Chemotaxis of human tonsil B lymphocytes to CC chemokine receptor ...

International Immunology, Vol. 14, No. 8, pp. 883±892

ã 2002 The Japanese Society for Immunology

Chemotaxis of human tonsil B lymphocytes to CC chemokine receptor (CCR) 1, CCR2 and CCR4 ligands is restricted to nongerminal center cells Anna Corcione1, Giuseppe Tortolina2, Raffaella Bonecchi3, Nicoletta Battilana1, Giuseppe Taborelli4, Fabio Malavasi5, Silvano Sozzani3, Luciano Ottonello2, Franco Dallegri2 and Vito Pistoia1 1Laboratory

of Oncology, G. Gaslini Institute, Largo G. Gaslini 5, 16148 Genova, Italy of Internal Medicine, University of Genova, Genova, Italy 3Department of Immunology, Mario Negri Institute, Milano, Italy 4Division of Otolaryngology, G. Gaslini Institute, Genova, Italy 5Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy 2Department

Keywords: B cell subsets, chemokines, chemokine receptors, locomotion Abstract We have investigated the effects of nine CC chemokines, i.e. macrophage in¯ammatory protein (MIP)-1a/CCL3, MIP-1b/CCL4, MIP-3a/CCL20, MIP-5/CCL15, monocyte chemotactic protein (MCP)1/CCL2, MCP-2/CCL8, MCP-3/CCL7, eotaxin/CCL11 and macrophage-derived chemokine (MDC)/ CCL22 on the locomotion of human tonsil B lymphocytes and their subsets. Upon isolation, B cells were poorly responsive, but, following short-term culture, they displayed statistically signi®cant chemotactic responses (P < 0.001) to MIP-1a, MIP-5, MCP-1, MCP-2, MCP-3 and MDC. CC chemokine receptor (CCR) 1 to CCR6 were up-regulated after culture. MIP-1b, MIP-3a and eotaxin did not stimulate B cell migration. Scattered information is available on B cell subset responses to chemokines. Therefore, we investigated the effects of MIP-1a, MIP-5, MCP1, MCP-2, MCP-3 and MDC on the in vitro locomotion of non-germinal center (GC) (CD38±) and GC (CD38+) B cells. All chemokines enhanced signi®cantly (P < 0.001) the migration of the former, but not of the latter, cells. CCR1, CCR2 and CCR4 were detected by ¯ow cytometry on non-GC (i.e. naive and memory) B cells, whereas they were absent (CCR1 and CCR2) or poorly expressed (CCR4) on GC B cells. Introduction During their life span, T and B lymphocytes continuously recirculate through different lymphoid and non-lymphoid tissue compartments (1,2). Cell migration depends on the expression of speci®c adhesion molecules that allow selective cell homing to different anatomical districts and on the delivery of chemotactic signals that trigger cell locomotion (1,2). Furthermore, differences in leukocyte migratory behavior may be related to their stage of maturation and/or functional differentiation (3±5). The latter concept is well exempli®ed by the re-circulation pathways of the major mature B lymphocyte subsets (4,5). Naive B cells migrate from the bone marrow to the peripheral lymphoid organs where, following re-circulation

through blood, they encounter speci®c antigen, undergo clonal expansion and colonize the primary lymphoid follicles (4,5). Here naive B cells differentiate into germinal center (GC) cells which ®rst proliferate, then somatically mutate Ig variable region genes and ®nally are positively selected according to the af®nity of surface Ig for antigen presented by follicular dendritic cells (6±9). GC B cells are a resident cell subset with poor propensity to migrate. Following positive selection, GC B cells differentiate into memory or effector cells (plasma cells) outside the lymphoid follicles (6±9). Memory B cells home to speci®c anatomical sites, such as the splenic marginal zone (10) or the tonsil subepithelial

Correspondence to: A. Corcione; E-mail: [email protected] Transmitting editor: L. Moretta

