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Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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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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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rolipram (racemate of 4-(3'-cyclopentyloxy-4'-methoxyphenyl)-2-pyrrolidone) was provided by Dr. Harald Dinter (Berlex Laboratories, Richmond, CA), Cilostamide (OPC 3689) was a gift from Dr. Vincent Manganiello Pulmonary-Critical Care Medicine Branch (PCCMB), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD). Fresh solutions of individual drugs were prepared for each experiment. The solvent for both drugs, DMSO (Sigma, St. Louis, MO), was used in 1/1000 dilution with T cell medium for the 10-µM concentration of drugs and at this concentration did not influence T cell proliferation when used as a negative control.
Myelin basic protein was prepared as previously described (17). The peptides Flu-HA306–318 (PKYVKQNTLKLAT) and tetanus830–843 (QYIKANSKFIGITQL) were synthesized by continuous flow, solid phase peptide synthesis on the basis of the F-moc/But strategy. Peptides were purified by HPLC, and their identities were tested using ion spray mass spectrometry.
The TCL were generated by an IL-7-modified primary proliferation assay, a method that allows the rapid expansion of Ag-specific T cells, including in vivo activated cells. Briefly, PBMC were isolated from fresh leukophereses by Ficoll density gradients and were seeded in 96-well U-bottom plates in T cell medium (IMDM, Life Technologies, Grand Islands, NY) containing 2 mM L-glutamine, 50 µg/ml gentamicin, 100 U/ml penicillin/streptomycin (all from BioWhittaker, Walkersville, MD), and 5% pooled human plasma at 1 x 105 cells/well, with addition of 10 ng/ml IL-7 (recombinant human IL-7; PeproTech, Rocky Hill, NJ). After 7 days (37°C and 5% CO2), cultures were split by transferring 100 µl of each cell culture into a daughter plate, which was pulsed with [3H]thymidine (Amersham, Arlington Heights, IL) at 1 µCi/well. The incorporated radioactivity (counts per minute) was measured by scintillation counting (Betaplate, Pharmacia LKB, Piscataway, NJ) 8 h later. Proliferation of cultures with Ag (25 µg/ml MBP or 5 µg/ml peptides) was compared with proliferation of negative control wells seeded without Ag. Positive cultures (stimulation index >2 and absolute counts per minute at least 3 SDs above the average counts per minute of negative control wells) were identified on the mother plates and were periodically restimulated. The Ag specificity was confirmed at the end of the second in vitro stimulation cycle in 48-h proliferation assays as previously described (18). All blood samples were collected according to an institutional review board-approved protocol, and informed consent was obtained before the study. None of the patients received any immunomodulatory or immunosuppressive treatment within 1 mo before blood collection.
Effects of PDE-4 and PDE-3 inhibitors on Ag-specific TCL proliferation and functional Ag avidity
The effects of PDE inhibitors on Ag-specific proliferation, cytokine production, and functional Ag avidity were assessed during the third stimulation cycle (days 22–25 ex vivo). Each drug was used alone (in three concentrations: 0.1, 1, and 10 µM) or in combination (0.05, 0.5, and 5 µM, concentrations of individual drugs in the mixture). The selected dose range included the concentrations achievable in vivo (for rolipram, 0.09 and 0.2 µM; Dr. Claus-Steffen Stuerzebecher, Schering, Berlin, Germany, personal communication). Each condition was tested in duplicate, including negative (no Ag) and positive (Ag, no drug) controls. T cells were plated in 96-well U-bottom plates at 2 x 104 T cells/well with irradiated autologous PBMC at 1 x 105 cells/well. The Ag was added at the seeding concentration (25 µg/ml MBP and 5 µg/ml peptides) or over a wide range of Ag concentrations for the functional Ag avidity assay. Supernatants from these assays were collected after 36 h of incubation and stored frozen until analysis. For the last 8 h of incubation, cells were pulsed with [3H]thymidine at 1 µCi/well, and the incorporated radioactivity was measured by scintillation counting.
