Neutrophil Retention in the Lungs of Patients with Chronic Obstructive Pulmonary Disease1- 3

C. SELBY, E. DROST, S. LANNAN, P. K. WRAITH, and W. MACNEE Introduction SUMMARY

In order to study neutrophil traffic In the lungs of humalls,

we harvested autologous

The presence of an imbalance between neutrophils and redlolabeled them with Indlum-111 prior to reinjection. The ~ ofthesa ['"lnlneuprotease activity, largely elastase, and trophlls through the pUlmonary vasculature was compared with that of [ 99mTclerythrocytes In norprotective antiproteases such as alpha-lmal elderly subjects and In patients with chronic obstructive pulmonary disease (COPO). Neutrophil protease inhibitor in the air spaces of the sequestration within the lungs of seven normal subjects, 10 min after reinjection, correlated with lungs is the central tenet of the current local erythrocyte transit times in the lungs ('t = 0.72, P< 0.001). This relationship was lost In patients with COPO. In seven patients studied during an acute exacerbation of COPO, neutrophil retention proteolytic theory of the pathogenesis of was higher during the first passage through the lungs (mean, 22.0 SO 14.1%) compared with 14 pulmonary emphysema (1, 2). The neupatients studied when their condition was stable (16.3 SO 3.4%, P < 0.001), or to the normal elderly trophil is a major source of elastase withsubjects (13.7 SO 7.0%, P < 0.001). In addition, the SUbsequent rete of neutrophil washout from in the lungs. Higher peripheral blood leuthe lungs was slower In patients with acute COPO (1.93 SO 0.66 x 10-'s-') than In those with stable kocytecounts occur in smokers (3), which disease (3.08 SO 1.8 x 10-'s·', P < 0.02). Neutrophil retention in the lungs correlated Inversely with correlates inverselywith the degree of airthe extent of emphysema, assessed quantitatively by CT scanning ('t = 0.68, P < 0.05). flow limitation (4). In addition, the deThus, patients presenting with acute exacerbations of COPO havean Increased neutrophil burden cline in FEV 1 in patients with chronic obIn the pulmonary vasculature with the potential for increased lung proteolysis. structive pulmonary disease (COPO) AM REV RESPIR DIS 1991; 143:1359-1364 correlates inversely with both the initial peripheral blood white cell count (5) and the change in white cell count over time, independent of smoking habit (6). Thus, dextrose anticoagulated whole blood using the specific radioimmunoassay method of the intravascular neutrophil burden in a a two-step, discontinuous 42%:65% Per- Plow (11). population of patients with COPO corre- colI/platelet-poor plasma gradient in lipoBacterial killing. The ability of harvested lates with the severity of their airflow polysaccharide-free reagents as described pre- neutrophils (1O"·ml-') to kill type I Streptococlimitation. viously (8). Contaminating erythrocytes were cus pneumoniae was measured by the techFurthermore, all of the circulating neu- lysed by brief exposure to hypotonic saline. nique of Catterall and associates (12). trophils pass through the pulmonary vasFunctional Integrity of the Neutrophil Kinetic Studies culature where their progress must be Harvested Neutrophils Patients/subjects. Twenty-one patients with delayed before migrating into the air spaces. Thus, knowledge of the factors Neutrophil morphology. Harvested neutro- severe, chronic airflow limitation were studthat influence the passage of these cells phils were stained with 10,70 acetic acid/O.01% ied. None had a greater than 10% improvement in FEV, in response to two puffs of a crystal violet and examined by light microsin the pulmonary circulation may ulti- copy. Aliquots of neutrophils were also fixed beta-2-agonist given by pressurized inhaler. mately be important in understanding the with 2% glutaraldehyde and then processed Spirometry and arterial blood gas values for cause of the increased numbers of neu- for transmission and scanning electron these patients are shown in table 1. They were trophils present in the distal air spaces microscopy. ,compared with a group of seven normal elderof cigarette smokers (7). Production of reactive oxygen intermedi- ly subjects with no clinical evidence of respiCigarette smoking delays the passage ates. Hydrogen peroxide (H 202) production ratory disease and normal spirometry. Fourteen patients with COPD were studof radiolabeled neutrophils in the lungs was measured by the method of Pick and of healthy subjects (8). Any delay in neu- Keisari (9). Generation of superoxide anion ied when clinically stable during the precedtrophil transit may increase the elastase (02 - ) was measured by the superoxideburden in the lungs and thus potentiate dismutase-inhibitable reduction of cytochrome C (10). (Received in original form August 6, 1990 and in lung proteolysis. The aims of this study All reagents were obtained from Sigma revised form January 7, 1991) were to compare the traffic of function- Chemical Co., (Poole, UK). The spontaneally intact neutrophils through the lungs ous release and the stimulated release by phorFrom the Unit of Respiratory Medicine, Rayne of patients with clinically stable or acute bol myristate acetate (PMA) (1 ug-ml") of Laboratory, Department of Medicine, City Hospiexacerbations of COPO and subjects H 202 and O2 - was measured using peripheral tal, Edinburgh, Scotland. 2 Supported by the Scottish Home and Health without cardiopulmonary disease, and blood neutrophils (2.5 x IOS·ml-')harvested Department, by the Norman Salvesen Emphysefrom healthy nonsmoking subjects. Reactive to relate such neutrophil kinetics to the oxygen species were also measured using neu- ma Research Trust, and by the Chest, Heart, and presence of emphysema in the lungs. Stroke Association. I

