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Up-Regulation of IL-7, Stromal-Derived Factor-1, Thymus-Expressed Chemokine, and Secondary Lymphoid Tissue Chemokine Gene Expression in the Stromal Cells in Response to Thymocyte Depletion: Implication for Thymus Reconstitution¹

Iryna Zubkova^*, Howard Mostowski and Marina Zaitseva^2,*

^* Division of Viral Products and Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Three in vivo adult mouse models were established to study which signals are required to restore the postnatal thymus. Single administration of dexamethasone, estradiol, or exposure to sublethal dose of gamma irradiation served as prototype thymus-ablating therapies. In all models, transient thymic atrophy was manifested due to the loss of the predominant portion of CD4^–CD8^– double negative and CD4⁺CD8⁺ double positive thymocytes and was followed by a complete regeneration of the thymuses. Acute atrophy/regeneration was observed in the dexamethasone and irradiation models; in the estradiol-treated animals, slow kinetics of atrophy and regeneration was observed. Importantly, in both acute and chronic models, high levels of IL-7 mRNA were detected in the thymuses isolated from mice during maximum atrophy. In addition, chemokine gene array analysis of involuted thymuses revealed high levels of mRNA expression of stromal-derived factor-1 (SDF-1), thymus-expressed chemokine (TECK), and secondary lymphoid tissue chemokine (SLC) but not of other chemokines. The levels of IL-7, SDF-1, TECK, and SLC mRNA inversely correlated with the kinetics of regeneration. RT-PCR analysis of stromal cells purified from involuted thymuses confirmed increased IL-7, SDF-1, and SLC gene expression in MHC class II⁺CD45^– epithelial cells and increased IL-7 and TECK gene expression in class II⁺CD45⁺CD11c⁺ dendritic cells. Thus, our data showed for the first time that expression of IL-7, SDF-1, TECK, and SLC mRNA is induced in the thymic stroma during T cell depletion and may play an important role in the reconstitution of the adult thymus.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Replenishment of T cells plays a critical role in immune recovery following bone marrow transplantation and the use of chemotherapeutic agents and during treatment of immune deficiency diseases, including HIV/AIDS. Peripheral expansion and thymic-dependent regeneration from the bone marrow progenitors are two main routes that contribute to the restoration of the T cell population (1). However, only the thymic-dependent pathway secures stable repopulation and diversity of the T cell repertoire. The function of the thymus is affected in various clinical situations. For example, HIV-1 infection, in vivo administration of estrogens, and synthetic analogues of cortisol were shown to induce thymic atrophy. Notably, clinical observations in humans and studies in animals have demonstrated that thymic function could be restored following the secession of treatment (steroids) or in the course of antiviral therapies (2, 3, 4). However, very few studies directly addressed the question of which salvage mechanisms are activated in the thymus in response to immunoablating therapies.

The thymus is primarily active during the prenatal period and in early life. In adults, the thymus undergoes age-dependent atrophy (5). However, involuted adult thymus has been shown to retain the ability to seed the periphery with new cells (6, 7). To support continuous development and release of T cells throughout life, the thymus periodically recruits bone marrow progenitor cells that circulate in the bloodstream (8, 9). Signals that attract circulating progenitors to the thymus are not known, although a feedback loop that is initiated in response to the migration of differentiating thymocytes from the sites of their original entry in the cortex has been suggested (10).

Thymic microenvironment, which is composed of the stromal cells of epithelial and nonepithelial origin, delivers signals that induce differentiation of the progenitor cells (defined as CD4/CD8 lineage double negative (DN)³ thymocytes) into the CD4⁺CD8⁺, double positive (DP) thymocytes (11, 12). The nonredundant signals that induce T cell lineage commitment are transmitted through the TCR, CD117 (c-kit), and Notch-1, which are expressed on the surface of the early thymocytes (13, 14, 15, 16). Thymic epithelial cells express IL-7 (17), which also plays a nonredundant role at early stages of T cell development in mice and in humans (18, 19, 20). IL-7 has been shown to stimulate proliferation of the DN thymocytes, development of T cells, and rearrangement of the TCR gene (21, 22, 23). However, the mechanism regulating IL-7 expression in the thymus has not been elucidated.

The signals for lineage commitment, for proliferation, and for death are delivered to differentiating thymocytes within the specific regions of the thymus. The movement of thymocytes into and within the thymus is governed by the interactions between chemokines produced by discrete stromal cells and chemokine receptors expressed by thymocytes (reviewed in Ref. 24). The switches in both chemokine receptor expression and chemokine response were shown to correlate with the transition between the stages of thymocyte maturation (25, 26). Specifically, the DN thymocytes migrate in response to the stromal-derived factor-1 (SDF-1; CXCL12) (27). The thymus-expressed chemokine (TECK; CCL25), along with other chemokines, has been implicated in the migration of DP thymocytes, and secondary lymphoid tissue chemokine (SLC; CCL21) has been shown to play a role in the migration of mature thymocytes (for review, see Ref. 28). Mouse cortical epithelial cells express SDF-1, in agreement with the location of the targeted population (27, 29), TECK is found in both cortical epithelial cells and medullary dendritic cells (DC) (30, 31), and SLC is found in the medullary region of the thymus (32). However, it is not known whether the expression of these chemokines is affected during regeneration of the postnatal thymus.

