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Neuroimmunology Unit, Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, we
wanted to determine whether inhibition of TNF could induce a reduction
of Th1 responses by its impact on systemic APCs. We found that soluble
TNFR p75-IgG fusion protein (TNFR:Fc) inhibited production of IFN-
by allo-Ag-activated blood-derived human CD4 T cells. We documented
reduced IL-12 p70 production by APCs in the MLR. By adding back
recombinant IL-12, we could rescue IFN- production, indicating that
TNFR:Fc acts on APC-derived IL-12. Consistent with an inhibition of the
Th1 polarization, we found a decreased expression of
IL-12R-ß2 subunit on the T cells. Furthermore, the
capacity of T cells to secrete IFN- upon restimulation when
previously treated with TNFR:Fc is impaired, whereas IL-2 secretion was
not altered. Our results define a TNF-dependent cytokine network that
favors development of Th1 immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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whereas Th2 cells produce IL-4 and IL-10. Proinflammatory Th1 T cells
are considered central to the development of target-directed autoimmune
inflammatory disorders including multiple sclerosis
(MS)3 and rheumatoid
arthritis (RA) (2, 3). In the animal model of MS, experimental
autoimmune encephalomyelitis (EAE), T cells recovered from the inflamed
central nervous system (CNS) are predominantly Th1-polarized cells (4).
In passive transfer experiments, EAE-inducing encephalitogenic T cell
lines or clones are Th1 polarized (5, 6). Th1 cells have an advantage
in crossing an endothelial barrier by binding to P- and E-selectin (7).
Simultaneous administration of Th2 cytokines (IL-10, IL-4) inhibits
disease development (8, 9, 10).
The APC-derived cytokine IL-12 is a potent activator of the Th1
phenotype in T cells. In contrast to the Th1 cytokines IFN- and TNF,
the APC-derived cytokine IL-12 is crucial for the induction of EAE
(11, 12, 13). Th1 cells express higher levels of IL-12R ß2
subunit, resulting in higher responsiveness to IL-12 compared with Th2
cells (14). IL-12 production by APC is itself tightly regulated (15, 16). IL-12 production in dendritic cells can be induced by cell to cell
contact with activated (CD154+) T cells (17), and IFN-
can further enhance IL-12 secretion. Our previous in vitro studies
using human adult CNS derived microglial cells as a source of IL-12
indicated that TNF- regulates IL-12 production via an autocrine
network (18). We found that LPS-stimulated human microglial cells
produce TNF- before IL-12 and that administration of TNFR:Fc or
anti-TNF Abs significantly inhibits IL-12 production. These
observations indicate that TNF should be considered among cytokines
that can drive Th1 responses.
EAE can be induced by active systemic immunization or passive T cell transfer, but not by direct intrathecal administration of myelin reactive cells, indicating a role of systemic immune regulation in the disease process and a rationale for systemic therapeutic intervention. TNFR:Fc and anti-TNF Abs, given systemically, have been used to ablate the development of EAE and have been used in clinical trials for RA and MS (7, 19, 20). Therapeutic effects in EAE were attributed to blocking the effector functions of TNF and lymphotoxin (LT); the effect on immunoregulatory functions was not considered (19). Monthly administration of a TNFR-Ig fusion protein containing the 50-kDa TNFR has been shown to be clinically efficacious in the treatment of RA (21). In contrast, a clinical trial for MS, in which TNFR:Fc was also used in the form of monthly systemic injections, was prematurely halted due to concerns regarding adverse impacts on the disease (22). The mechanism of action (or lack there of) in these clinical conditions was not established.
We wished to establish whether TNFR:Fc would inhibit IL-12 production
by systemic APCs and whether there was an associated reduction in
Th1 (IFN-) cytokine production by T cells responding to these APCs.
For our studies, we determined the effects of TNFR:Fc in an MLR assay,
as this system serves as an in vitro model for cell-mediated immunity.
Establishing the in vitro effects on immune reactivity of inhibiting
TNF/LT should help to predict what effects might be expected by
systemic TNF/LT-directed therapies involving molecules that may have
complicated in vivo pharmacokinetics.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN- and TNF- were obtained from Genzyme (Cambridge, MA),
IL-12 and anti-IL-12 mAbs from R&D Systems (Minneapolis, MN),
shu-TNFR was a generous gift from A. Troutt (Immunex, Seattle, WA).
