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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4434-4443
Differential Chemotactic Behavior of Developing T Cells in Response
to Thymic Chemokines
By
Chang H. Kim,
Louis M. Pelus,
John R. White, and
Hal E. Broxmeyer
From the Departments of Microbiology/Immunology and Medicine and the
Walther Oncology Center, Indiana University School of Medicine,
Indianapolis, IN; the Walther Cancer Institute, Indianapolis, IN; and
the Departments of Molecular Virology and Host Defense and of Molecular
Immunology, SmithKline Beecham Pharmaceuticals, Collegeville, PA, and
King of Prussia, PA.
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ABSTRACT |
Differentiation-dependent thymocyte migration in the thymus may be
important for T lymphopoiesis and might be regulated by thymic
chemoattractants. We examined modulation of chemotactic responsiveness
of thymocyte subsets during their early to late stages of development
in response to 2 thymus-expressed chemokines, SDF-1 and
CK -11/MIP-3/ELC. SDF-1 shows chemotactic preference for immature
thymocytes (subsets of triple negative thymocytes and double positive
[DP] subset) over mature single positive (SP) thymocytes.
CK-11/MIP-3/ELC shows low chemotactic activity on the immature
thymocytes, but it strongly attracts mature SP thymocytes, effects
opposite to that of SDF-1. SDF-1-dependent chemoattraction of immature
thymocytes is not significantly desensitized by a negative
concentration gradient of CK-11/MIP-3/ELC, and chemoattraction of
mature SP thymocytes to CK-11/MIP-3/ELC is not antagonized by
SDF-1, demonstrating that these two chemokines have different chemoattractant preferences for thymocyte subsets and would probably not inhibit each other's chemotaxis in the event of microenvironmental coexpression. The chemotactic responsiveness of thymocytes and mature T
cells to the 2 chemokines is respectively enhanced after selection
process and migration to the spleen. These studies demonstrate the
presence of thymocyte chemoattractants with differential chemotactic preference for thymocytes, a possible mechanism for thymocyte migration
in the thymus.
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INTRODUCTION |
CHEMOKINES ARE members of a family of
small proteins with molecular weight around 10 kD.1-5 There
are at least 4 chemokine subfamilies (CXC, CC, C, and
CX3C), depending on the number of cysteines and spacing
between the first 2 cysteines. Chemokines bind G-protein-coupled
receptors with 7-transmembrane domains.6-8 Although the
apparent functions of chemokines are to chemoattract diverse cell
types, it has been reported that these cytokines have many functions,
such as inhibition of human immunodeficiency virus (HIV) infection,
regulation of angiogenesis, regulation of immune reaction and
hematopoiesis, and antitumor effects.3 SDF-1 is a CXC
chemokine that is expressed ubiquitously in most tissues, including
thymus.9 SDF-1 has efficacious chemotactic activity for
hematopoietic progenitors,10,11 lymphocytes, and monocytes.12 Mice deficient in SDF-1 expression cannot
survive and die around birth.13 SDF-1 specifically binds
the CXCR4/fusin/LESTR/HUMSTR receptor and inhibits T-trophic HIV
infection.14,15 CK-11/MIP-3/ELC, a CC chemokine, is
highly expressed in thymus16,17 and has chemotactic
activity for T and B cells18 and a subset of hematopoietic progenitor cells (Kim and Broxmeyer, unpublished results).
It specifically binds to the CCR7/EBI1/BLR2 receptor.17
Both SDF-1 and CK-11/MIP-3/ELC are divergent from other CXC and
CC chemokines, showing low amino acid identities to other chemokines.
The chromosomal location for the SDF-1 and CK-11/MIP-3/ELC genes
is 10 and 9, respectively, in humans,17 unlike most CXC and
CC chemokines that have their genes, respectively, in human chromosomes
4 and 17.1,2
Stem cells or common lymphoid progenitor cells migrate from bone marrow
to the fetal thymic primordium to seed thymic T-cell hematopoiesis.19 Immature thymocytes undergo a multistep
differentiation pathway, forming an immune system able to distinguish
self from nonself and respond to most pathogens.20 T-cell
hematopoiesis in the thymus is highly organized. Immature double
negative (DN) thymocytes are found in the outer cortex of thymus,
whereas more mature double positive (DP) thymocytes reside in the
remainder of the cortex. Mature single positive (SP) thymocytes are
found in the medulla, where they migrate to the peripheral blood and become mature T cells.21,22 Specialized subcompartments of thymus have been identified based on epithelial cell distribution and
accessory cell composition.21 It is likely that migration of thymocytes at different stages of differentiation is a regulated process.
To study the possibility that different chemokines attract thymocytes
differentially and thus coordinate thymocyte migration within the
thymus, we examined the specific chemotactic activity of 2 thymus-expressed chemokines. Our results indicate that the chemokines
in thymus have different chemoattractant preferences for thymocyte
subsets and may selectively control the migration of these cells in the
thymus during T lymphopoiesis.
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MATERIALS AND METHODS |
Chemokines and antibodies.
