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Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University of Münster, Münster, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In keeping with the fundamental role of T cells in graft rejection, much of the development of immunosuppressive drugs has focused on targeting T cells. One of these immunosuppressive drugs is mycophenolate mofetil (MMF;3 RS61443). MMF has been successfully introduced in allogeneic transplantation medicine and, recently, in the treatment of autoimmune skin disorders such as bullous pemphigoid, pemphigus vulgaris, and pyoderma gangraenosum (3, 4, 5, 6). MMF is the morpholinoethyl ester prodrug of its active metabolite mycophenolic acid (MPA) (7). MPA is a noncompetitive, reversible inhibitor of the enzyme inosine 5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205), which plays a major role in the de novo synthesis of guanosine nucleotides (8). Unlike other cells that also use the salvage pathway for purine biosynthesis, proliferating B and T cells are singularly dependent on the de novo pathway for the generation of guanosine. By blocking IMPDH, B and T cells cannot synthesize the necessary levels of RNA and DNA to mount a proliferative response to Ags and mitogens. Thus, MMF exerts its immunosuppressive effects at a late stage of lymphocyte proliferation (7). This selective effect results in MMF having fewer side effects than other immunosuppressive drugs used in transplantation medicine, with the additional benefit of its effects being rapidly reversible upon discontinuation of administration (7).
The induction of primary immune responses does not solely rely on T
cells, but on a complex interaction between APC, in particular,
dendritic cells (DC) and T cells. DC, such as epidermal Langerhans
cells (LC), play a central role in Ag presentation by migrating from
sites of Ag capture to the draining lymph node and presenting Ag to T
cells within the lymph node. They furthermore have the unique capacity
to present Ag in the proper context of the required antigenic and
costimulatory molecules to prime naive T cells. Upon encountering an
Ag, DC initiate a differentiation process, referred to as maturation,
which results in decreased Ag-processing capacities and increased
Ag-presenting abilities as well as the up-regulation of costimulatory
molecules such as CD40, CD80/CD86, ICAM-1, and I-A (9, 10). Upon maturation, DC also acquire the ability to produce
IL-12, a cytokine necessary for the development of Th1 cells and
cell-mediated immunity (11). Upon ligation of CD40L on T
cells with CD40 on DC, DC are triggered to produce even higher
quantities of IL-12, thus consigning T cells to Th1 responses
(12). Recently, expression of Th1 cytokines, in particular
IFN-
, in a graft rejection model has been shown to be associated
with acute graft rejection (13). In contrast, the
predominant expression of Th2 cytokines, such as IL-4 and IL-10, was
implicated in long term survival of the allograft.
In the present work we show that MMF inhibits allospecific and hapten-specific immune responses not only by repressing T cell proliferation but also by directly exerting its inhibitory effects on APC. A decrease in the ability of MMF-treated mice to induce or elicit contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses was shown to be dependent on the dose of administered MMF. MMF did not induce immunotolerance in this model, as mice not further treated with MMF after the initial challenge mounted CHS responses similar to vehicle-treated mice when resensitized and rechallenged 2 wk later. The capacity of epidermal cells to stimulate allogeneic T cells was significantly decreased in mice chronically treated with MMF, although there was no difference in the number of I-A+ cells found in epidermal earsheets compared with that in control mice. Furthermore, flow cytometric analyses revealed a reduction of the expression of CD40, CD80, CD86, I-A, and ICAM-1 on bone marrow-derived DC treated with MMF. This was accompanied by a reduction of T cell stimulatory capacities in MLR, and a concurrent reduction of IL-12 production and a decrease in the capacity of DC to stimulate allogeneic T cells. These data suggest that MMF, in addition to affecting T lymphocytes, is able to suppress Ag presentation by attenuating functional maturation of DC, resulting in impaired immune responses. As IMPDH is the main target of the inhibitory effects of MMF, the results of this study also point to a possible role of IMPDH in DC development. Thus, this study elucidates another one of the underlying mechanisms resulting in the potent immunosuppressive effects of MMF in graft rejection and its beneficial effects on transplant maintenance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five- to 8-wk-old specific pathogen-free female BALB/c and C57/BL6 mice were obtained from Harlan-Winkelmann (Borchen, Germany) and housed according to institutional regulations.
