CD18 on alveolar macrophages in patients with active pulmonary tuberculosis

Tubercle and Lung Disease (1996) 77, 468-475 © 1996PearsonProfessionalLtd

Increased production of hydrogen peroxide and expression of CDllb/CD18 on alveolar macrophages in patients with active pulmonary tuberculosis H. P. Kuo, T. C. Ho, C. H. Wang, C. T. Yu, H. C. Lin Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan S U M M A R Y. Setting: Alveolar macrophages (AM) are important in host defense against Mycobacterium tuberculosis (TB). [32-integrins, especially CD11a/CD18 and CDllb/CD18, are implicated in leukocyte migration, antigen presentation, phagocytosis, and production of reactive oxygen species Objective: To explore the functional relevance of [~2-integrin expression to intracellular H202 capacity of AM in TB patients. Design: In a prospective study, AM retrieved from 18 active pulmonary TB patients and 18 normal subjects were assessed for [~2-integrin expression and intracellular H202 metabolism capacity by loading with antiCD11a/CD18, anti-CDllb/CD18 monoclonal antibodies and 2',7' dichlorofluorescein diacetate (DCFH-DA) respectively, and analyzed by flow cytometry. AM from 8 normal subjects were stimulated with tumor necrosis factor-alpha (TNF-~, l0 s units/ml) to examine the relationship between H20 ~ production and CDllb]CD18 expression. Results: The magnitude of DCFH oxidation and CDllb/CD18 expression of AM was higher in TB patients than in normal subjects. The CD11b/CD18 expression was related to the magnitude of DCFH oxidation, but not to lymphocyte numbers or subpopulations (CD4, CD8, CD25). Stimulation of AM with TNF-~ increased H202 production and CD11b/CD18 expression. Pretreatment with CDIlb/CD18 monoclonal antibodies inhibited TNF-~-induced H202. Conclusion: AM in TB patients possessed a higher capacity of oxidant metabolism. The increased CDllb/CD18 expression may be related to the increased respiratory burst response in AM against mycobacterial invasion. R E S U M E . Cadre: Les macrophages alv~olaires (AM) sont importants pour la d6fense le l'h6te contre Mycobacterium tuberculosis (TB). Les int6grines B2, surtout CD11a/CD18 et CDllb/CD18, sont impliqu~es dans la migration des leucocytes, la pr6sentation des antig~nes, le phagocytose et la production des esp~ces oxyg~nes r~actives. Objecttf: Explorer la pertinence fonctionnelle de rexpression de rint6grine B2 pour la capacit6 de production intracellulaire de H202 des AM chez les tuberculeux. Sch6ma: Dans une ~tude prospective, des AM provenant de 18 malades atteints d'une tuberculose pulmonaire active et de 18 sujets sains ont 6t6 6valu6s pour leur expression de l'int6grine B2 et leur capacit6 de production intracellulaire de H202 en les chargeant avec des anticorps monoclonaux anti-CDlla/CD18, avec des antiCD11b/CD18 et avec de la diac~tate dichloro-fluoresc6ine 2',7' (DCFH-DA), respectivement, et ont 6t~ analys~s par cytom6trie de flux. Des AM provenant de 8 sujets sains ont 6t6 stimulus par facteur de n6crose tumorale alpha (TNF-~, 105 unit~s/ml) afin d'examiner la relation entre la production de H202 et rexpression de CD11b/CD18. Rdsultats: La taille de l'oxydation DCFH et de l'expression de CDllb/CD18 des AM 6tait plus 61ev6e chez les malades tuberculeux que chez les sujets-t~moins. L'expression de CD11b/CD18 ~tait li6e h l'importance de l'oxydation du DCFH, mais non pas au comptage des lymphocytes ou aux sous-populations (CD4, CD8, CD25). La stimulation des AM avec TNF-~ a augrnent6 la production de H202 et l'expression de CDllb/CD18. Le pr~traitement par les anticorps monoclonaux CD11b/CD18 a inhib~ le H202 induit par TNF-ct.

Correspondenceto: Dr Han-PinKuo,Departmentof Thoracic Medicine,ChangGungMemorialHospital, 199 Tun-HwaN. Rd. Taipei,Taiwan.Tel: + 886-3-328 1200ext. 2286; Fax:+ 886-3-327 2474. Paper received 19 September 1995. Finalversion accepted8 April 1996. 468

Oxidant production and integrin expression in pulmonaryTB 469

Conclusion: Les AM chez les malades tuberculeux poss6daient une plus grande capacit6 m6tabolisme oxydant. L'expression plus 61ev6e de C D l l b ! CD18 pourrait ~tre li6e h la r6ponse accrue de la bouff6e respiratoire dans ies AM contre l'invasion mycobact6rienne.

