Abstract
Rationale: Patients with idiopathic pulmonary arterial hypertension (IPAH) present circulating autoantibodies against vascular wall components. Pathogenic antibodies may be generated in tertiary (ectopic) lymphoid tissues (tLTs).
Objectives: To assess the frequency of tLTs in IPAH lungs, as compared with control subjects and flow-induced PAH in patients with Eisenmenger syndrome, and to identify local mechanisms responsible for their formation, perpetuation, and function.
Methods: tLT composition and structure were studied by multiple immunostainings. Cytokine/chemokine and growth factor expression was quantified by real-time polymerase chain reaction and localized by immunofluorescence. The systemic mark of pulmonary lymphoid neogenesis was investigated by flow cytometry analyses of circulating lymphocytes.
Measurements and Main Results: As opposed to lungs from control subjects and patients with Eisenmenger syndrome, IPAH lungs contained perivascular tLTs, comprising B- and T-cell areas with high endothelial venules and dendritic cells. Lymphocyte survival factors, such as IL-7 and platelet-derived growth factor-A, were expressed in tLTs as well as the lymphorganogenic cytokines/chemokines, lymphotoxin-α/-β, CCL19, CCL20, CCL21, and CXCL13, which might explain the depletion of circulating CCR6+ and CXCR5+ lymphocytes. tLTs were connected with remodeled vessels via an ER-TR7+ stromal network and supplied by lymphatic channels. The presence of germinal center centroblasts, follicular dendritic cells, activation-induced cytidine deaminase, and IL-21+PD1+ follicular helper T cells in tLTs together with CD138+ plasma cell accumulation around remodeled vessels in areas of immunoglobulin deposition argued for local immunoglobulin class switching and ongoing production.
Conclusions: We highlight the main features of lymphoid neogenesis specifically in the lungs of patients with IPAH, providing new evidence of immunological mechanisms in this severe condition.
Work on chronic inflammatory disorders and autoimmune diseases suggests that pathogenic antibodies and T cells may be generated locally, in the targeted organ, in highly organized ectopic lymphoid follicles commonly called tertiary lymphoid tissues. Despite the importance of inflammatory influx in idiopathic pulmonary arterial hypertension (IPAH) lesions, lymphoid neogenesis has not been studied.
The presence of highly organized perivascular follicles in IPAH lungs argues for specific immune-adaptive mechanisms in the pathophysiology of the disease. It is highly important to understand how modulating factors that drive and maintain lymphoid neogenesis in IPAH lungs can contribute to disease progression.
Pulmonary arterial hypertension (PAH), a disease of the small pulmonary arteries (PAs), is characterized by vascular cell proliferation and remodeling. It results in a progressive increase in pulmonary vascular resistance, leading ultimately to right ventricular failure and death (1). Idiopathic/heritable PAH (IPAH) is diagnosed when no condition or exposure known to be associated with PAH is identified (1). Several studies have suggested a role for immune mechanisms in IPAH pathophysiology (2). Inflammatory cells and intense chemokine production have been detected within remodeled PAs, and vascular stromal cells have been shown to be sensitive to inflammatory stimuli (3). In addition, elevated circulating cytokine levels have been measured in IPAH (4, 5). PAH may also complicate the course of connective tissue diseases, and cases of reversible pulmonary vascular diseases have been reported in patients with PAH associated with lupus or mixed connective tissue disease treated with glucocorticosteroids and immunosuppressants (6, 7). Up to one-third of patients with IPAH have circulating autoantibodies against various vascular self-antigens (8–10). This suggests that the adaptive immune system, consisting of T and B lymphocytes, is involved, and indeed perivascular T and B lymphocytes have been detected in pulmonary vascular IPAH lesions (11). Stimulating autoantibodies are currently being discussed as possible mediators of PAH. Interestingly, functional autoimmunity directed at the angiotensin-1 receptor and endothelin receptor type A identifies a subset of patients with systemic sclerosis at particular risk for severe disease, pulmonary hypertension, and decreased survival (12). Work on chronic inflammatory disorders and autoimmune diseases suggests that pathogenic antibodies and T cells may also be generated locally, in the targeted organ, in highly organized ectopic lymphoid follicles commonly called tertiary lymphoid tissues (tLTs) (13). With respect to lymphoid follicles in the lung, some authors regarded these structures as part of the mucosal immune system and thus as so-called bronchus-associated lymphoid tissue (BALT). However, it is admitted that BALTs do not exist in normal human lungs (14), and thus any organized lymphoid tissue in the lung should be regarded as an ectopic lymphoid tissue resulting from local lymphoid neogenesis (15). The role of tLTs in chronic pulmonary diseases is gaining in importance, especially in chronic obstructive pulmonary diseases (16), in idiopathic pulmonary fibrosis (17), and in obliterative bronchiolitis (18). Despite the importance of inflammatory influx in IPAH lesions, lymphoid neogenesis has never been reported. In the present study, we show for the first time evidence of lymphoid neogenesis in explanted IPAH lungs. We furthermore analyze the cellular and molecular actors involved in the initiation, maintenance, and function of these ectopic lymphoid structures.
