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Cardiovascular Research 2004 63(2):347-356; doi:10.1016/j.cardiores.2004.04.004
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Copyright © 2004, European Society of Cardiology

Wild-type but not interferon-{gamma}-deficient T cells induce graft arterial disease in the absence of B cells

Yutaka Furukawaa,1, Sarah E Coleb, Ravi V Shahb, Yoshihiro Fukumotoa,2, Peter Libbya and Richard N Mitchell*,b

aLeducq Center for Cardiovascular Research, Cardiovascular Division, Department of Medicine, USA
bImmunology Research Division, Department of Pathology, Brigham and Women's Hospital, 77 Avenue Louis Pasteur, NRB 730D, Boston, MA 02115, USA

* Corresponding author. Tel.: +1-617-525-4303; fax: +1-617-525-4329. Email address: rmitchell{at}rics.bwh.harvard.edu

Received 7 January 2004; revised 25 March 2004; accepted 6 April 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Interferon-{gamma} (IFN-{gamma}), a cytokine produced primarily by T cells and by activated macrophages, plays a central role in the pathogenesis of graft arterial disease (GAD). This study investigated whether T cells can induce GAD in the absence of humoral alloresponses and whether activated macrophages or other host cell types can substitute as sources of IFN-{gamma} in GAD. Methods: Wild-type (WT), IFN-{gamma}–/–, or recombination-activating-gene-1–/– (RAG-1–/–; lacking mature T and B cells) mice received MHC II-disparate hearts. The grafts were harvested 8 weeks post-transplant and histological and immunohistochemical analyses, RNase protection assay (RPA), and flow cytometry were used to evaluate GAD lesions, infiltrating cell populations, and IFN-{gamma} expression by infiltrating cells. Results: Moderate-to-severe GAD developed in WT recipient allografts, associated with abundant IFN-{gamma} expression by both infiltrating T cells and macrophages. No GAD developed in IFN-{gamma}–/– or in RAG-1–/– hosts, nor was any IFN-{gamma} expression evident. RAG-1–/– hosts receiving naïve WT or IFN-{gamma}–/– T cells (107) after heart transplantation demonstrated no mature B cells but showed persistence of transferred T cells up to 8 weeks post-transplant. In the complete absence of B cells and alloantibody, transfer of WT T cells into RAG-1–/– recipients yielded GAD, with associated IFN-{gamma} expression by the transferred T cells and the host macrophages. Transfer of IFN-{gamma}–/– T cells induced neither GAD nor host macrophage IFN-{gamma} expression. Conclusions: T cells, even in the absence of B cells, suffice to induce GAD, and T cell-derived IFN-{gamma} plays a critical role in GAD pathogenesis.

KEYWORDS Transplantation; Leukocytes; Cytokines; Atherosclerosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Graft arterial disease (GAD) remains a major limiting factor for long-term survival of vascularized allografts [1]. Composed of smooth muscle cells and extracellular matrix admixed with host mononuclear inflammatory cells, GAD lesions occur diffusely throughout the graft arterial circulation [2,3]. The resulting concentric intimal hyperplasia causes progressive ischemia and eventually graft failure. Infiltrating host inflammatory cells produce numerous soluble and membrane-anchored immune mediators implicated in GAD pathogenesis (e.g., cytokines, growth factors, and costimulatory molecules) [2]. We previously demonstrated that complete T cell depletion prevents GAD [4], and that interferon-{gamma} (IFN-{gamma}), a predominantly (albeit not exclusively) T cell cytokine, is necessary to develop the arteriopathy [5]; congenital absence of host IFN-{gamma} or antibody neutralization of IFN-{gamma} markedly attenuates GAD in murine cardiac allografts [4–6]. However, those earlier studies did not address whether other sources of IFN-{gamma} can induce GAD or whether other cell types (e.g., B cells and/or macrophages) are secondarily required.

Activated host macrophages accumulate in long-term (8–12 weeks) murine cardiac allografts, typically forming a substantial perivascular infiltrate around GAD lesions [7,8]. Although CD4+ T helper cell type 1 (Th1) cells and CD8+ T cells likely provide much of the IFN-{gamma} in rejecting allografts [9], recent work demonstrates that activated macrophages also produce IFN-{gamma} in vitro [10,11]. Moreover, activated macrophages elaborate substantial IFN-{gamma} in vivo, thereby playing a critical role in host defense, e.g., against mycobacterial infections [12]. Although the perivascular macrophage infiltrate in GAD suggests a potential pathogenic role, it is uncertain to what extent macrophage IFN-{gamma} production contributes to GAD development. Because IFN-{gamma} potently activates macrophages [13,14], it is also germane to ask whether macrophages can produce IFN-{gamma} in the absence of T cell-derived IFN-{gamma}. Finally, since vascular smooth muscle cells (VSMC) (both in vitro and in GAD lesions) are capable of IFN-{gamma} production [15,16], it is apparent that multiple cell types in cardiac allografts can potentially contribute to the IFN-{gamma} necessary to induce GAD.

