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Expression of Chemokine Receptors by Lung T Cells from Normal and Asthmatic Subjects¹

James J. Campbell, Christopher E. Brightling^*, Fiona A. Symon^*, Shi Qin^§, Kristine E. Murphy^§, Mmarty Hodge^§, David P. Andrew^§, Lijun Wu^§, Eugene C. Butcher^2, and Andrew J. Wardlaw^2,3,*

^* Institute for Lung Health and Division of Respiratory Medicine, Leicester University School of Medicine, Leicester, United Kingdom; Harvard Medical School, Department of Pathology and Children’s Hospital, Division of Transfusion Medicine, Boston, MA 02115; Laboratory of Immunology and Vascular Biology, Department of Pathology and the Digestive Disease Center, Department of Medicine, Stanford University Medical School, Stanford, CA 94305, and the Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304; and ^§ Millenium Pharmaceuticals, Cambridge, MA 02142

	Abstract

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The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and ₄₇. No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.

	Introduction

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The lung is an important route of sensitization for airborne Ags. Consistent with this function, there is an extensive network of hilar and mediastinal lymph nodes that are anatomically and functionally connected to the lung. In addition, the normal adult human lung contains large numbers of lymphocytes. These are comprised largely of memory TCR T cells, which are found principally in two compartments, the bronchial lamina propria, where they form the major leukocyte subtype, and the peripheral alveolar and interstitial regions of the lung. In view of the importance of the lung as an immune organ, it is perhaps not surprising that many lung diseases are characterized by abnormal T cell function. For example, an overexuberant response to antigenic stimuli is associated with inflammatory lung diseases such as asthma, where there is a doubling of the number of CD4 T cells in the bronchial lamina propria (1). A number of inflammatory lung diseases of unknown etiology, such as sarcoidosis and cryptogenic organizing pneumonia, are associated with increased numbers of T cells in bronchoalveolar lavage (BAL)⁴ fluid. Impaired T cell function as seen in AIDS can lead to infectious diseases such as pulmonary tuberculosis and immunocompromised pneumonia. An important question in lung T cell biology is the mechanisms that control T cell migration into the lung in health and disease.

T cells recirculate from the blood to tissue and back to the blood. Naive T cells exclusively traffic through secondary lymphoid tissue such as lymph nodes and bronchus-associated lymphoid tissue (BALT) via specialized postcapillary venules called high endothelial venules (HEV) because of their distinctive plump appearance. In contrast, memory T cells, in addition to migrating into lymph nodes, can also transmigrate through the nonspecialized postcapillary venules of the systemic circulation. Whereas naive T cells do not appear to show any regional preference in their migratory habits, memory T cells preferentially return, or "home," to regions or microenvironments of the body similar to those where Ag was initially encountered (2). Lymphocyte homing is controlled by adhesion molecules and chemoattractant signals expressed by HEVs and postcapillary venular endothelium in an organ-specific manner. Interactions between homing T cells and the endothelium follow the now well-established multistep paradigm of leukocyte adhesion to endothelium, which creates the considerable combinatorial diversity necessary to direct the different patterns of leukocyte emigration that characterize various inflammatory responses (3). This involves an initial capture or tethering step mediated by selectins and their ligands as well as the ₄ integrins ₄₁ and ₄₇, an activation step mediated by chemoattractants, which for T cells appear to be largely chemokines, a firm arrest step mediated principally by ₂ integrins, and a transmigration step, which for T cells again is likely to be controlled principally by chemokines.

Chemokines are a large family of chemotactic peptides of 8–10 kDa molecular mass divided into at least four subfamilies based on the position of two adjacent, conserved cysteine residues (4, 5). Chemokines bind to one or more chemokine receptors (CR), which are members of the serpentine G protein-linked receptor superfamily (6). There is considerable diversity in the expression of CR between different lymphocyte subsets, depending in part on their state of maturity and activation. Therefore, the pattern of CR expression by PBLs is very heterogeneous. This heterogeneity makes chemokines and their corresponding receptors ideal candidates for directing the migration of distinct lymphocyte subsets depending on differential expression of chemokines in either an organ- or disease-specific fashion (7).

