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Eur Resplr J 1992, 5, 59-66

Lymphocyte interactions: peripolesis of human alveolar

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. • Beaumont Hospital D.J. Lyons, A. Gautam, J. Clark, M. G. Harries, E. Bruce Mitchell, J.S. Milledge, B.M. Dublin, Republic of Ireland. Ea/four. •• Dept of Medical Microbiology ABSTRACT: Perlpolesls Is a phenomenon In which a lymphocyte attaches it.self Stanford University to another cell, usually a macrophage or veiled cell, and proceeds to circle around Stanford CA it. In emperlpolesls, a related phenomenon, the Jymphocyte lnvaglnates the tar· USA. it get cell so deeply that appears to be Intracytoplasmic. t Clinical Research Centre cells In bronchoalveolar lavage nuids from 20 patients were observed In Horrow, Middlesex the living state and filmed. Perlpolesis of the alveolar macrophages was recorded UK. In six cases. These pallents Included one case each of of the , tt Black Rock Clinic , sarcoidosis and asthma, while two patients had no detectable lung Dublin disease. Five out of the six positive cases were females. In every instance there Republic of Ireland. 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 Correspondence: Dr B.M. Balfour minority of nlm sequences. The peripoJesing cells were also examined by trans· Clinical Research Centre Division of Immunological Medicine mission and scanning electron mlcroscopy. The lymphocyte was found to be Watford Road, Harrow closely attached to the surface of the macrophage, with no invagination and its Middlesex HA1 3U1 ultrastructure was that of a small lymphocyte. UK. Peripolesls is probably a physiological mechanism concerned with regulation of the Immune response in the lung. Keywords: Alveolar; lymphocytes; macrophages; peripolesis. Eur Respir J., 1992, 5, 59-66. Received: December 14, 1990; accepted after revision August 19 1991

The technique of bronchoalveolar lavage (BAL), now can be observed in a proportion of lung washings. The widely used in the diagnosis of pulmonary disease circling cells were lymphocytes and the peripolesed cells [1, 2] has made it possible to recover immunologically alveolar macrophages. active cells in the washing fluid, and to observe them in We did not observe peripolesis of dendritic cells, the living state. Lung macrophages are developmentally although animal experiments have shown that it does separated from gut and other tissue macrophage occur, apparently in connection with presenta­ systems [3] and include several types of cells varying tion [11 ]. In the lung the role of alveolar macrophages in size, content, motility and membrane mark­ is mainly down-regulation of the immune response, but ers [4]. Some lavage fluids contain veiled or dendritic they may acquire the ability to present antigen in antigen-presenting cells (APCs), which resemble conditions such as sarcoidosis [12]. macrophages but are not phagocytic or adherent and are This is the first proof of peripolesis in lung washings. also highly mobile [5]. The two types of APC have In the literature there are 'still' photographs showing different functions. The macrophages ingest particulate lung macrophages with attached lymphocytes [4]. , or large molecules, and after lysosomal deg­ However, contact between these cells is not always radation the fragments are returned to the cell mem­ followed by peripolesis, which can only be established brane, whereas dendritic cells deal with small antigens by cinematography or direct viewing of the living cells and digestion, if required, probably takes place in the at body temperature. cell membrane. There are also variable numbers of large and smalllymphocytes, neutrophilleucocytes and Materials and Methods ciliated bronchial epithelia. All these cells were ob­ served microscopically and their interactions recorded Patients by time-lapse cinematography. We also studied the same cells by transmission and scanning electron We studied an unselected group of 20 patients microscopy (TEM and SEM). In this paper we report undergoing diagnostic bronchoscopy, who gave that the cell interactions known as peripolesis [6-10] permission for an additional washing by the usual 60 D.J. LYONS ET AL.

Table 1. - Perlpolesls In bronchial lavage fluids: clinical correlations In six positive cases Sex Age Sm. Diagnosis Peripolesis of ComEosition of BAL fluid macrophages %Lympho %Macro %Neutro %Eosino Remarks (cinematography) yrs 4h 18 h M 30 Retronasal + • 42 53 3 2 Bronchoscopy and chest bleeding X-ray normal F 40 + Chest + + 22 74 4 <1 Thalassaemia; infection infection with haemoptysis chest X-ray normal complete recovery F 56 + Pulmonary + ND ND ND ND Asymptomatic tuberculosis Mycobacteria isolated F 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 + + 55 43 2 <1 Died carcinoma of bronchus 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: leucocytes; Eosino: leucocytes.