Although previous studies demonstrated a requirement for T cells in the development of GAD, it also remains controversial whether B cell alloantibodies and humoral immunity participate obligatorily in GAD. The few studies to date investigating the importance of humoral alloimmunity in GAD pathogenesis have been contradictory [17–20].

This study looked at whether B cell-induced humoral responses are required for GAD development, and also evaluated the relative importance of T cells versus macrophages and smooth muscle cells as sources of IFN-{gamma} in GAD pathogenesis. To accomplish this, Major Histocompatibility Complex class II (MHC II)-disparate cardiac allografts were transplanted into recombination-activating-gene-1-deficient mice (RAG-1–/–, lacking mature T and B cells [21]). Allograft recipients then received either wild-type (WT) IFN-{gamma}-sufficient T cells or T cells from IFN-{gamma}-deficient (IFN-{gamma}–/–) donors, producing alloresponses in the absence of B cells and in the presence of IFN-{gamma}-replete host macrophages and vascular smooth muscle cells (VSMC). In the group receiving T cells from IFN-{gamma}–/– donors, IFN-{gamma} could only derive from host and donor non-T cells, including macrophages and VSMC. The results demonstrate that in RAG-1–/– hosts lacking mature T and B cells, adoptively transferred WT T cells suffice to induce GAD, and that GAD development can occur in the complete absence of B cells and humoral alloimmunity. Moreover, the results show an essential role for T cell-derived IFN-{gamma} in GAD pathogenesis.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animals
Inbred male C57BL/6 (B/6, H-2b) mice were obtained from Taconic Farms, (Germantown, NY). B6.C-H-2bm12KhEg (bm12, H-2 bm12) mice, MHC class II-disparate from B/6, IFN-{gamma}–/– mice [22] and RAG-1–/– mice on the B/6 genetic background were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in the Harvard Medical School animal facilities and used at 9 to 12 weeks of age. All experimental protocols conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (Publication no. 85-23).

2.2. Antibodies and other reagents
Purified and fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, PerCP- or biotin-conjugated antibodies against mouse B220 (RA3-6B2), CD3e (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD11b (M1/70), CD16/CD32 (Fc-block; clone 2.4G2), I-Ab (25-9-17), IFN-{gamma} (XMG1.2) and NK-1.1 (PK136), and species- and isotype-matched IgG controls, allophycocyanin (APC)-conjugated streptavidin, and the Riboquant RNase protection assay (RPA) kits were obtained from PharMingen (BD Biosciences, San Diego, CA). Anti–I-Ab antibody (25-9-17) recognizes the B/6 MHC class II molecule but not the bm12 MHC class II protein. FITC-conjugated goat anti-mouse IgM and control goat IgG were from Southern Biotechnology Associates (Birmingham, AL). Mouse-adsorbed biotin-conjugated anti-rat IgG (H+L) antibody and an alkaline phosphatase-conjugated avidin–biotin complex kit (Vectastain ABC-AP kit) were from Vector Laboratories, (Burlingame, CA). FAST RED tablets (alkaline phosphatase substrate), hematoxylin, and collagenase I were from Sigma Chemical (St. Louis, MO). TRIZOL RNA extracting solution was from Gibco BRL/Life Technologies (Invitrogen, Carlsbad, CA). Ficoll lymphocyte separation medium was from Organon Teknika (bioMérieux, Durham, NC).

2.3. Heart transplantation
Heterotopic heart transplantation was performed as previously described [23]; donor and recipient mice were anesthetized by Metofane inhalation (Pittman-Moore, Terre Haute, IN). WT (n=14), IFN-{gamma}–/– (n=4), or RAG-1–/– (n=29) mice of B/6 background served as recipients of bm12 allografts. Grafts were harvested 8 weeks post-transplant and analyzed by histology, immunohistochemistry, flow cytometry of graft infiltrating cells, and RPA.