The molecular signals directing selective lymphocyte homing have been most clearly defined for peripheral lymph nodes, gut, and skin. For peripheral lymph nodes, the capture step is mediated by L-selectin, the peripheral lymph node homing receptor, which binds to a number of mucin-like ligands selectively expressed on HEVs. The activation step is mediated by the secondary lymphoid tissue chemokine (SLC) (6 C kine; CCL21 in the proposed new nomenclature (8)), which is preferentially expressed on HEVs and binds to CCR7, which is expressed by all naive T cells and a proportion of memory T cells (9). LFA-1 binding to ICAMs nonselectively mediates the firm arrest step. ₄₇ is the gut-homing receptor that binds to mucosal adressin cell adhesion molecule-1, which is selectively expressed on gut endothelium (10). Recent evidence implicates the thymus-expressed chemokine (CCL25), binding to CCR9 in either the activation or chemotaxis step of small-intestine-homing T cells (11, 12). The cutaneous lymphocyte Ag (CLA) is the skin-homing receptor binding to E-selectin, which is preferentially expressed on inflamed skin endothelium. CLA-positive T cells express the CR CCR4 that binds to the CC chemokine thymus activated and regulated chemokine (TARC) (CCL17), which is selectively expressed on skin endothelium and is thought to mediate the activation step for skin-homing T cells (13). The chemotaxis step may be mediated in part by the keratinocyte-derived chemokine cutaneous T cell-attracting chemokine (CTACK) (CCL27) (14), binding to the orphan CR G protein-coupled receptor 2, provisionally called CCR10 (Refs. 15, 16, 17 , and reviewed in Ref. 18).

The molecular signals that control T cell trafficking to the lung either during normal homeostasis or in disease are uncertain. The extent to which there are lung lymphocyte homing pathways distinct from other organs is also unclear. Traditionally, the lung has been grouped with the gut in a common mucosal system; however, what little is known about patterns of lung lymphocyte homing suggests that this is an oversimplification. For example, lung T cells are CLA^-, ₄₇^low (19), which is different from both skin- (CLA⁺) and gut-homing (₄₇^high) T cells. Moreover, in vivo homing studies suggest that ₄₁ and vascular VCAM-1 participate in lymphocyte homing to BALT (E. C. Butcher, manuscript in preparation) and lung (20), in contrast to the selective role of ₄₇ and mucosal adressin cell adhesion molecule-1 in gut lymphocyte homing.

We proposed the hypothesis that there is an organ-specific lung lymphocyte homing pathway. If this was correct, we would predict that lung T cells would express a distinctive pattern of CRs compared with that reported for other organs. Therefore, we investigated the expression of a panel of CRs on dispersed lung T cells from lung resection specimens and from BAL-derived T cells from normal volunteers. We also investigated expression of CR on BAL T cells from patients with asthma. We have found that lung T cells express a distinctive pattern of CRs consistent with the existence of a separate lung-homing pathway. We observed no differences between asthmatic and normal lymphocyte CCR expression, suggesting that the signals that control T cell migration in health and during Th2-type inflammatory responses are similar.

	Materials and Methods

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Lung resections

Lung tissue was obtained from six patients undergoing lung resection for operable nonsmall cell carcinoma of the bronchus in Glenfield Hospital (Leicester, U.K.). Tissue was processed anonymously, and no other clinical details were obtained.

Fiber-optic bronchoscopy (FOB)

Four asthmatic subjects undergoing FOB had a consistent clinical history and had either or both of airway hyperresponsiveness to inhaled methacholine (provocational concentration causing a 20% fall in forced expiratory volume in 1 s <8 mg/ml) and significant reversibility in forced expiratory volume in 1 s (>15%) to nebulized salbutamol. Subjects had mild disease, requiring intermittent inhaled 2 agonists (between daily and two to three times per week), but not inhaled steroids to control their disease. Four normal volunteers had no history of chest disease, were asymptomatic, and had normal lung function. All subjects were nonsmokers.

Ethical approval

The study had ethical approval from the Leicestershire Health Ethics Committee.

