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CHEMOKINES
From the Departamento de Inmunología y
Oncología, Centro Nacional de Biotecnología,
Universidad Autónoma de Madrid, Cantoblanco, and the Departamento
de Biología Celular, Facultad de Biología, Universidad
Complutense, Madrid, Spain.
Chemokines appear to have an important role in the seeding of
lymphoid progenitors in the thymus, the regulation of the coordinated movements of the maturing T cells within this organ, and the egress of
the resulting naive T cells to secondary lymphoid organs. CCR9, the
specific receptor for the T-cell development in the thymus starts
after bone marrow lymphoid multipotent precursors reach the fetal
thymic primordium, where thymocyte maturation is an organized
differentiation process that gives rise to a functional T-cell
compartment.1 During maturation, thymocytes migrate
sequentially through thymic regions, starting in the subcapsular region
where immature pre-T cells are found. Then they proceed to the cortex
and the medulla, where mature CD4+ and CD8+ T
cells reside.2,3 Pertussis toxin-sensitive mechanisms control the trafficking of lymphoid precursors to the
thymus4 as well as the thymocyte movement and distribution
within and outside the thymus,5-7 which is
consistent with the involvement of chemokine-dependent processes.
Chemokines are a family of small cationic proteins that exert a
highly specific control of the migration of leukocytes by
inducing their directional movement along chemokine gradients and by
activating adhesion molecules.8-10 Based on conserved
structural motifs, 4 chemokine subfamilies have been defined (CC, CXC,
C, and CX3C), with most proteins belonging to the CC and CXC
subfamilies. Here we use the recently proposed systematic nomenclature
for chemokines.10 Chemokines interact with their target
cells through specific receptors. The binding specificity and signaling
ability of 18 chemokine receptors have been described so far, all of
them G protein-coupled 7-transmembrane receptors that are inhibited by
pertussis toxin.11
A number of thymus-expressed chemokines are reported to participate in
the control of T-cell development. Trafficking of bone marrow
hematopoietic progenitors may be controlled by CXCL12/SDF-1 (stromal-cell-derived factor-1) (the CXCR4 ligand), a reported chemoattractant for human CD34+ hematopoietic progenitor
cells.12 Depending on their maturation stage, human and
murine thymocytes are chemoattracted by different chemokines; most
immature cells are responsive to CXCL12, whereas more mature T cells
respond to CCL4/MIP-1 Isolation of primary cell populations
For semiquantitative RT-PCR analysis, thymus, spleen, and lymph node
lymphocyte cells were purified into different subsets.
Thymocyte subsets.
Double-positive (DP) thymocytes were sorted as
CD4+CD8+ cells from a cell suspension after
double-staining with FITC-conjugated anti-CD4 and phycoerythrin
(PE)-conjugated anti-CD8. Total CD4+,
CD69+CD4+, and
CD69 Spleen and mesenteric lymph node lymphocytes.
B cells, CD4+ T cells, and CD8+ T cells were
sorted from spleen or mesenteric lymph node cell suspensions after
triple-immunofluorescent staining with FITC-conjugated anti-B220,
PE-conjugated anti-CD8, and tricolor-conjugated anti-CD4. Cells were
sorted on a fluorescence-activated cell sorter (FACS) (FACSort flow
cytometer; Becton Dickinson, Mountain View, CA). The sorted cell
populations were more than 98% pure on re-analysis (data not shown).
