CLINICAL PERSPECTIVE

This Article Abstract Full Text (PDF) All Versions of this Article: 114/15/1599    most recent CIRCULATIONAHA.105.597526v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Loosdregt, J. Articles by de Weger, R. A. Search for Related Content PubMed PubMed Citation Articles by van Loosdregt, J. Articles by de Weger, R. A. Pubmed/NCBI databases Medline Plus Health Information Coronary Artery Disease Heart Transplantation Related Collections Other heart failure Coronary circulation Mechanism of atherosclerosis/growth factors Other Vascular biology Growth factors/cytokines Smooth muscle proliferation and differentiation

(Circulation. 2006;114:1599-1607.)
© 2006 American Heart Association, Inc.

Transplantation

The Chemokine and Chemokine Receptor Profile of Infiltrating Cells in the Wall of Arteries With Cardiac Allograft Vasculopathy Is Indicative of a Memory T–Helper 1 Response

Jorg van Loosdregt, MSc; Matthijs F.M. van Oosterhout, MD, PhD; Annette H. Bruggink, MSc; Dick F. van Wichen, BSc; Joyce van Kuik, BSc; Erica de Koning, BSc; Carla C. Baan, PhD; Nicolaas de Jonge, MD, PhD; Frits H.J. Gmelig-Meyling, PhD; Roel A. de Weger, PhD

From the Department of Pathology (J.v.L., M.F.M.v.O., A.H.B., D.F.v.W., J.v.K., E.d.K., R.A.d.W.), Heart Lung Center Utrecht (N.d.J.), and Department of Immunology (F.H.J.G.-M.), University Medical Center Utrecht, Utrecht; and Department of Internal Medicine, Erasmus Medical Center, Rotterdam (C.C.B.), the Netherlands.

Correspondence to Roel de Weger, PhD, Molecular Pathology and Immunopathology, Department of Pathology (H04.312), University Medical Center Utrecht, Heidelberglaan 100, PO Box 85500, 3508 GA Utrecht, The Netherlands. E-mail r.deweger{at}umcutrecht.nl

Received October 25, 2005; revision received June 7, 2006; accepted June 21, 2006.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Despite improvement in short-term patient survival after heart transplantation (HTx), long-term survival rates have not improved much, mainly because of cardiac allograft vasculopathy (CAV). Cytokines and chemokines are considered to play an important role in CAV development.

Methods and Results— We focused on coronary arteries of HTx patients and made an inventory of the infiltrating cells and the expression of cytokines as well as chemokines and chemokine receptors (C+CR) in the different layers of the vessel wall with CAV. Tissue slides were stained for a variety of cell markers (CD3, CD4, CD8, CD20, CD68, CD79a), chemokines (monokine induced by interferon [MIG], interferon-inducible protein 10 [IP-10], interferon-inducible T cell- chemoattractant [ITAC], RANTES [regulated on activation normal T cell expressed and secreted], and fractalkine), and chemokine receptors (CXCR3, CCR5, and CX3CR1). In reference coronary arteries (not transplanted), almost no infiltrating cells were found, and in transplanted hearts with CAV (HTx+CAV), a large number of T cells were observed (CD4:CD8=2:1), mainly localized in the neointima and adventitia. Most of these T cells appeared to be activated (human leukocyte antigen DR positive). Coronary arteries from transplanted hearts without CAV (HTx–CAV), HTx+CAV, and references were also analyzed for cytokine and C+CR mRNA expression with the use of quantitative polymerase chain reaction. Interferon- was highly expressed in HTx+CAV compared with HTx–CAV. Interleukin-4 and interleukin-10 were expressed at the same level in both HTx groups and references. In HTx+CAV, all C+CR, but especially the T–helper 1 (TH1) C+CR, were more abundant than in the HTx–CAV and references. However, TH2 CCR4 expression did not differ significantly between both HTx groups.

Conclusions— In coronary arteries with CAV, most T cells are CD4⁺ and express human leukocyte antigen DR. These activated TH cells are mainly memory TH1 cells on the basis of their C+CR profile and cytokine expression.

Key Words: arteriosclerosis • cardiovascular diseases • chemokines • heart failure • immunology • pathology • transplantation

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Heart transplantation (HTx) is a last resort for patients with end-stage heart failure. Short-term patient survival of acute rejection has improved much over the last years owing to better immunosuppressive management, but long-term survival rates have not changed dramatically. This lack of progress is predominantly attributable to cardiac allograft vasculopathy (CAV). CAV is a concentric thickening of the blood vessel wall due to proliferation of smooth muscle cells (SMCs) in the intima layer (neointima) of the coronary arteries.^1,2 Consequently, the lumen of the arteries is narrowed, leading to untreatable cardiac ischemia.^3,4 On average, the incidence of CAV shows an increase of 10% every year.^5,6

