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Endothelin-1 (ET-1), a potent fibroblast/smooth muscle cells mitogen, has been implicated in the pathogenesis of systemic sclerosis lung disease (SSc). Since monocytes and macrophages are thought to be activated in SSc, we hypothesized that alveolar macrophages (AM) and their precursors blood monocytes from patients with SSc produced more ET-1 than cells from healthy subjects. ET-1 and big ET-1 concentrations were measured in plasma, in bronchoalveolar lavage (BAL) fluids and in cell culture supernatants from monocytes and alveolar macrophages derived from 13 patients with definite SSc with lung involvement and from 10 control subjects. Plasma and BAL fluid ET-1 and big ET-1 levels were similar in both controls and patients with SSc. ET-1 and big ET-1 concentrations in unstimulated alveolar macrophage supernatants were similar in both groups. In contrast, LPS-stimulated alveolar macrophages from patients with SSc secreted higher amounts of ET-1 and big ET-1 than control subjects. ET-1 and big ET-1 concentrations in monocyte supernatants (either LPS-stimulated or not) were not different in patients and controls. These results show that AM from patients with SSc are hyperresponsive to LPS in vitro in terms of ET-1 and big ET-1 production and suggest that AM could participate in vivo in the overproduction of this potentially profibrotic mediator in patients with SSc.
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Systemic sclerosis (SSc) is a collagen disease characterized by collagen overproduction by fibroblasts and endothelial cell injury, leading to specific clinical manifestations, in both skin and internal organs. It has been suggested that hyperactivity of fibroblasts and endothelial cell alterations are mediated by the secretory products of immune or inflammatory cells. Earlier studies have demonstrated chronic inflammation of the alveolar space as a component of the interstitial lung disease associated with SSc (1, 2). It has also been suggested that alveolar macrophages (AM) were activated, releasing increased levels of fibrogenic factors in vitro (1, 2).
Among these factors, endothelin-1 (ET-1), a peptide initially identified for its vasoconstrictor properties, has been shown to exert potent mitogenic effects on smooth muscle cells and fibroblasts (3, 4). Active mature ET-1 is derived from the 38-residue inactive intermediate big ET-1 by catalysis with an endothelin converting enzyme (ECE-1). ET-1 has been previously implicated in the pathogenesis of SSc. Indeed, increased ET-1 levels have been measured in the plasma, in the supernatant of dermal fibroblasts from patients with SSc (4) and in bronchoalveolar lavage (BAL) fluid from patients with SSc (5). It has been suggested that ET-1 could contribute to fibroblast mitogenic activity in the alveolar space (5). However, the cellular source of ET-1 in the lung of patients with scleroderma remains unknown.
ET-1 can be synthesized in various cell types: fibroblasts, endothelial and epithelial cells (9), as well as alveolar macrophages and their circulating precursors, monocytes (10, 11). The contribution of alveolar macrophage-derived endothelin-1 to the total alveolar ET-1 production has never been studied in SSc. We hypothesized that ET-1 production by AM was increased in patients with SSc. To test this hypothesis, we measured the concentration of ET-1 in both the BAL fluids and the AM cell culture supernatants from 13 patients with SSc and from 10 control patients. In addition to ET-1, we measured the concentration of its precursor, big ET-1, since activity of the ET pathway may be better assessed by measuring big ET-1 rather than ET-1, because the former has a longer serum half-life and may thus more accurately represent the relative physiological effect present at any given time (12). In order to evaluate the respective roles of alveolar cells and blood cells in ET-1 production, we also measured the ET-1 and big ET-1 concentrations in plasma and supernatants of cultured blood monocytes, the precursors of alveolar macrophages.
