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Original Studies |
Medical Research Council Reproductive Biology Unit (A.E.K., R.W.K.) and Department of Obstetrics and Gynecology (H.O.D.C.), University of Edinburgh, Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9ET; Department of Cell and Molecular Biology, Lund University (A.M.), S-221 00, Lund, Sweden; Department of Obstetrics and Gynecology, Karolinska Hospital (M.S.), S-171 76, Stockholm, Sweden; and University of Rochester Cancer Center (R.P.P.), Rochester, New York 14642
Address all correspondence and requests for reprints to: Dr. Anne King, Medical Research Council Reproductive Biology Unit, University of Edinburgh, Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9ET. E-mail: a.e.king-1{at}sms.ed.ac.uk
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Abstract |
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treatment (500 U/mL;
72 h). When activated with interferon- and CD40L, the
fibroblasts secreted increased amounts of IL-6, IL-8, and MCP-1. These
data suggest that the CD40-CD40L system may provide a link between the
resident structural cells of these reproductive tissues and the
infiltrating immune cells or activated platelets that may express
CD40L. The possible interaction of CD40 with CD40L may be particularly
important during events such as menstruation and cervical ripening,
where up-regulation of the proinflammatory molecules IL-6 and IL-8 is
viewed as critical for these processes. In addition, dysregulation of
this system may be a contributory factor to problems such as menstrual
dysfunction and preterm labor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cytokine interleukin-6 (IL-6) and the chemokines IL-8 (attracts mainly neutrophils) and monocyte chemoattractant peptide 1 (MCP-1; attracts mainly monocytes) are likely to be critical mediators in female reproduction. IL-8 and MCP-1 expression increases in the late secretory phase of the menstrual cycle (2), whereas the expression of IL-6 becomes apparent in the midsecretory phase and progressively increases in the late secretory and menstrual phases (3). This suggests a role for these mediators in menstruation. In the cervix IL-8 production is closely associated with ripening (4), and synergistic actions with PGE2 have been reported (5). Parturition also involves up-regulated expression of inflammatory mediators, and IL-6 in particular has been linked to preterm labor (6). Progesterone is likely to be involved in the control of inflammatory mediator expression in reproductive tissues, and a model of progesterone withdrawal has shown increased expression of chemokines in endometrium (7). However, the molecular mechanisms that cause this up-regulation remain unclear.
CD40 is a member of the tumor necrosis factor-
receptor family and
is expressed on several cell types, including B lymphocytes
(8), monocytes, vascular endothelial cells
(9), and some epithelial cells (10). CD40
ligand (CD40L), the endogenous ligand for CD40, is found on activated T
cells, mast cells (11), eosinophils (12), and
basophils (11). Interestingly, platelets have an
intracellular pool of preformed CD40L that is surface expressed upon
activation (13). The CD40-CD40L system is involved in B
cell-T cell signaling events (14) and has also been found
to be important in nonhemopoietic cell activation. For example, CD40
has recently been found on human fibroblasts from several sites, and
interferon- (IFN) treatment up-regulates CD40 expression on these
cells (15, 16). Activation of CD40 on some types of
fibroblast results in up-regulation of the proinflammatory cytokines
IL-6 and IL-8 (16, 17, 18).
The CD40-CD40L system has not previously been investigated in normal human endometrium and myometrium, although CD40 was recently detected in the squamous epithelium of cervical carcinoma (19). The presence of the CD40-CD40L system in reproductive tissues could provide a key link between the resident structural cells of the tissues and infiltrating immune cells. Such a link would be likely to be involved in the inflammatory-like responses associated with both physiological and pathophysiological reproductive events. This study investigates the expression of CD40 in human endometrium, myometrium, and cervix and the functional consequences of CD40 engagement on fibroblasts derived from these tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endometrial (n = 24), myometrial (n = 26), and nonpregnant cervical biopsies (n = 3) were collected from women undergoing gynecological procedures for benign conditions. All women had regular menstrual cycles (25–35 days) and had not received any form of hormonal treatment in the 3 months preceding biopsy. Endometrial biopsies were collected throughout the menstrual cycle (proliferative, n = 10; early to mid secretory, n = 8; late secretory, n = 3). Histological dating according to the criteria of Noyes et al. (20) and circulating progesterone levels were consistent with the date of the last menstrual period. Cervical biopsies were collected from the anterior lip region of the cervix. Cervical biopsies from pregnant women (n = 8) were collected from nulliparous patients during the first trimester of pregnancy.
