Download Muscle fibres and cultured muscle cells express the B7.1/2

DOI: 10.1093/brain/awg114
Brain (2003), 126, 1026±1035
Muscle ®bres and cultured muscle cells express the
B7.1/2-related inducible co-stimulatory molecule,
ICOSL: implications for the pathogenesis of
in¯ammatory myopathies
Heinz Wiendl,1 Meike Mitsdoerffer,1 Dagmar Schneider,1 Arthur Melms,1 Hanns Lochmuller,2
Reinhard Hohlfeld3 and Michael Weller1
1Department of Neurology, University of Tu
È bingen,
Medical School, TuÈbingen, 2Genzentrum, Friedrich-Baur
Institut, Department of Neurology and Ludwig Maximilians
University and 3Institute for Clinical Neuroimmunology,
Ludwig Maximilians University, Munich, Germany
Summary
Inducible co-stimulator ligand (ICOSL), a member of
the B7 family of co-stimulatory molecules related to
B7.1/2, regulates CD4 as well as CD8 T-cell responses
via interaction with its receptor ICOS on activated T
cells. Here we examined the expression and the functional relevance of ICOSL in human muscle cells in vivo
and in vitro. We investigated 25 muscle biopsy specimens from patients with polymyositis, dermatomyositis,
inclusion body myositis, Duchenne muscular dystrophy
and non-myopathic controls for ICOSL expression by
immunohistochemistry. Normal muscle ®bres constitutively express low levels of ICOSL. However, ICOSL
expression is markedly increased in muscle ®bres in
in¯ammatory myopathies. Cell surface staining was
most prominent in the contact areas between muscle
®bres and in¯ammatory cells, which in turn show
expression of ICOS as a marker of T-cell activation.
Muscle endothelial cells show constitutive expression of
ICOSL under normal and pathological conditions. We
also detected mRNA and cell surface protein expression
Correspondence to: Dr Heinz Wiendl, Department of
Neurology, University of TuÈbingen, Hoppe-Seyler-Strasse 3,
D-72076 TuÈbingen, Germany
E-mail: [email protected]
of ICOSL on myoblasts cultured from control subjects
and patients as well as in TE671 muscle rhabdomyosarcoma cells. ICOSL expression was upregulated by
tumour necrosis factor-a (TNF-a), whereas interferon-g
(IFN-g) had no such effect. Co-culture experiments of
major histocompatibility complex (MHC) class II-positive myoblasts with CD4 T cells together with superantigen demonstrated that the expression of musclerelated ICOSL has functional consequences: the production of Th1 (IFN-g) and Th2 cytokines [interleukin
(IL)-4 and IL-10] by CD4 T cells was markedly reduced
in the presence of a neutralizing anti-ICOSL monoclonal antibody (mAb HIL-131), thus showing the importance of ICOSL co-stimulation for T-cell activation.
Taken together, our results demonstrate that human
muscle cells express ICOSL, a functional co-stimulatory
molecule distinct from B7.1 and B7.2. ICOSL±ICOS
interactions may play an important role in in¯ammatory myopathies, providing further evidence for the
antigen-presenting capacity of muscle cells.
Keywords: co-stimulation; muscle immunobiology; B7 family; myositis; B7-H2
Abbreviations: ICOS = inducible co-stimulator; ICOSL = inducible co-stimulator ligand; IFN = interferon; MHC = major
histocompatibility complex; NCAM = neural cell adhesion molecule; PBMC = peripheral blood mononuclear cell;
PCR = polymerase chain reaction; SAg = superantigen; TNF = tumour necrosis factor
Introduction
A considerable body of evidence suggests that muscle
actively participates in local immune reactions (Hohlfeld
and Engel, 1994). Notably, upregulation of major histocompatibility complex (MHC) class I and II antigens under
Brain 126(5) ã Guarantors of Brain 2003; all rights reserved
pathological conditions (Karpati et al., 1988; Emslie-Smith
et al., 1989; Wiendl et al., 2000) is an essential prerequisite
for the interaction of muscle with CD8+ cytotoxic T cells and
CD4+ T helper cells, as observed in the idiopathic in¯amma-
Inducible co-stimulator ligand and muscle
tory myopathies (reviewed in Hohlfeld and Engel, 1994;
Dalakas, 1995, 2002). Human muscle cells can present
antigens to CD4+ T cells (Goebels et al., 1992) and express as
yet unidenti®ed co-stimulatory molecules associated with the
B7 family, but distinct from CD80 (B7.1) and CD86 (B7.2)
(Behrens et al., 1998; Murata and Dalakas, 1999).
Inducible co-stimulator ligand (ICOSL) is a member of the
B7 family of co-stimulatory ligands which shares 19±20%
sequence identity with CD80 and CD86 (reviewed in Coyle
and Gutierrez-Ramos, 2001; Carreno and Collins, 2002;
Liang and Sha, 2002; Sharpe and Freeman, 2002). In humans,
cell surface expression of ICOSL has been described on
B cells, dendritic cells, monocytes/macrophages, T cells and
endothelial cells (Coyle and Gutierrez-Ramos, 2001; Carreno
and Collins, 2002; Khayyamian et al., 2002; Liang and Sha,
2002). ICOSL mRNA expression has been detected in a
variety of lymphoid and non-lymphoid organs (Ling et al.,
2000), but the functional signi®cance of ICOSL on nonlymphoid cells has remained unclear. ICOSL binds to ICOS,
a T cell-speci®c co-stimulatory molecule homologous to
CD28 and CTLA-4 (Hutloff et al., 1999; Yoshinaga et al.,
1999; Beier et al., 2000). ICOS±ICOSL interactions have
strong impact on CD4 and CD8 T cell-mediated immune
responses, as shown in a number of in vivo studies using
ICOS knockout mice or by approaches blocking ICOS±
ICOSL interactions in animal models (reviewed in Sperling
and Bluestone, 2001; Carreno and Collins, 2002; Sharpe and
Freeman, 2002). In addition to their important role in T-cell
differentiation and T-cell±B-cell collaboration, ICOSL±
ICOS interactions play a prominent role in the co-stimulation
of CD4 as well as CD8 effector or memory T-cell responses
(Hutloff et al., 1999; Coyle et al., 2000; Sperling and
Bluestone, 2001; reviewed in Carreno and Collins, 2002;
Sharpe and Freeman, 2002).
