Changes in Interleukin-1 and Tumor Necrosis Factor Production by Peripheral Blood Monocytes after Specific Bronchoprovocation Test in Occupational Asthma 1- 4
ANDREA SIRACUSA, ANNA VECCHIARELLI, GIULIANA BRUGNAMI, ALESSANDRA MARABINI, DANIELA FELICIONI, and CARLA SEVERINI
Introduction
High-molecular-weight compounds can induce occupational asthma (OA) by producing specific IgE antibodies. The pathogenetic mechanisms of OA induced by low-molecular-weight compounds (LMWC) have not been well defined, and further studies are required to clarify the role of immunologic mechanisms (1). It is not clear how LMWC initiate the asthmatic reaction within the lung. Exposure to antigen is followed by peripheral blood monocyte (PBM) and alveolar macrophage (AM) activation in asthmatic patients (2-4). Furthermore, it has been shown that the number of monocytes in the airways increases at 48 h after antigen provocation in asthmatic patients (5). PBM produce numerous secretory products such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). IL-1 initiates T-lymphocyte activation (6), is a chemotactic factor for human T lymphocytes (7) and neutrophils (6), and acts synergistically with TNF in inflammatory reactions (8). T lymphocytes also seem to have an important role in asthma. Specialized T-cell subsets elaborate interleukin-4 (IL-4), an essential cofactor for IgE production, and interleukin-5 (IL-5), which brings about terminal differentiation and activation of eosinophils (9). Changes in T-cell subsets and circulating leukocytes have also been observed in the peripheral blood of patients with OA after bronchoprovocation tests with toluene diisocyanate (TDI) (10). To gain insight about the pathogenesis of OA due to LMWC, we evaluated IL-1 and TNF production by PBM, peripheral T-cell subpopulations, and leukocytes in 22 subjects with possible OA before and after specific bronchoprovocation test (SBPT). TNF secretion by PBM from patients with OA was in408
SUMMARY The pathogenetic mechanisms of occup.atlonalasthma (OA)due to low-molecular-weight compounds have been poorly defined, and further studies are requ Ired to clarify the role of Immunologic mechanisms In OA. Until now cellular mechanisms have been less Investigated than humoral ones. Wehave evaluated Interleukln-1 (IL-1)and tumor necrosis factor (TNF) production by peripheral blood monocytes (PBM), and peripheral T-cell subpopulatlons In 22 subjects with possible OA before and after specific bronchoprovocatlon test (SBPT). After SBPT, three SUbjects had an Immediate reaction, seven a late reaction, and two a dual reaction. Ten subjects had no asthmatic reaction to SBPT. Spontaneous release of 1L-1 from PBM did not change significantly after SBPT. TNF activity was Increased 48 h after SBPT In Immediate reactions, and 72 h after SBPT In late-dual reactions. These results suggest that exposure to occupational agents may Induce activation of PBM with Increased spontaneous release of cytoklnes, such as TNF. AM REV RESPIR DIS 1992; 146:408-412
creased 48 to 72 h after a positive SBPT, suggesting the participation of PBM in the mechanisms involved in OA. Methods Reagents and Media RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Eurobio Laboratories (Paris, France). Human recombinant interleukin-l was provided by Janssen Biochimica (Beerse, Belgium). Tumor necrosis factor-a (TNF-a) was purchased from Genzyme Corp. (Boston, MA). Endotoxic lipopolysaccharide from Escherichia coli 055:135 (LPS) was obtained from Difeo (Detroit, MI). [3H]-Thymidine (specificactivity 88 Ci/mmol) was purchased from Amersham International (Amersham, UK). Actinomycin D and concanavalin A (ConA) were obtained from Sigma Chemical Company (St. Louis, MO). FicollHypaque" solution was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). Subjects From February 1989 to May 1990 we carried out SBPT with suspected causative agents in 22 consecutive subjects referred to our institute because of possible OA. Possible OA was defined as a history of work-related asthmatic symptoms without objective assessments (SBPT and serial monitoring of peak expiratory flow rates for periods at work and away from work). SBPT was carried out in a challenge room as proposed by Pepys and Hutchcroft (11). The following tests wereperformed
on each subject at 8:00 A.M. On Day 1 a methacholine challenge test (MCT) was performed. On Day 2 subjects were exposed to control varnish (painting for 15 min) or dust (tipped from one receiver to another for 30 min). On Day 3 a MCT was performed again. On Day 4 subjects were exposed to TDI or synthetic varnishes (painting for 15 min) or wood dust (for 30 min). On Day 5 a MCT was performed for the third time. A controlled exposure (Day 2) with a control substance was followed in all subjects by negative reactions and, 24 h later, by insignificant changes in bronchial reactivity. After SBPT, three subjects had an immediate reaction, seven a late reaction, two a dual reaction, and 10no asthmatic reaction (table 1). The occupational agents used for SBPT were TDI in 14 sub-
(Received in original form September 11, 1991 and in revised form March 12, 1992) 1 From the Institute of Occupational Medicine and the Department of Experimental Medicine and Biochemical Sciences, Microbiology Section, University of Perugia, Perugia, Italy. 1 Supported by a grant from the Ministry of the University and Scientific Research and by Grant No. 9297/28-11-1989 from the Regionedell'Umbria. 3 Presented in part at the American Thoracic Society World Conference on Lung Health, Boston, May 1990. .. Correspondence and requests for reprints should be addressed to Prof. A. Siracusa, Institute of Occupational Medicine, Via E. Dal Pozzo, 06100 Perugia, Italy.
IL·1 AND TNF PRODUCTION BY MONOCYTES IN OCCUPATIONAL ASTHMA
jects, synthetic varnishes without TD I in six, and wood dust in two. The immediate reactions followed SBPT with synthetic varnish (two subjects) or Tanganyka aningre wood dust (one subject), late reactions were due to TDI (seven subjects), and dual reactions were due to TDI (one subject) or Mansonia altissima wood dust (one subject). Negative reactions followed TD I (six subjects) or synthetic varnishes (four subjects). FEV. was measured before exposure, after 30 min, hourly for 8 h and then 24 h after exposure. Blood samples for in vitro analyses were taken before exposure and 24, 48, and 72 h after exposure to occupational agents in the 22 subjects with possible OA.