Methods Neutrophil Harvesting Technique Neutrophils were harvested from acid-citrate-

trophils harvested from patients early during an acute exacerbation of COPD. Protease release. The spontaneous and PMA-stimulated release of neutrophil elastase from 2.5 x 105 cells-ml? was measured by

'Correspondence and requests for reprints should be addressed to Dr. C. Selby, Unit of Respiratory Medicine, Department of Medicine (RIE), City Hospital, Greenbank Drive, Edinburgh EHlO 5SB Scotland.

1359

1360

SELBY, DROST, LANNAN, WRAITH, ANO MACNEE

TABLE 1

beled cells. In addition, the migration of radiolabeled neutrophils into bronchoalveolar lavage of a further three chronic smokers undergoing diagnostic bronchoscopy was assessed 2 to 3 h after reinjection of ["lln]_ neutrophils.

PATIENTS/SUBJECTS

Normal Elderly

4 M, 3 F

Number Age, yr

FEV" L, OfoFEV,

eTPS

FVC, L, eTPS %FVC Pao" kPa Paco" kPa H+, nmollL

70-91 2.0 (0.5)' 82-120 2.8 (0.8) 70-100 10.6 (0.9)

4.8 (0.3) 37.8 (1.7)

Tmax WBC, x 10· V'

Acute Exacerbation of COPO

Stable

COPO

2 M, 5 F

10 M, 4 F 58-83 1.1 (0.7) 12-57 2.7(1.1) 34-113 7.9 (1.9) 6.8 (1.8)t 37.8 (3.4) 36-37 5-9.3

46-74 0.4 (0.2)t 10-23 1.9 (1.2) 29-82 6.3 {1.8)t 7.8 (1.7)t

45.2 (7.4) 36-38

7-14.5

Definition of abbreviations: H + = arterial blood hydrogen ion concentration; Tmax = highest oral temperature (centigrade) in 24 hr preceding kinetic study; wee = peripheral venous blood total leukocyte count at time of kinetic study. , Values are mean with SO shown in parentheses. t p > 0.05 by ANOVA.

ing 6 wk. Current cigarette smokers (n = 7) refrained from smoking for 2 h before and during the study. Drug therapy in these patients consisted of inhaled steroids and bronchodilators in all subjects, theophyllines in five, and diuretics in four. Seven patients with COPD were studied within 36 h of admission to hospital with an acute exacerbation. None of these patients was receiving steroid therapy orally or parenterally. Their drug therapy consisted of nebulized beta-z-agonists and/or ipatropium bromide, diuretic therapy (n = 2), and appropriate antibiotics (n = 2) for bacteriologically proved organisms in their sputum. No patient had clinical or radiographic evidence ofpneumonia or cor pulmonale. Seven healthy subjects, including two smokers, all with normal lung function, were studied for comparison. Medications in these subjects consisted of paracetamol (n = 3), imipramine (n = I), and replacement thyroxine (n = 1). All subjects/patients gave their informed, written consent to participate in this study, which had the approval of our local ethical committee.

Cell Labeling Technique Harvested neutrophils were labeled with 18 to 30 MBq of indium-lll oxine in phosphatebuffered saline, washed, and resuspended in platelet-poor plasma (8). The labeling efficiency was 69 ± 9070 (n = 28). Erythrocytes (RBCs) were labeled with 75 to UO MBq of technetium-99m using an in vitro/in vivo technique (13). These labeled cells were mixed together and flushed into the basilic vein as a bolus with sterile 0.9% saline. Imaging With the subject sernisupine, a large field of view gamma camera (Siemens-Gammasonics, Erlangen, Germany) was placed in an anterior projection. Data acquisition after the bolus injection was as a dual isotope dynamic

study, with energy windows of 140 ± 20 KeV for 99mTh and 247 ± 20 KeV for lllIn. The first passage of the injected bolus of radiolabeled cells through the central circulation and lungs was recorded as 60 x 500 ms timeframes. Thereafter, without repositioning the subject, image acquisition continued as 60-s time-frames for 29 min. Regions of interest (ROIs) were drawn around the right ventricle and the left lung, which was subdivided into three equal regions (upper, middle, and lower). The transit times for RBCs from the right ventricle to the left lung and its regions were obtained by deconvolution of the 99mTc time/activity curves obtained from these ROls (14). The first pass retention of neutrophils in the lung was calculated using the [' lIln]neutrophil and the [99mTc]RBC curves from the right ventricle and the left lung regions (8, 15). These ['IlIn]neutrophils were only temporarily retained in the lungs: a time/activity curve for the subsequent 29 min can then be acquired and fitted with the exponential equation: y

