Unité de Recherche, Centre de Pneumologie, Hôpital and Université Laval, Ste.-Foy, Québec, Canada G1V 4G5
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ABSTRACT |
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Alveolar macrophages (AM) from normal individuals suppress mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation, whereas cells from patients with hypersensitivity pneumonitis (HP) enhance PBMC. Because surfactant components can interfere with AM functions, we tested the effect of Survanta (a modified bovine surfactant) and surfactant fractions isolated from bronchoalveolar lavage of normal subjects and HP patients on AM-induced lymphoproliferation. Surfactant fractions were isolated from bronchoalveolar lavage fluids by differential centrifugation into total aggregates (TA) and large aggregates (LA). Surfactant preparations (200 µg/ml) from 10 normal subjects (N) or 12 HP patients or of Survanta were added to AM-PBMC cocultures stimulated with phytohemagglutinin (PHA) at 1:1 and 2:1 ratios. Results, expressed as percent of PHA-induced PBMC proliferation cocultures without surfactant, show that normal surfactant and Survanta decrease mitogen-induced proliferation of cells to a larger extent than surfactant from HP patients. For AM-to-PBMC ratios of 1:1, the results were as follows: N TA 10.58 ± 2.75% (mean ± SE), N LA 12.96 ± 2.78%, HP TA 43.09 ± 7.81%, HP LA 61.64 ± 7.77%, and Survanta 16.70 ± 2.95%. Similar data were obtained in 2:1 cocultures. Because surfactant components interact with AM, alterations of surfactant composition in lymphocytic diseases, mainly in the LA fraction, may account for the lack of suppressive activity in AM of HP patients and the observed alveolitis.
alveolitis; lymphocytes; lung immunology
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INTRODUCTION |
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THE EPITHELIAL SURFACE of the lung is exposed to microorganisms and other environmental pollutants. Although many immune effector cells, including macrophages and lymphocytes, are present within the alveolar spaces, clinically significant alveolitis is relatively rare. This indicates the presence of a highly effective local particle handling and downregulation of potentially harmful immune reactions in response to nonpathogenic antigens. Two main components of the air-tissue interface, alveolar macrophages (AM) and pulmonary surfactant, have immunosuppressive activities. AM, the main resident cell population on the epithelial wall of the alveoli, are capable of limiting lymphocyte proliferative responses both in vitro and in vivo (20). This suppressive activity requires cell-to-cell contact, and the mediator(s) responsible for this effect is (are) extractable in organic solvents, indicating that lipidic components might be involved in the suppression (22).
Alveolar cells bathe in pulmonary surfactant, a highly surface-active material lining the alveoli that is also involved in the downregulation of the host immune responses. Surfactant is synthesized and secreted into the air space by alveolar type II cells (30). Phospholipids make up ~90% of surfactant, and the remaining 10% is composed of specific proteins (12). The first described function of surfactant was its ability to stabilize the gas exchange surface during breathing. More recent studies have shown that surfactant components interact with alveolar cells and may modulate their function (1). Surfactant phospholipids have an inhibitory effect on lymphocyte proliferative responses to mitogens, alloantigens (21), and interleukin-2 (24), on immunoglobulin production by B cells (25) and on NK cell cytotoxicity (29). Surfactant phospholipids also interact with macrophages, inhibiting cytokine release (8, 26). On the other hand, protein components and, more specifically, surfactant protein (SP) A, the most abundant surfactant-associated protein, stimulate macrophage functions such as chemotaxis (31), act as an opsonin in the phagocytosis of bacteria and viruses (19), and enhance oxidative burst (27). SP-A also stimulates inflammatory cytokine release, immunoglobulin production (13), and mitogen-induced proliferation and seems to counteract the suppressive effect of phospholipids on lymphocyte proliferation (14). It is the current belief that the normal immune response in the lungs depends on the delicate balance between levels of phospholipids, having an inhibitory effect, and proteins, having more of an activating effect. Alterations in the composition or in the amount of surfactant components can modify the immunomodulatory activity of surfactant on alveolar cells. Such alterations occur in interstitial lung diseases involving lymphocyte recruitment and activation (28). In hypersensitivity pneumonitis (HP), for instance, changes in phospholipid composition (11, 16) and increase in SP-A content have been reported (5, 10).
We have previously reported that macrophages isolated from bronchoalveolar lavage (BAL) of patients with farmer's lung disease are not only unable to suppress the proliferative response of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA) but actually enhance this proliferation (6). Under the same conditions, AM from healthy individuals had a normal suppressive activity. Because surfactant components can affect the functions of AM, we hypothesized that alterations of surfactant in HP could be involved in the defective AM activity in this disease.
