Interleukin-10 regulates quartz-induced pulmonary inflammation
in rats
Kevin E.
Driscoll1,
Janet M.
Carter1,
Brian W.
Howard1,
Diana
Hassenbein1,
Marie
Burdick2,
Steven L.
Kunkel2, and
Robert M.
Strieter2
1 The Procter & Gamble Company,
Cincinnati, Ohio 45253; and
2 University of Michigan, Ann
Arbor, Michigan 48109-0602
 |
ABSTRACT |
Interleukin-10 (IL-10) can downregulate
expression of several proinflammatory cytokines including chemokines.
This study investigated the role of IL-10 in the acute response of the
rat lung to quartz particles. Intratracheal instillation of rats with 1 mg of quartz produced an inflammatory and cytotoxic response
demonstrated by increased bronchoalveolar lavage (BAL) fluid
neutrophils, lactate dehydrogenase, and protein. IL-10 was detected in
rat lung, but IL-10 levels were not altered by quartz. In contrast,
quartz increased lung levels of the chemokine macrophage inflammatory
protein-2 (MIP-2). Treatment with recombinant murine IL-10 (rmIL-10)
attenuated quartz-induced pulmonary inflammation and injury.
Pretreatment with anti-IL-10 antiserum enhanced inflammatory responses
to quartz. Consistent with effects on quartz-induced inflammation,
rmIL-10 and anti-IL-10 serum decreased and increased, respectively,
lung MIP-2 mRNA and protein in response to quartz. Additionally,
rmIL-10 reduced production of hydrogen peroxide, superoxide anion, and nitric oxide by BAL cells from quartz-exposed and control rats. These
results demonstrate that IL-10 is expressed in rat lung and
downregulates quartz-induced inflammation and cell activation. The
mechanism of the anti-inflammatory action of IL-10 after quartz administration involves, at least in part, attenuation of MIP-2 expression.
macrophage inflammatory protein-2; chemokine; lung; particulate
matter; hydrogen peroxide; nitric oxide
 |
INTRODUCTION |
SILICOSIS is a chronic interstitial lung disease
characterized by granulomatous inflammation and pulmonary fibrosis
resulting from inhalation of various crystalline forms of silica,
including quartz and cristobalite (22). The inflammatory component of silicosis appears to play a key role in the tissue injury associated with crystalline silica exposure (23). Studies (11, 13, 14) that used
animal models of silicosis have demonstrated that the recruitment and
activation of inflammatory cells in silica-exposed lungs result, at
least in part, from the activation of alveolar macrophages and lung
epithelial cells to produce proinflammatory cytokines, including tumor
necrosis factor-
(TNF-
), interleukin (IL)-1, and members of the
chemokine cytokine family such as the neutrophil chemoattractant
macrophage inflammatory protein-2 (MIP-2). Regarding the latter, lung
expression of MIP-2 increases rapidly after exposure of rats to quartz,
and pretreatment with neutralizing antibody to MIP-2 markedly
attenuates quartz-induced pulmonary inflammation (13, 16).
The degree and persistence of inflammation in the lung and other
tissues are influenced by the balance between proinflammatory factors
and other processes that serve to downregulate inflammation. In this
respect, a cytokine shown to exhibit an anti-inflammatory activity in a
number of in vitro and in vivo models is IL-10. IL-10 was initially
described as a Th2 lymphocyte-derived factor, which downregulated
synthesis of interferon-
by Th1 lymphocytes (18). Subsequently,
IL-10 was characterized as an 18-kDa protein produced by Th2
lymphocytes, macrophages, epithelial cells, and keratinocytes (27). In
vitro studies have shown that IL-10 can suppress synthesis of several
proinflammatory cytokines, including IL-1, TNF-
, IL-8, and MIP-1
,
by monocytes, macrophages, and/or neutrophils (1, 7).
Additionally, IL-10 can inhibit production of reactive oxygen and
nitrogen species by macrophages (1), suppress interferon-
production
by natural killer cells (27), stimulate monocyte
production of IL-1 receptor antagonist (4), and decrease expression of
major histocompatibility complex class II antigen on monocytes (7).
