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* From the Department of Pneumonology (Drs. Antoniou, Tsiligianni, Tzanakis, and Siafakas), University of Crete, Heraklion, Greece; the Department of Hematology (Dr. Kyriakou), University of Thessaly, Larissa, Greece; the Interstitial Lung Disease Unit (Dr. Tzouvelekis), Imperial College, Faculty of Medicine, London, UK; and the Department of Pneumonology (Dr. Bouros), Democritus University of Thrace, Alexandroupolis, Greece.
Correspondence to: Demosthenes Bouros, PhD, FCCP, Professor of Pneumonology, Head, Department of Pneumonology, Medical School University of Thrace, Alexandroupolis 68100, Greece; e-mail: bouros{at}med.duth.gr
Abstract
Background: Sarcoidosis is thought to be a T-helper type 1 cytokine-mediated disorder. Sputum induction has been proposed as a useful noninvasive method mainly for the assessment of airway diseases. However, it is unknown whether the balance of T-cytotoxic (Tc1) type 1 and Tc2 cells is altered in sarcoidosis.
Study objectives: The primary aim of this study was to characterize the CD8+ T lymphocyte subpopulations in induced sputum from sarcoidosis patients, and to compare these subpopulations to those found in BAL fluid (BALF) from sarcoidosis patients. To further investigate the mechanism of the cytotoxic activity of CD8+ lymphocytes, we measured their perforin expression. Additionally, two adhesion molecules (CD62 and CD71), which are expressed on CD8+ T cells and may serve as novel immunologic markers, were detected.
Settings: Department of Thoracic Medicine, University of Crete, and Department of Pneumonology, Democritus University of Thrace, Alexandroupolis, Greece.
Patients: We prospectively studied 22 patients with sarcoidosis (median age, 48 years; age range, 25 to 65 years) and 10 healthy subjects (5 female and 5 male; median age, 39 years; age range, 26 to 60 years).
Interventions: The stimulation of lymphocytes with phorbol 12-myristate 13-acetate was followed by the use of double immunocytochemical methods to identify CD8+ interferon (IFN)-
producing cells (ie, Tc1) and CD8+ interleukin-4 producing cells (ie, Tc2).
Measurements and results: We found a significant decrease in the prestimulation percentage of IFN--positive CD8+ T cells in the BALF (p = 0.001) and induced sputum (p = 0.001) of sarcoidosis patients compared to the number in samples from healthy control subjects. However, no significant difference was documented between lymphocyte subsets poststimulation. Decreased levels of perforin expression were found in BALF (p = 0.001) and induced sputum (p < 0.001) of sarcoidosis patients compared to those in control subjects. The adhesion molecules were significantly increased in both the BALF and induced sputum of the sarcoid population compared to those in healthy control subjects.
Conclusions: Our results suggest that inflammation could be effectively and noninvasively determined by using sputum induction in sarcoidosis patients. In addition, we have provided evidence suggesting the possibility that CD8+ lymphocytes might not play a major role in sarcoidosis.