PAH diagnosis and classification were confirmed by right-heart catheterization and additional tests performed according to European Society of Cardiology/European Respiratory Society guidelines (19). Our study was approved by the local ethics committee and patients gave informed consent to participate (Comité de Protection des Personnes Ile-de-France, Paris VII). Patient characteristics are described in Tables E1 and E2 in the online supplement.
Lung samples were from the tissue bank of the French Pulmonary Hypertension Referral Center (Université Paris-Sud and INSERM U999, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France). Twenty-one cases with confirmed IPAH and 5 cases with Eisenmenger syndrome (ES) were derived from explanted lungs. For each case, 15 paraffin blocks of lung parenchyma were randomly chosen. Twenty-one paraffin blocks of lung parenchyma derived from patients undergoing lobectomy for lung cancer, taken distal to the tumor, were used as controls (2 blocks per patient because of sample size limitations). After sectioning, mounting, and staining with hematoxylin–eosin–saffron, two experienced investigators (F.P. and P.D.) analyzed the samples separately. Results are expressed as tertiary follicles per square centimeter (tLT/cm2).
Immunofluorescence was performed on 6-μm-thick sections of frozen tissue. We used primary antibodies at the dilutions indicated in Table E3. Antibody binding was detected with the secondary antibodies listed in Table E4. Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Sections were viewed under an LSM 710 microscope (Carl Zeiss, Zaventem, Belgium) equipped with a Ti:sapphire tunable ultrafast two-photon laser (Coherent, Santa Clara, CA), and with 488-, 561-, and 633-nm lasers (Carl Zeiss). Images were recorded with ZEN software (Carl Zeiss) and analyzed with Imaris 4.2 software (Bitplane, Zurich, Switzerland). Because of technical issues, some stainings were applied on 4-μm-thick sections of paraffin blocks and detected by routine immunohistochemical techniques.
Peripheral blood mononuclear cells (PBMCs) were isolated with UNI-SEPmaxi+ tubes (Abcys, Paris, France), frozen in Cryo-SFM (PromoCell, Heidelberg, Germany), and stored at –80°C until use.
PBMCs were stained with DAPI and the antibodies listed in Table E5. Isotype-matched irrelevant antibodies were used as negative controls for nonspecific binding. Samples were analyzed on a FACSAria II (Becton Dickinson, Erembodegem, Belgium) flow cytometer. Events (106) were acquired with FACSDiva software (Becton Dickinson). Data were analyzed with FlowJo software (Tree Star, Ashland, OR).
We performed real-time polymerase chain reaction as previously described (20), using TaqMan gene expression assays (ID number in brackets) (β-actin [Hs99999903_m1], CCL19 [Hs00171149_m1], CCL20 [Hs00355476_m1], CCL21 [Hs99999110_m1], CXCL12 [Hs00930455_m1], CXCL13 [Hs00757930_m1], AID [Hs00757808_m1], and Bcl-6 [Hs00277037_m1]), on a StepOnePlus system (Applied Biosystems, Courtaboeuf, France).
Quantitative variables are presented as means ± SEM unless otherwise stated. We used Student t tests or Mann-Whitney tests to compare groups, depending on the normal or nonnormal nature of the distribution. We used the Kruskal-Wallis test to compare more than two groups. P values less than 0.05 were considered to indicate statistical significance.
We analyzed samples from 21 explanted lungs from patients with IPAH; from 5 patients with ES; and from 21 biopsies from patients with bronchial carcinoma, taken a distance from malignant foci. The clinical and pathological features of these patients are described in Table E1. Control lungs showed little detectable pulmonary accumulation of lymphocytes or inflammatory cells. By contrast, lung sections from patients with IPAH showed lymphoid aggregates of various sizes and degrees of organization (Figure 1), from small lymphoid aggregates (Figure 1A) to large accumulations of lymphocytes resembling highly organized lymphoid follicles (Figures 1B and 1C). tLTs were distributed throughout the pulmonary vasculature, from small distal remodeled arteries to larger pulmonary arteries (Figures 1A–1C). They actually spread along the pulmonary lymphatic vasculature from pulmonary arteries to septal veins. We found 0.76 ± 0.15 tLT/cm2 in IPAH lungs versus 0.04 ± 0.02 tLT/cm2 in control specimens (P < 0.0001), which represents about 20 times mores tLTs in patients with IPAH compared with control subjects (Figure 1D). Patients with ES were not different from control subjects regarding lung tLT density (0.26 ± 0.14). We found that the extent of the pulmonary lymphoid neogenesis in IPAH was not dependant on the size of IPAH lesions but on patient BMPR2 mutation status (Figure 1E).
