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
References
1 Butcher, E. C. and Picker, L. J. 1996. Lymphocyte homing and
homeostasis. Science 272:60.
2 Springer, T. A. 1995. Traf®c signals on endothelium for
lymphocyte recirculation and leukocyte emigration. Annu. Rev.
Physiol. 57:827.
B cell subset migration 891
3 Bowman, E. P., Campbell, J. J., Soler, D., Dong, Z., Manlongat,
N., Picarella, D., Hardy, R. R. and Butcher, E. C. 2000.
Developmental switches in chemokine response pro®les during
B cell differentiation and maturation. J. Exp. Med. 191:1303.
4 MacLennan, I. C. and Chan, E. 1993. The dynamic relationships
between B-cell populations in adults. Immunol. Today 14:29.
5 Liu, Y. J. and Bancherau, J. 1996. The paths and molecular
controls of peripheral B cell development. Immunologist 4:55.
6 Liu, Y. J., Joshua, D. E., Williams, G. T., Smith, C. A., Gordon, J.
and MacLennan, I. C. M. 1989. Mechanisms of antigen-driven
selection in germinal centers. Nature 342:929.
7 Berek, C., Berger, A. and Apel, M. 1991. Maturation of immune
responses in germinal centers. Cell 67:1121.
8 Leanderson, T., Kallberg, E. and Gray, D. 1992. Expansion,
selection and mutation of antigen-speci®c B cells in germinal
centers. Immunol. Rev. 126:47.
9 MacLennan, I. C. 1994. Germinal centers. Annu. Rev. Immunol.
12:117.
10 Liu, Y. J., Old®eld, S. and MacLennan, I. C. 1988. Memory B cells
in T cell-dependent antibody responses colonize the splenic
marginal zones. Eur. J. Immunol. 18:355.
11 Liu, Y. J., Barthelemy, C., de Buoteiller, O., Arpin, C., Durand, I.
and Banchereau, J. 1995. Memory B cells from human tonsils
colonize mucosal epithelium and directly present antigen to T
cells by rapid up-regulation of B7-1 and B7-2. Immunity 2:239.
12 Baggiolini, M. 1998. Chemokines and leukocyte traf®c. Nature
392:565.
13 Cyster, J. G. 1999. Chemokines and cell migration in secondary
lymphoid organs. Science 286:2098.
14 Kim, C. H. and Broxmeyer, H. E. 1999. Chemokines: signal lamps
for traf®cking of T and B cells for development and effector
function. J. Leuk. Biol. 65:6.
15 Sallusto, F., Lanzavecchia, A. and Mackay, C. R. 1998.
Chemokines and chemokine receptors in T-cell priming and Th1/
Th2-mediate responses. Immunol. Today 19:568.
16 Zlotnik, A., Morales, J. and Hedrick, J. A. 1999. Recent advances
in chemokines and chemokine receptors. Crit. Rev. Immunol.
19:1.
17 Rollins, B. J. 1997. Chemokines. Blood 90:909.
18 Bleul, C. C., Schultze, J. L. and Springer, T. A. 1998. B lymphocyte
chemotaxis regulated in association with microanatomic
localization, differentiation state, and B cell receptor
engagement. J. Exp. Med. 187:753.
19 Ma, Q., Jones, D. and Springer, T. A. 1999. The chemokine
receptor CXCR4 is required for the retention of B lineage and
granulocytic
precursors
within
the
bone
marrow
microenvironment. Immunity. 10:463.
20 Vicente Manzanares, M., Montoya, M. C., Mellado, M., Frade, J.
M., del Pozo, M. A., Nieto, M., de Landazuri, M. O., Martinez-A, C.
and Sanchez-Madrid, F. 1998. The chemokine SDF-1a triggers
the chemotactic response and induce cell polarization in human B
lymphocytes. Eur. J. Immunol. 28:2197.
21 Brandes, M., Legler, D. F., Spoerri B., Schaerli P. and Moser, B.
2000. Activation-dependent modulation of B lymphocyte migration
to chemokines. Int. Immunol. 12:1285.
22 Nagira, M., Imai, T., Yoshida, R., Takagi, S., Iwasaki, M., Baba, M.,
Tabira, Y., Akagi, J., Nomiyama, H. and Yoshie, O. 1998. A
lymphocyte-speci®c CC chemokine, secondary lymphoid tissue
chemokine (SLC) is a highly ef®cient chemoattractant for B cells
and activated T cells. Eur. J. Immunol. 28:1516.
