Download Leukemia Inhibitory Factor (LIF) is Produced by Myelin Reactive T

Leukemia Inhibitory Factor (LIF) is Produced by Myelin Reactive T cells
from Multiple Sclerosis Patients and Protects Against TNF-alpha Induced
Oligodendrocyte Apoptosis
Joris Vanderlocht1, Niels Hellings1, Jerome Hendriks1, Frank Vandenabeele1, Marjan
Moreels1 , Mieke Buntinx1 , Dick Hoekstra2 , Jack P. Antel3 and Piet Stinissen1
1
Hasselt University, Biomedical Research Institute and Transnationale Universiteit Limburg,
School of Life Sciences, Diepenbeek, Belgium
2
3
Department of Membrane Cell Biology, Faculty of Medical Sciences, University of
Groningen, Groningen, The Netherlands
Department of Neurology, Neuroimmunology Unit, Montreal Neurological Institute, McGill
University, Montreal, Canada
Running title: Production of LIF by immune cells in MS
Financial support:
‘Nationaal fonds voor wetenschappelijk onderzoek Vlaanderen (FWO)’
‘Bijzonder onderzoeksfonds’ Hasselt University
The Belgian WOMS foundation
The Belgian Charcot foundation
Transnational University Limburg
Correspondence to: Dr. Niels Hellings, Biomedisch Onderzoeksinstituut, Hasselt University,
Agoralaan building A, B-3590 Diepenbeek, Belgium
Email: [email protected]
Fax: +32 11 26 92 09
Tel: +32 11 26 92 02
ABSTRACT
In Multiple Sclerosis (MS), damage to oligodendrocytes is believed to be caused by an
aberrant immune response initiated by autoreactive T cells. Increasing evidence indicates that
these T cells are not exclusively detrimental, but may also exert protective effects. We report
for the first time that myelin reactive T cell clones from 8 MS patients (6/19) and 5 healthy
controls (4/11) produce leukemia inhibitory factor (LIF), a member of the neuropoietic family
of neurotrophins. In addition, T cell clones specific for tetanus toxoid, CD4+ and CD8+ T
cells, monocytes but not B cells secreted LIF. LIF producing T lymphocytes and macrophages
were also identified immunohistochemically in both active and chronic active MS lesions. We
further demonstrated dose-dependent protective effects of LIF on TNF-alpha induced
apoptosis of oligodendrocytes. In conclusion, our data demonstrate that peripheral and CNSinfiltrating T cells from MS patients produce LIF, a protective factor of oligodendrocytes.
This study emphasizes that secretion of LIF may contribute to the neuroprotective effects of
autoreactive T cells.
Keywords: multiple sclerosis, oligodendrocytes, leukemia inhibitory factor, myelin reactive
T cells
INTRODUCTION
The pathology of MS is characterized by focal areas of inflammatory infiltration and
demyelination in which oligodendrocytes, the myelin forming cells of the CNS, are depleted
(Lucchinetti et al., 1999). Although autoreactive T cells are considered to be key players in
the immunopathogenesis of MS (reviewed in (Hellings et al., 2002b)), the exact mechanisms
contributing to the immune mediated demyelination and oligodendrocyte loss remain elusive.
Since major histocompatibility complex (MHC) class II expression on oligodendrocytes is
lacking, cell death occurs in a MHC class II-unrestricted manner (Antel et al., 1998; Grenier
et al., 1989; Kim, 1985; Lee and Raine, 1989). Such mechanisms could involve immune
effector-target cell contact dependent mechanisms, such as MHC class I restricted lysis by
myelin specific CD8+ T cells (Jurewicz et al., 1998), killing by NK cells (Pouly et al., 2000),
NK-like killing by CD4+ T cells (Antel et al., 1994; Antel et al., 1998) or perforin/granzyme
mediated lysis by γδ T cells (Freedman et al., 1997; Zeine et al., 1998). Additional
mechanisms depending on free radicals, nitrogen oxide (van der Goes et al., 2001) and
proinflammatory cytokines released into the lesion sites may also be involved (D'Souza et al.,
1996). Convincing evidence links the proinflammatory cytokines TNF-α and IFN-γ to MS
pathology. The concentration of TNF-α, for example was not only correlated with the degree
of disability (Matsuda et al., 1994; Sharief et al., 1993), but it was also established that the
TNF-α concentration increases in blood cell cultures prior to exacerbation (Hellings et al.,
2002a; Philippe et al., 1996). Relapsing remitting MS patients showed periodic increases of
the TNF-α concentration (Huizinga et al., 1997) and several authors report direct cytotoxic
effects of TNF-α on oligodendrocytes (Mclaurin et al., 1995; Selmaj et al., 1991). Although
inflammation may play a crucial role in demyelination and axonal loss in the early phase of
MS, recent evidence challenges the simplistic view of inflammation as an exclusively
detrimental process (reviewed in (Martino et al., 2002)). TNF-α, for example, can have
differential effects depending on the signaling pathways that are activated. TNF receptor I
(TNFRI) signaling is considered to be detrimental rather than anti-inflammatory in the
initiation phase of experimental autoimmune encephalomyelitis (EAE) (Akassoglou et al.,
1998; Kassiotis et al., 1999; Korner et al., 1997), whereas beneficial actions of TNF are
predominantly mediated by TNFRII signaling (reviewed by (Kollias and Kontoyiannis,
2002)). In contrast to the well documented detrimental actions of TNF-α, mice with a knock
out mutation for TNF-R or TNF-α show increased susceptibility to CNS inflammation and
tissue injury (reviewed in (Buntinx et al., 2002)).
Several attempts have been undertaken to protect oligodendrocytes against the deleterious
effects of a proinflammatory environment by increasing trophic support. Recent reports
demonstrate that neuropoietins or neurokines such as leukaemia inhibitory factor (LIF) and
cilliary
neurotrophic
factor
(CNTF)
can
ameliorate
experimental
autoimmune
encephalomyelitis (EAE) and promote oligodendrocyte survival in vivo (Butzkueven et al.,
2002; Linker et al., 2002). Daily administration of LIF reduced clinical scores in both a
chronic and a relapsing-remitting mouse EAE model (Butzkueven et al., 2002). The favorable
clinical effect of LIF could be associated with increased oligodendrocyte survival in vivo and
was unrelated to effects on inflammation or immune responses. In CNTF-deficient mice, EAE
induction led to earlier disease onset and more severe clinical outcome and could be
associated with increased oligodendrocyte apoptosis and decreased numbers of proliferating
oligodendrocyte precursors (Ransohoff et al., 2002).
