PERSPECTIVE
What Is Eotaxin Doing in the Pleura?
Insights into Innate Immunity from Pleural Mesothelial Cells

Steve N. Georas, Lisa A. Beck, and Cristiana Stellato

Department of Medicine, Division of Pulmonary and Critical Care Medicine, and Division of Allergy and Clinical Immunology, The Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland

It was appreciated almost 20 years ago that pleural mesothelial cells (PMC) are a biologically active cell type capable of synthesizing a variety of extracellular matrix molecules (1). As such, PMC contribute to the integrity of the pleural membrane, which plays a vital role in facilitating lung expansion and deflation. Research in the past decade has uncovered an additional role for PMC in sensing and responding to signals within the pleural microenvironment. In particular, several recent studies have found that PMC synthesize a diverse array of chemotactic cytokines (or chemokines) in response to distinct extracellular stimuli. This suggests that PMC play an active role in the recruitment of inflammatory cells into the pleural space. Comparable functions have recently been ascribed to other mesodermally-derived cells, including peritoneal mesothelial cells (2).

Since the original observation that asbestos-induced pleural neutrophilia involved mesothelial-derived IL-8 production (3, 4), PMC have been shown to secrete both C-C and C-X-C chemokines in different experimental settings (Table 1). Interestingly, Nasreen and coworkers recently reported that PMC secrete IL-8 in a polarized fashion (from basal to apical), which would promote the transmesothelial migration of neutrophils into the pleural cavity (5). The cellular infiltrate in response to a chemokine gradient will depend on the expression of appropriate ligands on, and the state of, activation of target cells. Several recent reviews are available for this active area of research (6, 7). In general, the PMC-derived chemokines described to date can help explain the pleural neutrophilia or monocytosis that accompany different disease states. Thus, the concentration of IL-8 is increased in pleural diseases characterized by neutrophil influx (e.g., complicated parapneumonic effusion and empyema [8]), whereas macrophage inflammatory protein (MIP)-1 and monocyte chemoattractant protein (MCP)-1 are increased in monocyte-rich tuberculous effusions (8, 9).

In this issue of the AJRCMB, Katayama and coworkers report that normal human PMC secrete two potent eosinophil-active chemokines, namely, eotaxin and RANTES (10). Originally identified in BAL fluids in an animal model of allergic inflammation (11), eotaxin is now known to comprise a family of peptides that recognize the specific ligand chemokine receptor-3 (CCR3). The restricted expression of CCR3 to eosinophils, basophils, and mast cells helps explain the cell-type specific effects of eotaxin. RANTES binds in a more promiscuous fashion to CCR1-, CCR3-, and CCR5-expressing cells, and thus it is chemotactic for eosinophils as well as monocytes and lymphocytes in vivo (12).

Several features of the study by Katayama and colleagues are noteworthy (10). First, the authors studied primary human mesothelial cells obtained from the cellular fraction of pleural effusions and expanded by epidermal growth factor (EGF) for several weeks. Prior work by this group demonstrated that mesothelial cells obtained in this fashion were phenotypically identical to normal PMC, including staining positive for keratin and vimentin, and expressing surface microvilli (13). Second, the expression of eotaxin and RANTES by PMC was found to be enhanced by Th2 (IL-4) and Th1 (IFN-) cytokines, respectively, both of which acted in synergy with TNF-. This is in keeping with a recent study showing that mouse PMC express surface receptors for IL-4 and IFN- (14). Third, the authors dissected the molecular mechanisms of cytokine-dependent eotaxin and RANTES expression in PMC, and report that this involves both transcriptional and post-transcriptional gene regulation.

