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1-Acid Glycoprotein by Rat and Human Type II Alveolar Epithelial Cells1
*
Institut National de la Santé et de la Recherche Médicale (INSERM) U408 and
INSERM U327, Faculté de Médecine Xavier Bichat, and
Laboratoire de Biochimie A and
§
Laboratoire d’Anatomie-Pathologique, Hôpital Bichat, Assistance Publique-Hôpitaux de Paris, Paris, France
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Abstract |
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1-Acid glycoprotein (AGP) is a major acute phase
protein in rat and human. AGP has important immunomodulatory functions
that are potentially important for pulmonary inflammatory response. The
liver is the main tissue for AGP synthesis in the organism, but the
expression of AGP in the rat lung has not been investigated. We show
that AGP mRNA was induced in the lung of dexamethasone-, turpentine-,
or LPS-treated rats, whereas AGP mRNA was not detected in the lung of
control rats. In the lung of animals treated intratracheally with LPS,
in situ hybridization showed that AGP gene expression was restricted to
cells located in the corners of the alveolus, consistent with an
alveolar type II (ATII) cell localization. The inducible expression of
the AGP gene was confirmed in vitro with SV40 T2 cells and rat ATII
cells in primary culture: maximal expression required the presence of
dexamethasone. IL-1 and the conditioned medium of alveolar macrophages
acted synergistically with dexamethasone. Rat ATII cells secreted
immunoreactive AGP in vitro when stimulated with dexamethasone or with
a combination of dexamethasone and the conditioned medium of alveolar
macrophages. In vivo, in the human lung, we detected immunoreactive AGP
in hyperplastic ATII cells, whereas we did not detect AGP in the normal
lung. We conclude that AGP is expressed in the lung in cases of
inflammation and that ATII cells are the main source of AGP in the
lung. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 1-acid glycoprotein (orosomucoid,
AGP)3 is a typical acute
phase plasma protein in humans, rats, mice, and other species (2). AGP
is a single polypeptide with a molecular mass of 23 kDa plus three to
five highly sialylated carbohydrate side chains, the latter accounting
for 
45% of its total mass of 45 kDa (3). IL-1ß, TNF-, and
IL-6 have been shown to be the key cytokines controlling the hepatocyte
expression of AGP (1). Glucocorticoids stimulate AGP expression and act
synergistically with IL-1ß, TNF-, and IL-6 to induce AGP
expression by hepatocytes (1).
AGP has been shown to act in vitro and in vivo as an immunomodulatory
molecule. In vitro, AGP inhibits polymorphonuclear neutrophil
activation (4), modulates LPS-induced cytokine secretion by
monocytes/macrophages (5), and increases the secretion of an IL-1
inhibitor by murine macrophages, most probably the IL-1 receptor
antagonist (IL-1Ra) (6, 7). In vivo, AGP protects mice from
TNF--induced lethality (8). The marked increase in plasma AGP
concentration in the course of the acute phase response could therefore
act as a form of negative feedback aimed at limiting the extent of the
inflammatory reaction and its possible deleterious consequences. A
local expression of AGP, at the site of the initial acute phase
reaction, could also serve as a protection against the deleterious
effect of inflammation. This could be particularly important in the
alveolar space, an essential part of the lung, where gas exchange takes
place. Indeed, the integrity of this very delicate and specialized
structure is essential for the maintenance of the function of the
organ. Thus, any inflammatory reaction developing in the lung must be
tightly controlled to preserve the structure of the alveolar space.
There is a growing body of evidence that the acute phase response may take place in extrahepatic cell types, notably epithelial cells, and may be regulated by cytokines, as observed in hepatocytes (9, 10, 11, 12). Extrahepatic synthesis of AGP has been detected in vivo in the mouse kidney and the pregnant rat uterus (9, 13) and in the human prostate and myocardium (14, 15). However, AGP expression in the lung, either normal or inflamed, has never been detected.