R E S U M E N . Marco de referencia: Los macr6fagos alveolares (MA) son importantes en las defensas del hu6sped coutra Mycobacterium tuberculosis. Las 132-integrinas, especialmente CD11a/CD18 y CDllb/CD18 estfin implicadas en la migraci6n leucocitaria, en la presentaci6n de los antigenos, en la fagocitosis y e n la producci6n de especies oxigeno-reactivas. Objetivo: Explorar la importancia funcional de la expresi6n 132-integrina a la capacidad de producci6n intracelular de H202 de los MA en pacientes con tuberculosis. M~todo: En un estudio prospectivo, los MA obtenidos de 18 pacientes con tuberculosis p u l m o n a r y de 18 sujetos sanos fueron evaluados en cuanto a la expresi6n 132-integrina y a la capacidad metab61ica para la producci6n intracelular de H202, carg~indolos con anticuerpos monoclonales anti-CDlla/CD18, antiCD11b/CD18 y 2',7' diacetato de diclorofluorescelna (DCFH-DA) respectivamente y analiz~indolos por citometria de flujo. Los MA de 8 sujetos normales fueron estimulados con factor a de necrosis tumoral (TNF-~t, 105 unidades/ml) para examinar la relaci6n entre la producci6n de H202 y la expresi6n CDllb/CD18. Resultados: La magnitud de la oxidaci6n DCFH y de la expresi6n CDllb/CD18 de los MA fue mayor en los pacientes con tuberculosis q u e e n los sujetos normales. La expresi6n C D l l b / C D 1 8 fue puesta en relaci6n con la magnitud de la oxidaci6n DCFH pero no con el ndmero de linfocitos o con las subpoblaciones (CD4, CD8, CD25). La estimulaci6n de los MA con TNFI(Z aumenta la producci6n de I1202 y la expresi6n CDllb/CD18. El pretratamiento con anticuerpos monoclonales CDllb/CD18 inhibi6 la producci6n de H202 inducida por TNF-~. Conclusi6n: Los macr6fagos alveolares en los pacientes con tuberculosis poseen una mayor capacidad de metabolismo oxidante. El aumento de la expresi6n C D l l b / C D 1 8 puede estar relacionado con el aumento de la descarga respiratoria en los macr6fagos alveolares como respuesta contra la invasi6n micobacteriana.

INTRODUCTION Alveolar macrophages play a critical role in host defense against Mycobacterium tuberculosis (TB) invasion. Mycobacteria inhaled into the bronchial trees are phagocytosed by alveolar macrophages that are responsible for acquired immunologic responses by antigen presentation to T cells and by secreting active substances. 1,2Accumulation of monocytes and lymphocytes occurs at the sites of infection following cell adhesion to endothelial ceils and migration from blood vessels into tissues. Recently a number of cell surface molecules involved in cell adhesion, motility and directed migration have been recognizedo 3,4 Among these, ~2-integrins are involved in regulation of mononuclear cell function?,4 The 132integrins include three heterodimeric glycoproteins, namely CD1 la/CD18, CD1 lb/CD18, and CD1 lc/CD18. Each of these molecules is composed of different c~ subunits (CD1 la, b, c) and a common 132 subunit (CD18)) ,4 132 integrin adhesion molecules, especially CDlla/CD18 and CD1 Ib/CD18, play important roles in the extravasation of leukocytes and in other cellular functions such as cytotoxicity, phagocytosis, chemotaxis and induction of lymphocyte proliferation.4 The role of adhesion molecules may be moderated by both quantitative and qualitative changes in their expression? CD1 lb/CD18 has been shown to be upregulated in several inflammatory diseases, such as lupus erythematosus, sepsis and diabetes mellitus.4,5 Increased levels of expression of 132 integrins CD1 I/CD18 in the peripheral blood leukocytes, including monocytes, have been demonstrated in patients with sarcoidosis and TB.6,7The functional roles of these monocytes bearing increased levels of CD11/CD18 are not yet

clear. Increased CDllb expression has previously been associated with the increased phagocytic capacity of macrophages, 8 and CD1 la/CD18 is involved in antigen presenting capacity of alveolar macrophages as well as giant cell formation.3,4 It was thus hypothesized that the expression of 132 integrins on alveolar macrophages might be increased in patients with active pulmonary TB to establish cellular immunity against mycobacteriaI invasion. On interaction with particles or certain soluble stimuli, phagocytic cells strongly enhance their consumption of oxygen and release of superoxide anion, which rapidly undergoes dismutation into H202 .9 H202 is considered a major factor in macrophage-mediated cytotoxicity and parasite killing.1° The production of reactive oxygen species by leukocytes is not only essential for the destruction of most phagocytosed microorganisms, but also contributes to inflammatory injury in a variety of pathophysiologic conditions. 11 H202 production requires the coincident adherence to substrate and addition of a soluble stimuli. 1° 132-integrins, especially CDIlb/CD18, have been demonstrated to mediate hydrogen peroxide production from neutrophils in the peripheral blood? 2 Therefore, it is possible that CDllb/CD18 expression on alveolar macrophages may be associated with H20 z production and contribute to the intracellular killing of mycobacteria. The principal aim of this study was to quantify the expression of 132-integrin adhesion proteins and intracellular H202 capacity on the bronchoalveolar macrophages in patients with active pulmonary TB using flow cytometry, and thus seek the relationship between the expression of 132 integrins and the production of H202.

