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Institute of Rheumatology, Tokyo Women’s Medical University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, which is known to induce CD40 expression on various
types of cells, was also expressed on the MNCs in four of the PM and
four of the DM patients. Although cultured human myoblasts (SkMC 2859)
did not express CD40 constitutively, IFN- induced CD40 expression in
a dose-dependent manner. To clarify the functional roles of
CD40-mediated signals, the effects of a trimeric form of recombinant
human CD40L on cytokine production were studied in SkMC 2859 that were
prestimulated with IFN- to express CD40. Recombinant human CD40L
markedly increased the production of IL-6, IL-8, IL-15, and monocyte
chemoattractant protein-1 of SkMC 2859. The expression of these humoral
factors in muscle cells of PM and DM was demonstrated by
immunohistochemistry. These results suggest that interaction between T
cells and muscle cells via the CD40-CD40L system contributes to the
immunopathogenesis of PM/DM by augmenting inflammation via cytokine
production by the muscle cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to allogenic myoblasts upon
costimulation with anti-CD28 Ab (8). These results
suggest the importance of T cells in the pathogenesis of IIMs. On the
other hand, muscle cells of IIMs constitutively express various cell
surface molecules, such as CD54, CD106, MHC class I and II Ags, and
apoptosis-related molecules (i.e., CD95) (3, 9, 10, 11, 12). A
recent study (13) showed that muscle cells in IIMs express
BB-1, as do cultured myoblasts after stimulation with IFN-, TNF-
,
IL-4, or CD40L transfectant. Because these molecules are not expressed
on muscle cells of normal healthy controls and their counter-receptors
are expressed on infiltrating T cells in IIMs, they might mediate
interaction between T cells and muscle cells and provide signals for
regulation of muscle cells in the disorders.
CD40 is a 50-kDa type I cell surface molecule originally identified on
B cells and some epithelial carcinomas (14, 15) that
interacts with CD40L expressed on activated T cells (16, 17). In B cells, signals mediated by CD40-CD40L interaction
induce B cell proliferation, differentiation, and Ig production
(14, 15, 16, 17) and also rescue B cells from apoptosis
(18). Recent studies have shown that nonlymphoid cells
such as fibroblasts (19, 20, 21), epithelial cells
(22), and endothelial cells (23, 24, 25) also
express CD40. In vitro, CD40 ligation results in up-regulation of
several cell surface molecules and cytokine production in these cells.
In the present study we demonstrate that 1) muscle cells of PM/DM
express CD40 and muscle-infiltrating mononuclear cells (MNCs) express
CD40L; 2) CD40 is induced on cultured myoblasts by IFN- stimulation;
and 3) CD40 ligation increases IL-6, IL-8, IL-15, and monocyte
chemoattractant protein-1 (MCP-1) production by myoblasts. The results
are discussed in terms of the implication of the roles of CD40-CD40L
interaction in the immunopathogenesis of PM/DM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Muscle specimens were obtained from five patients with PM and
five patients with DM. The patients’ profiles are summarized in Table I
. All subjects fulfilled the diagnostic
criteria of Bohan and Peter (26). None was receiving any
immunosuppressive treatment at the time of muscle biopsy. As normal
controls, we chose three individuals who were suspected to have muscle
disorders, but whose biopsied muscle tissues showed normal histologic
findings. The ages of the PM patients, DM patients, and normal controls
were 57 ± 4.8, 56 ± 14.6, and 49 ± 7.8 years,
respectively.
