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Department of Anatomy, The Medical School, University of Birmingham, Birmingham, United Kingdom
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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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Evidence that chemotactic factors may be involved in this process has been provided by functional studies using transfilter migration assays that have demonstrated the ability of alymphoid fetal thymic lobes to attract stem cells from fetal liver fragments or other lymphoid fetal thymus lobes (6, 7, 8). However, the specific cell types responsible for the production of chemoattractant and the nature of such factors remain to be identified. In this context, mature T cells and thymocytes have been shown to respond to members of the chemokine family of cytokines that are known to mediate their activities via interaction with seven-transmembrane domain receptors coupled to pertussis toxin (PTX)3-sensitive heterotrimeric G proteins (9). Interestingly, the expression of a number of chemokines in the thymus has now been described, although a functional role for these chemokines in precursor recruitment has not been defined (10, 11, 12, 13, 14, 15).
Directed migration of cells in response to a gradient of chemotactic factors may also require the ability to interact with extracellular matrix (ECM) proteins. Matrix-disrupting enzymes, matrix metalloproteinases (MMPs), are believed to play an important role in the degradation of the ECM during cellular migration in a number of systems, and the production of these enzymes by T cell precursors could be important in facilitating their migration through the tissues surrounding the thymus (16). To date, the involvement of such enzymes in thymus colonization has not been established.
In this study, we have examined the role of chemokines and matrix-disrupting metalloproteinases in thymus colonization using a novel in vitro assay. Our data show that within the thymus, MHC class II+ epithelial cells are the source of chemoattractant factors for T cell precursors and that the response to these factors is dependent upon PTX-sensitive receptors on the responding cells, consistent with the factors being members of the chemokine family. Moreover, RT-PCR studies show that a number of chemokine receptors are expressed in populations of cells capable of thymic colonization, and that several chemokines are also expressed by embryonic thymic epithelial cells. However, these chemokines are also expressed in embryonic salivary gland rudiments that do not attract precursors, although the chemokine TECK is expressed at higher levels in thymic epithelial cells, and we show that it is chemotactic for isolated thymic precursors. Neutralizing Ab to TECK does not, however, inhibit precursor migration into thymic rudiments, suggesting that novel chemokines might be involved in this process.
In addition, we report that the ability of T cell precursors to respond to chemoattractant signals and migrate through the perithymic tissues is dependent upon MMP activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Embryonic material was obtained from BALB/c (Thy-1.2; H2d), AKR (Thy-1.1; H2k), and BALB/c nu/nu (Thy-1.2; H2d) mouse embryos at 14 and 15 days gestation. Timed matings were conducted, and the day of gestation was calculated by the plug date (= day 0). Mesenteric and inguinal lymph nodes from 4–6-wk-old adult BALB/c mice were used for the isolation of peripheral T cells.
Abs and immunoconjugates
For immunomagnetic selection, the following Abs were bound onto
either anti-rat Ig- or anti-mouse Ig-coated magnetic beads
(Dynal, Wirral, U.K.): anti-CD45 (clone M1/9; American Tissue
Culture Collection, Manassas, VA); anti-I-Ad (clone
MK-D6; Becton Dickinson, Cowley, U.K.); and anti-B220
(clone RA3-6B2; PharMingen, San Diego, CA). For flow cytometry, the
following Abs were used (all from PharMingen): anti-CD4-PE (clone
L3T4), anti-CD8-FITC (clone 53-6.7), anti-CD8-APC (clone
53-6.7), anti-Thy-1.2 FITC (clone 30-H12), and anti-CD3
FITC
(clone 145-2C11). For blocking experiments, anti-mouse MCP-1 (clone
2H5; PharMingen) and anti-mouse TECK (R&D Systems, Abingdon,
Oxford, U.K.) were used as azide-free preparations.
Chemokines
The mouse chemokine MCP-1 was purchased from PharMingen, and recombinant mouse TECK was purchased from R&D Systems. Chemokines were used at a range of dilutions, as indicated.
