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Abstract
Introduction
Materials & Methods
Results
Discussion
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There is evidence that, following exposure to crystalline silica, the release of several proinflammatory cytokines contributes to the induction of unbalanced inflammatory reaction leading to lung fibrosis. We have examined the potential contribution of interleukin-10 (IL-10), an anti-inflammatory cytokine, in the development of silicosis. In a mouse model of inflammatory lung reaction induced by intratracheal instillation of silica (0.5 mg and 5 mg DQ12/mouse), the levels of IL-10 protein (determined by ELISA) both in cells obtained after bronchoalveolar lavage (BAL) and in lung tissue homogenates were significantly increased when compared with controls. After in vitro lipopolysaccharide (LPS) stimulation (1 µg/ml), BAL cells obtained from silica-treated animals produced significantly more IL-10 protein and mRNA than cells obtained from control animals. To examine the role of IL-10 in the lung reaction induced by silica, IL-10- deficient animals were instilled with 5 mg of silica. Twenty-four hours after treatment, the amplitude of the inflammatory response (lactate dehydrogenase [LDH], protein and number of inflammatory cells in BAL) was significantly greater in IL-10-deficient animals than in the wild type. In contrast, the fibrotic response, evaluated by measuring lung hydroxyproline content and by histopathologic analysis 30 days after silica, was significantly less important in IL-10-deficient than in wild-type mice. Together, these data suggest that increased IL-10 synthesis induced by silica can limit the amplitude of the inflammatory reaction, but also contributes to amplify the lung fibrotic response.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Silicosis is the lung disease caused by the inhalation of damaging amounts of respirable free crystalline silica (1). This fibrotic disease is characterized by the persistence of pulmonary interstitial inflammation leading to increased proliferation of fibroblasts and exaggerated production of collagen (2). Alveolar macrophages (AM), but also other cell types such as fibroblasts and epithelial and endothelial cells, play a central role in the inappropriate inflammatory lung response and in the development of silicosis by releasing a variety of mediators such as eicosanoid metabolites (3), destructive proteolytic enzymes (4), oxidant molecules (5), proinflammatory cytokines (6), and factors that promote mesenchymal cell growth (7). However, a possible regulation of the inflammatory reaction by anti-inflammatory mediators and their contribution in the fibrotic disease has not yet been investigated.
Interleukin (IL)-10 has been reported to have an anti-inflammatory and protective role in different pathologies.
Like many other cytokines, IL-10 is produced by different
cell types and mediates several functions on various cell
types. In addition to T cells, IL-10 is also expressed by
stimulated B lymphocytes as well as monocytes-macrophages and other cell types such as keratinocytes, mast cells,
and epithelial cells (8). IL-10 inhibits several macrophage
functions, including antigen presentation to T cells, synthesis of several proinflammatory cytokines such as IL-1
and -
, IL-6, IL-8, tumor necrosis factor- (TNF-), granulocyte-macrophage colony-stimulating factor (GM-CSF),
and granulocyte colony-stimulating factor (G-CSF) (9,
10), and production of oxygen-free radicals and nitric oxide (11). In addition, IL-10 inhibits the production of Th1
cytokines by T lymphocytes (9) and stimulates the proliferation of activated B lymphocytes (12) and the production of immunoglobins (13). Therefore, IL-10 is generally
regarded as a cytokine which inhibits cell-mediated immunity and stimulates humoral immunity (8).
With regard to the lung, it has been clearly demonstrated that AM can produce significant amounts of IL-10
(14, 15). Several studies have shown that IL-10 reduces the
intensity of cellular recruitment in pulmonary inflammation and is an inhibitor of the induced release of several
proinflammatory cytokines such as TNF-, macrophage
inflammatory protein (MIP)-1, and MIP-2, supporting the
anti-inflammatory role of IL-10 in the lung (16). In
view of the anti-inflammatory activity of IL-10, especially
on the macrophage, it has been suggested that IL-10 must
be able to control not only inflammatory lung reactions
(19), but also the development of lung fibrosis (20). In a
mouse model of inflammatory reaction induced by silica,
we show a clear upregulation of IL-10 at both the mRNA
and protein levels. To assess the biological involvement of
IL-10 in the development of experimental silicosis, we
have examined the intensity of inflammatory and fibrotic
reactions in IL-10-deficient mice.