The purpose of this study was therefore to test the effect of various surfactant preparations on the altered immunosuppressive effect of AM from patients with HP. The effect of surfactant fractions isolated from BAL fluid of healthy subjects, of patients with HP, and of Survanta (bovine surfactant; Abbott Laboratories) was tested on PHA-induced proliferation of peripheral blood leukocytes from patients with acute HP when cocultured with autologous AM. In control experiments, some of these preparations were also tested on AM and PBMC from normal individuals.
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MATERIALS AND METHODS |
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Subjects. The study population included 12 patients with HP (10 men and 2 women, mean age 47 yr, range 31-71 yr). Nine patients had farmer's lung disease, 2 patients had bird's fancier disease, and 1 patient had humidifier's lung. The diagnosis was based on previously described criteria including exposure to appropriate environments, the presence of inspiratory crackles on physical examination, interstitial infiltration on chest X rays, and altered lung functions (23). Lung functions done were lung volume measurements by plethysmography, forced expired flows, and single-breath carbon monoxide diffusion capacity. BAL fluid and venous blood were obtained from all patients as part of the clinical diagnosis. All subjects had a lymphocytic alveolitis (see RESULTS). For the preparation of normal surfactant, 10 normal nonsmoking healthy subjects with a mean age of 26 yr (range 21-36 yr) underwent a bronchoscopy with the BAL procedure. Two additional normal volunteers were recruited to provide cells for the control studies of AM-PBMC cocultures. The study was approved by our institution's Ethics Committee, and all subjects signed an informed consent form.
BAL. BAL was carried out using a fiber-optic bronchoscope. A total volume of 300 ml of sterile, nonpyrogenic saline was instilled in 60-ml aliquots into the right middle lobe, gently withdrawn by aspiration, placed in 50-ml centrifugation tubes, and kept on ice until processing. The mean recovery volume was not significantly different between patients and control subjects. Recovered BAL fluids were centrifuged at 400 g for 10 min at 4°C to remove cells, and the supernatants were used for surfactant isolation.
Preparation of AM. Cells separated from BAL fluids were washed with Hanks' balanced salt solution (HBSS) and counted on a hemacytometer, and differential cell counts were performed after Diff-Quik staining on coverslip glass (15). The cell concentration was adjusted to 5 × 106 cells/ml in RPMI 1640 medium (GIBCO BRL) supplemented with 10% heat-inactivated fetal calf serum and 1% penicillin-streptomycin (complete medium; GIBCO BRL). AM were purified by adherence on plastic petri dishes for 1 h at 37°C in a 5% CO2-enriched humidified atmosphere. Nonadherent cells were removed by washing with warm HBSS. The adherent cells were covered with ice-cold HBSS, incubated at 4°C for 30 min, and finally detached by gently scraping with a sterile rubber policeman. Typically, this method yields a 95% viable AM-enriched cell preparation as indicated by esterase staining (26). Replating this purified population of adherent cells at the appropriate concentrations and additional washings further increased the purity of AM (>98%).
Preparation of PBMC. Heparinized blood was obtained from the same patients. PBMC were separated from whole blood by Ficoll-Hypaque (Pharmacia Biotech) density gradient centrifugation, washed three times in HBSS, and resuspended in complete RPMI medium at 0.5 × 106 cells/ml.
Quantification of endotoxin levels in BAL fluid. Endotoxin levels in normal and HP patient BAL fluids were measured using a Limulus amebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA).
Isolation of BAL surfactant. BAL surfactant was isolated as previously described (18). Approximately 80 ml of BAL fluid supernatant was centrifuged at 60,000 g for 1 h at 4°C, yielding a pellet containing total surfactant aggregates (TA). Because most of the active components, including large vesicular structures and SP-A, are present in the heavy fractions of surfactant, another 80 ml of BAL fluid were spun down at 40,000 g for 15 min at 4°C, and the pellet was designated as the large-aggregate surfactant fraction (LA). The pellets were resuspended in 1.5 ml of buffered saline (140 mM NaCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 2.5 mM CaCl2). Aliquots of 100 µl of TA and LA fractions were used for measures of phospholipid concentrations. The final concentration of extracted surfactant was adjusted to 1 mg of phospholipids/ml.
Measures of phospholipid contents. Lipid extraction of surfactant pellets was performed by organic solvent (4), and total phospholipids were determined using a modified Bartlett (2) procedure (3) for phosphate analysis. No significant differences in total phospholipid contents were observed between normal and HP surfactants.
AM-PBMC cocultures. AM were cultured
in complete medium with a fixed number of autologous PBMC (5 × 104/well) at ratios of AM to PBMC
of 1:1 and 2:1 in the presence or absence of PHA (1 µg/ml) in 96-well
microtiter plates. Some control wells contained PBMC alone, with or
without PHA, and other wells were seeded with AM only with or without
PHA to eliminate the possibility of contaminating alveolar lymphocytes.