CHS and DTH responses
CHS experiments were performed as previously described (14, 15). Briefly, mice were sensitized by painting 100 µl of 0.15% trinitrochlorobenzene (TNCB) or 2% oxazolone (Sigma, St. Louis, MO) in acetone/corn oil (4/1) onto the shaved back. For elicitation of CHS responses, 10 µl of 0.8% TNCB or 0.5% oxazolone, respectively, were painted on both sides of each ear 5 days after immunization. CHS was determined by the degree of ear swelling of the hapten-exposed ear compared to the ear thickness before challenge and was measured with a spring-loaded micrometer (Mitutoyo, Tokyo, Japan) 24 h after challenge. Mice that were ear challenged without prior sensitization served as negative controls. Groups of BALB/c mice (n = 5) were treated with 30 µg/g MMF i.p. in DMSO/PBS (1/10) 24, 12, and 1 h before immunization as well as 12 and 24 h after immunization. Another group received MMF 24, 12, and 1 h before challenge with oxazolone and 12 and 24 h after challenge. Control groups were treated accordingly with DMSO/PBS (1/10). In another experiment, mice were sensitized by s.c. injection of dinitrobenzene sulfonic acid (DNBS)-coupled DC and challenged with 12 µl of 0.3% dinitrofluorobenzene (DNFB) in acetone/olive oil (4/1) at the ear. DC were pulsed with DNBS by incubating DC with 1 mM DNBS in complete medium (CM; RPMI 1640 containing 5% FCS, 50 µM 2-ME, 2 mM glutamine, 0.1 mM nonessential amino acids, and 20 µg/ml gentamycin (all from PAA, Linz, Austria)) at 37°C for 90 min. DC were washed extensively with PBS to remove any unincorporated hapten, and 4 x 105 DC (suspended in 200 µl PBS) were then injected s.c. into the lower abdomen. Control groups received nonpulsed DC.
DTH experiments were performed by sensitizing mice by epicutaneous application of 100 µl of 0.3% DNFB onto the shaved lower back. One week after sensitization, DTH was elicited by challenging mice by injection of 4 x 105 haptenated DC or LC-depleted epidermal cells (EC; in 100 µl of PBS) into one hind footpad. Control groups were sensitized or challenged with nonpulsed DC or LC-depleted EC. The intensity of the DTH reaction was assessed as the degree of footpad swelling of the injected site compared with that of the untreated contralateral footpad and was measured with a micrometer 24 h after challenge.
Immunofluorescent staining of epidermal ear sheets
Epidermal sheets were stained essentially as previously described (16). Briefly, ears were mechanically split into dorsal and ventral sides and incubated in 20 mM EDTA (pH 7.3) for 3.5 h at 37°C. Epidermal sheets were collected, washed with PBS, and fixed in acetone at -20°C for 20 min. After washing, sheets were incubated in 1% BSA/PBS for 1 h. Staining with the Ab anti-I-A (clone M5/114; Roche Diagnostics, Mannheim, Germany) was performed overnight at 4°C. After washing, sheets were incubated with FITC-conjugated goat anti-rat IgG (PharMingen, Hamburg, Germany) for 1 h, washed extensively, mounted onto slides, and examined using a Zeiss Axiovert microscope.
