Immune and Inflammatory Cell Involvement in the ... - ATS Journals

Immune and Inflammatory Cell Involvement in the Pathology of Idiopathic Pulmonary Arterial Hypertension Rajkumar Savai1, Soni S. Pullamsetti1,2, Julia Kolbe2, Ewa Bieniek2, Robert Voswinckel1,2, Ludger Fink3, Axel Scheed4, Christin Ritter2, Bhola K. Dahal2, Axel Vater5, Sven Klussmann5, Hossein A. Ghofrani2, Norbert Weissmann2, Walter Klepetko4, Gamal A. Banat2, Werner Seeger1,2, Friedrich Grimminger2, and Ralph T. Schermuly1,2 1 Department of Lung Development and Remodelling, Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; 2Department of Internal Medicine and 3Department of Pathology, University of Giessen and Marburg Lung Center, Giessen, Germany; 4Department of Thoracic Surgery, University Hospital Vienna, Vienna, Austria; and 5NOXXON Pharma AG, Berlin, Germany

Rationale: Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and vascular remodeling. Recent studies have revealed that immune and inflammatory responses play a crucial role in pathogenesis of idiopathic PAH. Objectives: To systematically evaluate the number and cross-sectional distribution of inflammatory cells in different sizes of pulmonary arteries from explanted lungs of patients with idiopathic PAH versus healthy donor lungs and to demonstrate functional relevance by blocking stromal-derived factor-1 by the Spiegelmer NOX-A12 in monocrotaline-induced pulmonary hypertension in rats. Methods: Immunohistochemistry was performed on lung tissue sections from patients with idiopathic PAH and healthy donors. All positively stained cells in whole-lung tissue sections, surrounding the vessels, and in the different compartments of the vessels were counted. To study the effects of blocking SDF-1, rats with monocrotaline-induced pulmonary hypertension were treated with NOX-A12 from Day 21 to Day 35 after monocrotaline administration. Measurements and Main Results: We found a significant increase of the perivascular number of macrophages (CD681), macrophages/ monocytes (CD141), mast cells (toluidine blue1), dendritic cells (CD2091), T cells (CD31), cytotoxic T cells (CD81), and helper T cells (CD41) in vessels of idiopathic PAH lungs compared with control subjects. FoxP31 mononuclear cells were significantly decreased. In the monocrotaline model, the NOX-A12–induced reduction of mast cells, CD681 macrophages, and CD31 T cells was associated with improvement of hemodynamics and pulmonary vascular remodeling. Conclusions: Our findings reveal altered perivascular inflammatory cell infiltration in pulmonary vascular lesions of patients with idiopathic pulmonary arterial hypertension. Targeting attraction of inflammatory cells by blocking stromal-derived factor-1 may be a novel approach for treatment of PAH. Keywords: pulmonary arterial hypertension; pulmonary vascular remodeling; inflammatory cells; inflammation; chemokines

(Received in original form February 27, 2012; accepted in final form August 20, 2012) Supported by Universities of Giessen and Marburg Lung Center within the LOEWE program of the State of Hessen; DFG, Excellence Cluster Cardio Pulmonary System; and BMBF, German Lung Center. Author Contributions: Conception and design, R.S., S.S.P., W.S., R.T.S., A.V., and S.K.; analysis and interpretation R.S., S.S.P., J.K., E.B., R.V., L.F., A.S., C.R., B.K.D., N.W., W.K., and R.T.S.; drafting the manuscript for important intellectual content, F.G., R.S., S.S.P., H.A.G., G.A.B., W.S., and R.T.S. Correspondence and requests for reprints should be addressed to Ralph T. Schermuly, Ph.D., Pulmonary Pharmacotherapy, Universities of Giessen and Marburg Lung Center, Aulweg 130, 35392 Giessen, Germany. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 186, Iss. 9, pp 897–908, Nov 1, 2012 Copyright ª 2012 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201202-0335OC on September 6, 2012 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Immune and inflammatory responses have been shown to play an important role in the pathogenesis of pulmonary arterial hypertension. What This Study Adds to the Field

