University of Rochester Cancer Center and Departments of Microbiology and Immunology, Pediatrics, and Environmental Medicine, University of Rochester, Rochester 14642; Division of Molecular and Cellular Medicine, Department of Medicine, and Department of Biochemistry and Molecular Biology, Albany Medical College, and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208
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ABSTRACT |
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CD40 is an important signaling and activation antigen found on
certain bone marrow-derived cells. Recently, CD40 has also been shown
to be expressed by nonhematopoietic cells, including certain human
fibroblasts, but not others. Little is known about the function of CD40
on fibroblasts. The current study investigates the hypothesis that CD40
is expressed on orbital fibroblasts and represents a pathway for
interaction between these fibroblasts and CD40 ligand-expressing cells,
such as T lymphocytes and mast cells. We report here that orbital
connective tissue fibroblasts, obtained from normal donors and from
patients with severe thyroid-associated ophthalmopathy (TAO), express
functional CD40. CD40 is upregulated ~10-fold by interferon- (500 U/ml) treatment for 72 h. These fibroblasts become activated through
triggering of CD40 with CD40 ligand (CD40L). This is evidenced by
nuclear translocation of nuclear factor-
B and induction of the
proinflammatory and chemoattractant cytokines interleukin-6 and
interleukin-8, respectively. These data support the concept that
cognate interactions between orbital fibroblasts and infiltrating T
lymphocytes, via the CD40-CD40L pathway, may promote the tissue
remodeling observed in TAO and other inflammatory diseases of the
orbit. Disruption of the CD40-CD40L interaction may represent a
therapeutic intervention to reduce the inflammatory components of TAO,
which remains a vexing clinical problem.
fibroblasts; thyroid-associated ophthalmopathy; cellular activation; interleukin-6; interleukin-8
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INTRODUCTION |
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THE CONNECTIVE TISSUE of the human orbit can manifest dramatic inflammation in thyroid-associated ophthalmopathy (TAO) (30). TAO is synonymous with Graves' ophthalmopathy. In this disease process, tissues become infiltrated with activated T lymphocytes and mast cells and accumulate excessive amounts of the nonsulfated glycosaminoglycan hyaluronan (7). Hyaluronan is synthesized by many cell types. However, fibroblasts are a major source of this and other glycosaminoglycans (30). It is believed that orbital fibroblasts have a primary role in the pathogenesis of TAO. During the tissue remodeling associated with TAO, infiltrating T lymphocytes are thought to initiate, through unknown signaling pathways, biosynthesis of inflammatory mediators and hyaluronan, two cardinal features of TAO (22). It would therefore appear essential that conduits for molecular communication between orbital fibroblasts and infiltrating T lymphocytes be fully characterized if further insights are to be gained into the mechanism underlying the disease. Moreover, elucidation of the pathways utilized for lymphocyte-fibroblast cross talk could allow the formulation of specific therapeutic strategies designed to interrupt inflammation.
One candidate pathway for communication between orbital fibroblasts and
T lymphocytes is the CD40-CD40 ligand (CD40L) costimulatory pathway.
This system conveys signals for activation and differentiation between
hematopoietic cells. It has been implicated recently in T lymphocyte
costimulation (4, 28). Triggering through surface CD40, a 50-kDa member
of the tumor necrosis factor- (TNF-
) receptor gene superfamily,
is a powerful stimulus for B lymphocyte activation, proliferation,
immunoglobulin (Ig) production, and isotype class switching (6). CD40
is also expressed by non-B lymphocyte hematopoietic cells, including
interdigitating dendritic cells, Langerhans cells, dendritic cells, and
monocytes (4). CD40L, a member of the TNF-
cytokine gene
superfamily, is the counterreceptor for CD40 and is primarily expressed
on the surface of activated T lymphocytes and mast cells (34). In
addition to being an important signaling mechanism for B lymphocytes,
the CD40-CD40L intercellular ligand interaction can provide a necessary
"second signal" to help drive cytokine production and clonal
expansion of naive T lymphocytes (26).
Observations in patients with X-linked hyper-IgM syndrome provide evidence for the importance of the CD40-CD40L pathway in immune responses. These patients possess mutations in the gene encoding CD40L, resulting in the disruption of CD40-CD40L signaling. The loss of T lymphocyte "help" renders B cells functionally deficient, with abnormal Ig class switching and a profound inability to respond to bacterial antigens (2). The CD40-CD40L pathway has also been shown to play a major role in autoimmune diseases that have significant B lymphocyte involvement. For example, treatment with anti-CD40L antibodies are effective in preventing the onset of disease in murine models of collagen-induced arthritis, experimental autoimmune encephalomyelitis, and lupus nephritis (8, 24).
