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Intracellular cytokine profiles and T cell activation in pulmonary sarcoidosis
Cytokine 42 (2008) 289–292
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Short Communication
Intracellular cytokine profiles and T cell activation in pulmonary sarcoidosis T.A. Hill a,*, S. Lightman b, P. Pantelidis d, A. Abdallah c, P. Spagnolo c, R.M. du Bois d a
Department of Clinical Ophthalmology, Institute of Ophthalmology, University College London, London, UK Moorfields Eye Hospital, London, UK Imperial College of Science, Technology and Medicine, London, UK d Department of Medicine, National Jewish Medical and Research Center, Denver, Colarado, USA b c
a r t i c l e
i n f o
Article history: Received 3 September 2007 Received in revised form 11 March 2008 Accepted 31 March 2008
Keywords: Cytokines Flow cytometry Pulmonary Sarcoidosis T cells Intracellular
a b s t r a c t In granulomatous inflammatory lung diseases such as sarcoidosis, the balance of cytokine production by activated T cells in the lungs may influence clinical disease outcome. To investigate the potential of T lymphocytes to produce cytokines and contribute to this process, T cells from bronchoalveolar lavage (BAL) and PB from 19 patients with active lung disease were stimulated, stained, and analysed by flow cytometry for intracellular production of cytokines and expression of the activation marker CD69. Higher proportions of BAL cells expressed CD69 compared with PB, in the absence of in vitro stimulation. The expression of IFN-c was similar in unstimulated BAL and PB T cells, and there was no association between the expression of CD69 and IFN-c. Following stimulation, there were increased numbers of IFN-c+ T cells. A similar trend was found with IL-2+ T cells, but there were lower levels of IL-4+ T cells in BAL compared with PB, and similar levels of IL-10+ T cells. The presence of activated T lymphocytes in BAL samples from patients with sarcoidosis, with the potential to produce Th1 type 1 cytokines may contribute to the inflammatory processes in this granulomatous lung disease. The use of intracellular flow cytometry to investigate cytokine production by BAL T cells could help to indicate potential targets for future therapy. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction A feature of pulmonary sarcoidosis is the presence of lymphocytes in the interstitial inflammatory cell infiltrates, which are key effector cells in the development of granulomas. Progressive granuloma formation resulting from persistent T cell activation can lead to severe respiratory impairment. Bronchoalveolar lavage (BAL) samples used to clinically assess interstitial lung disease have also been used to investigate cellular infiltrates, and cytokine profiles of T cells. The type of cytokine profiles that follow an immune response could influence the clinical course of disease leading to either resolution or progression to end-stage pulmonary fibrosis. Analysis of T cell populations found in BAL fluid of patients with sarcoidosis has shown evidence of increased T cell activation as measured by expression of HLA-DR and CD25 [1] or cytokine and chemokine receptors [2]. Classification of T cell subsets is possible by characterisation of their cytokines [3]. A Th1 type profile is characterised by produc-
* Corresponding author. Present address: Department of Primary Care and Population Sciences, Royal Free and University College London, Royal Free Campus, Rowland Hill St. London NW3 2PF, UK. Fax: +44 (0)20 7794 1224. E-mail addresses: [email protected] (T.A. Hill), [email protected] (S. Lightman), [email protected] (R.M. du Bois). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.03.014
tion of pro-inflammatory cytokines such as IFN-c and IL-2, whereas IL-4 production influences the development of a Th2 response [4]. IL-10 is produced by Th1, Th2, and Th0 subsets [5] and is thought to have a role in regulation of Th1 responses [6]. The influence and contribution of T cell cytokines to the pattern of cytokine production in inflammatory lung diseases is becoming clearer, and studies investigating T lymphocytes from patients with pulmonary sarcoidosis at the single cell level have established that the cytokine profile of both CD4+ and CD8+ T cells is of a Th1 type [7–9]. Interleukin-10 is not often investigated as part of the cytokine profile, although one study found an increase in IL-10+ T cells in only the CD8 subset and only in patients with acute sarcoidosis [10]. In order to supplement current understanding of the phenotypic characteristics of BAL T cells [11], our approach was to use BAL samples collected as part of routine clinical assessment, and to focus specifically on T lymphocytes to measure intracellular production of IFN-c, IL-2, IL-4, and IL-10. In addition to this, we also measured the activation status of the T cells and investigated whether this was related to the potential of these cells to produce Th1 type cytokines. Furthermore, we examined whether the cytokine profiles of BAL T cells from patients with prior therapy were different to those from patients who had not received therapy.
