© 1995 American Heart Association, Inc.
Articles |
T Lymphocytes Affect Smooth Muscle Cell Phenotype and Proliferation
From the Centre for Research in Vascular Biology, Department of Anatomical Sciences, University of Queensland, Queensland, Australia.
Correspondence to Dr J.H. Campbell, Centre for Research in Vascular Biology, Department of Anatomical Sciences, University of Queensland, Queensland 4072, Australia.
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Abstract The effects of rabbit T lymphocytes on rabbit aortic smooth muscle cell (SMC) phenotype and proliferation were investigated in vitro. SMCs seeded at confluent density in primary culture had a volume fraction of myofilaments (Vvmyo) of 49.8±2.6% after 3 days of culture, not significantly different from that of freshly dispersed cells (Vvmyo, 54.1±2.1%). Sister cultures of SMCs to which Concanavalin A–activated T lymphocytes or T lymphocyte–conditioned medium was added had significantly lower Vvmyo (35.5±2.2% and 31.6±2.3%, respectively) at the same time point. We have previously shown that a decrease in Vvmyo could be induced by the heparan sulfate–degrading activity of living macrophages and by commercial preparations of heparinase. While activated T lymphocytes also completely degraded heparan sulfate–rich 35S-labeled extracellular matrix (an effect inhibited by the addition of 10 µg/mL heparin), no heparanase-like activity was detected in T lymphocyte–conditioned medium, indicating that for this cell type SMC phenotypic change is induced by a different mechanism. Incubation of the T lymphocyte–derived cytokine interferon gamma (IFN-
) with freshly isolated rat SMCs caused a significant
reduction in Vvmyo at day 2 in primary culture from
54.3±2.1% (control) to 35.4±3.0%. Furthermore, a neutralizing
antibody specific for IFN- removed the effect of T lymphocytes and
medium conditioned by them, thus positively identifying IFN- as the
T lymphocyte factor responsible for this activity. T
lymphocyte–conditioned medium was mitogenic for passaged
(low Vvmyo) SMCs. Although SMC proliferation was inhibited
by exogenous IFN-, two other T lymphocyte products,
granulocyte-macrophage–colony stimulating factor and tumor
necrosis factor–ß, were found to stimulate proliferation, while
interleukin-2 and interleukin-6 had no effect. It was concluded that T
lymphocytes, by inducing SMC phenotypic change and stimulating
proliferation, may play an important role in atherogenesis.
Key Words: smooth muscle cells • T lymphocytes • cytokines • phenotypic change • proliferation
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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T lymphocytes and macrophages are present in significant numbers at all stages of development of the human atherosclerotic lesion.1 2 3 In experimental models of this disease such as the cholesterol-fed rabbit, T lymphocytes and monocytes adhere to the endothelium of the artery wall within 7 days of the commencement of the experimental diet and penetrate the endothelium within 2 to 4 weeks.4 By 2 months a lesion is evident that resembles the fatty streak and consists mainly of macrophages and T lymphocytes.5 Medial smooth muscle cells (SMCs) migrate through the internal elastic lamina into the intima, where they proliferate and secrete extracellular matrix.6 Accompanying this process is the modulation of SMC phenotype, whereby there is a decrease in the volume fraction of myofilaments (Vvmyo).7 Studies in our laboratory have shown that in culture a decrease in the Vvmyo of SMCs can be triggered by living macrophages, which via membrane-bound or secreted proteases and a lysosomal endoglycosidase, degrade the heparan sulfate component of the SMC basal lamina.8 9 Macrophages also produce a number of growth factors for SMCs such as platelet-derived growth factor (PDGF).10
To date, little is known about the effect of T lymphocytes on SMC
phenotype and proliferation. Studies using animal models of
atherogenesis and vascular injury have yielded conflicting results,
with some suggesting that T lymphocytes inhibit SMC
proliferation11 12 and others that lesion development is
enhanced in the presence of T lymphocytes.13 Evidence that
T lymphocytes may have an indirect influence on SMC proliferation has
been presented by Wagner and coworkers,14 who show that
endothelial cell expression of SMC growth factors is increased by
exposure to alloreactive lymphocytes. However, the direct effects of T
lymphocytes on SMCs are uncertain. Most studies have concentrated on
the effect on SMCs of a single T lymphocyte product, interferon gamma
(IFN-), which inhibits SMC proliferation and collagen synthesis,
induces expression of class II MHC antigens, and decreases expression
of 
–smooth muscle actin in vitro.15 16 17
Myointimal thickening following arterial injury to the rat carotid
artery is also inhibited by IFN-.15 However, other T
lymphocyte products can influence SMC function; eg, tumor necrosis
factor–ß (TNF-ß) stimulates SMC proliferation18 and
induces expression of genes for interleukin-1 (IL-1) and
TNF.19 Ikeda et al20 report that IL-6
promotes SMC growth via the induction of endogenous PDGF, although an
earlier study by Loppnow and Libby21 failed to demonstrate
any effect.
