Specific pathogen-free. Wistar female rats ..... synthase, we used L-NMMA, a specific enzyme inhibi-. 2 tor (17). L-NMMA ..... S., R. M. J. Palmer, and E. A. Higgs.
Nitric oxide production by rat alveolar can be modulated in vitro by surfactant
macrophages protein A
HANNA BLAU, SHOSHANA RIKLIS, J. FREEK VAN IWAARDEN, FRANCIS X. McCORMACK, AND MOSHE KALINA Department of Cell Biology and Histology, Pulmonary Department, Children’s Medical Center of Israel, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; Department of Cell Biology and Immunology, Vrije Universiteit, Amsterdam 1081 BT, The Netherlands; and Divisions of Pulmonary I Critical Care Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Blau, Hanna, Shoshana Riklis, J. Freek Van Iwaarden, Francis X. McCormack, and Moshe Kalina. Nitric oxide production by rat alveolar macrophages can be modulated in vitro by surfactant protein A. Am. J. Physiol. 272 (Lung Cell. Mol. PhysioZ. 16): Lll98-Ll204, 1997.Alveolar macrophage and type II cells are known to generate nitric oxide, which is a highly reactive molecule that plays a role in host defense against pathogens, as well as tissue damage associated with inflammation in the lung. Both types of cells are known to generate the nitric oxide by inducible nitric oxide synthase (INOS). Surfactant-associated protein A @P-A) from various sources (human alveolar proteinosis, rat and recombinant rat) was found to upregulate nitric oxide production by alveolar macrophages in a concentrationand time-dependent manner, whereas type II cells were unresponsive to SP-A. The increase in nitric oxide production was associated with elevation in the expression of iNOS. However, only 30-50% of the cells responded by expressing iNOS, as was observed by immunofluorescence staining. The stimulatory effect of SP-A was found to be 3050% lower than the known nitric oxide agonists interferon-y (IFN-?I) and lipopolysaccharide (LPS). However, addition of the cytokines interleukin-l or granulocyte macrophage colony-stimulating factor elevated the levels of nitric oxide production to that of LPS and IFN-)I. Special attention was given to exclude the possibility that contaminating LPS in the various SP-A species stimulated nitric oxide production by the macrophages. Our results indicate that SP-Ais the agonist and not a contaminating LPS. The data presented in this report extend our knowledge regarding the nonsurfactant-related functions of SP-A.
various host defense functions in the lung (20). SP-A is the most abundant of the surfactant-associated proteins. Some of the nonsurfactant-related functions include augmentation of alveolar macrophage migration (30), enhancement of macrophage phagocytosis (23,27), and regulation of reactive oxygen species production (27). Recently, it was found that SP-A can stimulate both alveolar type II cells and macrophages to secrete granulocyte macrophage colony-stimulating factor (GM-CSF; see Ref. 1) and alveolar macrophages to secrete TNF-ar (12). The nonsurfactant-related function of SP-A encouraged us to test a possible modulatory effect of SP-A on nitric oxide production by alveolar macrophages and to compare it with that of IFN-y and LPS. Our results indicate that, in vitro, SP-A may indeed upregulate iNOS and nitric oxide production by alveolar macrophages. This extends our knowledge in regard to the multifunctional roles of SP-A in the lung. MlATERIALS
AND
METHODS
STUDIES have demonstrated that nitric oxide plays a role in host defense against pathogens, as well as in tissue damage associated with inflammation in the lung (3, 7, 8). One of its main sources in the lung is the alveolar macrophage, which is known to generate large quantities of nitric oxide by inducible nitric oxide synthase (iNOS; see Ref. 9). Upregulation of this enzyme in alveolar macrophages was obtained by inflammatory stimuli, such as interferon-y (IFN-7) and lipopolysaccharide (LPS) as well as tumor necrosis factor-a (TNF-CY) and interleukin (IL)-lf3 (4,11,X5,19). Recently, it was found that alveolar type II cells and some of their secretory products, including surfactantassociated protein A (SP-A) and D, have nonsurfactantrelated functions and are actively associated with