Antibodies

Directly conjugated mouse anti-human CD3-FITC (IgG1, clone UCHT1), CD4-APC (IgG1, clone RPA-T4), and anti-CD8-PE (IgG1, clone RPA-T8) were obtained from BD PharMingen (San Diego, CA). Unconjugated Abs against ₄₇ (clone ACT-1), CXCR3 (IC6), CXCR3R1 (IE5), CR1 (7G11), CCR3 (7B11), CCR4 (IG1), CCR5 (2D7), CCR7 (7H12), and CCR9 (96-1) were obtained from LeukoCite (Cambridge, MA). Abs against very late Ag (VLA)-4 (HP2/1), CD44 (J.173), and VLA-1 (HP2-B6) were obtained from Beckman-Coulter (Fullerton, CA). Abs against CXCR4 (12G5), CXCR5 (51505), CCR1 (53504), CCR2 (48607), and CCR6 (53103) were obtained from R&D Systems (Minneapolis, MN). Anti-L-selectin (Dreg-56) and CLA (HECA 452) were prepared in Dr. Butcher’s laboratory. All other unconjugated Abs including isotype controls were obtained from BD PharMingen.

Processing of dispersed lung T cells

Pieces of normal lung at some distance from the tumor were dissected away and washed in culture medium (RPMI 1640 with 10% bovine calf serum) to remove any blood. The tissue was then placed in a Petri dish containing buffer and finely minced into small fragments using scissors and scalpel. The tissue fragments and supernatant were then drained through gauze. The cell pellet was washed, and in some cases macrophages were depleted using the method of adherence to plastic for 1 h. The cell pellet was then frozen as if for a cell line in culture medium containing 20% DMSO, being placed at -80°C for 24 h before being placed in liquid nitrogen. The cells were then transported on dry ice from the United Kingdom to Stanford, CA, where the analysis took place. On the day of analysis, the cells were rapidly thawed by placing the pellet in a large volume of culture medium. The cells were then washed twice in culture medium before being immunostained and analyzed.

To test for the effects of freezing on CR expression, PBMC were Ficoll purified, washed, and resuspended in freezing medium. Cells in freezing medium were then placed in styrofoam to freeze slowly in a -80°C freezer. After 24 h, the vials were immersed in liquid nitrogen for at least 7 days. On the day of experiment, fresh PBMC were again purified from the same donor using the same method. The frozen cells were thawed and stained in parallel with the fresh cells. Both sets of PBMC were stained for CD3, CD4, and CD45RA, in addition to the CRs and their isotype controls. No appreciable difference in CR expression was observed between the frozen and freshly isolated cells. In addition, no difference in pattern of CR expression was observed in BAL cells that were freshly isolated or frozen.

FOB and processing of BAL fluid

FOB were performed in the United Kingdom. Subjects were premedicated with nebulized salbutamol before being lightly sedated with midazolam and having their upper airway anesthetized with 2% lignocaine. A total of 180 ml of normal saline were inserted through the bronchoscope into the right middle lobe and aspirated using gentle suction. Recovery was between 20 and 46%, with asthmatics having a mean of 9% less fluid returned. The BAL fluid was filtered through gauze to remove large pieces of mucus, and the cell pellet was washed twice in culture medium before immunostaining. In some cases, the cells were frozen as for the lung resection-derived cells and transported to Stanford.

Twenty milliliters of blood were also taken from the subjects undergoing FOB, and the mononuclear cell fraction was obtained by centrifugation on Ficoll. The mononuclear cells were then washed twice before immunostaining.

Analysis of expression by flow cytometry

Isolated lung cells either from lung tissue or BAL were resuspended in FACS buffer at a concentration of between 0.5 and 1 x 10⁶/ml depending on the number of cells available. Nonspecific Ab binding was blocked using horse IgG (Sigma, St. Louis, MO). Lung lymphocytes were stained with directly conjugated Abs against CD3, CD4, and where indicated, CD8, and with a large panel of up to 40 unconjugated Abs that were detected using a biotinylated horse anti-mouse IgG secondary Ab (Vector Laboratories, Burlingame, CA) and streptavidin-PerCP (BD PharMingen). Lymphocytes were gated for CD3 expression and then further subdivided by CD4 or CD8 expression. The majority of experiments were done using four-color flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA). Some of the experiments with BAL lymphocytes were done using three-color flow cytometry on a FACScan. In both cases, CellQuest software version 3.1 (BD Biosciences) was used.