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Materials and Methods |
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Introduction
Materials & Methods
Results
Discussion
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Mice
The animals were housed in positive pressure air conditioned units (25°C, 50% relative humidity) on a 12-h light/
dark cycle. Female NMRI mice weighing 20 to 25 g were
purchased from Iffa Credo (Brussels, Belgium). Interleukin-10-deficient mice (21) were obtained from B&K Universal Limited (Hull, UK). The homozygous mutants and
heterozygous and control mice were generated by intercrossing a large number of heterozygous IL-10 mice. All
animals were typed for the IL-10 mutation by polymerase
chain reaction (PCR) using DNA isolated from tail biopsies and extracted by the DNAzol procedure (Gibco BRL,
Paisley, UK) with specific primers encoding one sequence within the neomycin cassette (anti-sense, 5'-CCTGCGTGCAATCCATCTTG-3') and two adjacent sequences of the
IL-10 gene (sense 5'-TAGGCGAATGTTCTTCC-3'; anti-sense, 5'-CAGGCAGCATAGCAGTG-3') followed by
agarose gel separation and ethidium bromide staining of
the products (21, 22). A screening of 200 animals was necessary to obtain a homogeneous group of homozygous
(n = 14), heterozygous (n = 21) and wild type (n = 17)
mice. The selected animals did not display any symptoms of enterocolitis, and the average weights in the three
groups were not significantly different {(
/) = 21.5 ± 2.3 g; (+/) = 23.4 ± 3.4 g; (+/+) = 25.5 ± 3.5 g}.
Instillation Method
Silica (DQ12; d50, 2.2 µm) or saline (controls) was injected directly into the lungs by intratracheal instillation. All instillations (100 µl/mouse) were performed on anesthetized animals (sodium pentobarbital, 2 mg/mouse) after surgical opening of the neck. Silica was heated at 200°C for 4 h prior to use, to allow sterilization and inactivation of any trace of endotoxin.
Bronchoalveolar Lavage
At selected time intervals, treated mice were killed with sodium pentobarbital (20 mg/animals, i.p.) and bronchoalveolar lavage was performed by cannulating the trachea and infusing the lungs 6 times with 1.5 ml of NaCl 0.9%. The BAL fluid was centrifuged (1,000 × g, 10 min, 4°C) and the cell-free supernatant was used for the biochemical measurements. For each animal, the cell pellet was resuspended in 1 ml of phosphate-buffered solution (PBS; Gibco BRL) and the number of living cells was determined by the trypan blue exclusion method. The cell differentials were performed on cytocentrifuge preparations fixed in methanol and stained with Diff-Quik (Baxter, Lessines, Belgium; 200 cells counted).
Biochemical Analyses
Lactate dehydrogenase (LDH) activity was assayed spectrophotometrically by monitoring the reduction of NAD+ at 340 nm in the presence of lactate. Total proteins (TP) were determined by the Pyrogallol red staining method (Technicon RA system; Bayer Diagnostics, Domont, France).
Cell Culture
Twenty-four hours after treatment (5 mg silica or saline), bronchoalveolar lavage was performed with four volumes (1.5 ml) of sterile 0.9% saline. The lavage fluids were centrifuged and the cell pellets pooled for each treatment. Aliquots of the cell suspensions were used to determine cell number and viability. The cell suspensions were adjusted to a concentration of 0.5 × 106 BAL cells/ml of RPMI 1640 (Gibco BRL) containing lactalbumin hydrolysate (0.1%), and antibiotics (1%). Aliquots of 1 ml of the cell suspensions were seeded into 16-mm culture wells.