Cytokine secretion
Secretion of a Th1 (IFN-
) and a Th2 (IL-4) cytokine by
Ag-specific TCL was assessed by sandwich ELISA (Cyto-Sets from
BioSource International, Camarillo, CA) according to the
manufacturer’s recommendation. All standards and samples were tested
in duplicate.
Flow cytometry (FACS) analysis of the surface expression of costimulatory molecules
Fresh PBMC (1.2 x 106 cells/ml) were seeded in bulk cultures in 48-well plate with or without rolipram (10 µM). In addition to nonstimulated cells, the effect of rolipram was assessed upon nonspecific stimulation with LPS (2.5 µg/ml) or PHA-P (PHA; 5 µg/ml; both from Sigma). After 12-h incubation cells were washed with wash buffer (Dulbecco’s PBS with 1% heat-inactivated FCS and 0.1% sodium azide) and incubated with fluorescein-, PE-, or Cy-Chrome-conjugated Ab (HLA-DR,DP,DQ-FITC, CD19-FITC, CD14-FITC and -PE, CD80-FITC and -PE, CD86-FITC and -PE, and CD3-Cy-Chrome; all from PharMingen, San Diego, CA) at saturating concentrations for 30 min on ice, then washed three times and analyzed (FACScan, Becton Dickinson, CA) using Cell-Quest software. Isotype-matched mouse IgG negative controls were used for each staining. Monocytes were gated based on the size characteristics (forward and side scatter), and expression of CD14. Lymphocytes were identified by the size characteristics and differentiation between T and B lymphocytes was based on the expression of CD3 and CD19 molecules, respectively. Five thousand cells in the gated population were analyzed per sample.
Statistical analysis
The data were analyzed by a commercial software package (Sigma-Stat, SPSS, Chicago, IL). The effects of the drugs on biological functions of TCL was evaluated by one-way repeated measures ANOVA or, if normality failed, by Friedman’s repeated measure analysis on ranks. Statistically significant differences from repeated measures ANOVA were further analyzed by the Student-Newman-Keuls test, with p < 0.05 as a cut-off for statistical significance. The effect of rolipram on costimulatory molecules was assessed by the Mann-Whitney rank-sum test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined the effects of rolipram and cilostamide on the
proliferation of 47 Ag-specific TCL. Both drugs inhibited Ag-driven TCL
proliferation, but only rolipram and the combination of both drugs
expressed statistically significant inhibitory effects in a
dose-dependent manner (Fig. 1
). The
inhibition by rolipram (8.8–32% inhibition) was more pronounced than
that by cilostamide (6.31–22.61% inhibition). Combinations of both
drugs (1/1 ratio and half molar concentration of each drug (0.05, 0.5,
and 5 µM in the final mixture) proved to be most efficient in
inhibiting TCL proliferation (10.34–61.05%), exceeding the additive
effects of individual drugs. The observed differences between drugs and
their combination were statistically significant
(p < 0.05, by repeated measures ANOVA).
Because the above characteristics of the drug combination indicated
synergistic effects, we have quantified the degree of synergism between
rolipram based on the modified Berenbaum equation
(19):
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Based on the proliferation data in Fig. 1
we have estimated the
IC20 (the concentration of each drug that leads
to 20% inhibition of TCL proliferation; this was substituted for
IC50, as neither rolipram nor cilostamide reached
50% of inhibition) from the dose-response curves and calculated
Rrolipram+cilostamide = 0.1123. The
degree of synergism between rolipram and cilostamide is
1/R = 8.9. (Values <1 indicate antagonism, 1 indicates
additivity, and values >1 indicate synergism.)