SDF-1 was a kind gift from Dr Ian Clark-Lewis (University of British
Columbia, Vancouver, British Columbia, Canada). Human CK-11/MIP-3/ELC was expressed in Chinese hamster ovary (CHO) cells. Expression, purification, N-terminal analysis, and
matrix-assisted laser desorption ionized (MALDI) mass spectrometry for
recombinant human CK-11/MIP-3/ELC were performed as previously
described.18 This protein was previously demonstrated to be
chemotactically active for mouse cells.18 For purification
of lin thymocytes, the following antibodies were
used: anti-CD3e-biotin (clone 144-2C11), anti-CD4-biotin (clone
L3T4), anti-CD8a-biotin (clone Ly-2), anti-Gr-1-biotin (clone
RA3-6B2), anti-NK1.1-biotin (clone PK136), anti-Mac-1-biotin (clone
M1/70), and anti-B220-biotin (clone RA3-6B2) (obtained from
Pharmingen, San Diego, CA). For analysis of thymocyte migration, the
following antibodies were used: anti-CD3-phycoerythrin
(PE)1 (clone 144-2C11), anti-CD4-TriColor (clone L3T4),
anti-CD8a-fluorescein isothiocyanate (FITC) (clone Ly-2),
anti-CD25-PE (clone PC61), and anti-CD69-PE (H1.2F3) (Pharmingen). Anti-CD44-FITC (IM7.8.1) and Streptavidin-PE were purchased from Caltag Laboratories (South San Francisco, CA).
Animals and cell separation.
Four-week-old female BALB/C mice were purchased from Jackson Laboratory
(Bar Harbor, ME) and maintained in the Indiana University Medical
Center Laboratory Animal Resource Center in accordance with guidelines
of the Committee on Animals of the Indiana University School of
Medicine. Thymuses of 4- to 5-week-old female BALB/C mice were used to
prepare single cell suspensions of thymocytes. Thymuses were crushed on
an iron mesh. After debris, red blood cells (RBCs) and
polymorphonuclear cells (PMNs) were removed by centrifugation on lympholyte-M (Cedarlane, Hornby, Canada), thymocytes were depleted of lin+ non-T cells by a magnetic bead
depletion method using a cocktail of biotin-labeled antibodies to Gr-1,
B220, Mac-1, and NK1.1 according to the manufacturer's recommendation
(Pharmingen). Streptavidin-beads (Miltenyi Biotech, Auburn, CA) were
used to deplete lin+ non-T cells. This depletion method
routinely yielded non-T-cell-free thymocytes with purity greater than
97%. Triple negative (TN; CD3CD4CD8)
thymocytes were purified as described by others,23 except that Miltenyi Biotech streptavidin-beads were used to deplete antibody-coated cells in this study. Briefly, a cocktail of
biotinylated antibodies (anti-CD3, anti-CD4, anti-CD8, anti-B220,
anti-Gr-1, anti-Mac-1, and anti-NK1.1) was used to bind non-T cells
and T-lineage cells. Streptavidin-bead (Miltenyi biotec) was used to
deplete cells coated with the biotinylated antibodies. The purity of TN cells was routinely greater than 98%.
In vitro and organ chemotaxis assay.
Chemotaxis assays were performed using the Costar Transwell system
(6.5-mm diameter, 5-µm pore size, polycarbonate membrane) as
previously described.18 Briefly, purified thymocytes (5 × 105) were added to the upper chamber of each well,
and chemokines were added to the upper and/or lower chamber at
various concentrations. Cell migration was allowed to occur for 3 hours
and cells migrating to the lower chamber were harvested and either
directly counted by FACscan (Becton Dickinson, Mountain View, CA) for
30 seconds or counted after staining with fluorescent antibodies to CD4
and CD8, CD25 and CD44, or CD4, CD8, and CD69. Migrated cells were calculated as a percentage of the specifically phenotyped input cells.
For organ chemotaxis, thymuses were carefully removed from 4- to
5-week-old female BALB/C mice, and 1 thymus was put into the upper
chamber of the Transwell as a monolayer to cover the entire surface of
the 6.5-mm membrane. Diluted chemokines (final volume, 600 µL) were
added to the lower chamber, and chemotaxis medium (50 µL) without
chemokines was added to the upper chamber to form a chemotactic
gradient. Chemotaxis chambers were incubated for 4 to 5 hours at
37°C and 5% CO2 in a humidified incubator. Thymocytes
migrating into the lower chamber were stained with anti-CD4-TriColor
and anti-CD8-FITC. CD4+SP, CD4+8+
DP, CD48 DN, and CD8+
SP thymocyte populations were analyzed on the basis of CD4 and CD8
expression by FACscan (Becton Dickinson). Isolation and chemotaxis of
human CD4+ T cells and CD8+ T cells were
assayed as previously described.18 For pertussis toxin
inhibition of chemotaxis, pertussis toxin (Sigma Chemical Co, St Louis,
MO) at various concentrations (0, 10, 100, and 1,000 ng/mL) was used to pretreat thymocytes in the chemotaxis buffer (5 × 107 cells/mL) for 1 hour at 37°C and 5%
CO2 in a humidified incubator before chemotaxis.
Reverse transcription-polymerase chain reaction
(RT-PCR) analysis of CXCR4 and CCR7 mRNA distribution in
different thymocyte subsets.
Thymocytes were stained with anti-CD4-TriColor, anti-CD8-FITC, and a
cocktail of biotinylated antibodies (anti-B220, anti-Gr-1, anti-Mac-1, and anti-NK1.1), followed by second staining with streptavidin-PE. Non-T cells were excluded by gating only Lin (PE)-negative thymocytes. CD4+,
CD4+8+,
CD48, and CD8+
thymocytes ( 97% pure) were sorted using a FACStar plus
(Becton Dickinson). Total RNA was isolated from the sorted cells (5 × 105) with Trizol solution
(GIBCO-BRL/LifeTechnologies, Grand Island, NY) according to the
manufacturer's instructions. Single-strand cDNA was made from the 0.5 µg total RNA with SuperScript Preamplification System for First
Strand Synthesis (GIBCO-BRL/LifeTechnologies). Primers used to detect
CXCR4 mRNA were 5 -GTT CGA ATT CAA CCA CCA CGG CTG TAG
AG-3 for a forward primer and 5-GTC AGC CAT GGC ATC AAC
TG-3 for a reverse primer, which give PCR products of 349 bp.
