INTRODUCTION

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Keywords: chemokines; endothelial cells; primary pulmonary hypertension; pulmonary hypertension; RANTES

Pulmonary arterial hypertension (PAH) is characterized by an elevated mean pulmonary artery pressure  25 mm Hg at rest, with a normal pulmonary artery wedge pressure (1). A new diagnostic classification of PAH has been proposed at the 1998 World Health Organization (WHO) Pulmonary Hypertension Meeting held in Evian, France. This classification reflects recent advances in the understanding of pulmonary hypertensive diseases and recognizes the similarity between "unexplained" pulmonary hypertension (primary pulmonary hypertension [PPH]) and PAH of certain known causes such as collagen vascular diseases, human immunodeficiency virus (HIV) infection, portal hypertension, congenital systemic to pulmonary shunts, and anorexigen exposure (2). PPH is a rare disease, with an estimated incidence of 2 per million people (1). It can be either sporadic or clustered in families (1). The recent discovery that sporadic and familial PPH can be associated with germline mutations of genes encoding receptor members of the transforming growth factor-beta (TGF-) family (bone morphogenetic protein receptor type II [BMPR-II] and activin receptor-like kinase 1 [ALK1]) has focused much attention on cytokines and growth factors in pulmonary vascular disorders (3). Moreover, endothelial cells within PPH plexiform lesions harbor mutations permissive for clonal cell growth, including mutations of TGF- receptor type II (8).

In addition to abnormal TGF- signaling, altered expression and production of several cytokines and growth factors have been demonstrated in severe PAH, including interleukin-1 (IL-1), IL-6, and platelet-derived growth factor A (PDGF-A), highlighting the possible influence of inflammatory mechanisms in this condition (9, 10). This "inflammatory hypothesis" was further supported by the identification of perivascular cell infiltrates composed of macrophages and T cells and B cells in plexiform lesions of PPH (11). Leukocyte trafficking comprises successive events, including rolling, firm adhesion, and extravasation, presumably in response to a chemoattractant gradient where chemotactic cytokines (chemokines) are thought to play a critical role (12, 13). Chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) is an important chemoattractant for monocytes and T cells (13, 14) that constitute the main cell population within the perivascular infiltrates of PAH.

We therefore hypothesize that chemokine RANTES promotes cell recruitment in the lungs of patients displaying severe PAH. As a first step to test this hypothesis, evaluation of RANTES messenger RNA (mRNA) expression and protein production was performed using reverse transcriptase/polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry in lungs from patients displaying severe PAH, as compared with control lung biopsies.

	METHODS

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Subjects and Sample Processing

Ten nonsmoking patients with PAH (9 females, age 39 ± 4 yr) were included in the study. Eight suffered with sporadic PPH and one was HIV-seropositive. Baseline haemodynamics demonstrated severe PAH (mean pulmonary artery pressure = 66 ± 4 mm Hg, pulmonary artery wedge pressure = 8 ± 1 mm Hg, cardiac index = 2.09 ± 0.16 L · min¹ · m², total pulmonary resistance = 33.1 ± 3.2 mm Hg · L¹ · min¹ · m², mixed venous oxygen saturation = 58 ± 2%). All lung samples were obtained at the time of lung transplantation or open-lung biopsy. In parallel, we studied seven control subjects who underwent open lung biopsy for recurring pneumothoraces with a normal lung parenchyma excepting subpleural blebs (mean age, 32 yr; range, 22 to 43 yr; 5 males). Two control subjects smoked 10 pack-years or less. Lung tissue was embedded in ornithyl carbamyl transferase compound (Tissue-Tek; Sakura Finetek, Bayer Diagnostic, France), immediately snap-frozen in liquid nitrogen, and stored at 80° C, under ribonuclease (RNAse)-free conditions. Paraffin-embedded sections were produced in parallel.