Received 19 December 2001, accepted 3 May 2002

884 B cell subset migration criptae (11), where they settle until they interact with speci®c antigens. Thereafter, the majority of antigen-activated memory B lymphocytes re-circulate (9). Chemokines represent a group of chemotactic cytokines that mobilize subsets of effector leukocytes during in¯ammatory reactions or regulate the constitutive homing of B cells and T cells to peripheral lymphoid organs (12±17). The best characterized B cell tropic chemokines are stromal cell derived factor (SDF)-1 (18±21), secondary lymphoid tissue chemokine (SLC) (21,22), Epstein±Barr virus gene 1ligand chemokine (ELC), also known as macrophage in¯ammatory protein (MIP)-3b (21,23,24), and B cell-attracting (BCA)-1 chemokine, also known as B lymphocyte chemoattractant (BLC) (21,25,26). SDF-1 has been reported to be a potent chemoattractant for both normal and malignant human B lymphocytes (18±21,27). Furthermore, mice lacking the SDF-1 gene or the gene encoding the SDF-1 receptor, i.e. CXC chemokine receptor (CXCR) 4, show gross defects in B cell development (19,28,29). The SLC and ELC chemokines, expressed constitutively in the thymus, lymph nodes and other lymphoid tissues, have been shown to regulate lymphocyte homing (22,30). Mice de®cient for CC chemokine receptor (CCR) 7, which binds to both SLC and ELC, display a severely impaired migration of B cells to lymph nodes (LN) or Peyer's patches (PP) (31). BCA-1, expressed in lymphoid tissues at high levels, strongly attracts B lymphocytes (25,26). BCA-1 selectively binds to CXCR5, that is widely expressed on blood and tonsil B cells (32). Disruption of the CXCR5 gene leads to loss of B cell follicles and GC in the LN and PP, suggesting a crucial role of BCA-1±CXCR5 interactions in B lymphocyte recruitment to secondary lymphoid organs (33). Recently, it has been shown that thymus-expressed chemokine (TECK) attracts murine pre-pro-B cells and cells capable of generating pro-B colonies in the presence of IL-7 and ¯t3 ligand, whereas such response is lost in later stages of B cell development (3). In contrast, human peripheral blood B cells displayed low but consistent migratory responses to TECK (21). Finally, MIP-1a (34), IL-8 (35), growth-related oncogene (GRO)-a (35) and monocyte chemoattractant protein (MCP)-1 (36) have been reported to be chemotactic for human peripheral blood B cells and, in the case of MCP-1, also for human tonsil B cells (36). In this study we have investigated the chemoattractant activity of 9 CC chemokines [MIP-1a/CCL3, MIP-1b/CCL4, MIP-3a/CCL20, MIP-5/CCL15, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, eotaxin/CCL11 and macrophage derived chemokine (MDC)/CCL22] on human tonsil B lymphocytes, and, in particular, on the GC and non-GC subsets, since limited information exists on the latter issue (37). Methods Chemokines and antibodies MIP-1a, MIP-1b, MIP-3a, MIP-5, MCP-1, MCP-2, MCP-3, MDC and eotaxin were human recombinant molecules purchased from PeproTech (Rocky Hill, NJ). SDF-1, tested as positive

control at the ®nal concentration of 300 ng/ml (27), was also from PeproTech. The following mAb were used: CD19±FITC, CD3±FITC, CD68±phycoerythrin (PE), CD56±PE, CD38±FITC and antiHLA-DR±FITC (Becton Dickinson Immunocytometry Systems, San Jose, CA). Unconjugated CD3, CD56 and CD68 (Becton Dickinson) were used in cell separation experiments at the ®nal concentration of 1 mg/ml. The CD39±PE mAb was from PharMingen (San Diego, CA). The CD38 mAb was produced by one of us (F. M.) (38). The unconjugated anti-IgD mAb was purchased from Dako (Glostrup, Denmark) and used at the concentration of 1 mg/ml. The unconjugated CD39 mAb was obtained from Immunotech (Marseille, France) and used at the 1 mg/ml concentration. CCR were detected by staining B cells, either freshly isolated or 4 h cultured, in the absence of stimuli, with the following mAb: anti-CCR1±biotin (clone 53504.111), antiCCR2-biotin (clone 48607.211), anti-CCR3±PE (clone 61828.111) and anti-CCR6±PE (clone 53103.111) from R & D Systems (Minneapolis, MN). An unconjugated anti-CCR5 mAb (clone 2D7) was from PharMingen. The anti-CCR4 mAb (clone 305L) was kindly donated by Drs Carol Raport and Pat Gray (ICOS, Bothell, WA). A mAb to CCR2 (LS132) was donated by Dr Craig La Rosa (Millennium Pharmaceuticals, Cambridge, MA) and used in blocking experiments at the concentration of 1 mg/ml. Flow cytometric analyses were performed as previously reported (27). Controls for each of the above mAb were isotype-matched mAb of irrelevant speci®city conjugated with the same ¯uorochromes. All of the ¯ow cytometry experiments were performed using a FACScan (Becton Dickinson). B lymphocyte puri®cation Normal tonsils were obtained from patients undergoing tonsillectomy for in¯ammatory disorders. Mononuclear cells were isolated by Ficoll-Hypaque density gradients and depleted of lymphocytes forming rosettes with sheep red blood cells (T lymphocytes). Non-T cells were then incubated with the CD3, CD56 and CD68 mAb, treated with MACS goat anti-mouse IgG microbeads according to the instructions of the manufacturer (Milteny Biotec, Auburn, CA), and separated by applying a magnetic ®eld. Negatively selected cells contained on average 99% B cells, as assessed by staining for CD19 (27). Fractionation of tonsil B lymphocytes into GC and non-GC cells was performed as follows. Puri®ed B lymphocyte suspensions were incubated ®rst with the CD38 mAb for 30 min at 4°C and subsequently with MACS goat anti-mouse IgG microbeads. CD38+ (GC) and CD38± (non-GC) cells were separated by applying a magnetic ®eld. Naive B lymphocytes were isolated as IgD+ cells from total B lymphocyte suspensions by immunomagnetic bead manipulation. The IgD± B cell fractions were further separated into CD38+ (GC) cells and CD38± (memory) cells by the same technique (11). All of the above separation procedures were performed at 4°C in order to prevent spontaneous apoptosis of GC B cells. In some experiments, B lymphocytes were run on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient consisting of 100, 60, 50, 40 and 30% Percoll dilutions respectively from the bottom to the top of the tubes, as previously reported (6,39). Cells collected from the

