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Eur Resplr J
1992, 5, 59-66
Lymphocyte macrophage interactions: peripolesis of
human alveolar macrophages
D.J. Lyons*, A. Gautam**, J. Clarkt, M.G. Harriest, E. Bruce MitcheUtt,
J.S. Milledget, B.M. Balfourt.
Lymphocyte macrophage interactions: peripo/esis of human alveolar macrophages.
D.J. Lyons, A. Gautam, J. Clark, M. G. Harries, E. Bruce Mitchell, J.S. Milledge, B.M.
Ea/four.
ABSTRACT: Perlpolesls Is a phenomenon In which a lymphocyte attaches it.self
to another cell, usually a macrophage or veiled cell, and proceeds to circle around
it. In emperlpolesls, a related phenomenon, the Jymphocyte lnvaglnates the tar·
get cell so deeply that it appears to be Intracytoplasmic.
Lung cells In bronchoalveolar lavage nuids from 20 patients were observed In
the living state and filmed. Perlpolesis of the alveolar macrophages was recorded
In six cases. These pallents Included one case each of carcinoma of the bronchus,
tuberculosis, sarcoidosis and asthma, while two patients had no detectable lung
disease. Five out of the six positive cases were females. In every instance there
was a high number of lymphocytes l.n the washing. The perlpolesed macrophages
were not Injured, but temporary alteration of the cell membrane was noted in a
minority of nlm sequences. The peripoJesing cells were also examined by trans·
mission and scanning electron mlcroscopy. The lymphocyte was found to be
closely attached to the surface of the macrophage, with no invagination and its
ultrastructure was that of a small lymphocyte.
Peripolesls is probably a physiological mechanism concerned with regulation of
the Immune response in the lung.
Eur Respir J., 1992, 5, 59-66.
• Beaumont Hospital
Dublin, Republic of Ireland.
•• Dept of Medical Microbiology
Stanford University
Stanford CA
USA.
t Clinical Research Centre
Horrow, Middlesex
UK.
Black Rock Clinic
Dublin
Republic of Ireland.
tt
Correspondence: Dr B.M. Balfour
Clinical Research Centre
Division of Immunological Medicine
Watford Road, Harrow
Middlesex HA1 3U1
UK.
Keywords: Alveolar; lymphocytes;
macrophages; peripolesis.
Received: December 14, 1990; accepted
after revision August 19 1991
The technique of bronchoalveolar lavage (BAL), now
widely used in the diagnosis of pulmonary disease
[1, 2] has made it possible to recover immunologically
active cells in the washing fluid, and to observe them in
the living state. Lung macrophages are developmentally
separated from gut and other tissue macrophage
systems [3] and include several types of cells varying
in size, enzyme content, motility and membrane markers [4]. Some lavage fluids contain veiled or dendritic
antigen-presenting cells (APCs), which resemble
macrophages but are not phagocytic or adherent and are
also highly mobile [5]. The two types of APC have
different functions. The macrophages ingest particulate
antigens, or large molecules, and after lysosomal degradation the fragments are returned to the cell membrane, whereas dendritic cells deal with small antigens
and digestion, if required, probably takes place in the
cell membrane. There are also variable numbers of
large and smalllymphocytes, neutrophilleucocytes and
ciliated bronchial epithelia. All these cells were observed microscopically and their interactions recorded
by time-lapse cinematography. We also studied the
same cells by transmission and scanning electron
microscopy (TEM and SEM). In this paper we report
that the cell interactions known as peripolesis [6-10]
can be observed in a proportion of lung washings. The
circling cells were lymphocytes and the peripolesed cells
alveolar macrophages.
We did not observe peripolesis of dendritic cells,
although animal experiments have shown that it does
occur, apparently in connection with antigen presentation [11 ]. In the lung the role of alveolar macrophages
is mainly down-regulation of the immune response, but
they may acquire the ability to present antigen in
conditions such as sarcoidosis [12].
This is the first proof of peripolesis in lung washings.
In the literature there are 'still' photographs showing
lung macrophages with attached lymphocytes [4].
However, contact between these cells is not always
followed by peripolesis, which can only be established
by cinematography or direct viewing of the living cells
at body temperature.
Materials and Methods
Patients
We studied an unselected group of 20 patients
undergoing diagnostic bronchoscopy, who gave
permission for an additional washing by the usual
D.J. LYONS ET AL.
60
Table 1. - Perlpolesls In bronchial lavage fluids: clinical correlations In six positive cases
Sex
Age
Sm.