In addition to their pathogenic role in EAE, recent reports indicate that myelin reactive T cells
may also possess neuroprotective potential. MBP reactive T cells expand after non-specific
CNS insults such as virus infections, stroke and peripheral nerve trauma (Miller et al., 1997;
Olsson et al., 1993; Wang et al., 1992). In addition, systemic injection of MBP reactive T
cells resulted in an improved recovery after spinal cord contusion in rats (Fisher et al., 2001;
Hauben et al., 2000). The presence of autoreactive T cells in the peripheral immune cell
repertoire may therefore be considered as a naturally occurring second line of defence, next to
the buffering capacity of the CNS (Schwartz, 2001). This is in line with recent evidence that
myelin reactive T cells produce neurotrophic factors like brain derived growth factor (BDNF)
(Kerschensteiner et al., 1999).
We studied for the first time whether myelin reactive T cells from healthy controls and MS
patients are able to secrete LIF in vitro and whether LIF immunoreactive T cells could be
identified in the immune infiltrate of (chronic) active MS lesions. We also studied the in vitro
protective effects of LIF on TNF-α induced apoptosis in primary cultures of rat
oligodendrocytes. Our results show that autoreactive T cells are able to secrete LIF. This
further substantiates the neuroprotective effects of inflammatory infiltrates and thus may
provide
new
targets
for
increasing
trophic
support
in
the
inflamed
CNS.
MATERIALS AND METHODS
T cell cultures
MOG and MBP specific T cell lines and clones were generated from a group of 8 MS patients
and 5 healthy subjects as described earlier (Hellings et al., 2002a). Human MBP was purified
from white matter of human brain (Deibler et al., 1972). The peptides MOG (1-22), MOG
(34-56), MOG (64-86) and MOG (74-96) were synthesized and HPLC purified (>95% purity)
by Severn Biotech Ltd (Worcester, UK). T cell lines specific for tetanus toxoid (TT) were
used as a control. TT was obtained from the RIVM (Bilthoven, The Netherlands). Resulting
antigen specific T cell lines were cloned with phytohemagglutinin (PHA) in the presence of
allogeneic accessory cells. MBP-, MOG- and TT- reactive T cells were expanded by
successive rounds of restimulation with MBP/MOG/TT or PHA and autologous APC. The
stimulation index was assessed in a classical 3H thymidine uptake assay. Generated TCL were
classified as TH1, TH2, TH0, TH1/0 based on the ratio of IFN-γ/(IL-4 and/or IL-10). A ratio
> 5 was arbitrarily defined as TH1, a ratio < 0.5 as TH2, a ratio between 0.5 and 3 as TH0 and
a ratio between 3 and 5 as TH1/0. For 6 TCL, TH phenotype was assessed using intracellular
cytokine staining with monoclonal antibodies directed against IFN-γ (IgG2b, fluorescein
isothiocyanate (FITC) labeled, BD) and IL-4 (IgG1, phycoerythrin (PE) labeled, BD) as
described earlier (Van der et al., 2003). To generate NKT cell cultures, PBMC were cultured
in the presence of 100 ng/ml α-GalCer (Kirin Brewery Ltd, Japan) at a density of 7,5x105
cells per ml. After 7 days, cells were restimulated with irradiated autologous α-GalCer pulsed
PBMC and supplemented with 2 U/ml recombinant human IL-2 (Roche Diagnostics,
Brussels, Belgium)(Linsen et al., 2005). After one week, NKT cells were isolated using
Vα24+ magnetic isolation according to the manufacturer’s instructions (EasySep, Stemcell
technologies, Meylan, France). Purified NKT cells were stimulated with α-GalCer pulsed
irradiated PBMC and supernatant was collected 3 days later. CD4+CD25+ Treg cells were
isolated from peripheral blood by means of a negative, ’rosetting’ mediated (CD4+ T cells)
and positive immunobead mediated (CD25+ T cells) selection (Stem Cell Technologies).
Highly pure CD4+CD25+ T cells were subsequently stimulated with anti-CD3 antibody and
autologous feeders and supernatants were collected after 5 days. Immune cells were cultured
in RPMI 1640 (Life Technologies, Invitrogen), supplemented with 10% fetal calf serum
(FCS) (Hyclone Europe, Erembodegem, Belgium), 0.1 mM non essential amino acids, 1 mM
sodium pyruvate and the antibiotics penicillin (50 U/ml) and streptomycin (50 µg/ml).
Cell culture of oligodendrocytes
Rat oligodendrocytes were isolated from whole brain of mature rats as previously described
by Yong and Antel with some minor modifications (Yong and Antel 1992). Briefly, brain
tissue was subjected to enzymatic dissociation with trypsin (2.5%, Gibco, Canada) and DNase
I (50 µg/ml, Roche Diagnostics) and mechanical dissociation by passage through a 132 µm
mesh. Glial cells were separated from myelin and red blood cells using percoll gradient (30%)
centrifugation. The mixed glial cell fraction was suspended in DMEM supplemented with 5%
FBS and penicillin (50 U/ml) and streptomycin (50 µg/ml) (Life technologies, UK). The
cultures were selectively enriched for oligodendrocytes by means of differential adhesion to
plastic. The resulting oligodendrocyte suspension was plated on poly-L-lysin coated
recipients. The purity of the cultures was routinely evaluated by immunocytochemistry. The
resulting cultures were highly enriched for MBP+ cells (>90%) and a small variable
percentage of nestin+ oligodendrocyte precursors (data not shown). Contaminating astrocytes,
fibroblasts and endothelial cells constitute less than 5%. After approximately 10-14 days of
culture, oligodendrocyte cultures were stimulated with the indicated recombinant rat
cytokines purchased from Peprotech EC Ltd (Londen, UK) (TNF-α and CNTF) and from
Chemicon (Temecula, USA) (LIF).
Immune cell preparations
CD4+ T cells, CD8+ T cells, B lymphocytes and monocytes were isolated from PBMC by
immunomagnetic beads according to manufacturer’s instructions (Dynal Biotech, Oslo,
Norway). The purity of the collected cell fractions was >95% as determined by flow
cytometry. T lymphocyte fractions were stimulated with PHA (2 µg/ml; Sigma) in the
presence of irradiated autologous PBMCs. Monocytes were stimulated with E. coli
lipopolysaccharide (LPS) (10 ng/ml, Calbiochem, VWR, Leuven, Belgium) and B cells were
stimulated with Pansorbin (Staphylococcus aureus cells) (1/7500, Calbiochem, VWR,
Leuven, Belgium). Supernatants and cell pellets were collected after 3 days of stimulation.