Polymerase Chain Reaction Analysis
RNA from 1.5 × 106 BAL cells obtained from saline or silica-treated animals was isolated by the Tryzol method
(Gibco BRL). Reverse transcription was performed on total RNA with an oligo(dT) primer and amplification was
carried out for 30 cycles by PCR with specific primers as
follows: for IL-10, sense 5'-GAGACTTGCTCTTGCACTAC-3', antisense 5'-CCTGGAGTCCAGCAGACTCA-3'; for -actin, sense 5'-ATGGATGACGATATCGCTGC-3', antisense 5'-GCTGGAAGGTGGACAGTGAG-3'. An
aliquot of the PCR was run in a 1% agarose gel stained
with ethidium bromide.
Mimic Polymerase Chain Reaction (mPCR) Analysis
In order to achieve a greater accuracy, we used mPCR to compare mRNA levels in cultured BAL cells. Reverse transcription was carried out using Promega Kit A3500 (Promega, Madison, WI). A mix containing 5 mM MgCl2, 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100, 1 mM of each dNTP, 1 u/µl of rRNAse in ribonuclease inhibitor, 15 u/µg RNA of AMV reverse transcriptase (RT), 0.5 µg/µl RNA of oligo(dT)15 primer in a final volume of 40 µl was incubated at 42°C for 15 min. Reverse transcriptase was inactivated at 99°C for 5 min. For PCR, aliquots of a reverse transcription reaction were taken and then amplified for IL-10 and G3PDH in separate reaction tubes. Each reaction tube contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 0.2 nM of each dNTP, 1.5 mM MgCl2, 1 µM of each primer, and 0.025 u/µl of Taq polymerase. Reactions were spiked with a constant amount of the appropriate MIMIC DNA construct. The PCR was run for 30 cycles on a Hybaid Omnigene thermal cycler (Hybaid, Teddington, UK) with control tube. The thermal cycler was programmed as follows: denaturation at 95°C for 5 min, then 30 cycles of 1 s at 95°C, then 15 s at 54°C, and 1 min at 72°C. This was followed by a final 5 min at 72°C. Primer construction was as follows: IL-10 sense 5'-AAGGACCAGCTGGACAACATA-3', antisense 3'-CCCAAGGAATTCAAATGCTGC-5'. The expected products were 167 bp from cDNA, 552 bp from genomic DNA and 596 bp from the MIMIC construct. G3PDH sense 5'-ACCACAGTCCATGCCATCACT-3', antisense 3'-CACCCTGTTGCTGTAGCCATA-5'. The expected products were 452 bp from cDNA, 552 bp from genomic DNA and 596 bp from the MIMIC construct. The MIMIC was constructed from v-erb fragment following Clontech MIMIC construct kit K1700-1 (Clontech, Palo Alto, CA) using composite primers. PCR products were run on a 2% agarose gel in a submerged apparatus, photographed with Polaroid film (Sigma, Bornem, Belgium) and scanned with a Shimadzu CS 9301 Flying spot densitometer (Shimadzu, Kyoto, Japan).
Cytokine Determinations
The presence of IL-10 and TNF- proteins in BALF was
measured by cytokine specific ELISA obtained from Biosource International (Camarillo, CA). The detection limit
of these ELISA were 0.178 pg/ml and 3 pg/ml, respectively.
Lung Hydroxyproline Content
Collagen deposition was estimated by determining the hydroxyproline content of the right lung. The lung was excised, homogenized, and hydrolyzed in 6 N HCl overnight at 110°C. Hydroxyproline content was assessed by HPLC analysis (23). Data are expressed as µg of hydroxyproline/ lung.
Histopathology
The left lung was excised and fixed in Bouin solution (Merck, Darmstadt, Germany). Paraffin-embedded sections were stained with hematoxylin and eosin, Masson's trichrome or toluidine blue for light microscopic examination.
Statistics
Treatment-related differences were evaluated using a one-way ANOVA, followed by pairwise comparisons using the Newman-Keuls test. Statistical significance was considered at P < 0.05.
