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Hyporesponsiveness of Human Alveolar Leukocytes to Interferon-Alpha and Interferon-Gamma Inducers
Immunobiol., vol. 181, pp. 84-96 (1990)
1 Laboratory of Virology Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, and 2 Department of Lung Diseases, Clinical Military Hospital, Wroclaw, Poland
Hyporesponsiveness of Human Alveolar Leukocytes to Interferon-Alpha and Interferon-Gamma Inducers MONIKA CEMBRZYNSKA-NOWAK I, ANNA D. INGLOT I , EDWARD SZKLARZ2, and MIROSLAWA ALBINI Received November 29, 1989· Accepted in Revised Form May 9, 1990
Abstract Leukocytes were obtained from bronchoalveolar lavages (BAL) of 36 patients including 10 with lung cancer, 15 with inflammatory lung diseases and 11 healthy control patients undergoing diagnostic investigation. The entire alveolar cell population responded weakly to the classic interferon (IFN) inducers: Newcastle disease virus (NDV), phytohemagglutinin (PHA) and lipopolysaccharide (LPS). This refers mainly to normal healthy volunteers. Alveolar leukocytes from patients with inflammatory lung diseases and nonsteroid treated lung cancer responded better to the interferon inducers than did cells from other patients. The IFN-a or IFN-y response of whole blood leukocytes to the same inducers was 10 to 100-fold higher than that of the alveolar cells. Alveolar macrophages from 6 healthy individuals and 3 patients with inflammatory lung disease were cultured in vitro for 6 days. The IFN response to inducers appears to depend on the origin of the cultured cells. It increased in the initially hyporeactive macrophages from healthy subjects and decreased in the relatively reactive cells from the patients with inflammatory lung diseases. We suggest that the hyporeactivity to IFN induction is a physiological state of the alveolar leukocytes which are a specialized cell population having constant exposure to inhaled agents such as dust, smoke, microorganisms and their by-products. The hyporesponsiveness to IFN induction of the alveolar cells may have an important physiological role in protecting lungs against hyperproduction of cytokines involved in the inflammatory and allergic reactions.
Introduction The predominant cell type in alveolar leukocytes population are macrophages. Phagocytic and micro biocidal potential of these cells make them central in defending the lungs against the assaults of particles and pathogens in inspired air. Alveolar macrophages play a role that extends far beyond phagocytosis since they are secretory and regulatory cells. The cells have been shown to produce a wide variety of pro- and anti-inflammatory agents Abbreviations: IFN = interferon; BAL = bronchoalveolar lavage; PBL = peripheral blood leukocytes; PHA = phytohemagglutinin; PMA = phorbol-12-myristate-13-acetate; LPS= lipopolisaccharide; NDV= Newcastle disease virus; TNF= tumor necrosis factor; IL-J =interleukin-1; FCS=fetal calf serum.
Interferon in Human Alveolar Leukocytes . 85
including IFNs (1, 2). The capacity of IFN production of alveolar macrophages may have important implication for antiviral defense in the lung (3, 4) and may modulate certain pulmonary inflammatory and immune processes (5, 6). So far, only limited and discrepant data on IFN production by alveolar cells in health and disease are available. NUGENT et al. (7) demonstrated no difference between the levels of IFNs from stimulated peripheral blood monocytes and alveolar macrophages isolated from normal healthy volunteers. They also found that the nonstimulated alveolar macrophages did not spontaneously release detectable amounts of IFN. In contrast, PRIOR and HASLAM (8) observed that «spontaneous» IFN levels in the lungs were far above those in the serum of healthy volunteers. Of related interest are the observations (9) that in active pulmonary sarcoidosis, lung T lymphocytes and alveolar macrophages spontaneously release large amounts of IFN compared with very low levels of IFN obtained from cells of normal healthy individuals. The aim of this study was to evaluate IFN production by alveolar leukocytes obtained from healthy individuals and patients with disorders localized in lungs including inflammatory diseases and cancer. We have also compared IFN synthesis by macrophage cultures in vitro and the entire population of alveolar cells and the whole population of the blood cells.
Material and Methods Patients
Patients undergoing diagnostic investigations and who were free of any major infection or concomitant disease entered the study as healthy controls (n = 11, four females, age 45-57, and seven males, age 20-27). None of them were under medication; 4 were smokers and the rest were non-smokers. Patients had various inflammatory lung diseases such as pneumonia or chronic bronchitis (n= 15, one female, age 60 and fourteen males, age 21-35). Five patients were treated with antibiotics and ten men were not receiving any therapy at the time of bronchoscopy. Lung cancer patients (n=10, two females, age 60-61 and eight males, age 58-67) were diagnosed as having small cell lung cancer. All of them were smokers. Four of the patients were assessed before therapy and the others were undergoing steroid therapy. Six patients with advanced cancer received decadron phosphate, 12-14 mg per day for 10 days. All subjects gave informed consent to participating in the study. There were no complications due to the bronchoalveolar lavage procedures. Bronchoalveolar lavage
We performed BAL using the modified technique of DAVIS et al. (10). After premedication with dolargan and atropine sulfate, bronchoscopy was orally performed using a flexible fiberoptic bronchoscope (Olympus Type IT P10) under local anesthesia with 2 % lidocaine. The tip of the bronchoscope was wedged into a subsequental bronchus of the lingula or middle lobe. Once the bronchoscope was wedged, 40 ml of the 0.9 % sterile saline was inserted in the suction port by the use of a syringe. The fluid was immediately gently aspirated and collected in a glass bottle and then placed on ice. This process of lavage and suction was repeated four times. The second and further portions of BAL were used for cell differentiation and the
86 . MONIKA CEMBRZYNSKA-NoWAK et al. capability of an IFN response. In general, 30 % to 70 % of the infused fluid volume was recovered. Cell separation and culture