Written informed consent was received from all patients before biopsy collection. Ethical approval was received from Lothian research ethics committee and the ethics committee of the Karolinska Hospital.
Immunohistochemistry
Frozen tissue sections were lightly fixed in neutral buffered formalin for 10 min at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (BDH Laboratory Supplies, Poole, UK) in distilled water for 10 min at room temperature. Diluted normal horse serum (Vectastain 4002, Vector Laboratories, Inc., Peterborough, UK) was applied to all tissue sections for 20 min in a humidified chamber at room temperature. Tissue sections were incubated overnight at 4 C with 50 µL mouse anti-CD40 antibody (G28–5, diluted in horse serum; Dr. Ed Clark, Seattle, WA) (21, 22). The primary antibody was substituted with an equimolar concentration of mouse Ig (Vector Laboratories, Inc.) in negative control sections. Sections were incubated with biotinylated horse antimouse Ig (Vector Laboratories, Inc.) and then an avidin-biotin peroxidase detection system (both for 60 min at room temperature; Elite ABC 6101, Vector Laboratories, Inc.). The peroxidase substrate diaminobenzidine (Vector Laboratories, Inc.) was used to identify positive staining. Sections were counterstained with Harris’s hematoxylin (Pioneer Research Chemicals Ltd., Colchester, UK), dehydrated in ascending grades of alcohol, and mounted from xylene in Pertex (Cellpath, Hemel Hempsted, UK).
Derivation of fibroblast strains
Endometrial and myometrial explants were cultured in complete medium, i.e. RPMI 1640 (Sigma, Poole, Dorset, UK) supplemented with 10% FCS (Mycoplex, PAA Laboratories, Teddington, UK), penicillin (50 µg/mL; Sigma), streptomycin (50 µg/mL; Sigma), and gentamicin (5 µg/mL; Sigma). Cervical fibroblasts were established in MEM supplemented with 10% donor calf serum and then cultured as described above. Tissue was cut up into approximately 1-mm3 pieces. Explants were then placed under glass coverslips in 100-mm diameter tissue culture dishes (Corning, Inc./Costar, High Wycombe, UK) resulting in the outgrowth of fibroblasts from individual tissues as previously described (15, 18). On some occasions tissues were also digested in dispase II (2.4 U/mL; Roche Molecular Biochemicals, Lewes, UK) for 45 min at 37 C, and the resultant cell suspensions were cultured in 25-cm2 culture flasks (Corning, Inc./Costar). Both techniques yielded fibroblast strains (endometrial, n = 3; myometrial, n = 5; cervical, n = 3), and no differences were observed in terms of the efficiency of generation of fibroblast strains. Fibroblast strains were named E (endometrial), M (myometrial), or CX (cervical) to describe the tissue of origin and were subsequently numbered or lettered for research tracking purposes. Immunostaining showed the cells to be vimentin positive, but CD45 and cytokeratin negative. This is consistent with a fibroblast phenotype. The cells also expressed the fibroblast marker Thy-1. All cells used in experiments were from as early a passage as possible.
Flow cytometry and immunofluorescence
Fibroblasts were cultured with or without human IFN (500
U/mL; PeproTech, London, UK) for 72 h at 37 C. The cells were
lightly trypsinized, washed, and then resuspended in phosphate-buffered
saline supplemented with 0.1% azide and 1% BSA. Rapid treatment with
trypsin does not appreciably cleave CD40 from these cells
(17). The cells (1 x 106) were
then incubated with antihuman CD40 (G28–5) for 30 min on ice. Mouse
IgG and mouse IgG1 (Sigma) were used as negative controls.
The cells were washed and then incubated with fluorescein-conjugated
(FITC) goat antimouse Ig (1:100 dilution; Cappel, ICN Biomedical, Inc., Costa Mesa, CA) for 30 min on ice. After washing the cells
were resuspended in phosphate-buffered saline, 0.1% azide, and 1%
BSA. Analysis was performed on a Coulter model XL flow cytometer
(Hialeah, FL). Data analysis and subtraction were completed using an
Immuno-4 program (Coulter).
