Key words: allergic alveolitis; hypersensitivity pneumonitis; L-selectin; lymphocytes; selectin gene family; selectins

Transcription

1 Up-regulation of L-Selectin and E-Selectin in Hypersensitivity Pneumonitis* Carmen Navarro, MD; Felipe Mendoza, MSc; Lourdes Barrera, MSc; Lourdes Segura-Valdez, PhD; Miguel Gaxiola, MD; Ignacio Páramo, MSc; and Moisés Selman, MD, FCCP Background: Selectins are adhesion molecules that contribute to leukocyte recruitment into the tissue after an injury. Hypersensitivity pneumonitis (HP) is a lymphocytic alveolitis, and we hypothesized that the overexpression of selectins could play a role in this process. Patients and measurements: We studied 16 patients with HP and 7 healthy control subjects (HCs). Sera and BAL selectins and tumor necrosis factor- were determined by enzyme-linked immunosorbent assay, and cellular lung localization was determined by immunohistochemistry. Additionally, BAL L-selectin, and L-selectin-bearing T-lymphocytes analyzed by flow cytometry were evaluated in HP patients and in exposed but asymptomatic subjects (EAS). Setting: Tertiary referral center and immunohistochemistry laboratory. Results: Raised levels of E-selectin (mean [ SD], vs ng/ml, respectively; p < 0.001) and P-selectin (mean, vs ng/ml, respectively; p < 0.001) were detected in HP patient sera compared to control subjects, while L-selectin levels showed no differences between groups. Conversely, HP patients displayed a significant increase in levels of L-selectin found in BAL fluid compared with both HCs and EAS ( vs and ng/ml, respectively; p < 0.05). The levels of E-selectin found in BAL fluid were similar in patients from both groups, and P-selectin was not detected. Percentage of CD3 CD62 L lymphocytes was lower in HP patients compared with EAS ( vs , respectively; p 0.05). By immunohistochemistry, L-selectin was detected in interstitial macrophages and polymorphonuclear cells, and E-selectin was detected in endothelial cells. Conclusion: These findings demonstrate that L-selectin and E-selectin are up-regulated during the development of HP, suggesting that they may contribute to the increased traffic of lung inflammatory cells. (CHEST 2002; 121: ) Key words: allergic alveolitis; hypersensitivity pneumonitis; L-selectin; lymphocytes; selectin gene family; selectins Abbreviations: EAS exposed but asymptomatic; ELISA enzyme-linked immunosorbent assay; HC healthy control subject; HP hypersensitivity pneumonitis; IL interleukin; MIP macrophage inflammatory protein; PBS phosphate-buffered saline solution; TNF tumor necrosis factor The recruitment of inflammatory cells at the target site is mediated by interactions between leukocytes and endothelial cells through a group of cell-surface proteins known as adhesion molecules. So far, the following three families of adhesion molecules have been described that share a number of structural, functional, and genetic properties: the *From the Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, México DF, México. This work was partially support by Conacyt grant No M. Manuscript received March 15, 2001; revision accepted August 17, Correspondence to: Moisés Selman, MD, FCCP, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502; Col. Sección XVI, México DF, CP 14080, México; mselman@conacyt.mx selectin gene family; the Ig supergene family; and the integrin gene family. 1,2 Each group is involved in different steps of the leukocyte-endothelial cell rolling, tight adhesion, and migration stages. The most important role of the selectin family is the rolling of leukocytes along the vessel wall and the initial adhesion to endothelial cells. These molecules also are involved in the regulation of cell function. 3 Members of this family include L-selectin expressed on leukocytes, P-selectin expressed on platelets and endothelial cells, and E-selectin expressed on endothelial cells. Each of these proteins recognizes and binds to their carbohydrate ligands. Enzymatic cleavage from the membrane surface results in the presence of soluble forms in the circulation Clinical Investigations