BAL was strained through a double layer of sterile gauze and the volume was measured. Cells were pelleted at 1500 rpm for 10 min at +4°C and separated on Ficoll-Hypaque gradient (Gradisol, Polfa, Poland) into interface and sedimented fractions. Cell fractions were centrifuged at 500 rpm for 10 min and separated cells were suspended in 2 ml of McCoy medium containing 10 % heat inactivated human AB serum and 100 I.U.lml penicillin, 100 Itg/ml streptomycin, 50 Itg/ml gentamycin and 5 Itg/ml fungizone to viable cell concentration of about 6 x 106/ml. BAL cells were adhered to 96-well plastic culture plates (Nunc, Denmark) for 2 hat 37°C in a 5 % CO r 95 % air atmosphere. The non-adherent cells were removed and fresh medium was added to the adherent cells. The cells were cultured in McCoy medium with 15 % heatinactivated human AB serum for 6 days and sometimes for 10 to 21 days with refeeding every 72h. For counting, the adherent cells were removed by gentle scraping with a rubber policeman, followed by resuspension in the culture medium to obtain a desired concentration. Cells were counted in a Thoma chamber. Dead cells were identified by determining trypan blue exclusion (0.02 % solution in phosphate buffered saline). Macrophages were evaluated for purity by ingestion of latex beads. Cells at 5 x 106 per ml in McCoy medium with 20 % human serum were fed with latex beads (Sigma Chemicals). The ratio of cells to latex particles was 1:2. Latex beads were washed 3 times with phosphate buffered saline and stored at 4°C, at 109 latex beads/ml prior to use. Cells and latex were incubated at 37°C for 90 min with occasional shaking. Cytocentrifuged smears were prepared from total cells or cells that had ingested latex beads. Cytocentrifugation was carried out at 500 rpm for 10 min in Cytospin 2 (Shandon, England). Smears were stained with May-Griinwald-Giemsa and additionally for the nonspecific esterase according to KOSKI et al. (11). The morphological and staining characteristics of 200 cells were determined in every assay. Mean values and standard error were calculated. Interferon induction
IFN inducers were: NDV 108 EIDso/ml (640 HA units/ml), 10 Itg/ml PHA (Pharmacia Fine Chemicals) and 10 ng/ml PMA (Sigma) used in combination with PHA and 10 Itg/ml LPS from E. coli (Difco). The concentrations of the inducers used were found to be optimal. The suspension of 5-10 x 106 BAL cells in McCoy medium containing 2 % FCS with the inducers was incubated for 24h at 3rC in the CO 2 incubator. Whole blood assay was made as described by INGLOT et al. (12). Briefly, heparinized blood was mixed 1:10 with RPMI 1640 medium supplemented with penicillin (100 U/ml) and streptomycin (100 Itg/ml). The diluted blood was distributed in duplicates in one ml amounts into 24-well flat bottom plastic plates to which IFN inducers were added at indicated concentrations in volumes of 20 Itl!well. The leukocyte number per sample was approximately 105 cells/ml. The cultures were incubated at 37°C for 20 h, centrifuged and assayed for IFN activity. The IFN titers refer to 106 WBC/ml. IFN assay
IFN was assayed by inhibition of cytopathic effect of EMC virus in human lung adenocarcinoma cell line A549 (American Type Collection No 185) in monolayer cultures on microplates (Nunc, Denmark). The A549 cell line and EMC challenge virus has been used by WHO Scientific Committee on Standardization of IFNs (13). A standard IFN with known titer was used in each assay. The titer of interferon was defined as the dilution of an IFN sample that reduced the virus cytopathic effect by 50 %.
Interferon in Human Alveolar Leukocytes . 87 Antiinterferon sera
The antisera neutralizing various forms of IFNs were: sheep anti-HuIFN-a serum obtained from late DR. K. PAUCKER, USA, sheep anti-HuIFN-~ (No G028-501-568) from NIH, USA and rabbit anti-recombinant HuIFN-y (2891-569, Genentech Inc., USA).
Results
Characteristics of BAL cell populations The BAL cell yielded 7-27 x 106 cells and their viability ranged from 71-93 % depending on the disease. The best viability (93 %) was obtained during harvesting of cells from normal individuals. The viability of cells from lung cancer patients and inflammatory lung diseases was 71.3 % and 88.8 %, respectively (Table 1). The BAL leukocytes were characterized morphologically after staining with May-Griinwald-Giemsa, by determination of the nonspecific esterase activity and by estimation of phagocytosis of latex beads. The values of cells positive for the acid a-naphtyl acetate esterase activity as revealed by cytochemical staining were almost coincident with the results of phagocytosis. BAL of control patients contained approximately 93 % macrophages, 6 % lymphocytes and 1 % granulocytes. In patients with inflammatory lung diseases the number of macrophages was lower (54 %) and the number of lymphocytes and granulocytes was higher (44 % and 2 %, respectively). The differential counts of patients with lung cancer was variable but mean values appeared to be similar to that of controls (Table 1).
Interferon production by the whole BAL cell population Freshly collected whole BAL cells were divided into four samples. The first was unstimulated, whereas others were incubated with the following inducers: NDV, PHA + PMA or LPS. Results of IFN production after incubation of the cells for 24 h at 3rC are shown in Figure 1. A number of BAL cell cultures from either control or experimental patients produced low level IFN spontaneously. As expected, upon stimulation with inducers, the level of IFN significantly increased. In this study, several different batches of fetal calf serum in culture media were used. All of the batches were pre-selected in PBL cultures for IFN induction without PHA. Only sera with low spontaneous activity (:::; 10 UI ml) were used in the experiments. There were considerable variations in the spontaneous or induced IFN response between BAL cells from control and experimental patients, irrespective of the batch of serum used in the culture medium. The possible presence of small levels of LPS in the cultures of BAL cells cannot be excluded because the cells were harvested from the external environment. However, LPS in a concentration of 10 flg/ml was consistently found to be a weak IFN inducer in BAL cells (Fig. 1).
1.51 ± 0.4 5.57 ± 2.1 3.54 ± 1.1
7.5 ± 1.5
27.7 ± 2.1
17.7 ± 8.5
Inflammatory lung diseases n = 15
Healthy volunteers n = 11
± 11.8
93.5 ± 1.50 94
55.5 ± 7.8
88.8 ± 7.91 6
± 2.7
44.5 ± 14.3
10.05 ± 3.7
91.3 ± 4.6
71.3 ± 19.6
IFN titer was calculated to 106 BAL or WBC leukocytes per ml IFN from BAL vs IFN from WBC: statistically significant at p < 0.05
1.79 ± 0.64 4.20 ± 0.62
0.75 ± 0.66 1.64 ± 1.20
BAL WBC
inflammatory lung diseases
n = 15
0.57 ± 0.52 4.60 ± 0.78
0.61 ± 0.61 2.36 ± 0.65
BAL WBC
controls
n = 11
NDV
1.19 ± 0.71 2.06 ± 1.20
0.53 ± 0.62 3.40 ± 1.00
LPS
IFN titer loglO D/ml after induction with: None
Cells
Patients
Table 2. Interferon production by cells from bronchoalveolar lavages (BAL) and leukocytes in the whole blood cultures (WBC)
Lung cancer n = 10
0/0
Lymphocytes %
Differential Cell Counts Macrophages
Viability %
± 0.7
1.57 ± 1.03 4.41 ± 0.92
0.58 ± 0.75 4.52 ± 1.46
PHA + PMA
1.2 ± 0.3
2.5 ± 1.7
1.4
Granulocytes %
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Cellslml x 10-6
Patients
Total number of cells x 10-6
00 00
Table 1. Characteristics of cell populations in BAL
Interferon in Human Alveolar Leukocytes . 89
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Figure 1. Interferon activity in the unseparated population of bronchoalveolar leukocytes untreated or treated with inducers. Cells were obtained from x healthy individuals, 0 patients with inflammatory lung diseases, "" patients with lung cancer before therapy, and 6. patients with lung cancer receiving steroid therapy. BAL cells were incubated with one of the following inducers: NDV (640 HA units/ml), LPS (10 !!g/ml) or PHA + PMA (10 ng/ml, and 10 ng/ml, respectively). Spontaneous production refers to the BAL cells incubated without the inducers. Data are expressed as a single point for each individual. IFN units released by 106 cells are shown.