Thus IL-10 exhibits several bioactivities in vitro consistent with
downregulation of inflammatory responses. An anti-inflammatory function
for IL-10 has been demonstrated in vivo in studies in which rmIL-10 was
shown to decrease pulmonary inflammation in an immune complex model of
rat lung injury (31) and joint inflammation in collagen-induced
arthritis in rats (25). Additionally, a study (29) in humans
demonstrated that rmIL-10 can prevent pulmonary granulocyte
accumulation in response to a systemic endotoxin challenge. In the
rodent and human studies, treatment with rmIL-10 was associated with
decreased production of proinflammatory cytokines such as TNF-
,
IL-1, and members of the chemokine family.
Whereas several of the factors exerting a proinflammatory action after
exposure to quartz and other pneumotoxic particles have been
identified, little is known about factors that may attenuate inflammatory responses to particle exposure. Because there is evidence
from in vitro and in vivo studies that IL-10 can regulate cytokines
that contribute to particle-induced inflammation, we hypothesized that
this cytokine may influence the response of the lung to quartz. To
evaluate this possibility, we characterized inflammation and chemokine
expression in rats treated with either anti-IL-10 antibody or rmIL-10.
Our results indicate that IL-10 acts as a constitutive
anti-inflammatory factor in the rat lung and that its effect on
quartz-induced inflammation is mediated, in part, by attenuation of
MIP-2 gene expression.
 |
METHODS |
Treatment with anti-IL-10 antiserum, rmIL-10, and
quartz. Specific pathogen-free male Fischer 344 (F-344)
rats (Charles River Breeding Laboratories, Kingston, NY), 12-14 wk
old and ~200 g in body mass, were pretreated by intraperitoneal
injection with 1 ml of saline, nonimmune rabbit serum, or a rabbit
anti-murine IL-10 serum. The development of specific anti-IL-10
antisera was reported previously (32); a 1:1,000 dilution of the
anti-IL-10 antiserum neutralizes 30 ng of IL-10 protein. Two hours
after pretreatment, rats were intratracheally instilled with saline or
a saline suspension of 1 mg of quartz at a dose volume of 1 ml/kg body
wt as described previously in detail (14). The quartz particles
(Minusil 5; Pennsylvania Sand & Glass, Pittsburgh, PA) had
a median diameter of 0.9 ± 1.8 (geometric SD) µm and a surface area of 4.5 m2/g and were heated
for 2 h at 200°C for sterilization and inactivation of any
endotoxin present before use. Three days after quartz exposure, the
animals were killed by intraperitoneal injection of pentobarbital sodium (50 mg/kg) and exsanguinated via the abdominal aorta. The left
lung lobe was removed and frozen in liquid nitrogen for analysis of
MIP-2 mRNA and protein. Bronchoalveolar lavage (BAL) was performed on
the right lung lobe six times with 5 ml of Ca- and Mg-free phosphate-buffered saline (PBS; pH 7.2) as previously described (14).
The BAL fluid was centrifuged (300 g), and the acellular supernatant
from the first two washes were pooled and analyzed for total protein
and lactate dehydrogenase (LDH) as indicators of lung injury. Total
protein was determined with the Bio-Rad method (3), and LDH was assayed
on a Hitachi 705 autoanalyzer with commercially available kits
(Boehringer Mannheim). BAL fluid cells were quantified by hemocytometer
counting, and cell viability was determined by exclusion of trypan blue
dye. Cell differentials were performed on cytocentrifuge preparations
that were fixed in methanol and stained with Diff-Quik (Sigma, St.
Louis, MO).
To determine the effect of rmIL-10 protein on the acute lung response
to quartz, we intratracheally instilled specific pathogen-free male
F-344 rats with 1 mg of quartz as described above with and without 50 µg of rmIL-10 (Pepro Tech, Rocky Hill, NJ) added to the instillation
materials. Because of the short 2-h half-life of rmIL-10 (26),
responses were characterized 24 h after exposure. The left lung lobe
was removed and frozen in liquid nitrogen for analysis of MIP-2 mRNA
and protein or IL-10 protein. BAL was performed on the right lung lobe
six times with 5 ml of Ca- and Mg-free PBS (pH 7.2). The cellular and
acellular components of the BAL fluid were analyzed as described
above.
