscopy after 24 h. Homotypic adhesion was qualitatively scored as positive (all cells formed aggregates) and negative (no Homotypic adhesion of B-CLL cells transmitted via aggregates). CD19 To determine cell surface molecules involved in the mAb- induced homotypic adhesion, B-CLL cells were treated as In order to identify mechanisms regulating the adhesion above with mAbs for 24 h, washed, resuspended and allowed between B-CLL cells, we investigated aggregate formation of to reaggregate at 37°C for 24 h in media containing a second B-CLL cells induced by engaging cell surface antigens using set of different mAbs. Reduction of adhesion was determined a panel of different mAbs. Only mAbs against the CD19 anti- by comparison with reaggregations in media with isotype con- gen were able to provoke homotypic aggregation of B-CLL trol mAbs and was qualitatively scored as positive (inhibition cells. Antibodies against other antigens expressed on CLL cells of cluster formation) and negative (no inhibition of cluster did not induce homotypic adhesion (CD5, CD11a, CD20, formation). CD21, CD22, integrin ␤1, CD40, CD49c, integrin ␣4, ICAM- The involvement of intracellular pathways in mAb-induced 1, CD81 and ICAM-2). Cluster formation was detected in 32 Differential adhesion of B-CLL cells SI Behr et al 73 a

Figure 2 Differential expression of integrin ␣4 on B-CLL cells determines germinal center binding. Marked by the box diagram are the 10th, 25th, 50th (median), 75th and the 90th percentiles. Open circles indicate values below or above the 10th and 90th percentile, respectively. *P = 0.0017.

Figure 1 Binding of B-CLL cells to germinal centers of human ton- of 33 B-CLL cases after 24 h in vitro culture with anti-CD19. sil sections via integrin ␣4. B-CLL cells were incubated without prior Phenotypic analyses showed that lack of response to treatment treatment (a) and with prior treatment with anti-integrin ␣4 mAb (b) with anti-CD19 in one B-CLL sample was not due to low under rotation on dried tonsil sections. Binding and inhibition of expression levels of CD19 on these tumor cells (not shown). × adhesion was assessed by light microscopy ( 250). Interestingly, mAbs against CD21 and TAPA-1, which, together with CD19 and Leu-13, form a functionally important membrane complex on B cells,18 did not induce homotypic adhesion of B-CLL cells. In contrast to B-CLL cells, normal Table 1 Binding of B-CLL cells to the germinal centers of peripheral blood B cells did not show anti-CD19-speciﬁc human tonsils homotypic aggregation. Case Stage Age Sex Germinal center Next, we aimed at identifying the adhesion molecules that (Rai) (M/F) bindinga were involved in the anti-CD19-induced aggregation of B-CLL cells. Cellular clusters of anti-CD19-treated B-CLL cells were 1IV59M + dispersed and were examined for reduced reaggregation after 2IV84F + in vitro culture with mAbs. As seen in Table 2, reformation of 3IV45M + clusters was observed even in the absence of anti-CD19 mAb. 4 III 61 F + Among a large panel of different antibodies tested only mAbs + 5 III 69 M against integrin ␣ , ICAM-1 and CD21 were able to inhibit 6IV77F + L 7IV79F − cluster reformation of anti-CD19-treated B-CLL cells. Treat- 8II61M − ment with anti-CD19 mAb, however, did not lead to an 9 0 67 F − increased expression of the relevant adhesion molecules inte- − ␣ 10 0 64 F grin L, CD21 and ICAM-1 (not shown). When tested indi- 11 0 70 M − vidually, mAbs against integrin ␣ , ICAM-1 and CD21 blocked − L 12 I 50 M aggregation in more than half of the CLL cases examined 13 II 68 M − ␣ 14 I 80 M + (Table 2). Furthermore, combining anti-integrin L and anti- CD21 inhibited adhesion in all but one CLL case (95%; 18 aResults were obtained using morphometric analyses. Average of 19 cases tested) and anti-ICAM-1 together with anti-CD21 binding of cells: +, 986 ± 210/0.1 mm2; −,15± 5/0.1 mm2. interfered in 79% of the cases (15 of 19 cases examined). Difference between binding of CLL cases with high Rai stages (III To identify intracellular signals involved in the anti-CD19- and IV) and non-binding of cases with low stages (0–11): P = 0.029. induced homotypic aggregation of B-CLL cells, inhibitors of Differential adhesion of B-CLL cells SI Behr et al 74 Table 2 Inhibition of reaggregation of anti-CD19-treated B-CLL cells