Work in other lung cell types, including airway epithelial cells (15, 16), smooth muscle cells (17), and fibroblasts (18), has uncovered distinct patterns of chemokine secretion in response to Th1 and Th2 cytokines. In general, the Th2 cytokines IL-4 and IL-13 are potent inducers of eotaxin secretion, whereas IFN- (the prototypical Th1 cytokine) is a stronger inducer of RANTES expression. The effects of Th1 and Th2 cytokines on RANTES and eotaxin expression in these cells tend to be mutually antagonistic, and to synergize with the proinflammatory cytokine TNF-. Human PMC fit nicely into this paradigm (10). Taken together with similar findings in endothelial cells (19), these studies demonstrate that multiple cell lineages of distinct embryologic origin secrete eotaxin in a Th2-dependent manner. This helps explain the close association between Th2-driven immune responses (e.g., parasite infections and atopy) and eosinophilia. The evolutionary advantage conferred by this stereotypical and conserved response awaits further study.

Direct evidence that IL-4 is an inducer of pleural eotaxin production and eosinophilia is provided by a recent study by Larbi and coworkers (20) (Table 2). In these experiments, intrapleural injection of IL-4 led to a dose- and time-dependent accumulation of eosinophils within rat pleural cavities that was accompanied by the generation of eotaxin. Interestingly, unlike eosinophil recruitment into IL-4-challenged skin, recruitment into the pleura was independent of blockade of vascular cell adhesion molecule (VCAM)-1 (20). In contrast, neutralization of the VCAM-1 ligand very late antigen (VLA)-4 completely inhibited eosinophil accumulation in both tissues. Although the precise basis of VCAM-1-independent pleural eosinophil recruitment remains to be determined, this study provides an interesting example of a pleural-specific cell recruitment mechanism.

Pleural eosinophilia is a relatively uncommon diagnosis (see below). Interestingly, the presence of pleural eosinophilia is associated with a good prognosis in patients with chronic idiopathic effusions (21). Thus, pleural eosinophilia may be a marker of a more favorable immune response. A recent study from the Hirai research group found that eotaxin was readily detectable in human pleural effusions (22). Importantly, although eotaxin was detected in both transudates and exudates, eotaxin levels were higher in eosinophilic pleural effusions and correlated with eosinophil counts (22). The apparent constitutive eotaxin expression within the pleura is in keeping with studies of the human airway, where eotaxin is readily detectable in biopsies from normal subjects, but its expression is increased and correlates with tissue eosinophilia in individuals with atopic asthma (23).

It is interesting and instructive to compare immune responses occurring within the pleural space with those that occur within the airway. In allergic asthma, it is currently accepted that the driving force for airway eotaxin expression and eosinophilia is an allergen-specific Th2 immune response (24). In this model, inhaled allergens are processed by mucosal dendritic cells, which both initiate and maintain the activation of Th2 cells (25). Although the direct intrathoracic injection of allergen in sensitized animals leads to a robust pleural eosinophilia ("allergic pleurisy") (26, 27), this is not a typical feature of human allergy, likely due to the restricted access of inhaled allergens to the pleural space. Evidence that the pleura can mount a specific immune response is provided by an animal model of Staphylococcus aureus empyema, where chemokine production and neutrophil recruitment were found to be dependent on CD4⁺ T cells (28). It remains to be determined whether idiopathic eosinophilic pleural effusions are characterized by dysregulated Th2 immunity or other factors.

Airway eosinophilia in asthma differs from the most common causes of pleural eosinophilia, namely pneumothorax and hemothorax (29), which would not appear to involve adaptive, antigen-specific immunity. The mechanisms by which pneumo- and hemothorax lead to pleural eosinophilia remain to be determined. One possibility is that mechanical deformation of PMC is sufficient to induce their secretion of eotaxin and/or RANTES in a Th cell- independent (or innate) manner. This would be in keeping with the observations that: (i) physical injury induces PMC secretion of other chemokines (30), and (ii) eotaxin secretion is enhanced in endothelial cells subjected to shear stress (31). It will be interesting to determine whether resident airway cells secrete eotaxin or other chemokines in response to non-cognate environmental stimuli in a similar fashion.

