PERSPECTIVE
Intraepithelial Lymphocytes in the Lung
A Neglected Lymphocyte Population

David J. Erle and Reinhard Pabst

Lung Biology Center, Program in Immunology, and Cardiovascular Research Institute, University of California, San Francisco, California; and Center of Anatomy, Medical School of Hannover, Hannover, Germany

In the work presented in this issue, Goto and colleagues (1) have provided strong new evidence that a distinct population of lymphocytes resides within the bronchial epithelium. By following the fates of human bronchial xenografts after transplantation into SCID mice, these investigators were able to show long-term persistence (over 5 mo) of human intraepithelial lymphocytes (IEL) in grafts, even as the number of lymphocytes in the adjacent lamina propria declined precipitously. Although little is known about the function of bronchial IEL, their location suggests important roles in the control of immune and inflammatory reactions in the lung. Goto and coworkers' work should prompt further investigation into the roles of bronchial IEL in health and disease.

	Characteristics of IEL

Lymphocytes are easily identified within the epithelium of mucosal organs in the respiratory, gastrointestinal, and reproductive tracts, and in the epidermis of the skin. IEL are abundant cells (2). Biopsy sections from normal volunteers were found to have nearly 20 bronchial IEL/100 epithelial cell nuclei (3), a density similar to that reported for the human intestine (4). IEL are scattered along the length of the epithelium, usually near its basal surface, and generally do not form aggregates. There is a decrease in the number of IEL from the jejunum to the colon in humans (5), and it is not known whether a similar gradient exists from the trachea to small bronchi. We recently showed that the number and rate of proliferation of pig intestinal IEL depended on age and breeding conditions (germ-free, specified pathogen-free, or conventional) (6), and these parameters might also influence IEL in the bronchial tract.

The overwhelming majority of IEL in the bronchi and elsewhere are T cells. CD8⁺ cytotoxic/suppressor T cells usually predominate over CD4⁺ T-helper cells, although this predominance is less marked in bronchial epithelium (CD4/ CD8 ratio,  0.4) than in intestinal epithelium (CD4/CD8  0.1) (1, 3, 4, 7). In the intestine, a substantial fraction of IEL expresses T-cell receptors. Although T cells are commonly found in murine lung, almost all human bronchial IEL express T-cell receptors (1). Expression of a variety of cell-surface markers associated with T-cell activation or differentiation differs on IEL as compared with T cells at other sites. For example, the integrin E7 is expressed on nearly all intestinal IEL and on many bronchial IEL, but is rarely found on T cells at nonmucosal sites (8).

	Generation and Maintenance of the Bronchial IEL Population

As just previously discussed, IEL populations differ in several ways from populations of T cells found at other sites. These differences might arise by one or more of several mechanisms: preferential recruitment of subsets of circulating T cells to the epithelium, or selective survival, proliferation, or retention of subsets within the epithelium.

The recruitment of lymphocytes from the blood is regulated by multistep pathways, which involve adhesion molecules and chemoattractants, especially chemokines. Some lymphocyte adhesion molecules and chemokine receptors can direct selective migration (or "homing") of T-cell subsets to specific organs. For example, the endothelial surface glycoprotein MAdCAM-1 and the chemokine TECK are both expressed selectively in the intestine. T cells that express the MAdCAM-1 receptor (integrin 47) and the TECK receptor (CCR9) are preferentially recruited to the intestinal epithelium (as well as other compartments in the intestine) (9, 10). The lymphocyte adhesion molecule CLA and the chemokine CTACK promote migration of a distinct subset of T cells to the skin (11). Molecules that selectively promote migration of T cells to the airways have not yet been identified. However, it remains possible that selective migration to the airways could occur via as yet unidentified, novel lung-specific adhesion molecules or chemokines, or via multistep pathways involving lung-specific combinations of known adhesion molecules and chemokines. Furthermore, it has to be stressed that lymphocytes can reach the lung either via the pulmonary or the bronchial vasculature, which might express different adhesion molecules (reviewed in Reference 12).