The present study demonstrates that living T lymphocytes, T
lymphocyte–conditioned medium, and a T lymphocyte–derived cytokine
(IFN-) are potent inducers of SMC phenotypic change and that other T
lymphocyte products, granulocyte-macrophage–colony stimulating factor
(GM-CSF) and TNF-ß, stimulate SMC growth in vitro. This suggests that
T lymphocytes may play a wider role in atherogenesis than previously
suspected.
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Methods |
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Cytokines
Recombinant human IL-2 and IL-6 were purchased from Boehringer Mannheim, recombinant human GM-CSF and TNF-ß from Bachem Feinchemikalien, and recombinant rat IFN-
from Holland
Biotechnology. Except for IFN-, all cytokines tested
cross-react with rabbit cells. Because of its species specificity, rat
IFN- was tested for activity with rat SMCs. Monoclonal hamster
anti-murine IFN- neutralizing antibody, which binds mouse and rat
IFN-,22 was purchased from Genzyme.
Isolation of SMCs
Primary cultures of rabbit or rat SMCs were prepared by enzyme
digestion of the thoracic and abdominal aortic media from New Zealand
White rabbits (9 to 12 weeks old) or Wistar rats (8 to 10 weeks
old)23 (Central Animal Breeding House, University of
Queensland). Freshly dispersed rabbit or rat SMCs were seeded into
culture dishes at confluence (1.3x105
cells/cm2) in medium 199 (M199; Commonwealth Serum
Laboratories [CSL]) containing 5% fetal calf serum (FCS; PA
Biologicals) and 2 mmol/L glutamine (Sigma Chemical Co). Under these
confluent conditions, SMCs have been shown to maintain a high
Vvmyo for more than 2 weeks.24
For proliferation studies, rabbit SMCs in their first or second passage (seeded at 2.3x104 cells/cm2) were used. Rat SMCs were used in passages 5 through 10 and were seeded at the same density. All cells were maintained at 37°C in an atmosphere of 4% CO2 in air.
Isolation of T Lymphocytes
Rabbit or rat spleen cells were dispersed in Hanks' balanced
salt solution (HBSS; CSL), and viable mononuclear cells were separated
by Ficoll-paque (Pharmacia Fine Chemicals). The majority of
platelets were removed by centrifugation in HBSS
containing FCS at 1000 rpm for 5 minutes. The resulting cell
preparation was made up to a concentration of approximately
107 cells/mL in RPMI (CSL) containing 10 mmol/L HEPES (ICN
Biochemicals) and 10% FCS and was depleted of B lymphocytes by panning
on Petri dishes precoated with either anti-rabbit or anti-rat IgG
[goat affinity-purified F(ab')2 fragments to IgG, 10
µg/mL in 0.05 mol/L Tris-HCl, pH 9.2; Cappel Research Products].