Viability assay
Cell viability was analyzed after culturing oligodendrocytes for 5 days in the presence of the
indicated cytokines. The cells were harvested by trypsinization (0.25%) and washed with
phosphate buffered solution (PBS) containing 10% FBS. The annexin V-FITC/ propidium
iodide (PI) assay was performed to discriminate between viable, apoptotic and necrotic cells
(Bender Med systems, Vienna, Austria). The staining procedure was performed following the
manufacturer’s instructions. Briefly, after addition of 2.5 µl annexin V-FITC, the cells were
incubated for 10 min at RT, washed, centrifuged and resuspended in 100 µl binding buffer. PI
was added at a final concentration of 1 µg/ml prior to FACS analysis (FACSCalibur, BD
Biosciences). Data acquisition (2x104 events for each sample) and analysis were performed
using Cell Quest software. Results were compared using the student-t test. Differences were
considered significant when p<0.05.
Quantification of cytokines by ELISA
Supernatants of antigen specific T cell clones were collected after 3 days of stimulation with
autologous APC pulsed with their specific antigen, MBP, MOG or TT. Clones stimulated
with unpulsed APC were used as a control. IFN-γ, IL-4 and IL-10 levels in the supernatants
were measured using a sandwich ELISA based on commercially available Ab pairs (CytoSets,
biosource Europe, Nivelles, Belgium) as described earlier (Hermans et al., 1997). LIF
production was assessed using a commercially available sandwich ELISA for human LIF
according to the manufacturer’s instructions (Bender MedSystems, Vienna, Austria).
LIF RT-PCR analysis
Total RNA was extracted from cell pellets using the RNeasy kit (Qiagen, Hilden, Germany).
The mRNA was reverse transcribed into single stranded cDNA with AMV reverse
transcriptase using an oligo dT primer according to the manufacturer’s protocol (Promega,
Madison, USA). Primer pairs for the amplification of hLIF, hGAPDH and the constant region
of the T cell receptor beta chain (hTCR-β) were designed using Oligo 6.0 software (Molecular
Biological Insights Inc., Cascade, USA). All primer pairs were designed over intron-exon
boundaries to exclude amplification from contaminating genomic DNA. (hLIF F: 5’GCGGCAGGAGTTGTGCC-3’
hLIF R: 5’-CCCTGGGCTGTGTAATAGAG-3’, hGAPDH F: 5’-
GCTCTCCAGAACATCATCCCTGCC-3’ hGAPDH R: 5’-CGTTGTCATACCAGGAAATGAGCTT-3’
hTCR-β F: 5’-CCGAGGTCGCTGTGTTTGAGCCAT-3’ hTCR-β R: 5’-CTCTTGACCATGGCCATC3’). PCR was performed for 40 cycles (95°C 20 sec, 58°C for LIF and 55°C for GAPDH and
the constant region of the TCR-β 20 sec and 72°C 40 sec) and followed by 72°C for 7 min.
All PCR products were subcloned into a pCRII plasmid (Invitrogen, California, USA) and
their sequences were confirmed by dye terminator sequence analysis.
Immunostaining of human brain slices and rat oligodendrocyte cultures
Brain tissue samples were obtained from the Netherlands Brain Bank (Amsterdam) and the
Born Bunge Foundation (University of Antwerp, Belgium). MS tissue samples were taken
from lesions located in periventricular and subcortical white matter. Tissue samples contained
active (n=2) and chronic active (n=11) lesions from 12 MS patients. The normal appearing
white matter around lesions was used as a control.
Immunohistochemistry was performed on formalin-fixed paraffin-embedded brain tissue
using a sequential immunoenzymatic doublestaining technique (Envision Doublestain
System®, Dako Cytomation, Glostrup, Denmark) as described previously (De Bari et al.
2003). Prior to immunohistochemical staining, sections were microwaved in 10 mM citrate
buffer pH 6.0 and endogenous peroxidase activity was blocked with 0.5% H2O2 in methanol.
Next, sections were blocked with 3% goat serum and stained with mouse monoclonal
antibodies against human CD3 (1/60, Dako diagnostics, Heverlee, Belgium), CD4 (1/100,
CBT4.2 clone), CD8 (1/400, 2E7/B1) or CD68 (Dako Diagnostics, Heverlee, Belgium).
Immunostaining was visualized with peroxidase-conjugated secondary Ab. After blocking the
first staining (double stain block, Dako Diagnostics, Heverlee, Belgium), sections were
incubated with anti-human LIF Ab (1/500, 1F10 clone obtained from JF. Moreau (Taupin et
al., 1997)) as primary antibody and an alkaline phosphatase conjugated secondary Ab. For the
first staining, DAB was used as a chromogenic substrate, whereas the second staining was
visualized with Fast Red. Sections were counterstained using Mayers’ hematoxylin,
coverslipped in an aqueous mounting medium (Aquatex; Merck) and photographed using a
Zeiss photomicroscope equipped with an automated camera (Nikon).
Immunofluorescent double stainings were performed on cryo-preserved brain material.
Sections were incubated with the same mouse monoclonal antibodies (mAb) as above (antiCD3, anti-CD4, anti-CD8 and anti-CD68) and with an anti-MHCII antibody (1/100, BD
bioscience Pharmingen, San Diego, CA), in combination with a mouse IgG1 anti-LIF
antibody (1/500, 1-F10 clone obtained from J.F Moreau). Binding of primary antibodies
(CD3, CD4, CD8, MHCII and CD68) was visualised with a secondary biotine-coupled rabbitanti-mouse antibody in combination with avidin coupled to Alexa488. Binding of the anti-LIF
Ab was detected by a biotin-coupled rabbit anti mouse and avidin-rhodamin. Sections were
counterstained with Hoechst dye. All fluorescent detection tools were obtained from
molecular probes. Samples were analysed and photographed with a Nikon Eclipse 800
fluorescence microscope.
