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* Division of Developmental and Clinical Immunology, Departments of
Medicine,
Pediatrics,
Microbiology, and
¶ Pathology,
|| Howard Hughes Medical Institute, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell replenishment before 
T cell
reseeding, thereby indicating the complete recovery of thymic function
after a course of steroid treatment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The apoptotic effect of glucocorticoids is particularly notable for the immature thymocyte subpopulation denoted double-positive cells because they express both the CD4 and CD8 coreceptors (8). Accordingly, treatment with dexamethasone, a potent glucocorticoid agonist, may result in thymic involution (9). Although mature thymocytes and T cells in the periphery appear relatively resistant to the apoptotic effects of glucocorticoid hormones (10), in vivo appraisal of the peripheral T cell pool is complicated because of its mixed composition by recent thymic emigrants (RTE)3 and the more abundant established T cell residents which may have engaged a variety of immune stimuli.
Glucocorticoids have very similar apoptotic effects on thymic cells in avian and mammalian species, indicating the conservation of this steroid effect (10, 11). An avian model was used therefore in the present studies of steroid-induced thymic dysfunction to take advantage of the fact that the chicken T cell thymocyte Ag 1 (chT1) can be used as a direct marker for RTE in the peripheral T cell pool. Measurement of the chT1+ T cell levels can thus be used to accurately monitor thymic function in these gallinaceous birds (12). A comparable RTE marker in primates is presently unavailable, although the levels of TCR V(D)J gene rearrangement excision circles (TREC) in the peripheral T cell population provide a useful surrogate marker for estimating the numbers of RTE in both humans and birds (12, 13, 14, 15, 16, 17). These extrachromosomal DNA circles are stable, nonreplicating structures that are concentrated in the naive RTE population and diluted in the proliferating pool of mature T cells. TREC measurements thus have been used as surrogate markers to assess the functional status of the thymus in acquired and congenital immunodeficiency diseases (14, 17, 18, 19). In the present studies, we evaluated the effects of an intensive course of dexamethasone treatment on thymic function by direct assessment of the thymus and by monitoring the RTE subpopulation in the peripheral T cell pool through serial determination of chT1+ T cells and TREC levels. In parallel studies of thymectomized animals, we also evaluated the in vivo effects of glucocorticoids on the peripheral pool of mature T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fertilized eggs of the SC strain (Hy-Line International, Dallas City, IA) were incubated at 41°C and intermittently rotated in a humidified incubator. Chicks were maintained under conventional vivarium conditions. Intramuscular injections of dexamethasone (Elkins-Sinn, Cherry Hill, NJ) were initiated at 4 wk of age. Pilot studies were conducted to determine a dexamethasone dosage (5 mg/kg per day) that induced thymic involution and growth retardation, but which did not affect survival and general health status of the treated animals. Thymectomy or sham thymectomy surgical procedures were performed on 4-wk-old chicks anesthetized by i.m. injection of Nembutal (Abbott Laboratories, North Chicago, IL). The thymic lobes were removed through a dorsal incision in the neck as previously described (20).
Immunofluorescence analysis and cell sorting
Peripheral blood leukocytes, thymocytes, and splenic leukocytes
were prepared as single-cell suspensions (12). The
anti-chT1, CD3, CD4, CD8, TCR, and TCR (V1 plus
V2) mAbs (21, 22) were conjugated with either FITC or
PE by Southern Biotechnology Associates (Birmingham, AL). Cells were
stained with the mAbs and analyzed with a FACScan instrument using the
CellQuest software package (BD Biosciences, MountainView, CA).
and V1+ T cells were purified by
a FACS (FACStar; BD Biosciences) after staining blood lymphocytes with
the PE-conjugated anti-TCR and FITC-conjugated
anti-TCRV1 mAbs. Purity of the sorted cells in each experiment
was >99%.
