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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1419-1425, (2002)
© 2002 American Thoracic Society

Original Article

CX3C Chemokine Fractalkine in Pulmonary Arterial Hypertension

Karl Balabanian, Arnaud Foussat, Peter Dorfmüller, Ingrid Durand-Gasselin, Francis Capel, Laurence Bouchet-Delbos, Alain Portier, Anne Marfaing-Koka, Roman Krzysiek, Anne-Cécile Rimaniol, Gérald Simonneau, Dominique Emilie and Marc Humbert

INSERM U131, UPRES EA2705, Centre des Maladies Vasculaires Pulmonaires, Service de Pneumologie et Réanimation Respiratoire; and Service d'Hématologie, Institut Paris-Sud sur les Cytokines, Hôpital Antoine-Béclère, Université Paris-Sud, Assistance Publique-Hôpitaux de Paris, Clamart, France

Correspondence and requests for reprints should be addressed to Marc Humbert, Service de Pneumologie, Hôpital Antoine-Béclère, 157, Rue de la Porte de Trivaux, 92140 Clamart, France. E-mail: humbert{at}ipsc.u-psud.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Perivascular infiltrates composed of macrophages and lymphocytes have been described in lung biopsies of patients displaying pulmonary arterial hypertension (PAH), suggesting that circulating inflammatory cells can be recruited in affected vessels. CX3C chemokine fractalkine is produced by endothelial cells and promotes leukocyte recruitment, but unlike other chemokines, it can capture leukocytes rapidly and firmly in an integrin-independent manner under high blood flow. We therefore hypothesized that fractalkine may contribute to pulmonary inflammatory cell recruitment in PAH. Expression and function of the fractalkine receptor (CX3CR1) were studied by use of triple-color flow cytometry on circulating T-lymphocyte subpopulations in freshly isolated peripheral blood mononuclear cells from control subjects and patients with PAH. Plasma-soluble fractalkine concentrations were measured by enzyme-linked immunosorbent assay. Finally, fractalkine mRNA and protein expression were analyzed in lung samples by reverse transcriptase-polymerase chain reaction or in situ hybridization and immunohistochemistry, respectively. In patients with PAH, CX3CR1 expression and function are upregulated in circulating T-lymphocytes, mostly of the CD4+ subset, and plasma soluble fractalkine concentrations are elevated, as compared with control subjects. Fractalkine mRNA and protein product are expressed in pulmonary artery endothelial cells. We conclude that inflammatory mechanisms involving chemokine fractalkine and its receptor CX3CR1 may have a role in the natural history of PAH.

Key Words: chemokines • endothelium-derived factors • fractalkine • pulmonary hypertension • receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary arterial hypertension (PAH) is a severe condition leading to progressive right heart failure and ultimately death (1). This disease results from chronic obstruction of small pulmonary arteries, which is due at least in part to endothelial and vascular smooth muscle cell dysfunction and proliferation (2). The recent discovery that a significant proportion of patients with primary pulmonary hypertension is associated with germline mutations of genes encoding receptor members of the transforming growth factor-ß family (bone morphogenetic protein receptor type II [BMPR-II] and activin-receptor–like kinase 1 [ALK-1]) suggests that dysfunctional transforming growth factor-ß signaling could lead to abnormal proliferation of pulmonary vascular cells (3, 4). Although these major advances have improved our understanding of PAH, more information is needed to evaluate the possible involvement of additional factors in its pathogenesis.

Inflammatory mechanisms may play an important part in the genesis or progression of PAH. Animal models have demonstrated that proinflammatory cytokines and chemokines are involved in the genesis of pulmonary hypertension induced by monocrotaline (5, 6). The relevance of inflammation in humans displaying PAH is supported by additional data. First, PAH is a common complication of systemic inflammatory conditions such as scleroderma and systemic lupus erythematosus, emphasizing the possibility that PAH may be the consequence of an autoimmune vascular inflammation (7). Second, the course of other immunologic disturbances, including human immunodeficiency virus infection (7) or plasma cell dyscrasia with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (8), can be complicated with significant PAH. Third, a large proportion of so-called primary pulmonary hypertension patients has evidence of autoimmunity and/or active inflammation, including detection of circulating antinuclear antibodies (9), elevated serum levels of proinflammatory cytokines interleukin (IL)-1 and IL-6 (10), and increased pulmonary expression of platelet-derived growth factor (11) or macrophage inflammatory protein-1{alpha} (12). Fourth, antiinflammatory drugs (corticosteroids and immunosuppressants) have markedly improved the condition of some patients with PAH (13, 14). Last, identification of perivascular inflammatory cell infiltrates composed of T and B lymphocytes and macrophages has supported the concept that inflammatory cells may play some role in this condition (15). This study attempted to analyze the mechanisms leading to inflammatory cell recruitment in the lungs of patients displaying PAH.