A better understanding of the distribution and quantification of immune and inflammatory cells in pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension may aid in identifying potential therapeutic targets for prevention and treatment.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary vascular resistance that results in right heart dysfunction and right heart failure (1, 2). PAH is the collective term for a mixture of diseases that have several pathophysiologic, histologic, and prognostic features in common (3). Increased pulmonary arterial pressure in patients with PAH results from a combination of pulmonary vasoconstriction, inward vascular wall remodeling, in situ thrombosis, and inflammation. Mounting evidence suggests a link between the immune system and inflammatory mechanisms in the development of PAH, such as the association of idiopathic PAH (IPAH) and PAH with connective tissue diseases and HIV infection. Indeed, elevated circulating levels of the proinflammatory cytokines IL-1b, IL-6, macrophage inflammatory protein-1a, and P-selectin have been observed in patients (4–6). A role for inflammation in the development of IPAH is further supported by enhanced pulmonary expression of various cytokines and chemokines, such as fractalkine, CCL2, and CCL5, and their association with inflammatory cell infiltrates in severe PAH (7–9). Tuder and coworkers (10) described perivascular inflammatory cell infiltrates in plexiform lesions of PAH composed of T cells, B cells, and macrophages and the presence and correlation of perivascular inflammation with intima and media remodeling in PAH (11). In line with this, dendritic cell recruitment in vascular lesions of patients with IPAH (12) and the presence of tertiary ectopic lymphoid follicles associated with the presence of inflammatory cells (13) has been described recently. In addition, a higher number of circulating CD41CD25[1] high regulatory T (Treg) cells was described in IPAH, suggesting a role for T-cell regulation in disease pathogenesis (14). So far, however, most published studies have investigated the presence of inflammatory infiltrates, but the longitudinal and cross-sectional distribution, and the proportion,

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TABLE 1. DEMOGRAPHIC DATA OF PATIENTS WITH IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION Sex

Age

Mean PAP (mm Hg)

CI (L/min/m2)

NYHA class

Female Female Female Female Female Female Female Female Female Female Female Female Female Male Male Male Male Male Male Male Male Male

52 45 42 42 38 37 30 28 27 26 20 17 13 45 44 35 30 29 27 26 22 21

75 60 43 58 70 52 66 58 66 62 73 81 NA 47 72 56 62 56 52 34 62 44

1.6 2.74 2.6 2.1 1.2 1.94 2.1 2.3 3.23 2.1 1.56 NA NA 2.18 2.32 2.68 2.79 1.98 2.8 2.41 2.96 2.4

II–III III–IV III–IV III–IV II–III III–IV III–IV III–IV III–IV III–IV III III–IV III III III III II–III III III–IV IV III III

Definition of abbreviations: CI ¼ cardiac index; NA = information not available; NYHA ¼ New York Heart Association; PAP ¼ pulmonary arterial pressure.

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METHODS Patient Characteristics Human explanted lung tissues from subjects with IPAH (n ¼ 22) or donors (n ¼ 16) were obtained during lung transplantation. Samples of donor lung tissue were taken from lungs that had not been transplanted. A detailed description of how the donor lung explants were obtained is provided in the online supplement. The study protocol for tissue donation was approved by the ethics committee (Ethik Kommission am Fachbereich Humanmedizin der Justus Liebig Universität Giessen) of the University Hospital Giessen (Giessen, Germany) in accordance with national law and with Good Clinical Practice/International Conference on Harmonisation guidelines. Written informed consent was obtained from each individual patient or the patient’s next of kin (AZ 31/93). Clinical information on patients is summarized in Table 1. All lungs were reviewed for pathology, and the IPAH lungs were classified as grade III–IV according to Heath and Yacoub (17).

Tissue Harvest Explanted lungs were rinsed with ice-cold preservation buffer until free of blood. Tissue samples for immunohistochemistry were transferred to 4% phosphate-buffered paraformaldehyde and fixed for 24 hours at 48 C. The fixed samples were then dehydrated and paraffin embedded in a fully automated manner (Tissue processor ASP200; Leica, Heidelberg, Germany) (18).

Immunohistochemistry

of immune and inflammatory cells in the diseased pulmonary arteries is poorly understood. Here we systematically evaluated the number and crosssectional location of inflammatory cells in lungs from patients with IPAH in different-sized pulmonary arteries. To gain functional insight in the role of inflammation in pulmonary hypertension, monocrotaline (MCT)-injected rats were treated with the novel stromal-derived factor (SDF-1/CXCL12) binding and inhibiting Spiegelmer NOX-A12 (15). Some of the results of these studies have been previously reported in the form of an abstract (16).

