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*
Program in Immunology and Virology and Program in Molecular Medicine, and
Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655; and
Department of Pathology, University of Connecticut Health Center, Farmington, CT 06030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ßTCRlowB220+CD4-CD8-
phenotype was increased in DP peripheral lymphoid tissues when compared
with normal, nonlymphopenic diabetes-resistant (DR) BB rat tissues.
There was no evidence of DNA fragmentation in freshly isolated DP- or
DR-BB rat cells, but, after 24 h of culture, a higher proportion
of DP- than DR-BB splenic T cells underwent apoptosis. We then tested
the hypothesis that BB rat T cells with the
ßTCRlowB220+CD4-CD8-
phenotype accumulate and undergo apoptosis in the liver. Such cells
were observed undergoing apoptosis in both DP- and DR-BB rats, but
comprised 
80% of intrahepatic T cells in DP vs 20% in DR-BB
rats. Most
ßTCRlowB220+CD4-CD8-
cells in the liver were also Thy1+. The data suggest that T
cell apoptosis in the DP-BB rat is underway in peripheral lymphoid
tissues and is completed in the liver. Increased intrahepatic apoptosis
of recent thymic emigrants appears in part responsible for lymphopenia
in DP-BB rats and the concomitant predisposition of these animals to
autoimmunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DP-BB rats homozygous for lyp are deficient in
ßTCRhighCD4-CD8+ thymocytes
and export markedly reduced numbers of thymocytes to peripheral tissues
(12, 16, 17). Most DP-BB thymic emigrants undergo increased
proliferation and arrest of differentiation; they disappear from spleen
and lymph nodes within 7 days of export (17, 18). Very few ultimately
express the Thy1- RT6+ phenotype
characteristic of mature T cells in the rat (19). The mechanism by
which recent thymic emigrants disappear in the DP-BB rat is
unknown.
In mice, a subset of peripheral T cells localize to the liver, where they undergo apoptosis (20, 21, 22). These apoptotic T cells have an unusual cell surface phenotype indicative of down-regulation of the TCR, down-regulation of the CD4 and CD8 coreceptors, and up-regulation of the B cell lineage marker, B220 (20, 21, 22, 23).
In the present study, we demonstrated that, in BB rats as in mice, a population of peripheral lymphocytes undergoes apoptosis and clearance in the liver. In addition, the proportion of intrahepatic T cells undergoing apoptosis in the lymphopenic DP-BB rat was found to be much larger than that in the normal DR-BB rat. Most of these cells expressed the RT6- Thy1+ phenotype of recent thymic emigrants and the unusual TCRlow B220+ phenotype associated with intrahepatic mouse T cells undergoing apoptosis. These data suggest that increased intrahepatic apoptosis of T cells is in part responsible for the T cell lymphopenia of DP-BB rats and the concomitant predisposition of these animals to autoimmune diabetes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Viral Ab-free DP-BB and DR-BB rats were obtained from the National Institutes of Health sponsored colony at the University of Massachusetts Medical Center, Worcester, MA. They were certified as free of Sendai virus, pneumonia virus of mice, sialodacryoadenitis virus, rat corona virus, Kilham rat virus, H1 (Toolan’s virus), GD7, Reo-3, Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus, Hantaan virus, and Encephalitozoon cuniculi. Animals of either sex were used when 3 to 5 wk old. They were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996) and the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts Medical Center.
Antibodies
mAbs directed against ßTCR (clone R7.3), CD8 (clone
OX-8), CD4 (clone OX-35), B220 (CD45R, clone HIS24), CD90 (Thy1.1,
clone OX-7), and CD25 (IL-2 receptor -chain, clone OX-39) were
obtained from PharMingen (San Diego, CA). The DS4.23 hybridoma that
secretes an anti-RT6.1 mAb is maintained in our laboratory (11).
Anti-HSA (clone HIS50) mAb was obtained from Dr. Jan Rozing, University
of Groningen, The Netherlands. PE-, FITC-, and biotinylated mouse IgG1
and IgG2a isotype controls, and Red 670-conjugated streptavidin (SA)
were obtained from PharMingen.
Cell preparation
Rat donors were killed in an atmosphere of 100% CO2. Cervical and mesenteric lymph nodes, spleens, and thymi were removed, and single cell suspensions were prepared by gentle extrusion of tissues through stainless steel sieves into cold medium (17). Erythrocytes were lysed with hypotonic NH4Cl.