409
Preparation of PBM Heparinized venous blood, obtained from healthy donors or from asthmatic subjects, was diluted with RPMI 1640medium plus 50/0 FBS (hereafter referred to as cRPMI). Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation (14). Cells were washed twice in cRPMI, plated in cell culture petri dishes (Nunc, Naperville, IL) at a concentration ranging between 2 x 106 and 3 x 106/ml, and incubated at 37° C in 50/0 CO 2 for 1 h. Nonadherent cells were removed by washing the dishes three to five times with warm RPMI 1640 medium, and the adherent cells were carefully removed using a rubber policeman. The latter cellswere 95 to 98 % positive to esterase staining, and cell viability was assessed by trypan blue dye exclusion test (more than 980/0 viable).
maximal stimulation with a standard recombinant human IL-l preparation (Janssen) in a tritiated thymidine uptake test. TNF was determined as described (17),using actinomycin D-treated L-929 cells as target. Serial dilutions (0.1 ml) of supernatants were added to 4 x 104 L-929 cells seeded in 0.1 ml in flatbottomed microtiter plates. The determination of TNF activity was made in comparison with commercially available preparations (Genzyme) with known titers, and results were expressed as units per milliliter. In selected experiments TNF and IL-l activity was abrogated by monoclonal antibodies to IL-l (Janssen) and polyclonal antibodies to TNF-a (Genzyme).
Methacholine Challenge Test Data Analysis A breath-activated dosimeter (ME.FAR, BresThe data regarding clinical details (table 1), cia, Italy) was used. Double cumulative doses pulmonary function (table 2), and blood leuof methacholine were administered beginning Production of PBM Culture Supernatants kocytes and lymphocyte subpopulations (taand Assays for IL-l and TNF Activity with a dose of 25 J.1g (12). FEV. was recorded ble 3) are presented as mean ± SEM. Since 1 min later followed by the next dose. The Supernatants from PBM were obtained ac- IL-l and TNF activities are based on serial challenge sequence terminated when FEV. -: cording to a previously described method (15). twofold dilutions, the data of the activities had decreased by 200/0 or more or when the Briefly, PBM were seeded at a concentration of both cytokines (figures 1 and 2) were anamaximum cumulative dose (3.2 mg) had been of 5 x 106/ml of cRPMI for 24 h without lyzed as log, 0 f the values. Because there were given. The methacholine provocation dose, stimuli. Supernatants wereharvested, filtered, only two dual responders, we considered late necessary to decrease FEV. by 200/0 (PD 20 and stored at -80° C until assayed. IL-l was and dual responders together in the analysis. FEV.), was obtained from the dose-response measured as proliferative activity on freshly The result did not change when these two curve plotted on a semilogarithmic scale; the isolated thymocytes from C3H/HeJ male groups were considered separately. The total abscissa represents logarithmic methacholine mice, 4 to 8 wk old, obtained from the Jacknumber of leukocytes was correlated with that doses, and the ordinate reports the percentson Laboratory (Bar Harbor, ME). Briefly, of eosinophils, and the five lymphocyte subage decrease in FEV •. 1 x 106 thymocytes in 0.1 ml cRPMI were sets were also correlated to each other. For The challenge was performed using buf- incubated with 0.1 ml of twofold dilutions each of the three groups (immediate respondfered lyophilized methacholine (phosphate of supernatants in the presence of 1 ug/ml ers, late-dual responders, and no asthmatic buffer) (Lofarma, Milano, Italy); 0.20/0 and of ConA for 72 h at 37° C in 50/0 CO 2 (16). reaction), comparisons between baseline 2 % methacholine solutions were prepared by IL-l titers were calculated by comparing the values and values after SBPT were tested for diluting methacholine in distilled water. The dilution of the test supernatants that gave500/0 statistical significance using a multivariate t dosimeter deliveredmethacholine from a MB2 ampule (ME.FAR) by means of an air compressor (driving pressure 1.5 kg/ern") (12). Every inhalation delivered 25 or 250 J.1g methaTABLE 1 choline from 0.2 and 2 % concentrations, CLINICAL DETAILS* respectively. Preparation of PBM and Determination of IL-l and TNF Activities For preparation of PBM and assay of IL-l and TNF activities without stimuli, methods were used that have been previously described (13).IL-l and TNF activities were determined before SBPT and 24, 48, and 72 h after exposure to occupational agents. Blood Leukocytes and Lymphocyte Subpopulation Analysis Total numbers of leukocytes and eosinophils werecounted by routine methods. Lymphocyte subsets were evaluated by two-color immunofluorescence and fluorescence activated cell sorter (FACS) analysis (Becton-Dickinson, Mountainview, CAl using Becton-Dickinson anti-CD3 (Leu-4), anti-CD4 (Leu-3), antiCD8 (Leu-2), anti-CD57 (Leu-7), and antiClass II major histocompatibility complex (MHC) (HLA-DR) antibodies. Blood leukocytesand lymphocyte subpopulations weredetermined before and 24 h after SBPT with occupational agents.
Type of Asthmatic Reaction
Subjects (n)
Immediate Late alone Dual No asthmatic reaction
3
7 2 10
Duration of Exposure (yr)
Age (yr) 33.3 44.4 46.5 51.4
± ± ± ±
6.0 3.9 4.5 2.3
11.9 20.9 24.0 25.4
± ± ± ±
7.5 5.1 15.0 2.6
Duration of Symptomst (yr) 6.5 5.7 7.2 3.6
± ± ± ±
4.0 2.7 5.7 1.1
* Mean ± SEM.
t
Before diagnosis.