In some wells, surfactant fractions from normal subjects or patients
with HP were added at a final phospholipid concentration of 200 µg/ml. Survanta at concentrations of 50, 100, and 200 µg/ml was
also tested. The cultures were incubated for 72 h in a 5%
CO2-enriched incubator and were
pulsed with 1 µCi/well of tritiated thymidine for the last 18 h of
incubation. The cells were harvested onto glass fiber filters using a
microtiter cell harvester, and the radioactivity was counted in a
Packard Matrix -counter. The data were obtained as mean counts per
minute from quadruplicate values after subtracting the background
measured in unstimulated cultures. Results depicting the effect of
various ratios of AM on PBMC proliferation are expressed as a
percentage of PHA-stimulated PBMC without AM. Results describing the
effect of surfactant preparations are expressed as percent
proliferation of AM-PBMC cocultures without surfactant.
Statistical analysis. Results of graphic representations are expressed as mean values ± SE. For Fig. 5, a one-way analysis of variance was performed for each ratio to compare normal LA and TA with HP LA and TA and Survanta. Variance and normality assumptions were verified by valid tests. To analyze the percentage of proliferation, a univariate procedure was used to compare 1:1 and 2:1 ratios with the 0:1 ratio with the Bonferroni criteria to adjust the significance level to 0.05. All reported P values were two sided and were declared significant the 0.05 level.
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RESULTS |
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Lung functions of HP patients are given in Fig. 1. All patients had significant alterations in their lung functions; as expected, single-breath carbon monoxide diffusion capacity was the most severely decreased parameter. All normal subjects had normal lung functions (data not shown). Results of BAL cell characteristics are given in Table 1. All HP patients had an intense alveolitis (807 × 103 ± 94 cells/ml BAL fluid) with a high percentage of lymphocytes (60.4 ± 3.40%). Control subjects had a normal BAL cell profile. The levels of endotoxins were very low and similar in both groups (P = 0.575).
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The proliferative responses of PBMC from patients and normal subjects to the T cell-specific mitogen PHA cocultured with autologous AM at various AM-to-PBMC ratios are presented in Fig. 2. To compensate for differences in background levels of proliferation in the different experiments, results are expressed as percent proliferation of PHA-stimulated PBMC without AM (mean of 12 experiments for HP and 2 for normal subjects). A significant enhancement of the lymphocyte proliferative response to PHA was observed when AM from HP patients were cocultured with autologous PBMC (P > 0.0005), whereas, as expected, AM from normal subjects suppressed PHA-induced proliferation of autologous PBMC (23).
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Surfactant phospholipid content of the extracted fractions from normal and HP subjects are given in Fig. 3. A small but not significant (P = 0.82) decrease in phospholipid content was observed in reconstituted surfactant fractions from HP patients compared with controls.
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Figure 4 depicts representative experiments showing the effects of Survanta and normal and HP surfactant fractions on the AM-enhanced proliferative response of lymphocytes to PHA stimulation (1 µg/ml) in HP. Results are expressed in counts per minute of [3H]thymidine incorporation by PBMC (mean of quadruplicates). A concentration-dependent inhibition of proliferation was observed with increasing concentrations of Survanta (50-200 µg/ml) in cell cultures with or without AM (Fig. 4A). Like Survanta, normal surfactant total aggregates had a marked inhibitory effect on PBMC proliferation and showed the ability to abolish the AM-enhancing effect, thus restoring the normal suppressive effect of AM (Fig. 4, B and C). Pulmonary surfactant from HP patients also had an inhibitory effect on the lymphocyte proliferative response but was unable to significantly suppress the proliferation in AM-PBMC cocultures (Fig. 4B). LA (40 kg/15 min) had the same profile of activity as TA (Fig. 4C).
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When experiments with AM-PBMC cocultures from normal controls were performed (Fig. 5), a marked inhibition of lymphoproliferation to PHA was observed at a ratio of 2:1. The addition of normal surfactant preparations to the cocultures further increased this inhibition, whereas HP surfactant preparations did not.
