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B-Dependent T Cell Activation Abrogates Acute Allograft Rejection1
*
Laboratory of Molecular Immunology,
Pulmonary and Critical Care, Department of Medicine, and
Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and
Department of Medicine, Vanderbilt University, Nashville, TN 37232
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B(
N)-transgenic mice that express a transdominant inhibitor of
NF-B in T cells. Our results show indefinite prolongation of graft
survival in the I-B(N)-transgenic recipients. Interestingly, at
the time of rejection of grafts in wild-type recipients, histology of
grafts in the I-B(N)-transgenic recipients showed moderate
rejection; nevertheless, grafts in the I-B(N) recipients survived
>100 days. Analysis of acute phase cytokines, chemokine, chemokine
receptors, and immune responses shows that the blockade of NF-B
activation in T cells inhibits up-regulation of many of these
parameters. Interestingly, our data also suggest that the T cell
component of the immune response exerted positive feedback regulation
on the expression of multiple chemokines that are produced
predominantly by non-T cells. In conclusion, our studies indicate
NF-B activation in T cells is necessary for acute allograft
rejection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B comprises a family of NF-B/Rel transcription factors
that regulates the transcription of multiple inflammatory and immune
genes. NF-B family members are widely expressed in multiple cell
types and include p50, p52, RelA (p65), c-Rel, and RelB. In studies of
T cell activation, NF-B amplifies the expression of multiple
chemokine and cytokine genes (1, 2). Thus, NF-B is a
potent proinflammatory signal transduction molecule in T cells.
Relevant to clinical transplantation, corticosteroids, which are a
common treatment for allograft rejection, inhibit NF-B activation
(3, 4, 5). However, it remains undetermined whether the
crucial site of action of corticosteroids involves T cells or other
types of inflammatory cells.
In the resting state, NF-B is retained in the cytoplasm as a complex
bound by I-B. After activation, I-B is phosphorylated,
ubiquinated, and subsequently degraded in the proteosome, thus
facilitating NF-B translocation to the nucleus, where it functions
as a regulator of transcription (6, 7, 8). Mutations of the
phosphorylation site of I-B
can create dominant-negative mutants
that inhibit NF-B functions by blocking nuclear translocation
(9, 10, 11). For example, I-B(N), which contains a
deletion of the phosphorylation site, functions as a transdominant
inhibitor of NF-B activation. Transgenic mice that express an
I-B(N) transgene regulated by the T cell-specific proximal
lck promoter showed decreased cytokine production and
defective T cell proliferative responses that are not corrected with
the addition of exogenous IL-2 (12). Moreover, the
I-B(N)-transgenic mice exhibit decreased susceptibility to
allergic airway hyperresponsiveness and collagen-induced arthritis
(13, 14). Our current study analyzes allograft rejection
in I-B(N)-transgenic mice. In addition to multiple parameters of
rejection, our study investigated the role of NF-B-dependent T cell
activation on the expression of subsets of chemokines, cytokines, and
other immune genes up-regulated during the early vs late immune
response after transplantation. Our results demonstrate that the
inhibition of NF-B activation in T cells abrogates up-regulation of
chemokines and cytokines during the late phases of rejection, resulting
in indefinitely prolonged allograft survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine hearts were transplanted, as previously described (15). Briefly, hearts were harvested from freshly sacrificed donors and immediately transplanted into 8- to 12-wk-old recipients that were anesthesized via i.p. injection with 60 mg/kg pentobarbital sodium. The donor aorta was attached to the recipient abdominal aorta by end-to-side anastamosis, and the donor pulmonary artery was attached to the recipient vena cava by end-to-side anastamosis. All surgical procedures were completed in less than 60 min from the time that the donor heart was harvested. Donor hearts that did not beat immediately after reperfusion or stopped within 1 day following transplantation were excluded (>95% of all grafts functioned at day 2 following transplantation). Donor grafts were harvested at the indicated times following transplantation and divided into equal sections for preparation of RNA and tissue sections for histology. Clinical rejection was defined as the cessation of a palpable heartbeat and was confirmed by histology using the International Society for Heart and Lung Transplantation criteria (16).