Leukocyte trafficking involves successive events, including rolling, firm adhesion, and extravasation, in response to a chemoattractant gradient that is thought to involve chemokines (16). Chemokines are soluble secreted basic proteins that direct the migration of specific subsets of leukocytes (16, 17). Fractalkine (FKN/CX3CL1) is a unique chemokine because it exists in a soluble form (chemotactic protein), and in a membrane-anchored form on endothelial cells (cell-adhesion molecule) (18, 19). In healthy individuals, FKN is constitutively expressed by neurons and in several nonlymphoid tissues, mainly by epithelial and endothelial cells (19–23). In reactive lymph nodes, high endothelial venule, dendritic cells, follicular dendritic cells, and a few germinal center lymphocytes express FKN (21). FKN expression is increased in the brain of patients with human immunodeficiency virus–related encephalitis and in dendritic and plasma cells of lymph nodes of human immunodeficiency virus–infected patients (24, 25). Its actions are mediated by CX3CR1, a seven-transmembrane receptor that is expressed by monocytes, microglial cells, neurons, natural killer cells, mast cells, and subpopulations of T-lymphocytes (21, 22, 26–29). FKN promotes CX3CR1-expressing leukocyte recruitment, but unlike other chemokines, it can mediate the rapid capture, integrin-independent adhesion, and activation of circulating CX3CR1+ leukocytes under high blood flow (30–32). Several recent findings have reported a CX3CR1 gene polymorphism associated with a reduced risk of acute coronary artery disease, suggesting that FKN plays a critical role in monocyte/T cell recruitment to the vessel wall (33, 34).

To test the hypothesis that FKN contributes to cell recruitment in the lungs of patients with severe PAH, we studied CX3CR1 expression and function on circulating CD4+ and CD8+ T-lymphocytes, plasma concentrations of soluble FKN (sFKN), and pulmonary endothelial cell expression of FKN mRNA and protein product in patients with severe PAH and control subjects.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Material
Patients were referred to our institution with severe PAH. Healthy volunteers and patients displaying chronic thromboembolic pulmonary hypertension (CTEPH) were used as control subjects. They were all nonsmokers, were seronegative for human immunodeficiency virus, and were without evidence of cancer, congenital heart disease, connective tissue disorder, or chronic parenchymal lung disease. This study was approved by our local ethics committee, and all subjects gave informed consent to be included. Lung samples were obtained from five patients with PAH at the time of lung transplantation to study FKN mRNA and protein expression. They were compared with lung samples from seven control subjects who underwent surgery and open lung biopsy for recurring pneumothoraces with a normal lung parenchyma excepting subpleural blebs. Two control patients had a smoking history of 10 pack-years or less.

Expression and Function of CX3CR1 on T-Lymphocyte Subpopulations
Expression and function of the fractalkine receptor (CX3CR1) were studied on T-lymphocyte subpopulations of freshly isolated peripheral blood mononuclear cells from seven healthy individuals and seven patients with PAH (Table 1). Subsequently, seven additional healthy control subjects were compared with eight patients displaying CTEPH (Table 1). Expression and function of CX3CR1 were evaluated by triple-color flow cytometry (FACScan; Becton-Dickinson, Rungis, France) and by actin polymerization experiments, respectively, as previously described (21, 25, 35).


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  		TABLE 1. Pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension patient information

 
Plasma Concentrations of sFKN and Other Soluble Markers of Inflammation and/or Endothelial Dysfunction
Plasma concentrations of sFKN were measured by enzyme-linked immunosorbent assay in 29 patients with PAH and 26 control subjects, as previously described (25). This method allowed detection of 121.8 pg/ml (3.4 pM) of sFKN. Any values below this threshold were considered to be 120 pg/ml. Plasma levels of soluble CD25, soluble P-selectin, soluble E-selectin, soluble vascular cell adhesion molecule-1, and soluble intercellular adhesion molecule-1 were measured using enzyme-linked immunosorbent assay kits from R&D Systems (Abingdon, UK). Plasmatic soluble IL-6 and von Willebrand factor concentrations were performed with enzyme-linked immunosorbent assay kits purchased from Diaclone (Besançon, France) and Diagnostica Stago (Asnières, France), respectively.