Measurement of hydrogen peroxide, superoxide anion,
and nitric oxide. To characterize inflammatory cell
production of superoxide anion and hydrogen peroxide, cells obtained by
BAL were suspended in phenol red-free Earle's balanced salt solution
(GIBCO BRL, Gaithersburg, MD) and seeded into 96-well tissue culture
plates (Corning, Corning, NY) at 1 × 105 cells/well. Superoxide anion
levels were determined on the basis of the oxidation of cytochrome
c as described by Pick and Mizel (30).
Briefly, 100 µl of 50 µM cytochrome
c (Sigma) diluted in phenol red-free
Earle's balanced salt solution were added to the BAL cell cultures,
and at 15-min intervals over a 2-h period, the absorbance of each
culture well was determined at 550 nm with the use of an EL311 Biotek
microplate reader (Biotek Instruments, Winooski, VT). Control wells
consisted of BAL cells with cytochrome c and 100 U of superoxide dismutase
(Sigma). Superoxide anion concentrations were calculated with the
extinction coefficient for reduced and oxidized cytochrome
c (30). Hydrogen peroxide production
was determined by the horseradish peroxidase-dependent conversion of
phenol red by hydrogen peroxide into a compound with absorbance at 600 nm as described by Ding et al. (8). Briefly, 100 µl of phenol red
solution (in mM: 140 NaCl, 10 KH2PO4, 5.5 dextrose, and 0.56 phenol red; Sigma) containing 6 U/ml of type VI
horseradish peroxidase (Sigma) were added to cell cultures. Cells were
incubated for 2 h at 37°C, followed by addition of 10 µl of 1 N
NaOH. Absorbance at 600 nm was determined with an EL311 Biotek
microplate reader. Hydrogen peroxide concentrations were calculated
from a standard curve. To assess nitric oxide release, BAL cells were
suspended in RPMI 1640 medium and seeded at 1 × 105 cells/well in 96-well plates.
Cells were incubated overnight at 37°C in 5%
CO2, after which 100 µl of cell
supernatant were removed, and the cells were added to 100 µl of
Griess reagent [1% sulfanilamide and 0.1%
N-(1-naphthyl)ethylenediamine
dihydrochloride in 2.5% phosphoric acid] and incubated in the
dark for 10 min at room temperature (8). Absorbance was measured at 550 nm with an EL311 Biotek microplate reader, and concentrations were determined with a standard curve of sodium nitrite.
RT-PCR analysis of lung MIP-2 mRNA.
MIP-2 mRNA transcript levels were assessed by PCR amplification of the
MIP-2 cDNA as described by Driscoll et al. (13). Briefly, RNA was
extracted as described by Chomczynski and Sacchi (6) from the left lung
lobe of 2 animals/treatment group. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA was evaluated as an internal control. RNA
from the lungs of an untreated F-344 rat and from an F-344 rat
instilled with 10 µg of lipopolysaccharide and killed 6 h
after exposure was analyzed concurrently with unknown lung RNA samples
as negative and positive controls for chemokine mRNA expression. The
primers, designed from the published sequences for MIP-2 (12) and GAPDH (19), were as follows: MIP-2, 5'-GGCACATCAGGTACGATCCAG-3'
and 5'-ACCCTGCCAAGGGTTGACTTC-3'; and GAPDH,
5'-CAGGATGCATTGCTGACAATC-3' and
5'-GGTCGGTGTGAACGGATTTG-3'. PCR reactions were overlaid
with mineral oil. Amplification was carried out through 22-30
cycles of denaturation at 94°C for 1 min, oligo annealing at
55°C for 1 min, and extension at 72°C for 2 min. Reactions were
electrophoresed in 1.5% agarose gels containing ethidium bromide in
Tris-acetate-EDTA buffer to visualize the MIP-2 and GAPDH PCR products.
Confirmation that the PCR products obtained with the primer sequences
were MIP-2 or GAPDH was obtained by Southern analysis of the PCR
products and probing with oligonucleotide probes complementary to mRNA sequences internal to the PCR primer sequences used (data not shown).
