Cells by Rat and Human Type II Alveolar Epithelial

Inducible Expression of the α1-Acid Glycoprotein by Rat and Human Type II Alveolar Epithelial Cells

This information is current as Bruno Crestani, Corinne Rolland, Bernard Lardeux, Thierry of October 1, 2021. Fournier, Dominique Bernuau, Christian Poüs, Christiane Vissuzaine, Lin Li and Michel Aubier J Immunol 1998; 160:4596-4605; ; http://www.jimmunol.org/content/160/9/4596 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ␣ Inducible Expression of the 1-Acid Glycoprotein by Rat and Human Type II Alveolar Epithelial Cells1

Bruno Crestani,2* Corinne Rolland,* Bernard Lardeux,† Thierry Fournier,* Dominique Bernuau,† Christian Pou¨s,‡ Christiane Vissuzaine,§ Lin Li,* and Michel Aubier* ␣ 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 Downloaded from 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. The Journal of Immunology, 1998, 160: 4596–4605. http://www.jimmunol.org/ he host response to tissue injury, i.e., the “acute phase tion by monocytes/macrophages (5), and increases the secretion of response,” is a highly coordinated series of physiologic an IL-1 inhibitor by murine macrophages, most probably the IL-1 T reactions involving almost every major organ system. receptor antagonist (IL-1Ra) (6, 7). In vivo, AGP protects mice Marked changes in concentrations of plasma glycoproteins termed from TNF-␣-induced lethality (8). The marked increase in plasma “acute phase plasma proteins” occur in the course of the acute AGP concentration in the course of the acute phase response could phase of inflammation (1). The latter result from changes in the therefore act as a form of negative feedback aimed at limiting the expression of specific genes in the liver and are the consequences extent of the inflammatory reaction and its possible deleterious of the action of inflammatory mediators and hormones such as consequences. A local expression of AGP, at the site of the initial glucocorticosteroids on hepatocytes (1). acute phase reaction, could also serve as a protection against the by guest on October 1, 2021 ␣ 3 The 1-acid glycoprotein (orosomucoid, AGP) is a typical deleterious effect of inflammation. This could be particularly im- acute phase plasma protein in humans, rats, mice, and other species portant in the alveolar space, an essential part of the lung, where (2). AGP is a single polypeptide with a molecular mass of 23 kDa gas exchange takes place. Indeed, the integrity of this very delicate plus three to five highly sialylated carbohydrate side chains, the and specialized structure is essential for the maintenance of the ϳ ϳ latter accounting for 45% of its total mass of 45 kDa (3). function of the organ. Thus, any inflammatory reaction developing ␤ ␣ IL-1 , TNF- , and IL-6 have been shown to be the key cytokines in the lung must be tightly controlled to preserve the structure of controlling the hepatocyte expression of AGP (1). Glucocorticoids the alveolar space. ␤ stimulate AGP expression and act synergistically with IL-1 , There is a growing body of evidence that the acute phase re- ␣ TNF- , and IL-6 to induce AGP expression by hepatocytes (1). sponse may take place in extrahepatic cell types, notably epithelial AGP has been shown to act in vitro and in vivo as an immu- cells, and may be regulated by cytokines, as observed in hepato- nomodulatory molecule. In vitro, AGP inhibits polymorphonuclear cytes (9–12). Extrahepatic synthesis of AGP has been detected in neutrophil activation (4), modulates LPS-induced cytokine secre- vivo in the mouse kidney and the pregnant rat uterus (9, 13) and in the human prostate and myocardium (14, 15). However, AGP ex- *Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U408 and pression in the lung, either normal or inflamed, has never been †INSERM U327, Faculte´deMe´decine Xavier Bichat, and ‡Laboratoire de Biochimie A and §Laboratoire d’Anatomie-Pathologique, Hoˆpital Bichat, Assistance Publique- detected. Hoˆpitaux de Paris, Paris, France In view of the important immunomodulatory properties of AGP, Received for publication April 22, 1997. Accepted for publication January 8, 1998. we asked whether AGP is expressed in the rat lung in the course The costs of publication of this article were defrayed in part by the payment of page of different types of inflammatory response, and if so, how its charges. This article must therefore be hereby marked advertisement in accordance expression is controlled. We used two models of inflammation: 1) with 18 U.S.C. Section 1734 solely to indicate this fact. LPS administration (either intratracheal or i.p.) to mimic infection 1 This work was supported by a grant from Zeneca Pharma, Cergy, France. and 2) s.c. turpentine injection to induce a nonseptic localized 2 Address correspondence and reprint requests to Docteur Bruno Crestani, Unite´de inflammation. In this article, we show for the first time that AGP Pneumologie, Hoˆpital Bichat, 46 rue Henri Huchard, 75018 Paris, France. E-mail address: [email protected] mRNA is expressed in the rat lung in cases of inflammation or 3 ␣ steroid administration, that alveolar type II cells (ATII cells) are Abbreviations used in this paper: AGP, 1-acid glycoprotein; AM-CM, alveolar macrophage-conditioned medium; ATII cells, type II alveolar cells; rh, recombinant the primary site of AGP expression in the rat lung in vivo, and that human; rm, recombinant murine; GuSCN, guanidine thiocyanate; DMEM, Dulbec- co’s modified Eagle’s medium; GAPDH, glyceraldehyde-phosphate dehydrogenase; rat ATII cells in vitro express the AGP mRNA and secrete immu- RPA, ribonuclease protection assay; nt, nucleotide. noreactive AGP when stimulated with the secretory products of

