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*
Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033; and
Department of Dermatology, University of Innsbruck, Innsbruck, Austria
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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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In vitro, CCL21 has been shown to be chemotactic for thymocytes, naive and memory T cells, mature dendritic cells, and, to a lesser extent, B cells (3, 5, 6, 12). It binds to the chemokine receptors CCR7 (13) and CCR11 (14). Murine CCL21 also binds to CXCR3 (15). Genetic studies have provided evidence for an important role of CCL21 and its receptors in lymphocyte trafficking. For instance, deletion of the CCR7 receptor by targeted mutagenesis results in impaired lymphocyte migration into the secondary lymphoid organs (16). Abnormalities in T cell and dendritic cell migration and immune response have also been found in the mutant mouse strain, paucity of lymph node T cells (plt), which does not express CCL21 (17, 18). These abnormalities are likely to result from defects in the ability of lymphocytes to adhere to the endothelium, because it has been demonstrated that CCL21 induces integrin-dependent arrest of rolling lymphocytes and their binding to adhesion molecules (19, 20, 21, 22).
Our group and others have identified multiple copies of CCL21 genes in the murine genome. These genes encode two proteins that differ by one amino acid (serine or leucine) at position 65 (23, 24). The CCL21a gene encodes the serine form, whereas both CCL21b and CCL21c genes encode the leucine form. The plt mice lack the expression of the CCL21a gene in lymphoid organs due to deletion of this gene (23, 24). However, CCL21 message (presumably encoded by CCL21b and/or CCL21c) can still be found in nonlymphoid tissues of plt mice, suggesting that the CCL21a gene is expressed in lymphoid organs and that CCL21b and -c genes are expressed in nonlymphoid tissues. However, inferences on the expression pattern of CCL21 genes based on the analysis of the plt mutant may be inaccurate, because the entire locus may be dysregulated. To clearly define the expression patterns of both the CCL21a and CCL21b genes, we have used quantitative PCR. We report here that both genes are expressed in multiple tissues, with CCL21a being the predominant form in the lymphoid organs and CCL21b the predominant form in nonlymphoid tissues. In addition, we show here that both serine and leucine forms of CCL21 are biologically equivalent. When expressed in the pancreas of transgenic mice both CCL21a and CCL21b drive recruitment of lymphocytes into the pancreas and formation of lymph node-like structures. This property seems to be tissue dependent, because transgenic expression of CCL21b in the skin or brain (51) is not associated with lymphocyte recruitment or lymphoid neogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RCCL21a and RCCL21b. BstXI fragments of CCL21 genomic
DNA encoding CCL21a, 3.45 kb, or CCL21b, 3.55 kb, were isolated from
two independent bacterial artificial chromosome clones
(23) and subcloned into the
EcoRV/SalI sites of the RIP-TNF-
-pBS plasmid
containing rat insulin promoter II
(RIP)2
(25). The TNF- fragment in RIP-TNF--pBS was replaced
with the CCL21 genomic DNA. Transgenes were released from the vector by
restriction digest with NotI and ApaI restriction
enzymes.
KCCL21b. A 1-kb DNA fragment containing all four exons and three introns of mouse CCL21b genomic DNA was amplified by PCR from a bacterial artificial chromosome clone (6CKBAC1) (23) using the primers (forward, ccg aat tcg tcg aca cca cca tgg ctc aga tga tga ctc tg) and (reverse, ccg agc tcg cgg ccg ctg agg ctg gat cac ctt ttt). The KCCL21b transgene was generated by cloning this 1-kb fragment into the EcoRI-NotI sites of a vector containing a 2.7-kb promoter/enhancer elements of the human K14 gene (26). The transgene (KCCL21b) was isolated from the plasmid by restriction digest with EcoRI and NotI.
Separation of the transgenes from vector DNA was accomplished by zonal sucrose gradient centrifugation as previously described (27). Fractions containing the transgene were pooled, microcentrifuged through Microcon-100 filters (Amicon, Beverly, MA), and washed five times with microinjection buffer (5 mM Tris-HCl (pH 7.4), 5 mM NaCl, and 0.1 mM EDTA).
