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* Department of Cell Biology,
Section of Immunobiology, and
Ludwig Institute for Cancer Research, Yale University School of Medicine, and
Howard Hughes Medical Institute, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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by Th1 effector T cells. These studies suggest that CD4 T cells
are a predominant target for DSG and the immunosuppressive effects of
the drug may result from reduced CD4 T cell expansion and decreased
polarization into IFN--secreting Th1 effector T cells in the
induction of certain autoimmune disorders. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
(3, 4, 5). It is not clear how these molecular events induce
clinical immunosuppression although many genes involved in the immune
response have NF-B binding sites in their promoters. DSG has been
shown to have a variety of effects on immune responses including
decreased Ab production, inhibition of IL-6 and TNF-
production, and
inhibition of early T and B cell development (6, 7, 8).
Despite the fact that its mechanism of action remains unclear, some
studies indicate that DSG is more effective than other agents at
inducing immunosuppression. In one case report, DSG was shown to
reverse acute graft-vs-host disease that was nonresponsive to
cyclosporin A and methylprednisolone (MP) (9). In animal
studies, DSG was more effective than FK506 and cyclosporin A in
prolonging xenograft survival (10, 11). DSG has also been
used in combination therapy with other agents to increase allograft
survival time. Of particular interest, a study by Contreras et al.
(12) demonstrated that when a short course of DSG therapy
was given in combination with three doses of anti-CD3 immunotoxin
and MP, renal allograft survival in Rhesus monkeys was maintained for
up to 550 days. When DSG was excluded from the treatment regime, grafts
were rejected by day 50. A consistent finding in animals receiving DSG
was decreased detection of IFN- and TNF- in plasma as compared
with animals that received anti-CD3-immunotoxin and MP alone,
suggesting that DSG inhibits Th1 CD4 cells that produce these
proinflammatory cytokines.
DSG has also been effective in decreasing the severity of autoimmune diseases (13). Experimental autoimmune encephalomyelitis (EAE) is a prototypical animal model used to study Th1-mediated autoimmunity and is clinically similar to the human neurologic disease multiple sclerosis (14, 15). Disease in genetically susceptible strains of rodents can be induced either by injection of myelin-derived proteins or by adoptive transfer of preactivated encephalitogenic CD4 T cells. Immunization of (B10.PL x SJL/J)F1 mice with the NH2-terminal acetylated myelin basic protein (MBP) peptide (Ac1–11) results in the development of a chronic course of disease characterized by paralytic episodes with intermittent partial remission.
In this study, we investigated the effect of DSG on the course of this Th1-mediated autoimmune disease using a chronic relapsing model of EAE. In agreement with earlier DSG studies in the rat (16, 17), we found that treatment with DSG delayed the onset of clinical signs of EAE and decreased disease severity. Investigations conducted in vitro into the mechanism of DSG immunosuppression showed that activated, but not naive, T cells were inhibited by DSG. Our findings indicate that DSG inhibits CD4 T cells directly by blocking cell cycle progression and altering Th1 effector functions. Thus, at least one mechanism of DSG immunosuppression results from inhibiting the proliferation of Th1 effector cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.PL and SJL/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The MBP-TCR transgenic (tg) mice express a TCR transgene specific for the acetylated NH2-terminal peptide of MBP bound to I-Au and were generated as previously described (18). (B10.PL x SJL/J)F1 (H-2uxs) mice, as previously described (19), and MBP-TCR tg mice were bred and maintained in our colony at Yale University School of Medicine (New Haven, CT). All mice were used between 5 and 8 wk of age. Animal experiments were performed according to the Yale Animal Care and Use Center and national guidelines.
EAE induction and DSG treatment
The MBP peptide Ac1-11 (Ac-ASQKRPSQRHG) was synthesized and
HPLC-purified by the W. M. Keck Foundation Biotechnology Resource
Laboratory at Yale University. Female (B10.PL x
SJL/J)F1 mice were immunized with 75 µg of MBP
Ac1-11 emulsified in CFA containing 4 mg/ml heat-killed
Mycobacterium tuberculosis H37Ra (Sigma-Aldrich, St. Louis,
MO) s.c. in each internal flank. Pertussis toxin (200 ng; List
Biological Laboratories, Campbell, CA) in PBS was injected i.v. at the
time of immunization and again 48 h later. DSG (kindly provided by
Bristol-Myers Squibb, Lawrenceville, NJ) was administered i.p.
beginning 4 days following peptide challenge and was used at a dose of
3.75 mg/kg in PBS. Mice were treated with DSG daily for 5 days and then
for another 5 days following 1 day without treatment. Individual
animals were assessed daily for symptoms of EAE and were scored as
described (see Fig. 1
).