Detection of RANTES mRNA by Quantitative RT-PCR

Quantitative RT-PCR was performed as described previously using the following primers: RANTES antisense (5'-GGGTTGGCACACAC TTGGCG-3'), RANTES sense (5'-CATTCGTACTGCCCTCTGCG-3'), -actin antisense (5'-GGTCTCAAACATGATCTGGG-3'), and -actin sense (5'-GGGTCAGAAGGATTCCTATG-3') (15). RANTES and -actin RT-PCR from patients and control subjects were processed in parallel. The quantification of PCR products was performed using a colorimetric assay (Biomek 2000 automated workstation; Beckmann, Gagny, France) (15).

In Situ Hybridization and Immunohistochemistry

RANTES-specific sense and antisense probes were constructed by cloning a 411-base pair (bp) EcoRI-ApaI fragment of the human RANTES complementary DNA (cDNA) across the EcoRI-ApaI restriction sites of the Bluescript plasmid (Stratagene, La Jolla, CA). In situ hybridization was performed on 8-µm cryostat sections from eight patients and six control subjects, as described previously (16). Immunostaining was performed on 7-µm cryostat sections from eight patients and six control subjects, through the streptavidin-biotin complex/alkaline phosphatase method with a monoclonal IgG2b antibody directed against human RANTES (Peprotech clone VL-1 [immunoglobulin lambda light chain V-region]; Tebu, Le Perray, France) (1:70 dilution). Negative controls were produced by omitting the primary antibody or by substituting it with an irrelevant IgG2b antibody (anti-human chromogranin A; Dako, Trappes, France). The intensity of pulmonary artery RANTES staining (protein product and mRNA) was measured semiquantitatively by two investigators (P.D. and M.H.) who quoted whether endothelial cells and perivascular inflammatory cells were strongly (+++), moderately (++), mildly (+), or not () positive for RANTES.

Paraffin-embedded sections were used to identify RANTES+ and CD45+ cells in consecutive sections, using the Peprotech clone VL-1 (1:70 dilution) and the clone ubiquitin C-terminal hydrolase 1 (UCHL1) (anti-CD45RO, 1:100 dilution, Dako), respectively. Serial sections (4-µm) were deparaffinized in toluol, rehydrated in graded ethanols, and microwaved for 10 min in citrate buffer pH 6. Nonspecific antibody binding sites were blocked with 10% normal goat serum/5% human serum AB/Tris-buffered saline (TBS) for 30 min at room temperature. Incubation with anti-RANTES or anti-CD45RO monoclonal antibody was performed overnight at 4° C and detected by subsequently biotinylated goat anti-mouse IgG and alkaline phosphatase-conjugated streptavidin complex (Biogenex Super Sensitive, San Ramon, CA). Fast Red (Sigma, Steinheim, Germany) was used as the chromogen and Mayers hematoxylin as the counterstain.

Statistical Analysis

Data were analyzed using the Statview 4.5 Software (Abacus Concepts Inc., Berkeley, CA). The two-tailed Mann-Whitney U test and the unpaired Student's t test were used for between-group comparison. Mean values (± SEM) are presented in the text.

	RESULTS

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Detection of RANTES mRNA in Patients with PAH and Control Subjects

Quantitative RT-PCR allowed detection of chemokine RANTES mRNA in all samples from patients and control subjects. Elevated numbers of copies of RANTES mRNA relative to -actin mRNA were found in patients with PAH, as compared with control subjects (112 ± 30 versus 14 ± 4 RANTES mRNA molecules per 100 -actin mRNA molecules, p = 0.017) (Figure 1). In situ hybridization detected RANTES mRNA positive cells in the pulmonary arterial wall of patients displaying severe PAH (Figures 2A and 3). RANTES mRNA expression in pulmonary arteries of patients with PAH mainly derived from endothelial cells, a much lesser contribution arising from perivascular inflammatory cells (Table 1). RANTES expression was stronger in lesions displaying exuberant endothelial cell proliferation (plexiform lesions) (Figure 3). Those lesions showed larger inflammatory cell infiltrates, as compared with lesions with predominant medial hypertrophy or intimal fibrosis. Smooth muscle cells of muscular arteries were consistently negative. In control subjects, endothelial staining was weak or negative and there were no inflammatory cell infiltrates (not shown). All samples incubated with the sense probe were distinctly negative (Figure 2B).