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T.A. Hill et al. / Cytokine 42 (2008) 289–292
2. Materials and methods 2.1. Patient details Samples of BAL and peripheral blood (PB) from 19 patients with active sarcoidosis were analysed. At the time of lavage, 12 of the patients were known to be off treatment, and 4 were taking prednisolone. Details of treatment at the time of lavage for the remaining 3 patients were unavailable. Although BAL cells from healthy individuals or those with non-inflammatory conditions may contain enough cells for examination, we felt it would not enhance our study to additionally use healthy controls as a comparison, and it is also well documented that BAL cells from patients with sarcoidosis contain a higher proportion of cytokine expressing T cells compared with healthy controls [8,12]. 2.2. Preparation and stimulation of BAL and peripheral blood mononuclear cells (PBMC) BAL was performed as part of routine clinical evaluation, with bronchoscopy performed and BAL fluid processed as previously described [13]. Cells in BAL fluid were collected by centrifugation at 300g for 5 min at 4 °C, washed three times with cold minimal essential medium (MEM) containing 25 mM Hepes buffer, and adjusted to 1.0 106 cells/ml. PBMC were isolated by density gradient centrifugation on Histopaque (Sigma), washed and also adjusted to 1.0 106 cells/ml. BAL cells and PBMC were stimulated for cytokine production with 50 ng/ml PMA (Sigma), 1 lg/ml ionomycin (Sigma) and 20 lg/ml Brefeldin A (BFA) (Sigma) for 18 h at 37 °C in a 5% CO2 atmosphere. BFA disrupts intracellular protein transport [14] causing cytokines to accumulate within cells, producing an enhanced cytokine signal detectable by flow cytometry. 2.3. Intracellular cytokine staining and flow cytometry After stimulation, BAL cells and PBMC were transferred to microcentrifuge tubes, washed by adding RPMI and cells pelleted by centrifugation for 5 min at 600g. Cells were resuspended in 50 ll RPMI and aliquots stained for surface antigens and intracellular cytokine using fluorochrome-conjugated antibodies against cell surface antigens (CD3-PerCP or CD8-PerCP, Becton Dickinson (BD) Oxford, UK) for 15 min at room temperature in the dark. Following a wash with 200 ll FACSFlow (BD), the cell pellet was resuspended in 100 ll Cytofix/Cytoperm reagent (BD) and incubated for 20 min at 4 °C. After washing with Perm/Wash buffer (BD), cells were pelleted, resuspended in 50 ll Perm/Wash buffer, then labelled for 30 min at 4 °C with combinations of fluorochrome-conjugated anti-cytokine antibodies: anti-human IFN-c-FITC plus anti-human IL-10-PE and anti-human IL-4-FITC plus anti-human IL-2-PE (BD). Antibody to CD69 used to check for expression of an early cell activation marker was added to cells after permeabilization as BFA inhibits the cell surface expression of CD69 [15]. Following washing with Perm/ Wash buffer, the cells were resuspended in 200 ll FACSFlow for flow cytometric analysis. Controls included cells labelled with isotypematched irrelevant mAbs, and non-activated PBMCs stained with the same antibodies. As only small samples of BAL cells were available for this study (1 106 cells), we were limited to the following antibody and surface marker combinations: CD3/IFN-c/IL-10 and CD8/IL-4/IL-2. Cells from BAL samples and PB were analysed using a flow cytometer with Cell Quest software (FACScan, BD). 2.4. Statistical analysis Differences between BAL and PB samples, or between groups in the mean percentages of cytokine positive T cells were determined
by Wilcoxon or Mann–Whitney t-tests for non-parametric data as appropriate. Any association between the percentages of CD69+ and IFN-c+ T lymphocytes was determined by Spearman rank correlation. A p-value of <0.05 was considered significant. 3. Results The BAL samples were assessed microscopically to contain 39% lymphocytes (mean ± SD, 39 ± 13). When unstimulated BAL and PB T lymphocytes from patients with sarcoidosis were examined for CD69 expression, there was a significant difference between BAL and PB (BAL 30%, PB 9%, p = 0.01) (Fig. 1). In contrast with CD69+ T cells, there was no difference between unstimulated BAL and PB cells in the numbers of IFN-c+ T cells in these patients, and most samples contained <10% IFN-c+ T cells (data not shown). There was also no difference between BAL and PB in the proportion of CD3+/ CD8 T cells (mean + SD, BAL: 31 ± 28, PB: 36 ± 21, p = 0.30). To produce intracellular cytokine levels detectable by flow cytometry, BAL and PB T cells were stimulated with PMA and ionomycin in the presence of BFA. Fig. 2 illustrates results from stimulated samples from patients with sarcoidosis, and shows that there was a similar proportion of CD69+ T cells in BAL (79%) and PB cells (75%) from patients with sarcoidosis (p = 0.3). However, the proportion of T cells producing IFN-c was higher in BAL (71 ± 30%) compared with PB (29 ± 15%, p = 0.001). Levels of IL-2+ T cells were also higher in BAL (28 ± 26%) compared with PB (19 ± 20%), although this difference did not reach significance (p = 0.08). With IL-4+ T cells, it was found that these were lower in BAL (0.9 ± 0.7%) than in PB (1.7 ± 1.2%, p = 0.03). There was no difference in the levels of IL10+ T cells in BAL (0.9 ± 0.7%) and PB (1.3 ± 1.1%, p = 0.16). In the absence of in vitro stimulation, 30% of BAL T lymphocytes were shown to be activated by the expression of CD69, with 5/19 samples containing >75% CD69+ T cells. However, there was no association between expression of CD69 and IFN-c in BAL samples (r = 0.2, p = 0.4). Since BAL samples from sarcoidosis patients were shown to contain increased levels of IFN-c-producing T lymphocytes, the activation status of T lymphocytes together with their ability to produce IFN-c were examined in relation to whether or not the patients were on or off immunosuppressive treatment at the time of BAL. Although the number of sarcoidosis patients with treatment prior to BAL was small (n = 12), there was no apparent reduction in either the number of activated T lymphocytes, as judged by their expression of CD69, or the number of IFN-c+ T cells (data not shown). It is possible however, that the effect of any immunosuppressive drugs on cytokine production declined during
Fig. 1. CD69 expression on unstimulated BAL and PB T cells. Unstimulated and stimulated BAL and PB T cells from patients with sarcoidosis were examined for expression of CD69. Unstimulated BAL T cells contained a higher percentage of CD69+ T cells compared with paired PB (*p = 0.006). There was no such difference between stimulated BAL and PB samples. (, unstimulated BAL; e, unstimulated PB; d, stimulated BAL; s, stimulated PB.)
