Glucocorticoid Receptor mANA Expression in Pulmonary Alveolar Macrophages in Sarcoidosis* Olof Andersson, M.D.;t:j: Mikael Bronnegilrd, M.D ., Ph .D .;t Tomas Sonnenfeld, .\f.D ., Ph .D .;§ Birgitta Schmekel, M .D. , Ph .D .;II ]olum Lund, Ph .D .;t Erik Ripe, M.D . , Ph .D. ;:j: and jan-Ake Gustafsson, M.D., Ph.D.t
The presence of the glucocorticoid receptor was demonstrated by immunocytochemistry in pulmonary alveolar macrophages obtained by bronchoalveolar lavage. AJso, GR mRNA content was determined by solution hybridization in PAM from 12 healthy volunteers and in 6 patients with sarcoidosis. No significant differences with regard to GR mRNA expression was detected between the two groups examined. For comparison, lung tissue from three patients undergoing thoracic surgery was examined and found to contain GR mRNA levels in the same range. As an indication of GR function, we also determined the mRNA levels of a glucocorticoid-regulated gene, metallothionein IIA, during basal conditions and after in vitro incubation of PAM with dexamethasone. Neither the control sample nor the dexa-
methasone-stimulated MTII mRNA values in PAMs differed significantly between the two groups. Solution hybridization is a rapid, sensitive and convenient assay which enables accurate and specific quantitation of GR mRNA in PAM. The GR mRNA content and basal as well as dexamethasone-induced MID mRNA levels in PAM from patients with sarcoidosis is not significantly different from (Chest 1991; 99:1336-41) those in healthy volunteers.
have been widely used in the management of a variety of inflammatory and immunologic lung diseases. 1 It is, however, well known that considerable variations exist with regard to therapeutic response to treatment with glucocorticoids. Thus, in patients with idiopathic pulmonary fibrosis, there is a limited number of patients responding to therapy with glucocorticoids, 2 whereas the efficacy of glucocorticoids generally is high in sarcoidosis.-1 The molecular basis for interindividual differences, or differences between groups of patients, in glucocorticoid sensitivity is not known. Theoretically such differences could involve any step in the proposed mechanism of action of these hormones. Of fundamental importance at a subcellular level is the concept of a cytoplasmic receptor protein which, after interaction with its ligand, translocates to the nucleus. In vivo, the hormone-receptor complex thereafter interacts with specific DNA sequences, termed glucocorticoid responsive elements (GREs, 4 ) and elicits transcriptional activation or inhibition of target gene expression by a
largely unknown mechanism. s.s Obviously, another possible explanation for the lack of efficacy of the glucocorticoids in, for example, idiopathic pulmonary fibrosis, might be that these agents do not suppress the inflammatory processes that modulate the fibrosis. In view of this, the relation between the GR content in various immunocompetent and inflammatory cells in the lung and the in vivo effects of glucocorticoids on pulmonary lesions in patients with interstitial lung diseases has been the focus of much attention. The macrophage is a likely target cell for the effects of glucocorticoids due to its capacity to produce arachidonic acid metabolites7 of importance to evoke and sustain inflammation. These cells also participate in immune reactionsH known to be modified by glucocorticoid treatment. These properties of the alveolar macrophage, as well as the fact that the cell is easily harvested from the lung by means of bronchoalveolar lavage, explain the interest in this particular cell. Indeed, glucocorticoid receptors have been detected in lung tissues 9 · 10 or alveolar macrophages from animals. 11 •12 However, previous studies with ligand-based measurements of GR content in human bronchoalveolar lavage cells 13 or enriched pulmonary alveolar macrophages 14 have been somewhat contradictory. The high degree of nonspecific binding of tritiated ligand (synthetic glucocorticoid) to BAL cell cytosol makes precise quantitation of specific bindinr difficult and may account for some of the conflicting data.
Glucocorticoi~s
*From the tDepartment of Medical Nutrition, :j:Department of Lung Medicine and §De partment of Surgery, Karolinska Institute, Iluddinge University Hospital , Huddinge, Sweden, and IIDepartment of Clinical Explorative Research, Draco AB , Lund, Sweden. This study was supported hy grants 13X-2819, 19K-087 14 from the Swedish Medical Research Council , the Swedish Heart Lung Foundation, the Swedish Cancer Societv, and Draco AB. Manuscript received June 12; revision a~cepted November 6. RqHinl ll'lf!lests: Dr. AncU'!"s.wm, DqJClrtment of Medical Nutrition. H!lddingt• Unir,ersity Hospital. S-J.Il 86 Huddinge , Sweden
1336
GR=glucocorticoid receptor; PAM=pulmonary alveolar macrophages; MTII = metallothionein IIA; TNA =total nucleic acid; PAP= peroxidase/antiperoxidase; NRS =normal rabbit serum
Pulmonary Alveolar Macrophages in Sarcoidosis (Andersson et al)
The purpose of this investigation was primarily to unequivocally demonstrate the presence of the GR in human PAM, and secondly, to develop a suitable method for quantitative analysis of GR content in PAM. We, therefore, combined the use of a monoclonal antibody against GR, designated mab 7, 15 in immunocytochemical studies and a solution hybridization assay for measurements of GR mRNA levels in PAM . The metallothionein IIA gene is known to be ubiquitously expressed and regulated by glucocorticoids and heavy metals at the transcriptional level. 16 To obtain further information on the function of GR in PAM, studies of MTII mRNA levels during basal conditions and after in vitro incubations with dexamethasone or a heavy metal were included. MATERIALS AND METHODS
Study Population Bronchoalveolar lavage was performed in I2 healthy volunteers (eight smokers and four nonsmokers; age 3I ±8.8 years, range 2I to 47 years) and in six patients with biopsy proven sarcoidosis (all nonsmokers, four in stage II and two in stage I by traditional roentgenographic criteria; age 4I ± 8 .9 years, range 32 to 57 years). None of the patients had any evidence of mycobacterial, fungal, or parasitic infection or a history of exposure to organic or inorganic material known to cause granulomatous lung disease. For comparison, specimens of histologically-normal lung tissue were recovered from three patients undergoing thoracic surgery for lung cancer.
