Cell Origins and Diagnostic Accuracy of Interleukin 27 in Pleural Effusions

The objective of the present study was to investigate the presence of interleukin (IL)-27 in pleural effusions and to evaluate the diagnostic significance of pleural IL-27. The concentrations of IL-27 were determined in pleural fluids and sera from 68 patients with tuberculous pleural effusion, 63 malignant pleural effusion, 22 infectious pleural effusion, and 21 transudative pleural effusion. Flow cytometry was used to identify which pleural cell types expressed IL-27. It was found that the concentrations of pleural IL-27 in tuberculous group were significantly higher than those in malignant, infectious, and transudative groups, respectively. Pleural CD4+ T cells, CD8+ T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells might be the cell sources for IL-27. IL-27 levels could be used for diagnostic purpose for tuberculous pleural effusion, with the cut off value of 1,007 ng/L, IL-27 had a sensitivity of 92.7% and specificity of 99.1% for differential diagnosing tuberculous pleural effusion from non-tuberculous pleural effusions. Therefore, compared to non-tuberculous pleural effusions, IL-27 appeared to be increased in tuberculous pleural effusion. IL-27 in pleural fluid is a sensitive and specific biomarker for the differential diagnosing tuberculous pleural effusion from pleural effusions with the other causes.


Introduction
Pleural effusions (PEs) are a frequent clinical problem with more than 50 recognized causes including diseases local to the pleura or underlying lung, organ dysfunction, systemic conditions and drugs [1]. The development of PE is often associated with the accumulation of fluid enriched in proteins and cells in the pleural space [2]. There are many causes of PEs, and the precise pathophysiology of fluid accumulation varies according to underlying etiologies. It has been well documented that tuberculosis and cancer represent the two most frequent causes of exudative PEs with predominantly lymphocytes in pleural fluid; whereas infectious PEs, including empyema and parapneumonic effusion, are typically associated with an influx of neutrophils [3,4].
Interleukin (IL)-27, a member of IL-12 family, is a recently discovered heterodimeric cytokine consisting of Epstein-Barr virusinduced gene protein 3 and p28 subunits [5], and is mainly produced by activated antigen-presenting cells [6]. IL-27 has been found to be involved in malignancy [7] and in infection, including severe sepsis, bacteraemia, as well as tuberculosis [8,9]. This raises the possibility that IL-27 may play a role in the pathogenesis of PE, and may further be of diagnostic significance. We therefore performed the present study to: 1) determine whether PE IL-27 is produced in the pleural space; 2) identify the cell origins of pleural IL-27; 3) evaluate the diagnostic value of IL-27 in PEs.

Subjects
The study protocol was approved by our institutional review board for human studies of Tongji Medical College, China, and informed written consent was obtained from all subjects. One hundred and ninety-three consecutive patients with PEs of unknown causes were hospitalized for diagnostic investigation. Of these patients, 12 were excluded because they had received anti-tuberculosis treatment (n = 5) or anti-cancer chemotherapy (n = 7) prior to study, four were excluded because they had received corticosteroids or other nonsteroid anti-inflammatory drugs, and three were discharged from the hospital without a definitive diagnosis of PEs. Eventually, 174 patients were included in the current study (Table 1).
Sixty-eight anti-HIV Ab negative patients (age range: 16 to 76 yr) were proven to have tuberculous PE with, as evidenced by 1) presence of acid fast bacilli in pleural fluid specimen, growth of Mycobacterium tuberculosis (MTB) from pleural fluid, or demonstration of granulomatous pleurisy on closed pleural biopsy specimen in the absence of any evidence of other granulomatous diseases (n = 59); 2) an exudative lymphocytic effusion with an adenosine deaminase level of .40 U/L, along with a positive purified protein derivative skin test result and the exclusion of any other potential causes of pleurisy; after anti-tuberculosis chemotherapy, the resolution of pleural effusion and clinical symptoms was observed (n = 9).
Malignant PE was collected from 63 patients (age range: 37 to 84 yr) with newly diagnosed lung cancer with PE. Histologically, 44 cases were adenocarcinoma and 19 were squamous cell carcinoma. A diagnosis of malignant PE was established by demonstration of malignant cells in PE and/or on closed pleural biopsy specimen.
Twenty-two PE patients (age range: 38 to 74 yr) were classified as infectious PE (including 16 empyema and six parapneumonic effusion). Empyema was defined as an effusion that met one or more of the following criteria: presence of frank pus in the pleural space, purulent fluid on macroscopic examination, positive Gram stain and/or growth of organisms in culture, and PE pH ,7.2 or glucose ,3.3 mmol/L in association with pneumonia. Parapneumonic effusion was those with a glucose concentration .3.3 mmol/L and pH .7.2, and no organisms seen on Gram stain or found on PE culture in a patient with pneumonia.
Twenty-one patients (age range: 36 to 81 yr) with PE were classified as transudates on the basis of Light's criteria [10].