Comparison of the susceptibility to PDE-4 and -3 inhibition by autoreactive and foreign Ag-specific TCL and by TCL derived from MS patients and healthy donors
When the inhibitory effect of PDE inhibitors was assessed after
stratification of TCL based on their origin and the type of the
selecting Ag (TCL derived from MS patients (26 TCL, 17 autoreactive, 9
foreign-Ag-specific) vs. TCL derived from healthy donors (21 TCL, 5
autoreactive, 16 foreign-Ag-specific)), two interesting observations
emerged (Fig. 2). 1) The MS-derived TCL
were more susceptible to the effect of PDE inhibitors than were the
healthy donor-derived TCL (p < 0.05 for 10
µM rolipram, 10 µM cilostamide, and 0.l µM and 1 µM
concentrations of drug mixture; Friedman’s repeated measures analysis
on ranks). 2) Also, there appeared to be a differential sensitivity of
autoreactive TCL and foreign Ag-specific TCL to the effects of PDE4 and
PDE3 inhibition. Autoreactive TCL were inhibited by rolipram to a
greater extent than foreign Ag-specific TCL (p
< 0.05, through the tested concentration range of rolipram,
Friedman’s repeated measures analysis on ranks). Only stratified data
are depicted in Fig. 2. Due to the decreased power of a comparison of
divided data into individual patient groups, not all differences
reached statistical significance (p < 0.05, by
Friedman’s repeated measures analysis on ranks); these are marked with
an asterisk.
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We evaluated the effects of PDE inhibitors on Ag-driven
production of two cytokines, IFN- (prototypic Th1 cytokine) and IL-4
(prototypic Th2 cytokine; Fig. 3).
Although a significant variability was noted among individual TCL, the
average effect of PDE4 inhibition by rolipram and of PDE4 and PDE3
inhibition by the combination of drugs on IFN- production was
inhibitory. Rolipram (12.26–27.8% inhibition) and the combination of
both drugs (16.07–46.3%) inhibited IFN- synthesis in a
dose-dependent manner. The effect of cilostamide was mild and did not
reach statistical significance for any concentration (4.47–11.27%
inhibition). Differences among the drugs were again statistically
significant.
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production
paralleled those on proliferation, with the drug combination having
synergistic effects. The IL-4 production was largely unaffected despite
the demonstrated significant inhibition of TCL proliferation at the
drug concentrations tested. Effect of PDE inhibitors on the functional Ag avidity of TCL
To gain a better understanding of the mechanism of the PDE4 and
PDE3 inhibitor-induced bias of immune responses from a Th1 toward a Th2
phenotype, we decided to study their effects on the major components of
T cell priming. Apart from the influence on the cytokine milieu, which
was supported by the above experiments, we wanted to address the
question of whether these drugs influence the Ag dose needed for T cell
activation, i.e., antigen avidity, and costimulatory signals delivered
by APC. First, to address the question of whether the magnitude of the
immunomodulatory effect of PDE inhibitors on TCL varies depending on
the dose of Ag, we exposed a subgroup of TCL (15 TCL; selected as a
representative sample based on their phenotype, variable susceptibility
to PDE inhibition, and reactivity to either autoantigen or
environmental Ag; Table I) to a 1-µM
concentration of the individual drugs over a wide range of Ag
concentrations. The results of this functional Ag avidity assay for
three TCL (of Th1, Th0, and Th2 phenotypes) are summarized in Fig. 4. None of the autoreactive or foreign
Ag-specific TCL that we generated for this project expressed a clear
Th2 phenotype. We therefore included a Th2-like TCL specific for
copolymer-1 (Cop-1), an approved immunomodulatory drug for MS. The TCL
Th1:MBP and Th0:Flu-HA were representative of all other tested TCL and
illustrate the above-mentioned differential effect of PDE inhibition on
autoreactive vs foreign Ag-reactive TCL. All examples also demonstrate
the hierarchy of immunomodulation between individual drugs
(cilostamide < rolipram < rolipram + cilostamide). For
MBP-specific Th1-TCL, a combination of PDE4 and PDE3 inhibition
resulted in almost 100% inhibition of proliferation and IFN-
production. For Flu-HA-specific Th0-TCL, the inhibition of
proliferation and IFN- production was much less pronounced (
50%
with the combination of drugs for each Ag concentration), and the
production of IL-4 was either unaffected or even enhanced. For
Cop-1-specific Th2-TCL, the individual drugs had no effect on
Ag-specific proliferation or IL-4 production. Overall, the magnitude of
the effect of PDE inhibitors on individual TCL was similar through the
tested Ag concentration range. However, as demonstrated in Table I, the
PDE inhibition had a mild, but statistically significant, effect on the
EC50 (concentration of Ag that leads to 50%
maximal proliferation) of individual TCL (p =
0.009, Friedman’s repeated measures analysis on ranks).