This B lymphocyte–depleted preparation was incubated for a further 2
hours at 37°C in (uncoated) plastic Petri dishes to remove remaining
adherent cells. Immunofluorescence staining with
fluorescein isothiocyanate–conjugated anti-rabbit Ig
(Wellcome Diagnostics) or anti-rat Ig (Sera Lab Ltd) showed
that the resulting preparations contained less than 4% B
lymphocytes.
T lymphocyte–enriched preparations were activated by culturing
2x106 cells/mL for 48 hours at 37°C in RPMI
medium supplemented with 10 mmol/L HEPES, 5x10-5 mol/L
2-mercaptoethanol (ICN Biochemicals), 10% FCS, and 7.5 µg/mL
Concanavalin A (ConA, Boehringer Mannheim). Microscopic observation of
dishes after 48 hours of incubation showed <0.1% adherent cells.
Cells were then pelleted, and the supernatant medium (T
lymphocyte–conditioned medium) was collected (see below). The cells
were washed with RPMI containing –methyl mannoside (10 mg/mL;
Sigma) to remove ConA. T-blast cells were purified by
centrifugation on a cushion of Ficoll-paque for 20
minutes at 1500 rpm.25
Preparation of T Lymphocyte–Conditioned Medium
Supernatant was removed from activated T lymphocytes as
described above and mixed with two changes of Sephadex G-50
(Pharmacia Fine Chemicals). Each change lasted for 1 hour at
4°C (1 mL hydrated gel/mL T lymphocyte–conditioned medium) to remove
the ConA,26 and the conditioned medium was stored in small
volumes at -20°C until use. The conditioned medium was then desalted
on Sephadex G-25 columns (PD-10; Pharmacia) equilibrated with M199 and
passed through a 0.22-µm filter (Millipore Corp). As a control,
activation medium (RPMI containing 10 mmol/L HEPES,
5x10-5 mol/L 2-mercaptoethanol, 10% FCS, and 7.5 µg/mL
ConA) was processed in parallel. This medium had no effect on SMC
phenotype or proliferation.
Ultrastructural Morphometry
Freshly dispersed rabbit or rat SMCs in M199 with 5% FCS were
seeded at confluent density into 35-mm dishes. Rabbit SMCs were
incubated with either T lymphocytes (added on day 2 of culture) at a T
lymphocyte/SMC ratio of 1:5 for 24 hours or T lymphocyte–conditioned
medium (diluted 1 in 10 in M199 with 5% FCS, added on day 1) for 48
hours; control wells were incubated in medium alone. On day 3, SMCs
were washed and processed for electron microscopy.27 Test
media were added to rat SMCs on day 1 of primary culture and incubated
for 24 hours before processing for electron microscopy. Thin sections
were stained with 2% uranyl acetate and lead citrate and viewed under
the transmission electron microscope at a primary magnification of
x10 000. Photographs were taken of the cell closest to the
upper-right-hand corner of each grid square. The photographs were
analyzed by placing a lattice with 5-mm spacing over the
photograph and counting the number of lattice intersections falling
over the cytoplasm (Pcyt) and over filaments (Pmyo) (this included
myosin, actin, and 10-nm filaments and cytoplasmic- and
membrane-associated dense bodies). Vvmyo was then
calculated as a percentage of smooth muscle cytoplasm.
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Effect of Neutralization of IFN-
Medium conditioned by ConA-activated rat T lymphocytes
(diluted 1 in 10 in M199 with 5% FCS) was preincubated for 1 hour at
37°C with neutralizing antibody to IFN- (1 µg/mL). Anti–IFN-
(final concentration, 2 µg/mL) was added to activated T
lymphocytes. Following antibody treatment, conditioned medium or T
lymphocytes (T lymphocyte/SMC ratio, 1:5) were added to confluent
primary rat SMCs at day 1 of culture; sister cultures received M199
with 5% FCS alone, activated T lymphocytes, or T
lymphocyte–conditioned medium without antibody. Cells were incubated
for 24 hours and processed for ultrastructural morphometry as described
above.