470 Tubercleand Lung Disease

PATIENTS AND M E T H O D S

Giemsa staining. Informed consent was obtained from all patients.

Subjects Eighteen patients with pulmonary TB (12 men and six women with a mean of age of 46.2 _+ 5.8 years) were studied before anti-TB treatment. None were current smokers. In all patients, at least one recent sputum specimen was positive for acid-fast bacilli on microscopic examination and grew M. tuberculosis. The nutrition status of each patient was assessed, including measurement of body mass, height, triceps skin-fold thickness, mid-arm circumference, and serum albumin level. Patients with poor nutritional status (body mass < 90th percentile, or mid-arm circumference and triceps skinfold thickness < 25th percentile) were excluded from this study. None was taking corticosteroids or other immunosuppressants. Patients with systemic or local inflammatory diseases, such as lupus erythromatosus, sepsis, and lung infection, or patients with diabetes meltitus, were excluded from this study. The control group consisted of eighteen healthy subjects (10 men and eight women, with a mean age of 53.7 _+ 3.2 years), of whom eight presented to our hospital with hemoptysis. None had a history of lung disease or any evidence of lung disease based on physical, chest X-ray and bronchoscopic examinations. The remaining 10 voluntarily asked for check-ups and were free from any disease. None were current or ex-smokers, and none were taking medication at the time of evaluation. To examine whether the changes in alveolar macrophages of patients with pulmonary TB reflect an inflammatory response or are specifically associated with TB infection, five patients with bronchiolitis obliterans and interstitial pneumonitis (BOP) before treatment (three women and two men, with a mean age of 45.8 + 5.6 years), were recruited into the study for comparison. None were current or ex-smokers.

Preparation of lung cells Bronchoalveolar lavage (BAL) was performed using five aliquots (50 mi each) of 0.9% saline solution as described previously. 13Briefly, sterile saline solution was introduced into the right fourth or fifth subsegmental bronchus in normal subjects and patients with BOP. In cases of pulmonary TB, BAL was performed in involved bronchi. The lavage fluid was then recovered by gentle aspiration and pooled, and subsequently filtered through two layers of sterile gauze. Total cell count was evaluated on an aliquot of the pooled fluid using a hemocytometer. The BAL fluid was centrifuged at 600 g for 20 rain at 4°C. The cell pellet was then washed sequentially and resuspended in RPMI-1640 (GIBCO, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS, Flow Laboratories, Paisley, Scotland, UK) at 106 cells/ml. Cell viability was determined by trypan blue exclusion quantitated using a hemocytometer. Differential cell counts were determined from cytocentrifuge preparations with modified Wright-

Preparation of peripheral blood mononuclear cells (PBMC) PBMCs were isolated from heparinized venous blood by density centrifugation over Ficoll-Hypaque. A 50 ml sample of blood was drawn from each patient and layered over 50 ml of room temperature Ficoll-Hypaque (specific gravity 1.077) and centrifuged at 400 g for 20 rain. Cells at the plasma:Ficoll-Hypaque interface were recovered, washed once with RPMI-1640, and resuspended at 2 x 106/ml in RPMI-1640.

Measurement of oxidative metabolism capacity in alveolar macrophage and P B M C Individual alveolar macrophage or PBMC respiratory burst response was assessed by using 2',7' dichlorofluorescin diacetate (DCFH-DA) and flow cytometry. TM In brief, cell suspension was spun down and resuspended in phosphate buffered saline (PBS) containing 5 ~ glucose and 0.1% gelatin but lacking Ca 2+ and Mg 2+ (PBSg) at 105 cells/ml, and then loaded with DCFH-DA (1 gM) for 15 rain at 37°C with shaking. Cells were washed twice with PBSg and then incubated for further 20 rain at 37°C in PBSg containing 10% FCS with shaking to represent spontaneous oxidative metabolism. After incubation, cells were transferred to an ice bath and further incubated with Phycoerythrin (PE)-conjugated anti-CDlla/ CD18 and anti-CD11b/CD18 monoclonal antibodies for 20 rain at 4°C, and analyzed by flow cytometry after two extensive washings with cold PBSg containing 10% FCS.