Editorial p 1561

Clinical Perspective p 1607

The pathogenesis of CAV is related to hyperlipidemia, cytomegalovirus infection, hypertension, ischemia, and reperfusion injury, and the intensity of acute rejection and the development of CAV are related.^3,4,7 Still, the exact origin of CAV is not understood fully. Several studies indicate that interferon- (IFN-) plays a central role in its development.^8,9 It may activate inflammatory cells but also fibroblasts and SMCs to produce a variety of chemokines, eg, monokine induced by IFN (MIG), IFN-inducible T cell- chemoattractant (ITAC), and IFN-inducible protein 10 (IP-10).^10,11 These chemokines and others such as RANTES (regulated on activation normal T cell expressed and secreted) and fractalkine are directly linked with CAV development in animal studies.^12–14 Studies for the role of chemokines in human CAV development are limited to 2 immunohistochemical investigations^15,16 and studies on human coronary arteries transplanted in severe combined immunodeficiency mice reconstituted with human peripheral blood mononuclear cells (PBMCs).^17,18 CAV develops in rodents as early as 14 days after transplantation,¹² but its course in humans is relatively slow. Extrapolation of animal studies to the human situation seems treacherous and requires confirmation in human arteries. To obtain a better insight into the relation between infiltrating cells and CAV development, an inventory was made of infiltrating cells, cytokines, and chemokines and chemokine receptors (C+CR) in coronary arteries obtained from HTx patients at postmortem examination with the use of immunohistochemistry and quantitative polymerase chain reaction (PCR).

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Patients
Patient and coronary artery characteristics are summarized in Table 1. All patients were treated with a standard triple immunosuppressive therapy of cyclosporin A, azathioprine, and steroids. All coronary arteries were obtained at autopsy. Informed consent of all patients was obtained before transplantation. The HTx patients were subdivided into a group that developed severe CAV (n=8) and a group that did not (n=7). The severity of CAV was determined by the degree of intima thickening, amount of infiltrate, and degree of lumen occlusion. If the ratio [intima/(intima+lumen)] exceeded 50%, CAV was diagnosed (measurements were performed at 3 different positions in the vessel wall).

View this table: [in this window] [in a new window]   TABLE 1. Patient Characteristics

Histology
For routine pathology, all coronary arteries were analyzed with the use of hematoxylin-eosin (HE) or elastica van Gieson staining for detection of the lamina elastica. The total number of infiltrating leukocytes per area was determined with the use of a grid that corresponds to 10 000 µm². The mean amount of cells was determined at 5 different places in the same area.

Immunohistochemistry
Immunohistochemistry was performed as previously described by de Jonge et al.¹⁹ Briefly, 4-µm slides were cut from formalin-fixed, paraffin-embedded coronary arteries. The tissue slides were deparaffinized and rehydrated by subsequent washing in xylene, 100% EtOH, 70% EtOH, and distilled water, all for 5 minutes. Subsequently, endogenous peroxidase activity was blocked. Immunohistochemical procedures varied per antibody used; pretreatment, type of antibody, and procedure are summarized in Table 2.After incubation, all slides were stained with diaminobenzidine. Standard controls (isotype controls and controls that leave out the primary antibody) were tested on parallel tissue specimens. For control, all C+CR and marker antibodies were tested and titrated on lymphoid tissues¹⁹ (data not shown).

View this table: [in this window] [in a new window]   TABLE 2. Immunohistochemical Procedures

mRNA Isolation
mRNA was extracted from frozen sections of whole coronary arteries and isolated with the use of magnetic oligo(dT) beads (GenoPrep mRNA beads; GenoVision, Valencia, Calif), according to the manufacturer’s instructions. The mRNA was stored at –80°C.

cDNA Synthesis
One microliter of oligo(dT)(15) primers (0.50 µg; Promega Corporation, Phoenix, Ariz) and 1 µL of random primers (0.50 µg; Promega) were added to 18 µL mRNA derived from the mRNA isolation. This solution was heated in a closed Eppendorf tube for 5 minutes at 70°C, and the tube was cooled down to room temperature. Then 16 µL ribonuclease-free water, 12 µL 5X RT buffer (Invitrogen Corporation, Carlsbad, Calif), 6 µL 0.1 mol/L dithiothreitol, and 4 µL deoxy nucleotide triphosphate were added. After vigorous mixing, 1 µL RNasin (Promega) was added and mixed again, and finally 1 µL SuperScript RNase H-Reverse Transcriptase (Invitrogen) was added and heated in a closed Eppendorf tube for 1 hour at 42°C. The cDNA was stored at –20°C.

Real-Time Quantitative PCR
To study the expression levels of C+CR and cytokines, primer/probe combinations were custom-made by Applied Biosystems (Foster City, Calif). Per well, 12.5 µL Taqman Universal Master Mix was used, and 1.25 µL primer per probe, 6.25 µL Milli-Q, and 5 µL cDNA sample were added. The quantitative PCR reactions were performed by the Prism 7700 sequence detection system (Applied Biosystems). Thermal cycling constituted a denaturation step at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. All experiments were performed in duplicate; when the difference between duplicate experiments exceeded a mean threshold cycle (Ct) value of 0.8 (2xSD of all duplicates), the data were rejected and repeated.

To quantify the data, the comparative Ct method was used. Relative quantity was defined as 2^–Ct, in which Ct=Ct (target)–Ct (reference), Ct=Ct (sample)–Ct (calibrator). As reference, we used the gene PBGD, a housekeeping gene stably expressed in heart tissue.¹⁹ The calibrator was a batch placental mRNA used for normalization.

Laser Tissue Microdissection
Frozen tissue sections of 10 µm were cut on PEN-foil–mounted glass slides (PALM Microlaser Technologies, Bernried, Germany). The sections were air dried, stained with HistoGene Staining Solution (Arcturus, Mountain View, Calif), and rinsed with Milli Q water. From the coronary arteries, several layers were microdissected with the use of the Robot-Microbeam (Palm Microlaser Technologies), and mRNA was isolated.