Inhibitors
The synthetic MMP inhibitor, N4-hydroxy-N1-(1-(S)-methylaminocarbonyl-2-methyl-2-methylopropyl)-2-(R)-(2-methylpropyl)succinamide, designated CT1847 (17, 18) (gift from T. MacDonald, St. Bartholomew’s and Royal London School of Medicine and Dentistry, London, U.K.), was dissolved in DMSO at a stock concentration of 10 mM and added to cultures at final concentrations of 20 and 50 µM. PTX (Sigma, St. Louis, MO), dissolved in PBS, was used at final concentrations of 250 and 500 ng/ml.
Fetal thymus organ culture
Fetal thymic organ cultures were assembled on the surface of nucleopore filters supported on foam sponge rafts, as described previously (8). To deplete fetal thymus lobes of lymphoid and dendritic cells, isolated thymic rudiments were explanted into organ culture at fetal day (fd) 15 and cultured in 1.35 mM deoxyguanosine (dGuo) for 5 to 7 days (8).
Preparation of individual lymphoid and stromal cell subpopulations
Purified preparations of lymphoid and stromal cells were isolated by combinations of positive and negative selection using Ab-coated magnetic beads (Dynal), as described in detail previously (19, 20).
Fd14 precursors. Precursors were liberated from fd14 thymus lobes by teasing apart with fine cataract knives. When required, T cell precursors were further purified by removal of phagocytes based on Fc binding and/or bead ingestion by a round of depletion with anti-mouse Ig-coated beads, followed by positive selection on anti-CD45-coated beads to exclude any contaminating stromal cells. Rosetted cells were transferred directly to lysis buffer for RNA extraction.
Thymic stromal cells. Specific stromal cell populations were prepared from cell suspensions obtained from dGuo thymus lobes enzymatically disaggregated by incubation in 0.25% trypsin in 0.02% EDTA. Stromal cell suspensions were depleted of any residual cells of hemopoietic origin with anti-CD45-coated beads. When required, purified preparations of MHC class II+ epithelial cells were then prepared from CD45-depleted thymic stromal cell suspensions by positive immunomagnetic selection on anti-I-Ad-coated beads, as described in detail elsewhere (19).
Salivary gland cells. Nonthymic epithelial/mesenchymal cells were prepared by the enzymatic disaggregation of fd14 salivary gland rudiments. The resultant cell suspension was centrifuged, the remaining supernatant was discarded, and the pellet was snap frozen in liquid nitrogen in preparation for RNA extraction and RT-PCR.
Freshly isolated fetal thymus lobes. For the assessment of the expression of known chemokine genes in whole fetal thymus lobes freshly isolated from fd14 embryos, thymic rudiments were disaggregated enzymatically and the resultant cell suspension was snap frozen in liquid nitrogen in preparation for RNA extraction and RT-PCR.
Activated peripheral T cells.
Activated peripheral T cells were prepared from lymph node for use in
chemotaxis assays. Cell suspensions prepared by mechanical disruption
of mesenteric and inguinal lymph nodes from a 4–6-wk-old adult mouse
were aliquoted into a 96-well plate at a density of 1 x
106 cells/well in DMEM + 10% FCS supplemented with a final
concentration of 5 µg/ml Con A (Sigma). Cells were cultured at 37°C
for 48 h in a humidified atmosphere of 5% CO2 in air
before harvesting. Harvested cells were enriched for T cells by three
rounds of depletion of B cells and phagocytes with anti-B22O-coated
magnetic beads and were collected by centrifugation. The purity of the
resultant cell suspension, assessed by immunofluorescent labeling with
anti-CD3 and flow-cytometric analysis, was typically >99% for
all samples.