Immunohistochemistry and mast cell histochemical staining was performed on formalin-fixed paraffin-embedded blocks of lung tissue from patients with IPAH and healthy donors as described in the online supplement.

Cell Counting The positively stained cells were counted in whole-lung tissue sections from donors (n ¼ 16) and patients with IPAH (n ¼ 22) by light microscopy (Leica Instruments, Nussloch, Germany) using Leica QWin software (see Figure E1 in the online supplement). All positively stained cells surrounding the vessels and in the vessels were counted (see Figure E1). The categorization of vessels and the number of vessels counted are mentioned in the online supplement.

Figure 1. Morphologic analysis of pulmonary artery remodeling. Donor and idiopathic pulmonary arterial hypertension (IPAH) lung tissue sections were stained with hematoxylin and eosin (H&E; hematoxylin stains nuclei [blue] and eosin stains the cytoplasm [pink]), trichrome (stains collagen blue), van Gieson (stains connective tissue red), a-smooth muscle actin (a-actin; stains smooth muscle cells blue), von Willebrand factor (vWF; stains endothelial cells brown), and costaining with a-actin and vWF. Representative stained images of different-sized pulmonary arteries of donor and IPAH lungs are shown. Scale bar ¼ 20 mm.

Savai, Pullamsetti, Kolbe, et al.: Immune and Inflammatory Cells in PAH

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Figure 2. Staining and quantification of mast cells in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections were stained with toluidine blue to detect mast cells. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (purple). (B) Representative micrographs of negative and positive (dog mast cell tumor) control lungs. Scale bar ¼ 20 mm. Quantification of mast cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of mast cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 16 (donor) and n ¼ 22 (IPAH).

Data Analysis and Statistics All data are given as the mean 6 SEM. Graphical and statistical analyses were performed with GraphPadPrism5 (GraphPad, San Diego, CA). Statistical significance of differences between two groups was established (P , 0.05) using the unpaired t test with Welch correction. Differences between more than two groups (P , 0.05) were analyzed by one-way analysis of variance followed by the Student-NewmanKeuls post hoc test.

RESULTS Histomorphologic Appearance of IPAH Pulmonary Arteries

To characterize pulmonary vascular remodeling changes in different vascular categories (small, 20–50 mm; medium, 51–150 mm; and large, .150 mm) in donor versus IPAH lungs, lung sections were stained with hematoxylin and eosin, trichrome, and van Gieson. Hematoxylin and eosin staining showed an increased number of cells in all categories of pulmonary arteries in IPAH lungs compared with donor lungs. Trichrome and van Gieson staining showed increased collagen deposition in IPAH pulmonary arteries compared with donor lungs, and these changes were prominent in medium and large pulmonary arteries. Lung sections were also immunostained with antibodies against a-actin and von Willebrand factor, alone and in combination (Figure 1).

Analysis of Inflammatory and Immune Cells in IPAH Lung Tissues

Mast cells. There was a significant increase in the perivascular number of mast cells in IPAH vessels (Figures 2A–2E; see online supplement). CD681 macrophages. As shown in Figures 3A and 3C, the total number of CD681 cells was significantly higher in IPAH lungs compared with donor lungs (153.8 6 10.06 vs. 84.04 6 8.79 cells/mm2, respectively; P , 0.0003). Counting the number of vascular-infiltrated macrophages according to the diameter of pulmonary arteries showed significant changes. The number of CD681 cells was significantly higher in small pulmonary arteries (2.96 6 0.17 vs. 1.46 6 0.22; P , 0.0001), medium pulmonary arteries (7.06 6 0.29 vs. 2.15 6 0.24; P , 0.0001), and large pulmonary arteries (14.58 6 1.06 vs. 3.44 6 0.91; P , 0.0001) of IPAH lungs compared with the respective arteries of donor lungs (Figure 3D). This increase of CD681 cells was mainly confined to the adventitial layer of small, medium, and large pulmonary arteries of IPAH lungs (Figure 3E). CD141 monocytes and macrophages. As shown in Figures 4A and 4C, the total number of CD141 cells was significantly higher in IPAH lungs compared with donor lungs (20.08 6 4.38 vs. 9.73 6 2.13 cells/mm2, respectively; P , 0.0482). The representative immunohistochemical images show that very few CD141