Cells and culture conditions. Specific pathogen-free Wistar female rats (200 g) were used in most experiments. Alveolar macrophages were obtained from rat lung lavages as previously described (6). Rat lungs were lavaged eight times, each time with 8 ml salt solution containing (in mM) 140 NaCl, 5 KCl, 2.5 sodium phosphate buffer, 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 6 glucose, and 0.2 ethylene glycol-bis( P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, pH 7.40. The pooled lavage fluid was centrifuged, and the pelleted cells were plated in 96-well tissue culture plates (Corning, NY) at 1 X lo5 cells in Dulbecco’s modified Eagle’s medium (DMEM) and were allowed to adhere for 1 h at 37°C in 5% COB in air, followed by removal of nonadherent cells; then the cells were incubated for various lengths of time in DMEM supplemented with 10% fetal calf serum (FCS). The alveolar macrophage preparation was found to be 98% pure as was assessed by immunostaining with a specific rat macrophage antibody ED1 (details in Immunocytochemistry). Type II cells were isolated by tissue dissociation with elastase and immunoglobulin G panning by the method of Dobbs et al. (6). Cells were plated in 96-well tissue culture plates at 2 x lo5 cells per well in DMEM-FCS medium. The cells were 90 2 3% pure as was assessed by staining the cells for alkaline phosphatase. Pulmonary epithelial (PE) cells were obtained according to Kalina et al. (10) after low-density plating of primary type II cells cultured with conditioned media generated by alveolar macrophages and 0.5 pg/ml insulin. PE cells were proliferated in colonies and were harvested after 14 days. In the experiments testing nitric oxide release, the cells were re-
Lll98
the American
inducible nitric ride; interferon-y
oxide synthase;
type II cells; lipopolysaccha-
PREVIOUS
1040-0605/97
$5.00
Copyright
o 1997
Physiological
Society
SP-A AND
NITRIC
plated in 96-well tissue culture plates at 1 X lo5 cells/well in DMEM-FCS medium. Rat peritoneal macrophages and lung fibroblasts were obtained as previously described (10) and were plated at 1 X lo5 cells/well in 96-well plates. Measurement of nitrite in cell culture supernatant. Cells (1 X 105) in the 96-well plates were incubated for various lengths of time (as indicated) with and without the various additions (SP-A, LPS, cytokines, or growth factors). Nitric oxide, quantified by the accumulation of nitrite in the culture medium, was measured using the Greiss reaction; sodium nitrite was used as the standard (18). Briefly, 50 ~1 of culture medium aliquots were mixed with 50 ~1 of Greiss reagent and were incubated at room temperature for 10 min. The absorbency at 550 nm was measured in a microplate reader. In some experiments, nitrate was reduced to nitrite by nitrate reductase (EC 1.6-6.2) from Aspergillus species (Sigma, St. Louis, MO). Briefly, 0.1 units of reductase were added to 50 ~1 culture medium in the presence of 2.5 nM reduced NAD phosphate and were incubated for 3 h at 37°C (29). The products were then measured as nitrite; the combination of nitrite and reduced nitrate was designated nitrite-nitrate. Preliminary experiments indicated that added SP-A did not interfere with either the Greiss reaction or the reduction of nitrate to nitrite. Data from cultures are presented either as micromolar nitrite or as nanomoles nitrite per 1 X lo5 cells. The quantitative data are presented as means t SE. Statistical comparisons were made using a two-tailed Student’s t-test. Immunocytochemistry. Alveolar macrophages were grown on coverslips and were fixed in acetone-l% paraformaldehyde for 5 min. The cells were immunostained with the primary monoclonal antibody to murine iNOS (diluted 1:lOO). The cells were incubated overnight at 4°C washed in phosphatebuffered saline, and further processed by using a secondary rhodamine-labeled donkey anti-rabbit antibody (Jackson Laboratories, West Grove, PA). The purity of the macrophage preparation was assessed by immunostaining the cells with the specific rat macrophage monoclonal antibody ED1 (5). Cell preparations were examined and photographed with a Zeiss microscope equipped with epifluorescence. In some experiments, the fluorescent cells were counted (IO fields, 300 cells) and were expressed as percentage of stained cells from total cell number. Stimulation of the cells to produce nitric oxide. Macrophages were stimulated to produce nitric oxide by the various additives either immediately (after removal of the nonadherent cells) or after 18 h in culture with identical results. Therefore, most experiments with alveolar macrophages were conducted with immediate addition of the various additives, and then cells were incubated for 48 h unless otherwise stated. Type II and PE cells, peritoneal macrophages, and alveolar fibroblasts were stimulated after adhesion for 18 h in culture and then for an additional 48 h with the various additives. In some control experiments, the cells cultured for 48 h with the various additives were tested for their number and viability. The cells were removed from the wells with trypsin (0.25%)-EDTA (0.05%), stained with trypan blue, and counted. Both LPS (Escherichia coli, 55:135; Difco, Detroit, MI) and recombinant rat IFN-y (Genzyme, Cambridge, MA) were used to stimulate the cells to produce nitric oxide as positive controls to the effect of SP-A. Rough LPS from E. coli strain 35 (Rc mutant) and lipid A from E. coZi F583 (AC mutant; Sigma) were used in some experiments.
