Blockade of leucocyte function-associated antigen-1 (LFA-1) decreases lymphocyte trapping in the normal pulmonary vasculature: studies in the isolated buffer-perfused rat lung

Authors


Thomas Tschernig, Center of Anatomy 4120, Medical School of Hannover, 30623 Hannover, Germany.E-mail: Tschernig.Thomas@MH-Hannover.de

Abstract

Adhesion molecules regulate the migration of lymphocytes in lymphoid and non-lymphoid organs. In the lung, little is known about lymphocyte sticking and migration through the pulmonary vascular endothelium in physiological or pathological situations. Therefore the isolated buffer-perfused rat lung was used to investigate the mobilization of lymphocytes out of the normal lung into the venous effluent and to the bronchoalveolar space. The lymphocyte subset composition was characterized in the venous effluent, the lung tissue and the bronchoalveolar lavage (BAL) using immunocytology. Lymphocytes continuously left the normal lung at a total of 5·0 ± 0·7 × 106 cells within the first hour of perfusion. The injection of 200 × 106 lymphocytes via the pulmonary trunk increased the venous release of lymphocytes by 170%. To investigate the effect of LFA-1 and CD44 on the adhesion of lymphocytes to the pulmonary endothelium, lymphocytes preincubated with an anti-LFA-1 MoAb, which blocks the interaction of LFA-1 and intercellular adhesion molecule-1 (ICAM-1), or lymphocytes preincubated with an anti-CD44 MoAb, were injected. The injection of LFA-1-blocked lymphocytes led to an increase by 70% of injected cells recovered in the perfusate within the first hour, whereas anti-CD44 treatment of injected lymphocytes had no effect. The LFA-1-blocked lymphocytes showed higher numbers of T and B cells in the effluent. Thus, the present experiments demonstrate that LFA-1 influences the trapping of lymphocytes in the vasculature of the healthy rat lung.

Introduction

Lymphocytes are important participants in cellular immune reactions and are located in different lung compartments such as the pulmonary vascular bed, the lung interstitium and the bronchoalveolar space. In each compartment the lymphocyte subsets are composed differently [1,2]. This implies regulatory mechanisms by which a special subset composition is generated. But how do lymphocytes continuously enter the different lung compartments?

For neutrophils the mechanisms leading to migration through the pulmonary and bronchial endothelium have been studied in detail in the normal lung and several models of lung inflammation [3]. For lymphocytes these interactions have mainly been studied at specialized sites of the endothelium, the high endothelial venules (HEV) in lymph nodes and Peyer's patches [4–6]. While such structures are absent in the lung parenchyma, it has to be clarified whether lymphocyte migration to the lung is mediated through comparable mechanisms. Adhesion molecules, e.g. intercellular adhesion molecule-1 (ICAM-1) and ICAM-2, are expressed on arteriolar, capillary and venous endothelium in the human lung [7,8]. In a model of isolated lung perfusion it has been demonstrated in rats that ICAM-1 was expressed along venous and capillary but not arteriolar endothelium [9]. We focused on the role of LFA-1 and CD44 on the cell surface of lymphocytes and their interactions with pulmonary endothelium of healthy rat lungs. LFA-1 is expressed on all lymphocyte subsets in the marginal pool of rat lungs and could mediate cell trapping by interactions with ICAM-1 [10]. ICAM-1 is constitutively expressed on the pulmonary blood vessel endothelium of healthy humans and healthy mice [8,11]. During a T cell-mediated immune reaction after instillation of sheep erythrocytes in the bronchoalveolar space, an increase in ICAM-1 expression on the endothelium combined with an increased expression of LFA-1 on infiltrated lymphocytes was described [11]. This indicates the varying importance of adhesion molecules during a pulmonary immune response. In vitro the blockade of interactions between LFA-1 and ICAM-1 resulted in a saltatory movement of lymphocytes along a monolayer of lung microvascular endothelium under physiological flow [12]. The diameter of neutrophils and lymphocytes is on average larger than that of the capillaries, so that rolling along the endothelium might be impossible. In vivo experiments by Hamann et al. [13] showed that anti-LFA-1 treatment of normal mice resulted in a reduced number of 51Cr-labelled lymphocytes in the whole lung at 30 min and 2 h after injection of cells. These findings indicate that LFA-1/ICAM-1 plays a role in mediating interactions between lymphocytes and the endothelium. The CD44 molecule is expressed in high amounts on mature lymphocytes and has been thought to participate in lymphocyte binding to HEV mainly in Peyer's patches. It has been proposed that CD44 is not necessary for normal leucocyte recirculation but is required for leucocyte extravasation into an inflammatory site involving non-lymphoid tissue (reviewed in [6,14]). The isolated lung perfusion model used here has been shown to preserve the structure and function of rabbit and rat lungs well [15–17]. The rat lungs were perfused in an open, non-recirculating way under normothermic conditions and lung weight and perfusion pressure were monitored. The mobilization of lymphocytes out of the lung tissue was continuously determined in the effluent.