Mice
Eight- to 12-wk-old male wild-type BALB/cByJ (BALB/c)
(H-2d) and C57BL/6J (B6)
(H-2b) mice (The Jackson Laboratory, Bar Harbor,
ME) were used as donor and recipients, respectively, in the transplant
experiments. The I-B(N)-transgenic mice were also used as
recipients and produced by injecting an amino-terminally truncated form
of I-B, including aa 37–317 linked to the proximal
lck promoter plus the locus control region from the human
CD2 gene into C57BL/6 x DBA/2 zygotes (12) (see
Table I
). Founder mice were backcrossed with C57BL/6 mice for four
generations, and transgene expression was determined by Southern blot
analysis. Mice were maintained in virus Ab-free facility in accordance
with federal and state government regulations after Harvard Medical
School institutional approval.
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Chemokine, chemokine receptor, and CD marker expression was
analyzed by RNase protection assay
(RPA),3 as previously
described (17). Briefly, total RNA was isolated from
hearts using RNAzol and analyzed using the RiboQuant MultiProbe RPA
System (BD PharMingen, San Diego, CA). A total of 15 µg of RNA was
used per hybridization and RNase reaction with the templates mCK-5
(lymphotactin (Ltn), RANTES, eotaxin, macrophage-inflammatory
protein-1
(MIP-1), MIP-1, MIP-2, IFN-
-inducible protein-10
(IP-10), monocyte chemoattractant protein-1 (MCP-1), and macrophage
inhibitory factor), mCK2b (IL-1, IL-1, IL-1R antagonist
(IL-1ra), IL-6), mCK3b (TNF-), mCR-5 (CCR1, 1, 4, 5, and 2), a
custom template (CXCR2, 3, 4, and CCR6, 8, 8, and CXCR5 (BLR-1)),
and mCD-1 (TCR
, TCR, CD3
, CD4, CD8, CD8, CD19, CD14,
CD45) (BD PharMingen). The IP-10 template detects the C57BL/6 allele.
The protocol was modified to use 35S-labeled
probes to hybridize RNA. After RNase treatment and purification,
protected probes were electrophoresed on a denaturing 5%
polyacrylamide gel. The gels were exposed in a Molecular Dynamics
(Sunnyvale, CA) PhosphorImager. The identity of each protected fragment
was established by analyzing its migration distance against a standard
curve of the migration distance vs the log nucleotide length for each
undigested probe. Samples were normalized to the housekeeping gene,
GAPDH. Protected bands were quantitated by densitometry analysis using
ImageQuant software (Molecular Dynamics). All results represent a
minimum of two independent analyses.
Histology
Recipient native hearts and donor-transplanted hearts were harvested at the indicated times following transplantation and fixed in 10% neutral buffered Formalin. After dehydration and paraffin embedding, 5- to 6-µm-thick sections were routinely stained with H&E. Multiple sections were examined for each heart, and the extent of histological rejection (grade 0–4) and ischemia (grade 0–4, determined by the degree of healing ischemia) was quantified using a modified International Society of Heart and Lung Transplantation grading scale (16). Any samples with an ischemia score >1 were excluded from further analysis.
Mixed lymphocyte culture
A total of 2 x 105 responder spleen cells was stimulated with 4 x 105 stimulator spleen cells that had been irradiated with 2000 rad in 200 µl of RPMI 1640 plus 10% FCS. A total of 1 µCi of [3H]thymidine was added during the last 12 h of culture. After 96 h, cells were harvested and thymidine incorporation was determined, as described elsewhere (18). Cultures were performed in quadruplicate, and SEM were <10%.
ELISA
Cytokines TNF-, IL-6, and IL-1 were evaluated using a
Quantikine M immunoassay (R&D Systems, Minneapolis MN) per the
manufacturer’s directions. Briefly, the specific Ab for each cytokine
was precoated in microtiter wells. A total of 100 µl of serum sample
obtained by cardiac puncture or cytokine standard was incubated
overnight at 4°C. An enzyme-linked polyconal Ab specific for the
particular cytokine was added to the wells and incubated for 2 h.