Chemotaxis assays
Fd14 precursors and activated peripheral T cells, prepared as described, were independently assessed for their ability to mount chemotactic responses to recombinant mouse chemokines using a 24-well Transwell migration system (Costar, High Wycomb, U.K.) with 6.5-mm-diameter culture inserts with 5-µm pores. Chemokines diluted in assay medium (RPM1 1640 + 0.5% FCS) or medium alone were added to the lower compartment of 24-well tissue culture Transwell plates in a final volume of 600 µl. Fd14 precursors were resuspended in assay medium, and 2–2.5 x 105 cells in a 100 µl vol were loaded into the upper compartment. For the assessment of peripheral T cell responses, 7–5 x 105 cells resuspended in 100 µl of assay media were loaded in duplicate into the upper wells. Neutralizing anti-chemokine Abs diluted in assay media were added to the lower compartments where appropriate. The chambers were incubated for 4 h at 37°C in a 5% CO2 humidified atmosphere. After 4 h, the upper chambers were removed and the cells in each well bottom were counted using a hemocytometer (Weber, U.K.). Each experiment was repeated a minimum of three times and the data were expressed as a chemotactic index, calculated from the percentage of migrated cells detected in the test sample divided by the percentage of migrated cells detected with medium alone. To distinguish chemokinetic from chemotactic effects, replicate experiments were performed in which chemotactic factors were added at equal concentrations to both upper and lower wells.
RT-PCR
Gene expression in purified fd14 precursors or selected stromal
populations of thymic and nonthymic origin was assessed by RT-PCR and
compared by semiquantitative RT-PCR, performed as described in detail
elsewhere (21). To compensate for variable RNA and cDNA yields, cDNAs
were matched using the relative expression level of ß-actin as a
standard. RT-PCR reactions were performed in thin-walled tubes using a
PTC 200 Peltier Thermal Cycler (MJ Research, Cambridge, MA). PCRs were
sampled at the following cycle points: ß-actin, 17, 20, 23, 26, 29,
32; SDF-1 and TECK, 24, 28, 32, 36, 40, 44; MIP-1
, TCA4, RANTES, and
MCP-1, 26, 31, 36, 41, 46, 51. Size and/or DNA sequencing positively
identified PCR fragments visualized by agarose gel electrophoresis and
ethidium bromide staining. The intensity of the stained bands was
determined using a gel documentation system (Image Store 5000; Ultra
Violet Products, Cambridge, U.K.), followed by scanning densitometry
(Enhanced Analysis Systems, ultra violet products).
Reaggregate organ culture
Reaggregate cultures were prepared from anti-CD45-depleted thymic stromal cells or purified MHC class II+ epithelium prepared as described. Briefly, cell suspensions were pelleted by centrifugation and, after removal of supernatant, the cell pellet was vortexed and placed as a standing drop on the surface of a nucleopore filter in organ culture (19, 20). Intact rudiments reform from these standing drops within a few hours, providing an intact lobe for use in transfilter migration assays.
Transfilter migration assays
Transfilter migration assays (8) were a modification of those
used previously, and are illustrated in Fig. 1
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experiments, barrier filters coated with Matrigel matrix (Becton
Dickinson), which serves as a reconstituted basement membrane in vitro,
were used to provide a further physiological barrier. No more than
three donor/recipient combinations were placed on each 13-mm-diameter
filter. The efficiency of dGuo treatment was confirmed by the absence
of developing thymocytes in dGuo thymus lobes that had been cultured
without an underlying donor fragment. Transfilter cultures were
maintained for 6 h, after which time the recipient tissue was
removed for immediate analysis (Fig. 1b) or further culture
on a fresh filter to reveal the T cell potential of any precursors that
have migrated into the recipient lobes (Fig. 1a).
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a), recolonization was determined
by teasing individual lobes apart in a fixed volume using fine cataract
knives and examining the resultant cell suspension for the presence of
lymphoid cells. The number of lobes that had been recolonized out of
the total number of lobes was used to calculate the percentage of
recolonization. Cells recovered from successfully recolonized recipient
lobes were analyzed for T cell differentiation by immunolabeling with
Abs to CD4 and CD8, followed by flow cytometry. For Ab-blocking and inhibitor studies, recipient and donor tissues were independently preincubated for 2–3 h in organ culture with Ab/inhibitor at the appropriate concentrations. Transfilter cultures were then assembled in the continued presence (or absence) of Ab/inhibitor and left for 6 h to allow migration. Recipient tissues were recovered and washed for 1 h before being transferred to a fresh petri dish for a further 12-day culture in the absence of Ab/inhibitor. Possible toxic effects of Ab/inhibitors were tested by culturing fd14 thymus lobes in the presence/absence of Ab/inhibitor for a 6-h period, equivalent to that of the transfilter migration assay. After a further 12 days in culture, the development of precursors was assessed in these control thymus lobes by flow-cytometric analysis.