Cell lines and reagents
Generation of rabbit antimouse CCR9 polyclonal antibodies A peptide comprising murine CCR9 amino acids 3-22 was synthesized and coupled to KLH. Outbred New Zealand rabbits were injected intradermically in multiple sites with 300 µg peptide-KLH conjugate in complete Freund adjuvant. At weeks 4 and 7, intramuscular boosts were given using 150 µg of the same material in incomplete Freund adjuvant. Rabbit serum was collected 7-10 days after the last boost, antibody titers were determined by enzyme-linked immunosorbent assay (ELISA), and serum K629 was chosen for this study. Anti-CCR9 polyclonal antibodies (pAbs) were purified on a CCR9 peptide (3-22) affinity column, then biotinylated using EZ-link-Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL).Flow cytometry studies The following mAbs were used in this study (brand names in parentheses): antimouse B220-FITC (clone RA3.6B2), CD4-TC and CD4-PE (L3T4), CD8-FITC and CD8-TC (Ly2), CD69-FITC and CD69-PE (H1.2F3), and CD62L-PE (Mel-14) (Pharmingen); goat F(ab')2 antirabbit immunoglobulin G (IgG) (H+L)-RPE, antimouse B220-TC (RA3.6B2), and streptavidin-RPE (Southern Biotechnology, Birmingham, AL); CD4-Red613 (H129.19, Gibco); and antirabbit Ig-FITC (Amersham International, Little Chalfont, England). For staining, 5 × 105-106 cells were washed and resuspended in PBSst (PBS, 2% bovine serum albumin [BSA], and 0.05% sodium azide) with 20 µg/mL mouse IgG and then incubated for 20 minutes at 4°C. Primary antibodies were added, with blocking mouse IgG, followed by incubation at 4°C for 40 minutes. Following 2 washes in PBSst, subsequent antibodies were added, always with mouse IgG, followed by incubation for 30 minutes at 4°C. Four-color stainings were performed with the following combinations of mAbs and pAbs: affinity-purified rabbit antimouse CCR9 pAb, followed by antirabbit Ig-FITC, antimouse CD4-Red-613, CD8-TC, and CD69-PE or CD62L-PE. Three-color stainings were performed with purified rabbit antimouse CCR9 pAb, which was followed by antirabbit Ig-FITC, antimouse CD4-Red-613, and CD8-TC. Alternatively, 3-color experiments were done with biotinylated rabbit antimouse CCR9 followed by antimouse CD4-TC (or B220-TC), CD8-FITC, and streptavidin-RPE. Control stainings with pre-immune rabbit serum, an irrelevant affinity-purified rabbit serum raised against a mouse protein, or PBSst plus second antibody were performed routinely. All controls used gave a similar staining signal. Samples were analyzed in an EPICS XL flow cytometer (Coulter Electronics, Miami, FL), and events corresponding to approximately 2.5 × 105 cells were collected for each sample.Chemotaxis assays Cell migration was assayed in Transwell inserts (Costar, Cambridge, MA) with a 5-µm-pore diameter. Cells were resuspended in RPMI with 1% BSA and 25 mM HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) (107 cells per mL), and 100-µL aliquots were loaded into the upper inserts. Chemokine aliquots of 600 µL, prepared in the same medium, were placed in the lower wells. Two or more replicate wells were used for each point. After a 2-hour incubation, inserts were removed, and migrated cells were counted in an EPICS XL flow cytometer. To determine the migration of the lymphocyte subpopulations, the number of cells in each subset of the migrated cell population was determined by appropriate staining assays.Semiquantitative RT-PCR studies Total RNA from mouse tissues or FACS-sorted cell subsets was extracted using Tri-reagent (Sigma Chemical, St Louis, MO). A total of 5 µg RNA from tissue or the total amount obtained from 5 × 104-106 cells was reverse-transcribed with random hexamers and the Superscript enzyme (Gibco). Semiquantitative real-time fluorescence PCR was performed in a LightCycler instrument (Roche, Mannheim, Germany) using SYBR green as fluorogenic dye. Briefly, 3 serial dilutions of each complementary DNA (cDNA) were subjected to 35-45 PCR cycles for 1 second at 93°C, 25 seconds at 68°C, and 2 seconds at 80°C in a 15-µL mixture containing 1 × LightCycler-DNA Master SYBR Green I (Roche), 4.5 mM MgCl2, and 0.4 µM each of forward and reverse gene-specific primers. To reduce nonspecific amplification, the Taq-containing component (LightCycler-DNA Master SYBR Green I) was pre-incubated with an anti-Taq antibody (Clontech Laboratories, Palo Alto, CA) as recommended by the manufacturer. SYBR green fluorogenic emission was acquired at 80°C to further minimize the formation of low-temperature-melting nonspecific DNA products. Oligonucleotides used for the cDNAs were: mouse CCR9: 5'-CACCATGATGCCCACAGAAC-3' and 5'-GATGAGAAGCACACAGCTGTAG-3'; -actin: 5'-AGGCTCTTTTCCAGCCTTCCT-3' and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGGGCC-3'.