For the receptor stainings, 14 days old rat oligodendrocyte cultures on poly-L-lysin coated
glass coverslips were used. After fixation in 4% paraformaldehyde (Unifix®, Klinipath,
Duiven, The Netherlands) and blocking of non-specific binding sites with goat serum (Dako
cytomation, Glostrup, Denmark) cells were incubated with one of the following primary
antibodies: anti-rat gp130 (1/400), anti-rat LIFR-β (1/400), anti-rat TNFR I (1/200) or anti-rat
TNFR II (1/200) (Santa Cruz Biotechnology, Santa Cruz, California, USA). Prior to staining
with anti-LIFR-β, cells were permeabilised with triton. Binding of the primary antibodies was
visualized with peroxidase-conjugated secondary antibodies (Envision System®, Dako
Cytomation, Glostrup, Denmark) in combination with DAB as a chromogenic substrate. The
specimen were counterstained with haematoxylin, mounted on a coverslip and examined with
a light photo microscope (Zeiss) equipped with an automated camera (Nikon).
RESULTS
Myelin reactive T cells produce leukemia inhibitory factor (LIF)
Recent reports demonstrated that myelin reactive T cells produce neuroprotective factors
(Kerschensteiner et al. 1999). We therefore studied the possibility that myelin reactive T cells
may secrete LIF. Nineteen MBP and 11 MOG reactive T cell lines/clones (TCL) isolated from
8 MS patients and 5 healthy controls were stimulated with irradiated autologous antigen
presenting cells (APC) pulsed with their respective antigens. Cell supertanant was collected
after 72 h and LIF was measured by ELISA. This time point was based on a pilot experiment,
in which LIF secretion was evaluated at several time points (6, 12, 24, 48, 72 and 96 h after
activation, data not shown). A low level of LIF (12.7 pg/ml) was measured in culture medium
(containing FBS). This background level was subtracted from the LIF levels determined in T
cell supernatants. Table 1 provides an overview of the LIF levels in supernatant of the TCL
tested. Net LIF production of a stimulated TCL was calculated as: LIF level in supernatant
from antigen stimulated clone (cultured with antigen pulsed APC, +Ag) – LIF level in
supernatant from the unstimulated clone (cultured with unpulsed APC, -Ag). LIF production
is not a feature of all myelin reactive TCL and the amount of LIF produced is highly variable
between LIF secreting TCL. Ten out of 30 tested myelin reactive TCL (4/11 HC TCL, 6/19
MS TCL) were found to have a net LIF production of at least 10 pg/ml. Three TCL had a net
LIF production higher than 100 pg/ml. No correlation was found between stimulation index
and LIF production. In addition, no correlation was found between Th phenotype and the
capacity to produce LIF (Table 1). To confirm the LIF production at the mRNA level, a
specific reverse transcription (RT) PCR was developed. Again, as seen for the ELISA
measurements, not all myelin reactive TCL transcribed LIF mRNA (Fig. 1). To determine
whether LIF production is restricted to autoreactive T cells, supernatant of TT reactive TCL
isolated from a healthy donor was analyzed for LIF content. Four out of 5 TT reactive TCL
showed a net LIF production of at least 10 pg/ml (Table 1).
CD4+ and CD8+ T cells, monocytes and regulatory T cells produce LIF
We further studied the LIF production by various immune cell populations isolated from
blood mononuclear cells of two healthy subjects: B-cells, monocytes, CD4+ T cells and CD8+
T cells purified by immunomagnetic depletions. CD4+ and CD8+ T cells were activated by
PHA, B cells by Pansorbin (S. aureus extract) and monocytes by LPS. In addition, we tested
whether LIF is produced by two T cell populations with regulatory potential: CD4+CD25+
regulatory T (Tregs) cells and natural killer T cells (NKT cells). Tregs were isolated by
immunomagnetic depletions and stimulated with anti-CD3 antibody. These cells showed
significant in vitro suppressive capacity (data not shown). NKT cell lines were generated by
stimulation with alpha-GalCer. Figure 2 illustrates that NKT cells and purified CD4+ T cells
produce LIF at high levels upon activation (>150 ng/ml) while CD4+CD25+ Tregs and CD8+
T cells produce LIF at much lower levels (10-40 pg/ml). Monocytes produced a low level of
LIF upon stimulation (8 pg/ml), while no LIF production above background was measured in
supernatant of B lymphocytes (Fig. 2). LIF production of CD4+, CD8+ T cells and monocytes
was also confirmed by RT-PCR (data not shown).
LIF is produced by T cells and perivascular macrophages/microglia in early active and
chronic active MS lesions
To test whether the ability of immune cells to produce LIF in vitro is also maintained in situ,
we analyzed LIF expression by CD3-positive and CD68-positive cells in active MS lesions
(n=2 from 2 patients) and chronic active MS lesions (n=11 from 10 patients), using double
label immunohistochemistry (11 lesions) or immunofluorescence (2 lesions). Lesional staging
was determined on the basis of the presence of inflammatory cells and the extent of
astrogliosis (data not shown). Active lesions contained numerous perivascular and
parenchymal ‘foamy’ macrophages/microglia with strong CD68 or MHCII expression. In
addition, a variable number of CD3-positive lymphocytes was detected in perivascular spaces
and the plaque parenchyma. Chronic active lesions were characterized by a hypocellular
centre containing astroglial scar tissue with a small number of inflammatory cells, and a
hypercellular
margin
containing
perivascular
and
parenchymal
CD68-positive
macrophages/microglia and CD3-positive lymphocytes. Ongoing demyelination in the active
MS lesions and hypercellular margin of chronic active MS lesions was evidenced by Luxol
fast blue staining and the presence of MAG-positive and MBP-positive phagocytosed myelin
components in the ‘foamy’ macrophages/microglia (data not shown).
In all studied lesions (n=13) LIF immunoreactivity was present. Ten out of 13 lesions showed
T cells in the perivascular space and in four of these (both active and 2 chronic active lesions)
we also identified low numbers of CD3 positive cells in the parenchyma of the lesions.
Specific LIF immunoreactivity was detected on the majority of CD3 positive cells present in
the perivascular cuff (Fig 3A and 3C). Almost all infiltrating T cells in the parenchyma and at
lesion borders stained positive for LIF (Fig 3E). Additional double stainings showed the
presence of LIF immunoreactivity in both CD4+ and CD8+ perivascular T cells (data not
shown). LIF staining of CD68+ or MHCII+ cells in the parenchyma and at lesional borders
was weak and only observed in a small minority of CD68+ or MHCII+ cells in only 2 out of
13 lesions (Fig. 3F). However, strong LIF immunoreactivity was detected on CD68+ cells in
the perivascular space (10/13 lesions) (Fig. 3B and 3D). As previously described, neuronal
cells in grey matter as well as those included in (subcortical) plaques also stained positive for
LIF (data not shown) (Cheng and Patterson, 1997). No labeling was detected when primary
antibodies were omitted (control). In conclusion, these data indicate that LIF is produced by T
cells and perivascular macrophages in both active and chronic active lesions of MS patients.