2.4 Reconstitution of WT or IFN-{gamma}–/– T cells in RAG-1–/– recipients
Thy1.2+ T cells were purified from WT or IFN-{gamma}–/– B/6 splenocytes using MACS magnetic beads system (Miltenyi Biotec, Auburn, CA). By flow cytometry, approximately 95% of the recovered cells were CD3+ T cells. Ten million (107) WT (n=11) or IFN-{gamma}–/– (n=9) T cells were resuspended in cold phosphate-buffered saline (PBS) and transferred via tail vein to RAG-1–/– allograft recipients 24 h post-transplant. For controls, nine RAG-1–/– allograft recipients received PBS only.

2.5. Flow cytometry
Populations of B cells, and CD4+ or CD8+ T cells were analyzed by flow cytometry. Double staining of splenocytes from WT B/6, IFN-{gamma}–/– B/6, or RAG-1–/– B/6 recipients was performed using FITC-conjugated anti-CD4 and PE-conjugated anti-CD8, or FITC-conjugated anti-IgM and PE-conjugated anti-B220 antibodies.

2.6. Histologic evaluation
Explanted grafts were cut transversely into three portions. The basal section was fixed in 10% buffered formalin and histologic sections were stained with hematoxylin and eosin, or for elastin. The mid-transverse section was frozen in Tissue-Tek O.C.T. compound (Sakura Finetek USA, Torrance, CA) for immunohistochemistry (see below), and the apical transverse section was used for flow cytometry or RPA. The severity of parenchymal rejection and GAD was scored blindly by two independent observers (Y.F. and R.N.M.). Scores uniformly fell within a range of one grade for both observers, and were averaged. Parenchymal rejection was graded using a scale modified from the International Society for Heart and Lung Transplantation (0, no rejection; 1, mild interstitial or perivascular mononuclear inflammatory cell infiltrates without myocyte necrosis; 2, focal interstitial or perivascular infiltrates with necrosis; 3, multifocal infiltrates with necrosis; and 4, widespread infiltrate with hemorrhage and/or vasculitis) [5]. GAD scores were calculated as described previously, averaging the individual scores of ≥10 arteries in each specimen (individual vessels are scored: 0, no or minimal [<10%] vascular occlusion; 1, 10–25% occlusion; 2, 25–50% occlusion; 3, 50–75% occlusion; 4, 75–100% occlusion) [5].

2.7. RNase protection assay (RPA)
Total RNA was prepared using TRIZOL according to the manufacturer's recommendations. Ten micrograms of total RNA from each sample was subjected to RPA using multi-probe murine cytokine RPA kits mCK-2b, mCK-1, and/or mCK-5c following the manufacturer's instructions. The volume integrations of mRNA-incorporated 32P-labeled probe fragments were analyzed by bioimaging analyzer (Molecular Dynamics, Amersham Biosciences, Piscataway, NJ). The volume integrations of cytokine bands were normalized against GAPDH.

2.8. Immunohistochemical staining
Immunohistochemical staining was performed using avidin–biotin conjugates (Vectastain ABC-AP kit), as previously described [8]. PBS containing 4% paraformaldehyde (PFA) was used as fixative, and PBS with 1% bovine serum albumin and 10% normal goat or rabbit serum or 10 µg/ml of Fc-block (for direct staining of I-Ab and NK-1.1 with biotin-conjugated antibodies) was applied to reduce background staining. Alkaline phosphatase activity was visualized by incubating with FAST RED substrate; sections were counterstained with hematoxylin.

2.9. Intracellular IFN-{gamma} staining and flow cytometry
Intracellular staining and flow cytometry were performed as described previously [9]. Briefly, grafts were minced and digested for 2 h at 37 °C in borate-buffered saline containing 2% bovine serum albumin and 2 mg/ml collagenase. The mixture was strained through a 70-µm nylon cell strainer (BD Biosciences Discovery Labware, Bedford, MA). Mononuclear cells were recovered by centrifugation through Ficoll and washed twice in RPMI 1640; the resuspended cells were stimulated with 25 µM ionomycin and 10 ng/ml phorbol myristate acetate (Sigma) for 4 h at 37 °C in the presence of 10 µg/ml brefeldin A (Sigma). Cells were then fixed at RT for 10 min with 4% PFA in PBS and permeabilized with a PBS-saponin buffer (0.5% saponin and 1% bovine serum albumin) and incubated with Fc-block. For intracellular IFN-{gamma} staining, biotin-conjugated anti-IFN-{gamma} mAb or isotype-matched control antibody was used at 10 µg/ml. After 30-min incubation, cells were washed twice with saponin buffer and incubated with APC-conjugated streptavidin for a further 30 min. Cells were washed twice again with saponin buffer and once with PBS, and surface staining was performed using anti-CD11b-FITC, anti-CD8-PE, and anti-CD4-PerCP. Flow cytometry was performed on a four-color FACScan flow cytometer (BD Biosciences, Immunocytometry Systems, San Jose, CA) using CellQuest software. The threshold was adjusted to 5% of the background staining for isotype-matched control antibody staining. The percentage of positively-stained cells for each cytokine was calculated by subtracting 5 from the percentage of cells in the positive range.