View larger version (10K): [in this window] [in a new window]   Figure 1.   RANTES mRNA expression detected by competitive RT-PCR in lung biopsies from patients suffering from severe PAH and control subjects.

View larger version (90K): [in this window] [in a new window]   Figure 2.   (A) Endothelial cells expressing RANTES mRNA in a remodeled small muscular pulmonary artery from a patient displaying severe PAH (in situ hybridization; original magnification: ×200). (B) Sense control (serial section; original magnification: ×100).

View larger version (134K): [in this window] [in a new window]   Figure 3.   Endothelial cells expressing RANTES mRNA in a plexiform lesion from a patient displaying severe PAH (in situ hybridization; original magnification: ×200).

Localization of RANTES Protein Using Immunohistochemistry

Pulmonary arterial endothelium stained positive for RANTES in most cases of severe PAH (Table 2, Figures 4 and 5B). Small arteries of the muscular type were the main source of RANTES. Staining was clear, though of varying intensity in different arteries of the same patient. Arteries of small diameter and arterioles were mostly concerned. RANTES expression predominates in vascular lesions characterized by marked endothelial cell proliferation (Figure 5B). RANTES protein product expression in pulmonary arteries of patients with PAH mainly derived from endothelial cells, a much lesser contribution arising from perivascular inflammatory cells (Table 2). Therefore, RANTES production depended on the number of proliferating and presumably activated endothelial cells. Consecutive sections demonstrated that vessels characterized by endothelial cell-derived RANTES protein product expression were associated with CD45+ cell infiltrates (Figure 5). In control subjects, staining of endothelial cells was absent or weak and there were no inflammatory cell infiltrates (not shown). Negative controls using irrelevant antibodies did not give any signal (not shown).

View larger version (142K): [in this window] [in a new window]   Figure 4.   Endothelial cells expressing RANTES protein product in a remodeled small muscular pulmonary artery (immunohistochemistry; original magnification: ×200).

View larger version (162K): [in this window] [in a new window]   View larger version (125K): [in this window] [in a new window]   View larger version (124K): [in this window] [in a new window]   Figure 5.   Serial sections of a plexiform lesion from a patient displaying severe PAH (immunohistochemistry; original magnification: ×200). (A) Hematoxylin-eosin saffron staining. (B) Endothelial cells expressing RANTES protein product. (C ) Perivascular CD45+ inflammatory cells.

	DISCUSSION

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PAH is associated with vascular wall remodeling, including cell proliferation with medial hypertrophy, intimal thickening and plexiform lesions, concentric fibrosis with deposition of procollagen, in situ microthrombosis, and perivascular inflammatory infiltrates (11, 17). These changes predominate in muscular arteries of middle-to-small diameter (11, 17). Despite the recent evidence that abnormal TGF- signaling is a critical feature in many patients with familial as well as sporadic PPH (3), the pathophysiology of PAH remains poorly understood (1) and might also involve platelet (18, 19), smooth muscle (20), and endothelial cell dysfunction (21); imbalance between vasoconstrictors and vasodilatators (20); coagulation abnormalities (29); monoclonal endothelial cell proliferation (8, 30); and inflammation (9).

Inflammatory cells (T and B lymphocytes, and macrophages) have been described surrounding plexiform lesions in PAH (11). Local recruitment of circulating leukocytes requires simultaneous expression of adhesion molecules by endothelial and inflammatory circulating cells, allowing them to adhere to the vascular endothelium, and local production of chemoattractant factors which can promote transendothelial and subendothelial cells migration (12). Chemokines are a group of chemotactic cytokines promoting attraction and activation of granulocytes, monocytes, and lymphocytes (13). Chemokines foster tight adhesion of circulating leukocytes to the vascular endothelium by activating leukocytic integrins. Moreover, they guide leukocytes through the endothelial junctions and underlying tissue and activate leukocytes effector functions. We have recently shown that adhesion molecules (vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and E-selectin), as well as a member of the chemokine family, namely macrophage inflammatory protein-1 (MIP-1), could play a role in severe PAH (31, 32).

