| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
,§
Departments of
*
Surgery,
Pathology, and
Molecular Virology, Immunology, and Medical Genetics, and
§
Comprehensive Cancer Center, Ohio State University College of Medicine, Columbus, OH 43210
 |
Abstract |
|---|
 Top Abstract IntroductionMethods and MaterialsResultsDiscussionReferences |
|---|
C57BL/6 cardiac allograft recipients treated transiently with
gallium nitrate accept their grafts for >90 days and fail to display
DBA/2-reactive DTH responses. These DTH responses are restored when
anti-TGF-ß Abs are included at the challenge site, and cell
depletion studies showed that this DTH inhibition is mediated by
CD4+ cells. Real-time PCR analysis revealed that allograft
acceptor mice produce no more than background levels of TGF-ß mRNA at
DTH challenge sites. This suggests that DTH regulation in allograft
acceptor mice may involve TGF-ß activation, rather than TGF-ß
production. The protease, plasmin, can activate TGF-ß, and activated
T cells can express a receptor for the plasmin-producing enzyme
urokinase-type plasminogen activator (uPA), and can also produce both
uPA and tissue-type plasminogen activator (tPA). We observed that Abs
to tPA or uPA can replace anti-TGF-ß mAb for the restoration of
donor-reactive DTH responses in allograft acceptor mice. Histologic
analysis revealed that accepted cardiac allografts express uPA, tPA,
and active TGF-ß, whereas accepted cardiac isografts express only
tPA, but not uPA or activated TGF-ß. These data demonstrate that
local tPA and uPA contribute to DTH regulation in allograft acceptor
mice and suggest that these elements of the fibrinolytic pathway are
used to control donor-reactive cell-mediated immunity in allograft
acceptor mice. |
Introduction |
|---|
|
TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
|---|
A similar regulatory mechanism is revealed by the donor-reactive delayed-type hypersensitivity (DTH) responses of allograft acceptors. Tetanus toxoid-sensitized, allograft acceptor mice do not mount DTH responses when challenged with donor alloantigens, although they can mount prominent DTH responses when challenged with tetanus toxoid. However, allograft acceptor mice fail to mount DTH responses to mixtures of tetanus toxoid and donor alloantigens (6). Thus, allograft acceptor mice exhibit a regulatory mechanism that is donor alloantigen-dependent in its induction, but Ag nonspecific in its effect. As a result, immune responses to any Ags that happen to be in close proximity with donor alloantigens are effectively disrupted by the same regulatory mechanisms that disrupt acute allograft rejection and donor-reactive DTH responses. Because of this, these regulatory mechanisms are commonly referred to as "bystander suppression" or "linked Ag nonresponsiveness" (6, 7). The promiscuity of these regulatory mechanisms could be explained by the alloantigen-specific liberation of one or more immunoregulatory cytokines, such as TGF-ß, IL-10, or IL-4, which, in turn, influence immune functions in an Ag nonspecific manner. We tested this hypothesis, and in an earlier communication we reported that donor-reactive DTH responses in allograft acceptor mice could be restored if Abs to TGF-ß or IL10 were included at the DTH challenge site (8).
TGF-ß is especially interesting for a variety of reasons. In is a well-known immunosuppressive cytokine that is operative in several experimental models of immunity. These include both anterior chamber-associated immune deviation (ACAID) (9, 10, 11) and experimental autoimmune encephalomyelitis (12, 13). TGF-ß is unusual among cytokines in that it is usually found in a biologically inactive form as a complex with latent TGF-ß-binding protein (LTBP). To express its biologic activity, TGF-ß must be enzymatically dissociated from LTBP, thus liberating biologically active TGF-ß. Because of this, the regulation of TGF-ß activity is complex and occurs not only at the level of TGF-ß production but also at the level of TGF-ß activation.