Generation of transgenic mice
Transgenes were resuspended in microinjection buffer (5 mM Tris-HCl (pH 7.4), 5 mM NaCl, and 0.1 mM EDTA) to a final concentration of 1–5 ng/µl, microinjected into ((C57BL/6J x DBA/2)F2; The Jackson Laboratory, Bar Harbor, ME) eggs, and transferred into oviducts of ICR (Charles River Laboratories, Wilmington, MA) foster mothers, according to published procedures (28). At 10 days after birth, a piece of tail from the resulting animals was clipped for DNA analysis. Identification of transgenic founders was conducted by PCR analysis, as previously described (29). Identification of the transgenic mice was accomplished by PCR amplification of mouse tail DNA using specific primer sets. Specifically, the primers used for each transgene are listed as follows: KCCL21b: forward, 5'-cccgagcaccttctcttcactcagc-3'; and reverse, 5'-gtccccctccatcactgcctgcaag-3'); and RCCL21a and RCCL21b: forward, 5'-taagtgaccagctacagtcg-3'; and reverse, 5'-agccaggaccaggctaagga-3'. The endogenous ZP3 gene, used as an internal control, was amplified with the following primers: forward, 5'-cagctctacatcacctgcca-3'; and reverse, 5'- cactgggaagagacactcag-3'. The resulting transgenic animals were kept under pathogen-free conditions. PCR conditions were: 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s for both RCCL21 transgenes and 94°C for 30 s, 58°C for 30 s, and 72°C, 60 s for KCCL21b. For all transgenes 30 cycles of PCR were routinely performed. The resulting transgenic animals were kept under specific pathogen-free conditions. All animal experiments were performed following the guidelines of the Schering-Plough animal care and use committee.
Histological analysis
Tissues for light microscopic examination were fixed by
immersion in 10% phosphate-buffered Formalin and then processed for
paraffin sections. Routinely, 5-µm sections were cut and stained with
H&E. For immunohistostaining, fresh-frozen sections were fixed with
ice-cold acetone for 20 min, dried, and stored at -20°C. Slides were
stained and analyzed as described previously (30).
Purified primary Abs were incubated for 1 h at room temperature,
followed by incubation with the appropriate biotinylated secondary Abs
for 30 min. After incubation with an avidin-biotin-HRP complex
(Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) for 30
min, the tissue sections were stained with NovaRed (Vector
Laboratories) and counterstained with hematoxylin. Primary Abs used
were anti-m6CK (R&D Systems, Minneapolis, MN), anti-mucosal
addressin cell adhesion molecule-1 (MAdCAM), anti-peripheral lymph
node addressin (anti-PNAd), anti-TCR
, anti-CD4,
anti-CD11c, anti-pan NK, anti-B220 (BD PharMingen, San
Diego, CA), and anti-F4/80 (Serotec, Raleigh, NC). FITC-linked
anti-CD4, biotinylated anti-B220 (BD PharMingen), and
streptavidin-Cy3 (The Jackson Laboratory) were used in the
immunofluorescent double staining.
Quantitative PCR analysis
Quantitative PCR analysis is a newly developed technique in which the RT-PCR uses the 5' nuclease activity of the Taq DNA polymerase to cleave a fluorescently labeled gene-specific probe during PCR (31). The probe is labeled by a reporter dye on the 5' end and a quencher dye on the 3' end. The proximity of the two dyes allows the quencher dye to absorb the emission of the reporter dye. During polymerization, the Taq polymerase displaces and cleaves hybridized probes, leading to separation of the reporter dye from the quencher dye and allowing its fluorescence to be detected. This process produces and exponentially increases cleaved reporter dye emission so that the accumulation of PCR products can be monitored. Quantification is based on the early linear part of the reaction and by determining the threshold cycle (Ct) at which fluorescence above background is detected. For a specific target DNA, Ct values increase with decreasing target DNA quantity. The copy number or quantity of the RNA in each sample is calculated by comparison with a standard curve of known DNA, assuming it is amplified with the same efficiency.