Analysis of lung tissue for IL-10 and MIP-2
proteins. IL-10 and MIP-2 proteins were determined in
lung tissue homogenates as described by Standiford et al. (32).
Briefly, homogenates were prepared by suspending the left lung lobe in
a lysis buffer (PBS containing 2 mM phenylmethylsulfonyl fluoride and 1 mg/ml each of aprotinin, antipain, leupeptin, and pepstatin) with the use of a Polytron (Brinkmann Instruments, Westbury, NY). The
homogenates were centrifuged at 2,000 g for 10 min, and supernatants were filtered through a 0.2-µm Millipore filter. The level of
immunoreactive IL-10 protein was determined by double-ligand ELISA as
described in detail elsewhere (32). Immunoreactive MIP-2 protein was
determined with a commercially available MIP-2 ELISA (Biosource,
Camarillo, CA). The MIP-2 antisera did not cross-react with the
structurally related chemokines cytokine-induced neutrophil
chemoattractant, MIP-1
, MIP-1
, and IL-8.
Statistical analysis. Data were
analyzed by ANOVA, with group differences determined by the
Newman-Keuls test (38).
 |
RESULTS |
Expression of anti-inflammatory (IL-10) and
proinflammatory (MIP-2) cytokines during quartz-induced
inflammation. Lung IL-10 levels were determined in rat
lungs 6 h, 1 day, and 3 days after intratracheal instillation of saline
or 1 mg of quartz. Rat lung tissue contained levels of IL-10 protein
that ranged from 480 to 690 pg/ml lung homogenate. Quartz exposure had
no effect on rat lung IL-10 concentrations (Fig. 1).
Because a previous study (13) indicated that the chemokine MIP-2
contributes to quartz-induced neutrophil recruitment in rats, we
characterized lung MIP-2 levels in quartz-exposed and control rat
lungs. As shown in Fig. 1, lung MIP-2 protein increased
significantly after quartz exposure. A 12-fold increase in MIP-2 was
observed 6 h after exposure, with the levels of MIP-2 remaining
elevated approximately twofold over those in control lungs
3 days after exposure.

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Fig. 1.
Interleukin (IL)-10 protein (A) and
macrophage inflammatory protein (MIP)-2
(B) in rat lung homogenates after
quartz exposure. Cytokine protein concentrations in homogenates of left
lung lobe are shown 6, 24, and 72 h after intratracheal instillation
exposure of rats (n = 6) to saline or
saline suspension of quartz particles. Values are means ± SD.
* Significant difference from respective saline control group,
P < 0.05.
|
|
Treatment with anti-IL-10 augments quartz-induced
inflammation. Exposure of rats to quartz produced
pulmonary inflammation and tissue injury as indicated by significant
increases in BAL fluid neutrophils, LDH, and protein (Figs.
2 and 3). Pretreatment of rats with an
antiserum to IL-10 increased the severity of the lung response to
quartz as demonstrated by significantly greater increases in BAL fluid
neutrophils and LDH (Figs. 2 and 3). Saline or a nonimmune serum
pretreatment did not alter BAL fluid parameters after instillation of
saline or quartz.

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Fig. 2.
Number of macrophages (A) and
neutrophils (B) in bronchoalveolar
lavage (BAL) fluid from rats exposed to quartz and pretreated with
neutralizing anti-IL-10 antiserum. Rats
(n = 4) were pretreated with
intraperitoneal injection of saline, nonimmune serum, or anti-IL-10
serum 2 h before intratracheal instillation of saline ( ) or a saline
suspension of quartz particles (+). Responses were examined 3 days
after quartz exposure. Values are means ± SD. Significant
difference (P < 0.05) from:
* respective saline-instilled control group;
respective saline-instilled control group and
quartz-exposed saline- and nonimmune serum-pretreated groups.
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Fig. 3.