CD40 expression on fibroblasts was also examined in situ by immunofluorescence. Cells were cultured on eight-well chamber slides and then sequentially stained with antihuman CD40 and FITC goat-antimouse Ig as detailed above. Fibroblasts were examined by standard immunofluorescence on an Olympus Corp. Provis System (Melville, NY) and a Carl Zeiss LSM 510 laser scanning microscope (New York, NY).
IL-6, IL-8, and MCP-1 production
Fibroblasts were cultured in six-well culture plates
(Corning, Inc./Costar) in RPMI 1640 supplemented with 10%
FCS and antibiotics. After 24 h, culture medium was removed and
replaced with fresh medium containing 1% FCS. The cells were cultured
for an additional 72 h in the presence or absence of human IFN
(500 U/mL). The culture supernatant was removed and replaced with
medium containing 0.1% FCS. Four treatments were applied to the cells
for the final 24 h: control, no treatment; IFN alone (500
U/mL), CD40L alone (1:100 dilution), IFN (500 U/mL), and CD40L
(1:100 dilution). The CD40L was generated from insect cell membranes
expressing human CD40L as described previously (17, 18).
Sf21 insect cells were infected with the human CD40L baculovirus
expression vector or the glutathione-S-transferase (negative
control) vector for 2.5 days. Expression of human CD40L on the insect
cell membranes was confirmed by immunofluorescent staining with
phycoerythrin-conjugated antihuman CD40L antibody (clone 5c8). The
infected Sf21 cells were homogenized with a Polytron homogenizer
(Brinkmann Instruments, Inc., Westbury, NY), followed by
ultracentrifugation over a sucrose gradient. This allowed for the
separation and collection of the insect cell membrane-containing
fraction. Control membranes not containing CD40L fail to stimulate
CD40-bearing cells. Culture medium was removed and stored for
subsequent inclusion in the cytokine enzyme-linked immunosorbent assays
(ELISA).
IL-8 and MCP-1 were assayed by ELISA according to the methods of Denison et al. (23) and Ida et al. (24), respectively. IL-6 was measured by ELISA. Plates were passively coated overnight at 4 C with 100 µL/well of capture antibody (4 µg/mL; R and D Systems, Inc., Oxford, UK). After incubation, plates were washed in water, and 100 µL/well of blocking solution were added (2% polyvinyl pyrollidone, 5 mg/mL BSA, 5 mM ethylenediamine tetraacetic acid, and 50 mM Tris). After 30 min at room temperature, the solution was flicked out, and plates were air-dried and stored at 4 C. Before use plates were washed in water. Standards (R and D Systems, Inc.; top standard, 500 pg/mL) were diluted in ELISA buffer [100 mM Tris (pH 7.2), 150 mg/L 2-methylisothizalone (Roche Molecular Biochemicals, Indianapolis, IN), 150 mg/L bromonitrodioxane (Roche Molecular Biochemicals), 2 mg/mL BSA (Sigma, A3803), 300 µL 0.5% phenol red solution/L, 9 g/L NaCl, 2 mM ethylenediamine tetraacetic acid, and 0.05% Tween-20; final pH 7.2]. One hundred microliters per well of standard or sample were added. Plates were sealed, incubated overnight at 4 C, then washed four times in wash buffer (0.05% Tween-20, 9 g/L NaCl, and 100 mM Tris, pH 7–7.5). Detection antibody was added at 100 µL/well (50 µg/mL; R and D Systems, Inc.), and incubation was performed for 90 min on a plate shaker at room temperature. Plates were washed as described above. Streptavidin peroxidase (100 µL/well, 1:1000 dilution; Roche Molecular Biochemicals) was added, and plates were incubated for 20 min on a plate shaker at room temperature. Plates were washed as described above. Substrate (200 µL/well, 1:1:10; urea peroxidase, 0.3 g/50 mL 100 mM sodium acetate buffer (pH 6), 2 mg/mL tetramethylbenzidine in dimethylformamide, and 100 mM sodium acetate buffer, pH 6) were added. Plates were left for 20 min, quenched with 50 µL/well 2 N sulfuric acid, and then read on a plate reader at 450 nm. The intra- and interassay precisions were 8.3% and 9.8%, respectively. All samples were assayed in duplicate in the IL-6, IL-8, and MCP-1 ELISAs. Nonparametric statistical analysis was applied to the ELISA data.