Northern blot analysis Total RNA from mouse thymus was extracted with Tri-reagent (Sigma) and fractionated by electrophoresis on a denaturing formaldehyde-agarose gel. RNA samples were transferred to a Hybond-N (Amersham) membrane and ultraviolet cross-linked. The membrane was prehybridized and hybridized in Rapid Hyb buffer (Amersham) as recommended by the supplier.
The pAb K629 binds selectively to CCR9-expressing transfectant cells Rabbit pAb K629 raised against a peptide corresponding to CCR9 amino acids 3-22 was used to study CCR9 expression in different lymphoid subpopulations. To confirm specificity, pAb K629 was used in staining assays with Ba/F3 transfectants expressing different human and mouse chemokine receptors. Only Ba/F3 cells expressing mouse CCR9 bound K629 (Figure 1); there was no cross-reactivity with other Ba/F3-expressed chemokine receptors tested including hCCR9, which is 86% identical to CCR9.17 The immunizing peptide competed for CCR9 recognition by K629 (data not shown).
CCR9 is expressed mainly in CD4+CD8+ DP and
CD4  CD8 DN cells expressed the receptor,
almost the entire CD4+CD8+ DP thymocyte
subpopulation expressed CCR9. Among SP thymocyte subpopulations, both
CD4+CD8 and CD4CD8+
SP subsets showed 2 cell subpopulations that differed in their CCR9
expression levels. Differences were clearly observed between CD4+ and CD8+ cell subsets, however, when gated
cell subpopulations within the CD4 or CD8 lineages were analyzed for
CCR9 expression. The results showed that CCR9 expression was
progressively lost in maturing CD4+ cells, whereas more
mature CD8+ thymocytes showed higher CCR9 levels (Figure
2A).
The flow cytometry results were confirmed by a semiquantitative RT-PCR
procedure using cDNA obtained from FACS-sorted thymocyte subsets and
CCR9-specific primers (Table 1).
Migration assays using CCL25 as a chemoattractant were performed to
study whether the CCR9 protein detected in thymocytes was functional.
In agreement with the CCR9 expression levels detected in each
subpopulation, CD4+CD8+ DP thymocytes were the
subset showing the greatest CCL25-mediated chemoattraction (Figure 2B).
This migration was inhibited when the chemokine was present in
both upper and lower compartments of the migration device, confirming
that this response was mostly chemotactic (data not shown).
Anti-CD3 /) animals do not express
pre-TCR, and therefore their thymocyte maturation is blocked at the
CD44CD25+CD4CD8
DN stage. CD3 stimulation mimics pre-TCR signaling and is known to
promote the DN to DP transition in RAG-2-deficient
thymocytes. A recent report18 on the analysis of CCR9 mRNA
in RAG-2-deficient mice showed that it was up-regulated in
thymocytes when the animals were treated with a single i.p. injection
of the anti-CD3 mAb 2C11. We wanted to study whether the levels of
functional CCR9 protein were also increased following that mRNA
up-regulation. For this, the anti-CD3 mAb 2C11 was injected to
RAG-2-deficient mice, and total thymocyte samples were
prepared at different times after the treatment and stained with the
pAb K629. The results obtained were consistent with the results
reported by Norment et al18 for CCR9 mRNA; 24 hours after
the anti-CD3 treatment of RAG-2-deficient mice, an
increase in the CCR9 protein expression was already detected in the
thymocytes (Figure 3). Analysis 48 and 96 hours after the treatment showed that the amount of CCR9 protein
present in thymocyte membranes increased along that time interval.