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 4597 alveolar macrophages and dexamethasone. Moreover, we show esterase stain (Sigma). Conditioned medium, consisting of LPS-activated that hyperplastic alveolar epithelial cells express immunoreactive rat alveolar macrophages, was recovered after a 24-h incubation period. AGP in the human lung. Stimulation of AGP gene expression and AGP secretion by rat ATII cells in vitro Materials and Methods Rat ATII cells were used 24 h after isolation. Cells were incubated with Reagents complete DMEM. Rat CM (10% v/v) or cytokines (10 ng/ml) were added Ϫ5 Recombinant murine (rm)IL-1, rmTNF-␣, and recombinant human for the indicated times in the presence or absence of dexamethasone (10 (rh)IL-6 were purchased from Immugenex (Los Angeles, CA). Escherichia M). In these experiments, we stimulated rat ATII cells with murine recom- coli (strain 026; B6)-derived LPS was obtained from Difco (Detroit, MI). binant cytokines because rat products were not available. These cytokines TEMED, ammonium persulfate, urea, dexamethasone, and turpentine were have previously been shown to be active on rat cells (16, 17). Supernatants from Sigma (La Verpillie`re, France). Transcription reagents were pur- were recovered for the determination of AGP concentration. Cell mono- ␮ ϫ chased from Promega (Madison, WI). [␣-32P]UTP (400 Ci/mmol) was layers were scrapped in 500 l of PBS and centrifuged (5 min, 12,000 from Amersham (Les Ulis, France). RNase-free DNase I, RNase A, and T1 g, 4°C), and the pellet was solubilized in 5 M GuSCN, 0.1 M EDTA, pH ␮ ϫ 6 and brewer’s yeast tRNA were supplied by Boehringer (Mannheim, Ger- 7.0 (100 l per 2 10 pneumocytes) (18). The cell lysates were stored at Ϫ many). Acrylamide/bisacrylamide, phenol, and proteinase K were from 80°C until mRNA analysis. Appligene (Illkirch, France). Guanidine thiocyanate (GuSCN) was pur- In some experiments, we evaluated AGP gene expression by SV40 T2 chased from Fluka Chemie (Buchs, Switzerland). All restriction enzymes cells, a cell line derived from fetal rat ATII cells (a generous gift of Prof. were from New England BioLabs (Beverly, MA) or from Boehringer. Pro- A. Clement, Hoˆpital Trousseau, Paris, France) (19). SV40 T2 cells were 35 35 Mix (a mixture of L-[ S]methionine and L-[ S]cysteine) was obtained cultured until conﬂuence in complete DMEM and stimulated as indicated from Amersham (Buckinghamshire, U.K.). for rat type II cells. Cells lysates (prepared as indicated for ATII cells) were

Tissue culture media, supplements, and FBS were from Life Technol- used for the mRNA analysis. Downloaded from ogies (Cergy Pontoise, France). Tissue culture plasticware was from Costar (Cambridge, MA). Detection of AGP in rat ATII cell supernatants In vitro radiolabeling of AGP was obtained by incubating ATII cells for 30 Animals min in complete medium without L-methionine, then adding 100 ␮Ci/ml of Pro-Mix (14.3 mCi/ml) for 4 h. The cell culture supernatants were recov- Male Sprague Dawley rats weighing 220 to 250 g (Charles River Breeders, ered and adjusted to correspond to the immunoprecipitation buffer (20 mM St. Aubin les Elbeuf, France) were used within 4 days of arrival. Food and

