661
Clinical Science (1983) 65,661-664
Extracellular release of hydrogen peroxide by human
alveolar macrophages: the relationship to cigarette smoking
and lower respiratory tract infections
A. P. GREENING
AND
D. B. LOWRIE
Department of Medicine and MRC Unit for Laboratory Studies of Tuberculosis, Royal Postgraduate Medical School,
Hammersmith Hospital, London
(Received 20 December 198211 June 1983; accepted 23 June 1983)
summary
Introduction
1. It has been suggested that oxidants from
pulmonary inflammatory cells may contribute to
the development of emphysema by (i) direct tissue
toxicity and (i) inhibition of cul-antitrypsin, thus
diminishing protection of the lung from proteolytic
damage.
2. The extracellular release of hydrogen peroxide (H202)by human alveolar macrophages
(AM) has been measured. AM were obtained by
bronchoalveolar lavage and adherence from 24
smokers and 17 non-smokers.
3. Smokers’ AM released significantly more
H202(3.83 nmol h-’ pg-’ of DNA; SEM 0.44) than
those of non-smokers’ (2.33 nmolh-’pg-’ of DNA;
SEM 0.40) (P<0.05).
4. AM from donors with a recent lower respiratory tract infection released increased quantities
of H202(5.22 nmol h-’ pg-’ of DNA; SEM 0.72;
P<O.Ol) even when allowance was made for
smoking habits.
5. These findings are consistent with the hypothesis that H202of AM origin contributes to the
development of emphysema in smokers.
At present it is believed that emphysema develops
as a consequence of protease-antiprotease imbalance in the lung. This proteolytic theory has
evolved after the recognition of an association
between severe deficiency of&’-proteinase inhibitor
and the development of panacinar emphysema [ l ]
and the observations that emphysema can be
induced by instillation of elastolytic enzymes into
the lower respiratory tract in animals [2,3]. It is
thought that the alveolar structures are normally
protected from extracellular proteases, which can
originate from inflammatory cells, by an antiprotease screen. At the alveolar level al-proteinase
inhibitor is the principal antiprotease [4] but it
can be inhibited by oxidation of the methionine
that is part of the molecular structure in or near
the active site [5]. Thus, in the presence of
oxidants, a functional deficiency of cul-proteinase
inhibitor may occur in the lower respiratory tract,
disturbing the protease-antiprotease balance in
favour of protease activity. Cigarette smoking has
been shown to induce functional antiprotease
deficiency in the lower respiratory tract [6]. This
may be due to a direct action of oxidants in
cigarette smoke [7] but cigarette smoking also
enhances the oxidative metabolic response of
alveolar macrophages (AM) [8] and might, therefore, promote release from these cells of hydrogen
peroxide (H203 and other oxidants that are
important for the cells’ microbicidal functions [9].
Such release of AM-derived oxidants might lead to
Key words: alveolar macrophages, emphysema,
hydrogen peroxide, respiratory tract infections,
smoking.
Correspondence: Dr A. P. Greening, Department
of Respiratory Medicine, City Hospital, Greenbank
Drive, Edinburgh EHlO 5SB, Scotland, U.K.
A . P. Greening and D. B. Lowrie
662
functional inhibition of al-proteinase inhibitor in
the microenvironment of these cells. We have
therefore measured the extracellular release of
MzOz from human AM and examined the influences
of the macrophage donors’ smoking habits and
pulmonary diseases.
Methods
Alveolar macrophages
These were obtained by bronchoalveolar lavage
[ l o ] from 4 1 patients (Table 1) during diagnostic
bronchoscopy. Patients categorized as ‘recent lower
respiratory tract infection’ had all had parenchymal
shadowing on chest X-ray within the preceding
3 months. Two of these patients had bronchographically demonstrated bronchiectasis. Informed
patient consent and approval of Hammersmith
Hospital Ethics Committee were obtained. Lavage
of the lingula or middle lobe was performed with
two 120 ml volumes of sterile NaCl solution (154
mmol/l: saline) with immediate aspiration. For
patients with unilateral lung disease (bronchial
carcinoma or recent infection) the contralateral
lung was lavaged. Cells were recovered from the
lavage fluid by low speed centrifugation (60g for
15 min), resuspended at 2 x lo6 cellslml and AM
separated from other cell types by adherence and
maintenance in tissue culture as monolayers. AM
were cultured in 35 mm plastic Petri dishes (Nunc,
Gjbco Europe Ltd, Paisley, Scotland, U.K.) for
72 h in medium 199 (Gibco Europe Ltd), supplemented by 10% autologous serum, 4% bovine
embryo extract (Flow Laboratories, Irvine, Scotland, U.K.) and 1% liver fraction L (United
States Biochemical Corp., Cleveland, OH, U.S.A.).