Flow cytometric analysis Analysis was performed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and LYSYS II software (Becton Dickinson). All fluorescence was measured using logarithm amplification. Ten thousand events were collected for each sample. Cells were gated on the basis of forward scatter and side scatter into lymphocytes, macrophages and granulocytes. The ability of the gates to segregate cells was checked using CD14 and CD3 antibodies to determine the purity of the monocyte/macrophage and lymphocyte gates, respectively. Less than 5% of cells in the monocyte gate expressed CD3, and less than 0.4% of cells in the lymphocyte gate expressed CD14. In the case of dual-color analysis, overlaps of the green fluorescence and PE emission spectra was eliminated by electronic subtraction. Controls were used to give a measure of non-specific binding using PE-conjugated F(ab)2 fragments of rabbit anti-mouse immunoglobulin (Dako, Kyoto, Japan) against which results were expressed as a mean fluorescence intensity in arbitrary units transformed to a linear scale from the lOgl0 channel number

Oxidant production and integrin expression in pulmonary TB 471 of mean fluorescence, for a particular cell marker. Unlabeled cells and isotype controls (mouse or rat IgG2-PE, IgG2-unconjugated) were run in parallel. Spontaneous D C F H oxidation in the presence of unstimulated alveolar macrophages or PBMC was compared with that in the presence of DCFH-unloaded macrophages or PBMC, and the FACScan cytometer was operated at the same settings.

Tumor necrosis factor-alpha (TNF-o0 stimulation Bronchoalveolar cells retrieved from 8 normal subjects were prepared and loaded with D C F H - D A as above at 105 cells/ml in PBSg containing 10% FCS and then incubated with T N F - a (105 units/nil) for 0 min (as the baseline), 30 min and 2 h at 37°C with shaking. Control groups were incubated with vehicle (0.9% saline). After incubation, cell suspension was put in iced water (4°C) to stop the reaction and the adherent cells were carefully scraped off by a rubber policeman. The recovery rate after 2 h incubation was more than 90% in both treatment groups. Cell viability was examined b y trypan blue exclusion test. The cell suspension was further incubated with PE-conjugated anti-CDl lb/CD18 or a n t i - C D l l a / C D l l 8 monoclonal antibodies for 20 rnin at 4°C. The expressions of CD1 lb/CD18 or CD1 la/CD18 on alveolar macrophages were analyzed within 20 rain of incubation after two extensive washings with cold PBSg. In one subgroup, cells were oversaturated with unconjugated CD1 lb/CD18 monoclonal antibodies or IgG2 (10 gg/ml) 30 min at 4°C before TNF-o~ stimulation for 2 h at 37°C, and the changes in intracellular oxidative metabolism capacity were examined. To determine whether intracellular oxidation of D C F H is mediated by H202 generation by the cell, catalase (500 U/ml) or superoxide dismutase (SOD, 25 gg/ml) were added in some subgroups before T N F - ~ or saline control stimulation.

(SEM). Statistical analysis of results was performed by Student's t-test for unpaired data, or one-way analysis of variance followed by Bonferroni corrected t-test when multiple comparisons were made. For data with uneven variation, a Mann-Whitney U test or Wilcoxon signedrank test was used for unpaired or paired data respectively. The relationship between mean D C F fluorescence intensity, CD4/CD8 ratio and [32 integrins mean fluorescence intensity was sought by Spearman's rank correlation test. A P value < 0.05 was considered significant.

RESULTS Cellular profiles of BAL fluid in normal subjects, patients with TB The Table summarizes cell numbers and differentials of B A L fluid recovered from patients. Recovered volume of the lavage fluid, total cell number, and cell viability did not show any significant difference among the three groups of patients. The proportions of lymphocyte neutrophils were significantly higher in patients with TB (17.6 __ 1.9% and 19.5 _+ 7.3% respectively, n = 18, P = 0.01) than in normal subjects (7.8 _+ 1.2% and 1.2 _+ 0.5%, respectively, n = 18). Reciprocally, the proportion of macrophages was significantly lower in patients with TB (62.6 _+ 7.1%, n = 18, P = 0.01) than in normal subjects (91.0 _+ 1.4%, n = 18). The percentage of CD4 lymphocytes and CD4/CD8 ratio were higher in patients with TB (62.4 _+ 2.9% and 2.9 _+ 0.3, respectively, n = 18, P < 0.02) than in normal subjects (46.3 _+ 3.6% and 1.3 +_ 0.2, respectively, n = 18). The proportions of CD8 cells were significantly lower in patients with TB (26.7 +_ 4.8%, n = 18, P < 0.03) compared with normal subjects (42.9 __ 5.0%, n = 18). There was no significant difference in either the proportions or cell numbers of CD3 or CD25 cells between patients with TB and normal subjects.