The following questions were addressed: How is the mobilization of lymphocytes out of the pulmonary vascular bed characterized? How many and which lymphocytes are released? Is the interaction of lymphocytes and pulmonary endothelium dependent on CD44 or LFA-1/ICAM-1? Can lymphocytes migrate into the bronchoalveolar space during perfusion?

MATERIALS and METHODS

Animals

Congenic animals of the rat strains LEW.RT.7a (n = 17; male, body weight 320–380 g) and LEW.RT.7b (n = 14; male and female, body weight 250–380 g) were bred under specified pathogen-free conditions in the central animal laboratory (Medical School of Hannover, Hannover, Germany). Lungs of the LEW.RT.7a rats were used for ex vivo perfusion. Lymphocytes of the LEW.RT.7b strain were used as genetically labelled cells. These rats differ from congenic LEW.RT.7a only in the RT.7 allogeneic system, which is a polymorphism of the CD45 gene [18]. Cells of the LEW.RT.7b strain are detectable by a MoAb (His41). It has been shown in cell transfer experiments in vivo that the migration behaviour of lymphocytes of the two congenic strains is not different [19].

Lung isolation and perfusion

The isolation and perfusion of the lungs were performed as previously described [17]. In brief, LEW.RT.7a rats were anaesthetized with pentorbital-Na (100 mg/kg body weight i.p.). Following local anaesthesia with 2% xylocaine and median incision, the trachea was dissected and a tracheal cannula was inserted immediately. Lungs were ventilated with 4% CO2, 17% O2, 79% N2 (tidal volume 4 ml, frequency 65/min, end expiratory pressure 3 cm H2O). A median laparotomy was performed and subsequently the rats were anti-coagulated with 1000 U of heparin injected into the aorta caudal to the renal arteries. After midsternal thoracotomy the right ventricle was incised, a cannula was fixed in the pulmonary trunk, and the left ventricle was cannulated to obtain pulmonary venous outflow. Simultaneously, perfusion (13 ml/min) was commenced with cold buffer solution (4°C) containing 120 m m NaCl, 1·1 m m KH2PO4, 1·3 m m MgCl2, 4·3 m m KCl, 2·4 m m CaCl2, 25 m m NaHCO3, 13·3 m m glucose and 50 g/l hydroxyethylamylopectin ( Fig. 1). The pH of the perfusate (pH 7·35–7·4), the pulmonary arterial pressure (5–8 mmHg), the left ventricular pressure (1·5–2·0 mmHg), the ventilation pressure (10 cm H2O) and the organ weight were continuously monitored during the whole time of perfusion. Before the beginning of the experiments the system was equilibrated in a steady state period of 30 min, the temperature of the perfusate was raised to 36·5°C.

Figure 1.

(a) H–E-stained paraffin section of lung tissue 2 h after isolated perfusion of the organ, showing no pathological alterations. (b) Cryostat section of lung tissue stained for congenic lymphocytes (arrows). At 2 h after perfusion a high number of congenic lymphocytes is present in the lung. (c) Immunofluorescence staining of membrane-bound LFA-1 on cells in the venous effluent of the isolated lung 30 min after injection of LFA-1-blocked lymphocytes. The control staining omitting the secondary antibody is shown in the upper left corner.