Tris/HCl, pH 8.0, 1% (by vol) Triton X-100, 5 mM EDTA, 2 mM PMSF, http://www.jimmunol.org/ water were given ad libitum. 1 mM benzamidine, and 20 mM leupeptin). Isolated rat hepatocytes were Intratracheal LPS challenge. Rats were lightly anesthetized with ether similarly treated, and their supernatants were recovered to compare the and a midline incision was made above the sternum. The trachea was apparent m.w. of AGP secreted by rat ATII cells and by rat hepatocytes exposed by blunt dissection, a 28-gauge needle was inserted into the tra- (20). Immunoprecipitation of AGP was performed as described by Pou¨s et chea above the carina, and 0.5 ml of 9% NaCl containing 0.1 mg of LPS al. (20). Immunoprecipitates were analyzed by SDS/PAGE (separating gel, was instilled. Control animals received 0.5 ml of 9% NaCl alone. 7.5%; stacking gel, 4%) and exposed on Biomax Kodak films. Systemic LPS challenge. LPS was reconstituted in PBS (5 mg/ml). LPS Rat AGP concentration was measured in the supernatant of rat ATII (2.5 mg/kg) was administered i.p. in rats. Control rats received the same cells and confluent SV40 T2 cells using a “sandwich”-type ELISA (21, 22). volume of PBS. The cells were cultured for 24 h in 10-cm cell culture plates with 5 ml Turpentine. Turpentine injection was used to create a localized wound DMEM with antibiotics without FBS (FBS was avoided to limit the risk of known to stimulate the hepatic acute phase response. Rats received a s.c. interference with bovine AGP contained in FBS). The cells were stimulated by guest on October 1, 2021 injection with 1 ml of turpentine. Controls received the same volume of with dexamethasone (10 mM), with AM-CM (10% v/v), or with both. The saline. cell monolayers were trypsinized, and the cells were spun down by cen- For all of these experimental groups, the animals were sacrificed at trifugation and sonicated in 1 ml of PBS. Total cellular protein concentra- designated time intervals. The lungs were excised, immediately frozen in Ϫ tion was measured using the Bradford protein assay (23). The results are liquid nitrogen, and stored at 80°C until RNA extraction. expressed as ng of AGP secreted per mg of protein per 24 h. Effect of dexamethasone. In some experiments, we evaluated the effect of glucocorticoids on AGP expression. At time 0, the animals were chal- Northern blot analysis and RNase protection assay (RPA) lenged 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. AGP mRNA detection in the lung was performed by Northern blot analysis at time 0 and then 6 h later. The other animals received a similar volume using a 32P-labeled 0.8-kb cDNA probe as previously described (24). Total of PBS. The animals were killed 24 h after the first injection, and the lungs cellular RNA from lung and liver was isolated using RNAPlus (Bioprobe, were recovered, immediately frozen in liquid nitrogen, and stored at Montreuil, France) according to the instructions of the manufacturer. After Ϫ80°C until RNA extraction. In some experiments (three animals in each analysis, the membranes were dehybridized and rehybridized with a 32P- group), we evaluated the effect of 0.02, 0.2, and 2 mg/kg dexamethasone labeled cDNA rat glyceraldehyde phosphate dehydrogenase (GAPDH) administered i.p. at time 0 and 6 h later. Control rats received the same probe as a loading control (25). The blots were quantified through an elec- volume of PBS. tronic autoradiography device (Instant Imager, Packard, Groningen, The Netherlands). The ratio of the AGP mRNA signal to the corresponding Isolation of rat alveolar type II cells and preparation of rat GAPDH mRNA signal was calculated for each sample. alveolar macrophage-conditioned medium (AM-CM) 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 ATII cells were isolated from adult male pathogen-free Sprague Dawley previously described with minor modifications (18, 26). A cohybridization rats by enzymatic dissociation and purified by differential adherence to with AGP and GAPDH probes was performed. A 286-nucleotide (nt) AGP plastic as previously described (16). Cells (2 ϫ 106) were plated in each riboprobe was obtained from AGP cDNA inserts subcloned into pBlue- well of a six-well cell culture plate with 2 ml of Dulbecco’s modified script II SKϩ phagemid vector (18). Riboprobe synthesis was performed in Eagle’s medium (DMEM) containing 10% FBS, 105 U/L penicillin, 100 the presence of [␣-32P]UTP (50 ␮Ci, 400 Ci/mmol) and T3 RNA poly- mg/L streptomycin, and 0.25 mg/L amphotericin B (complete DMEM). merase after linearization of the vector by EcoRI digestion. The 185-nt After a 24-h period, nonadherent cells were removed by gently washing GAPDH riboprobe was synthesized from a XbaI-ApaI rat GAPDH cDNA twice with PBS, fresh complete medium was replaced, and the cells were insert (25) subcloned into pBluescript II SKϩ. used for the experiments. The viability of adherent cells was Ͼ98% as Cellular lysates (20 ␮l) were mixed with 2 ␮l of each radiolabeled probe assessed by the trypan blue exclusion test. (105 cpm/␮l). After overnight hybridization at 37°C, the samples were AM-CM was prepared as previously described (16). Rat alveolar mac- treated with RNases A and T1, then exposed to proteinase K. After ex- rophages recovered by bronchoalveolar lavage were resuspended in RPMI traction with phenol:chloroform:isoamyl alcohol (25:24:1), the protected containing 10% heat-inactivated FBS, antibiotics, and glutamine at a den- RNA:RNA hybrids (230 nt for AGP and 164 nt for GAPDH) were pre- sity of 106 cells/ml. Cells (106) were plated in each well of a 24-well cell cipitated and loaded on a 6% acrylamide:bisacrylamide (19:1), 7.8 M urea culture plate and allowed to adhere for 2 h, then nonadherent cells were denaturing gel. After a 1- to 2-h run, the gels were dried. Quantitative removed, and fresh medium containing 