RNA from various tissues was extracted using the Ultraspec RNA isolation kit from Biotecx (Houston, TX) following specifications from the manufacturer. cDNA was generated by RT using random hexamers (Promega, Madison, WI) and oligo(dT) primers (Life Technologies, Gaithersburg, MD). Quantitative PCR analysis was performed on an Applied Biosystems 7700 sequence detection instrument (TaqMan) following the manufacturer’s instructions. For TaqMan analysis, 25 ng cDNA was used along with primers at a 0.9 µM final concentration and a FAM-labeled diagnostic probe at a final concentration of 0.25 µM. Primers/probes sequences were as follows: CCL21a: forward, 5'-atggctcagatgatgactctgagc-3'; reverse, 5'-gtacttaaggcagcagtcctga-3'; probe, 5'-agcctggtcctggctctctgcatcc-3'; and CCL21b: forward, 5'-aggcagtgatggaggggga-3'; reverse, 5'-gcttagagtgcttccggggta-3'; probe, 5'-ttcttgcttcctatagcctcggacaatactgtagg-3'. Ribosomal RNA primers/probe (PE Applied Biosystems, Foster City, CA) were used as an internal control. Quantitative PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Plasmids containing the CCL21a and CCL21b genes were used as a standard, ranging from 1 ng to 1 fg. Data were analyzed using Sequence Detection Systems software version 1.7 (PE Applied Biosystems).
Flow cytometry and cell preparation from ears
To prepare single-cell suspension, the ears were first separated
into dorsal and ventral halves. The dorsal half was placed fur side up
in 8 ml of 0.25% trypsin and EDTA (Life Technologies) for 30 min at
37°C. The preparation was then diluted 1/4 with 1x PBS. The
epidermis and dermis were transferred with forceps to fresh 1x PBS and
then drawn up and down 
20 times with a 10-ml syringe. Following the
addition of 8 ml of 10% FBS in 1x PBS, the cell suspension was put
through a 100-µm pore size nylon mesh (Falcon; BD Biosciences,
Franklin Lakes, NJ). The cells were pelleted at 1000 rpm for 10 min and
resuspended at 1x 10E7 cells/ml in 1x PBS, 5% FBS, and 0.02% sodium
azide (FACS buffer).
Cells (105–106) were incubated with 5 µg/ml Fc block (BD PharMingen) and 300 µg/ml mouse IgG (Pierce, Rockford, IL). Cells were stained with directly conjugated primary mAbs in FACS buffer for 20 min at 4°C in the dark. mAbs to the following mouse surface markers were purchased from BD PharMingen: B220 (RA3-6B2), CD11c (HL3), MHC II (25-9-17), and TCRb (H57-597). To determine viability, samples were subsequently stained with 20 µl of 5 µg/ml propidium iodide (Calbiochem, La Jolla, CA). Events were acquired on a BD Biosciences FACScan and analyzed using CellQuest software.
Migration of Langerhans cells (LC)
The experiment was performed according to a published method (32). In brief, ear skin from transgenic and control mice was separated from the cartilage, and the ventral sides were floated on 1.5 ml of culture medium (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 0.05 mM 2-ME, and 50 µg/ml gentamicin) in 24-well plates. After 48 h, the explants were transferred onto fresh medium, and cells that had migrated into the medium during the first 48 h were counted in a hemocytometer, in which dendritic cells, dead cells, and other viable cells were morphologically distinguishable. Dendritic cells mature during migration and their shape is therefore typically hairy or veiled. Dendritic cells that migrated between 48 and 96 h after culture were counted and identified in the same way.
To determine the numbers of LC in epidermal and dermal sheets, FITC-labeled anti-I-Adiverse (clone 2G9; BD PharMingen) was used in immunofluorescent staining. In brief, epidermal and dermal sheets were prepared before the onset and at the end of culture. For this purpose ear skin was separated from the cartilage and floated on ammonium thiocyanate (0.5 M in phosphate buffer) for 30 min at 37°C. Afterward, epidermis and dermis were separated, cut into small pieces, and fixed for 20 min in acetone at room temperature. The following rinsing steps were performed: 2x PBS (20 min, room temperature) and 2x PBS/1% BSA (20 min, room temperature). Thereafter, epidermal and dermal sheets were stained overnight at 4°C, washed, and embedded in Vectashield mounting medium (Vector Laboratories).