Diagnosis
yrs
Peripolesis of
ComEosition of BAL fluid
%Lympho %Macro %Neutro %Eosino
macrophages
(cinematography)
4h
18 h
Remarks
Retronasal
bleeding
+
•
42
53
3
2
Bronchoscopy and chest
X-ray normal
+
Chest
infection
+
+
22
74
4
<1
Thalassaemia; infection
with haemoptysis
chest X-ray normal
complete recovery
+
Pulmonary
tuberculosis
+
ND
ND
ND
ND
Asymptomatic
Mycobacteria isolated
30
Sarcoidosis
+
+
25
72
3
<1
Active disease;
erythema nodosum
Hilar adenopathy
F
38
Asthma
+
45
52
3
<1
Past and family history
of asthma, urticaria and
eczema
F
74
Squamous
+
carcinoma of
bronchus
+
55
43
2
<1
Died
M
30
F
40
F
56
F
+
Mean age positive patients= 45 yrs. •: preparation fixed for scanning electron microscopy on day of lavage. BAL: bronchoalveolar
lavage; Sm.: smoking; ND: not done; Lympho: Lymphocytes; Macro: Macrophages; Neutro: Neutrophil leucocytes; Eosino:
Eosinophil leucocytes.
Table 2. - Clinical details In 14 patients showing no perlpolesis
Sex
Age
yrs
Sm.
M
M
F
61
63
57
65
45
+
+
+
+
53
+
+
F
F
M
M
M
M
M
M
F
M
M
77
47
66
70
+
+
29
74
31
73
+
%lympho
Diagnosis
Squamous carcinoma of bronchus
Oat cell carcinoma of bronchus
Squamous carcinoma of bronchus
Squamous carcinoma of bronchus
Pulmonary tuberculosis
Pulmonary tuberculosis
Squamous carcinoma of bronchus
Oat cell carcinoma of bronchus
Pulmonary embolus
Metaplasia and haemoptysis
Carcinoid
Pulmonary embolus with pleural effusion
Haemoptysis, nil found
Haemoptysis, nil found
10
12
8
23
7
16
ND
%Macro
80
76
91
69
66
77
ND
%Neutro
9
10
1
8
23
5
ND
%Eosino
1
2
<1
<1
4
2
ND
Mean age negative patients = 57 yrs. Sm.: smoking; ND: not done; Lympho: Lymphocytes; Macro: Macrophages; Neutro:
Neutrophil leucocytes; Eosino: Eosinophils leucocytes
technique [13]. There were 11 males and 9 females,
whose clinical details are given in tables 1 and 2; 60%
were smokers.
Bronchoalveolar lavage, tissue culture and filming
One hundred and twenty millilitres of prewarmed
phosphate buffered saline (PBS) was infused under
lignocaine anaesthesia and immediately recovered onto
ice. In cases of suspected lung tumour the extra lavage
was performed on the opposite side. After filtration the
cells were washed twice in PBS and resuspended at a
concentration of 5x105·ml·1 in culture medium (RPMI
1640) with 10% blood group AB serum or 10% fetal
calf serum (FCS) with added penicillin and
streptomycin. Fluids recovered from the lung are often
contaminated with yeasts and fungi. We attempted to
control this by adding mycostatic drugs including
PERIPOLESIS OF ALVEOLAR MACROPHAGE$
amphotericin B, flucytosine, miconazole, mycostatin
with gentamicin and econazole at recommended concentrations. These drugs were toxic for macrophages
and lymphocytes and none had the desired effect of
inhibiting the growth of contaminants.
At this time smears and cytospins were prepared from
the cell concentrates and stained with May-GrunwaldGiemsa. Five hundred cells were counted in each
preparation and the percentage of lymphocytes,
alveolar macrophages, neutrophil leucocyt es
and eosinophil leucocytes calculated. Ciliated epithelial cells and squamous cells were not included in the
total counts. The cells were transferred to a Petri dish
with a glass inset which was mounted on an inverted
phase contrast microscope, provided with a C0 2 gas
supply and enclosed in a hot box at 37°C (fig. 1) [14].
This system allows observation and recording of cellular movement by time-lapse filming for up to 48 h.
The lignocaine administered to the patients inhibited
the movement of the lavage cells and filming was
postponed until they had regained their motility,
usually 3-4 h. Filming was carried out at a speed of
one frame·3 s·1, i.e. speeded up 72 times, using 16
mm Eastman colour negative. If electron microscopy
(EM) studies were envisaged, the culture medium was
removed at the end of the film and replaced by 3%
glutaraldehyde.
61
be inserted into the stem unit of the electron microscope. A narrow copper strip was attached to the
undersurface of the coverslip in line with the reference
points and the glass was cut along the edge of the strip,
after which the cells were processed for SEM, i.e. critical point drying and gold coating.