10 ␮g/ml LPS was added. Adher- analysis of the radioactive protected bands was performed by direct count- ent cells were Ͼ98% alveolar macrophages as assessed by nonspecific ing of the gel (Instant Imager). AGP and GAPDH signals were corrected 4598 AGP SECRETION BY PNEUMOCYTES 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 FIGURE 1. Lung and liver AGP mRNA detection. Adult Sprague Daw- 4% paraformaldehyde for 1 h. Small fragments were cut using sterile blad- ley rats received 2.5 mg LPS/kg i.p. at time 0 (ϩ). Control animals re- der scissors. Fragments were washed overnight in fresh PBS, then embed- ceived the same volume of PBS i.p. (Ϫ). Lung and liver were recovered ded in paraffin (27). Tissue sections (2–3 ␮m thick) mounted on Superfrost 16 h after challenge, and AGP mRNA was detected by Northern blot. Plus slides (Consortium de Mate´riel pour Laboratoires, Nemours, France), Approximately 20 ␮g of total RNA was studied in each lane. This is a were deparaffinized, air dried, pretreated with 0.2 N HCl for 20 min, in- typical Northern blot representative of three different experiments. cubated in a proteinase K solution (2 ␮g/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 2ϫ SSC, 50% formamide, 1ϫ Denhardt’s solution, 10% dextran, 10 Since hepatic AGP mRNA is increased by the glucocorticoid Downloaded from mM dithiotreitol, 500 ␮g/ml yeast tRNA, 20 mM Tris-HCl, pH 8, 1 mM dexamethasone in rats (29), we asked whether dexamethasone EDTA, and the AGP RNA probe or a sense RNA probe (as a negative could modulate the expression of the AGP gene in the lung. Dexa- 35 6 control) radiolabeled with [ S]dCTP (10 cpm/slide). The riboprobes were methasone injected i.p. induced the expression of the AGP gene in synthesized as described above for the RPA. Slides were then washed sequentially with 5ϫ SSC at 50°C, 2ϫ SSC, 50% formamide at 65°C for the rat lung at levels similar to those elicited by the intratracheal 20 min and twice in NaCl, 0.5 M Tris-HCl, 10 mM-5 mM EDTA. To LPS challenge (Fig. 3). Moreover, dexamethasone potentiated the remove nonhybridized single-strand cRNA, slides were treated with RNase effect of intratracheally administered LPS, since the AGP mRNA http://www.jimmunol.org/ A (20 ␮g/ml) for 30 min at 37°C, washed in NaCl, 0.5 M Tris-HCl, 10 level was 11.5-fold that obtained with LPS alone (Fig. 3) when the ϫ mM-5 mM EDTA, then in 0.1 SSC at room temperature. Slides were animals received a combination of i.p. dexamethasone and dehydrated, air dried, and autoradiographed for 60 days at 4°C. After development and fixation, slides were counterstained with Giemsa stain. intratracheal LPS. In additional experiments, we evaluated the effect of lower Immunohistochemical analysis in the human lung dexamethasone doses. Rats (three animals in each group) received To determine whether human alveolar epithelial cells synthesized AGP in 2, 0.2, or 0.02 mg/kg dexamethasone at time 0 and at 6 h. AGP vivo, 4-␮m sections, embedded in paraffin, from normal lung biopsies (ob- mRNA expression in the lung was quantitatively analyzed at 24 h tained from two patients undergoing surgical lung resection for a localized lung tumor) and from diseased lung (obtained from two patients with id- (Fig. 4). While 0.2 mg/kg dexamethasone induced a small expres- iopathic pulmonary fibrosis, one patient with ruptured hydatid cyst, and sion of the AGP mRNA, 0.02 mg/kg dexamethasone had no effect. by guest on October 1, 2021 one patient with localized bronchiectasis) were stained for AGP with a Altogether, these results indicate that both inflammatory pro- monoclonal mouse anti-human AGP IgG1 (catalogue no. A5566, Sigma) cesses or corticosteroids administration induce the expression of (working dilution, 1/200). Paraffin-embedded liver biopsies were used as the AGP gene in the rat lung. 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 Ϯ SD. Statistical significance was assessed using the Kruskal-Wallis nonparametric test followed by the Mann-Whitney U test. Results AGP mRNA is inducible in the lung 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 FIGURE 2. Effect of intraperitoneal and intratracheal LPS on rat lung and was further increased after a systemic LPS challenge (Fig. 1). AGP mRNA. A, Intraperitoneal LPS challenge: adult Sprague Dawley rats The AGP mRNA was still detected in the lung 72 h after the received 2.5 mg LPS/kg i.p. at time 0. Control animals received the same challenge, but at very low levels (7% of the expression measured volume of PBS i.p. and AGP mRNA was detected 16, 24, and 36 h after 16 h after stimulation; Fig. 2). Similarly, an intratracheal LPS chal- i.p. LPS challenge. Minimal RNA expression was detected 72 h after challenge induced the expression of the AGP gene in the lung 24 and lenge. AGP mRNA was not detected in control rats. B, Intratracheal LPS 36 h after stimulation, while AGP mRNA was not detectable 72 h challenge: the animals received 0.1 mg LPS intratracheally at time 0. Con- trol animals received the same volume of saline i.p., and AGP mRNA was after the challenge. Control saline challenge, either intratracheal or detected 24 and 36 h after intratracheal LPS challenge but was not detect- i.p., did not induce any expression of the AGP gene. able 72 h after challenge. AGP mRNA was not detected in control rats. The Turpentine-induced acute phase inflammation also induced lungs were recovered at specified time points, total RNA was extracted, AGP gene expression in the lung as evidenced by Northern blot and Northern blot analysis was performed. GAPDH mRNA serves as an analysis (Fig. 3). Expression was lower than that induced by the internal standard. These are typical Northern blots representative of three intratracheal LPS challenge (8.5% of the intratracheal challenge). different experiments. The Journal of Immunology 4599