Table 2. - Clinical details In 14 patients showing no perlpolesis Sex Age Sm. Diagnosis %lympho %Macro %Neutro %Eosino yrs M 61 + Squamous carcinoma of bronchus 10 80 9 1 M 63 + Oat cell carcinoma of bronchus 12 76 10 2 F 57 + Squamous carcinoma of bronchus 8 91 1 <1 F 65 + Squamous carcinoma of bronchus 23 69 8 <1 F 45 Pulmonary tuberculosis 7 66 23 4 M 53 + Pulmonary tuberculosis 16 77 5 2 M 77 + Squamous carcinoma of bronchus ND ND ND ND M 47 Oat cell carcinoma of bronchus M 66 + Pulmonary embolus M 70 + Metaplasia and haemoptysis M 29 Carcinoid F 74 Pulmonary embolus with pleural effusion M 31 + Haemoptysis, nil found M 73 Haemoptysis, nil found Mean age negative patients = 57 yrs. Sm.: smoking; ND: not done; Lympho: Lymphocytes; Macro: Macrophages; Neutro: Neutrophil leucocytes; Eosino: leucocytes technique [13]. There were 11 males and 9 females, ice. In cases of suspected lung tumour the extra lavage whose clinical details are given in tables 1 and 2; 60% was performed on the opposite side. After filtration the were smokers. cells were washed twice in PBS and resuspended at a concentration of 5x105·ml·1 in culture medium (RPMI Bronchoalveolar lavage, tissue culture and filming 1640) with 10% group AB serum or 10% fetal calf serum (FCS) with added penicillin and One hundred and twenty millilitres of prewarmed streptomycin. Fluids recovered from the lung are often phosphate buffered saline (PBS) was infused under contaminated with yeasts and fungi. We attempted to lignocaine anaesthesia and immediately recovered onto control this by adding mycostatic drugs including PERIPOLESIS OF ALVEOLAR MACROPHAGE$ 61

amphotericin B, flucytosine, miconazole, mycostatin be inserted into the stem unit of the electron micro­ with gentamicin and econazole at recommended con­ scope. A narrow copper strip was attached to the centrations. These drugs were toxic for macrophages undersurface of the coverslip in line with the reference and lymphocytes and none had the desired effect of points and the glass was cut along the edge of the strip, inhibiting the growth of contaminants. after which the cells were processed for SEM, i.e. criti­ At this time smears and cytospins were prepared from cal point drying and gold coating. the cell concentrates and stained with May-Grunwald­ Giemsa. Five hundred cells were counted in each preparation and the percentage of lymphocytes, Results alveolar macrophages, neutrophil leucocytes and eosinophil leucocytes calculated. Ciliated epithe­ Peripolesis lial cells and squamous cells were not included in the total counts. The cells were transferred to a Petri dish Peripolesis was first observed 3.5 h after setting up with a glass inset which was mounted on an inverted cultures of lung washings and was also seen at 18-24 h. It occurred in 6 of the 20 preparations phase contrast microscope, provided with a C02 gas supply and enclosed in a hot box at 37°C (fig. 1) [14 ]. examined. Peripolesis was usually seen on the first and This system allows observation and recording of cellu­ second days but in one case it was observed after 18 h, lar movement by time-lapse filming for up to 48 h. but not on the first day, possibly due to a prolonged The lignocaine administered to the patients inhibited effect of the anaesthetic. Lymphocytes moved randomly, the movement of the lavage cells and filming was but in positive cultures when one came within reach postponed until they had regained their motility, of a macrophage it turned sharply towards the macro­ usually 3-4 h. Filming was carried out at a speed of phage, rapidly attached itself and began the character­ one frame·3 s·1, i.e. speeded up 72 times, using 16 istic circling movement, with extrusion of a mm Eastman colour negative. If electron microscopy lamellipodium in the direction of motion. Many (EM) studies were envisaged, the culture medium was complete circumnavigations were seen (fig. 2) lasting removed at the end of the film and replaced by 3% 3-8 min. Occasionally the lymphocyte changed glutaraldehyde. 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 C0 circulation, provided with spaced-out holes (not shown). The microscope with its attachments is kept in a bot box at [12). 1 37•c