Reverse Transcriptase-Polymerase Chain Reaction Studies
Lung biopsies from patients with severe PAH and control subjects were analyzed for the expression of the fractalkine gene by reverse transcriptase-polymerase chain reaction, as previously described (21, 36). Amplified products were subjected to 1.2% agarose gel electrophoresis, detected by ethidium bromide staining, and quantified by computed-assisted densitometry using the ScanAnalysis software (Biosoft, Cambridge, UK).

Detection of FKN mRNA and Protein by In Situ Hybridization and Immunohistochemistry
Lung samples from patients with severe PAH and control subjects were analyzed for FKN mRNA and protein expression by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, as previously described (21, 25).

Statistical Analysis
The two-tailed Mann–Whitney U test was used to compare the control and PAH groups. The Spearman test was used for correlations. Unless specified, mean values (± SEM) are presented in the text.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Function of CX3CR1 in Circulating T-Lymphocytes from Patients with PAH, Normal Healthy Control Subjects, and Subjects Displaying CTEPH
CX3CR1 expression was analyzed by flow cytometry on subsets of circulating T-lymphocytes. In all T-lymphocyte subpopulations, the fraction of cells expressing CX3CR1 was significantly higher in patients with PAH than in control subjects (Figure 1A) . Furthermore, the CX3CR1 labeling intensity of CD45RO+ and CD45RO- T cells, evaluated by the mean fluorescence intensity of positive lymphocytes, was also higher in patients with PAH than in control subjects (Figure 1B).



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  		Figure 1. Expression and function of CX3CR1 by T-lymphocytes from PAH patients. (A, B) Expression of CX3CR1 was analyzed by flow cytometry in memory (CD45RO+) and naive (CD45RO-) CD4+ and CD8+ T-lymphocytes. Results are expressed as the proportion of T-lymphocytes expressing CX3CR1 for healthy control subjects (n = 7, open circles) and PAH patients (n = 7, closed circles) (A) and are expressed as the mean fluorescence intensity (mean ± SEM) of CX3CR1+ cells for each T-cell subpopulation from healthy control subjects (open squares) and from PAH patients (closed squares) (B) (*p < 0.05 and **p < 0.005, respectively). (C) In the same individuals (closed squares: PAH patients, n = 7, and open circles: healthy control subjects, n = 7), the function of CX3CR1 was tested by monitoring actin polymerization. Results (mean ± SEM) show the kinetics of actin polymerization after FKN addition; 100% corresponds to the baseline level before the addition of FKN. The percentage mean fluorescence intensity modulation was calculated for each sample at each time point as follows: (mean fluorescence intensity after addition of FKN/mean fluorescence intensity before addition of FKN) x 100 (*p < 0.05). (D) In each individual, either healthy (open circles) or suffering from PAH (closed circles), the proportion of CX3CR1-expressing cells was compared with the intensity of response to FKN (peak at 15 seconds). The correlation between these two parameters was tested for the 14 individuals (#) and for the 7 patients with PAH ({dagger}).

 
CX3CR1 function in T-lymphocyte subpopulations was assessed by monitoring cytoskeleton rearrangement after the addition of FKN. Chemokine-dependent cell movement is thought to be driven mostly by the rapid, within seconds of receptor triggering, polymerization of actin monomers (G-actin) into filaments (F-actin) near the plasma cell membrane. Intracellular F-actin filaments can be easily quantified by flow cytometry using FITC-labeled phalloidin as a probe. Typically, the peak of F-actin polymerization response occurs spontaneously within 15 or 30 seconds. To determine whether CX3CR1 is functional in naive and memory T cells, we quantified the modulation in the intracellular F-actin content induced by FKN in naive and memory T cells from patients with PAH and from healthy individuals. For all naive and memory T-lymphocyte subsets tested, the intensity of FKN-triggered actin polymerization was significantly higher in patients with PAH than in control subjects (Figure 1C). The stronger responses to FKN stimulation were observed in naive and memory CD4+ T-lymphocytes. There was a significant correlation between CX3CR1 expression and response to FKN, especially in the CD4+ CD45RO+ memory T-helper cell population (Figure 1D), indicating that the limiting factor for response to FKN is the amount of expression of its receptor. Among patients with PAH, the intensity of deregulated CX3CR1 expression by CD4+ T-lymphocytes significantly correlated with the increased sensitivity to FKN. This correlation was not statistically significant for CD8+ T-lymphocyte subpopulations. CX3CR1 expression is, therefore, deregulated in T-lymphocytes in PAH, especially in naive and memory CD4+ T cells. This accounts for the increased sensitivity of these cells to FKN stimulation in this condition.