The epithelium is only one of several anatomically distinct lung compartments with a substantial population of lymphocytes (12, 13). Other compartments include the bronchial lamina propria, the lung interstitium, the bronchoalveolar lumen, the pulmonary vasculature, the bronchus-associated lymphoid tissue (BALT; not generally present in normal adult humans), and the bronchial lymph nodes. The IEL population differs in many respects from the lymphocyte populations in other compartments. For instance, the proportions of lymphocytes expressing CD8 and integrin E7 are higher in the epithelium than in other compartments. There is clearly some movement of lymphocytes between compartments, but it is not yet clear how frequently T cells move from adjacent compartments, e.g., lamina propria and bronchoalveolar space into the bronchial epithelium or vice versa.

Specialized pathways likely control the survival, proliferation, and retention of T cells within the epithelium. Two molecules that may have roles here are integrin E7, expressed on IEL, and its ligand E-cadherin, expressed on epithelial cells (14). This interaction can support IEL-epithelial cell adhesion in vitro and might help retain IEL in the epithelium in vivo. Ligation of E7 provides a costimulatory signal for T-cell proliferation (15), suggesting a mechanism that could promote the survival and proliferation of E7⁺ IEL. Mice deficient in E7 have reduced numbers of lymphocytes within the intestinal epithelium and lamina propria, but show no reduction of "peribronchial" or lung parenchymal lymphocytes (16). Although no functional role for E7 on bronchial IEL has been identified to date, the E7-deficient mice will provide an excellent tool for studying the role of this molecule in models of various diseases such as viral infections or asthma. In addition to the E7 ligand E-cadherin, epithelial cells produce other membrane-associated and soluble factors, which are also known to affect T-cell growth and differentiation (1). IEL might also play an important role in viral infections. After infecting mice with Sendai virus, a 12-fold increase of lymphocytes in the tracheal epithelium from Day 3 to Day 5 after infection was described with a 12 times higher increase of IEL (17).

Some IEL have lineages that are quite distinct from those of other T cells (4). Whereas T cells destined for other sites must undergo T-cell receptor rearrangement and selection in the thymus, a large fraction of intestinal IEL develops normally in athymic mice. This thymus- independent pathway results in the production of intestinal IEL bearing an unusual form of CD8 (CD8). Although IEL outside of the intestine do not arise via thymus-independent pathways, it remains possible that some bronchial IEL may arise from specialized precursor T cells. The data presented by Goto and associates (1) strongly suggest that bronchial IEL are long-lived T cells that survive within the epithelium for at least several months, in contrast to the short life span of lymphocytes in the lamina propria. An alternative possibility is that bronchial IEL are a self-renewing population. However, measurement of the proliferation marker Ki67 in IEL in the human small intestine suggests that these cells do not proliferate under normal conditions, even though considerable proliferation of both and IEL was seen in patients with celiac disease, an immune-mediated intestinal disorder (18).

	Potential Functions of Bronchial IEL

Lung lymphocytes clearly have essential functions in host defense against infection and in the pathogenesis of a variety of noninfectious disorders. Although phenotypic differences between IEL and other lung lymphocytes would suggest that IEL make unique contributions to airway immune responses, there is little, if any, direct experimental support for this concept. Although a variety of diseases, including asthma, chronic bronchitis, and bronchiectasis, have been associated with increases in the number and/or activation state of IEL, these changes are consistently associated with similar changes found in lymphocytes in other lung compartments.

Animal models of lung disease have been used to study the role of specific subsets of T cells. Mice deficient in T cells developed more severe disease than control mice after inoculation with Nocardia asteroides or exposure to ozone (19). Deficiency of T cells was associated with impairment in neutrophil recruitment and with development of more severe epithelial injury, suggesting that IEL may help protect epithelial surfaces. However, it is not yet known if there is a specific requirement for IEL versus T cells in other compartments. Even if IEL do have a unique role in the mouse, it is not at all clear that IEL in humans (which include few, if any, T cells) have similar involvement.

