MCP-1 modulates chemotaxis by follicular lymphoma cells

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British Journal of Haematology, 2001, 115, 554±562

MCP-1 modulates chemotaxis by follicular lymphoma cells Herve Husson, 1 , 2 Elizabeth G. Carideo, 2 Angelo A. Cardoso, 1 , 2 Serena M. Lugli, 1 , 2 Donna Neuberg, 3 Olivier Munoz, 1 , 2 Laurence de Leval, 4 Joachim Schultze 1 , 2 and Arnold S. Freedman 1 , 2 1 Department of Medicine, Harvard Medical School, Departments of 2 Adult Oncology and 3 Biostatistical Science, Dana-Farber Cancer Institute, Boston, MA, and 4 Department of Pathology, Massachusetts General Hospital, Boston, MA, USA Received 15 March 2001, accepted 9 July 2001

Summary. The localization and establishment of follicular lymphoma (FL) cells in distinct anatomic sites probably involves chemokine and adhesion receptors on the neoplastic cells and appropriate chemokines and adhesion receptor ligands in the microenvironment. Several chemokines play an important role in normal B-cell trafficking and differentiation. Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine that induces chemotaxis of a variety of lymphoid cells through its receptor CCR2. CCR2 is also expressed on B cells, and MCP-1 induces chemotaxis of normal B cells. In this report, we investigated expression and function of CCR2 on FL cells. We found FL cells as well as the t(14; 18)1 B-cell lymphoma line H2 expressed CCR2.

MCP-1 potentiated SDF-1-induced chemotaxis of FL cells and H2 cells, but MCP-1 alone did not induce chemotaxis. The specificity of the effects of MCP-1 and SDF-1 was demonstrated by antibody blocking studies. Because FL cells are generally associated with follicular dendritic cells (FDCs), FDCs may be an important source of chemokines. We found that cultured FDCs produced MCP-1, and this production was enhanced by tumour necrosis factor. These data implicate MCP-1 in the migration and localization of FL cells.

Follicular lymphoma (FL) cells are neoplastic counterparts of normal germinal centre (GC) B cells (Stein et al, 1982). These malignant cells circulate in the peripheral blood and localize within specific anatomic sites including lymph node, spleen, bone marrow, but generally not other extranodal sites. It is not known precisely what dictates the localization and establishment of FL cells in these sites but it probably involves chemokine and adhesion receptors on the neoplastic cells and appropriate chemokines and adhesion receptor ligands in the microenvironment. More importantly, once localized in these anatomic sites, these malignant cells receive growth and survival signals from their microenvironment in the form of soluble cytokines and cell±cell contact signals (Ghia et al, 1998). Normal lymphoid cell development and maturation occurs in distinct microenvironments and involves lymphocyte trafficking (Baggiolini, 1998). Recent studies support the role of chemokines in B-cell development and maturation (Bleul et al, 1996; FoÈrster et al, 1996; D'Apuzzo et al,

1997; Gunn et al, 1998; Kim et al, 1998; Legler et al, 1998; Ngo et al, 1998; Ghia et al, 1999). Normal B cells express several chemokine receptors including CCR2, CCR4, CCR6, CCR7, CXCR4 and CXCR5 (FoÈrster et al, 1996; D'Apuzzo et al, 1997; Frade et al, 1997; Kim et al, 1998; Legler et al, 1998; Ngo et al, 1998; Krzysiek et al, 2000). In addition, chemotaxis of normal B cells in vitro occurs in response to the chemokines SDF-1, ELC, SLC and BLC. Compelling evidence for the role of certain chemokines in B-cell trafficking and differentiation has come from mice lacking the genes for the chemokine SDF-1 and its receptor CXCR4, but also the chemokine receptor CXCR5. Mice lacking SDF-1 and CXCR4 genes are B lymphopenic (Nagasawa et al, 1996a; Ma et al, 1998). Mice deficient in the receptor CXCR5 lack inguinal lymph nodes (LNs) and have abnormal formation of GCs of secondary lymphoid follicles, and the B cells do not migrate into the B-cell areas of secondary lymphoid tissues (Legler et al, 1998). Malignant B cells express chemokine receptors and have been studied for responses to chemokines in vitro. SDF-1 stimulates proliferation of acute lymphoblastic leukaemia (ALL) cell lines, and induces their chemotaxis (Nagasawa et al, 1994, 1996a, b; D'Apuzzo et al, 1997; Ma et al, 1998; Honczarenko et al, 1999). A role for SDF-1 and its receptor CXCR4 has also been demonstrated in B-cell chronic lymphocytic leukaemia (B-CLL), FL, diffuse large cell and