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Figure 3. Immunostaining and quantification of macrophages in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with anti-CD68 to detect macrophages. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (brown). (B) Representative micrographs of negative and positive (human tonsil) controls. Scale bar ¼ 20 mm. Quantification of CD681 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of CD681 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 14 (donor) and n ¼ 22 (IPAH).

cells were present in donor pulmonary arteries (Figure 4A). In contrast, CD141 monocytes and macrophages were increased in all sizes of IPAH pulmonary arteries. The number of CD141 cells was significantly higher in small pulmonary arteries (0.52 6 0.09 vs. 0.27 6 0.03; P ¼ 0.009), medium pulmonary arteries (1.96 6 0.19 vs. 0.54 6 0.005; P , 0.0001), and large pulmonary arteries (.150 mm; 5.00 6 0.74 vs. 2.43 6 0.72; P ¼ 0.0131) of IPAH lungs compared with the respective arteries of donor lungs (Figure 4D). Further quantification of CD141 cell distribution in different vessel compartments of IPAH lungs demonstrated a higher number of CD141 cells in the adventitia of small, medium, and large pulmonary arteries (Figure 4E). Dendritic cells. To identify dendritic cells, we performed immunohistochemistry with a monoclonal antibody against CD209, a C-type lectin receptor. CD2091 cells were found in perivascular and parenchymal spaces of donor and IPAH lungs (Figure 5A). Quantification revealed a dramatic increase in the total number of CD2091 cells in lungs from patients with IPAH compared with donor lungs (16.39 6 4.42 vs. 3.07 6 0.92 cells/ mm2, respectively; P ¼ 0.0083) (Figure 5C). The number of infiltrating dendritic cells was significantly higher in small (0.78 6 0.11 vs. 0.11 6 0.03; P , 0.0001), medium (2.47 6 0.14 vs. 0.33 6 0.06; P , 0.0001), and large (8.03 6 0.70 vs. 3.24 6 1.22; P ¼ 0.0012) pulmonary arteries of IPAH lungs compared with donor lungs (Figure 5D). Infiltrating dendritic cells in IPAH lungs were primarily located in the adventitia of pulmonary arteries (Figure 5E).

CD31 T cells. The total number of CD31 cells was significantly higher in IPAH lungs compared with donor lungs (52.21 6 4.45 vs. 29.52 6 5.14 cells/mm2, respectively; P , 0.0027) (Figures 6A and 6C). The representative immunohistochemical images show that T cells were scant or virtually absent in donor pulmonary arteries (Figure 6A). In contrast, CD31 T cells were found in all sizes of IPAH pulmonary arteries. The number of CD31 cells was significantly higher in small pulmonary arteries (1.71 6 0.18 vs. 0.37 6 0.05; P , 0.0001) and medium pulmonary arteries (4.70 6 0.33 vs. 1.64 6 0.25; P , 0.0001) of IPAH lungs compared with the respective arteries of donor lungs (Figure 6D). However, the number of CD31 cells was not significantly increased in large pulmonary arteries of IPAH lungs (7.34 6 0.76 vs. 4.35 6 1.79; P ¼ 0.1372) (Figure 6D). Further quantification of CD31 cell distribution in different vessel compartments of IPAH lungs demonstrated a higher number of CD31 cells in adventitia of small, medium, and large pulmonary arteries (Figure 6E). Subsets of T cells. To define the subsets of T cells, lung sections were stained with anti-CD8 to detect cytotoxic T cells, anti-CD4 to detect helper T cells, and anti-FoxP3 to detect FoxP31 mononuclear cells. Cytotoxic T cells. Although T cells were scant or virtually absent in donor lungs, CD81 T cells were prominent in IPAH lungs (Figure 7A). The total number of CD81 cells was significantly higher in IPAH lungs compared with donor lungs (9.06 6 1.45 vs. 1.67 6 0.49 cells/mm2, respectively; P , 0.0003)


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Figure 4. Immunostaining and quantification of monocytes in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with anti-CD14 to detect monocytes. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (pink). (B) Representative micrographs of negative and positive (human tonsil) controls. Scale bar ¼ 20 mm. Quantification of CD141 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of CD141 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 14 (donor) and n ¼ 22 (IPAH).