After washing, the substrate tetramethylbenzidine plus hydrogen
peroxide was added and incubated 30 min at room temperature. After
adding a stop solution of dilute hydrochloric acid, the OD was measured
with an Emax microplate reader (Molecular Devices, Sunnyvale, CA) at
450 nm wavelength, along with a correction reading at 540 nm wavelength
for optical interference by the microtiter plate. Triplicate readings
of the control and serum samples and duplicate readings of each
standard were averaged after subtracting the background standard OD.
Final sample calculations were based on a regression analysis of the
log of the final OD vs the log of standard dilutions. The sensitivity
of detection is 3, 3.1, and 2 pg/ml for IL-1, IL-6, and TNF-,
respectively.
Statistics
Graft survival data were calculated as mean, and p
values were calculated using Kaplan-Meier/log rank test methods, and
differences were considered significant at p < 0.05.
Differential expression of mRNA determined by RPA on days 0, 1, 3, 5,
and 7 in the I-B(N) and wild-type recipients was analyzed by
two-factor ANOVA. Statistical significance of variances was calculated
for p < 0.05 using the F test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in T cells prolongs allograft
survival
In this study, we investigated the role of NF-B activation in T
cells in a murine model of vascularized heterotopic allogeneic heart
transplantation. To inhibit NF-B activation in T cells, we analyzed
I-B(N)-transgenic mice that express a transdominant inhibitor of
NF-B (I-B(N)) driven by the the T cell-specific proximal
lck promoter. Cardiac transplants were performed across a
complete MHC mismatch using BALB/c (H-2d) donors
and I-B(N) (in a C57BL/6 (H-2b) background)
recipients. In the wild-type group, BALB/c grafts were transplanted
into C57BL/6 (B6) (H-2b) recipients. All grafts
transplanted into the I-B(N) recipients survived >100 days
compared with 7.8 days in the wild-type B6 recipients
(p < 0.0001) (Table I
). Thus, expression of the transdominant
inhibitor of NF-B in T cells provided effective protection against
acute allograft rejection in our model.
To characterize acute rejection and the recruitment of inflammatory
cells, we analyzed graft histology at days 1 and 7 following
transplantation (Fig. 1). On day 1, the
I-B(N) recipients had no evidence of rejection (grade 0) with
focal mononuclear cell infiltration. By day 7, when the wild-type
control B6 recipients had grade 4 rejection including myocyte necrosis,
the I-B(N) recipients had developed grade 2 rejection with
moderate focal infiltrates, but without myocyte necrosis.
Interestingly, despite histological evidence of moderate rejection
(grade 2) in the I-B(N) recipients, all of the grafts survived
>100 days (Table I).
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B(N) recipients are
primarily macrophages
The lack of rejection despite the detection of moderate
infiltrates by histology could be due to differential composition of
the infiltrating cells. To analyze the types of infiltrating cells, we
performed RPA of CD markers, including TCR, TCR, CD3, CD4,
CD8, CD8, F4/80, and CD45 with RNA harvested on days 1, 3, 5, and
7 from graft hearts from I-B(N) and wild-type B6 recipients (Fig. 2). Quantification of these results by
densitometry showed that the infiltrating cells in the graft
I-B(N) recipients from days 1 to 7 predominantly expressed the
F4/80 macrophage and CD45 leukocyte markers (Fig. 2, A and
B). These results showed that grafts from both the
I-B(N) and wild-type B6 recipients developed macrophage
infiltration. However, the level of macrophage infiltration tended to
decrease in the I-B(N) recipients by days 5 and 7, whereas the
level increased in the wild-type B6 group. In addition, a low level of
T cell markers including TCR+ and CD8 was
detected on days 5 and 7, but the level of expression was less than 5%
of control GAPDH. In contrast, in the wild-type B6 group, there were
marked increases in
TCR+CD8+ T cells that
were detectable by day 5 and exceeded 20% of GAPDH by day 7 (Fig. 2, A and C). To determine the statistical
significance of these observations, variances were calculated for each
gene analyzed by RPA on days 0, 1, 3, 5, and 7 in the I-B(N) and
wild-type recipients, and significance was calculated as
p < 0.05 using the F test (Table II). Based on theseanalyses, the
grafts from I-B(N) recipients had a significant decrease in the
level of T cell infiltration compared with the wild-type B6
recipients.