CD40 expression is not limited to bone marrow-derived cells. It has been demonstrated to be expressed and functional on epithelial cells, such as human epidermal keratinocytes (15), thymic epithelium (13), and activated vascular endothelial cells (21). Very relevant to the current study is the observation of CD40 expression on nontransformed human fibroblasts derived from lung, foreskin, periodontal, and synovial tissue (12). CD40 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the protein was detected on cultured fibroblasts using flow cytometry. In tissue sections, immunostaining suggested fibroblast display of CD40. In contrast, some dermal fibroblasts failed to express CD40 (12).
The current studies were conducted to determine whether CD40 is
expressed by orbital fibroblasts derived from normal tissue and tissue
obtained from donors with TAO. We demonstrate here that CD40 is
expressed by orbital fibroblasts from either source and that these
fibroblasts can be activated through CD40 to mobilize nuclear
factor-B (NF-
B) and to express proinflammatory cytokines. We
hypothesize that the CD40-CD40L coreceptor system, described for B-T
lymphocyte communication, is an important pathway through which
communication between orbital fibroblasts and T lymphocytes and mast
cells can occur. This pathway may be relevant to the pathogenesis of
inflammatory diseases in the orbit, such as TAO. Moreover, the
demonstration of functional CD40 on fibroblasts represents a
potentially important target for therapy design.
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MATERIALS AND METHODS |
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Cell culture. Normal and TAO orbital primary fibroblasts were isolated from surgical explants as described in an earlier publication (33). TAO fibroblasts were initiated from surgical explants from individuals undergoing decompressive surgery for severe Graves' ophthalmopathy. Normal orbital strains were derived from individuals undergoing eye surgery for conditions not affecting the soft tissues of the orbit. Procurement of these tissues was approved by the Institutional Review Board of Albany Medical College. The fibroblasts were maintained in minimal essential medium with Eagle's salts (MEM; Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and gentamicin (50 µg/ml; Life Technologies). These cells are morphologically consistent with a fibroblast phenotype and express vimentin and collagen, two markers of fibroblasts. They do not, however, express CD45, factor VIII, or smooth muscle actin. Fibroblasts were passaged every 10-14 days and reseeded at 3 × 105 cells/75-cm2 flask (Costar, Cambridge, MA). Adherent monolayers of fibroblasts were dissociated with a 1:1 solution of sodium EDTA (0.1% wt/vol; Sigma Chemical) and trypsin (0.05% wt/vol; Worthington Biochemical, Freehold, NJ). All fibroblasts were maintained in a humidified 7% CO2 incubator. All experiments used cells between passages 2 and 13.
RT-PCR.
Fibroblasts were untreated or treated with recombinant human
interferon- (IFN-
, 500 U/ml; Genzyme, Cambridge, MA) for 48 h and
then scraped with a rubber policeman for extraction of total RNA with
Tri-Reagent (1 ml/106 cells;
Molecular Research Center, Cincinnati, OH), following the
manufacturer's protocol. RNA was solubilized in nuclease-free water by
heating to 55°C for 10 min. The concentration of total RNA was
quantitated by spectrophotometry, and 5 µg of RNA were reverse
transcribed using Moloney murine leukemia virus reverse transcriptase
(200 U/reaction; Life Technologies) and an oligo(dT) primer (Pharmacia,
Piscataway, NJ), as previously described (12). Reverse transcriptase
was withheld from replicate samples, which served as negative controls.
PCR reactions for human CD40 and
-actin consisted of cDNA obtained
above (2 µl), reaction buffer (Boehringer Mannheim Biochemicals),
deoxynucleotides (1 µM each), specific primers (1 µM each), and Taq
DNA polymerase (2.5 U; Boehringer Mannheim Biochemicals) in a total
volume of 50 µl. The human CD40 primer sequences were 3'-CGT
ACA GTG CCA GCC TTC TTC and 5'-ATG GTT CGT CTG CCT CTG CAG,
yielding a 330-base pair (bp) product; the human
-actin primer
sequences were 3'-CTC CTT AAT GTC ACG CAC GAT TTC and
5'-GTG GGG CGC CCC AGG CAC CA, which generated a 539-bp product.
Samples underwent 30 cycles of amplification in a PTC-200 DNA Engine
thermal cycler (M. J. Research, Watertown, MA), and each cycle included
denaturation at 94°C for 30 s, annealing at 63°C for 30 s, and
primer extension at 72°C for 60 s. The products were
electrophoresed on 2% agarose gels and visualized with ethidium bromide staining. A 100-bp ladder (Life Technologies) was used for size
determination.
