Immunology 1990 69 476-481

Selective attrition of non-recirculating T cells during normal passage through the lung vascular bed D. NELSON, D. STRICKLAND & P. G. HOLT Clinical Immunology Research Unit, Princess Margaret Hospital, Subiaco, Western Australia Acceptedfor publication 18 October 1989

SUMMARY Transient arrest of T lymphocytes in the lung vascular bed following infusion of cells subjected to in vitro manipulations has been recognized for many years as a troublesome 'artefact', and has generally been attributed to trauma-induced changes in lymphocyte surface membranes. However, a number of laboratories have reported that the trapping process also occurs under situations where lymphocyte surface damage is minimal or absent, suggesting that the phenomenon may represent an intrinsic component of normal lymphocyte circulation. Consistent with these suggestions, recent studies from our laboratory have demonstrated the presence of large numbers of T cells in collagenase digests of normal peripheral lung tissue, which cannot be removed by broncho-alveolar lavage or perfusion of the tissue vascular bed. In the present experiments we have characterized these lung T cells in SPF rats. The properties common to this population include hydroxyurea sensitivity, high CD8: CD4 ratio and high frequency of cells recently derived from the thymus, and saturation thymidine-labelling studies indicated that greater than 90% of the lung T cells had divided within a 14-day test period. Additionally, cloning studies under conditions of limiting dilution indicate markedly reduced capacity for proliferation, relative to T cells in blood or spleen. We interpret these data to indicate that selective trapping and subsequent down-regulation of non-recirculating T cells is a normal consequence of passage through the lung vascular bed.

The least studied of these compartments is the latter. However, the recent application of improved methods for removal of T cells from solid lung tissue (Holt et al., 1985, 1986), coupled with the use of precise techniques for determining the kinetics of lymphocyte transit through the lung vascular bed (Pabst et al., 1987), have demonstrated the presence of a much larger T-cell population in the lung wall than has hitherto been recognized. In a variety of animal species (Holt et al., 1985; Venaille et al., 1989; Pabst et al., 1987) and in man (Holt et al., 1986), the total number of T cells present in the lung wall at a static time-point exceeds the peripheral blood pool, and accordingly this population warrants detailed study. The origin(s) and fate of these lung wall T cells remain to be formally established. However, there are strong indications from the lymphocyte recirculation literature that they may be derived via temporary sequestration ofcells during their passage through the lung vascular bed, leading to transient margination and release back into the blood a few hours later (Hall, 1985; Pabst et al., 1987; Scheuermann, 1982). It is not known whether this process is random, or whether particular subsets of T cells are preferentially retained during circulation through the lung. We have addressed this latter question in the rat, and in addition have sought to define the

INTRODUCTION The epithelial surfaces of the upper and lower respiratory tract represent a large and fragile interface between the immune system and the environment, and the maintenance of a healthy steady-state requires precise control oflocal immuno-inflammatory processes, particularly those involving T lymphocytes (Holt & McMenamin, 1989). The study of T-lymphocyte regulation in different tissue microenvironments within the respiratory system is attracting increasing interest, as more precise techniques for the identification and characterization of these cells become available. The respiratory tract immune system comprises three major compartments: (i) a mucosal component including the BALT (bronchus-associated lymphoid tissue) associated with airway walls, which forms part of the common mucous-associated lymphoid tissue (MALT) system (Bienenstock & Befus, 1980); (ii) an intra-alveolar component accessible by broncho-alveolar lavage (BAL; Hunninghake et al. 1979); and (iii) lymphoid populations associated with the lung wall. Correspondence: Dr P. G. Holt, Clinical Immunology Research Unit, Princess Margaret Hospital, Thomas Street, Subiaco, Western Australia 6008.

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Non-recirculating T cells effects of transient sequestration in the lung vascular bed on the function(s) of individual T cells, employing limiting dilution analysis. MATERIALS AND METHODS Animals Female, specific pathogen-free (SPF) WAG and Brown Norway (BN) rats were used, aged 5-7 weeks old, except where specified (Animal Resource Centre, Murdoch University).