Primary rat oligodendrocyte cultures express TNFR-I, gp130 and LIFR-β.
To test the relevance of LIF in the CNS we optimized an in vitro culture system of primary rat
oligodendrocytes in order to test whether LIF protects oligodendrocytes against TNF-α
induced apoptosis. To determine whether cultured rat oligodendrocytes express receptors for
TNF-α and the neurokines LIF and CNTF, we studied the presence of TNFRI, TNFRII,
LIFR-β and gp130 on rat oligodendrocytes by means of immunocytochemistry.
Oligodendrocytes grown in GM were morphologically identified by their round nuclei
extending few processes, whereas the few contaminating astrocytes typically exhibited stellate
profiles. The cultured rat oligodendrocytes displayed weak but significant immunoreactivity
for TNFRI (Fig. 4C) and strong immunoreactivity for gp130 (Fig.4A) and LIFR-β (Fig. 4B).
Cells incubated with anti-TNFRII (Fig. 4D) or without primary antibody showed no
immunoreactivity. No up- or downregulation of these receptors was observed when cells were
stimulated with TNF-α, IFN-γ, LIF and the combination of LIF and TNF-α (data not shown).
All stainings occurred typically at cell surfaces. Taken together, these data demonstrate that
cultured rat oligodendrocytes express both common receptor subunits for neuropoietic factors
(gp130 and LIFR-β) and TNFRI but not TNFRII.
Dose dependent protective effects of LIF and CNTF on TNF induced oligodendroglial
apoptosis.
We and others have shown that TNF-α induces apoptotic cell death in human
oligodendroglial cell lines (Buntinx et al., 2004). To determine the cytokine concentration that
induces a maximal apoptotic response in the absence of non-specific toxicity, rat
oligodendrocytes were treated with different doses of recombinant TNF-α (between 1 and 500
ng/ml). The cells were treated with TNF-α for 5 days, stained with annexin V-FITC/PI and
analyzed by flow cytometry. A sharp rise in apoptotic cell death was observed reaching a
plateau at a dose of 200 ng/ml (data not shown). Maximal apoptosis (42.5%) was observed at
500 ng/ml. Only a minor increase in necrotic PI+ cells was observed in TNF-α treated
oligodendrocytes, which was not significantly different from control conditions (medium
only). All subsequent viability experiments were performed with a TNF-α concentration of
100 ng/ml, reaching 93% of the maximal TNF-α response (data not shown).
We subsequently studied the in vitro effects of LIF on TNF-α induced cell death of rat
oligodendrocyte cultures. Oligodendrocytes were incubated with different doses (ranging
from 1 to 60 ng/ml) of LIF for 2 h, and then TNF-α (100 ng/ml) was added for 5 days.
Apoptotic cell death was analyzed by means of annexin V FITC/ PI staining. In order to
evaluate the efficacy of LIF in these experiments, oligodendrocytes were also treated with the
same concentrations of CNTF, another member of the neuropoietic family of cytokines with
known protective effects on TNF-α induced oligodendrocyte apoptosis. Fig. 5 depicts the
percentage of apoptotic cells relative to the fraction of apoptotic cells observed in
oligodendrocytes treated with TNF-α alone (30.2% apoptotic cells). A background apoptosis
level of 15.4% corresponding to 53.8% of the maximal response (TNF-α alone) was observed
in oligodendrocytes cultured in medium only. Pretreatment with a low dose of CNTF or LIF
induced significant protection to TNF-α mediated apoptosis (Fig. 5A and 5B). A maximal
protection was observed at a concentration of 10-20 ng/ml for both neurokines. As seen in
figure 5, at a concentration of 15 ng/ml of LIF or CNTF, the percentage of apoptotic cells was
reduced to background values (no difference with % apoptotic cells in medium only).
Interestingly, pretreatment with higher doses of LIF (more than 30 ng/ml) or CNTF (more
than 45 ng/ml) led to increased apoptosis, indicating that neurokines are cytotoxic at these
concentrations.
We further tested whether a delayed addition of CNTF and LIF, after the start of TNF-α
treatment is still as protective. Figure 6 depicts the levels of inhibition of apoptotic cell death
when LIF or CNTF were added before or after the start of TNF-α treatment. Maximal
apoptosis in the presence of 100 ng/ml TNF-α alone (28.5 ± 4.5 %) and background apoptosis
in medium alone (14.3 ± 1.3 %) were set as 0% and 100% inhibition respectively.
Oligodendrocytes pretreated for 2 h with LIF or CNTF (15 ng/ml) before TNF-α addition
showed a significant (p<0.01) inhibition of TNF-α induced cytotoxicity in line with our
observations reported above (Fig. 6). The observed protection was almost complete since the
percentage of apoptotic cells did not significantly differ from the levels observed in control
conditions with medium alone (GM). When LIF or CNTF (15 ng/ml) were added 2 h after the
addition of TNF-α a similar level of protection was observed (Fig. 7). Note that the cells were
not washed after pretreatment, so TNF-α remained present during the complete culturing
period of 5 days. Interestingly, oligodendrocytes treated with LIF or CNTF alone showed an
increased level of apoptosis (23.6 ± 4.6 % for LIF and 17.1 ± 7.9 % for CNTF).
Our data show a moderate TNF-induced apoptotic response of rat oligodendrocytes. The
neurokines LIF and CNTF completely protect OL against this cytotoxicity. This protection is
however highly dependent on a critical dose.
DISCUSSION
Traditionally, the relative immune privilege of the CNS has been considered to protect
delicate neuronal networks from immune mediated reactions. Recent evidence however
suggests that lymphocytes may convey protective effects on neurons and glial cells after
trauma, mechanical nerve injuries and stroke (Olsson et al., 1993; Wang et al., 1992). This
report is the first to demonstrate that myelin reactive T cells isolated from MS patients and
healthy control subjects secrete leukemia inhibitory factor (LIF), a neuroprotective cytokine
(Murphy et al., 1991). LIF is not only produced by autoreactive T cells, but also by T cells
reactive against tetanus toxoid, and by purified CD4+ and CD8+ T cell subsets, monocytes and
the regulatory CD4+CD25+ and NKT cells. B lymphocytes do not secrete LIF. We also
identified LIF producing T cells in active and chronic active brain lesions of MS patients. LIF
producing T cells were mainly identified in perivascular regions. Occasionally, T cells were
detected within the parenchyma of the lesions. These infiltrating T cells also showed LIF
immunoreactivity. Both CD4+ and CD8+ T cells with LIF immunoreactivity could be detected
in the lesions. The relevance of LIF producing T cells in MS lesions is unclear. There is no
information on the antigen specificity or functional properties of the LIF producing T cells.