Although Kitayama and colleagues found that IL-4 was a selective inducer of eotaxin production by PMC, IL-4 plus TNF--costimulated cells secreted both eotaxin and RANTES (10). In fact, substantially more RANTES (ng/ml) than eotaxin (pg/ml) was produced under these conditions. Thus, it is interesting that in chemotaxis assays using conditioned supernatants from IL-4 plus TNF--stimulated PMC, an anti-RANTES antibody was ineffective in blocking cell migration when used alone or in combination with an anti-eotaxin antibody (which by itself inhibited eosinophil migration in a concentration-dependent manner). This may reflect the fact that eotaxin is a more potent inducer of eosinophil chemotaxis than RANTES. Alternatively, other soluble factors produced by PMC stimulated in this fashion may augment eotaxin responsiveness. One possible candidate in this regard is leukotriene B4 (LTB₄), which synergized with eotaxin to induce pleural eosinophilia in an animal challenge model (27). At present, however, direct evidence that PMC secrete LTB₄ or related lipid mediators is lacking.

Our knowledge of the molecular regulation of RANTES and eotaxin gene expression is rapidly growing. In other cell systems, the synergistic enhancement of RANTES expression by IFN- plus TNF- appears to occur in part at the transcriptional level by cooperative interactions between Stat (signal transducer and activator of transcription)-1 and NF-B proteins (32). Interestingly, Katayama and coworkers found that IFN- plus TNF- costimulation substantially prolonged the half-life of RANTES mRNA in actinomycin D-treated PMC (10), suggesting a major effect at the post-transcriptional level. Because actinomycin D can interfere with the expression of proteins involved in both the stabilization and destabilization of mRNA, these results must be interpreted with caution. Nonetheless, they are in keeping with the observations of Koga and colleagues that virus infection substantially prolonged RANTES mRNA half-life in airway epithelial cells (33). Interestingly, the molecular basis for this effect required distinct sequences within the RANTES coding region (33). Because the RANTES gene does not contain canonical AU-rich elements (AUREs) within its 3' untranslated region (3' UTR), it is likely that RANTES mRNA decay occurs independently of the recently-described exosome which degrades AURE-containing mRNA species in mammalian cells (34). It will be interesting in future studies to determine the role and identity of RNA-binding proteins in cytokine-induced RANTES mRNA stabilization.

In contrast to the induction of RANTES expression in PMC, actinomycin D experiments revealed that IL-4 plus TNF- enhanced eotaxin expression without affecting the decay of eotaxin mRNA. This suggests a predominant effect at the level of gene transcription. Recent work from our group (led by Dr. Robert Schleimer) helps explain this phenomenon. We reported that the eotaxin promoter contains overlapping NF-B and Stat6 sites which were required for TNF-- and IL-4-induced promoter transactivation in epithelial cells (35).

In summary, our knowledge of the mechanisms of cell recruitment into the pleura is rapidly growing. The key role played by the mesothelial cell in this process is now clear, and we are beginning to understand the molecular basis by which mesothelial cells sense and respond to extracellular stimuli. Several parallels can be drawn between the pleura and other tissues, including the inflamed airway. It will be most interesting to pursue the unique features of pleural-specific immune and inflammatory responses.

	Footnotes

Address correspondence to: Dr. Steve N. Georas, M.D., Rm. 4B.41, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: sgeoras{at}jhmi.edu

(Received in original form February 21, 2002).

	References

1. Rennard, S. I., M. C. Jaurand, J. Bignon, O. Kawanami, V. J. Ferrans, J. Davidson, and R. G. Crystal. 1984. Role of pleural mesothelial cells in the production of the submesothelial connective tissue matrix of lung. Am. Rev. Respir. Dis. 130: 267-274 [Medline].

2. Robson, R. L., R. M. McLoughlin, J. Witowski, P. Loetscher, T. S. Wilkinson, S. A. Jones, and N. Topley. 2001. Differential regulation of chemokine production in human peritoneal mesothelial cells: IFN-gamma controls neutrophil migration across the mesothelium in vitro and in vivo. J. Immunol. 167: 1028-1038 [Abstract/Free Full Text].