For CCR7 mRNA, 2 primers were used, ie, 5-GTT CGA ATT CAT CAG
CAT TGA CCG CTA CGT-3 for forward primer and 5-GCG TGC
CTG GAG CAA GGT ACG-3 for reverse primer, which give 318-bp PCR
products. PCR reactions were performed in the presence of 0.1 µL of
32PdCTP (Amersham, Arlington Heights, IL; 800 mCi/mmol/L/reaction) for 30 cycles (94°C for 1 minute, 55°C for
1 minute, and 72°C for 1 minute). The PCR products were resolved on
a 5% nondenaturing polyacrylamide gel. After drying, the filters were
analyzed by Molecular Imager (Bio-Rad, Hercules, CA) and exposed on
x-ray films. As an internal control, -actin mRNA was amplified using two primers: 5-ATG TTT GAG ACC TTC AAC AC-3 and
5-CACGTC ACA CTT CAT GAT GG-3.
Statistical analysis.
The Student's t-test was used to analyze data for significance
differences. P values less than .05 were regarded as
significant differences.
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RESULTS |
SDF-1 and CK-11/MIP-3/ELC are
chemoattractants for thymocytes.
Expression of SDF-1 mRNA was previously reported to be ubiquitous, with
a high level of mRNA expression detected in thymus.9 The
mRNA of CK-11/MIP-3/ELC is detected in thymus, lymph node, trachea, colon, small intestine, and lung.16,17
Interestingly, thymus is the organ expressing the highest level of
CK-11/MIP-3/ELC mRNA. These data suggest the possible roles of
these chemokines in thymocyte development and migration. We examined
the chemotactic activity of SDF-1 and CK-11/MIP-3/ELC for
thymocytes. SDF-1 and CK-11/MIP-3/ELC both attracted thymocytes
depleted of non-T cells in a dose-dependent manner
(Fig 1A). SDF-1 often provided a stronger
chemoattractant signal for total thymocytes than CK-11/MIP-3/ELC. Optimal chemokine concentrations for thymocyte chemotaxis were about
300 to 500 ng/mL for both SDF-1 and CK-11/MIP-3/ELC.
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| Fig 1.
Chemotactic preferences of SDF-1 and
CK-11/MIP-3/ELC for thymocyte subsets. Effects on total
thymocytes (A), CD4+ SP (B), DP (C), DN (D), and
CD8+ SP (E) cells are shown. Thymocyte isolation and
chemotaxis assays were performed as described in the Materials and
Methods. Thymocytes depleted of non-T cells were used as input cells
for chemotaxis. Numbers of cells migrating to the lower chamber were
expressed as the percentage of input thymocytes in the upper chamber at the start time of chemotaxis. Data are expressed as the mean
(±difference) of the percentage of cell migration obtained from
duplicated experiments and are representative of 5 independent
experiments. *Significant differences (P < .05) from
background migration.
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Differential chemotactic preference of SDF-1 and
CK-11/MIP-3/ELC.
Expression of CD4 and CD8 on thymocytes defines 4 populations of
thymocytes: most immature CD4CD8
DN thymocytes, immature CD4+CD8+ DP thymocytes,
and mature CD4+ and CD8+ SP T cells. To examine
the chemotactic activity of SDF-1 and CK-11/MIP-3/ELC on
thymocyte subpopulations, we isolated thymocytes depleted of non-T
cells, performed chemotaxis assays using the 2 chemokines at various
concentrations, and stained input and migrated cells with
anti-CD4-TriColor and anti-CD8-FITC. For the CD4+
thymocyte population, CK-11/MIP-3/ELC provided strong chemotactic activity, whereas SDF-1 demonstrated weak chemotactic activity (Fig
1B). The range of net CD4+ SP cell chemotaxis above
background level (shown as the percentage of input cell number) through
5 independent experiments was 6% to 16% for SDF-1 and 35% to 60%
for CK-11/MIP-3/ELC. For DP thymocytes, SDF-1 attracted these
cells far better than CK-11/MIP-3/ELC (Fig 1C). The range of net
chemotaxis of DP thymocytes through 5 independent experiments was 10%
to 19% for SDF-1 and 2% to 5% for CK-11/MIP-3/ELC. SDF-1 also
attracted DN thymocytes very well (17% to 21%; n = 5 experiments), whereas CK-11/MIP-3/ELC demonstrated weak
chemotactic activity (3% to 8%; n = 5 experiments) for this thymocyte
population (Fig 1D). Although both SDF-1 and CK-11/MIP-3/ELC
attracted CD8+ thymocytes, the chemotactic activity of
CK-11/MIP-3/ELC was far greater than that of SDF-1 (Fig 1E). The
range of net chemotaxis for CD8+ SP thymocytes through 5 independent experiments was 13% to 20% for SDF-1 and 33% to 47% for
CK-11/MIP-3/ELC. Thus, SDF-1 is more selective than
CK-11/MIP-3/ELC for chemotaxis of DP and DN thymocytes, and
CK-11/MIP-3/ELC is more selective than SDF-1 for the chemotaxis
of CD4+ and CD8+ thymocytes.