Electron Microscopy apparently by a shift of the lamellipodium to the opposite pole of the celL Sometimes the lymphocyte In order to identify peripolesing cells in fixed prepa­ abandoned the target cell and moved off; in other rations a map of the cells was made and reference points cases it contacted a neighbouring macrophage and marked on the end of the dish. continued to peripolese. Occasionally, two ­ For TEM the cells were embedded in a gel consisting ocytes attached themselves to the same cell, travelling of 10% bovine serum albumen (BSA) in PBS cross­ round in opposite directions, one cell passing over linked with 3% glutaraldehyde. Although alveolar the other. macrophages are strongly adherent they were easily In one instance, a macrophage developed elongated incorporated into the geL The position of the spiky processes, while being peripolesed. After the peripolesing group was marked on the surface, and the lymphocyte had moved away the membrane gradually gel was then cut up and processed in the usual way. recovered its normal appearance, a process which took Thin sections were examined in a Jeol (JEM 1200 EX) about half an hour (fig. 3). electron microscope. The proportion of macrophages subjected to For SEM the glass inset with attached macrophages peripolesis varied from 10-30% per field and over was cut down to approximately 6x2 mm so that it could 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 m in later, the lymphocyte moved away and the macrophage regained its normal appearance (x600). cultures, lymphocytes were seen to make short contacts produce a lamellipodium and finally moved off. At with macrophages. Such cells extended themselves no time did we observe emperipolesis with deep repeatedly away from the macrophage, but did not 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 Dendritic or veiled cells were seen in 3 of the included large, medium and small cells [1, 2]. Thirty 12 cultures recorded on film. These cells were easily percent had a distinct yellowish tinge. About 10% of identified in the living state. They were smaller the macrophages extended and retracted a fimbriated or than the macrophages, they moved at greater speed club-shaped process (fig. 4). This structure could have and continually extended long veils. Two of the been mistaken for an attached lymphocyte, but doe­ washings were from patients with carcinoma of the observations showed that it came out in several bursts, bronchus and contained 25 and 27% dendritic cells: of variable length and conformation, whereas attached no peripolesis was seen in these cultures. The lymphocytes extended and retracted themselves in a third washing from a patient with tuberculosis more repetitive manner. Sometimes a long thin process contained 10% dendritic cells. In this culture the made contact with another macrophage and was then macrophages were peripolesed, but the dendritic cells withdrawn. were not. 4(1) ... ,. \.; r 0 . ~ .; Q l.: •.,. ·o ;",(' ,. .Cl! c ~ 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 Figure SA shows a lymphocyte in the act widely. In stained preparations of the cell concentrates, of peripolesing a macrophage. The cells were filmed percentages ranged from 7-55%. In normal subjects and the clockwise direction of motion was correlated there are on average 10% using cytocentrifuge prepara­ with the EM appearance. The lamellipodium at the tions but up to 20% using an improved method (19]. advancing pole of the lymphocyte can be seen inter­ digitating with the ruffled surface of the macrophage. A Behind the lamellipodium the under-surface of the lym­ phocyte 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 gath­ ered 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 peri­ polesing cell. In this case the lymphocyte was moving anti-clockwise. The surface of the macrophage is cov­ ered with fairly large ruffles, but the lymphocyte appears smooth, except in the region of the lamellipodium.

Clinical correlations

In the group of six patients whose cells exhibited peripolesis there was one case of carcinoma of the bron­ chus, one of sarcoidosis, one of pulmonary tuberculosis and one of asthma; the remaining two patients, includ­ ing the only male, had slight bleeding and no lung dis­ ease was found (table 1). These six patients had a mean age of 45 yrs compared with 57 yrs for the nega­ B tive group, and 5 out of 6 were females. About half were smokers in each group. Peripolesis occurred in washings containing an increased proportion of lym­ phocytes. In stained preparations the percentage of lym­ phocytes ranged from 21.8-58.2 mean 35.6% (95% confidence interval 21.8-58.2). In six smears from neg­ ative 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 contamina­ tion. It is likely that the majority would have been negative at 18 h since, only one of the six cases show­ Fig. 5. - A) Transmission electronmicrograph (EM) of lymphocyte ing peripolesis became positive on the second day. peripolesing alveolar macrophage in clockwise direction. Washing was from female patient with non-recurrent haemoptysis. Lamellipodium at leading end and mitochondria behind nucleus. Discussion Area of contact between the cells is almost flat with small indenta· tions. Free surface of macrophage has processes, except in the area Previous investigations on cells in lung washings (1, immediately behind peripolesing lymphocyte. Bar=2 I'· B)- Scanning EM of peripolesis of alveolar macrophage. Washing 2] concentrated mainly on the morphology and secre­ was from male patient with non-recurrent haemoptysis. Lymphocyte tory activity of macrophages, the proportion of attached to lower part of macrophage was moving in anti-clockwise lymphocytes and the effects of smoking. More recently direction and lamellipodium can be seen on the right. Macrophage the phenotypes of the cells have been examined with covered with ruffles but lymphocyte has small ridges. Bar= 2~t. monoclonal (21-23]. PERIPOLESIS OF ALVEOLAR MACROPHAGES 65