2 Hypersensitivity pneumonitis (HP) represents a group of granulomatous interstitial lung disorders that are provoked by exposure to a variety of organic particles. 5 The disease is characterized by a marked influx and accumulation of inflammatory cells, primarily T-lymphocytes, which play a pivotal pathogenic role. 6 However, the mechanisms implicated in the inflammatory cell recruitment and traffic throughout the lungs in patients with HP are still unclear. Some chemokines, such as interleukin (IL)-8, and macrophage inflammatory protein (MIP)- have been shown to be increased in HP patients. 7,8 Several investigators have found an up-regulation of intercellular adhesion molecule-1, a cell surface glycoprotein that pertains to the Ig supergene family, on alveolar macrophages of patients with HP. 9 Also, increased levels of soluble intercellular adhesion molecule-1 in the sera of these patients have been reported. 10 The possible role of the selectin family in HP has not been explored. E-selectin and L-selectin actively participate in leukocyte/endothelial cell interactions. The members of the selectin family and the 4 integrins have been shown to mediate the initial contact between free-flowing leukocytes and the endothelium in vivo. 11,12 Likewise, P-selectin seems to play an important role for targeting neutrophil migration at endothelial borders. 13 However, the rolling of leukocytes on the endothelial selectins is not restricted to neutrophils, but it also has been demonstrated for bovine and human T cells. 14 Moreover, it has been demonstrated that the inefficient accumulation of T lymphocytes in an artificial site of inflammation is related to a lack of expression of the E-selectin ligand and L-selectin. 15 Since T lymphocytes are a major component of the pulmonary inflammatory process in patients with HP, the present study was designed to evaluate the expression of the three members of the selectin family in this disease. Levels of the soluble forms of selectins were determined in BAL fluid and serum by enzyme-linked immunosorbent assay (ELISA). L-selectin-bearing T lymphocytes were evaluated by flow cytometry, and lung tissue localization was performed by immunohistochemistry. In addition, we measured levels of tumor necrosis factor (TNF)-, a cytokine that plays a pivotal role in the expression of specific endothelial cell adhesion molecules and in the induction of the transendothelial migration of T lymphocytes. 16 Study Population Materials and Methods Sixteen nonsmoking female patients with subacute/chronic HP induced by avian antigens (ie, pigeon breeder s disease), aged 20 to 54 years (mean [ SD] age, years) were investigated. The diagnosis was based on a history of antigen exposure, the typical clinical manifestations of HP, chest radiograph, CT scans, pulmonary function tests, and BAL fluid features. All patients revealed positive specific serum antibodies, and the diagnosis was confirmed by histologic examination. 17 As control subjects, we studied seven healthy volunteers who were nonsmokers (three men and four women; mean age, years). They were sequentially enrolled, unrelated, healthy blood donors from the National Institute of Respiratory Diseases. The Transfusion Department of that institute serves the geographic area from which the HP patients were recruited. For BAL fluid and sera L-selectin measurements, we also included five asymptomatic subjects who had been exposed (EAS) to avian antigens (two men and three women; mean age, years). Serum samples were taken simultaneously from 9 of 16 patients with pigeon breeder s disease and the control groups. The study was approved by the Ethics Committee of the institute, and informed consent was obtained from each subject. BAL BAL samples were obtained by the modification of a previously described technique. 18 One of the subsegmental bronchi of the middle lobe was lavaged with six 50-mL aliquots of sterile saline solution. The recovered fluid was measured, strained through a surgical gauze to remove mucus and debris, and centrifuged at 400g for 10 min at 4 C. The cell pellet was resuspended in phosphate-buffered saline solution (PBS) in order to determine total cell counts and viability by trypan blue exclusion, followed by Wright-Giemsa staining for differential cell counts. The supernatants were stored at 70 C until use. Flow Cytometric Analysis of Surface L-Selectin on BAL Cells BAL cells ( ) were incubated in 50 L staining buffer (ie, PBS containing 1% bovine serum albumin and 0.1% sodium azide) with fluorescein isothiocyanate-conjugated anti-cd3 monoclonal antibody (Becton Dickinson; San José, CA) and phycoerythrin-conjugated anti-l-selectin monoclonal antibody (CD62 L; Becton Dickinson) for 30 min at 4 C. Mouse IgG antibodies of the same isotype and concentration were used for detection of the nonspecific binding of antibodies. After two washes, all cells were fixed in 1% paraformaldehyde. Flow cytometric analysis of cell-surface markers was performed using a flow cytometer (FACScan; Becton Dickinson) with CellQuest software (Becton Dickinson). Determination of L-selectin, P-selectin, E-selectin, and TNF- Sera and BAL fluid quantification of soluble L-selectin, P- selectin, E-selectin, and TNF- was performed by using a sensitive and specific commercial ELISA, following the instructions of the manufacturer (R&D Systems; Minneapolis, MN). Immunohistochemistry Lung tissues obtained by open lung biopsy were formalin-fixed and paraffin-embedded for conventional light microscopy. Lung sections from all patients were immunostained for L-selectin, P-selectin, and E-selectin, as previously described, 19 by a biotin/ streptavidin complex technique using a commercial kit (Vectastain Universal Quick Kit; Vector Laboratories, Inc; Burlingame, CA). In brief, sections were deparaffinized and hydrated through xylenes and graded alcohol series, were washed in PBS, CHEST / 121 / 2/ FEBRUARY,