TABLE 2 PULMONARY FUNCTION PD 20 FEV1 Methacholine (pg)
Immediate Late and dual No .asthmatic reaction
FEV1 (L)
FEV 1 (0/0 of predicted)
Before SBPT
After SBPT
3.68 ± 0.71 * 3.41 ± 0.25
94 ± 11* 99 ± 5
228 ± i t 1,067 ± 3
363 ± 2t 1,241 ± 1
747 ± 2
1,010 ± 2
3.31 ± 0.22
103 ± 7
Definitionof abbreviations: PD20 FEV t = methacholine provocative dose needed to decrease FEVt by 20%; SBPT =~pecific bronchoprovocation test. * Mean ± SEM. t Geometric mean ± geometric SEM.
SIRACUSA, VECCHIARELLI, BRUGNAMI, MARABINI, FELiCIONI, AND SEVERINI
410 TABLE 3 CHANGES IN PERIPHERAL BLOOD CELLS AFTER SPECIFIC BRONCHOPROVOCATION TEST' Type of Asthmatic Reaction
Late and Dual (n = 6)
No Asthmat ic Reaction (n 8)
7,250 ± 750 7,450 ± 1,150
6,962 ± 278 8,267* ± 627
7,000 ± 518 7,180 ± 843
150 ± 8 180 ± 17
115 ± 9 198* ± 22
124 ± 4 210 ± 25
66.5 ± 1.5 66.5 ± 4.5
69.2 ± 4.2 70.7 ± 2.7
69.8 ± 2.1 70.9 ± 2.1
38.0 ± 2.0 38.5 ± 0.5
42.9 ± 1.3 44.8 ± 1.2
45.0 ± 2.7 47.0 ± 3.4
39.0 ± 2.0 39.5 ± 0.5
33.8 ± 1.5 35.8 ± 1.9
34.1 ± 3.0 31.3 ± 3.0
22.0 ± 7.0 21.5 ± 4.5
16.0 ± 2.0 19.4 ± 2.8
20.4 ± 2.4 20.5 ± 1.8
20.5 ± 2.9 19.4 ± 2.9
23.6 ± 1.3 21.1 ± 1.5
20.5 ± 2.9 19.4 ± 2.9
Immediate (n
Leukocytes/mm' Baseline 24 hrt Eosinophils/mm' Baseline 24 h % CD3' T cells Baseline 24 h % CD4' Tcells Baseline 24 h % CD8' Tcells Baseline 24 h % HLA·DR' Baseline 24 h % Natural killer (Leu·7') cells Baseline 24 h
~
2)
=
'Mean ± SEM. That Is, 24 h after specific bronchoprovocatlon test. Significantly different from baseline values at p < 0.005 (Hotelling's l ' test for leukocytes and sinophils together).
t
*
eo-
8 7
6
E
Fig . 1. IL·l activity in supernatants of peripheral blood monocytes (PBM) from patientswith possible occupational asthma after specific bronchoprovocation test (SBPT). Data are mean ± SEM. Solid bars = negative ; hatched bars. immediate; shaded bars = late + dual.
:3
TIME (h) AFTER SBPT 8 7
*
6
E
Fig. 2. TNF activity in supernatants of PBM from patients with possible occupational asthma after SBPT.Data are mean ± SEM. ' Significantly different from baseline values , p < 0.001; t significantly different from baseline values, p < 0.01. Solid bars = negative; hatched bars = immediate; shaded bars = late + dual.
5
:3 LL
Z I-
24
48
TIME (h) AFTER S8PT
72
(Hotelling's P) test (18) twice, first for leukocytes and eosinophils considered together and then for the other five lymphocyte subsets. Comparisons betweenvalues of log, IL-l and log, TNF before and after SBPT were tested for statistical significanceusing the twotailed paired t test, with a < 0.05. Changes between baseline and 24, 48, and 72 h after SBPT values of log, IL-l and log, TNF were also tested separately using repeated-measures analysisof variance (19). In this analysis,using Fisher's test, we can evaluate the statistical significance of a set of comparisons simultaneously, taking into account possible correlations between the values for each subject. Because the occupational agent was TDI in 14of 22 cases, we analyzed the data separately for subjects with OA due to TDI, but the results did not change.
Results
SBPT was carried out in 22 subjects with possible OA. Before and after SBPT we evaluated FEV., spontaneous production of IL-l and TNF in culture supernatants of PBM , and peripheral T-cell subpopulations and leukocytes to study the activity of PBM and lymphocytes in OA. Baseline lung function measurements are listed in table 2. Mean values of FEV. were within the predicted values (20). Methacholine challenge test, carried out before SBPT, showed on the averagemild to moderate bronchial hyperreactivity, with trivial changes after SBPT with control materials (data not shown) and occupational agents. The maximal percentage decrease in FEV. was 35.5 for immediate reactions and 22.1 for late-dual (late component) reactions. In these subjects FEV. was still9.9OJo below the baseline value at 24 h. There was no correlation between the magnitude of the latedual asthmatic responses and the change in cytokine release by PBM 72 h after SBPT (for TNF r was -0.39, p = 0.52). Numbers of circulating leukocytes and eosinophils, tested together by HoteHing's T2test, increased 24 h after SBPT in latedual responders (p < 0.005) (table 3). Baseline values of the five lymphocyte subsets in the three groups and their variations 24 h after SBPT, tested together by the P test, were not significantly different. There were no differences in IL-l and TNF baseline values among the four groups (controls, nonresponders, and immediate and late-dual responders) (figures 1 and 2). After SBPT, IL-I activity increased slightly in both immediate (at 24 and 48 h) and late-dual reactions (at 72 h), but these differences were not significant (figure I). By t test it was shown that TNF activity increased 48 h
1L-1 AND TNF PRODUCTION BY MONOCYTES IN OCCUPATIONAL ASTHMA
In this study there is evidence of PBM activation in OA after airflow obstruction induced by occupational agents. It has been shown both in allergic asthmatic patients (5) and in mice (27) that after an inflammatory stimulus [allergen inhalation (5)and BCG injection (27)]there is a rapid migration of PBM that is transformed into AM. Moreover, monocytes and monokines have been found to increase in bronchoalveolar lavage fluid in symptomatic nonallergic asthmatic patients (28). Similarly, it is possible that in OA there is an influx of PBM in the lung that is transformed into AM after Discussion exposure to an occupational agent. This study shows that immediate, late, In disagreement with our results (taand dual reactions due to occupational agents are associated with an increase in ble 2), changes in bronchial responsivethe spontaneous production of TNF ness to methacholine have been observed released by PBM. In OA due to high- in OA after late or dual asthmatic reacmolecular-weight compounds, specific tions (29, 30). Significant changes have 19B antibodies to these occupational also been demonstrated at 3 and 8 h afagents can be detected (21). OA due to . ter SBPT (31, 32). The increase in reLMWC has not been clarified, and cel- sponsiveness may be transient, as at 24 h lular mechanisms have been underinves- it is less than at 8 h (31), and may be partially influenced by a reduction in tigated (10, 22). The studies regarding T-cell subsets in airway caliber (31). Moreover, some imthe peripheral blood of patients with provement in