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Results |
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Abstract
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Materials & Methods
Results
Discussion
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IL-10 Upregulation in NMRI Mice Treated with Silica
The expression of IL-10 in the lung inflammatory reaction induced by crystalline silica was examined by measuring this cytokine in tissue and BAL cell homogenates obtained 24 h after administration of 0.5 and 5 mg of DQ12 (Figure 1). A dose-dependent increase of IL-10 protein was observed both in total lung and in BAL cell homogenates. This was confirmed at the transcriptional level with a clear signal for IL-10 mRNA in BAL cells from silica-treated (5 mg) but not control mice (Figure 2). Curiously, we found a 50% reduction of IL-10 protein in the soluble BAL fraction of silica-treated animals compared with control animals (controls, 134 pg/ml; 0.5 mg DQ12, 74 pg/ml; 5 mg DQ12, 76 pg/ml).
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The in vitro production of IL-10 by BAL cells obtained from silica-treated and control animals was assessed after LPS stimulation (Figure 3). Significant amounts of IL-10 protein were only detected after 12 h in control cultures whereas BAL cells from silica-treated animals already produced significant amounts after 3 h. Whatever the time point considered, the cells obtained from silica-treated animals produced significantly more IL-10 protein than controls. When examined at the mRNA level by mRT-PCR, the IL-10 response to LPS decreased progressively with time in control cultures whereas it remained elevated up to 24 h in cultures obtained from silica-treated animals.
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Inflammatory and Fibrotic Responses in IL-10 Knockout Mice Treated with Silica
The role of IL-10 in the inflammatory and fibrotic lung reactions to silica was examined after 1 and 30 days, respectively, by comparing the response of wild type (+/+), heterozygous (/+), and homozygous (/) IL-10-deficient
animals after instillation of 5 mg of DQ12.
Inflammatory response. After 24 h, silica induced a significantly greater elevation of inflammatory markers in
BAL (LDH, total protein content and total cells) in (+/)
and (/) than in (+/+) mice (Figure 4). Neutrophils, but
not macrophage numbers in BAL, were higher in (+/)
and (/) than in (+/+) animals treated with silica. At
this stage, no lymphocyte was found in any group. No difference was found between (+/+), (+/) and (/) animals which were not treated with silica.
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Fibrotic response. After 30 days, no difference was
found between the three groups treated with silica with regard to LDH and protein content in BAL fluid (Figure 5).
The recruitment of lymphocytes in silica-treated animals
was 2.27 and 3.73 times greater in (+/) and (/) than
in (+/+) animals, respectively (Figure 5). Again, no significant difference was found between the groups which were
not treated with silica.
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Silica induced collagen accumulation, as measured by
lung hydroxyproline content, in the three groups, but the
amplitude of the phenomenon was significantly less pronounced in (/) than in (+/) and (+/+) animals (Figure 6). This was confirmed by histological analysis (Figure
7). In the wild type animals treated with silica, mature
granulomas were observed whereas the lungs of (/) animals displayed granulomas which were less organized and
less dense, as well as a scattered lymphocytic infiltration.
An intermediate response was observed in (+/) animals
treated with silica. No prominent histologic changes were
observed in control animals from the different groups (not
shown).
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Several experimental and human studies support the view
that the inflammatory and fibrotic responses of the lung to
crystalline silica are mediated by proinflammatory macrophage cytokines such as TNF- and IL-1 (24). However, little is known about the potential role of IL-10, an
anti-inflammatory cytokine. Our findings indicate that the
upregulation of IL-10 in response to silica can contribute to attenuate the intensity of the inflammatory response.
However, in the absence of IL-10 the amplitude of the fibrotic reaction was reduced, suggesting a pro-fibrotic effect of this cytokine in the lung response to silica.
Intratracheal instillation of silica induced, within 24 h, a
dose-dependent increase of both IL-10 protein and mRNA
expression in alveolar cells. The fact that the level of IL-10
protein was reduced in the soluble BAL fraction from silica-treated animals might indicate a possible redistribution
of the cytokine with a preferential cellular localization.