In general, BAL cells of normal controls or steroid-treated lung cancer patients were poor producers of IFN. The BAL cells from patients with inflammatory lung diseases produced more IFN either without inducers or after stimulation. Also, cancer patients who were not treated with steroids produced significantly more IFN than cancer patient undergoing steroid therapy (p < 0.01) (Fig. 1). There were no significant differences in the IFN response of BAL cells obtained from smokers and nonsmokers (data not shown). In the bronchoalveolar fluids, even when concentrated up to 10o-fold, an IFN antiviral activity was not detected (data not shown).
Comparison of IFN production of BAL cells and whole blood leukocytes Results in Table 2 show the IFN production of BAL cells and of whole blood cells that were unstimulated or treated with various inducers. The IFN titers were estimated as amounts of IFN produced per 106 cells. It should be mentioned that the two cell populations had completely different composition, and their cell number was not identical. BAL cell population
90 .
MONIKA CEMBRZYNSKA-NoWAK
et al.
contained 78-96 % macrophages, 4-12 % lymphocytes and 2.5-1.2 % granulocytes. In contrast, average differential white cell counts of whole blood culture were: 7 % monocytes, 24 % lymphocytes and 66 % granulocytes. Despite this restriction, it appears that WBC produce considerable more IFN than BAL cells. In other words, the BAL cells were found to be partially hyporesponsive to IFN induction. Even the relatively responsive BAL cells from patients with the inflammatory lung diseases produced less IFN than the whole blood cultures of the same patients (Table 2).
Interferon production by cultured alveolar macrophages The alveolar macrophages were separated from the whole BAL cell population by adherence to plastic plates. The adherent cells were cultured for 6 to 21 days. The freshly isolated macrophages as well as the cultured cells were identified by morphological criteria, their capacity to phagocytose latex beads and by esterase staining. According to these criteria, the freshly isolated adherent cells represented 98 % macrophages and 6-day-old cultures were found to be almost 100 % pure macrophages. The macrophages during culturing in vitro changed from the initially rounded forms into the elongated, spindle-shaped cells. A multiplication of macrophages
x
Z
LL
o n
=6
6
n =4
days of culture
Figure 2. Interferons induced in the normal alveolar macrophages cultured in vitro. Alveolar macrophages (6-10 x 106 cells/ml) were allowed to adhere to the 96-well flat bottom plates at 3rc for 2 h. Nonadherent cells were removed and macrophages were cultured in McCoy medium supplemented with 10 % human AB serum. IFN induction was carried out in the freshly prepared macrophages (day 0) and in the cells cultured for 6 days. 1m - NDV induction; § - PHA + PMA induction.
Interferon in Human Alveolar Leukocytes . 91
was not observed. The number of viable cells in the 6-day cultu~e was approximately 99 %. Interferon induction was carried out with 0 day macrophages and 6 daycultured cells. Day 0 indicated the freshly isolated cells immediately after placing them into culture wells. Results of IFN induction with NDV and PHA + PMA or LPS are shown in Figures 2 and 3. The freshly isolated alveolar macrophages from the control patients produced considerably lower levels of IFN after stimulation with NDV or PHA + PMA (Fig. 2) than cells cultured for 6 days at 3rC (p < 0.01). In contrast, alveolar cells separated from BAL of patients with inflammatory lung diseases were found to be more responsive to IFN inducers shortly after the isolation (at time 0) than after further culture for 6 days (Fig. 3). Only small cell numbers for in vitro culture were available from these patients. Therefore, it was not possible to assess all inducers simultaneously, and to study the kinetics of IFN production. Fragmentary data obtained from the study suggested that a 6-day culture is optimal for IFN production. After 8, 14 and 21 days, the inducible IFN levels decreased significantly (data not shown).
exp.1
exp.2
exp.3
o
o
x
Z
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-1
6
6
days of culture
Figure 3. Interferon induced in the inflammatory alveolar macrophages cultured in vitro. The BAL cell were obtained from patients with the inflammatory lung diseases including pneumonia and chronic bronchitis. For other explanations see Fig. 2. fillI- NDV induction; == PHA + PMA induction; m- LPS induction. Assays were done with the cells from individual patients.
92 .
MONIKA CEMBRZYNSKA-NOWAK
et al.
Table 3. Neutralization of antiviral activity in supernatants from alveolar cell or whole blood cultures treated with different IFN inducers Inducer nIFN-a NDV PHA+PMA LPS None'f
Detected
Reaction with antiserum to nIFN-B
rIFN-y
+ + + +
IFN-a IFN-y IFN-y IFN-y
'f Supernatants from control cells cultured without inducers n = natural r = recombinant
Antigenic identifi."cation of IFNs
The types of IFN produced by BAL cells and isolated macrophages were determined by neutralization with antibodies to IFN-a, IFN-~ and IFN-y (Table 3). The anti-HuIFN-a serum neutralized only the antiviral activity of IFN induced by NDV. The anti-HuIFN-y antibody completely inhibited antiviral activity of IFN from the alveolar macrophages induced with LPS or with PHA + PMA. The antiserum against IFN -~ had no effect on the IFN preparations studied. The IFN produced spontaneously without any exogenous inducers was neutralized by anti-IFN-y. However, the presence of small levels of other IFN types could not be excluded because their titers were too low. Discussion We have observed that macrophages which are a predominant component of the population of the bronchoalveolar cells, produce significantly lower levels of IFN-a and IFN-y than the peripheral blood leukocytes in the whole blood culture. This refers mainly to the cells from normal healthy individuals treated or untreated with inducers. Also BAL cells from patients with the inflammatory lung diseases and even with lung cancer produced relatively lower IFN levels than the blood leukocytes. Therefore, our consistent observation is partially discrepant with the results of NUGENT et al. (7) who described that normal human macrophages produced IFN after stimulation with classic inducers at the same level as blood monocytes. However, their finding that very low levels of IFNs were obtained from alveolar cells of normal healthy individuals (7, 9) corresponded well with our results. Furthermore, PRIOR and HASLAM (8) observed that «spontaneous» IFN levels in the lung were higher than that in the serum of healthy volunteers. Interferons as well as other cytokines are produced locally and they function in a paracrine fashion. Inability to detect IFN and other
Interferon in Human Alveolar Leukocytes . 93
cytokines in blood may be due to their modest spill-over in a rapidly turning-over pool (14). The inflammatory and immunological processes facilitate cell migration and activation connected with release of cytokines (15). Therefore, it is not surprising that BAL cells from patients with inflammatory lung diseases as well as those with untreated lung cancer produced more interferon than the normal control cells. This also refers to the amounts of IFN released without any treatment with exogenous inducers. Our results that the cells from lung cancer patients undergoing steroid therapy produced significantly lower level of IFN as compared to the cells from the untreated patients are consistent with the knowledge that steroids suppress the release of IFN-y by mononuclear cells (9, 16, 17). In that respect, it is noteworthy that lung mononuclear cells from patients with pulmonary sarcoidosis spontaneously released considerable amounts of IFN-y compared to normal (9). The limited ability to spontaneous release by interferon and the weak IFN induction in normal lung mononuclear cells which was observed by us and others (7, 9) prompted us to suggest that hyporeactivity of BAL leukocytes may be a physiological condition. BAL cells are constantly exposed to external inhaled stimuli such as dust, smoke, viruses, microorganisms and their products, especially to LPS. All of these factors may stimulate the cells to produce cytokines e.g. IFNs, IL-l, IL-2, TNF, IL-6, TGF-~, etc. (1). The resident alveolar macrophages with well-developed phagocytic activity and hyporeactive to IFN induction may protect the lungs against noxious stimuli, especially against allergens, and other immunologic stimulants. IFNs were found to enhance histamine release and to increase IgE binding to basophils. In view of the suggested pathogenic role of IFN in allergy, a low capacity for IFN synthesis appears to be a favorable condition (6, 18). Hyporesponsiveness to IFN induction may develop as a consequence of repeated exposure to various inducers. Approximately one week is required to overcome the induced hyporesponsiveness (19, 20). In our experiments, we observed an overcoming of the hyporeactivity of control alveolar macrophages during 6 days of culture in vitro. However, the BAL cells from patients with inflammatory lung diseases responded to the inducers more intensively immediately after isolation than after one week of culturing in vitro. This may be due to the elimination of the cells originating from the inflammatory exudate initially capable to relatively high cytokine production. It has been repeatedly suggested that the alveolar macrophages are metabolically different than the peritoneal, pleural and other phagocytes (21). This refers mainly to their strong suppressor activity in the mitogeninduced lymphocyte proliferation (22), and also in presenting antigens (23, 24).
