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1 Secretion In Vivo1
*
Neurological Diseases and
Cellular Immunology Sections, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892;
Institute of Experimental Internal Medicine, Department of Internal Medicine, Otto-von-Guericke University, Magdeburg, Germany;
Office of the Clinical Director, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; and
¶ Institute of Biochemistry, Department of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 in spinal cord tissue and plasma during acute EAE. In
vitro, while suppressing autoreactive T cell proliferation and TNF-
production, I40 consistently up-regulated TGF-1 secretion. A
neutralizing anti-TGF-1 Ab blocked the inhibitory effect of I40
on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not
generally immunosuppressive, neither eliminating encephalitogenic T
cells nor inhibiting T cell priming. These data suggest that DP IV
inhibition represents a novel and specific therapeutic approach
protecting from autoimmune disease by a mechanism that includes an
active TGF-1-mediated antiinflammatory effect at the site of
pathology. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-, and lymphotoxin are involved in the immunopathology
of organ-specific autoimmune disease (7, 8, 9). A role as
regulators has been suggested for Th2 cells (10, 11, 12) and
cells producing TGF- (13, 14, 15, 16). In addition, the
requirement for costimulatory signals in T cell activation and their
potential for modulating T cell-mediated autoimmunity have been clearly
established (17, 18). Recent evidence suggests that the
cell surface dipeptidyl peptidase IV (DP
IV,4 EC 3.4.14.5,
CD26) may have a role in T cell activation and homeostasis (19, 20).
DP IV is a highly conserved type II integral membrane protein,
constitutively expressed on a wide variety of epithelial, endothelial,
and lymphoid cell types (21). It corresponds to the
leukocyte differentiation Ag CD26. On CD4+ T
cells, CD26/DP IV is tightly regulated, depending on the state of
activation (22), and it is found on T cells activated in
vivo and memory T cells (23). DP IV acts as a serine
peptidase catalyzing the cleavage of N-terminal dipeptides from
peptides and proteins carrying proline, hydroxyproline, or alanine in
the penultimate position (24). A possible role of CD26/DP
IV in T cell-mediated immunity is suggested by: 1) its potent
costimulatory activity for T cells activated via the TCR
(20); 2) its capacity to interact with extracellular
matrix molecules (25, 26); and 3) the suggestion that
cleavage by DP IV might regulate the function of numerous
immunologically relevant peptides and proteins, including cytokines and
chemokines that carry an X-Pro-motif at the N terminus
(24). Clinical observations also link CD26/DP IV to
autoimmunity. Elevated numbers of
CD26+CD4+ T cells were
described in peripheral blood and cerebrospinal and synovial fluids
from patients with multiple sclerosis (27, 28, 29), and
clinically active rheumatoid arthritis (30, 31),
respectively. Recently, the reversible, competitive DP IV inhibitors,
Lys[Z(NO2)]-pyrrolidide (I40) and
Lys[Z(NO2)]-thiazolidide (I49) have been
extensively analyzed in vitro. They specifically and dose-dependently
suppress proliferation and secretion of various cytokines by human and
murine T cells (32, 33, 34). Interestingly, it is well
documented that these inhibitors also induce a 3- to 4-fold increase in
the secretion of latent TGF-1 by mitogen-stimulated murine and human
T cells (33, 35).
In this report, we address the role of CD26/DP IV in murine
experimental autoimmune encephalomyelitis (EAE), a well-characterized
CD4+ T cell-mediated autoimmune disease leading
to CNS inflammation and demyelination in susceptible strains of rodents
(1). We demonstrate for the first time that the signs of
EAE can be diminished by DP IV inhibition in vivo both in a preventive
and therapeutic fashion. CNS inflammation associated with acute EAE can
be reduced. Our data suggest that this therapeutic effect may be
mediated by up-regulation of the immunosuppressive cytokine TGF-1
and an inhibition of T cell effector functions in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and from the Frederick Cancer Research and Development Center (Frederick, MD). Mice were 7–14 wk of age when experiments were started. All procedures were conducted according to protocols approved by the ACUC of the National Institute of Neurological Disorders and Stroke.