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Results |
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Immunohistochemistry showed positive CD40 immunoreactivity in the
perivascular region of all endometrial biopsies studied (n = 21).
The area around the blood vessels that expressed CD40 was several cell
layers deep. These cells have been termed myofibroblasts
(25). Moderate immunostaining was also present in some
fibroblast-like cells, particularly in the basal and subglandular
regions of the stroma (Fig. 1A
). Very
faint epithelial staining and evidence of white blood cell CD40
expression were observed in some biopsies. No significant differences
in CD40 staining were observed during the menstrual cycle.
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Immunostaining of cervical biopsies (n = 8) also showed CD40 to be
localized around blood vessels and in some stromal cells. Additionally,
in contrast to endometrium, strong positive immunoreactivity was
present in the basal epithelium. Surface epithelium did not express
CD40 (Fig. 1e). This is similar to observations in human skin, where
both dermal fibroblasts and the basal epithelium express CD40
(10).
Expression of CD40 on endometrial, myometrial, and cervical fibroblasts
Human fibroblast lines were derived from endometrial (n = 3),
myometrial (n = 5), and cervical (n = 3) explants. The
resultant cells were morphologically consistent with a fibroblast
phenotype (Fig. 2) and expressed the
fibroblast markers Thy-1 and vimentin (data not shown). These cells
also failed to display the epithelial marker cytokeratin and CD45, a
marker of bone marrow-derived cells. They did not morphologically
resemble endothelial cells. All cells displayed the characteristics
detailed above, indicating that the cell populations were pure. Surface
CD40 expression was measured by flow cytometry and immunofluorescence.
Flow cytometric analysis revealed that all lines, with the exception of
myometrial fibroblast strain 9, displayed a small amount of CD40. No
consistent differences were observed in CD40 expression from fibroblast
strains derived from biopsies taken at various times during the
menstrual cycle. Figure 3 shows results
for endometrial 8 (E8), myometrial 2 (M2), and cervical NU (CX-NU)
fibroblast lines. Approximately 8% of the untreated endometrial
fibroblasts expressed CD40, whereas 4% of the myometrial fibroblasts
displayed the molecule. Twenty-two percent of the cervical fibroblasts
expressed CD40. These figures rose to approximately 36%, 22%, and
27%, respectively, after treatment with IFN for 72 h. In
general, the entire peaks shifted toward increased CD40 fluorescence,
which suggested that the majority of the fibroblasts were up-regulating
their display of CD40. Further evidence for this was obtained using
in situ immunofluorescence in concert with conventional and
confocal microscopy. Compared with non-IFN-treated cells, most of
the IFN-treated fibroblasts showed up-regulation of CD40, with a
surface punctate pattern of expression (Fig. 1, h and i).
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IL-6 was investigated because it is involved in the acute phase
response and is known to be produced by endometrium and to be involved
in infection-associated preterm labor. Four fibroblast strains were
cultured in the presence or absence of IFN for 72 h and then
stimulated with CD40L. Details for representative cell lines are
described in the text and figures. E8 showed a 25-fold increase in IL-6
production above control levels when stimulated with IFN plus CD40L.
A similar increase was found upon treatment with IFN alone.
Production of IL-6 by M2 was increased 6-fold in the presence of IFN
plus CD40L. IFN alone caused a 2-fold increase. CX-NU produced
17-fold more IL-6 in the presence of IFN plus CD40L. Treatment with
IFN alone or CD40L alone did not result in up-regulation of IL-6
production (Fig. 4). M9 had no detectable
surface CD40, and treatment with IFN plus CD40L did not result in
increased cytokine production (data not shown). The cytokine production
from four fibroblast strains was analyzed using nonparametric
statistics, and an effect (P < 0.05) was
demonstrated.
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IL-8 is a neutrophil chemoattractant. It is produced in the
perivascular region of endometrium and is found in the ripening cervix.