(Figure 7C). Furthermore, quantification of CD81 cells in all sizes of pulmonary arteries revealed a significantly higher number of CD81 cells in small pulmonary arteries of IPAH lungs compared with similar-sized donor pulmonary arteries (1.71 6 0.18 vs. 0.37 6 0.05; P , 0.0001) (Figure 7D). This increase was observed mainly in the adventitial layer of IPAH pulmonary vessels (Figure 7E). However, no significant increase was observed in medium (P ¼ 0.1077) and large (P ¼ 0.2751) IPAH pulmonary arteries compared with the respective donor pulmonary arteries (Figure 7D). CD41 helper T cells. As shown in representative immunohistochemical images (Figure 8), helper T cells were identified in the bronchioles and vessels of donor lungs. However, the number of CD41 T cells was higher in IPAH lungs (Figure 8A). Indeed, the total number of CD41 cells was significantly higher in IPAH lungs compared with donor lungs (13.09 6 2.35 vs. 4.13 6 0.57 cells/mm2, respectively; P ¼ 0.002) (Figure 8C). Furthermore, quantification of CD41 cells in all sizes of pulmonary arteries revealed a significantly higher number of CD41 cells in small pulmonary arteries (0.48 6 0.09 vs. 0.21 6 0.02; P ¼ 0.0037), medium pulmonary arteries (1.95 6 0.19 vs. 0.57 6 0.008; P , 0.0001), and large pulmonary arteries (3.62 6 0.64 vs. 1.40 6 0.08; P ¼ 0.0007) of IPAH lungs compared with similar-sized donor pulmonary arteries (Figure 8D). This increase was observed mainly in the adventitial layer of IPAH pulmonary arteries (Figure 8E).

FoxP31 mononuclear cells. A moderate to high number of FoxP31 cells was noted in donor lungs (42.35 6 4.97 cells per mm2), but the number was significantly lower in IPAH lungs (22.98 6 2.55 cells/mm2; P ¼ 0.0022) (Figure 9A, C). Furthermore, quantification of FoxP31 cells in all sizes of pulmonary arteries revealed low counts of FoxP31 cells in small pulmonary arteries (0.29 6 0.04 vs. 0.59 6 0.06, respectively; P , 0.0001) and medium pulmonary arteries (0.80 6 0.05 vs. 1.45 6 0.16; P , 0.0001) of IPAH lungs compared with donor lungs (Figures 9D and 9E). However, no significant changes were observed in large pulmonary arteries of IPAH compared with similar-sized donor pulmonary arteries (1.49 6 0.28 vs. 2.02 6 0.43; P ¼ 0.3045) (Figure 9D). We note that FoxP31 cells are distributed approximately evenly in the adventitia and intima, but are less in medial layer of small and medium IPAH arteries (Figure 9E). To localize FoxP31 cells to a particular cell group, we performed double (CD41FoxP31, CD81FoxP31, CD681FoxP31) and triple immunohistochemical staining (CD41CD251FoxP31). Double staining with CD4/FoxP3 antibodies demonstrated that not all FoxP31 cells were CD41 and only 20–25% of cells were double positive (CD41FoxP31) in IPAH lungs (see Figure E2). However, no other immunohistochemical staining worked convincingly. CD201 B cells. B-cell aggregates were found only in a few donor (3 of 16) and IPAH (5 of 22) lungs (Figure 10A). Quantification of these revealed a higher number of CD201 cells in

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Figure 5. Immunostaining and quantification of dendritic cells in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with anti-CD209 to detect dendritic cells. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (brown). (B) Representative micrographs of negative and positive (human tonsil) controls. Scale bar ¼ 20 mm. Quantification of CD2091 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of CD2091 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 15 (donor) and n ¼ 22 (IPAH).

IPAH lungs compared with donor lungs (1.04 6 0.36 vs. 0.07 6 0.05, respectively; P ¼ 0.0005) (Figure 10C). In contrast, we found only a few CD201 cells in IPAH vasculature, and we did not find any cells in donor vasculature (Figure 10D). The CD201 cells that were found were primarily in the adventitia of all sizes of IPAH pulmonary arteries (Figure 10E). Effects of Blocking SDF-1 on MCT-induced PH in Rats