In view of the important immunomodulatory properties of AGP, we asked whether AGP is expressed in the rat lung in the course of different types of inflammatory response, and if so, how its expression is controlled. We used two models of inflammation: 1) LPS administration (either intratracheal or i.p.) to mimic infection and 2) s.c. turpentine injection to induce a nonseptic localized inflammation. In this article, we show for the first time that AGP mRNA is expressed in the rat lung in cases of inflammation or steroid administration, that alveolar type II cells (ATII cells) are the primary site of AGP expression in the rat lung in vivo, and that rat ATII cells in vitro express the AGP mRNA and secrete immunoreactive AGP when stimulated with the secretory products of alveolar macrophages and dexamethasone. Moreover, we show that hyperplastic alveolar epithelial cells express immunoreactive AGP in the human lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant murine (rm)IL-1, rmTNF-, and recombinant human
(rh)IL-6 were purchased from Immugenex (Los Angeles, CA).
Escherichia coli (strain 026; B6)-derived LPS was obtained
from Difco (Detroit, MI). TEMED, ammonium persulfate, urea,
dexamethasone, and turpentine were from Sigma (La Verpillière,
France). Transcription reagents were purchased from Promega (Madison,
WI). [-32P]UTP (400 Ci/mmol) was from Amersham (Les
Ulis, France). RNase-free DNase I, RNase A, and T1 and brewer’s yeast
tRNA were supplied by Boehringer (Mannheim, Germany).
Acrylamide/bisacrylamide, phenol, and proteinase K were from Appligene
(Illkirch, France). Guanidine thiocyanate (GuSCN) was purchased from
Fluka Chemie (Buchs, Switzerland). All restriction enzymes were from
New England BioLabs (Beverly, MA) or from Boehringer. Pro-Mix (a
mixture of L-[35S]methionine and
L-[35S]cysteine) was obtained from Amersham
(Buckinghamshire, U.K.).
Tissue culture media, supplements, and FBS were from Life Technologies (Cergy Pontoise, France). Tissue culture plasticware was from Costar (Cambridge, MA).
Animals
Male Sprague Dawley rats weighing 220 to 250 g (Charles River Breeders, St. Aubin les Elbeuf, France) were used within 4 days of arrival. Food and water were given ad libitum.
Intratracheal LPS challenge. Rats were lightly anesthetized with ether and a midline incision was made above the sternum. The trachea was exposed by blunt dissection, a 28-gauge needle was inserted into the trachea above the carina, and 0.5 ml of 9% NaCl containing 0.1 mg of LPS was instilled. Control animals received 0.5 ml of 9% NaCl alone.
Systemic LPS challenge. LPS was reconstituted in PBS (5 mg/ml). LPS (2.5 mg/kg) was administered i.p. in rats. Control rats received the same volume of PBS.
Turpentine. Turpentine injection was used to create a localized wound known to stimulate the hepatic acute phase response. Rats received a s.c. injection with 1 ml of turpentine. Controls received the same volume of saline.
For all of these experimental groups, the animals were sacrificed at designated time intervals. The lungs were excised, immediately frozen in liquid nitrogen, and stored at -80°C until RNA extraction.
Effect of dexamethasone. In some experiments, we evaluated the effect of glucocorticoids on AGP expression. At time 0, the animals were challenged with either LPS or turpentine or were left untreated. Half of the animals in each group received 2 mg/kg of dexamethasone (4 mg/ml) i.p. at time 0 and then 6 h later. The other animals received a similar volume of PBS. The animals were killed 24 h after the first injection, and the lungs were recovered, immediately frozen in liquid nitrogen, and stored at -80°C until RNA extraction. In some experiments (three animals in each group), we evaluated the effect of 0.02, 0.2, and 2 mg/kg dexamethasone administered i.p. at time 0 and 6 h later. Control rats received the same volume of PBS.
Isolation of rat alveolar type II cells and preparation of rat alveolar macrophage-conditioned medium (AM-CM)
ATII cells were isolated from adult male pathogen-free Sprague Dawley rats by enzymatic dissociation and purified by differential adherence to plastic as previously described (16). Cells (2 x 106) were plated in each well of a six-well cell culture plate with 2 ml of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 105 U/L penicillin, 100 mg/L streptomycin, and 0.25 mg/L amphotericin B (complete DMEM). After a 24-h period, nonadherent cells were removed by gently washing twice with PBS, fresh complete medium was replaced, and the cells were used for the experiments. The viability of adherent cells was >98% as assessed by the trypan blue exclusion test.