They are possibly involved in the pathogenic inflammatory events, but may also be recruited
into the lesions as a protective response to the local insult. Interestingly, the LIF producing T
cells in active MS lesions may also have an immune regulatory role. In this light, this study
showed that regulatory CD4+CD25+ T cells and NKT cells produce high levels of LIF in vitro,
but there is currently no proof that these T cells infiltrate into the brain lesions of MS patients.
Together, these data indicate that various T cell subsets are able to produce the
neuroprotective cytokine LIF. Our data are in line with previous reports demonstrating that
autoreactive T cells secrete several neurotrophins of the NGF/NT family, such as BDNF
(Kerschensteiner et al., 1999), NGF, NT3 and NT4/5 (Moalem et al., 2000). Although the
mechanism of immune cell mediated neuroprotection remains to be determined, our data
further suggest that the neuroprotective properties of T cells are at least partially mediated by
their local production of neurotrophic factors. Interestingly, CD4+ T cells secrete higher
amounts of LIF upon stimulation as compared to CD8+ T cells. This corresponds with
previous findings indicating that CD8+ T cells also secrete lower levels of BDNF
(Kerschensteiner et al., 1999). The inherent lower production of neurotrophins such as BDNF
and LIF by CD8+ T cells together with the observation that CD8+ T cells outnumber CD4+ T
cells in MS brain tissue (Cabarrocas et al., 2003) points towards a more detrimental role of
these cells in MS lesions.
We also identified a subpopulation of CD68 positive cells within perivascular spaces that
showed strong immunoreactivity for LIF. It remains to be determined whether these
LIF+CD68+ cells in the perivascular space are recently migrated monocytes or perivascular
dendritic cells. Perivascular dendritic cells have recently been implicated in epitope spreading
and in the reactivation of encephalithogenic T cells in several animal models of MS (Greter et
al., 2005; McMahon et al., 2005). LIF expression by these cells could correlate with their
unique functional properties.
A second important observation of our study is that LIF significantly reduces cytokine
mediated cell death of primary rat oligodendrocyte cultures. In line with previous studies
using rodent or human oligodendrocytes (D'Souza et al., 1996; Selmaj et al., 1991),
oligodendrocytes in culture underwent apoptosis after TNF-α treatment. Although these data
are consistent with several other studies, it remains unclear why not all cells underwent TNF
mediated apoptosis. Because interference with NF-κB activation and blockade of the TRAF-2
response greatly enhances the susceptibility of primary oligodendrocytes to TNF-α mediated
cell death (Natoli et al., 1998), it is probable that TNF-α not only activates pro-apoptotic
programs in oligodendrocytes, but also initiates signaling events leading to the activation of
NF-κB transcription of protective genes. Indeed, previously we have reported the activation
of the NF-κB pathway following TNF-α treatment in the human oligodendroglioma (HOG)
cell line (Buntinx et al., 2004). Our data demonstrate that LIF is as efficient as CNTF in
reducing TNF-α mediated apoptosis of primary rat oligodendrocytes. Similar effects were
observed when these experiments were performed (with human recombinant cytokines) on the
human oligodendroglioma cell line (HOG) and the human MO3.13 cell line (data not
shown).This protective effect is most likely mediated by binding of the neurokines with their
specific receptors. We demonstrate that primary rat oligodendrocyte cultures display the
common receptor subunits for the neurokines (gp130 and LIFR-β) and the receptor for TNF-α
(TNFRI but not TNFRII) constitutively. .Immunohistochemical staining showed that human
oligodendrocytes also express low levels of gp130 (Cannella and Raine, 2004). In rats it has
been shown that oligodendrocytes express LIFR-β and this expression is upregulated after
injury (Butzkueven et al., 2002). Interestingly, protective effects of the neurokines were
observed when the oligodendrocytes were pre-treated with LIF or CNTF before TNF-α
administration, but also when the neurokines were added 2 h after the start of TNF-α
treatment. The latter approach better resembles a therapeutic design. It remains to be studied
whether LIF and CNTF could also protect against cytokine mediated apoptosis at a later time
point. Previously, CNTF has been shown to inhibit apoptotic cell death of human
oligodendrocytes following growth factor withdrawal or TNF-α treatment (Barres et al.,
1993; D'Souza et al., 1996; Louis et al., 1993). Other reports showed that LIF inhibits IFN-γ
induced cell death of rodent oligodendrocytes (Vartanian et al., 1995), while increased
oligodendrocyte survival was found in LIF treated EAE mice (Butzkueven et al., 2002).
Although there is still some controversy regarding the mode of oligodendroglial cell death in
MS, recent evidence confirms the importance of extensive oligodendroglial apoptosis in
newly formed lesions (Barnett and Prineas, 2004; Lucchinetti et al., 1999). Therefore, our
findings indicate that local neurokine administration is a possible therapeutic intervention to
reduce oligodendrocyte apoptosis in MS. The protective effect may however be highly
dependent on a critical dose. Indeed, we observed increased cytotoxicity at a high dose of
neurokines and also observed cytotoxic effects of the neurokines in the absence of TNF-α.
Interestingly, it has been reported that adenoviral delivery of large quantities of LIF in spinal
cords induces potent pro-inflammatory effects (Kerr and Patterson, 2004). It seems that the
protection mediated by LIF is also in vivo highly dependent on a critical dose. Apparently, a
critical balance between neuroprotective and pro-inflammatory cytokines mediates survival of
oligodendrocytes.