One of the enzyme systems that is capable of generating active TGF-ß is the serine protease, plasmin (14). Plasmin is generated from an ubiquitous serum protein, plasminogen that enters tissues whenever vascular integrity is lost, i.e., at sites of tissue injury or inflammation. There, two enzymes, tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) can catalyze its reduction to plasmin. As a serine protease, plasmin plays a major role in matrix remodeling and angiogenesis (15). More generally, it regulates the balance between thrombosis and fibrinolysis in damaged tissues. Although immunity is intimately associated with inflammation and tissue repair, little is known regarding the impact that immunity can have on these two processes. In this regard, it is interesting to note that activated T cells can produce uPA, its receptor uPAR, and tPA (16). This suggests that T cells can help to generate plasmin, and thus may promote tissue remodeling and angiogenesis at sites of inflammation.
It is also possible that vital components of inflammation and tissue repair may influence immunity. We have explored the possibility that one such component may be plasmin. We knew that the impairment of donor-reactive immune responses in allograft acceptor mice was under the control of TGF-ß, that plasmin was one of the proteases that can activate TGF-ß, and that one of the systems that generate plasmin is uPA/tPA. Hence, we tested the hypothesis that the regulation of donor-reactive alloimmunity in allograft acceptor mice depended, to some degree, on uPA/tPA.
|
Methods and Materials |
|---|
|
TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
|---|
C57BL/6 (B6, H-2b), DBA/2 (H-2d), and FVB/N (H-2q) mice were obtained from Taconic (Germantown, NY). All mice were housed and treated in accordance with Animal Care Guidelines established by the National Institutes of Health and the Ohio State University.
Cytokines and Abs
Porcine TGF-ß, polyclonal rabbit anti-human TGF-ß Abs, and control rabbit Ig were all obtained from R&D Systems (Minneapolis, MN). Murine IL-2 and monoclonal rat anti-mouse IL-2 Abs were obtained from PharMingen (San Diego, CA). Rabbit anti-mouse tPA and rabbit anti-rodent uPA were obtained from American Diagnostica (Greenwich, CT).
Murine cardiac transplantation
Heterotopic cardiac transplantation was performed as described by Corry et al. (17). In general, the native hearts from heparinized donor mice (DBA/2) were anastomosed to recipient B6 abdominal aorta and vena cava using microsurgical techniques. Graft survival was assessed by trans-abdominal palpation.
Immunosuppression with GN
As described previously (18), GN (Ganite, Fujisawa, Deerfield, IL) was administered as an initial s.c. bolus injection of 2.2 mg 24 h before graft implantation, followed by 28 days of continuous delivery via s.c. osmotic minipumps (model 2002; Alzet, Palo Alto, CA) which delivered 0.5 µl (12.5 µg GN) per hour. Circulating levels of GN fall to subtherapeutic levels within 7 days of pump removal (3).
Subcellular alloantigen
Subcellular alloantigen was prepared according to the method of Engers et al. (19). Briefly, fresh RBC-depleted DBA/2 splenocytes suspended in PBS were subjected to three rapid freeze/thaw cycles using liquid nitrogen, and spun at 13,000 rpm for 30 min to remove residual debris. The supernatant was adjusted to 3–5 mg protein/ml and used as the source of subcellular alloantigen. For DTH challenge, 25 µl (75–125 µg protein) of this solution was injected into murine pinna.
Transfer DTH assay
Cardiac allograft acceptor mice were tested for DTH responses between 60 and 90 days posttransplant using a transfer DTH assay. For this assay, 25 µl containing 8 x 106 syngeneic splenocytes from allograft acceptor mice plus challenge alloantigen were injected into the pinnae of naive B6 mice using a 30-gauge insulin syringe. Changes in ear thickness were measured both before injection and 24 h after injection using a dial thickness gauge (Swiss Precision Instruments, Carlstadt, NJ). For reference, changes in the range of 0–30 x 10-4 inches represent background swelling due to injection trauma, changes in the range of 40–60 x 10-4 inches represent moderate DTH responses, and changes in the range of 70–100 x 10-4 inches represent strong DTH responses.