responsiveness to methaasthma are not concordant. Some studies choline can result from hospitalization, have found an increase in activated T cells which reduces exposure to allergens (33). (HLA-OR+) in acute severe asthma (23, Finally, a recent retrospective study has 24). Others (10) have reported an increase demonstrated that changes in bronchial in suppressor/cytotoxic C08+ T cells48 h responsivenessafter late or dual reactions after SBPT with TOI in OA but no are not constant (34). Our study suggeststhat exposure to occhange in activated and helper T-cell subsets. In other studies, helper T cells of cupational agents may induce the actipatients with allergic asthma were re- vation of PBM with increased spontaneduced after SBPT with mixed grass ex- ous release of cytokines. Other authors tract (25) but were increased during the have shown the activation of T cells and pollen season (26). In our data on lym- AM in the airways of asthmatic subjects phocyte subsets before and after SBPT, (2, 5). Cytokines such as IL-l and TNF, there is no evidence in favor of or against released by activated monoeytes and AM, these contradictory results, with the pos- may play a role in the recruitment and sible exception of a nonsignificant in- activation of eosinophils and neutrophils crease in activated T cells in late-dual and in the formation of the inflammareactions (table 3). These differences tory cell infiltrate in OA. The role of procould, at least in part, be due to the het- inflammatory cytokines in the pathogenerogeneity of both study methods (vari- esis of asthma seems to be important; ous time intervals between exposure to their exact importance will be clarified precipitating factors and study of im- only by further studies. munocompetent cells) and pathogenetic Acknowledgment mechanisms. In the present study, subjects with im- The writers thank A. Forcina for statistical mediate and late-dual asthmatic reac- analysis of the data, A. Velardi for helpful tions exhibited different kinetics of in- criticism of the manuscript, and Eileen Macrease in TNF. In immediate reactions honey Zannetti for excellent editorial and secretarial assistance. this increase was significant at 48 hand in late-dual reactions it was significant References at 72 h (figure 2). These results may sug1. Chan-YeungM, Lam S. Occupational asthma. gest different mechanisms in immediate Am Rev Respir Dis 1986; 133:686-703. and late reactions or they may be a 2. Tonnel AB, Joseph M, Gosset P, Fournier E, consequence of bronchoconstriction, Capron A. Stimulation of alveolar macrophages delayed several hours in late responders. in asthmatic patients after local provocation test. after SBPT (versus baseline values) in immediate reactions (p < 0.001)and 72 h after SBPT (versus baseline values) in late-dual reactions (p < 0.01). By repeated-measures analysis of variance it was demonstrated that in immediate responders at 48 h and in late-dual responders at 72 h, considered simultaneously, TNF was significantly increased in comparison with baseline values (p < 0.005). All other comparisons with baseline values, considered simultaneously, were not significant.
r
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Lancet 1983; 1:1406-8. 3. Carroll MP, Durham SR, Walsh G, Kay AB. Activation of neutrophils and monocytes after allergen- and histamine-induced bronchoconstriction. J Allergy Clin Immunol 1985; 75:290-6. 4. Anderson CL, Spiegelberg HL. Macrophage receptors for IgE: binding of IgE to specific IgE receptors on a human macrophage cell line U937. J Immunol 1981; 126:2470-3. 5. Metzger WJ, Zavala D, Richerson HB, et ale Local allergen challenge and bronchoalveolar lavage of allergic asthmatic lungs. Am Rev Respir Dis 1987; 135:433-40. 6. Le J, Vilcek J. Thmor necrosis factor and interleukin 1:cytokines with multiple overlapping biological activities. Lab Invest 1987; 56:234-48. 7. Hunninghake GW, Glazier AJ, Monick MM, Dinarello CA. Interleukin-l is a chemotactic factor for human T lymphocytes. Am Rev Respir Dis 1987; 135:66-71. 8. Dinarello CA, Mier JW. Lymphokines. N Engl J Med 1987; 317:940-5. 9. Kay AB. Lymphocytes in asthma. Respir Med 1991; 85:87-90. 10. Finotto S, Fabbri LM, Rado V, Mapp CE, Maestrelli P. Increase in numbers of CD8 positive lymphocytes and eosinophils in peripheral blood of subjects with late asthmatic reactions induced by toluene diisocyanate. Br J Ind Med 1991; 48:116-21. 11. Pepys J, Hutchcroft BJ. Bronchial provocation tests in etiologic diagnosis and analysis ofasthmao Am Rev Respir Dis 1975; 112:829-59. 12. Cerveri I, Bruschi C, Zoia MC, et ale Smoking habit and bronchial reactivity in normal subjects. A population-based study. Am Rev Respir Dis 1989; 140:191-6. 13. VecchiarelliA, Dottorini M, Puliti M, Todisco T, Cenci E, Bistoni F. Defective candidacidal activity of alveolar macrophages and peripheral blood monocytes from patients with chronic obstructive pulmonary disease. Am Rev Respir Dis 1991; 143:1049-54. 14. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968; 21(Suppl 97:77-89). 15. Varesio L, Holden HT, Taramelli D. Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines. J Immunol 1980; 125:2810-7. 16. MizelSB.Production and quantitation of lymphocyte activating factor (interleukin-l). In: HerscowitzHB, Holden HT, Bellanti JA, Galfar A, eds. Manual of macrophage methodology. New York: Marcel Dekker, 1981; 407-16. 17. Rubin BY, Anderson SL, Sullivan SA, Williamson BD,Carswell BD,Old LY. Purification and characterization of human tumor necrosis factor. Proc Nat! Acad Sci USA 1985; 82:6637-41. 18. Chatfield C, Collins AJ. Introduction to multivariate analysis. London: Chapman and Hall, 1980; 114-39. 19. .SAS Institute Inc. SAS/STATuser's guide, version 6, 4th ed, vol 2. Cary, NC, 1990; 951-8. 20. Quanjer PH. Standardized lung function testing. Report working party. Standardization of lung function tests. Bull Eur Physiopathol Respir 1983; 19(suppl 5:1-95). 21. Grammer LC, Patterson R, Zeiss CR. Guidelines for the immunologic evaluation of occupationallung disease. J Allergy Clin Immunol 1989; 84:805-14. 22. SchliemerRP, Benenati SV, Friedman B, Bochner BS.Do cytokines playa role in leukocyterecruitment and activation in the lungs. Am Rev Respir Dis 1991; 143:1169-74. 23. Corrigan CJ, Hartnell A, Kay AB. T lympho-