Chemotaxis assays showed that the CCL25-induced migration of thymocytes
from RAG-2/-treated animals was also
progressively stronger, as expected from their increasing CCR9 protein
levels (data not shown). Control stainings showed that the anti-CD3
treatment caused RAG-2/ thymocytes to
progress from a CD44CD25+ to a
CD44CD25 phenotype, as expected (data
not shown).
CCR9 is expressed mainly in more mature CD8+, but not CD4+, SP thymocytes Four-color staining experiments were performed with SP thymocytes to analyze CCR9 expression in cells in distinct maturation stages, as indicated by CD4, CD8, CD69, and CD62L expression. Among CD4+ and CD8+ thymocytes, CD69,
CD69low, and CD69high subpopulations could be
distinguished. CCR9 expression was associated mainly with
CD69+ cells (Figure 4A). In
agreement with these data, CCL25-induced migration was higher for the
total CD69+ thymocyte subpopulation than for the
CD69 thymocyte subset (Figure 4B). Some differences were
detected between the CD4+ and the CD8+ SP cell
subsets; indeed, CCR9 expression in CD4+ SP cells was shown
mainly by CD4+CD69high thymocytes, whereas in
CD8+ SP cells, CCR9 expression could be detected in both
the CD8+CD69high and the
CD8+CD69low cell subpopulations. We confirmed
these results by semiquantitative PCR performed with FACS-sorted
CD69+ and CD69 subsets of both
CD4+ and CD8+ SP thymocyte subpopulations
(Table 1). SP thymocytes modulate their CD62L levels from
CD62Llow to CD62Lhigh before leaving the thymus
as naive T cells. We studied CCR9 expression on thymocytes during that
transition, and as Figure 4C shows, differences between the
CD4+ and CD8+ thymocyte subsets were again
detected. CCR9 expression was associated mainly to
CD4+CD62Llow cells, whereas for
CD8+ thymocytes, both CCR9+CD62Llow
and CCR9+CD62Lhigh cells were present in the
preparations.
CD8+ lymphocytes are the major CCR9-expressing cells in secondary lymphoid organs A semiquantitative RT-PCR procedure was employed to confirm CCR9 mRNA expression in different lymphoid tissues. The results obtained concurred with those obtained in Northern blot analysis17 and underscored that this-chemokine receptor is expressed
preferentially in thymus (Table 2). Low
CCR9 expression was also detected in lymph nodes, Peyer patches, and
spleen. A more detailed analysis of CCR9 protein expression on cell
surface in lymphocyte subpopulations from these secondary lymphoid
organs was carried out with pAb K629. The staining assays demonstrated
that all CD8+ lymphocytes showed moderate CCR9 expression
(Figure 5A). Conversely, CCR9+ lymphocytes less than 1% of total CD4+
cells in all secondary lymphoid organs studied, which is in agreement with the semiquantitative RT-PCR results (Figure 5A, Table 1). In
addition, approximately 1.5% to 3% of B220+ cells was
consistently found to be CCR9+ lymphocytes. Migration
assays performed with lymph node and spleen cells showed that
CD8+ cells were chemoattracted by 300 nM CCL25 (Figure
5B); in contrast, we were unable to detect clear migration by
B220+ cells. Consistent with their lack of CCR9 expression,
CD4+ T cells were unable to migrate.
CCR9 protein expression in thymus decreases in mature and old mice As the thymus involutes with age, we studied whether CCR9 was expressed in this lymphoid organ throughout murine life. Flow cytometry studies were thus performed with pAb K629 to analyze possible differences in the amount of CCR9 protein in thymocytes. Low CCR9 levels are expressed in thymocytes from day-15 fetuses, increase in thymocytes from day-17 fetuses, and reach maximum expression in newborn mice (Figure 6A). CCR9 expression decreased thereafter; this is illustrated by thymocytes from 1-month-old and 1-year-old animals, which showed lower, nearly similar CCR9 levels (Figure 6A). Semiquantitative RT-PCR analysis of CCR9 mRNA was performed in thymocytes from mice of different ages. Concurring with the results obtained with the pAb K629, CCR9 mRNA was detected in mouse thymocytes from day-15.5 fetuses, reached a maximum around birth, and then decreased with increasing animal age (Table 3). Indeed, 45-day-old animals showed 2- to 3-fold lower CCR9 mRNA levels than did newborn mice. We also studied the response of these thymocytes to CCL25 in migration assays. Thymocytes from newborn animals showed the greatest chemotactic response (Figure 6B). Interestingly, in spite of showing similar CCR9 levels, CCL25-mediated chemoattraction was clearly stronger in 1-month-old animals than in 1-year-old mice. To study CCL25 expression throughout murine life, Northern blots were prepared with total thymus RNA from mice of various ages (day-16.5 fetuses to 8-month-old adult animals) and were analyzed with a CCL25-specific probe. The results showed that CCL25 mRNA levels were similar in all animals analyzed, regardless of age (data not shown).