	Results

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Expression of adhesion and activation receptors by dispersed lung resection-derived T cells

In the dispersed lung leukocyte population 50% of cells in the lymphocyte gate (based on the PBL gate) were CD3⁺. Almost all the CD3⁺ cells fell within this gate. The great majority of the CD3⁺ cells were either CD4⁺ or CD8⁺. In all but one of the lung resection patients studied, (where there was a CD4/CD8 ratio of 1:4), there was a predominance of CD4 over CD8 T cells (mean ratio 2:1). More than 95% of the CD3⁺ cells in the lymphocyte gate were CD45RO⁺ and TCR⁺. In addition, as would be expected, all the CD3⁺CD4⁺ and CD3⁺CD8⁺ cells were CD19^- (B cells) and CD56^- (NK cells). Representative histograms showing expression of these receptors as well as adhesion and activation receptors from dispersed lung T cells is shown in Fig. 1A for CD4 cells and Fig. 1B for CD8 cells. A uniform shift in the histogram for ₄₇ suggested that the majority of lung T cells expressed this receptor, although expression was weak. When expression was compared on the same day with staining of blood-derived T cells (from a normal donor), the level of expression was intermediate between highly expressing putative gut-homing T cells and very weakly expressing naive cells (data not shown). CLA expression was negative (data not shown). CD44, CD29 (1), VLA-4, and LFA-1 were well expressed by all lung T cells. _E7 was bimodally expressed by both CD4 and CD8 T cells although the percentage of cells expressing this receptor was variable, ranging between 10.5 and 85.3%, with a mean of 46% and 49%, respectively, for each subset (n = 6). Lung T cells had an activated phenotype in that there was increased expression of CD69 (mean 72 ± 27% for CD4 and 65 ± 25% for CD8; n = 6) and VLA-1 (53 ± 24% and 55 ± 27%, respectively; n = 5) compared with peripheral blood T cells, where these receptors are not expressed. CD25 was only weakly expressed.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Expression of adhesion and activation receptors by lung T cells. Single-cell suspensions were obtained from the resected lung tissue as described in Materials and Methods and analyzed by four-color flow cytometry gating on small lymphocytes by forward and side scatter and then for CD3 positivity (A and B, upper left) and then for CD4+CD8-, (A, upper right), or CD4-CD8+ (B, upper right). A number of unconjugated Abs were used to analyze the phenotype of the cells (solid lines in each histogram panel) and overlaid on the appropriate isotype control (dotted lines in each panel). The histograms are representative of a total of six patients analyzed and findings were consistent among subjects. A and B are from different patients.

A representative histogram showing expression of CRs by lung resection-derived T cells is shown in Fig. 2, A (CD4) and B (CD8). Of the CXC receptors, both CD4 and CD8 T cells expressed CXCR3 and CXCR4 but not CXCR5. The neutrophil CRs CXCR1 and 2 were not expressed (data not shown). Of the CCR, dispersed lung T cells strongly expressed CCR5 in all cases. CD4 but not CD8 T cells expressed CCR6. Expression of CCR6 was bimodally distributed, with variability in the level of expression between subjects ranging from 1 to 37% (mean 21 ± 13%; n = 6). CD4 but again not CD8 T cells expressed weak to moderate levels of CCR4, with a unimodal distribution. Expression of CC, CRs 1, 2, 3, 7, and 9, as well as CR1, was either absent or negligible. A small and variable subset of both CD4⁺ (range 0–8%) and CD8⁺ (0–20%) (n = 6) T cells expressed the Fractalkine receptor CX₃CR1.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Expression of CRs by lung CD4 (A) and CD8 (B) T cells. Data was obtained in the same as way as described for Fig. 1. The subjects are the same as in Fig. 1 and are representative of a total of six patients.

