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Department of Pathology and Laboratory Medicine and the Jonsson Comprehensive Cancer Center (E.M.-R., K.D.), University of California, Los Angeles School of Medicine, Los Angeles, California 90025-1732; and the Research Center for Developmental Medicine and Biology (R.C.), Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand
Address all correspondence and requests for reprints to: Dr. Kenneth Dorshkind, Department of Pathology 173216, University of California, Los Angeles School of Medicine, 10833 Le Conte Avenue, Los Angeles, California 90095-1732. E-mail: kdorshki{at}pathology.medsch.ucla.edu
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Abstract |
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Introduction |
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The production of GH and IGF-I decline steadily with age (10, 11, 12, 13, 14, 15), which in turn, could be a factor that contributes to the decline of thymopoiesis. This possibility has formed the rationale for studies in which GH or IGF-I was administered to old individuals in an attempt to rejuvenate the involuted thymus. Although thymus cellularity increases in aged rodents treated with either GH or IGF-I, a consistent finding was that cell production was not restored to levels present in young animals. For example, whereas IGF-I treatment doubled the number of cells in the thymus of 9-month-old mice, the absolute number of thymocytes remained only 25% of that present in young animals (16). Similarly, GH treatment increased thymic cellularity in aged animals by only 10–20 million thymocytes (15, 17, 18). The similarity of GH and IGF-I effects is not surprising, because IGF-I is thought to mediate many GH actions (19). Higher increases in thymic cellularity were observed after transplantation of GH-secreting GH3 tumor cells into 18- to 24-month-old rats (20, 21), although the total thymocyte number still remained below numbers in young rats. Taken together, these findings suggest that there may be limits on the extent to which GH, IGF-I, or other thymopoietic hormones can stimulate thymopoiesis in old animals. One reason for this is that additional defects develop during aging.
Deficiencies that have been reported in the bone marrow T cell precursor compartment of old animals would be particularly relevant to this hypothesis. Sustained cell production in the thymus is thought to be dependent on the migration of T cell precursors from the marrow to thymus (22), and the frequency of bone marrow thymocyte precursors in old mice has been shown to be reduced by 40-fold. In addition, these cells display a reduced potential to colonize the thymus in vitro (23, 24, 25, 26) and in vivo (27). These observations raise the possibility that hematopoietic defects that accumulate during aging limit the degree to which hormones can rejuvenate the involuted thymus and suggest that treatment of aged mice with a combination of hormone therapy and young hematopoietic cells should be more effective at increasing thymus cellularity than administration of hormones alone.
The aim of the present study was to test this hypothesis in an in vivo model. Accordingly, 18-month-old mice were treated with IGF-I only or with IGF-I and young hematopoietic cells. The results indicate that thymus cellularity in the latter group of mice was significantly higher than in animals treated with hormone alone. In vitro studies, using the fetal thymic organ culture (FTOC) system, suggest that IGF-I acts by potentiating thymus colonization by bone marrow T cell precursors and/or some early event during T cell differentiation.
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Materials and Methods |
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Preparation of cell suspensions. Mice were killed by
cervical dislocation. Single-cell suspensions of thymocytes or
splenocytes were prepared in 
-MEM (GIBCO, Grand Island, NY),
containing 5% FCS (Hyclone, Logan, UT), by gently pressing the
organs through a fine-mesh screen. Viability was always greater than
95%. All cell counts were performed in a hematocytometer.
IGF-I Administration. Recombinant human IGF-I (100 µg/day; Genentech Inc., San Francisco, CA) or saline was administered continuously to mice via 14-day miniosmotic pumps implanted sc (Refs. 16, 28 ; ALZET no. 2002, Palo Alto, CA). Mice were anesthetized with Avertin, and pumps were implanted into animals through a small incision inferior to the scapulae. The preparation of recombinant human IGF-I used in these studies has been shown to be poorly immunogenic in rodents (Ref. 29 ; Clark, personal communication).
Bone marrow transplantation. In some experiments, old mice received a transplant of 5–10 x 106 young syngenic bone marrow cells by iv injection. Mice were preconditioned before transplantation with 400 R from a 137Cs irradiator (85.8 R/min; J. L. Shepherd, San Fernando, CA). This dose was chosen based on preliminary studies showing that higher doses were lethal to old mice.