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Patients
Thirteen patients with systemic sclerosis (SSc) with lung involvement were studied prospectively. One patient was studied twice. The study group was composed of two men and eleven women with a mean age of 43.6 ± 4.5 yr (range 18 to 77). They all fulfilled the preliminary criteria of the American Rheumatism Association for the diagnosis of progressive SSc (13). All patients had radiologic evidence of interstitial lung disease at examination: honeycomb lung in eight patients, increased reticulonodular markings in five patients. Two patients were current smokers while 11 patients never smoked. None had had significant environmental exposure to toxic gases or dust, or previous pulmonary disease. At examination, the therapeutics included corticosteroids in four patients, D-penicillamin in seven patients, colchicin in one patient, and nonsteroidal antiinflammatory drugs in two patients. Nine patients received diltiazem because of symptomatic Raynaud phenomenon. Clinical features of the patients with SSc are given in Table 1. The mean disease duration was estimated, defined as the time from clinical diagnosis to study entry. Lung function measurements FVC, FEV1, FEV1/FVC, and total lung capacity were determined in each patient. Carbon monoxide diffusion capacity of the lung was obtained by the single-breath method. The predicted values for each subject were obtained from standard tables. Pulmonary features and results of pulmonary function tests are given in Table 2.
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Ten patients (seven men) who required a fiberoptic bronchoscopy for the diagnosis of a unilateral lung nodule (n = 5), or the preoperative assessment of an esophageal or laryngeal tumor (n = 5) agreed to be submitted to a bronchoalveolar lavage. None of the patients had radiographic evidence of interstitial lung disease. These patients are considered as control subjects. Their mean age was 54.4 ± 5.0 yr (range 31 to 82). Five were smokers and five were nonsmokers. None of them received corticosteroids or nonsteroidal antiinflammatory drugs at the time of examination.
All patients gave their informed consent. The study protocol was approved by the local ethical committee of Bichat hospital.
Bronchoalveolar Lavage, Alveolar Macrophages Isolation and Culture
Alveolar macrophages (AM) were recovered by BAL. Bronchoscopy
and BAL were performed using a standardized technique described previously (14). Briefly, the tip of a flexible fiberoptic bronchoscope was wedged into the right middle lobe. Two hundred ml of sterile saline in four 50 ml aliquots were infused and removed by gentle suction. BAL cells were separated from the fluid by centrifugation at
1200 rpm for 10 min. The BAL supernatant was recovered and stored
with aprotinin, a potent protease inhibitor (Trasylol®; Bayer Pharma,
Sens, France) (33 International Pharmaceutical Federation Units
(IPFU)/ml of BAL) at 
20° C until ET-1 and big ET-1 assays. Cells
were resuspended in RPMI 1640 (GIBCO, Eragny, France) containing 2 mM L-glutamine, 105 U/l penicillin, 100 mg/l streptomycin, 0.25 mg/l amphotericin B and plated on 6-well cell culture plastic plates at
a concentration of 106 cells/well. Cytocentrifuge smears were prepared with an aliquot of AM population and stained with May-Grünwald-Giemsa for differential counts. After a 2 h incubation period at
37° C in a 5% CO2/95% air humidified incubator, nonadherent cells
were removed by washing twice with Hank's balanced salt solution
(HBSS). The adherent cell population was cultured during 24 h in
fresh RPMI 1640 either with or without 10 µg/ml Escherichia coli (strain
026: B6)-derived lipopolysaccharide (LPS) (Difco Laboratories, Detroit, MI) in order to assess the reactivity of AM to an ubiquitous activator. Culture medium was supplemented with 5% heat-inactivated
fetal bovine serum (FBS). 98% of the adherent cells were macrophages. Cell culture supernatants were recovered and stored at 20° C with aprotinin (33 IPFU/ml) until ET-1 and big ET-1 assay.
Plasma Recovery, Blood Monocyte Purification and Culture
Blood samples were collected in sterile heparine-tubes just prior to fiberoptic bronchoscopy. An aliquot was processed for monocyte culture as previously described (15) while the remainder was immediately separated by centrifugation (3000 rpm for 10 min) at 4° C. Plasma was stored with aprotinin (33 IPFU/ml) at 20° C until ET-1 and
big ET-1 assay. Blood mononuclear cells were recovered by centrifugation on Ficoll Hypaque gradients (Lymphoprep®; Flow Labs, Irvine
UK). Cells were washed twice in HBSS, resuspended at a concentration of 2.5 × 106/ml in RPMI 1640 supplemented with 2 mM
L-glutamine, 105 U/L penicillin, 100 mg/L streptomycin, 0.25 mg/l amphotericin B and 5% FBS (complete medium).
