Lipocortin I Production by Human Alveolar Macrophages Marty P. Ambrose, Chi-Ling C. Bahns, and Gary W. Hunninghake Pulmonary Division, Department of Medicine, Department of Veterans Affairs, and University of Iowa College of Medicine, Iowa City, Iowa

Lipocortin I, in some cells, may be a potent inhibitor of phospholipase A z activity. These studies evaluated the relative amounts of lipocortin I in human alveolar macrophages compared with blood monocytes, using a specific polyclonal antibody and the technique of Western analysis. Lipocortin I was detected in all isolates of human alveolar macrophages and had molecular masses of 37,000 and 33,000 D. Corticosteroids increased amounts of lipocortin I in these cells in a dose-dependent manner. This effect was specific for corticosteroids as related steroids had no effect. Blood monocytes, when compared with alveolar macrophages, contained relatively small amounts of lipocortin I. We conclude that lipocortin I is present in relatively large amounts in human alveolar macrophages and that amounts of the protein can be induced by corticosteroids. We further speculate that the relative amounts of lipocortin I within monocytes/macrophages may be a marker of differentiation.

Although the exact function of lipocortin I (also called annexin I) in various types of cells is still controversial (1), it may, in some instances, be one of the mediators of the immunosuppressive activity of corticosteroids (2, 3). In this capacity, it may act by inhibiting the actions of phospholipase A z (PLA z) on arachidonate-containing phospholipids (4, 5). Lipocortin I does not directly inhibit PLA z• Instead, it appears to bind to phospholipids, thereby inhibiting the release of arachidonic acid. Previous animal studies have demonstrated that the lung contains large amounts of lipocortin I, compared with other organs (6). In addition, peritoneal macrophages are a rich source of lipocortin I in the rat (6). Previous studies from our laboratory have shown that lipocortin I is a normal constituent on the alveolar surface of the human lung and that corticosteroids increase amounts of lipocortin I in these areas of the lung (7). These studies evaluated whether human alveolar macrophages, which are the most prominent cells on the alveolar surface of the human lung, could be a source of lipocortin I in the lung and whether corticosteroids might affect the amounts of lipocortin I present in these macrophages. We further determined whether alveolar macrophages contain more lipocortin I than do blood monocytes, which would suggest that amounts of this protein might be a marker of differentiation in monocytes/macrophages.

(Receivedin originalform September 21, 1990 and in revisedform June 17,

1991) Address correspondence to: Gary W. Hunninghake, M.D., Room C33G, GH, University of Iowa Hospitals and Clinics, Iowa City, IA 52242. Abbreviations: bronchoalveolar lavage, BAL; lactic dehydrogenase, LDH; polyacrylamide gel electrophoresis, PAGE; phospholipase A2, PLA2; sodium dodecyl sulfate, SDS. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp, 17-21, 1992

Materials and Methods Subjects After giving informed consent, normal, lifetime nonsmoking volunteers underwent bronchoalveolar lavage (BAL) for isolation of human alveolar macrophages. The subjects were 21 to 36 yr of age and had no previous history of pulmonary disease or recent viral respiratory illness. The protocol for BAL of normal volunteers was approved by the Committee for Investigations Involving Human Subjects at the University of Iowa. HAL

BAL was performed as previously described (8). The lavage fluid was strained through two layers of gauze and centrifuged at 500 X g to separate the cells from the lavage fluid. The supernatant was decanted from the cell pellet and frozen at -70 0 C for later use. The cells were rinsed twice in Hanks' balanced salt solution before resuspending them in RPM~ 1640 containing 1% glutamine and 100 ttg/ml gentamicm. The cells were counted using a hemacytometer (Reichert Diagnostics, Buffalo, NY). Cytospin slide preparations were made and differential cell counts were obtained as previously described (8). Isolation of Blood Monocytes Peripheral blood monocytes were isolated from the normal volun~eers just before their undergoing BAL. The monocytes were Isolated by counter flow centrifugal elutriation, according to the method of Sanderson and associates (9). Briefly, mononuclear cells were separated from heparinized blood obtained by venipuncture from normal volunteers utilizing a Ficoll-Hypaque gradient as described by Boyum (10). The monocytes were then separated from the remaining mononuclear cells by counter flow centrifugation, utilizing a San-




derson elutriation chamber (Beckman Instruments, Irvine, CA) within a JE 6B rotor (Beckman) at 2,500 rpm. Culture Conditions Human alveolar macrophages or blood monocytes were incubated at 3r C in a 5 % CO 2 atmosphere in flat-bottomed tissue culture plates (2-cm 2 surface area; Costar, Cambridge, MA) for 24 h. The cells were plated at a density of lQ6 cells/ml, They were cultured in serum-free medium (RPMI 1640), with and without various amounts of steroids (all from Sigma Chemical Corp., S1. Louis, MO). In some instances, the cells were cultured in medium with 5 % fetal calf serum. Supernatants were harvested and stored at -70 0 C for later use. Celllysates were obtained by placing the cells in 1.0 ml of 0.1% Triton X-100 for 30 min at 4 C. The lysates were then stored in the same fashion as the supernatants. Cell viability was determined by measuring release of lactate dehydrogenase (LDH) activity as previously described (11). Total protein was assayed using the Coomassie blue dye binding assay of Bradford (12). 0

Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Electrophoresis (pAGE) SDS-PAGE was performed as described by Laemmli (13). All samples were denatured at 95 0 C for 10 min and applied for electrophoresis. Resolving gels consisted of 15 % acrylamide (2.6% as bis acrylamide), 0.375 M Tris (pH 8.8), and 0.1% SDS. Stacking gels consisted of 3.75% acrylamide, 0.125 M Tris-HCl (pH 6.8), and 0.1% SDS. The gels were polymerized with ammonium persulfate and N,N,N',N' tetramethylethylenediamine. The SDS-PAGE sample buffer consisted of 0.0625 M Tris-Hel (pH 6.8), 10% glycerol, 5 % 2-mercaptoethanol, 2 % SDS, and 0.002 % bromophenol blue. The reservoir buffer consisted of 0.025 M Tris, 0.192 M glycine, and 0.1% SDS. The gels were electrophoresed with a 30-mA constant current until the dye front reached the bottom of the gel. Electroblotting After electrophoresis, the gels were electroblotted to 0.45p.mnitrocellulose (Bio-Rad Laboratories, Richmond, CA) at a constant voltage of 30 V overnight or 60 V for 5 h according to the method of Towbin and colleagues (14). The electroblotting buffer consisted of 0.025 M Tris, 0.192 M glycine, and 20% vol/vol methanol (pH 8.3). After electroblotting, lipocortin I was detected utilizing the method of Burnette (15). Briefly, the nitrocellulose membranes were blocked with 3 % gelatin, followed by exposure to a highly specific polyclonal anti-lipocortin I antibody (kindly provided by R. B. Pepinsky) (16, 17). Recombinant human lipocortin I and lipocortin III were also kindly provided by R. B. Pepinsky. The antibody-protein conjugates were visualized by washing the membranes with 125I-labeled protein A (New England Nuclear Products, Boston, MA) and subsequent e.xposure to X-ray film for 16 h at -70 C.







Figure 1. Western blotof separate alveolarmacrophage celllysates

for lipocortinI fromsix normalvolunteers. Lane A: cDNAderived lipocortinI standard(50 ng); lanes B through G: celllysates of human alveolar macrophages from six normal volunteers, loaded at 50 /Lg/well. titated, using a gamma counter (Gamma 5500; Beckman). Background emission was determined by evaluating a control piece of the same nitrocellulose of identical size. The data were expressed as a percentage increase in the cells exposed to corticosteroids compared with unstimulated cells.

Results Cell Populations For the alveolar macrophage suspensions, 92 to 95 % of the cells were macrophages and the rest were lymphocytes. For the monocyte suspensions, 85 to 90% of the cells were monocytes and the remainder of the cells were lymphocytes. Presence of Lipocortin I in Human Alveolar Macrophages Lipocortin I was detected in all isolates of human alveolar macrophages from normal subjects (Figure 1). Immunoreactive bands of lipocortin I were detected with molecular masses of 37,000 and 33,000 D, as have been previously described (19-21). Although the relative amounts of these two species of lipocortin I differed, alveolar macrophages from all subjects expressed both of these two species. Occasionally, a third band with a molecular mass of 36,000 D was identified (Figure 3). The antibody used to detect human lipocortin I was specific for this protein and did not crossreact with a closely related human protein, lipocortin III



Estimation of Relative Amounts of Lipocortin I To determine if corticosteroids increase amounts of Iipocortin I in alveolar macrophages, portions of the nitrocellulose that contained the immunoreactive bands oflipocortin I were carefully excised, and the amount of radioactivity was quan-

Figure 2. Specificity of the anti-lipocortin I antibody forhumanlipocortin I compared with lipocortin III. Lane A: lipocortin I standard (50 ng/well); lane B: lipocortin III standard (50 ng/well); lane C: macrophage cell lysate (100 /Lg protein/well).


Ambrose, Bahns, and Hunninghake: Lipocortin I and Human Alveolar Macrophages







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Dexamethasone log 1M)

Figure 3. Comparison of alveolar macrophage cell lysate products with bronchoalveolar lavage fluid proteins for lipocortin I. Lane A: lipocortin I standard; lanes B and C, D and E, F and G, and Hand I: matched pairs of macrophage celllysates (lanes B, D, F, and H) with lavage fluid proteins (lanes C, E, G, and I). Lanes loaded at 100 IJ.g protein/well.

(Figure 2). These results differ from studies in rats in which anti-lipocortin I antibodies cross-reacted with lipocortin III (21). Lipocortin I was released in small amounts from alveolar macrophages (data not shown). The same species of Iipocortin I was present in the celllysates and in the cell supernatants. Furthermore, the same relative amounts of the different species of lipocortin I were detected in alveolar macrophages and in BAL fluid, suggesting that these cells might be a source of the lipocortin I found on the alveolar surface of the human lung (Figure 3).

Figure 5. Dose-response curve demonstrating the expression of lipocortin I in alveolar macrophages as a function of dexamethasone dose. A significant dose-dependent increase in lipocortin I is observed. See text for discussion.

tin I in the cells in a dose-dependent manner (Figures 4 and 5). The effect was maximal at a concentration of 10-' M dexamethasone. At this concentration, dexamethasone increased the amounts of lipocortin I in the cells 286 ± 90 %, when compared with control levels. The increase observed at 10-' M was significantly greater than the increase observed at 10-9 M (P < 0.05). With a concentration of 10-3 M dexamethasone, no increases in amounts of lipocortin I were detected. This latter effect can be attributed to a toxic effect of dexamethasone at this concentration as it was associated with an increased release of LDH (71 ± 7.9% release ofLDH). An increase in cytotoxicity was not observed in macrophages exposed to 10-5 M dexamethasone when compared with LDH release of unstimulated macrophages

Effect of Corticosteroids Corticosteroids. when added to human alveolar macrophages under serum-free conditions, increased amounts of lipocor

Lipocortin I production by human alveolar macrophages.

Lipocortin I, in some cells, may be a potent inhibitor of phospholipase A2 activity. These studies evaluated the relative amounts of lipocortin I in h...
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