Analysis was performed with a standard fluorescence microscope. LC were identified by their expression of MHC class II. They were counted with a calibrated ocular grid (20–40 fields/sample; n = 3) at x40 magnification. Statistical analysis was performed using the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the expression pattern of the different
CCL21 genes, we used quantitative PCR analysis. Because
these genes are highly homologous, we first tested the specificity of
the primers on two different plasmid DNAs encoding the
CCL21a and the CCL21b genes, respectively. As
shown by the lower Ct values of CCL21a plasmid compared with those of
CCL21b plasmid in Fig. 1
A,
CCL21a-specific primers preferentially amplified the CCL21a plasmid.
For example, if 1 ng each of CCL21a and CCL21b template plasmids were
amplified together, the CCL21a-specific primers would have at least
10,000-fold higher amplification efficiency for the CCL21a than CCL21b
plasmid. Similarly, the CCL21b-specific primers showed a strong
preference toward the CCL21b plasmid (Fig. 1B). Additional
evidence for the specificity of the CCL21 primers was derived from
analysis of RNAs extracted from plt lymphoid tissues. No
amplification was detected in any plt tissues with
CCL21a-specific primers (not shown), consistent with deletion of this
gene in the plt mutant (23).
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C). On the other hand,
CCL21b was expressed in all tissues examined (Fig. 1D); the highest levels were found in lung and thymus. As
expected, no CCL21a expression was detected in any of the
plt tissues examined, while very low levels of
CCL21b (11–23.5 fg/25 ng cDNA) were detected in lymphoid
tissues of plt mice (data not shown). Generation of transgenic mice expressing CCL21a or CCL21b in the pancreatic islets
To study the biological function of the CCL21a and CCL21b in vivo,
we generated transgenic mice constitutively expressing either gene in
pancreatic islets. To this end we used a promoter, RIP (Fig. 2A) (25). These
transgenic mice are referred to as RCCL21a and RCCL21b, respectively. A
total of 7 and 10 founders was generated for RCCL21a and RCCL21b,
respectively. Five independent lines of mice were derived for each
transgene. All transgenic mice carrying either form of CCL21 developed
normally and were fertile.
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B, high
levels of CCL21 protein were expressed in the endocrine pancreas of
both RCCL21a and RCCL21b transgenic, but not control, mice. Next, we
examined the expression of the transgenes in kidney because this is an
organ occasionally targeted by the RIP promoter (33). We
did not detect transgene expression in the kidneys of any of the RCCL21
lines analyzed (n = 3; data not shown). Both CCL21a and CCL21b proteins promote recruitment of lymphocytes and dendritic cells into pancreatic islets
CCL21 has been shown to induce migration of lymphocytes and
dendritic cells in vitro (3, 5, 6, 12). To determine
whether CCL21 could induce lymphocyte recruitment in vivo, we examined
H&E-stained paraffin sections of transgenic pancreas microscopically.
Mononuclear infiltrates of varying sizes were found in the pancreatic
islets of both RCCL21a and RCCL21b transgenic mice (Fig. 3, A and B). The
large cellular infiltrates observed in the islets of both RCCL21a and
RCCL21b resembled those observed in insulitis. Despite this similarity,
none of the RCCL21a and RCCL21b mice examined (n = 26)
developed diabetes within the first year of life.
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C). CD8
T cells (Fig. 3D), CD11c+ cells (Fig. 3E), and very small numbers of F4/80+
cells (data not shown) were also found among the cellular infiltrates.
No NK+ cells were observed using the DX5 Ab that
reacts with the majority of NK cells (data not shown). Again, similar
immunohistochemistry results were observed for both RCCL21a and RCCL21b
transgenic mice. Lymph node-like structures were found in pancreatic islets of both RCCL21a and RCCL21b transgenic mice
As demonstrated by the previous results the cellular composition
of the pancreatic infiltrates was similar to that seen in secondary
lymphoid organs. To further define the nature of these aggregates we
examined a number of other parameters. We started by examining the
expression of adhesion molecules such as MAdCAM-1 and PNAd, adhesion
molecules that are specifically expressed in secondary lymphoid
structures. The expression of these two molecules was found in the
vessels present in the cellular infiltrates of both RCCL21a and RCCL21b
mice, but not in the islets of wild-type controls (Fig. 3, F
and G). Next we examined whether stromal reticulum could be
detected within the lymphoid aggregates. The presence of stromal
reticulum, as defined by the presence of ER-TR7-positive cells, was
detected in the pancreas of transgenic animals expressing both CCL21a
and CCL21b (Fig. 3H). No ER-TR7-positive cells were found in
the islets of wild-type controls. These results indicate that
expression of CCL21a or CCL21b in the pancreas is sufficient to induce
the formation of lymph node-like structures.