Figure 1. Lungs from patients with idiopathic pulmonary arterial hypertension (IPAH) show lymphoid follicles adjacent to remodeled vessels. (A–C) Hematoxylin and eosin (H&E) examination of sections of lung biopsy specimens from patients with IPAH shows lymphoid follicles close to remodeled vessels. Lymphoid aggregates were of various sizes and degrees of organization, from small lymphoid aggregates (A; scale bar, 100 μm) to large accumulations of lymphocytes resembling highly organized lymphoid follicles (B; scale bar, 100 μm; and C; scale bar, 200 μm). (D) The absolute number of lymphoid follicles, expressed as tertiary follicles per square centimeter, was increased in the lungs of patients with IPAH (n = 21) compared with control subjects (n = 21) and patients with Eisenmenger syndrome (ES) (n = 5). (E) Fourteen of the patients with IPAH who were tested had known BMPR2 mutation status (carriers, n = 7; noncarriers, n = 7). Noncarriers had significantly more tertiary lymphoid tissues (tLTs) per square centimeter. Immunostaining of control samples for (F) CD5 (T and B1 lymphocytes), (G) CD20 (B lymphocytes), and (H) podoplanin (lymphatic vessels) showed little positivity (arrows). (F–H) (scale bar, 100 μm), Representative stainings in which T lymphocytes, B1 lymphocytes, and lymphatic vessels were detectable. Five control samples were stained to produce these representative views. Ar = artery; DV = dilated vessel.
[More] [Minimize]tLTs displayed well-separated T-cell areas (mainly CD3+CD4+) (Figure 2A) supplied with multiple peripheral node addressin (PNAd)+ high endothelial venules (HEVs) (Figure 2F) surrounding B-cell follicles (CD19+CD20+) (Figure 2A) with Ki-M4+ follicular DCs (FDCs) (Figure 2B). Figure E1 depicts FDCs positive for CD23 and podoplanin in an area of Ki67+ germinal center (GC) cells. The presence of FDCs is a hallmark of functional lymphoid follicles, allowing B- and T-cell priming, clonal expansion, antigen retention (mostly as immune complexes), somatic hypermutation, affinity maturation, and immunoglobulin class switching (15). T-cell areas were highly enriched in CD11c+ DCs surrounded by CD4+ T cells (Figure 2C). The follicle backbone consisted of Erasmus University Rotterdam-thymic reticulum antibody-7 (ER-TR7)+ fibroblastic reticular cells (FRCs) and reticular fibers originating from α-actin+ PAs and organizing conduits in the tLTs (Figures 2D–2F). Confirming our previous observations (21), PAs were surrounded by numerous immature CD209 (DC-SIGN; dendritic cell–specific intercellular adhesion molecule 3-grabbing nonintegrin)+ DCs (Figure 2E). Pulmonary tLTs were often associated with loose dilated blood-free perivascular vessels (Figures 1B and 2G). These vessels were identified as lymphatics through the use of lymphatic vessel–specific markers: Lyve-1, podoplanin, and Prox-1 (Figure 2 and Figures E2A–E2D) (22). We highlighted a significant accumulation of c-Kit+ cells around remodeled pulmonary artery of explanted IPAH lungs (23). In the present work, we found that c-Kit+ cells coexpressing FcεRIα and tryptase, with a typical mast cell phenotype, were localized around the lymphatic vessels, suggesting a possible contribution to their development as suggested by Kunder and colleagues (24) (Figure 2G and Figures E2A–E2D). Some of these c-Kit+ cells displayed a smaller cytoplasm and lower c-Kit membrane expression together with OX40L expression (Figures 2H–2J). These cells could be the so-called lymphoid tissue-inducer cells that are thought to induce lymphatic tissue development by promoting the homing of cells to sites of newly formed lymphoid tissue (25). In accordance with our previous report (23), there was extensive development of CD34+ vessels around the remodeled PAs (Figure E2E), and these vessels were positive for vascular endothelial growth factor (VEGF)-C, a potent lymphangiogenic growth factor (Figure E2F). The endoluminal endothelium also stained for VEGF-C (Figure E2). Thus, lymphatic expansion associated with the presence of tLTs takes place around remodeled IPAH vessels, potentially providing the tLTs with pulmonary (self)-antigens, antigen-loaded DCs, and memory T cells.
Figure 2. Idiopathic pulmonary arterial hypertension (IPAH)–associated lymphoid follicles display canonical cellularity and structure of bona fide tertiary lymphoid tissues (tLTs). Sections of frozen lung samples from patients with IPAH were analyzed by immunofluorescence microscopy with antibodies against (A) α-actin, CD4, and CD20; (B) CD4 and follicular DCs (FDCs) (Ki-M4 staining); (C) α-actin, CD4, and CD11c; (D) reticular fibroblasts and reticular fibers (Erasmus University Rotterdam-thymic reticulum antibody-7 [ER-TR7] staining) and CD11c; and (E) α-actin, ER-TR7, and DC-SIGN. Scale bars, 100 μm. (F) Stainings with ER-TR7 revealed a reticular meshwork connecting the α-actin+ media to the perivascular tLTs and ending in the peripheral node addressin (PNAd)+ high endothelial venules (HEVs) (scale bar, 80 μm). (G) tLTs were supplied by expanded lymphatic vessels surrounded by c-Kit+ mast cells (scale bar, 100 μm). (H and I) Close to perivascular lymphatic vessels (podoplanin staining in white; scale bars, 50 μm), some of the c-Kit+ cells displayed a smaller cytoplasm and lower membrane expression of (H) c-Kit together with (I) OX40L expression; (J) merged stainings. These cells could be the so-called lymphoid tissue-inducer cells. All sections were counterstained with 4′,6-diamidino-2-phenylindole (blue). IPAH samples (n = 5) with mean tLT/cm2 values were analyzed by immunostaining to produce these representative views.