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Figure 6 summarizes the results of 12 experiments showing the effects of the different surfactant preparations (200 µg/ml), some used in more than one experiment, on PHA-induced PBMC proliferation in the absence of AM (Fig. 6A) or in presence of AM at ratios of 1:1 (Fig. 6B) and 2:1 (Fig. 6C) in both AM and PBMC from HP patients. Results are expressed as percent of PHA-stimulated PBMC proliferation. TA and LA from normal individuals markedly inhibited mitogen-induced cell proliferation (<10%). TA and LA from HP patients had a weaker inhibitory effect on PBMC proliferation (13.11 ± 5.80) but were not significantly different from normal TA (P = 0.39; Fig. 5A). However, when added to AM-PBMC cocultures, HP TA inhibited proliferation to a much lesser extent than normal TA (P = 0.0001) as demonstrated by a higher percent of proliferation (43.1 ± 6.4% at ratio 1:1 and 50.4 ± 6.1% at ratio 2:1; Fig. 5, B and C). This effect was even more evident in the LA (61.4 ± 7.8% at ratio 1:1 and 72.3 ± 7.8% at ratio 2:1; P = 0.0001). Survanta (200 µg/ml) had the same inhibitory effect on proliferation as normal surfactant (P = 0.0002).
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DISCUSSION |
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AM from HP patients enhance PHA-induced mononuclear cell proliferation, whereas normal AM suppress it (6). In the present study, we report the novel observation that normal pulmonary surfactant and Survanta, a modified bovine surfactant containing several different phospholipids and phospholipid-associated proteins (SP-B and SP-C but not SP-A or SP-D), are able to fully suppress mitogen-driven lymphocyte proliferation in HP patients in the presence of AM, whereas surfactant from HP patients is unable to do so. With AM and PBMC cocultures from normal subjects, the addition of normal surfactant preparations enhanced the suppression, whereas surfactant from HP patients did not.
Lung surfactant is a morphologically and functionally heterogenous material. Isolated surfactant preparations obtained by differential centrifugation (18) consist of ~90% lipids (phospholipids, cholesterol, and other neutral lipids) and 10% proteins, mainly serum proteins but also four surfactant-specific proteins, SP-A, SP-B, SP-C, and SP-D. Large-aggregate fractions (40,000-g pellet) consist of tubular myelin-rich forms and membrane lipid structures, with good surface tension properties and high SP-A content (17), whereas small aggregates (40,000-g supernatant) consist of smaller vesicular forms devoid of surface tension activity and SP-A. In the present study, because of the small amount of material obtained, we used reconstituted total surfactant (60,000 g/h) and large-aggregate fractions (40,000 g/15 min) to evaluate the effect of normal and HP surfactant on AM-PBMC proliferation. Whole PBMC were used in cocultures as previously described (6). This technique allows monocytes to provide the accessory function required for lymphocyte proliferation.
Normal pulmonary surfactant has been shown to be responsible for the suppressive activities of immunocompetent cells of the lung either by acting directly on lymphocytes or indirectly by modulating the functions of AM (21). Phospholipids, mainly phosphatidylcholine, were found to be responsible for this suppressive effect, which could be reproduced by Survanta.
In the present study, surfactant isolated from normal subjects and Survanta inhibited AM-induced enhanced lymphoproliferation in HP. Both total reconstituted surfactant and the large-aggregate fractions had the same inhibitory effect. Surfactant isolated from HP patients had a much lower inhibitory capacity on this altered AM function. We (5) and others (10) have previously shown that surfactant from HP patients has a higher SP-A content and other serum proteins. Guzman et al. (9) reported an increase in SP-A uptake by AM in HP (9). These proteins could alter macrophage immune responsiveness. Because of the limited amount of material, we could not measure SP-A levels in the surfactant aggregates used in the present study and therefore do not provide additional information on the possible role of this protein in the altered lymphoproliferation in HP. Previous studies have also shown that the percentage of different lipids is altered in HP (11, 16). Whether these alterations in composition could explain the findings of this study remains unknown.
It has previously been shown that AM in HP release proinflammatory
cytokines such as tumor necrosis factor- and interleukin-1 (7),
cytokines that may stimulate lymphocyte proliferation. Normal
surfactant components have an inhibitory effect on AM inflammatory cytokine release (26). One possible mechanism is that surfactant from
normal subjects or Survanta decreases cytokine release, thus limiting
lymphocyte stimulation, whereas altered HP surfactant has lost that
property.
In conclusion, the present study adds to our understanding of the immunologic reactions responsible for the lymphocytic alveolitis associated with HP by suggesting that alterations in surfactant immunosuppressive function are involved. Further studies are needed to better define the composition of surfactant fractions in HP and the mechanisms responsible for this dysfunction and to explore means to correct the defects as a potential treatment of HP.
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ACKNOWLEDGEMENTS |
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We recognize the contribution of Serge Simard for the statistical analysis and Carolle Bergeron for technical assistance.
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FOOTNOTES |
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This work was supported by the Respiratory Health Network of Centers of Excellence, Canada.
Address for reprint requests: Dr. Yvon Cormier, Centre de Pneumologie, Hospital Laval, 2725 Chemin Ste.-Foy, Ste.-Foy, Québec, Canada G1Y 1L4.
Received 10 October 1996; accepted in final form 4 September 1997.
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