Key Words: BAL fluid • CD62 • CD71 • induced sputum • pathophysiology • perforin • sarcoidosis • Tc1/Tc2 profile
Sarcoidosis is a chronic, systemic disease of unknown etiology, which is characterized by the formation of noncaseating granulomas in affected organs, most commonly the lung. According to the American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and Granulomatous Disorders statement on sarcoidosis,1 the diagnosis is based on clinical and radiologic findings, and on histologic evidence. The hallmark of pulmonary sarcoidosis is a mononuclear alveolitis, which is characterized by activated CD4+ memory lymphocytes.2 An imbalance in the expression of T-helper (Th) type 1 and Th2 type cytokines is thought to play an important role in the immunopathogenesis of this disease.123 There have been several studies45678 conducted regarding the Th1/Th2 balance in BAL fluid (BALF) or blood samples from sarcoidosis patients. Our study group has recently demonstrated predominant Th1 response in induced sputum. Additionally, we have detected a positive correlation between CD4+ interferon (IFN)--positive cells in induced sputum and BALF in the same group of sarcoidosis patients.9
On the basis of their cytokine profiles, CD8+ T cells (ie, T-cytotoxic [Tc] cells) are further classified into two distinct effector cell types, Tc1 and Tc2 cells.1011 Tc1 cells characteristically produce type 1 cytokines, whereas Tc2 cells secrete type 2 cytokines. Thus, a polarized Tc1/Tc2 dichotomy is thought to be a critical determinant of immune responses under certain pathologic conditions, as well as of an imbalance in the Th1/Th2 ratio. Increasing evidence suggests that the alteration of the Tc1/Tc2 balance could be implicated in the pathogenesis of different diseases.1213 Possibly, an imbalance of Tc1/Tc2 cells may also be involved in sarcoidosis. The first study investigating the CD8+ T-lymphocyte subpopulations in sarcoidosis was carried out by Baumer and coworkers.7 They reported that cloned CD8+ T cells that had been obtained from the lung parenchyma of sarcoid patients had no prevalence of type 1 or type 2 cytokine profiles, whereas CD4+ T-cell clones shifted to a type 1 profile.7 In agreement with this, a second study4 showed that the Tc1/Tc2 balance was not changed in the BALF and peripheral blood of patients with sarcoidosis. While studies47 in sarcoidosis patients have not identified a Tc1/Tc2 imbalance in tissue or BALF, the current study may still be a significant contributor because we aimed to investigate CD8+ cytotoxicity with the noninvasive method of sputum induction and by using different immunologic markers.
In detail, to further investigate the mechanism of the cytotoxic activity of CD8+ lymphocytes, we also measured their perforin expression in both biological fluids. Perforin-mediated cytotoxicity has been reported1415 to be involved in various disorders. The role of perforin has not yet been investigated in patients with pulmonary sarcoidosis. It has been reported16 that perforin-mediated apoptosis may be associated with the pathophysiology of lung injury and fibrosis. Furthermore, our group stated that the CD8+ lymphocytes of patients with COPD showed an increased expression of intracellular perforin compared to smokers without COPD and healthy nonsmokers.17
Additionally, the underlying mechanisms of lymphocyte accumulation in the lung are not fully understood. Although it is likely that a number of adhesion molecules are involved in this process, such as selectins (eg, L-selectin [CD62], which is a lymphocyte adhesion molecule, and transferrin receptor [CD71], which is a monocyte/macrophage marker), integrins, and the Ig superfamily.181920 In the current study, we aimed to explore the expression of the aforementioned CD8+ T-cell markers in BALF and induced sputum. L-selectin, which is a member of the selectin family, is a highly glycosylated protein of 95 to 105 kd on neutrophils and 74 kd on lymphocytes, and is constitutively expressed on most leukocytes.18 L-selectin has been reported1920 to be present in high concentrations in the serum and BALF of patients with sarcoidosis relative to those of healthy subjects and patients with idiopathic pulmonary fibrosis (IPF). However, in early disease there is a mononuclear cell alveolitis that is dominated both by T cells and macrophages. In order to further elucidate the suggested enhanced capacity of alveolar macrophages as efficient antigen presenters in sarcoidosis, transferrin receptor (ie, CD71) expression was investigated. Prominent changes in the alveolar macrophage phenotype were found in patients with active sarcoidosis showing lymphocytic alveolitis, with more frequent expression of CD54, KiM2, CD71, CD11b, and RFD9 in bronchoalveolar cells.21 In summary, we attempted to investigate T cell phenotype (ie, CD62 cells) in parallel with monocyte/macrophage phenotypic characteristics (ie, CD71 cells) in the CD8+ subpopulation in cells in both BALF and induced sputum.
The role of induced sputum has been investigated in patients with sarcoidosis, COPD, and, more recently, IPF by our study group.222324 We have previously shown that inflammation in sarcoidosis could be effectively and noninvasively investigated by the analysis of cell differential counts and T-lymphocyte subsets in induced sputum.22 In line with this, we have recently demonstrated9 a Th1-like cytokine pattern in CD4+ T lymphocytes in the BALF and induced sputum of sarcoidosis subjects.