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CD4 T cells were purified from spleens of MBP-TCR tg mice. RBC were removed by water lysis, and B cells were depleted by two rounds of panning with goat-anti-mouse IgA, M, and G (Zymed Laboratories, South San Francisco, CA). CD8 and remaining MHC class II-positive cells were removed with mAb and complement (Pel-Freez Biologicals, Rogers, AR). mAb used were 3.155 (rat anti-mouse Lyt2) purchased from American Type Culture Collection (ATCC; Manassas, VA) and maintained in our laboratory, 2.4G2 (rat anti-mouse FcR) produced locally (20), and Y3JP (mouse anti-mouse I-A), which has been described (21). Splenic APC were obtained from non-tg littermates by depleting T cells using 3.155, GK1.5 (rat anti-mouse CD4; ATCC), YCD3-1 (rat-anti-mouse Thy1) (21), and rabbit complement. APC were inactivated with mitomycin C (Sigma-Aldrich).
For cytokine analysis, 3 x 105 CD4 T cells from MBP-TCR tg mice were cultured with an equal number of inactivated APC and 5 µg/ml Ac1-11 in 48-well plates. Cells were cultured in the serum-free medium HL-1 (BioWhittaker, Walkersville, MD) supplemented with L-glutamine because polyamine oxidase activity present in FCS can result in the breakdown of DSG into a product that is nonspecifically toxic to cells (22). In some experiments, CD4 T cells were stimulated with 5 µg/ml plate-bound anti-CD3 (2C11; ATCC), 1 µg/ml soluble anti-CD28 (BD PharMingen, San Diego, CA) and 10% human rIL-2 (Hemagen, Columbia, MD). Preliminary proliferation assays were set up to determine the optimal dose of DSG to use in the in vitro studies (data not shown). As reported previously, we found that DSG inhibited proliferation of Ag-specific CD4 T cells in a dose-dependent manner (23). In all subsequent experiments, DSG was used at a concentration of either 5 or 10 µg/ml, doses that are believed to reflect levels achieved during in vivo treatment (24). DSG was added at the beginning of the culture or 48 h later. Where indicated, supernatants were collected from the primary culture following 24 h of stimulation. Otherwise, cultures were maintained for 5 days, collected, washed, rested for 24 h, and restimulated with an equal number of APC and Ac1-11 or with 5 µg/ml anti-CD3 and 1 µg/ml anti-CD28. Supernatants were collected at 24 h and cytokine levels were measured by ELISA (BD PharMingen).
For proliferation assays, 1 x 105 MBP-specific CD4 T cells were stimulated with an equal number of inactivated splenic APC and Ac1-11. DSG was added on day 0 or day 2 of culture. Cells were pulsed with 1 µCi of [3H]TdR at the times indicated and were harvested 16 h later. Alternatively, freshly isolated CD4 T cells were incubated with 0.5 µM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37oC and washed with PBS before culture. Proliferation was assessed by measuring mean fluorescence intensity using flow cytometry. In some cases, cells were incubated with PE-labeled annexin V (BD PharMingen) before FACS analysis to assess cell death.
In all experiments, data shown are representative of at least three experiments.
Cell surface staining and FACS analysis
FITC-conjugated anti-IL-2R and anti-CD69 were
purchased from BD PharMingen. Samples were analyzed on a FACSCalibur
(BD Biosciences, Mountain View, CA) using CellQuest software.