To examine the chemotactic activity of SDF-1 and CK-11/MIP-3/ELC
in the context of the thymus, we set up chemotaxis experiments using
whole thymuses. A whole murine thymus was placed on a polycarbonate membrane of the Transwell system so that the thymus tightly covered the
entire membrane surface. Chemokines, at optimal concentrations, were
added to the lower chamber and blank medium to the upper thymus-containing chamber forming a chemokine gradient from the lower
through the thymus and to the upper chamber. In this setting, SDF-1 and
CK-11/MIP-3/ELC attracted thymocytes out of thymus in the upper
chamber to the lower chamber (Fig 2). The
composition and number of mobilized thymocytes in response to SDF-1
were different from that attracted to CK-11/MIP-3/ELC or control
medium (background migration). Although SDF-1 attracted all thymocyte
subsets compared with control numbers, specific attraction of the DP
subset was more notable than other subsets. Although,
CK-11/MIP-3/ELC attracted notable numbers of SP and DP
thymocytes, it preferentially attracted more CD4+ and
CD8+ SP subsets than DP subset. This organ chemotaxis assay
supports the chemotaxis data shown in the Fig 1 for starting thymocytes in suspension culture.
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| Fig 2.
Chemoattraction of thymocytes from thymuses (organ
chemotaxis) by SDF-1 and CK-11/MIP-3/ELC. Thymocytes in thymus
before chemotaxis (A) and thymocytes migrating to control medium (B), CK-11/MIP-3/ELC (C), and SDF-1 (D) are shown. Total cell numbers per count for 30 seconds by FACscan and the percentage of
each subset in total thymocytes, representative of 4 experiments, are shown. Thymus organ chemotaxis assays were performed as described in
the Materials and Methods. Migrated thymocytes were stained with
anti-CD4-TriColor and anti-CD8-FITC, acquired for 30 seconds, and
analyzed by FACscan. The mean composition (±SD) of the 4 thymocyte subsets from the 4 experiments is 12.1 ± 0.6 (CD4+ SP),
83.8 ± 2.8 (DP), 2.8 ± 0.6 (DN), and 2.6 ± 0.7 (CD8+
SP) for control thymocyte suspension; 11.2 ± 1.9 (CD4+
SP), 80.2 ± 2.8 (DP), 5.6 ± 2.3 (DN), and 3.0 ± 0.7 (CD8+ SP) for those attracted to SDF-1; and 50.1 ± 9.8 (CD4+ SP), 30.7± 8.8 (DP), 2.4 ± 0.8 (DN), and 16.9 ± 3.2 (CD8+ SP) for those attracted to
CK-11/MIP-3/ELC. *Significant differences (P < .05) from control thymocyte subset composition.
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We performed calcium mobilization assay for thymocytes to study
differential signaling by SDF-1 and CK-11/MIP-3/ELC. However, SDF-1 and CK-11/MIP-3/ELC did not mobilize detectable levels of
calcium in thymocytes at concentrations up to 300 nmol/L, suggesting chemotactic activity is not necessarily correlated with
calcium-mobilizing activity in thymocytes (data not shown).
Expression of CXCR4 and CCR7 mRNA in different thymocyte subsets.
Chemokines induce chemotaxis by binding G-protein-coupled receptors
with 7-transmembrane domains. We examined mRNA expression of CXCR4, a
receptor for SDF-1, and CCR7, a receptor for CK-11/MIP-3/ELC, in
total thymocytes and thymocyte sub-populations. We sorted total thymocytes, CD4+ SP, DP, DN, and CD8+ SP
thymocyte populations depleted of lin+ non-T cells for
RT-PCR analysis of receptor mRNA expression. CXCR4 and
CCR7 mRNA was detected not only in total thymocytes, but also in all 4 subpopulations of thymocytes (Fig 3; 1 representative of 3 independent experiments). However, we consistently
observed that the mRNA expression level of CXCR4 in DP, DN, and
CD8+ thymocytes was greater than in CD4+
thymocytes (Fig 3). For CCR7 expression, the message level of CCR7 in
CD4+ and CD8+ thymocytes was higher than in DP
and DN thymocytes (Fig 3).
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| Fig 3.
RT-PCR analysis of CXCR4 and CCR7 mRNA expression in
thymocyte subsets. RT-PCR analyses were performed as described in the Materials and Methods using the same amount of total RNA obtained from
each sorted thymocyte subset. Data shown are representative of 3 independent experiments. -Actin was amplified as an internal control.
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Migration behavior of total thymocytes in response to complex
chemotactic environments formed by SDF-1 and
CK-11/MIP-3/ELC.