Flow cytometry. After lysis of red blood cells by hypotonic
shock in Tris-ammonium chloride, 106 cells were incubated
initially with an anti-Fc
-receptor antibody (anti-CD32/16;
Pharmingen, San Diego, CA) to block nonspecific binding of antibodies.
The samples were then incubated with anti-Thy-1, CD4, or CD8 antibodies
for 30 min at 4 C. Antibodies were conjugated to fluorescein,
phycoerythrin, or biotin. Biotinylated antibodies were revealed with
phycoerythrin-streptavidin (all from Pharmingen). The cells were then
washed and resuspended in cold calcium/magnesium free PBS and analyzed
on a FACScan (Becton-Dickinson, San Jose, CA). Dead cells were
excluded based on their low forward scatter and high side-scatter
profiles.
Cells were separated, on the basis of their CD4/CD8 phenotype, on a Becton-Dickinson FACStar. Purity of the population isolated was confirmed by reanalyzing an aliquot of it.
PCR. RNA from fluorescence-activated cell sorter-purified thymocytes was reverse-transcribed into complementary DNA and were amplified with the following IGF-I receptor (IGF-IR) primers: 5'- ATG CTG TTT GAA CTG CAG CGC ATG TGC TGG -3' and 5'- CCG CTC GAG CTT GCG GCC CCC GTT CAT -3'. The amplified product is 354 bp. Samples were also amplified for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) using the following primers: 5'- CCA TGG AGA AGG CTG GGC -3' and 5'- CAA AGT TGT CAT GGA TGA CC -3'. PCR amplification was performed in a final vol of 100 µl containing 10 mM Tris-HCL (pH 8.3); 2.5 mM MgCl2; 600 µM each of the deoxy-ATP, deoxy-GTP, deoxyuridine 5-triphosphate, and thymidine 5'-triphosphate; 1.0 µM and 0.5 µM of the IGF-IR and GAPDH primers, respectively; 50 mM KCl; and 2.5 U Taq polymerase (Perkin Elmer Cetus, Norwalk, CT). Cycles, repeated 35 times, consisted of 1 min denaturation at 94 C, 2 min annealing at 58 C, and 3 min polymerization at 72 C. The final polymerization step was extended an additional 15 min. A Perkin-Elmer Thermal Cycler was used for all reactions.
There is considerable identity between the insulin and IGF-IR sequences, and the IGF-IR primers selected in this reaction may also generate a 324-bp insulin fragment. Therefore, as described by Telford et al. (30), the identity of the IGF-IR product was confirmed by demonstrating that it does not contain a HincII restriction site present tin the 324-bp insulin fragment (data not shown).
FTOC. FTOC were initiated and maintained based on published protocols (31). Embryonic thymuses were harvested from timed pregnant Swiss Webster mice (Thy 1.1) at 14–15 days gestation (Taconic Farms). After 1500-R irradiation, to deplete endogenous thymocytes, individual thymic lobes were incubated with 105 donor bone marrow cells in hanging-drop cultures in Terasaki plates for 36–48 h. The lobes were then placed on filter/gelfoam rafts for 2 weeks, as described (31). Irradiated, unreconstituted thymic lobes were initiated as controls, and only a minimal number of cells were ever recovered from these cultures. Donor origin of the cells in the reconstituted lobes was determined by staining cells for the donor Thy 1.2 allotype. IGF-I was added in the culture media to some FTOC at the initiation of the hanging-drop phase and/or at each feeding during the subsequent 2-week culture period.
The reason for using thymic lobes from Swiss Webster mice was that this strain has consistently large litters. In general, BALB/c bone marrow cells were more efficient than C57BL/6 bone marrow cells at reconstituting Swiss Webster lobes. Thus, repopulation comparisons were made only for a given strain in a particular experiment.