Liver mononuclear cell suspensions were prepared using a minor modification of a technique previously described for use in the mouse (21). Briefly, livers were perfused in situ via the portal vein with 30 ml of PBS and then excised. Single cell suspensions were obtained by gentle extrusion of the organ through a 50-mesh metal cell strainer, washed once with 40 ml of RPMI 1640, and then incubated for 30 min at 37°C in 20 ml of RPMI 1640 containing 50 U/ml of collagenase IV (Sigma, St. Louis, MO) and 0.001% DNase I (Sigma). The digested cell suspensions were washed once in RPMI 1640 medium and centrifuged at 30 x g for 3 min to remove parenchymal hepatocytes and cell aggregates. After centrifugation at 400 x g for 10 min, the cell pellet was suspended in 2 ml of RPMI 1640 medium, and 1 ml of the suspension was then mixed with 4 ml of 30% metrizamide (Sigma) in PBS. The mixture was overlaid with 1 ml of PBS and centrifuged at 1500 x g for 20 min at 4°C in a 15-ml conical tube. Cells at the metrizamide/PBS interface were collected, washed twice with 20 ml of PBS, and counted manually using a hemocytometer and 1% trypan blue as previously described (21).
Standard flow microfluorometry
Two- and three-color flow cytometric analyses were performed as
previously described (17). Briefly, 1 x 106
viable lymph node, splenic, or intrahepatic T cells were reacted with a
mixture of FITC-, biotin-, and/or PE-conjugated mAbs for 20 min at
4°C. Cells were then washed, reacted with Red 670-conjugated SA to
visualize biotinylated mAbs, washed again, and fixed with 2%
paraformaldehyde. Controls for background staining were FITC- or
PE-conjugated mouse IgG1 or IgG2a, or Red 670-conjugated SA alone.
Labeled cells were analyzed using a FACScan instrument (Becton
Dickinson, Sunnyvale, CA). T cells were identified by their forward and
side light scatter profiles and the detection of ßTCR staining.
The composite phenotypes of T cell subsets for up to four antigenic
markers in addition to ßTCR were determined by combining the
results of multiple two- and three-color analyses of replicate samples
of lymphocytes, each incubated with a different combination of Abs. In
all instances, a minimum of 30,000 events was acquired for both DR and
DP animals.
Four-color flow microfluorometry with intravital dyes
Four color analyses were performed on intrahepatic T cells to
quantify the relative percentages of live, apoptotic, and dead cells
using a combination of two mAbs and two intravital dyes as described
(24). Briefly, liver mononuclear cells were isolated, reacted with
anti-ßTCR and anti-B220 mAbs, and suspended in 1 ml of
PBS-1% BSA that had been warmed to 37°C. Hoechst 33342 dye (HO342,
Molecular Probes, Eugene, OR) was then added to a final concentration
of 1 µg/ml, and the cells were incubated at 37°C for 7 min. The
second dye, 7-aminoactinomycin-D (7-AAD, Calbiochem, San Diego, CA.),
was then added to the cells at a final concentration of 1 µg/ml, and
the cell suspension was immediately placed on ice in the dark. Cells
were reacted with the 7-AAD for exactly 10 min, washed in PBS, and
analyzed within 1 h using a FACScan instrument.
Detection of DNA fragmentation by flow microfluorometry
Suspensions of thymocytes or splenocytes (20 x
106 cells/ml) were incubated in RPMI 1640 containing
10% lot-selected FBS for 24 h at 37°C in 5% CO2.
Replicate cultures were harvested at intervals, stained with
FITC-conjugated anti-ßTCR mAb as described above, fixed in
70% cold ethanol, and pelleted. Cells were suspended in a staining
solution containing propidium iodide (PI, 50 µg/ml) and RNase (100
U/ml), as previously described (17). Cell surface (FITC) and
intranuclear (PI) fluorescence intensity were analyzed simultaneously
using a FACScan instrument.
Purification of TCRlowB220+ liver cells
Intrahepatic cells were prepared and reacted with PE-conjugated
anti-ßTCR and FITC-conjugated anti-B220 mAbs as described
above. The TCRlowB220+ cell subset was
sorted on a Mo Flo cell sorter (Cytomation, Fort Collins, CO). Debris,
dead cells, and nonlymphoid cells were excluded by appropriate gating
based on forward and side scatter profiles.