BAL Bronchoalveolar lavage was performed as described earlier." Aliquots (5 ml) of BAL Ruid were removed, stained with May Criinewald-Ciemsa, and subjected to differential cell counting after cytocentrifugation. Examination of these cytospin preparations showed that approximately 90 percent of the recovered cells were alveolar macrophages among the healthy volunteers, whereas the sarcoidosis patients had higher levels oflymphocytes (5, 22, 3I, 46, I8, and 55 percent, respectively) in the BAL-Ruid as expected.
Cell Cultures The remaining lavage Ruid was separated from the cells by centrifugation, and the obtained BAL cells were allowed to adhere to plastic bottles containing serum-free medium supplemented with benzyl-penicillin (400 IU/ml), streptomycin (0.2 mwml), and Lglutamine (2 mM). The cells were cultured at 37"C in an atmosphere of 5 percent CO, in air. After 6 hours, nonadherent cells (mainly lymphocytes) were washed away. The remaining cells (PAMs) were refed with medium only (control) or medium containing dexamethasone (IO -' M) or CdSO, (Io -• M) for I2 hours. Prior to preparation of total nucleic acid, the cells were washed twice with PBS , and scraped into lxSET (I percent SDS, IO mM EDTA, and 20 mM Tris-HCI pH 7.5).
Preparation of TNA and RNA ForTNA preparation, the cells were scraped into lxSET, homogenized, and digested with 10 mwml proteinase K for 45 min at 45°C. The cells were then phenol-chloroform extracted, reextracted with chloroform and ethanol precipitated . The dried TNA was dissolved in 0.2xSET as described.•• The concentrations of the TNA samples were measured spectrophotometrically at 260 and 280 nm . The RNA from lung tissue specimens was prepared by the guanidinium thiocyanate method combined with C,Cl, centrifugation.••
Plasmids and Solution Hybridization Analysis For construction of the human CR probe, two mplimentary oligonucleotides corresponding to nucleotides 159-209 in the human CR eDNA'" were synthesized, annealed , and ligated into the Pst V Hind Ill sites in pCEM TMl. For construction of the human MTII probe, two complimentary oligonucleotides corresponding to nucleotides 406-456 in the human MTII gene" were synthesized and cloned into Pst 1/Hind Ill sites in pCEM TM1 as described above . The sequences of these inserts in pC EM TM I were confirmed by DNA sequencing using the dideoxy chain-termination method."' Northern blot analysis showed that the MTII probe hybridized to an RNA species of0.7-Q.8 kb and the CR probe to RNA species of 7.0 and 6.I kb.23 These results indicate that the probes used hybridized to RNA species similar to those reported for MTII and CR. 3 ' · " The plasmids designated phCR and phMTil were then used fi>r in vitro synthesis of eRN A radiolabelled with ·" S-UTP using SP6 RNA polymerase. The opposite mRNA strand was synthesized using T7 RNA polymerase . For solution hybridization," TNA samples were hybridized to 20,000 cpm of"'S-cRNA probe in 40 JLI of0.5 M NaCI, 20 mM Tris-HCI pH 7.5, 5 mM EDTA, 0.1 percent SDS, I mM dithiothreitol, and 25 percent formamide at 68°C for I2 to I6 h. The samples were then treated with RNase for 45 min by adding I ml of a solution containing 40 JLg RNase A and 2 JLg RNase Tl (Boehringer-Mannheim, CFR) and 100 JLg herring sperm DNA. Radioactivity protected against RNase digestion was precipitated by the addition of IOO JLI6M trichloroacetic acid and collected on a filter (Whatman CF/C). The hybridization signal of a sample was compared to a standard curve constructed from incubations with known amounts of the synthetic oligonucleotide mRNA . A standard curve (3-300 xlO - " mol mRNA/incubation) was included in each assay, and each TNA sample was analyzed in duplicate or triplicate. For each specific probe, the corresponding amount of sample mRNA was calculated from the linear part of the respective standard curve. Means and SD were calculated for the various experimental groups. Means were mmpared hy the Mann-Whitney U test, when appropriate . Differences between groups were considered significant for p values of 0.05 or less.
Immunocytochemistry Cytospin preparations were kept at -7ifC. The cells (immunocytochemistry experiments have only been carried out with cells obtained from healthy volunteers) were then fixed with 2 perr 15 min with 3 percent (v/v) normal rabbit serum, and the cells were then incubated with mab 7 at an optimal dilution of I:250 in PBS/I percent (v/v) NRS. The primary antibody was followed by an incubation with the bridge antibody, rabbit anti-mouse I.e (DAKOPATTS, Stockholm, Sweden) diluted I :SO in PBS/I percent (v/ v) NRS. After washing with PBS, the cells were incubated with a monoclonal mouse PAP complex diluted I:IOO in PBS/I per test the functional significance of the measured GR mRNA, we next wanted to measure a putatively receptor-mediated glucocorticoid response. For this purpose, the cells were put in primary culture overnight and incubated with medium (control) or medium containing dexamethasone (I0 - 7 M). No signs of toxicity were observed . Since the MTII gene is inducible both by glucocorticoids and heavy metals, the in vitro incubation of PAM with CdS0 4 provided a positive control. Unstimulated PAMs recovered from healthy volunteers and patients contained approximately equal concentrations ofMTII mRNA (Table 2), and treatment of the cells with dexamethasone caused an -1.9-fold 1338
Table 1-Erpression ofGR mRNA CelVfissue
GR mRNA (amoVJJ.g TNA)
Sarcoidosis- PAM* (n = 6) Healthy volunteers- PAM* (n = 121)
41±6 39±11
*PAMs obtained hv BAL were cultured in serum-free medium. The levels of GR mRNA in the TNA samples prepared from PAMs were determined hv solution hvhridization as described in Materials and Methods..Results oht~ned from healthy volunteers and patients "~th sarcoidosis (number of individuals indicated in brackets) presented as mean± SD. Each sample analyzed in duplicate or triplicate.