In this study, three in vivo animal models were used to investigate salvage mechanisms, which are activated in the thymus in response to immunoablating therapies. We demonstrate the induction of IL-7, SDF-1, TECK, and SLC mRNA expression in the involuted mouse thymus following in vivo administration of dexamethasone (Dex), estrogen, and following exposure to irradiation. Importantly, up-regulation of IL-7, SDF-1, TECK, and SLC mRNA correlated with the initiation of thymus reconstitution in adult mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice and protocol for in vivo treatments

Female BALB/c mice were purchased from the Charles River Laboratories and were used between 8 and 14 wk of age.

Transient thymic atrophy was induced in mice by injections with Dex in PBS or estradiol 17-valeriate (E2) in olive oil, both reagents from Sigma-Aldrich. Pilot studies were conducted to determine the dosage and route of administrations that induced thymic involution but that did not affect survival and general health status of the animals: 12.5 mg/kg i.p. and 5 mg/kg s.c. for Dex and E2, respectively. Age-matching control animals received PBS or olive oil alone in the Dex and E2 models, respectively. In separate experiments, transient thymic atrophy was induced in mice by subjecting them to sublethal (2 Gy) whole-body irradiation from a ¹³⁷Cs source (Gammacell-40 irradiator; Atomic Energy of Canada).

For detection of cell expansion in vivo, Dex was administered to mice at time 0, and the same animals were injected twice i.p. with 1 mg of BrdU solution (BD Biosciences) 20 and 16 h before sacrifice as described previously (33).

At indicated time points following in vivo administration of chemicals or irradiation, mice were euthanized by CO₂ asphyxiation, and their thymuses were excised, weighed, and used for RNA extraction and for the flow cytometry analysis. Three to seven mice were used at each time point for control and experimental groups, respectively. Mouse handling and experimental procedures were conducted in accordance with the National Institutes of Health Animal Research Committee guidance.

Isolation of stromal cells from the thymus

Digestions of the thymic tissues using collagenase were performed following a previously described protocol (34). In brief, thymuses were isolated from mice that were treated in vivo with Dex, E2, or that were exposed to irradiation or from age-matched control animals. Thymic tissues were first minced to allow release of thymocytes and then were incubated in 25 ml of RPMI 1640 on a magnetic stirrer twice for 15 min each at 4°C. The resulting thymic fragments were further separated with a 2-ml borosilicate glass pipette in 10 ml of the fresh RPMI 1640. Medium was changed three times, allowing thymic fragments to settle each time. Following mechanical disruption, thymic fragments were incubated in 5 ml of 0.125% (w/v) collagenase P (Roche) with 0.1% (w/v) DNase I (Ambion) in RPMI 1640 at 37°C three times for 15 min, each time in the fresh medium supplemented with enzymes with gentle pipetting every 5 min. Cells were collected during enzymatic digestions, washed with RPMI 1640, pulled, and used for FACS cell sorting.

Abs and flow cytometry

Abs used for staining and 7-aminoactinomycin D (7-AAD) dye were purchased from BD Biosciences. For evaluation of the thymic atrophy, cell suspensions were prepared from the thymuses of each mouse individually in the cold RPMI 1640 containing 5% FCS. Cells were stained with anti-CD4 mAb (clone GK1.5) conjugated to FITC and anti-CD8a mAb (clone 53-6.7) conjugated to PE in PBS supplemented with 0.5% BSA.

For the detection of BrdU incorporation, thymocytes were stained with anti-CD25 mAb (clone PC61) conjugated to PE and with anti-CD3 mAb (clone 145-2C11) conjugated to Cy5-PE, fixed, permeabilized, and tested for incorporated BrdU by intracellular staining using FITC BrdU Flow kit (BD Biosciences), according to the manufacturer’s instructions.

Twenty- to 50,000 viable cells were collected from each sample using FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences).

For cell sorting, cell suspensions obtained following collagenase/DNase I digestion were stained with a mixture of antibodies: anti-CD45 (clone 30-F11) conjugated to PE, anti-I-A^d (clone 39-10-8) conjugated to FITC, and anti-CD11c (clone HL3) conjugated to allophycocyanin. Cells were washed, resuspended in PBS supplemented with 5 mM EDTA, 1% FCS, and 7-AAD and were sorted on the FACSAria instrument using FACSDiVa software (BD Biosciences). Dead cells were excluded by gating on 7-AAD^– cells. CD45^–I-A^d+ epithelial cells and CD45⁺I-A^d+CD11c⁺ DC were collected into 1.5-ml vials (Sarstedt) containing 0.5 ml of RPMI 1640 supplemented with 20–30% FCS.