Sample Collection and Processing
The PE samples were collected in heparin-treated tubes from each subject, using a standard thoracocentesis technique within 24 h after hospitalization. Twenty milliliters of venous blood were drawn simultaneously. The PE specimens were immersed in ice immediately and were then centrifuged at 1,200 g for five min. The cell-free supernatants of PE and sera were frozen at -80uC immediately after centrifuge for later determining concentrations of IL-27, lactate dehydrogenase, and protein. Analyses of PEs for total nucleated cell and differentials counts were performed. Biochemical and cytological characteristics in PEs are summarized in Table in webappendix.

Flow Cytometry
The intracellular expression of IL-27 on various pleural cell types were determined by flow cytometry after surface or intracellular staining with anti-human-specific Abs conjugated with FITC, phycoerythrin, PerCP-cy5.5, or eFluor 660. These human Abs included anti-CD3, -CD4, -CD8, -CD14, -CD19, -CD56, -CD68, and -IL-27 mAbs, which were purchased from BD Biosciences (Franklin Lakes, NJ), eBioscience (San Diego, CA), or R&D systems (Minneapolis, MN). Intracellular staining for IL-27 was performed on pleural cells stimulated with phorbol myristate acetate (50 ng/ml; Sigma-Aldrich St. Louis, MO) and ionomycin (1 mM; Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences) for 4 h, and then stained with anti-IL-27 mAb conjugated with phycoerythrin. To identify pleural mesothelial cells, intracellular staining for calretinin was performed. Fixed and permeabilized mesothelial cells were primarily stained with mouse anti-human calretinin mAb (BD Biosciences), and then stained with FITC goat anti-mouse Igs (BD Biosciences). Appropriate species matched Abs served as isotype control. Flow cytometry was performed on a FACS Canto II (BD Biosciences) and analyzed using BD FCSDiva Software and FCS Epress 4 software (De Novo Software, Los Angeles, CA).

Measurement of IL-27
The concentrations of IL-27 in PEs, culture supernatants, and sera were measured by ELISA kit according to the manufacturer's protocol (BioLegend Inc., San Diego, CA, USA). The minimum detectable concentration of IL-27 was 11.0 ng/L. All samples were assayed in duplicate.

Statistical Analysis
Data were expressed as mean 6 SEM. Changes of IL-27 in PE were adjusted to that in serum by calculating either the IL-27 concentration ratio between PE and serum (PE/serum IL-27 ratio) or the difference between the concentrations of IL-27 between the two media (PE-serum IL-27D). Parametric tests were used since IL-27 data were normally distributed as determined by a normality test. Comparisons of data between different groups were performed using one-way analysis of variance or Student's t test. Comparisons of IL-27 levels in PEs and in corresponding sera were made using paired t test. The correlations between variables were determined by Pearson correlation coefficients. Receiver operating characteristic curve analyses were used to evaluate the capacity of IL-27 to differentiate tuberculous PE from PEs with the other etiologies. Analysis was done using SPSS version 16.0 Statistical Software (Chicago, IL, USA), a p-value ,0.05 was considered as statistically significant.