induction of MTII mRNA levels in both groups examined (Table 2). In PAMs recovered from healthy volunteers, CdS04 increased MTII mRNA levels fourfold, and this was in the same range as for PAMs from sarcoidosis. Thus, the extent of MTII expression and its induction by glucocorticoids and CdS04 was similar in the two study populations.
Immunocytochemistry The immunocytochemical experiments were included to obtain qualitative data on the cellular localization of the GR. The monoclonal antibody, designated mab 7, has been shown earlier to be monospecific for the GR and the staining technique including the permeabilization procedures was adopted from earlier experiments carried out in our laboratory. 15 ·2:; As can be seen from the experiments using cytospin preparations of BAL cells (done only in cytospin preparations from four healthy volunteers), there was an intense staining of PAM, predominantly in the cytoplasmic region (Fig IA). Occasionally, nuclear staining was observed in PAM, albeit at a lower intensity. Similar incubations, but where mab 7 Table 2-Erpression ofMTII mRNA in PAM from Healthy Volunteers and Patients with Sarcoidosis Cell Sarcoidosis-PAM* (n=6) Control DEX CdSO, Healthy volunteers-PAM* (n=5) Control DEX CdSO,
MTII mRNA (amoVJJ.g TNA)
Fold Induction
266± 127 495±305 1246±967
1.9,2.3,2.6,2.1,1.0,0.9 4.9,3.8,3.6,3.0,2.2,8.5
310±50 600± 120 1280± 150
1.4,2.6,1.8,2. 1, 1.9 3.8,4.4,4.9,4.0,3.7
*PAMs were treated with medium only (control) or medium containing 10 -' M dexamethasone or 10 -• M CdSO, as described in Materials and Methods. Levels of MTII mRNA in the TNA samples prepared from PAMs were determined by solution hybridization. Results obtained from healthy volunteers and patients with saR'lidosis (number of individuals indicated in brackets) presented as mean± SD. Each sample analyzed in duplicate or triplicate. Pulmonary Alveolar Macrophages in Sarcoidosis (Andersson at a/)
FIGURE 1 A and B. GR staining in PAM. Cytospin preparation of BAL-cells were fixed with 2 percent (v/ v} buffered formaldehyde . The GR was then stained with mab 7 and the PAP technique as described. Note the intense staining of PAM predominantly in the cytoplasmic region. No GR staining was obtained if mab 7 was substituted with ascites from a mouse injected with the myeloma cell line SP 210 {2} {original magnification x400).
had been replaced with an identical dilution of ascites from a mouse injected with the myeloma cell line SP 2/0, resulted in an almost total loss of staining (Fig lB) which further validated the specificity of the findings. DISCUSSION
In the present study, we demonstrated the expression ofGR mRNA and MTII mRNA in human alveolar macrophages and normal lung tissue, utilizing a solution hybridization assay. This technique showed a high sensitivity, and also, a high specificity for the mRNA studied, as indicated by previously performed Northern blot analysis. 23 The GR mRNA levels in human alveolar macrophages were seemingly in the same range as those in human fibroblasts and HeLa S3 cells but higher than in lymphocytes. 23 The levels of GR mRNA in lung tissue were in the same range as in PAMs found in this study. This comparison was based on the observation that RNA constitutes approximately 50 percent ofTNA in PAM . The GR mRNA levels in lung tissue could also be said to be in accordance with the findings in other species by Kalinyak et al 26 who reported relatively high GR mRNA levels in rat lung. Using the monoclonal anti-GR antibody (designated mab 7), the presence of GR in PAM was further studied. A strong staining of PAM in the cytospin preparations was seen, preferentially in the cytoplasmic compartment. The lack of GR staining after incubation with ascites from a mouse injected with the myeloma cell line SP 2/0 instead of incubation with the primary antibody further substantiated the specificity of these observations. In this study, no differences with regard to GR mRNA content in PAMs from healthy volunteers as
compared to patients with sarcoidosis were found. This appears to be in conflict with results from a recent investigation where the expression of GR was significantly increased in BAL cells in sarcoidosis. 2' It is, however, essential to consider that we measured GR mRNA levels in BAL cells that were almost exclusively macrophages since nonadherent cells (lymphocytes and the few neutrophils and eosinophils observed) had been separated from PAMs by adhesionpurification. In contrast, Sharma et aP' based their observations on unfractionated BAL cells. The authors also suggested the lymphocytes were the major contributors to the total GR content since a positive correlation between the percentage of lymphocytes and GR content was observed . In view of the fact that earlier investigations23 ·211 have found the GR content in lymphocytes from peripheral blood to he relatively low (both at the protein and the mRNA level), it seems unlikely that they could account for the high GR content in BAL cells seen in sarcoidosis. No Scathcard plot was presented by Sharma et aP' which would have been of great interest since Lacronique et al, 14 using similar ligand-binding techniques, were unable to saturate the putative receptors in the alveolar macrophages. Thus, it was demonstrated that the Scatchard plot, constructed from dexamethasone binding curves for normal human macrophages, did not show a straight line . 14 Instead, their nonlinear plots would suggest more than one binding entity in macrophage cytosol. The steady-state levels of a glucocorticoid regulated gene (MTII) were found to be similar in PAMs from the two study groups. The expression of MTII mRNA was shown to be induced both by cadmium sulphate (10 - 6 M) and by dexamethasone (IO - • M) to the same CHEST I 99 I 6 I JUNE. 1991