RNA isolation and RT-PCR analysis

Fragments of thymic tissues from individual mice were submerged in the lysis buffer from the RNeasy mini kit (Qiagen) and homogenized using a rotor-stator PCR Tissue Homogenizer (Omni International) to improve RNA extraction from the thymic stroma. Individually processed tissues were stored at –80°C. Total RNA was extracted and purified from the stored samples using RNeasy mini kit (Qiagen), according to the manufacturer’s instructions, and converted into the first-strand cDNA using oligo(dT) primers (Promega) and Moloney murine leukemia virus-reverse transcriptase (Invitrogen Life Technologies). Aliquots of cDNA were amplified by PCR using Taq polymerase (Applied Biosystems) under standard conditions.

For RT-PCR amplification of RNA following FACS sorting, cells collected in 1.5-ml microtubes were immediately pelleted on a tabletop Eppendorf centrifuge equipped with a swing rotor for 2 min at maximum speed. Supernatants were discarded, lysis buffer from the Cells-to-cDNA kit (Ambion) was added to the pellets (1 µl/400-1000 cells), and the cell lysates were incubated at 75°C for 15 min. Reverse transcriptase reactions were performed on samples stored at –80°C using Cells-to-cDNA Kit (Ambion), following the manufacturer’s instructions. Ten microliters of cDNA were amplified in each semiquantitative PCR using SuperTaq Plus polymerase (Ambion) and several different numbers of cycles to assure linear relationship between RNA concentration and PCR product.

The following primer pairs were used for PCR: IL-7 sense, 5'-GTCACATCATCTGAGTGCCACA-3', and antisense, 5'-GTAGTCTCTTTAGGAAACATGCATC-3' (Stratagene); -actin sense, 5'-GTGGGCCGCTCTAGGCACCAA-3', and antisense, 5'-CTCTTTGATGTCACGCACGATTTC-3' (BD Biosciences); SDF-1 sense, 5'-CAGTGACGGTAAACCAG-3', and antisense, 5'-ATATGCTATGGCGGAGT-3'; and SLC sense, 5'-CCTTAAGTACAGCCAGAAG-3', and antisense, 5'-ATGAGGTGGCTGCTTTG-3'. Both sets were obtained from SuperArray Bioscience. Primer pairs for TECK sense, 5'-TTTGAAGACTGCTGCCTGG-3', and antisense, 5'-GTCTTCTTCCTAACAAGCC-3', for GAPDH sense, 5'-TGCACCACCAACTGCTTAG-3', and antisense, 5'-GATGCAGGGATGATGTTC-3', for keratin-8 sense, 5'-CTCCGGCAGATCCATGAAGA-3', and antisense, 5'-GCTCGGCTGCGATTGG-3', and for keratin-14 sense, 5'-GGATGTGAAGACAAGGCTGGA-3', and antisense, 5'-AAGCCTGAGCAGCATGTAGCA-3' were synthesized by Integrated DNA Technologies, according to reported sequences (35, 36, 37, 38).

Amplified DNA fragments (321 bp for IL-7, 742 bp for TECK, 430 bp for SLC, 392 bp for SDF-1, 180 bp for keratin-8, 235 bp for keratin-14, 176 bp for GAPDH, and 540 bp for -actin) were resolved by electrophoresis in the 2% ethidium bromide-agarose gels and scanned using the AlphaImager Imaging System (Alpha Innotech). PCR products for IL-7 and chemokines were expressed relative to -actin or to GAPDH as indicated.

Targeted gene array analysis

Generation of labeled cDNA, hybridization, washings of the membranes, and analyses were performed using the mGEA Chemokine kit (SuperArray Bioscience), following the manufacturer’s instructions. In brief, radiolabeled cDNA was generated from 10 µg of total RNA using Moloney murine leukemia virus-reverse transcriptase enzyme (Promega) in the presence of RNase inhibitor (Promega) and [-³²P]dCTP (10 µCi/µl; Amersham Biosciences). Before hybridization, the array membranes were incubated with the prehybridization buffer in the presence of heat-denaturated salmon sperm-DNA (100 µg/ml) in a rotary hybridization oven at 68°C for 2 h. Membranes were hybridized with the ³²P-labeled cDNA for 16 h at 68°C with rotation. After hybridization, the membranes were washed with 2x SSC, 1% SDS, followed by 0.1x SSC, 0.5% SDS, three times with each buffer. The membranes were wrapped in Saran wrap, exposed to Imaging Plate (Fujifilm Medical Systems) for 1.5 h, and scanned on the PhosphorImager FLA-3000 (Fuji) at 100-µm resolution. Images were subsequently analyzed with ImageQuant Software (Fuji) and converted into a table of signal intensities. Further data analysis was performed using Microsoft Excel. A background signal derived from hybridization with the negative control of pUC18 DNA was subtracted from all samples.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Thymic atrophy in BALB/c mice induced by a single administration of Dex