Statistical Analysis
Quantitative PCR statistical analysis was performed with the use of the Mann-Whitney test (Prism GraphPad Software, San Diego, Calif). Correlation analysis was performed with Spearman correlation coefficients with the use of Prism. P<0.05 was considered statistically significant.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Cardiac Allograft Vasculopathy
Coronary arteries in normal individuals and in cardiac transplant recipients without CAV (HTx–CAV) contain an intima defined as a cellular layer (between endothelium and lamina elastica interna), mainly composed of SMCs (Figure 1a and 1b).

View larger version (81K): [in this window] [in a new window]   Figure 1. Cross section of the coronary artery wall. a, HE-stained coronary artery of a nontransplanted heart. b, -Smooth muscle actin staining of a nontransplanted heart. c, Coronary artery with CAV stained for -smooth muscle actin. d, Collagen type 1 staining on a coronary artery with CAV. e, Overview of a coronary artery with CAV (HE stained). Different areas are indicated by numbers: 1, endothelium; 2a, loose -smooth muscle actin–negative part of the neointima; 2b, -smooth muscle actin–positive part of the neointima; 3, tunica media; 4, adventitia; 5, accumulation of MNCs in the neointima; 6, accumulations of MNCs outside the vessel wall. The filled arrows indicate the lamina elastica interna, and the open arrows designate the lamina elastica externa.

For evaluation, the CAV vessels were divided into 6 areas (Figure 1e): endothelial layer (area 1), neointima (area 2), tunica media (area 3), adventitia (area 4), accumulations of mononuclear cells (MNCs) in the neointima (area 5), and accumulations of MNCs outside the vessel wall (area 6; Figures 1e and 2). Figures 1 and 2 clearly illustrate that the neointima in HTx+CAV is composed of 2 layers. One layer (area 2b) linked to the lamina elastica interna of the neointima is positive for anti–smooth muscle actin (Figure 1c). The other layer, next to the endothelium (area 2a), contains no SMCs but is composed of loose connective tissue, which is positive for anti–collagen type I (as is the total neointima; Figure 1d). Most infiltrating cells in the neointima were localized in area 2a, at the border of area 2a and 2b, or just beneath the endothelium. Many infiltrating cells were also observed in the adventitia. Signs of immigration of MNCs were observed at the site of the lumen of the coronary arteries in between the endothelial cells (Figure 2e and 2f) but also in the blood vessels in the adventitia (Figure 2e).

View larger version (155K): [in this window] [in a new window]   Figure 2. Coronary arteries with CAV. a, HE staining of 2 coronary arteries with CAV and surrounding accumulations of mononuclear cells (area 6). b, CD20 staining, coronary arteries almost devoid of CD20+ B cells but strong staining in area 6. c, CD3 staining, strong T-cell staining for CD3 in neointima and adventitia but also in area 6. d, HLA-DR staining, many HL-DR–positive MNCs in the infiltrate localized in the neointima. e, Lymphocyte migration from capillaries in the adventitia (arrow). f, Lymphocyte migration through the endothelium of the coronary arteries (arrow). L indicates lumen of the arteries.

In some coronary arteries atherosclerosis was observed, which was defined as focal lesions, more asymmetrical than in CAV, with or without a lipid core or calcification and with interruption of the lamina elastica interna. The infiltrates associated with atherosclerosis were predominantly located in the shoulder of the lesion. These areas with atherosclerosis are described separately.

Immunohistochemistry
Cell Markers and Cell Composition of Infiltrate
Because reference and coronary arteries without CAV contained few if any infiltrating cells, these were not analyzed by immunohistochemistry. For the evaluation of the infiltrating cells in HTx+CAV vessels, 8 paraffin-embedded coronary arteries were studied. A comparison was made between infiltrating cells in atherosclerotic plaques, infiltrating cells in the myocardium of the same allografted hearts, extravascular accumulations of MNCs (area 6), and infiltrating cells in the HTx+CAV coronary arteries (Table 3).

View this table: [in this window] [in a new window]   TABLE 3. Immunohistochemical Data of Coronary Arteries With CAV

All immunohistochemical data are summarized in Table 3 and partly illustrated in Figure 2. The majority of infiltrating cells were CD3⁺, and the CD4/CD8 ratio was 2. The percentage (indicated as percentage of total leukocytes) of B cells (CD20) was low (5% to 15%) in HTx+CAV vessels, except for the MNCs surrounding the coronary arteries (area 6); these contained about the same numbers of B and T cells. In the CAV areas 1 to 4, only low percentages of macrophages (CD68) were present (8% to 15%). In addition, in the MNC accumulations in and outside the CAV vessels, only small percentages of macrophages were observed. The number of macrophages in atherosclerotic plaques was much higher (up to 50% and more) than elsewhere. Human leukocyte antigen DR (HLA-DR) expression, almost absent in reference arteries, was high in most layers of the coronary arteries and heart tissue of the transplanted heart (Figure 2d). It was mainly expressed on infiltrating MNCs. The cell compositions in layers 1 (endothelium), 2 (neointima), and 4 (adventitia) were comparable (Table 3).