T.A. Hill et al. / Cytokine 42 (2008) 289–292
Fig. 2. Comparison of intracellular cytokine expression in sarcoidosis BAL and PB T cells. BAL and PB T cells from patients with sarcoidosis were stimulated and stained for intracellular cytokine expression. A higher percentage of BAL T cells were IFN-c+ (*p = 0.001), but a lower percentage were IL-4+ (**p = 0.03) compared with PB. Expression of intracellular IL-2 and IL-10 were similar in paired samples from sarcoidosis patients (, d, BAL; e, s, PB).
the time taken to prepare and stimulate BAL cells before detection of cytokines, or that the continual presence of drug is required to decrease cytokine production. 4. Discussion This study used intracellular flow cytometry to determine T cell cytokine profiles of routinely acquired BAL cells, thereby avoiding additional invasive clinical procedures. We aimed to extend previous findings which showed a predominantly type 1 cytokine profile of cells infiltrating the pulmonary interstitium of patients with sarcoidosis [8–10]. We also determined production of IL-10 by BAL and PB T cells, investigated cytokine production in relation to the activation status of T lymphocytes, and examined whether or not cytokine production was affected depending on a patient’s treatment prior to BAL. The investigation was designed to compare the activation and T cell cytokine profiles in the two different compartments, the lungs and peripheral blood. In patients with sarcoidosis it is known that CD69 on BAL T lymphocytes is over expressed compared with PB [11]. We found that BAL cells left unstimulated contained higher numbers of activated T cells compared with PB, indicating that it is feasible that these activated T cells could produce both inflammatory and possibly anti-inflammatory cytokines [5,16]. Our finding of increased proportions of IFN-c-producing BAL T cells from patients with sarcoidosis is in accordance with previous reports of elevated levels of IFN-c and IL-2 but not IL-4 or IL-5 in BAL fluid from sarcoidosis patients [17–20]. This finding indicates the selective entry into the lung of T cells with the potential to regulate inflammation, and the identification of T cells as a potential cellular source of the IFN-c found in BAL fluid. Production of IFN-c during a type 1 T cell response, at least in the earlier disease stages of sarcoidosis, would favour macrophage activation, the formation of granulomas and an inhibition of fibrogenetic processes [7,21]. Production of IFN-c may be beneficial as IFN-c levels in BAL fluid are inversely related to the progression of pulmonary fibrosis [22], although levels would have to be sufficient to balance and overcome the effects of any type 2 cytokines that might be produced, to influence the clinical outcome of pulmonary inflammation. We found relatively low but similar percentages of IL-10+ T cells in both BAL and PB, whilst a previous study demonstrated that BAL fluid IL-10 levels or cellular IL-10 mRNA levels were higher in sarcoidosis compared with healthy controls [12,23]. However, our study design was different in that it focussed on T cell cytokine production rather than measuring cytokines in BAL fluid or mRNA
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produced by a variety of cell types present in BAL fluid, which could account for the disparity in results. During the disease process, alteration of the balance of cytokine production may contribute to a more adverse outcome depending on the T cells present, and also on the ratio of cytokines that these and other cells secrete [24]. If the balance of T cells producing different cytokine profiles could be altered in favour of IL-10-producing T cells, then this could provide some beneficial regulation of the immune response [6]. The use of corticosteroid therapy in sarcoidosis is effective and can reduce levels of IFN-c in BAL fluid to undetectable levels after 2–3 months’ treatment [20]. This suggests that despite the infiltration or proliferation of type 1 T cells in the lung, corticosteroids are probably effective at regulating the immune response, and help to reduce the influence of type 1 T cell cytokines and may help restore the balance of type 1 and type 2 cytokine-producing T cells present in the lung during disease. Our study was unable to find any differences between patients off treatment at time of lavage and those receiving treatment prior to lavage in the proportions of either activated or IFN-c+ T cells, and further studies would be required to confirm this