Published ahead of print on August 23, 2007, doi:10.1164/rccm.200609-1347OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200609-1347OC
Dendritic Cells Accumulate in Human Fibrotic Interstitial Lung Disease1 Inserm, U 700, and Faculté de Médecine Paris-Nord, site Bichat, Université Paris 7, Paris, France; 2 Service de Pneumologie, Assistance Publique-Hôpitaux de Paris, Hôpital Bichat, Paris, France; and 3 Service d'Anatomo-Pathologie and 4 Service de Pneumologie, Assistance Publique-Hôpitaux de Paris, Hôpital Avicenne, Bobigny, France Correspondence and requests for reprints should be addressed to Paul Soler, Ph.D., Inserm U700, Faculté de Medicine Paris-Nord, site Bichat, 16 rue Henri Huchard, 75018 Paris, France. E-mail: soler{at}bichat.inserm.fr
Rationale: There is growing evidence that resident cells, such as fibroblasts and epithelial cells, can drive the persistent accumulation of dendritic cells (DCs) in chronically inflamed tissue, leading to the organization and the maintenance of ectopic lymphoid aggregates. This phenomenon, occurring through a chemokine-mediated retention mechanism, has been documented in various disorders, but not in fibrotic interstitial lung disorders in which the presence of organized lymphoid follicles has been documented. Objectives: To characterize the distribution of DCs in fibrotic lung, and to analyze the expression of the main chemokines known to regulate DC recruitment. Methods: Lung resection tissue (lungs with idiopathic pulmonary fibrosis; n = 12; lungs with nonspecific interstitial pneumonia, n = 5; control lungs, n = 5) was snap-frozen for subsequent immunohistochemical techniques on serial sections and reverse transcriptase–polymerase chain reaction analysis. Measurements and Main Results: Results were similar in idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia lungs, which were heavily infiltrated by immature DCs in established fibrosis and in areas of epithelial hyperplasia. Altered epithelial cells and fibroblasts, particularly in fibroblastic foci, frankly expressed all chemokines (CCL19, CCL20, CCL22, and CXCL12) susceptible to favor the recruitment of immune cells. Lymphoid follicles were infiltrated by maturing DCs, which could originate from the pool of DCs accumulating in their vicinity. Conclusions: These findings suggest that resident cells in pulmonary fibrosis can sustain chronic inflammation by driving the accumulation of DCs with the potential to mature locally within ectopic lymphoid follicles. Future strategies should consider DCs or chemokines as therapeutic targets in the treatment of pulmonary fibrosis.
Key Words: chronic inflammation • dendritic cells • epithelial cells • fibroblasts • idiopathic pulmonary fibrosis
We have recently shown that lymphoid follicles made of nonproliferating activated B cells and T cells and fully mature dendritic cells (DCs) are present in the lungs of patients with idiopathic pulmonary fibrosis (IPF) (1). These lymph node–like structures are organized close to blood vessels that strongly express the chemokines CCL19, CCL21, CCL22, and CXCL12. This suggests that IPF lymphoid follicles can persist in an autonomous fashion by recruiting circulating immune cells, such as maturing DCs and recently activated T cells. The following several arguments suggest, however, that DCs present within IPF lymphoid follicles might originate from a pool of DCs recruited to the fibrotic lung and maturing locally: (1) DCs are widely distributed throughout the normal lung (2); (2) they are increased in numbers in various respiratory disorders and they have been clearly involved in the pathogenesis of these disorders (2); (3) several inflammatory mediators are produced in IPF lung, susceptible to activate locally incoming DCs (3); and (4) there is growing evidence that fibrotic tissue may act as a "foster home" for immune cells through the inappropriate production of proretention inflammatory mediators (4, 5). From this point of view, fibroblasts and other stromal elements, including epithelial cells, define "stromal address codes" made of chemokines and cytokines that physiologically regulate leukocyte accumulation, differentiation, and survival in lymphoid organs, and in nonlymphoid organs during inflammatory responses (5). In chronic persistent inflammation, the stromal address code expressed locally is aberrantly produced after tissue repair, leading to the persistent accumulation of DCs and lymphocytes within inflamed tissue and their organization in ectopic lymphoid aggregates (5, 6). This phenomenon has been documented in various chronic inflammatory diseases, including rheumatoid arthritis, autoimmune liver or thyroid disease, and diabetes (5). It has not yet been described in IPF or in other fibrotic interstitial lung diseases, but the morphologic changes observed in these diseases strongly suggest that it might be involved as well; IPF and fibrosing nonspecific interstitial pneumonia (NSIP), the other major form of fibrotic lung disorders, are both characterized by the presence of lymphoid follicles and by the massive increase in numbers of altered alveolar epithelial cells, and of fibroblasts/myofibroblasts (7–9). These cell populations have the potential to modulate the recruitment and retention of immune cells, because they are known to produce various inflammatory mediators (3, 10). However, current information about the production of DC-attracting chemokines within fibrotic lung and about the presence and distribution of DCs outside lymphoid follicles is lacking. The aim of this study was to characterize in situ the phenotype and the distribution of DCs in human fibrotic interstitial lung disease, and to analyze fibroblastic and epithelial expression of the main chemokines known to regulate DC recruitment and migration. Results show that lung tissue from patients with IPF or NSIP is heavily infiltrated by immature DCs. Hyperplastic epithelial cells and fibroblasts, particularly fibroblasts present in IPF fibroblastic foci, strongly express all chemokines known to favor DC and lymphocyte recruitment and their organization in lymphoid-like structures (11).