Preliminary tests showed no change in H202
responses of AM between 2 4 and 72 h in culture.
Measurement of H202
release
H202release was estimated by the oxidation of
p-hydroxyphenylacetic acid, catalysed by horseradish peroxidase, to a fluorescent product [l I].
After 72 h in culture AM monolayers were incubated, with and without stimulation by phorbol
myristate acetate (PMA; lpg/ml), in 1 ml of
medium 199 containing 2.4 pmol of p-hydroxyphenylacetic acid and 5 p g of peroxidase. Standards were prepared by inoculating cell-free Petri
dishes with 2-20 nmol of H202together with the
test reagents in 1 ml of medium 199. After 60 min
incubation, 900p1 of each supernatant was removed into 1 ml of ice-cold borate buffer (0.2
mol/l, pH 10.4) and the mixture was allowed to
reach ambient temperature in the dark before
fluorescence measurement. To allow for any minor
variations in numbers of adherent AM in different
monolayer preparations the Hz02 release was
standardized to the DNA content of the monolayer. DNA was measured by an adaptation [12]
of the method of Giles & Myers [ 13J .
Statistical analyses
Comparison of H202release by smokers’ and
non-smokers’ AM was performed by using
Student’s t-test for unpaired data. Comparison of
H202 release by AM from donors with different
diseases, taking into account the donors’ smoking
habits, was performed by analysis of covariance.
Results
After 72 h culture Giemsa staining showed AM
monolayers to be free of other cell types. The AM
were >95% viable as judged by trypan blue
exclusion.
TABLE1. Release of hydrogen peroxide by human alveolar macrophages, stimulated with phorbol
myristate acetate, in relation to disease, smoking habit, sex and age o f the macrophage donors
Disease
No. of subjects
Smokers
No active pulmonary disease
Recent lower respiratory tract infection
Bronchial carcinoma
No n-bro nchial m ahg nancies
Sarcoidosis
Fibrosing alveolitis
* 1 X lo6 alveolar macrophages, 6.5 gg of DNA.
3
7
9
3
0
2
Nonsmokers
Men
2
1
2$
2
9
1
3
4
11
2
7
2
t P < 0.01, for comparison of these two groups against the other four.
$ Ex-smokers of
> 8 years.
Mean
age
(years)
43
50
62.5
52
41
51
H,O, (nmol h-’ fig-’
of DNA)*
Mean
SEM
2.42
5.221.
2.26
4.70t
2.16
3.21
0.75
0.72
0.59
0.69
0.44
0.67
Alveolar macrophages and hydrogen peroxide
101
l
0
N
PI
BrC
NbM
S
FA
FIG. 1. Extracellular hydrogen peroxide release
from alveolar macrophages obtained from smokers
and non-smokers (0)displayed according to
the disease category of the macrophage donor. N,
Normal subjects; PI, recent (<3 months) lower
respiratory tract infection; BrC, bronchial carcinoma; NbM, non-bronchial malignancies; S, sarcoidosis; FA, fibrosing alveolitis.