94 . MONIKA CEMBRZYNSKA-NOWAK et al.
Human alveolar macrophages have a limited capacity for interleukin-1 release as compared to peripheral blood monocytes (25, 26). The accumulation of mRNA for IL-1-~ was 20-fold greater in blood monocytes than in the alveolar macrophages (27). STRINGFELLOW et al. (19) and MONICK et al. (28) suggested that prostaglandins may play an important role in hypo reactivity of cells to IFN and IL-1 inducers. The role of eicosanoids in the described phenomenon remains to be determined. The relatively poor capacity of alveolar macrophages to produce IL-1 contrasts with the observation that alveolar macrophages had significantly greater TNF production in response to different inducers than peripheral blood monocytes (29-31). Although IFN s, IL-1 and TNF have a similar spectrum of activity, the capacity of alveolar macrophages to produce IFN sand IL-1 appears to be decreased and to produce TNF to be increased when compared with blood monocytes. It is generally believed that macrophages are producers of IFN -a/~ but not IFN-y (32). However, we have consistently found that the cultured alveolar cells produced, after stimulation with NDV, IFN-a and produced IFN-y following PHA + PMA or LPS stimulation. The observations are compatible with the findings of ROBINSON et al. (9), NUGENT et al. (7) and PRIOR and HASLAM (8) that freshly isolated alveolar macrophages may produce IFN-y spontaneously or after PHA or LPS induction. Variations in the IFN response of alveolar cells cannot be due to LPS only despite the fact that it is a ubiquitously contaminating agent. Our experiments showed the LPS at 10 flg/ml is a relatively weak IFN inducer in comparison to NDV or PHA (Fig. 1). We cannot exclude that our cultures contained small number of T lymphocytes or NK cells which contaminated apparently pure macrophages. Alveolar macrophages may also form syncytia with lymphocytes and the fused cells may respond to both NDV and PHA + PMA. Furthermore, a direct reaction of alveolar cells with PHA + PMA is also possible. All these hypotheses require further experimental verification.
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Interferon in Human Alveolar Leukocytes . 95 6. SKURKOVICH, S., B. SKURKOVICH, and J. A. BELLANTI. 1987. A unifying model immunoregulatory role of the interferon system: Can interferon produce disease in humans? Clin. Immunol. Immunopathol. 43: 362. 7. NUGENT, K. M., J. GLAZIER, M. M. MONICK, and G. W. HUNNINGHAKE. 1985. Stimulated human alveolar macrophages secrete interferon. Am. Rev. Respir. Dis. 132: 714. 8. PRIOR, c., and P. L. HASLAM. 1989. Interferon in lung. Lancet I: 1330. 9. ROBINSON, B. W. S., T. L. McLEMORE, and R. G. CRYSTAL. 1985. Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis. J. Clin. Invest. 75: 1488. 10. DAVIS, G. S., M. S. A. GIANCOLA, M. C. COSTANZA, and R. B. Low. 1982. Analyses of subsequential bronchoalveolar lavage samples from healthy human volunteers. Am. Rev. Respir. Dis. 126: 611. 11. KOSKI, I. R., D. G. POPLACK, and R. M. BLAESE. 1976. A nonspecific esterase stain for the identification of monocytes and macrophages. In: In Vitro Methods in Cell-Mediated and Tumor Immunity. BLOOM, B. R., and J. R. DAVID (eds.) Acad Press, New York, p 359 12. INGLOT, A. D., E. PIASECKI, B. SZULC, B. GROEHLICH, W. DOBRACKI, A. GLADYSZ, J. LEsZEK, E. SZKLARZ, and R. SCIBORSKI. 1988. Assessment of interferon production in patients with chronic nonviral diseases by whole blood or whole bone marrow techniques. Arch. Immunol. Ther. Exp. 36: 439. 13. WHO Expert Committee on Biological Standardization. 1988. Technical Report Series No 771, Annex 1. 37. 14. BOCCI, V. 1988. Roles of interferon produced in physiological conditions. A speculative review. Immunology 64: 1. 15. BITTERMAN, P. B., L. E. SALZMAN, S. ADELBERG, V. J. FERRANS, and R. G. CRYSTAL. 1984. Alveolar macrophages replication: one mechanism for the expansion of the mononuclear phagocyte population in the chronically inflamed lung. J. Clin. Invest 74: 460. 16. CESARIO, T., L. SLATER, H. KAPLAN, and G. GORSE. 1984. Alternations in gamma interferon production by antineoplastic agents. Clin. Res. 32: 365A. 17. ARYA, S., F. WONG-STAAL, and R. C. GALLO. 1984. Dexamethasone-mediated inhibition of human T cell growth factor and y-interferon messenger RNA. J. Immunol. 133: 273.