Cell depletions
Splenocytes were depleted of CD4+ cells, CD8+ cells, or both by magnetic cell sorting (Miltenyi Biotec, Auburn, CA). Cells were treated with CD4 (L3T4) or CD8a (LY-2) magnetic microbeads, and passed through a separation column, LS+/VS+, in a magnetic field (Miltenyi Biotec). The nonbound negative fraction of cells was collected and used for further analyses.
Real-time PCR
Total cellular RNA was isolated with RNAqueous RNA isolation
kits (Ambion, Austin, TX), and 3 µg were reverse transcribed into
cDNA with the Moloney murine leukemia virus reverse transcriptase. The
real-time PCR technique uses cytokine-specific oligonucleotide
hybridization probes that are labeled with a reporter fluorescent dye
(6-carboxy-fluorescein) at the 5' end, and with a quencher fluorescent
dye (6-carboxy-tetramethylrhodamine) at the 3' end. Before the start of
the PCR, when the probe is intact, the lack of reporter dye emission is
due to the physical proximity of the reporter and quencher fluorescent
dyes. During the extension phase of the PCR cycle, the nucleolytic
activity of the Taq DNA polymerase cleaves the hybridization
probe and releases the reporter dye from the probe. The resulting
relative increase in reporter fluorescent dye emission is monitored in
real time during PCR amplification using the Sequence Detection System
(ABI PRISM 7700 Sequence Detection System and software, PE Applied
Biosystems, Foster City, CA). A threshold cycle (Ct) value provides an
index of mRNA level. The 18S ribosomal RNA was used as an internal
standard to control for variability in amplification due to differences
in starting mRNA concentrations. The relative expression level of
cytokine mRNA was computed from the cytokine Ct and the 18S rRNA Ct
using the formula:
 |
.
|
Transplanted hearts were placed in OCT embedding medium and snap-frozen in superchilled isopentane. Tissues were sectioned at 6 µm and fixed in 2% paraformaldehyde. Immunohistochemistry was performed using the avidin-biotin complex methods. Sections were blocked with 10% normal serum and incubated overnight at 4°C with rabbit anti-rodent uPA (American Diagnostica), rabbit anti-rodent tPA (American Diagnostica), or chicken anti-human activated TGF-ß (R&D Systems). Tissues sections were incubated with a biotinylated then an alkaline phosphatase (ALP)-conjugated streptavidin. Slides were developed using the Vector Red ALP substrate kit and counterstained with methyl green. A Nikon Eclipse E400 microscope (Nikon, Melville, NY) was used to evaluate histologic sections. Images were captured using Pixera Communication Suite software version 2.0 (Pixera, Los Gatos, CA). Conversion of images from color to black and white (with adjustment of hue and saturation to optimize the signal-to-noise ratio) was performed using Aldus Photostyler version 2.0 software (Aldus, Seattle, WA).
Statistical analysis
DTH results were evaluated by unpaired Student’s t test. Differences between experimental and control data were considered significant if the analysis yielded p values <0.001.
|
Results |
|---|
|
TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
|---|
36B6 cardiac
allograft acceptor mice at 60 days posttransplant and transferred into
the pinnae of naive B6 mice along with subcellular DBA/2 alloantigen
and various concentrations of either neutralizing anti-TGF-ß mAb
or an isotype control Ab. DTH-like swelling responses were measured
24 h later. As shown in Fig. 1, the
TGF-ß mAb, but not the isotype control Ab, restored donor-reactive
DTH responses in a dose-dependent manner. A similar restoration of DTH
was observed when cardiac allograft acceptor mice were challenged
directly with donor alloantigens plus anti-TGF-ß mAb (data not
shown). The inset graphic demonstrates that the special
immunologic conditions are required for promotion of DTH responses by
anti-TGF-ß mAb. C57BL/6 mice sensitized to FVB/N
(H-2q) by splenocyte injection will make DTH
responses to FVB/N, but not DBA/2, alloantigens even if
anti-TGF-ß is present. Thus, cardiac allograft acceptor mice are
primed for donor-reactive DTH responses, but these DTH responses are
actively inhibited via a mechanism that involves TGF-ß.
|
, unseparated and
CD8-depleted acceptor cells challenged with donor alloantigens and Abs
to TGF-ß exhibited statistically significant DTH responses measured
compared to their challenge with donor Ag and control Abs
(p < 0.001). The removal of
CD8+ cells removed neither the donor-reactive DTH
activity nor the TGF-ß-mediated DTH regulatory activity, whereas
deletion of CD4+ cells removed both activities.