observation. We have demonstrated that intracellular flow cytometry can be applied to BAL cells harvested for routine clinical diagnostic purposes, to identify populations of cytokine-producing cells in the lung in pulmonary inflammation and fibrosis. Intracellular flow cytometry has the advantage of being able to determine both the phenotype and the cytokine profiles of relatively small numbers of BAL cells. This technique could be easily modified to further identify subpopulations of T lymphocytes or other leucocytes involved in the immune regulation of inflammatory lung diseases, or other Th1 type cytokines such as IL-12 or IL-18 [25]. Intracellular flow cytometry could also be used to determine patterns of T cell activation using different cellular markers as has been studied in pulmonary fibrosis associated with systemic sclerosis [26] and to relate this to cytokine profiles, disease stage and severity, which would require further study. We suggest that the intracellular flow cytometric method has the advantage of utilising routinely collected BAL samples to study cytokine profiles in the lung in sarcoidosis or other diseases where there is pulmonary inflammation and fibrosis. It can also be used to identify specific populations of cytokine-producing cells and to examine the activation status of cells involved in the immune response. We have successfully used this technique to specifically study and identify T lymphocytes as potential sources of cytokines that could contribute to and also regulate inflammation in the lung in sarcoidosis, and which could be targets for future therapeutic intervention. Acknowledgments This work was supported by the Arthritis Research Campaign and the Moorfields NHS R&D Support Levy. References [1] Spiteri MA, Johnson M, Epstein O, Sherlock S, Clarke SW, Poulter LW. Immunological features of lung lavage cells from patients with primary biliary cirrhosis may reflect those seen in pulmonary sarcoidosis. Gut 1990;31:208–12. [2] Katchar K, Eklund A, Grunewald J. Expression of Th1 markers by lung accumulated T cells in pulmonary sarcoidosis. J Intern Med 2003;254:564–71. [3] Mosmann TR, Coffman RL. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol 1989;7:145–73. [4] Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787–93. [5] Yssel H, de waal Malefyt R, Roncarolo M-G, Abrams JS, Lahesmaa R, Spits H, et al. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J Immunol 1992;149:2378–84.
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[6] Mosmann TR, Moore KW. The role of IL-10 in crossregulation of TH 1 and TH 2 responses. Immunol Today 1991;12:A49–53. [7] Minshall EM, Tsicopoulos A, Yasruel Z, Wallaert B, Akoum H, Vorng H, et al. Cytokine mRNA gene expression in active and nonactive pulmonary sarcoidosis. Eur Respir J 1997;10:2034–9. [8] Prasse A, Georges CG, Biller H, Hamm H, Matthys H, Luttmann W, et al. Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells. Clin Exp Immunol 2000;122:241–8. [9] Wahlström J, Katchar K, Wigzell H, Olerup O, Eklund A, Grunewald J. Analysis of intracellular cytokines in CD4+ and CD8+ lung and blood T cells in sarcoidosis. Am J Respir Crit Care Med 2001;163:115–21. [10] Mollers M, Aries SP, Dromann D, Mascher B, Braun J, Dalhoff K. Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis. Thorax 2001;56:487–93. [11] Wahlstrom J, Berlin M, Skold CM, Wigzell H, Eklund A, Grunewald J. Phenotypic analysis of lymphocytes and monocytes/macrophages in peripheral blood and bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis. Thorax 1999;54:339–46. [12] Idali F, Wikén M, Wahlström J, Mellstedt H, Eklund A, Rabbani H, et al. Reduced Th1 responses in the lungs of HLA-DRB0301 patients with sarcoidosis. Eur Respir J 2006;27:451–9. [13] Wells AU, Hansell DM, Haslam PL, Rubens MB, Cailes J, Black CM, et al. Bronchoalveolar lavage cellularity: lone cryptogenic fibrosing alveolitis of systemic sclerosis. Am J Respir Crit Care Med 1998;157:1474–82. [14] Klausner RD, Donaldson JG, Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 1992;116:1071–80. [15] Nylander S, Kalies I. Brefeldin A, but not monensin, completely blocks CD69 expression on mouse lymphocytes: efficacy of inhibitors of protein secretion in protocols for intracellular cytokine staining by flow cytometry. J Immunol Methods 1999;224:69–76. [16] Paliard X, de Waal Malefijt R, Yssel H, Blanchard D, Chretien I, Abrams J, et al. Simultaneous production of IL-2, IL-4 and IFN-gamma by activated human CD4+ and CD8+ T cell clones. J Immunol 1988;141:849–55.
