Nobuhiro Matsumoto MD, Third Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki, 889–1692, Japan. E-mail: email@example.com
To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of various chemokines including thymus and activation-regulated chemokine (TARC), eotaxin, RANTES and IL-8 in pleural effusion samples. Samples were obtained from 11 patients with Paragonimus westermani infection, six patients with pleural transudate, eight with tuberculous pleurisy and five with empyema. High percentages of eosinophils were detected in pleural fluid (range 9–100%, median 81%) of patients with paragonimiasis. TARC concentrations in pleural effusions of paragonimiasis were markedly higher than those of other groups. Eotaxin levels were also higher in pleural effusions of paragonimiasis patients, although significant difference was noted only against transudate samples. There was a significant correlation between TARC concentrations and percentages of eosinophils, and between TARC and eotaxin concentrations in pleural effusion. There were also significant correlations between TARC concentration and the titre of anti-P. westermani IgG and between eotaxin concentration and the titre of anti-P. westermani IgG. Our findings suggest that TARC contributes to the pathogenesis of eosinophilic pleural effusion in paragonimiasis.
Paragonimiasis is a parasitic disease caused by the trematode Paragonimus westermani. Infection occurs by ingestion of poorly cooked food contaminated with the encysted metacercarie. After being ingested, the metacercarie excyst and traverse the gastrointestinal tract of patients and migrate to their final destination, usually the lung, where they become adults and may live up to 20 years. It is a common parasitic zoonosis in Asia. The southern part of Kyusyu district, Japan has long been known as one of the major endemic areas of this disease [1–3]. In Japan, infection is acquired through eating freshwater crab, Eriocheir japonicus, the second intermediate host, or the flesh of wild boars, Sus scrofa leukomystax, a proven paratenic host [4,5]. Paragonimiasis is a disease associated typically with peripheral blood eosinophilia and a high incidence of eosinophilic pleural effusion [1,2]. However, the underlying mechanism of eosinophil accumulation into thoracic cavity of patients with paragonimiasis is still unclear. The immunological hallmarks of infection with parasitic helminthes-like paragonimiasis, namely eosinophilia, mastocytosis and increased IgE synthesis, appear to be induced by cytokines such as interleukin (IL)-4 and IL-5 from the T-helper 2 (Th2) subset of CD4+ T cells . High expression levels of Th2-type cytokine mRNAs were found in freshly isolated peripheral blood mononuclear cells from a patient with paragonimiasis . We recently reported elevated levels of IL-5 in pleural fluid of patients with paragonimiasis, which would account for eosinophilia in pleural effusion .
Chemokines are a large group of chemotactic cytokines that regulate leucocyte trafficking. Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine and is expressed constitutively in the thymus and transiently in stimulated peripheral blood mononuclear cells . TARC functions as a selective chemoattractant for Th2 cells . Th2 cells release cytokines such as IL-4 and IL-5 to promote humoral immunity and allergic responses . Thus, TARC appears to play an important role in Th2-type disease conditions. We hypothesized that selective recruitment of eosinophils to the thoracic cavity in patients with paragonimiasis might be mediated by locally produced chemokines. In the present study, we investigated the levels of TARC, eotaxin, regulated upon activation, normal T cell expressed and secreted (RANTES) and IL-8 in pleural fluid samples from patients infested with P. westermani. Patients with tuberculous pleurisy, which is considered as a Th1-type disease, pleural transudate and empyema were also included in the study in order to characterize the immunological mechanism of pleural effusion in paragonimiasis.