This indicates that the CD4+ subpopulation of
splenocytes in allograft acceptor mice contains not only cells that
promoted donor-reactive DTH response, but also cells that interfere
with donor-reactive DTH responses.
|
, the DTH challenge sites of allograft
acceptor mice produced no more TGF-ß mRNA than did normal,
nonchallenged, or DTH-challenged murine pinnae. In contrast, the DTH
challenge sites of allograft rejector mice produced about four times
more TGF-ß mRNA than the control tissues. This suggested that the DTH
responses in cardiac allograft acceptor mice may be regulated at the
level of TGF-ß activation, rather than TFG-ß production.
|
, acceptor cells challenged with donor
alloantigen and Abs to TGF-ß, tPA, or uPA exhibited statistically
significant DTH responses measured when compared to challenge with
third-party Ag and any of these Abs (p <
0.001). Abs to either tPA or uPA, but not their control Abs, were able
to restore DTH responses when DBA/2 alloantigens, but not FVB/N
(third-party) alloantigens, were used for DTH challenge. Similar tPA-
and uPA-mediated DTH restoration was observed when allograft acceptor
mice were challenged directly for donor-reactive DTH responses (data
not shown). These observations indicate that plasminogen activators are
involved in DTH inhibition, and imply that plasmin is the agent that
activates TGF-ß at challenge sites in cardiac allograft acceptor
mice.
|
, the provision of TGF-ß, but not
IL-2, overrides the reversal of DTH inhibition promoted by Abs to
either tPA or uPA, resulting in the DTH inhibition that is usually
observed in cardiac allograft acceptor mice. These data suggest that at
least one of the effects of tPA and uPA at DTH challenge sites in
cardiac allograft acceptors is to activate TGF-ß, presumably via the
production of plasmin. We next determined if elements of this
regulatory system were detectable in accepted cardiac allografts.
GN-treated DBA/2B6 cardiac allografts were harvested >60 days
posttransplantation and analyzed by immunohistochemistry for expression
of uPA, tPA, and activated TGF-ß. Controls included normal,
nontransplanted DBA/2 hearts and 60-day GN-treated DBA/2DBA/2
cardiac isografts. We observed that the accepted allografts, but not
the normal hearts or cardiac isografts, had prominent, widespread
expression of active TGF-ß and uPA (Fig. 6). All three types of cardiac tissues
displayed tPA (data not shown). In general, the tissue distribution of
active TGF-ß and uPA was quite similar, both being found throughout
the graft predominantly in areas of mononuclear cellular infiltration.
Thus, the regulatory elements uPA and activated TGF-ß are not only
operative at DTH sites, but also differentially present in graft sites
of cardiac allograft acceptor mice.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
|---|
B6 cardiac allograft recipients. In
extensive studies, we have found no substantial differences in the
immunologic phenotype of cardiac allograft acceptor mice produced with
any of these three immunomodulatory agents (3, 24). This
includes the observation that allograft acceptance in these mice is
mediated by a dominant-negative immunoregulatory mechanism that is
manifested by linked alloantigen nonresponsiveness (6),
i.e., a donor alloantigen-induced inhibitory mechanism that operates in
an Ag nonspecific manner. Such a regulatory mechanism would be consistent with the Ag-induced production of immunosuppressive cytokines, such as TGF-ß, by regulatory T cells. TGF-ß has been implicated in the regulation of a variety of cell-mediated immune responses, including ACAID (9, 10, 11) and experimental autoimmune encephalomyelitis (12, 13). In the ACAID model, it has been shown that the aqueous humor of the eye contains TGF-ß (10, 25), and that Ag presentation in the presence of TGF-ß, either in vivo or in vitro, can lead to systemic tolerance to the Ag, manifest as a loss of Ag-specific DTH responses. In this model, the immunoregulatory effects of TGF-ß were mediated by IL-10 (9). Importantly, studies by Cuturi and colleagues (26) have implicated TGF-ß in the mechanism of CsA-induced allograft acceptance in rats. Having observed that allograft acceptor mice fail to mount donor-reactive DTH responses, we recently demonstrated that DTH responses in allograft acceptor mice could be restored if either TGF-ß or IL10 were neutralized at the DTH challenge site (8). This indicates that allograft acceptor mice are fully allosensitized for donor-reactive DTH responses, but are prevented from making such responses by a mechanism that involves the immunomodulatory cytokines TGF-ß and IL-10. Interestingly, we could find no evidence that TGF-ß and IL-10 are functionally linked in this experimental system, and both appear to operate independently at the DTH challenge sites (8).