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Four-micron thick sections were air-dried and fixed in 2%
paraformaldehyde for 30 min at 4°C. Immunohistochemical staining was
performed using a commercial kit (Vectastain Universal Quick Kit,
Vector, Burlingame, CA). Briefly, all sections were incubated with
2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40
(Mab89, 10 µg/ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin
receptor agonist peptide 1 (TRAP1), 10 µg/ml, mouse IgG1; Coulter),
IL-15 (10 µg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1
(10 µg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for
IFN- (5 µg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5
µg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min. Mouse IgG1
(Dako), rabbit IgG (Dako), and goat IgG (Dako) were used as control
Abs. After being washed with PBS, the sections were incubated for 10
min with horse biotinylated secondary Ab, which commonly recognizes
mouse, rabbit, and goat IgG. After an extensive wash with PBS, all
sections were exposed to a streptavidin/peroxidase-preformed complex
for 5 min and then covered with diaminobenzidine tetrahydrochloride for
2 min. All sections were counterstained with hematoxylin.
Double staining was performed with biotinylated mouse mAb and nonlabeled mouse mAb. Briefly, sections were fixed, blocked with 2.5% normal goat serum, and incubated with 10 µg/ml of anti-CD40L Ab (TRAP1) or control mouse IgG1 for 30 min. They were then washed, incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (10 µg/ml; BioSource, Camarillo, CA) for 30 min, and visualized with phosphate substrate (Vector). Next, all sections were incubated first with 10 µg/ml of mouse IgG1 for 30 min to avoid nonspecific binding and then with biotinylated anti-CD4 mAb (10 µg/ml, mouse IgG1; Coulter), biotinylated anti-CD8 mAb (10 µg/ml, mouse IgG1; Coulter), or biotinylated control mouse IgG1 (Ancell, Bayport, MN). After washing, the sections were exposed to streptavidin/peroxidase complexes and colored with diaminobenzidene. All sections were counterstained with Methyl Green (Dako).
Myoblast culture
Myoblasts of human skeletal muscle (SkMC 2859) were obtained
from BioWhittaker (Walkersville, MD) and maintained in serum-free
culture medium (QBSF 51, Sigma, St. Louis, MO). SkMC 2859 were grown to
semiconfluence and detached from culture flasks or plates with 0.25%
trypsin-EDTA (Life Technologies, Frederick, MD). SkMC 2859 were
isolated by enzymatic digestion of ventral thigh tissue of a 22-wk-old
black male fetus according to the manufacturer’s data sheet. This cell
strain is positive for sacromeric myosin, -actinin (sacromeric), and
troponin-T.
Flow cytometry
At the end of culture, SkMC 2859 were detached from culture flasks by trypsinization. In all experiments, 1 x 106 cells were treated with human Ig to block nonspecific binding. Cells were then stained with anti-CD40 mAb or control mouse IgG1 for 30 min at 4°C, washed, and incubated with fluorescein-conjugated F(ab')2 goat anti-mouse IgG (Becton Dickinson, San Jose, CA) for 30 min at 4°C. The cells were washed and fixed with 2% paraformaldehyde, and the fluorescein intensity was measured with Cytoron Absolute (Ortho-Clinical Diagnostics, Tokyo, Japan).
RT-PCR
Total cellular RNA was isolated from SkMC 2859 with TRIzol (Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol. Total RNA was reverse transcribed to cDNA using Superscript II RT (Life Technologies). For PCR, 2 µl of RT product was used in a total volume of 50 µl containing the following reagents: 1.5 mM MgCl2, PCR buffer (1x, 50 mM KCl and 10 mM Tris-HCl, pH 8.3), 0.2 mM each of dNTPs, 1 U of AmpliTaq polymerase (Roche, Mannheim, Germany), and forward and reverse primers (20 µM each). The sequences of the primers were as follows: CD40 forward, 5'-ATGGTTCGTCTGCCTCTGCAG-3'; CD40 reverse, 5'-CTGGGCAGGGCTCGCAGATGG-3'; IL-6 forward, 5'-ATGAACTCCTTCTCCACAAGCCGC-3'; IL-6 reverse, 5'-GAAGAGCCCTCAGGCTGGACTG-3'; IL-8 forward, 5'-ATGACTTCCAAGCTGGCCGTGCT-3'; IL-8 reverse, 5'-TCTCAGCCCTCTTCAAAAACTTCTC-3'; IL-15 forward, 5'-TTGTATTGTAGGAGGCAT-3'; IL-15 reverse, 5'-AAGAGTTCATCTGATCCAAGG-3'; MCP-1 forward, 5'-TTGTGTGCCTGCTGCTCATA-3'; MCP-1 reverse, 5'-GGTTTGCTTGTCCAGGTGGT-3'; ß-actin forward, 5'-AAGAGAGGCATCCTCACCCT-3'; and ß-actin reverse, 5'-TACATGGCTGGGGTGTTGAA-3'. The thermocycle conditions were 35 cycles of 95°C for 1 min (denaturing), 60°C for 30 s (annealing), and 72°C for 1 min (extension). The PCR products were electrophoresed in a 2% agarose gel and visualized with ethidium bromide.