Media Dulbeccos A and B (DAB; Oxoid Ltd, Bucks, U.K.) were used throughout the experiments with 0-2% bovine serum albumen (BSA). Where stated RPMI-1640 (Gibco, Grand Island, NY) or phosphate-buffered saline (PBS) were used in the preparation of cell populations. Isolation of cell populations Lung cells were prepared as described by Holt et al. (1985). Briefly, the lung was perfused via the heart with DAB/BSA containing 10 U/ml heparin, until clear of blood. The lungs were then excised and the tissue sliced mechanically to 500 pm and suspended in 10 ml RPMI containing 10% fetal calf serum (FCS), 150 U/ml collagenase (Type 4197; Worthington, La Jolla, CA) and 50 U/ml DNAse-1 (Type IV; Sigma, St Louis, MO). After a 90-min incubation in a 370 shaking water bath, the digest mix was suspended using a wide bore Pasteur pipette. Large tissue fragments and dead cells were removed by rapid filtration through a thin cotton-wool mesh and the lung cells collected by centrifugation. Blood was collected into heparinized tubes and mononuclear cells isolated by differential centrifugation on FicollPaque (Pharmacia, Uppsala, Sweden) adjusted to specific gravity 1 088 with Hypaque (Winthrop Labs, Sydney). Spleen cells were collected by gently introducing cold DAB/ BSA into the spleen via a 5-ml syringe and applying simultaneous pressure with forceps to empty the spleen sac. The cells were resuspended using a wide-bore pipette, filtered rapidly through cotton-wool, collected by centrifugation and the red blood cells lysed with 0-83% ammonium chloride. Thymus and lymph nodes were minced, sieved and then passed through cotton-wool, and the cells were collected by centrifugation. Viable counts for the cell suspensions were performed employing typan blue exclusion.

Identification and purification ofmononuclear cell populations T cells and their subsets CD4+ and CD8+ were identified employing the mAb (monoclonal antibodies) OX 19/52, W3/25 and OX8 (Mason et al. 1983), provided by Professor Alan Williams and Dr Don Mason (Sir William Dunn School of Pathology, Oxford, U.K.). The mAb EDI (Dijkstra et al. 1985) was used to identify macrophages, and was supplied by Dr Christine Dijkstra (Vrije University, Amsterdam, The Netherlands). Where required, T-cell subset purifications employing these mAb were performed via the rosette depletion method described by Mason, Penhale & Sedgwick (1987). Cell suspensions were settled onto Alcian blue-treated slides for 20 min, alcohol-fixed and rehydrated. Immunoperoxidase detection of surface antigens on the cell suspensions was performed using a biotinylated sheep anti-mouse Ig (Amer-

477

sham, Amersham, Bucks, U.K.) and avidin-conjugated horseradish peroxidase. After washing, enzyme-linked antibody was stained by reacting with 3,3 diaminobenzidine and hydrogen peroxidase. The cells were counterstained with haematoxylin. Saturation labelling studies Osmotic pumps (Alzet, model 2ML2, California, CA; Shellito, Espaza & Armstrong, 1987) were employed to label cycling T cells in vivo with tritiated [3H]thymidine. The pumps were implanted subcutaneously to provide a continuous infusion of radiolabel over 7- and 14-day periods (1 microcurie/g body wt/ day). Cell suspensions from various organs were collected, adhered to Alcian blue-treated slides and stained with appropriate mAb via immunoperoxidase. The slides were then placed under stripping film (Kodak AR 10). The specimens were exposed for 3 weeks in the dark at -70°. The autoradiographs were developed in Kodak D-19 developer for 5 min, washed with distilled water and fixed. The cells were counterstained in

haematoxylin. Cells were scored positive when they displayed 2 three grains above the nucleus. A minimum of 200 cells was counted for each determination.

Intrathymic injections offluorescein isothiocyanate (FITC) Intrathymic (i.t.) injections of FITC were used to follow thymic migrants, based on the method developed by Scollay, Butcher & Weissman (1980). Briefly, the thymus was surgically revealed and 20-30 p1 FITC directly injected into each lobe. The lungs, spleen, lymph nodes and thymus were collected 2-4 hr later, and mononuclear cells were extracted and prepared for fluorescence microscopy.