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Finally, we wanted to assess the effect of PDE inhibition on the
third important component of T cell priming, the costimulatory signal.
We studied the effect of rolipram on the surface expression of
costimulatory molecules of human PBMC in the resting and in the
activated state. Freshly isolated PBMC were seeded ex vivo with or
without rolipram, in a resting state or upon activation with
nonspecific stimuli (PHA or LPS). After 12 h we analyzed these
cells by three-color flow cytometry. Rolipram consistently
down-regulated the surface expression of MHC class II both in the
resting state and after stimulation with PHA and LPS on monocytes (Fig. 5) and T and B lymphocytes (data not
shown). After 12-h stimulation of PBMC with LPS, rolipram-treated
cultures had decreased surface expression of CD80
(p < 0.001, by Mann-Whitney rank-sum test) and
increased surface expression of CD86 (p =
0.008; Fig. 5). Similar changes in the expression of costimulatory
molecules were observed on B and T lymphocytes or after stimulation
with PHA (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Few studies have addressed the question of the effect of PDE inhibitors
on human immune cells. A recent study from our laboratory examined the
expression of PDE4 and PDE3 enzymes in autoreactive MBP-specific TCL
(2) and demonstrated that these two families account for
the vast majority of PDE enzymatic activity in these cells. This is
consistent with our current data, demonstrating a significant
inhibition of proliferation at the highest concentration of the drug
combination (5 µM of both rolipram and cilostamide) for virtually all
tested TCL. Another study analyzed the influence of rolipram on the
functional characteristics of nine MBP-specific TCL, five derived from
MS patients and four from healthy donors (15), and raised
the issue of a differential susceptibility of individual TCC to the
immunomodulatory influence of rolipram. Although rolipram inhibited
TNF-
and -
as well as IL-10 production by TCL, the effect on
other cytokines (IFN-, IL-4, and IL-13) was inconsistent and did not
reach statistical significance. Moreover, Essayan et al
(16). suggested a higher susceptibility to PDE4 inhibition
by Th2-TCL compared with Th1-TCL in a limited number of Th1 and Th2 TCL
(total of four) derived from atopic and asthmatic patients. Although it
is difficult to compare the data derived from different experimental
systems, the reported inhibition of Th1-TCL derived from these
asthmatic patients did not reach the magnitude of the typical
inhibition of MS-derived Th1 TCL observed in our laboratory using the
same concentrations of drugs. Because asthmatic individuals and MS
patients have biased immune responses toward opposite Th phenotypes as
compared with unbiased responses of healthy donors, we asked whether
the explanation for these controversial data from human TCL lies in the
differential susceptibility to PDE inhibition between these different
patient groups. This hypothesis prompted us to examine the effect of
PDE inhibition on large numbers of TCL, derived from both healthy
donors and MS patients, and with specificity for two Ags, the
autoantigen MBP and the classical foreign recall Ags Flu-HA or tetanus
(22). Indeed, we were able to demonstrate a higher
susceptibility to PDE inhibition by MS-derived compared with healthy
donor-derived TCL. This finding has several important implications. It
explains how the same therapeutic agent could be considered for the
treatment of disorders with potentially different pathogenesis
(Th1-mediated autoimmune disorders vs. Th-2-mediated asthma and atopic
dermatitis). We and others recently demonstrated (23, 48)
that the immune system in MS patients is in a dysregulated state
characterized by an overshooting Th1 response not only to autoantigens,
but also to common environmental pathogens. A similar dysregulated
state, this time toward Th2 responses, is likely to exist in asthmatic
or atopic individuals (24). If such a dysregulation
involves abnormalities in the cAMP second messenger system, it would
render TCL derived from these individuals more susceptible to the
effects of PDE inhibition, thus at least in part explaining the
controversies between results obtained from MS and asthmatic patients.
