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*
Departments of Pathology and
Internal Medicine, Division of Pulmonary and Critical Care, University of Michigan Medical School, Ann Arbor, MI 48109; and
Department of Pathology, Veteran Affairs Medical Center, Ann Arbor, MI 48105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or IL-4 and TNF- (all at 10 ng/ml) for 24 h released five- to
eightfold more MCP-1 than similarly treated splenic macrophages. In
72-h coculture experiments, the synthesis of IL-4 by OVA-activated
CD4+ T cells added to lung fibroblasts or splenic
macrophages was significantly inhibited when endogenous MCP-1 was
neutralized using polyclonal anti-MCP-1 antiserum. In these same
cocultures, IFN-
levels were significantly enhanced. Similarly,
IFN- levels were significantly enhanced in 72-h cocultures of a
purified peptide derivative-activated CD4+ Th1 clone and
lung fibroblasts or splenic macrophages following immunoneutralization
of MCP-1. In separate experiments, the selective inhibition of MCP-1
synthesis by lung fibroblasts and splenic macrophages using an MCP-1
antisense oligonucleotide significantly enhanced the proliferation of
CD4+ T cells during a 96-h coculture. Taken together, these
data suggest that MCP-1 exerts an immunomodulatory effect on
CD4+ T cell-derived IL-4 and IFN- release and
CD4+ T cell proliferation during cell-to-cell interactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) exert potent effects on polymorphonuclear cells, whereas
the C-C chemokines predominately affect the movement of mononuclear
leukocytes such as monocytes and T lymphocytes. Examples of C-C
chemokines include macrophage chemotactic protein-1
(MIP-1),3 eotaxin,
C10, and monocyte chemoattractant protein-1 (MCP-1) (5). MCP-1 was first identified as a platelet-derived growth factor-inducible product (JE) from murine 3T3 fibroblasts (6, 7). Since this initial discovery, MCP-1 synthesis has been detected in synoviocytes, endothelium (8), epithelial cells (9, 10), fibroblasts (8, 11), monocytes/macrophages, vascular smooth muscle cells (12, 13), mesangial cells, and keratinocytes (14). MCP-1 has been implicated in various diseases in which infiltrating monocytes and/or lymphocytes exert a prominent effect during the inflammatory process (15), including rheumatoid arthritis (16), inflammatory bowel disease (17), and idiopathic pulmonary fibrosis (18, 19). In experimental models of disease, numerous studies have illustrated the contrasting pathogenic and protective role of MCP-1. For example, MCP-1 contributes to delayed-type hypersensitivity responses in rats (20), the inflammatory cell infiltration into the lungs of bleomycin-treated mice (21), and mediates vessel aortic smooth muscle injury (22). However, MCP-1 is necessary for the containment of Cryptococcus neoformans through its effect on the recruitment of CD4+ T cell and monocyte/macrophage into the lungs of infected mice (23). Thus, the regulated synthesis of MCP-1 is critical to the initiation and maintenance of inflammatory responses in various organs and tissues.
While chemokines clearly mediate leukocyte infiltration into injured or
inflamed tissue sites, recent findings also suggest that chemokines may
have additional immunoregulatory roles (3). For example, the systemic
overexpression of MCP-1 in transgenic mice demonstrated no overt
monocyte accumulation in organs, but these mice were susceptible to
infection by intracellular parasites (24). When MCP-1 was selectively
overexpressed in the murine lung, monocyte recruitment to the lung was
evident, yet lung injury was only observed when an inflammatory
stimulus was delivered (25). Separate studies have shown that MCP-1
contributes to the evolution of Th2 pulmonary granulomas, possibly
through MCP-1 modulation of lymphocyte IL-4 production (26).
Attenuation of experimental autoimmune encephalomyelitis was correlated
with increased MCP-1 synthesis and decreased MIP-1. In addition,
exogenous MCP-1 attenuated the transfer of disease in this model (27).
These studies support the hypothesis that MCP-1 may play an important
regulatory role in these and other inflammatory or immune responses via
novel effects on lymphocyte function.