Because of the paucity of cells compared with the lung resection-derived T cells, it was only possible to undertake a limited examination of the receptor phenotype of BAL T cells, focusing on the CRs that were of particular interest. A representative pattern of receptor expression for BAL T cells is shown in Figs. 3, A (CD4) and B (CD8) (normal donors), and 4, A and B (asthmatics). Some of these experiments, including the example shown, were done with three-color fluorescence omitting the CD8-conjugated Ab. The CD8 population was taken as the CD3⁺CD4^- cells. As for the dispersed cells, about 50% of the cells in the lymphocyte gate were CD3⁺. No major differences were observed between BAL T cells and dispersed lung T cells in the pattern of chemokine and activation receptor expression although generally greater expression of CCR7 was seen on BAL CD4 T cells compared with resection-derived cells. No difference was seen between asthmatic and normal BAL T cells. In particular, asthmatic T cells expressed the putative Th1-linked CRs CCR5 and CXCR3 but not the putative Th2 CR CCR3. The Th2-linked CR CCR4 was expressed to a similar extent by BAL T cells from both subject groups. CCR4 expression was 10-fold lower than that of CLA⁺ cells from patient-matched blood (not shown). CCR6 showed the same pattern of expression in the BAL T cells compared with the lung resection cells, although the degree of expression tended to be higher (CD4 asthma, 61 ± 4% and CD4 normal, 57 ± 17%; CD8 asthma, 21 ± 18% and CD8 normal, 20 ± 14%)(n = 4).

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Adhesion and CR phenotype of normal BAL-derived T cells. Single-cell suspensions were obtained from BAL fluid as described in Materials and Methods and analyzed by three-color flow cytometry, gating on small lymphocytes by forward and side scatter and then for either CD3+CD4+ (CD4 lymphocytes; A, upper panel), or CD3+CD4- (CD8 lymphocytes; B, upper panel) populations. Histograms are representative of a total of four patients, and expression was consistent among subjects.

	Discussion

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Traditional concepts of memory lymphocyte homing to extralymphoid (or tertiary lymphoid) sites have suggested two patterns of recirculation, the mucosal tissues of the gut, lung, and genitourinary tract and the "systemic" tissues such as liver and musculoskeletal system (21). The mucosal tissues have a specialized epithelium that is in direct contact with the outside environment, has a lamina propria rich in dendritic cells and lymphocytes, and has the capacity, through structures such as Peyer’s patches and BALT, to take up and process Ag directly through the epithelium without going via afferent lymphatics. The skin is generally included in the systemic system although it is in direct contact with the outside environment and has a specialized epithelium rich in dendritic cells. If this concept were correct, lung T cells might be expected to express ₄₇ and CCR9 in common with gut T cells.

However, this does not appear to be the case. Similar to previous reports (19), we found that lung T cells expressed only low levels of ₄₇. CCR9 was not expressed by normal or asthmatic lung T cells. It remains possible that there are homing characteristics shared between lung and large intestinal lamina propria lymphocytes because these do not express CCR9 to the same degree as do small-intestinal cells (11). Also, as reported by Picker and colleagues (19), we found that lung T cells do not express CLA. We have found that they express only low to moderate levels of CCR4. This is in contrast to the CLA⁺, CCR4^high skin-homing T cells. This data, suggesting different pathways of recirculation for the gut, skin, and lung, is consistent with experiments in animal models that also suggest the presence of a unique lung-homing pathway. For example, in the sheep, although gut-associated T cells were ₄₇^high, L-selectin^low, and T cells from peripheral lymph nodes were ₄₇^low, L-selectin^high, T cells from lung lymph were low for both receptors (22).