Cell cycle analysis
MBP-specific CD4 T cells (3 x 105) were cultured with an equal number of inactivated APC and Ac1-11 in 48-well plates. DSG was added to cultures on day 2. Cells were harvested on day 4 and dead cells were removed by density gradient centrifugation. Tissue culture supernatants were sterile-filtered to remove any remaining cells or debris. Live cells were replated at 3 x 105 cells/ml in their original supernatants to preserve any autocrine growth factors produced. For cell cycle analysis, cells were incubated with propidium iodide (PI)/citrate mix (50 µg/ml PI, 0.1% sodium citrate, 0.3% Nonidet P-40, 50 µg/ml RNase A) and analyzed by flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether treatment with DSG could alter the onset or
severity of EAE in mice, we actively immunized (B10.PL x
SJL/J)F1 mice with the MBP peptide Ac1-11
emulsified in CFA and administered DSG i.p. testing a variety of
treatment courses. We found that initiation of DSG treatment 2 days
before immunization challenge and continued for a total of 5 days did
not alter the onset or severity of EAE, even though a dose of 7.5 mg/kg
DSG was administered. However, if DSG was administered for a total of
10–14 days, the onset of EAE was delayed (data not shown). DSG also
suppressed the EAE disease course when it was given at a lower dose
(3.75 mg/kg), and when treatment for 10 days was initiated 4 days
following immunization (Fig. 1
). As shown
in Fig. 1, 3.75 mg/kg DSG treatment resulted in a substantial delay in
the earliest onset of EAE from days 13 to 21. Individual animals were
assessed daily for the severity of disease and mice treated with DSG
had a less severe disease course (Fig. 1). This milder disease course
was also not accompanied by signs of general malaise such as rough hair
coat or weight loss that were commonly seen in PBS-treated
mice.
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is a comparison of
disease parameters of the two groups of mice studied. The disease
parameters in the two groups of mice were quite different, as indicated
by a substantial delay in the average onset of disease from day 18 in
PBS-treated animals to day 31 when mice received DSG. This difference
was found to be highly statistically significant
(p < 0.0001). In addition, the administration
of DSG slightly reduced the incidence of EAE from 100 to 84%. Although
DSG delayed the onset and reduced the incidence of EAE, those mice that
succumbed to EAE did not have an altered early disease course compared
with PBS-treated mice. This is illustrated by a similar maximal disease
score recorded for the first disease episode in the two groups of mice
(Fig. 1). In addition, an unaltered early disease course is indicated
by similar average peak disease scores of 3.5 in PBS-treated mice and
2.7 in DSG-treated mice (Table I).
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). In addition, the average disease score 35 days
following onset was reduced from a disease score of 1.8 in the PBS
group to 0.9 in the DSG group (Fig. 1). A reduction in disease severity
was most clearly demonstrated by the absence of mortality in
DSG-treated mice, while mortality approached 28% in untreated mice
(Table I). These results indicate that while the immunosuppressive drug
DSG was not able to inhibit the onset of the disease EAE in mice, it
did have a beneficial affect in dampening the long-term severity of the
disease. These data suggest that DSG does not prevent the priming of
Th1 effector cells, but rather alters their functional capacity. DSG does not inhibit activation of naive MBP-specific CD4 T cells
Because the onset of EAE was delayed rather than inhibited in
DSG-treated mice, indicating that DSG did not prevent T cell priming,
we examined whether DSG had any effects on the priming and activation
of naive T cells in vitro. To test this, CD4 T cells were isolated from
MBP TCR tg mice and activated in vitro with Ac1-11 in the presence and
absence of DSG. In the MBP-TCR tg mouse, 95% percent of the CD4 T
cells expressed the transgenic TCR 
-chain, V8.2, of which only
1.2–1.5% are of the memory phenotype (21). Thus, any
response observed upon T cell activation should not have a substantial
contribution from memory Ac1-11-specific T cells. T cell activation was
determined by measuring the increased expression of two early
activation markers, the -chain of the IL-2R (IL-2R, CD25) and
CD69, on the cell surface. As shown in Fig. 2A, the increased expression
of both CD25 and CD69 was similar in DSG-treated and untreated cells
harvested at 24 h following activation as demonstrated by similar
percentages of cells staining positive and by similar mean channel
fluorescence intensities for the positive-staining cells in the two
groups. In addition, expression levels of CD25 remained similar after
72 h of culture in the two groups (Fig. 2B). Expression
of CD69 was consistently slightly higher (maximum 2-fold) on
DSG-treated cells than on control cells on day 4 (Fig. 2B).
Treatment with DSG also did not alter the expression pattern of CD44
(data not shown).