18. JANKOWSKA, R., H. SZALATY, and J. MROSs-NYCZKA. 1989. Interferon synthesis in peripheral blood leukocytes from bronchial asthma patients. Arch. Immunol. Ther. Exp. 37: 477. 19. STRINGFELLOW, D. A. 1981~1982. Interferon induction: hypo responsiveness and prostaglandins. Texas Rep. BioI. Med. 41: 116. 20. SZULC, B., A. D. INGLOT, and O. INGLOT. 1987. Tolerance of hyperreactivity to interferon induction by sodium salt of 9-oxo-10-acridineacetic acid and analogs in mice and in the mouse macrophage cultures. Arch. Immunol. Ther. Exp. 35: 389. 21. NIBBERING, P. H., P. C. LEIJH, and R. VAN FURTH. 1987. Quantitative immunocytochemical characterization of mononuclear phagocytes. I Monoblast, promonocytes, monocytes, and peritoneal and alveolar macrophages. Cell. Immunol. 105: 374. 22. SESTINI, P., A. TAGLIABUI, and D. BORASCHI. 1986. Modulation of macrophage suppressive comparison of alveolar, pleural and peritoneal macrophages. Clin. Exp. Immunol. 58: 573. 23. ULLRICH, S. E., and H. B. HERSCOWITZ. 1980. Immunological function of alveolar macrophages interaction with a soluble protein antigen and the immunogenicity of alveolar macrophage-associated antigen. J. Reticuloendothel. Soc. 28: 111. 24. WEINBERG, D. S., and E. R. UNANUE. 1981. Antigen-presenting function of alveolar macrophages: uptake and presentation of Listeria monocytogenes. J. Immunol. 126: 794. 25. BERNAUDIN, J. F., K. YAMAUCHI, M. D. WEWERS, M. J. TOCCI, V. J. FERRANS, and R. G. CRYSTAL. 1988. Demonstration by in situ hybridization of dissimilar IL-1 gene expression in human alveolar macrophages and blood monocytes in response to lipopolysaccharide. J. Immunol. 140: 3822.
96 . MONIKA CEMBRZYNSKA-NOWAK et al. 26. EDEN, E., and G. M. TURINO. 1986. Interleukin-l secretion from human alveolar macrophages in lung disease. J. Clin. Immunol. 4: 326. REED, R.J. LAMB, R. P. DANIELE, and P. C. NOWELL. 1987. Interleukin-l 27. KERN,J. A.,J. gene expression in blood monocytes and alveolar macrophages. Am. Rev. Resp. Dis. 135: A209. 28. MONICK, M.,J. GLAZIER, and G. W. HUNNINGHAKE. 1987. Human alveolar macrophages suppress interleukin-l (IL-l) activity via the secretion of prostaglandin E2. Am. Rev. Resp. Dis. 135: 72. 29. VALONE, S. E., E. A. RICH, R. S. WALLIS, and J. J. ELNERR. 1988. Expression of tumor necrosis factor in vitro by human phagocytes stimulated with whole Mycobacterium bovis BGG and mycobacterial antigens. Infec. Immunity 56: 3313. 30. MARTINET, Y., K. YAMAUCHI, and R. G. CRYSTAL. 1988. Differential expression of the tumor necrosis factor/cachectin gene by blood and lung mononuclear phagocytes. Am. Rev. Respir. Dis. 138: 659. 31. STRIETER, R. M., D. G. REMICK, J. P. LYNCH, R. N. SPENGLER, and S. L. KUNKEL. 1989. Interleukin-2-induced tumor necrosis factor - alpha (TNF-a) gene expression in human alveolar macrophages and blood monocytes. Am. Rev. Res. Dis. 139: 335. 32. KHAN, N. D. V., K. A. F. PULFORD, M. A. FARGUHARSAN, A. HOWATSON, C. STEWART, R. JACKSON, A. M. McNICOL, and A. K. FOULIS. 1989. The distribution of immunoreactive interferon-alpha in normal human tissues. Immunology 66: 201.
c.
Dr. MONIKA CEMBRZYNSKA-NoWAK, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Czerska 12, 53-114 Wroclaw, Poland
1 Laboratory of Virology Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, and 2 Department of Lung Diseases, Clinical Military Hospital, Wroclaw, Poland
Hyporesponsiveness of Human Alveolar Leukocytes to Interferon-Alpha and Interferon-Gamma Inducers MONIKA CEMBRZYNSKA-NOWAK I, ANNA D. INGLOT I , EDWARD SZKLARZ2, and MIROSLAWA ALBINI Received November 29, 1989· Accepted in Revised Form May 9, 1990
Abstract Leukocytes were obtained from bronchoalveolar lavages (BAL) of 36 patients including 10 with lung cancer, 15 with inflammatory lung diseases and 11 healthy control patients undergoing diagnostic investigation. The entire alveolar cell population responded weakly to the classic interferon (IFN) inducers: Newcastle disease virus (NDV), phytohemagglutinin (PHA) and lipopolysaccharide (LPS). This refers mainly to normal healthy volunteers. Alveolar leukocytes from patients with inflammatory lung diseases and nonsteroid treated lung cancer responded better to the interferon inducers than did cells from other patients. The IFN-a or IFN-y response of whole blood leukocytes to the same inducers was 10 to 100-fold higher than that of the alveolar cells. Alveolar macrophages from 6 healthy individuals and 3 patients with inflammatory lung disease were cultured in vitro for 6 days. The IFN response to inducers appears to depend on the origin of the cultured cells. It increased in the initially hyporeactive macrophages from healthy subjects and decreased in the relatively reactive cells from the patients with inflammatory lung diseases. We suggest that the hyporeactivity to IFN induction is a physiological state of the alveolar leukocytes which are a specialized cell population having constant exposure to inhaled agents such as dust, smoke, microorganisms and their by-products. The hyporesponsiveness to IFN induction of the alveolar cells may have an important physiological role in protecting lungs against hyperproduction of cytokines involved in the inflammatory and allergic reactions.
Introduction The predominant cell type in alveolar leukocytes population are macrophages. Phagocytic and micro biocidal potential of these cells make them central in defending the lungs against the assaults of particles and pathogens in inspired air. Alveolar macrophages play a role that extends far beyond phagocytosis since they are secretory and regulatory cells. The cells have been shown to produce a wide variety of pro- and anti-inflammatory agents Abbreviations: IFN = interferon; BAL = bronchoalveolar lavage; PBL = peripheral blood leukocytes; PHA = phytohemagglutinin; PMA = phorbol-12-myristate-13-acetate; LPS= lipopolisaccharide; NDV= Newcastle disease virus; TNF= tumor necrosis factor; IL-J =interleukin-1; FCS=fetal calf serum.
Interferon in Human Alveolar Leukocytes . 85
including IFNs (1, 2). The capacity of IFN production of alveolar macrophages may have important implication for antiviral defense in the lung (3, 4) and may modulate certain pulmonary inflammatory and immune processes (5, 6). So far, only limited and discrepant data on IFN production by alveolar cells in health and disease are available. NUGENT et al. (7) demonstrated no difference between the levels of IFNs from stimulated peripheral blood monocytes and alveolar macrophages isolated from normal healthy volunteers. They also found that the nonstimulated alveolar macrophages did not spontaneously release detectable amounts of IFN. In contrast, PRIOR and HASLAM (8) observed that «spontaneous» IFN levels in the lungs were far above those in the serum of healthy volunteers. Of related interest are the observations (9) that in active pulmonary sarcoidosis, lung T lymphocytes and alveolar macrophages spontaneously release large amounts of IFN compared with very low levels of IFN obtained from cells of normal healthy individuals. The aim of this study was to evaluate IFN production by alveolar leukocytes obtained from healthy individuals and patients with disorders localized in lungs including inflammatory diseases and cancer. We have also compared IFN synthesis by macrophage cultures in vitro and the entire population of alveolar cells and the whole population of the blood cells.