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heat-killed bacillus Calmette-Guerin. J Exp Med 1981; 154:235-52. 28. Mattoli S, Mattoso VL, Soloperto M, Allegra L, Fasoli A. Cellular and biochemical characteristics of bronchoalveolar lavage fluid in symptomatic nonallergic asthma. J Allergy Clin Immunol 1991; 87:794-802. 29. Lam S, Wong R, YeungM. Nonspecific bronchial reactivity in occupational asthma. J Allergy Clin Immunol 1979; 63:28-34. 30. Mapp CE, Dal Vecchio L, Boschetto P, De Marzo N, Fabbri LM. Toluenediisocyanate-induced asthma without airway hyperresponsiveness. Eur oJ Respir Dis 1986; 68:89-95. 31. Mapp CE, Polato R, Maestrelli P, Hendrick DJ, Fabbri LM. Time course of the increase in airway responsiveness associated with late asthmatic reactions to toluene diisocyanate in sensitized sub-
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ANDREA SIRACUSA, ANNA VECCHIARELLI, GIULIANA BRUGNAMI, ALESSANDRA MARABINI, DANIELA FELICIONI, and CARLA SEVERINI
Introduction
High-molecular-weight compounds can induce occupational asthma (OA) by producing specific IgE antibodies. The pathogenetic mechanisms of OA induced by low-molecular-weight compounds (LMWC) have not been well defined, and further studies are required to clarify the role of immunologic mechanisms (1). It is not clear how LMWC initiate the asthmatic reaction within the lung. Exposure to antigen is followed by peripheral blood monocyte (PBM) and alveolar macrophage (AM) activation in asthmatic patients (2-4). Furthermore, it has been shown that the number of monocytes in the airways increases at 48 h after antigen provocation in asthmatic patients (5). PBM produce numerous secretory products such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). IL-1 initiates T-lymphocyte activation (6), is a chemotactic factor for human T lymphocytes (7) and neutrophils (6), and acts synergistically with TNF in inflammatory reactions (8). T lymphocytes also seem to have an important role in asthma. Specialized T-cell subsets elaborate interleukin-4 (IL-4), an essential cofactor for IgE production, and interleukin-5 (IL-5), which brings about terminal differentiation and activation of eosinophils (9). Changes in T-cell subsets and circulating leukocytes have also been observed in the peripheral blood of patients with OA after bronchoprovocation tests with toluene diisocyanate (TDI) (10). To gain insight about the pathogenesis of OA due to LMWC, we evaluated IL-1 and TNF production by PBM, peripheral T-cell subpopulations, and leukocytes in 22 subjects with possible OA before and after specific bronchoprovocation test (SBPT). TNF secretion by PBM from patients with OA was in408
SUMMARY The pathogenetic mechanisms of occup.atlonalasthma (OA)due to low-molecular-weight compounds have been poorly defined, and further studies are requ Ired to clarify the role of Immunologic mechanisms In OA. Until now cellular mechanisms have been less Investigated than humoral ones. Wehave evaluated Interleukln-1 (IL-1)and tumor necrosis factor (TNF) production by peripheral blood monocytes (PBM), and peripheral T-cell subpopulatlons In 22 subjects with possible OA before and after specific bronchoprovocatlon test (SBPT). After SBPT, three SUbjects had an Immediate reaction, seven a late reaction, and two a dual reaction. Ten subjects had no asthmatic reaction to SBPT. Spontaneous release of 1L-1 from PBM did not change significantly after SBPT. TNF activity was Increased 48 h after SBPT In Immediate reactions, and 72 h after SBPT In late-dual reactions. These results suggest that exposure to occupational agents may Induce activation of PBM with Increased spontaneous release of cytoklnes, such as TNF. AM REV RESPIR DIS 1992; 146:408-412
creased 48 to 72 h after a positive SBPT, suggesting the participation of PBM in the mechanisms involved in OA. Methods Reagents and Media RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Eurobio Laboratories (Paris, France). Human recombinant interleukin-l was provided by Janssen Biochimica (Beerse, Belgium). Tumor necrosis factor-a (TNF-a) was purchased from Genzyme Corp. (Boston, MA). Endotoxic lipopolysaccharide from Escherichia coli 055:135 (LPS) was obtained from Difeo (Detroit, MI). [3H]-Thymidine (specificactivity 88 Ci/mmol) was purchased from Amersham International (Amersham, UK). Actinomycin D and concanavalin A (ConA) were obtained from Sigma Chemical Company (St. Louis, MO). FicollHypaque" solution was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). Subjects From February 1989 to May 1990 we carried out SBPT with suspected causative agents in 22 consecutive subjects referred to our institute because of possible OA. Possible OA was defined as a history of work-related asthmatic symptoms without objective assessments (SBPT and serial monitoring of peak expiratory flow rates for periods at work and away from work). SBPT was carried out in a challenge room as proposed by Pepys and Hutchcroft (11). The following tests wereperformed
on each subject at 8:00 A.M. On Day 1 a methacholine challenge test (MCT) was performed. On Day 2 subjects were exposed to control varnish (painting for 15 min) or dust (tipped from one receiver to another for 30 min). On Day 3 a MCT was performed again. On Day 4 subjects were exposed to TDI or synthetic varnishes (painting for 15 min) or wood dust (for 30 min). On Day 5 a MCT was performed for the third time. A controlled exposure (Day 2) with a control substance was followed in all subjects by negative reactions and, 24 h later, by insignificant changes in bronchial reactivity. After SBPT, three subjects had an immediate reaction, seven a late reaction, two a dual reaction, and 10no asthmatic reaction (table 1). The occupational agents used for SBPT were TDI in 14 sub-
(Received in original form September 11, 1991 and in revised form March 12, 1992) 1 From the Institute of Occupational Medicine and the Department of Experimental Medicine and Biochemical Sciences, Microbiology Section, University of Perugia, Perugia, Italy. 1 Supported by a grant from the Ministry of the University and Scientific Research and by Grant No. 9297/28-11-1989 from the Regionedell'Umbria. 3 Presented in part at the American Thoracic Society World Conference on Lung Health, Boston, May 1990. .. Correspondence and requests for reprints should be addressed to Prof. A. Siracusa, Institute of Occupational Medicine, Via E. Dal Pozzo, 06100 Perugia, Italy.
