Adhesion properties of human alveolar macrophages with respect to extracellular matrix components and chemotactic agonists

ELSEVIER

Immunology Letters 53 (1996) 121-124

Adhesion properties of human alveolar macrophages with respect to extracellular matrix components and chemotactic agonists J . Lundahl”** , A. Eklundb,

G. Halldkn”,

C.M. Sk61db

‘Department of Clinical Immunology and Transfusion Medicine. Karolinska Hospital. S-l 7176 Stockholm, Sweden bDepartment of Medicine, Division of Respiratory Medicine. Karolinska Hospital, S- 17176 Stockholm. Sweden

Received 1 July 1996; accepted 26 August 1996

Abstract Adherence has an essential impact on the differentiation and activation of tissue dwelling monocytes/macrophages. We have considered the effect of selected chemotactic agonists (fMLP, RANTES, IL-8) on the adhesion properties of human alveolar macrophages prepared by bronchoalveolar lavage. The macrophages were co-incubated in buffer alone or buffer supplemented with respective agonist, for different time points, in culture wells precoated with albumin, vi&one&in and fibronectin, respectively. The macrophages displayed a gradual increase in adhesion to all three surfaces and discriminated between the different matrix components, but did not respond to the selected agonists with increased adhesion. Kqvwords:

Adhesion; Alveolar macrophages;

Chemokines;

Matrix

1. Introduction Peripheral blood monocytes migrate in a random manner into various tissues where they undergo final differentiation into tissue macrophages, assuming tissue-specific properties. The outcome of this differentiation depends on the interplay with different factors in the microenvironment. The tissue resident population is constantly renewed by a steady influx of monocytes [1,2]. In the lung, alveolar macrophages constitute the main resident phagocytic cell population and, together with recruited neutrophils, exert an effective killing and clearance of invading microorganisms [3]. Once transmigrated through the endothelium, monocytes adhere to extracellular matrix, which comprises a network of various macromolecules with cell-adhesive properties [4]. This adherence has far-reaching effects on monocyte differentiation and function and it has been proposed that adherence has an essential impact

* Corresponding author. Tel.: + 46 8 7295930; fax: + 46 8 304887.

on the differentiation and activation of tissue dwelling monocytes/macrophages [5-71. The extracellular matrix associated form of vitronectin and fibronectin act as adhesive glycoproteins promoting adhesion and signal transduction of monocytes [8,9]. In addition, they also operate as non-immune opsonins enhancing TNF-lx secretion from macrophages [lo]. Both vitronectin and fibronectin are locally produced in the lung tissue by macrophages [l 1,121. The inflammatory response is a dynamic and complex process that comprises the generation of both proand anti-inflammatory chemotactic agonists. The chemokines form a group of chemotactic agonists that have been recognized as important mediators of inflammation and they have been divided into two families, the c(- and the p-family, respectively. Interleukin-8 (IL-8) is a member of the z-family and seems to have predominantly effect on neutrophils [ 131, whereas RANTES is a member of the p-family and has been recognized as a potent chemotactic agonist for monocytes and T lymphocytes [14]. In a murine model RANTES contributes to the accumulation of monocytes in

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J. Lundahl et al. I Immunology

the lung after administration of endotoxin [15]. Since RANTES is present in normal lung tissue, it may play a role in the steady state homing of monocytes, maintaining the population of resident macrophages [15]. The cellular source of RANTES seems to be a variety of resident cells in the lung tissue, including alveolar macrophages, which indicate that the recruitment of monocytes may be self-perpetuating [ 161. We have previously reported that peripheral monocyte adhesion to matrix proteins is selectively enhanced in the presence of chemotactic agonists [17]. In the present paper, we have addressed this question to alveolar macrophages. We sought to explore the effects of chemotactic agonists on the adhesion properties with respect to different extracellular matrix components: vitronectin, fibronectin and albumin, respectively. The chemotactic agonists were selected to represent a classical chemotactic agonist (fMLP) and agonists with preferential effect on monocytes (RANTES) and neutrophils (IL-8) respectively.

2. Material and methods All media used in this study contained no signs of endotoxins ( < 0.125 EU/ml) assayed using the gel-clot method. The study had the approval of the local Ethics Committee and informed consent was obtained.