To determine which signals are required for the reconstitution of the postnatal thymus, we sought to establish the in vivo model of transient thymic atrophy in adult mice. Following a single injection with Dex or with PBS, female BALB/c mice were sacrificed at the indicated times; their thymuses were removed and analyzed for the degree of atrophy by measuring thymic cellularity (Fig. 1A). The administration of Dex induced a dramatic reduction in the weight of thymuses (data not shown) and a 10-fold reduction in the total number of cells in the thymus 2–4 days posttreatment. Recovery of thymic cellularity started on day 5, and within 1 wk, thymic cell numbers reached control levels (Fig. 1A).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. In vivo model of transient thymic atrophy in adult mice following single administration of Dex. A, BALB/c mice were injected i.p. with 12.5 mg/kg Dex or PBS as control. Thymic cellularity was measured in Dex-treated mice and in the age-matched, PBS-treated control animals. B, Absolute number of DN and DP thymocytes in Dex-treated mice. FACS analysis was performed on thymocytes isolated from mice that received a single injection with Dex or PBS in control. DN and DP cell number was obtained by multiplying the percentage of CD4–CD8– thymocytes or CD4+CD8+ thymocytes by the total cellularity. Groups of three and seven mice were used at each time point in control and in treated groups, respectively. The error bars represent the mean values ± SD for individual mice. The data are representative of three separate experiments.

IL-7 mRNA expression in regenerating thymic tissues following Dex-induced atrophy

Earlier studies had demonstrated the critical role played by IL-7 in the early development of thymocytes in mice and humans (21, 40). To determine whether the levels of IL-7 mRNA expression are altered during transient thymic atrophy and during regeneration, RNA samples were extracted from thymuses of individual mice using mechanical homogenization and amplified by RT-PCR with IL-7- and -actin-specific primers (Fig. 2). Low levels of IL-7 mRNA were detected in the thymuses from control animals at all time points (Fig. 2A). However, 2.5- to 4-fold increases in IL-7 mRNA expression were detected in all animals on days 2 and 3 following Dex injection (Fig. 2B). IL-7 mRNA expression gradually returned to control levels between days 4 and 14 posttreatment. These data show that Dex administration induces transient increase in the IL-7 mRNA expression in the involuted thymuses.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Analysis of IL-7 mRNA expression in the thymuses of the Dex-treated mice. A, Thymic tissues were excised from control (lanes 1–3) or from Dex-injected (lanes 4–10) mice at indicated times. RNA was extracted following mechanical homogenization of the thymus, and RT-PCR was performed with IL-7- and with -actin-specific primer pairs for 24 and 30 cycles, respectively. Each band represents an individual mouse. B, Data were normalized using signals from -actin and were expressed as fold increases over the levels of IL-7 mRNA expression in PBS-treated, age-matched control animals. Mean of seven mice per group ± SD are shown.

To determine whether the increase in intrathymic IL-7 mRNA in Dex-treated involuted thymuses precedes expansion of the DN subset, the kinetics of cycling DN thymocytes was evaluated. Mice received a single injection with Dex and then two subsequent injections with BrdU at 20 and 16 h before sacrifice. The numbers of CD25⁺ DN thymocytes committed to T cell lineage (41) and the proportions of proliferating CD25⁺ thymocytes were determined by the immunofluorescence analysis of thymocytes using Abs recognizing CD25 Ag and BrdU (to mark DNA synthesis) (Fig. 3). To exclude from the analysis the CD3⁺CD4⁺ regulatory cells that express CD25 marker and that are present in the small numbers in the thymus (42), only CD3^–CD25⁺ thymocytes were evaluated for BrdU incorporation. Dex treatment induced a modest reduction in the percentage of cycling BrdU⁺CD25⁺ thymocytes on day 2 that was followed by a 4-fold increase in the proportion of cycling BrdU⁺CD25⁺ cells on day 3 and gradual return to control levels on days 5–7 (Fig. 3). At the same time, the absolute numbers of CD25⁺ thymocytes were reduced 10-fold on day 2 posttreatment, suggesting that a large portion of CD25⁺ thymocytes is susceptible to Dex-induced cell death. The numbers of CD25⁺ thymocytes gradually recovered between days 3 and 5. These data suggest that the increase in the proportion cycling CD25⁺ thymocytes contributed to the restoration of the CD25⁺ DN subset at the early stages of regeneration. The kinetics data further suggest that up-regulation of IL-7 mRNA expression in the involuted thymuses occurs before the expansion of the pool of cycling CD25⁺ thymocytes and precedes the recovery of their absolute numbers (compare Figs. 2 and 3).