23 Ngo, V. N., Tang, H. L. and Cyster, J. G. 1998. Epstein±Barr virusinduced molecule 1 ligand chemokine is expressed by dendritic
cells in lymphoid tissues and strongly attracts naive T cells and
activated B cells. J. Exp. Med. 188:181.
24 Kim, C. H., Pelus, L. M., White, J. R., Applebaum, E., Johanson, K.
and Broxmeyer, H. E. 1998. CKb-11 macrophage in¯ammatory
protein-3b
EB1-ligand
chemokine
is
an
ef®cacious
chemoattractant for T and B cells. J. Immunol. 160:2418.
25 Legler, D. F., Loetscher, R. S., Roos, R. S., Clark-Lewis, I.,
Biaggiolini, M. and Moser B. 1998. B cell-attracting chemokine 1,
a human CXC chemokine expressed in lymphoid tissues,
selectively attracts B lymphocytes via BLR1/CXCR5. J. Exp.
Med. 187:655.
26 Gunn, M. D., Ngo, V. N., Ansel, K. M., Ekland, E. H., Cyster, J. G.
and Williams, L. T. 1998. A B-cell-homing chemokine made in
lymphoid follicles activates Burkitt's lymphoma receptor-1. Nature
391:799.
27 Corcione, A., Ottonello, L., Tortolina, G., Facchetti, P., Airoldi, I.,
Guglielmino, R., Dadati, P., Truini, M., Sozzani, S., Dallegri, F. and
Pistoia, V. 2000. Stromal cell-derived factor-1 as a
chemoattractant for follicular center lymphoma B cells. J. Natl
Cancer Inst. 92:628.
28 Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa,
S., Kitamura, Y., Yoshida, N., Kikutani, H. and Kishimoto, T. 1996.
Defects of B-cell lymphopoiesis and bone marrow myelopoiesis in
mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635.
29 Ma, Q., Jones, D., Borghesani, P. R., Segal, R. A., Nagasawa, T.,
Kishimoto, T., Bronson, R. T. and Springer, T. A. 1998. Impaired Blymphopoiesis, myelopoiesis, and derailed cerebellar neuron
migration in CXCR4- and SDF-1-de®cient mice. Proc. Natl Acad.
Sci. USA. 95:9448.
30 Gunn, M. D., Tangemann, K., Tam, C., Cyster, J. G., Rosen, S. D.
and Williams, L. T. 1998. A chemokine expressed in lymphoid high
endothelial venules promotes the adhesion and chemotaxis of
naive T lymphocytes. Proc. Natl Acad. Sci. USA 95:258.
31 FoÈrster, R., Schubel, A., Breitfeld, D., Kremmer, E., Renner-Muller,
I., Wolf, E. and Lipp M. 1999. CCR7 coordinates the primary
immune response by establishing functional microenvironments in
secondary lymphoid organs. Cell 99:23.
32 FoÈrster, R., Emrich, T., Kremmer, E. and Lipp, M. 1994.
Expression of the G-protein-coupled receptor BLR-1 de®nes
mature, recirculating B cells and a subset of T-helper memory
cells. Blood 84:830.
33 FoÈrster, R., Mattis, A. E., Kremmer, E., Wolf, E., Brem, G. and Lipp,
M. 1996. A putative chemokine receptor, BLR-1, directs B cell
migration to de®ned lymphoid organs and speci®c anatomic
compartments of the spleen. Cell 87:1037.
34 Schall, T. J., Bacon, K., Camp, R. D. R., Kaspary, J. W. and
Goeddel, D. W. 1993. Human macrophage in¯ammatory protein a
(MIP-1a) and MIP-1b chemokines attract distinct populations of
lymphocytes. J. Exp. Med. 177:1821.
35 Jinquan, T., Moller, B., Storgaard, M., Mukaida, N., Bonde, J.,
Grunnet, N., Black, F. T., Larsen, C. G., Matsushima, K. and
Thestrup-Pedersen, K. 1997. Chemotaxis and IL-8 receptor
expression in B cells from normal and HIV-infected subjects. J.
Immunol. 158:475.