PCR determination of TCR gene rearrangement excision circles
Genomic DNA was isolated from FACS-sorted
TCRV1+ and
TCR+ T cells in blood samples of
dexamethasone-treated and age-matched control as described elsewhere
(12). Equal amounts of DNA from each sample were serially
diluted and used as templates for the PCR amplification of -actin,
V1-J1, and V1-D TREC. The primers, PCR conditions, and the
detection method for PCR products were the same as previously described
(12). PCR products separated on agarose gels were
transferred to Genescreen Plus nylon membranes (MEN Life science
Products, Boston, MA) and hybridized with
[-32P]ATP-labeled internal probes. After
autoradiography, the signal intensities of TREC PCR products of serial
dilutions of each sample were plotted. The midpoint was chosen and
normalized against that of -actin to compare the relative TREC
levels. TREC index indicates the signal intensity ratio of V1-J1
or V1-D TREC in the dexamethasone-treated and nontreated control
chickens.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the thymic effects of intensive glucocorticoid
treatment, 4-wk-old chicks were treated with 5 mg/kg dexamethasone
daily for 1 wk and on alternate days thereafter. Pilot experiments
indicated that while this steroid dosage induced thymic involution and
growth retardation, the treated birds appeared to be otherwise healthy.
The thymic mass was rapidly and persistently reduced by the treatment,
whereas thymic weight in the young controls increased over the ensuing
6 wk (Fig. 1
a). Thymocyte
numbers were decreased by 
100-fold after 1 wk of dexamethasone
therapy and remained depressed throughout the treatment period (Fig. 1b). The
CD4+CD8+TCRlow
subpopulation of thymocytes was most dramatically affected (Fig. 1c), with a 99.9% decline in cell numbers. The
CD4-CD8- double-negative
subpopulation and the CD4 and CD8 single-positive subpopulations of
T cells were also significantly reduced (24-, 8-, and 44-fold,
respectively) within the first week of steroid treatment. In parallel,
the numbers of + thymocytes were decreased
by 100-fold, indicating that this thymocyte sublineage is also
steroid sensitive. Among the residual thymocytes, the mature
TCRhigh T cells were relatively enriched in
treated animals. The differential effect of steroid treatment on the
mature CD4+ and CD8+
subpopulations was further manifested by an alteration of the CD4:CD8
ratio from 0.75:1 in controls to 4.4:1 in treated animals (Fig. 1c). These observations suggested a possible sparing effect
of the steroid treatment on mature CD4+ T
cells.
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T cells in the treated animals were 1/1,000, 1/30,000, 1/400,
1/500, 1/1,200, respectively, of control values (Fig. 1
b and
data not shown). Intensive steroid treatment thus compromises all
phases of thymocyte development, although the large
CD4+CD8+ subpopulation of
immature cortical thymocytes is the most profoundly depressed and
mature CD4+ T cells appear the least
affected. Steroid treatment impairs thymic output of naive T cells
The numbers of RTE marked by their transient expression of the
chT1 thymocyte Ag have been shown to progressively decline to
undetectable levels by 4 wk after thymectomy (12), and a
similar decline in circulating chT1+ T cells was
observed in the dexamethasone-treated chicks (Fig. 2a). The
chT1+ RTE levels were reduced by 50% by 10
days and >99% after 21 days of steroid treatment (Fig. 2b).
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1-J1 rearrangement were
examined in purified T cells, and V1-D TREC were assayed
in the T cell subpopulation of V1+
cells. The levels of TREC representing both types of rearrangements
were reduced by 50–60% after 3 wk of steroid treatment and were
10% of the control values by 5–7 wk of therapy (Fig. 2c). Notably, however, low levels of the V1-J1 TREC
and V1-D TREC were detectable well beyond the disappearance of
chT1+ T cells in the circulation (Fig. 2b) as further evidence that TREC levels are a less acute
marker of thymic function. Reversible steroid-induced impairment of thymic output
To evaluate whether the steroid treatment induced long-lasting
effects on thymic function, we examined the recovery of thymocyte
subpopulations after dexamethasone discontinuation. After 3 wk of
therapy, the steroid injections were discontinued in one experimental
subgroup. The cell numbers in each subset of
chT1+ thymocytes, including ,
double-positive, and single-positive T cells, increased rapidly
in the first 3 wk after discontinuation of the steroid treatment (Fig. 3 and data not shown). Interestingly,
T cell recovery preceded the T cell recovery. Recovery of
thymic function was also reflected by the increase in thymic weight to
normal levels by 1 mo after treatment cessation (data not shown).
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, a and b),
whereas in animals receiving continuous treatment the depletion of
chT1+ T cells persisted (Fig. 2b).
