Affiliation of authors: A. Corcione, P. Facchetti, I. Airoldi, R. Guglielmino, V. Pistoia, Laboratory of Oncology, G. Gaslini Institute, Genoa, Italy; L. Ottonello, G. Tortolina, F. Dallegri, Department of Internal Medicine, University of Genoa; P. Dadati, M. Truini, Service of Pathology, S. Martino Hospital, Genoa; S. Sozzani, Department of Immunology, Mario Negri Institute, Milan, Italy.
Correspondence to: Anna Corcione, Ph.D., Laboratory of Oncology, G. Gaslini Institute, Largo G. Gaslini, 5-16148 Genoa, Italy (e-mail: laboncologia{at}ospedale-gaslini.ge.it).
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ABSTRACT |
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INTRODUCTION |
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Follicular center lymphoma is a tumor of peripheral B lymphocytes representing the malignant counterparts of normal germinal center B cells (35). Histologically, follicular center lymphoma recapitulates the architecture and cytologic features of the normal secondary lymphoid follicle (5). According to the Revised EuropeanAmerican Classification of Lymphoid Neoplasms (R.E.A.L.), follicular center lymphoma is subdivided into three grades: grade I = predominantly small cleaved cells that resemble centrocytes; grade II = mixed small cleaved and large noncleaved cells, with the noncleaved cells resembling centroblasts; and grade III = predominantly large noncleaved cells (6).
Follicular center lymphoma B cells express surface immunoglobulin (sIg) and are CD19+, CD20+, CD10+/-, and CD5- (6,12). A large fraction of follicular center lymphoma cases (70%90%) have a characteristic translocation, t(14;18), involving rearrangement of the immunoglobulin (Ig) heavy chain locus and of the Bcl-2 oncogene, which results in overexpression of the Bcl-2 protein and inhibition of apoptosis, i.e., programmed cell death (1316).
Only limited information is available on the mechanisms controlling the migration of follicular center lymphoma B cells. For example, it has been shown that follicular center lymphoma B cells express the very late activation-4 ß1 integrin that binds to vascular cell adhesion molecule-1 expressed on follicular dendritic cells (17). These findings indicate that the neoplastic follicles use the same adhesive interactions involved in the localization of normal B cells to germinal centers (18). Chemoattractants that stimulate the locomotion of follicular center lymphoma B cells are unknown.
Various chemokines have been reported to enhance the in vitro locomotion of normal B lymphocytes [reviewed in (19)]. Four B-cell-tropic chemokines are of special interest, since they are expressed within secondary lymphoid organs, where they may influence the locomotion of different B-cell subsets. These chemokines are as follows: B-lymphocyte chemoattractant, which binds to the CXC chemokine receptor (CXCR) 5 (20,21); lymphoid tissue chemokine, which binds to the CC chemokine receptor (CCR) 7 (22); Epstein-Barr virus-induced molecule 1 ligand chemokine or macrophage inflammatory protein-3ß, which binds to CCR7 (23); and stromal cell-derived factor-1 (SDF-1), which binds to CXCR4 (24).
SDF-1, also known as pre-B-cell growth-stimulating factor, belongs to the CXC chemokine subfamily and is produced by stromal cells (25,26). Mice lacking the SDF-1 gene or the CXCR4 gene show defects in B-cell lymphopoiesis and myelopoiesis, as well as in heart and cerebellar development (27,28).
SDF-1 has been found to be chemotactic for human T lymphocytes, monocytes, CD34+ hematopoietic progenitor cells, dendritic cells, and megakaryocytes (24,2931). Recently, it has been shown that SDF-1 also attracts human B lymphocytes (3234).
Here we have investigated the effects of SDF-1 on the in vitro migration of neoplastic B cells purified from the invaded lymph nodes of patients with follicular center lymphoma as well as of their presumed normal counterparts, i.e., centrocytes and centroblasts, isolated from human tonsils.
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MATERIALS AND METHODS |
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This investigation was performed after approval by a local institutional review board. Mononuclear cells were isolated by FicollHypaque gradients from single-cell suspensions of lymph node biopsies performed at diagnosis. We studied four patients with grade I, four patients with grade II, and two patients with grade III follicular center lymphoma according to the R.E.A.L. Classification (6). Table 1 gives the basic information on the patients (6,12,35). In all cases, tumor cells expressed CD19, CD20, HLA-DR, Bcl-2, and sIg (6,12). In the different cases, CD10 expression was heterogeneous, ranging from a minimum of 12% to a maximum of 70% positive cells (6,12). Staining for
and
Ig light chains showed that, in the individual samples, the
/
or
/
ratio ranged from 8:1 to 20:1, indicating the monoclonal expansion of malignant B cells (4,6).