Antigens
Whole myelin basic protein (MBP) was prepared according to the method of Deibler et al. (36) from guinea pig spinal cords (Pel-Freez Biologicals, Rogers, AR). Peptide 139–151 from proteolipid protein (PLP), PLP139–151, was prepared by continuous flow solid phase synthesis according to the sequence for murine PLP (HSLGKWLGHPDKF) by the Protein and Nucleic Acid Facility, Beckman Center, Stanford University, Stanford, CA.
Induction of adoptive transfer EAE
Each recipient mouse was injected i.v. with 3 x 107 activated MBP- or PLP139–151-specific lymph node cells (LNC). Immunization (400 µg MBP or 200 µg PLP139–151) and the preparation of primary LNC cultures and short term T cell lines followed the method previously described (37). Mice were examined daily for signs of disease and graded on a scale of increasing severity from 0 to 5 as follows: 0, no signs; 0.5, partial tail weakness; 1, limp tail or slight slowing of righting from supine position; 1.5, limp tail and slight slowing of righting; 2, partial hind limb weakness or marked slowing of righting; 2.5, dragging of hind limb(s) without complete paralysis; 3, complete paralysis of at least one hind limb; 3.5, hind limb paralysis and slight weakness of forelimbs; 4, severe forelimb weakness; 5, moribund or dead. Treatment effects were assessed using the nonparametric Mann-Whitney rank sum test. All statistical tests were performed with SigmaStat software (Jandel, San Rafael, CA).
Histology
Selected mice were killed with CO2. CNS tissues were fixed in 10% PBS-buffered formalin. Paraffin sections (4 µm) were stained with hematoxylin-eosin or Luxol fast blue (American Histolabs, Gaithersburg, MD). At least two coronal sections from three brain levels and at least two longitudinal and coronal sections from cervical, thoracic, and lumbosacral levels of the spinal cord were evaluated in a blinded manner by an experienced neuropathologist.
Inhibitors and treatment
I40 and I49 and the noninhibitory compound Lys[Z(NO2)]-OH were provided in lyophilized form. For the experiments described, the inhibitors were dissolved in PBS at 10-2 M and adjusted to neutral pH. In treatment experiments, 1 mg I40 (M = 414.89) was injected from once every other day to three times daily, s.c. or i.p., as indicated below. Mice injected with equal amounts of PBS or Lys[Z(NO2)]-OH served as controls.
Determination of DP IV activity
The enzymatic activity of DP IV was determined according to the
method published by Schön et al. (38) using 1.6 mM
Gly-Pro-4-nitroanilide as substrate for DP IV. The resulting
4-nitroaniline strongly absorbs at 392 nm. The enzymatic activity at
37°C and pH 7.6 is expressed in picokatals. All measurements with
substrate and PBS controls were performed in duplicate. To measure DP
IV activity in serum, two to three mice per treatment group were bled
on the days indicated. Pooled sera were diluted 1:10 before the assay.
Brains and spinal cords (caudal from C2) of mice from treatment and
control groups (n = 5–6/group) were removed after
transcardial perfusion with PBS on the days indicated. Tissues were
carefully ground using 1% n-octyl--glucopyranoside
(Sigma, St. Louis, MO) in 10 mM HEPES, and a homogenate in a fixed
small volume of the detergent was obtained. After 60 min incubation on
ice, the homogenate was centrifuged at 100,000 x g and
4°C. The supernatant was diluted 1:10 and immediately used for the
assay. The amount of whole protein in the tissue was determined
according to the method of Bradford (Bio-Rad Protein Assay Kit II;
Bio-Rad, Richmond, CA), with BSA as standard. Sera and CNS tissue from
naive mice and from mice transferred with 3 x
107 keyhole limpet hemocyanin (KLH)-specific LNC
served as controls. The latter were obtained as described above for
MBP-specific LNC, using 100 µg KLH for immunization and 10 µg/ml
for the 96-h stimulation period in vitro. For the comparison of tissue
DP IV activities, a one-way ANOVA was performed.