Lymphocyte preparation

Lymphocytes were prepared from the mesenteric lymph nodes. Anaesthetized LEW.RT.7b rats were exsanguinated by puncturing the abdominal aorta caudal to the renal arteries, before the mesenteric lymph nodes were dissected. The tissue was completely disaggregated by passing through a 75-μm nylon mesh covered by a metal sieve. During the procedure the tissue was rinsed twice with 10 ml PBS. The cells were washed twice with PBS before they were used in the experiments.

Preincubation of lymphocytes with anti-LFA-1 and anti-CD44 MoAbs

The anti-LFA-1 MoAb WT.1 (IgG2a; diluted ascites fluid; Seikagaku Corp., Tokyo, Japan) is directed against the CD11a subunit of the LFA-1 heterodimer (CD11a/CD18). On rat lymphocytes MoAb WT.1 completely inhibits LFA-1-dependent cell aggregation in vitro and efficiently blocks lymphocyte binding to ICAM-1-coated wells [20]. Staining with WT.1 at a dilution of 1:500 results in a maximal fluorescence intensity in the lymphocyte population as tested by flow cytometry. Blocking of LFA-1 on the cell surface was performed using the WT.1 MoAb at a dilution of 1:200. For the incubation of lymphocytes with anti-CD44 MoAb, Ox49 (IgG2a; Biozol, Echingen, Germany) was used at a dilution of 1:10. After 30 min incubation at 4°C the cells were washed twice, resolved in 2 ml PBS and used in the experiments.

The binding of anti-LFA-1 or anti-CD44 MoAb on the cell surface of injected cells was checked by immunofluorescence staining. After preincubation the cells were incubated for 30 min with FITC-conjugated anti-IgG2a MoAb (Dianova, Hamburg, Germany) to determine membrane-bound WT.1 or Ox49. Using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) a minimum of 5000 cells was measured in an electronic gate set on the lymphocyte cluster. The events counted were analysed using CellQuest software (Becton Dickinson).

Injection of lymphocytes via the pulmonary artery and collection of cells in the venous effluent

After the steady state period 200 × 106 lymphocytes resolved in 2 ml PBS were injected into the afferent limb of the perfusion system within the first minute of perfusion. Simultaneously, the first sample (50 ml) of the venous effluent was collected. Every 10 min until the end of perfusion fractions of 50 ml were collected, centrifuged and resuspended. The cell numbers of each fraction were determined in a counting chamber. To perform cytospots 0·5–1·0 × 105 cells were transferred onto slides by centrifugation for 8 min at 400 g using a Cytospin3 cytocentrifuge (Shandon Scientific, Astmoor, Runcorn, UK). The slides were air-dried and stored at −20°C until immunocytological staining was performed.

Bronchoalveolar lavage and cryoconservation of the lung

At the end of the perfusion experiments the lungs were lavaged through the tracheal cannula by instillation of 5 ml cold (4°C) sodium chloride solution free of Mg/Ca2+ ions (0·9% NaCl; Braun Melsungen, Melsungen, Germany). The lavage fluid was retrieved by gentle aspiration and the lavage procedure was repeated four times. All lavage samples from the same lung were pooled and the volume recovered averaged > 90% of the instilled volume. The lavage fluid was centrifuged for 15 min at 400 g, the pellets were resuspended and the cell number was determined in a counting chamber.

After bronchoalveolar lavage (BAL), 5 ml of a cold (4°C) tissue preservation fluid (OCT/PBS; v/v 1:4; OCT-Tissue Tek; Miles, Elkhart, IN) were instilled into the lungs through the tracheal cannula before the lungs were frozen in liquid nitrogen. The specimens were stored at −70°C until cryosections were performed. Small pieces of the lungs were fixed in 4% formaldehyde for paraffin embedding. Sections of the right upper lobe of the lung were used for H–E staining and immunohistological staining. The sections were orientated from the lung hilum to the periphery.