In the current study, we focussed our attention on the contribution of
TGF-ß to DTH regulation in cardiac allograft acceptor mice. We first
determined that deletion of CD4+ splenocytes
eliminates both a subset of T cells that promotes donor-reactive DTH
responses and a subset of T cells that mediates TGF-ß-dependent
inhibition of donor-reactive DTH (Fig. 2
). Thus, the spleens of
allograft acceptor mice contain at least two functionally distinct
subpopulations of CD4+ T cells: 1) a
pro-inflammatory population capable of mediating donor-reactive DTH
responses, and 2) an anti-inflammatory population capable of
inhibiting DTH responses. Because they are revealed in DTH responses
directed at subcellular donor alloantigens, both subpopulations are
operating through the indirect, or self MHC-restricted, pathway of
alloantigen recognition. Thus, it appears that GN does not interfere
with acute allograft rejection by blocking the allosensitization or
clonal expansion of pro-inflammatory T cells. Rather, it operates at
the poorly understood level whereby allosensitized T cells cause the
failure of graft function. As it does so, it permits the emergence of a
second donor-reactive T cell population that is potently
anti-inflammatory, and appears to dominate the function of the
pro-inflammatory T cells by its production of TGF-ß. Whether these
two subpopulations are physically or phenotypically distinct remains to
be determined.
The role of CD8+ T cells in these allograft
acceptor mice remains obscure. We observed that deletion of
CD8+ splenocytes did not eliminate either
donor-reactive DTH responses or the DTH regulatory activity (Fig. 2).
The CD4-depleted splenocytes displayed no DTH activity, suggesting that
the CD8+ T cells may not be allosensitized for
DTH in this experimental system. It should be noted that allograft
rejection in this strain combination (DBA/2B6) is effectively
eliminated by treatment of the graft recipient with a depleting
anti-CD4 mAb (3), but is completely insensitive to
treatment with a depleting anti-CD8 mAb (A. A. Bickerstaff,
unpublished observations). It is possible that
CD8+ T cells are not recruited into the
donor-reactive alloimmune responses under the immunologic conditions of
these studies. Alternatively, the CD8+ T cells
may be capable of donor-reactive DTH, but are blocked from doing so by
a subpopulation of CD4-negative T cells that use a mechanism other than
TGF-ß to mediate inhibition. Thus, the CD8+ T
cell population in these allograft acceptor mice may contain 1) both
pro-inflammatory and dominant anti-inflammatory T cells (like the
CD4+ T cells), 2) only anti-inflammatory T
cells (which, by experimental design, would be undetectable in these
studies), or 3) neither pro-inflammatory nor anti-inflammatory T
cells. Experiments with positively selected CD4+
and CD8+ T cells are currently underway to
explore these possibilities, and to determine whether the function of
anti-inflammatory CD4+ T cells can influence
the immunologic behavior of alloreactive CD8+ T
cells, if and when they are generated in allograft recipients.