AM-CM was prepared as previously described (16). Rat alveolar macrophages recovered by bronchoalveolar lavage were resuspended in RPMI containing 10% heat-inactivated FBS, antibiotics, and glutamine at a density of 106 cells/ml. Cells (106) were plated in each well of a 24-well cell culture plate and allowed to adhere for 2 h, then nonadherent cells were removed, and fresh medium containing 10 µg/ml LPS was added. Adherent cells were >98% alveolar macrophages as assessed by nonspecific esterase stain (Sigma). Conditioned medium, consisting of LPS-activated rat alveolar macrophages, was recovered after a 24-h incubation period.
Stimulation of AGP gene expression and AGP secretion by rat ATII cells in vitro
Rat ATII cells were used 24 h after isolation. Cells were incubated with complete DMEM. Rat CM (10% v/v) or cytokines (10 ng/ml) were added for the indicated times in the presence or absence of dexamethasone (10-5 M). In these experiments, we stimulated rat ATII cells with murine recombinant cytokines because rat products were not available. These cytokines have previously been shown to be active on rat cells (16, 17). Supernatants were recovered for the determination of AGP concentration. Cell monolayers were scrapped in 500 µl of PBS and centrifuged (5 min, 12,000 x g, 4°C), and the pellet was solubilized in 5 M GuSCN, 0.1 M EDTA, pH 7.0 (100 µl per 2 x 106 pneumocytes) (18). The cell lysates were stored at -80°C until mRNA analysis.
In some experiments, we evaluated AGP gene expression by SV40 T2 cells, a cell line derived from fetal rat ATII cells (a generous gift of Prof. A. Clement, Hôpital Trousseau, Paris, France) (19). SV40 T2 cells were cultured until confluence in complete DMEM and stimulated as indicated for rat type II cells. Cells lysates (prepared as indicated for ATII cells) were used for the mRNA analysis.
Detection of AGP in rat ATII cell supernatants
In vitro radiolabeling of AGP was obtained by incubating ATII cells for 30 min in complete medium without L-methionine, then adding 100 µCi/ml of Pro-Mix (14.3 mCi/ml) for 4 h. The cell culture supernatants were recovered and adjusted to correspond to the immunoprecipitation buffer (20 mM Tris/HCl, pH 8.0, 1% (by vol) Triton X-100, 5 mM EDTA, 2 mM PMSF, 1 mM benzamidine, and 20 mM leupeptin). Isolated rat hepatocytes were similarly treated, and their supernatants were recovered to compare the apparent m.w. of AGP secreted by rat ATII cells and by rat hepatocytes (20). Immunoprecipitation of AGP was performed as described by Poüs et al. (20). Immunoprecipitates were analyzed by SDS/PAGE (separating gel, 7.5%; stacking gel, 4%) and exposed on Biomax Kodak films.
Rat AGP concentration was measured in the supernatant of rat ATII cells and confluent SV40 T2 cells using a "sandwich"-type ELISA (21, 22). The cells were cultured for 24 h in 10-cm cell culture plates with 5 ml DMEM with antibiotics without FBS (FBS was avoided to limit the risk of interference with bovine AGP contained in FBS). The cells were stimulated with dexamethasone (10 mM), with AM-CM (10% v/v), or with both. The cell monolayers were trypsinized, and the cells were spun down by centrifugation and sonicated in 1 ml of PBS. Total cellular protein concentration was measured using the Bradford protein assay (23). The results are expressed as ng of AGP secreted per mg of protein per 24 h.
Northern blot analysis and RNase protection assay (RPA)
AGP mRNA detection in the lung was performed by Northern blot analysis using a 32P-labeled 0.8-kb cDNA probe as previously described (24). Total cellular RNA from lung and liver was isolated using RNAPlus (Bioprobe, Montreuil, France) according to the instructions of the manufacturer. After analysis, the membranes were dehybridized and rehybridized with a 32P-labeled cDNA rat glyceraldehyde phosphate dehydrogenase (GAPDH) probe as a loading control (25). The blots were quantified through an electronic autoradiography device (Instant Imager, Packard, Groningen, The Netherlands). The ratio of the AGP mRNA signal to the corresponding GAPDH mRNA signal was calculated for each sample.