[More] [Minimize]Ectopic formation of secondary lymphoid tissue is initiated by the local attraction of naive T and B cells. Hence, the local production of homeostatic chemokines, such as CXCL13 and CCL19/CCL21 (15), is a critical event in the formation of ectopic lymphoid tissue. The instruction of stromal cells leads to the formation of specialized HEVs, and the coordinated production of chemokines organizes T cells and B cells within a discrete area (13). We determined mRNA levels for the chemokines CXCL12 (attracting CXCR4-expressing cells, such as naive B cells, monocytes, DCs), CXCL13 (attracting CXCR5-expressing cells, such as B cells, and subsets of T cells), CCL20 (attracting CCR6-expressing cells, B cells, subsets of T cells, and immature DCs), and CCL19 and CCL21 (attracting CCR7-expressing cells, such as mature DCs, naive T cells, and B cells) in lung samples from patients with IPAH and control patients. Lung mRNA levels for CCL20 and CXCL13 were significantly higher in patients with IPAH compared with control subjects (P < 0.023 and P < 0.0019, respectively) (Figures 3A and 3B), whereas CCL19, CCL21, and CXCL12 mRNA levels were not different (Figures 3C–3E). However, the measures in total pulmonary RNA do not reflect local ectopic expression of these chemokines. Indeed, CCL19 and CCL21 were expressed in lymphatic vessels and by podoplanin+ER-TR7+α-actin+ FRCs in extralymphatic positions in the T-cell area of the tLTs, where CCR7 expression was detected, mainly on CD3+ T cells (Figures 4A–4F). Immunostainings also revealed a tremendous network of interwoven CXCL13+ microtubules embedded in a matrix of fibronectin (FN), confining CXCR5+ lymphocytes, mainly B cells, in these ectopic lymphoid tissues (Figures 4G–4K). CXCL13 was also produced by follicular DCs (Figure 4I). All these lymphorganogenic chemokines are regarded as being under the control of lymphotoxin-β receptor (LTβR) signaling (26). Immunostainings for LTβR and for its two main ligands, lymphotoxin (LT)-α and LT-β, in IPAH-related tLTs gave the same pattern of positivity (Figures 5A–5D). All three stained some perifollicular vessels. Some isolated intrafollicular cells also expressed LT-β (Figure 5E). Hence, all the major lymphorganogenic signals are present in IPAH lungs, allowing the development of extensive pulmonary perivascular lymphoid neogenesis.
Figure 3. Lymphorganogenic chemokines are overexpressed in explanted idiopathic pulmonary arterial hypertension (IPAH) lungs. DNA-free RNA was extracted from control lungs (n = 10) or explanted lungs from patients with IPAH (n = 9). Extracted mRNA was reverse-transcribed and used for quantitative polymerase chain reaction to determine relative mRNA levels. The mRNA levels for each gene in each sample was normalized with respect to the β-actin RNA level. The (A) CCL20 and (B) CXCL13 genes were more strongly expressed in the lungs of patients with IPAH than in control lungs. (C) CCL19, (D) CCL21, and (E) CXCL12 mRNA levels were not significantly higher in IPAH lungs than in control lungs.
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Figure 4. Lymphorganogenic chemokines are expressed in idiopathic pulmonary arterial hypertension (IPAH)–related tertiary lymphoid tissues (tLTs). Sections of frozen lung samples from patients with IPAH were analyzed by immunofluorescence microscopy with antibodies against (A) α-actin and CCL19 (scale bar, 200 μm); (B) Lyve-1 (scale bar, 200 μm); (C) α-actin and CCL19 (scale bar, 50 μm); (D) Lyve-1, CCR7, and CD3; (E) podoplanin and CCL21; (F) α-actin, CCL21, and ER-TR7; (G) Ki-M4, CXCL13, and fibronectin (FN); (H) CXCL13, CD20, and CXCR5; and (I) CXCL13 and Ki-M4. Stainings of A–D, G, and H are done on serial sections. (C) Higher magnification of the outlined area of A. (J) Three-dimensional reconstruction after z-stacking of G at higher magnification. (K) Three-dimensional reconstruction after z-stacking of H without the fluorescence channel corresponding to CXCR5 staining. (J and K) The three-dimensional network of CXCL13+ microtubules. Arrows in F are pointing to high endothelial venules. Arrow in I is pointing to CXCL13+Ki-M4+ FDCs (yellow). Ar = artery; FRC = fibroblastic reticular cells; Ly = lymphatic vessel. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). IPAH samples (n = 5) with mean tLT/cm2 values were analyzed by immunostaining to produce these representative views.