This observation is reminiscent of human studies showing
that the respiratory epithelial lining fluid (ELF) from patients with cystic fibrosis contained significantly less soluble
IL-10 than that of healthy control subjects while, in contrast,
macrophages from the cystic fibrosis patients contained significantly more intracellular IL-10 than those from control subjects (28). This redistribution of the cytokine might be mediated through up-regulation of IL-10 receptors on target cells which might cause internalization of the majority
of the IL-10 protein secreted by effector cells. Alternatively,
it might also reflect, as previously shown for TNF- (29), a
degradation of the soluble cytokine by activated proteases
and/or neutrophils recruited at the site of the acute inflammatory reaction.
The upregulation of IL-10 in inflammatory alveolar cells was confirmed by comparing in vitro the LPS-induced production of the cytokine by BAL cells obtained from control and silica-treated animals. In control cultures, the IL-10 protein was only detected after 12 h of culture, which is in line with the relatively late expression of this cytokine (12-24 h) in human monocytes (10) and alveolar macrophages stimulated by LPS (15). Whatever the time point, the cells obtained from silica-treated animals produced more IL-10 protein than control cells. The apparent contradiction between enhanced in vitro production of IL-10 protein (at 3 and 6 h) in cultures obtained from silica-treated animals, and, at the same time point, similar transcript levels in control and silica cultures can be explained as follows. In control cells, LPS rapidly induced IL-10 mRNA expression (3 h) while the IL-10 protein was only released in the culture medium after a delay of at least 9 h (12th h). The fact that after silica treatment, the IL-10 protein was rapidly produced in the culture medium (3 h) denotes the presence of a prior in vivo stimulation by the silica particles which is confirmed by the higher expression of IL-10 in BAL cells obtained from silica-treated mice (Figures 1 and 2). The longer persistence of IL-10 mRNA after LPS stimulation in BAL cells from silica-treated animals could also be the reflection of this in vivo stimulation.
In order to examine the role of IL-10 in the pathologic process induced by silica, the inflammatory and fibrotic responses were analyzed in IL-10 knockout mice. Under conventional housing conditions, these animals show normal lymphocyte development and antibody response, but most animals suffer from chronic enterocolitis which leads to severe debilitation. By applying very stringent hygienic conditions in the animal facility, we were able to reduce the impact of these manifestations and to select a limited number of IL-10-deficient animals which showed no apparent sign of disease. With regard to the lung, in the absence of silica treatment, LDH and protein levels, cell numbers, and OH-proline content were not different between wild type and knockout animals. Histologic analysis of lung tissue confirmed the absence of pulmonary lesions in control IL-10-deficient animals. We may therefore reasonably exclude the possibility that a chronic inflammatory state in IL-10-deficient animals may have interfered with the lung response to silica.
The fact that in both (+/) and (/) IL-10-deficient
animals, the amplitude of the inflammatory response induced by silica (24 h after exposure) was increased compared with wild type animals is in agreement with the
strong anti-inflammatory properties of IL-10 and, together
with the upregulation of this cytokine observed in the
NMRI mouse, indicates that IL-10 is operative in the control of the acute lung inflammatory processes induced by
silica. The marked inflammation observed in the lungs of
(/) and (+/) mice was associated with an increased
influx of neutrophils. Two recent studies using mouse
models of lung inflammatory injury have shown that
blocking of IL-10 with antibodies resulted in an increased recruitment of neutrophils and increased TNF- levels in
lung homogenates, while administration of recombinant
IL-10 suppressed both the influx of neutrophils and the in
vivo formation of TNF- (30, 31). We have not found, after 24 h, any increased TNF- level in the BAL fluid of
(/) mice treated with silica (data shown), suggesting
that, in this model, the enhanced recruitment of neutrophils may also be driven by other proinflammatory cytokines such as IL-1, IL-8-like chemokines, MIP-1 or -2, and
other mediators such as leukotrienes (e.g., leukotriene B4)
or the fifth complement component (C5a) (19, 32, 33). The
increased recruitment of polymorphonuclear leukocytes
(PMNs) may however explain the higher degree of tissue
destruction and inflammatory reaction reflected by higher
LDH and protein levels in BAL fluid of (/) and (+/)
animals after 24 h. Furthermore, since the BAL responses to silica were similar in (+/) and (/) animals, it may
suggest that a slight deficit in IL-10 (42% of [+/+] cytokine level in the lung of [+/] animals) could already be
sufficient to influence the intensity of the lung inflammatory reaction.