Tissue Samples Lung tissue samples were obtained by open lung biopsy (n = 7) or at the time of lung transplantation (n = 5) from 12 patients with IPF (9 men, 3 women; median age 55, yr [range, 44–69 yr]; 8 smokers, 4 nonsmokers) and by open lung biopsy for 5 patients with fibrotic NSIP (4 idiopathic NSIP, 1 systemic sclerosis–associated NSIP; 2 men, 3 women; median age, 58 yr [range, 44–71 yr]; 2 ex-smokers, 3 nonsmokers). IPF and NSIP were diagnosed according to the American Thoracic Society/European Respiratory Society consensus criteria (12), including clinical, radiographic, and characteristic histopathologic features. Two patients with NSIP presented with a cellular and fibrosing histopathologic pattern. All patients showed a restrictive functional pattern. At the time of lung biopsy, two patients with IPF were treated with oral corticosteroids, and one patient was receiving low-dose hydrocortisone to treat adrenal insufficiency. Patients with NSIP received no treatment. Lung tissue samples obtained from five patients (4 men, 1 woman; median age, 60 yr [range, 52–69 yr]; 4 smokers, 1 nonsmoker) who underwent thoracic surgery for localized primary lung carcinoma were used as control subjects. Lung tissue was taken at a site distant from the lesion and was grossly normal. Microscopic examination demonstrated that lung tissue from the nonsmoking control subject was histologically normal, whereas biopsies from smokers showed the accumulation of pigment-laden macrophages and mild focal fibrotic changes associated with moderate alveolar epithelial cell hyperplasia. This study was approved by the Paris-Bichat University Hospital (Paris) ethics committee, and patients gave their informed consent before lung surgery.
Immunohistochemistry
Quantitative Analysis of Chemokine mRNA
Statistical Analysis
Immature DCs Accumulate in Large Numbers in Fibrotic Lung Different subsets of DCs were investigated on the basis of the expression of different markers that characterize immature (CD1a, CD1c, DC-specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN]) and mature (CD83, CD86, DC-lysosome-associated membrane protein [DC-LAMP]) DCs. DCs are professional antigen-presenting cells that are widely distributed, particularly at mucosal sites where they are specialized in internalizing foreign antigens. In these peripheral sites, intraepithelial DCs known as Langerhans cells (LCs) express CD1a and/or CD1c, whereas submucosal DCs express CD1c, and DCs residing within the surrounding connective tissue express DC-SIGN (14–17). On recognition of antigen and in the context of a danger signal, peripheral DCs undergo a process of maturation and migrate to the draining lymph nodes for interaction with specific T cells (14). During their migration, maturing DCs up-regulate the expression of costimulatory molecules (CD40, CD80, CD86) that are required for activation of T cells. Within the lymphoid organs, fully mature DCs express additional markers, such as CD83 and DC-LAMP. We used specific antibodies that recognize each of the markers described above to characterize the presence and the distribution of both immature and maturing/mature DCs in the fibrotic lung. Cells with a dendritic morphology expressing the markers of immature DCs were found to heavily infiltrate IPF and NSIP lesions. They were detected in all cases and were significantly increased in numbers compared with control lungs (Table 1). No differences were observed comparing patients with IPF and patients with NSIP, either with a fibrosing or a fibrosing and cellular pattern, or comparing smokers and nonsmokers (see below). As previously described, mature DCs expressing CD83, CD86, or DC-LAMP were detected within IPF lymphoid follicles mainly composed of T cells associated with variable numbers of B cells (1). Mature DCs expressing CD83, CD86, or DC-LAMP were also detected in NSIP lung samples, infiltrating lymphoid follicles similarly to those observed in IPF lung tissues (1). Lymphoid follicles were observed in all fibrotic lung samples examined. They were organized structures typical of lymphoid neogenesis, which is usually linked to chronic inflammation (11), eliminating a possible surinfection status in end stages of fibrosis. Within follicles, mature DCs were intercalated between T lymphocytes. They had a characteristic dendritic shape and were not stained with anti-CD20 antibody on adjacent sections, further confirming that they were not epithelial cells recently shown to express DC-LAMP (18), or CD83+ activated B lymphocytes. In control lungs, no cells with a dendritic shape expressing markers of mature DCs were identified because no lymphoid aggregates were observed associated with small airways.