The present study addressed the role of MCP-1 in the regulation of
CD4+ T cell activation during coculture with normal lung
fibroblasts or splenic macrophages. We examined the effects of MCP-1
released by lung fibroblasts and splenic macrophages on two aspects of
T cell activation, namely de novo IL-4 and IFN- generation and T
cell proliferation. These responses were tested using purified splenic
CD4+ T cells from mice sensitized to OVA in the presence of
either CFA-OVA or Schistosoma mansoni egg Ag (SEA-OVA). In
these experiments, isolated CD4+ T cells from both Ag
sensitization models were mixed in equal proportions to obtain a
CD4+ T cell population that generated IL-4 and IFN-
following OVA rechallenge. A 72-h coculture of CD4+ T cells
with lung fibroblasts or splenic macrophages demonstrated that
endogenous MCP-1 was necessary for IL-4 generation by the
OVA-stimulated CD4+ T cells. In the absence of MCP-1,
IFN- levels in these same cocultures were significantly increased.
Further, immunoneutralization of MCP-1 during a 72-h coculture of a
murine CD4 + Th1 clone reactive to purified-peptide derivative
(PPD) from Mycobacterium with lung fibroblasts or splenic
macrophages significantly enhanced IFN- levels. To address the
effect of MCP-1 on CD4+ T cell proliferation, MCP-1
synthesis by lung fibroblasts and splenic macrophages was inhibited by
using an MCP-1 antisense oligonucleotide during a 96-h coculture of
these cells and CD4+ T cells. In these experiments,
OVA-specific proliferative responses by CD4+ T cells were
significantly enhanced, not only during coculture with splenic
macrophages, but also during coculture with lung fibroblasts. These
data show a dichotomous influence of MCP-1 on IL-4 and IFN-
synthesis by CD4+ T cells, and a modulatory effect on the
proliferation of CD4+ T cells during cell-to-cell
interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female CBA/J mice (5–7 wk of age) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were housed under specific-pathogen-free conditions with access to food and water at all times. All procedures described herein were approved by the University Laboratory Animal Medicine facility at the University of Michigan Medical School. Before purification of OVA-sensitized CD4+ splenic T cells (see below), mice were injected i.p. with 400 µg of chicken egg albumin, fraction V (OVA; Sigma, St. Louis, MO) in combination with either 0.5 ml of CFA diluted 1:1 with normal saline (CFA-OVA) or 3000 freshly isolated S. mansoni eggs suspended in 0.5 ml of normal saline (SEA-OVA).
Purification of splenic CD4+ T lymphocytes from OVA-sensitized mice
Fourteen days after sensitization, spleens were aseptically
removed from either CFA-OVA or SEA-OVA mice and suspended in RPMI
containing 10% FBS (RPMI-10). RBC were lysed from the cell suspensions
using a hypotonic lysing buffer (150 mM NH4Cl; 10 mM
NaHCO3; 1 mM EDTA) for 2 min at 4°C and the remaining
cells were added to six-well tissue culture plates. After incubating
for 2 h at 37°C, the supernatants containing nonadherent
splenocytes were removed, and these cells were further purified to
yield cell preparations of CD4+ T cells. Briefly, the
nonadherent spleen cells were treated with anti-mouse Ly2 (CD8; YTS
169.4; Accurate, Westbury, NY) for 1 h at 4°C. CD4+
T cells were then recovered by eluting the Ab-treated spleen cells
through a Cedarlane Mouse CD4 Recovery Column (Accurate) with 20 ml of
RPMI-10 (37°C). According to flow cytometric analysis, 
85% of the
cells eluted from the CD4 recovery column were CD4+ T
cells. No CD8+, CD23+, or ED1+
cells were detected in cell suspensions of eluted cells.
Initial studies showed that CD4+ T cells purified from
CFA-OVA and SEA-OVA mice had polarized IFN- and IL-4, respectively,
following OVA rechallenge during a 72-h coculture with lung fibroblasts
or splenic macrophages. To obtain a CD4+ T cell population
from which IL-4 and IFN- production was consistently demonstrable,
CFA-OVA and SEA-OVA CD4+ T cells were mixed in equal
proportions before coculture experiments (see below). Mixed CFA-OVA and
SEA-OVA CD4+ T cells produced both IL-4 and IFN-
following OVA rechallenge in vitro.