Generation of transgenic mice expressing CCL21b in the skin
To further investigate the role of CCL21 in lymphocyte recruitment
in vivo, we generated transgenic mice expressing CCL21b in
keratinocytes. To this end, we used the human K14 promoter that targets
expression of transgenes to the basal cell layer of the epidermis (Fig. 4A). A total of 15 transgenic
founders were generated by microinjection, as determined by PCR
genotyping. These transgenic mice are referred to as KCCL21b
mice.
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B). However, expression of CCL21 in the epidermal cell
layer and the hair follicles was only found in transgenic, but not
wild-type control, mice. Two independent lines (lines 32 and 72) with
different levels of transgene expression (Fig. 4B) were
propagated for further analysis. No gross phenotypic abnormalities were
observed in the KCCL21b transgenic mice during development or in
adulthood. CCL21b does not promote recruitment of lymphocytes and dendritic cells to the skin
To investigate whether the K14-driven expression of CCL21b led to
leukocyte recruitment, we collected samples from dorsal skin, ear,
esophagus, stomach, vagina, and tongue and examined them by light
microscopy. No significant differences were observed between transgenic
and wild-type control tissues (Fig. 5, top panel). Tissue sections from both wild-type control and
KCCL21b transgenic mice were further analyzed by immunohistochemistry,
using anti-CD3e, anti-CD4, and anti-TCR Abs. As shown in
Fig. 5, the number of TCR-positive cells was comparable to that
found in the control. Similar results were obtained with either
anti-CD4 or anti-CD3e Abs (data not shown). Flow cytometry was
used next to study whether expression of CCL21b was associated with
changes in the numbers of dendritic cells in the skin. Single-cell
suspensions were prepared from ears of five transgenic and five age-
and gender-matched wild-type mice. Flow cytometric analysis did not
reveal changes in the number of TCR+,
B220+, or CD11c+ cells in
the transgenic ears compared with the control ears (Fig. 6). These results indicated that
expression of CCL21 is not sufficient to promote lymphocyte or
dendritic cell recruitment to the skin.
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CCL21 is normally expressed by the lymphatic endothelium in the
skin (Fig. 4B), but it is unclear what physiological role it
has in this tissue. Studies with anti-CCL21 Abs have suggested that
CCL21 may be involved in mediating the migration of skin-derived
dendritic cells into the draining lymph nodes (11). To
determine whether expression of CCL21b by keratinocytes would alter the
migration of dendritic cells from the skin, we tested the migration of
LC in ear organ cultures. Before culture, a small piece of the
epidermis was stained with an anti-MHC class II Ab to assess the
number of LC in the ear explants. In agreement with the results from
the flow cytometric analysis reported above, similar numbers of MHC
II-positive LCs were found in wild-type and KCCL21b ears (1260 ±
130 LC/mm2 in wild-type (Fig. 7A) vs 1240 ± 70
LC/mm2 in KCCL21b (Fig. 7B)). After
96 h of organ culture, the LC that remained in the epidermis were
counted. In wild-type ears, there was a dramatic decrease in LC
densities (90 ± 70 LC/mm2; Fig. 7C), whereas the numbers in KCCL21b ears were much less
reduced (850 ± 380 LC/mm2; Fig. 7D). This difference was highly significant
(p < 0.001; Fig. 7E). Notably the
LC of the KCCL21b mice were in an activated state and ready to leave
the epidermis, as suggested by their strong MHC class II expression and
their rounded shape (Fig. 7D).