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Figure 5. The lymphorganogenic receptor–ligand pair, that is, lymphotoxin-β receptor (LTβR)–lymphotoxin (LT), is expressed mainly in perifollicular vessels of idiopathic pulmonary arterial hypertension (IPAH) lungs. Serial sections of frozen lung samples from patients with IPAH were analyzed by immunofluorescence microscopy with antibodies against (A) LT-β (scale bar, 100 μm) and (B) LTβR (scale bar, 100 μm). Sections of paraffin-embedded lung samples from patients with IPAH were stained immunohistochemically with antibodies against LT-α (C, scale bar, 100 μm; D and E, scale bar, 50 μm). (D and E) Higher magnifications of C. (D) A perifollicular vascular pattern of positivity for LT-α, and (E) isolated intrafollicular cells also expressing LT-α (arrows). IPAH samples (n = 5) with mean tertiary lymphoid tissue (tLT)/cm2 values were analyzed by immunostaining to produce these representative views.
[More] [Minimize]We hypothesized that pulmonary lymphorganogenic chemokines that are overexpressed in IPAH lungs would have effects on the lymphocyte composition of circulating leukocytes, especially those positive for CXCR5 and CCR6, the membrane receptors for CXCL13 and CCL20, respectively. We determined the distribution of circulating lymphocyte subpopulations in the blood of 13 patients with IPAH and 14 healthy control subjects. Characteristics of patients with IPAH (age, functional and hemodynamic characteristics, BMPR2 mutation status, and treatments) are described in Table E2. Patients with IPAH had lower frequencies of circulating CD19+ B lymphocytes (which express both CXCR5 and CCR6) and CD4+ helper T (Th) lymphocytes than control subjects (5.75 ± 1.11% of PBMCs vs. 8.35 ± 0.55%, P = 0.03, and 21.65 ± 2.33% of PBMCs vs. 33.74 ± 2.09%, P < 0.0001, respectively) (Table 1). The cytokine receptors CD25 (IL-2Rα) and CD127 (IL-7Rα) can also be used to distinguish CD127+ effector Th cells from regulatory T (Treg) cells, which do not express CD127 but have high levels of CD25 (27). Using a strategy based on these markers (Figure E3), we found that patients with IPAH compared with control subjects had lower frequencies of CD4+CD127+CD25–CXCR5+CCR6– (P = 0.04), CD4+CD127+CD25–CCR6+CXCR5– (P < 0.01), and CD4+CD127+CD25–CCR6+CXCR5+ (P = 0.04) T cells. Inversely, circulating T cells negative for both CXCR5 and CCR6, expected to be nonresponsive to CXCL13 and CCL20, were enriched in circulating IPAH lymphocytes (P = 0.04). We excluded, by gating, CD4+CD127–CD25+ Treg cells that may also express CCR6 and CXCR5 (28) but have immunomodulatory properties. We observed no significant alteration of CD4+CD127–CD25+ Treg cell levels in the peripheral blood of patients with IPAH (P = 0.12) (Table 1).
| Phenotypic Markers | Population | Control Frequency (%) (n = 14) | IPAH Frequency (%) (n = 13) | P Value |
| CD19+ | B lymphocytes | 8.35 ± 0.55* | 5.75 ± 1.11* | 0.03 |
| CD3+CD4+ | Helper T lymphocytes | 33.74 ± 2.09* | 21.65 ± 2.33* | <0.0001 |
| CD4+CD127+CD25–CXCR5+CCR6– | Helper T effector subset, CXCL13 sensitive | 4.71 ± 0.50† | 3.05 ± 0.52† | 0.04 |
| CD4+CD127+CD25–CXCR5–CCR6+ | Helper T effector subset, CCL20 sensitive | 17.87 ± 1.39† | 8.95 ± 1.80† | <0.01 |
| CD4+CD127+CD25–CXCR5+CCR6+ | Helper T effector subset, CXCL13 and CCL20 sensitive | 4.47 ± 0.41† | 2.71 ± 0.73† | 0.04 |
| CD4+CD127+CD25–CXCR5–CCR6– | Helper T effector subset, CXCL13 and CCL20 nonsensitive | 58.87 ± 2.37† | 68.78 ± 3.93† | 0.04 |
| CD4+CD127–CD25+ | Regulatory T cells | 5.07 ± 0.41† | 6.17 ± 0.52† | 0.12 |
The high frequency of lymphoid follicles found in the lungs of patients with IPAH might be underpinned by survival factors that could be involved in tLT maintenance and lymphocyte survival. Studies in mice and humans have shown that IL-7 is involved in tLT formation and progression of chronic inflammation (29). IL-7 is produced by stromal cells and provides lymphoid cells with critical signals early in their development. The trophic response to IL-7 protects lymphoid progenitors from a death process resembling apoptosis. This protection is partly mediated by the induction of Bcl-2 by IL-7 (29). Consistent with these findings, we identified ER-TR7+ stromal cells and neighboring cells expressing IL-7 in IPAH-related tLTs (Figure 6A). Coinciding with this signal, we observed a Bcl-2+ mantle around Bcl-2– germinal centers (GCs) when present (Figure 6B). Indeed, most GC B cells are permissive to apoptosis, and only a few selected high-affinity B cells survive the selection process for affinity maturation. An important growth factor for stromal cells, platelet-derived growth factor (PDGF)-A, was also detected in pulmonary artery smooth muscle cells and within the tLTs (Figures 6C and 6D). Thus IL-7 and PDGF-A are likely important for tLT maintenance in IPAH lungs.
