Neutrophil function in chronic bronchitis
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Eur Resplr J
1991' 4, 536-543
Neutrophil function in chronic bronchitis
P. Venge*, S. Rak**, L. Steinholtz**, L. HAkansson*, G. Lindblad***
Neutrophil function in chronic bronchitis. P. Venge, S. Rak, L. Steinholtz, L. • Dept of Clinical Chemistry, University Hospital,
Hdkansson, G. Lindb/ad. Uppsala, Sweden.
ABSTRACT: This study was initiated with the question: Do defects In
neutrophil functions predispose patients with chronic bronchitis to their • • Dept of Lung Medicine, Central Hospital, Viister!s,
Sweden.
recurrent bacterial infections?
Forty flve patients with chronic bronchitis and recurrent bacterial infec- •• • Pbarmacia AB, Uppsala, Sweden.
tions were studied. A number of aspects of neutrophil functions reflecting
migratory and phagocytic functions and oxidative metabolism were meas- Correspondence: P. Venge, Laboratory for Inflamma-
ured in vitro, once in all 45 patients and 3 times in 22 patients over a tion Research, Dept of Clinical Chemistry, University
period of 6 months. Hospital, S-751 85 Uppsala, Sweden.
A great number of abnormalities was found on the first occasion with a
complete normalization for all variables except one at the end of the Keywords: Chemiluminescence; chemotaxis; chronic
observation period. The number of infectious exacerbatlons was bronchitis; infections; neutrophil; phagocyte funclion;
phagocytosis; polymorphonuclear neutrophil; smoking.
significantly higher (pNEUTROPHIL FUNCTION IN CHRONIC BRONCHITIS 537
this respect was missing for one patient. The smokers In brief, migration was assayed with purified cells
had smoked on average for 35:13 yrs (±sn) and on (1.5x106·ml· 1) suspended in Gey's solution or 10% fresh
average 14±7 cigarettes per day (:sn). Two patients were patient serum or pooled normal serum above the
treated regularly with inhalation steroids but none of the micropore filter (3 f..tm pore size) (MilJipore Corp.,
patients was treated with oral steroids. Lung function as Bedford, MA, USA). Incubation was allowed for 60 min
evaluated by forced expiratory volume in one second at 3rC. After fixation and staining of the filters the
(FEV1)was 79:22% of predicted (±sn) in the patients of leading-front was read on 3-5 microscopic fields and the
stage 2 and 50±30% of predicted (±sD) in the patients of results expressed as means of these readings on
stage 3. The patients were recruited to the study by duplicate filters. When random migration or
advertisement in the local press and a minority had been chemokinetic responses to pooled normal or patient's own
seeing a doctor regularly for their disease. The study was serum were assessed, Gey's solution was put under the
approved by the local Ethics Committee. filter. When chemotaxis was measured either 10%
Blood samples were drawn once from all 45 patients zymosan-activated serum or 100 nmoH1 FMLP was put
at the beginning of the study i.e. period I at October- under the filter and the cells were suspended in Gey' s
November. Twenty two of the patients were followed solution. The intra-assay coefficient of variations for the
during 32 weeks beginning in October-November and migration methods ranged from 8-12%.
ending April-May the following year, and from these
patients two additional blood samplings were made: one
at period II, i.e. January-February and one at period Ill, Measurement of neutrophil phagocytosis
i.e. at the end of study in April-May. These 22 patients
were chosen randomly and did not differ from the other Neutrophil phagocytosis was measured by a kinetic
half with respect to age, sex, smoking habits or numbers particle uptake method as described in detail previously
of anamnestic infectious exacerbations. Of these 22 [8]. The latex-particles were coated with either human
patients 10 were of stage 2 and 12 of stage 3. Blood immunoglobulin G (IgG) (Kabi, Sweden) or human IgG
samples were not drawn from any patient with obvious and fresh serum. The latter particle was designated
clinical signs of ongoing infection. The instructions were "C3b"-particles since we have demonstrated earlier that
also not to draw blood from a patient within 14 days the major additional opsonin on these particles is C3b
after a clinical infection. Infection episodes and the [9]. In brief, 0.5 ml of 4x10 6cells·mJ-1 were mixed with
number of infection days were monitored both by the 0.5 m! of 40xl06 particles·ml· 1• The mixture was kept at
patients through questionnaires and monthly by two 37°C under constant stirring. Aliquots of 100 f..ll were
researchers. Infection episodes were defined as infections removed every minute for the assessment by means of a
which indicated the use of antibiotics. The patients were thrombocounter (Coulter Electronics, USA) of free
medically treated according to their individual needs. latex-particles in !he suspension.