SMC Proliferation
Since preliminary experiments showed a good correlation between
increase in DNA synthesis and increase in cell numbers (data not
shown), SMC proliferation was measured by [3H]thymidine
incorporation. Rabbit SMCs (passage 1 or 2) or rat SMCs (passages 5
through 10) in M199 with 5% FCS were seeded into 24-well plates (Nunc)
at 2.8x104 cells/cm2. They were allowed to
adhere overnight and were then growth arrested by a medium change to
M199 with 0.5% FCS. After 48 hours, test media (diluted in M199 with
0.5% FCS) were added along with [3H]thymidine (0.5
µCi/well; Amersham); control wells received fresh M199 with 0.5% or
5% FCS. Cells were incubated a further 24 hours and harvested onto
glass-fiber filters, and the amount of [3H]thymidine
incorporated was determined by scintillation counting. Each assay was
performed a minimum of three times.
Assay for Degradation Products of 35S-Labeled
Proteoglycans
Heparan sulfate–rich
[35S]O42--labeled extracellular
matrix was prepared by using bovine aortic endothelial
cells as described by Savion et al.28 The cell-free
35S-labeled extracellular matrix was incubated for 24 hours
at 37°C with activated T lymphocytes
(1x106 cells/mL in M199 with 5% FCS), T
lymphocyte–conditioned medium (diluted 1 in 2 in M199 with 5% FCS),
medium alone, or heparitinase (0.001 U/mL; Seikagaku Kogyo Co). Some
experiments were performed in the presence of sodium heparin (10
µg/mL; Sigma). The resulting supernatants were collected and
centrifuged at 400g for 5 minutes to remove T
lymphocytes and debris followed by centrifugation at
10 000g for 5 minutes. Volumes of 0.5 mL were subjected to
gel filtration on a Sepharose 6B (Pharmacia) column (0.7x35 cm) that
had been equilibrated with phosphate-buffered saline (PBS) containing
0.01% sodium azide and calibrated with Pharmacia molecular-weight
standards. Fractions (0.4 mL) were collected at a flow rate of 10 mL/h,
and after the addition of scintillin (Readysafe, Beckman Instruments),
radioactivity was measured in a scintillation counter. Each experiment
was performed a minimum of three times, with variation in elution
profiles between runs less than 2%.
Assay for Degradation of Sulfated Proteoglycans in the SMC
Basal Lamina
Freshly dispersed rabbit aortic SMCs were plated at confluent
density in 35-mm dishes. After 7 days in primary culture,
Na2[35S]O42-
(Amersham) was added to a final concentration of 30 µCi/mL and
incubated for 24 hours at 37°C. Under these conditions, 87% of the
radioactivity is associated with the cell surface, 1% is
intracellular, and 12% is incorporated in the extracellular
matrix.9 The living cells were thoroughly washed with
several changes of Dulbecco's PBS and immediately incubated for 24
hours with either T lymphocyte–conditioned medium (diluted 1 in 10 in
M199 with 5% FCS) or M199 with 5% FCS alone. Control wells in which
SMCs had been killed by incubation for 1 hour in PBS containing 0.1%
sodium azide were run in parallel.
Statistical Analysis
Statistical comparisons were made by a one-way ANOVA using
Dunnett's method for comparison of test groups with the control group.
Values were considered statistically significant at
P<.05.
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Results |
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Ultrastructural Morphometry
The effect of T lymphocytes on SMC phenotype was investigated by ultrastructural morphometry. At day 3 of primary culture, rabbit SMCs that had been seeded at confluent density and incubated in M199 with 5% FCS had a Vvmyo of 49.8±2.6%, a value that was not significantly different from that for freshly dispersed SMCs (Vvmyo, 54.1±2.1%; Fig 1
).