AGP gene expression by rat ATII cells and SV40 T2 cells was evaluated
by RPA on cells solubilized in GuSCN without prior RNA extraction, as
previously described with minor modifications (18, 26). A
cohybridization with AGP and GAPDH probes was performed. A
286-nucleotide (nt) AGP riboprobe was obtained from AGP cDNA inserts
subcloned into pBluescript II SK+ phagemid vector (18).
Riboprobe synthesis was performed in the presence of
[-32P]UTP (50 µCi, 400 Ci/mmol) and T3 RNA
polymerase after linearization of the vector by EcoRI
digestion. The 185-nt GAPDH riboprobe was synthesized from a
XbaI-ApaI rat GAPDH cDNA insert (25) subcloned
into pBluescript II SK+.
Cellular lysates (20 µl) were mixed with 2 µl of each radiolabeled probe (105 cpm/µl). After overnight hybridization at 37°C, the samples were treated with RNases A and T1, then exposed to proteinase K. After extraction with phenol:chloroform:isoamyl alcohol (25:24:1), the protected RNA:RNA hybrids (230 nt for AGP and 164 nt for GAPDH) were precipitated and loaded on a 6% acrylamide:bisacrylamide (19:1), 7.8 M urea denaturing gel. After a 1- to 2-h run, the gels were dried. Quantitative analysis of the radioactive protected bands was performed by direct counting of the gel (Instant Imager). AGP and GAPDH signals were corrected for respective background measured on the same lane. For each experimental condition, the ratio of the corrected signals (AGP/GAPDH) was calculated. Relative changes were expressed vs control cells (unstimulated), the sensitivity of the Instant Imager allowing us to quantify extremely low levels of expression.
In situ hybridization
In situ hybridization was performed as previously described (27, 28) with some modifications. Rats challenged intratracheally with LPS or controls given PBS were anesthetized with ether 24 h after challenge and killed by exsanguination. The lungs were perfused for 15 min with cold 4% paraformaldehyde in PBS through a cannula placed in the pulmonary artery with constant flow (6 ml/min), then the lungs were postfixed in ice-chilled 4% paraformaldehyde for 1 h. Small fragments were cut using sterile bladder scissors. Fragments were washed overnight in fresh PBS, then embedded in paraffin (27). Tissue sections (2–3 µm thick) mounted on Superfrost Plus slides (Consortium de Matériel pour Laboratoires, Nemours, France), were deparaffinized, air dried, pretreated with 0.2 N HCl for 20 min, incubated in a proteinase K solution (2 µg/ml) at 37°C for 30 min, then immersed in 4% paraformaldehyde and further treated with 0.25% acetic anhydride. Then, the slides were dehydrated through graded ethanol and air dried. Hybridization was performed at 50°C overnight in a buffer containing 2x SSC, 50% formamide, 1x Denhardt’s solution, 10% dextran, 10 mM dithiotreitol, 500 µg/ml yeast tRNA, 20 mM Tris-HCl, pH 8, 1 mM EDTA, and the AGP RNA probe or a sense RNA probe (as a negative control) radiolabeled with [35S]dCTP (106 cpm/slide). The riboprobes were synthesized as described above for the RPA. Slides were then washed sequentially with 5x SSC at 50°C, 2x SSC, 50% formamide at 65°C for 20 min and twice in NaCl, 0.5 M Tris-HCl, 10 mM-5 mM EDTA. To remove nonhybridized single-strand cRNA, slides were treated with RNase A (20 µg/ml) for 30 min at 37°C, washed in NaCl, 0.5 M Tris-HCl, 10 mM-5 mM EDTA, then in 0.1x SSC at room temperature. Slides were dehydrated, air dried, and autoradiographed for 60 days at 4°C. After development and fixation, slides were counterstained with Giemsa stain.
