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Department of Tumor Immunology, University Medical Center, Nijmegen, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-1
, and by CD40
triggering. DC maturation involves a complex set of processes: 1)
migration from periphery into secondary lymphoid organs; 2) expression
of high numbers of MHC-molecules; 3) up-regulation of costimulatory
molecules, and 4) the release of cytokines and chemokines. DC are able
to recruit lymphocytes by producing various chemokines, such as Mip1
(CCL3), Mip1 (CCL4), RANTES (CCL5), MDC (CCL22), TARC (CCL17), and
MIP3 (CCL19) (1, 2). Recently, we have cloned a
DC-specific chemokine, DC-CK1 (CCL18 (3), expressed by DC
in the T cell zone and germinal centers (GCs) of tonsils. DC-CK1 (also
described as PARC) (4) preferentially attracts naive
CD45RA+ T lymphocytes, whereas
CD45RO+ T cells, monocytes, or DC are not
attracted by DC-CK1. Because of its DC-specific expression and its
selectivity for naive T cells, DC-CK1 may be very important in the
onset of primary immune responses. For initiating a humoral immune
response, the recruited and subsequently activated T cells have to
interact with Ag-specific B lymphocytes. As suggested by several groups
(5, 6), in humans this initiation would occur in a
multicell complex consisting of DC and T and B lymphocytes. In vitro
studies showed that DC can support B cell proliferation,
differentiation, and Ab production (7, 8, 9, 10). However, how B
cells are recruited by DC into such a cluster in vivo is poorly
understood. Several B cell-attracting chemokines expressed in secondary
lymphoid tissues have now been described, but these are involved in B
cell homing to secondary lymphoid organs (SDF-1 (CXCL12), SLC
(CCL21), MIP3 (CCL19)) (11, 12, 13, 14) or to B cell follicles
(BLC/BCA-1 (CXCL13)) (15, 16, 17, 18) rather than in recruiting B
cells to DC. In this study we define GCDC as the source of DC-CK1 synthesis in the GCs. In addition, we demonstrate that DC-CK1 acts as a chemoattractant for CD38-negative mantle zone (MZ) B lymphocytes, creating the conditions to establish DC-B cell and/or DC-T-B cell interactions beneficial for inducing a primary immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant DC-CK1 was produced in Escherichia coli strain X156F, transformed with the pOMP plasmid containing the DC-CK1 coding region as described previously (3). After lysis, the periplasmic fraction was purified on Q-Sepharose and S-Sepharose columns (Pharmacia LKB, Uppsala, Sweden) using a linear NaCl gradient (0–0.1 M). The DC-CK1-enriched fractions were loaded on a reverse-phase column and eluted using a linear gradient of 2–80% acetonitrile.
Immuno-labeling of cryosections
Six-micrometer-thick freshly prepared cryosections of human
tonsils were fixated for 15 min with 3% paraformaldehyde (in PBS) and
permeabilized for 10 min with 0.1% saponin (Sigma, St. Louis, MO).
Sections were stained with primary Abs for 1 h at 37°C, followed
by 30 min incubation with biotin-conjugated isotype-specific sheep
anti-mouse Abs (The Binding Site, Birmingham, U.K.), 30-min
avidin-biotin-AP or avidin-biotin-HRP complex (Vector Laboratories,
Burlingame, CA), and stained with Fast-red, Fast-blue substrate (for
alkaline phosphatase) (Vector Laboratories) or AEC substrate (Zymed,
San Francisco, CA). Sections were mounted in Kaiser’s glycerin-gelatin
solution (Merck, Darmstadt, Germany). For the detection of DC-CK1, we
used AZN-CK18 (mouse anti-DC-CK1, IgG1). This mAb specifically
reacts with DC-CK1 (CCL18) in direct and sandwich ELISA and does not
cross-react either with MIP1 (CCL3), which has the highest homology
with DC-CK1 (CCL18), or with MIP1 (CCL4), HCC-1 (CCL14), TARC
(CCL17), MDC (CCL22), MIP3 (CCL19), MCP-1 (CCL2), RANTES (CCL5), or
MCP-3 (CCL7) (E. Lindhout, R. Torensma, L. Guelen, N. van Berkum, D.