BAL fluid lactate dehydrogenase (LDH;
A) and total protein
(B) from rats exposed to quartz and
pretreated with neutralizing anti-IL-10 antiserum. Rats
(n = 4) were treated with
intraperitoneal injection of saline, nonimmune serum, or anti-IL-10
serum 2 h before intratracheal instillation with saline or saline
suspension of quartz particles. Amount of LDH and protein was
determined 3 days after quartz exposure as an indicator of lung injury
and edema. Values are means ± SD. Significant difference
(P < 0.05) from: * respective
saline-instilled control group; respective saline-instilled
control group and quartz-exposed saline- and nonimmune serum-pretreated
groups.
|
|
Inflammation and cellular oxidant production after
quartz is attenuated by rmIL-10. Treatment of rats with
rmIL-10 attenuated quartz-induced lung inflammation and tissue injury
(Figs. 4 and 5). rmIL-10 significantly
reduced the increases in BAL fluid neutrophil numbers after quartz
exposure, although the number of inflammatory cells remained elevated
over saline-treated control animals. Levels of BAL fluid LDH and
protein were significantly less in animals treated with rmIL-10 and
quartz than in animals treated with quartz alone.

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Fig. 4.
Effect of recombinant murine IL-10 (rmIL-10) on number of macrophages
(A) and neutrophils
(B) in BAL fluid 1 day after quartz
exposure. Rats (n = 3) underwent
intratracheal instillation of saline or a saline suspension of quartz
particles with or without rmIL-10. Values are means ± SD.
Significant difference (P < 0.05)
from: * respective saline-instilled control group;
respective saline-instilled control group and
rmIL-10-treated groups.
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Fig. 5.
Effect of rmIL-10 on BAL fluid LDH
(A) and total protein
(B) after quartz exposure. Rats
(n = 3) underwent intratracheal
instillation of saline or a saline suspension of quartz particles with
or without rmIL-10. LDH and protein levels were determined 1 day after
exposure as indicators of lung injury and edema. Values are means ± SD. Significant difference (P < 0.05) from: * respective saline-instilled control group;
respective saline-instilled control group and
rmIL-10-treated groups.
|
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A previous study (5) demonstrated that quartz in vitro and in vivo
activates lung inflammatory cells to produce reactive oxygen species,
and this is thought to contribute to the tissue injury associated with
quartz exposure. As shown in Fig. 6, instillation of
quartz resulted in a significant increase in the production of hydrogen
peroxide, superoxide anion, and nitric oxide by inflammatory cells
obtained by BAL. rmIL-10 treatment reduced basal production of hydrogen
peroxide and nitric oxide by BAL cells from control rats.
Coadministration of rmIL-10 with quartz significantly reduced production of reactive oxygen and nitrogen species by BAL cells.

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Fig. 6.
Effect of rmIL-10 on quartz activation of inflammatory cell hydrogen
peroxide
(H2O2;
A), nitric oxide (nitrate;
B), and superoxide anion
(O 2·;
C) production. Rats
(n = 3) underwent intratracheal
instillation of saline or a saline suspension of quartz particles with
or without rmIL-10. BAL was performed 1 day after exposure, and BAL
cells were cultured to determine release of reactive oxygen and
nitrogen species. Values are means ± SD. Significant difference
(P < 0.05) from: * respective
saline-instilled control group; respective saline-instilled
control group and rmIL-10-treated groups; saline control
group.
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|
IL-10 regulates quartz-induced expression of
MIP-2. To investigate whether IL-10 might influence
quartz-induced inflammation by influencing MIP-2 expression, we
determined the effect of anti-IL-10 antiserum or rmIL-10 on lung MIP-2
mRNA and protein. Treatment of rats with quartz increased MIP-2 mRNA,
with the increase in message appearing greater for rats pretreated with
anti-IL-10 antiserum (Fig.
7A). Minimal or no
MIP-2 message was detected in rats treated with saline or nonimmune
serum. Administration of rmIL-10 attenuated quartz-induced MIP-2 mRNA
(Fig. 8A). Consistent with the effects on gene expression, quartz increased MIP-2 protein in
BAL fluid, whereas pretreatment with antiserum against IL-10 resulted
in an increase in BAL fluid MIP-2 protein that was significantly greater than that seen with quartz alone (Fig.
7B). In contrast, rmIL-10 inhibited
the quartz-induced increases in BAL fluid MIP-2 protein (Fig.
8B).

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Fig. 7.