Immunohistochemical analysis in the human lung
To determine whether human alveolar epithelial cells synthesized AGP in vivo, 4-µm sections, embedded in paraffin, from normal lung biopsies (obtained from two patients undergoing surgical lung resection for a localized lung tumor) and from diseased lung (obtained from two patients with idiopathic pulmonary fibrosis, one patient with ruptured hydatid cyst, and one patient with localized bronchiectasis) were stained for AGP with a monoclonal mouse anti-human AGP IgG1 (catalogue no. A5566, Sigma) (working dilution, 1/200). Paraffin-embedded liver biopsies were used as positive controls. Monoclonal mouse anti-human thyroglobulin IgG1 (catalogue no. M781, Dakopatts, Glostrup, Denmark) was used as a control Ab (working dilution, 1/200). An avidin-biotin-peroxidase method was used. In some sections, the first Ab was omitted to assess the specificity of the immunolabeling.
Statistical analysis
The results are expressed as means ± SD. Statistical significance was assessed using the Kruskal-Wallis nonparametric test followed by the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AGP mRNA was not detected in the normal rat lung by Northern blot
analysis. However, 16 h after a systemic LPS challenge, high
levels of AGP mRNA were induced in the lung (Figs. 1
and 2). By contrast, AGP mRNA was
detected in the liver of unstimulated rats and was further increased
after a systemic LPS challenge (Fig. 1). The AGP mRNA was still
detected in the lung 72 h after the challenge, but at very low
levels (7% of the expression measured 16 h after stimulation;
Fig. 2). Similarly, an intratracheal LPS
challenge induced the expression of the AGP gene in the lung 24 and
36 h after stimulation, while AGP mRNA was not detectable 72
h after the challenge. Control saline challenge, either intratracheal
or i.p., did not induce any expression of the AGP gene.
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). Expression was lower than that
induced by the intratracheal LPS challenge (8.5% of the intratracheal
challenge).
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).
Moreover, dexamethasone potentiated the effect of intratracheally
administered LPS, since the AGP mRNA level was 11.5-fold that obtained
with LPS alone (Fig. 3) when the animals received a combination of i.p.
dexamethasone and intratracheal LPS.
In additional experiments, we evaluated the effect of lower
dexamethasone doses. Rats (three animals in each group) received 2,
0.2, or 0.02 mg/kg dexamethasone at time 0 and at 6 h. AGP mRNA
expression in the lung was quantitatively analyzed at 24 h (Fig. 4). While 0.2 mg/kg dexamethasone induced
a small expression of the AGP mRNA, 0.02 mg/kg dexamethasone had no
effect.
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Identification of the AGP expressing cell in the lung
To identify the cell(s) that expressed AGP mRNA in the lung in vivo, we performed an in situ hybridization analysis in normal rat lung and in the lung of LPS-challenged rats (intratracheal instillation 24 h before lung sampling).
AGP mRNA was never detected in the normal lung, whereas it was detected
in the lung of LPS-challenged animals. The specific signal was
exclusively observed in the alveolar region of the lung and was absent
in the bronchial epithelium and in the vessels. Positively labeled
cells were probably ATII epithelial cells because of their
characteristic location in the corner of the alveolus (30) (Fig. 5).
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To confirm that ATII cells are able to produce AGP mRNA, we
measured the steady state level of AGP mRNA in SV40 T2 cells, a cell
line originating from fetal rat type II cells (19) (Fig. 6). An extremely low signal could be
detected in unstimulated SV40 T2 cells by direct quantification of the
gel. This signal was not high enough to be detected by autoradiography.
The AGP/GAPDH ratio was increased 3.5-fold with CM, whereas
dexamethasone (10-5 M) increased the AGP/GAPDH ratio
15-fold over the control value. A maximal stimulation was obtained with
the synergistic combination of dexamethasone and CM (the AGP/GAPDH
ratio was increased 70-fold).
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We performed similar experiments with rat ATII cells in primary
culture (Fig. 7). A very low level of AGP
mRNA signal (not detected by autoradiography) could be detected in
unstimulated ATII cells by direct quantification of the gel. CM alone
did not increase the AGP/GAPDH mRNA ratio. A 3.3-fold increase of the
AGP/GAPDH ratio was induced with dexamethasone. Maximal stimulation was
obtained with the synergistic combination of dexamethasone and CM (the
AGP/GAPDH ratio was increased 9.3-fold). LPS (1 µg/ml) did not
increase the AGP/GAPDH mRNA ratio in the presence or absence of
dexamethasone (data not shown).
