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*
Department of Neuroimmunology, Max-Planck Institute of Neurobiology, D-82152 Martinsried, Germany; and
Department of Neurology, Klinikum Grosshadern, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or TNF-
, human myoblasts selectively express BB-1, but not
B7.1 or B7.2. BB-1 is detected by anti-BB-1, a mAb cross-reacting
with B7.1 (but not B7.2) and an as yet undefined costimulatory
molecule. The absence of B7.1 and B7.2 in BB-1-positive myoblasts was
confirmed by RT-PCR. The molecule detected by anti-BB-1 is
functional, because anti-BB-1 mAb and CTLA4Ig (but not
anti-B7.1- or anti-B7.2-specific mAbs) completely inhibit Ag
presentation by cytokine-induced myoblasts to HLA-DR-matched
Ag-specific CD4+ T cell lines. Stimulation of myoblasts
with IL-4 induces B7.1 and B7.2, as well as BB-1, but with different
time kinetics. Stimulation of CD40-positive myoblasts with
anti-CD40 mAb selectively induces BB-1, whereas stimulation with
CD40L-transfected mouse L cells induces BB-1 and B7.1, with different
kinetics. To assess whether BB-1 is expressed in muscle tissue, we
investigated 23 muscle biopsy specimens from patients with
polymyositis, dermatomyositis, inclusion body myositis, Duchenne
muscular dystrophy, and nonmyopathic controls by immunohistochemistry
and confocal laser microscopy. We found that, in all inflammatory
myopathy cases, but not in normal muscle, many muscle fibers strongly
react with anti-BB-1. In contrast, muscle fibers did not react with
B7.1- or B7.2-monospecific mAbs in any of the pathologic specimens or
in normal muscle. Our results demonstrate that human muscle cells can
be induced to selectively express BB-1, a functional costimulatory
molecule distinct from B7.1 and B7.2. This molecule may play an
important role in the immunobiology of muscle. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that muscle cells can actively participate in local immune reactions (1). Notably, human myoblasts can present Ags to CD4+ T cells (8). However, little is known about the costimulatory molecules expressed by muscle cells in vivo and in vitro. Here, we report that human myoblasts can be induced to selectively express BB-1, a functional CTLA4Ig3-binding costimulatory molecule that is distinct from B7.1 (CD80) and B7.2 (CD86) (9, 10, 11).
Our evidence for the differential expression and regulation of the different B7 molecules in muscle cells relies on functional, immunocytochemical and RT-PCR studies of cultured myoblasts. Furthermore, we investigated human muscle biopsy specimens for expression of the B7 family of costimulatory molecules, using mAbs specific for B7.1 (12) or B7.2 (13), and anti-BB-1 (9), a mAb that cross-reacts with B7.1 and a hitherto undefined member of the B7 family (10, 11). We observed that, whereas normal muscle fibers do not express B7 molecules, there is abundant expression of BB-1, but not B7.1 or B7.2, in different inflammatory myopathies. Based on these observations, we propose that BB-1 plays an important role in immune reactions in muscle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human myoblasts were isolated from muscle obtained for
diagnostic reasons from patients with suspected myopathy. For in vitro
experiments, myoblasts were obtained from nonmyopathic tissue.