Our results demonstrate that ICOSL is expressed in
muscle, and this co-stimulatory molecule affects functional
interactions between muscle cells and T cells.
Material and methods
Antibodies and reagents
The following primary antibodies were used: anti-human
ICOSL and anti-human ICOSL±phycoerythrin (PE) (HIL131; Khayyamian et al., 2002), anti-ICOS (F44), all generously provided by R.A. Kroczek (Robert-Koch-Insitut,
Berlin, Germany), anti-MHC I (W6/32), anti-MHC II
(L243), anti-CD3 (OKT3), anti-CD4 (RPA-T4), anti-CD8
(B9.11), anti-CD11b (BEAR1), anti-CD14 (B-A8), antiCD20 (B9E9), anti-CD56 (anti-NCAM, MY31), anti-CD80
(BB-1), anti-CD86 (B-T7) and anti-pan-a/b-T cell receptor
(TCR) (T10B9.1A-31) (all BD PharMingen, Heidelberg,
Germany). Secondary antibodies and isotype controls were:
goat
anti-mouse
F(ab¢)2±di-chlorotriazinyl-¯uorescein
(DTAF), goat anti-mouse-PE IgG (H+L) F(ab¢)2 fragment
(Dianova, Hamburg, Germany), mouse IgG1k (MOPC-21,
1027
Sigma, Saint Louis, MO, USA) and mouse IgG (Linaris,
Wertheim, Germany). All antibodies were titrated for ¯ow
cytometry. A ®nal concentration of 10±30 mg/ml acid-free
antibody was used for blocking experiments. Interferon-g
(IFN-g) and tumour necrosis factor-a (TNF-a) were from
Peprotech EC Ltd (London, UK). Normal goat serum
was from Dianova (Hamburg, Germany), human IgG
(Alphaglobinâ) was obtained from Grifols (Langen,
Germany), glatiramer acetate was obtained from Teva
Pharmaceutical Industries (Petah Tiqva, Israel), phytohaemagglutinin was from Biochrom KG (Berlin, Germany)
and CD3/CD28 beads were from Dynal Biotech (Hamburg,
Germany). Superantigens (staphylococcal enterotoxin B,
staphylococcal enterotoxin A, and toxic shock syndrome
toxin-1, all from Toxin Technology, Sarasota, FL) were used
as a SAg mix (1.5 pg/ml each).
Clinical material
Diagnostic muscle biopsy specimens were obtained from
patients with in¯ammatory myopathies (polymyositis, n = 7:
age range 51±89 years; dermatomyositis, n = 5: age range
61±70 years; inclusion body myositis, n = 6: age range
58±80 years) and non-in¯ammatory or non-myopathic controls (n = 7, age range: 20±62 years). Patients were not under
immunosuppressive therapy at time of the biopsy.
Immunohistochemical studies
Flash-frozen muscle biopsy specimens were cut into 8±10 mm
cryostat sections and analysed by immunohistochemistry.
Acetone-®xed, air-dried sections were blocked and incubated
with primary antibodies or corresponding non-immune IgG
isotype controls, diluted in phosphate-buffered saline (PBS)
containing 2% bovine serum albumin (BSA) for 45 min at the
concentrations optimized by titrating the respective primary
antibodies. The reaction product was visualized with the
streptavidin±biotin method (reagents from DAKO, Hamburg,
Germany) using diaminobenzidine (Serva, Heidelberg,
Germany) as an electron donor. Alternatively, antibody
binding was visualized by immuno¯uorescence microscopy
[Chromophore Cy3- or ¯uorescein isothiocyanate (FITC)labelled secondary antibodies; Dianova, Hamburg,
Germany]. Umbilical cord (endothelium of the vessels) was
used as a positive control.
Cell isolation and culture
Myoblasts were isolated from normal subjects or patients
with in¯ammatory myopathy, puri®ed by magnetic bead
separation and cultured as previously described (Wiendl et al.,
2002). Written consent was obtained from all donors, and
tissue sampling was approved by the local ethics committee
(Munich). Myoblasts stained >95% positive for the neural
cell adhesion molecule (NCAM) by ¯ow cytometry. TE671
cells were obtained from ATCC. Peripheral blood mono-
1028
H. Wiendl et al.
nuclear cells (PBMCs) were isolated from peripheral blood of
healthy volunteers by density gradient centrifugation using
Biocoll Separating Solution (Biochrom KG, Berlin,
Germany). Monocytes were depleted by adhesion to plastic
¯asks for 1 h. CD4 T cells were puri®ed with a negative
isolation kit (Dynal Biotech, Hamburg, Germany); only
>95% pure CD4 T cells were used in the experiments.