2.10. Statistical analysis
Values for GAD and parenchymal rejection scores and for relative gene expression of cytokines are expressed as mean±S.E.M.; comparison between groups used analysis of variance (ANOVA) followed by Fisher's protected least significant difference (PLSD) post hoc test. A p-value <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 GAD and parenchymal rejection in allografts in WT, IFN-{gamma}–/–, and RAG-1–/– recipients
All grafts analyzed in this study functioned well until harvest (8 weeks). Moderate to severe GAD developed in allografts in WT recipients by 8 weeks post-transplant (Figs. 1A and 2A)Go. GAD was almost completely absent in allografts in IFN-{gamma}–/– recipients (Figs. 1B and 2A)Go and in RAG-1–/– recipients receiving PBS injection (Figs. 1C and 2A)Go. Allografts in IFN-{gamma}–/– recipients showed mild to moderate parenchymal rejection, somewhat less than that seen in WT hosts (Fig. 2B); allografts in RAG-1–/– hosts receiving only PBS exhibited extremely sparse mononuclear cell infiltration (Fig. 2B).


Figure 1
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  		Fig. 1 Histologic sections from MHC II-mismatched allografts 8 weeks post-transplant (elastin stain; scale bar=50 µm). (A) WT host: allografts show severe intimal hyperplasia with perivascular mononuclear cell accumulation. (B) IFN-{gamma}–/– host: allografts show no intimal lesion and negligible perivascular mononuclear cell accumulation. (C) RAG-1–/– host receiving only PBS: allografts show no GAD or parenchymal rejection. (D) RAG-1–/– host following WT T cell transfer: allografts show restored parenchymal rejection and GAD. (E) RAG-1–/– host following IFN-{gamma}–/– T cell transfer: allografts show mild–moderate parenchymal rejection but no GAD. Overall, GAD occurred in allografts in hosts with WT T cells, while allografts in hosts lacking WT T cells (i.e., either IFN-{gamma}–/– hosts or RAG-1–/– hosts receiving PBS or IFN-{gamma}–/– T cells) demonstrated no or minimal GAD.

 

Figure 2
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  		Fig. 2 GAD and parenchymal rejection scores in MHC II-mismatched allografts 8 weeks post-transplant. Allografts were transplanted to WT, IFN-{gamma}–/– or RAG-1–/– recipients. PBS or 107 WT or IFN-{gamma}–/– T cells were transferred to each RAG-1–/– host 24 h post-transplant. (A) Bar graphs showing the GAD grading scores (mean±S.E.M.). (B) Bar graphs showing the parenchymal rejection grading scores (mean±S.E.M.).

 
3.2 Adoptive transfer of WT versus IFN-{gamma}–/– T cells into RAG-1–/– allograft recipients
Transfer of purified WT B/6 T cells to RAG-1–/– recipients resulted in allograft parenchymal rejection as well as vasculopathy, with GAD and rejection scores comparable to those seen in WT hosts (Figs. 1D and 2)Go. Conversely, GAD did not develop in allografts in RAG-1–/– recipients receiving purified IFN-{gamma}–/– T cells, despite the reconstitution of mild to moderate parenchymal rejection (Figs. 1E and 2)Go.

3.3 Persistence of transferred T cells and absence of B cells in RAG-1–/– recipients
As previously demonstrated [21], no B220+/IgM+, CD4+, or CD8+ cells were detectable in naïve RAG-1–/– spleens (data not shown). Flow cytometric analysis of spleens from RAG-1–/– allograft hosts 8 weeks post-transfer of either WT or IFN-{gamma}–/– T cells demonstrated persistence of comparable populations of CD4+ and CD8+ lymphocytes (Fig. 3). These spleens contained extremely rare mature (B220+/IgM+) B cells comparable to RAG-1–/– hosts receiving PBS alone, and indicated selective T cell reconstitution (Fig. 3). The presence of rare CD4+, CD8+, or B220+/IgM+ cells in spleens from RAG-1–/– allograft hosts receiving PBS only is attributable to donor passenger leukocytes in the cardiac allografts that cannot be eliminated by the RAG-1–/– recipient.