Results
Peripolesis
Peripolesis was first observed 3.5 h after setting up
cultures of lung washings and was also seen at
18-24 h. It occurred in 6 of the 20 preparations
examined. Peripolesis was usually seen on the first and
second days but in one case it was observed after 18 h,
but not on the first day, possibly due to a prolonged
effect of the anaesthetic. Lymphocytes moved randomly,
but in positive cultures when one came within reach
of a macrophage it turned sharply towards the macrophage, rapidly attached itself and began the characteristic circling movement, with extrusion of a
lamellipodium in the direction of motion. Many
complete circumnavigations were seen (fig. 2) lasting
3-8 min. Occasionally the lymphocyte changed
course from clockwise to anticlockwise, or vice versa,
A
F
E
Fig. 1. - Diagram of microscope attachment for observation and filming of cells from lung washings. A: Petri dish culture chamber reduced
in height so that microscope condenser can illuminate phase annulus; 8: thin glass floor to allow objective to be focused on cells; C: phase
objective of inverted microscope; D: cling film cover to prevent condensation; E: inlet for C01; F: cross-section of annular chamber for
C01 circulation, provided with spaced-out holes (not shown). The microscope with its attachments is kept in a bot box at 37•c [12).
Electron Microscopy
In order to identify peripolesing cells in fixed preparations a map of the cells was made and reference points
marked on the end of the dish.
For TEM the cells were embedded in a gel consisting
of 10% bovine serum albumen (BSA) in PBS crosslinked with 3% glutaraldehyde. Although alveolar
macrophages are strongly adherent they were easily
incorporated into the geL The position of the
peripolesing group was marked on the surface, and the
gel was then cut up and processed in the usual way.
Thin sections were examined in a Jeol (JEM 1200 EX)
electron microscope.
For SEM the glass inset with attached macrophages
was cut down to approximately 6x2 mm so that it could
apparently by a shift of the lamellipodium to the
opposite pole of the celL Sometimes the lymphocyte
abandoned the target cell and moved off; in other
cases it contacted a neighbouring macrophage and
continued to peripolese. Occasionally, two lymphocytes attached themselves to the same cell, travelling
round in opposite directions, one cell passing over
the other.
In one instance, a macrophage developed elongated
spiky processes, while being peripolesed. After the
lymphocyte had moved away the membrane gradually
recovered its normal appearance, a process which took
about half an hour (fig. 3).
The proportion of macrophages subjected to
peripolesis varied from 10-30% per field and over
90% were large or medium sized. In some negative
62
D.J. LYONS ET AL.
Fig. 2. - Frames from a time-lapse film of cells in a lung washing from a female patient with carcinoma of the bronchus. The sequence
shows the phenomenon of peripolesis. a) A mobile lymphocyte (arrow) with its lamellipodium extended in the direction of movement was
approaching an alveolar macrophage. b) The lymphocyte made contact with the macrophage, c, d, e and f) clockwise peripolesis was in
progress. In this case one complete cycle took 8 min (x600)
Fig. 3. - Frames from a time-lapse film of cells from the same washing a.s figure 2. The alveolar macrophage developed long spiky processes
and appeared to be injured while being subjected to peripolesis by a lymphocyte (arrow). The time interval between a and b was 3 min. c)
1 hour later, the lymphocyte detached itself. d) 25 min later, the lymphocyte moved away and the macrophage regained its normal appearance
(x600).
cultures, lymphocytes were seen to make short contacts
with macrophages. Such cells extended themselves
repeatedly away from the macrophage, but did not
produce a lamellipodium and finally moved off. At
no time did we observe emperipolesis with deep
invagination of the target cell [6, 15-18].
PERIPOLESIS OF ALVEOLAR MACROPHAGES
63
Alveolar macrophages
Other antigen-presenting cells
The macrophage population was heterogeneous and
included large, medium and small cells [1, 2]. Thirty
percent had a distinct yellowish tinge. About 10% of
the macrophages extended and retracted a fimbriated or
club-shaped process (fig. 4). This structure could have
been mistaken for an attached lymphocyte, but doeobservations showed that it came out in several bursts,
of variable length and conformation, whereas attached
lymphocytes extended and retracted themselves in a
more repetitive manner. Sometimes a long thin process
made contact with another macrophage and was then
withdrawn.