During the first week after steroid withdrawal, very few
chT1+CD3+ T cells could be
detected in the circulation, but by the 10th day the
chT1+ T cells began to reappear in the
blood. This was followed by a re-emergence of the
chT1+ T cells beginning on the 14th day of
the recovery period (Fig. 4a). The frequency of
chT1+ and T cells then increased
gradually to reach pretreatment levels 1 mo after steroid
discontinuation (Fig. 4a). An increase in the V1-J1
and V1-D TREC levels from the peripheral T cell pool also
reflected the recovery in thymic output of T cells. The V1-J1
TREC levels reached 60% of the normal control levels by 2 wk after
dexamethasone discontinuation, whereas recovery of the V1-D TREC
to this level required 4 wk (Fig. 4c). At this time, the
V1-J1 TREC levels were 20% higher than control levels (Fig. 4c), in agreement with the observation of a higher
chT1+:chT1- T
cell ratio in the steroid withdrawal group (data not shown).
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The intensive course of steroid treatment severely retarded growth
of the young chicks. The body weights, spleen weights, and total
splenocyte numbers were relatively static in the dexamethasone
administration period, whereas these growth parameters increased
progressively in untreated controls (Fig. 5, a and b, and
data not shown). Relative to the thymocyte population, however, mature
T cells in the periphery were much less affected by the intensive
steroid treatment. Following the onset of steroid treatment, the
numbers of splenic T lymphocytes increased only slightly in treated
chickens, while they continued to increase over the next 2 wk in
untreated controls undergoing normal growth (Fig. 5b and
data not shown). After 4 wk of treatment, the numbers of
CD4+ T cells in blood samples had increased
slightly, whereas CD8+ and T cell levels
were significantly decreased (Fig. 5d). Correspondingly the
CD4:CD8 ratio in the peripheral T cell pool was modified from 2:1 to
7:1. In parallel with this subpopulation distribution shift in the
periphery, a relatively severe depletion of CD8+
T cells was also observed in the thymus of steroid-treated animals.
Although not the primary focus of these studies, the steroid effects on
B cells were noted to be especially severe. B cell numbers were reduced
by >95% in the spleen and blood throughout the period of treatment.
The normal T:B cell ratio of 5:1 in the peripheral blood of untreated
birds was therefore increased to 40:1 in the steroid-treated group
(data not shown).
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50% reduction in levels of circulating T cells and an
80% reduction in T cells. Similar CD8+
and T cell levels were found in thymectomized animals
regardless of whether or not they were treated with steroids. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and T cells to the
periphery. Despite these severe deleterious effects, thymopoiesis was
immediately restored to normal levels when steroid treatment was
discontinued and this resulted in the resumption of T cell seeding to
the periphery. The remarkable poststeroid restoration of thymopoiesis
recapitulated the normal ontogenetic pattern with T cell
production preceding T cell repopulation. This rapid and
complete recovery of thymopoiesis suggests that intensive steroid
therapy for limited periods of time has no lasting effect on the T cell
compartment of the immune system.
The loss of RTE as a consequence of steroid-induced thymic involution
could be directly monitored in these studies by determining the levels
of T cells marked by chT1 cell surface expression in the chick model
and indirectly monitored by measuring the levels of TCR excision
circles. The RTE decline indicated by these parameters in the
steroid-treated birds resembled that observed following surgical
thymectomy (Fig. 2, b and c; Refs. 12, 13), although the steroid-induced decline was less acute. This
could reflect the transient supply of a limited number of relatively
mature, steroid-resistant thymocytes from the preexisting thymic
reservoir. In both steroid-treated and thymectomized birds, the decline
in TREC levels trailed the decline in the RTE subpopulation marked by
expression of the chT1 thymocyte Ag. Although chT1-marked T cells were
rarely found after 3 wk of steroid treatment, TREC could be
persistently detected among the peripheral and T cell
subpopulations, albeit in reduced levels. In thymectomized birds, the
t1/2 of chT1 expression by RTE is 3.5 days
vs a 2–3 wk t1/2 for TREC levels in the
peripheral T cell pool (12, 13). The difference in acuity
for gauging thymic impairment by the two parameters reflects the
stability of the TREC, the levels of which are reduced only through T
cell division and cell death. TREC measurements thus provide a less
precise means for monitoring thymic function than the assessment of RTE
using an identifying cell surface molecule.