Proliferation assays
MBP- or PLP139–151-specific proliferation
of primed LNC or short term T cell lines were measured as described
(37). To determine the effect of the inhibitors, varying
concentrations were added at a fixed antigenic concentration (25
µg/ml MBP or 10 µg/ml peptide). Wells without inhibitor or Ag,
respectively, were used as controls. Results are given as arithmetic
means from cultures set up at least in triplicate. In the
neutralization experiments, a purified chicken anti-human TGF-1
Ab or a chicken control Ab (both AB-101-C; R&D Systems, Minneapolis,
MN) were added at 10 µg/ml final concentration. The culture medium
was based on AIM-V (Life Technologies, Gaithersburg, MD), supplemented
with 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin.
Cytokine measurements
Cytokine secretion was measured by sandwich ELISA in culture
supernatants. LNC (4 x 106 or 8 x
106 per well) were incubated with varying doses
of the inhibitors in the presence or absence of Ag in 48- or 24-well
plates, respectively, using supplemented AIM-V medium as described
above. Cell-free supernatants were aliquoted and stored at -80°C
until measurements were performed. Duoset-ELISA kits for IFN-,
TNF-, and IL-4 were purchased from Genzyme (Cambridge, MA). For
IL-10, InterTest-4X kits were obtained (PharMingen, San Diego, CA).
Measurements from at least two dilutions per sample were performed in
duplicates according to the manufacturer’s instructions.
TGF-1 in culture supernatants, tissue homogenates, and plasma was
measured by ELISA as described previously (39). Brains and
spinal cords were processed as described above for determination of DP
IV activity. Plasma was obtained from deeply anesthetized mice by
drawing 0.5 ml cardiac blood into a syringe containing 50 µl citrate
as anticoagulant (ACD solution A; Becton Dickinson, Franklin Lakes,
NJ). For the ELISA, a mouse monoclonal anti-TGF-1, -2, -3
Ab (Genzyme) and a chicken anti-TGF-1 Ab (R&D Systems) were used
(40). To release latent TGF-1, samples were tested
before and after transient acidification (40). TGF-1
concentrations in CNS and plasma were compared by a one-way ANOVA and
the Tukey test for multiple pairwise comparisons (41).
Priming studies
Mice were immunized with 400 µg MBP s.c. as described above. On the day of immunization and again 2 days later, the mice were treated i.p. with either 0.5 mg I40 in PBS or an equal volume of PBS. Lymph nodes of treated mice were harvested on day 10, and proliferation from LNC cultures was determined as described above.
Ex vivo studies
In some experiments, spleen cells from recipient mice were serially transferred to test for encephalitogenicity. Single-cell suspensions from spleens harvested at the time points indicated were cultured at 8 x 106 per well with 25 µg/ml MBP in 24-well plates as described previously (37). After 4 days, 3 x 107 washed cells were injected i.p. into naive mice. Cells from each donor were transferred into two recipients. Proliferation assays and cytokine studies were performed in parallel as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adoptive transfer of activated MBP-specific LNC into naive SJL
mice induces acute EAE followed by partial recovery and one or more
relapses (Fig. 1
). To test the effect of
synthetic, reversible DP IV inhibitors on EAE in vivo, we treated
recipient mice with the inhibitor I40, starting on the day of the T
cell transfer (Fig. 1A). As a consequence, the disease
severity and incidence during the acute phase was significantly
reduced, whereas the onset of clinical signs was variably delayed. The
treatment effect of I40 was not permanent but consistently lasted for
2 mo as depicted in Fig. 1A. The control substance
Lys[Z(NO2)]-OH which is structurally related to
I40 but does not inhibit DP IV activity did not have a protective
effect on the development of the first disease exacerbation (Table I). We also performed additional
experiments in which treatment was initiated at the day mice in both
groups developed clinical signs. Again, the severity of clinical
disease was significantly ameliorated by in vivo blockade of DP IV
(Fig. 1B). Side effects from the treatment were not observed
except for mild injection site reactions. A neuropathological
evaluation was performed in mice treated with I40 or PBS at the time of
fully established disease in the control group. Although tissues from
control mice displayed moderate to severe, mainly mononuclear
inflammation and some myelin loss at the site of inflammation, no or
few inflammatory cells and no myelin loss were found in the cords from
I40-treated mice without clinical disease (Fig. 2). Lesions in I40-treated mice that
developed clinical disease were histologically indistinguishable from
lesions in control mice with the same disease severity. Demyelinating
lesions were seen in all animals moderately or severely affected,
irrespective of the treatment. These results demonstrate a strong
therapeutic potential of synthetic DP IV inhibitors for autoimmune
mediated CNS inflammation.
