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The Journal of Immunology, 2003, 171: 3823-3836.
Copyright © 2003 by The American Association of Immunologists

Cytokines Regulate the Pattern of Rejection and Susceptibility to Cyclosporine Therapy in Different Mouse Recipient Strains After Cardiac Allografting1

Hao Wang*,{dagger},§,¶, Karoline A. Hosiawa{ddagger}, Weiping Min, Jinming Yang, Xiaoxia Zhang, Bertha Garcia§, Thomas E. Ichim, Dejun Zhou, Dameng Lian, David J. Kelvin{ddagger},¶ and Robert Zhong2,*,{dagger},{ddagger},§,¶

* Multi-Organ Transplant Program, London Health Sciences Centre, London, Ontario, Canada; Departments of {dagger} Surgery, {ddagger} Microbiology and Immunology, and § Pathology, University of Western Ontario, London, Ontario, Canada; and Transplantation Group, Robarts Research Institute, London, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We determined the role of cytokines in regulating the pattern of rejection and recipient susceptibility to cyclosporine (CsA) in a mouse cardiac allograft model. Hearts from C3H mice transplanted into untreated BALB/c (Th2-dominant) and C57BL/6 (Th1-dominant) mice showed different patterns of rejection. C3H allografts in BALB/c mice showed typical acute vascular rejection (AVR) with strong intragraft deposition and high serum levels of anti-donor IgG with predominant IgG1, while C3H allografts in C57BL/6 mice showed typical acute cellular rejection (ACR) with massive intragraft infiltration of CD4+ and CD8+ lymphocytes and low serum levels of anti-donor IgG with predominant IgG2a. Elevated intragraft mRNA expression of IL-2, IFN-{gamma}, and IL-12 mRNA was present in C57BL/6 recipients, whereas allografts in BALB/c mice displayed increased IL-4 and IL-10 mRNA levels. CsA therapy completely inhibited ACR and induced indefinite allograft survival in C57BL/6 recipients, while the same therapy failed to prevent AVR, and only marginally prolonged graft survival in BALB/c recipients. In contrast, rapamycin blocked AVR, achieving indefinite survival in BALB/c recipients, but was less effective at preventing ACR in C57BL/6 recipients. The disruption of the IL-12 or IFN-{gamma} genes in C57BL/6 mice shifted ACR to AVR, and resulted in concomitant recipient resistance to CsA therapy. Conversely, disruption of IL-4 gene in BALB/c mice markedly attenuated AVR and significantly prolonged allograft survival. These data suggest that the distinct cytokine profiles expressed by different mouse strains play an essential role in regulating the pattern of rejection and outcome of CsA/rapamycin therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Organ transplantation is the preferred treatment for most patients with chronic organ failure. Although transplantation of kidney, liver, lung, and heart offers an excellent opportunity for rehabilitation as recipients return to a more normal lifestyle, it is limited by the medical/surgical suitability of potential recipients, an increasing shortage of donors, and premature failure of transplanted organ function. Rejection continues to be the single largest impediment to successful organ transplantation. Acute rejection episodes still occur in ~20–40% of organ transplant recipients, while chronic rejection and allograft dysfunction remain the most common causes of long-term graft loss (1). The most challenging factor for successful immunosuppressive therapy is the different susceptibility to immunosuppressive therapies among individuals. Many immunological and nonimmunological factors such as donor condition, MHC matching, recipient age, sex, race, and other conditions are affecting the outcome of treatment with immunosuppressive agents (2). Understanding how an individual patient responds to these drugs may significantly improve the outcome of current antirejection therapy.

Although cyclosporine (CsA)3 has been very effective at preventing acute graft rejection in most recipients, there are some individuals who are resistant to this therapy (3, 4). Little is known about the mechanisms dictating the differences in susceptibility to drugs among individuals. We hypothesize that such differences among recipients are primarily due to diversity of the recipient’s genetic background. In support of this concept, genetic susceptibility to certain immunosuppressive agents has been widely reported in experimental animal models. For example, the mice with C57BL/6 backgrounds are more resistant than other strains to tolerance induction by mAbs against CD4 and CD8 as well as by the simultaneous blockade of CD40 and CD28 signaling pathways (5, 6). It has been further suggested that the recipient’s cytokine profiles, as well as other factors, may play an important role in these models. The use of genetically well-defined inbred mouse strains may provide an ideal opportunity to elucidate the mechanisms underlying the heterogeneity of responses to antirejection therapy by studying strain-dependent susceptibility to immunosuppressive agents.

Support for the role of Th-derived cytokines in determining the direction of the immune response is evident in many experimental models. When challenged with pathogenic organisms, two strains of mice, C57BL/6 and BALB/c, develop opposing immune responses (7). For example, when challenged with Leishmania major, C57BL/6 mice respond by developing a vigorous cell-mediated immune response dominated by the production of classic Th1 cytokines (IL-12, IFN-{gamma}, IL-18) and fend off infection by this organism. BALB/c mice, in contrast, develop an immune response dominated by Th2 cytokines (IL-4, IL-10), thereby failing to clear the infection. In addition, strain-specific differences in mouse hepatic wound healing after liver injury have been proven to be mediated by divergent Th cytokine responses (8). Minimal fibrosis in C57BL/6 mice is consistent with elaboration of Th1 cytokines, such as IFN-{gamma}, whereas severe fibrosis in BALB/c mice is consistent with a Th2 cytokine response, such as IL-4. These studies not only highlight the importance of predetermining recipient cytokine expression patterns as an indicator of the onset and severity of graft rejection, but also suggest that modulation of the Th responses among individuals may provide a beneficial approach for inhibition of graft rejection or augmentation of the therapeutic effects of conventional immunosuppressive drugs.