Normal lung fibroblast cell cultures
Cultures of normal lung were isolated and cultured as previously
described in detail (11). Fibroblasts were grown at 37°C in a
humidified CO2 incubator and fed DMEM containing 1%
(v/v) antibiotic-antimycotic and 15% (v/v) FBS. These cells were
expanded in 175-ml tissue culture flasks, and after a minimum of two
passages, homogeneous populations of fibroblasts were transferred to
6-well or 96-well tissue culture plates for experiments. Cultured lung
fibroblasts were used in these experiments up to the sixth passage. For
immunocytochemical characterization, fibroblasts were transferred to
2-well Lab-Tek (Nunc, Naperville, IL) chamber culture slides. Cells
stained for -actin and desmin suggested a myofibroblast-type
phenotype, and these cultures were found to be completely free of
-naphthyl acetate esterase-positive macrophages (data not shown).
Five days before an experiment, each well in a 6-well and 96-well
tissue culture plate was initially seeded with 1 x
106 and 1 x 104 fibroblasts,
respectively.
Splenic macrophage cultures
Spleens were aseptically removed from nonsensitized mice,
dissociated, and suspended in RPMI-10. RBC were lysed from the cell
suspensions using a hypotonic lysing buffer for 2 min at 4°C and the
remaining cells were added to six-well tissue culture plates. After
incubating at 37°C for 2 h, the supernatants containing
nonadherent splenocytes were removed. The splenic macrophages were
maintained in DMEM growth medium for 5 days before the use of these
cells in an experiment. Each well of a six-well tissue culture plate
contained approximately 1 x 106 adherent cells,
of which approximately 95% were -naphthyl acetate esterase-positive
macrophages. -Naphthyl acetate esterase staining was performed
according to a protocol supplied by Sigma.
Lymphokine generation by purified CD4+ T cells or Th1 clones following a 72-h coculture with lung fibroblasts or splenic macrophages
The experimental protocol employing cocultures of purified
OVA-reactive CD4+ T cell and isolated lung fibroblasts
or splenic macrophages is summarized in Figure 1
. Lung fibroblasts and splenic
macrophages in six-well tissue culture plates were left untreated, or
exposed to murine IL-4 (R&D Systems, Minneapolis, MN), human TNF-
(Genzyme, Cambridge, MA), or IL-4 + TNF- (all cytokines at 10
ng/ml) in RPMI-10 containing 200 µg/ml of OVA for 24 h (step 1).
IL-4 and TNF- have previously been shown to be potent inducers of
MCP-1 synthesis by endothelial cells (28), lung fibroblasts (11), and
lung epithelial cells (9). Following a 24-h pretreatment of the lung
fibroblasts or splenic macrophages, a 500-µl aliquot was removed and
stored at -20°C for measurement of MCP-1 levels. These cells were
then thoroughly washed with buffered HBSS to remove all exogenous
cytokine and OVA (step 2), and 1 x 106 purified
CD4+ T cells in 1 ml of RPMI-10 containing 200 µg of OVA
were added to the cocultures (step 3). An equivalent mixture (i.e.,
5 x 105 of each) of CFA-OVA and SEA-OVA
CD4+ T cells was used in these experiments. Equivalent
numbers of CD4+ T cells were also added to wells lacking
lung fibroblasts or splenic macrophages. To address the role of MCP-1
on T cell lymphokine generation during the coculture, normal rabbit
serum or rabbit anti-MCP-1-neutralizing polyclonal Ab (11) was
added at the beginning of the coculture period (step 3). After 72
h in coculture, a cellfree supernatant was removed from each well (step
4) for chemokine and cytokine measurements (see below).
