Pulmonary Expression of Leukemia Inhibitory Factor Induces B Cell Hyperplasia and Confers Protection in Hyperoxia

Leukemia inhibitory factor (LIF) is produced by a large number of pulmonary cells in response to diverse stimuli. Exaggerated levels of LIF have also been detected in the adult respiratory distress syndrome and other disorders. The biologic effects of LIF in the lung, however, have not been elucidated. To define the respiratory effects of LIF, we generated transgenic mice in which human LIF was selectively targeted to the mature lung. In these mice, transgene activation caused an impressive increase in bronchoalveolar lavage (BAL) cellularity with a significant increase in BAL and tissue B lymphocytes. LIF also conferred protection in 100% O2 where it decreased alveolar-capillary protein leak and enhanced survival. This protective effect was associated with the induction of interleukin (IL)-6 mRNA and protein. LIF transgenic mice with a null mutation in IL-6 were more sensitive to the toxic effects of 100% O2 than LIF-transgenic animals with a wild-type IL-6 locus. These studies demonstrate that LIF induces B cell hyperplasia and confers protection in hyperoxic acute lung injury. They also demonstrate that LIF induces IL-6 and that the protective effects of LIF are mediated, in part, via this inductive event. LIF may be an important regulator of B cell-mediated responses and oxidant injury in the lung. Leukemia inhibitory factor (LIF) is produced by a large number of pulmonary cells in response to diverse stimuli. Exaggerated levels of LIF have also been detected in the adult respiratory distress syndrome and other disorders. The biologic effects of LIF in the lung, however, have not been elucidated. To define the respiratory effects of LIF, we generated transgenic mice in which human LIF was selectively targeted to the mature lung. In these mice, transgene activation caused an impressive increase in bronchoalveolar lavage (BAL) cellularity with a significant increase in BAL and tissue B lymphocytes. LIF also conferred protection in 100% O2 where it decreased alveolar-capillary protein leak and enhanced survival. This protective effect was associated with the induction of interleukin (IL)-6 mRNA and protein. LIF transgenic mice with a null mutation in IL-6 were more sensitive to the toxic effects of 100% O2 than LIF-transgenic animals with a wild-type IL-6 locus. These studies demonstrate that LIF induces B cell hyperplasia and confers protection in hyperoxic acute lung injury. They also demonstrate that LIF induces IL-6 and that the protective effects of LIF are mediated, in part, via this inductive event. LIF may be an important regulator of B cell-mediated responses and oxidant injury in the lung. Leukemia inhibitory factor (LIF) 1The abbreviations used are: LIF, leukemia inhibitory factor; BAL, bronchoalveolar lavage; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; ARDS, adult respiratory distress syndrome; rtTA, reverse tetracycline transactivator; hGH, human growth hormone; CMV, cytomegalovirus; tet-O, tetracycline operator; PBS, phosphate-buffered saline; RT, reverse transcription; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; gp, glycoprotein; dox, doxycycline; HALI, hyperoxic acute lung injury.1The abbreviations used are: LIF, leukemia inhibitory factor; BAL, bronchoalveolar lavage; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; ARDS, adult respiratory distress syndrome; rtTA, reverse tetracycline transactivator; hGH, human growth hormone; CMV, cytomegalovirus; tet-O, tetracycline operator; PBS, phosphate-buffered saline; RT, reverse transcription; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; gp, glycoprotein; dox, doxycycline; HALI, hyperoxic acute lung injury. is a highly glycosylated 38-kDa member of the IL-6-type cytokine family. It is produced by a large number of normal and neoplastic cells and mediates its effects by interacting with a diffusely distributed multimeric receptor complex made up of gp130 and gp190 molecules (1Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1730) Google Scholar, 2Kamohara H. Sakamoto K. Ishiko T. Mita S. Masuda Y. Abe T. Ogawa M. Res. Commun. Mol. Pathol. Pharmacol. 1994; 85: 131-140PubMed Google Scholar, 3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar). Under normal conditions, LIF production is tightly regulated with significant expression being detected only in the uterus (3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar, 4Brown M.A. Metcalf D. Gough N.M. Cytokine. 