Staining of injected cells, lymphocyte subsets, monocytes, natural killer cells, and granulocytes

Immunocytological characterization was performed by double staining procedure as previously described [21]. In brief, the injected congenic cells were identified by the MoAb His41 (Dianova) which recognizes leucocytes of the LEW.RT.7b strain only. Leucocyte subsets were characterized by MoAbs against T lymphocytes (R73), CD4+ cells (W3/25), CD8+ cells (Ox8), B lymphocytes (His14), natural killer (NK) cells (3.2.3), monocytes/macrophages (ED1) and granulocytes (RP1). The characterization of these MoAbs has previously been summarized [22,23]. All MoAbs were purchased from Camon (Wiesbaden, Germany). A double-staining technique combining alkaline phosphatase and peroxidase reactions was employed to identify the injected lymphocytes and the different subsets. Cytospots and sections were fixed in a mixture of methanol and acetone for 10 min at −20°C, incubated with the primary antibody for 30 min at room temperature, washed with TBS–Tween (0·05% Tween 20; Serva, Heidelberg, Germany), followed by 30 min incubation with a polyclonal rabbit anti-mouse immunoglobulin (Dako, Hamburg, Germany) functioning as a bridging antibody. Then the alkaline phosphatase–anti-alkaline phosphatase (APAAP) complex (Dako) was applied for 30 min. In order to enhance staining intensity, the incubation with the bridging antibody and the APAAP complex was repeated once more. Then the biotinylated His41 MoAb (Dianova) was added for 30 min, followed by 15 min incubation with streptavidin-peroxidase (Dako). Diaminobenzidine (Sigma, Deisenhofen, Germany) served as chromogen and fast blue (Sigma) as the substrate for alkaline phosphatase.

Fluorescence staining

To determine membrane-bound anti-LFA-1 MoAb on the cells in the venous effluent the cytospots were fixed in methanol and acetone for 10 min at −20°C and thereafter incubated with a Cy3-conjugated goat anti-mouse immunoglobulin (Dianova) for 30 min at room temperature. Confocal microscopy was performed using an inverted Zeiss Axiovert 35M microscope (Zeiss, Oberkochen, Germany) equipped with a BioRad MRC600 laser scanning subunit, an argon–krypton mixed gas laser and Comos software (version 6.0; BioRad, München, Germany). Data sets were processed using Lasersharp (version 1.02; BioRad) and Photoshop software (version 3.05; Adobe Systems, Edinburgh, UK).

Statistical analysis

To determine the percentage of congenic cells, at least 500 cells per animal were counted. The subset composition of the perfusate fractions was examined by analysing at least 200 cells per animal for each subset. The statistical package SPSS-Windows (version 6.0.1; SPSS Inc., Chicago, IL) was used to calculate means, s.e.m. and the level of significance. The Mann–Whitney U-test was used to compare the results of the groups. P < 0·05 was taken as statistically significant.

Results

Isolated perfused lung

Physiological parameters, e.g. pH of perfusate, organ weight, pulmonary arterial pressure, left ventricular pressure and ventilation conditions, were monitored throughout the experimental procedure in the isolated rat lung model. All these parameters remained within the normal range. H–E staining of a lung after 2 h of normothermic perfusion ( Fig. 1a) showed no pathological alterations—in particular no interstitial fluid—in the lung parenchyma.

Lymphocytes leave the normal isolated buffer-perfused lung

During normothermic perfusion of normal lungs, lymphocytes left the organ via the pulmonary veins. A total number of 5·0 ± 0·7 × 106 nucleated cells was recovered in the perfusate within the first hour. Lymphocytes were released continuously during the perfusion at a basal level ( Fig. 2). The subset composition of leucocytes in the perfusate 10 min after the end of the calibration time was determined: 35·6 ± 2·3% of the cells were T cells and 22·4 ± 2·0% B cells. The percentage of NK cells was 9·9 ± 0·9% and that of monocytes 9·7 ± 1·8%, while only 6·8 ± 1·8% were granulocytes.

Figure 2.

Absolute numbers of cells detected in the venous effluent of the isolated lung over time without injection (▪; n = 3), after injection of 200 × 106 lymphocytes (○; n = 5), or after injection of 200 × 106 LFA-1-blocked lymphocytes (▴; n = 5). The results of the three experimental groups are significantly different within the first 75 min of perfusion (P < 0·05; Mann–Whitney U-test). Data represent mean ± s.e.m.

Injection of lymphocytes increases the efflux of cells via the pulmonary veins

The congenic lymphocytes from mesenteric lymph nodes of LEW.RT.7b rats consisted of 60·3 ± 2·6% T cells and 40·5 ± 0·4% B cells. Only 0·2 ± 0·04% of the cells were NK cells, while no granulocytes or monocytes were found in the inoculum.