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).
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) allowed us to
detect one band with a molecular mass of 49.6-kDa in the supernatant of
cells cultured with a combination of dexamethasone and CM. No
radiolabeled protein could be detected in the supernatant of
unstimulated cells. In rat hepatocyte supernatants, a main 47.7-kDa
band was detected under control conditions, and its intensity increased
strongly after stimulation with a combination of dexamethasone and
CM.
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). We did not detect AGP in the
supernatant of control or CM-stimulated ATII cells.
Dexamethasone-stimulated cells secreted AGP, the higher levels being
measured in the supernatant of ATII cells stimulated with a combination
of CM and dexamethasone. When compared with rat ATII cells, AGP
secretion by SV40 T2 cells was 100-fold higher. CM alone induced AGP
secretion, but the maximal effect was obtained with the combination of
CM and dexamethasone.
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Among the mediators secreted by alveolar macrophages, the
cytokines IL-1, TNF-, and IL-6 play a key role in regulating the
expression of the acute phase proteins in the liver (1). We evaluated
the effect of rmIL-1, rmTNF-, and rhIL-6 (10 ng/ml each) on AGP mRNA
in SV40 T2 cells (Fig. 6). Without dexamethasone, all cytokines induced
a small increase in the AGP/GAPDH mRNA ratio, although at a very low
level compared with the effect of dexamethasone. The combination of
IL-1 and dexamethasone was highly synergistic and induced the
expression of the AGP gene at levels similar those obtained with the
combination of CM and dexamethasone (the AGP/GAPDH ratio was increased
63-fold over the control value). The combination of TNF and
dexamethasone was essentially additive and increased the AGP/GAPDH mRNA
ratio 20-fold. IL-6 did not increase dexamethasone-induced
stimulation.
Similar experiments were performed with rat ATII cells (Fig. 7). In the
absence of dexamethasone, stimulation of rat ATII cells with rmIL-1,
rmTNF-, or rhIL-6 (10 ng/ml each) did not increase the AGP/GAPDH
mRNA ratio (data not shown). In the presence of dexamethasone, TNF and
IL-6 had no effect, whereas IL-1 increased by twofold the AGP/GAPDH
mRNA ratio over the dexamethasone value.
Immunohistochemical detection of AGP in the human lung (Fig. 10)
We never detected immunoreactive AGP in normal human lung. By contrast, a strong positive specific signal was detected in areas of hyperplastic alveolar epithelium in all diseased lung samples that we studied. Immunoreactivity was never detected in bronchial epithelium or in endothelial cells. Some positive signal was observed in cells located in the alveolar lumen, consistent with desquamated alveolar epithelial cells or alveolar macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data provide the first evidence of inducible AGP expression in the lung. Although circulating AGP is essentially synthesized in the liver, extrahepatic expression of acute phase proteins is increasingly recognized (9, 10, 31). Local extrahepatic production of AGP has been detected in the myocardium (14), kidney (9), breast (32), prostate (15), and uterus (13). However, the regulation of extrahepatic AGP expression has never been explored in detail.
The regulation of the rat AGP gene expression by hepatocytes has been
extensively studied in vivo and in vitro. Hepatic AGP mRNA levels are
regulated in vivo both at the transcriptional (29) and
posttranscriptional (33) level by glucocorticoids and acute phase
mediators. It is now well established that the in vitro AGP gene
expression by rat hepatocytes and various rat hepatoma cell lines is
increased by IL-1 and TNF- (26, 34), IL-6 (and related cytokines)
(26, 35, 36), glucocorticoids (26, 29), and some exogenous molecules
such as phenobarbital (24). A positive interaction between
glucocorticoids and cytokines has been consistently found (37). In vivo
experiments with adrenalectomized rats indicate that there are
independent regulatory processes for induction by glucocorticoids and
by mediators of the acute phase response in the liver (29).