1339
degree in both groups. Some individuals showed only a modest induction of MTII mRNA levels upon dexamethasone stimulation, but due to the limited number of available cells in BAL, it was not possible to clarify the issue further by individual dose-response curves. It is clear, however, that there was no difference when comparing healthy volunteers to patients with sarcoidosis with regard to inducibility of a glucocorticoid-sensitive parameter, MTII mRNA in PAMs in vitro. Macrophages are cells that exert proinflammatory effects by releasing large quantities of prostaglandins and leukotrienes, 29 and administration of dexamethasone has been shown to suppress the release of PGE 2 from PAMs. 14 Thus, the unequivocal demonstration of glucocorticoid receptors in this putative target cell for glucocorticoids is of considerable importance. The mechanism of action of glucocorticoids as antiinflammatory agents that would justify their use in sarcoidosis is not known, although it is believed that the induction of phospholipase A 2 inhibitory proteins(s) would prevent the accumulation of products from the cyclooxygenase and lipooxygenase pathways. 30·31 Thus, it has been suggested that part of the antiinflammatory action of glucocorticoids may be mediated through the induction of synthesis of lipocortins. 32 ·33 However, since it has recently been found that lipocortin 1 mRN A levels are not regulated by glucocorticoids, 23 the role of these hormones in regulating phospholipase inhibitory proteins requires further study. Considering the results presented in this study and the discussion above, it is evident that the relation between GR content and function in BAL cells and the in vivo effects of glucocorticoids on pulmonary lesions in patients with inflammatory lung diseases merits further study. The rapid and convenient assessment of GR mRNA levels and of mRNA levels of a GR regulated gene in PAM by solution hybridization has provided the essential tools for examining larger groups of patients. In particular, patients with interstitial pulmonary fibrosis or asthma/chronic bronchitis would be of considerable interest since great interindividual variations in response to treatment with glucocorticoids have been observed in these patients. 34 ACKNOWLEDGMENTS: The authors are Sundher~ for excellent technical assistance.
~rateful
to Mrs. Maria
REFERENC ES
Fauci AS, Dale DC, Balow JE . Glut~>eortk,>id therapy: mechanisms of action and clinical considerations. Ann Intern Med 1976; 84:304-15 2 Watters L, Schwarz M, Cherniak R, Waldron J, Dunn Th. Stanford R. et al. Idiopathic pulmonary fibrosis : pretreatment hronchoalveolar lava~e cellular constituents and their relationships with lun~ histopatholo~ and clinical response to therapy. Am Rev Respir Dis 1987; 135:696 3 Sarcoidosis. In: Seaton A, Seaton D , Leitch G, eds. Crofton and Dou~las's respiratory diseases. Oxford : Blackwell Press,
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1989:630-59 4 Becker PB, Gloss B, Schmid W, Strahle U, Schlitz G . In vivo protein-DNA interactions in a glucocorticoid response element require the presence of the hormone . Nature 1986; 234:686-88 5 Yamamoto KR. Steroid receptor regulated transcription of specific genes and gene networks. Ann Rev Genet 1985; 19:20952 6 Gustafsson J-A, Carlstedt-Duke J, Poellinger L, Okret S, Wikstrom A-C, BronnegArd M, et al. Biochemistry, molecular biology, and physiology of the glucocorticoid receptor. Endocrine Rev 1987; 8:185-234 7 Hseuh W, Desai U, Gonzalez-Crussi F, Lamb R, Chu A. Two phospholipase pools for prostaglandin synthesis in macrophages. Nature 1981; 290:710-13 8 Schuyler MR, Todd LS. Accessory cell function of rabbit alveolar macrophages. Am Rev Respir Dis 1981; 123:53-57 9 Ballard PL, Ballard RA. Glucocorticoid receptors and the role of glucocorticoid in fetal lung development. Proc Natl Acad Sci USA 1972; 69:2668-72 10 Giannopoulos G. Glucocorticoid receptors in lung: specific binding of glucocorticoids to cytoplasmic components of rabbit fetal lung. J Bioi Chern 1973; 248:3876-83 11 Werb Z, Foler R, Munck A. Interaction of glucocorticoids with macrophages. Identification of glucocorticoid receptors in monocytes and macropha~es. J Exp Med 1978; 147:1684-94 12 George SC , Guckwicz PW, Jaffe RC . Glucocorticoid binding to normal and activated alveolar lung cells. Lung 1980; 157:57-64 13 Ozaki T, Nakayama T, Ishimi H, Kawano T, Yasuoka S, Tsubura