Cytocentrifuge smears were prepared with an aliquot of the mononuclear cell population and stained with May-Grünwald-Giemsa stain
for differential counts. About 30% of mononuclear cells were monocytes in both control subjects and patients with SSc. Cells were plated
on 6-dish cell culture plastic plates (2.5 × 106 cells/well), and monocytes were allowed to adhere for 2 h at 37° C with 5% CO2. Nonadherent cells were removed by washing twice. Adherent cells were cultured for 24 h in complete medium. More than 90% of adherent cells
were nonspecific stain esterase (NSE)-positive. Contaminating cells
were essentially lymphocytes. To evaluate the reactivity of blood
monocytes to an ubiquitous activator, 10 µg/ml of Escherichia coli
LPS was added to cells. After 24 h of culture, the resulting supernatants were collected and stored with aprotinin (33 IPFU/ml at 20° C
until assay.
ET-1 and Big ET-1 Measurements
Measurements of immunoreactive ET-1 and big ET-1 in BAL fluids, plasma and culture supernatants were assessed using a sandwich-type enzyme immunoassay developed by two of us (GL, JF) and adapted from Créminon and coworkers (16). For the ET-1 assay, the plates were coated with an anti ET-1 C-terminal monoclonal IgG (A gift of Dr. Créminon, Saclay, France). The conjugate antibody was an acetylcholinesterase-labeled anti-ET-1 loop domain mice monoclonal IgG1 kappa. The cross-reactivity of this immunoassay was 9% with ET-2 and 3% with ET-3. There was no cross-reactivity with big ET-1. The detection limit of the assay was 3 pg/ml. Synthetic ET-1 (Bachem, Voisins le bretonneux, France) was used as standard. The interassay and intraassay coefficients of variation were 13% and 10%, respectively.
The big ET-1 assay (17) was established by using a rabbit anti-big ET-1 C-terminal polyclonal IgG coated on plates. This antibody does not cross-react with ET-1, ET-2, and ET-3. The anti-ET-1 loop domain mice monoclonal antibody was used as the conjugate antibody since it recognizes bit ET-1. The cross-reactivity of this immunoassay was less than 0.5% with big ET-2 and big ET-3. Synthetic big ET-1 (France Biochem, Meudon, France) was used as standard. The detection limit of the assay was 3 pg/ml. The interassay and intraassay coefficients of variation were 12% and 8.5%, respectively.
ET-1 and big ET-1 extraction from plasma and BAL fluid was performed before assay according to the Nichols extraction method (NICHOLS Institute Diagnostic, San Juan Capistrano, CA) in order to remove interfering proteins and to concentrate ET-1 and big ET-1
levels in samples. Briefly, plasma and BALF were acidified with 4%
acetic acid and rinsed with distilled water. Samples (2 ml each) were
loaded onto a Sep-Pack Waters C18 reverse phase minicolumn (Millipore, Paris, France) previously prepared using 5-ml serial washes of
100% methanol, and 4% acetic acid in distilled water. Then, 25% ethanol in distilled water was applied to each column, before bound ET-1
was eluted with 4% acetic acid in 86% ethanol. Samples were dried by
evaporation at 37° C and reconstituted by adding 0.5 ml assay buffer
(RPMI 1640). Then, samples were stored at 20° C prior to the assay.
The extraction procedure yield was 85 to 100%. ET-1 and big ET-1
concentration in cell culture supernatants were measured without previous extractions.
For all samples, the results were expressed as the mean of duplicate assays. Results below the detection limit level were noted as not detectable (nd) and were not used for calculation of means. The person performing the assay was blinded to the different study groups.