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B(N) recipients
Chemokines are important mediators of cell recruitment to
inflammatory sites. Therefore, to characterize the cellular infiltrate
in the graft hearts, we analyzed the expression of a large panel of
chemokine receptors from both the CCR and CXCR families, including
CCR1, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, and CCR8, plus
CXCR2, CXCR3, CXCR4, and CXCR5 (Fig. 3).
CCR2, CCR5, and CXCR4 were moderately increased in the grafts from
I-B(N) recipients (Fig. 3A); however, the level of
expression of all of these chemokine receptors remained lower than in
the wild-type B6 recipients (Fig. 3B). ANOVA calculations
showed that these differences were highly significant for CCR2
(<0.004), CCR5 (<0.001), and CXCR4 (<0.041) (Table II). We also
found large increases in chemokine receptor expression in wild-type B6
recipients that includes CCR1, CCR3, and CCR4 in addition to the three
receptors up-regulated in the I-B(N) recipients. Thus, the
expression of some chemokine receptors was undetectable in
I-B(N), while detectable in the wild-type controls. Based on the
low level of T cell infiltration in the grafts from I-B(N)
recipients, these results suggest that the decreased expression of
chemokine receptors in these mice is due to decreased recruitment or
decreased cellular activation.
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B(N) recipients: allospecific
proliferation and cytokines
Because expression of the transgene is regulated by the T
cell-specific proximal lck promoter, our hypothesis was that
the global down-regulation of chemokine receptor expression was due to
a defect of T cell regulatory functions. To assess the allospecific
response of T cells from I-B(N) mice, we performed MLR (Fig. 4). These results showed a decreased
proliferative response to allogeneic stimulator cells by the
I-B(N) compared with wild-type B6 control responder cells. As
expected, the response to control syngeneic B6 stimulator cells was
comparable by both strains. We also assessed T cell responses in vivo
by analyzing the expression of cytokines, including IL-2, IFN-,
lymphotoxin (LT), and LT, which are produced, at least in
part, by T cells (Fig. 5). IFN- and
LT expression was markedly increased in the wild-type B6 control
grafts, whereas the level of expression remained low (<3% of GAPDH)
in the I-B(N) recipients. Both IL-2 and LT were expressed at
levels barely detectable by RPA at all time points analyzed in both
groups. ANOVA calculations showed that the increased expression was
significant for IFN- (<0.006) and LT (<0.042) (Table II). Taken
together, these results are consistent with decreased T cell responses
both in vitro and in vivo in the I-B(N) recipients.
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B(N) recipients: an initial
increase is followed by decreased expression
Our analysis of graft histology, the composition of the cellular
infiltrates, and chemokine receptor expression suggested decreased
positive feedback of the inflammatory response in the transgenic
I-B(N) recipients. Because the defect in the transgenic
recipients appears to be limited to the T cells, these observations
suggested that the defect was localized within T cell activity. To test
this hypothesis, we analyzed the acute phase response, which comprises
an aspect of the innate immune response including the cytokines
TNF-, IL-6, IL-1, and IL-1ra that are produced primarily by non-T
cells, but possibly regulated by T cell-dependent mechanisms. Analysis
of serum cytokines revealed that on day 1 after transplantation, both
IL-6 and IL-1 were increased in both the I-B(N) and wild-type
B6 groups, although to a lesser extent in the I-B(N) group (Fig. 6A). However, on day 7 after
transplantation, IL-6 levels are further increased in the wild-type B6
recipients, whereas expression in the I-B(N) group has decreased
to low levels (Fig. 6B).
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B(N) groups (Fig. 7). However, by day 7 following
transplantation, IL-6, IL-1, and IL-1ra are markedly increased in
the wild-type B6 group, but not the I-B(N) recipients. On days 3
and 5 posttransplantation, the levels of acute phase cytokines
decreased in the I-B(N), but increased in the wild-type B6
recipients (not shown). ANOVA calculations showed that the differential
expression was significant for IL-1 (<0.008) and IL-1ra (<0.013),
but not for TNF- or IL-6 (Table II). These results indicate that
although the acute phase cytokines are up-regulated on day 1 at the
level of mRNA in both the I-B(N) and wild-type B6 groups, a block
in NF-B activation in T cells can prevent further up-regulation of
acute phase cytokine levels by day 7.