OXIDE
L1199
PRODUCTION
SP-A from human and rat sources were used in the present study. Most of the experiments were conducted with human SP-A isolated from patients with alveolar proteinosis (AP SP-A) as previously described (31). Rat SP-A was isolated from rats 4 wk after intratracheal installation of silica and was purified as previously described (13). A recombinant wild-type rat SP-A that was expressed in a baculovirus expression system (16) was kindly provided by Dr. F. X. McCormack, Pulmonary/Critical Care Medicine, University of Cincinnati. The content of contaminating LPS in the SP-A was tested by using the Limulus amebocyte lysate (LAL) reagent and the kinetic methodology. The LAL-5000E automated endotoxin detection system (Atlas Bio-scan) was used for the detection. LPS content in the AP SP-A and rat SP-A was found to be ~25 pg/l pg SP-A. In another set of experiments, various cytokines were added to the incubated cells to test their effect on nitric oxide production. These include GM-CSF, IL-l (Advanced Magnetic, Cambridge, MA), and IL-6 (Genetic Institute, Boston, MA). In some experiments, polymyxin B (Sigma), which is known to inhibit LPS (22), was added at a concentration of IO pg/ml. In another set of experiments, an inhibitor of iNOS, NG-monomethyl+arginine (L-NMMA; Calbiochem, La Jolla, CA), was used at a concentration range of lo-500 PM. RESULTS
Alveolar macrophages obtained from rats and stimulated in vitro with SP-A from various sources showed a significant increase in nitric oxide production compared with unstimulated cells (Fig. 1). Data are presented in Fig. 1 as nitrite or as nitrite-nitrate after reduction of nitrate. Our data indicate that the ratio of nitrite to nitrate that resulted from stimulation with SP-A was similar to that observed with LPS and 1FN-y used as agonists. In most experiments, the ratio of nitrite to
A
B
C
D
E
Fig. 1. Nitric oxide production by alveolar macrophages (1 X lo5 cells/well) stimulated by various agonists and cultured for 48 h. A: control unstimulated cells; B: cells treated with lipopolysaccharide (LPS, 1 pg/ml); C: cells treated with interferon-y (IFN-y, 25 U/ml); D: cells treated with alveolar proteinosis (AP) surfactant protein A (SP-A, 1 pg/ml); E: cells treated wtih rat SP-A (1 pg/ml); and F: cells treated with recombinant rat SP-A (1 pg/ml). Supernatants were collected and analyzed for nitrite content (open bars) and after reduction of nitrate to nitrite (nitrite-nitrate content; filled bars). Each value is the mean + SE of triplicate samples from 1 of 5 similar experiments. All stimulated samples (B-F) are significantly different (P < 0.05) from control (A).