Immature DCs Are Specifically Distributed in Fibrotic Lung Immature DCs were distributed according to their surface phenotype and to the type of lesion. DCs expressing CD1a or CD1c antigens were essentially detected in areas characterized by hyperplastic alveolar epithelial changes. CD1a+ DCs were observed intercalated between hyperplastic epithelial cells, whereas CD1c+ DCs infiltrated the subepithelial connective tissue, sometimes in contact with epithelial cells (Figures 1A and B). Hyperplastic epithelium overlying fibroblastic foci contained few, if any, CD1a+ DCs, and within the foci, no CD1c+ DCs could be detected. As mentioned above, no differences were observed when comparing smokers and nonsmokers, reflecting the fact that alveolar epithelial hyperplasia is commonly observed in smokers (4 of 5 control patients in our series) and in all patients with fibrotic interstitial lung disease (15, 16, 19). On adjacent sections, the numbers of cells per millimeter of hyperplastic epithelium expressing CD1a or langerin were quite similar (IPF: 7 cells/mm [range, 1–11] and 6 cells/mm [range, 2–10], respectively; NSIP: 7 cells/mm [range, 5–8] and 6 cells/mm [range, 4–8], respectively). This strongly suggested that DCs infiltrating hyperplastic epithelia were CD1a+/langerin+ LCs committed in antigen uptake.
DC-SIGN+ DCs represented the most important population of DCs infiltrating fibrotic lung. In all cases, they were detected in large numbers in patients with IPF and NSIP compared with control subjects (Table 1). DC-SIGN+ cells were mainly observed in control lung in peribronchovascular and subpleural areas rich in connective tissue (Figure 1C). In fibrotic lung, unlike CD1a+/CD1c+ DCs, which were essentially distributed in areas of alveolar hyperplasia, DC-SIGN+ cells were scattered in the less cellular areas of established fibrosis and around blood vessels (Figure 1D). On adjacent sections, endothelial cells of blood vessels were found to strongly express intercellular adhesion molecule-2 (ICAM-2) (Figure 1E), the primary counterreceptor for DC-SIGN involved in DC trafficking (20, 21). In addition to their dendritic morphology, DC-SIGN+ cells were always CD68 negative as assessed using serial tissue sections (Figures 1F and 1G), further confirming that they were not inflammatory macrophages (22) but tissue resident immature DCs (17). Of note, DC-SIGN+ cells, likely corresponding to maturing DCs present in the T-cell area of lymph nodes (23), were also observed, intercalated between lymphocytes at the periphery of most lymphoid follicles associated with IPF or NSIP fibrotic lesions (Figure 1H). On adjacent sections, T-cell aggregates strongly expressed ICAM-3 (Figure 1I), the other ligand for DC-SIGN allowing immunologic synapse formation in secondary lymphoid organs (21). As for CD1c+ DCs, no DC-SIGN+ cells were observed within fibroblastic foci, whereas they were mobilized in adjacent fibrotic tissue. Likewise, whereas a moderate to important infiltration by lymphocytes was noted in all lesions, no lymphocytes infiltrated fibroblastic foci.