Chemokines and Chemokine Receptors on Infiltrating Cells
The C+CR expressions on MNCs (Table 3) in areas 1, 2, and 4 were comparable (indicated as percentage of total leukocytes) for RANTES (37% to 44%), MIG (29% to 37%), IP-10 (59% to 63%), ITAC (56% to 60%), fractalkine (59% to 66%), CCR5 (58% to 68%), CXCR3 (73% to 78%), and CX3CR1 (24% to 26%). The expression in area 3 (tunica media) was relatively low, as were the cell numbers. In the other areas of the CAV vessels, the composition of the C+CR differed: MNC accumulations in the CAV vessels (area 5) showed a relatively low expression of RANTES, ITAC, CXCR3, and CX3CR1, and the mononuclear cell accumulations outside the vessels (area 6) showed relatively low RANTES, MIG, and CX3CR1 expression. In both the myocardium of the transplanted heart and in atherosclerotic plaques, the occurrence of C+CR on infiltrating cells was low overall compared with the neointima (Table 3).

Coronary arteries with severe atherosclerosis contained a number of infiltrating cells, especially in the shoulders of fatty plaques. These infiltrates consisted of T cells (CD4⁺ as well as CD8⁺), plus a majority of macrophages. The number of infiltrating cells in atherosclerotic plaques was low compared with CAV coronary arteries. In atherosclerotic plaques in HTx+CAV, the infiltrates showed an expression pattern in between that of the intima of HTx+CAV and the nontransplanted atherosclerotic plaque, suggesting a mixture of both CAV and atherosclerosis. These infiltrates were therefore not evaluated further.

Real-Time Quantitative Polymerase Chain Reaction
Data on the expression of cytokines IFN-, interleukin-4 (IL-4), interleukin-10 (IL-10), and transforming growth factor-ß (TGF-ß) are shown in Figure 3a. IL-4 was weakly expressed, without significant differences between references, HTx–CAV, and HTx+CAV. In addition, the expression of IL-10 in these 3 groups did not differ, although it tended toward a higher expression in the HTx–CAV compared with the HTx+CAV group (P=0.19). By contrast, the IFN- expression in HTx+CAV was significantly higher (P=0.02) than the expression in reference vessels and HTx–CAV. Additionally, expression of IFN- was higher in HTx–CAV than in references. TGF-ß expression in coronary arteries after HTx was high compared with reference arteries, but coronary arteries after HTx with or without CAV did not differ significantly in this respect.

View larger version (37K): [in this window] [in a new window]   Figure 3. mRNA analysis by quantitative PCR. Mean and SE of mRNA analysis by quantitative PCR are depicted for the following: cytokines: IL-10, IL-4, IFN-, and TGF-ß (a); chemokines: MIG, IP-10, ITAC, RANTES, and fractalkine (b); and chemokine receptors: CXCR3, CCR5, CX3CR1, and CCR4 (c). *Significant (P<0.05) difference compared with the reference group. Significant difference between HTx–CAV and HTx+CAV.

The expression of the chemokines RANTES (P=0.01), ITAC (P=0.002), IP-10 (P=0.001), and MIG (P=0.001) in HTx–CAV was significantly higher than in references (Figure 3b). Fractalkine expression did not differ (P=0.73). In HTx+CAV, a significantly higher expression for RANTES (P=0.01), fractalkine (P=0.002), and ITAC (P=0.01) was observed compared with the HTx–CAV expression. The expression of MIG (P=0.41) and IP-10 (P=0.11) did not reach significance between the 2 HTx groups. All chemokines were more strongly expressed in HTx+CAV than in the reference vessels.

The expression of 4 chemokine receptors (CCR5, CCR4, CX3CR1, CXCR3) was only significantly different between the references and HTx–CAV for CXCR3 (P=0.002) and CCR4 (P=0.01; Figure 3c). The difference observed for CXCR3 (P=0.055) between HTx–CAV and HTx+CAV was nearly significant. A significantly higher expression for CCR5 (P=0.03) and CX3CR1 (P=0.001) was observed in the HTx+CAV group compared with the HTx–CAV group. Although in Figure 3c the expression of CCR4 seems to be lower in the HTx+CAV group than in the HTx–CAV group, the expression of CCR4 did not differ significantly between both HTx groups (P=0.42). The chemokine receptor expression was significantly higher for all receptors between the HTx+CAV and reference groups.

To exclude the possibility that the differences observed in C+CR expression were related to the time after HTx, the expressions of all C+CR in all HTx coronary arteries (both HTx–CAV and HTx+CAV) were plotted against time. In Figure 4 this is illustrated for CX3CR1 and CCR5; in none of the cases was a significant correlation observed. Likewise, no correlation was found between the C+CR expressions after HTx and (acute) rejection grade (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 4. mRNA expression correlated with time after HTx. Quantitative PCR analysis of mRNA expression correlated with time of the chemokine receptors CXCR3 (a) and CCR5 (b) is shown. No correlation of expression was found with time for CX3CR1 (P=0.08) and CCR5 (P=0.10).