Figure 6. Survival factors for lymphocytes are found in idiopathic pulmonary arterial hypertension (IPAH)–associated pulmonary tertiary lymphoid tissues (tLTs). Lung tLTs from patients with IPAH displayed locally produced trophic signals, such as IL-7 and platelet-derived growth factor (PDGF)-A, that could support tLT appearance and maintenance. IL-7 may be involved in the formation of tLTs in the lungs of patients with IPAH, by the induction of the antiapoptotic factor Bcl-2 in lymphoid cells. Consecutive sections of frozen lung samples from patients with IPAH were analyzed by immunofluorescence microscopy with antibodies against (A) α-actin, Erasmus University Rotterdam-thymic reticulum antibody-7 (ER-TR7), and IL-7; and (B) against α-actin, ER-TR7, and Bcl-2. Note the Bcl-2– germinal center (GC) in B. Scale bars, 70 μm. PDGF-A is produced in vascular smooth muscle cells and within IPAH-associated perivascular tLTs. Consecutive sections were (C) stained with hematoxylin and eosin (H&E), or (D) analyzed by immunofluorescence microscopy, with antibodies against PDGF-A, CD3, and ER-TR7. Scale bars, 80 μm. All slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). IPAH samples (n = 5) with mean tLT/cm2 values were analyzed by immunostaining to produce these representative views.
[More] [Minimize]In accordance with central Bcl-2– areas, the Ki-67 antigen was detected on small clusters of B cells in some follicles, suggesting a GC reaction. GC reactions are supported by follicular helper T (TFH) cells, which provide cognate help to B cells in the dynamic GC microenvironment, resulting in somatic hypermutation, class-switch recombination, and the selection of high-affinity B cells (30). TFH cells produce large amounts of IL-21, a cytokine essential for their generation and GC formation. TFH cells express the chemokine receptor CXCR5 and the activation molecule PD-1 (31). Lung sections from patients with IPAH contained CD4+, IL-21+, PD1+, and CXCR5+ TFH cells in the activated Ki-67+ GCs of the IPAH-associated tLTs (Figures 7A–7D).
Figure 7. Molecular and cellular germinal center (GC) inducers are found in idiopathic pulmonary arterial hypertension (IPAH)–associated tertiary lymphoid tissues (tLTs). Active Ki-67+ GCs of the tLTs contained numerous IL-21–producing PD-1+CXCR5+CD4+ follicular helper T (TFH) cells and were strongly immunoreactive for IL-21. Both IL-21 and Ki-67 have been shown to be involved in GC development and function, and autoimmunity. Consecutive sections of frozen lung samples from patients with IPAH were analyzed by immunofluorescence microscopy, with antibodies against (A) Ki-67; (B) CD4, IL-21, and α-actin; (C) PD-1, IL-21, and α-actin; and (D) PD-1 and CXCR5. Note: D shows a higher magnification of the outlined area in C. Scale bars, 100 μm. IPAH samples (n = 5) with mean tLT/cm2 values were analyzed by immunostaining to produce these representative views.
[More] [Minimize]The transcription factor B-cell lymphoma-6 (Bcl-6), the production of which is induced and maintained by IL-21 (32), functions as a molecular switch, controlling the differentiation of GC B cells. It also drives the development of TFH cells (31). The enzyme activation-induced cytidine deaminase (AID) controls the generation of high-affinity antibodies by somatic hypermutation and class-switch recombination (15, 32). Both AID and Bcl-6 mRNAs were produced in significantly larger amounts in the lungs of patients with IPAH than in control lungs (Figures 8A and 8B). Antigen-selected memory B cells and antibody-secreting plasma cells differentiate in the GC and then leave the GC, conferring long-lasting immunity. We observed a perivascular accumulation of CD138+ plasma cells in patients with IPAH, which was accompanied by strong immunoreactivity for immunoglobulin in the adventitial and endothelial compartments of the affected PAs (Figure 8C). These long-lived antibody-producing cells may provide a local source of pathogenic autoantibodies targeting pulmonary vascular fibroblasts and endothelial cells.