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Kinetics of BrdU labeling of the CD25+ thymocytes during thymic atrophy. Mice were injected with a single dose of Dex at time 0 followed by two subsequent BrdU injections at 20 and 16 h before indicated time points. The percentage of CD3–CD25+ thymocytes that incorporated BrdU in vivo was determined by FACS analysis of total thymocytes. The absolute number of CD3–CD25+ thymocytes was determined by multiplying the percentage of CD25+ thymocytes by the total cellularity. Each point and vertical bar indicate the mean ± SD of five mice per group. The data are representative of three separate experiments.

The increase in IL-7 mRNA could have occurred due to direct effect of steroid treatment or in response to thymocyte depletion. Therefore, two additional models of transient thymic atrophy were established. Mice were subjected to total body sublethal gamma irradiation (Fig. 4A) or received a single dose of E2 or olive oil as control (Fig. 4B); their thymuses were removed and evaluated for levels of atrophy. The kinetics of changes in the thymic cellularity (data not shown) and in the absolute numbers of DP and DN thymocytes in irradiated mice resembled those observed in Dex-treated animals (compare Figs. 4A and 1B). The acute reductions in the number of DP and DN thymocytes were observed between days 2 and 4 posttreatment (Fig. 4A). In contrast, in E2-injected mice, thymic cellularity (data not shown) and the numbers of DN and DP thymocytes were reduced at a later time points than in the Dex-treated or in the irradiated animals (compare Fig. 4B with Figs. 4A and 1B). In this model, the reduction in the numbers of DP and DN thymocytes was observed between days 5 and 12 and was followed by recovery between days 14 and 21, reaching control numbers by week 8.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. In vivo model of transient thymic atrophy induced in mice by sublethal irradiation or following administration of a single dose of E2. BALB/c mice were left untreated or exposed to gamma irradiation at 2Gy (A) or were injected s.c. with 5 mg/kg E2 or olive oil as control (B). The percentage of DN and DP thymocytes was determined by FACS analysis. DN and DP cell number was obtained by multiplying the percentage of CD4–CD8– thymocytes or CD4+CD8+ thymocytes by the total cellularity. Each point represents the mean ± SD of six mice per group. Representative data from three similar experiments are shown.

To determine whether irradiation or E2 injection affect intrathymic IL-7 mRNA expression, RNA samples were isolated from the thymic fragments of individual mice and were amplified by RT-PCR using IL-7-specific primers (Fig. 5). As was observed in the Dex model, transient increases in the levels of IL-7 mRNA expression were detected on days 2 and 3 in the thymuses of irradiated animals (Fig. 5A). E2 treatment induced increases in intrathymic IL-7 mRNA on day 5, which was followed by peak expression on day 7, after which the IL-7 mRNA expression started to diminish but remained at elevated levels throughout the observation period (Fig. 5B). These data demonstrate that intrathymic IL-7 mRNA expression is up-regulated during atrophy induced by three different thymic ablating treatments and that the levels of IL-7 mRNA expression inversely correlate with the levels of regeneration. The kinetics data also suggest that an increase in the IL-7 mRNA expression precedes restoration of thymic cellularity and normalization of the DN:DP ratio.

View larger version (12K): [in this window] [in a new window]   FIGURE 5. Up-regulation of IL-7 mRNA expression in mouse thymuses in the irradiation and in the E2 models. BALB/c mice were irradiated with 2Gy (A) or were injected with E2 (B). At indicated time points, thymuses were excised from mice. Total RNA was extracted from the thymic fragments and amplified in RT-PCR with IL-7- and -actin-specific primers. Data were normalized using a signal from -actin and were expressed as fold increases compared with age-matched untreated mice (A) or mice injected with olive oil (B). Each point represents the mean ± SD of five mice per group. Representative data from three experiments are shown.

To determine which signals in addition to IL-7 may be induced in response to thymic atrophy, targeted gene array experiments were performed. RNA samples were extracted from the thymuses of Dex-treated and E2-treated mice during maximum atrophy. Extracted RNAs were converted into radiolabeled cDNA probes for hybridization with nylon membranes expressing mouse chemokine genes (Fig. 6). The signal intensities of the majority of the chemokines were below detection (Table I). CCL11 (eotaxin), CX3CL1 (fractalkine), CXCL10 (IFN--inducible protein 10), CCL8 (MCP-2), CXCL9 (monokine induced by IFN-), and CCL5 (RANTES) chemokines were increased following treatment; however, the levels of their expression were still very low (Fig. 6 and Table I). In contrast, high levels of CXCL12 (SDF-1), CCL25 (TECK), and CCL21 (SLC) mRNA were present in control animals and were up-regulated (2.7- to 5.4-fold) in involuted thymuses (Fig. 6 and Table I). These data suggest that in addition to IL-7 the chemokines SDF-1, TECK, and SLC are expressed at elevated levels during transient atrophy.