Laser Tissue Microdissection
With the use of laser tissue microdissection, 4 distinct layers of the coronary arteries with CAV were isolated: the neointima (area 2a and area 2b separately), the tunica media (area 3), and the adventitia (area 4). mRNA from these layers was analyzed for IFN-, TGF-ß, ITAC, MIG, CXCR3, CX3CR1, and CCR4. Without exception, all these genes were expressed in the neointima layer (area 2a) and the adventitia, whereas the expression levels in the tunica media and the neointima (area 2b; containing predominantly SMCs) were very low or not detectable (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
CAV is a process that negatively affects survival after HTx. The concentric growth is generally believed to be caused by the influx and growth of SMCs originating in the tunica media of the coronary arteries. In this report we show that this neointima is composed of 2 layers: one adjacent to the lumen and composed of a loose connective tissue in which many infiltrating cells are present, and another next to the lamina elastica interna, which is composed of SMCs. A similar morphology was described by Atkinson et al.²¹ Many infiltrating cells are located at the border of these 2 layers in the neointima. Clearly, the neointima of all human coronary arteries obtained from nontransplanted hearts at autopsy already contains a considerable number of SMCs. This suggests that in the process of neointima formation, as observed in human CAV, the influx of SMCs is limited and the increase of intima volume is mainly owing to formation of connective tissue (filled with MNCs). This type of intima formation seems to differ from the more rapid neointima formation in experimental animals, which mainly contains SMCs.^4,22

Because T cells are important producers of cytokines and chemokines, we have focused on the role of T cells in the wall of coronary arteries. Most cells were present in the adventitia and in the neointima. The tunica media contained only a small number of MNCs. The cells seem to migrate from different sites. We speculate that the cells in the adventitia enter from the capillary network in the adventitia, whereas the cells in the neointima migrate directly into the arterial wall from the lumen of the coronary artery, through the endothelial layer. However, the composition of both populations did not differ in terms of lymphocyte subsets, chemokine production, and chemokine receptor expression (as determined by immunohistochemistry and mRNA expression after laser microdissection). This indicates that this population of cells in the blood encounters the same trigger for migration at the site of the adventitia and the neointima.

Most of the infiltrating MNC were T cells, at a CD4:CD8 ratio of 2. Because not all HLA-DR expression (45% to 55%) can be ascribed to macrophages (10% to 15%), at least part of the T cells must be HLA-DR positive (see also the morphology; Figure 2d). Consequently, many T cells are HLA-DR positive and apparently in an activated state. The numbers of macrophages and B cells are relatively small, particularly when the numbers were compared with those in the infiltrate in atherosclerotic plaques or in extranodular MNC accumulations surrounding the coronary arteries. The former consisted mainly of macrophages; the latter contained high numbers of B cells. Compared with the infiltrating cells in the myocardium, there is a clear difference in the percentages of CD8⁺ cells and C+CR expression. In brief, the composition of the T-cell population in the neointima is unique compared with adjacent infiltrates.

Chemokines and cytokines play an important role in both the process of acute heart allograft rejection^23,24 and neointima formation, as was shown in various experimental models.^11,25–27 Studies in human cardiac transplant recipients are scarce or fragmentary. In most human studies for CAV, heart biopsies or PBMCs were analyzed.^8,15,28 They have generally demonstrated an upregulation of ITAC, IP-10, and CXCR3 in CAV patients compared with controls, whereas MIG and RANTES did not change. In 2 studies, the presence of some chemokines in human CAV has been documented with the use of immunohistochemistry.^15,16 These studies showed a higher expression of RANTES and ITAC in coronary arteries with CAV compared with references, but IP-10 and MIG were completely absent.

In the present study, high numbers of infiltrating cells expressing cytokine receptors CCR5 and CXCR3 (50% to 75%) in the intima and adventitia were observed by immunohistochemistry. CX3CR1 was expressed less prominently but still on a relatively high percentage of leukocytes (25% compared with other infiltrates at 10% to 15%). In addition, the chemokines MIG (35% to 40%), IP-10 (55% to 60%), ITAC (55% to 60%), and fractalkine (55% to 65%) were expressed on a considerable number of MNCs. MIG and CCR5 are implicated in T-cell attraction and activation in CAV,^11,25–27 whereas CX3CR1 and IP-10 have been implicated in acute rejection in various animal studies.^24,28 This indicates that within the neointima and the adventitia, an active infiltrate of MNCs is present with a high chemoattractant potential. Within the T-cell population, CCR5 and CXCR3 are mainly expressed on T–helper 1 (TH1) cells.^28–32 CCR5 is also expressed on macrophages, but these are only present in low numbers. CCR5 and CXCR3 are also expressed on natural killer cells,³³ but these could not be detected in the CAV wall (data not shown). CX3CR1 can be expressed by natural killer cells, dendritic cells, and macrophages, but on T cells it is characteristic for memory cells.²⁹

To further evaluate the infiltrate in the arterial wall, quantitative PCR was performed. These data underscore the immunohistochemical data, a strong expression of the TH1 chemokines ITAC, MIG, and fractalkine, and a less prominent expression of RANTES and IP-10. Interestingly, the expression of all chemokines was stronger in the HTx+CAV vessels than in the HTx–CAV vessels. In addition, the TH1 chemokine receptors (CXCR3, CCR5, and CX3CR1) are highly expressed in the HTx+CAV group compared with the HTx–CAV group. Blockade of these receptors attenuates chronic rejection in experimental animals.^25,26 Despite the low number of leukocytes in HTx–CAV, there was a significant expression of IL-10, TGF-ß, MIG, and CCR4. This expression is most likely caused by macrophages, fibroblasts, or even SMCs.