Correspondence: Arnold S. Freedman, MD, Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA, USA. E-mail: address: [email protected] *Present address: DuPont Pharmaceuticals Company, Chestnut Run Plaza, Maple Run 1208, Centre Road, PO Box 80721, Wilmington, DE, USA.


Keywords: chemokine, MCP-1, B cell, lymphoma, follicular dendritic cell.

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555 Burkitt's lymphoma (Burger et al, 1999; Arai et al, 2000; Corcione et al, 2000). B-CLL cells also express CXCR3, and the cells undergo chemotaxis in response to its ligands, interferon-g inducible protein 10 (IP-10) and interferon-ginduced monokine (Mig) (Trentin et al, 1999; Jones et al, 2000). Other chemokine receptors have been reported to be expressed on distinct histological subtypes of non-Hodgkin's lymphoma (Trentin et al, 1999; Jones et al, 2000). The pattern of receptor expression may be important in the localization of specific disease entities. Monocyte chemoattractant protein-1 (MCP-1) is one of the C-C chemokines which induces chemotaxis of monocytes, memory T cells, as well as natural killer (NK) cells, dendritic cells, and basophils through its receptor CCR2 (Matsushima et al, 1989; Yoshimura et al, 1989; Allavena et al, 1994; Carr et al, 1994; Maghazachi et al, 1994; Myers et al, 1995; Oin et al, 1996). MCP-1 is produced by a large number of different cell types including fibroblasts, endothelial cells, mesothelial cells, and MCP-1 expression is increased by inflammatory mediators (Rollins, 1997). MCP-1 is also present in pathological states including inflammation, atherosclerosis, rheumatoid arthritis, asthma, and in tissues infiltrated with malignant cells such as melanoma and breast carcinoma. CCR2 is also constitutively expressed on B cells, and MCP-1 induces chemotaxis in tonsillar B cells and the B-cell line IM-9 (Frade et al, 1997) Within nodal and extranodal sites, including the bone marrow and liver, FL cells are enveloped in a network of follicular dendritic cells (FDCs) (Naiem et al, 1983). Previously, we have reported that FDC-like cells and FDC cell lines produce the chemokines SDF-1 and interleukin 8 (IL-8) (Husson et al, 2000). Furthermore, FL cells migrate in response to SDF-1 and towards lymph node stromal cells mediated by an SDF-1/CXCR4 interaction (Corcione et al, 2000). In the present study, we report that FL cells express functional CCR2 and cultured FDCs produce MCP-1. These data support the hypothesis that multiple chemokines are likely to direct the migration and homing of FL cells. MATERIALS AND METHODS Antibodies, chemokines and cytokines. The antibodies against CD4, CD8, CD14, CD56, IgD and CD44 used in the depletion were from Beckman Coulter (Brea, CA, USA). Anti-CCR2 (MAB150) and -CXCR4 (clone 12G5) monoclonal antibodies (mAbs) were purchased from R & D systems (Minneapolis, MN, USA) and Pharmingen (San Diego, CA, USA) respectively. Isotype-matched antibodies were purchased from Dako (Carpinteria, CA, USA). SDF-1a and MCP-1 were purchased from R & D systems, and tumour necrosis factor (TNF) from Peprotech (Rocky Hill, NJ, USA). Lymphotoxin (LT) a1b2 was generously provided by Dr J. Browning, Biogen, Cambridge, MA, USA (Browning et al, 1996). Cell culture and negative immunomagnetic bead depletion. FL cells were isolated from single cell suspension from digested lymph nodes or spleen that were histologically involved with follicular small cleaved cell lymphoma as described