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Analysis of chemokine mRNA expression in the thymic tissues using targeted gene arrays. Total RNA was extracted from the thymic tissues of mice following administration of Dex (PBS as control) or E2 (olive oil as control) on days 3 and 7 posttreatment, respectively. Radiolabeled cDNA was generated from the extracted RNA and hybridized with the array membranes containing duplicates of chemokine cDNA fragments. Colored boxes indicate location of individual chemokine cDNA fragments on the membranes. Representative digitized images of the four arrays analyzed in one experiment are provided. Experiment was performed three times.

Kinetics of intrathymic SDF-1, TECK, and SLC mRNA expression in the Dex- and E2-treated animals

To confirm that SDF-1, TECK, and SLC chemokines are up-regulated in involuted thymuses and to determine the kinetics of chemokine mRNA expression, RT-PCR was performed on RNA extracted from mouse thymuses at several times following in vivo treatments (Fig. 7). In agreement with our array data, 3- to 6-fold increases in the levels of all three chemokines were detected on days 2 and 5 in Dex- and E2-treated animals, respectively (Fig. 7). Elevated levels of SDF-1, TECK, and SLC mRNA were preserved during maximum atrophy (days 3 and 7 in Dex and E2 models, respectively), after which, gradual reduction in chemokine mRNA expression was observed (Fig. 7). These data demonstrate that the kinetics of SDF-1, TECK, and SLC expression correlates with the kinetics of IL-7 expression in the regenerating thymuses.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Kinetics of TECK, SDF-1, and SLC mRNA expression in mouse thymuses in the Dex and E2 models of thymic atrophy and regeneration. BALB/c mice were injected with Dex or PBS as control (A) or with E2 or olive oil as control (B). At indicated times, thymuses were excised from treated and from control animals. RNA were extracted from the thymic fragments and were amplified by RT-PCR with TECK ()-, SDF-1- ()-, and SLC ()-specific primers for 20, 25, and 26 cycles, respectively. Data were normalized using PCR amplification of the -actin mRNA and expressed as a fold increase over the values from control animals. Each point represents the mean ± SD of five mice per group. Representative data from three experiments are shown.

Increased expression of IL-7, SDF-1, and TECK mRNA in epithelial and DC sorted from involuted thymuses

To verify that the increases in IL-7, SDF-1, TECK, and SLC mRNA expression were mediated by increased gene expression, thymuses were isolated from experimental and from control animals, and the thymic fragments were subjected to collagenase/DNase I treatment. The single-cell suspensions were sorted on the FACS instrument (Fig. 8). MHC class II⁺CD45^– epithelial cells and MHC class II⁺CD45⁺CD11c⁺ DC were collected and tested for the IL-7, SDF-1, SLC, TECK, keratin-8, keratin-14, and GAPDH mRNA expression using semiquantitative RT-PCR with several different numbers of PCR cycles (Fig. 8). MHC class II⁺CD45^– cells sorted from the intact and involuted thymuses expressed keratin-8 and -14 mRNA confirming their epithelial origin. No or only modest differences were detected in the levels of keratin-14 and of keratin-8 expression in epithelial cells derived from control and experimental mice. In contrast, 4- to 6-fold higher levels of IL-7, 9- to 16-fold higher levels of SDF-1, and 11- to 14-fold higher levels of SLC mRNA were detected in the epithelial cells isolated from the thymuses of irradiated, Dex-treated, or E2-treated mice compared with control animals. TECK gene expression remained constant. MHC class II⁺CD45⁺CD11c⁺ DC isolated from the same involuted thymuses exhibited 5- to 6-fold increases in TECK mRNA, whereas IL-7 levels were up-regulated 2- to 4-fold in this cell type (Fig. 8). No keratin-8 or keratin-14 message was detected in RNA isolated from DC, suggesting that sorted DC were not contaminated with epithelial cells (data not shown). In addition, SDF-1 and SLC messages were not expressed in the intrathymic DC (data not shown). These data demonstrate that up-regulation of IL-7, SDF-1, TECK, and SLC mRNA is mediated by increased gene expression. Taken together, our data generated in three animal models suggest that increases in the levels of IL-7, SDF-1, TECK, and SLC mRNA expression in the thymic stromal cells are induced in response to the depletion of thymocytes and occur before thymic reconstitution.