DC-attracting Chemokines Are Strongly Expressed in Fibrotic Lung Results are summarized in Table 2. They were quite similar in both IPF and NSIP lung tissues: hyperplastic epithelial cells and fibroblasts, particularly fibroblasts in fibroblastic foci, the sites of active ongoing fibrosis in IPF lung, strongly expressed CCL19, CCL22, and CXCL12, and to a lesser degree CCL20 and CCL21 (Figures 2A–2E). Consistent with an afflux of DCs in fibrotic lung, endothelial cells in established fibrosis, unlike those in normal lung interstitium, expressed CXCL12 (Figure 2F), a very potent chemokine for transendothelial migration of DCs (20, 27). As previously described, lymphocytes organized in follicles similarly expressed these chemokines, and the numerous vascular structures associated with the follicles, including vessels resembling high endothelial venules, were found to be positive for CCL19, CCL21, CCL22, and CXCL12 (1). Characteristic thin lymphatic vessels strongly positive for CCL21 were present at the periphery of the follicles (Figure 3A), suggesting that neolymphangiogenesis was also associated with the follicles. On adjacent sections, DCs strongly positive for CCR7 were observed in lymphoid follicles in the same areas where mature DCs accumulated, suggesting DC recruitment via CCL21–CCR7 interaction (Figures 3B and 3C). Surprisingly, although epithelial cells frankly expressed CCL20 and were heavily infiltrated by CD1a+ DCs, no DCs expressing CCR6 were observed within hyperplastic epithelium, whereas CCR6+ lymphocytes were clearly present in the same sections (Figure 3D). CCL17 was only expressed by epithelial cells, whereas other cell types, including smooth muscle cells and alveolar macrophages, moderately expressed most of the chemokines examined (Figure 2). In control lung tissues, only airway epithelial cells and alveolar macrophages were found to express more or less frankly these chemokines (not shown).
To examine whether the immunodetection of chemokines in the lungs of patients with pulmonary fibrosis actually reflected a local production of these factors by lung tissue, we performed a real-time PCR analysis of chemokine mRNA expressed in fibrotic lung. Total RNA necessary for the analysis was available for patients with IPF and control patients only. As shown in Figure 4, mRNA coding for the different chemokines assessed by immunohistochemistry was detected in all samples examined. Although the levels of transcript expression were quite variable in patients with IPF, they tended to be higher than those of control patients for CCL17 and CCL22, and interestingly, they were significantly increased for CCL19, CCL21, and CXCL12, the main chemokines known to favor immune cell recruitment and accumulation (11, 25).
Although mRNA analysis could not be performed for patients with NSIP, altogether our results strongly suggested that the accumulation of DCs in fibrotic lung was a chemokine-mediated phenomenon driven by epithelial cells and fibroblasts.
The present study demonstrates for the first time that an important population of immature DCs infiltrate lung tissue in pulmonary fibrosis, with the potential to sustain chronic inflammation by maturing locally within ectopic lymphoid follicles. It also suggests that hyperplastic epithelial cells and fibroblasts, which are known to play a major role by driving pulmonary fibrogenesis, could play an additional key role by driving lymphoid neogenesis. These results offer new insights into the pathogenesis of fibrotic interstitial lung disease and reconcile previous contradictory models regarding the role of inflammation in the progression of IPF (9, 28). Different subsets of immature DCs were found to infiltrate fibrotic lung with a specific distribution. In line with previously published data on pulmonary DCs in smokers and in patients with fibrotic lung disorders (15, 16, 19), intraepithelial CD1a+/langerin+ DCs, corresponding to LCs committed in mucosal immunologic surveillance (2, 14), and subepithelial DCs expressing CD1c were observed in areas of alveolar hyperplasia. Pulmonary CD1c+ DCs, which are believed to give rise to intraepithelial LCs (15, 16, 29), are known to rapidly accumulate in inflammatory lung mucosa (30), strongly suggesting that DCs accumulating in areas of alveolar hyperplasia in fibrotic lung represented mucosal DCs mobilized at the sites of epithelial repair. The recruitment of DCs to inflamed mucosae is believed to be under the control of CCL20 overexpressed by epithelial cells and CCR6 expressed by DCs (14, 24). In the present study, although CCL20 was strongly expressed by hyperplastic epithelium, we failed to detect any intraepithelial CCR6+ DCs despite repeated efforts, whereas CCR6+ lymphocytes were observed in lymphoid follicles in the same sections. Very recently, human pulmonary DCs have been shown to express CCR6 in chronic obstructive pulmonary disease, but these cells were freshly isolated and assessed by RT-PCR and flow cytometry (31). This suggests that the expression of CCR6 at the surface of lung DCs is not yet easily detectable by immunohistochemistry, as stated by others in lung carcinoma (32), or that, in pulmonary fibrosis, immature DCs are attracted by chemokines other than CCL20 or by mediators other than chemokines (reviewed in Reference 33).