RT-PCR
RNA was prepared from cell suspensions by the guanidine thiocyanate-phenol, chloroform method and used for cDNA synthesis by reverse transcription (RT) as previously described (25, 26). TCRCß and ß-actin primers were synthesized by the Oligonucleotide Synthesis Core Facility at our institution based on published sequences as previously described (26). PCR were performed using a total volume of 50 µl PCR buffer (Promega, Madison, WI); 2 µl of cDNA were incubated with 1.25 U of Taq DNA polymerase (Promega), 0.5 mM deoxynucleotide triphosphates, and 1 µM sense and antisense primers. The linear range of amplification for each primer was established in independent preliminary studies. PCR reaction products were separated on an ethidium bromide gel and visualized in ultraviolet light.
Statistical analysis
Parametric data are presented as arithmetic means ± 1 SD. Pairs of means were compared by two-tailed t test using separate variance estimates and the Bonferroni adjustment for multiple comparisons, as required (27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many lymph node T cells in the DP-BB rat were found to exhibit an
atypical phenotype that was Thy1+ (56% of total T
cells) and TCRlow (24% of total T cells, Fig. 1
). In rats, the TCR+
Thy1+ phenotype is characteristic of cells that are recent
thymic emigrants (17, 18). Three-color flow cytometric analysis of
DP-BB rat lymph node cells for expression of ßTCR, Thy1, and
either CD4, CD8, B220, or HSA consistently revealed that 20% to 30%
of total T cells exhibited TCRlow CD4low,
CD8low, B220+, or HSA+ phenotypes
(Fig. 2, Table I). In addition, >65% of these unusual
T cells expressed the Thy1 Ag (Fig. 2). In contrast, analysis of DR-BB
rat lymph node cells revealed that the T cell phenotype of these
animals to be normal, with very low percentages of T cells expressing
the atypical phenotypes found in the DP-BB rat (Table I, Figs. 1 and 2). The ratio of TCRlow to TCRhigh lymph node
cells was comparable in all single, two-color, and three-color flow
cytometric analyses (Table I, Figs. 1 and 2). In their aggregate, the
data document the existence of a previously unrecognized minor
population of TCRlow lymph node cells in the DP-BB/Wor
rat.
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). Again, very low percentages of T
cells expressing the atypical phenotypes were found in the DR-BB
rat.
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8% and
as many as 16% of immature (Thy1+) peripheral T cells
in DP-BB rats express an ßTCRlow, CD4low,
(B220 or HSA)+ phenotype. Mouse T cells undergoing
apoptosis have previously been associated with this phenotype
(21). Increased frequency of apoptosis in cultured DP-BB rat splenic T cells
Suspensions of freshly isolated and cultured thymocytes and
splenic cells from DR-BB and DP-BB rats were stained with R7.3
anti-TCR mAb, permeabilized, and reacted with propidium iodide.
Cells were analyzed by flow cytometry for the presence of the
"subG1" peak indicative DNA fragmentation and apoptosis. Analysis
of thymocytes in both DR and DP animals revealed that the percentage of
apoptotic cells was initially low but increased with time (Table II). There were no statistically
significant differences between DR and DP thymocytes at any time point.
Analysis of freshly isolated spleen cells revealed no detectable
apoptotic T cells (<1%) in either rat strain (Table II). After
14 h of culture, apoptotic T cells were readily detectable, and
the proportion of DP-BB spleen cells undergoing apoptosis slightly
exceeded the proportion in DR-BB rats. After 24 h of culture, the
percentage of apoptotic T cells had increased further, and the
difference in percentage between DP-BB (21.3%) and DR-BB (9.6%) rats
was statistically significant (p < 0.05; Table II). Representative examples of the flow cytometric profiles for DR and
DP rat spleen cells at time 0 and after 24 h of culture are shown
in Figure 4.
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ßTCRlowB220+CD4-CD8-
T cells accumulate in the liver of both DP-BB and DR-BB rats
Cells with a surface phenotype predictive of apoptosis were
observed in a high percentage of DP-BB rat peripheral T cells (Figs. 2
and 3), but no evidence of DNA fragmentation was found in freshly
isolated cells (Fig. 4). To investigate the hypothesis that apoptosis
might be occurring in the liver (20, 21, 22), the number and cell surface
phenotype of intrahepatic T cells from DR and DP-BB rats were
determined. The number of lymphocytes present in the liver was found to
be similar in DR- and DP-BB rats, 31.7 ± 3.8 x
106 and 30.0 ± 3.6 x 106
(n = 3, p = NS) cells per liver,
respectively.