previously (Ghia et al, 1998). Tonsils were obtained from patients undergoing tonsillectomy. All samples were obtained according to appropriate Human Protection Committee validation and informed patient consent. Normal GC-B cells were enriched from single cell suspensions of tonsil by immunomagnetic bead depletion of T cells, monocytes, NK, naive B cells and non-GC-B cells using anti-CD4, -CD8, -CD14, -CD56, -IgD and -CD44, respectively, as previously described (Ghia et al, 1998). FL cells were isolated from single cell suspension from digested lymph nodes or spleen which were histologically involved with FL. These populations of cells were depleted of T cells, monocytes, NK cells as described above. The purity of the population was always . 95% CD201. The H2 FL cell line bearing the t(14 : 18) translocation was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS) (Harlan Bioproducts, Madison, WI, USA), 2 mmol/l l-glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml) (Life Technologies, Gaithersburg, MD, USA). The human follicular dendritic cell line HK (provided by Dr Y. Choi) was cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mmol/l l-glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml) as previously described (Kim et al, 1994). The isolation and culture conditions of FDC-LC was performed as previously described (Husson et al, 2000). Enzyme-linked immunosorbent assay (ELISA). Confluent FDC-LC and HK cells were incubated for 24 h in serum-free RPMI medium alone or in serum-free RPMI medium containing 10 ng/ml TNF or LTa1b2 as previously described (Husson et al, 2000). The supernatants were harvested and centrifuged to remove any cellular debris. The MCP-1 sandwich ELISA was performed according to the manufacturer's instructions (R & D Systems, Minneapolis, MN, USA) using the monoclonal capture anti-MCP-1 (MAB679) and the biotinylated polyclonal anti-MCP-1 (BAF279). Two-way analysis of variance (anova) was performed using STATA (Stata Corp., College Station, TX, USA). RNA extraction and real time quantitative (reverse transcription polymerase chain reaction (RT-PCR) analysis. Cells were resuspended in TRIzol reagent (Gibco BRL, Grand Island, NY, USA) according to the manufacturer's procedures. Contaminating genomic DNA was removed by treatment with DNAse I. Briefly, total RNAs were incubated at 0´5 mg/ ml in 1  NEB buffer 3 (New England Biolabs, Beverly, MA, USA) containing 50 U/ml DNAse I-RNAse free (Boehringer Mannheim, Indianapolis, IN, USA). The samples were incubated for 1 h, and then extracted with Phenol/Chloroform/Isoamylalcohol (25:24:1) and finally with Chloroform/Isoamyl-alcohol (24:1). Total RNAs were finally precipitated with 1/5 (v/v) 7´5 mol/l NH4 Acetate and 2´5 (v/v) 100% ethanol. Real time quantitative RT-PCR analysis was performed using an ABI PRISM 7700 Sequence Detection System instrument using the SYBRw Green I dye (PE Biosystems, Foster City, CA, USA). Briefly, direct detection of PCR product was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green I dye to

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Fig 1. Chemokine measurement using ELISA on TNF or LTa1b2 stimulation of FDCs. Confluent FDC-LC and HK cells were incubated for 24 h in serum-free RPMI medium alone (empty bars) or in serum-free RPMI medium containing 10 ng/ml TNF (grey bars) or LTa1b2 (black bars). Human MCP-1 was measured in supernatants by a specific sandwich ELISA for MCP-1 according to the manufacturer's instructions. Determinations were performed in duplicates, and results are expressed as mean ^ SD. Results are representative of three separate experiments.

double-stranded DNA by the Sequence Detection System directly into the reaction tube. The sequence detector software 1´7 calculates the threshold cycle number (CT) when signals reach 10 times the standard deviation of the base line. CT values have been shown to be directly proportionate to the mRNA levels of various genes tested. (Gibson et al, 1996; Nakao et al, 2000). DNase I-treated total RNA (1 mg) were reverse transcribed using the Advantagew RT-for-PCR kit (Clontech) following the manufacturer's instructions, and aliquoted to avoid freezing and thawing procedures. The appropriate primers were designed with the Primer Expresse 1´0 software (PE Biosystems). RT-reaction mixture (2´5 ml), in which the reverse transcriptase was omitted (to ensure the efficiency of the DNAse I treatment), or water (no template control) were amplified in triplicates using real time PCR in a final volume of 50 ml using the SYBRw Green Master Mix reagent at a final concentration of 1 (PE Biosystems). No amplification was observed in the RT reaction in which the reverse transcriptase was omitted. To ensure the specificity of the reaction, the size of the PCR product for each gene was verified using gel electrophoresis. The sequences of primers for CCR2 were: sense primer 5 0 AAGCTGAACAGAGAAAGT GGATTGA3 0 and antisense primer 5 0 AGAACGAGATGTGGA CAGCATGT3 0 . The optimum concentration for both primers was 0´3 mmol/l. Conditions were as follows: one cycle at 508C for 2 min, one cycle at 958C for 10 min, 40 cycles at 958C for 15 s, 608C for 1 min. Validation experiments to verify the efficiencies of amplification of the primers of target and reference genes were approximately equal. For each sample, CT values for b-actin and ribosomal protein S9 genes were generated to correct each sample for differences in RNA content. For each sample, CT values for b-actin was generated. A correction factor was calculated by dividing each b-actin CT value by the minimum CT value.