1994; 6: 300-309Crossref PubMed Scopus (58) Google Scholar). It is, however, stimulated by cytokines, calcium ionophore, phorbol ester, and endotoxin, and elevated levels of LIF have been detected in serum, cerebrospinal fluid, synovial fluid, and urine from patients with sepsis, meningitis, rheumatoid arthritis, and renal transplant rejection, respectively (3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar, 5Elias J.A. Zheng T. Whiting N.L. Marcovici A. Trow T.K. Am. J. Physiol. 1994; 266: L426-L435PubMed Google Scholar, 6Ulich T.R. Fann M.-J. Patterson P.H. Williams J.H. Samal B. Del Castillo J. Yin S. Guo K. Remick D.G. Am. J. Physiol. 1994; 267: L442-L446PubMed Google Scholar, 7Waring P. Wycherley K. Cary D. Nicola N. Metcalf D. J. Clin. Invest. 1992; 90: 2031-2037Crossref PubMed Scopus (99) Google Scholar). The roles of LIF in some of these settings have been investigated, and LIF is now known to regulate implantation in the uterus, myeloid cell differentiation, bone metabolism, adipogenesis, and neural homeostasis (3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar, 8Azari M.F. Galle A. Lopes E.C. Kurek J. Cheema S.S. Brain Res. 2001; 922: 144-147Crossref PubMed Scopus (31) Google Scholar, 9Lass A. Weiser W. Munafo A. Loumaye E. Fertil. Steril. 2001; 76: 1091-1096Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10Malaval L. Aubin J.E. J. Cell. Biochem. 2001; 81: 63-70Crossref Scopus (40) Google Scholar). In the majority of circumstances, the functions of LIF, however, are poorly understood. This is due, at least in part, to the perinatal lethality that is seen in mice with null mutations of the LIF/LIF receptor system (3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar, 11Ware C.B. Horowitz M.C. Renshaw B.R. Hunt J.S. Liggitt D. Koblar S.A. Gliniak B.C. McKenna H.J. Papayannopoulou T. Thoma B. et al.Development. 1995; 121: 1283-1299Crossref PubMed Google Scholar) and the lack of viscerally targeted overexpression transgenic systems that can be used to fully define the in vivo effector profile of this cytokine.The limitations in our understanding of the biology of LIF are nicely illustrated in the lung. LIF is produced during lung development and in mature respiratory tissues (12Fukada K. Korsching S. Towle M.F. Growth Factors. 1997; 14: 279-295Crossref PubMed Scopus (17) Google Scholar, 13Knight D.A. Lydell C.P. Zhou D. Weir T.D. Schellenberg R.R. Bai T.R. Am. J. Respir. Cell Mol. Biol. 1999; 20: 834-841Crossref PubMed Scopus (60) Google Scholar). Studies of the latter, from our laboratory and others, have demonstrated that basal epithelial cells, alveolar epithelial cells, smooth muscle cells, eosinophils, fibroblasts, and mast cells produce LIF in response to diverse stimuli including transforming growth factor-β, IL-1, and tumor necrosis factor-α, alone and in combination (5Elias J.A. Zheng T. Whiting N.L. Marcovici A. Trow T.K. Am. J. Physiol. 1994; 266: L426-L435PubMed Google Scholar, 13Knight D.A. Lydell C.P. Zhou D. Weir T.D. Schellenberg R.R. Bai T.R. Am. J. Respir. Cell Mol. Biol. 1999; 20: 834-841Crossref PubMed Scopus (60) Google Scholar, 14Knight D. McKay K. Wiggs B. Schellenberg R.R. Bai T. Br. J. Pharmacol. 1997; 120: 883-891Crossref PubMed Scopus (22) Google Scholar). IgE-mediated lung activation, endotoxin, and 10-μm sized air pollution molecules (PM10) also induce LIF elaboration (3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar, 4Brown M.A. Metcalf D. Gough N.M. Cytokine. 1994; 6: 300-309Crossref PubMed Scopus (58) Google Scholar, 13Knight D.A. Lydell C.P. Zhou D. Weir T.D. Schellenberg R.R. Bai T.R. Am. J. Respir. Cell Mol. Biol. 1999; 20: 834-841Crossref PubMed Scopus (60) Google Scholar, 15Fujii T. Hayashi S. Hogg J.C. Vincent R. Van Eeden S.F. Am. J. Respir. Cell Mol. Biol. 2001; 25: 265-271Crossref PubMed Scopus (204) Google Scholar), and elevated levels of LIF have been found in bronchoalveolar lavage fluids (BAL) from patients with the adult respiratory distress syndrome (ARDS) (16Gruson D. Hilbert G. Juzan M. Taupin J.L. Coulon V. Moreau J.F. Gualde N. Gbikpi-Benissan G. Intensive Care Med. 1998; 24: 366-368Crossref PubMed Scopus (2) Google Scholar, 17Jorens P.G. De Jongh R. Bossaert L.L. De Backer W. Herman A.G. Pollet H. Bosmans E. Taupin J.L. Moreau J.F. Cytokine. 