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, TNF-,
IL-4 + TNF-, or IFN- + TNF- (all at 10 ng/ml) for 24
h. Following the pretreatment, these cells were washed thoroughly, and
1 x 103 PPD-reactive Th1 cloned cells (kindly
provided by Dr. Dennis D. Taub, National Institute of Aging,
Gerontology Research Center, Baltimore, MD) suspended in 200 µl of
RPMI-10 containing 1 µg of PPD were added to the lung fibroblasts or
splenic macrophages. Either normal rabbit serum or
anti-MCP-1-neutralizing Ab was also added. Seventy-two hours later,
cellfree supernatants were removed for the measurement of IL-4 and
IFN- by ELISA. Effect of endogenous MCP-1 on the proliferation of purified CD4+ T cells following a 96-h coculture with lung fibroblasts or splenic macrophages
In experiments designed to address the role of MCP-1 in
CD4+ T cell proliferation, MCP-1 synthesis was
inhibited using purified phosphorothioated MCP-1 antisense
oligonucleotide (5'-AAG CGT GAC AGA GAC CTG CAT AGT GGT GG-3'; 10 nM
final concentration), which was added during the pretreatment and
coculture period. Purified phosphorothioated MCP-1 sense
oligonucleotide (5'-CCA CCA CTA TGC AGG TCT CTG TCA CGC TT-3'; 10 nM
final concentration) was added to other control cultures at the same
time points. To assess CD4+ T cell proliferation, these
cells were cocultured with fibroblasts or splenic macrophages in 6-well
tissue culture plates for 96 h. Equivalent numbers of
CD4+ T cells were also added to wells lacking lung
fibroblasts or splenic macrophages for the same time period. Changes in
proliferation by the Th1 clones were not examined due to a high
background of [3H]TdR incorporation by these cells. After
96 h, the CD4+ T cells were transferred to 96-well
tissue culture plates (Fig. 1
, step 5) and incubated at 37°C for
24 h in the presence of 1 µCi of [3H]TdR (ICN
Radiochemicals, Irvine, CA) and 200 µg of OVA (Fig. 1, step 6).
Fibroblast or splenic macrophage contamination of the transferred
CD4+ T cells was consistently below 1%. Finally, the
CD4+ T cells were suctioned from each well of the 96-well
tissue culture plates onto glass fiber filter strips (Cambridge
Technology, Watertown, MA) using a PhD Cell Harvester (Cambridge
Technology). Sections on the filter strips corresponding to each well
of the tissue culture plate were punched-out, and the pieces were
transferred to scintillation vials. [3H]TdR incorporation
by CD4+ T cells was then determined by liquid scintillation
counting on a Beckman counter (Beckman Instruments, Fullerton,
CA).
Chemokine and cytokine measurement
Murine MCP-1, IL-4, and IFN- levels were determined in
50-µl supernatant samples using a standardized sandwich ELISA as
previously described (19). Briefly, Nunc-immuno ELISA plates (MaxiSorp;
Naperville, IL) were coated with the appropriate cytokine capture Ab at
a dilution of 1 µg/ml of coating buffer (0.6 M NaCl, 0.26 M
H3BO4, 0.08 M NaOH, pH 9.6) for 16 h
at 4°C. The excess amount of capture Abs was washed away and each
plate was blocked for 90 min with 2% BSA-PBS at 37°C. After the
blocking period, each ELISA plate was washed with PBS Tween-20 (0.05%;
v/v), and samples (no dilution or 1:10; 50-µl vol) were added to
wells in duplicate for 1 h at 37°C. Recombinant murine MCP-1,
IL-4, and IFN- standard curves were used to calculate chemokine or
cytokine concentrations. The plates were then thoroughly washed and the
appropriate biotinylated polyclonal rabbit anti-cytokine Ab
(3.5 µg/ml) was added (11). After washing the plates 30 min
later, streptavidin-peroxidase (Bio-Rad, Richmond, CA) was added to
each well for 30 min, and each plate was thoroughly washed again.
Chromagen substrate (Bio-Rad) was added and plates were read on an
ELISA plate scanner at 492 nm. The limit of detection for each cytokine
and chemokine was consistently above 10 pg/ml.