E. Glucocorticoid receptors in bronchoalveolar cells from patients with idiopathic pulmonary fibrosis. Am Rev Respir Dis 1982; 126:968-71 14 Lacronique J, Rennard S, Bitterman P, Ozaki T, Crystal R. Alveolar macrophages in idiopathic pulmonary fibrosis have glucocorticoid receptors, but glucocorticoid therapy does not suppress alveolar macrophage release of fibronectin and alveolar macropha~e derived growth factor. Am Rev Respir Dis 1984; 130:450-56 15 Okret S, Wikstrom A-C , Wrange 0, Andersson B, Gustafsson J-A. Monoclonal antibodies against the rat glucocorticoid receptor. Proc Natl Acad Sci USA 1984; 81 :1609-13 16 Karin M, Haslinger A, Holtgreve H, Richards R, Krauter P, Westphal H, et al . Characterization of DNA-sequences through which cadmium and glucocorticoid hormones induce human metallothionein-11, gene. Nature 1984; 308:513-19 17 Lund J, Andersson 0 , Ripe E. Characterization of a binding protein for the PCB metabolite 4,4' -his (methylsulfonyl)-2,2' ,5'5tetrachlorobiphenyl present in bronchoalveolar lavage from healthy smokers and non-smokers. Toxicol Appl Pharmacol1986; 83:486-93 18 Durham OM , Palmiter RP. A practical approach for quantitating specific mRNA by solution hybridization. Anal Biochem 1983; 131:385-93 19 Maniatis T, Fritsch EF, Sambrook J. Molecular cloning-a laboratory manual . Cold Spring Harbor: Cold Spring Harbor Laboratory, 1982 20 Hollenber~ SM , Weinberger C , Ong ES, Cerelli G, Oro A, Lebo R, et al. Primary structure and expression of a functional human ~lucocortkoid receptor eDNA. Nature 1985; 318:635-41 21 Karin M, Richards R. Human metallothionein genes-primary structure of the metallothionein 11 gene and a related processed gene. Nature 1982; 299:797-802 22 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 1977; 74:546367 23 BronnegArd M, Andersson 0, Edwall D , Lund J, Norstedt G, Carlstedt-Duke J. Human calpactin 11 {lipocortin 1) messenger ribonucleic acid is not induced by ~lucocorticoids. Molecular Pulmonary Alveolar Macrophages in Sarcoidosis (Andersson et 81)
Endocrinol1988; 2:732-39 24 Mathews LS, Norstedt G, Palmiter RP. Regulation of insulinlike growth factor I expression by growth hormone. Proc Natl Acad Sci USA 1986; 83:9343-47 25 Wikstrom A-C, Bakke 0, Okret S, Bronneghd M, Gustafsson J-A. Intracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization. Endocrinology 1987; 120:1232-42 26 Kalinyak J, Dorin RI , HolTman AR, Perlman AJ. Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone. J Bioi Chern 1987; 262:10441-44 27 Sharma S, Verma U, Pande J, Murugesan K, Verma K, Guleria J. Glucocorticoid receptors in bronchoalveolar lavage Ruid in sarcoidosis. Chest 1988; 93:577-79 28 Lippman M, Barr R. Glucocorticoid receptors in purified subpopulations of human peripheral blood lymphocytes. J lmmunol1977; 118:1977-81 29 Goppelt-Stuebe M, Koerner CF, Hausmann G, Gemsa D,
30
31
32
33
34
Resch K. Control of prostanoid synthesis: role of reincorporation of released precursor fatty acids. Prostaglandins 1986; 32:373-76 Samuelsson B, Hammarstrom S, Borgeat P. Pathways of arachidonic acid metabolism . In: Weissman G, Samuelsson B, Paoletti R, eds. Advances in inflammation research. New York: Raven Press, 1979:405 Davies P, Baily PJ, Goldenberg MM. The role of arachidonic acid oxygenation products in pain and inflammation. Ann Rev Immunol 1984; 2:335-51 Di Rosa M, Flower RJ, Hirata F, Parente L, Russo-Marie F. Antiphospholipase proteins: nomenclature announcement. Prostaglandins 1984; 28:441-42 Hirata F, Notsu Y, Yamada R, Ishihara Y, Wano Y, Kunos I, et al. Isolation and characterization of lipocortin (lipomodulin). Agents Actions 1985; 17:263-66 Carmichael J, Paterson IC, Diaz P, Crompton GK, Kay AB, Grant IWB. Corticosteroid resistance in chronic asthma. Br Med J 1981; 282:1419-22
Plan to Attend ACcP·s
57th Annual Scientific Assembly ~ San Francisco ~ November 4-8, 1991
CHEST I 99 I 6 I JUNE, 1991
1341
The presence of the glucocorticoid receptor was demonstrated by immunocytochemistry in pulmonary alveolar macrophages obtained by bronchoalveolar lavage. AJso, GR mRNA content was determined by solution hybridization in PAM from 12 healthy volunteers and in 6 patients with sarcoidosis. No significant differences with regard to GR mRNA expression was detected between the two groups examined. For comparison, lung tissue from three patients undergoing thoracic surgery was examined and found to contain GR mRNA levels in the same range. As an indication of GR function, we also determined the mRNA levels of a glucocorticoid-regulated gene, metallothionein IIA, during basal conditions and after in vitro incubation of PAM with dexamethasone. Neither the control sample nor the dexa-
methasone-stimulated MTII mRNA values in PAMs differed significantly between the two groups. Solution hybridization is a rapid, sensitive and convenient assay which enables accurate and specific quantitation of GR mRNA in PAM. The GR mRNA content and basal as well as dexamethasone-induced MID mRNA levels in PAM from patients with sarcoidosis is not significantly different from (Chest 1991; 99:1336-41) those in healthy volunteers.
