Characterization of fractalkine (CX3CL1) and CX3CR1 in human coronary arteries with native atherosclerosis, diabetes mellitus, and transplant vascular disease

Cardiovascular Pathology 11 (2002) 332 – 338

Characterization of fractalkine (CX3CL1) and CX3CR1 in human coronary arteries with native atherosclerosis, diabetes mellitus, and transplant vascular disease Brian W.C. Wong, Donald Wong, Bruce M. McManus* UBC McDonald Research Laboratories/The iCAPTUR4E Centre, Department of Pathology and Laboratory Medicine, St. Paul’s Hospital/Providence Health Care - University of British Columbia, Vancouver, BC, Canada Received 19 December 2001; received in revised form 19 March 2002; accepted 6 May 2002

Abstract Background: Fractalkine is a novel chemokine that mediates both firm adhesion of leukocytes to the endothelium via CX3CR1 and leukocyte transmigration out of the bloodstream. Fractalkine has recently been shown to play a role in the pathogenesis of acute organ rejection. Since its expression is regulated by inflammatory agents such as LPS, IL-1, and TNF-a, fractalkine involvement in atherosclerosis and transplant vascular disease (TVD) is of particular interest. In this study, we characterized the presence of fractalkine and its receptor CX3CR1 in human coronary arteries from normal, atherosclerotic, diabetic, and TVD settings. Method: Polyclonal rabbit antibodies were used to immunostain human fractalkine and CX3CR1 to localize their presence in transverse sections of the proximal left anterior descending and/or right coronary arteries. Slides were scored in a blinded fashion for intensity of staining (0 to 4+) and for localization in vessel walls. Results: Normal coronary arteries showed no fractalkine staining. In atherosclerotic coronary arteries, staining was localized to the intima, media, and adventitia. Within the media, fractalkine expression was seen in macrophages, foam cells, and smooth muscle cells (SMCs). Diabetic vessels showed similar staining patterns to atherosclerotic coronaries, with much stronger staining in the deep intima. Transplanted coronaries showed staining in the endothelium, intima, and adventitia in early disease, and intimal, medial, and adventitial staining in late disease. CX3CR1 staining was seen in the coronary arteries of all cases, with specific localization to regions with fractalkine staining. Conclusion: The distinctive staining patterns in native atherosclerosis, diabetes mellitus with atherosclerosis, and TVD indicate that the expression of fractalkine and CX3CR1 may be important in the pathogenesis of these diseases. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Fractalkine; CX3CR1; Atherosclerosis; Diabetes mellitus; Transplant vascular disease; Chemokines

1. Introduction Atherosclerosis was initially viewed as a disease of lipid accumulation in the vessel wall, with the endothelium being affected secondary to the underlying degeneration. It is now clear that the endothelium is intimately involved in the initiation and progression of disease, with endothelial dysfunction being one of the earliest pathogenetic signs of atherosclerosis [1,2]. * Corresponding author. Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, University of British Columbia, 1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6. Tel.: +1-604-806-8586; fax: +1-604-806-8351. E-mail address: [email protected] (B.M. McManus).

Acute inflammation, via numerous pathways (including IL-1, TNF-a, complement activation, and viral infection), produces up-regulation of chemokines (e.g., IL-8, MCP-1, and fractalkine), which are involved in the chemotaxis of leukocytes and mononuclear cells and the up-regulation of selectins and integrin ligands that are part of the leukocyte adhesion cascade [3,4]. Such a cascade involves the immobilization of circulating leukocytes via selectin binding, followed by the process of firm adhesion, mediated by the binding of integrins to their ligands on leukocytes. In inflammatory processes, leukocyte adhesion leads to the release of hydrolytic enzymes, cytokines, chemokines, and growth factors at the site of adhesion, which can induce further damage and eventually lead to focal necrosis or apoptosis [3– 5]. Additionally, the infiltration and accumula-

1054-8807/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 4 - 8 8 0 7 ( 0 2 ) 0 0 111 - 4