In the experiments examining chemotactic activity of SDF-1 and
CK-11/MIP-3/ELC, only 1 chemoattractant had been added to the
chemotaxis assay system. To examine the chemotactic specificity and
selectivity of SDF-1 and CK-11/MIP-3/ELC in a more complex chemotactic environment, we set up experiments by adding the 2 chemokines to the upper and/or lower chambers in a
combinatorial manner. SDF-1 or CK-11/MIP-3/ELC in the upper
chamber respectively inhibited the chemotaxis by SDF-1 or
CK-11/MIP-3/ELC in the lower chamber, suggesting that there was
homologous desensitization/inhibition of chemotaxis
(Fig 4A, bar no. 2 v 4, bar no. 3 v 5). The homologous inhibition by SDF-1 was more complete than
that by CK-11/MIP-3/ELC (Fig 4A, bar no. 5 v 4). The
extent of cross or heterologous inhibition of chemotaxis between SDF-1
and CK-11/MIP-3/ELC was less than the homologous inhibition (Fig
4A, bar no. 4 v 7, bar no. 5 v 6). When SDF-1 and
CK-11/MIP-3/ELC were added to the lower chamber together, there
was an additive effect in the net migration above background (Fig 4A,
bar no. 8). Interestingly, either SDF-1 or CK-11/MIP-3/ELC in the
upper chamber inhibited the additive thymocyte migration by SDF-1 and
CK-11/MIP-3/ELC in a manner subtracting only the chemotactic
effect of either chemokine in the lower chamber (Fig 4A, bars no. 9 and
10). Homologous desensitization of chemotaxis by the 2 chemokines was
additive (Fig 4A, bar no. 11), but not complete enough to bring
chemotaxis to background, implying that the incomplete homologous
inhibition by CK-11/MIP-3/ELC seen in bar no. 4 of Fig 4A
contributed to this inhibition.
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| Fig 4.
Migration behavior of total thymocytes (A) and thymocyte
subsets, CD4+ SP (B), DP (C), DN (D), and CD8+
SP (E) subsets, in chemotactic environments formed by SDF-1 and CK-11/MIP-3/ELC. The 2 chemokines were added to upper or lower chambers as indicated in the Fig (+ and  denote the presence and
absence of indicated chemokines, respectively) at optimal concentrations (500 ng/mL for CK-11/MIP-3/ELC and 300 ng/mL for
SDF-1). Thymocytes depleted of non-T cells were used as input cells for
chemotaxis. Cell migration to lower chambers is expressed as the mean
percentage of migration (±differences of duplicated experiments) of
input thymocyte subsets added in the upper chamber at the start time of
chemotaxis. Significant differences (P < .05) were observed
between the following 2 bars: 1 and 2, 1 and 3, 2 and 4, 3 and 5, 4 and
7, 5 and 6, 2 and 8, 3 and 8, 8 and 10, and 9 and 11 (for [A]); 1 and
2, 1 and 3, 2 and 4, 3 and 5, 4 and 7, 3 and 8, 8 and 9, and 10 and 11 (for [B]); 1 and 2, 1 and 3, 3 and 5, 5 and 6, 2 and 8, and 9 and 11 (for [C]); 1 and 3, 3 and 5, 5 and 6, 2 and 8, and 9 and 11 (for
[D]); and 1 and 2, 1 and 3, 2 and 4, 3 and 5, 4 and 7, 5 and 6, 3 and
8, 8 and 9, 8 and 10, and 10 and 11 (for [E]).
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Chemotaxis of thymocyte subsets under complex chemotactic
environments formed by SDF-1 and
CK-11/MIP-3/ELC.
The thymocyte migration pattern seen in Fig 4A describes the chemotaxis
of total thymocytes in a chemotactic environment formed by SDF-1 and
CK-11/MIP-3/ELC. However, this is the mixed chemotactic behavior
of many thymocyte subsets. To examine the chemotactic behavior of each
thymocyte subset under these various chemotactic conditions, we tracked
migration of the each thymocyte subset by staining the input and
migrated cells with fluorescent anti-CD4 and anti-CD8 antibodies.
For the CD4+ SP thymocyte subset (Fig 4B), in addition to
the typical homologous desensitization of chemotaxis by
CK-11/MIP-3/ELC (bar no. 2 v 4) or SDF-1 (bar no. 3 v 5), there was heterologous desensitization of
SDF-1-dependent chemotaxis by CK-11/MIP-3/ELC (bars no. 6 and
7). However, the inhibition of CK-11/MIP-3/ELC-dependent chemotaxis by SDF-1 was marginal, suggesting the dominance of CK-11/MIP-3/ELC over SDF-1 on CD4+ thymocyte
migration (bars no. 7 and 10). Although
CK-11/MIP-3/ELC-dependent homologous and heterologous
desensitization was partial (bars no. 4, 9, and 11) in that it did not
reduce the chemotaxis to the background level, it was the most
influential factor in reducing the overall CD4+ SP cell
chemotaxis.
In contrast to the CD4+ subset, SDF-1 was a dominant
chemoattractant over CK-11/MIP-3/ELC for the DP subset (Fig 4C).
Efficient homologous desensitization was observed in SDF-1-dependent
chemotaxis for the DP subset (bar no. 3 v 5), whereas the
CK-11/MIP-3/ELC-dependent chemotaxis was weak and homologous
desensitization was not detectable (Fig 4C, bar no. 2 v 4).
CK-11/MIP-3/ELC in the upper chamber had no significant effect on
SDF-1-dependent chemotaxis (bar no. 6). SDF-1 in the upper chamber in
all conditions demonstrated a strong inhibitory effect on DP thymocyte
migration (bars no. 10 and 11).
Chemotaxis of DN thymocytes (Fig 4D) was very similar to that of the DP
thymocytes in that SDF-1 was a dominant chemoattractant for this
subset. CK-11/MIP-3/ELC showed a small but significant contribution not only in chemotaxis of DN thymocytes along with SDF-1
(bar no. 8), but also in homologous (bars no. 4 and 9) and heterologous
desensitization (bars no. 6, 9, and 11) of the chemotaxis. SDF-1 was
again dominant over CK-11/MIP-3/ELC in the desensitization of
chemotaxis (bar no. 9 v 10).