Several reports in the literature indicate that this may be the case.
Patients with MS, rheumatoid arthritis, or lupus were found to have low
intracellular cAMP levels (25, 26), decreased expression
and activity of G protein-coupled receptor kinases (27),
or deficient type I cAMP-dependent protein kinase A activity (28, 29). Similar abnormalities in cAMP signaling were suggested in
asthmatic patients (30, 31, 32).
It is more difficult to explain the suggested differential susceptibility to PDE inhibition between autoreactive and foreign Ag-reactive TCL. We did not find any significant skewing in the cytokine profiles between these two types of TCL that would account for the observed differential effect. This effect is not mediated by the need for antigenic processing of MBP compared with Flu-HA and tetanus peptides, because it was shown that MBP presentation by HLA-DR molecules does not require processing (33) and because we have noted a similar effect of PDE inhibition on seven MBP-specific TCL stimulated with the peptide epitope (data not shown). The two likely explanations are that either the signal delivered by the autoantigen may be qualitatively different (partial agonist vs full agonist signal) or the dysregulation in the cAMP system is more pronounced in autoreactive T cells. We are currently studying this issue in detail. Considering the therapeutic use of PDE inhibitors, the observed higher susceptibility of autoreactive TCL to PDE4 inhibition may widen the therapeutic window in the treatment of autoimmune disorders without inducing general immunosuppression.
To explore the possible cause of the PDE inhibitor-induced bias from
Th1 to Th2 phenotype, we decided to study the influence of these drugs
on T cell priming. There are three major components contributing to T
cell priming, which may influence the phenotype of the primed T cell:
the cytokine milieu, the dose and character of the Ag, and the
costimulatory signal. It was previously demonstrated by us and others
that selective PDE4 inhibitors or nonselective PDE inhibitors decrease
the secretion or expression of proinflammatory cytokines by human
mononuclear cells, favoring the cytokine milieu at the time of Ag
presentation toward an anti-inflammatory Th2 phenotype (6, 14, 34, 35). However, the influences of PDE inhibition on the
other components of T cell priming conditions were unknown. First, we
studied the PDE inhibitors in Ag dose-response assays, exploring their
effects on the dose of Ag required for T cell activation. The results
of these studies indicate that PDE inhibitors have two different
effects on the activation of TCL; they inhibit TCL proliferation
regardless of the Ag dose (Fig. 4). On the other hand, the effect of
PDE inhibitors on the EC50 indicates that a
higher Ag dose is necessary for the activation of TCL under their
influence. These data suggest that PDE inhibitors have complex
inhibitory effects on T cell activation, most likely by influencing
both proximal Ag-responsive events of T cell signaling
(36) as well as components of more downstream machinery
involved in T cell effector functions. The exact mechanisms of this
effect are currently under investigation in our laboratory. The
demonstrated influence of PDE4 and -3 on the Ag dose required for T
cell activation together with the data from the literature showing that
high Ag doses skew the developing immune response toward a Th1
phenotype, whereas low doses of Ag skew the response toward a Th2
phenotype (37) adds another mechanism for the observed
effect of these drugs on Th1/Th2 paradigm.