Isolation of peripheral blood-derived cells
PBMC were isolated from healthy adult volunteer donors by density gradient centrifugation using Ficoll-Hypaque (Pharmacia, Baie D’Urfe, Canada). For the isolation of enriched APCs, the PBMC were washed twice with PBS and cultured for 1 h in RPMI 1640 medium (Life Technologies, Burlington, ON, Canada) supplemented with 10% FCS, 2.5 mg/ml penicillin, 2.5 mg/ml streptomycin, and 2 mM glutamine (all from Life Technologies) in 75-cm2 tissue culture flasks (Falcon, VWR Scientific, Montreal, Canada). The nonadherent cells were removed by gentle shaking. The adherent cells consisted of 95% HLA-DR/B7-2 positive monocytes.
CD4+ T cells were isolated from PBMC using anti-CD4
mAbs conjugated to magnetic beads (Dynal, Great Neck, NY). The
beads were detached from the cells after isolation following the
supplied protocol. The cells were then washed with PBS and their purity
was of 
96% as assessed by flow cytometry (23).
Semiquantitative PCR analysis
Total RNA was isolated using TRIZOL Reagent (Life Technologies). To transcribe into cDNA, 3 µg RNA, 3.3 mM random hexamer primers (Boehringer Mannheim, Manheim, Germany), reverse transcriptase buffer, 3 mM dNTPs, 400 U Maloney murine leukemia virus reverse transcriptase (all from Life Technologies), 0.6 µl RNA guard, and 3 mM DTT (both from Pharmacia) were added to a total volume of 32 µl. The reaction mixture was incubated for 1 h at 42°C followed by a 10-min incubation at 75°C. Primers used for PCR were obtained from Life Technologies and had the following sequences: IL-12R ß2 forward, 5'-ACAGGACACACCTCCTGGAC-3'; reverse, 5'-AGAGGGACCTGTGTGTCACC-3'; and ß-actin forward, 5'-ATGCCATCCTGCGTCTGGACCTGGC-3'; reverse, 5'-AGCATTTGCGGTGCACGATGGAGGG-3'. The primers for IL-12R ß2 and ß-actin were constructed to generate fragments of 281 bp and 378 bp, respectively. cDNA 200 ng was added to the reaction mixture containing PCR buffer, 0.2 mM dNTPs (Life Technologies), 50 pMol of either primer set for IL-12R ß2 or ß-actin, and 0.5 µl Taq polymerase (Life Technologies). The reaction mixture was completed with H2O to a total volume of 50 µl. Samples were placed in a Gene Amp PCR system 9600 (Cetus, Perkin-Elmer, Norwalk, CT) for 25 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 1 min followed by a 10-min extension at 72°C. After amplification, 15 µl of each sample was electrophoresed on a 1.5% agarose gel (Life Technologies). The bands were visualized with ethidium bromide. For quantification purposes, 2.5 µCi of [32P]dCTP (DuPont/NEN, Mississauga, ON, Canada) was added to the reaction mixture before PCR. The gels were dried and the bands were analyzed using a phosphorimaging and Image Quant software (Molecular Dynamics, Sunny Valley, CA).
MLR
A total of 105 T cells was cocultured with either 2 x 104 or 5 x 104 allogeneic APC. For proliferation and cytokine assays, primary MLRs were conducted in 96-well plates. After 5 days, unless indicated otherwise, 1 µCi [3H]thymidine was added to the wells for 5 h. The cells were harvested and thymidine uptake was determined using a beta-scintillation counter. Culture medium was recovered from sister cultures to determine the cytokine concentrations. For secondary MLRs, T cells were recovered from the primary MLRs and cocultured with freshly isolated allogeneic APC from the same donor as in the primary MLR. Secondary MLRs were conducted for 3 more days at which time cytokine release was measured.
L929 cytotoxicity assay
L929 cells (104) were cultured in RPMI 1680
medium with 10% FCS. We added either 50 or 100 U/ml (1.7 or 0.85
ng/ml) of TNF- in the presence of different concentrations of
TNFR:Fc (Genzyme, Cambridge, MA) or carrier buffer. The cells were
incubated for 24 h and supernatants were analyzed for lactate
dehydrogenase (LDH) content as previously described (24).
Cytokine ELISA
IL-12 ELISA kits were obtained from R&D Systems. IL-2, IL-10,
and IFN- ELISA kits were obtained from BioSource International
(Camarillo, CA). Tissue culture supernatants were stored at -80°C
until analysis. ELISA assays were performed following manufacturers
instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated cytotoxicity
In initial studies, we established that TNFR:Fc could specifically
inhibit the cytotoxic effect of TNF- by exposing TNF-sensitive L929
cells to TNF- and TNFR:Fc or carrier control. Fig. 1
A shows the toxicity of
different concentrations of rTNF- on L929 cells as assessed by LDH
release assay. In a subsequent experiment, we have used 50 or 100 U
(0.85 or 1.7 ng/ml) of TNF- that results in maximum LDH
release. We can block cytolysis mediated by 100 U of recombinant
TNF- using 140 pg/ml of TNFR:Fc, indicating that engagement of
membrane TNFR on L929 cells is completely ablated by addition of
TNFR:Fc (Fig. 1B).