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We also investigated whether the treatment with I40 could suppress
DP IV activity in serum and in CNS tissue. Whereas serum DP IV activity
increased between days 2 and 10 after transfer in control mice, it
did not change significantly from baseline in I40-treated mice (Fig. 3). Spinal cord tissue was examined on
day 10 after transfer. In mice with EAE treated with either PBS or
Lys[Z(NO2)]-OH, we observed a 2- to 3-fold
increase in DP IV activity as compared with naive mice or mice
transferred with KLH-specific LNC, indicating that CNS inflammation
causes increased DP IV activity in situ (data not shown). I40 treatment
was able to partially suppress this increase (Table I
).
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1
DP IV inhibitors have been shown to up-regulate latent TGF-1
secretion by polyclonally stimulated murine T cells (33).
Many studies have suggested a protective role for endogenously
synthesized and therapeutically induced or administered TGF-1 in EAE
(42, 43, 44, 45). We therefore determined the amount of TGF-1
secreted into spinal cord tissues of treated mice. Remarkably, in
tissue from I40-treated mice, there was a significant increase in the
amount of latent TGF-1 secreted as compared with
Lys[Z(NO2)]-OH- or PBS-treated mice (Table I).
Further evidence for systemic TGF-1-up-regulation in vivo was
obtained by measuring latent TGF-1 concentrations in plasma. There
was a significantly higher TGF-1 concentration present in plasma
from I40-treated mice as compared with PBS-treated controls (Table I).
A clear association was observed among lack of clinical signs, paucity
of CNS inflammation, and high TGF-1 levels in I40-treated mice as
opposed to marked clinical disease and CNS inflammation and low
TGF-1 levels in PBS-treated mice. We then asked whether the
encephalitogenic T cells themselves might be the source of TGF-1.
Primed LNC were antigenically restimulated in vitro in the presence of
I40. Interestingly, during the first 48 h of antigenic
stimulation, we observed a modest but consistent increase in the amount
of secreted latent TGF-1 that was detectable after as early as
4 h. (Fig. 4). In most experiments,
the maximal effect was achieved by an inhibitor concentration 
10
µM.
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To analyze further the potential mechanism(s) of the treatment
effect, we examined the effect of DP IV blockade during stimulation of
MBP-primed LNC in vitro (Fig. 5).
Coincubation of primed LNC and Ag with I40 consistently resulted in a
dose-dependent inhibition of proliferation (Fig. 5A) and
secretion of the proinflammatory cytokine TNF- (Fig. 5B).
Inhibition of IFN- production was seen in two experiments but less
consistent in others. Similar results were obtained with a second
reversible DP IV inhibitor, I49 (data not shown). IL-10 and IL-4 were
usually not secreted in significant amounts by these LNC cultures. In
LNC cultures, concentrations of I40 up to 10 µM over 96 h did
not cause a significant loss of cell viability (data not shown) as
assessed by trypan blue staining.