As a regulatory cytokine, TGF-ß is unusual in that it is produced in
an inactive form, coupled to LTBP. LTBP can also bind to matrix
molecules, which effectively ties the inactive form of TGF-ß into the
extracellular matrix (27). Thus, TGF-ß production does
not imply biologic TGF-ß activity, because TGF-ß must be activated
before it can provide its regulatory function. Further, biologic
TGF-ß activity can be manifest in the absence of any ongoing TGF-ß
production. Having observed that serologic neutralization of TGF-ß
permits the recovery of donor-reactive DTH responses in allograft
acceptor mice (Fig. 1), we evaluated TGF-ß production at these DTH
sites using real-time PCR methodology to quantitate TGF-ß mRNA
expression, and were somewhat surprised to find that substantial
TGF-ß mRNA is produced at DTH sites of allograft rejector mice, but
that the DTH sites of allograft acceptor mice produced no more than
background amounts of TGF-ß mRNA (Fig. 3). This paradoxical
observation suggested that TGF-ß production may not be the primary
control point for TGF-ß-mediated DTH regulation. It should be noted,
however, that while no more than background production of TGF-ß mRNA
was detected at regulated DTH sites in allograft acceptor mice, this
background level of TGF-ß mRNA is substantial, even in normal,
noninflamed tissues. Hence, some degree of TGF-ß production occurs at
regulated DTH sites, and it is not clear whether infiltrating
leukocytes contribute to this TGF-ß production. Nevertheless, this
observation caused us to shift our attention to the mechanisms of
TGF-ß activation rather than mechanisms of TGF-ß production by
immunoregulatory cells.
Active TGF-ß can be liberated from its latent form in interstitial
depots by several proteases that are operative during inflammation,
including plasmin (14, 16) and thrombospondin (16, 28). Of these, plasmin is especially interesting because uPAR,
the receptor for the uPA protease that generates plasmin from
plasminogen, is also a late activation Ag on T cells
(29, 30, 31). In theory, the cell surface expression of
uPA/uPAR by activated T cells facilitates their degradation of the
extracellular matrix as they migrate through the interstitium at sites
of inflammation. This uPA/uPAR expression also endows these T cells
with the ability to activate either the TGF-ß that they may
themselves produce, or the latent TGF-ß that they encounter as they
digest the extracellular matrix. It should be noted that macrophages
can also produce uPA/uPAR (32, 33) as well as a second,
soluble form of plasminogen activator, tPA (34). Based on
this information, we determined whether uPA was involved in the
mechanism of DTH inhibition in cardiac allograft acceptor mice. As a
negative control, we determined whether tPA was similarly involved.
Unexpectedly, we obtained evidence that both tPA and uPA may be
actively involved the mechanism of DTH inhibition, because Abs to
either tPA or uPA can restore donor-reactive DTH responses by allograft
acceptor mice (Figs. 4 and 5). Interestingly, either set of plasminogen
activator-reactive Abs completely restores donor-reactive DTH
responses. It is not yet clear if both plasminogen activators are
actually operative, or if the Abs for tPA and uPA cross-react. Studies
with tPA knock-out (KO) or uPA KO mice are planned to resolve this
issue.
There are many ways in which tPA or uPA function might influence the
progression of events during DTH responses. Although it is difficult to
generate evidence for cause-and-effect in vivo, we were able to
demonstrate that the restorative effect of the anti-tPA and
anti-uPA Abs on DTH responses could be fully reversed by providing
activated TGF-ß to the DTH site (Fig. 5). This provides
circumstantial evidence that the tPA/uPA and TGF-ß activities may be
linked, presumably through the production of plasmin. We wanted to test
for this link, but were unable to identify Abs that could
differentially detect plasmin, but not its parent molecule,
plasminogen, a molecule that is ubiquitous at sites of inflammation.
Plasmin activity can be detected in tissues by zymography (35, 36). However, we plan to take an alternative approach that
utilizes plasminogen KO mice. These KO mice should be unable to
generate plasmin, and thus may not be able to activate TGF-ß in a way
that impairs donor-reactive cell-mediated immune responses at DTH sites
or in allografts. It is possible that plasminogen KO mice will 1) fail
to permanently accept cardiac allografts, 2) develop an alternative,
TGF-ß-independent mechanism of allograft acceptance, or 3) employ an
alternative mechanism of TGF-ß activation to promote allograft
acceptance. Any one of these outcomes will be informative.