Statistical analysis
Purified CD4+ T lymphocytes were used from a total of four OVA-sensitized mice (CFA-OVA and SEA-OVA), and all test conditions were completed in duplicate for cytokine and chemokine measurements and in triplicate for a proliferation assay. Results are expressed as mean ± SE of the mean of a minimum of three separate experiments. ANOVA and the Neuman-Keuls multiple comparison test were used to determine statistical significance between control and experimental groups; p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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De novo synthesis of MCP-1 by murine lung fibroblasts and splenic
macrophages is shown in Figure 2. After
24 h, 22 ± 6 ng/ml of MCP-1 was detected in cultures of
untreated lung fibroblasts whereas approximately four- to sixfold more
MCP-1 was measured in fibroblast cultures treated with either IL-4,
TNF, or both cytokines. No differences in MCP-1 levels were observed
between the cytokine treatment groups. When compared with untreated
fibroblasts, untreated splenic macrophages constitutively produced
markedly less MCP-1 (8 ± 1 ng/ml) over the same time period.
Further, MCP-1 synthesis by cytokine-treated splenic macrophages was
not increased after 24 h.
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In the investigations designed to assess the effect of MCP-1
released from lung fibroblasts or splenic macrophages on
CD4+ T cell lymphokine synthesis, we first assessed
the IL-4 levels in 72-h cocultures containing or lacking
anti-MCP-1-neutralizing Ab. When cellfree supernatants were
examined, immunoreactive MCP-1 was not detected in any of the cell
cocultures treated with the anti-MCP-1-neutralizing Ab (data not
shown). In these experiments an equivalent number of isolated
CD4+ T cells from CFA-OVA- and SEA-OVA-sensitized mice was
cocultured with lung fibroblasts or splenic macrophages. These
CD4+ T cell populations were mixed to simulate an equal
proportion of Th cytokine populations of CD4+ T cells,
since OVA rechallenge of pure populations of CFA-OVA or SEA-OVA
CD4+ T cells cocultured with lung fibroblasts for 72 h
polarized lymphokine synthesis toward IFN- and IL-4, respectively
(Table I).
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, step 2). Clear differences in IL-4 levels in cocultures of
CD4+ T cells and lung fibroblasts or splenic macrophages
were apparent between the control serum and anti-MCP-1 Ab treatment
groups at the 72-h time point (Fig. 3).
The nature of the cytokine treatment of the lung fibroblasts or splenic
macrophages exerted a direct effect on subsequent IL-4 generation
during the coculture period because the greatest levels of IL-4 were
detected in cocultures of CD4+ T cells and lung fibroblasts
(Fig. 3A) or splenic macrophages (Fig. 3B) treated with IL-4 and TNF-. Levels of IL-4
production by CD4+ T cells cocultured with fibroblasts were
similar to those generated by these T cells during coculture with
splenic macrophages (Fig. 3, A vs B).
However, significant reductions in IL-4 due to the presence of
anti-MCP-1 Ab were observed in cocultures of CD4+ T
cells and lung fibroblasts or splenic macrophages (Fig. 3B).
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production
It was next determined whether changes in IL-4 following
immunoneutralization of MCP-1 were associated with alterations to
CD4+ T cell-derived IFN-. Significantly higher
levels of IFN- were apparent when anti-MCP-1-neutralizing Ab was
included in cocultures of CD4+ T cells and cytokine-treated
lung fibroblasts (Fig. 4A). In addition,
significant elevations in IFN- levels were present in all cocultures
of mixed CFA-OVA and SEA-OVA CD4+ T cells and splenic
macrophages when MCP-1 was immunoneutralized (Fig. 4B). Specifically, fourfold increases in IFN- were
observed in cocultures of T cells and IL-4-treated splenic macrophages.
These data suggested that both IL-4 and IFN- levels were affected by
endogenous MCP-1 production during T cell coculture with either
fibroblasts or splenic macrophages.