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tion of mononuclear cells leads to their migration and proliferation in vessel walls, foam cell deposition, and lesion formation [1]. Transplant vascular disease (TVD) has been called an accelerated model of atherosclerosis [6,7]. Although transplant vasculopathy and native atherosclerosis are clinically and pathologically different entities, the pathogenesis of both diseases appears to involve common mechanisms. Both may be regarded as ‘‘responses to injury’’ within a broad concept of involvement of the immune system. In transplant vasculopathy, diffuse proteoglycan- and lipid-rich intimal thickening predominates early after transplantation [8,9] and focal atherosclerotic plaques become more common late after transplantation [6]. The human chemokine fractalkine (CX3CL1) is as a novel chemokine with a unique transmembrane chemokine/ mucin hybrid structure and is synthesized as a Type I transmembrane protein [10,11]. Fractalkine is regulated in vivo by inflammatory agents such as LPS, IL-1, IFN-g, and TNF-a [12,13]. A soluble form can be generated by proteolytic cleavage, mediated by TNF-a-converting enzyme, at the base of the mucin stalk [14]. Soluble fractalkine can mediate the chemotaxis of CX3CR1-expressing mononuclear cells, NK cells, and T-cells. Membrane-bound fractalkine can mediate the firm adhesion of CX3CR1-expressing cells to cardiac endothelial cells (ECs), smooth muscle cells (SMCs), and myofibroblasts [12,15]. In monocytes, fractalkine acts primarily in adhesion [16]. Mononuclear cell infiltration is a hallmark of the pathogenesis of atherosclerosis and results in foam cell deposition and plaque progression [1]. On NK cells, fractalkine has been shown to function as an adhesion molecule to ECs, enhancing the cytolytic activity in a dose- and timedependent manner, resulting in endothelial damage and vascular injury [17]. With respect to T-cells, the regulated expression of fractalkine on ECs has been shown to participate in an amplification circuit of polarized Type I responses [18]. In consideration of the potentially unique role of fractalkine in inflammatory events, the identification of fractalkine and CX3CR1 expression within the heart in the present study may provide insight into its role in atherosclerosis and TVD.

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2. Materials and methods 2.1. Cases Normal, non-atherosclerotic coronary artery tissues from individuals under the age of 35 who died as the result of acute trauma, were obtained from the Pathobiological Determinants of Atherosclerosis in Youth study (PDAY). These tissues were age- and sex-matched with the donors from the TVD cases. Patients with native atherosclerosis were selected on the criterion of greater than 25% luminal narrowing as examined at the time of autopsy. Patients in the diabetic group were selected based on diagnosis of both diabetes mellitus and coronary artery disease. All of the diabetic patients were diagnosed with non-insulindependent diabetes mellitus with a duration ranging from 3 to 23 years and all had their glucose levels under control by a combination of diet, antihypoglycemic drugs, or subclinical doses of insulin. Cases of native atherosclerosis and diabetes mellitus were also age- and sex-matched. The TVD cases were separated into early and late groups, where early TVD was classified as rejection or death less than 360 days post-transplantation and late TVD was classified as rejection or death greater than 360 days post-transplantation. All of the pathological cases of native atherosclerosis, diabetes mellitus, and TVD were obtained from the Cardiovascular Registry of the University of British Columbia (refer to Table 1 for details). 2.2. Antibodies Antibodies used in this study included purified polyclonal rabbit antihuman fractalkine directed against the chemokine domain of human fractalkine (gift from Dr. Thomas Schall, Chemocentryx, San Carlos, CA) and purified polyclonal rabbit antihuman CX3CR1 Ab (Torrey Pines Biolabs, San Diego, CA). Purified antihuman Factor VIII-related antigen, smooth muscle-a actin, CD3, and CD68 antibodies (Dako, Mississauga, ON) identified ECs, SMCs, total lymphocytes, and phagocytic mononuclear cells, respectively. The secondary antibodies used in this study were biotinconjugated goat antirabbit or antimouse IgG (Vector Laboratories, Burlingame, CA).

Table 1 Clinical features of the different patient groups n Normal Native atherosclerosis Diabetes mellitus with atherosclerosis Early TVD Late TVD

Mean Range of Mean implant Implant Mean age of Range of donor Males Females age ± S.D. (years) ages (years) duration ± S.D. (days) duration (days) donor ± S.D. (years) ages (years)