L1200
SP-A AND
NITRIC
nitrate was 1:0.8-1.1. The SP-A molecules used for stimulation were AP SP-A, rat SP-A, and recombinant rat SP-A. Although most of the experiments were conducted with AP SP-A, additional species of SP-A were tested to emphasize the general nature of the phenomenon. Levels of nitric oxide production after stimulation with SP-A were compared with those after stimulation with the known agonists for nitric oxide production, LPS and IFN-?I. Concentrations of 1 pglml LPS and 25 U/ml IFN-y used as agonists resulted in a maximum generation of nitric oxide by alveolar macrophages in the conditions of our assay (data not shown). The “spontaneous” release of nitric oxide from unstimulated macrophages was low, and the levels of nitrite found in the supernatants did not exceed 10 nmol./l X lo5 cells. Experiments in which the spontaneous release was higher (-15% of the rats) were discarded, as the lungs W vere considered inflamed, and the macroPha .ges we re already stimulated. Due to the possibility that cell number or viability could have changed with the various treatments, we carried out viability tests and cell counts after incubation with the various additives. Results shown in Fig. 2 indicate a minimal cell and viability loss with the various additives. Generation of nitric oxide by alveolar macrophages stimulated by SP-A was concentration and time dependent (Figs. 3 and 4). All experiments indicate that the maximal level of response was obtained at l-5 pg/ml SP-A and at 48 h of incubation. An increase in the concentration of SP-A up to 30 pg/ml did not alter this level of response. The maximal response to SP-A was always lower than the maximal response for LPS (1 pg/ml) or IFN-?/ (25 U/ml). These optimal levels of LPS and IFN-7 are similar to those previously described (21, 29). The concentration of SP-A that resulted in a maximal response (l-5 pg/ml) was similar
Fig. 2. Effect of various additives on cell number and viability of alveolar macrophages. Alveolar macrophages at 1 X lo5 cells/well were cultured for 48 h with various additives as follows: A: control; B: LPS (1 pg/ml); C: IFN-y (25 units); D: AP SP-A (1 pg/ml); E: AP SP-A (2.5 pg/ml); F: rat SP-A (1 pglml); and G: recombinant rat SP-A (1 pg/ml). Cells were removed from wells with trypsin-EDTA, stained with trypan blue, and counted. Results shown are means + SE of cells/well from 3 experiments, each performed in triplicate. No significant differences (P < 0.05) were found between various samples.
OXIDE
PRODI 50
CTION
1
I 4
I 2
0
I 8
I 6
I 10
p g SP-NmP
Fig. 3. Concentration-dependent stimulation of alveolar macrophage nitric oxide production by AP SP-A (0) and rat SP-A ( n ). Cells (1 -X 105/well) were cultured with the agonists for 48 h, when supernatants were collected and analyzed for nitrate content. Each value is the mean 2 SE of triplicate samples from 1 of 4 similar experiments.
to that described for SP-A in other biological systems. i.e., stimulation of type II cells and alveolar macrophages to secrete cytokines (1). To determine whether the increase in production of nitric oxide by SP-A and other agonists was due to alterations in cellular iNOS, we immunostained the macrophages with a monoclonal antibody to murine iNOS. Almost all of the cells were stained with EDl, a specific antibody to rat macrophages (Fig. 50). Control unstimulated cells were unstained (Fig. 5A), whereas cells stimulated with 25 U/ml IFN-y or AP SP-A (1 pg/ml) were stained (Fig. 5, B and C). It is important to note that only 30-50% (a range of staining in 3 experiments) of the cells were stained with both agonists. Thus not all of the macrophages express iNOS under these I
A
B
c
D
Fig. 4. Time-dependent generation of nitric oxide by alveolar macrophages (1 X lo5 cells/well) stimulated by IFN-y (A), LPS (B), AP SP-A (C), and rat SP-A (D). lime 0 is not presented, as no measurable nitrite was observed in the samples. Supernatants were collected and analyzed for nitrite content. Each value is the mean + SE of triplicate samples from 1 of 3 similar experiments. Open bars, 16 h; filled bars, 30 h; hatched bars, 48 h.
SP-A
AND
NITRIC
OXIDE
PRODUCTION
Fig. 5. Alveolar macrophages immunostained with antibody to inducible nitric oxide synthase (iNOS; A-C) and with the macrophage marker ED1 (0). Alveolar macrophages (1 x lo5 cells) were cultured on coverslips for 48 h with various additives. A: control unstimulated cells: B: cells stimulated with LPS (1 WE/ml); C: cells stimulated with AP SP-A(1 pug/ml); D: control cells immunostained with EDl. Note lack of fluorescence-in cells in A compared with B and C. Only a limited number of macrophages in B and C express iNOS compared with stained cell in D (immunostained with macrophage marker EDl). Bar = 20 PM.