Rescue assays
To define the specific cell type(s) responsible for production of thymic chemoattractants, recipient reaggregate thymus lobes of defined stromal cell populations were prepared as described above and used as recipients in transfilter migration assays. To reveal the presence of precursors that may have migrated in response to stromal cells that were able to attract them but not support their continued development, recipient tissue fragments recovered from transfilter cultures after 6 h were fused with an intact dGuo-treated "rescue" lobe to support continued development of any precursors present. This fused structure was cultured for an additional 12 days and analyzed for the presence of differentiated thymocyte populations, as described above.
The ability of nude thymus lobes, other fetal rudiments such as salivary gland, and agarose plugs conditioned with the secreted products of thymic stromal cells to attract T cell precursors was also investigated using this strategy.
Preparation of chemoattractant conditioned agarose plugs
To investigate whether thymic chemoattractants are soluble,
diffusible factors agarose plugs were conditioned with culture
supernatant from dGuo-treated thymus lobes and assessed for their
ability to attract T cell precursors. A 2% agarose gel was prepared in
a 35-mm Petri dish using sterile distilled water and, once set, plugs
were cut out using fine cataract knives. Each agarose plug was washed
in RF10-H and conditioned by placing it in organ culture overnight
surrounded by at least five dGuo thymus lobes. The conditioned agarose
plugs (or unconditioned control plugs) were used as recipients in the
transfilter migration assay (Fig. 1). After the 6-h transfilter culture
period, the 3-µm barrier filter, which supports the agarose plugs,
was removed and set up in a fresh petri dish with plugs remaining in
place. Fresh dGuo thymus lobes were placed adjacent to the agarose
plugs and cultured overnight to rescue any precursors that may have
migrated into the filter in response to thymic chemoattractants held in
the plugs. The dGuo thymus lobes were then set up in fresh culture for
an additional 12 days to reveal any rescued T cell precursors.
Flow cytometry
Two- and three-color labeling of thymocytes mechanically
isolated from recolonized thymi or reaggregate cultures was conducted
by incubation of the cells in a mixture of anti-CD4-PE and
anti-CD8-FITC or anti-CD4-PE, anti-CD8-APC, and
anti-Thy-1.2 FITC. To assess the purity of T cells isolated from
lymph node by immunomagnetic selection, cells were labeled with
anti-CD3 FITC. Analysis was performed using a Coulter Elite Dual
Laser machine (Coulter Electronics, Hialeah, FL) with forward and side
scatter gates set to exclude nonviable cells.
Statistical analysis
Statistical analysis, when required, was by ANOVA. Individual comparisons were made by the Tukey Pairwise Comparison test. The level of significance was defined as p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies have shown that T cell precursors in lymphoid fetal thymus lobes will migrate across a barrier filter into recipient dGuo-treated alymphoid thymus lobes when these are kept in association for 18–24 h (8). To establish the minimum time required for precursor migration, donor and recipient fetal thymus lobes associated in transfilter cultures were separated after varying periods of time, and the recipient tissues were cultured for an additional 12 days to reveal whether colonization by precursors had taken place.
Although recolonization of some lobes occurred within as little as
2 h (data not shown), a period of 6-h association was found to
result in a success rate of 85% recolonized recipient thymus lobes
(Fig. 2a), comparable with that seen after a
24-h association (data not shown). Such recolonized lobes supported a
pattern of T cell development comparable with that seen in age-matched
fetal thymus organ cultures (Fig. 2b). DGuo lobes not
exposed to donor tissue remained alymphoid, indicating a donor origin
of cells in colonized lobes. This was further confirmed using Thy-1.2
or Thy-1.1 donor and recipient combinations, in which >99% of
lymphoid cells in the lobes were shown to express the donor Thy-1
marker (Fig. 2c). These findings support earlier suggestions
that thymic stromal cells produce chemoattractant factors for T cell
precursors and further demonstrate that precursors can migrate rapidly
in response to such factors. In the light of these findings, a 6-h
association period was chosen for all subsequent experiments.