After injection of these lymphocytes via the pulmonary trunk, the number of cells leaving the lung with the perfusate during the first 75 min was significantly higher compared with the control experiments ( Fig. 2). During the first 60 min of perfusion an absolute number of 13·5 ± 1·2 × 106 cells containing 10·4 ± 1·2 × 106 congenic cells was recovered in the perfusate. Compared with the control experiments this was an increase of about 170%. Staining congenic cells on cryostat sections of the perfused lung showed a high number of lymphocytes localized in the lung tissue 2 h after injection ( Fig. 1b). However, on cryostat sections it is impossible to decide whether a lymphocyte is within a capillary lumen or in the interstitium.

Blocking LFA-1 increases the number of released lymphocytes, while anti-CD44 treatment has no effect

The binding of the antibodies to the injected cells was verified by flow cytometry, showing that 87·1% of the injected lymphocytes were LFA-1+ and that 82·6% had the anti-CD44 MoAb on the cell surface. After injection of LFA-1 blocked lymphocytes in the lung the number of cells in the perfusate increased within the first 75 min compared with the situation after injection of untreated lymphocytes ( Fig. 2). Using immunofluorescence staining, the binding of anti-LFA-1 MoAb was detected on cells in the perfusate 30 min after injecting LFA-1-blocked cells via the pulmonary trunk ( Fig. 1c), showing that the antibody was still present on the cell surface.

During the first hour after injecting LFA-1-blocked lymphocytes, an absolute number of 23·2 ± 3·4 × 106 cells containing 17·0 ± 2·8 × 106 congenic cells left the lung, representing 8·5 ± 1·4% of the injected cells. The recovery of injected cells was significantly lower after injection of untreated lymphocytes (5·2 ± 0·6%) or anti-CD44-treated lymphocytes (4·0 ± 0·6%) within the same period ( Fig. 3). As no differences were detected comparing the experiments with anti-CD44-treated or untreated lymphocytes, no further phenotyping of lymphocyte subsets was performed after injecting anti-CD44-treated lymphocytes.

Figure 3.

Percentage of injected cells recovered in the venous effluent within the first hour of perfusion after injection of 200 × 106 lymphocytes (□, n = 5), 200 × 106 anti-CD44-treated lymphocytes (hatched bar, n = 4) or 200 × 106 LFA-1-blocked lymphocytes (▪, n = 5). Data represent mean ± s.e.m. *Statistically significant result (P < 0·05, Mann–Whitney U-test).

Immediately after injection of lymphocytes about 80% of the cells in the perfusate showed a congenic phenotype. This percentage decreased slightly during the first hour of perfusion to about 75%, if congenic lymphocytes were injected, and to 64% after injection of LFA-1-blocked lymphocytes. There was a significantly diminished percentage of congenic cells in the perfusate 40–60 min after injection of LFA-1-blocked lymphocytes.

Leucocyte subsets of cells released from the lung

The percentage of T cells in the perfusate was comparable in experiments with and without blocking LFA-1 during the whole time of perfusion. Comparing the beginning and the end of the experiments, the percentage of T cells was significantly decreased after 60 min. Compared with the spontaneous release of lymphocytes (without injection of congenic cells) the percentages of all T cells recovered in the perfusate after injecting congenic lymphocytes were increased independent of the LFA-1 blockade ( Fig. 4a). The percentages of all B cells were increased only after supply of untreated cells, while after blocking LFA-1 and during the spontaneous release they did not differ ( Fig. 4b). Regarding NK cells, the percentages released after injection of lymphocytes were much lower than during experiments without adding cells to the perfusate ( Fig. 4c). The release of granulocytes was similar comparing the situation without injection of cells and after injecting untreated lymphocytes. In contrast, after injection of LFA-1-blocked lymphocytes the percentage of granulocytes was diminished within the first hour of perfusion ( Fig. 4d).

Figure 4.