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1
We next asked whether the inhibitory effect on T cell function
might be mediated by TGF-1. Indeed, a neutralizing anti-TGF-1
Ab completely blocked the effect of 10 µM I40 on the proliferation of
a PLP139–151-specific T cell line, whereas the
control Ab had no effect on inhibition by I40 (Fig. 5C).
Given that the T cell line was stimulated with peptide presented by
irradiated spleen cells, the source of the TGF-1 neutralized can
more likely be attributed to the antigenically stimulated T cells
rather than APCs. These results suggest that the effect seen in vivo
may be mediated in part by the down-modulation of TNF- secretion and
that TGF-1 up-regulation may contribute to the inhibitory effect on
T cell function.
Suppression, not elimination, of encephalitogenic T cells
Our in vivo studies had suggested that I40 treatment during the
induction phase does not indefinitely abrogate clinical disease;
indeed, after 2 mo, the clinical signs worsened in the group treated
with I40 and reached the severity displayed by the control group (Fig. 1A). Subsequently, as frequently seen in chronic murine EAE,
disease scores remained relatively stable, without apparent disease
activity. We therefore asked whether worsening of disease several weeks
after cessation of treatment was likely to be due to the survival of
encephalitogenic T cells after I40 treatment. We addressed this issue
by examining the MBP-specific proliferation, cytokine profile, and
encephalitogenicity of spleen cells taken after >30 days from animals
treated with I40 or PBS either during induction of disease or after
disease onset (Table II). In the absence
of the inhibitor in vitro, spleen cells from all animals proliferated
and secreted proinflammatory cytokines, regardless of whether they were
taken from healthy or diseased animals treated with I40 or from
PBS-treated control animals with EAE. Furthermore, spleen cells from
all mice examined induced typical relapsing EAE after transfer into
naive recipients. We conclude that potentially encephalitogenic cells
were still present even in animals that had never developed EAE signs.
These cells can be reactivated in vitro and produce disease in vivo,
suggesting that even after a prolonged period of in vivo treatment with
I40 these cells may eventually become susceptible again to antigenic
activation in vivo.
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We finally asked whether DP IV inhibition in vivo interferes with
Ag-specific priming responses. Mice were treated with I40 or PBS on the
day of immunization with MBP in CFA and again 48 h later. LNC from
both groups of mice proliferated equally well to MBP on secondary
stimulation suggesting similar priming efficacy (Fig. 6). These data suggest that during and
after treatment with I40, a disease perpetuation and/or exacerbation
may be possible via epitope spreading which can be considered as an
endogenous immunization by released myelin Ags (46)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Is the treatment effect DP IV specific? Several lines of evidence
suggest that DP IV is the specific target of I40 in vitro as well as in
vivo during the treatment of EAE. 1) We treated mice with
Lys[Z(NO2)]-OH, a truncated form of I40 that
lacks the pyrrolidide moiety, rendering it noninhibitory in vitro. This
substance did not have a significant effect on either the early
clinical course of EAE or the DP IV activity and latent TGF-1
secretion in spinal cord (Table I). 2) We observed an increase in serum
DP IV activity in control mice during the first 10 days after the
transfer of encephalitogenic T cells that did not occur in I40-treated
mice (Fig. 3). Our conclusion is further supported by a recent study in
a rat transplantation model (47). Treatment with the
irreversible DP IV inhibitor prodipine prevented the increase in serum
DP IV normally seen during the first days after cardiac
allotransplantation. Both allograft rejection and the concomitant
increase of DP IV activity in transplant tissue were delayed
(47). In another study, arthritis signs in rats could be
suppressed by several biochemically distinct DP IV inhibitors,
including I49, indicating that the effect was very likely to be due to
the specific inhibitory effect on DP IV (48). In vitro,
most studies found a correlation between the level of DP IV activity
and the effect of DP IV inhibitors on cellular activation (reviewed in
Ref. 20).
The inhibition seen in vitro is not a toxic effect toward T cells.
