Eur Resplr J 1992, 5, 739-747 Growth factor activity in the lung during compensatory growth after pneumonectomy: evidence of a role for IGF-1 R.J. McAnulty, D. Guerreiro, A.D. Cambrey, G.J. Laurent Growth factor activity in the lung during compensatory growth after pneumonectomy: Biochemistry Unit evidence of a role for IGF-1. R.J. McAnulty, D. Guerreiro, A.D. Cambrey, G.J. Dept of Thoracic Medicine Laurent. National Heart and Lung Institute ABSTRACT: Unilateral pneumonectomy In rats causes compensatory growth University of London London, UK of the remaining lung. During this growth, there are large Increases In the cell numbers and In the rates of collagen and non-collagen protein production. We Correspondence: R.J, McAnulty examined possible mechanisms by which these changes might occur. Biochemistry Unit Assessment of the effect of broncboalveolar lavage (BAL) nuld on fibroblasts National Heart and Lung Institute in vitro demonstrated the presence of stimulatory activity for flbroblast repll· Emmanuel Kaye Building cation In control animals. This activity was greatly Increased two and six Manresa Road days postpneumonectomy (115:26% and 75:18% above control values, respec· London SW3 6LR tively), but had returned to normal by 14 days. Preliminary characterization UK suggests that the activity is heat labile and consists of at least two moieties with Keywords: Bronchoalveolar lavage apparent molecular weights of 5-15 kD and 70-220 kD. The activity was extravascular albumin space partially blocked by antibodies to Insulin-like growth factor-1 (IGF-1), and levels fibroblast replication of IGF-1 were Increased by about 100% (p<0.001) two days after pneumonec· insulin-like growth factor-1 tomy compared with control values, pneumonectomy Examination of BAL cells demonstrated an early influx of Jeucocytes Into the remaining lung of pneumonectomized rats. At two days, about 25% of the Received: April 23 1991 lavageable cells were neutrophils, but macrophages were the predominant cell Accepted after revision January 25 1992 type at all times. The extravascular albumin space of the lung Increased by about 65% (p<0.01), six days after pneumonectomy. The Influx of circulatory proteins and cells are potential sources of the Increased mitogenic activity observed In the lung. Eur Respir J., 1992, 5. 739-747. The process of compensatory lung growth after par­ in culture. In conjunction with this we have investi­ tial pneumonectomy was first described more than a gated potential sources of such agents by examining century ago [1). The rapid growth includes large the cellular and protein influx into the right lung increases in cell number and total proteins, including after pneumonectomy. those of the extracellular matrix, of which collagen is the major component [2-4]. The mechanisms which control this growth are still poorly understood. Sev­ Methods eral factors have been implicated, including physical forces imposed by removal of lung tissue, changes in oxygen and carbon dioxide tension, altered blood flow Animals and hormonal effects [5-7). Mitogenic agents in serum have also been suggested to play a role in this Experiments were performed on male Lewis rats, an process. Enhanced levels of growth factors have been inbred strain originally derived from Wistar rats [10), described in the blood [7-9] but it is not known with weights of 192±2 (mean±SEM) g at the time of whether these compounds enter the circulation after operation. One group of rats was anaesthetized with production at a site remote from the lungs, or are pro­ Hypnorm (fentanyl citrate 0.315 mg·mi·l; fluanisone 10 1 1 duced in the lung and subsequently enter the circula­ mg·ml· ) at a dose of 1 ml·kg· body weight, admin­ tion. istered by intramuscular injection. The rats were intu­ In this study, we have investigated whether mito· bated with a 16 gauge catheter and ventilated by a genic agents are present in the lungs of normal ani· small animal respirator (Harvard Apparatus Ltd, mals, and in the right lung of animals that have Edenbridge, Kent, UK) at a rate of 54 breaths·min·1 undergone left pneumonectomy, by examining the and a stroke volume of 2.5 ml. The left side of the effects of bronchoalveolar lavage fluid on fibroblasts chest was opened at the fifth intercostal space, the 740 R.J. McANULTY ET AL. lung exposed, tied at the hilum and excised. The Fibroblast replication assay chest was sutured in layers, negative intrathoracic pressure re-established, and the animals allowed to The effect of BAL fluid on fibroblast replication was recover. assayed as described previously [11]. Preliminary ex­ Another group of rats was operated on in a similar periments were performed to determine the