Dendritic or veiled cells were seen in 3 of the
12 cultures recorded on film. These cells were easily
identified in the living state. They were smaller
than the macrophages, they moved at greater speed
and continually extended long veils. Two of the
washings were from patients with carcinoma of the
bronchus and contained 25 and 27% dendritic cells:
no peripolesis was seen in these cultures. The
third washing from a patient with tuberculosis
contained 10% dendritic cells. In this culture the
macrophages were peripolesed, but the dendritic cells
were not.
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.; ,.•
r
.
~
,.
Q
.,.
;",('
~
.Cl!
\.;
·o
c
0
l.:
fa
4(2)
Fig. 4. - a) Frames from a film of cells in a lung washing from a male patient with carcjnoma of the bronchus. Alveolar macrophage with
a fimbriated process (arrow). b) The same cell with process withdrawn (x600). c) Scanning electron micrograph of the macrophage illustrated
in a and b. The cell body lies above and behind a circular protrusion covered with ruffles located in the area from which a process was
extended and later withdrawn. Only a small proportion of macrophages possessed this parachute-like arrangement. The retracted process
looks superfici.ally like a lymphocyte, but we have proof from cine-viewing that no lymphocyte was present, also the deeply ruffled surface
is quite different from the ridged surface of the lymphocyte illustrated in figure 5B. Bar=2 11
64
D.J. LYONS ET AL.
Lymphocytes
TEM and SEM
The number of lymphocytes in the washings varied
widely. In stained preparations of the cell concentrates,
percentages ranged from 7-55%. In normal subjects
there are on average 10% using cytocentrifuge preparations but up to 20% using an improved method (19].
Figure SA shows a lymphocyte in the act
of peripolesing a macrophage. The cells were filmed
and the clockwise direction of motion was correlated
with the EM appearance. The lamellipodium at the
advancing pole of the lymphocyte can be seen interdigitating with the ruffled surface of the macrophage.
Behind the lamellipodium the under-surface of the lymphocyte is flat and closely applied to the macrophage,
which is here devoid of processes. There are a few
very short interdigitations between the cells in this area,
which suggests that the lymphocyte actively flattens the
macrophage membrane as it travels round. As usual,
during movement the lymphocyte organelles are gathered behind the nucleus. No large granules, or parallel
tubular arrays are present, therefore the peripolesing cell
was probably not a natural killer (NK) cell (20].
Figure 5B shows a scanning EM of another peripolesing cell. In this case the lymphocyte was moving
anti-clockwise. The surface of the macrophage is covered with fairly large ruffles, but the lymphocyte appears
smooth, except in the region of the lamellipodium.
A
Clinical correlations
B
Fig. 5. - A) Transmission electronmicrograph (EM) of lymphocyte
peripolesing alveolar macrophage in clockwise direction. Washing
was from female patient with non-recurrent haemoptysis.
Lamellipodium at leading end and mitochondria behind nucleus.
Area of contact between the cells is almost flat with small indenta·
tions. Free surface of macrophage has processes, except in the area
immediately behind peripolesing lymphocyte. Bar=2 I'·
B)- Scanning EM of peripolesis of alveolar macrophage. Washing
was from male patient with non-recurrent haemoptysis. Lymphocyte
attached to lower part of macrophage was moving in anti-clockwise
direction and lamellipodium can be seen on the right. Macrophage
covered with ruffles but lymphocyte has small ridges. Bar= 2~t.
In the group of six patients whose cells exhibited
peripolesis there was one case of carcinoma of the bronchus, one of sarcoidosis, one of pulmonary tuberculosis
and one of asthma; the remaining two patients, including the only male, had slight bleeding and no lung disease was found (table 1). These six patients had a
mean age of 45 yrs compared with 57 yrs for the negative group, and 5 out of 6 were females. About half
were smokers in each group. Peripolesis occurred in
washings containing an increased proportion of lymphocytes. In stained preparations the percentage of lymphocytes ranged from 21.8-58.2 mean 35.6% (95%
confidence interval 21.8-58.2). In six smears from negative cases it was significantly less, mean 11.6% (95%
confidence interval 7.2-18.5); two sample t-test (p=O.Ol).
Unfortunately the remaining negative smears were lost.
In 14 out of 20 washings no peripolesis occurred
and the range of diagnoses was similar to the group
showing the phenomenon (table 2). Eight of the
negative cultures were looked at continuously from
3.5-6 h and also at 18-21 h. The other six cultures
were negative on the day of lavage, but could not be
examined on the following day, owing to contamination. It is likely that the majority would have been
negative at 18 h since, only one of the six cases showing peripolesis became positive on the second day.
Discussion
Previous investigations on cells in lung washings (1,
2] concentrated mainly on the morphology and secretory activity of macrophages, the proportion of
lymphocytes and the effects of smoking. More recently
the phenotypes of the cells have been examined with
monoclonal antibodies (21-23].