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C). The absence of demonstrable effects of DSG
on expression of early activation markers or on production of IL-2
indicated that DSG did not inhibit the ability of naive CD4 T cells to
recognize or respond to Ag presented by APC. These data support the
observation that pretreatment of mice with DSG before induction of EAE,
and continued DSG treatment only for a short time course, had no affect
on the incidence or severity of EAE. DSG treatment of naive MBP-specific CD4 T cells results in decreased proliferation
Although DSG did not prevent the activation of naive CD4 T cells,
the drug may affect downstream activation events such as cell
proliferation and cytokine production. To examine this possibility, we
measured the level of cell proliferation in the presence and absence of
DSG following activation of naive CD4 T cells from MBP TCR tg mice.
Initial studies demonstrated that cell proliferation as measured by
[3H]TdR incorporation was similar in
DSG-treated and untreated cells stimulated with Ag after 48 h
(Fig. 3A). However, when
proliferation was examined at 72 and 96 h, the control group had a
time-dependent increase in cell proliferation while the DSG-treated T
cells proliferated at the same level as that observed after 48 h
(Fig. 3A). To examine the extent of cell proliferation in
more detail, proliferation was also measured by labeling the CD4 MBP
TCR T cells with CFSE before stimulation. CFSE is an amine-reactive dye
that is incorporated into cells and divided equally between daughter
cells during proliferation. The number of cell divisions can be
determined using flow cytometry to measure the mean fluorescence
intensity. As shown in Fig. 3B, only a small percentage of
CD4 T cells had undergone cell division after 48 h of stimulation
in either group. However, by 72 h, there was a detectable block in
proliferation of DSG-treated cells relative to control cells, with the
DSG-treated cells having undergone one less cell division (Fig. 3B). The block in cell division was even more dramatic after
96 h with the DSG-treated cells showing no additional cell
divisions beyond 72 h. In contrast, the T cells in the untreated
group continued to proliferate after 72 h, and the majority of the
cells had undergone one to four more cell divisions than the
DSG-treated T cells (Fig. 3B). These data are consistent
with the [3H]TdR incorporation data (Fig. 3A), that showed little difference in proliferation between
the DSG-treated and untreated T cells at 48 h. In addition, the
lack of continued cell proliferation with DSG treatment is reflected in
the absence of additional cell division after 72 h.
To determine whether the failure to undergo cell division after 72
h was due to enhanced cell death following DSG treatment, cells were
incubated with PE-labeled annexin V before FACS analysis at each of the
three time points. After 48 h, only a small fraction of cells
bound annexin V in either group (Fig. 3C, left
panel), indicating that DSG does not have overt toxicity. At
72 h, 37% of the untreated cells were undergoing cell death as
compared with 54% of the treated cells (Fig. 3C,
middle panel). By 96 h, there was a distinct bimodal
distribution in the control group with 54% of the cells binding
annexin V (Fig. 3C, right panel). In contrast,
75% of the cells treated with DSG for 96 h bound annexin V (Fig. 3C, right panel). The data for the control group,
shown in Fig. 3C, is consistent with reported kinetics of T
cell apoptosis following activation (25). DNA analysis of
both control and DSG-treated cells showed DNA fragmentation with ladder
formation characteristic of apoptosis at 96 h (data not shown).
These results suggest that there is an increase in cell death in cells
cultured with DSG. However, it is not clear whether enhanced cell death
results directly from the actions of DSG or is indirectly due to
a block in cell cycle progression.
DSG inhibits cell cycle progression of recently activated T cells
Our results indicated that the predominant effect of DSG was not
on primary stimulation of naive CD4 T cells, but rather on sustained
proliferation and/or survival of activated T cells. This is consistent
with our earlier finding that DSG was equally effective at inhibiting
the onset of EAE, whether treatment was initiated before or 4 days
following Ac1-11 challenge. To determine whether DSG could suppress
proliferation of T cells which had been recently activated, we measured
[3H]TdR incorporation by CD4 T cells that were
Ag-stimulated for 2 days before the addition of DSG. Addition of DSG to
cultures on day 2 inhibited proliferation of MBP-specific CD4 T cells
by 70% when measured at 96 h (Fig. 4A).
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B, the total number of control
cells increased daily. By day 7, a significant portion of the control
cells had died as indicated by trypan blue staining. In addition, the
control cells increased in total cell number between days 6 and 7
indicating that the cells were still undergoing proliferation at the
same time cell death increased. In comparison, DSG-treated cells did
not increase in total cell number after day 4, but underwent cell death
on day 6 and 7 in a similar manner as the control cells.