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synthesis by Th1
clones cocultured with lung fibroblasts or splenic macrophages
From the previous set of experiments, alterations in IFN-
synthesis by mixed CFA-OVA and SEA-OVA CD4+ T cells
appeared to be enhanced by the neutralization of MCP-1. To address this
observation more fully, we assessed whether MCP-1 had any direct effect
on the ability of a PPD-reactive Th1 clone to produce IFN- following
Ag rechallenge. When cultured in the absence of fibroblasts or splenic
macrophages, the Th1 clone did not produce detectable quantities of
IL-4 or IFN- following PPD rechallenge for 72 h (Table II). In addition, no IL-4 was detected
during PPD-rechallenge of the Th1 cell clone during a 72-h coculture
with untreated or cytokine-treated lung fibroblasts or splenic
macrophages. IFN- was detected in all cocultures of Th1 cells and
cytokine-treated lung fibroblasts except in cocultures of
TNF--treated fibroblasts and Th1 cells. In the presence of
anti-MCP-1 Ab, Th1-derived IFN- was significantly enhanced in
all cocultures after 72 h (Table II). In cocultures of the Th1
clone and cytokine-treated splenic macrophages, IFN- levels varied
between 0.17 and 1.7 ng/ml. The differences in IFN- generation
during the 72-h coculture appeared to be dependent on the nature of the
splenic macrophage pretreatment (Table II). Immunoneutralization of
MCP-1 significantly increased IFN- synthesis by Th1 cells
approximately 2-fold when these cells were cocultured with IL-4- or
IFN--treated splenic macrophages. IFN- levels were decreased in
cocultures of Th1 cells and TNF--treated splenic macrophages, but
levels of IFN- were increased 10-fold in cocultures of Th1 cells and
IL-4- + TNF--treated splenic macrophages. Similarly, IFN- levels
in cocultures of Th1 cells and IFN-- + TNF--treated splenic
macrophages were significantly enhanced at 72 h when MCP-1 was
immunoneutralized. Thus, these data suggested that MCP-1 modulated the
production of IFN- by Th1 clones during coculture with lung
fibroblasts or splenic macrophages.
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The proliferative response of CD4+ T cells during
their coculture with fibroblasts or splenic macrophages was next
examined. Although lung fibroblasts are not normally considered
professional APCs, it has been shown that lung fibroblasts express MHC
II and present Ag to T cells (29). Murine fibroblasts have also been
shown to express the costimulatory molecule CD80 (B7-1), which is
necessary for optimal T cell activation (30). Initial investigations
demonstrated that mixed CFA-OVA and SEA-OVA CD4+ T cells
showed the greatest proliferation responses to OVA rechallenge (data
not shown). The aim of these experiments was to determine the effect of
endogenous MCP-1 on the proliferation of CD4+ T cells
following a 96-h coculture with lung fibroblasts or splenic
macrophages. To this end, MCP-1 antisense oligonucleotide was used to
inhibit the synthesis of MCP-1. After 96 h, MCP-1 levels were
inhibited by approximately 46 to 61% in cocultures of fibroblasts and
CD4+ T cells and MCP-1 synthesis in cocultures of splenic
macrophages and CD4+ T cells was inhibited completely.
(Table III). The inhibitory effect of
MCP-1 antisense oligonucleotide was limited to MCP-1, since no effect
was observed on C10 or eotaxin generation by fibroblasts or splenic
macrophages (not shown). The proliferative responses of purified
CD4+ T cells during a 96-h coculture with untreated and
cytokine-treated fibroblasts or splenic macrophages in the presence of
MCP-1 sense or antisense oligonucleotide are shown in Figure 5, panels A and
B. CD4+ T cells proliferated poorly (i.e.,
incorporating approximately 1 x 103 cpm of
[3H]thymidine) when cultured alone, and their
proliferation responses during coculture with untreated or
cytokine-treated fibroblasts was marginally (i.e., <2-fold increase in
[3H]TdR uptake) affected by MCP-1 sense oligonucleotide.