In a murine model of colitis, Ag-specific TGF-ß-producing T cell clones have been shown to be immunosuppressive (37). Somewhat paradoxically, TGF-ß reportedly causes T cells to lose uPA/uPAR (30) and thus their ability to generate more activated TGF-ß. We suspect that this paradox can be resolved by considering the contributions of the APC to T cell behavior. In contrast to T cells, active TGF-ß reportedly stimulates macrophages to make uPA, uPAR, tPA, and TGF-ß (32, 33). Thus, by briefly generating active TGF-ß after Ag recognition, T cells could educate macrophages to perpetuate the process of TGF-ß production. Further, macrophages would continue to do so during their cognate interactions with all other T cells in the vicinity, including those T cells that are using the macrophage to visualize any nondonor Ags that may be present at the inflammatory site. This would provide a mechanism that explains the phenomenon of donor Ag-linked DTH nonresponsiveness to third-party Ags (6). Evidence of such APC education has come from the ACAID model, which also develops this dominant-negative form of DTH regulation (9). In that system, transfusion of naive mice with very small numbers of syngeneic, TGF-ß plus Ag-treated peritoneal exudate cells can completely mimic the ACAID-like systemic regulation of Ag-specific, cell-mediated immune responses.
Our studies suggest that accepted cardiac allografts may also use the
TGF-ß system, to some degree, for the control of local cell-mediated
immune responses. Using immunohistochemistry, we have identified uPA
and active TGF-ß in accepted murine cardiac allografts, but not in
long-term cardiac isografts or in normal nontransplanted hearts (Fig. 6). We are currently working to determine whether these molecules are
expressed by T cells, macrophages, or both. In preliminary experiments,
we were unable to compromise allograft acceptance by systemic treatment
of acceptor mice with anti-TGF-ß Abs. This could be due to an
insufficient amount of Ab arriving at the graft site, or to the
presence of backup regulatory systems that operate simultaneously at
the graft site. Indeed, we knew of at least one other regulatory
mechanism involving IL-10 production that is operative in allograft
acceptor mice (8). It is also possible that very different
regulatory mechanisms are used to establish alloantigen acceptance at a
tissue site (which can be monitored by DTH studies) vs maintaining
allograft acceptance after it has been established.
This information forms the basis of our working model of cell-mediated
immunity in cardiac allograft acceptor mice (Fig. 7). In general, there are two types of
alloreactive T cells, those that attack allogeneic cells (alloaggressor
T cells) and those that protect allogeneic cells (alloregulator T
cells). Alloaggressor T cells are generated routinely in most allograft
recipients, due to pro-inflammatory signals associated with ischemia
and reperfusion injury. In special cases, when the alloaggressive
responses are subverted by selected immunosuppressive agents, such as
anti-CD4 mAb or GN, the alloregulatory T cells can slowly emerge
over a 30- to 60-day period. It is not yet known if similar protective
mechanisms are engendered by immunosuppressive strategies that target
costimulator molecules. Like other T cells, including alloaggressive,
donor-reactive T cells, the regulatory T cells continuously infiltrate
the graft, and are rapidly mobilized to new sites of inflammation, such
as DTH sites. If they encounter donor alloantigen on local APC, they
utilize uPA/uPAR to generate plasmin, which liberates active TGF-ß.
This TGF-ß, in turn, educates the graft APCs to perpetuate the
TGF-ß production. Active TGF-ß interferes with the production of
the pro-inflammatory cytokine IFN-
by the alloaggressor T cells
(38, 39). Further, the locally produced plasmin, which is
necessary for TGF-ß activation, also enzymatically digests free
IFN- (40, 41). These mechanisms effectively subvert
aggressive alloimmunity at the site. Because they produce activated
TGF-ß, the TGF-ß-educated APC can interfere with the
pro-inflammatory behavior of any T cells with which they subsequently
have cognate interactions, regardless of their Ag specificity. This
results in the immunologic phenomena of "linked Ag
nonresponsiveness" or "bystander suppression" (6, 7, 42).