Generation and culture of bone marrow-derived DC
DC were generated as described by Inaba et al. (17) with some modifications. In brief, bone marrow cells were collected from murine tibias and femurs, suspended by vigorous pipetting, passed through a nylon mesh to remove debris, resuspended in CM, and cultured in tissue dishes for 2 h. Nonadherent cells were collected, and aliquots of 1 x 106 cells were placed in 24-well plates (Becton Dickinson, Heidelberg, Germany) containing 1 ml of CM supplemented with 150 U/ml GM-CSF (R&D Systems, Wiesbaden, Germany) and 75 U/ml IL-4 (PharMingen). After 2 days of incubation (37°C, 5% CO2), 600 µl of medium was removed, and the same volume of fresh CM containing 150 U/ml GM-CSF and 75 U/ml IL-4 was added. On days 5 and 7 of culture nonadherent cells were transferred into six-well plates containing CM supplemented with cytokines (3 x 106 cells/3 ml/well). After a total of 8 days of incubation, most of the nonadherent cells in culture had acquired typical dendritic morphology. These cells were harvested and used as a source of DC in subsequent experiments. At this time point LPS (0.1 µg/ml; Sigma) was added to the cell cultures in some experiments for overnight incubation. Subsequently, supernatants from these cultures were collected and stored at -20°C until they were used for cytokine quantifications. Unless otherwise noted, cells were cultured in the presence of MMF or the vehicle from day 1 on. MMF was added at every change of the culture medium.
Flow cytometry of DC
Expression of cell surface molecules was quantitated by flow cytometry as follows. Aliquots of 2 x 105 DC were incubated with mAb against BM8 (BMA, Augst, Switzerland), CD11b (clone M1/70; PharMingen), CD19 (ID3; PharMingen), CD40 (HM40-3; PharMingen), CD80 (1G10; PharMingen), CD86 (GL1; PharMingen), ER-HR3 (BMA), ER-TR9 (BMA), F4/80 (BMA), I-A (clone M5/114; Roche Diagnostics), ICAM-1 (clone YN1/1.7.4; American Type Culture Collection, Manassas, VA), NLDC145 (BMA), or rat IgG as an isotype control (Dianova, Hamburg, Germany) for 60 min on ice (1 µg/ml diluted in PBS/1.0% FCS (v/v)). The cells were washed with PBS/1.0% FCS (v/v) and labeled with FITC-conjugated goat anti-rat IgG or FITC-conjugated goat anti-hamster IgG (PharMingen or Dianova, respectively), diluted 1/50 in PBS/1.0% FCS (v/v)) for 30 min on ice. At the end of this incubation cells were washed, propidium iodide (100 µM; Sigma)/PBS was added, and the cells were subsequently analyzed in an EPICS-XL flow cytometer (Coulter, Krefeld, Germany). Propidium iodide fluorescence was used to detect dead cells, which were then gated out.
EC preparations
EC were prepared as previously described (16) from truncal skins of mice. Subcutaneous fat and panniculus carnosus were removed, after which the skins were floated dermal side down on 0.4% trypsin in Ca2+/Mg2+-free PBS for 60 min at 37°C. Epidermal sheets were collected and dissociated by gentle stirring for 20 min. The resulting EC were filtered through nylon gauze and washed. I-A+ cells were depleted by incubation on plates coated with anti-I-A mAbs (clone M5/114; Roche). Dead cells were removed by treatment with 0.05% trypsin and 80 µg of DNase/ml in Ca2+/Mg2+-free PBS for 5 min at room temperature. Aliquots were analyzed by flow cytometry before and after depletion.
Mixed epidermal cell-lymphocyte reactions (MELR)
Mice were treated with 30 µg/g/day MMF i.p. in DMSO/PBS (1/10) or vehicle alone for 30 days. The MELR was performed as described by Grabbe et al. (16). Briefly, 2 x 105 nylon-wool-enriched C3H splenic T cells were cocultured with varying numbers of freshly prepared EC from MMF-treated and untreated BALB/c mice. EC were enriched for I-A+ cells by depletion of Thy-1-bearing cells. EC were incubated in anti-Thy 1.2 mAb (clone NEI-001, Sigma) for 30 min on ice, followed by washing and subsequent incubation in low toxicity rabbit complement (Cedarlane Laboratories, Hornby, Canada) for 30 min at 37°C. Dead cells were removed by treatment with 0.05% trypsin and 80 µg of DNase/ml in Ca2+/Mg2+-free PBS for 5 min at room temperature. Cells were cultured in RPMI 1640 medium supplemented with 1.5% mouse serum/ml, 5 µg of indomethacin/ml, and 50 µM 2-ME. Serial dilutions of triplicate samples of epidermal cells enriched for I-A+ cells were mixed with a constant number (2 x 105) of allogeneic T cells. Cells were cultured for 6 days at 37°C in round-bottom 96-well plates and then pulse-labeled with 1 µCi of [3H]thymidine/ml for 24 h. Subsequently, cells were harvested, and the incorporation of [3H]thymidine was measured.