IL-8 production by E8 was up-regulated 9-fold over control levels in
the presence of IFN and was increased by a further 2-fold when
activated by IFN plus CD40L. M2 also produced 18-fold more IL-8 in
the presence of IFN plus CD40L. Secretion of IL-8 by CX-NU was
increased 6.5-fold by IFN plus CD40L treatment. Treatment with CD40L
alone did not result in up-regulation of IL-8 production (Fig. 5).
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MCP-1 attracts mainly monocytes and has been found to be produced
around the blood vessels of the endometrium. MCP-1 production by E8 was
up-regulated 3- and 9-fold by treatment with IFN alone and IFN
plus CD40L, respectively. Identical treatment of M2 produced 7- and
45-fold increases in MCP-1 secretion. Production by CX-NU was raised
approximately 2- and 8.5-fold (Fig. 6).
Each of the representative cell lines showed a modest up-regulation of
MCP-1 production in the presence of IFN alone, while CD40L treatment
alone did not increase production. Synergistic effects occurred upon
treatment with IFN plus CD40L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study IFN was used to increase surface CD40
expression before stimulation with CD40L. In vivo IFN or
another mediator may up-regulate CD40 display. IFN is produced by
activated T cells and natural killer cells. T cells present in
endometrial lymphoid aggregates are believed to produce IFN
(26). Interestingly, there is also evidence that
polymorphonuclear neutrophils in endometrium and from peripheral blood
secrete IFN (27). Neutrophils are closely associated
with menstruation (28), cervical ripening
(29) and active labor (30). This suggests the
presence of a positive feedback loop with neutrophils releasing IFN,
which would up-regulate CD40 expression, resulting in an enhanced
ability of resident fibroblasts to be activated and produce IL-8, the
key neutrophil chemoattractant.
The detection of CD40 in situ and on freshly derived
fibroblast strains in female reproductive tissues is particularly
interesting, as inflammatory mechanisms (with increased chemokine
release and infiltration of immune cells) are key elements in
reproductive physiology. We propose that the presence of CD40 allows
interaction of resident fibroblasts with immune cells. Conventionally,
the CD40-CD40L interaction is regarded as occurring via cellular
contact. However, our data do not exclude the possibility that in the
reproductive tract, the interaction is via soluble CD40L. In
endometrium, expression of IL-8 and MCP-1 increases in the perivascular
region before menstruation (2). Up-regulation of IL-8
messenger ribonucleic acid and protein is also observed in a model of
progesterone withdrawal (7). The detection of CD40 by
immunohistochemistry in the perivascular region suggests a mechanism
allowing up-regulation of these mediators. Although CD40 expression
remains constant throughout the menstrual cycle, it is possible that
the presence of CD40L in endometrium increases at the time of
menstruation. This would allow increased signaling via the CD40-CD40L
system. Additionally, COX-2 expression increases perivascularly in the
late secretory phase of the menstrual cycle (2) and after
progesterone withdrawal (7). CD40 activation may also be
responsible for this, as increased COX-2 and PGE2
expression occur upon stimulation of lung fibroblasts
(31). The expression of CD40 by perivascular cells also
suggests that the CD40-CD40L system may contribute to the tissue
remodeling that occurs during menstruation. The cells of the
endometrial perivascular area (predecidual stromal cells) are likely to
be of myofibroblast lineage, as they have been shown to express
-smooth muscle actin (25). Myofibroblasts have
contractile activity and are thought to be involved in wound healing.
Another interesting potential role for the CD40-CD40L system is in
cervical ripening and parturition. IL-8 release increases in the cervix
at the time of ripening (4, 32) and in the lower segment
myometrium during labor (30). As shown herein, activation
of human myometrial, cervical, and endometrial fibroblasts via CD40 is
a potent inducer of IL-8 synthesis. The increased production of IL-8,
possibly acting in synergy with PGE2 (5, 33), results in the recruitment and activation of neutrophils,
which are a prerequisite for the final rapid ripening of the cervix
before and during labor.