Mature T cells appear to be relatively resistant to
steroid-induced apoptosis (23), except for those in
splenic germinal centers, which exhibit thymocyte-like sensitivity
(24). Our assessment of the steroid effects on peripheral
T cells in thymectomized birds confirms their relative glucocorticoid
resistance. The CD4+ subpopulation of peripheral
T cells was slightly increased by the intensive dexamethasone
treatment. Although larger numbers of experimental animals are needed
to evaluate the significance of this enhancement,
CD4+ T cells were also found to be preponderant
among the residual thymocytes. These findings could reflect the ability
of glucocorticoids to accelerate the TCR-mediated induction of T cell
proliferation via the enhanced expression of the IL-2 receptor or other
cytokine receptors (25, 26). The steroid-induced
up-regulation of IL-7R and IL-7R expression observed in human
CD4+ T cells may also contribute to enhanced
survival and function of helper T cells (27). The
up-regulation of CD8 expression on the CD4+
T cells (28), that was also seen in our experiments, is
another indication of glucocorticoid enhancement of T cell
activation.
Dexamethasone effects on the peripheral T cell pool have previously
been examined in patients with systemic lupus erythematosus
(29) and in cattle (30). Although the
CD4+, CD8+, and T
cell levels were not significantly influenced by the dexamethasone
treatment, T cell levels in the circulation declined rapidly.
These findings suggested that either T cells are unusually
susceptible to glucocorticoid-induced apoptosis (29, 30)
or that steroids induce their redistribution from the circulation into
the lymphoid tissues (30). Our analysis of thymectomized
and nonthymectomized animals indicates that the dexamethasone-induced
decline in circulating T cells is primarily due to reduced
thymic output. Lacking the remarkable capacity that T cells
possess for peripheral expansion, T cell maintenance is
especially dependent upon continuous thymic output (31, 32).
Mature and immature T cells possess equivalent numbers of glucocorticoid receptors, the function of which is essential in the steroid-induced apoptosis pathway (9, 33). The sensitivity of lymphocytes to glucocorticoid-dependent apoptosis nevertheless may vary depending on lymphocyte differentiation status and availability of supportive factors (34). In this regard, the variation in sensitivity to glucocorticoid-induced apoptosis observed as a function of T cell differentiation is associated with changes in the levels of Notch and Bcl-2 expression (35, 36). Bcl-2, an antiapoptotic factor, is preferentially expressed by CD4+ and CD8+ thymocytes and peripheral T cells and not by the CD4+CD8+ thymocytes (37). Correspondingly, Notch is highly expressed in the progenitor subpopulation of CD4-CD8- thymocytes, absent in CD4+CD8+ double-positive thymocytes, and re-expressed at intermediate levels in single-positive thymocytes (38). This coordinate pattern of expression is due to the fact that activation of the Notch signaling pathway results in the up-regulation of Bcl-2 expression, thereby conferring resistance to glucocorticoids in the CD4+ and CD8+ single-positive thymocytes (36).
When thymopoiesis was resumed after discontinuation of the
dexamethasone treatment, exportation of T cells rapidly ensued. The
chT1+ and T cells began to reappear
in the circulation by the 10th and 14th days, respectively. Complete
recovery of thymic mass and levels of circulating
chT1+ RTE were achieved within 1 mo after the
final dexamethasone injection. These findings indicate that the
glucocorticoid-induced thymic involution has no lasting effects on
either the lymphoid progenitor population or the inductive thymic
microenvironment, both of which are crucial to normal thymic function
and regeneration of the T cell system (39, 40).
Interestingly, the regeneration of T cells during the recovery phase
recapitulated the normal ontogenetic pattern of and T
cell development. During ontogeny, T cells appear in the thymus
3 and 4 days before the T cells, and the two subpopulations
later begin migrating to the periphery in the same order
(41). The present analysis of a transient interruption of
thymopoiesis indicates that intrathymic development and export of
T cells from the thymus precedes that of T cells even in
adolescent animals. Studies in chicks and mice indicate that the
fledgling T cells have a relatively rapid turnover rate and
shorter period of intrathymic residence than do T cells,
possibly because the T cell subpopulation does not undergo
positive selection in the thymus (42, 43).