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In vivo studies in birds and mice (4, 5) have shown that
colonization of the thymic rudiment occurs in waves, suggesting that,
in some way, precursor recruitment is regulated. To determine whether
this is influenced by resident lymphoid cells, we compared the ability
of fd14 lymphoid and alymphoid fetal thymus lobes to attract T cell
precursors over a 6-h period using donor and recipient combinations
distinguishable by the expression of Thy-1.2 and Thy-1.1, respectively.
Individual recipient lobes were disaggregated after 6 h and
analyzed for the presence of migrant cells expressing the donor Thy-1.2
marker. As displayed in Fig. 3, donor-derived migrant
cells were readily detected in alymphoid thymus lobes, but few were
detected in lymphoid lobes, suggesting that the ability of thymus lobes
to attract T cell precursors may be regulated by their lymphoid
content.
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To obtain direct evidence that the attraction of precursors into
alymphoid thymus lobes involves the release of soluble, diffusible
factors, small agarose plugs were cocultured in close contact with
alymphoid thymus lobes to absorb any factors that were released. The
ability of these conditioned plugs to attract precursors from donor
thymus lobes was then compared with that of nonconditioned plugs.
Precursors that migrated into the filter in response to thymic
chemoattractants held in the plugs were revealed by the
addition of "rescue" dGuo thymus lobes to support their further
development. As shown in Fig. 4, a proportion of
conditioned plugs is able to attract precursors, indicating that they
have absorbed diffusible factors while in contact with the alymphoid
lobes. The reduced ability of these plugs to attract precursors as
compared with alymphoid fetal thymus lobes probably reflects the
limited amount of soluble chemoattractants they contain. This is less
likely to sustain a gradient over longer periods than alymphoid thymus
lobes capable of constant factor output. Nevertheless, the data provide
direct evidence for the release of diffusible factors with
chemoattractant properties for T cell precursors by the fetal thymus.
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To determine the specificity of chemoattractant production by the fetal thymic rudiment, we also examined the ability of a nonthymic epithelial/mesenchymal rudiment to attract T cell precursors. In these experiments, salivary gland rudiments from fd14 embryos, which contain both mesenchymal and epithelial components, were used. Salivary glands were assembled in transfilter cultures with donor fetal thymus lobes and cultured for 6 h before separation when any migrated precursors were rescued by fusion of the salivary gland rudiment with a dGuo-treated thymus lobe to support their further development. In contrast to the rescue experiment with factor-conditioned agarose plugs described in the previous section, no lymphoid cells were recovered from the rescue lobes associated with salivary rudiments (data not shown). This was not due to the failure of any precursors that had migrated to survive through the period before rescue, since isolated precursors cultured on a filter surface for 6 h remained viable and able to develop when a rescue lobe was added (data not shown). The failure of salivary rudiments to attract precursors in the rescue assay was further confirmed using the short-term transfilter assay. Thy-1.2+ migrant cells were not detected in salivary gland recipients in this assay (data not shown). Collectively, these data indicate that neither fetal mesenchymal cells nor the epithelial components of the salivary gland are able to produce T cell precursor chemoattractants.
MHC class II+ thymic epithelial cells are the source of thymic chemoattractants
Having established the production of diffusible chemoattractants
by thymic stromal cells, we next examined the specific cellular source
of these factors within the thymus. DGuo-treated alymphoid lobes
consist of a mixture of cells, including mesenchymal cells, MHC class
II+ epithelium, MHC class II- epithelium,
endothelial cells, and macrophages of hemopoietic origin. When such
lobes were disaggregated, depleted of CD45+ cells, and
reaggregated, the reaggregates retained the ability to attract
precursors, indicating that macrophages or any other residual cells of
hemopoietic origin surviving after dGuo treatment are not essential for
the production of chemoattractants (Fig. 5).
Importantly, when reaggregates consisting only of purified MHC class
II+ epithelium were used, these were also found to attract
precursors efficiently (Fig. 5). Since fibroblasts are required for
continued T cell maturation, the presence of precursors in reaggregates
of epithelial cells was revealed by the addition of a dGuo "rescue"
lobe after the migration period (22). However, nude thymic rudiments,
which are deficient in MHC class II+ epithelium (23), are
unable to attract precursors when used in rescue assays.