Figure 3
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  		Fig. 3 Flow cytometry of naïve wild-type (WT) C57BL/6 splenocytes and splenocytes from allograft recipients. Splenocytes from naïve mice or from WT allograft recipients contained mature (B220+/IgM+) B cells, as well as mature CD4+ and CD8+ T cells. Splenocytes from RAG-1–/– allograft hosts receiving PBS contained immature B cells (B220+/IgM–) and T cells (CD4–/CD8–); mature B and T cells were rarely detected. Splenocytes from RAG-1–/– recipients 8 weeks following transfer of purified WT T cells contained mature CD4+ and CD8+ T cells comparable to WT hosts, but no mature B cells.

 
3.4 Effects of WT versus IFN-{gamma}–/– T cell transfer on mononuclear cell infiltration and MCH II expression
In WT hosts, immunohistochemistry showed CD11b-positive monocyte/macrophages clustered in a perivascular distribution (Table 1), as seen previously [5,8]. Diffusely infiltrating CD4+ T cells substantially outnumbered CD8+ T cells, consistent with the graft-host MHC II disparity. Strong expression of recipient MHC II (I-Ab) co-localized with the macrophage infiltrates, demonstrating their host origin. Allografts in IFN-{gamma}–/– hosts showed reduced macrophage accumulation and markedly attenuated MHC II expression, as well as diminished CD4+ T cell infiltration. Rare CD4+ cells, presumably transferred with the allografts, were identified in transplanted hearts in RAG-1–/– hosts; infiltrating CD11b+ cells were infrequent. Extensive intragraft infiltration of CD4+, CD8+ and CD11b+ cells as well as host MHC II expression were completely restored in RAG-1–/– recipients of naïve WT T cells. Resembling allografts in IFN-{gamma}–/– hosts, allografts in RAG-1–/– hosts receiving IFN-{gamma}–/– T cells showed moderate CD4+ and CD8+ infiltration, with slightly increased CD11b+ cell accumulation and MHC II expression. Although WT allografts contained rare B cells, B cells were not identified in hearts transplanted into RAG-1–/– recipients, including hearts in recipients of transferred T cells.


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  		Table 1 Summary of immunohistochemistry

 
3.5. Expression of IFN-{gamma} and other cytokines in allografts
We determined relative mRNA expression of IFN-{gamma} and related cytokines in the various allografts (Fig. 4); IL-18 and IL-12 induce IFN-{gamma} expression, and macrophage activation by IFN-{gamma} can result in increased downstream expression of IL-1β, IL-12, IL-18, and IFN-{gamma} itself. RPA demonstrated strong expression of IFN-{gamma} gene transcripts in allografts in WT recipients at 8 weeks. IFN-{gamma} expression was absent in IFN-{gamma}–/– hosts and in control RAG-1–/– hosts receiving only PBS injection. Transfer of WT T cells fully restored IFN-{gamma} mRNA expression in allografts transplanted into RAG-1–/– hosts. However, transfer of IFN-{gamma}–/– T cells had a minimal effect on intragraft IFN-{gamma} mRNA expression in RAG-1–/– hosts (Fig. 4). Gene expression for two representative monocyte-derived cytokines, IL-12p40 and IL-1β, mirrored that seen for IFN-{gamma} mRNA. Indeed, in RAG-1–/– hosts receiving WT T cells (but not IFN-{gamma}–/– T cells), all measured cytokine mRNA levels in allografts increased in parallel without shift to a Th2-like pattern; IL-6, an inflammatory cytokine that can be expressed by a variety of cells including macrophages, T cells and cardiac myocytes, as well as a prototypical Th2 cytokine IL-10 also showed levels of mRNA expression that mirrored the patterns of IFN-{gamma} mRNA synthesis (Fig. 4). Chemokine mRNA expression in allografts (MCP-1, RANTES, IP-10, and MIP-1β) similarly increased in parallel when WT T cells (but not IFN-{gamma}–/– T cells) were adoptively transferred to RAG-1–/– hosts (data not shown).