A reliable marker of human RTE has not yet been identified, although TREC measurement can be used as a less direct estimate of thymic function in humans (14, 15, 16, 17). The RTE subpopulation can be identified as Thy1+CD45RC-RT6- T cells in rats (44, 45), whereas a suitable method for monitoring TREC levels is unavailable in mice. This leaves the chicken as the sole current model in which both methods of RTE assessment can be used to monitor thymic function (12, 13). It is likely, however, that the profound interruption of thymic function observed during intensive glucocorticoid treatment in this avian model also applies to steroid treatment in humans, since steroid-treated infants undergo a dramatic reduction in thymic mass (46). Moreover, human thymocytes treated with dexamethasone rapidly undergo apoptosis (47). These observations predict that comparable effects of steroid treatment on thymic function will be observed in humans. In this context, it should be emphasized that our studies were conducted during the height of thymopoietic activity in the chick model, and recovery from a comparable degree of steroid-induced involution of the human thymus would likely be limited by reduction in thymic regenerative capacity with increasing age (40, 48).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Max D. Cooper, University of Alabama, 378 WTI, 1824, 6th Avenue South, Birmingham, AL 35294-3300. E-mail address: max.cooper{at}ccc.uab.edu
3 Abbreviations used in this paper: RTE, recent thymic emigrant; chT1, chicken T cell thymocyte Ag 1; TREC, TCR gene rearrangement excision circle.
Received for publication January 25, 2002. Accepted for publication April 19, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell. Proc. Natl. Acad. Sci. USA 97:3919.. J. Immunol. 168:2212. chains on concanavalin A-activated rat CD4+ T cells: induction is inhibited by rat recombinant interleukin 4. J. Exp. Med. 176:1551. T cell receptor are susceptible to steroid-induced programmed cell death. Scand. J. Immunol. 41:504.[Medline]
T cells include exogenous cytokines, receptor ligation and in vivo priming. Eur. J. Immunol. 23:2230.[Medline]
T cells in the chicken. Semin. Immunol. 3:109.[Medline]
/ T cells. J. Exp. Med. 187:357.This article has been cited by other articles:
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  A R Lorenzi, T A Morgan, A Anderson, J Catterall, A M Patterson, H E Foster, and J D Isaacs Thymic function in juvenile idiopathic arthritis Ann Rheum Dis, June 1, 2009; 68(6): 983 - 990. [Abstract] [Full Text] [PDF]
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 R. Bianchini, G. Nocentini, L. T. Krausz, K. Fettucciari, S. Coaccioli, S. Ronchetti, and C. Riccardi Modulation of Pro- and Antiapoptotic Molecules in Double-Positive (CD4+CD8+) Thymocytes following Dexamethasone Treatment J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 887 - 897. [Abstract] [Full Text] [PDF]
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 R. M. Tempero, M. Hannibal, L. S. Finn, S. C. Manning, M. L. Cunningham, and J. A. Perkins Lymphocytopenia in Children With Lymphatic Malformation Arch Otolaryngol Head Neck Surg, January 1, 2006; 132(1): 93 - 97. [Abstract] [Full Text] [PDF]
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 I. Zubkova, H. Mostowski, and M. Zaitseva Up-Regulation of IL-7, Stromal-Derived Factor-1{alpha}, Thymus-Expressed Chemokine, and Secondary Lymphoid Tissue Chemokine Gene Expression in the Stromal Cells in Response to Thymocyte Depletion: Implication for Thymus Reconstitution J. Immunol., August 15, 2005; 175(4): 2321 - 2330. [Abstract] [Full Text] [PDF]
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 C Kayser, F L Alberto, N P da Silva, and L E. Andrade Decreased number of T cells bearing TCR rearrangement excision circles (TREC) in active recent onset systemic lupus erythematosus Lupus, December 1, 2004; 13(12): 906 - 911. [Abstract] [PDF]
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 A. Pazirandeh, M. Jondal, and S. Okret Glucocorticoids Delay Age-Associated Thymic Involution through Directly Affecting the Thymocytes Endocrinology, May 1, 2004; 145(5): 2392 - 2401. [Abstract] [Full Text] [PDF]
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 J. Norrman, C. W. David, S. N. Sauter, H. M. Hammon, and J. W. Blum Effects of dexamethasone on lymphoid tissue in the gut and thymus of neonatal calves fed with colostrum or milk replacer J Anim Sci, September 1, 2003; 81(9): 2322 - 2332. [Abstract] [Full Text] [PDF]
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