Evidence of AGP gene expression by SV40 T2 cells 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 (10Ϫ5 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 FIGURE 3. Effect of turpentine, intratracheal LPS, and dexamethasone on rat AGP mRNA in the lung. Rats received either a s.c. injection with 1 was increased 70-fold). ml turpentine (Tur) at time 0; 0.1 mg LPS intratracheally at time 0; 2 mg/kg dexamethasone (Dex, 4 mg/ml) i.p. at time 0 and 6 h later; or both intratracheal LPS and dexamethasone. The lungs were recovered 24 h after Evidence of AGP gene expression and AGP secretion by ATII stimulation, total RNA was extracted, and Northern blot analysis was per- cells in vitro formed. GAPDH mRNA serves as an internal standard. A, Northern blot We performed similar experiments with rat ATII cells in primary analysis. B, AGP/GAPDH mRNA ratio for three rats in each group (mean Ϯ SD). *, p Ͻ 0.05 compared with LPS and with Dex (Mann- culture (Fig. 7). A very low level of AGP mRNA signal (not de- Downloaded from Whitney U test). tected 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 stimu- Identification of the AGP expressing cell in the lung lation was obtained with the synergistic combination of dexameth-

asone and CM (the AGP/GAPDH ratio was increased 9.3-fold). http://www.jimmunol.org/ To identify the cell(s) that expressed AGP mRNA in the lung in LPS (1 ␮g/ml) did not increase the AGP/GAPDH mRNA ratio in vivo, we performed an in situ hybridization analysis in normal rat the presence or absence of dexamethasone (data not shown). lung and in the lung of LPS-challenged rats (intratracheal instil- We evaluated the time course of AGP gene expression by ATII lation 24 h before lung sampling). cells stimulated with the combination of dexamethasone and CM AGP mRNA was never detected in the normal lung, whereas it in vitro. The increase of the AGP/GAPDH mRNA ratio began 4 h was detected in the lung of LPS-challenged animals. The speciﬁc after stimulation and continued linearlly over a 72-h period (R2 ϭ signal was exclusively observed in the alveolar region of the lung 0.92, p ϭ 0.0001) (Fig. 8). and was absent in the bronchial epithelium and in the vessels. Immunoprecipitation of metabolically radiolabeled AGP from

Positively labeled cells were probably ATII epithelial cells be- by guest on October 1, 2021 rat SV40 T2 cell supernatants (Fig. 9) allowed us to detect one cause of their characteristic location in the corner of the alveolus (30) (Fig. 5). 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. We measured immunoreactive AGP in the supernatant of rat ATII cells in primary culture and SV40 T2 cells (Table I). 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 ϳ100-fold higher. CM alone induced AGP secretion, but the maximal effect was obtained with the combination of CM and dexamethasone.

Cytokine regulation of the AGP mRNA level by rat ATII cells in vitro Among the mediators secreted by alveolar macrophages, the cy- FIGURE 4. Dose-dependent effect of dexamethasone. Rats received 2, tokines IL-1, TNF-␣, and IL-6 play a key role in regulating the 0.2, or 0.02 mg/kg dexamethasone (4 mg/ml) i.p. at time 0 and 6 h later, or expression of the acute phase proteins in the liver (1). We evalu- they received the same volume of PBS (controls). The lungs were recov- ated the effect of rmIL-1, rmTNF-␣, and rhIL-6 (10 ng/ml each) on ered at 24 h, total RNA was extracted, and Northern blot analysis was performed. GAPDH mRNA serves as an internal standard. A, typical AGP mRNA in SV40 T2 cells (Fig. 6). Without dexamethasone, Northern blot. B, AGP/GAPDH mRNA ratio for three rats in each group all cytokines induced a small increase in the AGP/GAPDH mRNA (mean Ϯ SD). *, p Ͻ 0.05 when compared with controls (Mann-Whitney ratio, although at a very low level compared with the effect of U test). dexamethasone. The combination of IL-1 and dexamethasone was 4600 AGP SECRETION BY PNEUMOCYTES Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. In situ localization of AGP mRNA in the lung of control rats (A and B) or intratracheally LPS-challenged rats (C, D, E, and F). Sections A to D were hybridized to 35S-labeled antisense riboprobes, and sections E and F to 35S-labeled sense riboprobes. No specific signal was detected in control lungs (A and B). A specific hybridization signal was observed only in the alveolar areas of the lung and was detected neither on endothelial cells nor on bronchial epithelial cells. Positive cells were located in the corner of the alveolus (D). The sense sections (E and F) show no significant hybridization. (original magnification: A, C, and E, ϫ250; B, D, and F, ϫ400). highly synergistic and induced the expression of the AGP gene at Similar experiments were performed with rat ATII cells (Fig. 7). levels similar those obtained with the combination of CM and In the absence of dexamethasone, stimulation of rat ATII cells with dexamethasone (the AGP/GAPDH ratio was increased 63-fold rmIL-1, rmTNF-␣, or rhIL-6 (10 ng/ml each) did not increase the over the control value). The combination of TNF and dexameth- AGP/GAPDH mRNA ratio (data not shown). In the presence of asone was essentially additive and increased the AGP/GAPDH dexamethasone, TNF and IL-6 had no effect, whereas IL-1 in- mRNA ratio 20-fold. IL-6 did not increase dexamethasone-increased by twofold the AGP/GAPDH mRNA ratio over the dexa- duced stimulation. methasone value. The Journal of Immunology 4601