In both lymphopenic DP-BB and nonlymphopenic DR-BB rats, intrahepatic T
cells were observed to express two distinct phenotypes, one
TCRhighB220- (R2 gate) and the other
TCRlowB220+ (R3 gate, Fig. 5, upper panels). The
percentage of TCRhighB220- intrahepatic
lymphocytes in the DR-BB rat was sevenfold greater than in the DP-BB
rat (Table III). Conversely, the
percentage of TCRlowB220+ intrahepatic
lymphocytes in the DP-BB rat was threefold greater than in the DR-BB
rat. When the data are presented as the calculated percentage of
intrahepatic TCR+ T cells, only 18% of all intrahepatic T
cells in the DR-BB rat were TCRlowB220+ T cells
whereas, in the DP-BB rat, 81% of all intrahepatic T cells expressed
this unusual phenotype (Table III).
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). As expected,
TCRhigh T cells from the normal DR rat exhibited a narrow
range of cell sizes that was comparable to that of small resting
lymphocytes found in peripheral lymphoid tissues. In contrast,
TCRhigh T cells from the autoimmune DP-BB rat exhibited a
broad range of cell sizes; the great majority of the cells were larger
than those observed in the DR rat and consistent with the expression of
an activated phenotype. The TCRlow cells in both rat
strains also exhibited a broad range of cell sizes, consistent with the
presence of activated cells.
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ßTCRhigh (Fig. 7) or
B220+ (Fig. 8) phenotype. In
DR-BB rats, the TCRhigh intrahepatic T cell population
exhibited a phenotype consistent with that previously reported (9, 10, 12, 17) for peripheral T lymphocytes (Fig. 7, left
column). These cells exhibited a CD4:CD8 ratio of 2:1
and included a large number of RT6+ cells, only a small
number of Thy1.1+ cells, and a very small number of cells
expressing the IL-2 receptor (CD25) (Fig. 7, Table IV). In DP-BB rats, the
TCRhigh intrahepatic T cell population also exhibited a
phenotype consistent with that previously reported (9, 10, 12, 17) for
DP peripheral T lymphocytes (Fig. 7, right column). They were
predominantly CD4+CD8-. Approximately 50%
were Thy1.1+ and very few were RT6+. A
substantial number expressed the IL-2 receptor (CD25) (Fig. 7, Table IV).
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). These cells
expressed a CD4low/-, CD8low/-,
RT6low/-, and CD25low/- phenotype, and
90% expressed Thy1.1 (Fig. 8, Table IV).
TCRCß gene expression in
ßTCRlowB220+CD4-CD8-
intrahepatic cells
We next established that the
TCRlowB220+ subset observed in Figure 5
was, in fact, T cells expressing B220. cDNA was synthesized from total
RNA extracted from sorted TCRlowB220+ DP-BB
intrahepatic cells and from control DP-BB
TCRhighB220- sorted lymph node T cells. RT-PCR
analysis using ß-actin-specific primers was performed to control for
the integrity of the extracted mRNA and for the amounts of mRNA
assayed. As shown in Figure 9, TCRCß
mRNA was readily detected in cDNA prepared from both
TCRhighB220- lymph node cells and from
TCRlowB220+ intrahepatic cells.
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, upper panels), these results, together with
the data in Figures 7 and 8, indicate that a population of
TCRlowB220+CD4-CD8- T
cells accumulate in the liver of both DP-BB and DR-BB rats.
Intrahepatic
ßTCRlowB220+CD4-CD8-
T cells are apoptotic in both DP- and DR-BB rats
To test the hypothesis that the
ßTCRlowB220+CD4-CD8-
T cells in the livers of DR-BB and DP-BB rats (Fig. 5, upper
panels) are undergoing apoptosis, we used four-color flow
cytometry. We combined two-color analysis on the
TCRhighB220- (R2 gate, shown in red) and the
TCRlowB220+ (R3 gate, shown in blue, Fig. 5.)
populations of intrahepatic T cells with HO342 and 7-AAD staining.