On the basis of our previous reports, the primary aim of this study was to shed further light on the induced sputum CD8+ T-lymphocyte subpopulation compared to CD8+ cells in BALF samples from patients with pulmonary sarcoidosis. To this end, using immunocytochemical analysis and flow cytometry, we examined the production of the intracellular cytokines IFN- and interleukin (IL)-4, and the expression of the T-cell cytotoxicity marker, perforin, in CD8+ T cells. In addition, our secondary aim was to gain a better understanding of the pathogenetic mechanisms driving the granulomatous inflammation in sarcoidosis. We therefore measured the BALF and sputum levels of adhesion molecules (ie, CD62 and CD71) in CD8+ T cells, which, it has been suggested, serve as critical mediators of the interaction between T cells and macrophages.
Materials and Methods
Subjects
Twenty-two consecutive sarcoidosis patients, 10 male and 12 female (median age, 48 years; age range, 25 to 68 years) who were investigated in the sarcoidosis clinic of our hospitals, were enrolled into the study. Two patients were smokers, and 20 patients were nonsmokers. The American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and Granulomatous Disorders statement1 on sarcoidosis was followed for the diagnosis, which was based on medical history, clinical symptoms, standard chest radiography findings, CT scan findings, lung Ga67 scintigraphy findings, and the results of laboratory tests (eg, for serum angiotensin-converting enzyme [ACE] levels). All of them underwent transbronchial or open lung biopsy, showing histopathologic evidence of noncaseating epithelioid cell granulomas. According to the chest radiography classification of sarcoidosis, nine patients had stage I disease (ie, lymphadenopathy alone), seven patients had stage II disease (ie, lymphadenopathy and parenchymal opacities), and six patients had stage III disease (ie, only parenchymal opacities).
The control group included 10 healthy volunteers who were nonsmokers (5 female and 5 male; median age, 39 years; age range, 26 to 60 years) and were able to produce adequate sputum following sputum induction. Patients and control subjects who had experienced acute respiratory infections during the 6 weeks prior to enrolling in the study were excluded. The Hospital Ethical Committee approved the protocol, and all patients and control subjects gave their consent.
Spirometry
Spirometry was performed with a computerized system (Master Lab, 2.12; Jaeger; Würzburg, Germany). The measurement was performed using standard protocols according to American Thoracic Society guidelines.25
Sputum Induction and Processing
Sputum was induced by the inhalation of a hypertonic aerosol saline solution, which was generated by an ultrasonic nebulizer (Ultraneb 2000; DeVilbiss; Somerset, PA) according to the guidelines of previous reports.262728 Sputum was processed within 1 h after the termination of the induction. The standard method of sputum examination was used with some modifications.222324
BAL and Processing
Fiberoptic bronchoscopy with BAL was performed within 2 weeks from induced sputum examination, as part of routine clinical management, according to the recommended guidelines and previous reports.29 The BALF processing was previously described.22
Flow Cytometric Analysis of Induced Sputum and BALF
The samples (ie, sputum and BALF cells) were prepared as described above and were analyzed on a fluorescence-activated flow cytometer (Epics Elite; Coultronics; Louton, UK), as previously described.222324
Immunocytochemical Analysis
The double immunocytochemical analysis and T-cell determination were performed in two steps, as previously described.930 In the current study, the CD8+-IFN-/CD8+-IL-4 ratio was calculated.
Measurement of CD8+-IFN- and CD8+-IL-4 Producing T Cells, Perforin, and Adhesion Molecules
The following mouse-human monoclonal antibodies were used for labeling sputum and BALF lymphocytes: the primary anti-CD8+ mouse antihuman monoclonal antibody with secondary rabbit antimouse IgG-fluorescein isothiocyanate antibody and the primary anti-IFN- mouse antihuman monoclonal antibody (Caltag; Burlingame, CA) with secondary rabbit antimouse IgG-phycoerythrin-conjugated antibody were used. At least 500 CD8+ cells were counted to estimate the number of CD8+-IFN- cells. Also the same method was applied for staining CD8+-IL-4 cells with anti-IL4 antibody (Caltag).
In addition, we have used an unlabeled antihuman perforin for intracellular perforin detection. For intracellular perforin staining, CD8+ cells were subsequently incubated in a permeabilization reagent (Intraprep; Beckman Coulter; Fullerton, CA) for 10 min. As a control, unlabeled antiperforin at molar excess was used after permeabilization, and then antiperforin fluorescein isothiocyanate was added without washing and was incubated for 30 min at room temperature (competitive labeling). After staining, the samples were washed with phosphate-buffered saline solution plus 10% fetal calf serum and were immediately analyzed using flow cytometry. Furthermore, unlabeled antihuman CD62 and CD71 were used for intracellular detection of the adhesion molecules.