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Cell Fractionation
At the time of thawing, lymph node mononuclear cells were suspended in RPMI-1640 medium supplemented with 10% FCS and were incubated with monoclonal antibodies (MAbs) CD2, CD3, CD56, or CD68 or with MAb to the Ig light chain not expressed by the malignant cells in the individual cases. Subsequently, cells were incubated with magnetic beads coated with an anti-mouse Ig goat antiserum, according to the instructions of the manufacturer (Immunotech, Marseille, France). This procedure allowed the depletion of T lymphocytes, natural killer (NK) cells, macrophages, and the bulk of residual normal B cells. After magnetic separation, the purity of lymphoma B cells was higher than 95%, as assessed by the expression of CD19 and of monotypic Ig light chains.
Normal tonsils were obtained from patients undergoing tonsillectomy for inflammatory disorders. Tonsillar B lymphocytes were isolated from FicollHypaque-purified mononuclear cells by depletion of lymphocytes forming rosettes with sheep red blood cells (36). The purity of such B-cell-enriched fractions was, on average, 97%, as evaluated by staining for CD19. For the purification of the germinal center B cells, B-cell suspensions were fractionated on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient consisting of 2 mL each of 100%, 60%, 50%, 40%, and 30% Percoll dilutions from the bottom to the top of the tubes, respectively. Cells (4 x 107) were suspended in 2 mL of 100% Percoll and centrifuged at 1500g at 4°C for 15 minutes. Cells migrating into the low-density fractions of the gradient (30% and 40%) were collected, treated with CD39 and anti-IgD MAbs, and incubated with magnetic beads (36). B cells that did not bind to the beads were separated by applying a magnetic field. The unbound B cells represented a homogeneous population (>98%) of germinal center B cells, as shown by the expression of CD38 and by the negative staining for IgD and CD39 (36).
In some experiments, purified germinal center B cells were incubated for 4 hours at 37°C in RPMI-1640 medium containing 0.1% FCS in the presence or absence of a CD40 MAb (1 µg/mL) and of 10 ng/mL recombinant interleukin 4 (rIL-4) (Genzyme, Milan, Italy).
Monoclonal Antibodies
The following MAbs were used for surface staining according to the instructions of the manufacturer (Becton Dickinson Immunocytometry Systems, San Jose, CA): CD19fluorescein isothiocyanate (FITC), CD20phycoerythrin (PE), CD10PE, anti-HLA-DRFITC, CD38FITC, anti-Ig chainsFITC, anti-Ig
chainsPE, CD3FITC, CD68PE, and CD56PE. Anti-CD39PE was from Pharmingen (San Diego, CA). Controls for each of the above MAbs were isotype-matched MAbs of irrelevant specificity conjugated with the same fluorochromes. An unconjugated anti-IgD MAb was purchased from Dako (Glostrup, Denmark) and was used either for indirect staining by flow cytometry or for separation procedures at a concentration of 1 µg/mL. An unconjugated CD39 MAb was purchased from Immunotech and used in the separation and in the flow cytometry experiments at a concentration of 1 µg/mL. Cells incubated with unconjugated MAbs were subsequently exposed to an FITC-conjugated goat anti-mouse Ig antiserum and enumerated by flow cytometry. Controls were cells treated with unconjugated, isotype-matched MAbs of irrelevant specificity or with the goat antiserum alone. All of the flow cytometry experiments were carried out with the use of a FACScan instrument for fluorescence-activated cell sorting (Becton-Dickinson Immunocytometry Systems).
CD3, CD56, and CD68 unconjugated MAbs were obtained from Dako and used in the separation procedures at the same concentrations as those tested for flow cytometry. An unconjugated CD2 MAb was purchased from Dako and was used for the isolation of lymphoma B cells at the concentrations suggested by the manufacturer.
The 12G5 MAb against CXCR4 was purchased from Pharmingen and was used in blocking experiments at a concentration of 1 µg/mL. The unconjugated CD40 MAb, purchased from Immunotech, was used at a concentration of 0.5 µg/mL.