Elereld, M. Looman, T. Ruers, C. G. Figdor, and G. J. Adema, manuscript
in preparation). Other primary Abs used are: DRC-1 (mouse
anti-follicular DC (FDC), IgM; Dako, Glostrup, Denmark), PG-M1
(anti-CD68, IgG3; Dako), Edu-2 (mouse anti-CD4, IgG2a;
NovoCastra, Newcastle, U.K.), B-A1 (mouse anti-CD4, IgG2a; Diaclone
Research, Besançon, France), RPA-T4 (mouse anti-CD4, IgG1;
PharMingen, San Diego, CA).
B lymphocyte isolation
Purified tonsil B lymphocytes were isolated according to the method described by Falkoff et al. (19). Briefly, tonsillar cell suspensions were depleted of T cells by rosetting with 2-aminoethylisothiouronium bromide-treated (Sigma) SRBC. The rosetted cells were removed by centrifugation on Lymphoprep (1077 mg/ml; Nycomed, Oslo, Norway). The final cell population contained >98% CD20-positive cells (B cells) and <4% CD3-positive cells (T cells).
Low-density (LD) and high-density (HD) B cell fractions were obtained
according to the method of Koopman et al. (20). Briefly, B
cells were centrifuged (15 min, 1200 x g, 4°C) on a
Percoll gradient (Pharmacia LKB), consisting of four density layers
(1077/1067/1056/1043 mg/ml). Cells at the 1043/1056 interface LD B
cells and at the 1067/1077 interface HD B cells were used. LD B cell
fractions mainly consist of GC B cells (
70%
CD38+, 20% sIgD+ and
CD39+), HD B cell fractions contained both MZ and
GC B cells (50% sIgD+ and
CD39+, 40% CD38+).
Naive MZ B cells were obtained by depletion of CD38+ and IgG+ cells from the HD B cell fraction by incubating with anti-CD38 (T16; Beckman Coulter, Fullerton, CA) and anti-IgG (8a4; Beckman Coulter) followed by depletion of the labeled cells using sheep anti-mouse Ig-coated Dynabeads (Dynal, Oslo, Norway). Purified naive B cells fractions contained >95% IgM+, IgD+, and CD39+ B cells.
Purified GC B cells were obtained by incubating the LD B cell fraction with Abs against sIgD (JA11; Beckman Coulter) and anti-CD39 (AC2; Beckman Coulter) followed by depletion of the labeled cells using sheep anti-mouse Ig-coated Dynabeads (Dynal). Purified GC B cell fractions consisted of >98% CD38+ cells and <2% CD39+ and sIgD+ cells.
GCDC isolation
GCDC were purified as described by Grouard et al. (21). Briefly, tonsils obtained from children undergoing routine tonsillectomy were cut into small pieces and digested twice with collagenase IV (2 mg/ml; Sigma) and DNase I (0.04 mg/ml; Boehringer Mannheim, Mannheim, Germany) for 30 min at 37°C. Next the cells were washed, resuspended in PBS plus 2 mM EDTA plus 0.5% HSA (PBSe), and centrifuged over a Percoll (Pharmacia LKB) density gradient consisting of layers with densities of 1070, 1060, and 1030 mg/ml, respectively (15 min, 1200 x g). Cells on top of the 1060-mg/ml layer were harvested and CD3, CD14 CD19, and CD20-positive cells were depleted using Dynabeads (Dynal). Cells were labeled with FITC-conjugated mouse anti-CD1a (IQP, Groningen, The Netherlands), CD3 (Beckman Coulter), CD16 (Becton Dickinson), CD20 (Dako) and CD34 (Beckman Coulter), PE-conjugated anti-CD11c (Becton Dickinson) and Cy5-conjugated anti-CD4 (Beckman Coulter). FITC-negative, CD4-Cy5 and CD11c-PE double-positive cells were isolated using a Coulter Elite FACSort. Purified GCDC were used for cytospin preparation and mRNA isolation (12.000 GCDC/PCR).