View larger version (40K): [in this window] [in a new window]   FIGURE 8. Analysis of IL-7, SDF-1, and TECK mRNA expressions in the stromal cells isolated from the involuted thymuses. Mice were injected with PBS (lanes 1–3 and 13–15), were exposed to 2 Gy of gamma irradiation (lanes 4–6 and 16–18), or were injected with E2 (lanes 7–9 and 19–21) or with Dex (lanes 10–12 and 22–24). On day 3 following PBS, Dex, and irradiation treatments or on day 7 following E2 administration, thymic tissues were excised and were subjected to four-color FACS cell sorting. Dead cells were excluded using labeling with 7-AAD. Dot blot in the top left panel shows gating used to collect MHC class II+CD45– epithelial cells (lower gate). DC were collected from the subset of MHC class II+CD45+ cells (dot blot, upper gate) by additional gating on CD11c+ cells as shown in the histogram (right panel). Seven to 800 sort-purified epithelial cells and 2000 DC were used in RT-PCR. Wedges indicate PCR cycle number (from left to right) for epithelial cells, keratin-8 (K-8) (30 35 40 ), keratin-14 (K-14) (30 32 35 ) SDF-1 (34 36 38 ), SLC (28 30 33 ), TECK (23 25 28 ), IL-7 (28 33 38 ), and GAPDH (27 29 34 ); for DC, IL-7 (30 33 35 ), TECK (28 30 33 ), and GAPDH (28 30 33 ). Experiment was performed three times.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In AIDS patients, in patients with SCID, and in individuals with acute lymphoblastic leukemia an inverse correlation between the levels of IL-7 protein in the plasma and lymphopenia have been reported (46, 47, 48). Based on these observations it was postulated that IL-7 protein is accumulated in the plasma of lymphopenic patients as a result of reduced consumption (49). In our mouse experimental models, increased levels of IL-7 mRNA were detected during lymphopenia of the thymus. Importantly, we demonstrated that the increase in IL-7 mRNA was mediated by the increased IL-7 gene expression in the epithelial cells isolated from the involuted thymuses. Thus, we provide the first evidence in support of a feedback mechanism that is activated in response to T cell depletion and that regulates IL-7 gene transcription in vivo in the thymic epithelial cells. Our results suggest that up-regulation of IL-7 expression following T cell ablating treatments may represent a general compensatory mechanism that takes place not only in the thymus but also in the periphery.

A role for IL-7 in the development of DN immature thymocytes in mice and humans has been indicated by several earlier studies (21, 40). The DN thymocytes are not homogeneous and include cells at phenotypically and genetically different stages with so-called DN2 (CD25⁺CD44⁺) and DN3 (CD25⁺CD44^–) subsets comprising a pool of cells committed to T cell lineage. Cells at the DN2 stage were shown to require IL-7 for survival and proliferation (50, 51). In our experiments, a transient increase in the percentage of cycling CD25⁺ DN2/3 thymocytes was observed following in vivo Dex treatment and following irradiation (Fig. 3 and data not shown). These data are in agreement with earlier studies demonstrating that DN2 and DN3 are the most actively proliferating populations in the thymus (52). Notably, our kinetics experiments demonstrated that IL-7 mRNA up-regulation precedes expansion of CD25⁺ DN2/3 thymocytes (Figs. 2 and 3B). These data suggested that the increase in the intrathymic IL-7 expression might provide an important early signal to restart thymopoiesis. The increase in the numbers of CD44^–CD25^– DN4 was also detected following their transient depletion (data not shown). However, expansion of DN4 thymocytes might be mediated by factors other than IL-7 because it has been shown that DN4 thymocytes expressing low levels of IL-7R do not respond to IL-7 (53). Recent studies using gene-modified stromal cell as a cytokine delivery system demonstrated that the excess of intrathymic IL-7 could significantly improve early thymopoiesis by increasing the numbers of DN2 cells in the involuted thymuses of aged mice (54). However, the same study clearly showed that IL-7 alone was not sufficient to induce development of thymocytes beyond DN2/3 stage.

Which signals in addition to IL-7 are required to restart thymus reconstitution? Using targeted gene arrays, significant increases in the expression of SDF-1, TECK, and SLC chemokine mRNA were detected in the thymic tissues during profound atrophy. These data are in agreement with previous reports demonstrating intrathymic expressions of these chemokines and their role in the migration of thymocytes (28, 31, 55, 56, 57). Importantly, the up-regulation of SDF-1 expression detected in our experiments in adult mice provides additional support for the recently published observation on the critical role of SDF-1 in the migration of early thymocyte progenitors in the postnatal thymus (27, 58). In our study, analysis of the kinetics of chemokine expressions detected an accumulation of chemokines in the thymic tissues even before the peak atrophy in the acute and chronic models. Thus, chemokine up-regulation may represent one of the intrinsic compensatory mechanisms that secure the reinitiation of thymopoiesis. It is interesting to note that, in another study, only SDF-1, TECK, and SLC were detected in the alymphoid early thymic anlage and that fetal blood prothymocytes destined to colonize the thymus responded to the embryonic chemokines SDF-1 and TECK (59). These data together with the results of our study demonstrate a marked similarity between the signals that control thymic regeneration following transient atrophy in adult animals and the signals that control the population of the thymus in the embryo.