PERIPOLESIS OF ALVEOLAR MACROPHAGES
We set out to film the behaviour of Jiving cells and
this led to the observation of peripolesis, which had not
previously been described in alveolar cells, although
still photographs of lymphocyte and macrophage rosettes have been published [4, 24]. Peripolesis can be
studied without film or video equipment by looking at
the cells at 3rc for at least one hour. If nothing happens on the first day the cultures can be viewed again
the next day after incubation overnight.
Other workers have observed peripolesis of thyroid
epithe.lial cells [6, 25] and accessory cells (7-11].
Cytotoxic injury to the peripolesed cells was noted in
gut preparations made from patients with ulcerative
colitis [26), but in autoimmune thyroid no cell killing
was observed [6). In the present study of peripolcsed
alveolar macrophages, a small proportion developed a
spiky appearance suggestive of injury, but later recovered. This appearance was also seen in dendritic cells
after an attack by a killer cell and was followed by
death of the target cell (unpublished observation).
Peripolesis occurred in washings with high lymphocyte
counts (22-55%), whereas only one of the negative cases
had a borderline raised count of 23%. To see if blood
lymphocytes were capable of this behaviour we chose a
washing fluid with a low lymphocyte count and negative peripolesis and added up to 50% autologous peripheral blood lymphocytes to the culture. No peripolesis
was seen on the first or second days. This preliminary
experiment suggests Lhat lung lymphocytcs are a special population. The phenotype of the peripolesing
lymphocytes has not yet been determined. The cell
examined by TEM was a small lymphocyte without the
ultrastructural characteristics of NK cells which have
been implicated in emperipolesis of malignant cells [16,
17]. Peripolesis was more common in women (p=O.OS
Fishers exact test). In view of the close correlation
with the lymphocyte count one might expect females to
have higher counts. However, previous BAL studies on
normal subjects or smokers did not support this suggestion [1). Possibly in disease states the lung lymphocytes
increase more sharply in female patients. Obviously
our clinical sample is too small to detect any firm correlation between individual diseases and lung peripolesis.
Dendritic cells were found in three of the washings,
in one case the macrophages were peripolesed, but not
the dendritic cells, an interesting difference requiring
confirmation. In animal experiments veiled cells (precursors of dendritic cells) from the afferent lymph of
rabbits immunized with human gammaglobulin (HGG),
were pulsed with the antigen and mixed with autologous blood lymphocytes. Peripolesis of the veiled cells
was observed in two of the four cultures, but was not
seen in numerous preparations from rabbits immunized
with an unrelated antigen [11] . This suggested that
surface immunoglobulin might be the signal for the
phenomenon. Alveolar macrophages have numerous
receptors able to capture immunoglobulin [27) and the
flattening of the macrophage membrane during
lymphocyte movement would expose the Fc-receptorbearing part of the surface, which lies in the clefts
between the membrane ruffles [28], so ensuring
65
contact with the attached immunoglobulin. In HGG
cultures peripolesis of the veiled cells was sometimes
halted by the attachment of two more lymphocytes to
the peripolesing cell, producing a configuration typical
of antigen presentation by cells of this type, i.e. one
lymphocyte attached to the veiled cell and two more
attached to this cell [11]. Antigen-presenting macrophages form similar clusters [29, 30), but there are no
reports suggesting that peripolesis of the macrophages
precedes, or is associated with, Lhis type of activity. We
never observed the attachment of additional lymphocytes to the peripolesing cells in our BAL preparations,
but it might occur in advanced cases of sarcoidosis.
The main function of alveolar macrophages is
down-regulation of the immune response in the lung
[31] . Only one third of the macrophages were
peripolesed, which probably reflects the heterogeneity
of the population with respect to surface markers
[32-34] and the secretion of cytokines [31].
Mandatory investigations to clarify the role of
peripolesis in BAL fluids are as follows: to enrich the
cultures with lung lymphocytes as well as blood
lymphocytes in order to increase the incidence of
peripolesis. This would greatly facilitate the phenotyping
of the peripolesed macrophages which may represent a
non-secretory subgroup. The phenotypes of the
lymphocytes and their state of activation should also be
determined. Of further interest would be to study
precisely defined and larger groups of patients with
interstitial lung diseases in order to reveal possible
clinical correlations with disease state and activity.
Acknowledgements: The authors thank G.L.
Asherson for his support, J.L. Mackenzie for pre·
paring still photographs from the films, N. Webb
for the diagram of the apparatus and for the layouts
and N. Saunders for typing the manuscript.
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