FIGURE 7. Regulation of AGP gene expression by rat ATII cells in primary culture. Rat ATII cells were stimulated 24 h after isolation with AM-CM (CM, 10% v/v), dexamethasone (Dex, 10Ϫ5 M), or both (CM ϩ Dex). Recombinant murine cytokines (IL-1, TNF-␣, and IL-6, 10 ng/ml each) were tested in the presence of dexamethasone. ATII cells cultured without stimulant were used as controls (C). After a 24-h incubation period, the cells were scrapped, spun down, and solubilized in 5 M GuSCN as indicated in Materials and Methods. AGP and GAPDH mRNA were quantified using a RNase protection assay on cell lysates. For each exper- Downloaded from imental condition, the AGP/GAPDH mRNA ratio was calculated. Relative changes were expressed vs controls. A, one RNase protection assay blot from three different experiments. B, AGP/GAPDH mRNA ratio (mean Ϯ SD, n ϭ 3 experiments). *, p ϭ 0.04 vs dexamethasone. http://www.jimmunol.org/ and SV40 T2 cell line. Among the cytokines that we tested, IL-1 was the most consistent inducer of AGP gene expression and syn- FIGURE 6. Regulation of AGP gene expression by SV40 T2 cells. Con- ergized with dexamethasone. fluent SV40 T2 cells were stimulated with AM-CM (CM, 10% v/v), re- Our data provide the first evidence of inducible AGP expression combinant murine cytokines (IL-1, TNF-␣, and IL-6, 10 ng/ml each) in the in the lung. Although circulating AGP is essentially synthesized in presence or absence of dexamethasone (Dex, 10Ϫ5 M). Cells cultured with- the liver, extrahepatic expression of acute phase proteins is in- out stimulant were used as controls (C). After a 24-h incubation period, the creasingly recognized (9, 10, 31). Local extrahepatic production of cells were scrapped, spun down, and solubilized in 5 M GuSCN as indi- AGP has been detected in the myocardium (14), kidney (9), breast cated in Materials and Methods. AGP and GAPDH mRNA were quantified (32), prostate (15), and uterus (13). However, the regulation of by guest on October 1, 2021 using a RNase protection assay on cell lysates. For each experimental extrahepatic AGP expression has never been explored in detail. condition, the AGP/GAPDH mRNA ratio was calculated. Relative changes The regulation of the rat AGP gene expression by hepatocytes were expressed vs controls. A, one RNase protection assay blot from four has been extensively studied in vivo and in vitro. Hepatic AGP different experiments. B, AGP/GAPDH mRNA ratio (mean Ϯ SD, n ϭ 4 experiments). *, p Ͻ 0.05 when compared with dexamethasone. 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 Immunohistochemical detection of AGP in the human lung (Fig. gene expression by rat hepatocytes and various rat hepatoma cell 10) lines is increased by IL-1 and TNF-␣ (26, 34), IL-6 (and related cytokines) (26, 35, 36), glucocorticoids (26, 29), and some exog- We never detected immunoreactive AGP in normal human lung. enous molecules such as phenobarbital (24). A positive interaction By contrast, a strong positive specific signal was detected in areas between glucocorticoids and cytokines has been consistently found of hyperplastic alveolar epithelium in all diseased lung samples (37). In vivo experiments with adrenalectomized rats indicate that that we studied. Immunoreactivity was never detected in bronchial there are independent regulatory processes for induction by glu- epithelium or in endothelial cells. Some positive signal was ob- cocorticoids and by mediators of the acute phase response in the served in cells located in the alveolar lumen, consistent with des- liver (29). Characterization of the rat AGP gene has revealed both quamated alveolar epithelial cells or alveolar macrophages. separate and overlapping cis-acting elements for these factors, each element being composed of several interacting regulatory Discussion sequences (1). AGP is a typical acute phase protein in human and rodents. Our Our results indicate that regulation of the expression of the AGP results demonstrate for the first time the inducible expression of gene in the lung has some similarities with its regulation in the the AGP gene in the rat lung in vivo during localized or systemic liver. Indeed, the experimental conditions that induced the expres- inflammation as well as after dexamethasone administration and sion of the AGP mRNA in the lung in vivo (systemic LPS chal- the inducible production of immunoreactive AGP in the human lenge, turpentine challenge, dexamethasone administration) are lung in the course of local inflammatory reaction. Immunohisto- also conditions that are known to increase the expression of the chemistry and in situ hybridization results suggest that ATII cells AGP gene in the liver (1). Moreover, as previously shown for the are the primary source of AGP production in the human and rat liver, glucocorticoids play a key role in the expression of the AGP lung. In vitro, rat ATII cells consistently express the AGP gene and gene in the lung. In rat hepatocytes and hepatoma cell lines, the secrete immunoreactive AGP, particularly when stimulated with expression of the AGP gene has been shown to be highly depen- dexamethasone. Dexamethasone and the AM-CM act synergisti- dent on the presence of dexamethasone (26, 38, 39). We observed cally to increase the level of AGP mRNA on both rat the ATII cell that dexamethasone greatly increased the level of AGP mRNA in 4602 AGP SECRETION BY PNEUMOCYTES

Table I. Comparison of AGP protein expression by rat ATII cells and SV-40 T2 cells a

Conditions AGP (ng/mg Protein)

Control Dex CM CM ϩ Dex

Rat ATII cells 0 2.8 Ϯ 0.4 0 42.8 Ϯ 32.1 SV40-T2 cells 0 643.8 Ϯ 156.4 74.7 Ϯ 24.9 2666.1 Ϯ 205.7

a The results are presented as mean Ϯ SD for 3 experiments performed in dupli- cate. Dex, dexamethasone.