The mechanisms by which LIF and CNTF protect oligodendrocytes in an inflammatory
environment remain unresolved. This effect is most likely mediated by activation of specific
signaling pathways. Rat oligodendrocytes express the common neurokine receptors gp130 and
LIFR-β as shown in this report. The stoichiometry of the receptor complex (Zhang et al.,
1997), the presence of a third type of receptor subunit and the mode of binding to these
receptors vary between the different members of the neurokine family. However all
neurokines share common signaling components (Turnley and Bartlett, 2000). Binding of the
neurokine to its receptor complex may result in the activation of three different signaling
routes depending on the cell type. Activation of either of these signaling cascades may result
in increased survival of oligodendrocytes. The Ras-MAPK pathway is generally considered to
be a survival pathway and was shown to be activated following LIF stimulation of neuronal
cells (Segal and Greenberg, 1996). Activation of the PI3 kinase cascade by LIF leads to the
activation of NF-κB (Bonetti et al., 1999). Anti-apoptotic molecules, such as Bcl-xl and Bcl-2
are transcribed following STAT3 activation (Bowman et al., 2000). Activation of the JAKSTAT3 signaling cascade may also result in the expression of suppressors of cytokine
signaling (SOCS) genes. This recently discovered family of molecules act as negative
regulators of JAK/STAT signaling pathway. LIF induces CIS, SOCS2 and SOCS3 in bone
marrow cells (Turnley and Bartlett, 2000) and SOCS expression was shown to be inhibited by
TNF-α signaling (Morita et al., 2000). Recent insights demonstrate the important regulation
mechanisms of cytokine signaling via SOCS proteins (Alexander, 2002). This regulation
likely plays a crucial role in balancing the protective and deleterious consequences of
cytokine action. The desensitization of TNF-α signaling might explain the protective effects
of the neurokines, but also provides a possible explanation of the toxic effects of neurokines
at high concentrations and in the absence of proinflammatory cytokines. In the absence of
TNF-α, neurokine induced SOCS expression may also lead to desensitization of signaling
induced by growth factors, and could thus lead to cell death. Further research is required to
unravel the protective pathways that are upregulated in oligodendrocytes upon administration
of LIF in the presence of proinflammatory cytokines. Molecular approaches to activate these
pathways may prove to be effective as candidate therapies for MS.
Several previous studies have indicated a possible therapeutic role for CNTF and LIF.
Intravenous treatment of EAE mice with LIF resulted in a significantly improved clinical
outcome compared with placebo treated mice (Butzkueven et al., 2002). In addition to the
protective effects on oligodendrocytes, LIF administration also increases survival of both
sensory and motor neurons (Murphy et al., 1991), and LIF is a potent differentiation and
proliferation factor of neuronal progenitors (Carpenter et al., 1999). The beneficial effects of
LIF are most likely not mediated by an immunomodulatory effect as shown by Butzkueven
and collegues (Butzkueven et al., 2002). MS patients with a deficiency in the production of
CNTF have an early disease onset with predominant motor symptoms (Giess et al., 2002).
CNTF administration increases the proliferation of oligodendrocytes (Barres et al., 1996) and
enhances remyelination in vitro (Stankoff et al., 2002). The therapeutic application of CNTF
is hampered by the difficulties to reach the CNS if CNTF is administered systemically (Miller
et al., 1996). LIF has been shown to pass the blood brain barrier freely (Pan et al., 2000). Our
results indicate the possibility to enhance LIF production in the lesions by autoreactive T cells
that home to regions of inflammation in the CNS and are therefore able to deliver LIF locally.
If it would be possible to alter the pathogenic properties of these autoreactive T cells and also
increase the production of LIF in a controlled manner by for instance ex vivo gene transfer,
such approaches would be attractive for MS therapy. Our in vitro data however indicate that
LIF may only be therapeutically relevant in a relatively small dose window. If future animal
studies would confirm this in vivo, this would significantly limit the therapeutic potential of
LIF.
In summary, our data demonstrate that autoreactive T cells are able to secrete LIF, a cytokine
member of the neuropoietins. In addition, LIF expressing T cells and perivascular
macrophages are present in active and chronic active MS lesions. Production of neurotrophic
factors by immune cells may constitute an important mechanism for the protection of glial
and neuronal cells. This is supported by the in vitro observation that LIF, like CNTF is able to
protect oligodendrocytes against TNF-α induced apoptosis. Although this protective
mechanism is still operational in MS and can be observed in MS lesions, it is clearly
insufficient to alter the disease pathology. The concept of neuroprotective immunity has
profound implications regarding the use of nonselective immunosuppressive agents to treat
inflammatory diseases with an autoimmune component, such as MS. The identification of the
regulatory mechanisms governing the balance between the production of beneficial and
destructive factors by immune cells will provide new targets to boost the neurotrophic
properties of immune cells in a safe way at a well defined stage of the disease.
Acknowledgements
We thank E. Dreesen, A. Remels, C. Bocken, E. Smeyers, J. Bleus, I. Rutten, M.J. Sleypen, M.
Jans en W. Leyssens for excellent technical assistance and Prof. J. Raus, Dr. V. Somers, D.
Dumont and K. Baeten for critical reading and helpful discussions. We acknowledge Dr. R.
Medaer for the MS blood samples, the Born-Bunge foundation (Antwerp) and the Dutch Brain
Bank for the MS tissue. We also thank L. Linsen and K. Venken for providing supernatans of
regulatory T cell subsets, prof. JF Moreau and E. Bosmans for providing the anti-LIF antibody
and J. van Horssen for assistance with the immunofluorescent double stainings. This work was
supported by grants from the Belgian ‘Nationaal Fonds voor Wetenschappelijk Onderzoek
Vlaanderen (FWO)’, the Belgian Charcot foundation, the Belgian WOMS-foundation, Hasselt
University and the Transnational University Limburg. J.V. holds a fellowship from the ‘Bijzonder
Onderzoeksfonds’-Hasselt University.
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Figure 1: RT-PCR analysis of LIF transcription in myelin specific T cell lines.
RNA was extracted from five stimulated MS derived myelin reactive T cell lines (TCL 1-5)
and analyzed by RT-PCR for transcription of LIF (A) and of the constant region of the T cell
receptor β chain (TCR BC) as a control (B). -: water control, +: recombinant plasmid as
positive control.
Figure 2: LIF protein secretion by different immune cell populations.
CD4+ T cells, CD8+ T cells, CD4+CD25+ T cells, B lymphocytes and monocytes were isolated
from PBMC of two healthy donors by immunomagnetic separation. Resulting cell subsets
were stimulated with a specific stimulus (see materials and methods). NKT cells were isolated
from α-GalCer stimulated PBMC using Vα24+ immunomagnetic isolation. LIF secretion was
measured in supernatants of stimulated (+) and non stimulated (-) cell fractions by means
ELISA. NKT: NKT cells, M: monocytes, B: B-cells.