experimental conditions. An increase in the SP-A concentration (between 1 and 5 pglml) did not alter the percentage of responding cells. These results are in agreement with those presented by Warner et al. (29) that only part of the macrophages express iNOS after treatment with various agonists. To confirm that the alveolar macrophages were producing reactive nitrogen intermediates via nitric oxide synthase, we used L-NMMA, a specific enzyme inhibitor (17). L-NMMA was found to cause a dose-dependent inhibition of nitric oxide production by the alveolar macrophages at a concentration range of lo-500 JJM with all tested agonists (Fig. 6). In further studies, we analyzed the additive or synergistic effect to SP-A of various cytokines and growth factors that are known to modulate nitric oxide production in various cell types (4, 11, 15, 19). Results summarized in Table 1 show that both IL-lo and GM-CSF. at the concentrations used (l-10 r&ml and 50 U/ml; respectively), did not have any stimulatory effect by themselves but enhanced nitric oxide production when added together with SP-A. Addition of these cytokines together with SP-A increases nitric oxide production to the levels of LPS stimulation. IL-6 did not
75
50
I
2 2 2
25
0 A __
B
C
Fig. 6. Inhibitory effect of NC-monomethyl-L-arginine (L-NMMA) on nitric oxide production by alveolar macrophages. Cells (1 X 105/well) were cultured for 48 h with LPS (A). AP SP-A (B). and rat SP-A (C) together with iNOS inhibitor L-NMkA. Open bars, controls; filled bars, 100 ,GM L-NMMA, hatched bars, 500 PM L-NMMA. Supernatants were collected and analyzed for nitrite content. Each value is the mean + SE of triplicate samples from 3 similar experiments (P < 0.05). All samples from L-NMMA-treated wells were significantly different from controls.
L1202
SP-A
AND
Table 1. Effect of LPS and various cytohines or in combination with SP-A on nitric oxide production by type II cells Nitrite, Treatment
Medium LPS (1 pg/ml) IL-la (10 rig/ml) GM-CSF (50 U/ml) IL-6 (100 U/ml)
Control
5.6+ 1.9 125.5 * 9.5
4.520.8 4.221.2 3.920.7
nmol/l
NITRIC
OXIDE
PRODUCTION
alone
x lo5 cells SP-A
(1 pg/ml)
72.4-+2.5* 127 +- 10.3
128.2+7.5*j142.4&9.5*? 74.2+4.0*
Each value is the mean + SE of triplicate samples of a representative experiment from a total of 3 experiments. LPS, lipopolysaccharide; SP-A, surfactant protein A; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor. Alveolar macrophages (1 X lo5 cells/well) were incubated for 48 h with various stimulating agents added alone (control) or in combination with SP-A. Supernatants were collected and analyzed for nitrite content. ‘1:Significantly different from control experiment (P < 0.05). 7 Significantly different from experiment with SP-A alone (P < 0.05).
have any effect on nitric oxide production, either alone or combined with SP-A. A number of experiments were conducted to eliminate the possibility that contaminating LPS in our SP-A preparation is responsible for the stimulatory effect of nitric oxide production by alveolar macrophages. All of the SP-A preparations contained ~25 pg LPS/l lug SP-A as was indicated by the Limulus test (see MATERIALS AND METHODS). It was found that low concentrations (lo-100 pg/ml) of LPS did not stimulate the macrophages to produce nitric oxide above the level produced by control unstimulated cells (data not shown). Moreover, in some experiments, polymyxin B, a known inhibitor of LPS (22), was added together with SP-A to the cells. It was found that polymyxin B (10 pglml) blocked almost 80% of nitric oxide production after stimulation of alveolar macrophages by LPS, rough LPS (Rc mutant), and lipidA(Fig. 7). However, addition of polymyxin B to the various SP-A preparations had only a small inhibitory effect (
LPS
8.7kl.3 1.2450.7
68.3+5.2* l.0-t0.9
145.2+12.5* 24.4+5.5*
97.5+7.5* 10.4?2.4*
3.6-tl.2
2.4kl.O
148.0-+15.1*
59.0+4.5*
4.4kl.5
4.9k2.3
38.452.8'"
29.0t5.4'"
2.421.0
2.9kl.2
3.5kl.7
2.6kl.2
(1
pug/ml)
Each value is the mean +- SE of triplicate wells from 3 similar experiments. IFN-y, interferon-y; AP, alveolar proteinosis. Various cells (1 X lo5 cells/well) were cultured for 48 h either alone (control) or with various additives. Collected supernatants were analyzed for nitrite content. * Significant 1y different from control, corresponding cells cultured without additives (P < 0.05).