B cell subset migration 885 Checkerboard analysis Assays of cell migration with different doses of chemokines on both sides of the ®lter were performed. The results of these experiments were collected in checkerboard form by which chemokinesis (i.e. change in the intensity of random locomotion) and true chemotaxis (i.e. change in the directional response to the stimulus) were calculated according to Zigmond and Hirsch (40). Gene expression analysis by RT-PCR

Fig. 1. Dose±response curves of puri®ed tonsil B lymphocytes in response to a panel of CC chemokines. Puri®ed B lymphocytes were tested in a modi®ed Boyden chamber assay with or without the following chemokines: MIP-1a, MIP-1b, MIP-3a, MIP-5, MCP-1, MCP-2, MCP-3, MDC, eotaxin and MDC (concentration range from 0 to 1000 ng/ml) or SDF-1 (300 ng/ml), as positive control (27). Results are expressed as micrometers traveled and are means 6 SD from four different experiments for each chemokine.

low-density fractions of the gradient (30 and 40%) were treated with CD39 and anti-IgD mAb, and subsequently with anti-mouse IgG magnetic beads. Unbound B cells represented homogeneous populations (>98%) of GC B lymphocytes, as shown by expression of CD38, and by negative staining for IgD and CD39 (6,39). Migration assay Cell locomotion was studied using the leading front method in a modi®ed Boyden chamber assay (27,40). Duplicate tests were carried out in 48-well microchemotaxis chambers (Neuro Probe, Cabin John, MA) with an 8-mm pore size cellulose ester ®lter (SCWPO 1300, lot. no R4MM58776; Millipore, Milano, Italy) separating the cells (4 3 105) from the chemoattractant tested at different concentrations or from medium alone (control). Cells were cultured 2 h in RPMI 1640 medium (Seromed; Biochrom, Berlin, Germany) containing 0.1% human albumin and subsequently subjected to the migration assay in the presence of the chemoattractant at 37°C for 2 h. The ®lters were then removed, ®xed in ethanol, stained with Harris hematoxylin, dehydrated, cleared with xylene and mounted in Eukitt (Kindler, Freiburg, Germany). Duplicate chambers were run in each case and the distance (micrometers) traveled by the leading front of cells was measured at 3400 magni®cation (40). For blocking experiments, 2-h cultured B cells were incubated for 30 min at 4°C with the anti-CCR2 mAb (1 mg/ml) or with an isotype-matched control mAb, washed and tested for migration.