Characteristics in PEs
Biochemical and cytological characteristics in PEs are illustrated in Table 1. Subjects with lung cancer showed a large proportion of lymphocytes and macrophages in PE. Subjects with tuberculosis showed a marked elevation of total cell counts, and a large proportion of these cells were lymphocytes, with some neutrophils and macrophages. Absolute lymphocyte counts evidenced the highest values in tuberculous PE, showing a significant increase in comparison with malignant (p = 0.012), infectious (p = 0.009), transudative (p,0.001) PE, respectively. Also as expected, total cell counts in infectious PE were the greatest among four groups, and neutrophil was the most predominant cell type, the numbers of this kind of white cells were significantly greater than those in the PEs with any other causes (all p,0.001).

Concentrations of IL-27 in PEs
The concentrations of IL-27 in tuberculous PE were significantly higher than those in malignant, infectious, and transudative PE (all p,0.001) ( Figure 1A and Table 2). The difference also emerged from both PE/serum IL-27 ratio and PE-serum IL-27D that were much higher in tuberculous PE than in non-tuberculous PEs (all p,0.001) ( Table 2). On the other hand, concentrations of IL-27 in sera were not different with one another among four groups (all p.0.05) ( Figure 1B and Table 2). In addition, concentrations of IL-27 in both tuberculous and malignant PE were correlated positively with numbers of pleural mononuclear cells, including lymphocytes, macrophages, and mesothelial cells (r = 0.696; p,0.001 and 0.402; p = 0.001, respectively) (Figure 2), suggested that PE IL-27 might be produced by these local mononuclear cells.
The comparisons of IL-27 concentrations in PEs and sera in each group are also shown in Table 2. IL-27 concentrations were much higher in tuberculous PE than in the corresponding serum (p,0.001); there was no difference in IL-27 concentrations between PE and serum in malignant group (p = 0.091). IL-27 concentrations were lower in PEs than in sera from patients with infectious (p = 0.001), and transudative (p = 0.008) PE, respectively.

Cell origins of Pleural IL-27
Having known that IL-27 was significantly increased in tuberculous PE, and that PE IL-27 concentrations were correlated positively with numbers of pleural mononuclear cells, we then used flow cytometry to identify which pleural cell types could be the cell origins for IL-27 in PEs. The representative flow cytometric dotplots are presented Figure 3, and the summary data of percentages of pleural cells expressing IL-27 are presented in Table 3. Our data showed that CD4 + T cells, CD8 + T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells from both tuberculous and malignant PE expressed intracellular IL-27 at different extents, and that all the above pleural cells, but not NK cells, in tuberculous PE expressed much higher levels of IL-27 than their corresponding compartments in malignant PE did.
To explored effect of MTB antigen on IL-27 production, we isolated PBMCs from subjects with latent tuberculosis infection and cultured them in vitro in the presence of exogenous ESAT-6/ CFP-10. As shown in Figure 4, ESAT-6/CFP-10 is capable of inducing IL-27 production from PBMCs in a time-and dosedependent manner.

Diagnostic Value of IL-27
The capacity of IL-27 to differentiate tuberculous PE from PEs with the other etiologies was assessed with receiver operating characteristic curve analyses ( Figure 5). The area under curve when IL-27 was used to differentiate tuberculous from nontuberculous PEs (including malignant, infectious, and transudative