Chemotaxis Assays
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RNA was extracted from follicular center lymphoma B cells and tonsillar germinal center B lymphocytes with the use of Ultraspec (Biotech Laboratory Inc., Houston, TX) and retrot ranscribed into complementary DNA for polymerase chain reaction (PCR) amplification as previously described (40). Primer sequences and profiles of amplification were as follows: G3PDH (i.e., glyceraldehyde 3-phosphate dehydrogenase) sense ACA TCG CTC AGA ACA CCT ATG G, antisense GGG TCT ACA TGG CAA CTG TGA G, at 94°C for 1 minute, at 60°C for 1 minute, and at 72°C for 1 minute, 30 cycles; SDF-1 sense CGC CAT GAA CGC CAA GGT C, antisense CTT TAG CTT CGG GTC AAT GC, at 94°C for 1 minute, at 55°C for 1 minute, and at 72°C for 1 minute, 33 cycles. The PCR products (10 µL each) were subjected to electrophoresis through a 1.5% agarose gel with ethidium bromide to confirm the base-pair sequence length. Direct sequencing of PCR products was performed with the use of the Dye Terminator Cycle Sequencing Kit (ABI PRISM; Perkin-Elmer Applied Biosystem, Norwalk, CT). Sequences were resolved and analyzed on the ABI 373A Sequence Apparatus (Perkin-Elmer Applied Biosystem).
Statistical Analysis
Data are expressed as means ± 95% confidence interval. Data shown in Fig. 2 were analyzed by Wilcoxon signed rank test. All P values are two-sided and were considered statistically significant at P<.05.
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RESULTS |
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To investigate the chemotactic activity of SDF-1 versus follicular center lymphoma B cells, we isolated the latter cells from lymph node biopsy specimens taken from 10 patients by depletion of T cells, NK cells, macrophages, and residual normal B cells (average purity, 95%). Chemotaxis was tested by filter and collagen gel invasion assays.
In the filter assay, all but one of the follicular center lymphoma cell suspensions migrated significantly faster upon exposure to SDF-1 than under spontaneous conditions (P = .002, Wilcoxon test) (Fig. 2, A). The penetration of all follicular center lymphoma B-cell suspensions in the collagen matrix, which mimics closely the events occurring in vivo during cell migration, was augmented significantly by SDF-1 (P = .002, Wilcoxon test) (Fig. 2
, B). Notably, cells from patient 4 that did not migrate upon SDF-1 stimulation in the filter assay displayed an evident locomotory response to the chemokine in the collagen assay (Fig. 2
, A and B).
Since all of the follicular center lymphoma B-cell fractions migrated in response to SDF-1, no association could be established, in the individual cases, between the SDF-1-stimulated in vitro locomotory ability and the R.E.A.L. grade or clinical stage.
In subsequent experiments, the locomotion of follicular center lymphoma B cells in response to SDF-1 was investigated with the use of a checkerboard assay that assessed whether in vitro migration is the result of chemotaxis, chemokinesis, or both mechanisms. Table 2 shows the mean from two experiments performed with follicular center lymphoma B-cell fractions from patients 6 and 8. These data suggest that the large majority of SDF-1-triggered migration was attributable to true chemotaxis.
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The finding that neoplastic B cells from the lymph nodes of patients with follicular center lymphoma migrated in response to SDF-1 prompted us to investigate the surface expression of CXCR4 on the same cells. Malignant B lymphocytes were stained with an anti-CXCR4 MAb or with an isotype-matched irrelevant MAb and subsequently analyzed by flow cytometry. CXCR4 was expressed on all follicular center lymphoma samples in the range of 7%34% positive cells (Fig. 3). The fluorescence-activated cell sorter (FACS) profiles of three representative follicular center lymphoma B-cell suspensions with different levels of CXCR4 expression are shown. In the individual case subjects, no association was found between staining intensity for CXCR4 or number of CXCR4-positive cells and magnitude of in vitro migratory responses to SDF-1.
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The postulated normal counterparts of malignant B lymphocytes from follicular center lymphoma are germinal center B cells, both centrocytes and centroblasts (3,5,6). It was, therefore, of interest to compare the locomotory response of follicular center lymphoma B cells to SDF-1 with that of normal germinal center B lymphocytes. The latter cells were isolated from tonsillar B cells by a Percoll density gradient followed by removal of CD39+, IgD+ cells (36).
Tonsillar germinal center B cells, although expressing high levels of CXCR4 as assessed by flow cytometry (range from four different experiments: 35%60% positive cells), did not migrate when incubated with SDF-1 over a wide range of concentrations (3001000 ng/mL), either in the filter or in the collagen assays (not shown). Germinal center B-cell suspensions contained at least 80% viable cells as assessed by trypan blue dye exclusion both before and after chemotactic assays, suggesting that the failure of these cells to migrate upon exposure to SDF-1 was unrelated to massive spontaneous cell death.