Total RNA was extracted from puri®ed tonsil B lymphocytes by the guanidium thiocyanate method (41) and reverse transcribed into complementary DNA using Superscript Preampli®cation System (Gibco/BRL Life Technologies, San Giuliano Milanese, Italy). Primer sequences were as follows: hCCR1: 5¢-GGAAACTCCAAACACCACAGAGG-3¢ and 5¢-GCCTGGCATGGAAGCCAAGATG-3¢, amplifying a 502-bp product; hCCR2b: 5¢-CAGATATCATGCTGTCCACATCTCGTTCTCGG-3¢ and 5¢-CAGGATCCTTATAAACCAGCCGAGACTTCCTGC-3¢, amplifying a 1082-bp product; hCCR3: 5¢-ATATCTGCGGCCGCAATGACAACCTCACTAGATACAGTTG-3¢ and 5¢-TGAATCCTAAAACACAATAGAGAGTTCCGGC-3¢, amplifying a 1068-bp product; hCCR4: 5¢-ATATCTGCGGCCGCAATGAACCCCACGGATATAGCAGATAC-3¢ and 5¢-ATCGGATCCTACAGAGCATCATGAAGATCATG-3¢, amplifying a 1083bp product; hCCR5: 5¢ ATATCTGCGGCCGCGATGGATTATCAAGTGTCAAGTCCAA-3¢ and 5¢-ATC:GGATCCTCACAAGCCCACAGATATTTCCAGC-3¢, amplifying a 1058-bp product; hCCR6: 5¢-ATGAGCGGGGAATCAATGATTTC-3¢ and 5¢-TCACATAGTGAAGGACGACGCA-3¢, amplifying a 1124-bp product; CD3g: 5¢-GGTTCGGTACTTCTGACT-3¢ and 5¢-TGGTTTTGACTTGTTCTG-3¢ amplifying a 171-bp product; CD68: 5¢CATCCAACAAGCAATAGCA-3¢ and 5¢-CTGAGCCGAGAATGTCCACT-3¢ amplifying a 507-bp product; CD56: 5¢-AGGGCAGATGGGAGAGGA-3¢ and 5¢-AACCACCAGGAGCAGGAC-3¢ amplifying a 361-bp product; b-actin: 5¢-GGAGCAATGATCTTGATCTTC-3¢ and 5¢-AAGATGACCCAGATCATGTTTGAG-3¢ amplifying a 500-bp product. cDNAs were ampli®ed as follows. hCCR1 to hCCR6 and bactin genes: 1 cycle of 5 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 55, 58 or 60°C (depending on the primer pair) and 1 min at 72°C followed by one cycle of 10 min at 72°C. Ampli®cation conditions for the remaining primers were the following: CD3g, 1 min at 94°C, 1 min at 48°C, 1 min at 72°C, 32 cycles; CD68, 1 min at 94°C, 1 min at 54°C, 1 min at 72°C, 32 cycles; CD56, 1 min at 94°C, 1 min at 58°C, 1 min at 72°C, 32 cycles. For semiquantitative RT-PCR, cDNAs were co-ampli®ed using different experimental conditions to safeguard against non-linear ampli®cation: 1 cycle of 5 min at 94°C, 25 cycles (b-actin) and 30 cycles (CCR1, CCR3, CCR5 and CCR6) or 35 cycles (CCR2 and CCR4) of 1 min at 72°C, followed by one cycle of 10 min at 72°C. The PCR products were subjected to electrophoresis through 1% agarose with ethidium bromide to con®rm the base pair sequence length. In control experiments, RNA samples were subjected to PCR ampli®cation omitting the step of reverse transcription. These experiments were addressed at investigating whether genomic DNA possibly contaminating RNA was detected using the primers speci®ed above.

886 B cell subset migration Table 1. Checkerboard assay of puri®ed tonsillar B cells in response to CC chemokines Chemokine (ng/ml) above the ®lter

Chemokine (ng/ml) below the ®lter

MIP-1a 0 10 100 MDC 0 30 100 MIP-5 0 30 100 MCP-1 0 100 300 MCP-2 0 100 300 MCP-3 0 100 300

0 60

10 77 (65) 74

0 78

30 107 (84) 91

0 73

30 93 (77) 81

0 62

100 79 (64) 66

0 41

100 67 (43) 47

0 41

100 59 (45) 57

100 91 80 80 100 112 107 102 100 108 83 89 300 86 68 73 300 72 60 63 300 75 68 59

(68) (77) (90) (97) (80) (85) (67) (69) (47) (52)

Fig. 2. CCR gene expression in human tonsil B lymphocytes. Total RNA was extracted from tonsil B cells, reverse transcribed and subjected to RT-PCR by the use of primers speci®c for CCR1 to CCR6. From the left to the right: negative control in which RT-PCR was performed in the absence of cDNA; three different tonsil B cell samples, indicated as donor 1±3; positive control, represented by human monocytes for CCR1 to CCR5 and by dendritic cells derived from cultured CD34+ progenitor cells for CCR6.

(46) (58)

Results are expressed as distance migrated (micrometers in 2 h) by the leading front of cells (mean of two experiments, SD omitted for sake of clarity). Figures in parentheses show the calculated distance migrated if cells would have not responded to the cytokine gradient, but only to the absolute concentration (64).

Statistical methods Data are expressed as mean 6 SD. Differences were determined by repeated measures ANOVA followed by the Bonferroni multiple comparisons post test. Differences were accepted as signi®cant when P < 0.05. Results Migration of tonsil B lymphocytes in response to CC chemokines Tonsil B cells were isolated (99% average purity) and tested in a modi®ed Boyden chamber assay for their locomotory responses to a panel of CC chemokines after 2 h culture in the absence of stimuli, as previously reported (Fig. 1). The following chemokines were tested: MIP-1a/CCL3, MIP-1b/ CCL4, MIP-3a/CCL20, MIP-5/CCL15, MCP-1/CCL2, MCP-2/ CCL8, MCP-3/CCL7, eotaxin/CCL11 and MDC/CCL22, concentrations ranging from 0 to 1000 ng/ml (Fig. 1). A statistically signi®cant (P < 0.001) dose-dependent locomotion to MIP-1a, MIP-5, MCP-1, MCP-2, MCP-3 and MDC with the typical bell-shaped curve was observed (Fig. 1). MIP1b, MIP3a and eotaxin did not increase the spontaneous motility of tonsil B cells at any concentration tested (Fig. 1). The same cell fractions migrated to SDF-1, tested as positive control (18±21,27) (Fig. 1). Based upon these results, the following chemokine concentrations were used for further