Chemotaxis assays
B cell migration was measured using either 48-well chemotaxis chambers (Neuroprobe, Pleasanton, CA) or 5-µm pore size bare filter Transwell inserts (Costar, Cambridge, MA). Briefly, chemokines in RPMI 1640 were added to the lower chamber and were separated from 105 B cells in RPMI 1640/10% FCS by a 5-µm PVP-free polycarbonate membrane (Costar). After incubation for 1 h at 37°C, the membrane was removed and the upper side washed with PBS, scraped to remove residual cells, and washed again. After methanol fixation and staining with Field’s A and Field’s B (BDH Chemicals, Poole, U.K.), the number of migrated cells was counted microscopically in 5 high power fields (x400) per well. Each experiment was performed in triplicate. Inhibition of DC-CK1 induced migration was done by treatment with pertussis toxin (PTX; Sigma). For PTX-treatment, cells were preincubated with PTX (100 ng/ml) for 2 h, 37°C.
The transwell method was done according to Nagira et al. (14). Briefly, B lymphocytes (106 per 100 µl, total cell fraction) in RPMI 1640/10% FCS were added to the upper compartment. The lower compartments contained 600 µl RPMI 1640 with or without chemokines. Cells were allowed to migrate for 4 h at 37°C and migrated cells were harvested, counted, and labeled for FACS.
RT-PCR
Total RNA was extracted using Trizol Reagent (Life Technologies, Breda, The Netherlands). Reverse transcription was performed using random hexamers and Mo-MLV reverse transcriptase (Life Technologies). The PCR was performed in 50 µl Taqman buffer A with cDNA of 25 to 50 ng total RNA, 1.25 U AmpliTaq Gold polymerase (Perkin-Elmer Applied Biosystems U.K., Warrington, U.K.), 5 mM MgCl2, 250 µM dNTPs, 600 nM sense, and 600 nM antisense primer using ABI/PRISM 7700 (Perkin-Elmer Applied Biosystems U.K.). Primers used: DC-CK1: forward 5'CCTCTGCTCCTGTGCACAAGT-3' and reverse 3'TGCAGCTCAACAATAGAAATCAATT-5', amplifying a 424-bp product and GAPDH: forward 5'GAAGGTGAAGGTCGGAGT-3' and reverse 5'GAAGATGGTGATGGGATTTC-3', amplifying a 200-bp product. The DC-CK1 primers were designed as such that they span an intron and do not cross-react with any other chemokine.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Staining of tonsil sections with the anti-DC-CK1 mAb AZN-CK18
demonstrates the same expression pattern as previously observed with in
situ hybridization (ISH); DC-CK1-positive cells were present in both T
and B cell areas of tonsils (Fig. 1
b). The specificity of the
AZN-CK18 mAb was further supported by the absence of reactivity against
chemokines to which DC-CK1 is most homologous in an indirect ELISA
(data not shown). In resting lymph node, expression of DC-CK1 was
observed in the T cell area (Fig. 1c). In spleen, some
DC-CK1-positive cells were found in the PALS (Fig. 1d).
However, DC-CK1-positive cells were most abundant in highly inflamed
tonsils. The number of DC-CK1-positive cells observed by
immunohistology varied significantly between tonsils from different
donors as was previously observed for DC-CK1 RNA by ISH, suggesting a
strictly regulated expression of DC-CK1 in vivo. Even within one
tonsil, a remarkable difference in DC-CK1-positive cells between
adjacent GCs was observed (Fig. 1b).