The intensity of the fibrotic lesion was markedly less
important in (/) than in (+/+) and (+/) animals. We
can therefore conclude that IL-10 does not block fibrogenesis. The reduction of the disease in IL-10 (/) animals
was associated with an important influx of lymphocytes in
the lung. The association of an increased lymphocytic population with reduced fibrotic response is reminiscent of the
antifibrotic role attributed to T lymphocytes, in some recent studies. In a mouse model of asbestosis (34) or bleomycin-induced fibrosis (35), lung collagen synthesis levels
were significantly higher in nude than in euthymic mice.
However, other studies with athymic mice have concluded
that neither T cells nor the cells they influence affect the
ultimate amount of collagen deposition after silica instillation (36) and bleomycin exposure (37). These discrepancies might be explained by the fact that, depending on the
strain used, nude mice may retain the capacity to produce T-cell cytokines such as interferon-
(IFN-) and IL-2
(38), which may be sufficient to control the fibrotic
process. Furthermore, most human studies on lymphocyte
numbers and responsiveness to minerals report suppressive effects of lymphocytes (especially T helper) on the fibrotic response (41). Taken together, these data are in
agreement with our finding that an excessive influx of lymphocytes is correlated with a reduction of experimental silicosis, which however does not formally demonstrate a
causal relationship. The comparison of the fibrotic response in (+/) and (/) animals suggests that the protective role of lymphocytes may only be observed when
their recruitment reaches a sufficient level (> 20% lymphocytes). The mechanism by which lymphocytes can influence the fibrotic response is poorly understood, but it is
very likely that, in the absence of IL-10, Th1 cytokines such as IFN- and IL-2 are markedly overproduced after
silica administration and that, as demonstrated for IFN-
(45), these lymphokines inhibit collagen production
and fibroblast proliferation. Importantly, the fact that inflammatory markers (LDH, protein and PMNs) were not
different between (+/+), (+/), and (/) animals at day
30, indicates that at this stage of the disease the main target of IL-10 is no more the AM which mainly controls the
inflammatory reaction, but lymphocytes or parenchymal
cells accumulated in the alveoli.
In conclusion, this study demonstrates that the fibrotic reaction is not only determined by the degree of the inflammatory reaction, but is also dependent on the lymphocyte response, which is influenced by IL-10. It is clear that IL-10 exerts a significant control on the intensity of the inflammatory reaction induced by silica, but with regard to the fibrotic process, other mechanisms are operative and probably predominant including the action of this cytokine on the lymphocytes. Therefore, these results support the hypothesis that according to the type of inflammatory cells involved (macrophages/neutrophils or lymphocytes), IL-10 mediates different types of lung responses. In addition, the finding that IL-10 is anti-inflammatory but exerts a pro-fibrotic activity, indicates that, when looking at human data, the stage of the pulmonary disease needs to be carefully taken into account to interpret changes in IL-10 levels.
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Footnotes |
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Address correspondence to: Dominique Lison, M.D., Ph.D., Industrial Toxicology and Occupational Medicine Unit, School of Medicine, UCL, 30.54 clos chapelle-aux-champs, 1200 Brussels, Belgium. E-mail: lison{at}toxi.ucl.ac.be
(Received in original form January 28, 1997 and in revised form April 2, 1997).
Acknowledgments: This work was supported by the Commission of the European Communities (Directorate General XII-Research and Technological Development-Environment EV5V-CT94-0399). The writers thank M. Bouyer, C. Nicolas, D. Boesmans, and M. P. Scott for their excellent technical assistance.
Abbreviations AM, alveolar macrophages; BAL, bronchoalveolar lavage; ELF, epithelial lining fluid; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; mPCR, mimic polymerase chain reaction; TNF, tumor necrosis factor; TP, total proteins.
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References |
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Materials & Methods
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References
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