Effect of anti-IL-10 antiserum treatment on quartz-induced MIP-2 mRNA
and MIP-2 protein in rat lung homogenates. Rats underwent
intraperitoneal injection of saline, nonimmune serum, or anti-IL-10
serum 2 h before intratracheal instillation with saline or a saline
suspension of quartz particles. Responses were characterized 3 days
after exposure. A: RT-PCR products for
MIP-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs
amplified from rat lung RNA; n = 2 rats/treatment. B: MIP-2 protein in
homogenates of left lung lobe. Values are means ± SD;
n = 3 rats. Significant difference
(P < 0.05) from: * respective
saline-instilled control group; respective saline-instilled
control group and quartz-exposed saline- and nonimmune serum-pretreated
groups.
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Fig. 8.
Effect of rmIL-10 on quartz-induced lung MIP-2 gene expression and
MIP-2 protein in rat lung homogenates. Rats underwent intratracheal
instillation of saline or a saline suspension of quartz particles with
or without rmIL-10. Responses were characterized 1 day after exposure.
A: RT-PCR products for MIP-2 and GAPDH
mRNA amplified from rat lung RNA; n = 2 rats/treatment. B: MIP-2 protein
concentrations in homogenates of left lung. Values are means ± SD; n = 3 rats. Significant
difference (P < 0.05) from:
* respective saline-instilled control group;
respective saline-instilled control group and
quartz-exposed saline- and nonimmune serum-pretreated groups.
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 |
DISCUSSION |
Inhalation of quartz can result in lung injury and fibrosis secondary
to the inflammatory response elicited by this mineral dust (20, 21).
Several studies (11, 13, 14) have shown that members of the chemokine
cytokine family, including MIP-2, contribute to quartz-induced
inflammatory cell recruitment. In addition to proinflammatory
chemokines, cytokines such as IL-10, IL-4, and transforming growth
factor-
, exist with the potential to exert an anti-inflammatory
effect on the response of the lung to inhaled particles (27, 28, 34).
Regarding IL-10, this cytokine was reported to attenuate pulmonary
inflammation elicited by immune complexes or endotoxin (28, 29, 31).
Here we show that IL-10 can downregulate the rat lung inflammatory and
cell activation response to quartz by mechanisms that involve
suppression of quartz-induced expression of the chemokine MIP-2. These
observations support a key role for IL-10 and MIP-2 as anti- and
proinflammatory cytokines regulating inflammation within the lower
respiratory tract during quartz-induced lung injury.
The rat lung contained basal levels of IL-10 that were not altered by
quartz exposure. In contrast, quartz markedly increased lung MIP-2
protein levels. This apparent quartz-induced imbalance between pro- and
anti-inflammatory cytokines in the rat lung is likely a factor in the
marked pulmonary inflammatory response elicited by this mineral dust.
That IL-10 can downregulate the response of the lungs to quartz was
demonstrated by administration of exogenous IL-10, which attenuated
quartz-induced pulmonary inflammation, cytotoxicity, and inflammatory
cell oxidant production. That endogenous IL-10 exerts an
anti-inflammatory action toward quartz responses was shown by the
greater than threefold increase in quartz-induced inflammation in rats
passively immunized with a neutralizing anti-IL-10 antiserum.
Apparently, however, the levels of IL-10 were insufficient to fully
compensate for the potent proinflammatory action of quartz. In this
respect, our observations suggest that variations in basal lung IL-10
levels can influence susceptibility to inhaled materials. In this
respect, cystic fibrosis patients have been reported to have lower
levels of IL-10 in the respiratory tract fluids than normal subjects (2). Similar to silica responses in rats treated with anti-IL-10 antiserum, the depressed IL-10 levels in the lungs of cystic fibrosis patients may result in enhanced local inflammatory responses to inhaled
pathogens.
In contrast to our observations with quartz, increases in tissue IL-10
have been described during the course of inflammatory responses
elicited by immunogenic stimuli. For example, in a murine model of
pneumococcal pneumonia, IL-10 levels increased in the lung over a
period of 3 days after infection (33), and in mice exposed systemically
to endotoxin, plasma IL-10 was observed to increase 2 and 6 h after
exposure (32). Also, in collagen-induced arthritis in the rat, IL-10 in
joint tissue increased by 44 days after the initial immunization (25).