Cytokine induction and ¯ow cytometry
For analysis of surface molecules and co-culture experiments,
myoblasts or TE671 cells were cultured in the presence of
500 U/ml IFN-g and/or 500 U/ml TNF-a for the times
indicated. Adherent cells were detached non-enzymatically
using cell dissociation buffer (InvitrogenÔ, Heidelberg,
Germany). All cells were washed with FACS (¯uorescenceactivated cell sorting) buffer containing PBS with 0.1% BSA
and 0.1% sodium azide. To minimize binding to Fc receptors,
PBMCs, puri®ed T cells and myoblast cells were blocked
with human immunoglobulins at 1.25 mg/ml for 10 min at
4°C. After one washing step, the unlabelled or labelled ®rst
antibody was added. Isotype control monoclonal antibodies
(mAbs) were used at the same concentration as the primary
antibody. Incubation was done on ice for 45 min, followed by
two washes. Goat anti-mouse IgG [F(ab)2±PE (5 mg/ml,
Sigma)] was used as secondary antibody. Histograms of
stained cells were analysed by calculating the speci®c
¯uorescence index (speci®c geometric mean:geometric
mean of isotype). For labelled PBMCs, quadrant analysis of
dot blots was performed. Unspeci®c isotype binding was
substracted for each sample. Surface expression was quanti®ed by ¯ow cytometry on a ¯uorescence-activated cell
sorter (FACS Calibur cytometer, Becton Dickinson BD,
Heidelberg, Germany). Data were analysed using CellQuestâ
software.
Co-culture experiments of myoblasts and T cells
Myoblasts were plated in six-well plates (Costar, Bodenheim,
Germany) at a density of 5 3 105 and cultured in the absence
or presence of IFN-g (500 U/ml) and TNF-a (500 U/ml) for
24±48 h to induce MHC II expression. After washing the
cells, 5 3 105 puri®ed CD4 T cells isolated with a negative
isolation kit (Dynal Biotech) were added in the presence of a
SAg mix (see above). Cells subsequently were co-cultured in
RPMI1640 supplemented with 1% glutamine (Gibco Life
Technologies, Paisley, UK), 10% foetal calf serum (Fetal
Calf Serum Gold, PAA Laboratories, Linz, Austria) and
penicillin (100 IU/ml)/streptomycin (100 mg/ml) (Gibco).
The purity of the isolated T-cell populations used in the
experiments was >95%. Where indicated, anti-MHC II
(L243), anti-ICOSL (HIL-131) or an isotype control antibody
(MOPC-21; 15 mg/ml each) were added to the co-cultures.
For measurement of cytokine production at the indicated
times, T cells alone or T cells plus myoblasts were suspended
in Trizolâ and subjected to RNA isolation.
RNA extraction, cDNA synthesis and
polymerase chain reaction (PCR)
Total RNA extraction was performed using peqGOLD Tri
FastÔ isolation reagent (peqLab, Erlangen, Germany). For
®rst strand cDNA synthesis, 2.5 mg of total RNA were
dissolved in 21.5 ml of diethylpyrocarbonate (DEPC)-treated
double-distilled water (H2Odd). A 2 ml aliquot of random
hexamers (200 ng/ml) was added to each sample prior to
incubation at 70°C for 10 min. Samples were cooled on ice
and subjected to a mixture consisting of 53 M-MLV reverse
transcriptase (RT) buffer (10 ml/sample, Promega, Madison,
WI, USA), dNTPs (10 mM, 10 ml/sample), RNasin (40 U/ml,
0.25 ml/sample, Promega), M-MLV RT (200 U/ml, 1 ml/
sample, Promega) and DEPC-treated H2Odd (5.25 ml/sample).
This mixture of 26.5 ml was added to each RNA/random
hexamer solution. Samples were mixed and incubated for
10 min at room temperature, then for 50 min at 42°C and
®nally for 15 min at 70°C.
For conventional PCR, 1 ml of cDNA was used to amplify
the products of interest in a 20 ml standard PCR; 18S RNA
was ampli®ed as a control. Speci®city of ampli®cation was
con®rmed by direct DNA sequencing of extracted bands after
reampli®cation.
For quantitative real-time PCR, measurement of gene
expression was performed utilizing the ABI prism 7700
Sequence Detection System (Applied Biosystems, Foster
City, CA). The HUSAR Genius software package
(http://genius.embnet.dkfz-heidelberg.de, DKFZ, Heidelberg,
Germany) was used to design the primers for ampli®cation.
Primers (BioChip Technologies GmbH, Freiburg, Germany)
were designed when possible to span exon±exon junctions in
order to prevent ampli®cation of genomic DNA and to result
in amplicons of <150 bp to enhance the ef®ciency of PCR
ampli®cation. Relative quanti®cation of speci®c gene expression was performed by two-step real-time PCR using cDNA
as a template. Templates were multiplied using PE Applied
Biosystems (Perkin Elmer) SYBRâ Green PCR Master Mix
[containing hot-start-AmpliTaqGoldâ, SYBRâ Green PCR
buffer (23), MgCl2 and dNTPs]. RT±PCR of cDNA specimens was conducted in a total volume of 15 ml with 13 Taq
Man Master Mix (Perkin Elmer) with primers at optimized
concentrations. Thermal cycler parameters were 2 min at
50°C, 10 min at 95°C and 40 cycles of denaturation at 95°C
for 15 s followed by annealing/extension at 60°C for 1 min.
The ¯uorescence resulting from binding of SYBRâ Green
dye to double-stranded DNA was measured directly in the
PCR tube. Data were analysed with the ABI PRISMâ
Detection System using the comparative CT (threshold
cycle) method (PE Applied Biosystems, User Bulletin).