3. Boylan, A. M., C. Ruegg, K. J. Kim, C. A. Hebert, J. M. Hoeffel, R. Pytela, D. Sheppard, I. M. Goldstein, and V. C. Broaddus. 1992. Evidence of a role for mesothelial cell-derived interleukin 8 in the pathogenesis of asbestos-induced pleurisy in rabbits. J. Clin. Invest. 89: 1257-1267 .

4. Griffith, D. E., E. J. Miller, L. D. Gray, S. Idell, and A. R. Johnson. 1994. Interleukin-1-mediated release of interleukin-8 by asbestos-stimulated human pleural mesothelial cells. Am. J. Respir. Cell Mol. Biol. 10: 245-252 [Abstract].

5. Nasreen, N., K. A. Mohammed, J. Hardwick, R. D. Van Horn, K. L. Sanders, C. M. Doerschuk, J. W. Hott, and V. B. Antony. 2001. Polar production of interleukin-8 by mesothelial cells promotes the transmesothelial migration of neutrophils: role of intercellular adhesion molecule-1. J. Infect. Dis. 183: 1638-1645 [Medline].

6. Baggiolini, M.. 2001. Chemokines in pathology and medicine. J. Intern. Med. 250: 91-104 [Medline].

7. D'Ambrosio, D., M. Mariani, P. Panina-Bordignon, and F. Sinigaglia. 2001. Chemokines and their receptors guiding T lymphocyte recruitment in lung inflammation. Am. J. Respir. Crit. Care Med. 164: 1266-1275 [Free Full Text].

8. Antony, V. B., S. W. Godbey, S. L. Kunkel, J. W. Hott, D. L. Hartman, M. D. Burdick, and R. M. Strieter. 1993. Recruitment of inflammatory cells to the pleural space. Chemotactic cytokines, IL-8, and monocyte chemotactic peptide-1 in human pleural fluids. J. Immunol. 151: 7216-7223 [Abstract].

9. Mohammed, K. A., N. Nasreen, M. J. Ward, K. K. Mubarak, F. Rodriguez-Panadero, and V. B. Antony. 1998. Mycobacterium-mediated chemokine expression in pleural mesothelial cells: role of C-C chemokines in tuberculous pleurisy. J. Infect. Dis. 178: 1450-1456 [Medline].

10. Katayama, H., A. Yokoyama, N. Kohno, K. Sakai, K. Hiwada, H. Yamada, and H. K. Hirai. 2002. Production of eosinophilic chemokines by normal pleural mesothelial cells. Am. J. Respir. Cell Mol. Biol. 26: 398-403 [Abstract/Free Full Text].

11. Jose, P. J., D. A. Griffiths-Johnson, P. D. Collins, D. T. Walsh, R. Moqbel, N. F. Totty, O. Truong, J. J. Hsuan, and T. J. Williams. 1994. Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation. J. Exp. Med. 179: 881-887 [Abstract/Free Full Text].

12. Beck, L. A., S. Dalke, K. M. Leiferman, C. A. Bickel, R. Hamilton, H. Rosen, B. S. Bochner, and R. P. Schleimer. 1997. Cutaneous injection of RANTES causes eosinophil recruitment: comparison of nonallergic and allergic human subjects. J. Immunol. 159: 2962-2972 [Abstract].

13. Fujino, S., A. Yokoyama, N. Kohno, and K. Hiwada. 1996. Interleukin 6 is an autocrine growth factor for normal human pleural mesothelial cells. Am. J. Respir. Cell Mol. Biol. 14: 508-515 [Abstract].

14. Mohammed, K. A., N. Nasreen, M. J. Ward, and V. B. Antony. 1999. Helper T cell type 1 and 2 cytokines regulate C-C chemokine expression in mouse pleural mesothelial cells. Am. J. Respir. Crit. Care Med. 159(5, Pt. 1):1653-1659.

15. Stellato, C., L. A. Beck, G. A. Gorgone, D. Proud, T. J. Schall, S. J. Ono, L. M. Lichtenstein, and R. P. Schleimer. 1995. Expression of the chemokine RANTES by a human bronchial epithelial cell line. Modulation by cytokines and glucocorticoids. J. Immunol. 155: 410-418 [Abstract].