In the present study, the absence of any apparent increase in IL-10 in
quartz-exposed rat lungs may reflect an inherent difference between
quartz and the eliciting agents used in the other studies. In this
respect, it is tempting to speculate that the lack of IL-10 induction
after quartz may be a factor that contributes to the progressive nature of quartz-induced lung disease (15, 22).
Rat MIP-2 is a member of the C-X-C chemokine family and is structurally
and functionally related to the human GRO chemokines (12). MIP-2 is a
potent neutrophil chemoattractant and a mitogen for epithelial cells,
and it plays a significant role in mediating the rat lung inflammatory
response to quartz as well as to other inhaled agents (e.g., asbestos,
ozone, and endotoxin; Refs. 9, 10, 12, 13, 17, 33, 37). Consistent with
previous observations (10, 13) and a key role of MIP-2 in
quartz-induced inflammation (13), the present study demonstrates that
this chemokine increases greater than sixfold shortly after quartz exposure and remains elevated throughout a 3-day postexposure period.
Importantly, rmIL-10 markedly attenuated quartz-induced increases in
both lung MIP-2 mRNA and protein, whereas passive immunization with
anti-IL-10 antiserum increased lung MIP-2 protein levels above those
seen with quartz alone. These results indicate downregulation of MIP-2
is responsible, at least in part, for the effects of IL-10 on
quartz-induced inflammation. At present, the mechanism(s) by which
IL-10 inhibits quartz activation of MIP-2 is uncertain. However, it is
noteworthy that IL-10 can inhibit nuclear translocation of a
transcription factor known as nuclear factor-
B (35) that regulates
activation of MIP-2 gene transcription (36).
In addition to modulating neutrophilic inflammation, treatment with
rmIL-10 or anti-IL-10 serum decreased or increased lung injury,
respectively, after quartz exposure. Also, rmIL-10 significantly attenuated production of reactive oxygen and nitrogen species by
inflammatory cells after quartz exposure. Whereas the mechanism of
rmIL-10-mediated decreases in hydrogen peroxide, superoxide anion, and
nitric oxide was not investigated, previous studies (1, 21) have
demonstrated that IL-10 can attenuate production of hydrogen peroxide
and nitric oxide by macrophages. Additionally, MIP-2 can stimulate an
oxidative burst by rat neutrophils (20), and the ability of rmIL-10 to
attenuate expression of this chemokine may contribute to decreased
oxidant generation in quartz-exposed animals. The concurrence among
quartz-induced inflammation, oxidant production, and lung injury
further supports a key role for inflammatory cells in the tissue damage
resulting from quartz exposure. This observation is consistent with
that of Henderson et al. (23), who demonstrated that neutrophil
depletion in rats results in a significant attenuation in lung injury
from subsequent quartz exposure. Collectively, these
findings indicate that a significant component of the tissue damage
occurring after quartz exposure occurs secondary to MIP-2 expression
and the recruitment and activation of inflammatory cells.
In summary, the present study demonstrates that treatment with rmIL-10
attenuates quartz-induced pulmonary inflammation, tissue injury, and
production of reactive oxygen and nitrogen species in rat lungs,
whereas passive immunization with anti-IL-10 serum enhances the
inflammatory response to quartz. The modulation of quartz-induced
responses by IL-10 was associated with decreases in MIP-2 mRNA and
protein, indicating the anti-inflammatory action of IL-10 was mediated,
at least in part, by a downregulation of MIP-2 gene expression. Basal
levels of IL-10 were detected in the rat lung; however, IL-10 did not
increase after quartz exposure. These observations suggest that IL-10
acts as a constitutive anti-inflammatory factor serving to attenuate
the inflammatory response to quartz. The absence of any detectable
induction of IL-10 in response to quartz may be a factor contributing
to the progressive nature of quartz-induced lung disease in rodents and
humans.
 |
ACKNOWLEDGEMENTS |
These studies were partially supported by National Heart, Lung, and
Blood Institute Grant P50-HL-56402 (to S. L. Kunkel and R. M. Strieter).
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: K. E. Driscoll, Procter & Gamble
Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery
Rd., Mason, OH 45040-9462.
Received 9 February 1998; accepted in final form 10 June 1998.
 |
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