have been widely used in the management of a variety of inflammatory and immunologic lung diseases. 1 It is, however, well known that considerable variations exist with regard to therapeutic response to treatment with glucocorticoids. Thus, in patients with idiopathic pulmonary fibrosis, there is a limited number of patients responding to therapy with glucocorticoids, 2 whereas the efficacy of glucocorticoids generally is high in sarcoidosis.-1 The molecular basis for interindividual differences, or differences between groups of patients, in glucocorticoid sensitivity is not known. Theoretically such differences could involve any step in the proposed mechanism of action of these hormones. Of fundamental importance at a subcellular level is the concept of a cytoplasmic receptor protein which, after interaction with its ligand, translocates to the nucleus. In vivo, the hormone-receptor complex thereafter interacts with specific DNA sequences, termed glucocorticoid responsive elements (GREs, 4 ) and elicits transcriptional activation or inhibition of target gene expression by a
largely unknown mechanism. s.s Obviously, another possible explanation for the lack of efficacy of the glucocorticoids in, for example, idiopathic pulmonary fibrosis, might be that these agents do not suppress the inflammatory processes that modulate the fibrosis. In view of this, the relation between the GR content in various immunocompetent and inflammatory cells in the lung and the in vivo effects of glucocorticoids on pulmonary lesions in patients with interstitial lung diseases has been the focus of much attention. The macrophage is a likely target cell for the effects of glucocorticoids due to its capacity to produce arachidonic acid metabolites7 of importance to evoke and sustain inflammation. These cells also participate in immune reactionsH known to be modified by glucocorticoid treatment. These properties of the alveolar macrophage, as well as the fact that the cell is easily harvested from the lung by means of bronchoalveolar lavage, explain the interest in this particular cell. Indeed, glucocorticoid receptors have been detected in lung tissues 9 · 10 or alveolar macrophages from animals. 11 •12 However, previous studies with ligand-based measurements of GR content in human bronchoalveolar lavage cells 13 or enriched pulmonary alveolar macrophages 14 have been somewhat contradictory. The high degree of nonspecific binding of tritiated ligand (synthetic glucocorticoid) to BAL cell cytosol makes precise quantitation of specific bindinr difficult and may account for some of the conflicting data.
Glucocorticoi~s
*From the tDepartment of Medical Nutrition, :j:Department of Lung Medicine and §De partment of Surgery, Karolinska Institute, Iluddinge University Hospital , Huddinge, Sweden, and IIDepartment of Clinical Explorative Research, Draco AB , Lund, Sweden. This study was supported hy grants 13X-2819, 19K-087 14 from the Swedish Medical Research Council , the Swedish Heart Lung Foundation, the Swedish Cancer Societv, and Draco AB. Manuscript received June 12; revision a~cepted November 6. RqHinl ll'lf!lests: Dr. AncU'!"s.wm, DqJClrtment of Medical Nutrition. H!lddingt• Unir,ersity Hospital. S-J.Il 86 Huddinge , Sweden
1336
GR=glucocorticoid receptor; PAM=pulmonary alveolar macrophages; MTII = metallothionein IIA; TNA =total nucleic acid; PAP= peroxidase/antiperoxidase; NRS =normal rabbit serum
Pulmonary Alveolar Macrophages in Sarcoidosis (Andersson et al)
The purpose of this investigation was primarily to unequivocally demonstrate the presence of the GR in human PAM, and secondly, to develop a suitable method for quantitative analysis of GR content in PAM. We, therefore, combined the use of a monoclonal antibody against GR, designated mab 7, 15 in immunocytochemical studies and a solution hybridization assay for measurements of GR mRNA levels in PAM . The metallothionein IIA gene is known to be ubiquitously expressed and regulated by glucocorticoids and heavy metals at the transcriptional level. 16 To obtain further information on the function of GR in PAM, studies of MTII mRNA levels during basal conditions and after in vitro incubations with dexamethasone or a heavy metal were included. MATERIALS AND METHODS
Study Population Bronchoalveolar lavage was performed in I2 healthy volunteers (eight smokers and four nonsmokers; age 3I ±8.8 years, range 2I to 47 years) and in six patients with biopsy proven sarcoidosis (all nonsmokers, four in stage II and two in stage I by traditional roentgenographic criteria; age 4I ± 8 .9 years, range 32 to 57 years). None of the patients had any evidence of mycobacterial, fungal, or parasitic infection or a history of exposure to organic or inorganic material known to cause granulomatous lung disease. For comparison, specimens of histologically-normal lung tissue were recovered from three patients undergoing thoracic surgery for lung cancer.
BAL Bronchoalveolar lavage was performed as described earlier." Aliquots (5 ml) of BAL Ruid were removed, stained with May Criinewald-Ciemsa, and subjected to differential cell counting after cytocentrifugation. Examination of these cytospin preparations showed that approximately 90 percent of the recovered cells were alveolar macrophages among the healthy volunteers, whereas the sarcoidosis patients had higher levels oflymphocytes (5, 22, 3I, 46, I8, and 55 percent, respectively) in the BAL-Ruid as expected.
Cell Cultures The remaining lavage Ruid was separated from the cells by centrifugation, and the obtained BAL cells were allowed to adhere to plastic bottles containing serum-free medium supplemented with benzyl-penicillin (400 IU/ml), streptomycin (0.2 mwml), and Lglutamine (2 mM). The cells were cultured at 37"C in an atmosphere of 5 percent CO, in air. After 6 hours, nonadherent cells (mainly lymphocytes) were washed away. The remaining cells (PAMs) were refed with medium only (control) or medium containing dexamethasone (IO -' M) or CdSO, (Io -• M) for I2 hours. Prior to preparation of total nucleic acid, the cells were washed twice with PBS , and scraped into lxSET (I percent SDS, IO mM EDTA, and 20 mM Tris-HCI pH 7.5).