MLR
DC were cultured in the continuous presence of various concentrations of MMF in CM, washed extensively with PBS, resuspended in MLR medium (RPMI containing 10% FCS, 100 µM HEPES, 1 mM pyruvate, 50 µM 2-ME, 2 mM glutamine, 0.1 mM nonessential amino acids, and 20 µg/ml gentamycin (all from PAA)), and applied in graded doses to 2 x 105 allogeneic T cells in 96-well round-bottom plates. T cells were obtained from spleen cells of C57/BL6 mice by nylon-wool adherence and subsequent elimination of residual contaminating cells with Ab-coated T cell isolation columns (Cellect mouse T cell immunocolumns, Biotex, Alberta, Canada). The resulting cell preparation contained <0.1% I-A+ cells. After 4 days, T cell proliferation was measured by adding 1 µCi of [3H]thymidine followed by an overnight incubation period and quantification of incorporated [3H]thymidine by subsequent liquid scintillation counting.
Detection of IL-12
IL-12 production by DC was determined by solid phase sandwich ELISA assays (Laboserv, Giessen, Germany) according to the manufacturer’s specifications. This kit allows the detection of natural IL-12 (p70) in addition to the free p40 subunit of IL-12 with a minimum detectable dose of <2 pg/ml. Supernatants from 106 DC/ml propagated in the presence of 0.1, 0.01, and 0.001 µM MMF or in DMSO were assayed after 8 days of culture and overnight stimulation with LPS (0.1 µg/ml; Sigma). Recombinant cytokines were used to generate standard curves, with concentrations of IL-12 determined using the linear portion of the curve. The background values were determined by replacing the recombinant cytokines with DMSO or tissue culture medium. All experiments were performed in triplicate.
Statistical evaluation
The significance of differences between the mean values obtained for CHS, DTH, and MLR experiments was assessed by two-tailed Student’s t test for unpaired data; p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As MMF has a direct influence on T cells, we were interested in
investigating its effects on murine CHS responses. The effects of MMF
on CHS responses to the hapten oxazolone were evaluated. Groups of
BALB/c mice were treated with MMF at different time points before
immunization with oxazolone as well as after immunization. The dosage
of MMF used was ascertained by preliminary experiments based on the
dosage in various animal models described previously (7, 18, 19). Multiple injections were performed, as MMF is rapidly
converted to MPA and MPA has an apparent half-life of 17.9 h
(7). Another group received MMF before and after challenge
with oxazolone. Control groups were treated accordingly with the
vehicle only. As a measure of CHS responses, ear swelling was assessed
24 h after challenging the ear skin. The results of a
representative experiment are presented in Fig. 1
A. In comparison to
vehicle-treated control animals (immunized and challenged), the groups
of mice treated with MMF during the elicitation of CHS exhibited
reduced ear swelling, indicating that the afferent phase of CHS is
impaired by MMF. Surprisingly, MMF also reduced CHS responses when
administered upon initial hapten application, indicating that the
sensitization phase of CHS was impaired. Compared with their respective
vehicle-treated control groups, MMF significantly inhibited the
induction (32%) and elicitation (38%) of CHS. Similar results were
obtained when TNCB was used as a contact allergen (data not shown).
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B). Hence, MMF applied before sensitization did not induce
hapten-specific immunotolerance.
To define whether the systemic effects of MMF on cell types other than
APC were responsible for the impaired CHS responses or whether MMF had
had a direct effect on APC, we employed CHS and DTH experiments with
MMF-treated haptenated DC. To investigate the effect of MMF on
DC-mediated Ag presentation to naive T cells, DC were cultured in the
continuous presence of MMF, DNBS coupled, and s.c. injected into the
lower back of naive syngeneic mice for hapten sensitization. One week
later CHS responses were evaluated by administering the hapten to the
ear and measuring ear swelling (Fig. 2).