Statistical analysis. Data were analyzed using a single-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, thymocyte numbers in all mice
increased approximately 2-fold. The finding that the thymus in
IGF-I-treated mice was markedly larger than in the saline-treated
control animals was consistent with these findings (Fig. 1). No significant effect of IGF-I on the
distribution of CD4- and/or CD8-expressing thymocyte subpopulations was
observed. However, even though thymus cellularity in 6- and
18-month-old mice was increased by IGF-I treatment, total thymocyte
numbers in these animals remained significantly lower than in
1-month-old animals.
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In initial experiments, IGF-I containing osmotic pumps were implanted into mice and 24 h later, animals were irradiated. The mice then received an injection of 107 syngenic bone marrow cells from young donors. Mice were irradiated to facilitate engraftment of the injected bone marrow cells. Under these conditions, no increase in thymic cellularity was observed 2.5 weeks after treatment (data not shown). One explanation for this result was that the time interval between bone marrow transplantation and IGF-I treatment might have been too short for the grafted T cell precursors to expand and/or for the thymus to recover from irradiation-induced damage.
Therefore, a protocol was adopted in which 18-month-old mice were
irradiated and received an injection of 107 syngenic bone
marrow 4 h later. These mice were allowed to recover for 1 week to
permit engraftment of the young donor cells. At that time, the mice
were given a second injection of syngenic bone marrow without further
irradiation to provide an additional input of young stem/progenitor
cells. Three weeks after this second transplantation, IGF-I- or
saline-containing minipumps were implanted into the mice and their
level of thymopoiesis was assessed 2.5 weeks later. Although there was
considerable mouse-to-mouse variation, the number of thymocytes in
18-month-old mice that received young bone marrow cells and IGF-I was
significantly higher than in age-matched mice that received young bone
marrow cells only or IGF-I treatment only (Fig. 2). The frequencies of CD4+
and CD8+ cells were normal in the bone marrow/IGF-I-treated
old mice (Table 2).
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, a PCR fragment of 354 bp,
characteristic of the IGF-IR, was detected in all subpopulations of
murine thymocytes. This is in agreement with results showing that human
(32) and rat (33) thymocytes also express the IGF-IR. Taken together
with the observation that thymic stromal cells can also express the
IGF-IR (34), these results raised the possibility that IGF-I has direct
effects on the thymus. The fetal thymus organ culture system (FTOC) was
used to investigate this issue.
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demonstrate that,
after 14 days of culture, IGF-I had no effect on steady-state
thymopoiesis in fetal thymic lobes from which endogenous precursors had
not been depleted. This same result was obtained when thymic lobes were
examined at 1 week after culture initiation (data not shown). However,
addition of IGF-I to thymic lobes depleted of endogenous thymocytes and
seeded with bone marrow from young mice resulted in a significant
increase in thymic cellularity. As shown in Table 4, IGF-I significantly increased the
number of cells recovered from the lobes, when tested after 2 weeks of
culture. Additional thymic lobes in three of these experiments were
repopulated in parallel with bone marrow cells from 18-month-old mice.
As shown in Table 5, IGF-I also
potentiated the ability of these old bone marrow cells to colonize the
thymus. However, the number of thymocytes harvested from fetal thymic
lobes repopulated with old bone marrow cells was always less than when
young donor cells were used (compare values in Tables 4 and
5).
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As shown in Table 6, donor cell recovery
from cultured thymic lobes was maximal when they were incubated with
IGF-I during both the hanging-drop phase and the subsequent 2 weeks of
culture. Thymocyte recovery was slightly reduced when thymic lobes were
incubated with IGF-I, either during the hanging-drop phase or the
subsequent 2-week culture period. Nevertheless, the number of donor
cells recovered from thymic lobes grown in these latter two conditions
was over twice that obtained from fetal thymic lobes cultivated in the
absence of IGF-I (Table 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The absolute number of thymocytes in 9-month-old mice treated with IGF-I remained significantly below that in young animals (16), suggesting that the ability of hormone alone to restore thymus cellularity is limited. To investigate the effects of IGF-I on thymus involution in more detail, mice ranging in age from 1–18 months were treated with IGF-I, and thymopoiesis was examined 2 weeks later. Results of this analysis indicated that, although IGF-I nearly doubled thymocyte numbers in all of these mice, the increase in absolute numbers of thymocytes in the older mice was comparatively lower as a function of age. Thus, even after 2 weeks of IGF-I treatment, the absolute number of thymocytes in 18-month-old mice remained only 15% of that in 1-month-old animals.