experiments: 100 ng/ml for MIP-1a, MIP-5 and MDC; 300 ng/ ml for MCP-1, MCP-2 and MCP-3. To investigate the nature of the CC chemokine-dependent increase in B cell locomotion, the checkerboard analysis was performed. As shown in Table 1, MIP-1a, MDC, MIP-5, MCP-1, MCP-2 and MCP-3 stimulated both the rate of cell locomotion and the true chemotaxis. In two different experiments, taking into account spontaneous migration (i.e. in the absence of chemokines above and below the ®lter), the following individual concentrations above and below the ®lter (chemokinetic conditions) enhanced cell migration as indicated (mean percent increment 6 SD): MIP-1a (100 ng/ml; 33.7 6 14.8), MDC (100 ng/ml; 31.9 6 14.1), MIP-5 (100 ng/ml; 21.1 6 2.1), MCP-1 (300 ng/ml; 17.8 6 4.2), MCP-2 (300 ng/ml; 56 6 21.3) and MCP-3 (300 ng/ml; 42.8 6 1.4). On the other hand, compared with B cell migration in the absence of CC chemokines above and below the ®lter (i.e. spontaneous migration), the above chemokine concentrations placed only below the ®lter (chemotactic conditions) augmented cell migration as follows (mean increment 6 SD): MIP-1a (50.5 6 5.9%), MDC (45 6 20.5%), MIP-5 (46.7 6 8.6), MCP-1 (25,5 6 6.6%), MCP-2 (109.5 6 20) and MCP-3 (77.9 6 41.4). On the contrary, cell migration in negative gradients was consistently lower than that calculated on the basis of the expected response to absolute concentrations alone. In conclusion, MIP-1a, MDC, MIP-5, MCP-1, MCP-2 and MCP-3 stimulated both true chemotaxis and chemokinesis, the former being predominant over the latter. CCR expression in tonsil B lymphocytes The CC chemokines here investigated interact with CCR1 to CCR6 (12±17). Therefore, the expression of CCR1 to CCR6 mRNA was investigated in tonsil B lymphocytes by RT-PCR. Figure 2 shows the results obtained in three different experi-

B cell subset migration 887 Locomotion of tonsil B lymphocyte subsets in response to CC chemokines

Fig. 3. Functional role of CCR2 in the MCP-1-, MCP-2- and MCP-3triggered B cell migration. Puri®ed tonsil B lymphocytes were incubated with an anti-CCR2 blocking mAb or with an isotypematched irrelevant (control) mAb. Thereafter, cells were tested in a modi®ed Boyden chamber assay in the presence of MCP-1, MCP-2, MCP-3 or medium alone. Results are means from three experiments. Asterisks indicate statistically signi®cant differences in the migration of B cells exposed to chemokines in the presence versus absence of the anti-CCR2 mAb. *P < 0.01; **P < 0.001.

ments. CCR1 to CCR6 transcripts were consistently detected both in B cells and in control cells (i.e. monocytes for CCR1 to CCR5, dendritic cells differentiated from CD34+ hemopoietic progenitors for CCR6). Control experiments ruled out that genomic DNA contaminated B cell RNA. Furthermore, the B cell suspensions tested did not express CD3g, CD56 or CD68 mRNA, thus ruling out the presence of contaminant T cells, NK cells or macrophages respectively (not shown). Tonsil B cells were found to express CCR1 (7±20%), CCR2 (10±24%), CCR4 (4±35%), CCR5 (7±10%) and CCR6 (20± 40%), whereas CCR3 was detected on a minor proportion of cells (3±6%) (data not shown). Studies carried out with different human leukocyte subsets (T and NK lymphocytes, eosinophils, basophils, monocytes and dendritic cells) have demonstrated that CCR2 binds to MCP-1, MCP-2 and MCP-3 [reviewed in (42)]. However, MCP-2 and MCP-3 may utilize alternative CCR (42). Therefore, the respective contribution of MCP-1, MCP-2 and MCP-3 to the triggering of B cell-associated CCR2 was studied. Puri®ed tonsil B cells were pre-incubated with an anti-CCR2 blocking mAb or with an isotype-matched irrelevant mAb and were subsequently tested in the modi®ed Boyden chamber assay in the presence of MCP-1, MCP-2 or MCP-3. In three different experiments, blocking of CCR2 inhibited signi®cantly B cell migration in response to MCP-1 (P < 0.001) and to MCP2 (P < 0.01), but not to MCP-3 (P > 0.05) (Fig. 3).