Monoclonal antibodies Anti-Leu-4/CD3 Fluorescein isothioocyanate (FITC), anti-Leu-3a/CD4 FITC, anti-Leu-2a/CD8 PE, and antiLeu-4/IL2R PE (Becton Dickinson) were used to give the proportions of T lymphocytes and CD4, CD8, CD25 T cell subpopulations respectively. Monoclonal antibodies against lymphocyte function antigen (LFA-1) (CD1 la/CD18) are mouse monoclonal antibodies (IgG2a, clone 38) obtained from R & D Systems Inc. (Minneapolis, USA), and antimacrophage ( M a c - l ) ( C D l l b / C D 1 8 ) monoclonal antibodies are rat IgG2b (M1/70HL) obtained from Boehringer Mannheim (Mannheim, Germany). As irrelevant isotype-matched antibodies, mouse IgG2 (for CD 1 l a / C D 18) and rat IgG2 (for CD1 lb/CD18) were used. Both were obtained from Boehringer Mannheim.

Statistical analysis Results are expressed as means _+ standard error of mean

Intracellular oxidative metabolism and [~2-integrin expression The mean D C F fluorescence intensity of alveolar macro-

Table Findings of BAL fluid in normal subjects, patients with pulmonary TB and bronchiolitis obliterans with interstitial pneumonifis (BOP) Tuberculosis Normal subjects BOP (n = 18) (n = 18) (n = 5) Total cell number, ×107 Recovered volume, % Cell viability AM, % Neutrophils, % Lymphocytes, % CD3, % CD4, % CD8, % CD4/CD8 CD25, %

4.4 ± 1.5 62.8 -+ 3.3 81.9 ± 2.2 62.6 ± 3.1" 19.5 _+4.3* 17.6 _.+1.9" 89.8 +_2.4 62.4 ± 3.9# 26.7 ± 4.8# 2.9 +_0.4* 8.5 -+ 1.4

2.0 + 0.3 75.1 -+ 2.1 90.3 + 1.9 91.0± 1.4 1.2 + 0.5 7.8 _+1.2 87.6 _ 1.8 46.3± 2.6 42.9 + 5.0 1.3 ± 0.2 9.3 -+ 1.4

2.6 ± 0.3 58.5 + 6.4 80.4 ± 3.2 61.4 -+ 8.2* 15.9 _+5.3* 22.7 _+7.7* N/A N/A N/A N/A N/A

AM, alveolar macrophages; N/A, not available. Values represent means + SEM; *P < 0.01; #P < 0.03 compared with normal subjects.

472 Tubercleand Lung Disease phages, representing spontaneous oxidative metabolism, in patients with active pulmonary TB (763.9 +__94.1, n = 18) was significantly higher than that in normal subjects (243.0 _+ 20.1, n = 18, P = 0.0001) (Fig. 1). The mean D C F fluorescence intensity of P B M C (274.6 _+ 18.3, n = 18) in TB patients was not significantly different from that of P B M C in normal subjects (224.5 _ 31.9, n = 18). The levels of C D l l b / C D 1 8 on alveolar macrophages in patients with TB (942.9 _+ 257.6, n = 8) were higher than with normal subjects (242.3 _+ 22.3, n = 18, P < 0.01) (Fig. 2). There was also an increase in C D l l b / C D 1 8 expression on P B M C in patients with TB (704.4 _ 167.3, n = 18) compared with normal subjects (263.0 _+ 32.2, n = 18, P < 0.01). The expression of C D l l a / C D 1 8 on alveolar macrophages was not significantly different between TB patients (558.6 _+ 90.4, n = 7) and normal subjects (595.4 +_ 96.8, n = 12) (Fig. 2). The mean D C F fluorescence intensity was significantly related to the levels o f C D l l b / C D 1 8 (r = 0.70, n = 33, P < 0.01) (Fig. 3) and CD4/CD8 ratio (r = 0.63, n = 21, P < 0.01), but not to CD1 la/CD18 expression (r = 0.12, n = 20, P > 0.1), or the proportions o f CD4 (r = 0.39, n = 32, P > 1000

800

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p<0.01

o

i

i

i

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1000

2000

3000

4000

5000

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Fig. 3---Correlation between the mean extent of spontaneous dichlorofluorescein (DCFH) oxidation and the expression of CD1 lb/CD18 in alveolar macrophages (n = 33). A significant correlation is indicated. 0.05), CD8 (r = 0.36, n = 32), CD25 cells (r = 0.007, n = 32). The levels of C D l l b / C D 1 8 were not related to the proportions of CD4 (r = 0.06, n = 32), CD8 (r = 0.23, n = 32), CD3 (r = 0.12, n = 32), CD25 cells (r = 0.15, n = 32), or CD4/CD8 ratio (r = 0.18, n = 32). In patients with BOP, the level o f C D l l b / C D 1 8 on alveolar macrophages (460.6 _+ 59.3, n = 5, P < 0.05) was significantly higher than that in normal subjects. There was a slight, but not statistically significant, increase in the mean D C F fluorescence intensity o f alveolar macrophages in patients with B O P (371.6 _+ 62.4, n = 5) when compared with normal subjects.