It is tempting to speculate about (as yet unproven) unique functions for bronchial IEL. These cells are located very near the mucosal surface, an initial site of contact with pathogens and other antigens. Epithelial cells themselves, as well as dendritic cells trafficking through the epithelium, express a variety of molecules that play key roles in antigen presentation, raising the possibility that inhaled antigens may be presented rapidly and directly to IEL within the airway wall. The ability of bronchial IEL to persist within the epithelium for long periods of time may provide a pool of memory cells primed to respond rapidly to antigens most commonly encountered in the airways. IEL might also serve in the maintenance of tolerance to nonpathogenic environmental allergens encountered in the airways.

Emerging evidence indicates that cytokines and other substances produced by T cells can have significant, direct effects on epithelial function, and this is likely to be important in the pathogenesis of airway diseases such as asthma. The cytokine IL-9, for instance, is produced by T cells during allergic airway responses and induces the production of mucin by epithelial cells (20). It also seems evident that at least some of the major effects of the type 2 cytokines IL-4 and IL-13 on the airway involve direct effects on epithelial cells (21, 22). IEL would seem to be ideally positioned to produce substances that act on epithelial cells. Conversely, epithelial cells can modulate the function of lymphocytes: the cytokine TGF-, which can be produced and locally activated by epithelial cells (23), has been shown to promote expression of integrin E7 in vitro (24). Evidence also shows that epithelial cells can selectively downregulate proliferation and cytokine production by intestinal IEL but not by spleen T cells, suggesting that specialized IEL-epithelial cell interactions might be important in the induction of tolerance (25). It is therefore apparent that some of the special properties of IEL derive from their interactions with adjacent epithelial cells.

	Challenges for the Future

The work of Goto and coworkers (1) should motivate investigators to consider the likelihood that bronchial IEL may function differently than T lymphocytes at other sites in the airways, even those that reside in other compartments only microns away. While some important insights into IEL function have come from studies of intestinal IEL, it is now clear that these insights cannot be extrapolated to bronchial IEL without direct experimental proof.

The complex anatomic arrangement of lung compartments obviously poses a considerable technical obstacle for those who wish to study bronchial IEL function. Many studies of lung lymphocyte function have involved analysis of lymphocytes isolated by bronchoalveolar lavage (BAL). BAL fluid lymphocytes commonly express E7 (26), suggesting that at least some of these cells are derived from the intraepithelial and lamina propria compartments; but the relationship between BAL T cells and IEL is not understood.

The population of cells recovered by BAL differs from the IEL population in important ways. For example, CD4/CD8 ratios are usually > 1.0 in BAL fluid but < 1.0 in the epithelium. Further complicating the issue, the recovery of IEL in BAL fluid might be altered in asthma or during viral infections. Lymphocytes can also be isolated from the lung by mincing or enzymatic digestion, but these methods clearly result in pooling of T cells from more than one compartment.

The development of methods for the purification of bronchial IEL would be a crucial step. Methods for purifying intestinal IEL are well established and have been widely used to help characterize the function of these cells. Because of the complex branching structure of the airways, these methods are not readily transferable to studies of bronchial IEL. Identification of a unique surface marker (or combination of markers) for bronchial IEL would certainly facilitate the isolation of these cells, but it is not at all clear that such markers exist. The development of newer techniques, such as laser microdissection, could facilitate the isolation of pure populations of bronchial IEL. Although the yield of such a method would be quite limited, techniques that now exist or are in development should allow for the analysis of lymphocyte activation, cytokine production, cytotoxic effects, gene expression, and other phenomena, even when very few IEL are obtained.

	Footnotes

Address correspondence to: David J. Erle, M.D., UCSF Box 0854, San Francisco, CA 94143-0854. E-mail: erle{at}itsa.ucsf.edu

(Received in original form January 29, 2000).

Acknowledgments: The authors' own work was supported in part by NIH grants HL50024 and DK54212 (D.J.E.) and German Research Foundation grant DFG Pa 240/8-1 (R.P.).

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