After 96 h of coculture with I40, an increased percentage of
trypan blue-staining T cells was only observed at a concentration of 50
µM or higher. The inhibitory effects on proliferation and cytokine
production, however, were already seen at lower concentrations
(IC50 10 µM). The up-regulation of TGF-1
was even maximal at concentrations of 10 µM. Moreover, the
antiproliferative effect of 10 µM I40 on an autoreactive T cell line
was completely blocked by a neutralizing anti-TGF-1 Ab. Various
techniques to determine cell viability or apoptosis induction,
respectively, have previously shown that these inhibitors do not
adversely affect cell viability in inhibitory doses up to 10 µM
(19). Three further observations exclude depletion of
lymphocytes in vivo as a major mechanism of peripheral tolerance under
effective treatment conditions: 1) T cells may be primed during
I40-treatment (Fig. 6); 2) autoreactive T cells capable of transferring
EAE after stimulation in vitro were recovered from mice that were
previously injected with encephalitogenic T cells and treated with I40
(Table II); 3) animals developed EAE weeks after treatment with I40 had
been stopped (Fig. 1A).
What is the mechanism of autoimmune disease suppression? We suggest
that the protective effect of DP IV inhibition is caused by a
modulation of T cell effector function. In a biochemical assay, we
found DP IV activity on the cell surface of all autoreactive T cell
clones examined, on resting as well as on activated T cells. The DP IV
inhibitors I40 and I49 had strong antiproliferative effects in vitro on
the T cell clones examined, on both an encephalitogenic Th1 clone and a
nonencephalitogenic Th2 clone (data not shown). I40 and I49 also
suppressed the proliferation of LNC and, importantly, their secretion
of TNF- and, to a lesser extent, IFN-. These data confirm and
extend earlier reports showing that DNA synthesis and the secretion of
IL-2, IL-6, and IL-10 from mouse spleen cells and thymocytes
(33) are suppressed by DP IV inhibitors. Likewise, in
human T cells proliferation and the secretion of various cytokines
including TNF- and IFN- were suppressed (34, 35). It
is widely accepted that EAE can be mediated by Th1
CD4+ T cells typically secreting IFN-,
TNF-, and lymphotoxin. Various therapeutic approaches that target
Th1 cytokines in vivo have been found to be effective in EAE (reviewed
in Refs. 7, 8, 9 and 49). We therefore conclude
that the inhibition of T cell proliferation and effector functions
including proinflammatory cytokine secretion may in part be responsible
for the in vivo effect observed.
More importantly, we found an up-regulation of latent TGF-1
production in vivo, both in spinal cord tissues and in plasma, from
I40-treated mice as compared with mice treated with PBS or
Lys[Z(NO2)]-OH. We demonstrate furthermore
that I40 increases the secretion of latent TGF-1 by Ag-stimulated
LNC populations. Recently, it has been demonstrated in vitro that DP IV
inhibition induces a severalfold increase in TGF-1 mRNA and protein
in polyclonally stimulated T cells (33, 35). Anti-TGF-1
neutralized the effect of DP IV inhibition on the proliferation of
PWM-stimulated human T cells (35). We extend these
findings, demonstrating for the first time that anti-TGF-1 can
also block the effect of DP IV inhibition on Ag-specific T cell
proliferation. Our in vitro data suggest that the cell types induced to
secrete latent TGF-1 by I40 treatment include T cells. One can
assume that TGF-1 secretion is increased both by the
encephalitogenic T cells initiating the infiltrate and by T cells that
are attracted and activated during the later stages of lesion formation
in a bystander fashion. Not surprisingly, macrophages and microglia, in
addition to a majority of T cells, appear to produce TGF-1 in acute
EAE lesions (50). Whether those or other cell types are
susceptible to regulation of DP IV remains to be investigated.