Statistical Analysis
All analyses were performed using a statistical software package (StatsDirect for Windows, version 2.4.1; Camcode; Cambridge, UK). The results are expressed as the mean ± SD or median (range), unless otherwise indicated. The Shapiro-Wilk W test for normality was applied to assess normality. Differences between sarcoidosis patients and control subjects were tested using the t test for normally distributed data, and using the Mann-Whitney U test for nonnormally distributed data. Differences between BALF and sputum within groups were tested using the paired Student t test for normally distributed variables and the Wilcoxon signed rank test for nonnormally distributed variables. A p value of < 0.05 was considered to be statistically significant.
Results
The demographic and clinical characteristics of the patients and control subjects are shown in Table 1 . Patients with sarcoidosis were older than healthy control subjects and had lower FVC and FEV1 values, but the differences were not statistically significant. Sputum induction was well-tolerated by all subjects, without any adverse events.
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producing CD8+ T cells was significantly lower in patients with sarcoidosis than in healthy control subjects (11.8 ± 6.7% vs 23.7 ± 2.3%, respectively) before the stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Fig 1
). Similarly, in induced sputum the mean percentage of IFN- producing CD8+ T cells was significantly lower in patients with sarcoidosis than in healthy control subjects (6.0 ± 2.6% vs 13.4 ± 5.6%, respectively; p = 0.001) before the stimulation (Fig 2
).
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To the best of our knowledge, this is the first study to investigate the role of CD8+ lymphocytes and their subtypes, Tc1 and Tc2 cells, in induced sputum cells of patients with pulmonary sarcoidosis. Our novel finding is a down-regulation of perforin expression in two different biological fluids, whereas the levels of both adhesion molecules were significantly elevated in sarcoid cases compared to those of healthy subjects.
In the present study, a method of inducing sputum was used to investigate CD8+ T lymphocytes in sarcoidosis patients and to compare them with those of healthy control subjects. We have demonstrated that a Tc1-like cytokine pattern was significantly decreased in cells in BALF and induced sputum before stimulation with PMA/ionomycin in comparison with those in healthy subjects. Inui and coworkers4 also detected small percentages of the cytokine-producing cells, the proportions of which in BALF samples were not statistically significant between sarcoidosis patients and healthy subjects.
We subsequently stimulated lymphocytes with PMA and ionomycin in order to obtain precise information on the immune status of patients with sarcoidosis. This method enabled us to study cells with little in vitro incubation, decreasing the potential of artifacts arising from prolonged culture.931 PMA is a potent stimulator of CD8+ cells, which enhances their ability to express IL-4 or IFN-.32 Thus, it is possible to detect important effector differences in cytokine regulation that are not detectable otherwise.43133 On stimulation with PMA/ionomycin, no difference was found in the proportions of cytokine-producing CD8+ T cells (Tc1 and Tc2) either in induced sputum or BALF, between the patients and healthy subjects. In agreement with our findings, previous studies in lung parenchyma,7 BALF, and peripheral blood4 showed that the Tc1/Tc2 balance was not changed in patients with pulmonary sarcoidosis. To the best of our knowledge, this is the first time that these parameters have been evaluated in induced sputum. Our results are that Tc1 cells might not play a major role in sarcoidosis.
Moreover, the cytotoxicity of CD8+ T lymphocytes, as reflected by the perforin expression profile, was investigated. We have previously shown in COPD patients that perforin expression in CD8+ cells was significantly higher in smokers with COPD compared to those without COPD (p = 0.01) and to healthy nonsmokers (p = 0.001),17 indicating that cigarette smoking could up-regulate perforin. On the other hand, there is a known negative association between cigarette smoking and sarcoidosis.3334 Further studies should explore the expression of perforin in smokers with pulmonary sarcoidosis in order to elucidate the possible protective effect of smoking in sarcoidosis patients. Unfortunately, this was not possible in the current study, as our sarcoidosis population included only two smokers.