Myoblasts were cultured as previously described (8) with some
modifications. Briefly, muscle specimens were mechanically dissociated
and passed through a steel sieve (Tissue Grinder Kit, Sigma,
Deisenhofen, Germany). The homogenate was digested in
trypsin-EDTA-solution (Life Technologies, Eggenstein, Germany) for 30
min at 37°C. The resulting suspension was centrifuged (5 min, 1500
rpm) and washed with PBS. The cells were transferred into plastic
tissue culture flasks (Falcon, Heidelberg, Germany) and incubated in
skeletal muscle cell growth medium (modified MCDB 120 supplemented with
5% FCS, 10 ng/ml epidermal growth factor (EGF), 1 ng/ml basic
fibroblast growth factor (bFGF), 0.5 mg/ml fetuin, 0.1 mg/ml insulin,
0.4 µg/ml dexamethasone, 50 µg/ml gentamicin sulfate, 50 ng/ml
amphotericin B; PromoCell, Heidelberg, Germany). After 30 min,
nonadherent cells were removed and seeded in new tissue flasks coated
with poly-L-lysine (Sigma) and laminin (kindly provided by
Dr. H. Neumann, MPI of Neurobiology, Martinsried, Germany) and cultured
at 37°C at 5% CO2 in skeletal muscle cell growth medium
(see above). The myoblasts were repeatedly purified during the culture
period and 1 wk before experiments, using a magnetic cell separation
system (Dynal, Hamburg, Germany) and anti-CD56 mAb (8)
(NCAM/Leu-19; Becton Dickinson, Heidelberg, Germany). Purity was
checked by FACS analysis. Only >95% pure cultures were used in all
experiments. The cultured myoblasts do not contain professional APC
(macrophages or dendritic cells), since they are are negative for
CD11b, CD14, and VCAM by flow cytometry and do not express B7.1 or B7.2
by RT-PCR. However, a small proportion (<5%) of NCAM-negative
fibroblasts may be present. To exclude the possibility that fibroblasts
also express BB-1, we isolated and expanded the NCAM-negative cell
population and treated them with IFN- or TNF-, alone or in
combination. The fibroblasts remained negative for B7.1, B7.2, and BB-1
by flow cytometry and did not transcribe B7.1 or B7.2 mRNA by RT-PCR.
The HLA-DR type of the myoblasts was determined by Drs. E. Albert and S. Scholz (Labor für Immungenetik, Ludwig-Maximilians-Universität, München, Germany), using PCR and sequence-specific oligonucleotide primers (Dynal).
Culture of CD40L-transfected mouse fibroblasts
CD40L-transfected Ltk cells (kindly provided by Dr. S. Lebecque, Schering-Plough, Dardilly, France) were cultured in plastic tissue culture flasks (Falcon) at 5% CO2 as described previously (14). The culture medium consisted of 45% DMEM, 45% F10-Nutrient Mix (HAM) with Glutamax-I, 10% FCS, 10 mM HEPES, and 50 µg/ml penicillin/streptomycin (all from Life Technologies). For coculture experiments, 3 x 105 myoblasts were incubated with the same number of irradiated (100 Gy) CD40L transfectants. Expression of surface molecules was determined by FACS analysis at 24, 48, and 72 h after initiation of cultures. The expression of CD40L on transfected L cells and CD40 on myoblasts was checked and confirmed in all experiments. The CD40L transfectants do not express B7.1, B7.2, or CD40. Myoblasts are CD40L negative (data not shown).
Monoclonal Abs
The source and working concentrations of the mAbs used as primary Abs for flow cytometry are as follows: anti-CD11b and anti-CD14 (IgG; 10 µg/ml; Becton Dickinson); anti-VCAM (IgG; 10 µg/ml; Novacastra, Dossenheim, Germany); anti-NCAM (Leu-19; IgG; 1:100; Becton Dickinson); anti-MHC class II (L243; hybridoma supernatant; 10 µg/ml; American Type Culture Collection (ATCC), Manassas, VA); anti-B7.1 (L307; IgG; 10 µg/ml; M. Azuma, Tokyo, Japan (12)); anti-B7.2 (IT.2; IgG; 10 µg/ml; M. Azuma or PharMingen, Hamburg, Germany (13)); anti-BB-1 (IgM; 10 µg/ml; NatuTec, Frankfurt, Germany or PharMingen); anti-CD40 (Mab89; IgG; 10 µg/ml; S. Lebecque); and anti-CD40 CEA-5; IgG; 10 µg/ml; NatuTec). For controls, isotype-matched irrelevant mAbs were substituted at identical concentration (mouse anti-DNP; IgG or IgM; PharMingen). Dichlorotriazinyl-fluoresceine (DTAF)-conjugated goat F(ab')2 anti-mouse IgG and IgM were used as secondary Ab (1:150; Immunotech, Hamburg, Germany). Fusion protein human (h) CTLA-4 fused with mouse IgG2a (Ancell, Läufelfingen, Switzerland) was used at 10 µg/ml. This protein was detected with FITC-conjugated goat anti-mouse Ig heavy and light chain (Immunotech).