Cytokines were not evaluated immunohistochemically because the stainings were not unequivocal. However, the quantitative PCR data showed that the TH1 cytokine IFN- was significantly elevated in the HTx+CAV group compared with the HTx–CAV group. IFN- has been indicated in various experimental and human studies as one of the most important mediators of CAV induction.^17,18 Burns et al³⁰ performed similar techniques on human coronary arteries transplanted in severe combined immunodeficiency mice reconstituted with human PBMCs. They also described a major involvement of TH1 cells producing IFN-, which were localized mainly in the neointima or adventitia. The TH2-linked cytokine IL-4 is only expressed at low levels in all groups, and the TH0/TH2-associated cytokine IL-10 is relatively abundantly expressed in the HTx–CAV group compared with the nontransplanted and HTx+CAV group. Expression of TGF-ß was significantly higher in all HTx coronary arteries compared with references, but there was no significant difference between the HTx–CAV and HTx+CAV groups.

In summary, coronary arteries with CAV contain an abundant T-cell infiltrate. Most T cells are CD4⁺ and express HLA-DR. These activated T lymphocytes appear to be TH1 cells on the basis of their C+CR profile and cytokine expression (IFN- versus IL-4, CCR4); most likely they are memory cells (CX3CR1⁺).^29,31–33 A TH1 response is often associated with a strong inflammatory (cytotoxic) response. Although low numbers of TH2 cells are present, this study suggests that the activated TH1 cells in the intima are able to induce a matrix proliferative response, perhaps by activating macrophages (TGF-ß production), which leads to an increase of the neointima.^32,33

	Acknowledgments

 
Disclosures

None.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Segovia J. Update on cardiac allograft vasculopathy. Curr Opin Organ Transplant. 2002; 7: 240–251.[CrossRef]
   
2. Weis M, von Scheidt W. Coronary artery disease in the transplanted heart. Annu Rev Med. 2000; 51: 81–100.[CrossRef][Medline] [Order article via Infotrieve]
   
3. Weis M, von Scheidt W. Cardiac allograft vasculopathy: a review. Circulation. 1997; 96: 2069–2077.[Medline] [Order article via Infotrieve]
   
4. Waller J, Brook NR, Nicholson ML. Cardiac allograft vasculopathy: current concepts and treatment. Transpl Int. 2003; 16: 367–375.[CrossRef][Medline] [Order article via Infotrieve]
   
5. Costanzo MR, Naftel DC, Pritzker MR, Heilman JK 3rd, Boehmer JP, Brozena SC, Dec GW, Ventura HO, Kirklin JK, Bourge RC, Miller LW. Heart transplant coronary artery disease detected by coronary angiography: a multiinstitutional study of preoperative donor and recipient risk factors: Cardiac Transplant Research Database. J Heart Lung Transplant. 1998; 17: 744–753.[Medline] [Order article via Infotrieve]
   
6. Julius BK, Attenhofer Jost CH, Sutsch G, Brunner HP, Kuenzli A, Vogt PR, Turina M, Hess OM, Kiowski W. Incidence, progression and functional significance of cardiac allograft vasculopathy after heart transplantation. Transplantation. 2000; 69: 847–853.[CrossRef][Medline] [Order article via Infotrieve]
   
7. Yamani MH, Yousufuddin M, Starling RC, Tuzco M, Ratliff NB, Cook DJ, Abdo A, Crowe T, Hobbs R, Rincon G, Bott-Silverman C, McCarthy PM, Young JB. Does acute cellular rejection correlate with cardiac allograft vasculopathy? J Heart Lung Transplant. 2004; 23: 272–276.[CrossRef][Medline] [Order article via Infotrieve]
   
8. Zhao DX, Miller GG, Luster AD, Mitchell RN, Libby P. Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines, IFN-inducible protein 10, monokine induced by IFN, and IFN-inducible T cell alpha chemoattractant in human cardiac allografts: association with cardiac allograft vasculopathy and acute rejection. J Immunol. 2002; 169: 1556–1560.[Abstract/Free Full Text]
   
9. Nagano H, Libby P, Taylor MK, Hasegawa S, Stinn JL, Becker G, Tilney NL, Mitchell RN. Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-gamma. Am J Pathol. 1998; 152: 1187–1197.[Abstract]
   
10. Shimizu K, Mitchell RN. Chemokine-mediated recruitment of inflammatory and smooth muscle cells in transplant-associated arteriosclerosis. Curr Opin Organ Transplant. 2003; 8: 55–63.
    
11. Yun JJ, Fischbein MP, Whiting D, Irie Y, Fishbein MC, Burdick MD, Belperio J, Strieter RM, Laks H, Berliner JA, Ardehali A. The role of MIG/CXCL9 in cardiac allograft vasculopathy. Am J Pathol. 2002; 161: 1307–1313.[Abstract/Free Full Text]
    
12. Yun JJ, Fischbein MP, Laks H, Fishbein MC, Espejo ML, Ebrahimi K, Irie Y, Berliner J, Ardehali A. Early and late chemokine production correlates with cellular recruitment in cardiac allograft vasculopathy. Transplantation. 2001; 69: 2515–2524.
    