[17] Robinson BW, McLemore TL, Crystal RG. Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis. J Clin Invest 1985;75:1488–95. [18] Moller DR, Forman JD, Liu MC, Noble PW, Greenlee BM, Vyas P, et al. Enhanced expression of IL-12 associated with Th1 cytokine profiles in active pulmonary sarcoidosis. J Immunol 1996;156:4952–60. [19] Hoshino T, Itoh K, Gouhara R, Yamada A, Tanaka Y, Ichikawa Y, et al. Spontaneous production of various cytokines except IL-4 from CD4+ T cells in the affected organs of sarcoidosis patients. Clin Exp Immunol 1995;102:399–405. [20] Milburn HJ, Poulter LW, Dilmec A, Cochrane GM, Kemeny DM. Corticosteroids restore the balance between locally produced Th1 and Th2 cytokines and immunoglobulin isotypes to normal in sarcoid lung. Clin Exp Immunol 1997;108:105–13. [21] Agostini C, Facco M, Chilosi M, Semenzato G. Alveolar macrophage-T cell interactions during Th1-type sarcoid inflammation. Microsc Res Tech 2001;53:278–87. [22] Kuroki S, Ohta A, Sueoka N, Katoh O, Yamada H, Yamaguchi M. Determination of various cytokines and type III procollagen aminopeptide levels in bronchoalveolar lavage fluid of the patients with pulmonary fibrosis: inverse correlation between type III procollagen aminopeptide and interferon-c in progressive fibrosis. Br J Rheum 1995;34:331–6. [23] Meloni F, Caporali R, Marone Bianco A, Paschetto E, Morosini M, Fietta AM, et al. BAL cytokine profile in different interstitial lung diseases: a focus on systemic sclerosis. Sarcoidosis Vasc Diffuse Lung Dis 2004;21:111–8. [24] Katsikis PD, Cohen SBA, Londei M, Feldmann M. Are CD4+ Th1 cells proinflammatory or anti-inflammatory? The ratio of IL-10 to IFN-c or IL-2 determines their function. Int Immunol 1995;7:1287–94. [25] Antoniou KM, Tzouvelekis A, Alexandrakis MG, Tsiligianni I, Tzanakis N, Sfiridaki K, et al. Upregulation of Th1 cytokine profile (IL-12, IL-18) in bronchoalveolar lavage fluid in patients with pulmonary sarcoidosis. J Interferon Cytokine Res 2006;26:400–5. [26] Bresser P, Jansen HM, Weller FR, Lutter R, Out TA. T-cell activation in the lungs of patients with systemic sclerosis and its relation with pulmonary fibrosis. Chest 2001;120:66S–8S.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Short Communication
Intracellular cytokine profiles and T cell activation in pulmonary sarcoidosis T.A. Hill a,*, S. Lightman b, P. Pantelidis d, A. Abdallah c, P. Spagnolo c, R.M. du Bois d a
Department of Clinical Ophthalmology, Institute of Ophthalmology, University College London, London, UK Moorfields Eye Hospital, London, UK Imperial College of Science, Technology and Medicine, London, UK d Department of Medicine, National Jewish Medical and Research Center, Denver, Colarado, USA b c
a r t i c l e
i n f o
Article history: Received 3 September 2007 Received in revised form 11 March 2008 Accepted 31 March 2008
Keywords: Cytokines Flow cytometry Pulmonary Sarcoidosis T cells Intracellular
a b s t r a c t In granulomatous inflammatory lung diseases such as sarcoidosis, the balance of cytokine production by activated T cells in the lungs may influence clinical disease outcome. To investigate the potential of T lymphocytes to produce cytokines and contribute to this process, T cells from bronchoalveolar lavage (BAL) and PB from 19 patients with active lung disease were stimulated, stained, and analysed by flow cytometry for intracellular production of cytokines and expression of the activation marker CD69. Higher proportions of BAL cells expressed CD69 compared with PB, in the absence of in vitro stimulation. The expression of IFN-c was similar in unstimulated BAL and PB T cells, and there was no association between the expression of CD69 and IFN-c. Following stimulation, there were increased numbers of IFN-c+ T cells. A similar trend was found with IL-2+ T cells, but there were lower levels of IL-4+ T cells in BAL compared with PB, and similar levels of IL-10+ T cells. The presence of activated T lymphocytes in BAL samples from patients with sarcoidosis, with the potential to produce Th1 type 1 cytokines may contribute to the inflammatory processes in this granulomatous lung disease. The use of intracellular flow cytometry to investigate cytokine production by BAL T cells could help to indicate potential targets for future therapy. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction A feature of pulmonary sarcoidosis is the presence of lymphocytes in the interstitial inflammatory cell infiltrates, which are key effector cells in the development of granulomas. Progressive granuloma formation resulting from persistent T cell activation can lead to severe respiratory impairment. Bronchoalveolar lavage (BAL) samples used to clinically assess interstitial lung disease have also been used to investigate cellular infiltrates, and cytokine profiles of T cells. The type of cytokine profiles that follow an immune response could influence the clinical course of disease leading to either resolution or progression to end-stage pulmonary fibrosis. Analysis of T cell populations found in BAL fluid of patients with sarcoidosis has shown evidence of increased T cell activation as measured by expression of HLA-DR and CD25 [1] or cytokine and chemokine receptors [2]. Classification of T cell subsets is possible by characterisation of their cytokines [3]. A Th1 type profile is characterised by produc-