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, a and
b). The immunohistological finding that FDC do not express
DC-CK1 was further supported by the finding that FDC, like B and T
cells, do not express DC-CK1 mRNA (3) (data not shown). As
expected, the DC-CK1 producing cells in the T cell areas do not express
CD68 (data not shown). Staining of serial sections with AZN-CK18 and
CD4, which is expressed by both GCDC and T cells inside GCs, suggested
that GCDC produce DC-CK1. Because CD4/DC-CK1 double staining appeared
technically impossible, we isolated GCDC from tonsils by extensive
negative selection followed by positive selection for CD4 and the DC
marker CD11c, and analyzed them for DC-CK1 expression at protein and
mRNA level. Staining of cytospin preparations confirmed that isolated
GCDC indeed react with anti-DC-CK1 mAb AZN-CK18 but not with an
isotype-matched control Ab (Fig. 2, c and d).
Moreover, RT-PCR analysis of mRNA isolated from purified GCDC
demonstrated the presence of DC-CK1 mRNA in the sorted GCDC-fraction
(Fig. 2e). Therefore, these data define GCDC as the DC-CK1
producing cells in the GC.
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Previously, DC-CK1 was shown to be a potent chemoattractant for
freshly isolated naive (CD45RA+) T lymphocytes
(3), which is consistent with its expression in T cell
areas of tonsil and lymph nodes. Because DC-CK1 is also expressed in B
cells areas (GCs) of secondary lymphoid organs, we investigated its
chemotactic activity toward B lymphocytes. Therefore, B lymphocytes
were isolated from human tonsils and analyzed for their ability to
migrate in response to different concentrations of DC-CK1 in chemotaxis
assays. The results showed that DC-CK1 chemoattracts B cells in a
dose-dependent manner (Fig. 3a). To investigate whether a
particular subpopulation of B cells is preferentially attracted by
DC-CK1, isolated B cells were (negatively) separated into MZ and GC B
cell subsets and subjected to chemotaxis assays. Strikingly, we
observed that DC-CK1 preferentially attracts MZ B cells
(CD38-, IgG-) at an
optimal concentration of 0.1–1 ng/ml DC-CK1 but not the GC B cells
(CD39-, IgD-) (Fig. 3b). Furthermore, the specific migration of B cells to
DC-CK1 was completely abolished after pretreatment of the B cells with
PTX, indicating the involvement of a
Gi-coupled receptor
(Fig. 3c). Checkerboard analysis demonstrated that the
effect of DC-CK1 on B cells is chemotactic rather than chemokinetic
(data not shown).
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a). On average 1.2% of the
total B cells migrated into the lower compartment under influence of
DC-CK1 (3% of CD38-negative cells) compared with a nonspecific
migration of 0.3% (Fig. 4b). These data demonstrate that
DC-CK1, next to being chemoattractive for naive T cells, is a
chemoattractant for CD38-negative MZ B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
indicating that within one organ the expression of DC-CK1 in GCs is
likely depending on the activation and/or progression stage of the GC
reaction. In addition, in spleen and lymph node, expression of DC-CK1
could easily be detected. However, the number of positive cells was
much lower than in inflamed tonsils. This indicates that next to a
basal constitutive level, DC-CK1 expression is highly increased under
inflammatory conditions. This conclusion is further supported by the
finding that DC-CK1 secretion increases 30-fold upon maturation of in
vitro generated immature DC (32). A recent article by
Reape et al. (22), showing the presence of DC-CK1 mRNA in
atherosclerotic plaques, also supports this finding.
Inside GCs, the DC-CK1-expressing cells were negative for DRC-1 and
CD68 (Fig. 2), implying that in contrast to the suggestion of Hieshima
et al. (4), FDC do not express DC-CK1. Furthermore, also
tingible body macrophages and macrophages in the T cell area do not
express DC-CK1. Because staining of serial sections for DC-CK1 and CD4
suggested that GCDC are the DC-CK1-producing cells in GCs, we purified
GCDC from tonsils. Analysis of isolated GCDC indicated that they indeed
express DC-CK1 at both the RNA and protein level (Fig. 2). Considering
the recently described function of GCDC in (naive) B cell
proliferation, isotype switching, and Ab production (23),
we investigated the ability of DC-CK1 to attract B lymphocytes. Using
two different migration assays, DC-CK1 showed to be a (PTX-dependent)
chemoattractant for B lymphocytes with a preference for naive
(CD38-, IgM+) B cells
(Figs. 3 and 4). The unresponsiveness of GC B cells to DC-CK1 could be
due to down-regulation of the putative DC-CK1R. A more likely
explanation is a markedly impaired signaling via
GI proteins in GC B
cells, due to up-regulation of the regulator of G protein signaling 1
protein (24). Together, the findings that DC-CK1 is
produced by GCDC and attracts MZ B cells further strengthen the idea
that GCDC provide key signals to B cells. This might also relate to the
observed variation in expression levels of DC-CK1 in GCs. High
expression levels of DC-CK1 would be expected during the onset of a GC
reaction, the time point that primary activated MZ B cells have to
interact with GCDC to boost the onset of a GC reaction.