The activation of CD40 in vivo relies on the presence of CD40L-expressing cells at the local site. Several cell types present in reproductive tissues may express CD40L. In endometrium there are resident T cells, macrophages, and mast cells throughout the menstrual cycle, with the appearance of eosinophils premenstrually and uterine natural killer cells in the mid to late secretory phase (34, 35). Myometrium is infiltrated by T cells and macrophages at the onset of labor. Mast cells are present at low levels throughout pregnancy, with higher levels in the nonpregnant state (30). In the ripening cervix, mast cells and eosinophils (36) are present, and macrophages are present from late pregnancy onward (37). Activated platelets may also be present in reproductive tissues at times of infection and bleeding.
In addition to a seminal role in physiological reproductive events, there are pathophysiological problems that may also involve the CD40-CD40L system. Dysfunctional menstrual bleeding is likely to involve aberrant expression of uterine mediators. For instance, those associated with the perivascular region of endometrium and myometrium may contribute to menorrhagia, and it seems likely that dysregulation of the CD40 system could be involved. The importance of the CD40 system in menstrual pathology will depend on identification of the relevant source of the ligand. Further studies are necessary to determine whether T cells or platelets can activate the system in endometrium. However, it is clear that the extravasation of blood at the initiation of menstruation will deliver large numbers of platelets close to the site of CD40 expression. These platelets may express CD40L, allowing activation of CD40 (13). The resultant cytokine expression could modulate vessel permeability and thus act as a negative feedback regulator.
Premature labor is associated with increased expression of proinflammatory cytokines in the uterine cavity, particularly during infection (6, 38). The CD40-CD40L system may be activated during uterine infection and therefore be involved in the up-regulation of cytokines. Recently, CD40 has been detected in cervical carcinoma, with CD40L present on infiltrating T cells. CD40-CD40L interactions were suggested as a possible regulator of chemokine expression in carcinoma cells, indicating that the CD40-CD40L system may be involved in reproductive cancers (19).
Currently, disruption of the CD40-CD40L system using biologics has proven extremely successful in blunting transplant rejection (39), pulmonary injury (40, 41), and autoimmune disease (42). Disruption of the CD40-CD40L system in the female reproductive tract may also prove valuable in treating reproductive disorders.
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Acknowledgments |
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Footnotes |
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Received November 17, 1999.
Revised April 14, 2000.
Revised May 23, 2000.
Accepted September 29, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J Immunol. 162:4140–4147. in
human endometrium. Ann NY Acad Sci. 734:1–6.[Medline]
is produced by polymorphonuclear
neutrophils in human uterine endometrium and by cultured peripheral
blood polymorphonuclear neutrophils. J Immunol. 160:5145–5153.This article has been cited by other articles:
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  E. Malmstrom, M. Sennstrom, A. Holmberg, H. Frielingsdorf, E. Eklund, L. Malmstrom, E. Tufvesson, M. F. Gomez, G. Westergren-Thorsson, G. Ekman-Ordeberg, et al. The importance of fibroblasts in remodelling of the human uterine cervix during pregnancy and parturition Mol. Hum. Reprod., May 1, 2007; 13(5): 333 - 341. [Abstract] [Full Text] [PDF]
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 L. Koumas, T. J. Smith, S. Feldon, N. Blumberg, and R. P. Phipps Thy-1 Expression in Human Fibroblast Subsets Defines Myofibroblastic or Lipofibroblastic Phenotypes Am. J. Pathol., October 1, 2003; 163(4): 1291 - 1300. [Abstract] [Full Text] [PDF]
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 L. Koumas and R. P. Phipps Differential COX localization and PG release in Thy-1+ and Thy-1- human female reproductive tract fibroblasts Am J Physiol Cell Physiol, August 1, 2002; 283(2): C599 - C608. [Abstract] [Full Text] [PDF]
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L. Koumas, A. E. King, H. O. D. Critchley, R. W. Kelly, and R. P. Phipps Fibroblast Heterogeneity : Existence of Functionally Distinct Thy 1+ and Thy 1- Human Female Reproductive Tract Fibroblasts Am. J. Pathol., September 1, 2001; 159(3): 925 - 935. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
) or an equimolar concentration of mouse IgG1
(isotype control; 
). Low levels of CD40 were detected on untreated
fibroblasts compared with those incubated with mouse IgG1 (see
percentages in boxes). Treatment with IFN