Preparation of cell extracts
SkMC 2859 were detached by trypsinization, washed three times in PBS, and lysed in 100 µl of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% sodium deoxycholate, and 1% Nonidet P-40, containing 1 mM PMSF (Sigma) and 0.2 U/ml aprotinin (Sigma). The extracts were centrifuged at 5000 x g, and the supernatants were used for ELISA.
ELISA
To determine the concentrations of cytokines in the culture supernatants or cell extracts, a specific ELISA was developed for each cytokine using specific Abs. For IL-6, IL-15, and MCP-1, ELISA plates (Costar, Cambridge, MA) were preabsorbed with specific capture Abs for IL-6 (1 µg/ml, rat IgG; PharMingen, San Diego, CA), IL-15 (2 µg/ml, mouse IgG; R & D Systems) or MCP-1 (2 µg/ml, mouse IgG; PharMingen, San Diego, CA). After overnight incubation at 4°C, the plates were washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 10% FCS for 2 h at room temperature. After more washing, serial dilutions of recombinant cytokines or samples were applied to the plates in duplicate. Recombinant IL-6, IL-15, and MCP-1 were purchased from R&D Systems. After overnight incubation at 4°C, the plates were washed three times, then incubated with biotin-conjugated Abs for IL-6 (rat monoclonal, 0.5 µg/ml; PharMingen), IL-15 (mouse monoclonal, 200 ng/ml; R&D Systems), or MCP-1 (rabbit polyclonal, 1 µg/ml; PharMingen) for 45 min at room temperature. All plates were then incubated with avidin-peroxidase (10 mg/ml, Sigma) for 30 min at room temperature. An ABTS peroxidase substrate mixture (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to each well, and the absorbance was measured with an ELISA plate reader (Immuno Mini NJ-2300, Nippon Intermed, Tokyo, Japan) with a test wavelength of 414 nm and a reference wavelength of 490 nm. The sensitivities of the assay were 7.81, 7.81, and 31.3 pg/ml for IL-6, IL-15, and MCP-1, respectively.
For assessment of IL-8, ELISA plates were coated with anti-IL-8 mAb (1 µg/ml, mouse IgG; R&D Systems) overnight at 4°C. The plates were blocked with Tris-HCl containing 2% BSA (Sigma) for 2 h, and then rhIL-8 (R&D Systems) or diluted samples were applied. After an overnight incubation at 4°C, the plates were incubated with polyclonal rabbit anti-IL-8 Ab (2 µg/ml; Endogen, Woburn, MA) for 2 h at room temperature. Plates were then incubated with a 1/2000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (BioSource) for 1 h and reacted with a phosphate substrate (1.0 mg/ml of p-nitrophenylphosphate; Sigma). Absorbance was read at 405 nm (test wavelength) to 620 nm (reference wavelength). The sensitivity of the IL-8 ELISA was 15.6 pg/ml.