The same procedure was performed for ribosomal protein S9. The CT value for CCR2 was then corrected for its RNA content by the average of the corrected b-actin and ribosomal protein S9 CT values. Flow cytofluorometry. Cells (5  106 cells/ml) were washed in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) (w/v; PBS-A) and then blocked with human purified IgG at 0´2 mg/ml (Sigma, St Louis, MO, USA) for 15 min at room temperature. Cells (0´5  106) were incubated with the indicated mAbs at 10 mg/ml for 45 min at 48C, and washed subsequently twice in cold PBS-A. In control experiments, an irrelevant isotype-matched control mAb was used. Secondary antibodies included phycoerythrin R-conjugated goat F(ab 0 )2 anti-mouse IgG2a or IgG2b (Southern Biotechnology Associates Inc., Birmingham, AL, USA) diluted in PBS-A, for 30 min at 48C. Flow cytometry was performed on a FACScan (Becton Dickinson, San Jose, CA, USA). Chemotaxis assays. The chemotaxis assays were performed in 24-well plates containing 5 mm porous polycarbonate membrane inserts (Costar, Cambridge, MA, USA). Inserts were equilibrated for 1 h in AIM-V medium before adding the cells at 378C, 5% CO2. Cells were washed in AIM-V medium containing BSA (Gibco), resuspended at 1  106 cells/ml and serum starved for 1 h at 378C, 5% CO2. Chemokines were added in the bottom wells (300 ml total volume) and 100 ml (1  105) of cells with or without chemokines were loaded on to the inserts. Cells migrating to the bottom well were collected after 4 h and counted using trypan blue exclusion. The results are expressed as the total number of cells that migrated. No dead cells were observed during the 4 h incubation as measured by trypan blue exclusion. To block SDF-1-induced chemotaxis, the cells were incubated after the starvation procedure for 0´5 h on ice with 5 mg/ml antiCXCR4 or an isotype-matched irrelevant antibody Dako (Carpinteria, CA, USA). Anti-CXCR4 or isotype-matched irrelevant antibody was also present during the chemotaxis assay. To block the MCP-1-induced chemotaxis, anti-MCP-1 or an isotype-matched irrelevant antibody was preincubated for 0´5 h on ice before equilibration of the inserts. Pertussis toxin (PTX) (Sigma) was preincubated with the cells overnight at 0´1 mg/ml as well as during the chemotaxis assay. RESULTS Chemokine production by FDC-LC and effects of TNF family members Within secondary lymphoid organs, FDCs may be an important source of chemokines and cytokines, directing B cells within lymphoid follicles and providing B cells with maturation and survival signals (Clark et al, 1995; Skibinski et al, 1998; Schwarz et al, 1999;Ansel et al, 2000; Choe et al, 2000; Li et al, 2000; Tsunoda et al, 2000). It has been shown that stromal cells including FDCs constitute an important source of SDF-1, directing normal and neoplastic B cells within lymphoid follicles (Bleul et al, 1996; Arai et al, 2000; Corcione et al, 2000; Husson et al, 2000). We examined supernatants from cultured FDCs (FDC-LC) and the FDC cell line HK for MCP-1 secretion by ELISA. Because