To test the effects of MMF on Ag presentation to primed T cells, mice
were sensitized by application of DNFB to the shaved lower back and
challenged 1 wk later by injection into the hind footpad of DC that had
been propagated in MMF and then coupled to DNBS (Fig. 3). Footpad swelling was subsequently
assessed. In all experiments DC that had not been treated with MMF
served as the control. As a further control experiment, untreated
I-A-depleted EC (<1% I-A+ as assessed by flow
cytometric analysis) or I-A-depleted EC that had been exposed to hapten
were injected. There were no significant differences in DTH responses
when uncoupled or hapten-coupled I-A-depleted EC instead of DC were
used for sensitization, indicating that DC were not just an unspecific
vector of the hapten (data not shown). In both experimental approaches
there was a significant reduction of the hapten-specific immune
response, indicating that MMF had had a direct and dose-dependent
effect on the Ag-presenting function of the DC.
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As the impaired immune responses could also be attributed to a reduction in the numbers of APC as a result of MMF treatment, mice were chronically treated with 30 µg/g for 30 days. Subsequently, the numbers of LC, the resident DC of the skin, were evaluated after immunofluorescent staining of earsheets with I-A. There were no significant differences in terms of numbers of LC between the MMF-treated mice (718 ± 32/mm2) and the control mice (704 ± 23/mm2).
To test the functional ability of epidermal cells derived from
MMF-treated mice to stimulate T cells, epidermal cells were prepared
from mice chronically treated with MMF or vehicle and enriched for LC
content. These EC were used to stimulate allogeneic T cells in MELR. As
demonstrated in Fig. 4, EC of MMF-treated
mice were significantly impaired in their ability to stimulate T cells,
pointing to a functional impairment of Ag-presenting capacities of
their LC.
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Although it is well known that MMF affects lymphocyte
proliferation, the inhibition of induction of CHS was unexpected. These
data prompted us to study the effects of MMF on APC in vitro. As only
mature, not immature, DC are capable of activating naive T cells, it
was conceivable that the impairment of the Ag-presenting capacities of
DCs propagated in the presence of MMF may be due to alterations in the
expression of costimulatory and MHC molecules. To investigate cell
surface expression of immunomodulatory molecules, DC were prepared and
cultured in the continuous presence of various concentrations of MMF
(0.001, 0.01, and 0.1 µM). The expression of the cell surface
molecules CD40, CD80, CD86, ICAM-1, NLDC-145 (DEC205), and I-A (MHC
class II), all of which are up-regulated upon DC maturation (9, 10), was then assessed by flow cytometry. In the presence of
MMF, DC expressed lower amounts of these molecules, reflecting that MMF
inhibits DC differentiation from bone marrow precursors (Fig. 5). Although expression of these markers
was somewhat reduced at a concentration of 0.001 µM MMF, it did not
differ markedly from that of the vehicle-treated controls. These
effects were not owing to an increase in the percentage of dead cells,
as determined by trypan blue exclusion or flow cytometry with propidium
iodide. The number of cells was only slightly reduced in cultures
containing 0.1 µM MMF compared with vehicle-treated or untreated
cultures. In a typical experiment cultures from untreated or
vehicle-treated DC contained 8.1 x 106
DC/ml, whereas DC from 0.1 µM MMF-treated cultures contained
7.65 x 106 DC/ml.
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To assess whether a normal functional maturation would occur after
termination of MMF exposure, DC were cultivated in the presence of 0.01
and 0.1 µM MMF or vehicle alone or the cells were left untreated to
day 7. At this point, the cultures were split and half of the
respective cultures was cultivated without MMF, whereas the other half
was further cultivated in the presence of MMF or vehicle alone. DC were
subjected to flow cytometric analysis of the cell surface Ags CD40,
CD80, CD86, and I-A 2 days later. There was a distinct increase in
costimulatory molecule expression in DCs that had been propagated as of
day 7 without MMF compared with the respective cells that had been
further cultured with MMF (data not shown). A similar trend was
observed when MMF was used at a concentration of 0.01 µM.