* Corresponding author. Present address: Department of Primary Care and Population Sciences, Royal Free and University College London, Royal Free Campus, Rowland Hill St. London NW3 2PF, UK. Fax: +44 (0)20 7794 1224. E-mail addresses: [email protected] (T.A. Hill), [email protected] (S. Lightman), [email protected] (R.M. du Bois). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.03.014
tion of pro-inflammatory cytokines such as IFN-c and IL-2, whereas IL-4 production influences the development of a Th2 response [4]. IL-10 is produced by Th1, Th2, and Th0 subsets [5] and is thought to have a role in regulation of Th1 responses [6]. The influence and contribution of T cell cytokines to the pattern of cytokine production in inflammatory lung diseases is becoming clearer, and studies investigating T lymphocytes from patients with pulmonary sarcoidosis at the single cell level have established that the cytokine profile of both CD4+ and CD8+ T cells is of a Th1 type [7–9]. Interleukin-10 is not often investigated as part of the cytokine profile, although one study found an increase in IL-10+ T cells in only the CD8 subset and only in patients with acute sarcoidosis [10]. In order to supplement current understanding of the phenotypic characteristics of BAL T cells [11], our approach was to use BAL samples collected as part of routine clinical assessment, and to focus specifically on T lymphocytes to measure intracellular production of IFN-c, IL-2, IL-4, and IL-10. In addition to this, we also measured the activation status of the T cells and investigated whether this was related to the potential of these cells to produce Th1 type cytokines. Furthermore, we examined whether the cytokine profiles of BAL T cells from patients with prior therapy were different to those from patients who had not received therapy.
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2. Materials and methods 2.1. Patient details Samples of BAL and peripheral blood (PB) from 19 patients with active sarcoidosis were analysed. At the time of lavage, 12 of the patients were known to be off treatment, and 4 were taking prednisolone. Details of treatment at the time of lavage for the remaining 3 patients were unavailable. Although BAL cells from healthy individuals or those with non-inflammatory conditions may contain enough cells for examination, we felt it would not enhance our study to additionally use healthy controls as a comparison, and it is also well documented that BAL cells from patients with sarcoidosis contain a higher proportion of cytokine expressing T cells compared with healthy controls [8,12]. 2.2. Preparation and stimulation of BAL and peripheral blood mononuclear cells (PBMC) BAL was performed as part of routine clinical evaluation, with bronchoscopy performed and BAL fluid processed as previously described [13]. Cells in BAL fluid were collected by centrifugation at 300g for 5 min at 4 °C, washed three times with cold minimal essential medium (MEM) containing 25 mM Hepes buffer, and adjusted to 1.0 106 cells/ml. PBMC were isolated by density gradient centrifugation on Histopaque (Sigma), washed and also adjusted to 1.0 106 cells/ml. BAL cells and PBMC were stimulated for cytokine production with 50 ng/ml PMA (Sigma), 1 lg/ml ionomycin (Sigma) and 20 lg/ml Brefeldin A (BFA) (Sigma) for 18 h at 37 °C in a 5% CO2 atmosphere. BFA disrupts intracellular protein transport [14] causing cytokines to accumulate within cells, producing an enhanced cytokine signal detectable by flow cytometry. 