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Fig 2. CCR2 expression measurement in normal GC-B cells and FL cells by real time quantitative RT-PCR and flow cytometric analysis. (A) Real time quantitative RT-PCR was performed with appropriate primers for CCR2 using SYBRGREEN method as described in the Materials and Methods. Amplification results were visualized using the sequence detector 1´7 software. CT represents the threshold cycle at which fluorescence is first detected above background. Each condition corresponds to the average of triplicate determinations performed simultaneously, normalized as described in Materials and Methods ^ SD. Quantified RT-PCR was performed on cDNAs from six normal patients (GC, empty symbols) and six patients with FL (FL, black symbols). (B) FL cells from six patients were purified as described in Material and Methods, and then stained with anti-CCR2 (solid line), CXCR4 as positive control (dotted line) or irrelevant-matched control mAb (black profile) followed by the respective anti-isotype phycoerythrin R-conjugated goat F(ab 0 )2 anti-mouse Ab.

TNF and LT augment MCP-1 production in other cell types, we also examine supernatants of cytokine stimulated cells (Cuff et al, 1998). As shown in Fig 1, MCP-1 was detected in unstimulated HK cells but not in FDC-LC. After stimulation of FDC-LC and HK cells with TNF, the production of MCP-1

was significantly increased (P , 0´001 and P ˆ 0´001 respectively). An increase in MCP-1 secretion by FDC-LC and HK cells with LTa1b2 was also observed in three independent experiments, but was not statistically significant (P ˆ 0´58 and P ˆ 0´22 respectively).

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Fig 3. Chemotaxis assay of FL patient cells in response to SDF-1 and MCP-1. Transwells (5 mm porous membrane) were pre-equilibrated for 1 h in AIMV medium alone or containing SDF-1 (10 ng/ml) and/or MCP-1 (100 ng/ml) as described in the figure before the purified FL cells (105 cells per condition) were applied. The cells were incubated for 4 h in duplicate. Cells present in the bottom chamber were harvested and counted using the Trypan blue exclusion method. Results are expressed as the absolute number of cells that migrated and are representative of three independent determinations.

Follicular lymphoma and normal GC-B cells express CCR2 (MCP-1 receptor) Because CCR2 has been shown to be expressed on normal B cells, we examined FL cells and normal GC-B cells for CCR2 expression (Frade et al, 1997). To ensure that we were monitoring the CCR2 expression on purified normal GC or FL cells, we depleted the cell suspensions of monocytes, T cells and NK cells using immunomagnetic beads. Furthermore, the normal GC-B cells were depleted of IgD1 (naõÈve) and CD441 (non-GC) cells using immunomagnetic beads. We compared the mRNA expression level for CCR2 using real time quantitative RT-PCR using the SYBR Green method for six normal individuals as well as six FL patients. As shown in Fig 2A GC-B cells and FL cells expressed similar levels of CCR2 mRNA (P ˆ 0´78). As seen in Fig 2B, using flow cytometry, FL cells expressed cell surface CCR2. As a positive control, the FL cells also express the SDF-1 receptor CXCR4. MCP-1 potentiates SDF-1-induced chemotaxis of follicular lymphoma cells and the t(14; 18)1 B-cell line H2 MCP-1 is a chemotactic factor for normal B cells (Frade et al, 1997). We first evaluated the capacity of FL cells isolated from involved tissues to respond to MCP-1 in an in vitro chemotaxis assay. As seen in Fig 3, FL cells did not migrate