Figure 4
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  		Fig. 4 Cytokine mRNA expression in allografts by RPA. (A) Representative bioimager gel patterns from cardiac allograft mRNA. (B) Bar graphs showing densitometric analysis from RPA gels for IFN-{gamma}, IL-12p40, IL-18, IL-1β, IL-6 and IL-10 gene expression in allografts 8 weeks post-transplant (n=4–6 for each group). IFN-{gamma} mRNA was expressed in all allografts in WT recipients; IFN-{gamma} was absent in IFN-{gamma}–/– hosts and in RAG-1–/– hosts receiving PBS only. IFN-{gamma} mRNA expression in allografts in RAG-1–/– hosts was restored by WT T cell transfer, while IFN-{gamma}–/– T cell transfer only minimally restored intra-graft IFN-{gamma} mRNA expression. Following WT T cell transfer, gene transcripts for IL-12p40, IL-18, IL-1β, IL-6 and IL-10 showed increased expression comparable to that seen for IFN-{gamma} mRNA. In IFN-{gamma}–/– hosts and in RAG-1–/– hosts receiving PBS injection, only minimal expressions of IL-18 and IL-1β were observed. Ten-micrograms RNA from each sample was analyzed; values are mean±S.E.M. of relative cytokine mRNA expression normalized against GAPDH.

 
To identify the cell types expressing IFN-{gamma} in allografts, we extracted graft-infiltrating cells and performed flow cytometry following intracellular staining for IFN-{gamma} (Fig. 5). As previously demonstrated [24], the majority of graft-infiltrating CD4+ and CD8+ cells in WT hosts (60–70%) produced IFN-{gamma}. In addition, a smaller percentage of allograft-infiltrating CD11b+ macrophages also produced IFN-{gamma} (~30%). In allografts from RAG-1–/– recipients of WT T cells, graft-infiltrating CD4+ and CD8+ cells as well as host-derived graft-infiltrating CD11b+ cells showed similar IFN-{gamma}-producing capability. However, neither IFN-{gamma}–/– CD4+ and CD8+, nor IFN-{gamma}+/+ CD11b+ graft-infiltrating cells in RAG-1–/– recipients of IFN-{gamma}–/– T cells produced any IFN-{gamma}. Thus, by both RPA and intracellular cytokine staining analyses, IFN-{gamma} expression in graft-infiltrating macrophages occurs only in the presence of WT T cells.


Figure 5
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  		Fig. 5 Intracellular IFN-{gamma} staining and flow cytometry of allograft-infiltrating cells. Infiltrating cells extracted from allografts in WT hosts or in RAG-1–/– hosts following transfer of purified WT T cells revealed IFN-{gamma} expression in 50 to 70% of CD4+ or CD8+ T cells, and in ~30% of CD11b+ macrophages. In allografts in RAG-1–/– hosts receiving IFN-{gamma}–/– T cells, graft-infiltrating CD11b+ (IFN-{gamma}+/+) macrophages, as well as transferred IFN-{gamma}–/– T cells showed negligible IFN-{gamma} production.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In this paper we have investigated the role of B cells, as well as the role of T cell-derived versus non-T cell-derived IFN-{gamma} in the pathogenesis of GAD. The present study employed a well-characterized mouse cardiac allograft model where genetic backgrounds of the donor and recipient strains differ at the MHC II locus [5,7]. All allografts in WT B/6 recipients functioned 8 weeks post-transplant without immunosuppression, and developed parenchymal rejection with moderate to severe GAD. Although most donor/recipient allogeneic disparities in clinical heart transplantation involve both MHC I and MHC II as well as minor histocompatibility antigens, we and others have shown entirely comparable results in the MHC II-mismatched allograft model. The characteristics of the GAD lesions and the tempo of their development in MHC II-mismatched grafts are also comparable to total allomismatched grafts prevented from failing by monoclonal antibody administration [4,5,25]. The thickened intima of early GAD lesions (2 to 4 weeks post-transplant in mice) shows increased cellularity, predominantly composed of mononuclear inflammatory cells; late GAD lesions (8 to 12 weeks post-transplant) contain more extracellular matrix and VSMC with diminished inflammation. These histologic changes depend on the maturity of the lesions rather than allogeneic disparities [5,7,26]. In addition, the temporal changes in inflammatory cytokine mRNA expression in allografts up to 7 days post-transplant do not differ between MHC I-, MHC II- and total allomismatch grafts [27], suggesting common cytokine-mediated mechanisms for the initial immunologic injury that triggers GAD. Finally, we and others have demonstrated comparably diminished GAD in the setting of host IFN-{gamma} deficiency for both MHC II-disparate grafts and for total allomismatch grafts whose survival was prolonged by immunosuppression [4,5,28].