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F). These results show that expression of
CCL21b in the keratinocytes inhibited migration of LC from the
skin. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because CCL21 is expressed normally in several tissues and has been shown to chemoattract lymphocytes and dendritic cells in vitro, it has been suggested that it may be an important regulator of their trafficking in vivo. The discovery of at least three murine CCL21 genes (23, 24) encoding two different proteins prompted a series of questions, among them, what are the patterns of expression of these genes and what are their biological functions. Before this study it was known that expression of CCL21 could be detected by Northern blot analysis in multiple murine tissues (3, 23). Furthermore, it was known that CCL21 expression could be detected in nonlymphoid tissues of plt mice, which had a deletion of the CCL21a gene, suggesting the expression of either CCL21b or CCL21c (which are identical in their coding region) in these tissues (23, 24). However, it was unclear whether the analysis of CCL21 expression in plt mice would reflect the physiological regulation of the locus. Formally it could be argued that this pattern of expression could have resulted from an abnormal regulation of the mutated locus. Another unresolved question was whether the CCL21a gene would be expressed in lymphoid as well as nonlymphoid tissues. Using primers specific for the two forms of the CCL21 protein (CCL21a and CCL21b/c), we demonstrate here that CCL21a is expressed in most tissues examined and that its highest levels of expression are found in lymphoid structures. The levels of expression of CCL21a in the nonlymphoid tissues are low. In contrast, the CCL21b gene is expressed in all nonlymphoid organs (especially the lung) and at very low levels in spleen and mesenteric lymph nodes. Taken together, these results indicate that the closely linked CCL21 genes are differentially regulated at the transcriptional level.
Leukocyte trafficking from blood vessels into tissue parenchyma is a multistep process (34, 35). Genetic evidence supporting a critical role for chemokines in this process has accumulated in the recent years (36). For instance, tissue-specific expression of the murine CXC chemokine KC in thymus, skin (26), brain (37), lung (38), or heart (S. A. Lira, unpublished observations) induces neutrophil recruitment into tissues. Similarly, the expression of the murine CC chemokine JE in thymus, brain (39), pancreas (40), lung (41), or heart (42) is associated with macrophage recruitment. Recently, two studies have demonstrated tissue-specific accumulation of lymphocytes upon expression of chemokines in transgenic mice. Luther et al. (43) have demonstrated accumulation of B and T cells in mice expressing BLC (CXCL13) in the pancreas. In addition, Fan et al. (44) reported the generation and analysis of animals expressing CCL21 in pancreas. Similar to what has been reported by Fan et al., we observed that expression of CCL21 in pancreas induces the recruitment and stereotypical arrangement of lymphocytes in the pancreatic islets, with T cells being surrounded by B cells, the presence of low number of dendritic cells, and the expression of adhesion molecules. Here we extend these observations by showing that both forms of CCL21 (Leu or Ser) can induce the formation of lymph node-like structures in the pancreas. Furthermore, we suggest a tissue-specific requirement for the biological activity of these molecules in the pancreas, because the expression of CCL21 in skin or brain (51) does not induce the same phenomenon.
What could account for the tissue-specific ability of CCL21 proteins to
induce the formation of lymph node-like structures? One hypothesis
would be that CCL21 induces a permissive "endothelial" environment
for initial recruitment of lymphocytes and subsequent development of
these structures. CCL21 could directly or indirectly induce the
expression of the requisite adhesion molecules (such as MAdCAM-1 and
PNAd), which could then facilitate initial infiltration of lymphocytes.
At least one of the CCL21 receptors, CXCR3, has been identified in
vascular structures (45, 46) and could in theory mediate
this effect. Alternatively, CCL21 could act on a bystander cell to
induce the production of factors that would affect the expression of
adhesion molecules in the endothelium. Such a bystander cell could be
located within the pancreatic islet or be one of the initially
recruited leukocytes. In transgenic mice expressing BLC in the
pancreas, induction of the adhesion molecules is dependent on the
presence of lymphotoxin
12 (43).