The vital dye HO342 stains apoptotic cells with greater intensity than nonapoptotic cells. The dye 7-AAD stains dead cells that lack membrane integrity but is excluded from live cells (24). The results obtained using this dye combination were analyzed as follows: HO342low,7-AAD- populations were categorized as live cells; HO342intermediate,7-AAD- populations were categorized as apoptotic, and HO342high,7-AAD+ cells were categorized as dead. The method does not permit discrimination between cells that died as a result of apoptosis and those that died as the result of damage and necrosis.
The results of the four-color flow cytometric analysis of DP- and DR-BB
intrahepatic T cells are shown in the lower panels of Figure 5. The
TCRhighB220- intrahepatic T cell subset in
both DR-BB and DP-BB rats (R2 gate, red) exhibited predominantly the
living cell phenotype (HO342low, 7-AAD-). The
TCRlowB220+ intrahepatic T cell subset in both
the DR-BB and DP-BB rats (R3 gate, blue) exhibited predominantly the
apoptotic (HO342intermediate, 7-AAD-) and dead
cell phenotypes (HO342high, 7-AAD+).
In the case of the DR-BB rat, the data in Table III had demonstrated
that the majority (82%) of intrahepatic DR-BB T cells exhibited a
TCRhighB220- phenotype, whereas most (81%)
intrahepatic DP-BB T cells exhibited a
TCRlowB220+ phenotype. The results shown in
lower panels of Figure 5 recapitulate these proportions. The
majority of intrahepatic DR-BB T cells were live cells whereas most
DP-BB intrahepatic T cells were either undergoing apoptosis or
dead.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis of intrahepatic T cells in normal rats and mice
Our first finding is based on studies of the intrahepatic T cell
population in DR-BB rats. Although this rat strain is known to possess
a genetic predisposition to the generation of autoreactive T cells (2, 3), they are phenotypically normal. In the absence of experimental
manipulation they never develop spontaneous autoimmune disease (2, 3).
As expected, very few peripheral T cells in the normal DR-BB rat
expressed an apoptotic phenotype. In contrast, we observed that 20%
of the total intrahepatic T cell population in these animals expressed
an
ßTCRlowB220+CD4-CD8-
phenotype and were undergoing apoptosis. This phenotype is similar to
that of apoptotic intrahepatic T lymphocytes in the mouse
(20).
The present studies extend previous observations made in the mouse by
identifying the phenotype of the intrahepatic apoptotic T cell
population in the rat as Thy1+RT6-. Previous
reports have provided strong evidence that rat T cells expressing this
phenotype are predominantly immature recent thymic emigrants(19, 28, 29). Our inference that the Thy1+RT6-
intrahepatic T cell population undergoing apoptosis in the rat is
comprised of recent thymic emigrants was confirmed in a preliminary
study that showed that, 24 h after an intrathymic injection of
FITC, 10% of TCRlow intrahepatic T cells undergoing
apoptosis in the rat were FITC+ (D. L. Greiner,
unpublished observations). Both CD4+ßTCR+
and CD8+ßTCR+ recent thymic emigrants in
the rat express an immature Thy1+RT6-
phenotype (18, 19). Within 3 to 7 days of their release from the
thymus, the majority of these cells become
Thy1-RT6+, the phenotype associated with
mature peripheral T cells in the rat (12, 17, 18). More than 90% of
the intrahepatic apoptotic T cell population in the normal DR-BB rat
expressed the phenotype of recent thymic emigrants.
Our data do not permit us to identify the coreceptor that was expressed
on any given apoptotic intrahepatic T cell. Both CD4 and CD8, in
addition to ßTCR, are down-regulated in cells that up-regulate
B220 and undergo apoptosis (20). Whether the
CD4-CD8- cells being destroyed in the rat
liver originally expressed CD4 or CD8 cannot be determined.
In the mouse, it was initially hypothesized that apoptotic peripheral T cells were taken up and rapidly eliminated by local tissue macrophages (30). This hypothesis predicted that apoptotic cells should not be readily detectable unless the salvage system was saturated. This prediction was tested in the mouse and found to be valid in the case of apoptotic peripheral CD4+ T cells. After the administration of superantigens, which leads to extensive, rapid depletion of peripheral CD4+ T cells, apoptotic cells become readily detectable among freshly isolated spleen cells and in situ in peripheral lymphoid tissues (23, 31, 32).