Albumin Determination
As a reference for BAL ET-1 and big ET-1 concentrations, albumin concentration was determined in BAL fluid using an automated immunoturbidimetric assay with a rabbit antihuman albumin antibody (Behring, Rueil-Malmaison, France) as previously described (14).
Statistical Analysis
All data are presented as mean SEM. Analyses were performed using the Mann-Whitney nonparametric U test. The Wilcoxon signed-rank test was used to evaluate the effect of LPS. A standard linear regression analysis was used for all correlations, and p values of less than 0.05 were considered significant. Calculations and statistical analysis were performed using the StatView SETM II statistical package (Abacus Concept, Berkeley, CA).
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Bronchoalveolar Lavage Characteristics
The main results of BAL fluid analysis are presented in Table 3. The amount of cells per ml tended to be higher in patients with SSc than in control patients without reaching statistical significance (p = 0.08). Patients with scleroderma had a higher absolute lymphocyte count/ml and neutrophil count/ml than controls. BAL fluid albumin levels tended to be higher in patients with SSc than in control subjects without reaching statistical significance (p = 0.08).
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Big ET-1 and ET-1 Levels in Plasma (Fig. 1)
ET-1 and big ET-1 were detectable in all the samples tested. Patients with SSc and control subjects had similar levels of plasma ET-1 and plasma big ET-1.
Big ET-1 and ET-1 Levels in BAL Fluid (Fig. 1)
In BAL fluid, big ET-1 was below the detection limit level in six control and nine SSc samples. Mean values were similar in the two groups. When BAL fluid big ET-1 was expressed with respect to albumin, the concentration of big ET-1 in BAL fluid from patients (0.05 ± 0.03 ng big ET-1/mg albumin) was not different from control levels (0.10 ± 0.09 ng big ET-1/mg albumin, p = 0.96).
ET-1 was detectable in all the samples tested. Patients with SSc and control subjects had similar levels of BAL fluid ET-1. When BAL fluid ET-1 was expressed with respect to albumin, the concentration of ET-1 in BAL fluid from patients with SSc (0.8 ± 0.2 ng ET-1/mg albumin) tended to be lower than control levels (1.5 ± 0.6 ng ET-1/mg albumin) without reaching statistical significance (p = 0.13).
Correlation Between Big ET-1 and ET-1 Concentrations in Plasma or in BAL Fluid
ET-1 and big ET-1 concentrations in plasma were positively correlated in patients with SSc (R = 0.77; p = 0.002) and in controls (R = 0.83; p = 0.003) or in patients and controls taken as a group (R = 0.79; p = 0.0001) (Figure 2). By contrast, we found no correlation between ET-1 and big ET-1 levels in BAL fluid, neither in patients with SSc nor in controls, probably because of the high number of BAL samples with undetectable big ET-1. BAL fluid ET-1 concentrations did not correlate with plasma ET-1 concentrations. Similarly, BAL fluid big ET-1 concentrations did not correlate with plasma big ET-1.
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Spontaneous Big ET-1 and ET-1 Secretions by Monocytes
Mean big ET-1 (Figure 3A) and ET-1 concentrations (Figure 3B) detected in the supernatant of unstimulated monocytes were similar in controls (18.8 ± 5.3 pg/ml and 6.5 ± 0.7 pg/ml, respectively) and in patients with SSc (22.2 ± 4.5 pg/ml and 6.2 ± 1.0 pg/ml, respectively). Big ET-1 and ET-1 concentrations were positively correlated (R = 0.82, p = 0.006, patients and controls taken as a group). We found no correlation between concentrations in the supernatant of unstimulated monocytes and plasma concentrations.
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Big ET-1 and ET-1 Secretions by LPS-stimulated Monocytes
LPS stimulation increased big ET-1 concentration (Figure 3A) in monocytes supernatants from controls and patients with SSc. By contrast, LPS had no reproducible effect on ET-1 concentration in monocytes supernatants (Figure 3A and B). There was no correlation between ET-1 and big ET-1 concentrations in LPS-stimulated monocytes supernatants. Mean ET-1 and big ET-1 concentrations in the supernatant of LPS-stimulated monocytes were not statistically different in control patients and in patients with SSc.