~J =

200

TNF-c~ stimulation of alveolar macrophages

normal

subjects

TB

patients

Fig. 1--The extent of spontaneous dichlorofluorescein (DCFH) oxidation by alveolar macrophages in patients with pulmonary tuberculosis (n = 18) and normal subjects (n = 18). Values are means _+SEM. **P < 0.01 compared with normal subjects.

1200

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600 o

=

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400 "-T--

200

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CDlla/CD18

I

CD11b/CD18

Fig. 2---Expression of adhesion molecules, CD1 la/CD18 and CD 1 l b/CD18 in alveolar macrophages in patients with pulmonary tuberculosis (n = 18, hatched bars) and normal subjects (n = 18, open bars). The values represent mean - SEM. **P < 0.01 compared with normal subjects.

T N F - ~ stimulation o f alveolar macrophages retrieved from 8 normal subjects induced a significant increase in the magnitude of C D l l b / C D 1 8 expression from 198.2 _+41.8 (n = 8) at baseline to 313.5 +_ 77.5 (P < 0.01) at 30 min and 263.1 _+ 57.2 (P < 0.01) at 2 h (Fig. 4). Incubation with vehicle did not significantly change the expression of C D l l b / C D 1 8 at the corresponding time points (201.7 __ 39.5, 221.6 _+ 51.9 and 203.8 -+ 65.8, n = 7 respectively). TNF-(x did not induce any significant change in the expression o f C D I la/CD18 at 30 min (611.6 __ 68.6, n = 8) or at 2 h (625.7 _+ 67.9, n = 8) compared with the corresponding time controls (614.0 _+ 66.9 and 628.3 __ 67.7, n = 7, respectively). The cell viability after incubation for 2 h was not significantly different between two groups: 85.4 _+5.3% (n = 8) with T N F - ~ and 87.3 +_ 4.8 (n = 7) with vehicle incubation. Stimulation with TNF-o~ for 2 h also induced a concomitant increase in mean intracellular D C F fluorescence intensity compared to baseline b y 691.8 _+ 87.9% (n = 8) which was significantly higher than corresponding saline control (by 369.4 + 25.2%, n = 8, P < 0.01) (Fig. 5). In the presence of catalase or SOD, the effect of T N F - ~ on D C F H oxidation was significantly inhibited by catalase (435.2 _+ 56.1% above baseline, n = 7, P < 0.01), but not by SOD (714.8 _+ 64.2% above baseline, n = 7) when com-

Oxidant production and integfin expression in pulmonary TB

400 It

,It

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300

.=.

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T

200

T

100 ¢=

0

0

0.5 incubation

2

time

(hours)

Fig. & - T i m e course for tumor necrosis factor-c~ (105 unit/ml, solid bars, n = 8) or its vehicle (0.9% saline, open bars, n = 7)-induced changes in expression of CD1 lb/CD18 on alveolar macrophages recovered from 8 normal subjects. Values are means + SEM. **P < 0.01 compared with time 0.

pared with TNF-~ stimulation alone. Pre-incubation with CDllb/CD18 monoclonal antibodies significantly inhibited the response to TNF-a (481.4 + 41.0% above baseline, n = 8, P < 0.03) compared with saline control group or nonspecific IgG2 pretreatment group (639.6 _+ 59.4% above baseline) (Fig. 5). DISCUSSION In the present study, we demonstrate that intracellular spontaneous oxidative metabolism and the expression of 132-integrin CDllb/CD18, but not CDlla/CD18, are increased in alveolar macrophages in patients with active pulmonary TB before treatment. The oxidative metabolism capacity was closely related to the level of CDllb/CD18 expression on macrophages. These findings suggest that the upregulation of CDllb/CD18 on 800

6oo

#

o

400

200 o

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0 Control

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TNF+IgG

TNF+Mac-1

Fig. 5--The extent of spontaneous dichlorofluorescein (DCFH) oxidation (Control) or induced by tumor necrosis factor-co (TNF-c~ 105 unit/ml) in the absence or presence of Mac-1 monoclonal antibodies (Mac-l) or IgG2 (IgG) in alveolar macrophages retrieved from 6 normal subjects. Values are percent increase above baseline (means --- SEM). *P < 0.01 compared with Control; #P < 0.03 compared with TNF-(x alone.