Flow cytometry analysis. Fibroblasts were prepared for flow cytometry by washing a suspension of cells in phosphate-buffered saline (PBS) with 0.1% sodium azide and 1.0% bovine serum albumin (BSA). Cells were incubated with anti-human CD40 monoclonal antibody (G28-5, 100 µl of hybridoma supernatant; American Type Culture Collection) or anti-HLA-DR (L243, 100 µl of hybridoma supernatant; American Type Culture Collection) for 30 min on ice. After the cells were washed, fluorescein isothiocyanate-conjugated goat anti-mouse Ig (1:50 dilution; Cappel Research Products, Durham, NC) was added for 30 min on ice. Samples stained with only the secondary antibody served as negative controls. Once washed, the cells were resuspended in PBS with 0.1% sodium azide and 1.0% BSA and analyzed on a flow cytometer (Elite, Coulter, Hialeah, FL). We have demonstrated that trypsin treatment, under the conditions used to disrupt fibroblast monolayers, fails to alter the detection of surface CD40 or HLA-DR (data not shown). Viable cells were gated on the basis of forward light scatter, and the data were analyzed with the Cytologo software program (Coulter). All experiments were repeated a minimum of three times, and representative results are presented.
Electrophoretic mobility shift assays.
Orbital fibroblasts were allowed to proliferate to confluence in 100-mm
tissue culture dishes covered with MEM supplemented with 10% FBS. The
fibroblasts were pretreated with IFN- (500 U/ml, Genzyme) for 72 h.
The cultures were then washed extensively and incubated overnight with
MEM containing 1% FBS. The cells were then stimulated for 2 h at
37°C with medium alone, control insect cell membranes containing
glutathione S-transferase (GST), or
insect cell membranes containing human CD40L. Membranes were generously
provided by Dr. Marilyn Kehry (Boehringer Ingelheim, Ridgefield, CT)
and were prepared as described previously (19). Recombinant human
TNF-
(50 U/ml; Genzyme) was used to trigger the cells as a positive
control for NF-
B mobilization. The cells were harvested, nuclear
extracts were prepared as described by Andrews and Faller (1), and
protein concentrations were normalized by the bicinchoninic acid
protein assay (Pierce, Rockford, IL). To assess NF-
B binding
activity, 25 µg of nuclear extract were incubated with
32P-labeled double-stranded
oligonucleotide probe representing the consensus sequence for the
NF-
B binding sites following the supplier's instructions (Promega,
Madison, WI). Samples were electrophoresed on 4% polyacrylamide gels,
and the DNA-protein complexes were visualized by autoradiography.
Cytokine production.
Orbital fibroblasts were seeded into 96-well tissue culture plates
(5,000 cells/well) and, once established, were untreated or treated
with IFN- (500 U/ml) for 72 h in MEM containing 10% FBS. The
fibroblast monolayers were then washed extensively with fresh culture
medium at 37°C and incubated in triplicate for 72 h with medium
alone, control insect cell membranes containing GST, or insect cell
membranes containing human CD40L. Culture medium was harvested, and the
concentration of IL-6 and IL-8 was determined by specific enzyme-linked
immunosorbent assay (ELISA). Media samples were appropriately diluted
with sample buffer so as to fall within the linear sensitivity range of
the ELISAs.
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RESULTS |
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CD40 mRNA expression by normal and TAO orbital fibroblasts.
Primary human orbital fibroblasts were isolated to examine the
expression and function of CD40. Previous reports have demonstrated that fibroblasts from human lung tissue constitutively express CD40
mRNA and protein. In addition, it was shown that the proinflammatory cytokine IFN- induces hyperexpression of this surface antigen (28).
RT-PCR analysis was performed on total RNA extracted from normal and
TAO fibroblasts treated without or with IFN-
(500 U/ml) for 48 h.
Equivalent amounts of RNA were reverse transcribed, and the resulting
cDNA was then amplified for 30 cycles with primers specific for CD40
and the housekeeping gene
-actin. Samples prepared without reverse
transcriptase failed to yield any CD40 or
-actin product (data not
shown). A representative ethidium bromide-stained gel showing the
amplification products from normal orbital fibroblast RNA
(±IFN-
) is shown in Fig.
1A.
The amplification products from TAO orbital fibroblast RNA
(±IFN-
) are depicted in Fig.
1B. It is clearly demonstrated that
normal and TAO orbital fibroblasts express CD40 mRNA constitutively,
and the intensity of the bands suggests that the amount of steady-state
CD40 mRNA is elevated in the IFN-
-treated samples, using
-actin
expression as a control. From the results shown and multiple
repetitions of this experiment, it does not appear that there is a
significant difference in the level of CD40 mRNA expression between
normal and TAO orbital fibroblasts, under control or IFN-
-treated
conditions.