[3H]TdR incorporation by CD4+ T cells during
a 96-h coculture with splenic macrophages in the presence of MCP-1
sense oligonucleotide was enhanced >25-fold above control (i.e.,
CD4+ T cells cultured alone) only when the splenic
macrophages were treated with TNF- alone. While MCP-1 antisense
oligonucleotide treatment did not enhance CD4+ T cell
proliferation when these cells were cultured alone or cocultured with
untreated fibroblasts, [3H]TdR incorporation by these
cells typically exceeded 5 x 103 cpm during coculture
with cytokine-treated fibroblasts. The greatest CD4+ T cell
proliferative response was observed on IL-4-treated fibroblasts, in
which [3H]TdR incorporation approached 1 x
105 cpm (Fig. 5). Markedly enhanced CD4+ T cell
proliferation was also observed when MCP-1 antisense oligonucleotide
was included during the coculture of CD4+ T cells and IL-4-
and IL-4 + TNF--treated splenic macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis by CD4+ T cells was augmented, most notably
during their coculture with splenic macrophages. This finding was
consistent with the increased IFN- levels measured in cocultures of
PPD-reactive Th1 cells and IL-4-treated fibroblasts or splenic
macrophages. Inhibition of MCP-1 synthesis by lung fibroblasts and
splenic macrophages by 
46% using an MCP-1 antisense oligonucleotide
enhanced the proliferation of mixed CD4+ T cells during
their coculture with either adherent cell type. These data suggest that
MCP-1 production by stromal cells such as lung fibroblasts could be
important in the generation of Th2-type responses characterized by IL-4
synthesis (37, 38), and it may be one regulating factor that prevents
inappropriate T cell activation in the lung, spleen, and other
organs.
The present findings build on our previous observations that MCP-1
exerts a dichotomous effect on Th cell cytokine synthesis in the
inflamed lung and in the spleen. Recent investigations have shown that
MCP-1 contributes to experimental granulomatous inflammation in the
lung dictated by Th2-type cells. From those studies, levels of MCP-1
were higher in the Th2 granuloma than in the Th1 type, and depletion of
MCP-1 reduced the size of the Th2 lesion without affecting the size of
the Th1-type lesion. In other studies, we showed that MCP-1 directly
contributed to IL-4 synthesis in mixed spleen cell cultures (26).
Karpus et al. (39) have confirmed these observations by showing that
the presence of MCP-1 during OVA-specific TCR-transgenic T cell
activation enhances IL-4 production. It is well appreciated that
fibroblasts express receptors for IL-4 and respond to IL-4 by
increasing extracellular matrix protein production (40, 41). In
contrast to the potentiating effects of MCP-1 on IL-4 synthesis by
CD4+ T cells, the reduction of MCP-1 levels was shown
to enhance IFN- generation by these T cells and a Th1 clone during
their coculture with lung fibroblasts or splenic macrophages. Studies
are currently being performed to examine the exact regulatory mechanism
through which MCP-1 regulates IL-4 and IFN- generation by T cells.
This mechanism conceivably occurs at the gene transcription level,
although posttranslational mechanisms may also be operations since
the Th1 clone could not be "redirected" toward a Th2 phenotype
or IL-4 production by the presence of endogenous MCP-1. The same was
shown with the addition of exogenous MCP-1 to cocultures of the Th1
clone and splenic macrophages where IFN- levels were decreased, but
IL-4 was not detected (our unpublished observations). Thus, the present
data suggest that MCP-1 has a direct role in T cell activation and may
be particularly important in the modulation of Th2-type responses
through a currently undefined mechanism.