above the basal level in response to MCP-1 alone. In contrast, SDF-1-induced cell migration of FL cells, as previously described. However when MCP-1 was combined with SDF-1 at suboptimal concentrations (10 ng/ml), we observed a potentiation of the SDF-1-induced chemotaxis (Fig 3). When SDF-1 was present in the upper and lower compartments of the Transwell, background levels of migration was observed. When MCP-1 was present in the upper compartment with SDF-1 and MCP-1 in the lower compartment, the migration was the same as when SDF-1 was present alone in the lower compartment. To further investigate the effect of MCP-1 on FL cells, we used the t(14; 18)1 B-cell lymphoma line H2. H2 cells express both chemokine receptors CCR2 and CXCR4 (insert, Fig 4A). As was observed with cells isolated from patients, MCP-1 potentiated the SDF-1-induced chemotaxis of H2 cells (Fig 4A). This occurred in a dose-dependent manner up to a concentration of MCP-1 of 62´5 ng/ml. Above that concentration, the potentiating effect was no longer observed, consistent with the biphasic curve seen for migration assays with other chemokines such as SDF1(D'Apuzzo et al, 1997). To determine the specificity of the SDF-1 and MCP-1 effects, we performed blocking antibody studies. As seen in Fig 4B, anti-MCP-1 mAb inhibited the potentiating effect of MCP-1 on H2 chemotaxis. No effect on SDF-1-induced chemotaxis was seen. Anti-CXCR4 mAb inhibited chemotaxis in response to SDF-1 and the combination of SDF-1 and MCP-1. Finally as a control, PTX completely inhibited all chemotaxis. DISCUSSION FL cells generally localize in distinct anatomic sites, including lymph nodes, spleen and bone marrow. Within these sites, FL cells reside in structures that resemble normal germinal centres, and they are associated with FDCs. Analogous to the interactions of FDCs with normal GC-B cells, FDCs probably provide FL cells with chemotactic, adhesive and survival signals (Choe et al, 2000; Ghia & Caligaris-Cappio, 2000). This hypothesis is supported by the recent finding that SDF-1 produced by lymph node stromal cells was a major chemotactic factor directing FL cells (Arai et al, 2000). Recently, we showed that FDCs isolated from tonsils produced chemokines including both SDF-1 and IL-8 (Husson et al, 2000). We also showed that inflammatory molecules that are found in high levels in FL cells, such as TNF, down regulate SDF-1. In the search for other potential chemotactic factors directing FL cells towards secondary lymphoid organs, we found that cultured FDCs secreted MCP-1. MCP-1 receptor, CCR2, was found expressed on FL cells isolated from involved tissues and MCP-1 enhanced SDF-1-induced FL cell chemotaxis. These findings support the hypothesis that MCP-1/CCR2 interaction may be an additional system which contributes to the migratory pattern of FL cells. FL cells bind to FDCs through adhesion receptor±ligand interactions, predominantly a4b1- and aLb2-integrins binding to vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) respectively

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Fig 4. CXCR4 and CCR2 expression, and chemotaxis assay of H2 B-cell lymphoma line. (A) Insert: H2 cells were stained with anti-CXCR4, anti-CCR2 or an isotypematched irrelevant antibody and analysed using flow cytometric analysis as described in the Materials and Methods. Transwells were equilibrated as described in Fig 3 for 1 h in AIM-V medium alone or containing SDF-1 (10 ng/ml) and/or increasing concentrations of MCP-1 before the cells were applied. (B) Cells were preincubated for 30 min on ice with either control (control Ig), anti-MCP-1 or anti-CXCR4 antibodies and also during the chemotaxis assay. For pertussis toxin (PTX) blocking experiment, the cells were incubated overnight as well as during the chemotaxis assay with 0´1 mg/ml PTX. The migration assay was then performed and analysed as described in Fig 3. Results are depicted as in Fig 3 and represent the mean value of triplicate determinations.

(Freedman et al, 1992). It is not known whether FL cells migrate toward FDCs, but production of SDF-1 and IL-8 by FDCs suggests that they might induce FL cell migration. There is evidence in murine systems that the chemokine BLC induces B cell migration into secondary follicles (Ansel et al, 2000). Our finding that cultured FDCs produce MCP-1 may provide another mechanism that directs migration of FL cells in lymphoid tissues. It is not known whether FDCs in FL tissues express MCP-1. However, MCP-1 mRNA is present in lymphoma-involved tissue (Luciani et al, 1998). We and colleagues (Luciani et al, 1998) have attempted to demonstrate MCP-1 expression in lymphoma as well as normal hyperplastic tissue using immunohistochemistry. To date, the results have been inconsistent. The difficulties with documenting protein production may be caused by a low level of expression, instability of the protein and/or lack of the appropriate monoclonal antibodies. FL cells have been reported to express the chemokine receptors CXCR4 (SDF-1 receptor) and CXCR5 (BLC receptor) (Jones et al, 2000). Furthermore, FL cells migrate in response to SDF-1. FL cells express CCR2 by quantitative RT-PCR and flow cytometric analysis but do not migrate in response to MCP-1 as unique chemoattractant. Interestingly, MCP-1 enhanced SDF-1-induced chemotaxis. The