2.3. Intracellular cytokine staining and flow cytometry After stimulation, BAL cells and PBMC were transferred to microcentrifuge tubes, washed by adding RPMI and cells pelleted by centrifugation for 5 min at 600g. Cells were resuspended in 50 ll RPMI and aliquots stained for surface antigens and intracellular cytokine using fluorochrome-conjugated antibodies against cell surface antigens (CD3-PerCP or CD8-PerCP, Becton Dickinson (BD) Oxford, UK) for 15 min at room temperature in the dark. Following a wash with 200 ll FACSFlow (BD), the cell pellet was resuspended in 100 ll Cytofix/Cytoperm reagent (BD) and incubated for 20 min at 4 °C. After washing with Perm/Wash buffer (BD), cells were pelleted, resuspended in 50 ll Perm/Wash buffer, then labelled for 30 min at 4 °C with combinations of fluorochrome-conjugated anti-cytokine antibodies: anti-human IFN-c-FITC plus anti-human IL-10-PE and anti-human IL-4-FITC plus anti-human IL-2-PE (BD). Antibody to CD69 used to check for expression of an early cell activation marker was added to cells after permeabilization as BFA inhibits the cell surface expression of CD69 [15]. Following washing with Perm/ Wash buffer, the cells were resuspended in 200 ll FACSFlow for flow cytometric analysis. Controls included cells labelled with isotypematched irrelevant mAbs, and non-activated PBMCs stained with the same antibodies. As only small samples of BAL cells were available for this study (1 106 cells), we were limited to the following antibody and surface marker combinations: CD3/IFN-c/IL-10 and CD8/IL-4/IL-2. Cells from BAL samples and PB were analysed using a flow cytometer with Cell Quest software (FACScan, BD). 2.4. Statistical analysis Differences between BAL and PB samples, or between groups in the mean percentages of cytokine positive T cells were determined
by Wilcoxon or Mann–Whitney t-tests for non-parametric data as appropriate. Any association between the percentages of CD69+ and IFN-c+ T lymphocytes was determined by Spearman rank correlation. A p-value of <0.05 was considered significant. 3. Results The BAL samples were assessed microscopically to contain 39% lymphocytes (mean ± SD, 39 ± 13). When unstimulated BAL and PB T lymphocytes from patients with sarcoidosis were examined for CD69 expression, there was a significant difference between BAL and PB (BAL 30%, PB 9%, p = 0.01) (Fig. 1). In contrast with CD69+ T cells, there was no difference between unstimulated BAL and PB cells in the numbers of IFN-c+ T cells in these patients, and most samples contained <10% IFN-c+ T cells (data not shown). There was also no difference between BAL and PB in the proportion of CD3+/ CD8 T cells (mean + SD, BAL: 31 ± 28, PB: 36 ± 21, p = 0.30). To produce intracellular cytokine levels detectable by flow cytometry, BAL and PB T cells were stimulated with PMA and ionomycin in the presence of BFA. Fig. 2 illustrates results from stimulated samples from patients with sarcoidosis, and shows that there was a similar proportion of CD69+ T cells in BAL (79%) and PB cells (75%) from patients with sarcoidosis (p = 0.3). However, the proportion of T cells producing IFN-c was higher in BAL (71 ± 30%) compared with PB (29 ± 15%, p = 0.001). Levels of IL-2+ T cells were also higher in BAL (28 ± 26%) compared with PB (19 ± 20%), although this difference did not reach significance (p = 0.08). With IL-4+ T cells, it was found that these were lower in BAL (0.9 ± 0.7%) than in PB (1.7 ± 1.2%, p = 0.03). There was no difference in the levels of IL10+ T cells in BAL (0.9 ± 0.7%) and PB (1.3 ± 1.1%, p = 0.16). In the absence of in vitro stimulation, 30% of BAL T lymphocytes were shown to be activated by the expression of CD69, with 5/19 samples containing >75% CD69+ T cells. However, there was no association between expression of CD69 and IFN-c in BAL samples (r = 0.2, p = 0.4). Since BAL samples from sarcoidosis patients were shown to contain increased levels of IFN-c-producing T lymphocytes, the activation status of T lymphocytes together with their ability to produce IFN-c were examined in relation to whether or not the patients were on or off immunosuppressive treatment at the time of BAL. Although the number of sarcoidosis patients with treatment prior to BAL was small (n = 12), there was no apparent reduction in either the number of activated T lymphocytes, as judged by their expression of CD69, or the number of IFN-c+ T cells (data not shown). It is possible however, that the effect of any immunosuppressive drugs on cytokine production declined during
Fig. 1. CD69 expression on unstimulated BAL and PB T cells. Unstimulated and stimulated BAL and PB T cells from patients with sarcoidosis were examined for expression of CD69. Unstimulated BAL T cells contained a higher percentage of CD69+ T cells compared with paired PB (*p = 0.006). There was no such difference between stimulated BAL and PB samples. (, unstimulated BAL; e, unstimulated PB; d, stimulated BAL; s, stimulated PB.)
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Fig. 2. Comparison of intracellular cytokine expression in sarcoidosis BAL and PB T cells. BAL and PB T cells from patients with sarcoidosis were stimulated and stained for intracellular cytokine expression. A higher percentage of BAL T cells were IFN-c+ (*p = 0.001), but a lower percentage were IL-4+ (**p = 0.03) compared with PB. Expression of intracellular IL-2 and IL-10 were similar in paired samples from sarcoidosis patients (, d, BAL; e, s, PB).