Material and Methods Patients
Patients undergoing diagnostic investigations and who were free of any major infection or concomitant disease entered the study as healthy controls (n = 11, four females, age 45-57, and seven males, age 20-27). None of them were under medication; 4 were smokers and the rest were non-smokers. Patients had various inflammatory lung diseases such as pneumonia or chronic bronchitis (n= 15, one female, age 60 and fourteen males, age 21-35). Five patients were treated with antibiotics and ten men were not receiving any therapy at the time of bronchoscopy. Lung cancer patients (n=10, two females, age 60-61 and eight males, age 58-67) were diagnosed as having small cell lung cancer. All of them were smokers. Four of the patients were assessed before therapy and the others were undergoing steroid therapy. Six patients with advanced cancer received decadron phosphate, 12-14 mg per day for 10 days. All subjects gave informed consent to participating in the study. There were no complications due to the bronchoalveolar lavage procedures. Bronchoalveolar lavage
We performed BAL using the modified technique of DAVIS et al. (10). After premedication with dolargan and atropine sulfate, bronchoscopy was orally performed using a flexible fiberoptic bronchoscope (Olympus Type IT P10) under local anesthesia with 2 % lidocaine. The tip of the bronchoscope was wedged into a subsequental bronchus of the lingula or middle lobe. Once the bronchoscope was wedged, 40 ml of the 0.9 % sterile saline was inserted in the suction port by the use of a syringe. The fluid was immediately gently aspirated and collected in a glass bottle and then placed on ice. This process of lavage and suction was repeated four times. The second and further portions of BAL were used for cell differentiation and the
86 . MONIKA CEMBRZYNSKA-NoWAK et al. capability of an IFN response. In general, 30 % to 70 % of the infused fluid volume was recovered. Cell separation and culture
BAL was strained through a double layer of sterile gauze and the volume was measured. Cells were pelleted at 1500 rpm for 10 min at +4°C and separated on Ficoll-Hypaque gradient (Gradisol, Polfa, Poland) into interface and sedimented fractions. Cell fractions were centrifuged at 500 rpm for 10 min and separated cells were suspended in 2 ml of McCoy medium containing 10 % heat inactivated human AB serum and 100 I.U.lml penicillin, 100 Itg/ml streptomycin, 50 Itg/ml gentamycin and 5 Itg/ml fungizone to viable cell concentration of about 6 x 106/ml. BAL cells were adhered to 96-well plastic culture plates (Nunc, Denmark) for 2 hat 37°C in a 5 % CO r 95 % air atmosphere. The non-adherent cells were removed and fresh medium was added to the adherent cells. The cells were cultured in McCoy medium with 15 % heatinactivated human AB serum for 6 days and sometimes for 10 to 21 days with refeeding every 72h. For counting, the adherent cells were removed by gentle scraping with a rubber policeman, followed by resuspension in the culture medium to obtain a desired concentration. Cells were counted in a Thoma chamber. Dead cells were identified by determining trypan blue exclusion (0.02 % solution in phosphate buffered saline). Macrophages were evaluated for purity by ingestion of latex beads. Cells at 5 x 106 per ml in McCoy medium with 20 % human serum were fed with latex beads (Sigma Chemicals). The ratio of cells to latex particles was 1:2. Latex beads were washed 3 times with phosphate buffered saline and stored at 4°C, at 109 latex beads/ml prior to use. Cells and latex were incubated at 37°C for 90 min with occasional shaking. Cytocentrifuged smears were prepared from total cells or cells that had ingested latex beads. Cytocentrifugation was carried out at 500 rpm for 10 min in Cytospin 2 (Shandon, England). Smears were stained with May-Griinwald-Giemsa and additionally for the nonspecific esterase according to KOSKI et al. (11). The morphological and staining characteristics of 200 cells were determined in every assay. Mean values and standard error were calculated. Interferon induction
IFN inducers were: NDV 108 EIDso/ml (640 HA units/ml), 10 Itg/ml PHA (Pharmacia Fine Chemicals) and 10 ng/ml PMA (Sigma) used in combination with PHA and 10 Itg/ml LPS from E. coli (Difco). The concentrations of the inducers used were found to be optimal. The suspension of 5-10 x 106 BAL cells in McCoy medium containing 2 % FCS with the inducers was incubated for 24h at 3rC in the CO 2 incubator. Whole blood assay was made as described by INGLOT et al. (12). Briefly, heparinized blood was mixed 1:10 with RPMI 1640 medium supplemented with penicillin (100 U/ml) and streptomycin (100 Itg/ml). The diluted blood was distributed in duplicates in one ml amounts into 24-well flat bottom plastic plates to which IFN inducers were added at indicated concentrations in volumes of 20 Itl!well. The leukocyte number per sample was approximately 105 cells/ml. The cultures were incubated at 37°C for 20 h, centrifuged and assayed for IFN activity. The IFN titers refer to 106 WBC/ml. IFN assay
IFN was assayed by inhibition of cytopathic effect of EMC virus in human lung adenocarcinoma cell line A549 (American Type Collection No 185) in monolayer cultures on microplates (Nunc, Denmark). The A549 cell line and EMC challenge virus has been used by WHO Scientific Committee on Standardization of IFNs (13). A standard IFN with known titer was used in each assay. The titer of interferon was defined as the dilution of an IFN sample that reduced the virus cytopathic effect by 50 %.
Interferon in Human Alveolar Leukocytes . 87 Antiinterferon sera
The antisera neutralizing various forms of IFNs were: sheep anti-HuIFN-a serum obtained from late DR. K. PAUCKER, USA, sheep anti-HuIFN-~ (No G028-501-568) from NIH, USA and rabbit anti-recombinant HuIFN-y (2891-569, Genentech Inc., USA).
Results
Characteristics of BAL cell populations The BAL cell yielded 7-27 x 106 cells and their viability ranged from 71-93 % depending on the disease. The best viability (93 %) was obtained during harvesting of cells from normal individuals. The viability of cells from lung cancer patients and inflammatory lung diseases was 71.3 % and 88.8 %, respectively (Table 1). The BAL leukocytes were characterized morphologically after staining with May-Griinwald-Giemsa, by determination of the nonspecific esterase activity and by estimation of phagocytosis of latex beads. The values of cells positive for the acid a-naphtyl acetate esterase activity as revealed by cytochemical staining were almost coincident with the results of phagocytosis. BAL of control patients contained approximately 93 % macrophages, 6 % lymphocytes and 1 % granulocytes. In patients with inflammatory lung diseases the number of macrophages was lower (54 %) and the number of lymphocytes and granulocytes was higher (44 % and 2 %, respectively). The differential counts of patients with lung cancer was variable but mean values appeared to be similar to that of controls (Table 1).