DCs expressing DC-SIGN, scattered in fibrotic tissue, represented the most important population of DCs infiltrating fibrotic lung. DC-SIGN, by interacting with ICAM-2 and by regulating CXCL12-induced transendothelial migration of DCs, is central to the unusual trafficking capacity of DCs (19, 21, 27). In the present study, DC-SIGN+ DCs were frequently observed around blood vessels, and on adjacent sections, blood vessels were found to express both ICAM-2 and CXCL12, suggesting an enhanced recruitment of immature DCs from the vascular compartment. The local microenvironment, known to be rich in IL-10 and transforming growth factor- Once within lymphoid organs, DC-SIGN helps maturing DCs form contacts with T cells via interactions with ICAM-3, leading to the full maturation of DCs (16, 36). In agreement with this, lymphoid follicles were strongly positive for ICAM-3, and they were infiltrated by fully mature DCs expressing CD83, CD86, and DC-LAMP, suggesting that DCs clustered with T cells were functionally communicating. Interestingly, T cells populating the lung are memory cells that remain fully competent with respect to effector cytokine production (37, 38). This strongly suggests that reactivation of memory T cells accumulating in pulmonary fibrotic lesions by DCs maturing locally likely plays a central role in sustaining chronic inflammation. Interaction of mature CD4+ T cells with DCs has been recently shown to trigger the development of ectopic lymphoid structures in the thyroid of mice (39). A similar functional approach is clearly needed to demonstrate a pathogenic role for DCs in pulmonary fibrosis. Finally, our results suggested that hyperplastic epithelial cells and fibroblasts, particularly those present in fibroblastic foci, could drive the recruitment and accumulation of DCs in fibrotic lung. These cell populations were found to express CCL19, CXCL12, and CCL21, all chemokines known to mediate DC and lymphocyte recruitment and to orchestrate the formation of both normal and pathologic lymphoid structures (11, 25, 26, 40). This is in complete agreement with recent insights suggesting that the persistent production of lymphoid chemokines by fibroblasts and other resident cells plays a critical role in accumulation and retention of immune cells in inflamed tissues and in the formation of organized infiltrates (4, 5, 11). As discussed above, functional studies are again clearly needed to demonstrate a specific role for chemokines in DC recruitment in the course of pulmonary fibrosis, and to attribute a pathogenic role to them. Surprisingly, although they expressed most chemokines known to attract DCs and lymphocytes, fibroblastic foci were devoid of immune cells, yet present in their vicinity. This could be due to the deficient microvascularization in fibroblastic foci preventing the direct afflux of immune cells (41). This could also be due to a particular microenvironment within fibroblastic foci limiting the migration of immune cells. For example, multiple signals have been described that are able to regulate DC migration, including nonchemokine chemotactic factors, lipid mediators, and membrane proteins (33). More recently, matrix metalloproteases and their tissue inhibitors have been shown to be up-regulated in fibroblastic foci, which could interfere with DC migration (42). In conclusion, our study demonstrates that DCs accumulate in human fibrotic interstitial lung disease independently of the type of pneumonia, representing a general reaction associated with pulmonary fibrosis. It also suggests that epithelial cells and fibroblasts, which can orchestrate ongoing fibrogenesis by producing a complex network of profibrotic inflammatory cytokines and chemokines (43), also have the potential to sustain DC recruitment and lymphoid neogenesis. Although these data obtained using human biopsy material are necessarily mainly descriptive, they offer new pathogenic concepts that now can be tested in experimental systems.
Supported by a grant from the Chancellerie des Universités de Paris (Legs Poix). Y.U. is the recipient of a grant from the Fondation pour la Recherche Médicale. S.M.-A. is the recipient of a grant from the Fondation pour la Recherche Médicale (Prix Mariane Josso). P.S. is the recipient of a Contrat d'Interface Inserm-Assistance Publique/Hôpitaux de Paris. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200609-1347OC on August 23, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 20, 2006; accepted in final form August 16, 2007
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ABSTRACT
AT A GLANCE COMMENTARY
(MIP-3
), CCL21 (6Ckine), and CXCL12 stromal cell-derived factor (SDF-1), all chemokines known to favor immune cell recruitment and accumulation in the course of lymphoid tissue development (