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For mature lymphoid and myeloid cells, recruitment and migration
are known to be regulated by members of the chemokine family (24).
Chemokines induce chemotaxis via interaction with seven-transmembrane
domain receptors coupled to PTX-sensitive heterotrimeric G proteins
(9). To determine whether the migration of T cell precursors in
response to thymic chemoattractants involved interactions with a G
protein-coupled receptor, we examined the effects of PTX on thymus
colonization using the transfilter migration assay. Concentrations of
PTX 2.5 to 5 times higher than those shown to inhibit chemokine-induced
migration of thymocytes in other systems were used to ensure saturation
of fetal tissue (14). The majority of recipient thymus lobes exposed to
250 and 500 ng/ml PTX during a 6-h colonization period failed to become
lymphoid upon subsequent culture (Fig. 6a).
In contrast, the cellularity of fd14 lymphoid lobes cultured for the
same period following a 6-h exposure to the same concentration of PTX
was unaffected as compared with untreated lobes, indicating that PTX
inhibits the initial migration of T cell precursors rather than their
subsequent proliferation. This was confirmed by direct analysis of the
number of Thy-1.2 donor cells recolonizing dGuo-treated alymphoid
Thy-1.1 recipient lobes during a 6-h association. As shown in Fig. 6b, the presence of 500 ng/ml PTX led to a marked reduction
in the number of T cell precursors migrating into the recipient lobes.
Collectively, these data suggest that chemotaxis of T cell precursors
in response to thymic stromal chemoattractants is mediated via a G
protein-coupled receptor, consistent with the factor being a member of
the chemokine family.
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To explore further the possible role of a chemokine/chemokine
receptor interaction in precursor recruitment to the thymus, we next
examined thymic and stromal cells and thymic precursors for the
expression of these molecules. To date a number of chemokine receptors
have been cloned and classified into two groups, CC chemokine receptor
and CXC chemokine receptor (24). RT-PCR analysis of purified fd14
thymic precursors revealed the expression of mRNA for CXC chemokine
receptor 4 and the CC chemokine receptors 1, 2a and 2b, 4, 5, and 8,
indicating that these receptors are potentially available to
participate in precursor recruitment. Consistent with this, a growing
number of chemokines have been shown to possess potent T cell and
thymocyte chemoattractant activity (24). Thus, we compared the
expression of chemokine mRNA in fetal thymic rudiments with known
ability to attract precursors with that in salivary gland rudiments
that lack this ability. Using RT-PCR, we revealed that a number of
chemokines were absent (MCP-5, lymphotactin) or expressed at very low
levels (MIP-1
, MIP-1ß, and MIP-2) in MHC class II+
thymic epithelium, but were expressed in nonattracting salivary
rudiments (Table I). Others, such as MIP-1, RANTES,
MCP-1, and SDF-1, were readily detected in both nonattracting and
attracting tissues (Table I) and in freshly isolated thymus lobes,
ruling out the possibility that chemokine expression is a consequence
of culture. Interestingly, the level of expression of MIP-1, RANTES,
MCP-1, and SDF-1 in attracting thymic epithelial cells did not exceed
that in nonattracting salivary epithelium (data not shown). In
contrast, the recently described thymus-associated chemokine TECK (14)
was differentially expressed at high levels in MHC class
II+ thymic epithelial cells, as compared with a
nonattracting tissue (Fig. 7). Moreover, this expression
of TECK mRNA detected in the thymus corresponded to protein production,
as shown by immunolabeling of tissue sections (data not shown). This
pattern of expression of TECK is consistent with a possible role for
this chemokine in thymus colonization.
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To determine whether TECK had chemoattractant properties for fd14
precursors, functional studies using modified Boyden chamber chemotaxis
assays were employed. Previous studies have examined the effect of TECK
on the migration of unfractionated murine thymocytes, but did not
determine its effect on immature precursor cells (14). As displayed in
Fig. 8a, fd14 precursors responded to TECK in
a biphasic fashion, a feature characteristic of chemokines, with
optimal migration obtained at 10 ng/ml TECK. A polyclonal Ab raised
against TECK blocked TECK-induced migration of fd14 precursors.