Interferon production by the whole BAL cell population Freshly collected whole BAL cells were divided into four samples. The first was unstimulated, whereas others were incubated with the following inducers: NDV, PHA + PMA or LPS. Results of IFN production after incubation of the cells for 24 h at 3rC are shown in Figure 1. A number of BAL cell cultures from either control or experimental patients produced low level IFN spontaneously. As expected, upon stimulation with inducers, the level of IFN significantly increased. In this study, several different batches of fetal calf serum in culture media were used. All of the batches were pre-selected in PBL cultures for IFN induction without PHA. Only sera with low spontaneous activity (:::; 10 UI ml) were used in the experiments. There were considerable variations in the spontaneous or induced IFN response between BAL cells from control and experimental patients, irrespective of the batch of serum used in the culture medium. The possible presence of small levels of LPS in the cultures of BAL cells cannot be excluded because the cells were harvested from the external environment. However, LPS in a concentration of 10 flg/ml was consistently found to be a weak IFN inducer in BAL cells (Fig. 1).
1.51 ± 0.4 5.57 ± 2.1 3.54 ± 1.1
7.5 ± 1.5
27.7 ± 2.1
17.7 ± 8.5
Inflammatory lung diseases n = 15
Healthy volunteers n = 11
± 11.8
93.5 ± 1.50 94
55.5 ± 7.8
88.8 ± 7.91 6
± 2.7
44.5 ± 14.3
10.05 ± 3.7
91.3 ± 4.6
71.3 ± 19.6
IFN titer was calculated to 106 BAL or WBC leukocytes per ml IFN from BAL vs IFN from WBC: statistically significant at p < 0.05
1.79 ± 0.64 4.20 ± 0.62
0.75 ± 0.66 1.64 ± 1.20
BAL WBC
inflammatory lung diseases
n = 15
0.57 ± 0.52 4.60 ± 0.78
0.61 ± 0.61 2.36 ± 0.65
BAL WBC
controls
n = 11
NDV
1.19 ± 0.71 2.06 ± 1.20
0.53 ± 0.62 3.40 ± 1.00
LPS
IFN titer loglO D/ml after induction with: None
Cells
Patients
Table 2. Interferon production by cells from bronchoalveolar lavages (BAL) and leukocytes in the whole blood cultures (WBC)
Lung cancer n = 10
0/0
Lymphocytes %
Differential Cell Counts Macrophages
Viability %
± 0.7
1.57 ± 1.03 4.41 ± 0.92
0.58 ± 0.75 4.52 ± 1.46
PHA + PMA
1.2 ± 0.3
2.5 ± 1.7
1.4
Granulocytes %
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Interferon in Human Alveolar Leukocytes . 89
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Figure 1. Interferon activity in the unseparated population of bronchoalveolar leukocytes untreated or treated with inducers. Cells were obtained from x healthy individuals, 0 patients with inflammatory lung diseases, "" patients with lung cancer before therapy, and 6. patients with lung cancer receiving steroid therapy. BAL cells were incubated with one of the following inducers: NDV (640 HA units/ml), LPS (10 !!g/ml) or PHA + PMA (10 ng/ml, and 10 ng/ml, respectively). Spontaneous production refers to the BAL cells incubated without the inducers. Data are expressed as a single point for each individual. IFN units released by 106 cells are shown.
In general, BAL cells of normal controls or steroid-treated lung cancer patients were poor producers of IFN. The BAL cells from patients with inflammatory lung diseases produced more IFN either without inducers or after stimulation. Also, cancer patients who were not treated with steroids produced significantly more IFN than cancer patient undergoing steroid therapy (p < 0.01) (Fig. 1). There were no significant differences in the IFN response of BAL cells obtained from smokers and nonsmokers (data not shown). In the bronchoalveolar fluids, even when concentrated up to 10o-fold, an IFN antiviral activity was not detected (data not shown).
Comparison of IFN production of BAL cells and whole blood leukocytes Results in Table 2 show the IFN production of BAL cells and of whole blood cells that were unstimulated or treated with various inducers. The IFN titers were estimated as amounts of IFN produced per 106 cells. It should be mentioned that the two cell populations had completely different composition, and their cell number was not identical. BAL cell population
90 .
MONIKA CEMBRZYNSKA-NoWAK
et al.
contained 78-96 % macrophages, 4-12 % lymphocytes and 2.5-1.2 % granulocytes. In contrast, average differential white cell counts of whole blood culture were: 7 % monocytes, 24 % lymphocytes and 66 % granulocytes. Despite this restriction, it appears that WBC produce considerable more IFN than BAL cells. In other words, the BAL cells were found to be partially hyporesponsive to IFN induction. Even the relatively responsive BAL cells from patients with the inflammatory lung diseases produced less IFN than the whole blood cultures of the same patients (Table 2).
Interferon production by cultured alveolar macrophages The alveolar macrophages were separated from the whole BAL cell population by adherence to plastic plates. The adherent cells were cultured for 6 to 21 days. The freshly isolated macrophages as well as the cultured cells were identified by morphological criteria, their capacity to phagocytose latex beads and by esterase staining. According to these criteria, the freshly isolated adherent cells represented 98 % macrophages and 6-day-old cultures were found to be almost 100 % pure macrophages. The macrophages during culturing in vitro changed from the initially rounded forms into the elongated, spindle-shaped cells. A multiplication of macrophages
x
Z
LL
o n
=6
6
n =4
days of culture
Figure 2. Interferons induced in the normal alveolar macrophages cultured in vitro. Alveolar macrophages (6-10 x 106 cells/ml) were allowed to adhere to the 96-well flat bottom plates at 3rc for 2 h. Nonadherent cells were removed and macrophages were cultured in McCoy medium supplemented with 10 % human AB serum. IFN induction was carried out in the freshly prepared macrophages (day 0) and in the cells cultured for 6 days. 1m - NDV induction; § - PHA + PMA induction.
Interferon in Human Alveolar Leukocytes . 91
was not observed. The number of viable cells in the 6-day cultu~e was approximately 99 %. Interferon induction was carried out with 0 day macrophages and 6 daycultured cells. Day 0 indicated the freshly isolated cells immediately after placing them into culture wells. Results of IFN induction with NDV and PHA + PMA or LPS are shown in Figures 2 and 3. The freshly isolated alveolar macrophages from the control patients produced considerably lower levels of IFN after stimulation with NDV or PHA + PMA (Fig. 2) than cells cultured for 6 days at 3rC (p < 0.01). In contrast, alveolar cells separated from BAL of patients with inflammatory lung diseases were found to be more responsive to IFN inducers shortly after the isolation (at time 0) than after further culture for 6 days (Fig. 3). Only small cell numbers for in vitro culture were available from these patients. Therefore, it was not possible to assess all inducers simultaneously, and to study the kinetics of IFN production. Fragmentary data obtained from the study suggested that a 6-day culture is optimal for IFN production. After 8, 14 and 21 days, the inducible IFN levels decreased significantly (data not shown).
exp.1
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o
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Figure 3. Interferon induced in the inflammatory alveolar macrophages cultured in vitro. The BAL cell were obtained from patients with the inflammatory lung diseases including pneumonia and chronic bronchitis. For other explanations see Fig. 2. fillI- NDV induction; == PHA + PMA induction; m- LPS induction. Assays were done with the cells from individual patients.