For inhibition experiments, azide-free anti-MHC II, anti-B7.1, anti-B7.2, anti-BB-1, and CTLA4Ig (Ancell) were used at 5–50 µg/ml. Mouse IgM anti-DNP mAb was used as an isotype control for anti-BB-1.
For immunohistochemistry, anti-BB-1 was used at 10 µg/ml. Anti-MHC class I (W6/32; IgG; hybridoma supernatant; ATCC) and class II mAbs were used at 2 µg/ml. Cy3 F(ab')2 goat anti-mouse IgG and IgM were used at 2 µg/ml as the secondary reagent.
Cytokine induction
For analysis of inducible surface molecules and coculture
experiments, myoblasts were cultured in the presence of 100 U/ml
rIFN- (Boehringer, Mannheim, Germany) and/or 250 U/ml TNF-
(provided by Drs. U. Traugott and M. Müller-Neumann,
BASF/Knoll, Ludwigshafen, Germany) or 500 U/ml rIL-4 (PromoCell) for
the time indicated. These concentrations were found to induce maximal
expression of inducible surface Ag. Expression was monitored at 24, 48,
and 72 h following cytokine induction. For functional studies in
cocultures, myoblasts were induced for 72 h with IFN- and
TNF-, or for 24 h with IL-4.
Ag-specific T cell lines
PBMC were stimulated with a synthetic peptide of human myelin basic protein (MBP; sequence 86–105; synthesized by Luis Moroder, MPI for Biochemistry, Martinsried, Germany), and CD4+ Ag-specific T cell lines were isolated using the split-well cloning technique as described previously (8). Every 2 wk, T cell cultures were restimulated with Ag plus irradiated (50 Gy) autologous or HLA-DR-matched PBMC. Ag-specific proliferation was measured by [3H]TdR incorporation. For coculture experiments with myoblasts, the MBP-specific T cell lines SS014 and SS018 were used between days 13 and 15 after the last restimulation with peptide Ag.
Coculture experiments of myoblasts and T cells
Myoblasts were plated in 96-well flat-bottom plates (Costar,
Bodenheim, Germany) at a density of 2 x 104
cells/well and induced for 72 h with the indicated cytokines. In
previous experiments, a cell density of 1–2 x 104
myoblasts per flat-bottom well was found optimal for T cell
stimulation, because the myoblasts grow essentially confluent at this
seeding density (8). After induction, myoblasts were irradiated with 50
Gy, washed in RPMI 1640 (Life Technologies) and cultured in the
presence or absence of Ag and 2 x 105 HLA-DR-matched
or autologous Ag-specific T cells. Irradiated PBMC, isolated from blood
using standard density gradient centrifugation (Lymphoprep, Nycomed,
Oslo, Norway), were used as professional APC in control experiments. T
cell proliferation was measured by [3H]TdR incorporation
in a gas scintillation counter (Matrix 96 Direct Beta Counter, Packard,
Frankfurt, Germany). It should be noted that the absolute counts
measured by this "dry" scintillation system are only 
20% of the
counts obtained by liquid scintillation (15); variability and
proportions of the measured values are identical with both methods. Ag
was used at a concentration of 5 µg/ml. Abs were used at the
indicated concentrations (see mAb section).