In contrast, apoptosis of peripheral mouse CD8+ T cells has not been observed either in situ in the spleen or in freshly isolated spleen cells (21). To account for the behavior of the CD8+ T cells, a second mechanism for elimination of apoptotic peripheral T cells was recently proposed. It was hypothesized that apoptotic peripheral CD8+ T cells home to nonlymphoid tissues such as the liver for elimination (20, 21, 22). This prediction was tested and confirmed using MHC class I-restricted anti-SV40 large T Ag TCR-transgenic mice (21). Peptide-induced peripheral deletion of most peripheral CD8+ TCR-transgenic T cells resulted in their rapid disappearance from the lymph node and spleen and their accumulation in the liver. This mechanism appears not to apply to CD4+ T cells (W.Z. Mehal and I.N. Crispe, unpublished observations).
Based on these results, we interpret our results as demonstrating that the majority of rat intrahepatic T cells undergoing apoptosis are naive, immature peripheral T cells. As a corollary, we would also predict that a subset of the CD8+ cells undergoing intrahepatic apoptosis in the mouse may be recent thymic emigrants. The liver, in this view, would function as a site of both activation-induced cell death (as shown in the mouse) and programmed cell death (as shown in the present study) of newly released naive T cells.
Massive apoptosis of intrahepatic lymphocytes in the DP-BB rat
In normal DR-BB rats, 4 to 6% of peripheral T cells, but 20%
of intrahepatic T cells, expressed an apoptotic phenotype. In contrast,
in the DP-BB rat an
ßTCRlowB220+CD4-CD8-
phenotype was observed in 25% of peripheral T cells and 80% of
intrahepatic T cells. In addition, although the apoptotic
phenotype
(ßTCRlowB220+CD4lowCD8low)
was present in both the periphery and in the liver, apoptotic cell
death (evidenced by the presence of a "subG1" peak indicative of
the DNA fragmentation present in cells that are in the final stages of
apoptotic cell death) could be documented only in freshly isolated
intrahepatic T cells. We interpret our findings as evidence that the
process of T cell apoptosis is greatly amplified in the lymphopenic,
autoimmune DP-BB rat where it is underway in the periphery and
completed in the liver.
The present data derived from the DR-BB rat suggest that intrahepatic T
cells undergoing apoptosis are both immature
(Thy1+RT6-) and activated. In the DP-BB
rat, 30 to 50% of peripheral T cells are known to express an immature
phenotype (12, 17, 33). In addition, 15% of Thy1+
peripheral T cells in BB rats are in the S/G2/M phase of cell
cycle, indicative of an activated phenotype and proliferation (17). The
presence of such cells in abundance in the periphery is consistent with
the large number of such cells observed to be undergoing apoptosis in
the liver in the present study.
It has been argued that only mature or effector T cells are capable of homing to nonlymphoid tissues (12), but our observation that most apoptotic intrahepatic T cells in DP-BB rats are Thy1+ immature T cells suggests that there are exceptions to that rule. The present data do not permit us to determine whether the increase in intrahepatic T cell apoptosis in the DP-BB rat is the consequence of premature death of recent thymic emigrants or the result of an abnormal intrahepatic process that is eliminating such cells. Previous analyses of irradiated BB rats reconstituted with normal rat bone marrow have, however, demonstrated normal donor T cell development in the chimeric animal (34). This observation would argue against an intrinsic abnormality of the DP-BB rat liver leading to T cell elimination.
As was the case with the DR-BB rat, our data do not permit us to determine whether the CD4-CD8- intrahepatic T cells undergoing apoptosis were derived from CD4+ or CD8+ peripheral T cells. As in the mouse, DP-BB rat CD4+ T cells (which are the predominant peripheral T cell subset in these animals) may be eliminated in the periphery by resident macrophages (23, 31, 32) whereas DP-BB CD8+ T cells may be eliminated in the liver (21). It will be important to determine whether the intrahepatic T cells undergoing apoptosis in the DP-BB rat include both immature CD4+ and immature CD8+ T cells.