Furthermore, removal of MMF from the cultures on day 4 further enhanced
the expression of the cell surface Ags compared with DC that had been
further propagated in the presence of MMF (data not shown). To confirm
the maturation of DC after discontinuation of the immunomodulatory
effects of MMF on the functional level of T cell stimulation, the DC
cultivated to days 7 and 9 were used in MLR. As shown in Fig. 6, the results of the flow cytometric
analyses were corroborated by the results of the MLR, in that DC
cultivated with MMF to day 7 had no inhibition of T cell-stimulating
capacities, whereas DC cultivated in MMF until day 9 were significantly
impaired. This time course dependency further supports the conclusion
that MMF impairs DC maturation.
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IL-12 is produced in large amounts by mature, but not by immature,
DC and therefore is a suitable surrogate marker for DC differentiation.
To determine whether IL-12 production is altered in DC treated with
MMF, the production of IL-12 was measured by ELISA. Supernatants were
collected from LPS-triggered DC propagated in the presence of MMF. The
results of a representative experiment are shown in Fig. 7. Pre-exposure of developing DC to 0.1
µM MMF considerably reduced their subsequent ability to secrete IL-12
upon stimulation by LPS. Doses of 0.1 µM MMF reduced IL-12 production
by almost 40% compared with vehicle-treated DC, whereas concentrations
of 0.01 and 0.001 µM MMF had little effect on IL-12 production.
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To investigate the capacity of DC cultivated in the presence of
MMF to stimulate allogeneic T cells in vitro, DC were cultured in the
presence of MMF and used to stimulate allogeneic T cells in a MLR.
Graded numbers of DC were cultivated with a fixed number of allogeneic
T cells. The results of a representative experiment are shown in Fig. 8. Preincubation of DC with MMF resulted
in a dose-dependent inhibition of their ability to stimulate allogeneic
T cell proliferation. Approximately twice the number of DC treated with
0.1 µM MMF were needed to attain a similar amount of thymidine
incorporation as that in untreated control groups. DC treated with
0.001 µM MMF did not differ from vehicle-treated DC in their T cell
stimulatory abilities.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects MMF exerts on Ag presentation were studied 1) in vivo using CHS and DTH assays as a measure of functional immune response, and 2) in vitro using bone marrow-derived DC as an example for APC to assess changes in their functional properties. Consistent with other studies, in which impairment of T cell functionality has been reported (23, 24), CHS responses after applying MMF in the T cell-dependent elicitation phase were reduced. Application of MMF before sensitization also resulted in a reduction of CHS responses, although Ag-specific immunotolerance was not induced in this model. As the interaction of T cells with APC is essential at this stage, these data are suggestive of a regulatory effect of MMF on APC function. These results were further corroborated by 1) the decrease in the allostimulatory capacity of EC in MELR, and 2) the reduction of hypersensitivity responses evoked by hapten-coupled DC following ex vivo treatment with MMF. Furthermore, this approach excluded any systemic effects MMF may have had on other cell types in vivo following i.p. injection of MMF.
Immune responses leading to allograft rejection are initiated by T cell-dependent recognition of foreign Ags displayed on allogeneic histocompatibility complexes on APC originating from the allograft (1). The complex interactions resulting in the modulation of T cell-mediated immune responses are directly dependent on Ag presentation, cognate interactions between T cells and APC, and the concomitant production of the necessary costimulatory molecules by allogeneic APC and T cells (reviewed in Refs. 9 and 25). This is also the fundamental mechanism underlying MLR, namely clonal expansion of alloresponsive T cells following priming by APC. MLR assays can be used to make a prognosis on graft survival, with intense proliferation of T cells indicating an incompatible donor (26, 27, 28). DC cultivated in the presence of MMF exhibited a dose-dependent reduction in their capacity to stimulate T cell proliferation in MLR assays, indicating a direct effect of MMF on DC. This conclusion was further substantiated using flow cytometric analyses, which revealed a dose-dependent reduction in the expression of immunomodulatory molecules crucial for T cell activation, namely CD40, CD80, CD86, ICAM-1, and I-A.