92 .
MONIKA CEMBRZYNSKA-NOWAK
et al.
Table 3. Neutralization of antiviral activity in supernatants from alveolar cell or whole blood cultures treated with different IFN inducers Inducer nIFN-a NDV PHA+PMA LPS None'f
Detected
Reaction with antiserum to nIFN-B
rIFN-y
+ + + +
IFN-a IFN-y IFN-y IFN-y
'f Supernatants from control cells cultured without inducers n = natural r = recombinant
Antigenic identifi."cation of IFNs
The types of IFN produced by BAL cells and isolated macrophages were determined by neutralization with antibodies to IFN-a, IFN-~ and IFN-y (Table 3). The anti-HuIFN-a serum neutralized only the antiviral activity of IFN induced by NDV. The anti-HuIFN-y antibody completely inhibited antiviral activity of IFN from the alveolar macrophages induced with LPS or with PHA + PMA. The antiserum against IFN -~ had no effect on the IFN preparations studied. The IFN produced spontaneously without any exogenous inducers was neutralized by anti-IFN-y. However, the presence of small levels of other IFN types could not be excluded because their titers were too low. Discussion We have observed that macrophages which are a predominant component of the population of the bronchoalveolar cells, produce significantly lower levels of IFN-a and IFN-y than the peripheral blood leukocytes in the whole blood culture. This refers mainly to the cells from normal healthy individuals treated or untreated with inducers. Also BAL cells from patients with the inflammatory lung diseases and even with lung cancer produced relatively lower IFN levels than the blood leukocytes. Therefore, our consistent observation is partially discrepant with the results of NUGENT et al. (7) who described that normal human macrophages produced IFN after stimulation with classic inducers at the same level as blood monocytes. However, their finding that very low levels of IFNs were obtained from alveolar cells of normal healthy individuals (7, 9) corresponded well with our results. Furthermore, PRIOR and HASLAM (8) observed that «spontaneous» IFN levels in the lung were higher than that in the serum of healthy volunteers. Interferons as well as other cytokines are produced locally and they function in a paracrine fashion. Inability to detect IFN and other
Interferon in Human Alveolar Leukocytes . 93
cytokines in blood may be due to their modest spill-over in a rapidly turning-over pool (14). The inflammatory and immunological processes facilitate cell migration and activation connected with release of cytokines (15). Therefore, it is not surprising that BAL cells from patients with inflammatory lung diseases as well as those with untreated lung cancer produced more interferon than the normal control cells. This also refers to the amounts of IFN released without any treatment with exogenous inducers. Our results that the cells from lung cancer patients undergoing steroid therapy produced significantly lower level of IFN as compared to the cells from the untreated patients are consistent with the knowledge that steroids suppress the release of IFN-y by mononuclear cells (9, 16, 17). In that respect, it is noteworthy that lung mononuclear cells from patients with pulmonary sarcoidosis spontaneously released considerable amounts of IFN-y compared to normal (9). The limited ability to spontaneous release by interferon and the weak IFN induction in normal lung mononuclear cells which was observed by us and others (7, 9) prompted us to suggest that hyporeactivity of BAL leukocytes may be a physiological condition. BAL cells are constantly exposed to external inhaled stimuli such as dust, smoke, viruses, microorganisms and their products, especially to LPS. All of these factors may stimulate the cells to produce cytokines e.g. IFNs, IL-l, IL-2, TNF, IL-6, TGF-~, etc. (1). The resident alveolar macrophages with well-developed phagocytic activity and hyporeactive to IFN induction may protect the lungs against noxious stimuli, especially against allergens, and other immunologic stimulants. IFNs were found to enhance histamine release and to increase IgE binding to basophils. In view of the suggested pathogenic role of IFN in allergy, a low capacity for IFN synthesis appears to be a favorable condition (6, 18). Hyporesponsiveness to IFN induction may develop as a consequence of repeated exposure to various inducers. Approximately one week is required to overcome the induced hyporesponsiveness (19, 20). In our experiments, we observed an overcoming of the hyporeactivity of control alveolar macrophages during 6 days of culture in vitro. However, the BAL cells from patients with inflammatory lung diseases responded to the inducers more intensively immediately after isolation than after one week of culturing in vitro. This may be due to the elimination of the cells originating from the inflammatory exudate initially capable to relatively high cytokine production. It has been repeatedly suggested that the alveolar macrophages are metabolically different than the peritoneal, pleural and other phagocytes (21). This refers mainly to their strong suppressor activity in the mitogeninduced lymphocyte proliferation (22), and also in presenting antigens (23, 24).
94 . MONIKA CEMBRZYNSKA-NOWAK et al.
Human alveolar macrophages have a limited capacity for interleukin-1 release as compared to peripheral blood monocytes (25, 26). The accumulation of mRNA for IL-1-~ was 20-fold greater in blood monocytes than in the alveolar macrophages (27). STRINGFELLOW et al. (19) and MONICK et al. (28) suggested that prostaglandins may play an important role in hypo reactivity of cells to IFN and IL-1 inducers. The role of eicosanoids in the described phenomenon remains to be determined. The relatively poor capacity of alveolar macrophages to produce IL-1 contrasts with the observation that alveolar macrophages had significantly greater TNF production in response to different inducers than peripheral blood monocytes (29-31). Although IFN s, IL-1 and TNF have a similar spectrum of activity, the capacity of alveolar macrophages to produce IFN sand IL-1 appears to be decreased and to produce TNF to be increased when compared with blood monocytes. It is generally believed that macrophages are producers of IFN -a/~ but not IFN-y (32). However, we have consistently found that the cultured alveolar cells produced, after stimulation with NDV, IFN-a and produced IFN-y following PHA + PMA or LPS stimulation. The observations are compatible with the findings of ROBINSON et al. (9), NUGENT et al. (7) and PRIOR and HASLAM (8) that freshly isolated alveolar macrophages may produce IFN-y spontaneously or after PHA or LPS induction. Variations in the IFN response of alveolar cells cannot be due to LPS only despite the fact that it is a ubiquitously contaminating agent. Our experiments showed the LPS at 10 flg/ml is a relatively weak IFN inducer in comparison to NDV or PHA (Fig. 1). We cannot exclude that our cultures contained small number of T lymphocytes or NK cells which contaminated apparently pure macrophages. Alveolar macrophages may also form syncytia with lymphocytes and the fused cells may respond to both NDV and PHA + PMA. Furthermore, a direct reaction of alveolar cells with PHA + PMA is also possible. All these hypotheses require further experimental verification.
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Dr. MONIKA CEMBRZYNSKA-NoWAK, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Czerska 12, 53-114 Wroclaw, Poland