Thirty apparently healthy laboratory employees served The rate of disappearance of the latex-particles was
as controls, age range 20-62 yrs (mean age 32 yrs). used as a measure of the phagocytic rate and expressed
Twenty one were women and 9 men. Six of the controls, as min. The intra-assay coefficient of variation (CV) of
were smokers. At least one control was assayed on the the phagocytosis method was 8-10%.
same day as a patient. Of the controls, 8 were assayed
repeatedly i.e. 2-4 times, during the study. There was Measurement of neutrophil oxidative metabolism
no seasonal variation with respect to neutrophil functions
in the control group. The oxidative metabolism of neutrophils was measured
by means of chemiluminescence. The production of
chemiluminescence was assayed in a Biocounter® M2010
Methods (Lumac B.V., The Netherlands). The detailed method
has been described previously [10]. In brief, 100 f..ll of
Venous blood for neutrophil function testing was drawn cells (1x106 ·ml·1) were mixed with 100 f..ll of luminol 25
in heparinized evacuated blood tubes (Venoject, Terumo, f..lg·ml· 1 (Merck, Darmstadt, West Germany) or 100 f..ll of
Japan). Venous blood for the preparation of serum was Jucigenin 100 f,.lg·ml·1 (Sigma Chemical Company, USA).
drawn in evacuated blood tubes without any additives After this 100 f..ll of stimulus was added and the reaction
(Becton-Dickinsson, New Jersey, USA). White blood cell recorded continuously on a recorder. As stimuli serum-
counts and differentials were made by means of the opsonized zymosan 4 mg·mt·1, phorbol myrisate acetate
Technicon Hl analyser (Technicon Instruments Corpora- nmol (PMA) 10 ng·ml·1 or FMLP 100 nmol·mJ'l were
tion, Tarrytown, NY, USA). used. The activity was expressed in relative light units
Neutrophil granulocytes were isolated as described [6]. (RLU) from the peak of the curve. The intra-assay
variations of the methods ranged from 7-12% (CV).
Measurement of neutrophil migration
Measurement of whole blood chemiluminescence
Neutrophil migration was assayed by a modified
leading-front technique as described by WlLKINSON [7]. Whole blood chemiluminescence was measured as
The detailed method has been described previously [6]. described in detail previously [11]. In brief, 50 !!I538 P. VENGE ET AL.
heparinized blood was mixed with 400 !!1 of Gey's indicated. For calculations of the statistics the personal
solution. To the cells 100 !!l of luminal (1 !!g·ml-1) or 100 computer program Statgraphics, STSC, USA was
!!l lucigenin (0.5 !!g·ml-1) was added and allowed to used.
preincubate for 5 min at 37°C in the cuvette in the meas-
uring chamber of the Biocounter. After this preincubation
period, 100 !!l of serum-opsonized zymosan particles were Results
added and the reaction recorded. The peak activity was
used for the calculation and the results were expressed as Neutrophil migration
RLU·l0· 6 neutrophils in the blood. The activity was
corrected for the quenching caused by haemoglobin in Table 1 shows the results for the whole group of
the blood. The intra-assay variation of the methods ranged patients. As compared to the control population two
from 7%-9% (CV). variables were different: random migration was on average
16% higher in the patient group (pNEUTROPHIL FUNCTION IN CHRONIC BRONCHITIS 539
The follow-up of half of the patients showed MIn
significant variations over time of random migration
(p540 P. VENGB BT AL.
RLU The follow-up study showed a significant (pNEUTROPHIL FUNCTION IN CHRONIC BRONCHITIS 541
Table 5. - Number of infectious episodes for each 2 As shown in table 6, migration of patient neutrophils
month period during the 6 month observation period in the presence of patient's own serum was significantly
lower (p542 P. VENGE ET AL.
We also show that active smoking adversely affects to postulate that the mobilization of myeloperoxidase from
several aspects of neutrophil functions, such as chemo- the neutrophils is enhanced and compensates the defect
kinesis and luminol-enhanced chemiluminescence, and found with lucigenin. This is because lucigenin gener-
may in fact prevent the normal response to the bacterial ates a signal independent of myeloperoxidase. Luminol,
challenge. The enhanced migratory activity of the on the other hand, is strictly dependent on the presence
neutrophils at period I was most obvious when the of peroxidases in the reaction [19]. Consequently, the
capacity to migrate randomly and towards a chemotactic reduction in luminol-enhanced chemiluminescence, which
signal such as activated serum (presumably C5fr) was was observed in smokers, must be explained by an
measured. These results are in keeping with a previous adverse effect of smoking on the mobilization or activity
report on patients with chronic bronchitis and emphysema of myeloperoxidase. The observation of a reduced activ-
[3]. In contrast, the response to chemokinetic signals ity in this respect in smokers is opposite to the findings
was unaffected at that period but increased at period 11 of others, who reported an increment as a consequence
when the other variables started to be normalized. We of smoking [20]. These increments were only seen in
have shown earlier that chemokinetic signals are produced patients with high white blood cell counts which may
in large amounts during active inflammation [10, 15, 16] explain the seeming discrepancies.