16 9 17 11 15 8

7 6 7

22.0 ± 5.9 65.2 ± 17.0 64.6 ± 9.6

17 – 34 24 – 87 39 – 85

– – –

– – –

– – –

– – –

12 9

5 4

42.3 ± 16.0 38.6 ± 17.0

15 – 60 16 – 67

126.8 ± 109.9 670.5 ± 329.7

13 – 311 360 – 1432

24.6 ± 9.9 30.0 ± 10.5

16 – 44 17 – 47

7 5

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2.3. Immunohistochemistry

3. Results

Formalin-fixed tissues were paraffin-embedded, sectioned, and mounted on slides. Sections were dewaxed and rehydrated, then immunohistochemical staining for fractalkine and CX3CR1 was performed using Shandon disposable immunostaining coverplates (Thermo Shandon, Pittsburgh, PA). Factor VIII-related antigen, CD3, smooth muscle-a actin, and CD68 staining was performed on the Ventana ES IHC Staining System (Ventana Medical Systems, Tucson, AZ). Antigen retrieval was performed for only the CX3CR1 run, using the autoclave method for 7 min in 5% urea in 0.1 M Tris – HCl, pH 9.50. Slides were incubated with primary antibody overnight. Biotinylated goat antirabbit IgG (Vector) and StreptABComplex/AP (Dako) were incubated sequentially at room temperature. Antibodies were localized using the Chromagen Vector Red (Vector), followed by counterstaining with hematoxylin. Human tonsil tissue was used as the positive test control tissue. Species and isotype-matched IgG (Dako) and primary antibody omission slides were used as negative controls on both control and test tissues.

3.1. Controls In the human tonsil tissues, specific fractalkine staining was present in ECs of the vessels. CX3CR1 staining was specific for mononuclear cells, with faint staining of capillaries. In all the coronary arteries, no staining was seen in either the isotype-matched IgG or primary antibody omission control slides. 3.2. Normal (non-atherosclerotic) None of the 16 normal case materials showed staining for fractalkine in the coronaries (Fig. 1A). It is noted that coronary arteries showed diffuse cytoplasmic staining for CX3CR1 in SMCs (Fig. 2A). Of note, this constitutive expression in coronary SMCs was confirmed using immunocytochemistry and Western blotting in cultured human coronary artery SMCs (Clonetics, Guelph, ON; data not shown). There was little to no immune cell infiltration identified in our PDAY tissues. 3.3. Native atherosclerosis

2.4. Analysis Slides were scored in a blinded fashion for intensity of immunostaining (0 to 4+) in the intima, media, and adventitia of the coronary artery. Positivity for fractalkine and/or CX3CR1 was then related to lesion features.

Of the 17 cases with native atherosclerosis, 6 showed staining for fractalkine. All cases that showed fractalkine staining exhibited greater than 50% cross-sectional area occlusion of the coronary artery and were all over the age of 75 years (76 – 87 range). There was localization of

Fig. 1. Immunostaining for fractalkine in human coronary arteries. No fractalkine staining was seen in any of the normal cases (A). In native atherosclerosis (B), fractalkine was seen in the intima, media, and adventitia. In diabetes mellitus (C), fractalkine was localized to the deep intima and media, with some expression in the microvessels of the adventitia. In TVD (D), fractalkine was localized to the endothelium, intima, and adventitia at an early time-point (102 days post-transplantation). In contrast, at a later time-point (360 days post-transplantation), fractalkine was localized primarily to the deep intima, media, and adventitia in TVD (E). Insets: Movat’s pentachrome staining illustrates the presence of the internal and external elastic laminae in the coronary arteries. In normal cases (A), the intima of the coronary artery is thin. In comparison, in native atherosclerosis (B), diabetes mellitus (C), and TVD (D), the intima is noticeably thicker. At later time-points, the intima of transplanted arteries were noticeably thicker (E), with a considerable concentric lipid core. Scale bars represent 50 mm.

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Fig. 2. Immunostaining for CX3CR1 on human coronary arteries. CX3CR1 staining was performed on serial sections with those shown in Fig. 1. Diffuse staining for CX3CR1 is seen in all the normal cases (A). In native atherosclerosis (B), specific staining for CX3CR1 is localized in the media and adventitia. In diabetes mellitus (C), CX3CR1 staining is localized in the deep intima and adventitia. TVD cases at an early time-point (D) showed staining localized to the endothelium, intima, and adventitia. At a later time-point (E), fractalkine localized throughout the vessel, including the cells of endothelium, intima, media, and adventitia, as well as the lipid core. Scale bars represent 50 mm.

fractalkine staining to cells in the intima, media, and adventitia, but minimal staining of ECs in the coronary arteries (Fig. 1B). All of the cases with native atherosclerosis showed staining for CX3CR1. In the cases that showed fractalkine staining, CX3CR1 staining was localized to fractalkine positive areas and appeared to stain with increased intensity (Fig. 2B). T-cells and mononuclear cells

localized to the atherosclerotic plaque, shoulder region, and adventitia of vessels (Fig. 4). 3.4. Diabetes mellitus Of the 15 cases diagnosed with diabetes mellitus and atherosclerosis, all showed staining for fractalkine (Fig. 1C).

Fig. 3. Cellular localization of fractalkine and CX3CR1 using IHC. Fractalkine (A) and CX3CR1 (B) are seen up-regulated in the endothelium (arrowheads) and immediately subendothelial cells (arrows) of transplanted coronary arteries as identified by Factor VIII-related antigen (C). Also, fractalkine (D) and CX3CR1 (E) immunoreactivity localizes to SMCs (F), macrophages, and foam cells (arrows) in the superficial and deep intima and in the media. Scale bars represent 20 mm.