Letters

53 (1996) 121-124

2.2. Adhesion assay The adhesion assay was performed as previously described [18]. Briefly, culture wells, with a growth area of 3.83 cm2 (art. no 25815-12, Corning Lab. Science, New York), were precoated with human albumin (10 mg/ml, KABI Vitrum, Stockholm, Sweden) diluted in RPM1 1640 medium for 30 min under culture conditions (at + 37°C 5% CO,-95% air atmosphere). The wells were rinsed with PBS-EDTA and air-dried. The alveolar macrophages were thereafter added (500 ~1, total cell count approximately 0.5 x 106) and incubated under culture conditions (at 37°C 5% CO,-95% air atmosphere) for different time points stated below. The non-adherent cells were recovered by gently rinsing with 500 ~1 RPM1 1640 medium at + 37°C. Finally, the percentage of adherent cells was calculated from the initial number of added cells and the number of nonadherent cells recovered. The number of cells was analysed by flow cytometry (see below). The viability, determined by trypan blue exclusion, did not differ between the adherent and nonadherent populations, excluding the possibility that adherence is mainly related to viability. The cell recovery rate (number of adhered cells + number of nonadhered cells/ number of initial cells added) was greater than 95%. The intra-assay variation for the adhesion experiments was less than 10%.

2.1. Preparation of alveolar macrophages

2.3. In vitro activation of alveolar macrophages

Alveolar macrophages were recruited from healthy smokers (n = 16) (age ranging from 21 to 43 years, median 28 years). Their mean cigarette consumption was 11.4 + 4.9 (mean f S.D.) pack-years and the present consumption exceeded 10 cigarettes/day for the last 5 years. All subjects had a normal chest X-ray and were free of medication. A routine physical examination was unremarkable. Bronchoalveolar lavage (BAL) was performed by wedging the fiberbronchoscope (Olympus BF, type P20, Olympus, Tokyo), in a middle lobe bronchus. Five aliquots of phosphate buffered saline, 50 ml each, were then instilled, gently aspirated and collected in a siliconized bottle kept on ice, which immediately was transported to the laboratory. The BAL fluid was strained through a layer of Dacron nets (Millipore, Cork, Ireland) and the cells were pelleted by centrifugation at 400 x g at 4°C for 10 min. Smears for differential counts were prepared by cytocentrifugation at 500 rpm for 3 min (Cytospin 2 Shandon, Southern Products, Runcorn, U.K.). Smears were stained with May-Griinwald Giemsa and 500 cells were counted. Alveolar cells were finally suspended in RPM1 1640 (Northumbria Biologicals, Crankington, UK) medium to a cell concentration of 1 x 106/ml.

To obtain in vitro activation of the alveolar macrophages in the adhesion assay, we used three different inflammatory mediators. These mediators were N-formyl-methionyl-leucyl-phenyl alanin (fMLP) (final concentration lo-’ M) (Sigma), IL-8 (final concentration 5 x lop8 M) (Pepro Tech, Rocky Hill, NJ) and RANTES (final concentration 1 pg/ml)(Pepro Tech NJ). The alveolar macrophages were co-incubated with respective agonist at 37°C for 15 min.

2.4. Flow cytometry The cells were finally resuspended in 0.5 ml PBSEDTA and analysed in Epics XL flow cytometer (Coulter, Hialeah, FL). Alveolar macrophages were represented by a well separated cluster in a twoparameter scatter plot histogram with logarithmic amplification and a discrimination gate was placed around the alveolar macrophage cluster, as previously described [ 181. The absolute number of alveolar macrophages in a sample was calculated from analysing 20 ,ul. The instrument was daily calibrated with Immunocheck (Coulter, FL).

J. Lundahl et al. I Invnunology Letters 52 (1996) l-71-124

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2.5. Statistics

Results are expressed as median (range). Data were analysed using a non-parametric method (Mann-Whitney U-test) and differences were considered statistically significant at P less than 0.05.

100

80

m

Vltmnectin

0

Albumin

m

Fibronectin

*

I

3. Results 3.1. General lavage data

The viability of BAL cells, estimated by trypan blue exclusion, was greater than 95%. The differential cell count, described as per cent and range, was as follows: alveolar macrophages 89-99%; lymphocytes 1- 10%; neutrophils l-3%; eosinophils O-2%; basophils O0.2%. 3.2. Time course of alveolar macrophage adhesion to albumin, vitronectin andJibronectin We analysed the adhesion of resting alveolar macrophages to albumin, vitronectin and fibronectin precoated surfaces at three time points (5, 15 and 60 min) and found a gradual increase in adhesion to all three surfaces. The alveolar macrophage adhesion increased to all surfaces at 15 min compared with 5 min (P < 0.05 for vitronectin and albumin, P < 0.01 for fibronectin). At 60 min incubation there was a further increase in the adhesion (P < 0.05 for vitronectin and albumin, P < 0.01 for fibronectin vs. 15 min). Adhesion to fibronectin was significantly higher at all time points compared with the adhesion to albumin (P < 0.01, 0.01 and 0.05, respectively) and at 5 and 60 min compared to the adhesion to vitronectin (P < 0.01 and 0.05, respectively) (Fig. 1). 3.3. The ejjkcts of chemotactic agonists on alveolar macrophage adhesion to albumin, vitronectin and fibronectin To further explore the adhesion properties of alveolar macrophages to different extracellular matrix components, the adhesion to albumin, vitronectin and fibronectin was tested in the presence of fMLP, IL-8 and RANTES. The alveolar macrophages were incubated for 15 min which was based on previous data (Fig. l), that indicated that the alveolar macrophages had an ability to further increase their adhesion at this time point. However, we did not find any significant increase in the adhesion properties when alveolar macrophages were incubated in buffer supplemented with either NLP, IL-8 or RANTES than in buffer alone (Table 1).