CD40 MAb with or without rIL-4 has been previously shown to activate germinal center B cells and to rescue them from apoptosis (36,39,41,42). Therefore, we investigated whether, in our experimental conditions, a 3-hour culture of tonsillar germinal center B cells with CD40 MAb and rIL-4 would endow the same cells with the ability to migrate upon exposure to SDF-1. To this end, freshly isolated germinal center B lymphocytes were first cultured in the presence or absence of CD40 MAb and rIL-4. Chemotaxis was subsequently tested in the presence or absence of SDF-1 in the filter or collagen invasion assays. As shown in Fig. 5, panels A and B, germinal center B cells pretreated with CD40 MAb and rIL-4 displayed a statistically significantly higher locomotion in the presence than in the absence of SDF-1 in both assays. The migration of germinal center B lymphocytes preincubated with medium alone was unaffected by their subsequent exposure to SDF-1 in either assay (Fig. 5
, A and B).
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Expression of the SDF-1 Gene in Follicular Center Lymphoma and Germinal Center B Lymphocytes
In subsequent experiments, we investigated the expression of the SDF-1 gene in follicular center lymphoma cells and in tonsillar germinal center B cells. These experiments were based on the hypothesis that expression of the SDF-1 gene in follicular center lymphoma cells might generate paracrine and/or autocrine loops whereby the in vivo migration of neoplastic B cells would be stimulated (43).
As shown in Fig. 6, A, SDF-1 transcripts were detected by reverse transcription (RT)PCR in the samples from four patients with follicular center lymphoma as well as in four germinal center B-cell fractions (Fig. 6
, B). To confirm the specificity of the amplified bands, we sequenced the products of PCR reactions carried out with follicular center lymphoma cells from patient 6, one germinal center B-cell sample, and human fibroblasts tested as positive control (Fig. 6
, C). The sequences obtained from the three sources were virtually identical to each other, thus demonstrating that the expected PCR bands were indeed SDF-1 transcripts (GenBank accession No. L36033).
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DISCUSSION |
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Here we report for the first time, to our knowledge, that malignant B lymphocytes purified from the lymph nodes of patients with follicular center lymphoma migrate in vitro in response to SDF-1. This conclusion is supported by experiments carried out in samples from 10 patients with the use of two different locomotory assays, i.e., the filter and the collagen invasion tests. Checkerboard analysis showed that the locomotory response of follicular center lymphoma B cells to SDF-1 was attributable predominantly to chemotaxis.
Follicular center lymphoma B cells were found to express the SDF-1 receptor CXCR4, and their pretreatment with a blocking antibody to CXCR4 abrogated locomotory response to SDF-1.
The presumed normal counterparts of follicular center lymphoma B cells, i.e., germinal center B cells (3,5,6) freshly isolated from tonsils, did not show any locomotory response to SDF-1 either in collagen or in filter assays, although they expressed CXCR4. These findings are consistent with the previous demonstration that tonsillar germinal center B lymphocytes, at variance with naive and memory B cells, did not migrate upon exposure to SDF-1 in a filter assay because of delayed internalization of the SDF-1CXCR4 complex (32).
A major difference between neoplastic and normal B cells of germinal center origin is the constitutive expression of the Bcl-2 protein in the former (14) as opposed to the latter (48) cells. Seventy percent to 90% of follicular center lymphoma cases are characterized by t(14;18), whereby the Bcl-2 gene at 18q21 recombines with the Ig heavy chain-joining segment on chromosome 14, leading to constitutive Bcl-2 expression (1316).
Although it has been held that Bcl-2 expression accounts for follicular center lymphoma B-cell resistance to apoptosis, a recent study (49) has demonstrated that enhanced expression of the Bcl-xL death-suppressor protein is indeed the main determinant of the prolonged survival of follicular center lymphoma B cells. Analogously, CD40-stimulated germinal center B cells are rescued from apoptosis primarily through an early increase in the expression of the Bcl-xL protein (50), followed by late expression of the Bcl-2 protein (50,51). These findings point to strong similarities between follicular center lymphoma and germinal center B lymphocytes with regard to the mechanisms involved in cell survival.
We, therefore, reasoned that short-term prestimulation of tonsillar germinal center B cells with CD40 MAb and rIL-4 (36,39,41,42) might render them competent to respond to SDF-1. After pretreatment with CD40 MAb and rIL-4, germinal center B cells migrated upon exposure to SDF-1 in the same manner as follicular center lymphoma B cells did. Although the biochemical mechanisms of the phenomenon were not investigated, it is likely that increased expression of Bcl-xL is involved in the enhanced locomotion of prestimulated germinal center B cells in response to SDF-1.