Samples were normalized to 18S rRNA, in order to account
for the variability in the initial concentration of the total RNA
and conversion ef®ciency of the RT reaction. Product
speci®city of the PCR products was con®rmed by agarose
gel electrophoresis or dissociation curve analysis. The
internal reference dye ROX included in the PCR buffer was
Inducible co-stimulator ligand and muscle
1029
Fig. 1 Expression of ICOSL and its receptor ICOS in muscle tissue specimens in vivo. Detection of ICOSL (HIL-131) was performed by
immunohistochemistry. Transverse serial cryostat sections from a non-myopathic control stained with anti-ICOSL mAb HIL-131 (B) and
isotype control antibody (A) show low levels of constitutive ICOSL expression of muscle ®bres and clear staining of capillaries
(arrowhead). (C) A cryostat section from one patient with polymyositis (patient 1) stained with anti-ICOSL mAb HIL-131 shows
enhanced expression of ICOSL that was found predominantly in areas of in¯ammatory cell invasion and in®ltration. Fibres surrounded or
invaded by in¯ammatory cells (asterisks) stain more intensely for ICOSL. A transverse section from another patient with polymyositis
(patient 2) stained with ICOSL mAb HIL-131 (D) shows enhanced ICOSL expression of muscle ®bres and blood vessel endothelium
(arrowhead) (compare with B). (E) A section from a patient with dermatomyositis shows strong ICOSL expression on endothelial cells of
larger blood vessels within the tissue specimen (asterisks). (F) Endothelial cells of the umbilical cord venule served as a positive control
for ICOSL staining with HIL-131. (G) Immuno¯uorescence shows the expression of ICOS on T cells (green) lying in apposition to
muscle ®bres (asterisks), suggesting that T cells are activated. Magni®cations 3200 (A, B, D, E and F), 3300 (C) and 3400 (G).
1030
H. Wiendl et al.
used to check for ¯uorescence ¯uctuations caused by changes
in concentration or volume. All PCR assays were performed
in duplicate.
The following oligonucleotides were used in this study.
18S: 18s-for, 5¢-CGGCTACCACATCCAAGGAA; 18s-rev,
5¢-GCTGGAATTACCGCGGCT; ICOSL (B7H2): ICOSLfor, 5¢-CGTGTACTGGATCAATAAGACGG; ICOSL-rev,
5¢-TGAGCTCCGGTCAAACGTGGCC (Ling et al., 2000);
ICOSL (GL50): GL50-for, 5¢-CTTGTGGTCGTGGCGGTG;
GL50-rev, 5¢-TCACGAGAGCAGAAGGAGCAGGTTCC
(Ling et al., 2000);
IFN-g: IFN-g-for, 5¢-TTCAGCTCTGCATCGTTTTG; IFN-grev, 5¢-CTTTCCAATTCTTCAAAATGCC; interleukin-4:
IL-4-for, 5¢-CTTTGAACAGCCTCACAGAGC; IL-4-rev,
5¢-AACTGCCGGAGCACAGTC; IL-10: IL-10-for, 5¢-GTTTTACCTGGAGGAGGTGATG; IL-10-rev, 5¢-GGCCTTGCTCTTGTTTTCAC.
Results
Analysis of ICOSL expression in muscle biopsy
specimens by immunohistochemistry
To assess whether the B7 family co-stimulatory molecule
ICOSL is expressed in muscle tissue, we analysed 25 muscle
biopsy specimens from patients with polymyositis, inclusion
body myositis, dermatomyositis, non-in¯ammatory myopathic controls (Duchenne muscular dystrophy) and nonmyopathic controls by immunohistochemistry using mAbs
against ICOSL (HIL-131) (Khayyamian et al., 2002), MHC I
(W6/32) and MHC II (L243) molecules, and T-cell markers
(anti-CD4, RPA-T4; anti-CD8, B9.11, anti-CD3, OKT3). As
a positive control for ICOSL expression, we used human
umbilical cord, where strong endothelial expression was
observed (Khayyamian et al., 2002). HLA class I and class II
antigens are not detectable on normal muscle ®bres in situ,
but HLA class I, and in some instances HLA class II, antigens
are strongly upregulated on muscle ®bres in in¯ammatory
myopathies (Karpati et al., 1988; Emslie-Smith et al., 1989;
Wiendl et al., 2000; and data not shown). Low levels of
constitutive ICOSL expression were observed on the surface
of muscle ®bres in our muscle biopsy specimens from nonmyopathic controls (Fig. 1A and B) and in non-in¯ammatory
myopathies (data not shown). The staining intensity of
ICOSL was markedly enhanced on muscle ®bres in in¯ammatory myopathies, especially polymyositis (seven of seven
specimens) (Fig. 1C and D). In polymyositis, muscle-related
expression of ICOSL was strongest in areas where in¯ammatory cells surrounded or invaded muscle ®bres. Some
regenerating or degenerating muscle ®bres also showed
ICOSL staining of the cytoplasm (Fig. 1C). It is of note that
muscle capillaries showed constitutive expression of ICOSL,
regardless of the origin of the muscle biopsy specimens (nonmyopathic or myopathic) (Fig. 1B±E). Further, some of the
mononuclear cells within the in¯ammatory tissue sections
also stained positive for ICOSL, most probably re¯ecting
ICOSL-expressing B cells and monocytes (Fig. 1C and D).
The potential relevance of ICOSL±ICOS interactions for
disease pathogenesis of in¯ammatory myopathies was delineated further by detecting the expression of ICOS on T cells
in some polymyositis specimens (Fig. 1G). Since ICOS is
known to be expressed on CD4 or CD8 T cells upon
activation (Hutloff et al., 1999), our ®ndings suggest that
muscle-derived ICOSL may interact directly with ICOS on
activated T cells. Further, ICOSL expression might be
upregulated in the presence of in¯ammatory cytokines
released by in¯ammatory cells or destroyed muscle ®bres.