Figure 3: LIF expression by CD3+ and CD68+ cells in MS lesions.
Immunoperoxidase double staining was performed on formalin-fixed paraffin-embedded
brain tissue containing active MS lesions (n=2) and chronic active MS lesions (n=11) of 12
MS patients. No labeling was detected when primary antibodies were omitted (control).
Sections were counterstained with hematoxylin. A. Chromogenic double staining of CD3
(visualized with DAB) and LIF (visualized with Fastred). B. Chromogenic double staining of
CD68 (DAB) and LIF (Fastred). Immunofluorescent double stainings of active MS lesions
(n=2) were performed on cryopreserved braintissue. Sections were counterstained with
Hoechst dye. C. Fluorescent double staining of LIF (visualised with rhodamin) and CD3
(visualised with alexa488) in a perivascular region. D. Fluorescent double staining of CD68
(Alexa488) and LIF (rhodamin) in a perivascular region. E. Double staining of CD3
(Alexa488) and LIF (rhodamin) in the lesion parenchyma. F. Fluorescent double staining of
MHCII (Alexa488) and LIF (rhodamin) in the lesion parenchyma.
Figure 4: Immunocytochemical receptor staining of rat oligodendrocyte cultures.
Cells were cultured for 13 days in growth medium, subsequently fixated and incubated with
anti-gp130 (A), anti-LIFR-β (B), anti-TNFRI (C) and anti-TNFRII (D). Binding of primary
antibodies was visualized using secondary anti-mouse antibodies conjugated with peroxidase
and DAB solution. Cells were counterstained with hematoxylin.
Figure 5: Annexin V/PI analysis of primary rat oligodendrocytes.
Rat oligodendrocytes were cultured in growth medium and incubated with increasing
concentrations of CNTF or LIF in combination with a constant dose of TNF-α (100 ng/ml).
After 12hr of incubation the viability was assessed using flow cytometry. The y-axis depicts
the percentage of apoptotic cells relative to the apoptotic percentage observed in
oligodendrocytes treated with TNF-α alone. The curves represent the mean of three
independent experiments with duplicate measurements. The continuous horizontal line
represents the maximal apoptotic response (TNF-α alone), whereas the dotted lines represent
the background apoptotic levels (growth medium).
Figure 6: Percentage inhibition of TNF-α induced apoptosis.
Rat oligodendrocytes stimulated for 5 days with TNF-α were pre- (e.g. CNTF + TNF) or
post-treated (e.g. TNF + CNTF) for 2 hrs with the respective neurokine. Apoptotic
percentages were scored by means of annexin V/PI staining combined with flow cytometry.
Maximal apoptosis in the presence of 100 ng/ml TNF-α alone and background apoptosis in
medium alone were set as 0% and 100% inhibition respectively. The graph represents the
mean of 5 independent experiments with double measurements for each experiment.
Table 1. LIF secretion of myelin reactive T cells clones.
Subject
Clone
Antigen
Specificity
Stimulation
Index
LIF secretion1
- Ag
+ Ag
Cytokine secretion (net values)2
Net
IFN-γ
IL-10
IL-4
Th fenotype3
Healthy controls
HC1
HC2
HC3
HC4
HC5
HC6
2G10-4
2C5-16
2B6-19
2B5-1
MC6-1
MD7-1
C2-3
2G3-16
MB9-7
BM9-5
1B2-1
2F2-1
4G2-5
4B4-2
4B5
2D8
MOG
MOG
MOG
MOG
MBP
MBP
MOG
MOG
MBP
MBP
MOG
TT
TT
TT
TT
TT
10.7
2.0
82.2
3.0
2.0
NT
2.6
25.9
NT
NT
NT
80.4
25.2
129.1
3.5
4.3
24
23
0
0
43
0
24
27
0
0
0
1
0
0
128
7
34
25
0
0
541
0
57
86
0
2
0
88
1
12
265
26
10
2
0
0
498
0
33
59
0
2
0
87
1
12
137
19
NT
NT
NT
NT
12273
5053
NT
NT
1182
3126
69
NT
NT
NT
NT
NT
NT
NT
NT
NT
466
375
NT
NT
5
24
0
NT
NT
NT
NT
NT
NT
NT
NT
NT
176
61
NT
NT
16
136
135
NT
NT
NT
NT
NT
NT
NT
TH1*
NT
TH1*
TH1*
TH1*
TH0*
TH1
TH1
TH2
NT
NT
NT
NT
NT
3B1-3D3
1G5-1
2E2-2
2D6-15
2D2-5
2C8-10
2F7-2B9
1E3-3C5
2E4-17
2C7-11
E4
2E5-6
E5
1C1-1
1C1-11
1E3-10
1B7-4
MD7-8
E9-27
MBP
MBP
MBP
MBP
MBP
MBP
MBP
MBP
MBP
MBP
MBP
MBP
MBP
MOG
MOG
MOG
MBP
MBP
MOG
41
42
11
2.4
7.9
20
217
150
2
53
2.3
2.7
89
42
71
7
209
NT
NT
19
18
18
35
17
17
18
18
17
17
20
20
17
18
23
18
17
17
24
45
19
19
135
17
17
23
18
18
21
25
28
85
31
951
21
18
20
35
26
1
1
100
0
0
5
0
1
5
5
8
68
13
928
3
1
3
11
1200
0
1280
2440
340
1090
1420
1180
NT
375
2253
5519
7540
NT
NT
NT
240
2000
8705
6
5
4
0
0
4
22
27
NT
8
118
36
185
NT
NT
NT
11
0
14
134
0
0
6
0
0
0
33
NT
0
0
6
0
NT
NT
NT
0
0
292
TH1
TH2
TH1
TH1
TH1
TH1
TH1
TH1
NT
TH1
TH1
TH1
NT
TH1*
NT
NT
TH1
TH1
TH1
MS patients
MS1
MS2
MS3
MS4
MS5
MS6
MS7
MS8
1
LIF concentrations in supernatants of unstimulated (-Ag) and Ag-stimulated (+Ag) T cell lines. Net secretion was calculated as: LIF levels in stimulated cells – LIF
levels of unstimulated cells. Net secretion was considered significant when higher than 10 pg/ml (bold). 2 Net values of cytokine secretion measured in supernatants by
means of ELISA.3 T cell lines were classified based on the ratio of IFN-γ/(IL-4 and/or IL-10) as described in materials and methods. Helper phenotypes marked with
an asterisk (*) were determined by intracellular FACS. NT: not tested.