The effects in Fig 4E demonstrating the migration behavior of
CD8+ SP thymocytes were similar to that of the
CD4+ thymocytes in Fig 4B suggested the dominance of
CK-11/MIP-3/ELC over SDF-1 in induction of chemotaxis and
inhibition of chemotaxis. Although the chemotactic activity of SDF-1 is
weaker than that of CK-11/MIP-3/ELC for this subset, it was
significantly high, and SDF-1 showed homologous and heterologous
desensitization of CD8+ thymocyte chemotaxis (bars no. 5, 7, 10,and 11).
Chemotactic activity of SDF-1 and
CK-11/MIP-3/ELC for TN thymocyte
subsets.
Lymphoid common progenitors migrate from bone marrow to fetal thymus to
seed and repopulate the thymus. The earliest thymic lymphoid
progenitors have a
CD117+CD44+CD25CD4lo
phenotype.24 This population is rare and represents only
0.05% of all thymocytes. It is difficult to examine the chemotactic activity of SDF-1 and CK-11/MIP-3/ELC on this rare thymocyte population. Instead, we isolated TN
(CD3CD4CD8)
thymocytes that were depleted of committed T cells and non-T cells. We
tracked chemotaxis of 4 thymocyte populations defined by expression of
CD44 and CD25.25 The phenotypic flow of TN thymocyte
differentiation is CD25CD44+  CD25+CD44+ CD25+CD44 CD25CD44. SDF-1 attracted all 4 TN thymocyte populations (Fig 5), which was
predictable from the data in the earlier figures showing strong chemotactic activity of SDF-1 on DN thymocytes. CK-11/MIP-3/ELC had no significant chemotactic activity for
CD25+CD44+,
CD25+CD44, and
CD25CD44 TN thymocyte subsets. It
did demonstrate some chemotactic activity for the
CD25CD44+ thymocyte population over
background level, but this was not a statistically significant effect.
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| Fig 5.
Chemotactic activity of SDF-1 and CK-11/MIP-3/ELC
for TN thymocyte subsets, CD25CD44+
(A), CD25+CD44+ (B),
CD25+CD44 (C), and
CD25CD44 (D) subsets. Isolated TN
thymocytes were added to the upper chambers, and the 2 chemokines were
added to the lower chambers as indicated in the figure. Data are
expressed as the mean percentage of migration (±differences of
duplicated experiments) for each subset. *Significant differences
(P < .05) from background migration.
|
|
Effects of selection processes on chemotactic responsiveness of DP
thymocytes to SDF-1 and
CK-11/MIP-3/ELC.
The specificity and affinity of T-cell antigen receptors (TCR)
on DP thymocytes determine the fate of T-cell
development.26-29 The negative selection process eliminates
potentially harmful DP T cells that are reactive with self-antigens,
whereas positive selection saves DP T cells with TCR that recognizes
self-MHC. Expression of CD69 is induced after initiation of the
selection on DP thymocytes.30,31 We examined the
chemotactic activity of SDF-1 and CK-11/MIP-3/ELC to test whether
chemotactic responsiveness of developing thymocytes is regulated by the
selection process. The 2 chemokines attracted CD69+ DP
cells better than CD69 DP cells
(Fig 6). SDF-1 showed stronger chemotactic
activity for both CD69 DP and CD69+ DP
thymocytes than CK-11/MIP-3/ELC. CK-11/MIP-3/ELC attracted mainly CD69+ DP thymocytes, but not much
CD69 DP thymocytes, suggesting it has a preference
for positively selected DP thymocytes. CD69+ DP cells
accounts for 12% of total DP thymocytes (data not shown). After
chemotaxis, the CD69+ DP cells were enriched to account for
24% of total DP thymocytes in response to SDF-1 and 43% in response
to CK-11/MIP-3/ELC, suggesting that the thymic selection process
modulates the chemotactic responsiveness of DP thymocytes to the
chemokines (data not shown).
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| Fig 6.
Chemotactic activity of SDF-1 and CK-11/MIP-3/ELC
for DP thymocyte subsets defined by CD69 expression. Total thymocytes were added to the upper chambers and optimal concentrations of SDF-1
(300 ng/mL) and CK-11/MIP-3/ELC (500 ng/mL) were added to the
lower chambers. Input and migrated cells were stained with antibodies
to CD4, CD8, and CD69.
CD4+CD8+CD69+ cells and
CD4+CD8+CD69 cells were
counted by FACscan for 30 seconds. Data are shown as the net mean
percentage of migration after background subtraction for each subset
(±SD of triplicated experiments). *Significant migration (P < .05) from background migration.
|
|
Mature splenic CD4+ SP T cells are more responsive to
CK-11/MIP-3/ELC and SDF-1 than their thymic
counterparts.