Treatment of MCT rats with NOX-A12 significantly decreased inflammatory cell infiltration as shown by a reduction of perivascular CD681 macrophages, CD31 T cells, and mast cells (Figures 11A–11D). This was associated with a reduction of pulmonary vascular remodeling (proportion of fully muscularized pulmonary arteries decreased from 65.4 6 4.5% to 35.71 6 4.71% and proportion of nonmuscularized arteries increased from 1.25 6 0.49% to 31.91 6 3.16%) (Figure 11E) and a decrease in RV/(LV1S) ratio (0.61 6 0.03 vs. 0.48 6 0.04) (Figure 11F). The right ventricular systolic pressure decreased from 67.8 6 5.1 to 51.2 6 3.1 mm Hg (Figure 11G) and total pulmonary resistance from 2.2 6 0.2 to 1.49 6 0.1 mm Hg min/ml 100 g bodyweight compared with placebo-treated animals (Figure 11H).

DISCUSSION Inflammatory processes are prominent in various forms of PAH and are increasingly recognized as major pathogenic components

of pulmonary vascular remodeling in IPAH or PAH related to more classical forms of inflammatory syndromes, such as connective tissue diseases, HIV, or other viral diseases (19). This inflammatory hypothesis is supported by the identification of perivascular cell infiltrates composed of macrophages, T cells, and B cells in plexiform lesions of PAH (10–13). In our study, we provide for the first time comprehensive characterization of quantitative and qualitative distributions of various inflammatory cells throughout the pulmonary vasculature. Additionally, the cross-sectional distribution of these cells in small to medium sized pulmonary arteries was analyzed. We observed an overall accumulation of immune and inflammatory cells, except FoxP31 cells, in the adventitial layer, suggesting their involvement in IPAH pathogenesis. Mechanistically, we demonstrate that therapeutic inhibition of SDF-1, a key chemokine for recruitment of inflammatory cells (20), blocks pulmonary vascular infiltration with CD68 1 cells, CD3 1 cells, and mast cells in established model of MCT-induced PAH. This reduction of inflammatory cells affects pulmonary vascular remodeling highlighting the functional role of these cells in PAH. Therefore, we believe that our study provides unique and important information to the field of PAH. We observed an increased number of mast cells in medium and large pulmonary arteries of IPAH lungs. A detailed discussion of our findings with regard to mast cells is given in the online supplement. We also found accumulation of macrophages, monocytes,


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Figure 6. Immunostaining and quantification of T cells in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with antiCD3 to detect T cells. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (pink). (B) Representative micrographs of negative and positive (human spleen) controls. Scale bar ¼ 20 mm. Quantification of CD31 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of CD31 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 16 (donor) and n ¼ 22 (IPAH).

and dendritic cells in vascular lesions of IPAH. Macrophage and monocyte accumulation and activation usually leads to the release of cytokines, vasoactive molecules, such as endothelins and eicosanoids, and reactive oxygen species (20, 21), all of which have been implicated in pulmonary vascular remodeling. Activated macrophages also secrete matrix metalloproteinases (9 and 19) and serine proteases, such as urokinase-like plasminogen activator. These proteases degrade basement membranes and extracellular matrix and seem to aid leukocyte migration, adventitial fibroblast activation, or transdifferentiation (22, 23). Finally, activated macrophages may also play a role in presenting antigens to T cells (24). However, a recent study in mice with hypoxia-induced pulmonary hypertension suggests that macrophages acquire an alternatively activated phenotype (M2) in response to hypoxia, and they express genes involved in the inflammatory response (25), suggesting a role of microenvironment in deciding macrophage phenotype. Future studies need to address the status of macrophage polarization and their influence on pulmonary vascular remodeling. In agreement with dendritic cell accumulation, Perros and colleagues demonstrated immature dendritic cell infiltration in vascular lesions of patients with IPAH and in experimental pulmonary hypertension (12). However, the number of circulating monocyte-derived dendritic cells was lower in patients with IPAH than in control subjects (18), suggesting trafficking to the