ICOSL protein expression in cultured human
myoblasts and TE671 rhabdomyosarcoma cells
To con®rm that muscle cells express ICOSL and to identify
potential regulatory cytokines for ICOSL expression, we
analysed several lines of cultured myoblasts for ICOSL
expression using mAb HIL-131 (Khayyamian et al., 2002). In
all experiments, myoblasts were >95% positive for NCAM
(CD56), which serves as a reliable marker for myoblasts (Illa
et al., 1992; Fig. 2A). As reported previously, myoblasts
constitutively express MHC I. MHC II is induced by IFN-g
but not TNF-a (Hohlfeld and Engel, 1990a; Goebels et al.,
1992; Fig. 2A). There were low levels of constitutively
expressed ICOSL on six of eight tested myoblast cultures
derived from different donors (Fig. 2A). No differences were
observed between myoblasts derived from patients with
in¯ammatory myopathies and non-myopathic control subjects. Speci®city of staining was demonstrated in some
experiments by cold blocking: unlabelled anti-ICOSL antibody was added prior to staining with the PE-labelled HIL131
at 1000-fold excess, and abolished staining (data not shown).
We next investigated the effects of in¯ammatory cytokines
on the expression of ICOSL. Myoblasts were incubated for
24, 48 or 72 h with IFN-g, TNF-a or both. ICOSL expression
was upregulated by TNF-a, whereas IFN-g had no such effect
(Fig. 2 and data not shown). Further, we investigated TE671
muscle rhabdomyosarcoma cells for ICOSL expression.
TE671 cells constitutively express MHC I but no MHC II.
These cells exhibited strong constitutive expression of
ICOSL which was not modulated further by treatment with
in¯ammatory cytokines (Fig. 2B and data not shown).
ICOSL RNA expression in cultured human
myoblasts and muscle biopsy specimens
Two splice variants of human ICOSL have been described
and designated B7-H2/B7RP-1/hLICOS (Brodie et al., 2000;
Wang et al., 2000; Yoshinaga et al., 2000) and hGL50 (Ling
et al., 2000). To corroborate our results and to identify the
splice variants expressed in muscle, we analysed total RNA
for ICOSL transcripts by RT±PCR using primers that allow
the ampli®cation of B7-H2 or hGL50. Direct visualization of
the PCR products showed B7-H2 transcripts in unstimulated
Inducible co-stimulator ligand and muscle
1031
Fig. 2 ICOSL is expressed in cultured muscle cells in vitro. Flow cytometric analysis of ICOSL cell surface expression on cultured
myoblasts. Puri®ed unstimulated NCAM+ myoblasts express MHC I (mAb W6/32) and low levels of ICOSL (mAb HIL-131). Surface
expression of ICOSL was enhanced by TNF-a but not by IFN-g, as shown for myoblasts 48 h after induction. Histograms show staining
with the designated antibodies (shaded) underlaid with the isotype controls for ICOSL (mIgG1). (B) Flow cytometric analysis of ICOSL
surface expression on TE671 human rhabdomyosarcoma cells. Histograms show staining with the designated antibodies (shaded)
underlaid with the isotype controls for ICOSL (mIgG1). (C) RT±PCR of ICOSL transcript B7-H2 shows basal expression in unstimulated
cultured human myoblasts (myoblasts I, II and III; myoblast III corresponds to myoblasts shown in (A). After stimulation with TNF-a for
48 h, the B7-H2 transcript was upregulated. A moderate RNA upregulation was observed also in response to IFN-g. 18S RNA was used as
an internal control for the quality of cDNA (lower panel). Myoblasts I, II and III are derived from three different donors and are shown as
representative examples for these experiments. B cells and monocytes were used as positive controls for B7-H2. NTC = no template
control.
1032
H. Wiendl et al.
expression under any conditions in cultured human myoblasts
or TE671 cells (data not shown). It is of note that we
investigated total RNA derived from muscle biopsy specimens for expression of ICOSL and found the B7-H2 isoform
in 15 of 17 tested samples derived from donors without
pathological abnormalities and donors with in¯ammatory
myopathies (data not shown).
ICOSL on myoblasts co-stimulates Th1 and Th2
cytokine mRNA synthesis of CD4 T cells
To demonstrate that the expression of ICOSL on muscle cells
has functional consequences, we performed co-culture
experiments with allogeneic CD4 T cells and assessed the
modulation of T-cell cytokine mRNA synthesis. Myoblasts
were pre-induced to express MHC II by addition of IFN-g and
to upregulate ICOSL by TNF-a, and subsequently cocultured with puri®ed CD4 T cells and SAg in the absence
or presence of the inhibitory anti-ICOSL mAb HIL-131. The
production of Th1 (IFN-g) and Th2 cytokines (IL-4 and IL10) was measured by assessing RNA synthesis via quantitative real-time PCR. HIL-131 inhibited the production of Th1
as well as Th2 cytokines between 52 and 61% (IFN-g,
61 6 12%; IL-4, 57 6 12%; IL-10, 52 6 11%) (Fig. 3). In
comparison, L243, an anti-MHC II antibody used to block the
interaction of the trimolecular complex (MHC±SAg±TCR),
reduced cytokine production by ~90% (81±92%). It is of note
that no relevant cytokine mRNA synthesis was observed
when myoblasts were cultured together with SAg in the
absence of CD4 T cells.
Discussion
Fig. 3 Inhibition of cytokine production in myoblast and T-cell cocultures by blocking ICOSL±ICOS interaction. Myoblasts (Myo)
were cultured in the presence of IFN-g (500 U/ml)/TNF-a (500 U/
ml) to upregulate MHC II and ICOSL expression, and co-cultured
with SAg and puri®ed CD4 T cells (T) in the presence of an
isotype control (MOPC-21), anti-ICOSL mAb (HIL-131) or antiMHC class II mAb (L243). CD4 T cells plus SAg without
myoblasts (T + SAg) and myoblasts plus SAg (Myo + SAg) were
used as controls. Cytokine mRNA synthesis by T cells was
assessed by quantitative real-time PCR after 24 h of co-culture.