Characterization of the rat AGP gene has revealed both separate and
overlapping cis-acting elements for these factors, each
element being composed of several interacting regulatory sequences
(1).
Our results indicate that regulation of the expression of the AGP gene in the lung has some similarities with its regulation in the liver. Indeed, the experimental conditions that induced the expression of the AGP mRNA in the lung in vivo (systemic LPS challenge, turpentine challenge, dexamethasone administration) are also conditions that are known to increase the expression of the AGP gene in the liver (1). Moreover, as previously shown for the liver, glucocorticoids play a key role in the expression of the AGP gene in the lung. In rat hepatocytes and hepatoma cell lines, the expression of the AGP gene has been shown to be highly dependent on the presence of dexamethasone (26, 38, 39). We observed that dexamethasone greatly increased the level of AGP mRNA in the lung in vivo and in ATII cells in vitro, as well as acting synergistically with inflammatory mediators (rmIL-1 and AM-CM) to induce the expression of the AGP gene by ATII cells in vitro, whereas the cytokines had either a small effect (SV40 T2) or no effect at all (ATII cells) in the absence of dexamethasone.
The main difference between lung and liver expression of the AGP gene is that the AGP mRNA is not detected in the lung in healthy animals, whereas it is detected in the liver, although we cannot totally exclude the possibility that a very low level of expression of the AGP gene, not detectable by Northern blot or in situ hybridization analysis, is present in the lung of healthy animals. Moreover, cytokine regulation of the expression of the AGP gene by rat ATII cells in vitro differs somewhat from what is known concerning the hepatocytes. Indeed, in vitro we observed that rhIL-6 had no effect on rat ATII cells in primary culture, slightly increased the expression of the AGP gene by SV40 T2 cells, and did not potentiate the effect of dexamethasone either on rat ATII cells or on SV40 T2 cells. It is unlikely that the lack of effect of IL-6 was due to the use of rhIL-6 for the stimulation of rat ATII cells, since human IL-6 is highly efficient for the stimulation of AGP expression by rat hepatocytes (26). Among the cytokines that we tested, rmIL-1 was the most consistent inducer of AGP gene expression. Indeed, rmIL-1 induced AGP expression and acted synergistically with dexamethasone to stimulate the expression of AGP by SV40 T2 cells, to reach a level similar to that obtained with the combination of AM-CM and dexamethasone. Moreover, rmIL-1 potentiated the effect of dexamethasone on rat ATII cells. It is interesting to note that the combination of IL-1 and dexamethasone induced a 60-fold increase in the AGP mRNA level in SV40 T2 cells, a level of induction similar to that measured in rat hepatoma cells (38). Thus, IL-1 has a prominent effect on the expression of the AGP gene by rat ATII cells, whereas IL-6 seems to have a very limited action. This is in contrast with normal rat hepatocytes, since IL-1 and IL-6 have been shown to exert almost similar effects on AGP gene expression in the presence of dexamethasone (26).
A most important finding of this study is that ATII cells not only
express the AGP gene in vitro, but they also secrete immunoreactive AGP
in their supernatant when stimulated with dexamethasone or
the combination of dexamethasone and AM-CM. However, the AGP levels
measured in dexamethasone plus CM-stimulated ATII cell
supernatants are about 0.5% of those previously measured in the cell
culture supernatants of unstimulated rat hepatocytes by one of us (40).
Furthermore, it is worth noting that the apparent m.w. of secreted AGP
was slightly higher in SV40 T2 supernatants than in rat hepatocyte
supernatants. The difference could be due to a difference in
glycosylation, as we have previously shown for
1-antitrypsin secreted in vitro by rat ATII cells
(41).
In the present study, in situ hybridization and immunohistochemistry results suggest that ATII cells are the main source of AGP in the rat lung (at least after intratracheal LPS administration) and in the diseased human lung. Synthesis of AGP by epithelial cells has been the subject of only a few reports in the literature. In the decidual cells of the pregnant rat uterus, there is a burst of AGP synthesis 1 to 5 days postimplantation; by parturition, the level of uterine AGP synthesis declines to near zero (13). Immunoreactive AGP in the human prostate and in vitro synthesis of AGP by human breast epithelial cells have been demonstrated (15, 32).