In contrast, the intensity and kinetics of FKN-triggered actin polymerization were similar in all T-lymphocyte subpopulations from CTEPH patients and control subjects (Figure 2) . Memory CD4+ CD45RO+ T-helper lymphocytes from normal control subjects and CTEPH patients responded similarly to FKN with a maximal response at 15 seconds. In contrast, FKN did not trigger any actin polymerization in naive CD4+ CD45RO- T-lymphocytes in both control and CTEPH patient populations. Memory CD45RO+ CD8+ T-lymphocytes responded similarly to FKN in terms of intensity and kinetics. Finally, no or low responses were detected in CD45RO- CD8+ T-lymphocytes from normal control subjects and CTEPH patients. Therefore, in contrast to patients with PAH, CX3CR1 function is not altered in naive and memory T-cell subpopulations in CTEPH patients, as compared with healthy individuals.



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  		Figure 2. Function of CX3CR1 by T-lymphocytes from CTEPH patients. The response of memory (CD45RO+) and naive (CD45RO-) CD4+ and CD8+ T-lymphocytes after addition of FKN was evaluated by determining the kinetics of actin polymerization in eight CTEPH patients (closed triangles) and in seven healthy individuals (open circles). Results are expressed as the mean (± SEM) variation of fluorescence intensity induced by chemokine (FKN) addition in function of time, as compared with baseline value (100%), before chemokine addition.

 
Increased Concentration of Circulating sFKN and Other Markers of Inflammation and/or Endothelial Dysfunction in PAH
Plasma sFKN concentrations were significantly higher in patients with PAH than in control subjects (2,050.0 ± 691.1 pg/ml versus 381.6 ± 149.3 pg/ml, corresponding to 57.9 ± 19.9 pM versus 10.8 ± 4.2 pM, p = 0.007) (Table 2). Plasma sFKN amounts measured in patients with PAH were also elevated when compared with the concentrations detected in patients with CTEPH (295.6 ± 92.6 pg/ml, corresponding to 8.5 ± 2.6 pM, p = 0.01). sFKN concentrations were similar in patients with PAH treated with conventional (n = 16) or continuous intravenous epoprostenol (n = 13) therapy (data not shown). As compared with normal healthy control subjects, patients with PAH showed higher plasma amounts of soluble CD25, soluble P-selectin, soluble E-selectin, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, soluble IL-6, and von Willebrand factor (Table 2). No correlation was found between plasma sFKN concentrations and any other biologic (soluble CD25, soluble E-selectin, soluble P-selectin, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, soluble IL-6, and von Willebrand factor) or hemodynamic (mean pulmonary artery pressure, mean right atrial pressure, cardiac index, and total pulmonary vascular resistance) parameters (data not shown).


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  		TABLE 2. Plasma concentrations of soluble markers in patients with pulmonary arterial hypertension and in control subjects

 
Pulmonary Artery Endothelial Cell Expression of FKN in PAH and Normal Lungs
Reverse transcriptase-polymerase chain reaction studies allowed detection of fractalkine mRNA in all samples from patients with PAH and control subjects (data not shown). However, FKN mRNA expression was significantly increased in patients with PAH as compared with control subjects (p = 0.05) (Figure 3) . ISH and IHC experiments confirmed that normal pulmonary artery endothelial cells express FKN, as previously reported (21) (Figures 4A and 5A) . A similar pattern of FKN expression was observed in patients with PAH (Figures 4C and 5C). No labeling was seen in negative controls, which consisted of a sense probe in ISH experiments (Figures 4B and 4D) and a nonimmune rabbit serum in IHC experiments (Figures 5B and 5D).