=

A

+ Be-Ct

(1)

where parameter C is the rate of "washout." From this fitted curve a percentage sequestration at 10 min after reinjection can be calculated with 100% lung activity at 1 min. Time-activity curves were also obtained from ROI over the liver and spleen. These curves can also be fitted with a monoexponential curve of the form

CT Scanning Nine of the patients with stable COPD had a CT scan ofthe thorax (General Electric 9000 scanner, 8-s scan time, 3-cm slice thickness). The scan of the left lung was divided into three equal regions: an upper, middle, and lower zone to correspond with the divisions of the radionuclide image. From each region the frequency distribution of tissue density values for pixels within the lung fields were obtained (as EMI numbers) (16). From these cumulative density histograms (on a scale of -500 corresponding to air, zero to water, and + 500 to bone), the EMI number that characterized the mean and lowest fifth percentile was obtained. We have previously shown that CT lung density measured in this way correlates with quantitative morphometric measurements of the size of-distal air spaces in the lungs - a defining characteristic of emphysema (16). Statistics Differences between group means were assessed using one-way ANOVA with both Tukey and Scheffe corrections for multiple comparisons. Linear regression analysis was performed with Kendall's rank correlation coefficient r (17). Results

Neutrophil Purity Using the harvesting technique described above in 28 subjects, 7.42 ± 3.86 x 107 neutrophils (94.0 ± 2.6070 pure and < 2070 erythrocytes) were harvested from a 30ml sample of whole blood, representing a mean recovery of 55%. These cells were > 98% viable as assessed by trypan blue exclusion.

Morphologic Examination Examination of neutrophils by both light and electron microscopy demonstrated single cells with a normal structure and no shape change.

where C is the rate of ''washin'' to these organs.

Production oj Reactive Oxygen Intermediates and Protease Release Spontaneous production of H 2 0 2 and

Neutrophil Migration In Vivo The ability of harvested and reinjected [1I1In]neutrophils to migrate into the air spaces was assessed in sputum and bronchoalveolar lavage fluid. Cell-associated 1I1In in the sputum of five patients with chronic purulent sputum production was measured at intervals over 48 h after reinjection of radiola-

O 2 - from harvested neutrophils was minimal, but it increased on stimulation with PMA (p < 0.01) (table 2). Neutrophils harvested from patients with an acute exacerbation of COPD were no different from those of normal subjects (table 2). Release of immunoreactive human neutrophil elastase was

y

=

A -

Be-Ct

(2)

NEUTROPHIL RETENTION IN LUNGS OF PATIENTS WITH COPO

1361

TABLE 2

100

IN VITRO FUNCT ION OF HARVESTED NEUTROPHILS

Spontaneous Release Number

(mean)

(SD)

! z

PMA Stimulated Release (1 Jlg'ml- ') • (mean)

0

i= -e a:

...

Ul

w ::>

aw

(SD)

50

Ul

H,O" nmo1/2.5 x 10' PMN/2 h Healthy nonsmokers Patients with acute COPO 0 ,-, nmoU2.5 x 10' PMN/2 h Healthy nonsmokers Patients with acute COPD

CI

20 4

4.8 4.3

3 .5 3 .0

25.67 22.7

z

::>

10.2 9.1

oJ

z

::l; 0.

20 4

1.36 1,06

0 .6 0 .9

15.5 13.6

6.2 8.3

0 0

10

ERYTHROCYTE TRANSIT TIME (s)

below the limit of detection « 1.2 ng-rnl"), but it increased with PMA stim ulation to 15.6 ± 3.6 ng-ml? (n = 6, p < 0.01).

after reinjection. This compares with a similar percent recovery reported in preliminary studies in humans by other workers (18).

Bacterial Killing

Studies of Neutrophil Kinetics

Harvested neutrophils reduced pneumococ cal colony formation by 37 to 96070 after 5 min of incubation. In Vivo Neutrophil Function One hour after reinjection, 95.2 ± 1.1% of the 111 In activity in peripheral venous blood was still neutrophil-bound (n = 7). Cell-associated 111 In activity (representing 200 to 1,000 cells-h") was pres ent in the sputum collected from five subjects with chronic sputum production from 2 h until 48 h after reinjection (fig ure 1). In addition, these cells migrated rapidly in vivo to sites of infection. At diagnostic fiberoptic bronchoscopy with bronchoalveolar lavage, between 0.004 and 0.5% of the total injected ' l' In was recovered as cell-pellet-associated activity in bronchoalveolar lavage 2 to 3 h

...::>~ "'"

12

10

~

Fig. 2. The relationsh ip between neutroph il lung sequestration at10 min (100% at 1 min) and local erythrocyte transit time for each of three lung regions in seven normal elderly subjects. The regression line (~ = 0.72, P < 0.001) and 95% confidence intervals are shown.


absence of any significant relationship iii -450 zw between erythrocyte transit time and the first-pass neutrophil retention in the "Z::>..J lungs. TIME (min) ...0 -400 The relationship between erythrocyte ..J Fig. 4. The difference in rates of washout of the time/actransit time and lO-minneutrophil reten