Tonsil B lymphocytes are comprised of three major subpopulations which are distinguished according to immunophenotype, anatomic location and functional features (6±9,43,44). These B cell subsets, named GC, naive and memory cells, are found in the GC, in the follicular mantle and in the subepithelial areas of the tonsil respectively (9±11,45). The CD38 surface marker allows us to separate GC (CD38+) from non-GC (CD38±), i.e. naive and memory, B lymphocytes (6,9,11,43). In subsequent experiments, puri®ed tonsil B cells were fractionated into the GC and non-GC subpopulations by incubation with CD38 mAb followed by immunomagnetic bead manipulation. These B cell subsets were tested for their migratory responses to MIP-1a, MIP-5, MCP1, MCP2, MCP3 and MDC, in comparison with unfractionated B cells from the same tonsils. Positive control for the latter cells was SDF-1. As shown in Fig. 4, non-GC B cells, as well as unfractionated B cells, migrated signi®cantly faster in the presence that in the absence of the above CC chemokines (P < 0.001 for all chemokines). Furthermore, as expected, the same cell fractions displayed a signi®cantly increased locomotion in response to SDF-1 (P < 0.001) (Fig. 4). In contrast, the spontaneous migration of GC B cells was not enhanced by incubation with MIP-1a, MIP-5, MCP1, MCP2, MCP3 or MDC (Fig. 4). Since GC B cells had been positively selected for CD38, the possibility existed that the in vitro migration of GC B cells was down-regulated by CD38 triggering. To test this hypothesis, GC B lymphocytes were puri®ed using an alternative method based on their enrichment by Percoll density gradients followed by depletion of CD39+, IgD+ B cells (6,39). When tested in the Boyden chamber assay, the latter GC B cell fractions did not migrate upon exposure to any CC chemokine (data not shown), thus con®rming the results shown in Fig. 4. Control experiments ruled out that the failure of GC B lymphocytes to migrate in vitro in response to the above chemokines was related to their propensity to undergo apoptosis (9). Thus, all the separation procedures were conducted at 4°C to prevent spontaneous apoptosis, and freshly isolated GC B cells contained consistently >90% viable lymphocytes, as assessed by Trypan blue staining. An equivalent proportion of viable cells was detected after 2 h incubation for the locomotory assays. Next, the expression of CCR1 to CCR6 was investigated by ¯ow cytometry on naive (IgD+), GC (CD38+, IgD±) and memory (CD38±, IgD±) B cells isolated by immunomagnetic bead manipulation. Figure 5 shows the results from three experiments. CCR1 and CCR2 were detected on naive (26±45% for CCR1 and 31±40% for CCR2) and memory (28±44% for CCR1 and 16±40% for CCR2) B cells, but not on GC B cells. CCR4 was expressed on the majority of naive (56±70%) and memory (40±73%) B cells, whereas it was found on 6±12% GC B cells (Fig. 5). Naive and memory B cells expressed CCR3 (6±10% for naive B cells and 5±12% for memory B cells) and CCR5 (9±10% for naive B cells and 3±13% for memory B cells), while these receptors were detected on 6±8% of GC B cells for CCR3 and 3±8% of the same cells for CCR5 (Fig. 5). Finally,

888 B cell subset migration CCR6 was detected on 18±30% of naive B cells, 20±28% of memory B cells and 2±4% of GC B cells (Fig. 5). Since GC B cells had been positively selected, control experiments were carried out to exclude that the failure to detect surface expression of some CCR (e.g. CCR1 and CCR2) was attributable to non-speci®c down-regulation of such receptors upon cell incubation with the CD38 mAb. Thus, selected CD38+ tonsil B cells were stained with CD19 or with anti-HLA-DR mAb, two pan-B cell markers, following overnight incubation in the absence of stimuli. In four different experiments, all viable cultured cells stained positively for CD19 and HLA-DR (not shown). In the same experiments, expression of CD38 was heterogeneous, ranging from a minimum of 15% to a maximum of 80% (data not shown). These studies demonstrate that positive selection of GC B cells as CD38+ cells did not affect CD19 and HLA-DR expression. In contrast, the results with CD38 staining may be related to the variable kinetics of CD38 surface re-expression after antibody-induced internalization. The results of ¯ow cytometry experiments shown in Fig. 5 raised the possibility that CCR1 and CCR2 genes were not expressed in GC B cells. Therefore, tonsil B cells were separated into the CD38+ GC and the CD38± naive and memory subsets; these cell fractions were subjected to RNA extraction and semiquantitative RT-PCR for CCR1 to CCR6 gene expression. As shown in Fig. 6, the transcripts of all receptors were detected in both CD38+ and CD38± B cells. The CCR2, CCR3 and CCR4 ampli®ed bands were of comparable intensity in the two cell fractions, whereas CCR1, CCR5 and CCR6 mRNA were more expressed in CD38± than in CD38+ cells (Fig. 6).

Discussion

Fig. 4. CC chemokine-driven locomotion of GC and non-GC B lymphocytes. GC and non-GC B cells were isolated from tonsil B cell suspensions by incubation with CD38 mAb followed by immunomagnetic bead manipulation. CD38+ (GC) and CD38± (nonGC) B cell subsets were subsequently tested in a modi®ed Boyden chamber assay, together with unfractionated B cells isolated from the same tonsils, in the presence (®lled bars) or absence (open bars, nil) of MIP-1a, MIP-5, MCP-1, MCP-2, MCP-3 or MDC. Positive control for unfractionated B cells was SDF-1 (300 ng/ml). Results are means from ®ve experiments. All chemokines induced a statistically signi®cant increase of the migration of non-GC and unfractionated B cells (P < 0.001).