The physical architecture of the thymus provides an adequate
environment for T-cell development by means of a complex and organized
process involving sequential chemokine-controlled movement of maturing
thymocytes through this lymphoid
organ.1-3,5- 7,13-15 We recently described
human and murine CCR9 as the specific receptor for the In thymocytes, CCR9 is expressed at all stages of T-cell maturation,
with maximum expression in CD4+CD8+ DP cells
(Figure 2). Because most DN thymocytes do not express CCR9, it is
tempting to speculate that the CCL25/CCR9 axis is not essential for the
arrival of bone marrow lymphoid progenitors to the thymus. In
consonance with this, thymus recolonization by T-cell precursors is
reported to occur in the presence of a neutralizing antibody to
CCL25.4 Altogether, these data suggest that other
chemo-attractants are also involved in this process. Thymocytes from
RAG-2-deficient mice treated with anti-CD3 Positive selection in the thymus is a multistage process that involves transition through a CD4+CD8+CD69+ intermediate phase and subsequent maturation phases that lead to the SP thymocyte stage.19 SP thymocytes then decrease their CD69 levels and increase expression of the homing receptor CD62L (L-selectin); these more mature cells are the major source of thymic emigrants.20 CCR9 expression in SP thymocytes was mainly associated to cells undergoing positive selection, as defined by their levels of the CD69 activation marker. This was also functionally corroborated, as the CD69+ thymocyte subset showed the highest CCL25-induced chemoattraction (Figure 4). A recent report showed that initiation and subsequent events in the positive selection of thymocytes are critically dependent on their sustained interaction with thymic epithelium, although interaction with MHC molecules is necessary only during the initiation stage.21 CCR9 is expressed by thymocytes undergoing positive selection, and its ligand, CCL25/TECK, is expressed by thymic epithelial cells.22 It is therefore tempting to suggest that in thymus, the CCL25/CCR9 axis delivers signals that play a role in the positive selection process of thymocytes. Concurring with the results obtained with pAb K629, semiquantitative
RT-PCR experiments showed that concomitantly with the CD69+
to CD69 We found that in secondary lymphoid organs, CCR9 is expressed mainly by CD8+ lymphocytes. These CD8+ T cells migrate in response to CCL25 (Figure 5). It therefore appears that CD8+ thymocytes still express CCR9 when they abandon the thymus, and CCR9 is also functionally expressed in CD8+ T cells from secondary lymphoid organs; this suggests a possible role for CCR9 in that migration process. As substantial CCL25 levels have not been found in organs other than thymus and small intestine,16,22 this putative role for CCR9 may be mediated by a distinct ligand produced in these lymphoid organs. CCR7 is a chemokine receptor with a very important role in T-cell migration to lymphoid organs. Studies in CCR7 null mice nevertheless showed that although greatly impaired, some T-cell migration to lymphoid organs still existed in these animals,23 which suggests that other chemokine receptors may contribute to this process. From our data it can be speculated that CCR9 could be one of these chemokine receptors. The situation is markedly different for CD4+ cells, as the more mature CD4+ SP thymocytes express very low levels of CCR9, which is then practically undetectable in lymph node, Peyer patches, and spleen CD4+ T cells. In addition to CD8+ lymphocytes, a small subset of B220+ cells was consistently found to express CCR9. Nevertheless, these B lymphocytes were not clearly chemo-attracted by 300 nM CCL25 in migration assays (Figure 5). Similar results have been reported for a CCR9-expressing B-cell subpopulation from human peripheral blood lymphocytes.24 It is presently not possible