Materials and methods
Patients and diagnostic categories
Patients examined in this study were 11 cases of paragonimiasis caused by P. westermani, comprising eight men and three women with a mean age of 45·6 ± 14·6 years (± s.d.). We also included patients with other pleural diseases including six cases with pleural transudate (two men and four women; age, 71·3 ± 20·1 years), eight cases of tuberculous pleurisy (five men and three women; age, 69·8 ± 13·3 years) and five cases of empyema (five men; age, 76·8 ± 11·5 years). Infestation with P. westermani was confirmed by immunodiagnosis. Briefly, a multiple-dot enzyme linked immunosorbent assay (ELISA) was used for routine primary screening of parasitic diseases, and binding inhibition ELISA and/or Ouchterlony's method were used for identification of the pathogens. Details of the immunodiagnostic methods have been described previously . Concurrent infestation with P. westermani in other patients was ruled out by microplate ELISA titration of P. westermani-specific IgG antibody. Details of the microplate ELISA titration have been described previously . Briefly, each of the wells of a 96-well microtitre plate was coated with 10 µg/ml of crude somatic extract of P. westermani adult worms and incubated overnight at 4°C. Wells were washed with phosphate-buffered saline (PBS) containing 0·05% Tween 20, blocked with blocking buffer (1% casein in 20 mm Tris HCl, pH 7·6), and then incubated with a serum sample from the patient, diluted 1 : 10000, at 37°C for 1 h. After washing, peroxidase-labelled rabbit antihuman IgG (gamma chain specific; Dako Corporation, Carpinteria, CA, USA) was added to each well and further incubated at 37°C for 1 h. 2, 2′-Azino-di-[3-ethylbenzthiazoline 6-sulphonate] peroxidase substrate (1-component type; Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was added to each well and incubated at 37°C for 15 min. The optical density was read at 405 nm in an ELISA reader (Multiskan Biochromatic, Thermo Labsystems, Helsinki, Finland). No parasites were found in the pleural effusion of patients with paragonimiasis. Pleural transudate was associated with congestive heart failure, nephrotic syndrome, or liver cirrhosis, and was defined based on the following criteria: (i) pleural fluid protein to serum total protein ratio < 0·5, and (ii) pleural lactate dehydrogenase (LDH) to serum LDH ratio < 0·6. Patients with tuberculous pleural effusions were defined as those with (i) growth of Mycobacterium tuberculosis in cultures from pleural fluid or biopsy specimens and (ii) growth of M. tuberculosis in sputum cultures. In patients with empyema, the pleural fluid was characterized by (i) neutrophilia (ii) growth of bacteria on microbiological culture of pleural fluid or (iii) organisms seen on Gram-staining of pleural fluid. Pleural fluid samples were obtained from each patient by thoracocentesis. The sample was centrifuged at 500 g for 10 min and the supernatants were stored at −20°C until analysis [8,13]. The study protocol was approved by the Human Ethics Review Committees of Miyazaki Medical College and a signed consent form was obtained from each subject.
Measurement of chemokine concentrations in pleural fluid
Concentrations of TARC, eotaxin, RANTES, and IL-8 in pleural fluid were measured using commercial ELISA kits (R&D Systems Inc., MN, USA; R&D Systems, BioSource International Inc., Camarillo, CA, USA and TFB, Tokyo, Japan, respectively) and the measurements were performed as described in the kit manuals. The detection limits were 8·9, 7·8, 15·6 and 6·25 pg/ml for TARC, eotaxin, RANTES and IL-8, respectively. Samples with concentrations below the lower limit of detection were considered to contain no cytokines.
Data were expressed as mean ± s.d. or median and range. Comparisons of paired data were carried out by using Kruskal–Wallis anova followed by the SchefféF-test. Correlations between two variables were examined by using the Pearson correlation coefficient. Statistical comparisons of the leucocyte differential counts of in pleural effusion were performed by using the Mann–Whitney U-test. Differences with P-values of less than 0·05 were considered significant.
Characteristics of pleural effusion and patients
Table 1 summarizes the differential counts of pleural effusion cells and Table 2 presents the characteristic of participating patients. Eosinophilia was seen only in pleural effusions of patients with paragonimiasis (P < 0·001). The percentage of neutrophils in empyema was significantly higher than other groups (P < 0·05). No significant differences were found in the percentages of monocytes and lymphocytes among all disease groups (Table 1). Titre of anti-P. westermani IgG was elevated only in patients with paragonimiasis (Table 2).
Table 1. Cell differentials in the pleural fluid
Cell differentials (%) (median, range)
P < 0·05 compared with each of the other groups.
P < 0·0001 compared with each of the other groups.
Table 2. . Clinical characteristics of participating patients
Duration of illness (days)
Total protein in pleural fluid (g/dl)
LDH in pleural fluid (g/dl)
Titre of Paragonimuswestermani- specific IgG
Transudate (n = 6)
70·5 ± 20·1
2·0 ± 1·8
188·8 ± 56·7
0·001 ± 0·001
Tuberculosis (n = 8)
69·5 ± 13·3
263·8 ± 296·9
4·3 ± 1·5
804·4 ± 835·0
0·002 ± 0·002
Emyema (n = 5)
76·8 ± 11·5
4·1 ± 0·9
4883·0 ± 6588·7
0·0 ± 0·0
Paragonimiasis (n = 11)
45·6 ± 14·6
31·3 ± 23·1
5.6 ± 0·8
3293·9 ± 2398·5
0·826 ± 0·382
Chemokine concentrations in pleural effusions
To investigate the possible role of chemokines in each lung disease, we determined the concentrations of four chemokines (TARC, eotaxin, RANTES and IL-8) in the pleural effusion. TARC concentrations in pleural effusion of patients with paragonimiasis were significantly higher than other groups (P < 0·0005; Fig. 1a). Furthermore, there was a significant positive correlation between TARC concentrations and percentages of eosinophils in pleural effusion of all disease groups (r = 0·802, P < 0·0001; Fig. 2a). Levels of eotaxin, a potent and eosinophil-specific chemoattractant, were also higher in pleural effusion from patients with paragonimiasis, although statistical significance was seen only against transudates (P < 0·05; Fig. 1b). No significant differences were noted among all disease groups in the levels of RANTES, an eosinophilotactic chemokine (Fig. 1c). There was a significant positive correlation between the concentrations of TARC and eotaxin in pleural effusion samples (r = 0·55, P < 0·005; Fig. 2b), but no such correlation between the concentrations of TARC and RANTES (data not shown). Moreover, there were significant positive correlations between TARC concentration and the titre of anti-P. westermani IgG (r = 0·697, P < 0·0001; Fig. 3a), and between eotaxin concentration and the titre of anti-P. westermani IgG (r = 0·581, P < 0·01, Fig. 3b). There was no significant difference in the levels of IL-8 among disease groups (Fig. 1d), but there was a significant correlation between IL-8 concentrations and the percentages of neutrophils in pleural effusion samples (data not shown).