In this study, MIP-1a/CCL3, MIP-5/CCL15, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7 and MDC/CCL22 were found to enhance the in vitro locomotion of tonsil B cells, whereas MIP1b/CCL4, MIP-3a/CCL20 and eotaxin/CCL11 were ineffective (21,46,47). All the biologically active chemokines stimulated B cell locomotion predominantly through a chemotactic effect. mRNA of CCR1 to CCR6 was detected in puri®ed tonsil B cells and, accordingly, ¯ow cytometric analyses showed the expression of the corresponding proteins on the cell surface. Some of the CC chemokines tested bind to a single CCR, whereas others share different receptors. For example, MCP-1 binds exclusively to CCR2; this receptor binds also to MCP-2 and MCP-3. Both of the latter chemokines interact with CCR1 and CCR3; furthermore, MCP-2 interacts with CCR5 (42). In this study, treatment of B cells with a CCR2-blocking mAb inhibited signi®cantly B cell migration induced by MCP-1 and MCP-2, but not that triggered by MCP-3. These results indicate that not only MCP-1, but also MCP-2-driven B cell migration largely depends on CCR2 engagement. It is conceivable that MCP-3 interacted predominantly with CCR1 to stimulate B cell motility, since eotaxin, that binds exclusively to CCR3 (42), was ineffective. The same considerations apply to MIP-5, a ligand of CCR1 and CCR3 (48), that likely enhanced B cell migration by engaging CCR1.

B cell subset migration 889 MDC is known to interact exclusively with CCR4 (49). In this study, we provide the ®rst evidence that MDC attracts human B lymphocytes and that CCR4 is abundantly expressed on the surface of freshly isolated B cells. Since MDC is produced in the T cell areas of secondary lymphoid organs, these observations may suggest novel B cell-tropic functions of MDC, in addition to its postulated role in attracting activated T cells and keeping them in close contact with dendritic cells (50). MIP-1a binds to CCR5 (51±53) and CCR1 (54±56); since MIP-1b, that binds predominantly to CCR5 (50±52), did not enhance the locomotion of peripheral blood (46) or tonsil (this study) B cells, it is conceivable that MIP-1a increased B cell migration through CCR1 activation. CCR6 binds to MIP-3a only and vice versa (57). In our experiments, CCR6 was strongly expressed on the surface of tonsil B cells (21,37,47), but its ligand MIP-3a had no effect on B lymphocyte migration. This result is consistent with some (21,47), but not other (37) reports; notably, in the latter study (37), MIP-3a was found to attract naive and memory, but not GC, B lymphocytes isolated from human tonsils. Taken together, our ®ndings suggest that the key receptors involved in tonsil B cell mobilization by MIP-1a, MIP-5, MCP-1,

MCP-2 and MCP-3 are CCR1 and CCR2, while CCR4 binds to MDC only. A recent study has addressed chemokine responsiveness of murine B cells at all stages of differentiation (3), providing important information on this issue. Some of the chemokines herein investigated were among those tested in such study: MIP-1a, MIP-1b, MCP-1 (JE), MCP-3, MIP3a and eotaxin (3). MIP-1a, MIP-1b, MCP-1 (JE) and MCP3 were found to attract early progenitor murine B cells, but not peripheral B cells; eotaxin was completely inactive versus any B cell fraction and, ®nally, MIP-3a stimulated the locomotion of all peripheral B cell subsets, but not that of bone marrow B cells (3). The results on murine, mature B cells differ remarkably from those obtained in this study with human B lymphocytes. Other discrepancies between the two experimental systems are (i) the failure of freshly isolated human GC B cells to migrate to SDF-1 (18,27), as opposed to their murine counterparts (3), and (ii) the failure of TECK to attract murine (3), but not human (21), mature B lymphocytes. Collectively, these ®ndings raise a note of caution in extrapolating murine data to the human system or vice versa.

Fig. 5. Flow cytometric analysis of CCR expression on naive, GC and memory B lymphocytes. Puri®ed tonsil B cells were fractionated into the naive (IgD+), GC (CD38+, IgD±) and memory (CD38±, IgD±) cell subsets, and stained with mAb against CCR1 to CCR6. The results of three different experiments are shown. The relative cell number on the ordinate axis is plotted versus the ¯uorescence intensity (log scale) on the abscissa. Controls were isotype-matched mAb of irrelevant speci®city conjugated with the same ¯uorochromes as test mAb. The percentage of positive cells is shown in the upper right side of each histogram.