Cytokine mRNA synthesis of the isotype control was set to 100%
and data are expressed as percentages of mRNA relative to the
isotype control. Error bars represent the means 6 SEM of three
experiments. nm, not measurable.
cultured myoblasts (Fig. 2C). Corresponding to the protein
expression data, RNA expression was upregulated by
addition of TNF-a, and to a lesser extent by IFN-g
(Fig. 2C). In contrast to B cells and monocytes, which were
used as a positive control, direct visualization of RT±PCR
products did not result in the detection of hGL50 mRNA
Muscle ®bres and myoblasts can actively participate in
immune cell interactions (e.g. Hohlfeld and Engel, 1990b;
Goebels et al., 1992; Wiendl et al., 2003) and thus probably
play an important role in initiating or perpetuating muscledirected immune responses (reviewed in Hohlfeld and Engel,
1994; Nagaraju, 2001; Dalakas, 2002). (i) Cultured muscle
cells induce primary immune responses in allogeneic situations (Curnow et al., 1998) and are killed ef®ciently by
cytotoxic T cells and natural killer (NK) cells (Hohlfeld and
Engel, 1990b; Wiendl et al., 2003). (ii) In polymyositis and
sporadic inclusion body myositis, CD8+ T cells and macrophages invade initially non-necrotic muscle ®bres (Arahata
and Engel, 1988). The expression of MHC I in all of the
invaded muscle ®bres and some non-invaded ®bres has been
taken as evidence for an HLA class I-restricted cytotoxic T
cell-mediated response against surface antigens expressed on
muscle ®bres (reviewed in Hohlfeld and Engel, 1994;
Nagaraju, 2001; Dalakas, 2002). (iii) Myoblasts inducibly
express MHC II molecules and have the capability to present
antigens to CD4 T cells (Goebels et al., 1992; Curnow et al.,
2001).
Inducible co-stimulator ligand and muscle
The muscle cells armamentarium with co-stimulatory
molecules has been the subject of ongoing debate. Presence
of 37.5 (CD80/86) is believed to be a prerequisite for antigenpresenting cells to initiate immune responses according to the
two-signal model of T-cell activation (Lafferty and
Woolnough, 1977). However, muscle ®bres and cultured
myoblasts do not express detectable levels of B7.1 (CD80) or
B7.2 (CD86) protein under normal or in¯ammatory conditions (Behrens et al., 1998; Bernasconi et al., 1998; Murata
and Dalakas, 1999; Nagaraju et al., 1999), but nonetheless are
capable of augmenting antigen-speci®c T-cell responses.
Muscle cells therefore have been postulated to express other
co-stimulatory molecules including an as yet unidenti®ed
B7-related co-stimulatory protein (BB-1) that interacts with
CD28/CTLA-4 and believed to exhibit strong co-stimulatory
function (Behrens et al., 1998; Murata and Dalakas, 1999).
The important advance of our present work is the
demonstration of a recently identi®ed and molecularly
de®ned member of the B7 family, ICOSL, functionally
expressed in muscle under physiological and pathological
conditions in vivo as well as in cultured human myoblasts
in vitro. Despite the existing data showing that muscle cells
can act as potent antigen-presenting cells, the lack of CD80/
86 has questioned the potential of muscle to prime naive T
cells per se and maintained the debate on how and when
muscle cells would interact with immune cells during the
initiating or perpetuating events of in¯ammatory myopathies.
In this view, the characterization of novel members of the B7
family of co-stimulatory molecules that exhibit a broader
tissue distribution than B7.1/2 importantly advances our
understanding of immune reactions directed against or
derived from non-lymphoid tissues (reviewed in Coyle and
Gutierrez-Ramos, 2001; Carreno and Collins, 2002; Liang
and Sha, 2002; Sharpe and Freeman, 2002). ICOSL is a
member of the B7 family of co-stimulatory ligands that has
strong impact on T cell-mediated immune responses by
interacting with ICOS on T cells (reviewed in Coyle and
Gutierrez-Ramos, 2001; Carreno and Collins, 2002; Liang
and Sha, 2002; Sharpe and Freeman, 2002). Two splice
variants of human ICOSL have been described and designated hGL50 (Ling et al., 2000) and B7-H2/B7RP-1/hLICOS
(Brodie et al., 2000; Wang et al., 2000; Yoshinaga et al.,
2000). hGL50 showed a more lymphoid-restricted expression
pattern (spleen, lymph node), whereas B7-H2/B7RP-1/
hLICOS mRNA was expressed in a broader variety of organs
tested (Ling et al., 2000). We report here B7-H2 mRNA and
protein expression in muscle tissue, cultured myoblasts and
TE671 rhabdomyosarcoma cells. ICOSL expression was
enhanced in muscle cells under in¯ammatory conditions
in vivo and in vitro, and the in¯ammatory cytokine TNF-a
augmented cell surface expression of ICOSL on cultured
myoblasts. We further showed that ICOSL expression on
cultured muscle cells is functional in that it modulates the
cytokine production by activated T cells. Thus, since ICOSL±
ICOS interactions have strong impact on CD4+ and CD8+
1033
T-cell responses, this pathway may be of critical relevance for
immune reactions under different conditions in muscle.