mals. 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 Downloaded from 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 http://www.jimmunol.org/ acted synergistically with dexamethasone to stimulate the expression of AGP by SV40 T2 cells, to reach a level similar to that FIGURE 8. Time-dependent AGP gene expression by rat ATII cells. obtained with the combination of AM-CM and dexamethasone. Monolayers of rat ATII cells were stimulated at time 0 with the combina- Moreover, rmIL-1 potentiated the effect of dexamethasone on rat Ϫ5 tion of dexamethasone (10 M) and AM-CM (10%, v/v). At different time ATII cells. It is interesting to note that the combination of IL-1 and points after stimulation, the cells were scrapped, spun down, and solubi- dexamethasone induced a 60-fold increase in the AGP mRNA lized in 5 M GuSCN as indicated in Materials and Methods. AGP and GAPDH mRNA were quantified using a RNase protection assay on cell level in SV40 T2 cells, a level of induction similar to that mea- lysates. For each experimental condition, the AGP/GAPDH mRNA ratio sured in rat hepatoma cells (38). Thus, IL-1 has a prominent effect was calculated. Relative changes were expressed vs controls (time 0). A, on the expression of the AGP gene by rat ATII cells, whereas IL-6 by guest on October 1, 2021 RNase protection assay blot from two different experiments. B, Linear seems to have a very limited action. This is in contrast with normal regression analysis of the AGP/GAPDH mRNA ratio. 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). the lung in vivo and in ATII cells in vitro, as well as acting syn- A most important finding of this study is that ATII cells not only ergistically with inflammatory mediators (rmIL-1 and AM-CM) to express the AGP gene in vitro, but they also secrete immunoreac- induce the expression of the AGP gene by ATII cells in vitro, tive AGP in their supernatant when stimulated with whereas the cytokines had either a small effect (SV40 T2) or no dexamethasone or the combination of dexamethasone and AM- effect at all (ATII cells) in the absence of dexamethasone. CM. However, the AGP levels measured in dexamethasone plus The main difference between lung and liver expression of the CM-stimulated ATII cell supernatants are about 0.5% of those AGP gene is that the AGP mRNA is not detected in the lung in previously measured in the cell culture supernatants of unstimu- healthy animals, whereas it is detected in the liver, although we lated rat hepatocytes by one of us (40). Furthermore, it is worth cannot totally exclude the possibility that a very low level of ex- noting that the apparent m.w. of secreted AGP was slightly higher pression of the AGP gene, not detectable by Northern blot or in in SV40 T2 supernatants than in rat hepatocyte supernatants. The situ hybridization analysis, is present in the lung of healthy ani- 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 FIGURE 9. Immunoprecipitation of radiolabeled AGP. Rat hepatocytes uterine AGP synthesis declines to near zero (13). Immunoreactive (cultured in control conditions (C) or stimulated with dexamethasone (Dex)) and SV40 T2 cells (cultured in control conditions (C) or stimulated AGP in the human prostate and in vitro synthesis of AGP by hu- with dexamethasone plus conditioned medium (MCϩDex)) were cultured man breast epithelial cells have been demonstrated (15, 32). with radiolabeled amino acids, and secreted AGP was immunoprecipitated The physiologic role of local AGP production by alveolar epi- from supernatants. Immunoprecipitates were analyzed by SDS/PAGE and thelial cells is potentially important. In vitro data suggest that AGP fluorography. could modulate the degree of activation of inflammatory cells in The Journal of Immunology 4603 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 10. AGP immunoreactivity in human lung tissue. Immunoreactivity to AGP is shown in the normal lung (A), the acutely inflamed lung (C), and the fibrotic lung (E). Immunoreactivity to the isotype-matched control Ab is shown in the normal lung (B), the acutely inflamed lung (D), and the fibrotic lung (F). Original magnification: ϫ250. No specific staining was detected in the normal human lung. Hyperplastic alveolar epithelial cells (arrows) are strongly immunostained with the anti-AGP Ab in the fibrotic lung and in the acutely inflamed lung. Some desquamated alveolar epithelial cells or alveolar macrophages (arrowheads) are also stained in the air spaces. the alveolus, such as neutrophils, lymphocytes, or alveolar mac- peritoneal macrophages and human blood monocytes (5). Tilg and rophages. Indeed, AGP is a potent inhibitor of neutrophil chemo- coworkers extended these results, showing that blood mononuclear taxis and oxidative metabolism in vitro (4) and inhibits in vitro cells incubated with AGP synthesized large quantities of IL-1Ra, mitogen-induced lymphoproliferation (42). Boutten and col- 5- to 10-fold more than the amount of IL-1␤ produced by these leagues showed that AGP increased the in vitro secretion of IL-1␤, cells, and that AGP was synergistic with low concentrations of TNF-␣, and IL-6 by human alveolar macrophages stimulated with endotoxin in the induction of IL-1Ra and IL-1␤ synthesis (7). The E. coli-derived LPS. Similar results were obtained with human preferential induction of IL-1Ra by AGP may contribute to its 4604 AGP SECRETION BY PNEUMOCYTES anti-inflammatory effect (7). Recently, AGP has been shown to 9. Kalmovarin, N., W. Friedrichs, H. O’Brien, L. Linehan, B. Bowman, and F. Yang. 1991. Extrahepatic expression of plasma protein genes during inflam- inhibit the apoptosis of hepatocytes by TNF/galactosamine, while mation. Inflammation 15:369. similar apoptosis of hepatocytes induced by anti-Fas remained un- 10. Molmenti, E., T. Ziambaras, and D. Perlmutter. 1993. Evidence for an acute affected (43). Whether these properties are relevant in vivo in the phase response in human intestinal epithelial cells. J. Biol. Chem. 268:14116. lung is currently unknown; however, the protective effect of AGP 11. Molmenti, E. P., D. H. Perlmutter, and D. C. Rubin. 1993. Cell-specific expression of alpha-1-antitrypsin in human intestinal epithelium. J. Clin. Invest. 92: has been demonstrated in vivo in a model of sepsis induced by 2022. TNF or endotoxin in mice (8). 12. Cichy, J., J. Potempa, R. Chawla, and J. Travis. 1995. Stimulatory effect of Given these properties, the expression of AGP in the alveolar inflammatory cytokines on alpha1-antichymotrypsin expression in human lung- derived epithelial cells. J. Clin. Invest. 95:2729. space in cases of acute inflammation may exert a local protective 13. Thomas, T., S. Fletcher, G. Yeoh, and G. Schreiber. 1989. The expression of effect by limiting the inflammatory reaction and its potentially del- alpha-1-acid glycoprotein mRNA during rat development. J. Biol. Chem. 264: eterious effect on alveolar structures. This hypothesis is further 5784. 14. Siegel, R., M. Fishbein, J. Said, Z. Tokes, and W. Shell. 1985. Localization of supported by the fact that ATII cells appear to be the main source alpha-1-acid glycoproteins in human myocardium. Lab. Invest. 52:107. of AGP expression in the lung, both in rat and humans, pointing 15. Dube, J., G. Paradis, B. Tetu, and R. Tremblay. 1989. Synthesis of alpha-1-acid out once again the fundamental role of ATII cells in the control of glycoprotein by the human prostate. Prostate 15:251. the integrity of the alveolar space. ATII cells synthesize and se- 16. Crestani, B., P. Cornillet, M. Dehoux, C. Rolland, M. Guenounou, and M. Aubier. 1994. Alveolar type II epithelial cells produce interleukin-6 in vitro crete surfactant, control the volume and composition of the epi- and in vivo. J. Clin. Invest. 94:731. thelial lining fluid, and proliferate and differentiate into type I al- 17. Chin, Y., J. Cai, and K. Johnson. 1990. Lymphocyte adhesion to cultured Peyer’s veolar epithelial cells after injury to maintain the integrity of the patch high endothelial venule cells is mediated by organ-specific homing recep-