Hydroxyurea administration The turnover of T cells was investigated using hydroxyurea (HU; Calbiochem, La Jolla, CA), which is selectively toxic to cycling cells (Rocha, Frietas & Coutinho, 1983; Philips et al., 1967). Preliminary dose-response experiments were performed to ascertain an effective dosage for rats, and based on these results a dosage of 15 g/kg body weight (dissolved in PBS) was administered in two separate injections within a 7-hr period. Various organs were collected 24 hr later for enumeration of surviving T cells. The percentage of T cells killed by HU administration was determined by comparison ofT-cell yields to control animals injected with PBS. Limiting-dilution analysis of T cells T-cell subsets were prepared from blood, spleen and lung digests by rosette depletion, employing appropriate mAb (Mason et al., 1987). Following assessment of purity by staining with FITCconjugated mAb, they were seeded into wells of 96-well roundbottomed microtest plates at densities of 32, 16, 8, 4, 2, 1 and 0 5 T cells per well in a total volume of 200 p1 RPMI medium containing 15% FCS, 20% crude T-cell growth factor (concanavalin A-activated spleen cell supernatant), 5 x 10-5 M 2-mercaptoethanol, 5 pg/ml Con A, 50 U/ml recombinant IL-2 (Cetus Corporation, Emeryville, CA) and 2 x 105 feeder cells. The feeder cells were thymocytes prepared from 6-week-old female rats, and were X-irradiated (2000 rads) in suspension. Control wells containing feeders alone were included in each experiment. The cloning plates were incubated at 370 in 5% C02,

D. Nelson, D. Strickland & P. G. Holt

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,a 80

and the frequency of wells containing growing T-cell clones was determined by visual inspection at Days 7-9. Immunocompetent T-cell precursor frequencies were calculated from these data, as described previously (Holt et al. 1988).

T

60

Lungs 1* Blood

00

40

RESULTS

I (n

0

+l 20

Characterization of cells in collagenase digests of rat lung tissue Figure 1 shows pooled data from groups of 10 rats of each strain. An average of 2-5 x 107 T cells was recovered per gram of lung tissue. As reported previously (Holt et al., 1985), the digests also contain large numbers of macrophages stained by the mAb ED1, together with cells staining for Ia. While the latter have not been formally characterized here, earlier immunohistochemical data imply that a significant proportion are dendritic cells (Holt & Schon-Hegrad, 1987). We have also noted that overall T-cell yields per gram lung are identical in tissue segments from the periphery or the hilar region of the lung (data not shown); the peripheral segments contained little discernible BALT, indicating that BALT T cells did not contribute significantly to overall T-cell yields from the whole lung. Hence, it is likely that the bulk of the latter are derived from the population we have previously demonstrated in frozen lung sections, distributed randomly as single cells in alveolar septal walls (Holt & Schon-Hegrad, 1987). Figure 2 contains estimates of the relative contribution of CD8+ cells to the overall T-cell population in lungs and blood. These figures were arrived at indirectly as follows (two-colour flow cytometry is not currently available to us). Because the CD4 marker in rat recognized by the mAb W3/25 is also expressed on a high > 50% of macrophages (Mason et al., 1983), CD4: CD8 T-cell ratios cannot be directly calculated from the W3/25: OX8 data in Fig 1. In contrast, the only significant nonT-cell population present in the lung digest which stains with the anti-CD8 mAb OX8 are large granular lymphocytes (LGL; putative NK cells), which we estimate on the basis of this and earlier (Holt et al., 1985) studies at approximately 10% of the OX8 + lung pool. On this basis, the contribution of CD8 + T cells to the overall T-cell population stained with the mixture of pan T-cell mAb OX19 and OX52 can be estimated with reasonable

OD 0

Figure 2. T-cell subsets in lung versus blood. The contribution of the CD8 + subset to overall T-cell recoveries in lung and blood from groups (n >20) of WAG and BN rats was determined as detailed in the Materials and Methods. Data shown are group means + SE. Table 1. Thymic migrants in the lungs and other organs Rat no.

Lungs

Spleen

MLN

1 2 3 4 5

10%* 11-5% 23% 23% ND

5-7% 1-6% 2-4% 2-3%

0% 0%

5.4%

07%

X

16-9%

3-3%

0 3%

060%0 0%

Cells were extracted quantitatively from each organ 2-4 hr after intrathymic injection of FITC, and prepared either for fluorescence microscopy or immunoperoxidase staining. Control animals included were injected i.p. or i.v. with FITC, an in both cases fluorescent cells comprised