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Fig. 4. Localization of leukocyte infiltration with fractalkine and CX3CR1 immunopositivity. Within the adventitia of coronaries with native atherosclerosis, leukocytic infiltration identified as CD3-positive T-cells (A) and CD68-positive mononuclear cells (B) is seen. These infiltrates localized to areas of fractalkine (C) and CX3CR1 (D) staining. Scale bars represent 25 mm.

Fractalkine (Fig. 3D) and CX3CR1 (Fig. 3E) immunoreactivity co-localized to SMCs (Fig. 3F), mononuclear cells, and foam cells in the deep intima and media. Similar staining patterns were also observed in the vaso vasora. Staining was seen for CX3CR1 in all the diabetic cases with expression in the same cells as those expressing fractalkine (Fig. 2C). T-cells and mononuclear cells localized to the atherosclerotic plaque, shoulder, and adventitia of coronaries, similar to native atherosclerosis. 3.5. Transplant vascular disease Within the early TVD group, 2 of 12 cases showed staining for fractalkine. Staining was localized to the endothelium, intima, and adventitia (Fig. 1D). Fractalkine (Fig. 3A) and CX3CR1 (Fig. 3B) are particularly prominent on the endothelium and immediately subendothelial cells of the intima of transplanted coronary arteries, as identified by staining for Factor VIII-related antigen (Fig. 3C). CX3CR1 staining was seen in areas of fractalkine localization (Fig. 2D). There was T-cell and mononuclear cell localization to the endothelium and adventitia early in TVD. Within the late TVD group, eight of nine cases stained positive for fractalkine. Staining was localized to foam cells and SMCs of the deep intima underlying the lipid-rich core of the vessel, as well as in the cells of the media and vaso vasorum in the adventitia (Fig. 1E). Again, CX3CR1 was seen in areas of fractalkine localization (Fig. 2E). There was

T-cell and mononuclear cell localization to the intimal medial junction and adventitia late in TVD.

4. Discussion Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiological flow that is not inhibited by pertussis toxin, EDTA/EGTA, or anti-integrin antibodies, indicating an integrin independent firm adhesion [19,20]. This is significant in that up-regulation of fractalkine will not only promote chemotaxis, but also mediate firm adhesion. It has been shown by Foussat et al. [21] that CX3CR1 is expressed on human T-lymphocyte subpopulations, specifically CD8+ T-lymphocytes in both CD45RO and CD45RO+ cells, and also CD4+ T-lymphocytes mainly in CD45RO+ cells. These findings suggest that fractalkine may contribute to the recruitment of effector T helper lymphocytes in peripheral tissues. Here, we show for the first time in normal coronary sections that there is expression of CX3CR1 throughout the vessel (Fig. 2A). In conjunction, we are able to identify constitutive expression of CX3CR1 in cultured human coronary artery SMCs (Clonetics) using the same antibody (Torrey Pines) via both immunocytochemistry and Western blotting. These novel findings may suggest a possible role for fractalkine signaling within SMCs.

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The role of fractalkine and its receptor CX3CR1 in cardiac allograft rejection was studied in mouse models by Robinson et al. [22]. Their findings show that the fractalkine – CX3CR1 pathway has a nonredundant role in allograft rejection: there is enhanced fractalkine expression in rejecting allografts (around vessels and in myocytes). As well, they showed that enhanced expression on mouse ECs activated by TNF-a promotes increased leukocyte adhesion via fractalkine and inhibition of the fractalkine– CX3CR1 signaling pathway with an anti-CX3CR1 antibody significantly prolonged the survival of mouse cardiac allografts in a vigorous model of acute rejection in the absence of immunosuppression [22]. More recently, Haskell et al. [23] have shown using a heterotopic mouse cardiac allograft model that CX3CR1 / mice had significantly increased survival times in the presence of cyclosporine (CsA). In addition, a reduction of macrophages, NK cells, and other leukocyte infiltration was seen in grafts receiving CsA. In the present study, we demonstrate fractalkine staining in several examples of coronary artery inflammatory disease. In normal, non-atherosclerotic coronaries, fractalkine staining was absent in all cases. In native atherosclerosis, staining was localized in the intima, media, and adventitia of the vessel, with the trend toward greater expression in individuals greater than 75 years of age and diagnosed with greater than 50% luminal narrowing. Adventitial expression may have particular relevance in later stages of disease when the atherosclerotic plaque may block the infiltration of immune cells through the vessel wall to the intima/ media, and a secondary route of passage through the adventitia is required. This hypothesis is supported by the localization of numerous infiltrating T-cell and mononuclear cells seen within the adventitia in areas of fractalkine and CX3CR1 staining (Fig. 4). These results are in correlation with those published by Greaves et al. [24], who showed fractalkine and other linked chromosome 16q13 chemokines are expressed in human carotid artery atherosclerotic lesions, implicating them in mononuclear cell recruitment. Diabetes mellitus is a risk factor that accelerates and intensifies the pathogenesis of atherosclerosis [25,26]. In comparison to native atherosclerosis cases, the diabetic cases showed a greater number of positive cases, with more intense staining that localized to the deep intima, media, and adventitia. Fractalkine expression in diabetes appeared to be independent of degree of occlusion and age as cases diagnosed with less than 25% occlusion and patients as young as 39 years of age exhibited immunopositivity. CX3CR1 expression in diabetes was diffuse, with intense, specific localization to areas of fractalkine staining. At early time-points in TVD, staining was localized primarily to the coronary endothelium and the vaso vasora of the adventitia. This may reflect the early immune response targeting leukocytes to ECs early in the pathogenesis of atherosclerotic disease. This is in line with recent reports of intimal inflammation and immune cell infiltration