It is known that SDF-1 and CK-11/MIP-3/ELC are potent
chemoattractants for human peripheral blood lymphocytes and T
cells.12,18 However, SDF-1 was a weaker, whereas
CK-11/MIP-3/ELC was a stronger chemoattractant for murine thymic
SP T cells in this study. It is possible that the chemotactic
responsiveness of SP T cells to SDF-1 would be upregulated after
migration to peripheral blood or secondary lymphoid tissues. To test
this possibility, we compared the chemotactic activity of SDF-1 and
CK-11/MIP-3/ELC on mouse thymic and spleen CD4+ and
CD8+ SP T cells. Spleen CD4+ T cells were more
responsive than their thymic counterparts to SDF-1 and
CK-11/MIP-3/ELC (Fig 7A). In mice,
SDF-1 was less potent than CK-11/MIP-3/ELC in chemotaxis of
thymic and spleen CD4+ T cells (Fig 7A). CD8+ T
cells from mouse spleen and thymus were equally responsive to
CK-11/MIP-3/ELC (Fig 7B). Although SDF-1 appeared to show higher
chemotactic activity for mouse spleen CD8+ SP than their
thymic counterparts, this was not a statistically significant
difference (Fig 7B).
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| Fig 7.
Comparison of chemotactic activity of SDF-1 and
CK-11/MIP-3/ELC for splenic T cells with that for thymic T cells.
Murine CD4+ (A) and CD8+ (B) SP T cells
from thymus and spleen were assayed for their chemotactic
responsiveness to SDF-1 and CK-11/MIP-3/ELC. Input and migrated
cells were stained with fluorescent antibodies to human or mouse CD4
and CD8, acquired for 30 seconds, and analyzed by FACscan. Cell
migration to lower chambers is expressed as the mean percentage
(±differences of duplicated experiments) of input subset cell number
added in the upper chamber at the start time of chemotaxis. Background
migration was subtracted from the mean migration to show net migration.
Significant differences were observed between the following 2 peak
points: A and B, A and C, A and D, B and C, B and D, and C and D (for
[A]); and A and D and B and D (for [B]).
|
|
Differential sensitivity of SDF-1- and
CK-11/MIP-3/ELC-dependent chemotaxis to
pertussis toxin.
Pertussis toxin specifically inhibits only Gi/Go proteins among other
G-proteins such as Gq, Gs, or G12/G13 proteins.32 Both
SDF-1- and CK-11/MIP-3/ELC-dependent chemotaxis were sensitive to pertussis toxin (Fig 8). However,
SDF-1-dependent chemotaxis was more sensitive to pertussis toxin than
that of CK-11/MIP-3/ELC. Pertussis toxin at 10 ng/mL was
effective in inhibiting SDF-1-dependent chemotaxis, whereas higher
concentrations (~1,000 ng/mL) were required for complete inhibition
of CK-11/MIP-3/ELC-dependent chemotaxis (Fig 8). This result
supports that the 2 chemokines use different receptors (CXCR4 receptor
for SDF-1 and CCR7 receptor for CK-11/MIP-3/ELC). It also
suggests that these 2 receptors have different sensitivities to
pertussis toxin, possibly by using different G-proteins for signaling.
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| Fig 8.
Chemotactic activity of SDF-1 and CK-11/MIP-3/ELC
on thymocytes is sensitive to pertussis toxin. Low-density total
thymocytes were preincubated with pertussis toxin at indicated
concentrations for 1 hour and used for chemotaxis to SDF-1 (300 ng/mL)
and CK-11/MIP-3/ELC (500 ng/mL). Data are expressed as the mean
percentage of inhibition (±differences of duplicated experiments).
Complete inhibition (100%) means that chemotaxis to chemokines is
inhibited to background or lower than background levels, and no
inhibition (0%) represents maximum chemotaxis to chemokines observed
without pertussis toxin treatments. Partial inhibition is between 0%
and 100%. *Significant inhibition (P < .05) from controls
(no pertussis toxin treatment).
|
|
| |
DISCUSSION |
In this study, we examined the activity of 2 thymus-expressed
chemokines, SDF-1, a CXC chemokine, and CK-11/MIP-3/ELC, a CC
chemokine, for chemoattraction of total and subsets of thymocytes. The
thymus is organized into subcompartments supporting the proliferation and differentiation of thymocytes at different stages of maturation. Thymocyte migration between compartments correlates with the
differentiation stage of thymocytes. Thymic chemoattractants may be
involved in thymocyte migration within the thymus and also in thymocyte
differentiation. One way to understand migration of thymocytes would be
to investigate selective chemoattraction of thymocyte subsets by
multiple thymic chemoattractants. This model would require multiple
thymic chemoattractants that have specificity for each or a group of
thymocyte subsets. Alternatively, thymic anatomy is designed such that
thymocytes migrate in a unidirectional manner according to their
differentiation stages by simple overflow of thymocytes from one
microenvironment to another by chemoattraction to a nonselective
chemoattractant(s). These 2 models are not necessarily incompatible.
The preferences of the 2 chemokines, SDF-1 and CK-11/MIP-3/ELC,
for selective chemoattraction of thymocyte subsets demonstrated in this
study provide evidence for the first model.
In the present study, we consistently observed the modulation of
chemotactic responsiveness of thymocyte subsets to SDF-1 and
CK-11/MIP-3/ELC during their development
(Fig 9). SDF-1 maintains its chemotactic
potency on thymocytes from the early stages of TN thymocytes
(CD25CD44+,
CD25+CD44+,
CD25+CD44, and
CD25CD44) to intermediary DP
thymocytes (before and after initiation of thymic selection). After DP
thymocytes become SP CD4+ T cells, SDF-1 shows reduced
chemotactic activity for SP thymocytes. In contrast,
CK-11/MIP-3/ELC shows minimal chemotactic activity for the TN and
CD69 DN thymocyte subsets and begins to enhance
chemotactic activity for CD69+ DP thymocytes.