1996; 8: 873-876Crossref PubMed Scopus (23) Google Scholar). LIF has pro- and anti-inflammatory properties (3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar). It can also augment eosinophil activation, enhance epithelial phospholipase A2 production, inhibit smooth muscle hyperplasia, and enhance tachykinin production and airway smooth muscle tachykinin responses (3Knight D. Pulm. Pharmacol. Ther. 2001; 14: 169-176Crossref PubMed Scopus (39) Google Scholar, 18Ikezono T. Wu T. Yao X.L. Levine S. Logun C. Angus C.W. Shelhamer J.H. Biochim. Biophys. Acta. 1997; 1355: 121-130Crossref PubMed Scopus (14) Google Scholar, 19Moran C.S. Campbell J.H. Campbell G.R. J. Vasc. Res. 1997; 34: 378-385Crossref PubMed Scopus (12) Google Scholar). Little else is known, however, about the effector properties of LIF in the lung. In addition, it is not known if LIF production is a feature of disease pathogenesis or an appropriate healing response elicited by lung injury (17Jorens P.G. De Jongh R. Bossaert L.L. De Backer W. Herman A.G. Pollet H. Bosmans E. Taupin J.L. Moreau J.F. Cytokine. 1996; 8: 873-876Crossref PubMed Scopus (23) Google Scholar).To define the in vivo effector properties of LIF in the lung, we used an externally regulatable overexpression transgenic system to inducibly express LIF in the mature murine lung. These studies demonstrate that LIF is a potent stimulator of B cell accumulation. They also demonstrate that LIF has protective effects in oxidant-induced pulmonary injury and demonstrate that these effects are mediated, at least in part, via the ability of LIF to induce IL-6 elaboration.EXPERIMENTAL PROCEDURESGeneration of Transgenic Mice—We used an externally regulatable, dual construct overexpression transgenic system to generate CC10-rtTA-LIF mice. The constructs used in this system have been described previously by our laboratory (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (358) Google Scholar). The CC10-rtTA-hGH construct contains the CC10 promoter, the reverse tetracycline transactivator (rtTA), and human growth hormone (hGH) intronic and polyadenylation sequences (Fig. 1). The rtTA is a fusion protein made up of a mutated tetracycline repressor and the herpes virus VP-16 transactivator. The tet-O-CMV-LIF-hGH construct contains a polymeric tetracycline operator (tet-O), minimal CMV promoter, human LIF cDNA, and hGH intronic and polyadenylation signals (Fig. 1). In this system, the CC10 promoter directs the expression of rtTA to the lung. In the presence of doxycycline (dox), rtTA is able to bind in trans to the tet-O, and the VP-16 transactivator activates LIF gene transcription. In the absence of dox, rtTA binding occurs at very low levels, and only low level gene transcription is noted. The preparation of the CC10-rtTA construct has been described previously (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar). The tet-O-CMV-LIF-hGH construct was prepared by replacing the IL-11 cDNA in the construct tet-O-CMV-hIL-11-hGH described previously by our laboratory (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar) with the human LIF cDNA. This construct was checked for correct insert orientation by restriction enzyme digestion and sequencing. Both constructs were purified, linearized, separated by electrophoresis through agarose, and purified as described previously (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (358) Google Scholar). Transgenic mice were prepared in (CBA × C57BL/6)F2 eggs by mixing and simultaneously injecting the constructs into pronuclei as described previously (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (358) Google Scholar). The mice were bred for over 10 generations onto a C57BL/6 background before being used in these studies.Documentation of Transgene Status—The presence or absence of the transgenes was initially evaluated using Southern blot analysis and later by PCR. Southern analysis was performed as described previously using cDNA encoding human LIF or rtTA (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (358) Google Scholar). PCR for rtTA was also performed using protocols described by our laboratory (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (358) Google Scholar). PCR for the LIF-containing construct was undertaken using the following primers: upper primer, 5′ GGC ATC CCG CGG TTC CTC CAA GGC CCT CTG 3′; lower primer, 5′ AGC ACT GGA TCC GAC CTC CTG CTG 3′. The cycling conditions were 1 cycle at 94 °C for 5 min, 35 cycles of 94 °C for 1 min, followed by 60 °C for 1 min and 72 °C for 1 min, 1 cycle at 72 °C for 5 min, and 1 cycle at 4 °C