What is the potential relevance of these phenomena in vivo? Germinal center B lymphocytes have little or no propensity to migrate, likely because, in the germinal center microenvironment, they must undergo hypermutation of Ig variable region genes and selection of cells bearing high-affinity antigen receptors [reviewed in (5254)]. Most germinal center B cells die by apoptosis, and only a minority of them differentiate into memory cells or plasma cells (5254). It may be hypothesized that selected germinal center B cells acquire locomotory capacities to complete their differentiation outside the germinal centers. In this perspective, SDF-1 responsiveness would be a property of late germinal center B lymphocytes that are in the process of leaving the germinal centers. It is conceivable that chemotactic signals other than SDF-1 contribute to stimulating the locomotion of germinal center B cells.
In search of paracrine and/or autocrine loops controlling neoplastic B-cell migration (43), we investigated whether follicular center lymphoma B lymphocytes expressed the SDF-1 gene. Such expression was detected by RTPCR in four follicular center lymphoma cases as well as in four preparations of freshly isolated germinal center B cells.
Although these experiments did not prove that the SDF-1 protein was produced by either malignant or normal B cells, it may be hypothesized that low amounts of the chemokine are synthesized in vivo under appropriate microenvironmental conditions. Future studies will address to what extent endogenously produced SDF-1 may contribute to the stimulation of B-cell locomotion.
Previously, the SDF-1 gene was shown to be expressed by in situ hybridization only in tonsillar reticulum cells located at the periphery of the lymphoid follicles (32). The apparent discrepancy between the latter results and our findings may be explained by the higher sensitivity of the RTPCR procedure employed here as compared with in situ hybridization methods. Another possibility is that, during the separation procedure, germinal center B cells were activated by nonspecific stimuli to express SDF-1 messenger RNA; this hypothesis, however, appears unlikely, since all of the steps for germinal center B-lymphocyte isolation were carried out at 4°C.
The structure of the invaded lymph nodes in follicular center lymphoma roughly resembles that of normal lymph nodes (3,5,6). At diagnosis, enlarged lymphoid follicles of uniform size with a prominent germinal center and a mantle zone of variable thickness are usually observed (55). The interfollicular tissue containing fibroblastic reticular cells, the predominant stromal cell type in the lymph node cortex (56), is variably compressed among the expanding follicles, as demonstrated by silver impregnation (55). Nonetheless, a certain amount of interfollicular tissue is detected in follicular center lymphoma lymph nodes over time, suggesting that stromal cells (32) may favor the spread of malignant B lymphocytes from one follicle to the adjacent ones by generating a chemotactic gradient of SDF-1. This loop might be reinforced by SDF-1 produced by follicular center lymphoma B cells themselves.
Studies carried out in transgenic mice bearing a fusion Bcl-2Ig minigene that mimics the t(14;18) of human follicular center lymphoma have shown that these animals develop a long-lasting polyclonal follicular hyperplasia, but the condition in only some of them progresses to high-grade lymphoma after acquisition of a c-myc rearrangement (57). These findings suggest that t(14;18) prolongs the survival of germinal center B cells but is not sufficient per se to induce malignancy. This hypothesis may be supported by the identification of t(14;18) in the peripheral blood or in the secondary lymphoid organs from normal individuals (58,59).
This study demonstrates that normal germinal center B cells display a migratory behavior similar to that of their malignant counterparts, follicular center lymphoma B cells, provided that the former cells are rescued from apoptosis. Such an observation, coupled with the conclusions of the studies with t(14;18) transgenic mice (57), may lead to the speculation that the locomotory responsiveness of follicular center lymphoma B cells to SDF-1 is a feature acquired at the early stages of transformation, when t(14;18) has already occurred but the target cell is not yet fully malignant.
Our results may be relevant for a better understanding of the mechanisms whereby follicular center lymphoma B cells disseminate in vivo.
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NOTES |
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We thank Dr. Carlo Grossi (Department of Experimental Medicine, University of Genoa, Italy) for helpful discussion and revision of the manuscript and Dr. Silvio Roncella (Laboratory of Pathology, La Spezia Hospital, Italy) for help in the immunophenotyping of follicular lymphoma cells.
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Manuscript received August 5, 1999; revised January 31, 2000; accepted February 16, 2000.
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