Consistent with previous results [4,5], allografts placed in IFN-{gamma}–/– recipients developed little or no GAD but did show ongoing parenchymal rejection; allografts in RAG-1–/– recipients developed neither GAD nor parenchymal rejection. However, transfer of naïve purified WT T cells reproducibly restored both parenchymal rejection and GAD in allografts transplanted into RAG-1–/– hosts. In contrast, transfer of IFN-{gamma}–/– T cells restored only parenchymal rejection but not GAD. Transferred WT or IFN-{gamma}–/– CD4+ or CD8+ T cells were comparably present in recipient spleens and allografts 8 weeks after transplant.

It is noteworthy that GAD developed in allografts in RAG-1–/– recipients following WT T cell transfer, and did so in the absence of mature B cells. Flow cytometry of RAG-1–/– host spleens 8 weeks after adoptive transfer of either WT or IFN-{gamma}–/– T cells showed that the B cells present were immature cells of host origin (B220+/IgM–) with extremely rare mature (B220+/IgM+) B cells present. In addition, RAG-1–/– recipients of adoptively transferred T cells had very low serum immunoglobulin levels (<5 µg/ml) as measured by ELISA, comparable to immunoglobulin levels in naïve RAG-1–/– mice (data not shown). The data clearly establish that GAD can develop in the absence of mature B cells and alloantibody.

Such results agree with those reported previously [19,20] but contradict other works suggesting that host alloantibodies are important for GAD pathogenesis; in the latter studies, GAD was attenuated in allografts in immunoglobulin-deficient recipients in murine carotid arterial or cardiac transplants [17,18]. Moreover, human clinical analyses have shown that serum anti-donor HLA antibody titers correlate with GAD incidence [29]. The discrepancies may result from species differences, or differences in experimental preparations, i.e., arterial grafts versus solid organ grafts. In particular, the relevance of findings in animal end-to-side arterial interposition grafts to human GAD remains speculative. Nevertheless, a potentially unifying hypothesis is that multiple immunologic effector pathways—including T cell cytokine- and/or humoral-mediated mechanisms—are all capable of inciting GAD development. Any initial cell- or humoral-mediated injury could subsequently induce common downstream pathways to recruit and activate the macrophages and smooth muscle cells that form GAD [26,30].

IFN-{gamma} is a prototypical Th1 cytokine with a variety of activities that potentiate allograft rejection. It stimulates effector mechanisms by directly activating macrophages and by promoting cytotoxic T cell generation [31,32]. IFN-{gamma} also induces the expression of a number of chemokines [33], and enhances antigen presentation via professional antigen-presenting cells by augmenting MHC II and costimulatory molecule expression [34]. Originally proposed as an important mediator of GAD in 1989 [2], IFN-{gamma} has subsequently been shown to be a major effector cytokine in both atherogenesis and GAD pathogenesis [5,35]. Systemic neutralization with monoclonal antibody or genetic deficiency of recipient IFN-{gamma} results in marked attenuation of allograft GAD [5,6]. Moreover, by directly acting on vascular smooth muscle cells to potentiate growth-factor-induced mitogenesis, IFN-{gamma} alone induced GAD development in arterial xenografts transplanted in SCID/beige mice lacking T, B, and NK cells [30].

CD4+ and CD8+ T cells, as well as NK cells, serve as major sources of IFN-{gamma} in a variety of inflammatory disorders [9]. Nevertheless, macrophages can also express and secrete IFN-{gamma} after stimulation by a combination of IL-12 and IL-18, or by IFN-{gamma} itself [10,11]; in vivo data also support the importance of macrophage-derived IFN-{gamma} in murine host defenses [12]. Consequently, it is increasingly recognized that macrophages are important not only as antigen-presenting or terminal effector cells but also as potent regulators of host immune reactions. More recently, IL-18-stimulated VSMC have also been shown to produce IFN-{gamma}, particularly in combination with IL-12 [15]. Histopathologic evaluation demonstrated IFN-{gamma} mRNA and/or IFN-{gamma} immunoreactivity in human atherosclerotic plaque and in murine cardiac GAD lesions co-localizing with smooth muscle {alpha}-actin [15,16]. Such observations suggest that, besides T cells, multiple other cell types can contribute to IFN-{gamma} production during GAD pathogenesis.