Checkerboard analysis demonstrated that the effect of TECK on fd14
precursors was chemotactic rather than chemokinetic in nature (Fig. 8b). In contrast, activated peripheral T cells were found to
be unresponsive to TECK, although they were able to mount a chemotactic
response to MCP-1 (Fig. 8c), suggesting that the effect of
TECK in migration is specific to immature T cells.
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Colonization of the fetal thymus by T cell precursors is dependent upon MMP activity
Precursors migrating from the pharyngeal vessels to the
thymic rudiment move through the perithymic mesenchyme and across the
basement membrane surrounding the epithelial rudiment, suggesting that
they are capable of degrading ECM components. To test this directly, we
used barrier filters coated with reconstituted basement membrane
material (Matrigel) occluding the pores in the filter in transfilter
cultures. As shown in Fig. 9a, this did not
prevent recolonization of the majority of recipient thymus lobes,
suggesting that the migrating precursors have the ability to move
through matrix proteins. In this context, the family of
matrix-disrupting enzymes, MMPs, is believed to play a critical role in
the degradation of the ECM during cellular migration and has been shown
to play a role in the migration of mature T cells (16). We therefore
investigated the effects of an inhibitor of MMPs, CT1847 (17, 18), on
thymus colonization. Exposure to CT1847 during a 6-h transfilter
association resulted in a dose-dependent reduction in the proportion of
recipient lobes that had become lymphoid upon subsequent culture (Fig. 9b). The cellularity of fd14 lymphoid lobes exposed to
CT1847 for the same period, and then cultured further, was unaffected,
suggesting that the effect of the inhibitor on MMP activity did not
result from nonspecific toxicity. These data support the notion that
thymus colonization is dependent upon MMP activity, a conclusion
further reinforced by the observation that fd14 precursors analyzed by
RT-PCR express gelatinase B (MMP-9) mRNA (Fig. 9c).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo studies in birds and mice have demonstrated that the thymic rudiment is colonized in discrete waves, suggesting that thymus colonization is a regulated process (3, 4, 5). In mice, the first wave of colonizing precursors enters the thymic rudiment between 10.5 and 12 days of gestation and gives rise to the first generation of thymocytes up until the end of the first week after birth (5). After a short refractory period, a second wave of precursors invades the thymus around birth giving rise to a second generation of thymocytes during the second week after birth (5). In our experiments, recipient lymphoid thymus lobes were taken at day 14 of gestation, during the refractory period following the first wave of colonization. We show that such lymphoid lobes attract precursors much less efficiently than alymphoid lobes, arguing that the output of thymic chemoattractants is regulated by the size and/or composition of the intrathymic lymphoid pool, with output increasing as this changes, so initiating the second wave of recruitment. It remains to be determined whether this reflects a decrease in chemoattractant synthesis or secretion in response to thymocyte epithelial cell interaction or a decrease in local consumption of chemoattractants as stem cell numbers fall, leaving sufficient to establish a gradient for further recruitment.
The PTX sensitivity of precursor migration in our assay indicates the involvement of G protein-coupled receptors that is characteristic of chemokine-mediated responses. Thus, the thymus chemoattractants may belong to this family of molecules. Chemokines are known to play a role in regulating the migration of mature T cells and adult thymocytes (24), and the recent demonstration that SDF-1 is important in B cell development in mice (28) suggests potential roles for chemokines in early lymphoid development. In support of this, we have been able to demonstrate the expression of mRNA for both CC and CXC chemokine receptors in fd14 thymic precursors. However, further studies will be required to define membrane expression of these receptors on those cells within the total 14 day precursor pool that are capable of migrating into an alymphoid lobe, as well as on the earliest cells to enter the thymus.