Globerson et al. (23, 24, 25, 26) have reported that the number of
bone marrow T cell precursors in old mice is reduced by approximately
40-fold and that these cells have reduced thymus colonization ability.
This conclusion was based on data showing that bone marrow cells from
old mice do not compete effectively with young ones at generating T
cells in fetal thymic lobes (23). The deficiency of old bone marrow
cells in this assay was only manifest in cocultures and not upon
seeding cells from donors of each age group separately into different
individual thymic lobes (23). These results contrast with the findings
presented in Tables 4
and 5 showing that, even when seeded separately
in the presence or absence of IGF-I, bone marrow cells from
18-month-old mice do not repopulate thymic lobes to the same level as
do cells from young animals. The reason why the FTOC protocol employed
in the present studies would directly detect the deficiency in thymus
colonization of old bone marrow cell is unclear at the moment.
Nevertheless, the data herein are in fundamental agreement with the
results of Globerson et al. (23) that defects accumulate in
the bone marrow T cell compartment with aging. In addition, the results
show that these defects accumulate gradually, given that the effects of
IGF-I on total thymus cellularity in 6-month-old mice was greater than
in 18-month-old mice and less than in 1-month-old animals.
These defects in the bone marrow T cell precursor pool in aging animals would explain why the ability of IGF-I, or other hormones such as GH, to increase the absolute number of thymocytes in 18-month-old mice is limited. Taken together with observations that marrow from aged donors has a diminished capacity to repopulate the thymus of an irradiated host (27), these findings provided the rationale for testing whether optimal rejuvenation of the involuted thymus was dependent on providing aging animals with a source of young hematopoietic cells in addition to hormones. Thus, groups of mice were treated either with hormone only, bone marrow cells from young mice, or a combination of these treatments.
Bone marrow transplantation alone failed to increase cellularity of the
involuted thymus in 18-month-old mice. This finding is consistent with
a preliminary report (36) and data in Fig. 2 showing that no
significant increase in thymocyte numbers was observed in old mice
injected with young bone marrow cells. Also, no significant increase in
thymic cellularity was observed in mice that received implants of
IGF-I-containing osmotic pumps, irradiation, and bone marrow
transplantation within a 24-h period (data not shown). Optimal
improvement of thymopoiesis in old mice was observed when irradiation
and bone marrow transplantation were performed before the initiation of
IGF-I treatment. This protocol may have permitted the bone marrow cells
to engraft, as well as the thymus to undergo repair of irradiation
damage, before initiation of IGF-I treatment. Further studies will be
needed to refine this protocol further and to determine whether the
level of thymopoiesis in the bone marrow transplanted/hormone-treated
mice is sustained.
In vitro studies, using the FTOC system, were used to investigate the mechanism of IGF-I action on thymic rejuvenation. No enhancing effect of IGF-I pretreatment of bone marrow cells on thymocyte recovery in the FTOC was observed under these conditions. This result indicated that actions of IGF-I on the bone marrow alone are not sufficient to account for its thymopoietic effects and suggested that the hormone had some effect on thymic colonization and/or intrathymic differentiation.
To investigate this possibility, FTOC in which IGF-I was present during all stages of culture, or only during either the initial hanging-drop phase or the subsequent 2-week culture period, were initiated. Optimal recovery of donor cells was observed when IGF-I was present at all stages of the culture. Though cell recovery from fetal thymic lobes incubated with IGF-I during one or the other stages of the culture was slightly reduced, it still was over twice that observed in cultures maintained in the absence of IGF-I. Taken together, these data suggest that IGF-I may act during the initial colonization phase of thymic lobes by bone marrow, as well as during intrathymic development.
This latter observation is surprising, considering the results shown in
Table 3, demonstrating that IGF-I had no significant effect on thymus
cellularity in intact fetal thymic lobes. However, the thymic
microenvironment present in an intact thymus may differ dramatically
from that in the irradiated, bone marrow-reconstituted thymus, and the
effects of IGF-I may be most demonstrable only under these latter
conditions.