(0)
Unstimulated AM released minimal quantities
of H202 (<0.2 nmol h-' pg-' of DNA). However,
on stimulation with PMA this was markedly enhanced (0.2-8.73nmol h-' pg-' of DNA). PMAstimulated AM from smokers released more H202
(3.83 nmol h-' pg-' of DNA; SEM 0.44; n = 24)
than PMA-stimulated AM from non-smokers (2.33
nmol h-' pg-' of DNA; SEM 0.40;n = 17). The difference was significant (P< 0 .05). Age and sex of
the AM donors had no influence in the quantity of
Hz02released. However, some diseases of the
donors did affect the H202 release, even when
allowance was made for smoking habits (Table 1;
Fig. 1). AM from patients with a recent lower
respiratory tract infection released more H202
(5.22 nmol h-' pg-' of DNA) on stimulation as did
AM from patients with non-bronchial malignancies
(4.70 nmol h-' pg-' of DNA). The difference was
significant (P<0.01) in both instances.
Discussion
Our results show that AM from cigarette smokers,
when stimulated, released significantly greater
quantities of H202into the extracellular environment than AM from non-smokers. These results
accord with those of other workers who, by
measuring release of superoxide, have shown similar
enhancements of AM oxidative metabolism for
young asymptomatic cigarette smokers [8]and for
smokeexposed rats [14].The unstimulated AM
663
released minimal quantities of H20z, indeed, for
AM from all but three patients (all smokers) the
basal levels of release were below the lower limit
of sensitivity of our assay (<0.2 nmol h-' pg-' of
DNA); thus it was not possible to determine
whether unstimulated AM from smokers released
greater quantities of H202 than the AM of nonsmokers. Nevertheless, in vivo, AM will be subject
to frequent phagocytic stimulation. We chose
PMA as our receptor-mediated [15] phagocytic
stimulus [16] in view of its degree of cellular
stimulation, repeatability of results and convenience of use.
Our interpretation of the data for H202release
from stimulated AM is made cautiously since
numbers of smokers and non-smokers in the various
disease groups were not matched (Fig. 1). However, even if the disease group that contributes
most to the high value for the smokers (recent
lower respiratory tract infection) is removed
from analysis, smokers' AM still release significantly greater quantities of H202 than the AM of
non-smokers (3.1 k 0.4 nmol h-' pg-' of DNA vs
2.3 f 0.4). This implies that the effects of smoking
and infection might be cumulative but whether
infection would have an. effect in non-smokers
cannot be assessed from the data. The mechanisms
by which the enhancement of release of H202
from smokers' macrophages occurs are not known.
Clearly one or more components of cigarette smoke
may directly affect the AM. A more intriguing
possibility is that smokers' AM have been primed
for enhanced responses by pulmonaly lymphocytes or lymphocyte products as part of a cellmediated immune response. In support of this
hypothesis two of the five patients with nonbronchial malignancies (one Hodgkin's disease and
one disseminated thyroid carcinoma) and one
patient with bronchiectasis had significant elevation of the percentage of lymphocytes in their
bronchoalveolar cells (24.0, 45.6 and 33.6% respectively). We have shown [17] that AM, in
vitro, can be primed by lymphokines for an enhanced H202release on stimulation. There would
be ample opportunity in vivo for cell-mediated
immunity to be developed against antigenic components of cigarette smoke or bacterial antigens.
We suggest that in vivo priming of AM bypulmonary lymphocytes also explains the enhanced H202
release by cells from donors with recent lower
respiratory tract infections or with non-bronchial
malignancies (an heterogeneous group including
disseminated nasopharyngeal and thyroid carcinomata and pulmonary lymphomas). In these groups
the presumptive cell-mediated immunity would be
in response to microbial and tumour antigens
respectively.
664
A. P. Greening and D. B. Lowrie
Irrespective of the mechanisms underlying the
macrophage activation the enhanced release of
H202 may result in a functional deficiency of
al-proteinase inhibitor in the microenvironment
around AM, thus locally favouring uninhibited
protease activity [18,19]. In addition Hz02may
itself be directly tissue-toxic [20]. Thus our findings sustain the possibility that H202
of macrophage
origin might contribute to the development of
emphysema by smokers.
Acknowledgments
A.P.G. was in receipt of a Medical Research Council Training Fellowship and gratefully acknowledges
this support. We thank Mr V. R. Aber for statistical
analyses and Alma Dixon for secretarial assistance.
We thank our colleagues for referral of patients in
this study and in particular Dr N. B. Pride for his
encouragement and support.
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