Discussion
In the previous studies, we have demonstrated that regulatory T cells and IL-172producing CD4 + T cells play important immunoregulatory roles in the pathogenesis of lymphocytic PEs [11][12][13][14]. It has been reported that IL-27 controls the development of regulatory T cells and IL-172producing CD4 + T cells [15][16][17]. We therefore speculated that IL-27 might be involved in the development of PE.
The first identified biological function for IL-27 was its ability to increase the proliferation of naïve CD4 + T cells [18]. Initial studies with IL-27 receptor deficient mice indicated that IL-27 can   promote the generation of Th1-cell responses [19,20]. It is well accepted that IL-27 regulates various immune diseases through its dual proinflammatory and anti-inflammatory effects on immune responses [21]. In the present study, our data have shown for the first time that IL-27 could be detected in PEs, and that IL-27 concentrations in tuberculous PE were significantly higher than those in malignant, infectious, and transudative PE, suggesting that more pleural sources of IL-27 exist in tuberculous patients.
Our results also favored the concept of a local production of IL-27 in tuberculous PE, rather than a passive diffusion of this cytokine from plasma to the pleural compartment, since IL-27 concentration in serum was much lower than that in tuberculous PE. The fact that increased IL-27 was found in tuberculous PE compared with malignant, infectious, or transudative PE suggested that IL-27 might be of particular interest in tuberculosis. Animal studies have demonstrated that mice deficient in IL-27 or its receptor chains are able to reduce microbial burdens during MTB infection; on the other hand, IL-27 limits the pathological sequelae of chronic inflammation [22,23]. Moreover, IL-27 can enhance the ability of MTB-specific Th1 cells to inhibit intracellular mycobacterial growth in human macrophages [24]. The precise pathophysiological roles of IL-27 in tuberculous PE need further investigation.
Although few studies have directly addressed the events that lead to the production of IL-27, it is known that IL-27 is mainly produced by activated antigen-presenting cells [6]. To reveal the cell origins of PE IL-27, flow cytometry was performed to determine the expression of IL-27 on pleural cells. Our data showed that CD4 + T cells, CD8 + T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells from both tuberculous and malignant PE expressed intracellular IL-27, and that all these pleural cell types, but not NK cells, in tuberculous PE expressed much higher IL-27 than their corresponding compartments in malignant PE. We therefore inferred that PE IL-27 might be derived from pleural CD4 + T cells, CD8 + T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells, and that all these pleural cell types, but not NK cells, were further responsible for the increase in IL-27 in tuberculous PE. The mechanism by which pleural cells expressed more IL-27 in tuberculous than in malignant PE might be associated with MTB antigen stimulation, since we observed in the present study that MTB antigen stimulated IL-27 production from PBMCs in vitro in a time-and dose-dependent manner.  The presence of MTB antigens in pleural space elicits an intense immune response, initially by macrophages and neutrophils [25,26], followed by Th1 cells [27], resulting in a lymphocytepredominant exudative effusion. Accompanying the infiltrate of immune cells is the accumulation of cytokines, chemokines, growth factors, and other soluble mediators in PEs, some of them have been proposed to be helpful in diagnosis of PEs [28]. Of all the cytokines, interferon-c has been the most studied. The evidence for use of pleural interferon-c for diagnosis of tuberculous PE has been reviewed in our previous meta-analysis [29]. Our analysis showed that the summary estimate of sensitivity of interferon-c was 89% (95% confidence interval, 87-91%) and of specificity was 97% (96-98%) [30], which seemed to be somehow better than the diagnostic accuracy of adenosine deaminase [30].
To our knowledge, the current study was the first one to investigate the diagnostic value of IL-27 in differential diagnosing tuberculous PE from PEs with the other etiologies (Panel). Using the receiver operating characteristic curve, we noted that the area under curve was 0.994 for IL-27 to diagnose tuberculous PE. The cut off value for IL-27 was selected on the basis of the highest diagnostic accuracy (true positive + true negative/true positive + true negative + false positive + false negative) with the highest sensitivity, which was determined to be 1,007 ng/L. With this cut off, sensitivity and specificity of IL-27 for tuberculous PE were 92.7% and 99.1%, respectively. Our data also revealed a high positive likelihood ratio of 98.21 and a low negative likelihood ratio of 0.07, further confirming IL-27 was a good diagnostic parameter. It should be noted that although IL-27 has a good diagnostic performance, it is a biomarker of the inflammatory process in the pleural space and does not confirm the etiologic agent.
An obvious limitation of this study was the relatively low diversity of PE etiologies. For example, we failed to recruit patients with exudates secondary to collagen vascular diseases, abdominal inflammatory disorders, drugs, etc. When a new marker is considered for diagnosing PE, one always needs to include patients with exudates of different origins. On the other hand, the receiver operating characteristic analysis in a first study such as this might always be more sensitive/specific than seen in real practice, as the cut off was derived from the subjects assessed. It should therefore be mentioned that further prospective studies are necessary to verify the diagnostic value of IL-27 in a larger number of patients with PEs of diverse etiologies.
In conclusion, our data showed that compared to nontuberculous PEs, IL-27 was increased in tuberculous PE, and that pleural CD4 + T cells, CD8 + T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells were cell sources of PE IL-27. We further described for the first time that IL-27 appeared to distinguish well between tuberculous and nontuberculous PEs, especially malignant PE.