Spontaneous Big ET-1 and ET-1 Secretion by AM
Spontaneous secretion of big ET-1 (Figure 4A) and ET-1 (Figure 4B) by adherent AM was similar in controls (63.6 ± 25.6 pg/ml and 16.9 ± 3.5 pg/ml, respectively) and in patients with SSc (78.8 ± 11.0 pg/ml and 16.7 ± 4.6 pg/ml, respectively). ET-1 concentrations in AM supernatants were positively correlated with BAL fluid ET-1 concentrations in controls (R = 0.96; p = 0.002) but not in SSc patients (R = 0.26; p = 0.4). No correlation was found between big ET-1 concentrations in AM supernatants and BAL fluid whatever the group tested, probably because of the high number of BAL samples with undetectable big ET-1.
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Big ET-1 and ET-1 Secretion by LPS-stimulated AM
LPS increased in vitro big ET-1 (Figure 4A) and ET-1 secretions (Figure 4B) by AM. A 2-fold increase was measured in controls, and a 3.5-fold increase in SSc patients. Mean big ET-1 and ET-1 concentrations in LPS-stimulated AM supernatants were significantly higher in patients with SSc (274.3 ± 43.3 pg/ml and 55.9 ± 7.3 pg/ml, respectively) than in controls (150.8 ± 46.9 pg/ml and 36.7 ± 6.0 pg/ml, respectively) (Figure 4A and B). We found no correlation between Spontaneous ET-1 and big ET-1 secretion by AM whereas we found a positive correlation between ET-1 and big ET-1 secretion by LPS-stimulated AM (R = 0.71; p = 0.001, patients and controls taken as a group) (Figure 5).
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Correlation Between ET-1 and Big ET-1 Concentrations, and Clinical or Paraclinical Characteristics Among SSc Patients
We found no correlation between ET-1 and big ET-1 concentrations in any biological fluid, and pulmonary function tests (TLC, FEV1, FVC, FEV1/FVC ratio, DLCO), arterial blood gas values or BAL fluid cytologic data,
Moreover, we found no difference in ET-1 and big ET-1 concentrations and treatment regimen, smoking status, or the radiographical pattern among SSc patients. However, the number of patients examined may have been too low to detect any difference.
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Fibrosing alveolitis is a common complication of SSc, and endothelins have been implicated in the pathogenesis of this disease. In this study, we show that (1) ET-1 and big ET-1 concentrations in plasma, BAL fluid, monocyte cell culture supernatants, and unstimulated adherent AM supernatants are not different in patients with SSc and in controls, whereas (2) ET-1 and big ET-1 concentration in LPS-stimulated alveolar macrophages are increased in patients with SSc.