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, MIP-1, MCP-1, and MIP-2 in I-B(N)
recipients: initial increase, followed by decrease
Chemokines were up-regulated by day 1 posttransplantation in both
the I-B(N) (Fig. 8A) and
wild-type B6 recipients (Fig. 8B), although the magnitude of
the increase was already greater on day 1 in the control recipients. A
kinetic analysis of expression showed that the levels of expression
tended to decrease through days 3–7 following transplantation in the
I-B(N) recipients. In contrast, the level of expression markedly
increased on days 5 and 7 following transplantation in the control
group. ANOVA calculations showed that the differential expression was
significant for MIP-1 (<0.001), MIP-1 (0.005), MIP-2 (<0.005),
and MCP-1 (<0.030) (Table II). These results suggest that chemokines
that are inducible by innate immunity can be further up-regulated by
adaptive responses, presumably by T cell-dependent mechanisms. However,
inhibition of NF-B activation in T cells prevents the amplification
of the chemokine expression.
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B(N) recipients
In our analysis of the I-B(N) recipients, we found minimal
increases of Ltn, RANTES, and IP-10 chemokines detected from days 1
through 7 following transplantation in the I-B(N) recipients
(Fig. 9a); however, consistent
with our previous results, all four of these chemokines were highly
induced by days 5 and 7 in the control B6 recipients (Fig. 9b). ANOVA calculations showed that the differential
expression was highly significant for Ltn (<0.003), RANTES (<0.001),
and IP-10 (<0.001) (Table II). These results indicate that
up-regulation of this subset of chemokines, which is dependent upon the
adaptive immune response, is not up-regulated in the I-B(N)
recipients that have deficient NF-B activation in T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel family of transcription factors has pleiotropic
functions involving the up-regulation of multiple inflammatory and
immune genes (19). Previous studies in transplantation
investigating NF-B have focused on the role of p50; these studies
showed modest prolongation of graft survival (20).
Although NF-B/Rel proteins are widely expressed in most cell types,
in this study we focused on the role of NF-B activation in T cells
in a model of vascularized heterotopic heart transplantation. To
inhibit NF-B activation in T cells, we used I-B(N)-transgenic
mice that express a transdominant inhibitor of NF-B in T cells
(12). Previous studies reported detection of the
I-B(N) transgene in peripheral lymphoid tissue, but not T
cell-depleted spleen cells, confirming predominant T cell expression of
the transgene (14). As expected, our studies showed
decreased T cell responses determined in vitro by mixed lymphocyte
reactions and in vivo by decreased cytokines, including reduced levels
of IFN- and LT. These results are consistent with previous
studies showing decreased production of IFN- in vivo in the
I-B(N)-transgenic mice in a model of allergic pulmonary
inflammation (13).
In this study, cardiac transplants in I-B(N) recipients all
survived until the recipients were sacrificed after >100 days compared
with a mean survival time of 7.8 days in wild-type B6 recipients. Thus,
inhibition of NF-B in T cells was sufficient to inhibit acute
allograft rejection. However, histological analysis of allografts in
the I-B(N) recipients at day 7 following transplantation, a time
immediately preceding acute rejection in the wild-type B6 recipients,
showed grade 2 rejection with mononuclear cell infiltration compared
with grade 0 in syngeneic control grafts. Characterization of the
phenotype of the infiltrating cells demonstrated that the composition
of the infiltrate consisted predominantly of
F4/80+ macrophages and
CD45+ leukocytes; however, only extremely low
levels of T or B cell markers were detected. Decreased detection of
lymphocytes could be due to decreased migration into the graft,
decreased proliferation, or increased apoptosis. As expected, the
control wild-type B6 recipients had evidence of infiltrating T cells in
addition to F4/80+ macrophages and leukocytes.