IL·1 AND TNF PRODUCTION BY MONOCYTES IN OCCUPATIONAL ASTHMA
jects, synthetic varnishes without TD I in six, and wood dust in two. The immediate reactions followed SBPT with synthetic varnish (two subjects) or Tanganyka aningre wood dust (one subject), late reactions were due to TDI (seven subjects), and dual reactions were due to TDI (one subject) or Mansonia altissima wood dust (one subject). Negative reactions followed TD I (six subjects) or synthetic varnishes (four subjects). FEV. was measured before exposure, after 30 min, hourly for 8 h and then 24 h after exposure. Blood samples for in vitro analyses were taken before exposure and 24, 48, and 72 h after exposure to occupational agents in the 22 subjects with possible OA.
409
Preparation of PBM Heparinized venous blood, obtained from healthy donors or from asthmatic subjects, was diluted with RPMI 1640medium plus 50/0 FBS (hereafter referred to as cRPMI). Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation (14). Cells were washed twice in cRPMI, plated in cell culture petri dishes (Nunc, Naperville, IL) at a concentration ranging between 2 x 106 and 3 x 106/ml, and incubated at 37° C in 50/0 CO 2 for 1 h. Nonadherent cells were removed by washing the dishes three to five times with warm RPMI 1640 medium, and the adherent cells were carefully removed using a rubber policeman. The latter cellswere 95 to 98 % positive to esterase staining, and cell viability was assessed by trypan blue dye exclusion test (more than 980/0 viable).
maximal stimulation with a standard recombinant human IL-l preparation (Janssen) in a tritiated thymidine uptake test. TNF was determined as described (17),using actinomycin D-treated L-929 cells as target. Serial dilutions (0.1 ml) of supernatants were added to 4 x 104 L-929 cells seeded in 0.1 ml in flatbottomed microtiter plates. The determination of TNF activity was made in comparison with commercially available preparations (Genzyme) with known titers, and results were expressed as units per milliliter. In selected experiments TNF and IL-l activity was abrogated by monoclonal antibodies to IL-l (Janssen) and polyclonal antibodies to TNF-a (Genzyme).
Methacholine Challenge Test Data Analysis A breath-activated dosimeter (ME.FAR, BresThe data regarding clinical details (table 1), cia, Italy) was used. Double cumulative doses pulmonary function (table 2), and blood leuof methacholine were administered beginning Production of PBM Culture Supernatants kocytes and lymphocyte subpopulations (taand Assays for IL-l and TNF Activity with a dose of 25 J.1g (12). FEV. was recorded ble 3) are presented as mean ± SEM. Since 1 min later followed by the next dose. The Supernatants from PBM were obtained ac- IL-l and TNF activities are based on serial challenge sequence terminated when FEV. -: cording to a previously described method (15). twofold dilutions, the data of the activities had decreased by 200/0 or more or when the Briefly, PBM were seeded at a concentration of both cytokines (figures 1 and 2) were anamaximum cumulative dose (3.2 mg) had been of 5 x 106/ml of cRPMI for 24 h without lyzed as log, 0 f the values. Because there were given. The methacholine provocation dose, stimuli. Supernatants wereharvested, filtered, only two dual responders, we considered late necessary to decrease FEV. by 200/0 (PD 20 and stored at -80° C until assayed. IL-l was and dual responders together in the analysis. FEV.), was obtained from the dose-response measured as proliferative activity on freshly The result did not change when these two curve plotted on a semilogarithmic scale; the isolated thymocytes from C3H/HeJ male groups were considered separately. The total abscissa represents logarithmic methacholine mice, 4 to 8 wk old, obtained from the Jacknumber of leukocytes was correlated with that doses, and the ordinate reports the percentson Laboratory (Bar Harbor, ME). Briefly, of eosinophils, and the five lymphocyte subage decrease in FEV •. 1 x 106 thymocytes in 0.1 ml cRPMI were sets were also correlated to each other. For The challenge was performed using buf- incubated with 0.1 ml of twofold dilutions each of the three groups (immediate respondfered lyophilized methacholine (phosphate of supernatants in the presence of 1 ug/ml ers, late-dual responders, and no asthmatic buffer) (Lofarma, Milano, Italy); 0.20/0 and of ConA for 72 h at 37° C in 50/0 CO 2 (16). reaction), comparisons between baseline 2 % methacholine solutions were prepared by IL-l titers were calculated by comparing the values and values after SBPT were tested for diluting methacholine in distilled water. The dilution of the test supernatants that gave500/0 statistical significance using a multivariate t dosimeter deliveredmethacholine from a MB2 ampule (ME.FAR) by means of an air compressor (driving pressure 1.5 kg/ern") (12). Every inhalation delivered 25 or 250 J.1g methaTABLE 1 choline from 0.2 and 2 % concentrations, CLINICAL DETAILS* respectively. Preparation of PBM and Determination of IL-l and TNF Activities For preparation of PBM and assay of IL-l and TNF activities without stimuli, methods were used that have been previously described (13).IL-l and TNF activities were determined before SBPT and 24, 48, and 72 h after exposure to occupational agents. Blood Leukocytes and Lymphocyte Subpopulation Analysis Total numbers of leukocytes and eosinophils werecounted by routine methods. Lymphocyte subsets were evaluated by two-color immunofluorescence and fluorescence activated cell sorter (FACS) analysis (Becton-Dickinson, Mountainview, CAl using Becton-Dickinson anti-CD3 (Leu-4), anti-CD4 (Leu-3), antiCD8 (Leu-2), anti-CD57 (Leu-7), and antiClass II major histocompatibility complex (MHC) (HLA-DR) antibodies. Blood leukocytesand lymphocyte subpopulations weredetermined before and 24 h after SBPT with occupational agents.