13. Yun JJ, Fischbein MP, Laks H, Irie Y, Espejo ML, Fishbein MC, Berliner JA, Ardehali A. RANTES production during development of cardiac allograft vasculopathy. Transplantation. 2001; 71: 1649–1656.[CrossRef][Medline] [Order article via Infotrieve]
    
14. Umehara H, Bloom E, Okazaki T, Domae N, Imai T. Fractalkine and vascular injury. Trends Immunol. 2001; 22: 602–607.[CrossRef][Medline] [Order article via Infotrieve]
    
15. Kao J, Kobashigawa J, Fishbein MC, MacLellan WR, Burdick MD, Belperio JA, Strieter RM. Elevated serum levels of the CXCR3 chemokine ITAC are associated with the development of transplant coronary artery disease. Circulation. 2003; 107: 1958–1961.[CrossRef][Medline] [Order article via Infotrieve]
    
16. Pattison JM, Nelson PJ, Huie P, Sibley RK, Krensky AM. RANTES chemokine expression in transplant-associated accelerated atherosclerosis. J Heart Lung Transplant. 1996; 15: 1194–1199.[Medline] [Order article via Infotrieve]
    
17. Wang Y, Burns WR, Tang PC, Yi T, Schrechner JS, Zerwes HG, Sessa WC, Lorber MI, Pober JS, Tellides G. Interferon-gamma plays a nonredundant role in mediating T cell-dependent outward vascular remodeling of allogeneic human coronary arteries. FASEB J. 2004; 18: 606–608.[Abstract/Free Full Text]
    
18. Koh KP, Wang Y, Yi T, Shiao SL, Lorber MI, Sessa WC, Tellides G, Pober JS. T cell-mediated vascular dysfunction of human allografts results from IFN-gamma dysregulation of NO synthase. J Clin Invest. 2004; 114: 846–856.[CrossRef][Medline] [Order article via Infotrieve]
    
19. de Jonge N, van Wichen DF, van Kuik J, Kirkels H, Lahpor JR, Gmelig-Meyling FH, van der Tweel JG, de Weger RA. Cardiomyocyte death in patients with end-stage heart failure before and after support with a left ventricular assist device: low incidence of apoptosis despite ubiquitous mediators. J Heart Lung Transplant. 2003; 22: 1028–1036.[CrossRef][Medline] [Order article via Infotrieve]
    
20. van Hoffen E, van Wichen DF, Leemans JC, Broekhuizen RA, Bruggink AH, De Boer M, De Jonge N, Kirkels H, Slootweg PJ, Gmelig-Meyling FH, De Weger RA. T cell apoptosis in human heart allografts: association with lack of co-stimulation? Am J Pathol. 1998; 153: 1813–1824.[Abstract/Free Full Text]
    
21. Atkinson C, Horsley J, Rhind-Tutt S, Charman S, Phillpotts CJ, Wallwork J, Goddard MJ. Neointimal smooth muscle cells in human cardiac allograft coronary artery vasculopathy are of donor origin. J Heart Lung Transplant. 2004; 23: 427–435.[CrossRef][Medline] [Order article via Infotrieve]
    
22. Atkinson C, Southwood M, Pitman R, Phillpotts C, Wallwork J, Goddard M. Angiogenesis occurs within the intimal proliferation that characterizes transplant coronary artery vasculopathy. J Heart Lung Transplant. 2005; 24: 551–558.[CrossRef][Medline] [Order article via Infotrieve]
    
23. Hancock WW, Gao W, Csizmadia V, Faia KL, Shemmeri N, Luster AD. Donor-derived IP-10 initiates development of acute allograft rejection. J Exp Med. 2001; 193: 975–980.[Abstract/Free Full Text]
    
24. Whiting D, Hsieh G, Yun JJ, Banerji A, Yao W, Fishbein MC, Belperio J, Strieter RM, Bonavida B, Ardehali A. Chemokine monokine induced by IFN-gamma/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector cytokine production. J Immunol. 2004; 172: 7417–7424.[Abstract/Free Full Text]
    
25. Gao W, Faia KL, Csizmadia V, Smiley ST, Soler D, King JA, Danoff TN, Hancock WW. Beneficial effects of targeting CCR5 in allograft recipients. Transplantation. 2001; 72: 1199–1205.[CrossRef][Medline] [Order article via Infotrieve]
    
26. Yun JJ, Whiting D, Fischbein MP, Banerji A, Irie Y, Stein D, Fishbein MC, Proudfoot AE, Laks H, Berliner JA, Ardehali A. Combined blockade of the chemokine receptors CCR1 and CCR5 attenuates chronic rejection. Circulation. 2004; 109: 932–937.[CrossRef][Medline] [Order article via Infotrieve]
    
27. Horiguchi K, Kitagawa-Sakakida S, Sawa Y, Li ZZ, Fukashima N, Shirakura R, Matsuda H. Selective chemokine and receptor gene expressions in allografts that develop transplant vasculopathy. J Heart Lung Transplant. 2002; 21: 1090–1100.[CrossRef][Medline] [Order article via Infotrieve]
    
28. Melter M, Exeni A, Reinders MEJ, Fang JC, McMahon G, Ganz P, Hancock WW, Briscoe DM. Expression of the chemokine receptor CXCR3 and its ligand IP-10 during human cardiac allograft rejection. Circulation. 2001; 104: 2558–2564.[Abstract/Free Full Text]
    
29. Fraticelli P, Sironi M, Bianchi G, D’Ambrosio D, Albanesi C, Stoppacciaro A, Chieppa M, Allavena P, Ruco L, Girolomoni G, Sinigaglia F, Vecchi A, Mantovani A. Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses. J Clin Invest. 2001; 107: 1173–1181.[Medline] [Order article via Infotrieve]
    