16. Stellato, C., S. Matsukura, A. Fal, J. White, L. A. Beck, D. Proud, and R. P. Schleimer. 1999. Differential regulation of epithelial-derived C-C chemokine expression by IL-4 and the glucocorticoid budesonide. J. Immunol. 163: 5624-5632 [Abstract/Free Full Text].

17. John, M., S. J. Hirst, P. J. Jose, A. Robichaud, N. Berkman, C. Witt, C. H. Twort, P. J. Barnes, and K. F. Chung. 1997. Human airway smooth muscle cells express and release RANTES in response to T helper 1 cytokines: regulation by T helper 2 cytokines and corticosteroids. J. Immunol. 158: 1841-1847 [Abstract].

18. Teran, L. M., M. Mochizuki, J. Bartels, E. L. Valencia, T. Nakajima, K. Hirai, and J. M. Schroder. 1999. Th1- and Th2-type cytokines regulate the expression and production of eotaxin and RANTES by human lung fibroblasts. Am. J. Respir. Cell Mol. Biol. 20: 777-786 [Abstract/Free Full Text].

19. Shinkai, A., H. Yoshisue, M. Koike, E. Shoji, S. Nakagawa, A. Saito, T. Takeda, S. Imabeppu, Y. Kato, N. Hanai, H. Anazawa, T. Kuga, and T. Nishi. 1999. A novel human CC chemokine, eotaxin-3, which is expressed in IL-4- stimulated vascular endothelial cells, exhibits potent activity toward eosinophils. J. Immunol. 163: 1602-1610 [Abstract/Free Full Text].

20. Larbi, K. Y., A. R. Allen, F. W. Tam, D. O. Haskard, R. R. Lobb, P. M. Silva, and S. Nourshargh. 2000. VCAM-1 has a tissue-specific role in mediating interleukin-4-induced eosinophil accumulation in rat models: evidence for a dissociation between endothelial-cell VCAM-1 expression and a functional role in eosinophil migration. Blood 96: 3601-3609 [Abstract/Free Full Text].

21. Rubins, J. B., and H. B. Rubins. 1996. Etiology and prognostic significance of eosinophilic pleural effusions. A prospective study. Chest 110: 1271-1274 [Abstract/Free Full Text].

22. Yokoyama, A., N. Kohno, M. Ito, M. Abe, K. Hiwada, H. Yamada, K. Matsushima, and K. Hirai. 2000. Eotaxin levels in pleural effusions: comparison with monocyte chemoattractant protein-1 and IL-8. Intern. Med. 39: 547-552 [Medline].

23. Ying, S., D. S. Robinson, Q. Meng, J. Rottman, R. Kennedy, D. J. Ringler, C. R. Mackay, B. L. Daugherty, M. S. Springer, S. R. Durham, T. J. Williams, and A. B. Kay. 1997. Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma: association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells. Eur. J. Immunol. 27: 3507-3516 [Medline].

24. Nickel, R., L. A. Beck, C. Stellato, and R. P. Schleimer. 1999. Chemokines and allergic disease. J. Allergy Clin. Immunol. 104(4, Pt. 1):723-742.

25. Lambrecht, B. N., M. De Veerman, A. J. Coyle, J. C. Gutierrez-Ramos, K. Thielemans, and R. A. Pauwels. 2000. Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation. J. Clin. Invest. 106: 551-559 [Medline].

26. Larangeira, A. P., A. R. Silva, R. N. Gomes, C. Penido, M. G. Henriques, H. C. Castro-Faria-Neto, and P. T. Bozza. 2001. Mechanisms of allergen- and LPS-induced bone marrow eosinophil mobilization and eosinophil accumulation into the pleural cavity: a role for CD11b/CD18 complex. Inflamm. Res. 50: 309-316 [Medline].