Type of Asthmatic Reaction
Subjects (n)
Immediate Late alone Dual No asthmatic reaction
3
7 2 10
Duration of Exposure (yr)
Age (yr) 33.3 44.4 46.5 51.4
± ± ± ±
6.0 3.9 4.5 2.3
11.9 20.9 24.0 25.4
± ± ± ±
7.5 5.1 15.0 2.6
Duration of Symptomst (yr) 6.5 5.7 7.2 3.6
± ± ± ±
4.0 2.7 5.7 1.1
* Mean ± SEM.
t
Before diagnosis.
TABLE 2 PULMONARY FUNCTION PD 20 FEV1 Methacholine (pg)
Immediate Late and dual No .asthmatic reaction
FEV1 (L)
FEV 1 (0/0 of predicted)
Before SBPT
After SBPT
3.68 ± 0.71 * 3.41 ± 0.25
94 ± 11* 99 ± 5
228 ± i t 1,067 ± 3
363 ± 2t 1,241 ± 1
747 ± 2
1,010 ± 2
3.31 ± 0.22
103 ± 7
Definitionof abbreviations: PD20 FEV t = methacholine provocative dose needed to decrease FEVt by 20%; SBPT =~pecific bronchoprovocation test. * Mean ± SEM. t Geometric mean ± geometric SEM.
SIRACUSA, VECCHIARELLI, BRUGNAMI, MARABINI, FELiCIONI, AND SEVERINI
410 TABLE 3 CHANGES IN PERIPHERAL BLOOD CELLS AFTER SPECIFIC BRONCHOPROVOCATION TEST' Type of Asthmatic Reaction
Late and Dual (n = 6)
No Asthmat ic Reaction (n 8)
7,250 ± 750 7,450 ± 1,150
6,962 ± 278 8,267* ± 627
7,000 ± 518 7,180 ± 843
150 ± 8 180 ± 17
115 ± 9 198* ± 22
124 ± 4 210 ± 25
66.5 ± 1.5 66.5 ± 4.5
69.2 ± 4.2 70.7 ± 2.7
69.8 ± 2.1 70.9 ± 2.1
38.0 ± 2.0 38.5 ± 0.5
42.9 ± 1.3 44.8 ± 1.2
45.0 ± 2.7 47.0 ± 3.4
39.0 ± 2.0 39.5 ± 0.5
33.8 ± 1.5 35.8 ± 1.9
34.1 ± 3.0 31.3 ± 3.0
22.0 ± 7.0 21.5 ± 4.5
16.0 ± 2.0 19.4 ± 2.8
20.4 ± 2.4 20.5 ± 1.8
20.5 ± 2.9 19.4 ± 2.9
23.6 ± 1.3 21.1 ± 1.5
20.5 ± 2.9 19.4 ± 2.9
Immediate (n
Leukocytes/mm' Baseline 24 hrt Eosinophils/mm' Baseline 24 h % CD3' T cells Baseline 24 h % CD4' Tcells Baseline 24 h % CD8' Tcells Baseline 24 h % HLA·DR' Baseline 24 h % Natural killer (Leu·7') cells Baseline 24 h
~
2)
=
'Mean ± SEM. That Is, 24 h after specific bronchoprovocatlon test. Significantly different from baseline values at p < 0.005 (Hotelling's l ' test for leukocytes and sinophils together).
t
*
eo-
8 7
6
E
Fig . 1. IL·l activity in supernatants of peripheral blood monocytes (PBM) from patientswith possible occupational asthma after specific bronchoprovocation test (SBPT). Data are mean ± SEM. Solid bars = negative ; hatched bars. immediate; shaded bars = late + dual.
:3
TIME (h) AFTER SBPT 8 7
*
6
E
Fig. 2. TNF activity in supernatants of PBM from patients with possible occupational asthma after SBPT.Data are mean ± SEM. ' Significantly different from baseline values , p < 0.001; t significantly different from baseline values, p < 0.01. Solid bars = negative; hatched bars = immediate; shaded bars = late + dual.
5
:3 LL
Z I-
24
48
TIME (h) AFTER S8PT
72
(Hotelling's P) test (18) twice, first for leukocytes and eosinophils considered together and then for the other five lymphocyte subsets. Comparisons betweenvalues of log, IL-l and log, TNF before and after SBPT were tested for statistical significanceusing the twotailed paired t test, with a < 0.05. Changes between baseline and 24, 48, and 72 h after SBPT values of log, IL-l and log, TNF were also tested separately using repeated-measures analysisof variance (19). In this analysis,using Fisher's test, we can evaluate the statistical significance of a set of comparisons simultaneously, taking into account possible correlations between the values for each subject. Because the occupational agent was TDI in 14of 22 cases, we analyzed the data separately for subjects with OA due to TDI, but the results did not change.