No inhibition of reaggregation by mAbs against:a Inhibition of reaggregation by mAbs against:

b c negative control, CD5, CD9, CD11b Integrin ␣L (CD11a) 60% 15/25

CD11c, CD15, CD16, CD18, CD20 CD21 61% 16/26

CD22, CD23, CD26, CD41, integrin ␣2–␣6 ICAM-1 (CD54) 52% 13/25

␣ CD51, CD61, CD69, CD70 Integrin L and CD21 95% 18/19

CD72, CD77, CD103, CD106 ICAM-1 and CD21 79% 15/19

an = 9. bPercentages of B-CLL cases in which anti-CD19-induced aggregation could be inhibited. cNumber of B-CLL cases in which anti-CD19-induced aggregation could be inhibited/total number of cases examined

deﬁned intracellular pathways were used. Aggregation triggered by anti-CD19 mAb was blocked by an inhibitor of protein synthesis (cycloheximide), whereas inhibiting protein kinases (staurosporine), protein kinase C (H7), tyrosine kinases (genistein and herbimycin) or the cytoskeleton (cytochalasin B) did not impair homotypic adhesion. These observations indicated that protein synthesis was involved in the anti- CD19-triggered homotypic aggregation of B-CLL cells. Furthermore, to determine the inﬂuence of anti-CD19 on the biology of CLL cells, the effect of anti-CD19-treatment on stimulation and apoptosis of B-CLL cells was investigated. However, the effect of anti-CD19 on these parameters did not differ from that of control antibodies (Figure 3).