Th1-type cells are particularly involved in acute allograft rejection
(13). Studies on patients receiving MMF immediately after
transplantation or after the onset of rejection reactions revealed
diminished rejection of the transplant (7). This would
coincide with the similar effects evidenced by the inhibition of the
elicitation of CHS in the animal model used in this study and the
decrease in the expression of IL-12, a key mediator of T cell
differentiation into Th1-type effector cells, by DC propagated in the
presence of MMF. A number of groups have reported the absence of
Th1-type cytokines, such as IFN-, in allograft recipients receiving
tolerizing therapies (29, 30, 31). Recently, Lui et al.
(22) reported a dose-dependent reduction of IFN-
production, as assessed by mRNA levels, after treatment of mice with
MMF. Furthermore, MPA, the bioactive metabolite of MMF, has been shown
to have little effect on the expression of the Th1-immunosuppressive
cytokine IL-10 in mouse spleen cells (18). Insufficient
amounts of IL-12 and/or CD80 production by APC during Ag presentation
have also been implicated in anergy and tolerance, both of which render
T cells functionally unresponsive (32). Although the
expression of these molecules was reduced when cells were treated with
MMF, administration of MMF did not induce Ag-specific immunotolerance
in vivo in the model used in this study.
The direct stimulation of recipient T cells by donor APC originating from the graft has long been considered to be critical in graft rejection. In the past several years evidence has accumulated that presentation of donor Ags originating from the transplant and being presented by MHC on the recipients APC, termed indirect recognition, is a mechanism involved in chronic rejection of allograft transplants (1, 33, 34). Wecker et al. (35) demonstrated that MHC class II-deficient skin grafts were able to elicit chronic rejection in SCID mice reconstituted with CD4+ T cells via MHC class II-restricted Ag recognition. Another study also confirmed that allograft rejection is linked to the indirect pathway of allorecognition (36). MMF has been shown to attenuate chronic rejection of transplants in rats and humans (37, 38, 39). In addition, graft rejection by DTH-like reactions by Th1 cells has been inferred as playing a role in chronic rejection (1, 40). Interestingly, MMF was also shown to decrease DTH-like responses using hapten-coupled MMF-treated DC in the mouse model employed in this study. Taking not only the inhibitory effects of MMF on T cells into account but also the role of APC in chronic rejection, this study sheds new light on additional beneficial effects of MMF in transplantation therapy.
Taken together, these results highlight a novel facet of MMF action, namely not only does MMF exert its immunosuppressive effects by suppressing T cell proliferation but also by impairing APC function, as assessed using bone marrow-derived DC as an exemplary APC cell type. Our studies indicate that this effect may be based on the retardation of DC maturation, because the expression of costimulatory cell surface markers is directly correlated to the duration and the concentration of MMF treatment. Although the molecular events leading to the immunosuppressive effects of MMF on DC remain to be resolved, one possible mechanism mediating these effects may well be the inhibition of IMPDH. If this were the case, as has been reported for T and B cells, DC and other cells of the immune system may also depend on the de novo synthesis of guanosine. This would allow new strategies and alternative implementations of immunosuppressive regimens, thus expanding therapeutic flexibility in treating immune disorders and transplant rejection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefan Beissert, Department of Dermatology, University of Münster, Von Esmarch Strasse 56, D-48149 Münster, Germany.
3 Abbreviations used in this paper: MMF, mycophenolate mofetil; CHS, contact hypersensitivity; CM, complete medium; DC, dendritic cells; DNBS, dinitrobenzene sulfonic acid; DNFB, dinitrofluorobenzene; DTH, delayed-type hypersensitivity; EC, epidermal cells; IMPDH, inosine 5'-monophosphate dehydrogenase; LC, Langerhans cells; MELR, mixed epidermal cell-lymphocyte reaction; MnX, mean fluorescence intensity; MPA, mycophenolic acid; TNCB, trinitrochlorobenzene.