The physiologic role of local AGP production by alveolar epithelial
cells is potentially important. In vitro data suggest that AGP could
modulate the degree of activation of inflammatory cells in the
alveolus, such as neutrophils, lymphocytes, or alveolar macrophages.
Indeed, AGP is a potent inhibitor of neutrophil chemotaxis and
oxidative metabolism in vitro (4) and inhibits in vitro mitogen-induced
lymphoproliferation (42). Boutten and colleagues showed that AGP
increased the in vitro secretion of IL-1ß, TNF-, and IL-6 by human
alveolar macrophages stimulated with E. coli-derived LPS.
Similar results were obtained with human peritoneal macrophages and
human blood monocytes (5). Tilg and coworkers extended these results,
showing that blood mononuclear cells incubated with AGP synthesized
large quantities of IL-1Ra, 5- to 10-fold more than the amount of
IL-1ß produced by these cells, and that AGP was synergistic with low
concentrations of endotoxin in the induction of IL-1Ra and IL-1ß
synthesis (7). The preferential induction of IL-1Ra by AGP may
contribute to its anti-inflammatory effect (7). Recently, AGP has
been shown to inhibit the apoptosis of hepatocytes by
TNF/galactosamine, while similar apoptosis of hepatocytes induced by
anti-Fas remained unaffected (43). Whether these properties are
relevant in vivo in the lung is currently unknown; however, the
protective effect of AGP has been demonstrated in vivo in a model of
sepsis induced by TNF or endotoxin in mice (8).
Given these properties, the expression of AGP in the alveolar space in
cases of acute inflammation may exert a local protective effect by
limiting the inflammatory reaction and its potentially deleterious
effect on alveolar structures. This hypothesis is further supported by
the fact that ATII cells appear to be the main source of AGP expression
in the lung, both in rat and humans, pointing out once again the
fundamental role of ATII cells in the control of the integrity of the
alveolar space. ATII cells synthesize and secrete surfactant, control
the volume and composition of the epithelial lining fluid, and
proliferate and differentiate into type I alveolar epithelial cells
after injury to maintain the integrity of the alveolar wall (44).
Moreover, we and others have shown that ATII cells may have an
immunoregulatory role in the lung through the secretion of mediators
such as cytokines (16, 45), prostaglandins (46), or nitric oxide (47).
We have also previously shown that ATII cells in vitro secreted
1-antitrypsin, thereby contributing to the intraalveolar
antiproteases shield (41).
In summary, the present study demonstrates the inducible expression of AGP, a typical acute phase protein with potent immunomodulatory properties, by alveolar epithelial cells in vitro and in vivo and illustrates the potential for effective communication between macrophages and epithelial cells in the alveolar space.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Docteur Bruno Crestani, Unité de Pneumologie, Hôpital Bichat, 46 rue Henri Huchard, 75018 Paris, France. E-mail address:
3 Abbreviations used in this paper: AGP, 1-acid glycoprotein; AM-CM, alveolar macrophage-conditioned medium; ATII cells, type II alveolar cells; rh, recombinant human; rm, recombinant murine; GuSCN, guanidine thiocyanate; DMEM, Dulbecco’s modified Eagle’s medium; GAPDH, glyceraldehyde-phosphate dehydrogenase; RPA, ribonuclease protection assay; nt, nucleotide.
Received for publication April 22, 1997. Accepted for publication January 8, 1998.
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1-acid glycoprotein gene. Mol. Cell. Biol. 5:3634.1-acid glycoprotein in release of macrophage inhibitor of interleukin-1 activity. Inflammation 14:315.[Medline]
1-acid glycoprotein gene transcription. J. Biol. Chem. 260:15386.1-acid glycoprotein RNA during inflammation. J. Biol. Chem. 262:12826., IL-1ß, and hepatocyte growth factor cooperate in stimulating specific acute phase plasma protein genes in rat hepatoma cells. J. Immunol. 151:4248.[Abstract]
1-acid glycoprotein by recombinant human interleukin-1 in rat hepatoma cells. J. Biol. Chem. 263:7141.This article has been cited by other articles:
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