Figure 8. Immunoglobulin (Ig) class switching occurs in tertiary lymphoid tissues (tLTs). Idiopathic pulmonary arterial hypertension (IPAH) was associated with increased expression of activation-induced cytidine deaminase (AID) and Bcl-6 in the lungs of patients with IPAH. Stronger relative mRNA levels of the genes encoding (A) the enzyme AID and (B) the transcription factor Bcl-6 were present in the lungs of patients with IPAH as compared with control lungs, demonstrating GC formation with immunoglobulin class switching and somatic hypermutation in the patients’ lungs. This was associated with the accumulation of immunoglobulin and immunoglobulin-producing CD138+ plasma cells around the remodeled pulmonary arteries of patients with IPAH. (C) Sections of frozen lung samples from patients with IPAH were analyzed by immunofluorescence microscopy with antibodies against human immunoglobulin, CD138, and α-actin. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Note the vascular adventitial and luminal deposition of immunoglobulin. Scale bars, 100 μm. IPAH samples (n = 5) with mean tLT/cm2 values were analyzed by immunostaining to produce these representative views.
[More] [Minimize]Inflammatory elements are commonly described in pulmonary vascular lesions of patients with IPAH (33). Our present comprehensive analysis provides the first demonstration that perivascular lymphocytic infiltrates can form bona fide highly organized tLTs (see Figure 9 for a schematic overview of histological organization of perivascular tLTs in IPAH), supporting the hypothesis that local adaptive immune responses could occur in IPAH lungs. Moreover, we found no difference between the lungs of control subjects and those of patients with flow-induced PAH (Eisenmenger syndrome), arguing for the relevance of pulmonary lymphoid neogenesis in the pathophysiology of IPAH.
Figure 9. Schematic overview of the histological organization of idiopathic pulmonary arterial hypertension (IPAH)–associated tertiary lymphoid tissues (tLTs). We hypothesize that anti-fibroblast and anti-endothelial autoantibodies frequently detected in patients with IPAH arise from the positive selection and activation of self-reactive B cells within the tLTs developing in the vicinity of remodeled pulmonary arteries (PAs) in IPAH lungs, providing an optimal microenvironment for the generation of pathogenic autoantibodies. These tLTs comprise separated B- and T-cell areas with Bcl-2–Ki-67+ germinal centers (GCs). Vascular-derived self-antigens may reach these tLTs by Erasmus University Rotterdam-thymic reticulum antibody-7 (ER-TR7)+ conduits formed by reticular fibroblasts and by expanded perivascular lymphatic elements, and may be presented to self-reactive T and B cells by myeloid and follicular dendritic cells (FDCs), respectively. tLT-constitutive inflammatory cells may accumulate from the vasa vasorum developing in the adventitia of the remodeled PA in the lungs of patients with IPAH. Naive T cells may also enter the tLTs via well-differentiated high endothelial venules (HEVs), subsequently becoming polarized toward a Th17 phenotype likely to induce autoimmunity. Moreover, follicular helper T cells may provide cognate help to the B cells of the GCs (centrocytes), resulting in somatic hypermutation, immunoglobulin class-switch recombination, and the selection of high-affinity B cells. The dilated afferent adventitial lymphatic and vasa vasorum network may provide the tLTs with pulmonary (self)-antigens, antigen-loaded DCs, and memory T cells. Finally, mast cells may also promote lymphatic and lymphoid structure development.
[More] [Minimize]Lymphoid neogenesis in the target organ is considered to be a hallmark of autoimmune diseases (13). As such, tLTs have also been found in the pancreas of mice with autoimmune diabetes (34) and in the salivary glands in Sjögren syndrome (13). In humans, tLTs have been observed in the joints and lungs of patients with rheumatoid arthritis (13), around the airways of patients with chronic obstructive pulmonary disease (15, 35), and in the thyroid gland of patients with Hashimoto thyroiditis (15). Several studies suggest that lymphoid neogenesis correlates with local autoantibody production, for example, in patients with pulmonary manifestations of rheumatoid arthritis and in patients with Sjögren syndrome (13). Hence, the presence of pulmonary tLTs in IPAH lungs could provide a structural basis for a local autoimmune response occurring in this disease. Accumulation of long-lived antibody-secreting CD138+ plasma cells together with immunoglobulin deposits we observed around pulmonary vascular lesions is consistent with locally produced (auto)-antibodies, a prominent feature of IPAH (8–10).