That is, under these culture conditions, SMCs maintained a high
Vvmyo 3 days after isolation. However when SMCs were
cocultured for 24 hours (between days 2 and 3) with activated T
lymphocytes in M199 with 5% FCS (T lymphocyte/SMC ratio, 1:5), the
Vvmyo at day 3 was significantly lower (35.5±2.2%) than
that of the control SMCs grown in M199 with 5% FCS alone
(P<.01), indicating that they had undergone a change in
phenotype. This change in phenotype was also induced by
incubation of primary SMCs with T lymphocyte–conditioned medium
(diluted 1 in 10 in M199 with 5% FCS, corresponding to a T
lymphocyte/SMC ratio of 1:2) for 48 hours (between days 1 and 3). For
these cells, the Vvmyo at day 3 was 31.6±2.3%, also
significantly lower than that of the control SMCs (P<.01).
Thus, activated T lymphocytes can induce a change in SMC
phenotype that is mediated by a releasable T lymphocyte
product.
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SMC Proliferation
To determine whether soluble T lymphocyte product(s) can also
influence SMC proliferation, T lymphocyte–conditioned medium was
tested for its effect on incorporation of [3H]thymidine
by passaged (low Vvmyo) SMCs. SMCs were growth arrested for
48 hours and then incubated for 24 hours with conditioned medium
(10-fold dilutions of 1 in 10 to 1 in 1000 in M199 with 0.5% FCS).
Control wells were incubated with medium alone. As shown in Fig 2, T lymphocyte–conditioned medium was equally
mitogenic at all concentrations tested.
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In light of these results, possible mechanisms by which T lymphocytes (and medium conditioned by them) can induce SMC phenotypic change and proliferation were investigated.
Degradation of Sulfated Proteoglycans
Enzymes that degrade heparan sulfate proteoglycan from the basal
lamina of SMCs, including a macrophage-derived heparanase,
induce SMC phenotypic change.9 Since T lymphocytes also
produce such enzymes,28 they may be responsible for the
induction of SMC phenotypic change observed in the present study. T
lymphocytes and medium conditioned by them were therefore tested for
their ability to degrade 35S-labeled extracellular matrix.
When 35S-labeled extracellular matrix was incubated for 24
hours at 37°C with M199 (with or without FCS), only
high-molecular-weight material was released. Fractionation of the
resulting supernatant on Sepharose 6B resulted in a single peak of
radioactivity eluting at the void volume (Fig 3).
Incubation of T lymphocytes with 35S-labeled extracellular
matrix for 24 hours resulted in the release of low-molecular-weight
degradation products with approximately 48% of the total
radioactivity eluting with a Kav
0.84, similar
to the peak of degraded material obtained by incubation of matrix with
heparitinase (0.001 U/mL). The degradative activity of T lymphocytes
was specific for heparan sulfate proteoglycan since no degradation
products were liberated in the presence of sodium heparin (10
µg/mL).
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), T
lymphocytes (106 cells/mL)+sodium heparin (10
µg/mL; 
), T lymphocyte–conditioned medium (diluted 1 in 2;
), heparitinase (0.001 U/mL; 
), or medium alone (
).In contrast, incubation of labeled matrix with T lymphocyte–conditioned medium yielded an elution profile similar to that obtained with medium alone. Thus, while T lymphocytes have heparanase activity that may promote SMC phenotypic change, this activity is cell associated and cannot account for the phenotype-modulating activity of T lymphocyte–conditioned medium. Other (releasable) T lymphocyte products must be responsible for inducing SMC phenotypic modulation.
Degradation of Smooth Muscle Basal Lamina
SMCs with a high Vvmyo are thought to maintain their
phenotype by continuously internalizing and degrading the
heparan sulfate from their own basal lamina.9 We therefore
investigated the possibility that a soluble T lymphocyte product
may interfere with this process by either inhibiting uptake or
enhancing the rate of degradation and in so doing, induce a change in
phenotype. When living SMCs whose basal lamina had been labeled
with 35S were incubated with M199 and 5% FCS,
low-molecular-weight degradation products
(Kav0.84) were released into the medium.