Discussion

The present study describes cellular mechanisms regulating lymphoid tissue binding and homotypic adhesion of B-CLL cells. B-CLL cells specifically bound to germinal centers of tonsils via integrin ␣4␤1/VCAM-1 and triggering via CD19 induced homotypic aggregation of CLL cells via integrin ␣ L/ICAM-1 and CD21. Defining these adhesion pathways might provide help in exploring the pathophysiology of the CLL disease. A frozen section assay11–16,19,20 was used to determine whether human B-CLL cells bind to distinct histologic compartments of human tonsils. Previously, clinically relevant data were provided with this assay by demonstrating that the in situ binding pattern of lymphoma cells was an indicator for the capacity of the tumor cells to metastasize in vivo.21 In addition, adhesion of non-Hodgkin’s lymphoma cells to HEV was found to correlate with the clinical manifestation of the disease.19 The current study demonstrates that in vitro B-CLL cells employ the adhesion molecule integrin ␣4␤1 to bind to Figure 3 Treatment with anti-CD19 mAb has no effect on prolifer- VCAM-1 within germinal centers of normal human tonsils. ation or apoptosis of B-CLL cells. Effect of treatment with mAbs and Germinal centers of secondary lymphoid organs are filled with with TPA on proliferation of CLL cells (a). Apoptosis of CLL cells after incubation with mAbs and with dexamethasone (b). a dense network of antigen presenting follicular dendritic cells (FDCs)13,22–24 which have been identified as ligands for binding of B and T cells in vitro and in situ.13–16,20,25,26 the binding, we cannot completely rule out the possibility that Interestingly, in situ binding of CLL cells was not mediated these or other adhesion molecules were involved in the in situ ␣ ␤ 14,16 by integrin L 2, although this antigen was differentially adhesion process. Consistent with previous reports, the expressed by these cells and has been shown to mediate ger- result of the binding assay appears to be determined by the minal center binding of activated T cells.16 Possibly, the affin- extent of the antigen expression and by the affinity of the ␣ ␤ ity of integrin L 2 on CLL cells towards their respective adhesion molecules. Determining the antigen expression ligands was too low to be detected by this assay. Therefore, alone, therefore, does not appear to be sufficient for predicting although mAbs against other integrins did not interfere with the adhesion pattern in situ. Differential adhesion of B-CLL cells SI Behr et al 75 It may be tempting to speculate that the observed in vitro anti-CD21 but not anti-CD23 impaired the anti-CD19- binding of CLL cells to FDCs of normal lymphoid tissues might induced aggregation of B-CLL cells, CD21 appears to employ reflect the capacity of these tumor cells to invade normal ger- a ligand different from CD23 for this homotypic adhesion. minal centers via integrin ␣4␤1/VCAM-1. However, histologi- Treatment with anti-CD19 did not lead to an increased cal evaluation of the lymph nodes affected by CLL do not expression of the relevant adhesion molecules. It seems plaus- demonstrate infiltrations of germinal centers. Furthermore, the ible, therefore, that this mAb initiated adhesion by increasing ␣ pattern of rearrangement of the immunoglobulin (Ig) heavy the affinity of the molecules integrin L, ICAM-1 and CD21. and light chain genes and the lack of somatic mutations of This interpretation is supported by previous findings that inte- the Ig-V region in CLL cells argue against the interpretation grin-mediated adhesion is mediated by a rapid increase of the that these cells migrate into germinal centers. Yet, VCAM-1 is affinity of these molecules towards their respective also selectively expressed by endothelial cells in adult and ligands.14,47 However, the fact that the anti-CD19-induced fetal hematopoietic organs27 where it mediates the in vitro aggregation could be blocked by cycloheximide demonstrated adherence of normal and leukemic cells.28–30 Furthermore, that initiation of homotypic adhesion also depended on pro- binding on to bone marrow fibroblasts via integrin tein synthesis. The activity of integrin molecules is influenced ␣4␤1/VCAM-1 supported survival of B cell lineage acute lym- by a large number of regulatory proteins (reviewed in Ref. 48). phoblastic leukemia (ALL) cells.31 We speculate, therefore, It is possible, therefore, that aggregation of B-CLL cells trig- that the observed germinal center binding of B-CLL cells via gered by anti-CD19 also involves an increase in the integrin ␣4␤1/VCAM-1 might mirror the capacity of these expression level of proteins regulating the affinity of integrin ␣ cells to use this ligand pair for their migration and proliferation L, ICAM-1 and CD21. within the microenvironment of hematopoietic organs. Could the in vitro observed homotypic aggregation of B- The relevance of the integrin ␣4␤1/VCAM-1-mediated CLL cells be relevant for the biology of these cells in vivo? adherence for the pathophysiology of the CLL disease is Adhesion of normal B cells and of B cell lines through integrin ␣ further strengthened by our observation that expression of L/ICAM-1 leads to proliferation and inhibits apoptotic cell integrin ␣4 and germinal center binding via this molecule cor- death.49–52 In contrast, CD19 is able to initiate cell cycle related with advanced disease stages (P = 0.029). Consistent arrest53 and apoptosis54,55 in B cell lines. However, in this with this are reports that patients with B-CLL cells expressing study we did not find evidence that anti-CD19 had a detect- ␤1, ␤2 and ␤3 integrin chains presented with a poor prognosis able impact on the proliferation or apoptosis of CLL cells. It and with splenomegaly.2 seems obvious that in contrast to homotypic aggregation The present study identified the molecules on B-CLL cells induced by anti-CD19 alone, the ultimate fate of B-CLL cells that influence the intercellular binding of these cells. Among a is determined by more complex processes that require large panel of mAbs tested, only mAbs against CD19 induced additional signalling. homotypic aggregation of B-CLL cells. Our observation that Our descriptions of the mechanisms that regulate hetero- the clone of anti-integrin ␣4 that was used (HP2/1) did not typic and homotypic adhesion of B-CLL cells provide ground induce aggregation, was consistent also with the observations for hypothetical models that may help to explain some aspects of Campanero et al.32 CD19, together with complement of the B-CLL disease. We propose that the observed germinal receptor CD21 (CR2), target of an antiproliferative antibody- center binding of B-CLL cells via integrin ␣4␤1/VCAM-1 1 (TAPA-1, CD81) and Leu-13, forms a functionally important reflects the capacity of these cells to migrate and proliferate membrane complex on B cells.33–35 Each component of this within the hematopoietic organs. CD19-mediated homotypic ␣ complex independently regulates the activation, proliferation aggregation of B-CLL cells via integrin L/ICAM-1 and CD21 and function of B cells18,36 and is able to initiate homotypic might supply additional signals that modulate the fate of these and heterotypic adhesion.37–39 Aggregation of B cells could tumor cells. Future studies will address the question of also be triggered by other cell surface molecules.40 Our find- whether these adhesion pathways are valuable targets for ing that homotypic adhesion of B-CLL cells was induced only novel immunotherapeutic strategies to treat patients with by anti-CD19 and not by mAbs against other B cell antigens B-CLL. indicated, in contrast, that CD19 may have a unique role in initiating aggregation of B-CLL cells. The finding that peripheral blood B cells did not show anti-CD19-induced homotypic Acknowledgements aggregation is consistent with earlier reports indicating that normal B cells need appropriate stimuli to exert binding in This work was supported by a grant of the Wilhelm Sander vitro.13,14 ␣ Stiftung (94.042.1). Folke Schriever is supported by a research We identified the adhesion pair integrin L/ICAM-1 and fellowship of the DFG (Schr 318/3-1) CD21 as the molecules that mediate the anti-CD19-induced aggregation of B-CLL cells. 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