Enhanced IFN- synthesis by CD4+ T cells is potentially
deleterious to tissues such as the lung, since it has been shown that
fibroblasts may perpetuate CD4+ T cell activation following
exposure to IFN- through the expression of MHC II and CD80 (B7-1)
(29, 30). The present findings concur with these previous observations
in that in the absence of endogenous MCP-1 production by fibroblasts,
mixed CFA-OVA and SEA-OVA CD4+ T cells had a greater
propensity for proliferation after a 96-h coculture. This enhancement
was also apparent in cocultures of CD4+ T cells and splenic
macrophages, which are well-described professional APCs. Extensive
research has shown that T cell proliferation requires a complex array
of cell-to-cell signals in addition to interaction between the TCR-MHC
II bound to processed Ag, but numerous adhesion and/or soluble
molecules have been shown to supply a secondary or costimulatory signal
(42) necessary for T cell proliferation. More recent work by Taub et
al. (43) has shown that C-C chemokines such as MIP-1, macrophage
inflammatory protein-1ß, RANTES, and MCP-1 are also costimulatory
candidates in that these chemokines appear to enhance mitogen or
Ag-mediated human T cell activation. While maximal human T cell
proliferation in these studies occurred at chemokine concentrations
below 10 ng/ml (43), we observed inhibitory effects on murine T cell
proliferation when MCP-1 concentrations exceeded 20 ng/ml. Our data
coincide with a previous report by Zhou et al. (44), who demonstrated
that pretreatment of murine T cells with MIP-1 inhibited IL-2
transcription and translation typically induced by an anti-CD3 mAb
stimulus. Thus, T cell activation leading to proliferation is affected
by chemokines, and this effect is probably dependent not only on the
concentration of the chemokine but the presence or absence of other
costimulatory signals.
Structural-type cells in the lung and elsewhere have been shown to be a
source of MCP-1 (8, 9, 11, 28). Data from this study and previous
studies support the concept that all of the MCP-1 synthesis observed in
these cocultures was attributable to fibroblasts or splenic
macrophages, since CD4+ T cells cultured alone did not
release detectable (i.e., amounts less than 50 pg/ml) quantities of
MCP-1. In addition, it was apparent that lung fibroblasts produce more
MCP-1 constitutively than splenic macrophages, and are responsive to
cytokines such as IL-4 and TNF-. This may be explained by the fact
that the spleen plays a far more important role in immune activation
than the lung, and as a consequence MCP-1 production in the spleen may
compromise this function. Conversely, T cell activation in the lung
could have many deleterious consequences. Accordingly, the synthesis of
MCP-1 by lung fibroblasts may exert an important immunomodulatory role
on the T cell and, for example, prevent excess IFN- production.
However, it should be noted that augmented IL-4 may also have negative
effects on the architecture of the lung since this cytokine can
activate fibroblasts to produce collagen (41). Fibroblast interaction
with T cells is not a passive event, but this interaction is
conceivably balanced by a number of regulatory mechanisms that prevent
the excessive activation of either cell type. MCP-1 appears to be one
soluble factor produced by structural cells such as fibroblasts that
participates in the regulatory balance of T cell activation.
In conclusion, the present study documents the regulatory role of MCP-1 on T cell activation during cell-to-cell interactions with normal lung fibroblasts or splenic macrophages. Taken together, these findings suggest that endogenous MCP-1 supplies an important immunomodulatory signal to immune cells such as CD4+ T cells. The modulatory effect of MCP-1 may also dictate the cytokine profile associated with a Th response. These data are impetuses to further explore the role that other C-C chemokines play in regulating immune cell/nonimmune cell interactions within various tissues.
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2 Address correspondence and reprint requests to Dr. Steven L. Kunkel, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602.
3 Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1; CCR2, chemokine receptor-2; MCP-1, monocyte chemoattractant protein-1; PPD, purified peptide derivative; RPMI-10, RPMI-1640 medium containing 10% fetal bovine serum; SEA, Schistosoma mansoni egg antigen.
Received for publication June 24, 1997. Accepted for publication January 8, 1998.
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by inflammatory granuloma fibroblasts. Am. J. Pathol. 144:711.[Abstract]
. J. Immunol. 146:1192.[Abstract]
plus TNF- induce regulated expression of CD80 (B7-1) but not CD86 (B7-2) on murine fibroblasts. J. Immunol. 158:4921.[Abstract]
and IL-4 on the in vitro development of human Th1 and Th2 clones. J. Immunol. 148:2142.[Abstract]
rapidly modulates its receptor and inhibits the anti-CD3 mAb-mediated proliferation of T lymphocytes. J. Immunol. 151:4333.[Abstract]
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0.05 compared with control fibroblasts or splenic macrophages in the
normal serum treatment group.