to end. All CC10-rtTA-LIF lineage animals were evaluated for the presence of both the rtTA and LIF-containing transgenic constructs. Comparisons were undertaken of mice with both transgenic constructs (transgene (+) mice) and mice with neither construct (transgene (–) mice)Dox Water Administration—CC10-rtTA-LIF animals were maintained on normal water until 4–6 weeks of age. At that time they were randomized to receive normal water or water containing dox (0.5 mg/ml) as described previously (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (358) Google Scholar).Bronchoalveolar Lavage and Quantification of LIF Levels—Mice were killed; the trachea was isolated by blunt dissection, and small caliber tubing was inserted and secured in the airway. Two volumes of 1.0 ml of PBS with 0.1% bovine serum albumin were then instilled and gently aspirated and pooled. Each BAL sample was centrifuged, and the supernatants were stored at –70 °C until used. The levels of LIF were determined using a commercial ELISA (R & D Systems Inc., Minneapolis, MN) as per the manufacturer's instructions.Histologic Analysis—Animals were euthanized, a median sternotomy was performed, and right heart perfusion was accomplished with calcium and magnesium-free PBS to clear the pulmonary intravascular space. The heart and lungs were then removed en bloc, inflated to 25 cm with neutral-buffered 10% formalin, fixed overnight in 10% formalin, embedded in paraffin, sectioned at 5 mm, and stained. Hematoxylin and eosin, Congo red, Mallory's trichrome, periodic acid-Schiff with diastase, and Alcian blue stains were performed in the Research Histology Laboratory of the Department of Pathology, Yale University School of Medicine.mRNA Analysis—mRNA levels were assessed using reverse transcription (RT)-PCR as described previously by our laboratories (22Yoon H.J. Zhu Z. Gwaltney Jr., J.M. Elias J.A. J. Immunol. 1999; 162: 7461-7469PubMed Google Scholar, 23Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Invest. 1999; 103: 779-788Crossref PubMed Scopus (1481) Google Scholar). In these experiments, total cellular RNA from lungs or a variety of other mouse tissues were obtained using Trizol reagent (Invitrogen) as per the manufacturer's instructions. In the RT-PCR assays, RNA samples were reverse-transcribed, and gene-specific primers were used to amplify selected regions of the LIF target moiety. To verify that equal amounts of undegraded RNA were added in each RT-PCR, β-actin was used as an internal standard. Amplified PCR products were detected using ethidium bromide gel electrophoresis, quantitated electronically, and confirmed by nucleotide sequencing. Ribonuclease protection assays were performed using the RiboQuant kit purchased from Pharmingen. These assays were performed according to instructions provided by the manufacturer.ELISA Evaluations—The levels of LIF and IL-6 protein in murine BAL were quantitated by ELISA using commercial assays (R & D Systems Inc., Minneapolis, MN) as per the manufacturer's instructions.Fluorescence-activated Cell Sorting (FACS) Analysis—Transgenic and non-transgenic mice were treated for 2 weeks with dox or normal water as described above. The animals were then sacrificed. The pulmonary vascular tree was perfused with calcium- and magnesium-free PBS (pH 7.4) via a right heart catheter, and lung lymphocytes were prepared using collagenase III digestion, mechanical tissue disruption, and Ficoll density centrifugation. The resulting cells were prepared for three-color staining by incubating with phycoerythin-labeled anti-mouse CD3, FITC-labeled anti-mouse CD8, CyChrome-labeled anti-CD4, and FITC-conjugated anti-B220 (Pharmingen) for 1 h on ice. Analysis was performed on a FACSCalibur flow cytometer (BD Biosciences). Data are displayed as dot plots of FITC (x axis) versus phycoerythin- or CyChrome (y axis) fluorescence (log scales). Quadrant markers were positioned to include ≥99% of control Ig-stained cells in the left lower quadrant.Exposure to 100% O 2 —Mice were exposed to 100% oxygen in a 50 × 30 × 30-cm airtight chamber as described previously (24Ward N.S. Waxman A.B. Homer R.J. Mantell L.L. Einarsson O. Du Y. Elias J.A. Am. J. Respir. Cell Mol. Biol. 2000; 22: 535-542Crossref PubMed Scopus (186) Google Scholar, 25Waxman A.B. Einarsson O. Seres T. Knickelbein R.G. Warshaw J.B. Johnston R. Homer R.J. Elias J.A. J. Clin. Invest. 