3 and were incubated in blocking serum for 10 min. Prior to the immune reaction, antigen retrieval with 0.1 M citrate buffer (ph, 6.0) was performed. The serum was drained, and sections were incubated with the polyclonal antibody anti-human E-selectin (R&D Systems) or with monoclonal antibodies anti-human L- selectin or P-selectin (Zymed Laboratory, Inc; San Francisco, CA) overnight at 4 C. After three 5-min washes in PBS, the slides were incubated with biotinylated universal secondary antibody for 10 min, were washed in PBS, and were incubated with streptavidin/peroxidase complex for 5 min. Sections were washed in PBS and were incubated with peroxidase substrate solution until staining developed. Slides were counterstained with hematoxylin, cleared, and mounted. Three macroscopic and microscopic healthy lung specimens that were obtained from lobectomies were used as controls. Statistical Analysis All data are expressed as the mean SD. Comparisons were made using a Student s t test for paired observations. Values of p 0.05 were considered to be statistically significant. The relationships among L-selectin from BAL fluid, TNF- from BAL fluid, and L-selectin from BAL with BAL lymphocytes were assessed using Spearman s correlation coefficient. Results Table 1 Baseline Characteristics of the Study Population* Characteristics Values Patients, No. 16 Age, yr Gender Female 16 Male 0 Time elapsed to first visit, mo FVC, % predicted FEV 1, % predicted FEV 1 /FVC Pao 2,mmHg Paco 2,mmHg *Values given as mean SD, unless otherwise indicated. Normal values in Mexico City (altitude, 2,240 m): Pao mm Hg; Paco mm Hg. The baseline characteristics of patients with HP are summarized in Table 1. All patients showed clinical and functional evidence of interstitial lung disease, with variable degrees of dyspnea, reduced lung capacities, and hypoxemia at rest that worsened during exercise. The differential cell count in BAL fluid was characterized by a marked lymphocytosis, usually 50%, which was significantly higher when compared with both control groups (Table 2). Patients were defined as having subacute/chronic HP when they complained of at least 3 months of persistent symptoms, primarily progressive dyspnea on exercise. The patients exhibited predominant ground-glass attenuation on CT scans and a noteworthy increase in BAL fluid lymphocytes, supporting the presence of active disease. In addition, a lung biopsy showed diffuse lung inflammation with poorly formed granulomas that were located mainly in terminal and respiratory bronchioles, but also in the alveolar walls. Soluble Selectins in Serum Figure 1 illustrates E-selectin and P-selectin sera findings. The level of soluble E-selectin (Fig 1, A) was measured in the serum of 9 of 16 HP patients and in 7 healthy control subjects (HCs). The HP patient group displayed a significant increase in serum levels of E-selectin compared to control subjects ( vs ng/ml, respectively; p 0.001). Likewise, serum levels of soluble P-selectin (Fig 1, B) were significantly higher in HP patients than in those found in healthy subjects ( vs ng/ml; p 0.001). By contrast, the level of soluble L-selectin in the HP patient group showed no significant difference from the control group (1, vs 1, ng/ml, respectively). There was no correlation between serum levels of E-selectin and P-selectin and the number or type of inflammatory cells found in BAL fluid. L-Selectin, E-Selectin, and P-Selectin in BAL Fluid The levels of selectins in BAL fluid were determined in HP patients, HCs, and EAS subjects. Soluble L-selectin was detected in the BAL fluid of all 16 HP patients, and the levels were significantly higher than those in both control groups of EAS individuals ( ng/ml vs and ng/ml, respectively; p 0.05; Fig 2). By contrast, the levels of soluble E-selectin displayed no significant differences (data not shown), and soluble P-selectin was not detected in the BAL fluid of either control group or in the HP patient group. Additionally, TNF- was only revealed in the BAL fluid of HP patients (mean level, pg/ml). Although a tendency for correlation between L- selectin and TNF- levels was suspected because there were two higher coincident points for both molecules, no significant correlation was revealed (r 0.47; p 0.06). Flow Cytometric Analysis of Surface L-Selectin in Cells in BAL Fluid In addition to soluble L-selectin in BAL fluid, we examined the expression of this adhesion molecule on the surface of lymphocytes from BAL fluid in eight HP patients and the five EAS individuals. A marginal but significant decrease in the percentage 356 Clinical Investigations