tors and can be regulated by cytokines. J. Immunol. 145:3669. Downloaded from alveolar wall (44). Moreover, we and others have shown that ATII 18. Kaabache, T., B. Barraud, G. Feldmann, D. Bernuau, and B. Lardeux. 1995. cells may have an immunoregulatory role in the lung through the Direct solution hybridization of guanidine thiocyanate-solubilized cells for quan- secretion of mediators such as cytokines (16, 45), prostaglandins titation of mRNAs in hepatocytes. Anal. Biochem. 232:225. 19. Clement, A., J. Campisi, S. Farmer, and J. Brody. 1990. Constitutive expression (46), or nitric oxide (47). We have also previously shown that ATII of growth-related mRNAs in proliferating and non-proliferating lung epithelial ␣ cells in vitro secreted 1-antitrypsin, thereby contributing to the cells in primary culture: evidence for growth-dependent translational control. intraalveolar antiproteases shield (41). Proc. Natl. Acad. Sci. USA 87:318.

20. Pou¨s, C., A. Drechou, J.-D. Rouzeau, J. Guibourdenche, A. El Moujahed, and http://www.jimmunol.org/ In summary, the present study demonstrates the inducible ex- G. Durand. 1992. Differential rates of glycoprotein secretion by isolated rat hepa- pression of AGP, a typical acute phase protein with potent immu- tocytes studied in terms of concanavalin A binding. Eur. J. Biochem. 203:277. nomodulatory properties, by alveolar epithelial cells in vitro and in 21. Biou, D., M. Daveau, O. Rigal, M. Hiron, D. Porquet, and J. Lebreton. 1989. An enzyme-linked immunoassay for the measurement of rat alpha-1-acid glycopro- vivo and illustrates the potential for effective communication be- tein synthesized by cultured hepatocytes. J. Immunol. Methods 125:1. tween macrophages and epithelial cells in the alveolar space. 22. Drechou, A., N. Perez-Gonzalez, D. Biou, J. Rouzeau, J. Feger, and G. Durand. 1989. One-step purification of rat plasma alpha-1-acid glycoprotein by antibody affinity chromatography: application to normal and inflammed rat sera. J. Chro- matogr. 489:273. Acknowledgments 23. Bradford, M. 1976. A rapid and sensitive method for the quantitation of micro-

gram quantities of protein utilizing the principle of protein-dye binding. Anal. by guest on October 1, 2021 We thank Paul Soler Institut National de la Santeét de la Recherche Me´di- Biochem. 72:248. cale ((INSERM) U82, Paris, France) for his helpful advice concerning in 24. Fournier, T., N. Mejdoubi, C. Lapoumeroulie, J. Hamelin, J. Elion, G. Durand, situ hybridization and immunohistochemistry, Dr. Brigitte Escoubet (IN- and D. Porquet. 1994. Transcriptional regulation of rat alpha-1-acid glycoprotein gene by phenobarbital. J. Biol. Chem. 269:27175. SERM U428, Paris, France) for her generous gift of the GAPDH probe, 25. Fort, P., L. Marty, M. Piechaczyk, S. El Sabrouty, C. Dani, P. Jeanteur, and and Mrs. Monique Dehoux and Nathalie Seta (Laboratoire de Biochimie A, J. Blanchard. 1985. Various rat adult tissues express only one major mRNA Hoˆpital Bichat) for continuous support to this work. 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