CK-11/MIP-3/ELC demonstrates potent chemotactic activity for
mature SP thymocytes. These data allow us to speculate that SDF-1 may
be a major chemoattractant regulating migration of immature DN and DP
thymocytes at premedullar stage (cortex) migration, whereas
CK-11/MIP-3/ELC regulates migration of mature thymocytes from the
medullar compartment into peripheral blood. There was no significant
desensitization or inhibition of chemotaxis to the major
chemoattractant by the minor chemoattractant, suggesting that, in the
event of microenvironmental coexpression, the preferential chemotaxis
by the major chemoattractant may not be significantly affected by
coexisting minor chemoattractants. Unfortunately, the spatial
expression of SDF-1 and CK-11/MIP-3/ELC proteins in different
murine thymic microenvironments is not yet known. Thymic SP T cells
enter the peripheral blood or lymphatic system and migrate to a
secondary lymphoid system such as the spleen. Both SDF-1 and
CK-11/MIP-3/ELC demonstrate enhanced chemotactic activity for
these spleen T cells. These results suggest that chemokines in
different lymphoid organs (thymus v spleen) may have different
roles to help achieve differential organ function. On the other hand,
cells in different developmental stages or organs appear to actively
modulate their responsiveness to chemokines to achieve the same goals.
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| Fig 9.
Modulation of chemotactic responsiveness to SDF-1 and
CK-11/MIP-3/ELC during T-cell development from early TN stage to
mature CD4+ T cells in secondary lymphoid tissues.
|
|
The thymus is a site of massive cell death or apoptosis as a result of
thymic selection processes.29 These selection processes occur with thymocytes at the DP stage. If chemokines are important in
these processes, they can facilitate apoptosis by recruiting macrophages and apoptotic thymocytes together in specialized thymic compartments. The majority of DP thymocytes cannot survive the selection processes, and only a small fraction of DP cells are allowed
to contribute to the total T-cell repertoire.27 It is also
possible that thymic chemoattractants may be involved in the rescue of
selected DP thymocytes by inducing the migration of selected thymocytes
away from phagocytic macrophages. SDF-1 and CK-11/MIP-3/ELC may
be appropriate chemokines for such functions because they attract
CD69+ DP thymocytes.
CXCR4, the receptor for SDF-1, is expressed on human peripheral blood
lymphocytes, neutrophils, T cells, and blood monocytes.33 CCR7, the receptor for CK-11/MIP-3/ELC, is expressed in activated peripheral blood lymphocytes, Epstein-Barr virus-positive B-cell lines,
and most lymphoid tissues.34,35 We have demonstrated herein
that the mRNA for these receptors is also expressed in most subsets of
thymocytes. Recently, it was reported that CXCR4 is expressed at a
higher level on immature DP than mature CD4+ SP human
thymocytes, suggesting a possible role of the CXCR4 coreceptor for HIV
infection of immature thymocytes.36 This differential CXCR4
expression level among human thymocyte subsets is similar to what we
have seen on mouse thymocytes. Taken together, the murine and human
data imply that there are potential roles for thymic chemokine
receptors and their ligands in thymocyte migration and differentiation.
Expression of CXCR4 mRNA appears higher in DP, DN, and CD8+
SP thymocytes than in CD4+ SP thymocytes, whereas CCR7 mRNA
expression appears higher in SP cells than in immature DP and DN
thymocytes. Overall, the receptor message levels correlate with the
chemotactic activity and selectivity of SDF-1 and CK-11/MIP-3/ELC
for thymocyte subsets.
Recently, several chemokines have been reported to have chemotactic
activity for thymocytes. Thymus-expressed chemokine (TECK) and
lymphotactin have been reported to attract thymocytes, although the
precise chemotactic specificity of each chemokine is not
known.37 Interestingly, TECK shows chemotactic activity for
thymic dendritic cells, suggesting that this chemokine might regulate
thymic dendritic cell migration within or to thymus.37 It
is known that peripheral blood T cells recirculate to the
thymus.38,39 These peripheral blood T cells, especially
CD4+ T cells, negatively regulate CD4+ T-cell
generation from DP thymocytes through unknown mechanisms to maintain
homeostasis of T lymphopoiesis.40 Thymic chemoattractants such as SDF-1 and CK-11/MIP-3/ELC may have the potential to regulate thymopoiesis by recruiting appropriate amounts of peripheral blood T cells into the thymus. The discovery and characterization of
thymic chemoattractants would potentially provide further information on intrathymic thymocyte migration or migration of thymocytes and
accessory cells into/out of thymus, information that would further our
understanding of T lymphopoiesis.
| |
FOOTNOTES |
Submitted February 9, 1998;
accepted March 23, 1998.
Supported by US Public Health Service Grants No. R01 HL 56416 and R01
HL 54037 and by a project in P01 HL 53586 from the National Institutes
of Health to H.E.B.
Address reprint requests to Hal E. Broxmeyer, PhD, Department of
Microbiology/Immunology and the Walther Oncology Center, Indiana
University School of Medicine, Bldg R4, Room 302, 1044 W Walnut St,
Indianapolis, IN 46202.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Edward Appelbaum, Kyoung Johanson, Donna Cusimono,
Joyce Mao, Stephanie Van Horn, Laura Grayson, Gil Scott, and Dean
McNulty (SmithKline Beecham) for the production and analytical characterization of CK-11. We also thank Rebecca Miller and Cindy Booth (Indiana University) for assistance in the preparation of this
manuscript.
| |
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Abstract
Introduction
Abstract