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Surface expression of CD40 and HLA-DR by normal and TAO orbital
fibroblasts.
The next step in the analysis of CD40 expression by normal and TAO
orbital fibroblasts was to verify the findings regarding expression of
CD40 mRNA by flow cytometric detection of basal and IFN--induced
surface CD40 protein expression. Fibroblasts were cultured in the
presence or absence of IFN-
for 72 h and then stained with a
monoclonal antibody specific for CD40. Histograms showing background
staining (control) and CD40 expression by untreated and IFN-
-treated
normal orbital fibroblasts are presented in Fig.
2A. The
log fluorescence intensity of staining is plotted vs. cell number for
each sample analyzed. CD40 is constitutively expressed on the surface
of the fibroblasts compared with background staining profiles.
Stimulation with IFN-
results in a dramatic 10-fold increase in the
expression of CD40 on normal orbital fibroblasts. Figure
2B shows the flow cytometry histograms
obtained for CD40 staining of TAO orbital fibroblasts treated
similarly. TAO fibroblasts also display detectable basal levels of CD40
comparable to those of normal orbital fibroblasts and are also
stimulated by IFN-
to upregulate CD40 expression.
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CD40L triggers mobilization of NF-B in orbital
fibroblasts.
To address whether CD40 on orbital fibroblasts is functional, its
signal transduction capacity was investigated. Although little is known
about CD40 signaling in nonhematopoietic cell types, CD40 on B
lymphocytes is known to mobilize the ubiquitous transcription factor
NF-
B (23). Therefore, electrophoretic mobility shift analysis of
NF-
B binding activity in nuclear extracts from IFN-
-treated
normal and TAO orbital fibroblasts was performed. The cells were
stimulated for 2 h with control insect cell membranes containing GST
(control) or insect cell membranes containing human CD40L. Nuclear
proteins were extracted from the treated cells, and equivalent amounts
of protein were incubated with
32P-labeled double-stranded
oligonucleotide probe representing the consensus sequence for the
NF-
B binding site. An induction of NF-
B binding activity (arrow)
was observed after stimulation with CD40L of normal and TAO orbital
fibroblasts (Fig. 3). Supershift studies
with anti-p65 antisera confirmed that the upper band is a
p65-containing NF-
B complex (data not shown). A constitutively shifted lower band was detected in all extracts. This band is a
putative p50/p50 homodimer thought to be transcriptionally inactive. The specificity of the protein-DNA complex was confirmed by incubation of the nuclear extract with 200-fold excess unlabeled NF-
B probe before addition of 32P-labeled
probe (data not shown). The detected shift in CD40L-treated cells is
similar to that seen after treatment with the potent NF-
B-mobilizing
agent TNF-
(Fig. 3) (3). Densitometric analysis of the shifted bands
vs. the basal level of NF-
B binding activity revealed no significant
difference between the normal and TAO fibroblasts (data not shown).
These results indicate that orbital fibroblasts, primed with the
proinflammatory cytokine IFN-
to optimize CD40 expression, display
functional CD40 molecules on their surface that can be activated with
CD40L to translocate NF-
B into the nucleus.
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Cross-linking CD40 on orbital fibroblasts activates production of
IL-6 and IL-8.
To determine the functional consequence of CD40-mediated signal
transduction in the orbital fibroblasts, we investigated IL-6 and IL-8
expression after triggering of CD40 with CD40L. IL-6 and IL-8 are
potent proinflammatory cytokines, the promoter regions of which possess
NF-B binding sites. IL-6 is a pleiotropic cytokine, often expressed
at high levels at sites of inflammation, and has been shown to be an
autocrine growth factor for mouse fibroblasts (11). Confluent
monolayers of orbital fibroblasts were pretreated for 72 h in medium
alone or with IFN-
to upregulate surface CD40 expression. The cells
were then stimulated for 72 h with fresh medium, control insect cell
membranes, or membranes containing human CD40L. Media samples were then
harvested for cytokine-specific ELISA. Induction of IL-6 after
activation of CD40 in orbital strains from three normal and three TAO
tissues (±IFN-
) is shown in Fig. 4.
The concentration of cytokine detected in the conditioned medium was
corrected for the background secretion of cytokine observed in
untreated fibroblasts. Orbital fibroblasts express low levels of IL-6
and have detectable IL-6 mRNA under basal culture conditions (data not
shown). Fibroblasts stimulated with CD40L, without IFN-
, expressed
higher levels of IL-6 than did controls. A far more robust induction of
IL-6 production occurred when the fibroblasts were primed with IFN-
before CD40 engagement (Fig. 4A).