CD40 ligation on SkMC 2859
Semiconfluent SkMC 2859 were stimulated with 100 U/ml of IFN-
(PharMingen) for 48 h to induce CD40 expression. Then, the plates
were washed three times with PBS, and the medium was changed to contain
various concentrations of trimeric human CD40L-leucine zipper fusion
protein (rhCD40L, provided by Immunex, Seattle, WA). Forty-eight hours
after CD40 ligation, the culture supernatants and cell extracts were
subjected to ELISA. To confirm the specificity of rhCD40L, an
anti-human CD40L mAb, TRAP1, or control mouse IgG1 (Cappel, Aurora,
OH) was added to the culture of IFN--treated SkMC 2859 along with
rhCD40L.
Statistical analysis
Comparison of data, presented as the mean ± SEM, was performed using unpaired Student’s t test as indicated. p < 0.05 was considered significantly different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in PM/DM
Immunohistochemistry revealed the sarcolemma of muscle cells in
inflammatory foci to express CD40 in all the PM/DM patients except
patient 10 (Fig. 1A and Table II). The percentage of fibers positive
for CD40 was 11.6 ± 7.6% in PM and 15.1 ± 15.6% in DM
(Table II). Infiltrating MNCs and endothelial cells of capillaries and
venules were also stained by the anti-CD40 Ab (Fig. 1A).
CD40L was expressed on infiltrating MNCs surrounding muscle cells in
all cases of PM/DM, but not in the muscle fibers (Fig. 1B
and Table II). In patient 10 the MNCs infiltrating muscle tissue showed
relatively mild staining for CD40L, and the serum CK (creatine kinase)
level at biopsy was low (Table I
), which may explain the absence of
CD40 expression on muscle fibers in this case. The percentages of
CD40L-positive MNCs was 19.5 ± 15.4% in PM and 16.8 ±
12.4% in DM. IFN- is known to induce or up-regulate CD40 expression
on various types of cells. As shown in Fig. 1C and Table II,
IFN- was expressed on infiltrating MNCs in endomysial and
perivascular areas in 8 of the 10 PM/DM patients. Serial section
analysis showed that these IFN--expressing MNCs localized close to
CD40-positive muscle cells and CD40L-expressing MNCs (data not shown).
The percentage of MNCs expressing IFN- was 6.9 ± 12.7% in PM
and 10.2 ± 12.5% in DM. In normal individuals, CD40 was detected
on some endomysial MNCs, but not on muscle cells. Neither CD40L nor
IFN- was expressed in the control tissues. These CD40L and/or
IFN--expressing MNCs were localized predominantly in endomysial
areas in both PM and DM, although a small number was also detected in
perivascular areas in DM patients (data not shown).
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Double staining was performed in two representative cases (patient
2 with PM and patient 6 with DM) to clarify which cell populations
expressed CD40L (Fig. 2). At least 100
CD40L-postive MNCs were analyzed for each case. Of the CD40L-positive
MNCs, 78.8 ± 9.68% (three different high power fields) were
CD4+ T cells and 8.9 ± 3.5% were
CD8+ T cells in patient 2 (PM). In patient 6
(DM), 85.2 ± 7.8% were CD4+ T cells and
5.3 ± 4.9% were CD8+ T cells.
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induced CD40 on SkMC 2859
RT-PCR revealed that unstimulated as well as IFN--stimulated
SkMC 2859 constitutively expressed CD40 mRNA (Fig. 3A). To investigate the cell
surface expression of CD40, semiconfluent SkMC 2859 were stimulated
with various concentrations of IFN-, TNF- (R&D Systems) or
IFN- plus TNF- for 48 h, stained with anti-CD40 Ab or
control mouse IgG1, and analyzed by flow cytometry. Without
stimulation, SkMC 2859 expressed marginal levels of CD40 (Fig. 3B). IFN- induced CD40 expression in a dose-dependent
manner (Fig. 3B). The effect of IFN- became apparent at
12 h after stimulation and reached a plateau at 24 h (data
not shown). TNF- alone only weakly induced the expression of CD40,
but it synergistically enhanced CD40 expression on SkMC 2859 in
combination with IFN- (Fig. 3B). Neither IL-1,
IL-1ß, IL-4, IL-6, nor GM-CSF induced CD40 expression on SkMC 2859
(data not shown).