What does the CR expression of lung T cells tell us about the signals that control migration of lymphocytes into the lung? There is increasing evidence that chemokines control the precise tissue localization of T cells both during ontogeny and in trafficking in peripheral secondary and tertiary lymphoid tissue. They do this either by tissue-specific expression on endothelial cells, where they trigger the activation step in the lymphocyte adhesion cascade, or by localized expression within different organs or specific sites within the same organ, where they mediate chemotaxis of distinct lymphocyte subsets (18, 23). Triggering activation in the adhesion cascade appears to require higher levels of expression of the receptor than are required for chemotaxis (24). The only receptor expressed to a high level on lung T cells, and which therefore might be a candidate for an adhesion-triggering receptor, is CCR5, whose ligands include RANTES (CCL5), macrophage inflammatory protein (MIP)1 (CCL3), and MIP1 (CCL4). RANTES is expressed by a number of cell types in the lung, including the epithelium and smooth muscle. However, expression in normal lung tissue is weak and not obviously endothelial. In addition, all tissue T cells appear to highly express CCR5 (J. J. Campbell and E. J. Kunkel, unpublished observation), so CCR5 expression is not organ specific. A number of CRs were expressed to a lower level, in particular, CXCR3, CXCR4, CCR4, and CCR6. CXCR3 binds IFN-inducible T cell chemoattractant (CXCL11), IFN-inducible protein of 10 kDa (CXCL10), and monokine induced by IFN- (CXCL9), which have been shown to be expressed by activated but not resting lung epithelial cells, suggesting a role in T cell recruitment during inflammation, but not during normal homeostasis (25). Again, expression of CXCR3 is common to all tissue-homing cells. CXCR4, the ligand for stromal cell-derived factor (CXCL12),is expressed on all T cells in both peripheral blood and tissue. CCR4 might be involved in chemotaxis of lung T cells via either TARC or monocyte-derived chemokine (CCL22). Moderate levels of CCR4 are expressed by CLA⁺, ₄₇^low memory T cells in the peripheral blood (J. J. C. and E. J. Kunkel, unpublished observation), and it is possible that this could represent a lung-homing population. The extent to which TARC and monocyte-derived chemokine are expressed in the lung has not been reported. CCR6, which binds MIP3 (CCL20), was expressed on a variable subpopulation of lung T cells. The original reports of MIP3 detected mRNA expression for this chemokine in the lung. However, CCR6 is also expressed by T cells from other tissues. There are a number of chemokines and CRs that have not yet been characterized except at the level of orphan receptors and expressed sequence tags, and it remains possible that other as yet unknown receptors play a paramount role in normal lung T cell trafficking.

Unlike most CRs on lung T cells, CCR6, and to a lesser extent the Fractalkine receptor CX₃CR, had a bimodal distribution. One explanation for this would be differential expression by T cells from separate lung compartments. T cells migrate into the lung either through the bronchial postcapillary venules into the bronchial lamina propria, which is part of the systemic circulation, or through the pulmonary capillary and venular bed into the alveolar compartment, a process that appears to be less dependent on the adhesion cascade because of the lower shear stresses involved. We used peripheral lung for our lung resection material, and the majority of the T cells in BAL are probably largely from the small airways and alveolar compartments. It is possible that bronchial T cells have homing requirements different from those of alveolar T cells in a manner similar to the small and large gut, in which case there could be differences in CCR6 expression on T cells from the bronchial mucosa and alveolar tissue. Alternatively, CCR6 expression may be a marker for functionally distinct subsets of lung T cells. CD8 T cells did not express either CCR6 or CCR4, suggesting that there may be differences in the mechanism of recruitment of CD4 and CD8 T cells.