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Alice Bickerstaff, Department of Surgery/Transplant, Ohio State University, N944 Doan Hall, 410 West, 10th Avenue, Columbus OH 43210.
3 Abbreviations used in this paper: GN, gallium nitrate; DTH, delayed-type hypersensitivity; ACAID, anterior chamber-associated immune deviation; LTBP, latent TGF-ß-binding protein; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator; KO, knock out.
Received for publication October 21, 1999. Accepted for publication March 3, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
|---|
production. Proc. Natl. Acad. Sci. USA 93:3193.. J Interferon Cytokine Res. 15:557.[Medline]
by plasmin and miniplasmin. J. Interferon Res. 9:517.[Medline]
This article has been cited by other articles:
|
|
 |
|
  R. E. Cone, S. Chattopadhyay, R. Sharafieh, Y. Lemire, J. O'Rourke, R. A. Flavell, and R. B. Clark T cell sensitivity to TGF-{beta} is required for the effector function but not the generation of splenic CD8+ regulatory T cells induced via the injection of antigen into the anterior chamber Int. Immunol., May 1, 2009; 21(5): 567 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. T. H. Roelofs, K. M. A. Rouschop, G. J. D. Teske, G. T. M. Wagenaar, N. Claessen, J. J. Weening, T. van der Poll, and S. Florquin Endogenous tissue-type plasminogen activator is protective during ascending urinary tract infection Nephrol. Dial. Transplant., March 1, 2009; 24(3): 801 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Stojakovic, L. I. Salazar-Fontana, Z. Tatari-Calderone, V. P. Badovinac, F. R. Santori, D. Kovalovsky, D. Sant'Angelo, J. T. Harty, and S. Vukmanovic Adaptable TCR Avidity Thresholds for Negative Selection J. Immunol., November 15, 2008; 181(10): 6770 - 6778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Bogunovic, M. Stojakovic, L. Chen, and M. Maric An Unexpected Functional Link between Lysosomal Thiol Reductase and Mitochondrial Manganese Superoxide Dismutase J. Biol. Chem., April 4, 2008; 283(14): 8855 - 8862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Ilkovitch, M. E. Handel-Fernandez, L. M. Herbert, and D. M. Lopez Antitumor Effects of Mucin 1/sec Involves the Modulation of Urokinase-Type Plasminogen Activator and Signal Transducer and Activator of Transcription 1 Expression in Tumor Cells Cancer Res., April 1, 2008; 68(7): 2427 - 2435. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Cook, A. A. Bickerstaff, J.-J. Wang, T. Nadasdy, P. Della Pelle, R. B. Colvin, and C. G. Orosz Spontaneous Renal Allograft Acceptance Associated with "Regulatory" Dendritic Cells and IDO J. Immunol., March 1, 2008; 180(5): 3103 - 3112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-H. Sonoda, T. Nakamura, H. A. Young, D. Hart, P. Carmeliet, and J. Stein-Streilein NKT Cell-Derived Urokinase-Type Plasminogen Activator Promotes Peripheral Tolerance Associated with Eye J. Immunol., August 15, 2007; 179(4): 2215 - 2222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yamaguchi, K. Yamada, T. Suzuki, Y.-P. Wu, K. Kita, S. Takahashi, M. Ichinose, and N. Suzuki Induction of uPA release in human peripheral blood lymphocytes by [deamino-Cysl,D-Arg8]-vasopressin (dDAVP) Am J Physiol Endocrinol Metab, November 1, 2004; 287(5): E970 - E976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Bishop, S. C. Wood, E. J. Eichwald, and C. G. Orosz Immunobiology of Allograft Rejection in the Absence of IFN-{{gamma}}: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions J. Immunol., March 1, 2001; 166(5): 3248 - 3255. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