However, incubation of these SMCs with T lymphocyte–conditioned medium
had no significant effect on either the amount of label released into
the medium or the size of the degradation products (Fig 4).
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Effect of T Lymphocyte Cytokines
Activated T lymphocytes produce a number of protein
mediators, some of which have been shown to influence the behavior of
vascular cells.29 We therefore investigated whether known
cytokines may be responsible for the activity observed in T
lymphocyte–conditioned medium. Since IFN- causes a decrease in
-actin expression by SMCs,16 we determined whether
IFN- could reduce the Vvmyo of primary SMCs. For this
experiment, rat IFN- (100 U/mL in M199 with 5% FCS) was added to
densely seeded primary rat SMCs at day 1 of culture and incubated for
24 hours; the use of rat cells was necessitated by the species
specificity of IFN- activity. Cells were fixed at day 2 and
analyzed by ultrastructural morphometry. There was a reduction
in Vvmyo (P<.01) from 54.3±2.1% for the
control cells (incubated in M199 with 5% FCS) to 35.4±3.0%
for cells exposed to IFN- (Fig 5, top). To determine
whether IFN- was responsible for the phenotype-modulating
activity in T lymphocyte releasates, conditioned medium was
preincubated with neutralizing antibody to IFN-. While T
lymphocyte–conditioned medium induced a decrease in Vvmyo
compared with control cells (36.3±2.5% versus 54.3±2.1%,
P<.05), preincubation of this medium with neutralizing
antibody completely removed its effect (Vvmyo, 51.4±2.3%;
Fig 5, top).
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In separate experiments, we determined whether IFN- is solely
responsible for the ability of T lymphocytes to induce SMC phenotypic
change or whether other cell-associated mechanisms (eg, heparanase
activity) also play a role. In these experiments, day-2 rat SMCs that
had been incubated with T lymphocytes for 24 hours (T lymphocyte/SMC
ratio, 1:5) had a Vvmyo of 38.0±2.4%, significantly
lower than that of control cells (Vvmyo, 59.0±3.1%,
P<.05; Fig 5
, bottom). The higher control values in this
set of experiments, though not significant, are probably due to
differences in the ages of the animals used as the source of SMCs. In
the presence of neutralizing antibody to IFN-, T lymphocytes had no
effect on Vvmyo (63.8±2.2%, NS versus control), thus
implicating IFN- as the sole mechanism by which T lymphocytes induce
change in SMC phenotype.
Several cytokines produced by T lymphocytes (IFN-, IL-2,
IL-6, TNF-ß, and GM-CSF) were tested for their ability to influence
SMC proliferation. Rabbit SMCs (passage 2) or rat SMCs (passages 5
through 10) were growth arrested for 48 hours before incubation with
the appropriate cytokine (in the presence of
[3H]thymidine) for 24 hours. With the exception of
IFN- (which was diluted in M199 with 5% FCS and incubated with rat
SMCs), cytokines were diluted in M199 with 0.5% FCS and tested
(at 10-fold dilutions) over their known range of biological activity
with rabbit SMCs; control wells were incubated in medium alone. IFN-
(50 U/mL) in the presence of 5% FCS inhibited
[3H]thymidine incorporation by 34±8%
(P<.05), a result similar to that described by
others.15 16 18 In contrast, GM-CSF stimulated SMC
proliferation in the presence of 0.5% FCS, giving a bell-shaped
dose-response curve, with maximal stimulation at 0.1 to 1.0 ng/mL (Fig 6). Incubation of SMCs with TNF-ß (0.5 to 50 ng/mL)
also produced a small though significant increase in
[3H]thymidine incorporation (45±23% at 50 ng/mL, n=6;
P<.01). IL-2 (1 to 100 U/mL) and IL-6 (0.5 to 50 U/mL) had
no effect (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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SMCs are maintained in the high-Vvmyo phenotype by the presence of heparan sulfate proteoglycans in their basal lamina, and commercial heparan sulfate–degrading enzymes (eg, heparinase) specifically induce a change to the low-Vvmyo phenotype.9 Living macrophages also degrade the heparan-sulfate component of the SMC basal lamina (via proteases and a lysosomal endoglycosidase) and trigger the modulation of SMC phenotype, processes that are inhibited by the addition of heparin. In the present study, both T lymphocytes and T lymphocyte–conditioned medium induced decreases in the Vvmyo of cultured SMCs. However, the mechanism by which T lymphocytes were effective was not directly through degradation of SMC heparan sulfate proteoglycan, since T lymphocyte–conditioned medium had no heparan sulfate–degrading activity. Furthermore, neutralizing antibody to IFN-