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-treated SkMC 2859
We next analyzed the function of CD40 on SkMC 2859.
IFN--treated SkMC 2859 were incubated with rhCD40L as described in
Materials and Methods. Forty-eight hours after CD40
ligation, the culture supernatants and cell extracts were collected,
and the concentrations of cytokines were measured by ELISA. As shown in
Fig. 4A, unstimulated SkMC
2859 constitutively secreted low levels of IL-6, IL-15, and MCP-1, but
not IL-8. Recombinant hCD40L showed marginal effects on the production
of IL-6, IL-8, IL-15, and MCP-1 by untreated SkMC 2859. IFN- alone
augmented IL-6, IL-8, IL-15, and MCP-1 production. The effects of
IFN- on the production of these cytokines were dose dependent (data
not shown). Ligation of CD40 further increased the production of these
cytokines by IFN--treated SkMC 2859 in a dose-dependent manner (Fig. 4A). It was interesting that >50% of IL-15 was detected in
the cell extracts regardless of the pretreatment with IFN-. We also
measured IL-1, IL-1ß, IL-10, GM-CSF, and TNF- in the same
culture supernatants, but their concentrations were below the
sensitivity of the ELISA (data not shown). mRNAs of IL-6, IL-8, IL-15,
and MCP-1 were detected by RT-PCR not only in IFN-- plus
rhCD40L-stimulated SkMC 2859 (Fig. 4B), but also in
unstimulated and IFN--stimulated SkMC 2859 (data not shown).
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Immunohistochemical staining for IL-6, IL-8, IL-15, or MCP-1 was
performed in four representative patients (patients 1, 2, 6, and 7) and
three normal individuals. Some of the muscle cells were stained with
anti-IL-6 mAb or anti-MCP-1 mAb in the four patients with
PM/DM, but not in the controls (Fig. 5,
A, C, F, and H).
Infiltrating MNCs also expressed IL-6 and MCP-1 in the patients. The
cytoplasm of muscle cells was strongly stained with anti-IL-15 mAb
in the four patients, although that of normal controls was only
marginally stained (Fig. 5, B and G). Anti-IL-8
Ab stained muscle cells in neither patients nor controls (data not
shown).
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To assess the specificity of the effect of rhCD40L,
IFN--treated SkMC 2859 were stimulated with 0.2 mg/ml of rhCD40L in
the presence of anti-human CD40L Ab (TRAP1) or control mouse IgG1.
After 48 h, culture supernatants and cell extracts were collected,
and the concentrations of cytokines were measured. As shown in Fig. 6, TRAP1 inhibited rhCD40L-stimulated
IL-6 production in a dose-dependent manner, while control IgG1 showed
no effect. TRAP1 (10 µg/ml) almost completely abrogated the effects
of rhCD40L. Similar results were obtained with IL-8, IL-15, and MCP-1
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have shown that CD40 is expressed on a broad range of
cells and has various physiological roles, although it was initially
thought to be expressed only on B cells and have limited functions
(27). The cross-linking of CD40 on monocytes results in
cytokine production (28) and nitric oxide synthesis
(29). Cells of nonlymphoid origin, including fibroblasts,
endothelial cells, epithelial cells, and myoblasts, also express CD40
molecules (19, 20, 21, 22, 23, 24, 25). Ligation of CD40 on fibroblasts
enhances the expression of CD54 and CD106 (19), the
production of IL-6 (19) and IL-8 (21), and
the proliferation of the cells (19). In endothelial cells,
ligation of CD40 also up-regulates the expression of CD54, CD106, and
CD68 (E-selectin) (24, 25) and increases the production of
IL-8 and MCP-1 (25). These reports suggest that CD40-CD40L
interaction induces chemotaxis and leukocyte adhesion to inflammatory
sites in vivo. There are also reports that CD40-CD40L interaction is
involved in chronic inflammatory diseases such as rheumatoid arthritis.