lung. Accumulation of these professional antigen-presenting cells may involve the presentation of antibodies to endothelial cells, fibroblasts, and nuclear antigens that are found in the serum of patients with IPAH and collagen vascular disease–associated PAH (19, 20). They may also be involved in contacting and restimulating T cells to mediate disease (21). Recently, the presence of tertiary lymphoid tissues with B-cell follicles was demonstrated by a broad spectrum of techniques (CD5, CD20, CD19, podoplanin) in IPAH (13). We found only few CD201 cells and few B-cell aggregates (5 out of 22 IPAH lungs) in the vessels of patients with IPAH, whereas we did not find any cells in vessels from healthy donors. The description of tertiary lymphoid tissues was not the focus of this study but the initiation of lymphoid neogenesis is poorly understood. It may be caused by the need of prolonged cytokine production or the lymphoid chemokine expression to induce lymphoid neogenesis. This may also be caused by plasticity of tertiary lymphoid follicles. It is becoming apparent that they can be turned off or that they resolve after removal of the initial stimulus or after therapeutic intervention (26). We observed an increased number of total T cells in remodeled pulmonary vasculature. Moreover, in line with the recent report by Austin and coworkers (27), we found that the CD81 T cell formed a prominent component of vascular lesions. Little is known about the role of this cell type in PAH pathogenesis.

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Figure 7. Immunostaining and quantification of cytotoxic T cells in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with anti-CD8 to detect cytotoxic T cells. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (pink). (B) Representative micrographs of negative and positive (human spleen) controls. Scale bar ¼ 20 mm. Quantification of CD81 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of CD81 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 12 (donor) and n ¼ 15 (IPAH).

The perivascular CD81 T cell infiltration may be associated with impaired endothelial cell apoptotic-antiapoptotic signaling as observed in subjects with pulmonary hypertension and congenital heart disease (28). In addition, infiltrating T cells can release vascular endothelial growth factor and can increase angiogenesis (29, 30). Importantly, nuclear factor of activated T cells, a transcription factor that initiates T-cell cytokine gene transcription, is activated and contributes to the remodeling of mitochondrial membrane potential, and the inflammatory response in PAH (31). We further investigated the changes in different subsets of T cells (cytotoxic T cells, helper T cells, and FoxP31 cells). We observed that cytotoxic T cells and helper T cells accumulated in remodeled pulmonary vasculature. In corroboration, the helper T (type 2) cell immune response regulates signals for cell proliferation, differentiation of smooth muscle actin–positive cells, thus resulting in a severely remodeled arterial wall (32). Treg cells are emerging as important immune cells because they tightly regulate the immune responses of helper T (type-1 and type-2) cells and are critical for maintaining immunologic tolerance (33). In line, FoxP31 Treg cells have been reported to be functionally or quantitatively deficient in many autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus, type-I diabetes, or rheumatoid arthritis (34–36). Interestingly, we found a decreased FoxP31 cell count in the lungs of patients with IPAH. Our finding is consistent with the immune

dysregulation proposed for IPAH. Indeed, there may be an immune dysregulation that occurs in this disease, as evident from the autoantibodies detected in patients with IPAH (37, 38). Therefore, impaired function or absence of Treg cells is likely among the reasons for the local inflammation and proinflammatory response in IPAH. A more detailed discussion of our findings with regard to FoxP31 cells is given in the online supplement. Importantly, we observed accumulation and infiltration of inflammatory and immune cells in the adventitial layer of IPAH lungs, supporting in part the “outside-in” hypothesis of pulmonary vascular remodeling (39). Accordingly to this hypothesis, vascular inflammation because of immune cells recruitment occurs early and persists in the adventitia. Immune cell activation, along with fibroblast activation, and progenitor cell accumulation and retention lead to remodeling not only of the adventitia, but cause subsequent changes in the media and ultimately even the intima. The cellular mechanisms contributing to chronic inflammatory responses remain unclear. Perivascular inflammation may be perpetuated by activated adventitial fibroblasts, which, through sustained production of proinflammatory cytokines and chemokines and adhesion molecules, induce accumulation, retention, and activation of immune and inflammatory cells (39). A potential limitation of this work is the descriptive component that is inherent to the nature of these studies on explanted


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Figure 8. Immunostaining and quantification of helper T cells in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with anti-CD4 to detect helper T cells (pink). Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (pink). (B) Representative micrographs of negative and positive (human spleen) controls. Scale bar ¼ 20 mm. Quantification of CD41 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of CD41 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 12 (donor) and n ¼ 19 (IPAH).