1998; 101: 1970-1982Crossref PubMed Scopus (152) Google Scholar). The fractional inspired O2 concentration in the chamber was monitored with an in-line oxygen analyzer (Vascular Technology, Inc., Chelmsford, MA) and maintained with a constant flow of gas (∼3 liters/min). The mice were fed food and water ad libitum and kept on a 12-h dark-light cycle at sea level and at room temperature. The animals were monitored at intervals for signs of respiratory difficulty and euthanized if there were signs of respiratory distress.BAL Protein Quantitation—BAL was performed as described above, and BAL protein was assayed as an index of lung injury and capillary leak. Protein quantification was accomplished using the method of Lowry.Generation of LIF (+)/IL-6 (–/–) Mice—C57BL/6 mice with a null mutation in IL-6 (IL-6 (–/–)) and IL-6 sufficient (IL-6 (+/+)) controls were obtained from The Jackson Laboratories (Bar Harbor, ME). CC10-rtTA-LIF mice with wild-type and null IL-6 loci were generated by breeding of the CC10-rtTA-LIF mice with the IL-6 (–/–) animals. As described previously, PCR was used to define the transgenic status of all offspring, using primers that detected rtTA, the junction region of our LIF-human growth hormone construct, and IL-6.Statistical Analysis—Data are expressed as the mean ± S.E. unless otherwise indicated. Data were assessed for significance using Student's t test or analysis of variance as appropriate.RESULTSGeneration of Transgenic Mice—To characterize the effects of LIF in the adult lung, we used an externally regulatable, dual construct overexpression transgenic system previously described by our laboratory (20Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (358) Google Scholar). The constructs required for these transgenics (Fig. 1) were prepared, purified, and simultaneously microinjected. Tail biopsies were obtained from potential founder animals; DNA was isolated, and the presence or absence of LIF and rtTA transgenic sequences was determined via PCR. Three dual positive founder animals were obtained. They were subsequently back-crossed with C57BL/6 mice to generate transgene (–) and transgene (+) progeny.Regulation and Organ Specificity of LIF Production—Transgene (–) and transgene (+) mice were kept on normal water until they were 4–6 weeks of age. They were then randomized to normal water or dox water. In the absence of dox administration, BAL LIF levels ≤10 pg/ml were appreciated. Dox administration caused a significant increase in BAL LIF. This increase was noted within 48 h of dox administration, persisted for extended intervals, and returned to base line within 4 days of removing dox from the animal's drinking water. The different transgenic animals had BAL LIF levels between ∼200 and 500 pg/ml after 1–4 weeks of dox administration.To determine whether LIF was appropriately targeted to the lung, RNA was obtained from the lungs and a variety of other tissues from transgene (+) mice that had received dox water for 2 weeks. LIF mRNA was then evaluated via RT-PCR analysis. Impressive levels of LIF mRNA could be appreciated in lungs from transgene (+) mice on dox water. In contrast, transgene-induced LIF mRNA was not noted, and histologic abnormalities were not appreciated in a variety of visceral tissues from transgene (+) animals (Fig. 2 and data not shown). This demonstrates that our CC10-driven system appropriately targeted LIF to the lungs of these transgenic animals.Fig. 2Organ specificity of LIF mRNA expression. RNA was isolated from lungs and a variety of other organs from 6-week-old transgene (+) mice that received dox water for 2 weeks. The levels of mRNA encoding human LIF were evaluated by RT-PCR as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of LIF on BAL Cellularity—Transgene (+) and transgene (–) mice were kept on normal water until they were 4–6 weeks of age. They were then placed on normal water or dox water and maintained on this regimen for an additional 1–3 months. At intervals, BAL cellularity and lung histology were evaluated. The cell recovery and cellular differentials of the BAL from transgene (–) mice on normal water and dox water and transgene (+) mice on normal water were almost identical at all time points that were evaluated (Fig. 3). In contrast, LIF induction caused an impressive increase in BAL cell recovery (Fig. 3). This increase could be appreciated after 1 month and was even more striking after 3 months of dox administration to transgene (+) mice (Fig. 3). This increase was largely the result of an increase in mononuclear cells with an increase in the percentage of BAL cells that were lymphocytes and an increase in lymphocyte and macrophage recovery (Fig. 3 and data not shown). Abnormalities in neutrophil recovery and eosinophil recovery were not noted.Fig. 3BAL cellularity of LIF transgenic mice. Transgene (+) and transgene (–) mice were randomized to normal water or dox water at 4 weeks of age and maintained on this regimen for up to 3 months. BAL cellularity was assessed after 1 and 3 months of dox administration (*, p < 0.05 versus other 4 groups).