RNA extraction, cDNA synthesis, and PCR
Total cellular RNA was extracted using an RNA extraction kit
(Qiagen, Hilden, Germany). The RNA was subsequently treated with DNase
I (Boehringer) for 15 min at 37°C to digest any potentially
contaminating genomic DNA and afterward heated to 95°C for 5 min to
inactivate the enzyme. RNA (100 ng) was reverse transcribed using
random hexamer primers (Boehringer) and SuperScript TM Reverse
Transcriptase (Life Technologies). Single-stranded cDNA was amplified
in a thermal cycler (Biometra Personal Cycler, Biotron,
Göttingen, Germany). All reactions were conducted in a total
volume of 50 µl containing 2.5 U Taq Polymerase
(Perkin-Elmer, Applied Biosystems, Weiterstadt, Germany), 200 µM of
each dNTP (Roth), and 50 pmol of each primer. The individual cycles of
hot start PCR, after an initial denaturation of 5 min at 93°C,
consisted of 1 min denaturation at 94°C, 1 min annealing at 60°C or
63°C (B7.1a), and 1 min extension at 72°C, followed by a 10-min
extension step at 72°C. RNA from normal PBMC, which was not reverse
transcribed, was used as a negative control in all PCR experiments. A
25-µl aliquot of the PCR reaction was eletrophoretically separated on
a 1.7% agarose gel (Roth) using standard conditions. UV-induced
fluorescence of ethidium bromide was used for visualization of
amplified products. The correct size of the bands was determined by
comparison with a DNA mass standard (
-X 174, Life Technologies; and
pUC8, MBI Fermentas, St. Leon-Rot, Germany). All PCR products were
sequenced (Medigene, Martinsried, Germany).
The primer sequences were as follows: ß-actin (accession no. M10277 (16)) forward, 5'-CCACACCTTCTACAATGAGC-3' (exon 3, position 1485–1504); ß-actin reverse, 5'-ACAGCCTGGATAGCAACGTA-3' (exon 4, position 2062–2081).
The primers for B7.1, B7.2, and CD40 (accession nos. M27533, L25259, and HSCDW40) were designed using published cDNA sequences (17, 18), as follows: B7.1a forward, 5'-GTGGCAACGCTGTCCTGTGGT-3' (cDNA-position 450–471); B7.1a reverse, 5'-CCAGGAGAGGTGAGGCAC-3' (cDNA-position 842–825); B7.2 forward, 5'-CCAAAGCCTGAGTGAGCTAGT-3' (cDNA position 270–291); B7.2 reverse, 5'-CTTAGGTTCTGGGTAACCGTG-3' (cDNA position 630–610); CD40 forward, 5'-TGGGGCTGCTTGCTGACCGC-3' (cDNA-position 78–97); CD40 reverse, 5'-CCAAAGCCGGGCGAGCATGA-3'(cDNA-position 436–417).
To rule out the presence of an unusual splice variant of B7.1 in myoblasts, two additional primer pairs (B7.1b and -c) were used for amplification of B7.1: B7.1b forward, 5'-ACTGGCAAAAGGAGAAGAAA-3' (cDNA position 511–530); B7.1b reverse, 5'-ATACAGGGCGTACACTTTCC-3' (cDNA position 1182–1163) (19); B7.1c forward, 5'-CCTAAGAATTCGAAGCCATGGGCCACACACCGAGG-3' (cDNA position 301–335); B7.1c reverse, 5'-TGGGCGCAGAGCCAGGATCA-3' (cDNA position 642–622) (20). Also, with these additional primers, only PBMC but not myoblasts were found to transcribe B7.1. The PCR products obtained from PBMC were confirmed as B7.1 by sequence analysis.
Muscle biopsy specimens for immunohistochemistry
Diagnostic muscle biopsy specimens were obtained from patients with inflammatory myopathies (six polymyositis, five dermatomyositis, six inclusion body myositis) and degenerative muscle disease (three Duchenne muscular dystrophy); there were three nonmyopathic controls.
Confocal laser microscopy
Twenty-micrometer serial cryostat muscle sections were analyzed. Optical sections were acquired in 0.5-µm steps along the z-axis with a confocal laser scanning microscope equipped with x40 oil objectives (Leica TCS 4D, Nussloch, Germany). Baseline labeling was revealed with irrelevant mAbs and secondary fluorochrome Cy3-conjugated goat anti-mouse Ig. Fluorescence intensity signals were converted into different colors.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and/or
TNF-, b) with IL-4, and c) with cross-linking anti-CD40 mAb or
CD40L-transfected cells. In all cases, B7 transcription and protein
expression were investigated by RT-PCR and FACS analysis. The
functional role of B7 molecules induced in myoblasts was investigated
in cocultures of myoblasts with MHC-class II-matched Ag-specific
CD4+ T cell lines. All functional experiments were
performed with highly purified NCAM-positive human myoblasts (8) (see
Materials and Methods).