T cell maturation, intrahepatic T cell apoptosis, and lyp
It is known that severe lymphopenia in the DP-BB rat (9, 10, 12, 17) results from homozygosity for a gene of unknown function designated lyp (13). The high frequency of T cells undergoing apoptosis in the livers of these animals suggests that intrahepatic apoptosis contributes to the lymphopenia and may be a direct consequence of lyp. How the lyp gene might lead to the amplification of intrahepatic T cell apoptosis in the rat is not known, but interference with positive selection is an attractive hypothesis.
It is clear that the immature T cells in DP-BB peripheral tissues have undergone positive selection, a process involving TCR signaling, because the RAG-1 and RAG-2 transcripts are down-regulated in these cells (35). We hypothesize that the lyp gene may induce abnormal TCR signaling during positive selection in the thymus, resulting in a chronic state of T cell activation, apoptosis, and a deficiency of single positive thymocytes. Consistent with this hypothesis, intrathymic maturation of CD8+ T cells is decreased (9, 10, 12), thymocyte export is reduced (17), and the thymic emigrants that do reach the periphery are abnormal. Most of the peripheral T cells are proliferating, activated, immature cells that fail to develop into mature Thy1- T cells; the few Thy1- T cells that do develop appear normal (17).
Apoptosis and autoimmunity in the DP-BB rat
The DP-BB rat is both susceptible to autoimmune diabetes and severely lymphopenic (2, 3). The majority of its T cells are short-lived and disappear from the peripheral lymphoid tissues soon after their release from the thymus (12, 17, 18). These observations lead to an intriguing question as to the identity and life span of the autoreactive T cell population that mediates the autoimmune diabetes.
It has been demonstrated that a population of CD4+ peripheral T cells in the DP-BB rat resists apoptosis in the presence of a superantigen (36). It has also been observed that the induction of insulin-dependent diabetes mellitus in both BB rats (37, 38, 39) and nonobese diabetic (NOD) mice (40, 41, 42, 43) appears to depend on the presence of both CD4+ and CD8+ T cell populations. In the DP-BB rat, a minor population of CD8+ T cells has been detected and hypothesized to include the autoreactive effector cell (10, 12). The present data suggest that a small subset of either CD8+ T cells, CD4+ T cells, or both must be resistant to the apoptotic effects of the lyp gene and that these apoptosis-resistant T cells are likely to be the mediators of DP-BB rat diabetes. Understanding the basis for the resistance of these minor populations of T cells to lyp-induced apoptosis should provide insight into the biology of the autoreactive effector T cell population active in autoimmune diabetes.
Whatever the underlying mechanisms are, the biologic consequences of the T cell lymphopenia resulting from increased intrahepatic apoptosis of T cells in the DP-BB rat are clear. The deficiency of mature RT6+ peripheral T cells results in a relative imbalance between effector and regulatory T cells and leads to spontaneous expression of autoimmune diabetes and thyroiditis (2, 3, 4). The DP-BB rat model represents an attractive object for further study because it is a naturally occurring example of autoimmunity associated with increased intrahepatic apoptosis. In other models of abnormal intrahepatic apoptosis (21, 44), the apoptotic event is induced by peptide administration to TCR-transgenic mice. Understanding both the cell biology of its immune system and the molecular biology of its lyp locus should provide new insights into the role of apoptotic pathways in the maintenance of homeostatic balances between regulatory and effector T cells relevant to autoimmune disease states.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dale L. Greiner, Diabetes Division, University of Massachusetts Medical School, Two Biotech, 373 Plantation Street, Suite 218, Worcester, MA 01605. E-mail address:
3 Abbreviations used in this paper: DP, diabetes-prone; BB, BioBreeding; DR, diabetes-resistant; PI, propidium iodide; 7-AAD, 7-aminoactinomycin-D; PE, phycoerythrin; SA, streptavidin; HSA, heat-stable Ag; Wor, Worcester.