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of LIF on Lung Histology—Inflammation was not seen in the hematoxylin and eosin stains of the lungs from transgene (–) mice on normal or dox water. Similarly, inflammation was not noted in histologic sections from transgene (+) mice on normal water at all time points that were evaluated. In contrast, inflammation was noted in the lungs from transgene (+) animals. This response was made up almost entirely of lymphocytes. It was mild, occasionally nodular, and noted in peribronchiolar locations (Fig. 4). The periodic acid-Schiff with diastase stains and Alcian blue stains did not reveal mucus metaplasia or an alteration in histologically apparent mast cells. In addition, the trichrome stains and hematoxylin and eosin stains did not reveal airway wall or alveolar thickening, fibrosis, or remodeling in any of the transgenic animals (data not shown). When viewed in combination, these studies demonstrate that LIF elaboration in the lung causes a lymphocytic infiltrate without mucus metaplasia, eosinophilia, or airway or alveolar remodeling.Fig. 4Histology of LIF transgenic mice. Four-week-old transgene (–) and transgene (+) mice were randomized to normal water or dox water and maintained on this regimen 3 months. Hematoxylin and eosin stains were then used to evaluate the histologic features of these animals.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of the Lymphocytic Infiltrate—Studies were next undertaken to define the lymphocytic infiltrate that was present in lungs from dox-treated transgene (+) mice. This was done by isolating the parenchymal cells from transgene (+) and transgene (–) mice that had been randomized for 3 months to normal or dox water. FACS was then used to compare the surface markers on these cells. As can be seen in Fig. 5, differences in CD3, CD4, and CD8 cells were not readily appreciated. In contrast, an impressive increase in B220+ cells was appreciated in lungs from transgene (+) mice on dox for 3 months. These studies demonstrate that the majority of the lymphocytes in the lungs from the LIF transgenic mice are B lymphocytes.Fig. 5FACS analysis of cells in LIF transgenic mice. Four-week-old transgene (–) and transgene (+) mice were randomized to normal water or dox water and maintained on this regimen for 3 months. Lung cells were then isolated, and lymphocyte populations were characterized by FACS analysis as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of LIF in Hyperoxic Acute Lung Injury (HALI)—To investigate the contribution(s) that LIF makes in ARDS, we compared the survival in 100% O2 of transgene (–) and transgene (+) mice on normal water and dox water. As can be seen in Fig. 6, transgene (–) mice on normal water and dox water died after 3–5 days of 100% O2 exposure. In contrast, transgene (+) mice on dox water manifested a markedly enhanced survival with 100% of the animals living for at least 6 days and >50% living for 8 days or more in these hyperoxic conditions (p < 0.002). Interestingly, transgene (+) mice on normal water lived for a slightly longer interval than transgene (–) mice on normal or dox water. This difference, however, was not statistically significant.Fig. 6Effects of LIF on HALI. Four-week-old transgene (–) and transgene (+) mice were normalized to normal water or dox water and maintained on this regimen for 2 weeks. They were then placed in 100% O2, and survival and alveolar-capillary protein leak were assessed at intervals thereafter. A, we compare the survival of transgene (+) mice on dox water (solid circles), transgene (+) mice on normal water (open circles), transgene (–) mice on dox water (open triangles), and transgene (–) mice on normal water (closed triangles). Each curve illustrates the survival of at least eight animals (*, p < 0.002 versus the other three groups). B, the levels of BAL protein are evaluated. Each point represents the mean ± S.E. of evalua