Profound T cell depletion resulted in the higher proportion of non-T cells in the involuted thymuses. As a consequence, RNA samples prepared from these tissues were enriched in RNA derived from the thymic stroma. Therefore, it was important for us to confirm up-regulation of IL-7, SDF-1, TECK, and SLC mRNA in the stromal cells purified from involuted thymic tissues. In agreement with previous reports (17, 29, 30, 32, 60), IL-7 and TECK mRNA were detected in the sorted epithelial cells and in DC, whereas SDF-1 and SLC mRNA were found in epithelial cells only. Importantly, the mRNA of IL-7, SDF-1, and SLC were significantly up-regulated in epithelial cells following thymic ablating treatments. Because cortical (keratin 8⁺14^–) and medullary (keratin 8^–14⁺) epithelial cells express class II molecules (34), it is not clear whether in our experiments cortical or medullary epithelial cells in regenerating thymuses up-regulated IL-7, SDF-1, and SLC. Recent studies have shown that under normal conditions IL-7-expressing cells are located throughout the medulla and at the corticomedullary junction of the thymuses in the adult animals and in the subcapsular region in the newborns (61). Studies are underway to determine the location of IL-7-expressing cells in the regenerating postnatal thymuses.

No differences were detected in the levels of TECK mRNA in isolated epithelial cells between experimental and control animals. These data suggested that it is possible that the signals that regulate IL-7, SDF-1, and SLC production in intrathymic epithelial cells differ from those that control TECK or that different types of class II⁺ epithelial cells express IL-7, SDF-1, SLC, and TECK.

Our sorting experiments demonstrated increased TECK gene expression in DC sorted from involuted thymuses suggesting that DC play an important role in regulating restoration of the DP subset during regeneration (28). In addition, IL-7 mRNA was expressed in purified DC, in agreement with previous studies in humans (62, 63). The increase in the IL-7 expression observed in DC isolated from involuted thymuses may play an important role in providing necessary milieu for differentiation of regenerating DP thymocytes into mature CD8⁺CD4^– single positive thymocytes in the medulla (64). Thus, our data demonstrate for the first time the activation of IL-7, SDF-1, SLC, and TECK gene expression in the stromal cells of the involuted thymuses.

Although some of the signals that govern the migration and differentiation of the progenitors and early thymocytes are already described, it is not known what mechanisms regulate their initial activation. A recent elegant study demonstrated that occupation of the hypothetical stromal niches in the thymic cortex by the DN2/3 cells prevented colonization of the thymus with precursors, and the release of stromal niches from DN2/3 induced reconstitution (10). These data suggested that DN2/3 thymocytes play a major role in controlling the ability of the stromal cells to recruit progenitors to the thymus and to regulate their intrathymic differentiation through a hypothetical feedback reaction (10). It is not clear from our experiments the depletion of which individual subset (DN2, DN3, DN4, or DP) initiated feedback reaction. However, because DN2/3 thymocytes were shown previously to play a major role in controlling thymic cellularity (10), it is possible that in our experimental system up-regulation of IL-7, SDF-1, TECK, and SLC gene expressions in the stromal cells was induced by depletion of DN2/3 subsets, although the role of DN4 and DP thymocytes cannot be formally excluded.

It is important to note that in vivo administrations of rIL-7 have been shown to improve the rate of T cell immune reconstitution following bone marrow transplantation and chemotherapy in mice (49). However, the potential of developing anticytokine Abs cannot be excluded, as this was documented for cancer patients receiving rGM-CSF therapies (65, 66). Our data suggest that, in the absence of ablating treatments, the intrinsic capacity of the stromal cells to generate feedback signals is limited by resident thymocytes. The mechanism of this T cell-mediated impediment to produce IL-7 and chemokines is not known and could be mediated by various types of lymphostromal interactions. Our results indicate that this impediment could be removed, and endogenous productions of thymus recovery signals could be induced in vivo. Development of treatments that activate these signals in the thymic stroma could provide a new strategy in the design of therapies for immune reconstitution.

	Acknowledgments

 
We express our gratitude to Drs. Hana Golding, Keith Peden, and Remy Bosselut for carefully reviewing the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a grant from the Office of Women’s Health, Food and Drug Administration (FDA), and by Counter Terrorism funds from Center for Biologics Evaluation and Research, FDA.

² Address correspondence and reprint requests to Dr. Marina Zaitseva, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Building 29B, Room 4NN06, 8800 Rockville Pike, Bethesda, MD, 20892. E-mail address: zaitseva{at}cber.fda.gov

³ Abbreviations used in this paper: DN, double negative; DP, double positive; SDF-1, stromal-derived factor-1; TECK, thymus-expressed chemokine; SLC, secondary lymphoid tissue chemokine; DC, dendritic cell; Dex, dexamethasone; E2, estradiol 17-valeriate; 7-AAD, 7-aminoactinomycin D.

Received for publication December 8, 2004. Accepted for publication May 26, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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