It is unknown at this point whether CCL21 induces lymphotoxin
12, and to what
extent it may contribute to the findings reported here. Regardless of
the site of CCL21 action (endothelium vs bystander cell), one would
have to propose that the activation of these mechanisms would take
place preferentially in the pancreas, because no induction of adhesion
molecules has been found in skin or brain upon overexpression of CCL21
(our unpublished observations). These differences could be due to the
different vascular beds serving these tissues. The pancreatic islets
are highly vascularized, and the functional capacity of this
endothelium may differ from that of other vascular beds
(47). Finally, it is theoretically possible that
lymphocytes may be recruited by an as yet unidentified mechanism driven
by CCL21 presented in the surface of the endothelium. In this case the
differences in the recruiting properties of CCL21 would depend on the
ability of the endothelium of the different tissues to present it to
the circulating lymphocytes.
Mice lacking CCR7 and plt mice (which lack both CCL21 and CCL19 expression in lymphoid organs) have reduced numbers of dendritic cells in lymph nodes. Mature dendritic cells express CCR7 (11, 48, 49), which could mediate their migration toward the lymphatics, a site of abundant expression of CCL21. Thus, it would be expected that ectopic (basal cell layer of the epidermis) expression of CCL21 would dysregulate dendritic cell numbers or migration. In this study, we observed that epidermal CCL21 overexpression does not change the number of dendritic cells in the skin, but it significantly reduced the mobility of dendritic cells from this tissue. The overexpression of CCL21 in the epidermis may have disrupted a chemokine gradient generated by expression of CCL21 in dermal lymphatics. This chemokine gradient may be essential for normal migration of LC and dermal dendritic cells, particularly into the dermal lymphatics. Evidence for this has recently been corroborated in skin explant culture models (11, 16, 50). Alternatively, the ectopic expression of CCL21 in the epithelium may have disrupted the ability of these cells to migrate by desensitizing CCR7 (9, 21). Epidermal CCL21 overexpression does not affect the number of dendritic cells in the skin in the steady state, but may be important during infection or inflammation. Experiments to further address the mechanistic basis of these findings are currently underway.
In conclusion, we have observed that the ability of CCL21 to induce lymphocyte recruitment is context dependent. This property is in clear contrast to that shown by neutrophil and macrophage chemoattractant chemokines such as KC and JE, which are sufficient to promote recruitment of target cells to multiple tissues. These findings highlight further complexity in the chemokine system and suggest that the constitutively expressed homeostatic chemokines (such as CCL19 and CCL21) have specific requirements for their biological activity. Understanding the nature of these requirements will certainly lead to a better understanding of the lymphoid system and contribute to the development of CCL21-based therapies.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: RIP, rat insulin promoter II; Ct, threshold cycle; LC, Langerhans cells; MAdCAM-1, mucosal addressin cell adhesion molecule-1; PNAd, peripheral lymph node addressin.
Received for publication August 13, 2001. Accepted for publication November 7, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine, thymus-derived chemotactic agent 4, with activity on T lymphocytes and mesangial cells. J. Immunol. 159:5671.[Abstract]
47-mediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) under flow. J. Immunol. 161:952. in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells. J. Exp. Med. 184:1963.