and it is therefore likely that such signals were produced Whole blood chemiluminescence is a complicated
in the studied patients during their active periods of variable and the results are consequences of the activity
disease. Possibly, therefore, the neutrophils at period I of several cells and their relative proportions in the blood.
were desensitized to further such signals in vitro whereas There is some evidence that the lucigenin-enhanced
at period 11, when the disease activity was somewhat activity primarily reflects monocyte activity [11] and the
reduced and the production of chemokinetic signals enhanced activity at period I would therefore indicate an
consequently lower, the cells had regained their increased activity of these cells with a subsequent nor-
responsiveness. The subsequent complete normalization malization at periods 11 and Ill.
at period Ill of all functions reflecting the capacity of the We conclude from this study that at active ongoing
neutrophils to move and respond to various signals disease a number of neutrophil functions are altered in a
suggests that individuals suffering from chronic bronchitis manner anticipated from the study of neutrophil functions
and recurrent infection do not have a constitutional during episodes of acute bacterial infection. Most such
predisposition to recurrent infections as a consequence abnormalities are normalized in parallel to the reduction
of defects in the migratory capacity of their neutrophils. of the disease activity which could indicate that
The phagocytic activity of the neutrophils was seem- constitutional defects in neutrophil functions are rare
ingly unaffected and normal at all three periods, causes of the recurrent bacterial infections in these
probably excluding any constitutional defects in this patients. However, the reduced capacity of the
function, although single patients indeed had subnormal neutrophils to generate 0 2-metabolites may be an
activities in this respect. These results agree well with exception to this general conclusion and in addition to
those of other authors [4]. In other infection-prone the adverse effects of smoking on some other functions
individuals, defects in the capacity to phagocytose IgG- these defects, constitutional or acquired, may add to the
coated particles are fairly common findings [10, 16]. In predisposition of these individuals to their recurrent
contrast to the normal phagocytic activity of the cells, bacterial infections. It should also be emphasized that
the capacity of patient serum to opsonize zymosan- these data are derived from in vitro studies of neutrophil
particles was clearly abnormal. Thus, at both periods I functions and may not necessarily reflect what is ongo-
and 11 this capacity was considerably enhanced. A similar ing in the lung.
enhancement is a general finding in acute infections (to
be published Pauksens and Venge) and probably reflects Acknowledgements: The technical assistance of A.
activation of the complement system during this period. Hull and A-K. Pettersson was greatly appreciated.
The subsequent normalization at period Ill is probably
another indication of a reduction in the disease activity
of the group as a whole. References
The oxidative metabolism of cells can be measured by
means of chemiluminescence. Defects in oxidative 1. Sachs FL. - Chronic bronchitis. Clin Chest Med, 1981,
metabolism of neutrophils is a common cause of increased 2, 79-89.
infection susceptibility. Complete deficiencies are typical 2. Taylor JC, Mittman C, eds. - Pulmonary emphysema
findings in patients suffering from chronic granulomatous and proteolysis: 1986. Academic Press, New York, 1987.
disease, a severe life-threatening condition [17]. How- 3. Burnett D, Chamba A, Hill SL, Stockley RA.
ever, relative deficiencies have also been associated with Neutrophils from subjects with chronic obstructive lung disease
an increased susceptibility to infections in children [18]. show enhanced chemotaxis and extracellular proteolysis. Lancet,
1987, ii, 1043-1046.
In the present patients the purified cells demonstrated a 4. Fietta A, Bersani C, De Rose V, Grassi FA, Mangiarotti
significant reduction in lucigenin-enhanced chemilumi- P, Uccelli M, Grassi C. - Evaluation of systemic host defense
nescence at periods 11 and Ill which may suggest a mechanisms in chronic bronchitis. Respiration, 1988, 53,
constitutional defect in handling 0 2 • However, to 37-43.
explain the fact that chemiluminescence enhanced with 5. Medical Research Council. - Committee on the etiology
luminol was seemingly normal at these periods one has of chronic bronchitis. Lancet, 1965, i, 775-779.NEUTROPHIL FUNCfiON IN CHRONIC BRONCHITIS 543 6. H~kansson L, Venge P.- The influence of serum on ran- 19. Williams AJ, Cole PJ. - The onset of polymorphonuclear dom migration and chemotaxis of polymorphonuclear leukocyte membrane-stimulated metabolic activity. Immunology, leukocytes. Methodological evaluation using sera from infection- 1981, 43, 733-739. prone patients and normals. Scand J Immunol, 1980, 11, 20. Ludwig PW, Hoidal JR. - Alterations in leukocyte 271-282. oxidative metabolism in cigarette smokers. Am Rev Respir Dis, 7. Wilkinson PC. Chemotaxis and inflammation. 1982, 126, 977-980. Churchill Livingstone, Edinburgh and London, 1974. 8. HM
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