As noted previously, IGF-I is thought to mediate many of the effects of GH (19). Therefore, it is not surprising that the above results parallel those obtained in studies of GH effects on thymopoiesis. GH was also shown to have no effect on steady-state T cell production in intact fetal thymic lobes, but it increased cell recovery in reconstituting ones (17). It has been proposed that GH and IGF-I may act by potentiating engraftment of bone marrow T cell precursors (37, 38, 39, 40). The precise developmental status of this latter population is unclear, but it could include the pluripotential hematopoietic stem cell or more differentiated bone marrow progenitors with T cell developmental potential. These T cell progenitors, which may express low levels of Thy-1, high levels of Sca-1, and CD44 (22, 41, 42, 43), are thought to migrate into the thymus at a low rate and contribute to T cell production throughout adult life.
Although treatment with IGF-I and young bone marrow cells resulted in remarkable recovery of T cell production in the old animals, thymocyte numbers were not consistently restored to levels present in young mice. One reason for this could be that intrinsic structural and/or functional defects in the involuted thymus might present an additional barrier to engraftment by colonizing T cell precursors. This, in turn, could further compromise T lymphopoiesis, because the maintenance of a normal thymic architecture is dependent on ongoing thymopoiesis (44, 45). To investigate this, it will be of value if assays could be developed to test the ability of the old thymic microenvironment to support T cell development from young bone marrow. Attempts to use old thymic tissue, instead of fetal thymuses, in the organ culture system, and to inject young bone marrow cells intrathymically into old mice, have not been successful (unpublished observations).
In addition to their expression in hematopoietic tissues, GH and IGF-I receptors are widely distributed on cells in multiple organs (19), which is consistent with their role as systemic mediators, rather than specific regulators of hematopoiesis or a particular blood cell lineage (46). Accordingly, one would predict that a decline in circulating levels of GH and/or IGF-I during aging should have systemic effects and that administration of GH or IGF-I to aging individuals should result in effects on multiple targets, in addition to effects on blood-forming tissues described herein and by others (4, 5, 6, 7, 8, 20, 21, 27, 47). This seems to be the case, as evidenced by the fact that, in addition to effects on the thymus, a decline in muscle and bone mass is also observed during aging. Moreover, increases in kidney and overall weight gain of aging mice (16) and an increase in lean body mass, skin thickness, and bone density occur in elderly men after IGF-I or GH administration (14, 48).
In conclusion, the data suggest that hormone treatments alone are limited in their capacity to rejuvenate the involuted thymus and that highest increases in cell numbers are achieved only when multiple defects, such as those that occur in both the aging endocrine and hematopoietic systems, are addressed. Based on these observations, devising an efficient therapy in humans would be quite difficult and costly. Therefore, though considerable focus has been placed on restoring thymic cellularity, a more important issue may be whether or not the relatively modest increase in T cell production induced by IGF-I treatment in old animals is sufficient to restore cell-mediated immune responses. In this regard, Clark et al. (16) have demonstrated that, in 9-month-old mice, IGF-I treatment alone significantly augmented immune function; and LeRoith et al. (49) reported that IGF-I enhanced immune function in aging primates. Further studies are required to investigate the role of hormones in the restoration of T cell function in very old animals, in view of recent reports showing that T cells that have matured in an old thymic environment are functionally defective (2).
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Footnotes |
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Received January 13, 1998.
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) received 107 bone marrow
cells from young, syngenic donors 4 h after irradiation with 400
R. One week later, a second inoculum of young bone marrow was injected
into them without any additional irradiation. Three weeks later, IGF-I-
or saline-containing pumps were then implanted into the mice, and
thymic cellularity was assayed after 2.5 weeks. The mice represented by
closed bars (
) were treated exactly as above, except
that saline-containing pumps were implanted after bone marrow
transplantation. The mice represented by hatched bars
(
) received no bone marrow but were irradiated and received an
IGF-I-containing osmotic pump. Data for the first two groups were
obtained in two independent experiments, and each bar
represents an individual mouse. The mean cellularity ± SD
for each group is indicated above the histograms. Total
thymocyte numbers differ significantly (*, P <
0.0025; #, P < 0.0025) for the indicated groups.