Preparation of TNA and RNA ForTNA preparation, the cells were scraped into lxSET, homogenized, and digested with 10 mwml proteinase K for 45 min at 45°C. The cells were then phenol-chloroform extracted, reextracted with chloroform and ethanol precipitated . The dried TNA was dissolved in 0.2xSET as described.•• The concentrations of the TNA samples were measured spectrophotometrically at 260 and 280 nm . The RNA from lung tissue specimens was prepared by the guanidinium thiocyanate method combined with C,Cl, centrifugation.••
Plasmids and Solution Hybridization Analysis For construction of the human CR probe, two mplimentary oligonucleotides corresponding to nucleotides 159-209 in the human CR eDNA'" were synthesized, annealed , and ligated into the Pst V Hind Ill sites in pCEM TMl. For construction of the human MTII probe, two complimentary oligonucleotides corresponding to nucleotides 406-456 in the human MTII gene" were synthesized and cloned into Pst 1/Hind Ill sites in pCEM TM1 as described above . The sequences of these inserts in pC EM TM I were confirmed by DNA sequencing using the dideoxy chain-termination method."' Northern blot analysis showed that the MTII probe hybridized to an RNA species of0.7-Q.8 kb and the CR probe to RNA species of 7.0 and 6.I kb.23 These results indicate that the probes used hybridized to RNA species similar to those reported for MTII and CR. 3 ' · " The plasmids designated phCR and phMTil were then used fi>r in vitro synthesis of eRN A radiolabelled with ·" S-UTP using SP6 RNA polymerase. The opposite mRNA strand was synthesized using T7 RNA polymerase . For solution hybridization," TNA samples were hybridized to 20,000 cpm of"'S-cRNA probe in 40 JLI of0.5 M NaCI, 20 mM Tris-HCI pH 7.5, 5 mM EDTA, 0.1 percent SDS, I mM dithiothreitol, and 25 percent formamide at 68°C for I2 to I6 h. The samples were then treated with RNase for 45 min by adding I ml of a solution containing 40 JLg RNase A and 2 JLg RNase Tl (Boehringer-Mannheim, CFR) and 100 JLg herring sperm DNA. Radioactivity protected against RNase digestion was precipitated by the addition of IOO JLI6M trichloroacetic acid and collected on a filter (Whatman CF/C). The hybridization signal of a sample was compared to a standard curve constructed from incubations with known amounts of the synthetic oligonucleotide mRNA . A standard curve (3-300 xlO - " mol mRNA/incubation) was included in each assay, and each TNA sample was analyzed in duplicate or triplicate. For each specific probe, the corresponding amount of sample mRNA was calculated from the linear part of the respective standard curve. Means and SD were calculated for the various experimental groups. Means were mmpared hy the Mann-Whitney U test, when appropriate . Differences between groups were considered significant for p values of 0.05 or less.
Immunocytochemistry Cytospin preparations were kept at -7ifC. The cells (immunocytochemistry experiments have only been carried out with cells obtained from healthy volunteers) were then fixed with 2 perr 15 min with 3 percent (v/v) normal rabbit serum, and the cells were then incubated with mab 7 at an optimal dilution of I:250 in PBS/I percent (v/v) NRS. The primary antibody was followed by an incubation with the bridge antibody, rabbit anti-mouse I.e (DAKOPATTS, Stockholm, Sweden) diluted I :SO in PBS/I percent (v/ v) NRS. After washing with PBS, the cells were incubated with a monoclonal mouse PAP complex diluted I:IOO in PBS/I per test the functional significance of the measured GR mRNA, we next wanted to measure a putatively receptor-mediated glucocorticoid response. For this purpose, the cells were put in primary culture overnight and incubated with medium (control) or medium containing dexamethasone (I0 - 7 M). No signs of toxicity were observed . Since the MTII gene is inducible both by glucocorticoids and heavy metals, the in vitro incubation of PAM with CdS0 4 provided a positive control. Unstimulated PAMs recovered from healthy volunteers and patients contained approximately equal concentrations ofMTII mRNA (Table 2), and treatment of the cells with dexamethasone caused an -1.9-fold 1338
Table 1-Erpression ofGR mRNA CelVfissue
GR mRNA (amoVJJ.g TNA)
Sarcoidosis- PAM* (n = 6) Healthy volunteers- PAM* (n = 121)
41±6 39±11
*PAMs obtained hv BAL were cultured in serum-free medium. The levels of GR mRNA in the TNA samples prepared from PAMs were determined hv solution hvhridization as described in Materials and Methods..Results oht~ned from healthy volunteers and patients "~th sarcoidosis (number of individuals indicated in brackets) presented as mean± SD. Each sample analyzed in duplicate or triplicate.
induction of MTII mRNA levels in both groups examined (Table 2). In PAMs recovered from healthy volunteers, CdS04 increased MTII mRNA levels fourfold, and this was in the same range as for PAMs from sarcoidosis. Thus, the extent of MTII expression and its induction by glucocorticoids and CdS04 was similar in the two study populations.
Immunocytochemistry The immunocytochemical experiments were included to obtain qualitative data on the cellular localization of the GR. The monoclonal antibody, designated mab 7, has been shown earlier to be monospecific for the GR and the staining technique including the permeabilization procedures was adopted from earlier experiments carried out in our laboratory. 15 ·2:; As can be seen from the experiments using cytospin preparations of BAL cells (done only in cytospin preparations from four healthy volunteers), there was an intense staining of PAM, predominantly in the cytoplasmic region (Fig IA). Occasionally, nuclear staining was observed in PAM, albeit at a lower intensity. Similar incubations, but where mab 7 Table 2-Erpression ofMTII mRNA in PAM from Healthy Volunteers and Patients with Sarcoidosis Cell Sarcoidosis-PAM* (n=6) Control DEX CdSO, Healthy volunteers-PAM* (n=5) Control DEX CdSO,
MTII mRNA (amoVJJ.g TNA)
Fold Induction
266± 127 495±305 1246±967
1.9,2.3,2.6,2.1,1.0,0.9 4.9,3.8,3.6,3.0,2.2,8.5
310±50 600± 120 1280± 150
1.4,2.6,1.8,2. 1, 1.9 3.8,4.4,4.9,4.0,3.7
*PAMs were treated with medium only (control) or medium containing 10 -' M dexamethasone or 10 -• M CdSO, as described in Materials and Methods. Levels of MTII mRNA in the TNA samples prepared from PAMs were determined by solution hybridization. Results obtained from healthy volunteers and patients with saR'lidosis (number of individuals indicated in brackets) presented as mean± SD. Each sample analyzed in duplicate or triplicate. Pulmonary Alveolar Macrophages in Sarcoidosis (Andersson at a/)
FIGURE 1 A and B. GR staining in PAM. Cytospin preparation of BAL-cells were fixed with 2 percent (v/ v} buffered formaldehyde . The GR was then stained with mab 7 and the PAP technique as described. Note the intense staining of PAM predominantly in the cytoplasmic region. No GR staining was obtained if mab 7 was substituted with ascites from a mouse injected with the myeloma cell line SP 210 {2} {original magnification x400).