Although DSG appeared to inhibit the proliferation of activated CD4 T
cells, PI staining demonstrated that there was little difference in the
percentage of control (50%) and treated cells (52%) in S and
G2 before replating on day 4 (Fig. 4C
and Table II). Moreover, a larger
percentage of DSG-treated cells remained in S and
G2 on days 6 and 7 compared with control cells
(Fig. 4C and Table II). These data indicate that the failure
of cells to proliferate in the presence of DSG results from a block in
cell cycle progression. They further suggest that treatment with DSG
can block cell cycle progression without directly inducing cell
death.
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by direct action on the CD4
T cell
We next sought to determine whether DSG could also block the
differentiation of naive T cells into polarized effector cells in
addition to blocking cell division and cell cycle progression. Previous
studies have shown that DSG can inhibit the production of IFN- both
in vivo and in vitro (12, 26). This finding could be due
to a direct effect of DSG or result from the block in proliferation
leading to fewer cytokine-secreting cells. To determine whether DSG
could directly inhibit cytokine production, naive MBP-specific CD4 T
cells were stimulated with APC and Ac1-11 in primary culture in the
presence or absence of DSG. DSG was added to the primary cultures on
days 0 or 2, and the cells were cultured for 5 days. The cells were
then rested for 24 h and equivalent numbers of treated or
untreated cells were restimulated with Ag. DSG was added to primary
cultures on day 0 to assess the effect on cytokine production by naive
cells or on day 2 to assess the effect on cytokine production by cells
that have started to polarize to a Th1 phenotype (27).
Supernatants were collected following 24 h of restimulation in the
secondary culture and were analyzed for the production of IFN- and
IL-2. To determine whether any observed block in cytokine production
resulted from a direct action of DSG on the T cell, highly purified
MBP-specific CD4 T cells were activated in parallel in an
Ag-independent manner with anti-CD3, anti-CD28, and IL-2. For
secondary culture, the cells were restimulated with anti-CD3 and
anti-CD28 in the absence of IL-2.
CD4 T cells stimulated with APC and Ac1-11 in the absence of DSG
produced high levels of IFN- upon restimulation (Fig. 5A, left panel). In
contrast, little or no IFN- was produced by cells that had been
treated with DSG on days 0 or 2 in the primary culture. Addition of
IL-12 and anti-IL-4 to the primary culture induced higher levels of
IFN- production (208 ± 11 ng/ml) by control cells upon
restimulation (data not shown). In comparison, IFN- production
remained lower in similar cultures in which DSG was added on day 2
(47 ± 10 ng/ml), indicating that IL-12 is not sufficient to
overcome the inhibitory effects of DSG (data not shown).
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in secondary culture, and the
addition of DSG to the primary culture either on day 0 or 2 inhibited
IFN- production by 80% (Fig. 5A, right
panel). The effect of DSG treatment on IL-2 production was much
less dramatic. The addition of DSG to Ag-activated cells on day 0 of
the primary culture resulted in only 50% inhibition in IL-2 production
and treatment on day 2 inhibited production by only 27% (Fig. 5B, left panel). Furthermore, there was little
difference in the level of IL-2 produced by control cells and cells
treated with DSG on day 2 in the Ag-independent culture. In several
experiments, the addition of DSG to the primary cultures on day 0
resulted in slightly higher levels of IL-2 production upon
restimulation (Fig. 5B, right panel). Little IL-4
was detected in any of the cultures (data not shown) suggesting that
the lack of IFN- production in the DSG-treated groups is not due to
an IL-4-induced skewing toward a Th2 response. These data suggest that
the inhibition in IFN- production observed following DSG treatment
results from a direct action of DSG rather than from a decrease in the
number of cells producing IFN- in the cultures. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. The DSG-treated cells were able to
undergo several rounds of cell division in the absence of overt
apoptotic cell death suggesting that the delay in EAE onset that we
observed was due to the slower accumulation of MBP-specific Th1 cells
required for the induction of EAE. The overall severity of the EAE
disease course in DSG-treated mice was significantly reduced as
compared with control mice, suggesting that DSG was able to permanently
alter the Ag-specific T cells themselves, perhaps by altering the level
of cytokine production thought to be important in EAE pathogenesis.