The expression patterns, inducibility characteristics in vitro
and receptor interactions of ICOSL make it unlikely that this
molecule represents the postulated B7-related co-stimulatory
molecule BB-1 in muscle cells, for the following reasons
(Behrens et al., 1998; Murata and Dalakas, 1999): the
putative B7 family co-stimulatory molecule BB-1 shows no
constitutive expression of ICOSL on cultured myoblasts nor
on capillaries (Behrens et al., 1998); further, BB-1 has been
suggested to interact with CTLA-4 (Behrens et al., 1998;
Murata and Dalakas, 1999), a condition that does not hold
true for ICOSL interacting with ICOS on activated T cells.
Current models propose that ICOSL±ICOS interactions
play a more prominent role in the co-stimulation of effector or
memory T-cell responses (Hutloff et al., 1999; Coyle et al.,
2000), whereas CD28 co-stimulates primary T-cell functions.
It is tempting to speculate that muscle-associated ICOSL
augments the production of Th1 and Th2 cytokines by
interaction with CD4 effector/memory T cells also in vivo,
thus augmenting the muscle-directed antigen-speci®c immune responses in in¯ammatory myopathies. Further,
ICOSL±ICOS interactions in¯uence the clonal expansion
and the cognate destruction of tumour cells by CD8 cytotoxic
lymphocytes, thus extending the immunological role of
ICOSL±ICOS interactions to CD8 T-cell functions (Liu
et al., 2001; Wallin et al., 2001). This may be of particular
importance for MHC I-expressing muscle cells presenting the
target antigen(s) on the muscle ®bre surface to cytotoxic T
cells in polymyositis or inclusion body myositis. We also
observed ICOSL expression on endothelial cells in muscle. It
is therefore conceivable that ICOSL is of relevance for the
immune pathogenesis of dermatomyositis where endothelial
cells represent the primary target structure for the autoimmune process. ICOSL could play an important role in the
(re)activation of effector/memory T cells on the endothelium
either controlling the entry of immune cells into in¯amed
tissue or augmenting antibody production by B cells.
It should be noted that the signi®cance of ICOSL
expression in muscle reaches beyond its implications for
the pathogenesis and treatment of in¯ammatory myopathies.
Muscle is the site for many other immune reactions, including
muscle infections, graft-versus-host disease, conventional
intramuscular vaccination and, intramuscular gene transfer by
injection of genetically engineered myoblasts or naked DNA
(Hohlfeld and Engel, 1994; Blau and Springer, 1995; Miller
and Boyce, 1995; Pardoll and Beckerleg, 1995). In all these
reactions, the expression of ICOSL on muscle ®bres could
play an important role.
Acknowledgements
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Wi 1722/2-1 to H.W., SFB571 A-1
to R.H., and Lo 549/2-3 to H.L.) and the IZKF TuÈbingen
(M.W.). The Institute for Clinical Neuroimmunology is
1034
H. Wiendl et al.
supported by the Hermann and Lilly Schilling Foundation.
Human myoblast cultures were obtained from the Muscle
Tissue Culture Collection at the Friedrich-Baur-Institute
(Department
of
Neurology,
Ludwig-MaximiliansUniversity, Munich, Germany). The Muscle Tissue Culture
Collection is supported by the `Association Francaise contre
les Myopathies' (Paris, France) and the German Ministry for
Education and Research (BMBF; MD-NET, project S1).
Emslie-Smith AM, Arahata K, Engel AG. Major histocompatibility
complex class I antigen expression, immunolocalization of
interferon subtypes, and T cell-mediated cytotoxicity in
myopathies. Hum Pathol 1989; 20: 224±31.
References
Arahata K, Engel AG. Monoclonal antibody analysis of
mononuclear cells in myopathies. IV: cell-mediated cytotoxicity
and muscle ®ber necrosis. Ann Neurol 1988; 23: 168±73.
Hohlfeld R, Engel AG. Lysis of myotubes by alloreactive cytotoxic
T cells and natural killer cells. Relevance to myoblast
transplantation. J Clin Invest 1990b; 86: 370±4.
Behrens L, Kerschensteiner M, Misgeld T, Goebels N, Wekerle H,
Hohlfeld R. Human muscle cells express a functional costimulatory
molecule distinct from B7.1 (CD80) and B7.2 (CD86) in vitro and
in in¯ammatory lesions. J Immunol 1998; 161: 5943±51.
Beier KC, Hutloff A, Dittrich AM, Heuck C, Rauch A, Buchner K,
et al. Induction, binding speci®city and function of human ICOS.
Eur J Immunol 2000; 30: 3707±17.
Bernasconi P, Confalonieri P, Andreetta F, Baggi F, Cornelio F,
Mantegazza R. The expression of co-stimulatory and accessory
molecules on cultured human muscle cells is not dependent on
stimulus by pro-in¯ammatory cytokines: relevance for the
pathogenesis of in¯ammatory myopathy. J Neuroimmunol 1998;
85: 52±8.
Blau HM, Springer ML. Muscle-mediated gene therapy. N Engl J
Med 1995; 333: 1554±6.
Brodie D, Collins AV, Iaboni A, Fennelly JA, Sparks LM, Xu XN,
et al. LICOS, a primordial costimulatory ligand? Curr Biol 2000;
10: 333±6.
Goebels N, Michaelis D, Wekerle H, Hohlfeld R. Human myoblasts
as antigen-presenting cells. J Immunol 1992; 149: 661±7.
Hohlfeld R, Engel AG. Induction of HLA-DR expression on
human myoblasts with interferon-gamma. Am J Pathol 1990a;
136: 503±8.
Hohlfeld R, Engel AG. The immunobiology of muscle. Immunol
Today 1994; 15: 269±74.
Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R,
Anagnostopoulos I, et al. ICOS is an inducible T-cell co-stimulator
structurally and functionally related to CD28. Nature 1999; 397:
263±6.