to suggest a clear role for CCR9 in peripheral B cells. Consistent with the CCL25 expression reported in small intestine, we found CCR9 expression in a small lymphocyte subpopulation from Peyer patches (Figure 5, Table 2) and in intra-epithelial lymphocytes (IELs) (data not shown), although the CCR9 expression detected in mice is clearly lower than that reported for human IELs.24 Analysis of bone marrow showed that CCR9 was expressed by B220low lymphocytes from this primary lymphoid organ (data not shown). Interestingly, a recent report on the chemotactic responses of B cells as they progress through maturation in bone marrow showed that the cells respond to CCL25/TECK during an early B-lineage stage.25 Our finding of CCR9 expression in bone marrow B cells is thus in consonance with those results and suggests that as for T cells in the thymus, CCR9 plays a role in B-lymphocyte maturation in the bone marrow. Advancing age is accompanied by a series of alterations in the immune
system including thymic involution.19 Examination of CCR9
protein and mRNA levels showed that they were higher in newborns than
in older animals or fetuses. This concurs with thymocyte population
dynamics as described in the mouse, during which nearly all mouse
thymocytes are CD4 In summary, we report here a detailed characterization of mouse CCR9
expression. Our data suggest that this
We would like to thank Dr J. Gutiérrez for critical reading and comments on the manuscript and L. Gómez and M. Lozano for excellent technical assistance. We also thank M. C. Moreno-Ortíz and I. López-Vidriero for help with flow cytometry analysis, Dr J. P. Albar and F. Roncal for providing the peptide used in immunization, and C. Mark for editorial assistance. The Departamento de Inmunología y Oncología was founded and is supported by the Spanish Research Council (CSIC) and by Pharmacia Corporation.
Supported in part by grant 08.1/0018/1998 (C.A.) from the Comunidad Autónoma de Madrid, Spain.
Submitted June 21, 2000; accepted October 18, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Gabriel Márquez, Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain; e-mail: gmarquez{at}cnb.uam.es.
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S. Singh, U. P. Singh, J. K. Stiles, W. E. Grizzle, and J. W. Lillard Jr. Expression and Functional Role of CCR9 in Prostate Cancer Cell Migration and Invasion Clin. Cancer Res., December 15, 2004; 10(24): 8743 - 8750. [Abstract] [Full Text] [PDF]
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A. Misslitz, O. Pabst, G. Hintzen, L. Ohl, E. Kremmer, H. T. Petrie, and R. Forster Thymic T Cell Development and Progenitor Localization Depend on CCR7 J. Exp. Med., August 16, 2004; 200(4): 481 - 491. [Abstract] [Full Text] [PDF]
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S. Uehara, A. Grinberg, J. M. Farber, and P. E. Love A Role for CCR9 in T Lymphocyte Development and Migration J. Immunol., March 15, 2002; 168(6): 2811 - 2819. [Abstract] [Full Text] [PDF]
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M.-A. Wurbel, M. Malissen, D. Guy-Grand, E. Meffre, M. C. Nussenzweig, M. Richelme, A. Carrier, and B. Malissen Mice lacking the CCR9 CC-chemokine receptor show a mild impairment of early T- and B-cell development and a reduction in T-cell receptor gamma delta + gut intraepithelial lymphocytes Blood, November 1, 2001; 98(9): 2626 - 2632. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-chemokine receptor is modulated in
thymocyte differentiation and is selectively maintained in
CD8+ T cells from secondary lymphoid organs
Top
Abstract
Introduction
),
human CXCR4 (--), human CCR9 (--), and the void vector pCIneo
(.....).
). The chemotactic component of this
migration was estimated by placing CCL25 in the upper and lower
chambers of the Transwell (
). Results are representative of 3 independent experiments performed.