Constitutive expression of a number of chemokines within the thymus has also been reported, consistent with a role for chemokines in T cell precursor recruitment (24). Indeed, we revealed the expression of mRNA for a range of chemokines in the fetal thymus, and more specifically in MHC class II+ epithelial cells. However, most of the chemokines studied were also expressed, at least at the mRNA level, in fetal rudiments that lack the ability to attract precursors. If mRNA levels provide a reflection of protein secretion, the data suggest that these chemokines are unlikely to be primarily involved in thymus colonization. An exception to this is the recently described thymus-specific chemokine TECK (14) that is expressed at high levels in MHC class II+ epithelium, but at much lower levels in fetal tissues that lack the ability to attract precursors. Expression of TECK in the murine thymus was previously reported to be restricted to thymic dendritic cells (14), although our evidence clearly shows that it is also a product of MHC class II+ epithelium, at least in the fetal organ. TECK was shown recently to possess potent chemotactic activity for unfractionated murine thymocytes, but not for peripheral T cells (14). We have extended this observation to show that TECK induces migration of isolated immature fd14 thymic precursors, and that this is also specifically inhibited by an anti-TECK Ab. The expression of TECK in the fetal thymus and its ability to induce directed migration of thymic precursors make it a strong candidate for a role as a thymic chemoattractant factor. However, our inability to block recolonization of alymphoid lobes with a neutralizing Ab at concentrations up to 8 times greater than those shown to inhibit migration of isolated cells suggests that TECK does not have an exclusive role in T cell precursor recruitment to the fetal thymus. Even at fd14 the precursor population is heterogeneous (29), and it is possible that only the most immature precursors are responsible for recolonization in transfilter assays and that these cells respond to chemotactic factors, other than TECK, produced by alymphoid thymus lobes. However, a role for TECK in regulating the intrathymic behavior of responsive precursors remains possible, and deletion of the TECK gene in genetically modified mice will provide insights into its precise role in the thymus.
Histological analysis of the developing thymus rudiment shows that, in addition to responding to chemoattractants, colonizing precursors require the ability to move through cell matrix and basement membrane material (2). Mature T cells have been shown to utilize gelatinase type MMPs to disrupt tissue matrix (30, 31, 32). Our functional evidence that thymus-colonizing precursors can move through matrix material coupled with the expression of gelatinase B in thymic precursors and the inhibition of colonization by an MMP inhibitor strongly suggests that similar mechanisms are important in the colonization of the fetal thymus by stem cells. In this context, adhesive interactions between precursors, endothelial cells, and matrix proteins may also be important in facilitating their migration out of blood vessels and through the perithymic mesenchyme during thymus colonization. Precursor migration from the perivascular space along chemoattractant gradients toward the thymus rudiment may require ECM proteins as anchoring points (33), or for presentation of chemokines as a bound gradient (34). Some reports have also shown that chemokines can activate adhesive mechanisms in mature T cell responses (35, 36, 37). It will be interesting to investigate the complex relationships between chemoattractants, MMPs, and adhesion molecules on precursors colonizing the fetal thymus.
In conclusion, we have shown that the migration of T cell precursors into the early fetal thymus, in common with other migratory responses, involves response to a gradient of chemotactic factors and the ability to disrupt tissue matrix. Our findings argue strongly that the chemotactic factors involved are members of the chemokine family, suggesting that a continued search for novel thymus-specific chemokines will be important. Recent molecular cloning techniques and the use of expressed sequence tag (EST) databases are identifying a number of candidate molecules. Of particular interest will be novel members of the group of newly identified human chemokines that include thymus and activation regulated chemokine, EBI1 ligand chemokine, secondary lymphoid tissue chemokine, liver and activation regulated chemokine (LARC), and pulmonary and activation regulated chemokine, which, with the exception of LARC, are expressed at constitutively high levels in the thymus and other lymphoid tissues (24). The in vitro approaches used in this study provide convenient functional assays to investigate the roles of such novel chemokines in thymus colonization and development.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Beverley Wilkinson, Department of Immunology, IMM-8, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: PTX, pertussis toxin; dGuo, deoxyguanosine; ECM, extracellular matrix; fd, fetal day; MCP, monocyte-chemotactic protein; MIP, macrophage-inflammatory protein; MMP, matrix metalloproteinase; PE, phycoerythrin; RANTES, regulated upon activation, normal T cell expressed and secreted; SDF, stromal cell-derived factor; TECK, thymus expressed chemokine.
Received for publication September 18, 1998. Accepted for publication December 23, 1998.
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