had been replaced with an identical dilution of ascites from a mouse injected with the myeloma cell line SP 2/0, resulted in an almost total loss of staining (Fig lB) which further validated the specificity of the findings. DISCUSSION
In the present study, we demonstrated the expression ofGR mRNA and MTII mRNA in human alveolar macrophages and normal lung tissue, utilizing a solution hybridization assay. This technique showed a high sensitivity, and also, a high specificity for the mRNA studied, as indicated by previously performed Northern blot analysis. 23 The GR mRNA levels in human alveolar macrophages were seemingly in the same range as those in human fibroblasts and HeLa S3 cells but higher than in lymphocytes. 23 The levels of GR mRNA in lung tissue were in the same range as in PAMs found in this study. This comparison was based on the observation that RNA constitutes approximately 50 percent ofTNA in PAM . The GR mRNA levels in lung tissue could also be said to be in accordance with the findings in other species by Kalinyak et al 26 who reported relatively high GR mRNA levels in rat lung. Using the monoclonal anti-GR antibody (designated mab 7), the presence of GR in PAM was further studied. A strong staining of PAM in the cytospin preparations was seen, preferentially in the cytoplasmic compartment. The lack of GR staining after incubation with ascites from a mouse injected with the myeloma cell line SP 2/0 instead of incubation with the primary antibody further substantiated the specificity of these observations. In this study, no differences with regard to GR mRNA content in PAMs from healthy volunteers as
compared to patients with sarcoidosis were found. This appears to be in conflict with results from a recent investigation where the expression of GR was significantly increased in BAL cells in sarcoidosis. 2' It is, however, essential to consider that we measured GR mRNA levels in BAL cells that were almost exclusively macrophages since nonadherent cells (lymphocytes and the few neutrophils and eosinophils observed) had been separated from PAMs by adhesionpurification. In contrast, Sharma et aP' based their observations on unfractionated BAL cells. The authors also suggested the lymphocytes were the major contributors to the total GR content since a positive correlation between the percentage of lymphocytes and GR content was observed . In view of the fact that earlier investigations23 ·211 have found the GR content in lymphocytes from peripheral blood to he relatively low (both at the protein and the mRNA level), it seems unlikely that they could account for the high GR content in BAL cells seen in sarcoidosis. No Scathcard plot was presented by Sharma et aP' which would have been of great interest since Lacronique et al, 14 using similar ligand-binding techniques, were unable to saturate the putative receptors in the alveolar macrophages. Thus, it was demonstrated that the Scatchard plot, constructed from dexamethasone binding curves for normal human macrophages, did not show a straight line . 14 Instead, their nonlinear plots would suggest more than one binding entity in macrophage cytosol. The steady-state levels of a glucocorticoid regulated gene (MTII) were found to be similar in PAMs from the two study groups. The expression of MTII mRNA was shown to be induced both by cadmium sulphate (10 - 6 M) and by dexamethasone (IO - • M) to the same CHEST I 99 I 6 I JUNE. 1991
1339
degree in both groups. Some individuals showed only a modest induction of MTII mRNA levels upon dexamethasone stimulation, but due to the limited number of available cells in BAL, it was not possible to clarify the issue further by individual dose-response curves. It is clear, however, that there was no difference when comparing healthy volunteers to patients with sarcoidosis with regard to inducibility of a glucocorticoid-sensitive parameter, MTII mRNA in PAMs in vitro. Macrophages are cells that exert proinflammatory effects by releasing large quantities of prostaglandins and leukotrienes, 29 and administration of dexamethasone has been shown to suppress the release of PGE 2 from PAMs. 14 Thus, the unequivocal demonstration of glucocorticoid receptors in this putative target cell for glucocorticoids is of considerable importance. The mechanism of action of glucocorticoids as antiinflammatory agents that would justify their use in sarcoidosis is not known, although it is believed that the induction of phospholipase A 2 inhibitory proteins(s) would prevent the accumulation of products from the cyclooxygenase and lipooxygenase pathways. 30·31 Thus, it has been suggested that part of the antiinflammatory action of glucocorticoids may be mediated through the induction of synthesis of lipocortins. 32 ·33 However, since it has recently been found that lipocortin 1 mRN A levels are not regulated by glucocorticoids, 23 the role of these hormones in regulating phospholipase inhibitory proteins requires further study. Considering the results presented in this study and the discussion above, it is evident that the relation between GR content and function in BAL cells and the in vivo effects of glucocorticoids on pulmonary lesions in patients with inflammatory lung diseases merits further study. The rapid and convenient assessment of GR mRNA levels and of mRNA levels of a GR regulated gene in PAM by solution hybridization has provided the essential tools for examining larger groups of patients. In particular, patients with interstitial pulmonary fibrosis or asthma/chronic bronchitis would be of considerable interest since great interindividual variations in response to treatment with glucocorticoids have been observed in these patients. 34 ACKNOWLEDGMENTS: The authors are Sundher~ for excellent technical assistance.
~rateful
to Mrs. Maria
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Plan to Attend ACcP·s
57th Annual Scientific Assembly ~ San Francisco ~ November 4-8, 1991
CHEST I 99 I 6 I JUNE, 1991
1341