Although F4/80+ macrophages were present, the
lack of high levels of TNF- and IL-1, cytokines commonly produced
by macrophages, suggests that the macrophages may not be fully
activated. Also, we analyzed chemokine receptor levels in the graft
tissue. These results showed decreased levels of multiple chemokine
receptors of both the CCR and CXCR families, including CCR1, CCR1,
CCR2, CCR3, CCR4, CCR5, and CXCR4. Although these receptors can be
expressed by multiple cell types, all have been reported to be
expressed by T cells (20), which could partially account
for the decreased levels in the I-B(N) recipients following
transplantation. Taken together, these studies indicate that T cells
expressing the transgene were inefficient at infiltrating
allografts.
To investigate mechanisms of rejection that were defective in the
I-B(N) recipients, we focused on kinetic analyses of cytokine and
chemokine expression starting at time points 24 h following
transplantation. The acute phase response is an important component of
innate immunity mediated by cytokines, including IL-1, IL-6, and
TNF- in response to infection, stress, or injury (21).
Interestingly, our analysis of the acute phase reactants, in particular
IL-6 and IL-1, indicates an increase at day 1 after transplantation
in both the I-B(N) and wild-type B6 recipients at the levels of
both serum cytokines and graft heart mRNA. In contrast, at day 7 after
transplantation, the wild-type B6 recipients expressed increased levels
of IL-6 in the serum and IL-6, IL-1, and IL-1ra mRNA, whereas in the
I-B(N) recipients all of these acute phase reactants were
decreased. Both groups express similar levels of TNF- mRNA at both
time points without marked increases in the serum, suggesting that
TNF- may be functioning locally within the graft at these time
points. Interestingly, in the wild-type B6 group, increased IL-6 mRNA
correlates with the serum cytokine level at both days 1 and 7; however,
although IL-1 mRNA is increased at both time points, at day 7 serum
IL-1 levels are low. One interpretation is that IL-1 is produced
by multiple sites, including locations peripheral to the graft.
Alternatively, at day 7 after transplantation, IL-1-converting enzymes
could be activated, leading to the degradation of IL-1 in the serum.
In contrast, the levels of IL-6 mRNA and serum protein correlate at
both time points, suggesting that IL-6 may be produced, at least
predominantly, by cells within the graft.
The chemokines MIP-1, MIP-1, MIP-2, and MCP-1 were increased 1
day following transplantation in both the I-B(N) and wild-type B6
groups, although the level expressed was lower in the I-B(N)
mice. Using lymphocyte-deficient RAG knockout mice, we have found that
innate immune responses were sufficient to induce these same four
chemokines in the absence of lymphocytes 1 day following
transplantation (data not shown). These observations suggest the
possibility that the lower chemokine levels observed in this study were
due to the lack of positive feedback regulation from activated T cells
in the I-B(N)-transgenic mice. This hypothesis is supported by
our kinetic analysis indicating decreased chemokine levels in the
I-B(N) recipients at days 3 through 7, whereas chemokine levels
were markedly increased by days 5–7 in the wild-type B6 recipients.
This conclusion is further supported by previous reports indicating
that the major producers of these four chemokines are non-T cells
(22, 23). In contrast, increased production of TGF-1
and TGF-3 is not inhibited in the I-B(N) recipients.
A common paradigm of immunity is that activation of innate immunity is
important, or even a prerequisite, for the initiation of an adaptive
immune response (24). In this paradigm, innate responses
produce chemokines and cytokines and up-regulate costimulatory ligands
and adhesion molecules, creating a milieu that promotes and regulates
the activation of adaptive immunity. Interestingly, our results suggest
an additional reciprocal pattern of regulation in which adaptive
immunity produces positive feedback regulation of the innate response.
Furthermore, our studies indicate that NF-B-dependent activation of
the T cell component of adaptive immunity is required for the positive
feedback.
In our current experiments, levels of the chemokines RANTES, Ltn, and
IP-10 were also markedly decreased in the I-B(N) recipients.