Next, we wanted to assess the influence of rolipram on the third
component of T cell priming, the costimulatory signals. It has been
suggested that costimulation by CD80 preferentially drives the T cell
differentiation toward Th1 responses, whereas CD86 costimulation biases
T cell priming toward Th2 responses (38). Although some
concerns were raised regarding the general validity of this dichotomy
of the roles of CD80 and CD86 (39), several reports
indicate that the CD80/CD86 costimulatory system is altered in MS
patients. Specifically, higher numbers of CD80+ B
lymphocytes in the cerebrospinal fluid (40, 41) increased
serum levels of CD80+ lymphocytes in patients
during MS exacerbation (42), and low expression of CD86 on
cerebrospinal fluid T cells (41) have been reported in
patients with MS. Our data indicate that rolipram down-regulates CD80
expression and up-regulates CD86 expression on monocytes and B and T
lymphocytes upon nonspecific activation with PHA or LPS. The likely
explanation for this observation is the differential kinetic of
induction of these costimulatory molecules on APC; CD80 is expressed
later than CD86; therefore, rolipram may be preventing the switch from
CD86 to CD80 expression, an issue that merits further study. We also
demonstrated that rolipram down-regulates MHC class II expression, both
in resting conditions and after induction by proinflammatory signals.
This finding is in agreement with the observation in a murine system,
where increases in intracellular cAMP inhibit the IFN--mediated
induction of class II MHC genes (43). Together, these
changes in the costimulatory profile on APC favor T cell priming from
Th1 toward a Th0 or Th2 phenotype and may limit the effective
presentation of autoantigen in inflammatory MS lesions. Indeed,
rolipram was shown to reduce the number of IFN--secreting cells upon
priming of human mononuclear cells in bulk cultures to the autoantigen
MBP, while the numbers of IL-4- or IL-10-secreting cells were
unaffected (44).
The above data demonstrate a favorable drug profile of PDE4 and PDE4
combined with PDE3 inhibitors for the treatment of Th1-mediated
autoimmune disorders; however, the question remains of whether this
immunomodulatory effect is expressed within a concentration range that
is achievable in humans in vivo. The concentrations of rolipram
achievable in healthy volunteers following the administration of 0.75
mg three times daily and 1.5 mg three times daily were 24 and 53 ng/ml
(0.09–0.2 µM; Dr. Claus-Steffen Stuerzebecher, unpublished
observations) Our data, summarized in Fig. 6, indicate that rolipram and especially
the combination of rolipram and cilostamide have a mild
immunomodulatory effect at the concentration of 0.1 µM. However, due
to the immunomodulation at multiple levels (influence on T cell priming
conditions, Ag-specific proliferation, and cytokine production) the in
vivo effect is likely to be more prominent. The combination of PDE4 and
PDE3 inhibitors expresses a high degree of synergism that is, to our
experience, achievable even at much lower concentrations of cilostamide
in the final mixture (data not shown). Therefore, the combination of
PDE4 inhibitors with relatively small amounts of PDE3 inhibitors may
represent a way to broaden the therapeutic window in the treatment of
human disorders and should be considered for future drug development.
The molecular mechanism of this synergy is not known. It has been
reported that PDE3 inhibitors alone have little effect on the total
intracellular cAMP levels, and they do not further enhance the cAMP
accumulation induced by rolipram (45). However, it has
been suggested, that PDE3 (predominantly localized to the particulate
cellular fraction) and PDE4 (predominantly cytosolic) may regulate
different pools of cAMP (46, 47). It is conceivable that
intracellular signaling can partially adapt to the effects of PDE4
inhibition by diverting critical pathways blocked by high cytosolic
cAMP concentrations and activation of PKA to the alternative pathways,
which, in turn, may be affected by PDE3 inhibition. Elucidating the
molecular mechanism of this synergy between PDE4 and PDE3 inhibition
will enhance our understanding of cAMP second messenger signaling.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: MS, multiple sclerosis; PDE, phosphodiesterase; MBP, myelin basic protein; TCL, T cell line.
Received for publication July 8, 1999. Accepted for publication October 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression, inhibition of Th1 activity, and amelioration of collagen-induced arthritis by rolipram. J. Immunol. 159:6253.[Abstract]
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