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production in an allogenic MLR
To analyze the effect of TNFR:Fc on immunoregulatory functions, we
have performed MLRs in the presence of TNFR:Fc. CD4+ T
cells were isolated from healthy donors and mixed with allogeneic APC
at different ratios (5:1–2:1) in 96-well plates. TNFR:Fc or irrelevant
IgG1 mAb was added to the reaction. The MLR was conducted for 5 days,
and T cell proliferation was assessed by [3H]thymidine
uptake (23). Supernatants from sister cultures were harvested and
analyzed for IFN- by ELISA.
As shown in Fig. 2A, addition
of TNFR:Fc to the primary MLRs inhibited IFN- production in a
dose-dependent fashion. Maximum inhibition was first observed with 140
pg/ml of TNFR:Fc. For subsequent experiments we used 2.8 µg/ml of
TNFR:Fc and 4 µg/ml of irrelevant IgG1 in vehicle buffer (ctrl).
After 5 days, TNFR:Fc inhibited IFN- production by 76 ± 7%
SEM, on average (Table I). As shown in
Fig. 2B, levels of IL-2 and IL-10 were not altered by
TNFR:Fc. We did not detect IL-4 or IL-5 under any culture condition
used. In time course studies, we observed that TNFR:Fc inhibits IFN-
production from the earliest time point this cytokine could be detected
(Fig. 2C). TNFR:Fc does not alter proliferation at the
concentrations used over the 5-day time period of the primary MLR.
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production
Because TNF can be produced by either APCs or T cells, and both
cell types are responsive to TNF, subsequent studies were designed to
determine whether TNFR:Fc-mediated inhibition of IFN- production
involved either an APC-dependent network and/or a direct effect on the
T cells. Regarding the former, we have previously shown that TNFR:Fc
can inhibit IL-12 production by activated-human adult microglial cells
(18). When microglial cells are activated with LPS, they produce
TNF- before IL-12. When we then block the action of autocrine
TNF- by using TNFR:Fc, we could significantly inhibit IL-12
production. In the current study, the levels of IL-12 p70 production in
the MLR were at the lower levels of detectability (7.8 pg/ml). To
overcome this limitation and to directly determine whether IL-12 p70
production is dependent on TNF, we added anti-CD3 mAbs (0.1
µg/ml) to the MLR to activate nonalloresponsive T cells, which in
turn results in more robust IL-12 levels. Table II shows the decrease in IL-12 p70
production by APC in MLRs treated with TNFR:Fc measured by ELISA for
IL-12 p70.
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in
an allogenic MLR. We added human rIL-12 (R&D Systems) to the cells, to
determine whether IFN- production can be recovered. When 100 pg of
IL-12 was added to the cultures, we were able to completely
recover IFN- production by the T cells (Fig. 3). Addition of IL-1ß did not rescue
IFN- secretion (data not shown). We could also mimic the effect of
TNFR:Fc and decrease IFN- production by the addition of 0.5 µg/ml
anti-IL-12 mAbs (Fig. 3).
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during a secondary MLR in the absence of the antagonist, we
isolated and extensively washed the T cells after the initial 5-day
primary MLR. These cells were then incubated with fresh allogeneic APC
for 3 more days, at which time the culture medium was collected and
analyzed for IFN-. Fig. 4 shows that T
cells isolated from TNFR:Fc-treated MLR cultures produce significantly
less IFN- than control cultures when restimulated by fresh APCs
under regular culture conditions. As expected, the levels of IFN-
were generally higher in the secondary response. The levels of IL-2 in
secondary MLRs were also increased compared with primary MLR cultures
but not altered due to the depletion of TNF in the primary MLR (data
not shown).
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To assess the direct effects of TNFR:Fc on T cells in the absence
of APC, we stimulated CD4 T cells for 24 h by polyclonal
activation with anti-CD3 mAbs in the presence of TNFR:Fc. Fig. 5A shows inhibition of IFN-
production by activated T cells. The inhibitory effect is less
pronounced than in MLRs. Addition of TNF- to these cultures restores
normal IFN- levels, indicating specificity of TNFR:Fc. In contrast
to the MLR studies, we could not mimic the effect of TNFR:Fc by the use
of anti-IL-12 mAbs (not shown). However, when recombinant IL-12 was
added, we could again increase IFN- levels (Fig. 5B).
These results demonstrate that exogenous IL-12 can override the
inhibitory effect of TNFR:Fc on IFN- production.