It has been suggested that the acquisition of IFN- production
following activation of naive T cells is linked to cell cycle number,
with a higher percentage of T cells producing IFN- with increasing
cell cycle numbers (29, 30, 31). Thus, the lack of IFN-
production (Fig. 5) we observed following DSG treatment could be
directly related to the block in cell cycle progression observed by
both CFSE (Fig. 3) and PI (Fig. 4) staining. However, it is also
possible that the block in cell cycle progression and inhibition of
IFN- production we observed are independent events. In support of
this are two recent studies that demonstrated that noncycling T cells
have the capacity to produce IFN-, even when blocked in
G1 (32, 33). In addition, the study
by Ben-Sasson et al. (33) showed that the ability of a T
cell to produce IFN- was not regulated by the number of cell
divisions alone. They further showed that naive CD4 T cells stimulated
with peptide Ag for 2 days that had undergone 0, 1, or 2 cell divisions
produced IFN-, with 43, 85, and 91% of the cells producing IFN-,
respectively. Thus the inability of T cells treated with DSG to
progress past two to three cell divisions would not necessarily lead to
the profound inhibition of IFN- production that we observed. Also it
is known, using IFN--/- mice that EAE
disease is more severe rather than less (34, 35), as would
be predicted. This could reflect the ability of IFN- to kill
proliferating T cells (36).
Our results demonstrating that DSG inhibited production of IFN- by
direct action on CD4 T cells, without an apparent link to the block in
cell cycle progression, indicates that DSG may target several different
cellular processes. The only known targets of DSG are hsc70 and hsp90
(3, 4). Because it was also shown that DSG partially
inhibits NF-B transport into the nucleus, one model that has been
proposed is that hsc70 acts as a chaperone for NF-B translocation
and that by binding hsc70, DSG inhibits its nuclear transport
(8). However, hsp interact with a large number of
proteins. hsp90-binding agents, such as the benzoquinoid ansamycins
geldanamycin and herbimycin A, have been shown to inhibit a variety of
protein tyrosine kinases (PTK) suggesting a role for hsp in maintaining
signal transduction pathways (37, 38). It is possible that
DSG similarly inhibits PTK resulting in the immunosuppression observed
in our study. PTKs are involved in transducing many intracellular
signals including the activation of the mitogen-activated
protein (MAP) kinase pathway (39). Two MAP kinase
kinases (MKK), MKK4 and MKK7, activate c-Jun
NH2-terminal kinases (JNK1 and JNK2), and these
molecules in turn influence Th differentiation and cytokine production
(39, 40, 41). JNK2 has been reported to be critical for
IFN- production and for polarization of CD4+ T
cells to a Th1 phenotype (40, 41). Thus, it is conceivable
that the inhibition of IFN- production that we observed in our
studies could result from a block in JNK2 activity or the activity of
an upstream protein kinase.
Although there are no reports describing a direct role for hsp in the activation of MKK4/7 or JNK, it has recently been demonstrated that hsp90 and hsp70 are detected in a multimolecular signaling complex which includes the recently described kinase suppressor of Ras (KSR) and the MAP kinase kinases, MEK1 and MEK2 (42). Geldanamycin decreases the half-life of KSR, presumably by interfering with its association with hsp90 (42). Both MEK1 and MEK2 have been implicated in maintaining cell cycle progression through G1, M, and G2 (43, 44). Thus, one can speculate that DSG may block cell cycle progression by interfering with the signaling complex formed between KSR and MEK1/2.
Odaka et al. (45) have recently reported that a DSG analog, methyldeoxyspergualin, induced apoptosis in rapidly dividing T cell hybridomas while slower growing hybridomas and naive splenic T cells were more resistant. They also found that mitochondrial transmembrane potential was reduced in cells treated with methyldeoxyspergualin, suggesting that the drug may induce apoptosis directly by interfering with mitochondrial respiratory function. In keeping with Odaka’s findings, we found that DSG had no obvious effects on the survival of unstimulated MBP-specific CD4+ T cells (data not shown), but that increased cell death accompanied the block in proliferation of naive CD4 T cells following activation. In contrast, when cells were stimulated for 2 days before treatment with DSG, cell cycle progression was effectively blocked without enhanced cell death. There are two possible explanations for these findings. First, DSG may directly induce death of CD4 T cells only at selective stages of their activation. Alternatively, the predominant effect of DSG may be a block in cell cycle progression that subsequently leads to cell death in some instances. Cells that have received prior activation may be sensitive to the DSG-induced block in cell cycle progression, but remain more resistant to cell death due to enhanced expression of survival factors upon stimulation.