Received for publication June 26, 1997. Accepted for publication February 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 p40 mRNA increase with age in both diabetic and insulin-treated nondiabetic BB rats. J. Immunol. 156:1315.[Abstract]
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 S. Schnell, C. Demolliere, P. van den Berk, and H. Jacobs Gimap4 accelerates T-cell death Blood, July 15, 2006; 108(2): 591 - 599. [Abstract] [Full Text] [PDF]
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D. A. Murray and I. N. Crispe TNF-{alpha} Controls Intrahepatic T Cell Apoptosis and Peripheral T Cell Numbers J. Immunol., August 15, 2004; 173(4): 2402 - 2409. [Abstract] [Full Text] [PDF]
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D. J. Todd, E. M. Forsberg, D. L. Greiner, J. P. Mordes, A. A. Rossini, and R. Bortell Deficiencies in Gut NK Cell Number and Function Precede Diabetes Onset in BB Rats J. Immunol., May 1, 2004; 172(9): 5356 - 5362. [Abstract] [Full Text] [PDF]
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 H. J. Hosea, E. S. Rector, and C. G. Taylor Zinc-Deficient Rats Have Fewer Recent Thymic Emigrant (CD90+) T Lymphocytes in Spleen and Blood J. Nutr., December 1, 2003; 133(12): 4239 - 4242. [Abstract] [Full Text] [PDF]
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 M. Pandarpurkar, L. Wilson-Fritch, S. Corvera, H. Markholst, L. Hornum, D. L. Greiner, J. P. Mordes, A. A. Rossini, and R. Bortell Ian4 is required for mitochondrial integrity and T cell survival PNAS, September 2, 2003; 100(18): 10382 - 10387. [Abstract] [Full Text] [PDF]
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 L. Hornum, J. Romer, and H. Markholst The Diabetes-Prone BB Rat Carries a Frameshift Mutation in Ian4, a Positional Candidate of Iddm1 Diabetes, June 1, 2002; 51(6): 1972 - 1979. [Abstract] [Full Text] [PDF]
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D. J. Todd, D. L. Greiner, A. A. Rossini, J. P. Mordes, and R. Bortell An Atypical Population of NK Cells That Spontaneously Secrete IFN-{gamma} and IL-4 Is Present in the Intraepithelial Lymphoid Compartment of the Rat J. Immunol., October 1, 2001; 167(7): 3600 - 3609. [Abstract] [Full Text] [PDF]
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W. Z. Mehal, F. Azzaroli, and I. N. Crispe Antigen Presentation by Liver Cells Controls Intrahepatic T Cell Trapping, Whereas Bone Marrow-Derived Cells Preferentially Promote Intrahepatic T Cell Apoptosis J. Immunol., July 15, 2001; 167(2): 667 - 673. [Abstract] [Full Text] [PDF]
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T. Dao, M. Exley, W. Z. Mehal, S. M. A. Tahir, S. Snapper, M. Taniguchi, S. P. Balk, and I. N. Crispe Involvement of CD1 in Peripheral Deletion of T Lymphocytes Is Independent of NK T Cells J. Immunol., March 1, 2001; 166(5): 3090 - 3097. [Abstract] [Full Text] [PDF]
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E. Seung, N. Iwakoshi, B. A. Woda, T. G. Markees, J. P. Mordes, A. A. Rossini, and D. L. Greiner Allogeneic hematopoietic chimerism in mice treated with sublethal myeloablation and anti-CD154 antibody: absence of graft-versus-host disease, induction of skin allograft tolerance, and prevention of recurrent autoimmunity in islet-allografted NOD/Lt mice Blood, March 15, 2000; 95(6): 2175 - 2182. [Abstract] [Full Text] [PDF]
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W. Z. Mehal, A. E. Juedes, and I. N. Crispe Selective Retention of Activated CD8+ T Cells by the Normal Liver J. Immunol., September 15, 1999; 163(6): 3202 - 3210. [Abstract] [Full Text] [PDF]
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D. Y. Wu and I. Goldschneider Cyclosporin A-Induced Autologous Graft-Versus-Host Disease: A Prototypical Model of Autoimmunity and Active (Dominant) Tolerance Coordinately Induced by Recent Thymic Emigrants J. Immunol., June 1, 1999; 162(11): 6926 - 6933. [Abstract] [Full Text] [PDF]
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S. Ramanathan and P. Poussier T Cell Reconstitution of BB/W Rats After the Initiation of Insulitis Precipitates the Onset of Diabetes J. Immunol., May 1, 1999; 162(9): 5134 - 5142. [Abstract] [Full Text] [PDF]
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B. J. Whalen, P. Weiser, J. Marounek, A. A. Rossini, J. P. Mordes, and D. L. Greiner Recapitulation of Normal and Abnormal BioBreeding Rat T Cell Development in Adult Thymus Organ Culture J. Immunol., April 1, 1999; 162(7): 4003 - 4012. [Abstract] [Full Text] [PDF]
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