36 Frade, J. M. R., Mellado, M., del Real, G., Gutierrez-Ramos, J. C.,
Lind, P. and Martinez, C. 1997. Characterization of the CCR2
chemokine receptor: functional CCR receptor expression in
B cells. J. Immunol. 159:5576.
37 Krzysiek, R., Lefevre, E. A., Bernard, J., Foussat, A., Galanaud, P.,
Louache, F. and Richard, Y. 2000. Regulation of CCR6 chemokine
receptor expression and responsiveness to macrophage
in¯ammatory protein-3a/CCL20 in human B cells. Blood 96:2338.
38 Malavasi, F., Caligaris-Cappio, F., Dellabona, P., Richiardi, P. and
Carbonara, A. O., 1984. Characterization of a murine monoclonal
antibody speci®c for human early lympho-hemopoietic cells. Hum.
Immunol. 9:9.
39 Corcione, A., Baldi, L., Zupo, S., Dono, M., Rinaldi, G. B.,
Roncella, S., Taborelli, G., Truini, M., Ferrarini, M. and Pistoia, V.
1994. Spontaneous production of granulocyte-macrophage
colony-stimulating factor in vitro by human B-lineage
lymphocytes is a distinctive marker of germinal center cells. J.
Immunol. 153:2868.
40 Zigmond, S. H. and Hirsch, J. G. 1973. Leukocyte locomotion and
chemotaxis. New methods for evaluation, and demonstration of a
cell-derived chemotactic factor. J. Exp. Med. 137:387.
41 Chomczynsky, P. and Sacchi, N. 1987. Single step method of
RNA isolation by acid guanidium thiocyanate±phenol±chloroform
extraction. Anal. Biochem. 162:156.
42 Van Coillie, E., Van Damme, J. and Opdenakker, G. 1999. The
MCP/eotaxin subfamily of CC chemokines. Cytokine Growth
Factor Rev. 10:61.
43 Lagresle, C., Bella, C. and Defrance, T. 1993. Phenotypic and
functional heterogeneity of the IgD± B cell compartment:
892 B cell subset migration
44
45
46
47
48
49
50
51
52
53
54
55
identi®cation of two major tonsillar B cell subsets. Int. Immunol.
5:1259.
Pascual, V., Liu, Y. J., Magalski, A., de Bouteiller, O., Banchereau,
J. and Capra, J. D. 1994. Analysis of somatic mutation in ®ve B
cell subsets of human tonsil. J. Exp. Med. 180:329.
Szakal, A. K. H., Kosko, M. and Tew, J. G. 1989. Microanatomy of
lymphoid tissues during humoral immune responses: structure
function relationships. Annu. Rev. Immunol. 7:91.
Schall, T. J., Bacon, K., Camp, R. D. R., Kaspari, J. W. and
Goeddel, D. V. 1993. Human macrophage in¯ammatory protein-a
(MIP-1a) and MIP-1b chemokines attract distinct population of
lymphocytes. J. Exp. Med. 77: 1821.
Fang, L., Rabin., R. L., Smith, C. S., Sharma, G., Nutman, T. B. and
Farber, J. M. 1999. CC-chemokine receptor 6 is expressed on
diverse memory subsets of T cells and determines
responsiveness to macrophage in¯ammatory protein 3a. J.
Immunol. 162:186.
Coulin, F., Power, C. A., Alouani, S., Peitsch, M. C., Schroeder, J.
M., Moshizuki, M., Clark-Lewis I. and Wells, T. N. 1997.
Characterisation of macrophage in¯ammatory protein-5/human
CC cytokine-2, a member of the macrophage-in¯ammatoryprotein family of chemokines. Eur. J. Biochem. 248:507.
Imai, T., Chantry, D., Raport, C. J., Wood, C. L., Schweickart, V. L.,
Epp, A., Smith, A., Syine, J. T., Walton, K., Tjoelker, L., Godiska, R.
and Gray, P. W. 1998. Macrophage-derived chemokine is a
functional ligand for the CC chemokine receptor 4. J. Biol. Chem.
273:1764.
Melchers, F., Rolink, A. G. and Schaniel, C. 1999. The role of
chemokines in regulating cell migration during humoral immune
responses. Cell 99:351.
Alkhatib, G., Combadiere, C., Broder, C. C., Feng, Y., Kennedy, P.
E., Murphy, P. M. and Berger, E. A. 1996. CC CKR5: a RANTES,
MIP-1a, MIP-1b receptor as a fusion cofactor for macrophagetropic HIV-1. Science 272:1955.