Myoblasts stimulated with IFN- or TNF- selectively express
BB-1, a CTLA4Ig-binding costimulatory molecule distinct from B7.1 and
B7.2
For detection of B7 molecule expression, we used mAbs
against B7.1, B7.2, and an mAb named BB-1, which cross-reacts with B7.1
and an as yet undefined costimulatory molecule (9, 10, 11). Without
cytokine treatment, myoblasts did not express B7.1 or B7.2 (Fig. 1
). Myoblasts treated with TNF- or
IFN-, alone or in combination, remained negative for B7.1 and B7.2,
as analyzed by flow cytometry and RT-PCR (Fig. 2, A and B). In
contrast, BB-1 was strongly induced after treatment of myoblasts with
TNF- or IFN-, alone or in combination (Fig. 2A).
Induction of BB-1 was maximal after 72 h of cytokine treatment.
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C. Also, with the additional
B7.1 primers, a PCR product was obtained only from PBMC. Sequencing of
the PCR products confirmed that they correspond to B7.1. Together with
results from another group who used yet another set of B7.1 primers
(21), our data suggest that B7.1 or B7.1 splice variants are absent in
myoblasts. Myoblasts induced with IL-4 express B7.1, B7.2, and BB-1 with different time kinetics
Next we assessed whether treatment with IL-4 can induce B7.1 or
B7.2 in myoblasts. IL-4 has previously been reported to induce B7.1 or
B7.2 in B cells, Langerhans cells, and keratinocytes (22, 23, 24). FACS
analysis showed that IL-4 induces different members of the B7 family on
myoblasts with different kinetics (Fig. 3A). After 24 h,
anti-B7.1, anti-B7.2, anti-BB-1, and CTLA4Ig fusion protein
all bound to myoblasts. After 72 h, B7.1 and B7.2 expression was
reduced to background, whereas BB-1 and CTLA4Ig still bound strongly
(Fig. 3A). Consistent with the FACS results, RT-PCR analysis
showed that IL-4-induced myoblasts transcribe mRNA for B7.1 and B7.2
(Fig. 3B). Transcription was detected after 24 and 72
h. The FACS data provide further evidence for differential regulation
of the different B7 family members in human myoblasts.
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B,
these muscle-derived fibroblasts do not transcribe B7.1 or B7.2 and are
therefore not the source of the B7 message detected in our myoblasts by
RT-PCR. Anti-CD40 stimulation differentially induces B7 molecules in myoblasts
B7 expression is known to be influenced by CD40-CD40L signaling
(25, 26, 27, 28). We therefore asked whether human myoblasts express CD40 and,
if so, whether CD40 stimulation would affect B7 expression in
myoblasts. As shown in Fig. 4,
NCAM-positive myoblasts constitutively and uniformly express CD40.
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A shows that stimulation with
mAb EA-5 induces BB-1 but not B7.1 or B7.2 after 24 and 72 h. In
contrast, a nonstimulatory anti-CD40 mAb (Mab89) (29) failed
to induce any of the B7 molecules after 24, 48, or 72 h (data not
shown). The results of RT-PCR analysis of myoblasts stimulated with mAb
EA-5 are consistent with the FACS data (Fig. 5B).
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A). B7.2 was not
expressed. BB-1 expression was maximal after 72 h. A similar
staining pattern was seen with CTLA4Ig. The PCR results are consistent
with the FACS data (Fig. 6B). Human control fibroblasts did
not transcribe B7.1 or B7.2 (Fig. 6B). The CD40L-transfected
mouse fibroblasts also did not yield a PCR signal for human B7.1 or
B7.2 (not shown).