Previous studies have shown that RANTES and Ltn, although produced by
multiple cell types, are highly up-regulated in T cells. Thus, these
results suggest that inhibition of NF-B in T cells may directly
decrease production of RANTES and Ltn. In contrast, IP-10 is produced
primarily by monocytes, endothelial cells, keratinocytes, and
fibroblasts, but not T cells. However, IP-10 is induced by IFN- and
TNF-, two cytokines that can be produced by Th1 T cells. Thus,
decreased production of IP-10 in the I-B(N) recipients may be due
to reduced secretion of IFN- or TNF- by T cells. Previous studies
have shown that transcription of TNF- is regulated, at least in
part, by NF-B (25, 26). These observations indicate
that the T cell component of adaptive immunity is crucial for the
up-regulation of these chemokines during days 5 to 7 of allograft
rejection. Thus, in the I-B(N) recipients of allografts, the
expression of RANTES, Ltn, and IP-10 is blocked.
In this study, we have analyzed allograft survival, T cell functions,
and levels of genes, including cell surface markers, chemokine
receptors, chemokines, and cytokines. Using this approach, we have
assessed the NF-B-dependent components of an immune response in a
model of transplantation. Future studies will be required to analyze
the function of individual genes in the process of rejection. Our
results demonstrate that NF-B activation in T cells is necessary for
multiple parameters of in vitro and in vivo T cell activation and for
acute rejection of vascularized cardiac allografts. In addition, our
results indicate that the T cell component of the adaptive immune
response exerts positive feedback regulation on the early inflammatory
response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David L. Perkins, Brigham and Women’s Hospital, PBB-170, 75 Francis Street, Boston, MA 02115. E-mail address: dperkins{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: RPA, RNase protection assay; IP-10, IFN--inducible protein-10; LT, lymphotoxin; Ltn, lymphotactin; MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein; IL-1ra, IL-1R antagonist.
Received for publication June 18, 2001. Accepted for publication August 31, 2001.
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  J. Koreth, K. E. Stevenson, H. T. Kim, M. Garcia, V. T. Ho, P. Armand, C. Cutler, J. Ritz, J. H. Antin, R. J. Soiffer, et al. Bortezomib, tacrolimus, and methotrexate for prophylaxis of graft-versus-host disease after reduced-intensity conditioning allogeneic stem cell transplantation from HLA-mismatched unrelated donors Blood, October 29, 2009; 114(18): 3956 - 3959. [Abstract] [Full Text] [PDF]
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 K. Bunting, S. Rao, K. Hardy, D. Woltring, G. S. Denyer, J. Wang, S. Gerondakis, and M. F. Shannon Genome-Wide Analysis of Gene Expression in T Cells to Identify Targets of the NF-{kappa}B Transcription Factor c-Rel J. Immunol., June 1, 2007; 178(11): 7097 - 7109. [Abstract] [Full Text] [PDF]
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P. Zhou, S. J. Balin, M. Mashayekhi, K. W. Hwang, D. A. Palucki, and M.-L. Alegre Transplantation Tolerance in NF-{kappa}B-Impaired Mice Is Not Due to Regulation but Is Prevented by Transgenic Expression of Bcl-xL J. Immunol., March 15, 2005; 174(6): 3447 - 3453. [Abstract] [Full Text] [PDF]
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 K. Sun, L. A. Welniak, A. Panoskaltsis-Mortari, M. J. O'Shaughnessy, H. Liu, I. Barao, W. Riordan, R. Sitcheran, C. Wysocki, J. S. Serody, et al. Inhibition of acute graft-versus-host disease with retention of graft-versus-tumor effects by the proteasome inhibitor bortezomib PNAS, May 25, 2004; 101(21): 8120 - 8125. [Abstract] [Full Text] [PDF]
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S. A. McCracken, E. Gallery, and J. M. Morris Pregnancy-Specific Down-Regulation of NF-{kappa}B Expression in T Cells in Humans Is Essential for the Maintenance of the Cytokine Profile Required for Pregnancy Success J. Immunol., April 1, 2004; 172(7): 4583 - 4591. [Abstract] [Full Text] [PDF]
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D. Artis, S. Shapira, N. Mason, K. M. Speirs, M. Goldschmidt, J. Caamano, H.-C. Liou, C. A. Hunter, and P. Scott Differential Requirement for NF-{kappa}B Family Members in Control of Helminth Infection and Intestinal Inflammation J. Immunol., October 15, 2002; 169(8): 4481 - 4487. [Abstract] [Full Text] [PDF]
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B6 graft heart day 1 (b),
BALB