Samson, M., Labbe, O., Mollereau, C., Vassart, G. and
Parmentier, M. 1996. Molecular cloning and functional
expression of a new human CC-chemokine receptor gene.
Biochemistry 35:3362.
Combadiere, C., Ahuja, S. K., Tiffany, H. L. and Murphy, P. M.
1996. Cloning and functional expression of CC CKR5, a human
monocyte CC chemokine receptor selective for MIP-1(alpha),
MIP-1(beta), and RANTES. J. Leuk. Biol. 60:147.
Gao, J. L., Kuhns, D. B., Tiffany, H. L., McDermott, D., Li, X.,
Francke, U. and Murphy P. M. 1993. Structure and functional
expression of the human macrophage in¯ammatory protein 1
alpha/RANTES receptor. J. Exp. Med. 177:1421.
Neote, K., DiGregorio, D., Mak, J. Y., Horuk, R. and Schall, T. J.
56
57
58
59
60
61
62
63
64
65
66
1993. Molecular cloning, functional expression and signalling
characteristics of a CC chemokine receptor. Cell 72:415.
Wu, L., Paxton, W. A., Kassam, N., Ruf®ng, N., Rottman, J. B.,
Sullivan, N., Choe, H., Sodroski, J., Newman, W., Koup, R. A. and
Mackay, C. R. 1997. CCR5 levels and expression pattern correlate
with infectability by macrophage-tropic HIV-1, in vitro. J. Exp.
Med. 185:1681.
Baba, M., Imai, T., Nishimura, M., Kakizaki, M., Takagi, S.,
Hieshima, K., Nomiyama, H. and Yoshie, O. 1997. Identi®cation of
CCR6, the speci®c receptor for a novel lymphocyte-directed CC
chemokine LARC. J. Biol. Chem. 272:14893.
Corcione, A., Ottonello, L., Tortolina, G., Tasso, P., Ghiotto, F.,
Airoldi, I., Taborelli, G., Malavasi, F., Dallegri, F. and Pistoia, V.
1997. Recombinant tumor necrosis factor enhances the
locomotion of memory and naive B lymphocytes from human
tonsils through the selective engagement of the type II receptor.
Blood 90:4493.
Ottonello, L., Corcione, A., Tortolina, G., Airoldi, I., Albesiano, E.,
Favre, A., D'Agostino, R., Malavasi, F., Pistoia, V. and Dallegri, F.
1999. rC5a directs the in vitro migration of human memory and
naive tonsillar B lymphocytes: implications for B cell traf®cking in
secondary lymphoid tissues. J. Immunol. 162:6510.
Streeter, P. R., Berg, E. L., Rouse, B. M., Bargatze, R. F. and
Butcher, E. C. 1988. A tissue-speci®c endothelial cell molecule
involved in lymphocyte homing. Nature 331:41.
Gallatin, W. M., Weissman, I. L. and Butcher E. C. 1983. A cell
surface molecule involved in organ-speci®c homing of
lymphocytes. Nature 304:30.
Komai-Koma, M. and Willkinson, P. C. 1997. Locomotor properties
of human germinal centre B cells: activation by anti-CD40 and IL-4
allows chemoattraction by anti-immunoglobulin. Immunology
90:23.
Harris, E. D., Jr. 1990. Rheumatoid arthritis: pathophysiology and
implications for therapy. N. Engl. J. Med. 332:1277.
Moutsopoulos, H. M. and Youinou, P. 1991. New developments in
SjoÈgren's syndrome. Curr. Opin. Rheumatol. 3:815.
Krzysiek, R., LefeÂvre, E. A., Zou, W., Foussat, A., Bernard, J.,
Portier, A., Galanaud, P. and Richard, Y. 1999. Antigen receptor
engagement selectively induces macrophage in¯ammatory
protein-1a (MIP-1a) and MIP-1b chemokine production in
human B cells. J. Immunol. 162:4455.
Schaniel, C. E., Pardali, E., Sallusto, F., Speletas, M., Ruedl, C.,
Seidl, T., Anderson, J., Melchers, F., Rolink, A. G. and Sideras, P.
1998. Activated murine B lymphocytes and dendritic cells
produce a novel CC chemokine which acts selectively on
activated T cells. J. Exp. Med. 188:451.