Percentage of (a) T cells, (b) B cells, (c) natural killer (NK) cells, and (d) granulocytes in the perfusate without injection of cells (▪; n = 2–3) and after injecting 200 × 106 cells with (▴; n = 5) or without (○; n = 5) blockade of LFA-1. Data represent mean ± s.e.m.

Influence of LFA-1 blockade on percentage and absolute number of injected B and T cells

During the first hour of perfusion the percentage of the injected T cells was higher after LFA-1 blockade ( Fig. 5a). The opposite was true for the injected B lymphocytes detected in the venous effluent (data not shown). Comparing the absolute number of the two subsets, the number of B cells was two-to-three-fold increased, while a three-to-four-fold increase in the absolute number of injected T cells was detected after blocking LFA-1 ( Fig. 5b). Since few NK cells and no granulocytes were present in the inoculum, it was not possible to differentiate the release of the injected cells of these subsets in the perfusate.

Figure 5.

Percentage of (a) congenic T cells recovered in the perfusate after injecting 200 × 106 cells with (▴; n = 5) or without (○; n = 5) blockade of LFA-1. Absolute number of (b) congenic T cells in the perfusate. Data represent mean ± s.e.m. *Statistically significant result (P < 0·05, Mann–Whitney U-test).

Detection of injected cells in the bronchoalveolar lavage

Only extremely rarely were cells of the inoculum detected in the BAL at the end of the perfusion. In both experiments after supply of cells with or without blocking LFA-1, only 0·1 ± 0·02% cells in the BAL were of the injected origin. Due to the low number of injected cells recovered in the BAL no further phenotyping was performed.

Discussion

The experimental set up of the isolated buffer-perfused rat lung provides a unique system in which the initial interactions of lymphocytes with the pulmonary endothelium can be studied in the intact working organ. As the cells are not recirculating as they would in vivo, they pass the endothelium of a single organ once, avoiding any influence of other organ-specific endothelial structures. The model is well established and often used for studies investigating the release of mediators regulating homeostasis in lung perfusion and ventilation [24–26]. H–E-stained tissue showed no alterations such as oedema or necrosis. In previous ultrastructural studies the structure of the rabbit lung was shown to be intact after isolated perfusion in the same system [16]. The spontaneous release of leucocytes from the lung free of blood is not surprising, since this has been shown in pigs [27] and rabbits [16] and termed marginal pool of leucocytes in the lung vascular bed. Other investigations have shown the existence of a marginal vascular pool of lymphocytes in the lungs of rats [1]. As the lung has to be perfused initially with cold buffer solution and thereafter warmed up and equilibrated in a steady-state period, no exact quantification of the marginal pool was performed in this study. In peripheral lymph nodes it has been demonstrated by in vivo videomicroscopy that repetitive sticking and release of many cells causes the circulating and the marginating pool of lymphocytes to mix [28,29]. For that reason no clear cut discrimination can be made between lymphocytes of the marginal vascular pool and the peripheral blood pool. The injection of 200 × 106 leucocytes into the afferent vessel is equivalent to twice the blood pool of an adult rat [30]. This almost led to a doubling of the cell numbers released from the lung. The circumstance that overall only a small percentage of the added cells was recovered in the venous effluent is on the one hand in accordance with in vivo findings, where temporary sticking in the lung after injection is well known [31,32]. On the other hand, this effect might be enhanced by using cell preparations from lymph nodes, thereby injecting cells which are not migrating in the in vivo situation they have been taken from. Furthermore, it has to be taken into account that the lung was perfused during the steady-state period in which cells are continuously being detached and leaving the lung with the perfusate, so that the injected cells might have a better chance to attach to the endothelium. The use of heparin during the preparation of the lung is negligible, since it was washed out or extremely diluted. Heparin can influence the endothelial ICAM-1 expression and inhibit the ligand binding to the integrin Mac-1 [33,34], but any effect would be the same for each group.