Although levels of IL-6 varied in all six fibroblast strains tested,
the pattern of induction remained the same (Fig.
4B).
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DISCUSSION |
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CD40 has been recently detected on nonlymphoid cells, such as keratinocytes, endothelial cells, and fibroblasts; however, certain primary human fibroblasts, such as those from the skin of the abdominal wall, do not express CD40 (15, 16, 21). Here we have examined the expression of CD40 by orbital fibroblasts. Normal and TAO orbital fibroblasts were found to display this surface antigen. Moreover, CD40 engagement with its natural ligand resulted in the activation of intracellular signaling and a dramatic upregulation of IL-6 and IL-8 expression. We hypothesize that orbital fibroblasts interact with infiltrating T lymphocytes via the CD40-CD40L costimulatory pathway. This in turn promotes fibroblast activation, which can result in the tissue remodeling observed in orbital inflammatory conditions such as TAO.
RT-PCR analysis of total RNA extracted from normal and TAO orbital
fibroblasts revealed a detectable basal level of CD40 mRNA expression
(Fig. 1). Similar to pulmonary, gingival, and foreskin fibroblasts,
there was an apparent increase in the steady-state level of CD40 mRNA
when the orbital fibroblasts were stimulated with IFN- (12). This
finding was confirmed by flow cytometry (Fig. 2). In addition to
enhancing CD40 expression, IFN-
treatment also induced expression of
HLA-DR on the orbital fibroblasts. This finding is consistent with our
earlier studies (18). Despite being isolated from tissues in two very
different states (inflamed vs. noninflamed), the two types of
fibroblasts have similar basal levels of CD40 and HLA-DR is absent.
Moreover, IFN-
induces HLA-DR expression and relatively similar
levels of CD40. It can therefore be inferred that any increase in
surface expression of CD40 and HLA-DR on orbital fibroblasts in situ is
not an imprinted characteristic induced by the disease process but,
rather, represents a transient activation of these fibroblasts in the
setting of an inflammatory mircroenvironment.
Orbital fibroblasts are heterogeneous with respect to expression of the surface antigen Thy-1. Thy-1 is expressed on 54-71% of the cells in primary cultures of orbital fibroblasts, with the remainder of the cells staining negatively (32). This expression profile is stable in culture, and representative clones of orbital fibroblasts can be isolated that faithfully retain Thy-1-positive and Thy-1-negative expression status. Thy-1 heterogeneity indicates a complexity of orbital tissue, which may provide insight into the mechanisms of tissue remodeling associated with inflammatory diseases of the orbit. Interestingly, normal and TAO orbital fibroblasts homogeneously express CD40. This suggests that all fibroblasts in the orbit are capable of receiving and transducing signals via CD40. CD40 expression is therefore not a criterion for rationalizing functional heterogeneity observed in orbital fibroblasts (32). However, CD40-mediated activation may be a key signaling conduit for orbital fibroblasts. The signal may be processed independently or in conjunction with one or more other signals to elicit a desired cellular response. This would then provide a mechanism for fibroblast subpopulations to serve distinct functions in the orbit.
The ubiquitous transcription factor NF-B has been identified as a
major signaling pathway utilized in CD40-mediated events in
hematopoietic cells, such as induction of proinflammatory cytokine synthesis (3, 23). Little is known about CD40-mediated cell signal
transduction and cellular activation in fibroblasts. It was
hypothesized that NF-
B is mobilized after fibroblasts are triggered
through CD40. Indeed, we have verified that CD40 engagement of orbital
fibroblasts with insect cell membranes containing human CD40L results
in the translocation of an NF-
B complex to the cell nucleus. These
findings are significant, because nuclear mobilization of NF-
B is a
conduit for cellular activation relevant to the transcriptional
upregulation of many genes involved in inflammatory and specific immune
responses (3).
Our results suggest that the cytokine milieu surrounding the orbital
fibroblast in situ may represent a critical determinant of the cellular
responses mediated through the CD40-CD40L pathway. Of particular
relevance would appear to be the tissue concentration of IFN-. This
cytokine is known to upregulate cellular adhesion molecules such as
intercellular adhesion molecule 1 and vascular cell adhesion molecule
1, which can promote fibroblast adhesion to T lymphocytes (10, 14, 25).