SP-A
AND
NITRIC
The increase in nitric oxide production was associated with elevation in the expression of iNOS after the exposure of alveolar macrophages to SP-A. However, only 30-50% of the alveolar macrophages responded by expressing iNOS, as was observed by the immunofluorescence staining. The percentage of the responding cells was similar to that observed with LPS or IFN-y and was similar to the results presented recently by Warner and colleagues (29) using the same agonists either in vitro or in vivo. These authors found a similar pattern of limited cell responsiveness to various agonists with in vivo- and in vitro-stimulated type II cells. At the present time, the reason for this phenomenon is unclear. Special attention was given to exclude the possibility that contaminating LPS in the various SP-A species stimulated nitric oxide production by alveolar macrophages. Our results indicate that SP-A is the agonist and not contaminating LPS. 1) The Limulus test indicates that ~25 pg/l lug SP-A of LPS is in all of the SP-A preparations, a level that was not sufficient to stimulate a measurable production of nitric oxide by the cells. 2) The LPS inhibitor polymyxin B inhibited alveolar macrophage nitric oxide production by ~20% when cells were stimulated with SP-A. Over 80% inhibition occurred when cells were stimulated with LPS or when LPS derivatives [rough LPS (Rc mutant) and lipid A] were used as agonists. The limited inhibitory effect of polymyxin B in the SP-A-induced alveolar macrophages may be related to a low level of LPS contamination in the SP-A preparations. These low levels of LPS do not have any stimulatory effect by themselves but may have a synergistic effect with SP-A that is eliminated by the inhibitor polymyxin B. 3) Type II cells, PE cells, and peritoneal macrophages were stimulated by LPS to produce nitric oxide but not by the various species of SP-Aused as agonists. SP-A is the most abundant of the surfactantassociated proteins. Mounting evidence supports a central role for SP-A in nonsurfactant host defenserelated functions. These include stimulation of chemotaxis of alveolar macrophages (30), regulation of reactive oxygen species production by stimulating alveolar macrophages and circulating neutrophils (27), and enhancement of macrophage phagocytosis of opsonized or nonopsonized bacteria (23, 27) or viruses (26). Recently, it was suggested that SP-A plays a part in the cytokine network in the lung. It was found that SP-A can stimulate alveolar macrophages and type II cells to secrete colony-stimulating factors, such as GM-CSFand IL-3-like factors (1). SP-A was also found to stimulate secretion of TNF-a by alveolar macrophages (12). Peritoneal macrophages did not generate nitric oxide in response to SP-A probably because of the lack of a known receptor. However, alveolar type II cells are capable of binding SP-A either via the specific receptors for SP-A or the carbohydrate-binding domain on the SP-A molecule (14, 24). It is therefore surprising that type II cells did not respond by nitric oxide generation after stimulation by SP-A but responded to LPS and IFN-y, which were used as agonists. Similarly, PE cells
OXIDE
L1203
PRODUCTION
produced nitric oxide in response to LPS and IFN-y but not in response to SP-A. In contrast, both type II and PE cells were previously found to respond to SP-A by secreting cytokines (1). It therefore may suggest specific interaction(s) between SP-A and alveolar macrophages to induce iNOS but not with the other cell types studied. However, the nature of such an interaction(s) is unknown at the present time. Little is known about the alveolar defense system of the lung, which includes cells (alveolar macrophages, type II cells, and lymphocytes), surfactants (lipids and proteins), and a complex network of cytokines (for review, see Ref. 20). The relative contribution of the various components of the defense system is unknown. In vitro, SP-A can upregulate a variety of alveolar macrophage functions, including nitric oxide production. There are a limited number of studies in vivo, in either humans or animal models, that describe the immunoregulatory roles of SP-A (2). In vivo, alveolar macrophages are continuously in contact with surfactant and SP-A and yet are not permanently activated. The low level of macrophage activation in normal rats can be observed in the present communication, as the levels of spontaneous production of nitric oxide in control unstimulated cells are very low (X0 nmol/105 cells). Moreover, the unstimulated alveolar macrophages did not express iNOS after immunostaining. Warner and colleagues (29) found that spontaneous release of nitric oxide and iNOS expression in alveolar macrophages was found in cells obtained from inflamed but not from normal lungs. It seems that in vivo nitric oxide production by alveolar macrophages is suppressed by components of the alveolar defense system that prevent permanent activation of these cells under normal conditions. The nature of such a suppression is unknown at present and is currently being investigated by us. This study was supported by a grant from the Chief Scientist, Ministry of Health, Israel. Address for reprint requests: M. Kalina, Dept. of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel. Received
28 November
1995;
accepted
in final
form
7 March
1997.