We set out to film the behaviour of Jiving cells and contact with the attached immunoglobulin. In HGG this led to the observation of peripolesis, which had not cultures peripolesis of the veiled cells was sometimes previously been described in alveolar cells, although halted by the attachment of two more lymphocytes to still photographs of lymphocyte and macrophage ro­ the peripolesing cell, producing a configuration typical settes have been published [4, 24]. Peripolesis can be of by cells of this type, i.e. one studied without film or video equipment by looking at lymphocyte attached to the veiled cell and two more the cells at 3rc for at least one hour. If nothing hap­ attached to this cell [11]. Antigen-presenting macro­ pens on the first day the cultures can be viewed again phages form similar clusters [29, 30), but there are no the next day after incubation overnight. reports suggesting that peripolesis of the macrophages Other workers have observed peripolesis of precedes, or is associated with, Lhis type of activity. We epithe.lial cells [ 6, 25] and accessory cells (7-11]. never observed the attachment of additional lympho­ Cytotoxic injury to the peripolesed cells was noted in cytes to the peripolesing cells in our BAL preparations, gut preparations made from patients with ulcerative but it might occur in advanced cases of sarcoidosis. colitis [26), but in autoimmune thyroid no cell killing The main function of alveolar macrophages is was observed [6). In the present study of peripolcsed down-regulation of the immune response in the lung alveolar macrophages, a small proportion developed a [31]. Only one third of the macrophages were spiky appearance suggestive of injury, but later recov­ peripolesed, which probably reflects the heterogeneity ered. This appearance was also seen in dendritic cells of the population with respect to surface markers after an attack by a killer cell and was followed by [32-34] and the secretion of [31]. death of the target cell (unpublished observation). Mandatory investigations to clarify the role of Peripolesis occurred in washings with high lymphocyte peripolesis in BAL fluids are as follows: to enrich the counts (22-55%), whereas only one of the negative cases cultures with lung lymphocytes as well as blood had a borderline raised count of 23%. To see if blood lymphocytes in order to increase the incidence of lymphocytes were capable of this behaviour we chose a peripolesis. This would greatly facilitate the phenotyping washing fluid with a low lymphocyte count and nega­ of the peripolesed macrophages which may represent a tive peripolesis and added up to 50% autologous pe­ non-secretory subgroup. The phenotypes of the ripheral blood lymphocytes to the culture. No peripolesis lymphocytes and their state of activation should also be was seen on the first or second days. This preliminary determined. Of further interest would be to study experiment suggests Lhat lung lymphocytcs are a spe­ precisely defined and larger groups of patients with cial population. The phenotype of the peripolesing interstitial lung diseases in order to reveal possible lymphocytes has not yet been determined. The cell clinical correlations with disease state and activity. examined by TEM was a small lymphocyte without the ultrastructural characteristics of NK cells which have Acknowledgements: The authors thank G.L. been implicated in emperipolesis of malignant cells [16, Asherson for his support, J.L. Mackenzie for pre· 17]. Peripolesis was more common in women (p=O.OS paring still photographs from the films, N. Webb for the diagram of the apparatus and for the layouts Fishers exact test). In view of the close correlation and N. Saunders for typing the manuscript. 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 sugges­ References tion [1). Possibly in disease states the lung lymphocytes 1. Reynolds HY. Bronchoalveolar lavage. Am Rev increase more sharply in female patients. Obviously Respir Dis, 1987, 135, 250-263. our clinical sample is too small to detect any firm cor­ 2. 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