Macrophage-like synovial cells in the lining region strongly express
CD40, and the infiltrating T cells express CD40L (30, 31, 32).
Ligation of CD40 on synovial cells in primary culturesenhances the
production of TNF- (32) and also induces the
proliferation of synovial fibroblasts (30). Thus,
CD40-CD40L interaction should be important for the initiation and
perpetuation of chronic inflammatory disorders.
Because the function of CD40 on myoblasts remains unknown in PM/DM, we set out to investigate the effects of CD40 ligation on cytokine production by cultured normal myoblasts. For CD40 engagement, we chose a trimeric form of CD40L-leucine zipper fusion protein. This rhCD40L has been demonstrated to have biological functions equivalent to those of its membrane-bound forms (33). In our study the production of IL-6, IL-8, IL-15, and MCP-1 was enhanced by CD40 ligation. We performed CD40 ligation using myoblasts of adult human isolated from muscle tissues in the manner described in Materials and Methods and obtained results similar to those obtained with SkMC 2859 (data not shown). Hence, the results of the experiments using SkMC 2859 probably reflect real properties of myoblasts in vivo.
IL-8 and MCP-1 are cytokines that mainly induce chemotaxis of neutrophils and monocytes, respectively (34, 35). Recent studies have revealed that these chemokines induce the expression of integrins such as LFA-1 on circulating leukocytes (36, 37), which results in leukocyte adhesion to the vascular wall, extravasation, and infiltration of inflammatory foci. Specific Abs against IL-8 or MCP-1 have been shown to inhibit leukocyte infiltration in vivo (38, 39). According to our data, it is plausible that the T cells infiltrating inflammatory foci in PM/DM stimulate the production of these chemokines by myoblasts via CD40-CD40L interaction, which further induces infiltration by MNCs around target muscle cells. This positive feedback loop may be relevant to the progression and perpetuation of inflammation in PM/DM.
Another remarkable observation was that CD40 ligation induces the
production of IL-15 by myoblasts. IL-15 is a 14- to 15-kDa molecule and
a member of the 4-helix bundle cytokine family, with biological
functions similar to those of IL-2 despite the absence of significant
amino acid sequence homology. IL-15 and IL-2 share multiple biological
functions, including stimulation of the proliferation and activation of
T cells and NK cells, induction of cytotoxic effector cells, T cell
chemoattraction and proliferation, and Ig synthesis by B cells
(40, 41, 42). In contrast to IL-2, IL-15 mRNA is expressed in
various nonlymphoid tissues and cells, such as placenta, skeletal
muscle, kidney, lung, heart, fibroblasts, and monocytes, but not in
resting or activated T cells (40). IL-15 also possesses
biological functions not shared by IL-2. On muscle cells, IL-15 is
reported to exert its effect as an anabolic cytokine (43)
and to promote differentiation (44). Although IL-15 mRNA
is widely expressed, it has been difficult to demonstrate IL-15 at the
protein level in supernatants of various kinds of cell culture
(45). We established a sensitive ELISA system for IL-15
and proved for the first time that myoblasts secrete this molecule. It
is of interest that, in contrast to IL-6, IL-8, and MCP-1, >50% of
IL-15 remained intracellularly. As shown in Fig. 5, the cytoplasm of
muscle cells was strongly stained by anti-IL-15 mAb, but
cytoplasmic staining was marginal in normal muscle cells; this suggests
the presence of intracellular IL-15 in vivo in PM/DM. Increased
production of IL-15 by muscle cells due to CD40 ligation might
contribute to further T cell migration and activation, as discussed for
IL-8. Further studies should attempt to clarify the molecular structure
and biological function of intracellular IL-15.