In this study, we were not able to show any difference between controls and SSc patients concerning plasma and BAL
fluid ET-1 and big ET-1 levels. There are no data in the literature concerning plasma and BAL fluid levels of big ET-1 in
patients with SSc. However plasma (4, 7, 8, 18, 19) and BAL
fluid (5) concentrations of ET-1 have been reported to be increased in patients with SSc, although this remains an open
debate since Vancheeswaran and colleagues recently reported
that circulating ET-1 levels were normal in patients with SSc
(20). We do not believe that the lack of difference between patients and controls in our study was secondary due to an inappropriate storage of samples since all of the study samples were
stored at 20° C with aprotinin, a potent antiprotease, and for
a limited period of time (not more than 6 mo). Furthermore,
plasma ET-1 and big ET-1 concentrations measured in control
subjects were in the expected range according to the literature
(4, 7, 21). It is possible that the difference between our results
and previously published results is due to different assays or
different technical procedures. Most of the previous studies
(4, 5, 8, 18) used a radioimmunoassay method and most radioimmunoassays do not adequately distinguish among the mature isoforms of endothelin and are also cross reactive with big
ETs (22). Therefore, some previous studies could have overestimated circulating ET-1 levels by evaluating big ET-1 in addition to Et-1. Moreover, the difference could be due to different study populations. Indeed, Cambrey and colleagues showed
that BAL fluid ET-1 concentrations in SSc patients varied according to the pattern of lung disease. Namely, BAL ET-1 levels were higher in SSc patients with normal lung computed tomography (CT) scan than in patients with abnormal CT lung
scan (5). This suggests than ET-1 might be involved in the earlier phase of lung involvement with SSc. Since all the patients
with SSc that we studied had evidence of interstitial lung disease on CT scan and since the mean disease duration of SSc in
our patients was longer than that reported by Cambrey and
colleagues (7.0 yr and 2.5 yr, respectively), this could be an explanation. However the degree of lung function impairment
(as assessed by pulmonary function tests) was similar in our
study and in the study of Cambrey and colleagues thus suggesting that the degree of lung fibrosis was similar.
The treatments received by our patients might participate in the low ET-1 concentrations that we measured. Seven patients were receiving D-penicillamin and nine patients received diltiazem (a calcium antagonist) as a long term treatment. However, we found no difference in ET-1 and big ET-1 concentrations in any biological fluid according to the treatment, and we found no data in the literature concerning the influence of these pharmacologic agents on ET-1 production in vitro or in vivo, although calcium antagonists are known to modulate some of the effects of ET-1. Cambrey and colleagues gave no data concerning the treatments received by the patients they studied.
We found a strong correlation between ET-1 and big ET-1 levels in plasma, for controls and SSc patients, as previously reported by others (23). The analysis could not be adequately performed on BAL samples because of numerous samples with low big ET-1 concentrations. It is worth noting that ET-1 concentrations in BAL fluid were much higher than in plasma, despite the dilution inherent to the BAL procedure. This suggests a local production of ET-1 in the alveolar space rather than a passive transfer from the plasma. The sources of ET-1 in the lung are controversial. In the normal lung, endothelial cells, and epithelial and neuroendocrine cells of the airways are probably the main local producers of ET-1 (24, 25). In pathological conditions such as pulmonary fibrosis, increased expression of immunoreactive ET-1 and prepro ET-1 mRNA by alveolar epithelial cells (26) and alveolar macrophages (3) has been demonstrated. In view of these data, we predicted that alveolar macrophages obtained from patients with SSc and interstitial lung disease would secrete elevated levels of ET-1 and big ET-1 compared with controls in vitro. However, we measured similar concentrations of ET-1 or big ET-1 in the supernatants of adherent unstimulated alveolar macrophages from controls and SSc patients, after a 24-hr culture period. Most importantly, we noted a strong positive correlation between ET-1 concentrations in alveolar macrophage supernatants and ET-1 concentrations in BAL fluid in control patients. This suggests that under normal conditions, alveolar macrophages could participate in some way to the total burden of ET-1 in the alveolar space. By contrast, considering the SSc group of patients, we found no correlation between these values. It is possible that in this pathological condition ET-1 production by alveolar macrophages has a relatively minor contribution to the local production of ET-1 when compared with other cell types. It is well known that hyperplastic alveolar epithelial cells in fibrotic lung contain prepro ET-1 mRNA and immunoreactive ET-1 in the fibrotic lung (26), whereas normal alveolar epithelial cells do not (26). Endothelial cells are also an extensively studied source of ET-1 within the lung vasculature. In vitro studies indicate that 80% of the total amount of ET-1 synthesized by human vein endothelial cells is found in the basolateral compartment (27) thus suggesting a potential role for endothelial cells in ET-1 production in the alveolar air space. Immunohistochemistry and in situ hybridization studies to not support a significant role for lung fibroblasts in the alveolar production of ET-1 in the course of lung fibrosis (26) although it has been shown that dermal fibroblasts from involved skin overproduced ET-1 in patients with SSc (6).