5 min

60 mln

15min

Incubation time Fig. 1. The time course of alveolar macrophage as ‘%, adherent cells) to vitronectin, albumin culture wells. The experiment was performed 2. Boxes enclose interquartiles with medians Results are based on seven experiments, *, Statistics are only presented within each time

adherence (expressed and fibronectin coated as described in Section and ranges marked. P < 0.05; **. P < 0.01. point,

4. Discussion

In this paper we demonstrate that alveolar macrophages discriminate between different matrix components in the context of adhesion properties. However, alveolar macrophages do not respond to selected chemotactic agonists used in this study, with increased adhesion. The inflammatory cascade is characterized by a coordinated expression of adhesion molecules and production of chemotactic agonists, e.g. chemokines. Combinatorial use of these chemokines plays an essential role in the regulation of the leukocyte recruitment thereby determining the cellular profile as well as the differentiation and activation of inflammatory leukocytes at the inflammatory spot [19]. Since adhesion to extracellular matrix components is suggested to be an important event in the differentiation and activation of monocytes into macrophages [6], we wanted to evaluate Table 1 The effect of chemotactic agonists on alveolar macrophage adhesion (expressed as ‘%I adherent cells) to albumin. vitronectin and fibronectin

Albumin Fibronectin Vitronectin

Results marked.

are based

Buffer

fMLP

IL-8

RANTES

58 (55564) 72 (67-78) 67 (63-73)

60 (59964) 73 (70 -76) 67 (67-71)

61 (65 73) 75 (73379) 71 (69 74)

65 (57 67) 72 (69- 74) 68 (64 71)

on nine

experiments,

with

medians

and

ranges

124

J. Lundahl et al. / Immunology Letters 53 (1996) 121-124

the adhesion properties of alveolar macrophages with respect to their ability to discriminate between different extracellular matrix components as well as different chemotactic agonists. We have recently reported that the adhesion of peripheral monocytes is ranked in relation to extracellular matrix components and that the adhesion is enhanced preferentially in the presence of RANTES and fMLP [17]. Here we have extended these experiments to alveolar macrophages. In accordance to the findings in monocytes, we found a gradual increase in the adhesion to vitronectin, fibronectin and albumin, with adhesion to fibronectin being consistently higher than adhesion to vitronectin and albumin. This indicates that during in vivo maturation of monocytes into alveolar macrophages, the ability to discriminate between different substrates is retained. However, our data suggest that alveolar macrophages, in contrast to monocytes, have lost the ability to respond to RANTES and fMLP in the context of increased adhesion. These findings are consistent with the findings by Audran et al., who found a reduced chemotactic response of monocyte-derived macrophages towards fMLP despite a sustained response with respect to up-regulation of the CDllc/CD18 antigen [20]. In addition, we have previously reported that alveolar macrophages have a reduced responsiveness towards fMLP in terms of adhesion and up-regulation of the adhesion molecule CD1 lb/CD18 [21]. In this paper we used alveolar macrophages from smokers, as in-house experiments have shown no differences in in vitro adhesion properties between alveolar macrophages from smokers and non-smokers. This is in agreement with a previous study by McGowan et al. [22]. The presence of an additional environmental stimulus can prime the cells for augmented activity [23]. An awareness of this is important since contaminating endotoxins introduced in vitro can influence the results. Our experiments were carried out under accurate defined endotoxin-free conditions. It has been suggested that alveolar macrophages have reached a more final stage of maturation due to a history of phagocytosis in the alveolar space. Our data imply that alveolar macrophages, compared to monocytes, have a reduced repertoire of biological responsiveness. In addition, our results indicate that the biological outcome of the presence of selected chemotactic agonists, in the context of monocyte/macrophage adhesion, depends on the stage of maturation.

Acknowledgements

We thank Marie Hallgren for excellent technical assistance. This study was supported by the Swedish Heart Lung Foundation, the Swedish Council for Work Life, the Swedish Medical Research Council (grant 16x-105), the National Association for the Prevention of Asthma and Allergy, the Swedish Society of Medicine and the Karolinska Institute.

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