CD40-CD40L interaction also induced IL-6 production by
IFN--stimulated SkMC 2859. IL-6 has multiple biological functions,
such as B cell differentiation and T cell activation (46).
IL-6 production by muscle cells in PM/DM may activate T cells at
inflammatory foci and contribute to autoantibody production in these
disorders.
Because CD40 ligation induces CD80 and CD86 expression on macrophages (27), we examined its effect on the expression of these molecules on SkMC 2859. Recombinant hCD40L did not induce the expression of either cell surface CD80 or CD86, although it did induce the expression of CD80 mRNA in SkMC 2859 (data not shown). Behrens et al. used anti-CD40 mAb (EA-5) or CD40L-transfected mouse fibroblasts for CD40 ligation (13). CD40L transfectant induced weak expression of CD80 and strong expression of BB-1 in myoblasts, although EA-5 stimulated BB-1 expression alone. It is possible that CD40-mediated signals induce CD80 expression in myoblasts in vitro, but not in vivo, because we cannot find CD80-expressing muscle cells in IIMs (13) (our unpublished observation).
Anti-human CD40L Ab inhibited rhCD40L-stimulated cytokine production in
SkMC 2859 (Fig. 6). Thus, blocking of the CD40-CD40L interaction, which
is reported to ameliorate several autoimmune disorders such as
collagen-induced arthritis (47) and lupus-like nephritis
in animal models (48, 49), may also have therapeutic value
in PM/DM.
Regarding the inducers of CD40 on muscle cells in PM/DM, IFN- would
be a prime candidate. We demonstrated that infiltrating MNCs expressed
IFN- in eight of the 10 PM/DM patients, and in all these cases,
muscle cells expressed CD40. Expression of IFN- in infiltrating
cells was also shown by other investigators using immunohistochemistry
and RT-PCR (50, 51, 52), consistent with our data.
Furthermore, IFN- induced CD40 expression on cultured SkMC 2859 in a
dose-dependent manner (Fig. 3B), as reported for B cells,
endothelial cells, macrophages, fibroblasts, and synovial cells of RA
by other investigators (27). These results support the
hypothesis that IFN- is an inducer of CD40 on muscle cells in PM/DM.
Another candidate for a CD40 inducer in PM/DM is TNF-, although its
effect is not as marked as that of IFN- (Fig. 3B).
Expression of TNF- has also been reported in PM/DM
(53). Because TNF- in combination with IFN- had a
synergistic effect on CD40 expression on SkMC 2859 in vitro (Fig. 3B), the two molecules, IFN- and TNF-, may
synergistically stimulate CD40 expression on muscle cells in
vivo.
CD40L is transiently expressed on activated T cells. The T cells that express CD40L are predominantly CD4+ T cells, with some CD8+ T cells also expressing the molecule in vitro (54). This agrees with our immunohistochemical data, wherein most of CD40L-positive cells are CD4+ T cells and some populations are CD8+ T cells in both PM and DM. Thus, CD40L-expressing CD4+T cells are in their activated state and play a central role in the CD40-CD40L interaction in inflammatory lesions of PM/DM.
In conclusion, our results indicate that cross-talk between the inflammatory cytokines and the CD40-CD40L system contributes to T cell recruitment and activation in PM/DM and to perpetuation of the inflammatory process. Blocking of CD40-CD40L interaction may have therapeutic value in PM/DM.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yasushi Kawaguchi, Institute of Rheumatology, Tokyo Women’s Medical University, 10–22 Kawada-cho, Shinjuku-ku, Tokyo 162-0054, Japan.
3 Abbreviations used in this paper: IIM, idiopathic inflammatory myopathy; PM, polymyositis; DM, dermatomyositis; CD40L, CD40 ligand; MNC, mononuclear cell; MCP, monocyte chemoattractant protein-1; rh, recombinant human; TRAP1, thrombin receptor agonist peptide 1.