27. Klein, A., A. Talvani, P. M. Silva, M. A. Martins, T. N. Wells, A. Proudfoot, N. W. Luckacs, and M. M. Teixeira. 2001. Stem cell factor-induced leukotriene B4 production cooperates with eotaxin to mediate the recruitment of eosinophils during allergic pleurisy in mice. J. Immunol. 167: 524-531 [Abstract/Free Full Text].

28. Mohammed, K. A., N. Nasreen, M. J. Ward, and V. B. Antony. 2000. Induction of acute pleural inflammation by Staphylococcus aureus. I. CD4+ T cells play a critical role in experimental empyema. J. Infect. Dis. 181: 1693-1699 [Medline].

29. Light, R. W. 1995. Pleural Diseases. Williams & Wilkins, Baltimore, MD.

30. Nasreen, N., K. A. Mohammed, G. Galffy, M. J. Ward, and V. B. Antony. 2000. MCP-1 in pleural injury: CCR2 mediates haptotaxis of pleural mesothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L591-L598 [Abstract/Free Full Text].

31. Cuvelier, S. L., and K. D. Patel. 2001. Shear-dependent eosinophil transmigration on interleukin 4-stimulated endothelial cells: a role for endothelium-associated eotaxin-3. J. Exp. Med. 194: 1699-1709 [Abstract/Free Full Text].

32. Ohmori, Y., R. D. Schreiber, and T. A. Hamilton. 1997. Synergy between interferon-gamma and tumor necrosis factor-alpha in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor kappaB. J. Biol. Chem. 272: 14899-14907 [Abstract/Free Full Text].

33. Koga, T., E. Sardina, R. M. Tidwell, M. Pelletier, D. C. Look, and M. J. Holtzman. 1999. Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization. Proc. Natl. Acad. Sci. USA 96: 5680-5685 [Abstract/Free Full Text].

34. Chen, C. Y., R. Gherzi, S. E. Ong, E. L. Chan, R. Raijmakers, G. J. Pruijn, G. Stoecklin, C. Moroni, M. Mann, and M. Karin. 2001. AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell 107: 451-464 [Medline].

35. Matsukura, S., C. Stellato, J. R. Plitt, C. Bickel, K. Miura, S. N. Georas, V. Casolaro, and R. P. Schleimer. 1999. Activation of eotaxin gene transcription by NF-kappa B and STAT6 in human airway epithelial cells. J. Immunol. 163: 6876-6883 [Abstract/Free Full Text].

36. Antony, V. B., J. W. Hott, S. L. Kunkel, S. W. Godbey, M. D. Burdick, and R. M. Strieter. 1995. Pleural mesothelial cell expression of C-C (monocyte chemotactic peptide) and C-X-C (interleukin 8) chemokines. Am. J. Respir. Cell Mol. Biol. 12: 581-588 [Abstract].

37. Nasreen, N., D. L. Hartman, K. A. Mohammed, and V. B. Antony. 1998. Talc-induced expression of C-C and C-X-C chemokines and intercellular adhesion molecule-1 in mesothelial cells. Am. J. Respir. Crit. Care Med. 158: 971-978 [Abstract/Free Full Text].

38. Tanaka, S., N. Choe, A. Iwagaki, D. R. Hemenway, and E. Kagan. 2000. Asbestos exposure induces MCP-1 secretion by pleural mesothelial cells. Exp. Lung Res. 26: 241-255 [Medline].

39. Miller, E. J., O. Kajikawa, S. Pueblitz, R. W. Light, K. K. Koenig, and S. Idell. 1999. Chemokine involvement in tetracycline-induced pleuritis. Eur. Respir. J. 14: 1387-1393 [Abstract].

40. Penido, C., H. C. Castro-Faria-Neto, A. Vieira-de-Abreu, R. T. Figueiredo, A. Pelled, M. A. Martins, P. J. Jose, T. J. Williams, and P. T. Bozza. 2001. LPS induces eosinophil migration via CCR3 signaling through a mechanism independent of RANTES and eotaxin. Am. J. Respir. Cell Mol. Biol. 25: 707-716 [Abstract/Free Full Text].