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As previously reported, IFN-/TNF--induced human myoblasts
can act as facultative APC in vitro and present Ag to CD4+
Ag-specific T cell lines (8). To assess the functional role of BB-1 in
this system, we cocultured IFN-/TNF--double-induced myoblasts
with autologous or HLA-DR-matched Ag-specific CD4+ T cell
lines and measured Ag-induced proliferation in the presence of CTLA4Ig
or mAbs against MHC class II, B7.1, B7.2, BB-1, or MHC class I (Fig. 7C). CTLA4Ig (10 µg/ml) was
completely inhibitory (Fig. 7C). Furthermore, Ag
presentation was completely blocked in the presence of anti-MHC-II
mAbs or anti-BB-1, but not in the presence of B7.1- or
B7.2-specific mAbs (Fig. 7C). Mouse IgM anti-DNP, which
was used as an IgM isotype control for anti-BB-1, was not
inhibitory (Fig. 7C).
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A) and by myoblasts treated for 24
h with IL-4 (Fig. 7B). These functional data are consistent
with our observation that IL-4-induced myoblasts transiently express
B7.1 and B7.2 (Fig. 3A).
To corroborate the results of the functional experiments, we repeated
the experiment with the same TCL (SS014) and with another MBP-specific
CD4+ TCL (SS018). Mouse IgM anti-DNP was used as an
isotype control for anti-BB-1. As shown in Table I, the results are consistent with those
shown in Fig. 7, B and C.
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To assess whether muscle fibers express BB-1 or any other B7
molecule in muscle tissue, we treated cryostat sections of 23 muscle
biopsy specimens from patients with polymyositis, dermatomyositis,
inclusion body myositis, and Duchenne muscular dystrophy and from
nonmyopathic controls with mAbs against B7.1, B7.2, and the BB-1 Ag for
immunohistochemistry. B7.1 and B7.2 were detectable only on mononuclear
cells in inflammatory infiltrates, but never on muscle fibers. In
contrast, BB-1 was expressed on many muscle fibers in all inflammatory
myopathy cases (six polymyositis, five dermatomyositis, six inclusion
body myositis). In one case of Duchenne muscular dystrophy, BB-1 was
expressed on a few scattered inflammatory cells, but not in muscle
fibers. BB-1 was absent in nonmyopathic controls. In the inflammatory
cases, the distribution of BB-1 was similar to HLA class I (Fig. 8). (It should be noted that HLA class I
and II Ags are not detectable on normal muscle fibers in situ (1, 4)).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The costimulatory molecules B7.1 (CD80) and B7.2 (CD86) are members of the Ig superfamily (30, 31, 32, 33, 34). They are expressed mainly on professional APCs and T cells. Although their precise function(s) and relationship with the ligands CD28 and CTLA-4 are still not completely understood, there is universal agreement that the B7 molecules provide critical costimulatory signals to T cells and play an essential role in normal and pathologic immune reactions (30, 31, 32, 33, 34, 35).
The genes for murine and human B7.1 and B7.2 have been cloned (13, 17, 18, 36). It has long been suspected, however, that additional functional members of the B7 family exist. Indeed, discordant expression of BB-1 and B7 has been noted for activated human B lymphocytes (10) and keratinocytes (37). Furthermore, an alternative CTLA-4-binding molecule was identified on mouse B cells (38). Our present results provide strong additional support for the existence of at least one additional functional member of the B7 family of costimulatory molecules. Presently, our evidence for the differential expression and regulation of the different B7 molecules in muscle cells relies on the results of immunocytochemical and RT-PCR studies, and, perhaps most significantly, on functional experiments. Our results will provide a basis for future experiments to identify the postulated B7/BB-1 molecule at the molecular level. One problem that needs to be overcome is that only relatively small numbers of human myoblasts can be expanded from diagnostic muscle biopsy tissue, whereas large amounts of purified, cytokine-induced myoblasts will be needed for immunoprecipitation, Western blotting, and protein sequencing studies.