Received for publication June 17, 1999. Accepted for publication April 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß variable and joining gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies. J. Immunol. 15:2569.
secretion and cytotoxicity. J. Neuroimmunol. 86:53.[Medline]
of synovial tissue cells from patients with rheumatoid arthritis: a novel mechanism of activation of synoviocytes. J. Rheumatol. 26:1035.[Medline]
in muscles in polymyositis. J. Neurol. Sci. 146:45.[Medline]
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 S. E. Riechman, G. Balasekaran, S. M. Roth, and R. E. Ferrell Association of interleukin-15 protein and interleukin-15 receptor genetic variation with resistance exercise training responses J Appl Physiol, December 1, 2004; 97(6): 2214 - 2219. [Abstract] [Full Text] [PDF]
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 D. Daoussis, A. P. Andonopoulos, and S.-N. C. Liossis Targeting CD40L: a Promising Therapeutic Approach Clin. Vaccine Immunol., July 1, 2004; 11(4): 635 - 641. [Full Text]
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 J Yazdany and J Davis The role of CD40 ligand in systemic lupus erythematosus Lupus, May 1, 2004; 13(5): 377 - 380. [Abstract] [PDF]
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 A.-J. Ruth, A. R. Kitching, T. J. Semple, P. G. Tipping, and S. R. Holdsworth Intrinsic Renal Cell Expression of CD40 Directs Th1 Effectors Inducing Experimental Crescentic Glomerulonephritis J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2813 - 2822. [Abstract] [Full Text] [PDF]
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 Y. Ogawa, M. Kuwana, K. Yamazaki, Y. Mashima, M. Yamada, T. Mori, S. Okamoto, Y. Oguchi, and Y. Kawakami Periductal Area as the Primary Site for T-Cell Activation in Lacrimal Gland Chronic Graft-Versus-Host Disease Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 1888 - 1896. [Abstract] [Full Text] [PDF]
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 T. Sugiura, M. Harigai, Y. Kawaguchi, K. Takagi, C. Fukasawa, S. Ohsako-Higami, S. Ohta, M. Tanaka, M. Hara, and N. Kamatani Increased IL-15 production of muscle cells in polymyositis and dermatomyositis Int. Immunol., August 1, 2002; 14(8): 917 - 924. [Abstract] [Full Text] [PDF]
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 U. Schonbeck and P. Libby CD40 Signaling and Plaque Instability Circ. Res., December 7, 2001; 89(12): 1092 - 1103. [Abstract] [Full Text] [PDF]
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O. Benveniste, P. Cherin, T. Maisonobe, R. Merat, O. Chosidow, L. Mouthon, L. Guillevin, A. Flahault, M.-C. Burland, D. Klatzmann, et al. Severe Perturbations of the Blood T Cell Repertoire in Polymyositis, But Not Dermatomyositis Patients J. Immunol., September 15, 2001; 167(6): 3521 - 3529. [Abstract] [Full Text] [PDF]
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 D. M. Wuttge, P. Eriksson, A. Sirsjo, G. K. Hansson, and S. Stemme Expression of Interleukin-15 in Mouse and Human Atherosclerotic Lesions Am. J. Pathol., August 1, 2001; 159(2): 417 - 423. [Abstract] [Full Text]
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 S. Stemme Plaque T-Cell Activity : Not So Specific? Arterioscler. Thromb. Vasc. Biol., July 1, 2001; 21(7): 1099 - 1101. [Full Text] [PDF]
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A. BASOK, A. SHNAIDER, L. MAN, C. CHAIMOVITZ, and A. DOUVDEVANI CD40 Is Expressed on Human Peritoneal Mesothelial Cells and Upregulates the Production of Interleukin-15 and RANTES J. Am. Soc. Nephrol., April 1, 2001; 12(4): 695 - 702. [Abstract] [Full Text] [PDF]
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, without IFN-
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