TGF-1 is a cytokine with powerful immunoregulatory effects (reviewed
in Ref. 51). A protective role for TGF-1 in EAE has
been clearly established. The endogenous TGF-1 production is
up-regulated in the CNS and presumably plays a down-modulatory role
during the recovery phase of acute EAE (43, 44). Although
anti-TGF-1 Ab treatment in vivo aggravates EAE severity
(43, 45, 52, 53, 54), TGF-1 treatment can prevent EAE and
suppress disease already established (42, 55). Weiner et
al. (45) showed that the protective effect of oral
administration of myelin Ags is abrogated by anti-TGF-1. Oral
low dose feeding of myelin Ags induced a specific regulatory and
protective population of Th3 cells secreting TGF-1, IL-10, and IL-4
(13, 15). Treatment of myelin Ag-specific T cells in vitro
with TGF-1 suppresses their proliferation, secretion of TNF- and
IFN-, and capacity to induce EAE (55). Finally,
TGF-1 has antiinflammatory effects in situ by suppressing the
production of NO, and TNF- by microglia and of cytokine-induced MHC
class II, TNF-, and ICAM-1 expression by rodent astrocytes
(56). A recent study showed that adoptive transfer of
activated MBP-specific Th1 clones transduced to secrete latent TGF-1
delayed and ameliorated EAE signs in mice immunized with PLP
(57). Taken together, up-regulation of latent TGF-1
production by DP IV inhibition may be an important mechanism of
autoimmune disease suppression. It may represent TGF-1-mediated
bystander suppression that also appears to be one of the protective
mechanisms of oral tolerance (14) and of transduced
self-reactive T cells as shown by Chen et al. (57).
The partial inhibition of DP IV activity in vivo and the moderate
up-regulation of TGF-1 may explain why we were not able to
completely abrogate EAE by increasing the amount of I40 administered,
especially in a therapeutic setting. However, the transduced T cells
producing considerably higher amounts of latent TGF-1 in vitro than
our I40-treated LNC did not completely abrogate EAE either
(57). Indeed, results from others and our own preliminary
data indicate a complex role of DP IV inhibition and TGF-1-mediated
disease modulation, respectively, in acute vs chronic EAE. Different
treatment effects have been demonstrated depending on the exact time
the animals were exposed to TGF-1 (52). Furthermore, it
appears that TGF-1-mediated treatment effects are temporally and
spatially confined to the inflammatory infiltrate in the CNS,
explaining the lack of general immunosuppression during treatment with
I40. In particular, DP IV inhibition in vivo did not eliminate
encephalitogenic T cells (Table II). In addition, despite the
antiinflammatory effects of I40 on LNC in vitro (Fig. 5), incidence and
severity of EAE were similar when equal numbers of DP IV inhibitor- and
PBS-pretreated lymph node cells were injected into naive mice (data not
shown). Finally, DP IV inhibition during antigenic priming did not
suppress secondary Ag-specific T cell proliferation (Fig. 6). Because T
cell priming is not affected by DP IV inhibition, epitope spreading may
still occur which has been involved in the clinical progression of EAE
induced with PLP139–151-specific T cells
(46, 58). Currently, we are investigating systematically
the effects of long term treatment with DP IV inhibitors on chronic
disease.
In conclusion, our findings show that the DP IV activity associated with CD26 plays an important role in the activation of autoreactive T cells. Inhibition of DP IV activity in vivo provides a new approach to down-modulate tissue-specific autoimmunity. These results could have important implications with regard to the treatment of human diseases thought to be mediated by T cell-mediated autoimmune mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andreas Steinbrecher, Department of Neurology, University of Regensburg, Universitaetsstrasse 84, 93053 Regensburg, Germany.
3 Current address: Department of Pathology, Hadassah Medical School, The Hebrew University, P.O. Box 12272, Jerusalem 91120, Israel.
4 Abbreviations used in this paper: DP IV, dipeptidyl peptidase IV; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; PLP, proteolipid protein; LNC, lymph node cells; Z(NO2), 4-nitrobenzyloxycarbonyl; I40, Lys[Z(NO2)]-pyrrolidide; I49, Lys[Z(NO2)]-thiazolidide; KLH, keyhole limpet hemocyanin.