During the inflammation and tissue remodeling associated with TAO,
large numbers of infiltrating T lymphocytes have been detected in
orbital tissue. In addition, T lymphocytes with cytolytic properties
have been isolated from TAO tissue (7, 22). Little is known about the
cytokine milieu present in TAO; however, one study demonstrated by
immunostaining the presence of IL-1
, TNF-
, and IFN-
(17). The
actual concentrations of these cytokines were not determined. These
observations, in conjunction with our results, suggest that an
inflammatory environment rich in IFN-
-producing infiltrating cells,
such as T lymphocytes, may provide a priming effect for interstitial
fibroblasts and elevate CD40 expression on fibroblasts in inflamed
tissue. This might in turn condition the fibroblasts to be
hyperresponsive to CD40 engagement and, therefore, result in the
amplified induction of proinflammatory cytokines.
IL-6, the human promoter of which contains an identifiable NF-B
site, is of significant interest with regard to TAO. A recent report by
Salvi and colleagues (27) demonstrated elevated serum levels of IL-6 in
patients with Graves' disease compared with normal subjects. These
authors also observed higher levels of serum soluble IL-6
receptor in patients with active inflammatory TAO than in
patients with inactive orbital disease. Thus IL-6 may have a pathogenic
role in TAO. The degree of T lymphocyte infiltration detected in TAO
tissue (7, 30) suggests that a chemoattractant signal for T
lymphocytes, such as IL-8, might emanate from orbital cells. We
hypothesized that activation of CD40 on orbital fibroblasts may
represent an important mechanism for the upregulation of local IL-6 and
IL-8 expression in the orbit. Data presented here support this
hypothesis by clearly demonstrating that engagement of CD40 on orbital
fibroblasts by CD40L results in the induction of IL-6 and IL-8 proteins
(Figs. 4 and 5). No difference was observed in the induction of either cytokine in normal and TAO fibroblasts.
Human orbital fibroblasts exhibit a set of phenotypic attributes in culture that distinguish them from many types of extraorbital fibroblasts. They are particularly susceptible to certain actions of proinflammatory cytokines with regard to the induction of plasminogen activator inhibitor type 1 (5, 20, 29), hyaluronan synthesis (31, 33), and the upregulation of prostaglandin endoperoxide H synthase 2 (35). The latter may represent the molecular basis for the intense inflammatory response observed in certain orbital disorders such as TAO (30). It is therefore critical to compare the magnitude of CD40L-dependent cytokine expression in orbital fibroblasts with that in fibroblasts derived from other tissues. Future studies will examine whether CD40 signaling influences the expression of prostaglandin endoperoxide H synthase 2 and the synthetic rate of hyaluronan, independently and in conjunction with proinflammatory cytokines.
In conclusion, data in this report support the hypothesis that the fibroblast CD40 is an important mediator of cognate interactions between infiltrating CD40L-expressing immunocompetent cells and fibroblasts. These interactions may play a pivotal role in regulating normal tissue function and may underlie many aspects of remodeling associated with inflammation in the orbit. Anti-CD40L antibodies have been shown to be effective in preventing the onset of disease in murine models of collagen-induced arthritis, experimental autoimmune encephalomyelitis, and lupus nephritis (8, 24). We propose that strategies utilizing recombinant fusion proteins or monoclonal antibodies, targeting the disruption of the CD40-CD40L interactions between fibroblasts and lymphocytes, may represent powerful therapeutic strategies for TAO and other autoimmune diseases.
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ACKNOWLEDGEMENTS |
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We thank Chantal K. Turner for expert technical assistance.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants CA-11198, DE-11047, AG-56002, EY-08976, EY-11708, and P30-ES-01247, a Merit Review Award from the Department of Veterans Affairs, and the Rochester Area Pepper Center. G. D. Sempowski is supported by National Institute of Dental Research Grant DE-07202.
Present address of G. D. Sempowski: Duke University Medical Center, Box 3258, Durham, NC 27710.
Address for reprint requests: R. P. Phipps, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 704, Rochester, NY 14642.
Received 7 May 1997; accepted in final form 12 November 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Andrews, N. C.,
and
D. V. Faller.
A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells.
Nucleic Acids Res.
19:
2499,
1991[Medline].
2.
Aruffo, A.,
M. Farrington,
D. Hollenbaugh,
X. Li,
A. Milatovich,
S. Nonoyama,
J. Bajorath,
L. S. Grosmaire,
R. Stenkamp,
M. Neubaure,
R. L. Robers,
R. J. Noelle,
J. A. Ledbetter,
U. Francke,
and
H. D. Ochs.
The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome.
Cell
72:
291-300,
1993[Medline].
3.
Baeuerle, P.,
and
T. Henkel.
Function and activation of NF-B in the immune system.
Annu. Rev. Immunol.
12:
141-179,
1994[Medline].
4.
Banchereau, J.,
F. Bazan,
D. Blanchard,
J. P. Briere,
C. Galizzi,
C. Van Kooten,
Y. J. Liu,
F. Rousset,
and
S. Saeland.
The CD40 antigen and its ligand.