30. Burns WR, Wang Y, Tang PCY, Ranjbaran H, Iakimov A, Kim J, Cuffy M, Bai Y, Pober JS, Tellides G. Recruitment of CXCR3+ and CCR5+ T cells and production of interferon--inducible chemokines in rejecting human arteries. Am J Transplant. 2005; 5: 1226–126.[CrossRef][Medline] [Order article via Infotrieve]
    
31. Loetscher M, Gerber B, Loetscher P, Jones SA, Piali L, Clark-Lewis I, Baggiolini M, Moser B. Chemokine receptor specific for IP10 and MIG: structure, function, and expression in activated T-lymphocytes. J Exp Med. 1996; 184: 963–996.[Abstract/Free Full Text]
    
32. Sallusto F, Lanzavecchia A, Mackay CR. Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol Today. 1998; 19: 568–574.[CrossRef][Medline] [Order article via Infotrieve]
    
33. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004; 25: 677–686.[CrossRef][Medline] [Order article via Infotrieve]
    

---

 

CLINICAL PERSPECTIVE

Cardiac allograft vasculopathy is an untreatable concentric enlargement of the intima of coronary arteries in the donor heart after heart transplantation. This intima enlargement (neointima formation) causes ischemia and finally failure of the transplanted heart. In contrast to the current opinion on cardiac allograft vasculopathy or chronic rejection, this study shows that this intima enlargement is not caused by smooth muscle cell infiltration and proliferation but is merely a volume increase caused by extracellular matrix formation (collagen I and fibroblasts). This luminal layer of the neointima is in most cases populated by a large number of T cells. These T cells have an activated helper 1 phenotype and are most likely either directly or indirectly via macrophages responsible for the induction of the intima proliferation. These data can lead to a more specific form of therapeutic approach of chronic rejection, which is an increasingly difficult problem in long-term survival of transplanted organs. The medication should therefore be directed against matrix formation rather than against smooth muscle cell infiltration and proliferation. Possible targets for this therapeutic approach may be the cytokine production of the T cells (eg, inhibition of growth factors like transforming growth factor-ß) or the infiltration of these T cells in the intima. This may explain the positive effect of medication with immunosuppressive drugs like everolimus and sirolimus.

This article has been cited by other articles:

				D. Schmauss and M. Weis Cardiac Allograft Vasculopathy: Recent Developments Circulation, April 22, 2008; 117(16): 2131 - 2141. [Abstract] [Full Text] [PDF]

				A. Lombardi, G. Cantini, E. Piscitelli, S. Gelmini, M. Francalanci, T. Mello, E. Ceni, G. Varano, G. Forti, M. Rotondi, et al. A New Mechanism Involving ERK Contributes to Rosiglitazone Inhibition of Tumor Necrosis Factor-{alpha} and Interferon-{gamma} Inflammatory Effects in Human Endothelial Cells Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 718 - 724. [Abstract] [Full Text] [PDF]

				S. Arora, P. A. Jenum, P. Aukrust, H. Rollag, A. K. Andreassen, S. Simonsen, E. Gude, A. E. Fiane, O. Geiran, and L. Gullestad Pre-Transplant Toxoplasma gondii Seropositivity Among Heart Transplant Recipients Is Associated With an Increased Risk of All-Cause and Cardiac Mortality J. Am. Coll. Cardiol., November 13, 2007; 50(20): 1967 - 1972. [Abstract] [Full Text] [PDF]

				M. L. Rose Interferon-{gamma} and Intimal Hyperplasia Circ. Res., September 14, 2007; 101(6): 542 - 544. [Full Text] [PDF]

				Y. Wang, Y. Bai, L. Qin, P. Zhang, T. Yi, S. A. Teesdale, L. Zhao, J. S. Pober, and G. Tellides Interferon-{gamma} Induces Human Vascular Smooth Muscle Cell Proliferation and Intimal Expansion by Phosphatidylinositol 3-Kinase Dependent Mammalian Target of Rapamycin Raptor Complex 1 Activation Circ. Res., September 14, 2007; 101(6): 560 - 569. [Abstract] [Full Text] [PDF]

				D. M. Brainard, A. M. Tager, J. Misdraji, N. Frahm, M. Lichterfeld, R. Draenert, C. Brander, B. D. Walker, and A. D. Luster Decreased CXCR3+ CD8 T Cells in Advanced Human Immunodeficiency Virus Infection Suggest that a Homing Defect Contributes to Cytotoxic T-Lymphocyte Dysfunction J. Virol., August 15, 2007; 81(16): 8439 - 8450. [Abstract] [Full Text] [PDF]

				G. Tellides and J. S. Pober Interferon-{gamma} Axis in Graft Arteriosclerosis Circ. Res., March 16, 2007; 100(5): 622 - 632. [Abstract] [Full Text] [PDF]

				G. Tellides Th1 adaptive immune responses in cardiac graft arteriosclerosis: deleterious or beneficial? Circulation, October 10, 2006; 114(15): 1561 - 1564. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 114/15/1599    most recent CIRCULATIONAHA.105.597526v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Loosdregt, J. Articles by de Weger, R. A. Search for Related Content PubMed PubMed Citation Articles by van Loosdregt, J. Articles by de Weger, R. A. Pubmed/NCBI databases Medline Plus Health Information Coronary Artery Disease Heart Transplantation