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  		Figure 3. Detection of FKN mRNA by reverse transcriptase-polymerase chain reaction in lung biopsies from control individuals and patients displaying severe PAH. Expression of the fractalkine gene was evaluated by reverse transcriptase-polymerase chain reaction in lung biopsies from patients with severe PAH (n = 5) and healthy individuals (n = 7). Each sample, containing 2.0 x 105 molecules of ß-actin mRNA, was analyzed for FKN mRNA expression by polymerase chain reaction. Amplified FKN products were then quantified by densitometry. Results are expressed as the mean ± SEM of arbitrary units, quantifying amplified FKN products in healthy control subjects (open squares) and in PAH patients (closed squares).

 


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  		Figure 4. FKN mRNA expression in lung biopsies from control individuals and patients displaying severe PAH. FKN mRNA expression was tested by ISH with an antisense probe (A, C) in lung samples from control subjects (A) and from patients with severe PAH (C). A sense probe was used as negative control in ISH experiments performed in lung biopsies from control subjects (B) and from PAH patients (D). Original magnification: x200 in A-D.

 


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  		Figure 5. FKN protein production in lung biopsies from control individuals and patients displaying severe PAH. FKN protein production was determined by IHC with an antifractalkine antibody (A, C) in lung samples from control subjects (A) and from patients with severe PAH (C). A nonimmune rabbit serum was used as negative control in IHC experiments performed in lung samples from control subjects (B) and from PAH patients (D). Original magnification: x200 (A, B) and x400 (C, D).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chemokines are cytokines orchestrating the traffic of leukocytes throughout the body (16, 17). Unlike other chemokines except CXCL16 (37), FKN is produced as a membrane-bound form presented at the cell surface by a mucin-like stalk. FKN can be released as a soluble chemotactic form after proteolytic cleavage (18). In both cases, its actions are mediated by CX3CR1, a seven-transmembrane receptor that is mainly expressed on monocytes, T cells, mast cells, and natural killer cells (21, 22, 26–29). Leukocytes expressing the CX3CR1 receptor rapidly and firmly adhere to cells expressing the membrane-bound form of FKN (mainly endothelial cells) in an integrin-independent manner under high blood flow (30–32). This activity, together with its expression on endothelial cells, suggests that FKN might mediate adhesion of leukocytes to the endothelium during inflammation.

This study demonstrates the following: (1) CX3CR1 is upregulated in circulating CD4+ and CD8+ T-lymphocytes from patients with PAH, as compared with control subjects. (2) This deregulation of CX3CR1 expression accounts for the increased sensitivity of these cells, especially in the CD4+ T-lymphocyte subset. (3) The abnormal response of T-lymphocytes to FKN is not the mere consequence of pulmonary hypertension, as it is not present in patients with pulmonary hypertension secondary to CTEPH. (4) Elevated sFKN plasma concentrations are measured in patients with PAH, as compared with CTEPH patients and normal control subjects. (5) Lung samples from patients with PAH show an increased FKN mRNA expression, as compared with control subjects, and pulmonary artery endothelial cells from patients with PAH express FKN. Moreover, we confirm that patients displaying PAH have evidence of persistent inflammation and endothelial cell dysfunction, as demonstrated by elevated plasma amounts of CD25, IL-6, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E- and P-selectins, and von Willebrand factor.

The relevance of FKN in inflammatory conditions has been already demonstrated. Increased FKN expression has been shown in lymph nodes of human immunodeficiency virus–infected patients (25), in the vessel wall of patients with atherosclerosis (38), in psoriatic plaques (39), in rheumatoid arthritis joints (40), and in crescentic glomerulonephritis (41). Immunoneutralization of CX3CR1 attenuates cardiac transplant rejection and leads to a marked inhibition of leukocyte recruitment and subsequent crescentic glomerulonephritis in rodent models of acute renal injury (32, 42). In addition, a CX3CR1 gene polymorphism (I249) has been associated with a reduced risk of acute coronary events such as atherosclerosis (33, 34), suggesting that FKN plays a critical role in inflammatory cell recruitment to the vessel wall. Our present results suggest that PAH should be added to these FKN-mediated inflammatory conditions. FKN might be involved in a multiple step mechanism involving the local production of proinflammatory cytokines, growth factors, adhesion molecules, and chemokines leading to inflammatory cell infiltrates in diseased vessels (10–12, 43, 44, and this study). In addition to FKN, chemokine RANTES (regulated upon activation, normal T cell expressed and secreted/CCL5) is an important chemoattractant for monocytes and T cells and may promote cell recruitment (17, 44, 45). In this context, endothelial cell-derived FKN could promote the firm capture to the vascular endothelium under high blood flow of circulating leukocytes expressing CX3CR1. Then other chemoattractant factors, such as RANTES would promote transendothelial and subendothelial cell migration in lung tissues. This multistep model of leukocyte–endothelial cell interactions, involving the chemokines FKN and RANTES may account for the selective accumulation of cell infiltrates in lung tissues in PAH.