Although it is clear that DSG strongly inhibits CD4 T cell function, short-term treatment with DSG alone was not sufficient to completely abrogate the onset of clinical signs of EAE. This is in contrast to the long-term inhibition in graft rejection observed when DSG was given in combination therapy to kidney graft recipients in a primate model of transplantation (12). This may be due to the failure of Th2-mediated responses, which were observed in the transplantation study, to be induced in the EAE model used in this study. We speculate that although DSG does not drive polarization to Th2-type CD4 T cells, Th2 cells may be more resistant to the effects of DSG and maintain inhibition of Th1-mediated responses upon cessation of treatment. We have started to address this issue by comparing the effect of DSG on CD4 T cells from tg mice expressing a TCR specific for pigeon cytochrome C stimulated in vitro and polarized to a Th1- or Th2-type phenotype. In preliminary experiments, we have found that DSG inhibits cell cycle progression of both subsets of Th cells, but that cells polarized to a Th2 phenotype are somewhat less susceptible to cell death (our unpublished data). This is not surprising because IL-4 is known to be a survival factor, and appears to act in part by enhancing survival proteins such as Bcl-2 and Bcl-xL (46). However, the presence of IL-4 alone does not appear to be sufficient for overcoming the effects of DSG, as addition of IL-4 to MBP-specific CD4 T cells enhanced survival only marginally (data not shown), suggesting that other, undefined, mechanisms are involved in the enhanced survival of Th2-type CD4 T cells.
Taken as a whole, our data suggest that the clinical immunosuppressive
effects of DSG observed in both human and animal models is a direct
affect of DSG on recently activated T cells. In addition,
immunosuppression by DSG appears to occur at multiple levels, with cell
cycle progression and cytokine production being affected. DSG very
likely prevents the accumulation of autoreactive or alloreactive cells
due to their inability to proliferate, leading to a less severe immune
response. In addition, reduced IFN- production may result in a
dampened inflammatory response. Thus, immunosuppression with DSG seems
well suited to those clinical situations when T cell suppression would
be of most benefit.
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Acknowledgments |
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Footnotes |
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2 Current address: Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02138
3 Address correspondence and reprint requests to Dr. Bonnie N. Dittel at the current address: Blood Research Institute, Blood Center of Southeast Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178. E-mail address: bdittel{at}bcsew.edu
4 Abbreviations used in this paper: DSG, 15-deoxysergualin; hsp, heat shock protein; hsc, heat shock cognate protein; MP, methylprednisolone; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; tg, transgenic; PI, propidium iodide; PTK, protein tyrosine kinase; MAP, mitogen-activated protein; MKK, MAP kinase kinase; JNK, c-Jun NH2-terminal kinase; KSR, kinase suppressor of Ras; MEK, MAP kinase kinase.
Received for publication March 6, 2002. Accepted for publication August 29, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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light chain expression in 70Z/3 pre-B cells by blocking lipopolysaccharide-induced NF-B activation. J. Immunol. 155:2427.[Abstract]
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or IL-4. J. Immunol. 166:112. gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156:5.[Abstract]
plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
plays a critical role in induced cell death of effector T cell: a possible third mechanism of self-tolerance. J. Exp. Med. 172:1735.This article has been cited by other articles:
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) and 16 mice were treated with
DSG (•) for 10 days beginning 4 days after Ag challenge in groups of
three to five mice. Individual mice were scored daily for 55 days for
severity of EAE using the following scale: 0, no disease; 1, limp tail
or mild head tilt; 2, hind limb paresis or moderate head tilt; 3, front
limb or hind limb paralysis; 4, front and hind limb paralysis; and 5,
death. The daily scores were averaged from all mice in each
group.
)
or presence (
) of DSG after 24 h of culture and IL-2 was
measured by ELISA.
) cell numbers from control and DSG-treated cultures were
compared. Live and dead cells were distinguished using trypan blue.
C, Cell cycle analysis of control and DSG-treated cells
incubated with PI were compared by FACS analysis immediately following
live cell recovery on day 4 (top histograms) and then 2
days (middle histograms) and 3 days (bottom
histograms) after replating the cells.
, DSG added day 0.