modulation by MCP-1 of the response of FL to SDF-1 supports the hypothesis that lymphoma cell migration is a complicated process, in which there is integration of multiple signals in a network of chemokines. This may be a mechanism for cells to continue to migrate in the face of suboptimal concentrations of SDF-1 in tissue. MCP-1 is produced in lymphoid tissues by endothelial cells (Tedla et al, 1999), and MCP-1 expression is increased by TNF, IL-1 and other inflammatory mediators. We found that MCP-1 production by cultured FDCs also increased after stimulation with TNF, whereas SDF-1 production by these FDCs decreased. Therefore, because FL cells produce TNF, the downregulation of SDF-1 production in FDCs may be compensated by increased production of MCP-1. These data suggest that the downregulation of SDF-1 by TNF is necessary to reveal a migratory capacity of B cells to MCP-1 in the presence of low levels of SDF-1. MCP-1 has been found to exert effects on leucocytes besides chemotaxis. In T cells, MCP-1 induces adhesion to fibronectin through activation of a4b1- and a5b2-integrins and activation of b2-integrins in monocytes (Carr et al, 1996; Weber et al, 1999). MCP-1 also enhances cytotoxicity of NK and T cells (Taub et al, 1996). MCP-1 stimulates mesothelial cell proliferation and haptotaxis, the migration

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of cells along a gradient of substrate bound chemoattractants (Nasreen et al, 2000). In our studies, MCP-1 did not induce chemotaxis by itself, but MCP-1 enhanced SDF-1induced migration. The mechanism for this is not clear, but was not caused by augmented expression of the SDF-1 receptor CXCR4 by MCP-1 or increased expression of CCR2 by SDF-1 (data not shown). A potentiating effect of Ca12 and MCP-1 on macrophage chemotaxis has been reported. However, the mechanism of this effect is not known presently (Olszak et al, 2000). Our data suggests that multiple chemokines can co-operate in regulating malignant B-cell chemotaxis. The demonstration that the microenvironment provides viability signals and directs the localization of lymphoma cells in model systems suggests that interruption of these pathways may limit the growth and dissemination of these diseases (Wakabayashi et al, 1995; Wang et al, 1998; Ghia & Caligaris-Cappio, 2000). Interfering with the ability of FL cells to localize to a favourable environment may be a novel approach to treating these diseases and limiting disease dissemination. A number of approaches are being taken to perturb these pathways including small molecules that inhibit chemokine±chemokine receptor interactions and inhibitors of chemokine production (Donanz et al, 1997; Gong et al, 1997; Donzella et al, 1998; Liu et al, 1999; Romano et al, 2000). Because TNF plays an important role in stimulating MCP-1 production and TNF is produced by FL cells, (Mapara et al, 1994; Gruss & Dower, 1995; Salles et al, 1996; Warzocha et al, 1997) interruption of the TNF±TNF receptor pathway may be yet another way to modulate FL cell homing and adhesion (Maini et al, 1999; Weinblatt et al, 1999). Further investigation into the signals that direct FL cell migration will provide important insights into the disseminated nature of this disease and the potential for novel therapeutic strategies. ACKNOWLEDGMENTS The authors thank Joyce Lavecchio for her technical assistance. This work was supported in part by NIH grant CA66996, The Leukemia and Lymphoma Society of America and the Norman Hirschfield Foundation. H.H. was supported by the `Cure for Lymphoma Foundation'. REFERENCES Allavena, P., Bianchi, G., Zhou, D., van Damme, J., Jilek, P., Sozzani, S. & Mantovani, A. (1994) Induction of natural killer cell migration by monocyte chemotactic protein-1-2 and -3. European Journal of Immunology, 24, 3233±3236. Ansel, K.M., Ngo, V.N., Hyman, P.L., Luther, S.A., Forster, R., Sedgwick, J.D., Browning, J.L., Lipp, M. & Cyster, J.G. (2000) A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature, 406, 309±314. Arai, J., Yasukawa, M., Yakushijin, Y., Miyazaki, T. & Fujita, S. (2000) Stromal cells in lymph nodes attract B-lymphoma cells via production of stromal cell-derived factor-1. European Journal of Haematology, 64, 323±332. Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature, 392, 565±568.

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