Illa I, Leon-Monzon M, Dalakas MC. Regenerating and denervated
human muscle ®bers and satellite cells express neural cell adhesion
molecule recognized by monoclonal antibodies to natural killer
cells. Ann Neurol 1992; 31: 46±52.
Karpati G, Pouliot Y, Carpenter S. Expression of immunoreactive
major histocompatibility complex products in human skeletal
muscles. Ann Neurol 1988; 23: 64±72.
Khayyamian S, Hutloff A, Buchner K, Grafe M, Henn V, Kroczek
RA, et al. ICOS-ligand, expressed on human endothelial cells,
costimulates Th1 and Th2 cytokine secretion by memory CD4+ T
cells. Proc Natl Acad Sci USA 2002; 99: 6198±203.
Lafferty KJ, Woolnough J. The origin and mechanism of the
allograft reaction. Immunol Rev 1977; 35: 231±62.
Carreno BM, Collins M. The B7 family of ligands and its receptors:
new pathways for costimulation and inhibition of immune
responses. Annu Rev Immunol 2002; 20: 29±53.
Liang L, Sha WC. The right place at the right time: novel B7 family
members regulate effector T cell responses. Curr Opin Immunol
2002; 14: 384±90.
Coyle AJ, Gutierrez-Ramos JC. The expanding B7 superfamily:
increasing complexity in costimulatory signals regulating T cell
function. Nat Immunol 2001; 2: 203±9.
Ling V, Wu PW, Finnerty HF, Bean KM, Spaulding V, Fouser LA,
et al. Cutting edge: identi®cation of GL50, a novel B7-like protein
that functionally binds to ICOS receptor. J Immunol 2000; 164:
1653±7.
Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, Manning S,
et al. The CD28-related molecule ICOS is required for effective
T cell-dependent immune responses. Immunity 2000; 13:
95±105.
Curnow SJ, Willcox N, Vincent A. Induction of primary immune
responses by allogeneic human myoblasts: dissection of the cell
types required for proliferation, IFNgamma secretion and
cytotoxicity. J Neuroimmunol 1998; 86: 53±62.
Curnow J, Corlett L, Willcox N, Vincent A. Presentation by
myoblasts of an epitope from endogenous acetylcholine receptor
indicates a potential role in the spreading of the immune response. J
Neuroimmunol 2001; 115: 127±34.
Dalakas MC. Immunopathogenesis of in¯ammatory myopathies.
Ann Neurol 1995; 37 Suppl 1: S74±86.
Dalakas MC. In¯ammatory myopathies. Recent advances in
pathogenesis and therapy. Adv Neurol 2002; 88: 253±71.
Liu X, Bai XF, Wen J, Gao JX, Liu J, Lu P, et al. B7H
costimulates clonal expansion of, and cognate destruction of
tumor cells by, CD8(+) T lymphocytes in vivo. J Exp Med
2001; 194: 1339±48.
Miller JB, Boyce FM. Gene therapy by and for muscle cells. Trends
Genet 1995; 11: 163±5.
Murata K, Dalakas MC. Expression of the costimulatory molecule
BB-1, the ligands CTLA-4 and CD28, and their mRNA in
in¯ammatory myopathies. Am J Pathol 1999; 155: 453±60.
Nagaraju K. Immunological capabilities of skeletal muscle cells.
Acta Physiol Scand 2001; 171: 215±23.
Nagaraju K, Raben N, Villalba ML, Danning C, Loef¯er LA, Lee E,
et al. Costimulatory markers in muscle of patients with idiopathic
in¯ammatory myopathies and in cultured muscle cells. Clin
Immunol 1999; 92: 161±9.
Inducible co-stimulator ligand and muscle
1035
Pardoll DM, Beckerleg AM. Exposing the immunology of naked
DNA vaccines. Immunity 1995; 3: 165±9.
Hohlfeld R, et al. An autoreactive gd TCR derived from a
polymyositis lesion. J Immunol 2002; 169: 515±21.
Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nature Rev
Immunol 2002; 2: 116±26.
Wiendl H, Mitsdoerffer M, Hofmeister V, Wischhusen J, Weiss EH,
Dichgans J, et al. The non-classical MHC molecule HLA-G protects
muscle cells from immune-mediated lysis: implications for
myoblast transplantation and gene therapy. Brain 2003; 126:
176±85.
Sperling AI, Bluestone JA. ICOS costimulation: it's not just for
TH2 cells anymore. Nat Immunol 2001; 2: 573±4.
Wallin JJ, Liang L, Bakardjiev A, Sha WC. Enhancement of CD8+
T cell responses by ICOS/B7h costimulation. J Immunol 2001; 167:
132±9.
Wang S, Zhu G, Chapoval AI, Dong H, Tamada K, Ni J, et al.
Costimulation of T cells by B7-H2, a B7-like molecule that binds
ICOS. Blood 2000; 96: 2808±13.
Wiendl H, Behrens L, Maier S, Johnson MA, Weiss EH, Hohlfeld
R. Muscle ®bers in in¯ammatory myopathies and cultured
myoblasts express the nonclassical major histocompatibility
antigen HLA-G. Ann Neurol 2000; 48: 679±84.
Wiendl H, Malotka J, Holzwarth B, Weltzein HU, Wekerle H,
Yoshinaga SK, Whoriskey JS, Khare SD, Sarmiento U, Guo J,
Horan T, et al. T-cell co-stimulation through B7RP-1 and ICOS.
Nature 1999; 402: 827±32.
Yoshinaga SK, Zhang M, Pistillo J, Horan T, Khare SD, Miner K,
et al. Characterization of a new human B7-related protein: B7RP-1
is the ligand to the co-stimulatory protein ICOS. Int Immunol 2000;
12: 1439±47.
Received October 29, 2002. Revised November 27, 2002.
Accepted November 11, 2002