Annu. Rev. Immunol.
12:
881-922,
1994[Medline].
5.
Cao, H. J.,
M. G. Hogg,
L. J. Martino,
and
T. J. Smith.
Transforming growth factor- induces plasminogen activator inhibitor type-1 in cultured human orbital fibroblasts.
Invest. Ophthalmol. Vis. Sci.
36:
1411-1419,
1995[Abstract].
6.
Clark, E. A.,
and
J. A. Ledbetter.
Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50.
Proc. Natl. Acad. Sci. USA
83:
4494-4498,
1986[Abstract].
7.
De Carli, M.,
M. M. D'Elios,
S. Mariotti,
C. Marcocci,
A. Pinchera,
M. Ricci,
S. Romagnani,
and
G. del Prete.
Cytolytic T cells with Th1-like cytokine profile predominate in retroorbital lymphocyte infiltrates of Graves' ophthalmopathy.
J. Clin. Endocrinol. Metab.
77:
1120-1124,
1993[Abstract].
8.
Durie, F. H.,
T. M. Foy,
and
R. J. Noelle.
The role of CD40 and its ligand (gp39) in peripheral and central tolerance and its contribution to autoimmune disease.
Res. Immunol.
145:
200-205,
1994[Medline].
9.
Endo, H.,
T. Akahoshi,
K. Takagishi,
S. Kashiwazaki,
and
K. Matsushima.
Elevation of interleukin-8 (IL-8) levels in joint fluids of patients with rheumatoid arthritis and the induction by IL-8 of leukocyte infiltration and synovitis in rabbit joints.
Lymphokine Cytokine Res.
10:
245-252,
1991[Medline].
10.
Fries, K. M.,
T. Blieden,
R. J. Looney,
G. D. Sempowski,
M. R. Silvera,
R. A. Willis,
and
R. P. Phipps.
Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.
Clin. Immunol. Immunopathol.
72:
283-292,
1994[Medline].
11.
Fries, K. M.,
M. E. Felch,
and
R. P. Phipps.
Interleukin 6 is an autocrine growth factor for murine fibroblast subsets.
Am. J. Respir. Cell Mol. Biol.
11:
552-560,
1994[Abstract].
12.
Fries, K. M.,
G. D. Sempowski,
A. A. Gaspari,
T. Blieden,
R. J. Looney,
and
R. P. Phipps.
CD40 expression by human fibroblasts.
Clin. Immunol. Immunopathol.
77:
42-51,
1995[Medline].
13.
Galy, A.,
and
H. Spits.
CD40 is functionally expressed on human thymic epithelial cells.
J. Immunol.
149:
775-782,
1992
14.
Gao, J. X.,
and
A. C. Issekutz.
Expression of VCAM-1 and VLA-4 dependent T-lymphocyte adhesion to dermal fibroblasts stimulated with proinflammatory cytokines.
Immunology
89:
375-383,
1996[Medline].
15.
Gaspari, A. A.,
G. D. Sempowski,
P. Chess,
J. Gish,
and
R. Phipps.
Human epidermal keratinocytes are induced to secrete IL-6 and costimulate T-lymphocyte proliferation by a CD40-dependent mechanism.
Eur. J. Immunol.
26:
1371-1377,
1996[Medline].
16.
Hess, S.,
A. Rensing-Ehl,
R. Schwabe,
P. Bufler,
and
H. Engelmann.
CD40 function in nonhematopoietic cells: nuclear factor B mobilization and induction of IL-6 production.
J. Immunol.
155:
4588-4595,
1995[Abstract].
17.
Heufelder, A. E.,
and
R. S. Bahn.
Detection and localization of cytokine immunoreactivity in retro-ocular connective tissue in Graves' ophthalmopathy.
Eur. J. Clin. Invest.
23:
10-17,
1993[Medline].
18.
Heufelder, A. E.,
T. J. Smith,
C. A. Gorman,
and
R. S. Bahn.
Increased induction of HLA-DR by interferon- in cultured fibroblasts derived from patients with Graves' ophthalmopathy and pretibial dermopathy.
J. Clin. Endocrinol. Metab.
73:
307-313,
1991[Abstract].
19.
Hodgkin, P. D.,
L. C. Yamashita,
R. L. Coffman,
and
M. R. Kehry.
Separation of events mediating B cell proliferation and Ig production by using T cell membranes and lymphokines.
J. Immunol.
145:
2025-2034,
1990
20.
Hogg, M. G.,
C. H. Evans,
and
T. J. Smith.
Leukoregulin induces plasminogen activator inhibitor type 1 in human orbital fibroblasts.