We isolated and cultured human myoblasts from adult muscle biopsy tissue, using standard protocols (8, 39). Myoblasts express the neural cell adhesion molecule NCAM, which serves as a convenient selection marker for magnetic bead sorting (8, 40). Our myoblast preparations are >95% NCAM positive. They do not contain professional APC (macrophages or dendritic cells), since they are negative for CD11b, CD14, and VCAM by flow cytometry and do not constitutively express B7.1 or B7.2 by RT-PCR and FACS analysis. However, a small proportion of NCAM-negative fibroblasts may be present. Therefore, we isolated and expanded the NCAM-negative cell population and used them as a control. After stimulation with cytokines, anti-CD40 mAb, or CD40L-transfected L cells, the human fibroblasts remained negative for B7.1, B7.2, and BB-1 by flow cytometry and did not transcribe B7.1 or B7.2 mRNA by RT-PCR. Therefore, the B7 transcripts and protein detected in our myoblast preparations could not have been derived from potentially contaminating NCAM-negative fibroblasts.
Our study reveals that, in myoblasts, B7.1, B7.2, and BB-1 are
differentially regulated and expressed. Stimulation with IFN- and/or
TNF- selectively induces BB-1, but not B7.1 or B7.2. Recently, the
absence of expression of B7.1 and B7.2 in human myoblasts has also been
reported by other groups (21, 41). To exclude the possibility that BB-1
and CTLA4Ig recognize a splice variant of B7.1 on cytokine-induced
myoblasts, we used three different primer pairs for RT-PCR
amplification of B7.1. In all cases, B7.1 was absent from myoblasts
(Fig. 2, B and C). These results argue against
the expression of an alternative splice form of B7.1 and support the
notion that myoblasts can be induced to express a hitherto undefined B7
molecule recognized by anti-BB-1 and CTLA4Ig.
In contrast, stimulation with IL-4 induces B7.1 and B7.2, as well as BB-1, but with different kinetics. This is consistent with previous reports that IL-4 can induce B7.1 and B7.2 in B cells, Langerhans cells, and keratinocytes (Refs. 22–24 and 42; reviewed in 30 .
The CD40/CD40L pathway is known to play an important role in controlling B7.1 and B7.2 expression (Refs. 25–28; reviewed in Refs. 29, 43, and 44). Because we observed that myoblasts constitutively express CD40, we were interested to see whether stimulation with anti-CD40 mAb or CD40L-transfected cells can induce BB-1 or other B7 molecules in myoblasts. Again, we found evidence for selective expression and transcription of the different B7 molecules. Stimulation with anti-CD40 mAb selectively induces BB-1, but not B7.1 or B7.2. In contrast, stimulation with CD40L-transfected cells induces B7.1 and BB-1, but with different kinetics.
As we have shown previously, myoblasts treated with IFN- can present
different protein Ag to Ag-specific CD4+ HLA-DR-matched T
cell lines (8). We used this system to investigate the functional role
of the B7 molecules inducible on myoblasts. The results show that BB-1,
which is selectively induced by treatment of myoblasts with
IFN-/TNF-, provides a costimulatory signal to T cells.
Proliferation of Ag-specific CD4+ T cells is completely
blocked in the presence of anti-BB-1 mAb or CTLA4Ig, but not
anti-B7.1 or -B7.2 mAbs. Of importance, the IFN-/TNF--induced
myoblasts do not express B7.1 and B7.2 by FACS analysis (Fig. 2A), nor do they transcribe B7.1 or B7.2 mRNA by RT-PCR
(Fig. 2, B and C). The selective expression of
BB-1 allows functional studies of BB-1 in isolation, without
coexpression of B7.1 and B7.2 as it is observed in most other systems.
By comparison, myoblasts treated with IL-4 for 24 h coexpress
B7.1, B7.2, and BB-1 (Fig. 3). In this case, Ag presentation to
CD4+ T cells was inhibited not only by anti-BB-1 mAb,
but also by anti-B7.1 and anti-B7.2 mAbs (Fig. 7B).