completely removed the SMC
phenotype–modulating effect of both T lymphocytes and T
lymphocyte–conditioned medium, while exogenous addition of IFN- to
SMCs induced a decrease in their Vvmyo. Thus, while T
lymphocytes do exhibit protease30 and heparanase activity,
these enzymes do not play a significant role in the modulation of SMC
phenotype by T lymphocytes, at least under the experimental
conditions used in this study. Instead, all the activity can be
attributed to IFN-. This is consistent with an earlier
observation of Hansson et al16 that IFN- reduces SMC
-actin expression.
IFN- is known to alter SMC expression of several genes, including
-actin, class II MHC antigens, and collagen types I and
III.15 16 17 Its effect on SMC phenotype is most
likely mediated by such alterations, although the genes responsible
have not been identified. Since cytokines can alter
proteoglycan metabolism,31 another possibility
is that IFN- may either inhibit SMC capacity to degrade
phenotype-controlling heparan sulfate proteoglycans or alter
their rate of synthesis. For example, some cytokines are known
to induce the synthesis and activation of proteases31 that
may enhance degradation of heparan sulfate proteoglycans by SMCs,
possibly by producing a more accessible substrate for heparanases. In
the present study, however, we were unable to demonstrate any
effect of T lymphocyte–conditioned medium on degradation of
35S-labeled proteoglycans by SMCs themselves.
Alternatively, IFN- may induce qualitative changes in
glycosaminoglycan synthesis, similar to those reported for TNF- and
IL-1.32 33 The effect of IFN- (or other soluble T
lymphocyte products) on glycosaminoglycan synthesis by SMCs has not
yet been determined.
IFN- is also an effective inhibitor of SMC
proliferation.15 16 18 Thus, it can induce a change in SMC
cytodifferentiation to a state in which proliferation can occur
but then maintains the cells in a quiescent state. However, T
lymphocyte–conditioned medium can stimulate SMC proliferation,
suggesting that other T lymphocyte products also influence SMC
biology. Indeed, the present study identifies two
cytokines, GM-CSF and TNF-ß, that are mitogenic
for SMCs. While one study has identified TNF-ß as an SMC
mitogen,18 this is the first report that GM-CSF is
mitogenic for SMCs. Although GM-CSF acts primarily on the
hemopoietic system, receptors for GM-CSF have been found on various
cell types,34 and GM-CSF influences the migration and
proliferation of endothelial cells and fibroblasts and
is chemotactic for SMCs.35 36
In conclusion, the present in vitro studies predict that invasion
of the artery wall by T lymphocytes promotes myointimal thickening by
inducing a change in SMC phenotype. The modulation of SMC
phenotype is effected through IFN-, which also acts to
inhibit proliferation of SMCs. However, the cells become receptive to
mitogenic stimulation by other factors, including the T
lymphocyte products GM-CSF and TNF-ß. This complexity is
reflected in the conflicting reports on the effects of T lymphocytes in
atherogenesis or following vascular injury.11 12 13
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Acknowledgments |
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This work was supported by a grant from the National Health and Medical Research Council of Australia. The authors wish to thank G. Hansson for valuable discussion, A. Thomas for her assistance with computer graphics, and P. Bretherton and N. Lee for typing the manuscript.
Received February 2, 1995; accepted April 21, 1995.
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References |
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