Our lung resection tissue was not entirely normal, being from patients with lung cancer, most of whom will have smoked and will have a degree of smoking-related airways disease. In addition, it is not known whether the recruitment of T cells during inflammation is mediated by the same signals as homeostatic lung trafficking. To address these questions, we studied the phenotype of BAL T cells from normal and asthmatic subjects. The pattern of adhesion, activation, and CR expression was very similar between lung resection T cells, normal BAL, and asthmatic BAL. In particular, we saw no bias in the asthmatics toward the putative Th2-linked CRs CCR3 and CCR4. CCR3 expression was negative, and expression of CCR4 was the same as in the normal BAL. In addition, as was the case in the normal BAL, asthmatic T cells expressed the putative Th1 receptors CXCR3 and CCR5. Although it has become accepted that extrinsic asthma is a disease mediated by activation of Th2 lymphocytes, the number of Th2 cells in asthmatic BAL fluid is uncertain. We are aware of two studies that have addressed cytokine production by BAL T cells in clinical asthma in humans (26, 27). Robinson et al. studied mRNA expression using in situ hybridization and found that a mean of 73% of the CD3⁺ T cells produced IL-4 and so could be designated Th2. The number of T cells expressing IFN- was not quantified. In contrast, in the paper by Krug et al. using intracellular flow cytometry, a mean of 75% of T cells expressed IFN- in asthma compared with a mean of only 2% expressing IL-4 (50 and 1% in normal subjects, respectively). Therefore, there is a clear discrepancy in the assessment of Th2 status, depending on whether there is mRNA or protein expression, the reasons for which are yet to be explained. About 10% of BAL T cells in our study are CXCR3^-CCR5^-. If the number of Th2 cells in asthma BAL is <10%, as suggested by Krug et al., then it is possible that these are the CCR5^-CXCR3^- cells, although this would not be possible if the assessment by in situ hybridization were correct. It should be noted that our asthmatics were similar in terms of severity and treatment both to those studied by Krug et al. and those studied by Robinson et al. We have attempted to dual-stain BAL T cells for CCR5/CXCR3 against IL-4 and IFN- (using intracellular flow cytometry after stimulation with PMA and ionomycin in the presence of brefeldin) to try and answer more definitively the relationship between CR expression and cytokine production. However, stimulation of the T cells resulted in a marked increase in expression of CCR5 and CXCR3, so that they became 100% positive for these receptors (data not shown). Therefore, we are not able to answer this question conclusively. However, the observed pattern of CR expression by the BAL T cells makes it difficult to see how CR expression and Th1 vs Th2 cytokine production could be linked in the way that has been proposed. Together with data showing no relation between cytokine expression and the expression of CCR4 in CLA⁺ skin-homing T cells (28), it suggests that the Th1/Th2 polarization of CR expression seen in vitro is not straightforwardly reflected in vivo.

A potential problem with lung-derived T cells is that they can be contaminated by peripheral blood T cells. However, the profile of receptor expression by lung T cells, both from the dispersed and BAL populations, strongly argues against significant contamination. Thus, the lung T cells were virtually all CD45RO⁺. They had a uniform expression of CCR5 and CXCR3, unlike the heterogenous pattern seen in peripheral blood. They expressed high levels of CD69 and _E7, which are not expressed by the great majority of peripheral blood T cells. To exclude the possibility that _E^- cells in the lung were derived from peripheral blood we examined the receptor phenotype of _E⁺ and _E^- cells and found that both populations were >95% memory T cells (data not shown). This suggests that if there was any blood contamination it must be very minor and would not influence the interpretation of the data. As has been noted previously, the lung T cells had an activated phenotype in that they expressed high levels of CD69, VLA-1, and _E7 compared with blood T cells (29). Interestingly, expression of another putative activation marker, CD25, was relatively low, with no clear difference between the BAL and dispersed lung cells, and expression did not correlate with the other activation-related receptors. CD25 is a marker of a subset of TGF-producing regulatory CD4 cells (30), and it is intriguing to speculate that CD25⁺ cells in the lung also have this function.

In summary, we have characterized the expression of known CRs by lung T cells and have found that they express a pattern of receptors distinct from gut- and skin-homing T cells. This suggests the existence of a separate lung-homing population. However, it is likely that the chemokines and CRs that control organ-specific lung T cell homing remain to be determined.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Adhesion and CR expression by BAL-derived T cells from subjects with mild asthma. Data was obtained as described for Fig. 3, and histograms are representative of four asthmatics, with expression being consistent among subjects. (A (CD4) and B (CD8)).

	Footnotes

² E.C.B. and A.J.W. were joint principal investigators in this study.

³ Address correspondence and reprint requests to Dr. Andrew J. Wardlaw, Glenfield Hospital, Groby Road, Leicester LE3 9QP, U.K.

⁴ Abbreviations used in this paper: BAL, bronchoalveolar lavage; BALT, bronchus-associated lymphoid tissue; HEV, high endothelial venule; FOB, fiber-optic bronchoscopy; CLA, cutaneous lymphocyte Ag; CR, chemokine receptor; VLA, very late Ag; MIP, macrophage inflammatory protein; TARC, thymus activated and regulated chemokine.

Received for publication July 24, 2000. Accepted for publication November 16, 2000.

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