890 B cell subset migration

Fig. 6. Semiquantitative CCR mRNA expression in CD38+ and CD38± tonsil B cells. Total RNA was extracted from CD38+ and CD38± tonsil B cells, reverse transcribed, and subjected to semiquantitative RT-PCR by co-ampli®cation with primers speci®c for b-actin and for CCR1, CCR2, CCR3, CCR4, CCR5 or CCR6. For each transcript, semiquantitative evaluation was obtained by normalization to b-actin mRNA. Lane (±) no template; lane (+) cDNA plasmid control. One representative experiment out the three performed with similar results is shown.

Separation of tonsil B lymphocytes in the GC and non-GC subpopulations showed that non-GC cells only were attracted by MIP-1a, MIP-5, MCP-1, MCP-2, MCP-3 and MDC. Accordingly, CCR1, CCR2 and CCR4 were detected on both naive and memory B cells. Non-GC B cells re-circulate physiologically through blood, lymph and secondary lymphoid organs (4,5). These cells, freshly isolated from human tonsils, have been shown to migrate in vitro in response to various chemoattractants, such as tumor necrosis factor (57), C5a (58) and SDF-1 (18). The present results are therefore in line with the well-characterized locomotory activity of non-GC B cells, both in vivo (4,5) and in vitro (18,58,59). In contrast, most GC B lymphocytes are resident cells that complete their life cycle in the GC, where they die by apoptosis (5±9). The poor propensity of human GC B cells to migrate has been related to the low expression of CD62 ligand (43), that is instrumental for cell interaction with high endothelial venules (60), and of CD44 (43,61). Furthermore, in vitro studies have demonstrated that freshly isolated GC B cells are not attracted by various stimuli (18,58,59,62). In human GC B cells, two different patterns of chemoattractant receptor expression have been described: (i) GC B cells do not express such receptors at the cell surface, as in the case of TNF or C5a receptors, and therefore are not responsive to their speci®c ligands (58,59); and (ii) GC B cells express chemoattractant receptors, such as CXCR4 that binds to SDF-1 but does not trigger cell migration due to delayed internalization of the receptor±ligand complex (18). Consistently with this scheme, staining of GC B cells for CCR1 and CCR2 was negative (although their transcripts were detected in the same cells), whereas CCR3, CCR4, CCR5 and CCR6 were on a minor proportion of cells. Thus, the failure of GC B cells to migrate in response to MIP-1a, MIP-5, MCP-1,

MCP-2 and MCP-3 is related to the absence of the relevant receptors, i.e. CCR1 and CCR2. In contrast, CCR4, although expressed on a minor but sizeable proportion of GC B cells, was ineffective at delivering stimulatory signals to the same cells following interaction with MDC. In conclusion, this study demonstrates that, in addition to chemokines synthesized constitutively in the secondary lymphoid tissues, inducible chemokines produced at the periphery may also stimulate B cell locomotion. A major functional difference between the two groups of chemokines is that the former regulate B cell homing to lymphoid follicles, whereas the latter mobilize effector cells to sites of in¯ammation (12). In pathological conditions, human B lymphocytes may cross the vascular endothelium and migrate to in¯amed tissues. For example, in rheumatoid arthritis and SjoÈgren's syndrome, B cells in®ltrate the synovial membrane or the salivary glands respectively, where they often cluster in newly formed lymphoid follicles (63,64). The present results support the hypothesis that some CC chemokines synthesized on demand at the site of an in¯ammatory process generate chemotactic gradients that contribute to promote extravasation of B cells, in particular of the memory subset, and their tissue localization. Notably, the B cells themselves can produce certain CC chemokines, such as MIP-1a (65) and MDC (66), which may amplify B cell recruitment to in¯ammatory foci through paracrine and/or autocrine interactions. Acknowledgements The Authors wish to thank Drs Carol Raport and Pat Gray (ICOS) for the generous gift of the anti-CCR4 mAb, Dr Greg LaRosa (Millenium Pharmaceuticals) for the gift of the anti-CCR2 mAb, and Dr Alberto Mantovani for encouragement and discussion. The authors also acknowledge the excellent secretarial assistance of Mrs Eliana Campochiaro. This work has been supported by grants from Associazione Italiana per la Ricerca sul Cancro and Ministero della SanitaÁ, Progetti di Ricerca Corrente and Progetti di Ricerca Finalizzata 1997 to V. P., and from the University of Genova to F. D.

Abbreviations BCA BLC CCR CXCR ELC GC GRO LN MCP MDC MIP PP SDF SLC TECK

B cell-attracting B lymphocyte chemoattractant CC chemokine receptor CXC chemokine receptor Epstein±Barr virus gene 1-ligand chemokine germinal center growth-related oncogene lymph node monocyte chemoattractant protein macrophage-derived chemokine macrophage in¯ammatory protein Peyer's patch stromal cell derived factor secondary lymphoid tissue chemokine thymus-expressed chemokine

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