In the present study, we extend this observation to chemokine RANTES, an important chemoattractant for monocytes and T cells (13, 14). RANTES presumably plays a key role in a number of arterial inflammatory processes such as glomerulonephritis (33), Kawasaki disease (34), and Takayasu arteritis (35). In addition, successful antagonization of RANTES has been reported in animal models of inflammatory disease (36- 38). RANTES may also play an indirect role in PAH through the induction of endothelin-converting enzyme-1 and endothelin-1, a potent endothelium-derived factor with strong vasoconstrictive and mitogenic action (39). Indeed, elevated endothelin-1 expression has been detected in PAH (22) and novel therapeutic approaches include endothelin-1 receptor antagonists (40, 41). Lastly, a link between the TGF- pathway and chemokine RANTES has been suggested by the fact that TGF-₁ regulates chemotaxis of human monocyte-derived dendritic cells through regulation of chemokine RANTES receptor expression (42).

In the absence of double staining procedures, we cannot define precisely the cellular origins of RANTES in this study. Possible sources include epithelial and endothelial cells as well as inflammatory cells such as macrophages and T lymphocytes (12, 13). Our present data indicate that RANTES expression predominates in vascular lesions characterized by marked endothelial cell proliferation and that endothelial cells are the main source of RANTES immunostaining in the pulmonary arteries of PAH patients, a much lesser contribution arising from perivascular inflammatory cells. Therefore, RANTES production presumably depended on the number of proliferating endothelial cells. Endothelial cells are an important source of chemokine RANTES, particularly in response to proinflammatory cytokines (43). There is now strong evidence that endothelial cell dysfunction is a hallmark of severe PAH: endothelial cell-derived production of endothelin-1, thromboxane A₂, and von Willebrand factor is increased whereas nitric oxide and prostaglandin I₂ production is reduced (21). Moreover, Lopes and colleagues have shown that circulating von Willebrand factor antigena marker of endothelial cell dysfunctionis a predictor of short-term prognosis in PAH (26, 27). In addition, we have recently shown that von Willebrand factor overproduction is reduced during continuous infusion of epoprostenol (28), a potent pulmonary vasodilator that produces substantial and sustained hemodynamic and symptomatic responses as well as improved survival in severe PAH refractory to conventional medical therapy (44). Therefore, we hypothesize that abnormal chemokine production may be another marker of endothelial dysfunction in severe PAH. Lastly, RANTES may also derive from inflammatory cells themselves, leading to a self-perpetuating mechanism amplifying inflammatory mechanisms (45).

Our results are consistent with the hypothesis that immune mediators and recruited inflammatory cells could play a part in the pathophysiology of PAH. It will be of interest to confirm our data with novel biologic techniques, including microarray gene analysis, which should provide information pertinent to a better characterization of the pathobiology of PAH (46). It will also be of great interest to screen the microarray data with respect to the expression of RANTES in familial and sporadic PPH as well as in PAH associated with various conditions, including autoimmune and other immunologic diseases (46). We hypothesize that chemokine RANTES is a member of a multiple step process involving proinflammatory cytokines (6), adhesion molecules (31), and chemokines (32 and this study), leading to inflammatory cell infiltrates in diseased vessels (11). This hypothesis is supported by serial sections of pulmonary arteries from patients displaying PAH demonstrating that endothelial cells expressing RANTES are associated with perivascular CD45+ inflammatory cells. Animal models of PAH have already demonstrated that proinflammatory cytokines and chemokines are involved in the genesis of monocrotaline-induced pulmonary hypertension (47). The relevance of inflammatory mechanisms in some individuals with severe PAH has been further suggested by the clinical improvement of a subset of patients after corticosteroids or immunosuppressive therapy (50). Our present data support the concept that inflammatory mechanisms could indeed play some role in the natural history of PAH.