Received for publication August 4, 1999. Accepted for publication June 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but not transforming growth factor ß inhibits tumor antigen presentation by epidermal antigen-presenting cells. J. Invest. Dermatol. 102:67.[Medline]
mRNA levels and by strong accumulation of major histocompatibility transcripts in the graft. Transplantation 54:219.[Medline]
in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy. Transplantation 55:1112.
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 Y. Ji, J. Gu, A. M. Makhov, J. D. Griffith, and B. S. Mitchell Regulation of the Interaction of Inosine Monophosphate Dehydrogenase with Mycophenolic Acid by GTP J. Biol. Chem., January 6, 2006; 281(1): 206 - 212. [Abstract] [Full Text] [PDF]
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 J A Hardin Dendritic cells: potential triggers of autoimmunity and targets for therapy Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv86 - iv90. [Full Text] [PDF]
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 C. Lagaraine, C. Hoarau, V. Chabot, F. Velge-Roussel, and Y. Lebranchu Mycophenolic acid-treated human dendritic cells have a mature migratory phenotype and inhibit allogeneic responses via direct and indirect pathways Int. Immunol., April 1, 2005; 17(4): 351 - 363. [Abstract] [Full Text] [PDF]
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 K. P. A. MacDonald, V. Rowe, A. D. Clouston, J. K. Welply, R. D. Kuns, J. L. M. Ferrara, R. Thomas, and G. R. Hill Cytokine Expanded Myeloid Precursors Function as Regulatory Antigen-Presenting Cells and Promote Tolerance through IL-10-Producing Regulatory T Cells J. Immunol., February 15, 2005; 174(4): 1841 - 1850. [Abstract] [Full Text] [PDF]
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L. Quemeneur, L. Beloeil, M.-C. Michallet, G. Angelov, M. Tomkowiak, J.-P. Revillard, and J. Marvel Restriction of De Novo Nucleotide Biosynthesis Interferes with Clonal Expansion and Differentiation into Effector and Memory CD8 T Cells J. Immunol., October 15, 2004; 173(8): 4945 - 4952. [Abstract] [Full Text] [PDF]
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P.-H. Chiang, L. Wang, C. A. Bonham, X. Liang, J. J. Fung, L. Lu, and S. Qian Mechanistic Insights into Impaired Dendritic Cell Function by Rapamycin: Inhibition of Jak2/Stat4 Signaling Pathway J. Immunol., February 1, 2004; 172(3): 1355 - 1363. [Abstract] [Full Text] [PDF]
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 A. M. Woltman and C. van Kooten Functional modulation of dendritic cells to suppress adaptive immune responses J. Leukoc. Biol., April 1, 2003; 73(4): 428 - 441. [Abstract] [Full Text] [PDF]
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L. Quemeneur, M. Flacher, L.-M. Gerland, M. Ffrench, J.-P. Revillard, and N. Bonnefoy-Berard Mycophenolic Acid Inhibits IL-2-Dependent T Cell Proliferation, But Not IL-2-Dependent Survival and Sensitization to Apoptosis J. Immunol., September 1, 2002; 169(5): 2747 - 2755. [Abstract] [Full Text] [PDF]
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S. Gregori, M. Casorati, S. Amuchastegui, S. Smiroldo, A. M. Davalli, and L. Adorini Regulatory T Cells Induced by 1{alpha},25-Dihydroxyvitamin D3 and Mycophenolate Mofetil Treatment Mediate Transplantation Tolerance J. Immunol., August 15, 2001; 167(4): 1945 - 1953. [Abstract] [Full Text] [PDF]
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H. Hackstein, A. E. Morelli, A. T. Larregina, R. W. Ganster, G. D. Papworth, A. J. Logar, S. C. Watkins, L. D. Falo, and A. W. Thomson Aspirin Inhibits In Vitro Maturation and In Vivo Immunostimulatory Function of Murine Myeloid Dendritic Cells J. Immunol., June 15, 2001; 166(12): 7053 - 7062. [Abstract] [Full Text] [PDF]
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