Results
SBPT was carried out in 22 subjects with possible OA. Before and after SBPT we evaluated FEV., spontaneous production of IL-l and TNF in culture supernatants of PBM , and peripheral T-cell subpopulations and leukocytes to study the activity of PBM and lymphocytes in OA. Baseline lung function measurements are listed in table 2. Mean values of FEV. were within the predicted values (20). Methacholine challenge test, carried out before SBPT, showed on the averagemild to moderate bronchial hyperreactivity, with trivial changes after SBPT with control materials (data not shown) and occupational agents. The maximal percentage decrease in FEV. was 35.5 for immediate reactions and 22.1 for late-dual (late component) reactions. In these subjects FEV. was still9.9OJo below the baseline value at 24 h. There was no correlation between the magnitude of the latedual asthmatic responses and the change in cytokine release by PBM 72 h after SBPT (for TNF r was -0.39, p = 0.52). Numbers of circulating leukocytes and eosinophils, tested together by HoteHing's T2test, increased 24 h after SBPT in latedual responders (p < 0.005) (table 3). Baseline values of the five lymphocyte subsets in the three groups and their variations 24 h after SBPT, tested together by the P test, were not significantly different. There were no differences in IL-l and TNF baseline values among the four groups (controls, nonresponders, and immediate and late-dual responders) (figures 1 and 2). After SBPT, IL-I activity increased slightly in both immediate (at 24 and 48 h) and late-dual reactions (at 72 h), but these differences were not significant (figure I). By t test it was shown that TNF activity increased 48 h
1L-1 AND TNF PRODUCTION BY MONOCYTES IN OCCUPATIONAL ASTHMA
In this study there is evidence of PBM activation in OA after airflow obstruction induced by occupational agents. It has been shown both in allergic asthmatic patients (5) and in mice (27) that after an inflammatory stimulus [allergen inhalation (5)and BCG injection (27)]there is a rapid migration of PBM that is transformed into AM. Moreover, monocytes and monokines have been found to increase in bronchoalveolar lavage fluid in symptomatic nonallergic asthmatic patients (28). Similarly, it is possible that in OA there is an influx of PBM in the lung that is transformed into AM after Discussion exposure to an occupational agent. This study shows that immediate, late, In disagreement with our results (taand dual reactions due to occupational agents are associated with an increase in ble 2), changes in bronchial responsivethe spontaneous production of TNF ness to methacholine have been observed released by PBM. In OA due to high- in OA after late or dual asthmatic reacmolecular-weight compounds, specific tions (29, 30). Significant changes have 19B antibodies to these occupational also been demonstrated at 3 and 8 h afagents can be detected (21). OA due to . ter SBPT (31, 32). The increase in reLMWC has not been clarified, and cel- sponsiveness may be transient, as at 24 h lular mechanisms have been underinves- it is less than at 8 h (31), and may be partially influenced by a reduction in tigated (10, 22). The studies regarding T-cell subsets in airway caliber (31). Moreover, some imthe peripheral blood of patients with provement in responsiveness to methaasthma are not concordant. Some studies choline can result from hospitalization, have found an increase in activated T cells which reduces exposure to allergens (33). (HLA-OR+) in acute severe asthma (23, Finally, a recent retrospective study has 24). Others (10) have reported an increase demonstrated that changes in bronchial in suppressor/cytotoxic C08+ T cells48 h responsivenessafter late or dual reactions after SBPT with TOI in OA but no are not constant (34). Our study suggeststhat exposure to occhange in activated and helper T-cell subsets. In other studies, helper T cells of cupational agents may induce the actipatients with allergic asthma were re- vation of PBM with increased spontaneduced after SBPT with mixed grass ex- ous release of cytokines. Other authors tract (25) but were increased during the have shown the activation of T cells and pollen season (26). In our data on lym- AM in the airways of asthmatic subjects phocyte subsets before and after SBPT, (2, 5). Cytokines such as IL-l and TNF, there is no evidence in favor of or against released by activated monoeytes and AM, these contradictory results, with the pos- may play a role in the recruitment and sible exception of a nonsignificant in- activation of eosinophils and neutrophils crease in activated T cells in late-dual and in the formation of the inflammareactions (table 3). These differences tory cell infiltrate in OA. The role of procould, at least in part, be due to the het- inflammatory cytokines in the pathogenerogeneity of both study methods (vari- esis of asthma seems to be important; ous time intervals between exposure to their exact importance will be clarified precipitating factors and study of im- only by further studies. munocompetent cells) and pathogenetic Acknowledgment mechanisms. In the present study, subjects with im- The writers thank A. Forcina for statistical mediate and late-dual asthmatic reac- analysis of the data, A. Velardi for helpful tions exhibited different kinetics of in- criticism of the manuscript, and Eileen Macrease in TNF. In immediate reactions honey Zannetti for excellent editorial and secretarial assistance. this increase was significant at 48 hand in late-dual reactions it was significant References at 72 h (figure 2). These results may sug1. Chan-YeungM, Lam S. Occupational asthma. gest different mechanisms in immediate Am Rev Respir Dis 1986; 133:686-703. and late reactions or they may be a 2. Tonnel AB, Joseph M, Gosset P, Fournier E, consequence of bronchoconstriction, Capron A. Stimulation of alveolar macrophages delayed several hours in late responders. in asthmatic patients after local provocation test. after SBPT (versus baseline values) in immediate reactions (p < 0.001)and 72 h after SBPT (versus baseline values) in late-dual reactions (p < 0.01). By repeated-measures analysis of variance it was demonstrated that in immediate responders at 48 h and in late-dual responders at 72 h, considered simultaneously, TNF was significantly increased in comparison with baseline values (p < 0.005). All other comparisons with baseline values, considered simultaneously, were not significant.
r
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