473

alveolar macrophages in active pulmonary TB might be functionally related to cellular immunity against mycobacterial invasion. Alveolar macrophages engulf invading infectious agents, invaginating the plasma membrane to become a phagosome which fuses with a lysosome. 15 Within this structure, concurrent with the phagocytic process, oxidative processes are harnessed in an attempt to kill the microorganism by transferring an electron to the terminal NADPH oxidase in the alveolar macrophage surface membrane, which results in the production of a variety of oxygen radicals, such as superoxide anion, hydrogen peroxide, hydroxyl radical and single oxygen. 15 The production of reactive oxygen species by leukocytes is not only essential for the destruction of most phagocytosed microorganisms, but also contributes to inflammatory injury in a variety of pathophysiologic conditions. 11 H202 is considered a major factor in macrophagemediated cytotoxicity and parasite killing. I° Several assays have been developed to measure the capacity of phagocytic cells to undergo a respiratory burst response, including quantitation of SOD inhibitable reduction of ferricytochrome C 9, and H20 z production quantified by numerous fluorimetric and spectrophotometric methods. 16'17 TO concomitantly measure the expression of adhesion molecules on an individual alveolar macrophage and its intracellular oxidative metabolism, the capacity for H20 z production was here determined by loading cells with DCFH-DA and flow cytomeUy. DCFH-DA, a stable lipid soluble compound, is readily taken up by leukocytes, deacetylated and trapped within the cytoplasm. The resultant non-fluorescent product DCFH is rapidly oxidized to a fluorescent compound dichlorofluorescein (DCF) in the presence of H202. DCFH oxidation can therefore be quantified by single-cell analysis using flow cytometry.14 Thus, the measurement of DCFH oxidation fluorescence changes represents the intracellular capacity of oxidant metabolism. The intracellular DCFH oxidation was inhibited by catalase, but not SOD. SOD destroys superoxide and prevents formation of other reduced oxygen species, such as hydroxyl radicals or single oxygen. Our results therefore support the role of H202 and counter the participation of other oxygen derivatives in the oxidation of DCFH. The mean magnitude of spontaneous DCFH oxidation in alveolar macrophages was significantly enhanced in patients with active pulmonary TB compared with normal subjects, indicating that those alveolar macrophages were activated to increase the production of H202 against mycobacterial invasion. The functional role of increased production of H202 in pulmonary TB is yet to be elucidated. In a recent study in murine macrophages, reactive nitrogen intermediates rather than H202 were essential for killing virulent mycobacteriumY Whether the increased production of H202 in human alveolar macrophages is implicated in damaging mycobacterium deserves future investigation. Increased H202 production in macrophages has been reported to produce platelet-activating factor~9and arach-

474

Tubercle and Lung Disease

idonic acid products 2° which are potent chemotactic factors for neutrophil and may contribute to the increased numbers of neutrophil sequestrated in BAL in TB patients found in this study. H202 and other oxygen radicals released from macrophages or recruited neutrophils are capable of causing tissue injury and promoting tissue inflammation. Thus, the increased production of H202 in alveolar macrophages may be involved in the pathogenesis of pulmonary inflammatory responses to mycobacterial invasion. Effective host defense against mycobacteria involves collaboration between mononuclear phagocytes and lymphocytes. The dominance of CD4 ceils and CD4/CD8 in our patients with active TB is consistent with previous reports, 2~,22 and supports a central role of CD4 lymphocytes in antimycobacterial responses. Antigen-specific CD4+ lymphocytes elaborate several cytokines, such as TNF-(x, IFN-'f, IL-2, and IL-4. 23 These cytokines play a critical role in the modulation of macrophage activation for inhibition of intracellular mycobacterial growth? 3 In the present study, TNF-c~ was shown to increase intracellular oxidative metabolism in alveolar macrophages. Thus, it is possible that TNF-cz and other cytokines produced from activated T-lymphocyte in the bronchoalveolar spaces may stimulate macrophages to increase their intracellular oxidative metabolism and contribute to the high level of spontaneous DCFH oxidation by alveolar macrophages. In addition, the high correlation between the level of spontaneous oxidative metabolism and the CD4/CD8 ratio was demonstrated in our results, suggesting the importance of CD4 cells in regulating the capacity of alveolar macrophage for intracellular killing of microorganisms. The recruitment of macrophages and lymphocytes from blood vessels to the sites of infection requires cell adhesion to endothelial cells and migration into tissues? Increased levels of expression of the 132 integrins CD11/ CD 18 have been associated with increased cell adhesion and migration both in vivo and in vitro. 3 CD11b/CD18 is found on monocytes, macrophages, granulocytes, and NK cells, and binds the complement components iC3b, as well as ICAM-1, factor X, fibrinogen, and polysaccharides. Recently, the expression of CDllb/CD18 on the peripheral blood monocytes was reported to be increased in patients with TB. 7 Increased levels of the adhesion proteins intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin have also been reported in the serum of patients with TB. 24,2sThe present results further demonstrate increased expression of CDllb/CD18 on the alveolar macrophages as well as PBMC in patients with pulmonary TB before treatment. Therefore, it is possible that the alveolar macrophages bearing increased levels of CDllb/CD18 may be due to an increase in influx of monocyte-like cells to the lung parenchyma. The higher level of DCF oxidation in alveolar macrophages than in PBMC in TB patients may suggest that monocytes/ macrophages are further stimulated during the course of transmigration into alveolar space.