optimum way except that the left lung was not tied or removed assay conditions described below. The BAL fluid con­ (sham-operated controls). A third group of rats was centrates, were thawed, appropriate aliquots were cen­ used as unoperated normal controls. Animals were trifuged at 9,000xg for 2 min (Beckman microfuge B; studied in groups of at least five, at various times Beckman - RIIC Ltd, High Wycombe, Bucks, UK) and between 2-28 days after operation. serial twofold dilutions in DMEM were made. Assays were carried out in flat-bottomed 96 well tissue cul­ ture plates (Nunc Cat. No. 1-67008 96F; Nunc, Bronchoalveolar lavage (BAL) of rats and processing Kamstrup, Denmark). The outermost wells, rows of the fluid obtained A and H and columns 1 and 12, were not used for assay and were filled with 100 J.ll of DMEM contain­ Rats were deeply anaesthetized with intramuscular ing 2% NCS. This left the central 10x6 matrix of Hypnorm (1.5 mHg·1) and exsanguinated by opening wells for the assay. The wells in columns 2 and 11 the peritoneal cavity and cutting the aorta. A ventral were filled with 50 J.ll of DMEM for use as medium midline incision was made from the neck to the dia­ controls. Aliquots of 50 J.ll of diluted sample were phragm, the left main bronchus ligated and a cannula added to each of the six wells in columns 4 to 10. passed through the trachea into the right bronchus. Plates of confluent Rat 2 cells were then trypsinized, Normal saline at room temperature was instilled slowly transferred to a sterile polypropylene 50 ml centrifuge into the right lung, via the cannula, in 4 ml aliquots tube and centrifuged at 300xg for 5 min. The cells over a period of 15 s, left in the lung for 30 s, with­ were resuspended in DMEM containing 4% NCS at a 4 drawn over 15 s and collected in a 50 ml polypro­ concentration of 8x10 cells·ml·1• Aliquots (50 J.ll) of pylene centrifuge tube which was kept on ice. A total this cell suspension were added to each of the wells of 40 ml of saline was used and in all cases >95% in the inner 10x6 matrix of the assay plate, to give was recovered. 4,000 cells·well·1 in 100 ~-tl of DMEM with a final The BAL fluids were centrifuged at 4°C for 10 min concentration of 2% NCS. Cells were incubated for at 300xg. Supernatants were retained for further 48 h, after which the cell layer was washed with phos­ analysis. The cell pellets were resuspended in 10 ml phate buffered saline and fixed by adding 100 ~-tl of of Dulbecco's modified minimum essential medium 10% formol saline. (DMEM) and recentrifuged. The supernatant was dis­ Staining and spectrophotometric assessment of cell carded and the cell pellet resuspended in 1 ml of numbers were carried out exactly as described previ­ DMEM. An aliquot of 20 ~-tl was taken for a total ously [11]. Briefly, after fixing, the forrnol saline was cell count in an improved Neubauer counting cham­ removed and the cells stained by addition of 100 J.ll ber, after mixing with 20 ~-tl of a solution of 1% crys­ of methylene blue (1% in 0.01 M borate buffer, pH tal violet in 1% acetic acid. 8.5). Thirty minutes later the excess dye was removed Aliquots of 100 J.ll of the cell suspension were used by flicking and rinsing the stained monolayer four for cytocentrifuge preparations. These were centri­ times in 0.01 M borate buffer (pH 8.5). The dye was fuged for 10 min at 450 rpm (Cytospin 2, Shandon eluted from the cells with 100 ~-tl of a 1:1 solution of Southern Products Ltd, Runcorn, Cheshire, UK). The ethanol and 0.1 M HCl and the absorbance measured slides with the adhering cells were allowed to dry in at 650 nm using a microplate spectrophotometer. Val­ air and then stained with May Griinwald Giemsa stain. ues for cells incubated in the presence of BAL fluid The cell free BAL supernatants were exhaustively concentrates were compared with those for cells alone. dialysed against distilled water using a dialysis mem­ This method depends on the binding of methylene blue brane with a nominal molecular weight cut-off of 3.5 dye to negatively charged moieties within cells. kD. The dialysates were lyophilized, redissolved in Although these moieties are predominantly in poly­ 2 ml of DMEM and frozen at -40°C before analysis. nucleotides within the nucleus, some cytoplasmic mol­ ecules also bind the dye. Therefore, an in~rease in dye uptake could be effected by hypertrophy as well Fibroblast cultures as hyperplasia of cells. For this reason, changes in fibroblast replication observed using this method were A cloned rat embryonic fibroblast cell line was used confirmed by direct counting of cell numbers. for these experiments (Rat 2, obtained from the Ameri­ can Type Culture Collection, Rockville, Maryland, USA).