Many details are known about the adhesion of neutrophils to the pulmonary and bronchial endothelium of the normal lung (reviewed in [3]). It has been demonstrated that neutrophil emigration shows organ-specific differences, since distinct adhesion molecules are required for adhesion to different organs [35], and even for dendritic cell precursors it has recently been described that their number is enriched in the lung vasculature [36]. Furthermore, a number of investigators have studied the adhesion and migration properties of neutrophilic and eosinophilic granulocytes in the lung during various pathological alterations such as asthma [37,38], endotoxin-induced pneumonia [39] or endotoxin treatment [40]. However, hardly any attention has been paid to lymphocyte behaviour. In contrast to the enormous number of studies on the role of adhesion molecules in the interaction of lymphocytes and the endothelial wall in lymphoid organs (reviewed in [6,14]), only few investigations have focused on leucocyte–endothelial interactions in the lung [9,12,13]. Using the ex vivo system of the isolated buffer-perfused rat lung we studied the interactions in the working organ, avoiding any distribution phenomena of the leucocytes in the entire body and a continuous mixing of lymphocytes, which are arrested in the pulmonary vascular bed. In accordance with the literature which proposed that CD44 plays no role in normal trafficking of lymphocytes into the non-inflamed lung, the injection of anti-CD44-treated lymphocytes in the isolated perfused lung showed no differences from the situation after injecting untreated lymphocytes. These results underline that CD44 has no effect on lymphocyte adhesion to the pulmonary endothelium of the healthy rat lung.

The overall effect of blocking LFA-1 on the injected cells was an increase of the released cells as a result of decreased adhesion of the leucocytes to the lung vascular endothelium. As the LFA-1 treatment increased the number of released cells within the first hour after single application of lymphocytes, this effect might be magnified in the in vivo situation over time and due to the fact that lymphocytes might pass the pulmonary endothelium several times. Li et al. [12] found in an in vitro system, focusing on firm adhesion and rolling of lymphocytes along microvascular endothelium, that the blockade of LFA-1 on lymphocytes led to decreased adhesion and to increased rolling of these cells along an endothelial monolayer. It has been shown in vivo that simultaneous injection of lymphocytes and anti-LFA-1 MoAb in normal mice reduced the recovery of radiolabelled lymphocytes to a small but significant extent in the whole lung within the first 30 min after injection [13]. However, using in vivo videomicroscopy in a model of isolated lung perfusion, different mechanisms of cell adhesion have been shown in arterioles, venules and capillaries [9]. Interestingly, blocking ICAM-1 had no effect in that system, whereas LFA-1 was not examined. The advantage of the present study was that it was possible to focus on the dynamics of the cellular release, the localization of the cells and their subset composition, reflecting only the interactions between lymphocytes and the pulmonary vascular bed. After blocking the LFA-1/ICAM-1 interaction on injected lymphocytes the number of T cells, and especially of T cells of the injected origin, is increased in the perfusate. As to the percentages of injected B cells detected in the perfusate, the percentage was lower after injecting LFA-1-blocked cells compared with the unblocked situation. This decrease in percentage is a result of the increased percentage of injected T cells recovered in the perfusate. Regarding the absolute number of each subset, both the B and T cells were increased after blocking LFA-1. While the increase is more dominant in the T cell compartment, the adhesion of T cells might be mediated by a higher percentage of LFA-1 in contrast to the adhesion of B cells. Preliminary experiments have shown that a significantly higher percentage of T cells of the mesenteric lymph node are LFA-1+ than B cells of the same origin (U. Bode, unpublished data). Thus, the adhesion of lymphocytes to the vascular endothelium might be regulated differently for each distinct lymphocyte subset.

The cellular composition of the BAL is an important factor in clinical diagnostics, although the migration time of lymphocytes from the blood to the bronchoalveolar space is still unclear. Our results show that even without any treatment of the injected lymphocytes, a very low number of injected cells were able to reach the BAL within 2 h of perfusion.

The increase in number of released leucocytes after blocking the LFA-1/ICAM-1 interaction demonstrates a major involvement of this adhesion mechanism in the lung vasculature. The very small number of injected cells in the BAL indicates a perfusion time longer than 2 h for transmigration of lymphocytes from the lung vessels into the bronchoalveolar space. Adhesion and transmigration of lymphocytes in different compartments of the lung involve further unknown mechanisms.

Acknowledgments

The authors thank Annette Weiss and Karin Westermann for excellent technical assistance. Help with the English text of Sheila Fryk and in preparing the figures by Marita Peter is gratefully acknowledged. The study was supported by the Deutsche Forschungsgemeinschaft (Pa 240/7-3).

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