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seen within juvenile atherosclerosis from the PDAY study [27], although our PDAY cases were not positive for fractalkine. Similar to a report by Robinson et al. [22] in a murine model of cardiac allograft rejection, expression of fractalkine in human TVD was seen in endothelium and vascular tissues in acute rejection progressing to expression in vessels and cardiac myocytes in chronic rejection. At later time-points in TVD, staining for fractalkine and CX3CR1 differentially localizes to the deep intima, media, and adventitia of transplanted coronary arteries. Recently, a polymorphism in the fractalkine receptor CX3CR1 has been demonstrated to associate independently with both prevalence and severity of coronary artery disease, relating to endothelial dysfunction [28]. Moatti et al. [29] showed that heterozygosity in the CX3CR1 halotype (I249) was associated with a markedly reduced risk of acute coronary events, independent of established coronary risk factors such as smoking and diabetes. In addition, Robinson et al. [22] showed that anti-fractalkine and anti-CX3CR1 antibodies significantly inhibited peripheral blood mononuclear cell binding, indicating that a large proportion of mononuclear cell binding to endothelium may occur via the fractalkine and CX3CR1 adhesion interaction. It was also shown that treatment with anti-CX3CR1 antibody with no additional immunosuppression in mouse models significantly prolonged graft survival [22]. These findings indicate an important role for the fractalkine– CX3CR1 pathway in promoting allograft rejection. In consonance with published studies, our observations on fractalkine and CX3CR1 in immune-mediated conditions like atherosclerosis and TVD suggest a significant role in pathogenesis. In both murine models and human cardiac allograft rejection, fractalkine is up-regulated, specifically in the endothelium and other vascular cells [22]. We have specifically shown in humans with native atherosclerosis and accelerated atherosclerosis (diabetes mellitus), that fractalkine expression is prominently expressed in the deep intima, media, and adventitia of vessel walls, localizing to SMCs, mononuclear cells, and foam cells. In transplanted human coronaries, fractalkine expression is up-regulated in the endothelium, intima, and adventitia early in TVD, and up-regulated in the deep intima, media, and adventitia at later time-points. The localization of fractalkine in these atherosclerotic diseases provides novel insight into how these diseases may be clinically and pathologically similar.

5. Summary Fractalkine is a novel chemokine that mediates both the chemotaxis and firm adhesion of T-lymphocytes, monocytes, and NK cells. Distinctive staining patterns indicate that the specific expression of fractalkine on ECs and other vascular cells may mediate vascular damage and contribute to the pathogenesis of native atherosclerosis, diabetes mellitus, and TVD.

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Acknowledgments We would like to thank the International Society for Heart and Lung Transplantation, the Heart and Stroke Foundation of BC and Yukon, and the Canadian Institutes of Health Research for funding this research. We also thank Ms. Amrit Mahil, Mr. Albert Lee, Ms. Linda Hughes, Ms. Julie Chow, Ms. Agripina Suarez, Mr. Stuart Greene, Ms. Ellie Wong, and Ms. Janet WilsonMcManus for their excellent technical assistance. The purified polyclonal rabbit antihuman fractalkine used in this study was generously donated by Dr. Thomas Schall (Chemocentryx, San Carlos, CA). Normal arterial segments were kindly provided by the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study.

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