Received for publication December 7, 1999. Accepted for publication November 6, 2000.
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  A. C. C. Girardi, L. E. Fukuda, L. V. Rossoni, G. Malnic, and N. A. Reboucas Dipeptidyl peptidase IV inhibition downregulates Na+-H+ exchanger NHE3 in rat renal proximal tubule Am J Physiol Renal Physiol, February 1, 2008; 294(2): F414 - F422. [Abstract] [Full Text] [PDF]
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 V. Preller, A. Gerber, S. Wrenger, M. Togni, D. Marguet, J. Tadje, U. Lendeckel, C. Rocken, J. Faust, K. Neubert, et al. TGF-beta1-Mediated Control of Central Nervous System Inflammation and Autoimmunity through the Inhibitory Receptor CD26 J. Immunol., April 1, 2007; 178(7): 4632 - 4640. [Abstract] [Full Text] [PDF]
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 D. Perez-Tilve, L. Gonzalez-Matias, M. Alvarez-Crespo, R. Leiras, S. Tovar, C. Dieguez, and F. Mallo Exendin-4 Potently Decreases Ghrelin Levels in Fasting Rats Diabetes, January 1, 2007; 56(1): 143 - 151. [Abstract] [Full Text] [PDF]
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 S. Wrenger, J. Faust, D. Friedrich, T. Hoffmann, R. Hartig, U. Lendeckel, T. Kahne, A. Thielitz, K. Neubert, and D. Reinhold Attractin, a dipeptidyl peptidase IV/CD26-like enzyme, is expressed on human peripheral blood monocytes and potentially influences monocyte function J. Leukoc. Biol., September 1, 2006; 80(3): 621 - 629. [Abstract] [Full Text] [PDF]
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 M. Togni, K. D. Swanson, S. Reimann, S. Kliche, A. C. Pearce, L. Simeoni, D. Reinhold, J. Wienands, B. G. Neel, B. Schraven, et al. Regulation of In Vitro and In Vivo Immune Functions by the Cytosolic Adaptor Protein SKAP-HOM Mol. Cell. Biol., September 15, 2005; 25(18): 8052 - 8063. [Abstract] [Full Text] [PDF]
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 N. Busso, N. Wagtmann, C. Herling, V. Chobaz-Peclat, A. Bischof-Delaloye, A. So, and E. Grouzmann Circulating CD26 Is Negatively Associated with Inflammation in Human and Experimental Arthritis Am. J. Pathol., February 1, 2005; 166(2): 433 - 442. [Abstract] [Full Text] [PDF]
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 S. Adams, G. T. Miller, M. I. Jesson, T. Watanabe, B. Jones, and B. P. Wallner PT-100, a Small Molecule Dipeptidyl Peptidase Inhibitor, Has Potent Antitumor Effects and Augments Antibody-Mediated Cytotoxicity via a Novel Immune Mechanism Cancer Res., August 1, 2004; 64(15): 5471 - 5480. [Abstract] [Full Text] [PDF]
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 A M A. El-Asrar, S Struyf, S A Al-Kharashi, L Missotten, J Van Damme, and K Geboes Expression of T lymphocyte chemoattractants and activation markers in vernal keratoconjunctivitis Br. J. Ophthalmol., October 1, 2002; 86(10): 1175 - 1180. [Abstract] [Full Text] [PDF]
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U. Aytac, F.-X. Claret, L. Ho, K. Sato, K. Ohnuma, G. B. Mills, F. Cabanillas, C. Morimoto, and N. H. Dang Expression of CD26 and Its Associated Dipeptidyl Peptidase IV Enzyme Activity Enhances Sensitivity to Doxorubicin-induced Cell Cycle Arrest at the G2/M Checkpoint Cancer Res., October 1, 2001; 61(19): 7204 - 7210. [Abstract] [Full Text] [PDF]
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