4 Table 2 Cell Profile in BAL Fluid* Cells Patients (n 16) Control Subjects (n 7) EAS (n 5) p Value BAL fluid total cells Macrophages, % Lymphocytes, % Neutrophils, % NS Eosinophils, % NS *Values given as mean SD, unless otherwise indicated. NS not significant. No. of cells given as of CD3 CD62 L lymphocytes was observed in the HP patients compare to control subjects ( vs , respectively; p 0.05). Figure 3 illustrates a representative comparison between a single HP patient and a single EAS. Immunohistochemistry Tissue from HP patients showed positive signals for E-selectin and L-selectin, whereas P-selectin was not detected. L-selectin was found mainly on the surface of interstitial mononuclear cells (Fig 4, A), primarily macrophages (Fig 4, B [upper inset]), and in some polymorphonuclear cells (Fig 4, B [bottom inset]). E-selectin was expressed in the lungs of HP patients by endothelial cells primarily from small blood vessels (Fig 4, C). Healthy lungs were usually negative for both selectins, as exemplified for L-selectin in Figure 4, D. Control samples incubated with nonimmune sera were negative (Fig 4, E). Discussion HP represents a form of T-cell alveolitis provoked by exposure to a variety of organic particles. Thus, lung histology and the BAL fluid cell profile typically exhibit increased numbers of activated lymphocytes Figure 1. Serum levels of soluble E-selectin (A) and P-selectin (B) in nine HP patients and seven HCs, measured by ELISA as described in the Materials and Methods section. Values are expressed as the mean SD. Figure 2. An increased concentration of soluble L-selectin was found in the BAL fluid obtained from HP patients compared with HCs and EAS individuals. The data represent the mean SD. CHEST / 121 / 2/ FEBRUARY,

5 Figure 3. Flow cytometry dot plots of lymphocytes in BAL fluid from a representative patient (left) and an EAS control subject (right). The number in each upper right quadrant denotes the percentage of CD3 lymphocytes that were positive for L-selectin. with a dominance of CD8 T cells at the onset of disease. 20,21 However, the mechanisms accounting for the accumulation of lymphocytes and other inflammatory cells in the lung parenchyma have not been elucidated. Two different mechanisms appear to be implicated in this process. A polyclonal and oligoclonal local T-cell expansion in the pulmonary microenvironment, probably mediated by the IL-2 system, has been proposed. 22 The detection of the compartmentalization of T cells bearing discrete V Figure 4. L-selectin and E-selectin immunolocalization in the lungs of HP patients and HCs. A and B: lungs of HP patients exhibiting inflammatory cells immunolabeled for L-selectin (A, original 20; B, original 40). B: upper inset, macrophage (original 100); bottom inset, polymorphonuclear cell (original 100). C: E-selectin immunoreactive endothelial cells in the lung of an HP patient (original 20). D: healthy lungs were usually negative for L-selectin (original 10) and E-selectin (not shown). E: negative control omitting the primary antibody (original 20). 358 Clinical Investigations

6 gene products in the lungs of HP patients suggests that the proliferation of specific T-cell subsets probably occurs as a result of local triggering by a specific antigen. 23 Alternatively, increased traffic and the migration of lymphocytes from the blood to the lung could account for the intense alveolitis observed in these patients. Usually, this cellular influx to an injured site is mediated by the production of a number of chemotactic agents and by the expression of adhesion molecules in endothelial and inflammatory cells. There is evidence of increased synthesis of the chemokines IL-8 and MIP-1 in the lungs of HP patients, which may be implicated in neutrophil chemoattraction and in macrophage activation. 7 Also, MIP-1 appears to attract CD8 T lymphocytes, which are pivotal cells in the pathogenesis of HP. 24 The migration of these cells into lung tissue in response to the stimuli depends on the expression of a variety of adhesion molecules. Studies of this process in HP are scanty. The recruitment of inflammatory cells from the bloodstream is a very rapid process, and selectins represent a class of cell adhesion molecules that are specialized for this purpose. 