In the present study, we identified the mechanisms responsible for the formation, perpetuation, and function of IPAH-linked pulmonary tLTs. First, we investigated the mechanisms of lymphoid neogenesis, by measuring total levels of lymphorganogenic chemokines expressed in explanted lungs from patients with IPAH. Indeed, the size and complexity of tLTs in autoimmune tissues have been shown to correlate with local levels of homeostatic cytokines and chemokines, including CXCL13, CCL19, CCL21 (36), and CCL20 (37). We found strong CXCL13 and CCL20 mRNA and protein expression in the lungs and perivascular tLTs of patients with IPAH, which was also associated with a lower proportion of circulating CXCR5+ and CCR6+ cells (B cells and several CD4+ Th populations). We also observed LT-α/β and LTβR expression in some perifollicular vessels. This vascular pattern of expression was not surprising. Indeed, LTβR blockade reduces homeostatic VEGF levels and endothelial cell proliferation in lymph nodes, and LTβR stimulation of murine fibroblast–type cells up-regulates VEGF expression, suggesting that LTβR signaling regulates lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation (38). Moreover, LT-α1β2 and LTβR signals control proper development and maintenance of the mature marginal sinus structure and implicate mucosal vascular addressin cell adhesion molecule (MAdCAM)-1 in the structuring of the marginal sinus endothelial cells, which is important for the movement of immune cells within the spleen (39). Our results are in favor of an autocrine production of LT-α/β by some vascular endothelial cells to promote the vascular remodeling necessary for lymphoid tissue development.
Second, we observed immunoreactivity within tLTs for IL-7, which is involved in the maintenance of ectopic follicles in patients with rheumatoid arthritis (40). In the T-cell area of IPAH-related tLTs, we found accumulation of CD11c+ DCs, which have been reported to play a crucial role in the development and maintenance of these structures (41), promoting the differentiation of endothelial cells into HEVs and lymphocyte survival, via an IL-7–dependent pathway (41). In accordance with our previous report (20), we also detected PDGF-A in pulmonary artery smooth muscle cells and within the tLTs. The production of PDGF-A at all stages of B-lymphocyte differentiation suggests an important role in B-cell differentiation and proliferation (42). PDGF-A is also an important trophic factor for the FRC organizing follicle structure (43).
Third, we detected IL-21–producing CD4+CXCR5+PD-1+ TFH cells in the GCs of IPAH-related tLTs, and pulmonary overexpression of crucial genes for somatic hypermutation, affinity maturation and immunoglobulin class switching (Bcl-6 and AID) (13). Thus, we identified most of the main recognized cellular actors and molecular signal of tLT formation in IPAH lungs.
Th17 cells have attracted considerable attention, because of their presence in the blood and affected tissues of patients with autoimmune disorders, including connective tissue diseases, in which they control tissue-specific inflammatory responses (12). Moreover, evidence indicates that IL-17 produced by CD4+ T cells is essential for the formation of BALT (26). All IL-17–producing Th17 cells express CCR6, regardless of their tissue tropism, and CCR6+ T-cell sorting yielded populations with a high proportion of IL-17–producing T cells (15). Our findings, showing the depletion of circulating CD4+CD127+CD25– non-Treg, CCR6+, presumably IL-17–producing cells and an overexpression of CCL20, the ligand of CCR6, in the lungs explanted from patients with IPAH, provide some evidence for the recruitment of IL-17–producing cells to IPAH lungs. We show in Figure E4 that CCL20 immunoreactivity was strong in IPAH-related pulmonary tLTs, and associated with the massive accumulation of CCR6+CD4+ and IL17+CD4+ T cells. Last, we detected local expression of the orphan nuclear receptor RORγt (retinoic acid receptor–related orphan receptor γt), a key transcription factor orchestrating the differentiation of the Th17 lineage (28) in CD4+ and IL-17+ T cells, in the follicles of lungs from patients with IPAH. Thus, an IL-17–driven response may participate in tLT formation and function in IPAH lungs.
In conclusion, despite the limits inherent to studies in humans, we provide new and important insight into the pathogenesis of IPAH and the factors driving and maintaining lymphoid neogenesis.
The authors thank Prof. Gérald Simonneau, Prof. Phillipe Dartevelle, and Prof. Elie Fadel (Antoine Béclère and Marie Lannelongue Hospitals, Université Paris-Sud 11) for facilitating access to lung samples from well-phenotyped patients and control subjects.
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Supported in part by the Legs Poix, the Université Paris-Sud, INSERM, an Actelion Endothelin Research Award (AERA 2007-010), and a grant from the Fondation pour la Recherche Médicale (FRM; DEQ20100318257). The tissue bank was supported in part by the Legs Poix, Chancellerie des Universités de Paris. F.P. was supported by a long-term fellowship grant from the European Respiratory Society (ERS fellowship LTRF 171), and then by the FRM (grant DEQ20100318257). D.M. and P.D. are supported by a grant from the Association HTAP France. B.N.L. is supported by an Odysseus grant from the Flemish government (FWO).
Author Contributions: Conception and design: F.P., B.L., M.H., and S.C.K.; acquisition of data: F.P., P.D., H.H., W.W., B.G., N.R., O.M. and S.M.; analysis and interpretation: F.P., P.D., D.M.; drafting the manuscript for important intellectual content: F.P., B.L., S.C.K., P.D. and M.H.
This article has an online supplement, which is available from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201105-0927OC on November 22, 2011
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