32, CCR2–V64I, and SDF1–G801A related to pathogenesis of AIDS. Clin. Chem. 46:24. are potent chemoattractants for in vitro- and in vivo-derived dendritic cells. J. Immunol. 162:3859.This article has been cited by other articles:
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  F. Barone, M. Bombardieri, M. M. Rosado, P. R. Morgan, S. J. Challacombe, S. De Vita, R. Carsetti, J. Spencer, G. Valesini, and C. Pitzalis CXCL13, CCL21, and CXCL12 Expression in Salivary Glands of Patients with Sjogren's Syndrome and MALT Lymphoma: Association with Reactive and Malignant Areas of Lymphoid Organization J. Immunol., April 1, 2008; 180(7): 5130 - 5140. [Abstract] [Full Text] [PDF]
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 A. P. Martin, M. G. Grisotto, C. Canasto-Chibuque, S. L. Kunkel, J. S. Bromberg, G. C. Furtado, and S. A. Lira Islet Expression of M3 Uncovers a Key Role for Chemokines in the Development and Recruitment of Diabetogenic Cells in NOD Mice Diabetes, February 1, 2008; 57(2): 387 - 394. [Abstract] [Full Text] [PDF]
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 H. Unsoeld, K. Mueller, U. Schleicher, C. Bogdan, J. Zwirner, D. Voehringer, and H. Pircher Abrogation of CCL21 chemokine function by transgenic over-expression impairs T cell immunity to local infections Int. Immunol., November 1, 2007; 19(11): 1281 - 1289. [Abstract] [Full Text] [PDF]
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B.-G. Yang, T. Tanaka, M. H. Jang, Z. Bai, H. Hayasaka, and M. Miyasaka Binding of Lymphoid Chemokines to Collagen IV That Accumulates in the Basal Lamina of High Endothelial Venules: Its Implications in Lymphocyte Trafficking J. Immunol., October 1, 2007; 179(7): 4376 - 4382. [Abstract] [Full Text] [PDF]
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 P. J. Holst, C. Orskov, K. Qvortrup, J. P. Christensen, and A. R. Thomsen CCR5 and CXCR3 Are Dispensable for Liver Infiltration, but CCR5 Protects against Virus-Induced T-Cell-Mediated Hepatic Steatosis J. Virol., September 15, 2007; 81(18): 10101 - 10112. [Abstract] [Full Text] [PDF]
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 J. Rangel-Moreno, J. E. Moyron-Quiroz, L. Hartson, K. Kusser, and T. D. Randall Pulmonary expression of CXC chemokine ligand 13, CC chemokine ligand 19, and CC chemokine ligand 21 is essential for local immunity to influenza PNAS, June 19, 2007; 104(25): 10577 - 10582. [Abstract] [Full Text] [PDF]
|
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 J. R. Kocks, A. C. M. Davalos-Misslitz, G. Hintzen, L. Ohl, and R. Forster Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue J. Exp. Med., April 16, 2007; 204(4): 723 - 734. [Abstract] [Full Text] [PDF]
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G. C. Furtado, T. Marinkovic, A. P. Martin, A. Garin, B. Hoch, W. Hubner, B. K. Chen, E. Genden, M. Skobe, and S. A. Lira Lymphotoxin beta receptor signaling is required for inflammatory lymphangiogenesis in the thyroid PNAS, March 20, 2007; 104(12): 5026 - 5031. [Abstract] [Full Text] [PDF]
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 U. E. Hopken, A. M. Wengner, C. Loddenkemper, H. Stein, M. M. Heimesaat, A. Rehm, and M. Lipp CCR7 deficiency causes ectopic lymphoid neogenesis and disturbed mucosal tissue integrity Blood, February 1, 2007; 109(3): 886 - 895. [Abstract] [Full Text] [PDF]
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T. Raine, P. Zaccone, P. Mastroeni, and A. Cooke Salmonella typhimurium Infection in Nonobese Diabetic Mice Generates Immunomodulatory Dendritic Cells Able to Prevent Type 1 Diabetes J. Immunol., August 15, 2006; 177(4): 2224 - 2233. [Abstract] [Full Text] [PDF]
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) and CCL21b (
) plasmids with
either CCL21a-specific (A) or CCL21b-specific
(B) primers was shown at plasmid DNA concentrations from
1 ng to 1 fg. Ct values correspond to the cycle number at which the
amplification plot crosses the defined fluorescence threshold. RNAs
isolated from lymphoid and nonlymphoid tissues of B6D2 analyzed with
CCL21a-specific (C) or CCL21b-specific
(D) primers in PCR using a TaqMan instrument. Th,
thymus; ML, mesenteric lymph node; Sp, spleen; Li, liver; SI, small
intestine; LI, large intestine; St, stomach; He, heart; Lu, lung; Ki,
kidney; Mu, muscle; Te, testis.
). On the
average, 68 ± 28% LC were still found in the epidermis of
KCCL21b transgenic mice after organ culture compared with only 7
± 6% LC in the epidermis of wild-type mice. The reciprocal picture
emerges when the numbers of dendritic cells that emigrated into the
culture medium (between 0 and 48 h and between 48 and 96 h)
were counted (F). Combined data from three independent
experiments with three mice (six ears) per group in each experiment are
shown. Substantially fewer dendritic cells could be retrieved from the
culture medium in KCCL21b mice (