The recruitment of T and B lymphocytes toward DC is crucial for the initiation of an immune response. During an infection, Ag is initially presented by DC in the T cell area, where T and B cells carrying receptors specific for the infecting Ag may encounter their Ag and are activated through a series of cognate interactions (5). Next to the GCDC, also DC in the T cell area express DC-CK1. This implicates that DC in the T cell area are able to attract B cells as well as naive T cells. Therefore, the production of DC-CK1 may be an important first step in establishing the physical interaction between DC, naive T cells, and Ag-specific B cells, leading to the induction of an immune response. Colocalization of B lymphocytes and interdigitating cells in human tonsils has indeed been demonstrated (9). Furthermore, several reports also provide evidence for the importance of DC-B cell interaction in the primary activation of B lymphocytes in vitro. Interaction of DC and B cells results in enhanced B cell proliferation; production of IgM, IgG, and IgA; and promotes the differentiation of naive B cells into plasma cells (7, 8, 25, 26). These data indicate that besides the interaction of B cells with CD4-positive T cells in the MZ area, as was recently reported in the murine system, in the human setting B lymphocytes also interact directly with DC in the T cell areas. The relative contribution of either pathway remains to be elucidated but may well depend on the chemokine microenvironment in the lymphoid organs. B cells express the receptors for BCA-1 (CXCR5) (27) and for C5a (CD88) (28) and are responsive to DC-CK1. Furthermore, fractalkine is expressed by FDC and CD40L (CD154)-stimulated B cells and DC, whereas T cells are able to up-regulate the fractalkine-receptor CX3CR and the BCA-1R (CXCR5) after activation (29, 30). Ultimately, both T and B cells have to localize inside B cell follicles to initiate a GC reaction. Recruitment of preactivated B and T lymphocytes into the follicle can be guided by BCA-1, DC-CK1, the classic chemoattractant C5a (28, 31), fractalkine (CX3CL1), and maybe by other yet unknown chemokines expressed in follicles. In this way, both activated Ag-specific T and B cells are able to migrate toward follicles, where they interact with GCDC and FDC resulting in the development of GC reactions and memory B cell formation (21, 23).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell(s); GC, germinal center; MZ, mantle zone; FDC, follicular DC(s); LD, low density; HD, high density; PTX, pertussis toxin; ISH, in situ hybridization.
Received for publication July 24, 2000. Accepted for publication December 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/LD78 and chemotactic for T lymphocytes, but not for monocytes. J. Immunol. 159:1140.[Abstract]
-11/macrophage inflammatory protein-3/EBI1-ligand chemokine is an efficacious chemoattractant for T and B cells. J. Immunol. 160:2418. signaling responses of B lymphocytes. J. Immunol. 164:1829.This article has been cited by other articles:
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) vs GC (
) B
cells (b) are indicated as the number of migrated cells
per 5 high power fields minus the number of migrated cells in the
medium control vs the amount of chemokine added to the lower well of a
modified Boyden migration chamber. The medium control values indicated
as the mean of duplicates for the total B cells, the CD38-
and CD38+ B cells are 19, 24, and 7 per cells 5 high power
fields, respectively. Five-micrometer pore size filters were used.
c, Migration of MZ B cells to 1 ng/ml DC-CK1 (CCL18) is
inhibited by pretreatment with PTX. Results are shown as mean of
duplicates from a representative experiment of five.