Our previous work showed that host cells are the critical source of the IFN-{gamma} responsible for inducing GAD development; IFN-{gamma}–/– grafts transplanted into WT hosts developed GAD, whereas WT hearts placed in IFN-{gamma}–/– hosts did not [5]. Thus, IFN-{gamma} produced by VSMC and/or resident macrophages in donor organs alone cannot trigger GAD lesions. Although recipient T cells are almost certainly important origins of intragraft IFN-{gamma}, it was still conceivable that VSMC or infiltrating macrophages of host origin could also be critical IFN-{gamma} sources. Recent reports demonstrating the host origin of intimal VSMC in murine GAD lend some credence to the hypothesis [36,37]. However, the results of the present study clearly demonstrate that host T cells are indispensable sources of IFN-{gamma} in initiating GAD development, and that other host cells cannot compensate for T cells that lack IFN-{gamma}.

Immunohistochemistry showed that long-term MHC II-disparate allografts in WT hosts contained predominantly CD11b+ cells, with scattered CD4+ T cells, fewer CD8+ T cells, and extremely rare B cells and NK cells. Intracellular staining and flow cytometry showed that infiltrating T cells and CD11b+ cells from grafts in WT hosts, as well as from grafts in RAG-1–/– hosts receiving WT T cells, all expressed IFN-{gamma}. The CD11b+ cells also express F4/80 [8], confirming their identity as macrophages.

RPA revealed comparable mRNA expression of IL-12p40 and IL-18 in allografts in WT hosts and in RAG-1–/– hosts receiving WT T cells. Although IL-18 and native IL-12, a heterodimer of IL-12p35 and IL-12p40, potently induce IFN-{gamma} in T cells, the role of IL-12 in this model is likely to be negligible, since the grafts consistently lacked IL-12p35 transcripts and the IL-12p40 homodimer acts as an IL-12 antagonist [38]. The data also indicated that mRNA expression for multiple cytokines and chemokines was comparable between WT hosts and RAG-1–/– hosts receiving WT T cells; expression was negligible for IFN-{gamma}–/– hosts and RAG-1–/– hosts lacking T cell-derived IFN-{gamma}. It is important to note that the effects of specific cytokines on GAD development are not necessarily predictable from in vitro data. Thus, IL-10 is frequently designated an anti-inflammatory Th2 cytokine, yet IL-10 has paradoxical proinflammatory effects in vivo and exacerbates GAD [24]. Moreover, the roles of these cytokines (e.g., IL-6, IL-10) in GAD development are probably less important overall, since their intragraft mRNA expression was substantially less prominent than, e.g., IL-1β, even in the WT hosts.

IL-1β was strongly expressed in the allografts in WT hosts, consistent with a previous report showing persistent IL-1β gene expression in long-term mouse cardiac allografts with GAD [39]. In allografts in IFN-{gamma}–/– hosts or in RAG-1–/– hosts receiving IFN-{gamma}–/– T cells, mRNA expression for these cytokines diminished dramatically. In view of the reduced macrophage accumulation observed by immunohistochemistry, this decreased cytokine expression can be attributed in part to the decreased number of macrophages. Moreover, the absence of T cell-derived IFN-{gamma} would likely limit macrophage activation, and subsequently reduce macrophage cytokine expression. This conclusion is supported by the flow cytometry and RPA data, indicating that host macrophages only contribute to IFN-{gamma} production in the presence of T cell-derived IFN-{gamma}; the overall IFN-{gamma} expression in grafts becomes negligible in the absence of IFN-{gamma} production by T cells.

In conclusion, T cells alone can induce GAD in the absence of B cells or alloantibody. In addition, IFN-{gamma} produced by T cells plays a critical role in the pathogenesis of GAD, likely involving secondary macrophage accumulation and activation.


   Acknowledgements
 
We thank Ms. Eugenia Shvartz, Ms. Elissa Simon-Morrissey and Ms. Karen E. Williams, Brigham and Women's Hospital, for their skillful assistance.

This study was supported by National Institutes of Health grant RO1 HL-43364 to P.L. and R.N.M.


   Notes
 
1 Current address: Dr. Yutaka Furukawa, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawaharacho, Sakyo-ku, Kyoto 606-8097, Japan. Back

2 Current address: Dr. Yoshihiro Fukumoto, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medicine, Fukuoka, Japan. Back

Time for primary review 41 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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