FKN is constitutively expressed by several cell types, including endothelial cells (21). The detection of FKN in pulmonary endothelial cells from control subjects supports the concept that this chemokine has a role in the trafficking of immune cells throughout the lungs of healthy individuals (21). Unlike other chemokines, FKN can be released in a soluble form after proteolytic cleavage (18). Under normal conditions, the action of sFKN is not yet well defined. However, a role for sFKN released from the anchored-bound form has been suggested in inflammatory diseases, such as rheumatoid arthritis. Elevated sFKN amounts have been measured in synovial fluid of patients with rheumatoid arthritis, as compared with patients with other forms of arthritis, suggesting a role for FKN in inflammatory cell chemotaxis in the rheumatoid arthritis joint (40). In this study, we report a significantly increased concentration of plasma sFKN in patients with PAH. This may be due to a raised production and/or an increased cleavage of the molecule. IHC experiments demonstrated that if increased cleavage occurs, it does not result in the disappearance of FKN from endothelial cells. Additional experiments are needed to evaluate whether FKN is expressed more intensely locally in the vessel wall of pulmonary arteries of patients displaying severe PAH, as compared with control subjects.

A protease responsible for the conversion of FKN from a membrane-bound adhesion molecule to a soluble form has been recently identified: TACE/ADAM17 (tumor necrosis factor-{alpha} converting enzyme/a disintegrin and metalloproteinase 17) presents a metalloproteinase-like domain and is upregulated in a variety of inflammatory conditions in vivo (46). It is tempting to speculate that metalloproteinase-mediated cleavage of FKN from the endothelial cell surface may regulate FKN function in PAH. Initially, FKN may act predominantly as an adhesion molecule for circulating leukocytes. Subsequently, FKN cleavage might be necessary to modify the high-affinity interactions reported between endothelial-expressed FKN and its receptor during leukocyte recruitment. sFKN may induce leukocyte adhesion by upregulating integrin avidity, as already reported for freshly isolated monocytes (47). Another possibility is that FKN bound to the endothelial cell surface may act in fact as a proform for sFKN that can be released by cleavage. Finally, FKN may act as a dual-function protein acting both as an adhesion molecule and a soluble chemoattractant chemokine. At this stage, one should discuss the possibility that sFKN may antagonize adherence of CX3CR1+ leukocytes to the endothelial membrane-bound form of FKN. The range of reported dissociation constants (Kd) for FKN/CX3CR1 (30–740 pM) suggests that it has a higher affinity than the other chemokine/receptor pairs (26, 27, 48–50). The wide range of Kd values probably reflects differences in experimental systems and assay methods but the most convergent Kd value reported for FKN and its receptor is approximately 100 pM. Our results show that plasma concentrations of sFKN were significantly higher in patients with PAH than in control subjects (57.9 ± 19.9 pM versus 10.8 ± 4.2 pM), suggesting that sFKN may partially antagonize the membrane-bound form of FKN but not totally.

Detection of sFKN in the plasma of patients with PAH could be regarded as another marker of endothelial cell dysfunction in this condition (51, 52). As we discussed, sFKN may be released in response to an inflammatory stimuli in our patient population, emphasizing the possibility of an inflammatory component in PAH. Ultimately, in vivo models will help in defining the importance of FKN and its receptor in PAH. FKN- and CX3CR1-deleted mice have now been generated (53, 54). These animals were both viable and did not exhibit specific abnormalities. Exposure of these animals to conditions promoting PAH will be of great interest.


   Acknowledgments
 
This study was supported by grants from Legs Poix, Université Paris-Sud, AFM, and INSERM.

Received in original form June 6, 2001; accepted in final form January 22, 2002


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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