Here TNF-(z increased the expression of C D l l b ! CD18 on alveolar macrophages retrieved from normal subjects, indicating that the upregulation of C D l l b / CD18 in patients with TB may arise from a local inflammatory response. TNF-c~ and other cytokines released from activated T lymphocytes have been reported to upregulate the expression of CDll/CD18 on monocytes 26 and may contribute to the increased expression of CD11b/CD18 on alveolar macrophages in our patients with active pulmonary TB. Thus, the upregulation of CDllb/CD18 on alveolar macrophages in patients with pulmonary TB may be related to T celt activation. However, we failed to find a relationship between the level of CD11/CD18 expression and T-lymphocyte activation in terms of CD4/CD8 ratio, IL-2R receptor expression (CD25) or CD4, CD3 cell numbers. Macrophages also produce modulatory cytokines in an autocrine fashion after direct interaction with various mycobacterial products. TNF-~, IL-113, prostaglandin E2 and TGF-~ by macrophages can be induced by mycobacterial polysaccharides, proteins and intact bacilli. 27-29 Thus, it is also possible that the expression of CDllb/CD18 on alveolar macrophages may be increased after direct interaction between mycobacteria and macrophages and independent of T-lymphocyte activation. However, the upregulation of CDllb/CD18 is not specific to TB infection. We also found alveolar macrophages in patients with BOP bearing a high level of CD11b/CD18. In other inflammatory lung diseases, such as sarcoidosis and idiopathic pulmonary fibrosis,3° the expression of C D l l b / CD18 on alveolar macrophages is also found to be enhanced. Therefore, the increased level of C D l l b / CD18 on alveolar macrophages in patients with active pulmonary TB may reflect a response to inflammation. The functional significance of the increased C D l l b / CD18 expression on alveolar macrophages is not clear. CDllb/CD18 is involved in phagocytosis and cytokine production from monocytes? 1 Recently, CDllb/CD18 was shown to mediate HzO 2 production from neutrophils in the peripheral blood. 12 H202 production requires coincident adherence to substrate and addition of soluble stimuli? 2The good correlation between the magnitude of intracellular DCFH oxidative metabolism capacity and the levels of CD11b/CDI8 expression may suggest a possible functional relevance. Our results also showed that pretreatment with monoclonal antibodies against CD1 lb/CD18 inhibited TNF-c~-induced intracellular oxidative metabolism, suggesting that C D l l b / CD18 may be essential for alveolar macrophage to produce H20 2 against mycobacterial invasion. Leukocyte integrins are also required for neutrophil and T-cell function. Anti-CD1 la/CD18 antibodies inhibit T-cell proliferation in response to cell adhesion in vitro, and prevent transplant rejection in vivo.4 T-cell stimulation by antigen presentation requires binding between CD11a/CD18 on antigen presenting cells and its ligand ICAM-1 on T cells, and/or the reciprocal case, C D l l a / CD18 on T-cells binding to ICAM-1 on macrophages.

Oxidant production and integrin expression in pulmonary TB

Cytokine-induced generation of multinucleated giant cells from monocytes also requires CD1 la/CD18. 33Therefore, increased levels of CDlla/CD18 on alveolar macrophages were expected in patients with active pulmonary TB, since the antigen-presentation capacity of alveolar macrophages was reported to be enhanced. 34 To our surprise, there was no significant difference in C D l l a / CD1 8 expression on alveolar macrophages between TB patients and normal subjects. The reason for this is not clear. Unlike CDllb/CD18, there was no significant change in CDlla/CD18 expression on alveolar macrophages after TNF-c~ stimulation. That CDlla/CD18 mediates the homotypic adhesion of leukocytes has been demonstrated to depend on qualitative rather than quantitative changes in the cell-surface expression of adhesive molecules. 35Stimulating resting T-lymphocytes with phorbol esters or crosslinking the T-cell antigen receptors with monoclonal antibodies converts cellular CD1 1a/CD18 from a low to a high-avidity state, with no change in surface density. 35 Thus, no significant change in CD lla/CD 18 density on alveolar macrophages, either at rest or after TNF-~ stimulation, cannot exclude the functional role of CD 1 la/CD 18 in the regulation of cellmediated immunity against mycobacterial infection. In conclusion, we have shown elevated CD1 lb/CD18 expression and enhanced spontaneous oxidative metabolism in alveolar macrophages from patients with active pulmonary TB. Increased CDllb/CD18 expression may arise from activation by cytokines or microbial products, and may be associated with enhanced intracellular production of H 2 0 2 to induce pulmonary inflammatory responses to mycobacterial invasion.

Acknowledgement Supported by CMRP 414 and NSC83-0412-B-182-053, 011.

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