4 We hypothesize that these selectins, particularly up-regulated L-selectin, act as a lymphocyte-homing receptor and play a critical role in the entry of these cells into inflamed tissues. The results of a recent study 25 suggested that the inhibition of selectin binding to ligands suppressed the inflammatory response and consequently reduced the number of lymphocytes in the lung interstitium in experimental HP induced by Saccaropolyspora rectivirgula. To date, there are no studies in the human disease. Our results confirmed an increase of soluble L- selectin in BAL fluid and an up-regulation in lung tissue in patients with HP. This selectin is expressed mainly by macrophages but also by polymorphonuclear cells. Additionally, a low percentage of L- selectin-bearing T lymphocytes in the BAL fluid of HP patients compared with EAS HCs was found. Similar findings recently have been reported 26 in active pulmonary sarcoidosis, which is also a T- lymphocyte granulomatous alveolitis. Kaseda et al 26 showed raised levels of soluble L-selectin in the BAL fluid of sarcoidosis patients compared with those in idiopathic pulmonary fibrosis patients and healthy subjects. However, a lower percentage of L-selectinpositive T lymphocytes in BAL fluid also was observed, suggesting that the shedding of L-selectin occurs when T lymphocytes migrate from the circulation into the lungs of sarcoidosis patients. 26 In HP patients, we found that E-selectin and Figure 5. Hypothetical scheme of the cellular and molecular events taking place during lung inflammation. The synchronized increased expression of proinflammatory cytokines (TNF- ), chemotactic factors (IL-8 and MIP- ), and adhesion molecules (selectins) enhanced the traffic of T cells from the circulation to the interstitial and alveolar spaces. P-selectin in BAL fluid displayed no differences with the levels found in HCs. The absence of expression of lung endothelial P-selectin may be due at least partially to the chronic state of disease in the patients we studied. P-selectin plays a major role in the early inflammatory response and is primarily responsible for targeting neutrophil transmigration at the endothelial borders. 13 This member of the selectin family might be up-regulated in acute cases of HP in which there is also a noteworthy increase in the number of neutrophils. 27 A small but significant increase in the number of neutrophils is also observed in patients with subacute/chronic HP. 18 L-selectin also has been implicated in leukocyte sequestration throughout lung capillaries. 28 Conversely, the levels of P-selectin and E-selectin were increased in the serum of patients with subacute/chronic HP, while L-selectin showed no increase. These increases in the levels of P-selectin and E-selectin may reflect endothelial activation. E-selectin was found in the endothelial cells of small vessels from the tissue of HP patients, suggesting that E-selectin also may participate in the recruitment of inflammatory cells from the bloodstream. L-selectin is lost rapidly from the surface of normal leukocytes after cellular activation, yielding a soluble fragment. Soluble L-selectin can be detected in the serum of healthy individuals at high concentrations and may increase or decrease in certain disease states. 4 The physiologic significance of shedding remains unclear but may be a mechanism of down-regulating adhesion following firm attachment to the endothelium, 4 or L-selectin may regulate the leukocyte rolling velocity. 29 CHEST / 121 / 2/ FEBRUARY,

7 Finally, although a tendency for correlations among the levels of soluble L-selectin, the levels of TNF-, and the percentage of lymphocytes in BAL fluid was observed, the results did not reach significance, perhaps because of the small number of patients. It is known that TNF- promotes the adhesion, activation, and migration of circulating leukocytes by inducing the expression of adhesion molecules and chemoattractants. 30 In summary, our results suggest that during the development of HP there is an up-regulation of lung L-selectin, which may contribute to the persistence of lymphocytic inflammation observed in patients with HP (Fig 5). ACKNOWLEDGMENT: The authors thank Miss Guadalupe Hiriart for her excellent technical support. References 1 Frenette PS, Wagner DD. Adhesion molecules: part I. N Engl J Med 1996; 334: Frenette PS, Wagner DD. Adhesion molecules: part II. Blood vessels and blood cells. N Engl J Med 1996; 335: Vestweber D, Blanks JE. 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