One of our important results is the finding that alveolar
macrophage increase their in vitro ET-1 production when
stimulated with E. coli LPS, whereas blood monocytes do not.
By contrast, both alveolar macrophages and blood monocytes
increase their big ET-1 secretion when stimulated with LPS.
Moreover, we found a strong positive correlation between big
ET-1 and ET-1 concentrations in LPS-stimulated AM supernatants whereas no correlation was found in LPS-stimulated
monocyte supernatants. This suggests that ET-1 production is
differentially regulated in monocytes and alveolar macrophages. Ehrenreich and colleagues (28) previously showed that
LPS and PMA increased in vitro ET-1 secretion by AM 6-fold and 10-fold, respectively, whereas TGF
had no effect. In endothelial cells, however, ET-1 was not increased by PMA (29),
and TGF was a potent stimulator of preproET-1 message
(30). Thus, the signals for production of this peptide may vary
among different cell types. Different mechanisms could be involved in this phenomenon. ET-1 concentrations in cell culture supernatants is the result of a balance between ET-1 synthesis (from its precursors preproET-1 and big ET-1) and ET-1
degradation. Since both ET-1 and its precursor big ET-1 increased in AM supernatants with LPS stimulation, we suspect
that increased big ET-1 production is the main mechanism for
increased ET-1 concentrations in AM supernatants.
Concerning blood monocytes, we observed that LPS increased big ET-1 concentration in monocytes supernatants, whereas ET-1 concentrations were unchanged. A relative inhibition of endothelin converting enzyme activity or an increased degradation of ET-1 in monocyte supernatants, or both, could explain these results.
ET-1 and big ET-1 concentrations in LPS-stimulated AM supernatants were higher in SSc patients than in controls whereas baseline levels were similar in the two groups. We and others have shown that alveolar macrophages from patients with SSc are activated in vivo and release factors such as IL-8, capable of attracting and activating neutrophils to the alveolus (15), or capable of stimulating fibroblast migration, replication, and excessive collagen deposition such as fibronectin (1, 2, 31) or alveolar macrophage-derived growth factor for fibroblasts (1).
Whether higher ET-1 production by stimulated AM plays a physiopathological role in SSc is unknown. However, ET-1 has been shown to have profibrotic properties. In addition to its chemoattractive activity, ET-1 is a mitogen for fibroblasts that acts synergistically to amplify the response of these cells to other growth factors (9). ET-1 is known to stimulate collagen expression in normal fibroblasts, mainly through activation of protein kinase C (4, 9). Furthermore, increased ET-1 is localized to sites of collagen deposition during bleomycin-induced pulmonary fibrosis in rats (32) and an endothelin receptor antagonist (bosentan) has been shown to have protective properties in a model of acute inflammatory lung disease in rats (33). In humans, high levels of ET-1 have been measured in BAL fluid from patients with fibrotic lung diseases (5, 34). Although these studies suggest that ET-1 may act as a mitogenic or growth factor when the parenchymental lung fibrotic process is going on, impaired growth response to ET-1 in scleroderma skin fibroblasts has been demonstrated, and linked to a reduced expression of the ETA receptor (35).
In conclusion, our results show that AM from patients with SSc are hyperresponsive to LPS in vitro in terms of ET-1 and big ET-1 production and suggest that AM could participate in vivo in the overproduction of this potentially profibrotic mediator in patients with SSc.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Bruno Crestani, Unité de Pneumologie, Hôpital Bichat, 46 rue Henri Huchard, 75877 Paris cedex 18, France.
(Received in original form November 1, 1996 and in revised form June 5, 1997).
Acknowledgments: The authors thank Naïma Viires and Thierry Fournier for critical reading of the manuscript and Sylvain Roger for photographs.
This study was supported by a grant from the Délégation à la Recherche Clinique, Assistance Publique-Hôpitaux de Paris (n° AOB 94-002).
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References |
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Mathew, V., and
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