the time taken to prepare and stimulate BAL cells before detection of cytokines, or that the continual presence of drug is required to decrease cytokine production. 4. Discussion This study used intracellular flow cytometry to determine T cell cytokine profiles of routinely acquired BAL cells, thereby avoiding additional invasive clinical procedures. We aimed to extend previous findings which showed a predominantly type 1 cytokine profile of cells infiltrating the pulmonary interstitium of patients with sarcoidosis [8–10]. We also determined production of IL-10 by BAL and PB T cells, investigated cytokine production in relation to the activation status of T lymphocytes, and examined whether or not cytokine production was affected depending on a patient’s treatment prior to BAL. The investigation was designed to compare the activation and T cell cytokine profiles in the two different compartments, the lungs and peripheral blood. In patients with sarcoidosis it is known that CD69 on BAL T lymphocytes is over expressed compared with PB [11]. We found that BAL cells left unstimulated contained higher numbers of activated T cells compared with PB, indicating that it is feasible that these activated T cells could produce both inflammatory and possibly anti-inflammatory cytokines [5,16]. Our finding of increased proportions of IFN-c-producing BAL T cells from patients with sarcoidosis is in accordance with previous reports of elevated levels of IFN-c and IL-2 but not IL-4 or IL-5 in BAL fluid from sarcoidosis patients [17–20]. This finding indicates the selective entry into the lung of T cells with the potential to regulate inflammation, and the identification of T cells as a potential cellular source of the IFN-c found in BAL fluid. Production of IFN-c during a type 1 T cell response, at least in the earlier disease stages of sarcoidosis, would favour macrophage activation, the formation of granulomas and an inhibition of fibrogenetic processes [7,21]. Production of IFN-c may be beneficial as IFN-c levels in BAL fluid are inversely related to the progression of pulmonary fibrosis [22], although levels would have to be sufficient to balance and overcome the effects of any type 2 cytokines that might be produced, to influence the clinical outcome of pulmonary inflammation. We found relatively low but similar percentages of IL-10+ T cells in both BAL and PB, whilst a previous study demonstrated that BAL fluid IL-10 levels or cellular IL-10 mRNA levels were higher in sarcoidosis compared with healthy controls [12,23]. However, our study design was different in that it focussed on T cell cytokine production rather than measuring cytokines in BAL fluid or mRNA
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produced by a variety of cell types present in BAL fluid, which could account for the disparity in results. During the disease process, alteration of the balance of cytokine production may contribute to a more adverse outcome depending on the T cells present, and also on the ratio of cytokines that these and other cells secrete [24]. If the balance of T cells producing different cytokine profiles could be altered in favour of IL-10-producing T cells, then this could provide some beneficial regulation of the immune response [6]. The use of corticosteroid therapy in sarcoidosis is effective and can reduce levels of IFN-c in BAL fluid to undetectable levels after 2–3 months’ treatment [20]. This suggests that despite the infiltration or proliferation of type 1 T cells in the lung, corticosteroids are probably effective at regulating the immune response, and help to reduce the influence of type 1 T cell cytokines and may help restore the balance of type 1 and type 2 cytokine-producing T cells present in the lung during disease. Our study was unable to find any differences between patients off treatment at time of lavage and those receiving treatment prior to lavage in the proportions of either activated or IFN-c+ T cells, and further studies would be required to confirm this observation. We have demonstrated that intracellular flow cytometry can be applied to BAL cells harvested for routine clinical diagnostic purposes, to identify populations of cytokine-producing cells in the lung in pulmonary inflammation and fibrosis. Intracellular flow cytometry has the advantage of being able to determine both the phenotype and the cytokine profiles of relatively small numbers of BAL cells. This technique could be easily modified to further identify subpopulations of T lymphocytes or other leucocytes involved in the immune regulation of inflammatory lung diseases, or other Th1 type cytokines such as IL-12 or IL-18 [25]. Intracellular flow cytometry could also be used to determine patterns of T cell activation using different cellular markers as has been studied in pulmonary fibrosis associated with systemic sclerosis [26] and to relate this to cytokine profiles, disease stage and severity, which would require further study. We suggest that the intracellular flow cytometric method has the advantage of utilising routinely collected BAL samples to study cytokine profiles in the lung in sarcoidosis or other diseases where there is pulmonary inflammation and fibrosis. It can also be used to identify specific populations of cytokine-producing cells and to examine the activation status of cells involved in the immune response. We have successfully used this technique to specifically study and identify T lymphocytes as potential sources of cytokines that could contribute to and also regulate inflammation in the lung in sarcoidosis, and which could be targets for future therapeutic intervention. Acknowledgments This work was supported by the Arthritis Research Campaign and the Moorfields NHS R&D Support Levy. References [1] Spiteri MA, Johnson M, Epstein O, Sherlock S, Clarke SW, Poulter LW. Immunological features of lung lavage cells from patients with primary biliary cirrhosis may reflect those seen in pulmonary sarcoidosis. Gut 1990;31:208–12. [2] Katchar K, Eklund A, Grunewald J. Expression of Th1 markers by lung accumulated T cells in pulmonary sarcoidosis. J Intern Med 2003;254:564–71. [3] Mosmann TR, Coffman RL. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol 1989;7:145–73. [4] Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787–93. [5] Yssel H, de waal Malefyt R, Roncarolo M-G, Abrams JS, Lahesmaa R, Spits H, et al. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J Immunol 1992;149:2378–84.
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