REFERENCES Blau, H., S. Riklis, V. Kravtsov, and M. Kalina. Secretion of cytokines by rat alveolar epithelial cells: possible regulatory role for SP-A. Am. J. Physiol. 266 (Lung CeZZ. Mol. Physiol. 10): Ll48-Ll551994. Crapo, J. D., and J. R. Wright. SP-A deficiency-a cause or consequence of inflammation in bronchopulmonary dysplasia. Am. J. Respir. Crit. Care Med. 151: 595-596,1995. Dawson, V L., T. M. Dawson, E. D. London, D. S. Bredt, and S. H. Snyder. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. NatZ. Acad. Sci. USA 88: 63866371,199l. Denis, M. Tumor necrosis factor and granulocyte macrophagecolony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium auium and to kill avirulent M. auium. Killing effector mechanism depends on the generation of reactive nitrogen intermediates. J. Leukoc. BioZ. 48: 380-387,199l. . Dijkstra, C. D., E. A. Dopp, P. Joling, and G. Kraal. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulation in the rat recognized by the
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monoclonal antibodies EDl, ED2, and ED3. Immunology 54: 589-601,1985. Dobbs, L., G. R. Gonzalez, and M. C. Williams. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134: 141-145, 1986. Gaston, B., J. M. Drazen, J. Loscalzo, and J. S. Stamler. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 149: 538-551,1994. Ialenti, A., A. Ianar, S. Moncada, and M. Dir Rosa. Modulation of acute inflammation by endogenous nitric oxide. Eur. J. Pharmacol. 211: 177-182,1992. Jorgens, P. G., F. J. Van Overveld, H. Bult, P. A. Vermeire, and A. G. Herman. L-Arginine-dependent production of nitrogen oxides by rat pulmonary macrophages. Eur. J. Pharmacol. 200: 205-209,199l. Kalina, M., S. Riklis, and H. J. Blau. Pulmonary epithelial cell proliferation in primary culture of alveolar type II cells. Exp. Lung Res. 19: 153-175,1993. Klebanoff, S. J. Oxygen metabolites from phagocytes. In: Inflammation -Basic Principles and Clinical Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1992, p. 541-588. Kremlev, S. G., and D. S. Phelps. Surfactant protein A stimulation of inflammatory cytokine and immunoglobulin production. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L712-L719,1994. Kuroki, Y., R. J. Mason, and D. R. Voelker. Pulmonary surfactant apoprotein A structure and modulation of surfactant secretion by rat alveolar type II cells. J. BioZ. Chem. 263: 3388-3394,1988. Kuroki, Y., R. J. Mason, and D. R. Voelker. Alveolar type II cells express a high affinity receptor for pulmonary surfactant protein A. Proc. NatZ. Acad. Sci. USA 85: 5566-5570,1988. Lavnikova, M., J. C. Drapier, and D. L. Laskin. A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity. J. Leukoc. BioZ. 54: 322-328,1993. McCormack, F. X., H. Calvert, D. L. Smith, and D. R. Voelker. Surfactant protein A modified genetic deletion of the carbohydrate and collagen-like domains regulates the secretion of surfactant from type II cells (Abstract). FASEB J. 6: A371, 1992. Moncada, S., R. M. J. Palmer, and E. A. Higgs. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Reu. 43: 109-142,199l. Nicholas, D. J. D., and A. Nason. Nitrite determination by diazotization and coupling reactions. In: Methods in Enzymology, edited by S. P. Colowick and N. 0. Kaplon. New York: Academic, 1957, vol. 3, p. 983-984. 1957. Pendino, J. J., J. D. Laskin, R. L. Shuler, C. J. Punjabi, and D. L. Laskin. Enhanced production of nitric oxide by rat alveolar macrophages following inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase. J. Immunol. 151: 7196-7205,1993.
OXIDE 20.
21.
22.
23.
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25.
26.
27.
28.
29.
30.
31.
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