TARC is a specific chemotactic factor for Th2 cells  and its expression in the lung is up-regulated in a murine model of allergic asthma [14,15]. Furthermore, treatment with anti-TARC antibody attenuates the accumulation of eosinophils and lymphocytes into the lung in a mouse model of asthma . In addition, the bronchial epithelium of patients with asthma expresses TARC more intensely than normal subjects  and serum levels of TARC in atopic dermatitis are higher than in healthy subjects . Thus, TARC appears to play an important role in eosinophilic inflammation in patients with allergic diseases.
In the present study, we found extremely high levels of TARC in pleural effusion fluid samples from patients with paragonimiasis compared with patients with other pleural diseases (Fig. 1a). Our results also showed that TARC concentrations in pleural effusion correlated well with those of eotaxin level (Fig. 2b) and percentages of eosinophils in pleural effusion (Fig. 2a). Moreover, there were significant positive correlations between TARC/eotaxin concentrations and the titre of anti-P. westermani IgG in pleural effusion (Fig. 3a,b). These results suggest that the level of exposure to P. westermani correlates with local production of TARC and eotaxin and that TARC plays an important role in the development of eosinophilic pleural effusion in patients with paragonimiasis. Th2 type cells that accumulate in the thoracic cavity in response to TARC stimulation may produce excess amounts of Th2 type cytokines, such as IL-4 and IL-5. This conclusion is based on the results of our previous study, which demonstrated the presence of extremely high levels of IL-5 in pleural effusion of patients with paragonimiasis . IL-4 has been reported to induce eotaxin production , and IL-5 is known to stimulate the proliferation and activation of eosinophils . Eosinophils may transmigrate from peripheral blood into the thoracic cavity based on the signals of these cytokines and chemokines, although the main source of TARC in the thoracic cavity and the regulatory mechanism involved in its production remain to be defined. In tuberculous effusion, TARC and eotaxin levels were elevated compared to those in the transudate, although the differences were not significant (Fig. 1a,b). These results are in agreement with previously reported findings in both animal and human studies. For example, animal studies have shown a high expression of TARC mRNA in the airways of mice injected with a Mycobacteria-purified protein derivative and human studies reported no increase in eotaxin levels in the airways of patients with pulmonary tuberculosis [20,21]. In this study, a role of TARC and eotaxin in the tuberculous pleurisy is unclear.
In patients with paragonimiasis, percentages of monocytes and lymphocytes were lower than those of patients with tuberculosis and transudate, although the difference was not significant (Table 1). There were too many eosinophils in the pleural fluid and, thus, the proportion of monocytes and lymphocytes in the fluid may be too small to allow meaningful statistical analysis.
RANTES is also known as a chemotaxin for eosinophils, but it is less potent than eotaxin . In patients with paragonimiasis, we also demonstrated the presence of large amounts of eotaxin in the pleural effusion, while RANTES concentrations were not significantly elevated (Fig. 1b,c). These results indicate that eotaxin plays more important role in the development of eosinophilic pleural effusion in patients with paragonimiasis compared with RANTES.
IL-8 is considered as the most potent chemoattractant factor for polymorphonuclear leucocytes . In our study, IL-8 concentrations were the highest in empyema group, although they were not significantly different from those of other groups due to the high variability (Fig. 1d). IL-8 concentrations correlated with the percentages of neutrophils in pleural effusion (data not shown), which adds further support to the results of previous studies  and suggests that IL-8 plays a role in neutrophilic pulmonary inflammation.
In conclusion, the present study suggests a possible role for TARC in the pathogenesis of eosinophilic pleural effusion in pulmonary paragonimiasis. Further studies are required to refine our understanding of the role of TARC in the development of eosinophilic pleural effusion associated with paragonimiasis.
We thank Mrs S. Tajiri for the excellent technical assistance.