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or IL-12 can restore
higher levels of this receptor (14). After stimulation for 24 h
with anti-CD3, we performed a radiolabeled semiquantitative PCR for
IL-12R ß2. The gel was analyzed by phosphorimaging. Fig. 6 shows that anti-CD3-activated T
cells in TNFR:Fc-treated cultures express significantly less IL-12R
ß2 message then control cultures, indicating that
TNF- can mimic some of the functions ascribed to IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by CD4 T cells. TNFR:Fc does
not alter IL-2 or IL-10 levels. This finding supports our conclusion
that treatment with TNFR:Fc selectively inhibits IFN- secretion
without impacting on general lymphocyte function and survival or
driving Th2 polarization. We have previously shown that TNF- and
IFN- are important costimuli for the induction of IL-12 expression
in APC (18). IL-12 is the most potent soluble factor driving the
development of a Th1 profile by T cells (16, 25). Here, we demonstrate
a significant reduction of IL-12 p70 production by APCs in
allogeneic MLRs supplemented with TNFR:Fc. Addition of rIL-12 to
the TNFR:Fc-treated cultures restores normal IFN- levels, indicating
that the dominant effect of TNFR:Fc on this system is the inhibition of
IL-12 production by APCs.
The APC-dependent mechanism may not be the only modulator of IFN-
production by T cells. We show that in the absence of APCs,
polyclonally activated pure CD4+ T cells decrease
IFN- production when treated with TNFR:Fc. The effect is less
pronounced than in APC-stimulated T cells. It is feasible that TNF acts
directly on T cells to maintain a Th1 phenotype by enhancing IL-12
responsiveness (via IL-12-Rß2) and up-regulating IFN- production.
The direct inhibitory effect of TNFR:Fc on T cells can be overridden by
exogenous IL-12. The latter finding supports the hypothesis, that the
inhibition of IFN- production by TNFR:Fc is predominantly achieved
via its effect on APC, in particular, IL-12 production. Ultimately,
IFN- itself can also contribute to this cytokine network in a
feedback fashion by further stimulating IL-12 production in activated
APC.
TNFR:Fc has now been used therapeutically both in experimental and
human autoimmune inflammatory disorders. TNF levels produced by
monocytes in MS are increased and correlate with disease severity (26).
Animals treated with TNFR:Fc after immunization with myelin basic
protein or proteolipid protein but before onset of clinical symptoms do
not develop disease but have continued inflammation as assessed by the
number of CNS infiltrating leukocytes (19). The authors concluded that
the therapeutic effect of TNFR:Fc reflected blocking the effector
functions of TNF. TNF-/- mice also display a delayed
onset of clinical symptoms (29), an effect that could be explained by
either regulatory or effector functions. Our data indicate that it is
also possible that treatment with TNFR:Fc may alter the cytokine
polarization of the infiltrates by lowering the levels of IL-12 and
subsequently IFN-. Reduced IFN- production would result in
reduced activation of bystander cells such as macrophages and
microglia. Patients with MS also are reported to have elevated
cerebrospinal fluid and serum IL-12 levels (27, 28).
Our in vitro study provides insight as to how TNFR:Fc can influence cytokine networks that regulate the polarization of cytokine patterns. Immune therapy in RA with TNFR:Fc has been shown to be efficacious for clinical symptoms. However, when patients with MS were treated with TNFR p50-IgG fusion protein, there was a reported increase in the relapse rate lesion formation as assessed by magnetic resonance imaging (22). Neither study provided data on whether there was skewing of the T cell cytokine response, in a manner demonstrated in our study. To understand the in vivo effects of TNF/LT-directed therapy, one needs to consider the complicated pharmacokinetics and the pleiotrophic nature of the cytokines TNF and LT. Our current study may provide an approach to determine that desired in vivo effects are occurring and minimize the risk of clinical toxicity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Burkhard Becher, Montreal Neurological Institute, 3801 University, Montreal, QC, H3A 2B4, Canada. E-mail address:
3 Abbreviations used in this paper: MS, multiple sclerosis; CNS, central nervous system; EAE, experimental allergic encephalomyelitis; RA, rheumatoid arthritis; TNFR:Fc, soluble human p75 TNF receptor-IgG:Fc fusion protein; LT, lymphotoxin; LDH, lactate dehydrogenase.
Received for publication March 25, 1998. Accepted for publication September 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor fusion protein prevents experimental auto-immune encephalomyelitis and demyelination in Lewis rats: an overview. J. Neuroimmunol. 72:163.[Medline]
messenger RNA expression in patients with relapsing-remitting multiple sclerosis is associated with disease activity. Ann. Neurol. 37:82.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or 4 µg/ml irrelevant IgG1 isotype control in
carrier buffer (
). Supernatants were analyzed for IFN-
) or
TNFR:Fc + IL-12 (100 pg/ml) (
) or 
).