In addition, it has been shown16 that perforin-mediated apoptosis may be associated with the pathophysiology of lung injury and fibrosis. The differential expression of perforin in patients with sarcoidosis and IPF could partially explain the major differences regarding the pathogenesis, prognosis, and clinical course of the aforementioned disorders. Future investigations are warranted to elucidate the pivotal role of T-cell cytotoxicity markers in the immunomodulatory cascade of sarcoidosis and to define whether this event is disease-specific or is associated with other interstitial lung diseases.
It has been speculated that the granulomatous inflammation in sarcoidosis is driven by the coordinated interplay between T cells and macrophages.35 Several mediators of cell-cell and cell-matrix adhesion, including selectins, integrins, and members of the Ig family, have been proposed to function as contributors in the process of T-lymphocyte alveolitis in sarcoidosis patients.192036 Ishii and Kitamura36 reported elevated serum and BALF concentrations of soluble adhesion molecules in sarcoidosis patients, which was further supported by two other groups of investigators.1920 In addition to this, lymphocytic alveolitis in patients with active sarcoidosis is associated with prominent changes of the alveolar macrophage phenotype, and the more frequent expression of CD71 and other molecules.213738 However, the underlying mechanisms of the lymphocyte accumulation in the lung and the formation of noncaseating granulomas are still under consideration and deserve further investigation. Based on the same perspective idea and to gain a holistic view in the local expression of surface molecules, we examined for the first time the activation of a lymphocyte adhesion molecule (L-selectin CD62) and a monocyte/macrophage marker (transferrin receptor CD71) both in BALF and sputum samples of sarcoidosis patients. Intriguingly, we found increased levels of both markers in BALF and induced sputum, corroborating and further extending previous findings. Nonetheless, our study demonstrated no significant association of L-selectin and CD71 concentrations with clinical laboratory parameters of sarcoid disease activity, including ACE concentrations. However, this observation does not diminish the value of our data since they are in agreement with those of earlier studies,1920 and the number of parameters used to assess sarcoid disease activity is conventional and exhibits major limitations.39 Future studies should investigate whether local levels of adhesion factors probe a crucial aspect of inflammatory activity and therefore reflect the disease severity.
Today’s attitude and the opinion of researchers is that sputum induction is a promising technique in assessing interstitial lung diseases, but its diagnostic role has not yet been clarified.4041 Taking into consideration the data of this study and previously mentioned studies,922 we suggest that inflammation as well as the immunologic response in sarcoidosis patients could be effectively and noninvasively determined by using the method of sputum induction. This technique should be added to those for the evaluation and follow-up of patients with sarcoidosis.
In conclusion, our results suggest that the balance of Tc1/Tc2 cells in the induced sputum and BALF of sarcoidosis patients is not altered in those with pulmonary sarcoidosis, as is reflected in our poststimulation data. Furthermore, we demonstrated that perforin is down-regulated and that both lymphocytic and macrophage adhesion molecules are overexpressed in sarcoidosis patients, indicating a potential involvement of these parameters in T lymphocyte alveolitis in sarcoidosis subjects. These findings provide us with novel pathogenetic insights, illuminating the role of T cells and macrophages in sarcoidosis and highlighting potential therapeutic targets. Future studies are required to validate these findings in different stages of disease activity, accelerating the molecular profiling of sarcoidosis and elucidating the regulatory immune mechanisms that govern matrix modifications in patients with this disease.
Footnotes
Abbreviations: ACE = angiotensin-converting enzyme; BALF = BAL fluid; IFN = interferon; IL = interleukin; IPF = idiopathic pulmonary fibrosis; PMA = phorbol 12-myristate 13-acetate; Tc = T-cytotoxic type; Th = T-helper
Received for publication July 2, 2005. Accepted for publication December 16, 2005.
References
This article has been cited by other articles:
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  K. M. Antoniou, A. Tzouvelekis, M. G. Alexandrakis, K. Sfiridaki, I. Tsiligianni, G. Rachiotis, N. Tzanakis, D. Bouros, J. Milic-Emili, and N. M. Siafakas Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis. Chest, October 1, 2006; 130(4): 982 - 988. [Abstract] [Full Text] [PDF]
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