To investigate whether our in vitro observations with cultured myoblasts are relevant in vivo, we studied the expression of B7 molecules on mature muscle fibers in muscle biopsy tissue. Muscle fibers are highly differentiated large syncytial cells that contain hundreds of nuclei. It would therefore not be surprising if the regulation of surface molecules such as B7 was different in muscle fibers as compared with mononuclear myoblasts. For example, whereas cultured human myoblasts constitutively express MHC class I (8), mature muscle fibers in muscle tissue are MHC class I negative, as assessed by immunohistochemical staining (1, 4, 5, 45). However, MHC class I molecules are strongly up-regulated on muscle fibers in inflammatory myopathies (1, 4, 5, 45).
This group of diseases includes dermatomyositis, different forms of polymyositis, and inclusion body myositis (1, 4, 5, 45, 46, 47). In dermatomyositis, muscle fiber injury is thought to be secondary to an Ab- or immune complex-mediated response against a vascular-endothelial component. In polymyositis and inclusion body myositis, CD8+ T cells and macrophages invade and destroy MHC-class I-positive muscle fibers. The autoaggressive CD8+ T cells are clonally expanded (48). They harbor granules, containing perforin, that aggregate near the contact zone with the target muscle fiber, suggesting a perforin- and secretion-dependent mechanism of muscle fiber injury (49).
We investigated B7 expression in 23 muscle biopsy specimens from
patients with polymyositis, dermatomyositis, inclusion body myositis,
and Duchennne muscular dystrophy and from nonmyopathic controls, using
mAbs directed against B7.1, B7.2, and BB-1 for immunohistochemistry and
confocal laser microscopy. B7.1 and B7.2 were expressed on inflammatory
cells in inflammatory myopathy tissue, but not on muscle fibers. In
contrast, BB-1 was strongly expressed on many muscle fibers in the
inflammatory myopathies, but not in nonmyopathic control tissue. The
distribution of BB-1 resembles that of MHC class I (Fig. 8).
The induced and selective expression of BB-1 in inflammatory myopathy tissue is remarkably consistent with the selective induction and regulation of BB-1 in cultured myoblasts. Since many BB-1+ fibers are contacted by inflammatory cells, it is likely that both MHC class I and BB-1 are up-regulated under the influence of proinflammatory cytokines secreted by inflammatory T cells and macrophages. It seems that BB-1 plays an important role in the immunobiology of muscle and, hence, may be a target for therapeutic intervention in autoimmune muscle diseases.
It should be noted, however, that the significance of BB-1 expression in muscle reaches beyond its implications for the pathogenesis and treatment of inflammatory myopathies. Muscle is the site for many other immune reactions, including muscle infections, graft-vs-host disease, conventional intramuscular vaccination, and, recently, intramuscular gene transfer by injection of genetically engineered myoblasts or naked DNA (1, 3, 6, 7, 50). In all these reactions, the induced expression of BB-1 on muscle fibers should play an important role.
Note added in proof. After submission of our manuscript it was
reported that mAb BB-1 reacts with MHC class II-associated invariant
chain CD74 (51). Our results with human muscle cells cannot be
explained by the CD74 cross-reactivity of BB-1. First, B7.1/B7.2
double-negative myoblasts stained with BB-1 and CTLA4Ig (Fig. 2).
Second, the costimulation of cultured T cells by B7.1/B7.2
double-negative, BB-1-positive myoblasts was inhibited not only with
BB-1 but also with CTLA4Ig (Fig. 7). Third, the anti-CD74 mAb BU-45
(kindly provided by Drs. G. Moldenhauer and G. J. Hämmerling,
Deutsches Krebsforschungszentrum, Heidelberg, Germany) did not stain
cultured myoblasts in FACS analysis or muscle fibers in tissue
sections, whereas BB-1 did (our unpublished results).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Reinhard Hohlfeld, Department of Neuroimmunology, Max-Planck Institute of Neurobiology, D-821521 Martinsried, Germany. E-mail address:
3 Abbreviations used in this paper: CTLA4Ig, CTL-associated protein 4 Ig; MBP, myelin basic protein; NCAM, neural cell adhesion molecule; TCL, T cell line.
Received for publication April 23, 1998. Accepted for publication July 30, 1998.
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secretion, and cytotoxicity. J. Neuroimmunol. 86:53.[Medline]
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