Am. J. Physiol.
269 (Cell Physiol. 38):
C359-C366,
1995
21.
Hollenbaugh, D.,
N. Mischel-Petty,
C. P. Edwards,
J. C. Simon,
R. W. Denfield,
P. A. Kiener,
and
A. Aruffo.
Expression of functional CD40 by vascular endothelial cells.
J. Exp. Med.
182:
33-40,
1995[Abstract].
22.
Hufnagel, T. J.,
W. F. Hickey,
W. H. Cobbs,
F. A. Jakobiec,
T. Iwamoto,
and
R. C. Eagle.
Immunohistochemical and ultrastructural studies on exenterated orbital tissues of a patient with Graves' disease.
Ophthalmology
91:
1411-1414,
1984[Medline].
23.
Lalmanach-Girard, A.,
T. C. Chiles,
D. C. Parker,
and
T. L. Rothstein.
T cell-dependent induction of NF-B in B cells.
J. Exp. Med.
177:
1215-1219,
1993[Abstract].
24.
Mohan, C.,
Y. Shi,
J. Laman,
and
S. Datta.
Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis.
J. Immunol.
154:
1470-1480,
1994
25.
Piela, T. H.,
and
J. H. Korn.
Lymphocyte-fibroblast adhesion induced by interferon-.
Cell. Immunol.
114:
149-160,
1988[Medline].
26.
Ramsdell, F.,
M. S. Seaman,
K. N. Clifford,
and
W. C. Fanslow.
CD40 ligand acts as a costimulatory signal for neonatal thymic T cells.
J. Immunol.
152:
2190-2197,
1994
27.
Salvi, M.,
G. Girasole,
M. Pedrazzoni,
M. Passeri,
N. Giuliani,
R. Minelli,
L. E. Braverman,
and
E. Roti.
Increased serum concentrations of interleukin-6 (IL-6) and soluble IL-6 receptor in patients with Graves' disease.
J. Clin. Endocrinol. Metab.
81:
2976-2979,
1996[Abstract].
28.
Sempowski, G. D.,
P. R. Chess,
and
R. P. Phipps.
CD40 is a functional activation antigen and B7-independent T cell costimulatory molecule on normal human lung fibroblasts.
J. Immunol.
158:
4670-4677,
1997[Abstract].
29.
Smith, T. J.,
A. Ahmed,
M. G. Hogg,
and
P. J. Higgins.
Interferon- is an inducer of plasminogen activator inhibitor type 1 in human orbital fibroblasts.
Am. J. Physiol.
263 (Cell Physiol. 32):
C24-C29,
1992
30.
Smith, T. J.,
R. S. Bahn,
and
C. A. Gorman.
Connective tissue, glycosaminoglycans, and diseases of the thyroid.
Endocr. Rev.
10:
366-491,
1989[Medline].
31.
Smith, T. J.,
R. S. Bahn,
C. A. Gorman,
and
M. Cheavens.
Stimulation of glycosaminoglycan accumulation by interferon- in cultured human retroocular fibroblasts.
J. Clin. Endocrinol. Metab.
72:
1169-1171,
1991[Abstract].
32.
Smith, T. J.,
G. D. Sempowski,
H. S. Wang,
P. J. Del Vecchio,
S. D. Lippe,
and
R. P. Phipps.
Evidence for cellular heterogeneity in primary cultures of human orbital fibroblasts.
J. Clin. Endocrinol. Metab.
80:
2620-2625,
1995[Abstract].
33.
Smith, T. J.,
H. S. Wang,
and
C. H. Evans.
Leukoregulin is a potent inducer of hyaluronan synthesis in cultured human orbital fibroblasts.
Am. J. Physiol.
268 (Cell Physiol. 37):
C382-C388,
1995
34.
Spriggs, M. K.,
W. C. Fanslow,
R. J. Armitage,
and
J. Belmont.
The biology of the human ligand for CD40.
J. Clin. Immunol.
13:
373-380,
1993[Medline].
35.
Wang, H. S.,
H. J. Cao,
V. D. Winn,
L. J. Rezanka,
Y. Frobert,
C. H. Evans,
D. Sciaky,
D. A. Young,
and
T. J. Smith.
Leukoregulin induction of prostaglandin-endoperoxide H synthase-2 in human orbital fibroblasts. An in vitro model for connective tissue inflammation.
J. Biol. Chem.
271:
22718-22728,
1996
36.
Yoshimura, T.,
K. Matsushima,
J. J. Oppenheim,
and
E. J. Leonard.
Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin-1 (IL-1).
J. Immunol.
139:
788-793,
1987