lungs of patients with PAH (11). In contrast to other diseases, taking biopsies in patients with PAH during the course of the disease is obsolete because of high complication rates. Therefore, we can only learn about pathologic changes from transplanted (end-stage) tissue. The functional role of inflammatory cells on pulmonary vascular remodeling has mainly been addressed by cell culture experiments or the use of transgene or chimeric animals. To get more insight into the functional role of inflammatory cells on pulmonary vascular remodeling we blocked SDF-1, a general chemoattractant for proinflammatory cells by a highly specific Spiegelmer. Spiegelmers are a new class of oligonucleotide therapeutics made from the L-stereoisomer of RNA with high biologic stability and no immunostimulatory properties. The Spiegelmer NOX-A12 is a 45-nucleotide L-RNA oligonucleotide conjugated with 40-kD polyethylene glycol to increase plasma half-life to several hours. NOX-A12 binds and inhibits SDF-1 of human mouse and also rat (15). NOX-A12 decreased the accumulation of monocytes and macrophages, T lymphocytes, and mast cells in the established animal model of MCT-induced pulmonary hypertension. As a consequence, there was a reduction in pulmonary vascular remodeling and improvement of hemodynamics and right heart hypertrophy. In this line, a neutralizing antibody of SDF-1 has been shown to attenuate neonatal hypoxia-induced PH (40), whereas other studies investigated the effects of the CXCR4 antagonist

AMD3100 (plerixafor) (41, 42). Most interestingly, beside CXCR4, SDF-1 is also signaling by CXCR7, a receptor that can signal downstream by the proproliferative phospholipase-MAPK pathway (43). Although CXCR7 can be blocked by the small molecule investigational drug CCX771, the alternative signaling mechanism by CXCR4 can antagonize this inhibition suggesting that effective neutralization of the ligand SDF-1 is the most effective strategy. Against the background that NOX-A12 finished phase 1 trials (www.clinicaltrials.gov, NCT00976378 and NCT01194934), proof-of-concept studies in PAH are a desirable next step to extend the current treatment options by a drug with a novel mechanism of action. Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors thank Christina Vroom, Marianne Hoeck, and Stephanie Viehmann for help with immunohistochemical analysis; Stefan Vonhoff; and the chemistry group of NOXXON Pharma AG for providing NOX-A12.

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Figure 9. Immunostaining and quantification of FoxP31 mononuclear cells in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with anti-FoxP3 to detect FoxP31 mononuclear cells. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (pink). (B) Representative micrographs of negative and positive (human spleen) controls. Scale bar ¼ 20 mm. Quantification of FoxP31 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of FoxP31 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 12 (donor) and n ¼ 19 (IPAH).

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Figure 10. Immunostaining and quantification of B cells in donor and idiopathic pulmonary arterial hypertension (IPAH) lungs. (A) Paraffin lung tissue sections of donor and IPAH lungs were stained with anti-CD20 to detect B cells. Shown are representative micrographs of lung sections and pulmonary arteries from donors and patients with IPAH. Arrowheads indicate the positively stained cells (pink). (B) Representative micrographs of negative and positive (human spleen) controls. Scale bar ¼ 20 mm. Quantification of CD201 cells in lungs (C) and in different sizes of pulmonary arteries (D) from donors and patients with IPAH. (E) Quantification of CD201 cells in intima, media, and adventitia of different-sized IPAH pulmonary arteries. n ¼ 15 (donor) and n ¼ 22 (IPAH).

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Figure 11. Effects of NOX-A12 on monocrotaline (MCT)-induced pulmonary hypertension. Rats received placebo or NOX-A12 from Days 21–35 after MCT injection. The rat lung sections were stained with anti-CD68, anti-CD3, and toluidine blue to detect macrophages, T cells, and mast cells, respectively. (A) Representative photomicrographs and quantification of (B) CD681 cells, (C) CD31 cells, and (D) mast cells in lungs obtained from the experimental groups. Arrowheads indicate the positively stained cells. Scale bar ¼ 20 mm. The rat lung sections were immunostained for von Willebrand factor, a-smooth muscle actin, and a total of 80–100 intraacinar vessels and were analyzed by vascular morphometry. (E) Proportion of nonmuscularized (N), partially muscularized (P), or fully (F) muscularized vessels, as percentage of total pulmonary artery cross-section (sized 20– 50 mm) are given. Each bar represents mean 6 SEM (n ¼ 8–10). (F) Right ventricle (RV) to left ventricle (LV) plus septum (S) ratio [RV/(LV1S)]. (G) Right ventricular systolic pressure (RVSP) and (H) total pulmonary resistance (TPR). *P , 0.05 versus healthy control, #P , 0.05 versus placebo.

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