In this study, we used two well-defined mouse strains, BALB/c (Th2 dominant) and C57BL/6 (Th1 dominant), as recipients of C3H cardiac allografts, and compared rejection patterns, cytokine profiles, and susceptibility to CsA and rapamycin (RAPA). We have shown that C3H allografts transplanted in C57BL/6 recipients developed acute cellular rejection (ACR), dominated by production of Th1 cytokines such as IL-2, IFN-{gamma}, and IL-12. In this context, CsA therapy effectively induced long-term graft survival. In contrast, BALB/c recipients of C3H allografts developed acute vascular rejection (AVR), dominated by Th2 cytokines such as IL-4 and IL-10 and resistance to CsA therapy. In addition, AVR was induced in CsA-treated C57BL/6 recipients by passive transfer of serum from untreated BALB/c mice with rejected C3H allografts, but not from untreated C57BL/6 mice undergoing rejection. We have also shown that C57BL/6 recipients with disrupted Th1 cytokine genes such as IL-12 or IFN-{gamma} underwent a shift in the pattern of rejection, from ACR to AVR, resulting in recipient resistance to CsA. Conversely, disruption of Th2 cytokine genes such as IL-4 attenuated AVR in BALB/c mice, resulting in recipient susceptibility to CsA. Although CsA seems to be more effective in the context of Th1 immunity, we used RAPA as a control immune suppressant based on its ability to inhibit Th2 responses. Accordingly, RAPA effectively inhibited AVR in BALB/c mice, but was less effective at preventing ACR in C57BL/6 mice. These data suggest that cytokine predisposition of the recipients regulates the pattern of rejection and efficacy of immune suppressive therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and immunosuppressive drug

Male adult C3H (H-2k) mice were used as donors. Male adult wild-type BALB/c (H-2d) mice, wild-type C57BL/6 (H-2b) mice, IFN-{gamma}-/- C57BL/6 mice (C57BL/6-IFN-{gamma}tm1Ts) (9), IL-12p40-/- C57BL/6 mice (C57BL/6-IL-12btm1jm) (10), and IL-4-/- BALB/c mice (BALB/c-IL-4tm2Nnt) (11), weighing 25–30 g, were used as recipients (The Jackson Laboratory, Bar Harbor, ME). Each group included eight animals. In the groups receiving immunosuppression, the recipients were injected s.c. with CsA 15 mg/kg/day, daily from day 0 to endpoint rejection or until day 100, or with RAPA orally, 4 mg/kg/day, from day 0 to postoperative day (POD) 13. Whole blood CsA trough levels were measured on days 7, 16, and 100 using standard RIA with mAb kits supplied by Novartis (Basel, Switzerland) (12). All mice were housed in a conventional facility, except knockout mice, which were kept in pathogen-free conditions at the John P. Robarts Research Institute Barrier Facility (London, Ontario, Canada) for 1 wk before surgery. All animals were cared for in accordance with the guidelines established by the Canadian Council on Animal Care (13).

Heterotopic cardiac transplantation

Intra-abdominal heterotopic cardiac transplantation was performed, as previously described by Corry et al. (14). Briefly, a median sternotomy was performed on the donor, and the heart graft was slowly perfused in situ with 1.0 ml of cold heparinized Ringer’s lactate solution through the inferior vena cava and aorta before the superior vena cava and pulmonary veins were ligated and divided. The ascending aorta and pulmonary artery were transected, and the graft was removed from the donor. The graft was then revascularized with end-to-side anastomoses between the donor’s pulmonary artery and the recipient’s inferior vena cava as well as the donor’s aorta and the recipient’s abdominal aorta using 11-0 nylon suture. The beating of the grafted heart was monitored daily by direct abdominal palpation. The degree of pulsation was scored as: A, beating strongly; B, noticeable decline in the intensity of pulsation; or C, complete cessation of cardiac impulses. When cardiac impulses were no longer palpable, the graft was removed for routine histology, immunohistochemistry, and RT-PCR. Serum samples were collected for flow cytometric analysis or adoptive transfer.

Graft histology

Tissue samples were removed at necropsy and fixed in 10% buffered formaldehyde. Specimens were then embedded in paraffin, and sectioned for H&E staining or Martius scarlet blue staining. The microscopic sections were examined in a blinded fashion for severity of rejection by a pathologist (B. Garcia). Criteria for graft rejection included the presence of vasculitis, thrombosis, fibrin deposition, hemorrhage, and lymphocyte infiltration. These changes were scored as: 0, no change; 1, minimum change; 2, mild change; 3, moderate change; or 4, marked change.

Immunohistochemistry

Four micrometer sections were cut from tissue samples embedded in Tissue-Tek O.C.T. (Optimum Cutting Temperature gel; Skura Finetek, Torrance, CA) mounted on gelatin-coated glass microscope slides and stained by a standard indirect avidin-biotin immunoperoxidase staining method using an Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Specimens were stained for CD4+ and CD8+ cells with biotin-conjugated rat anti-mouse CD4 mAb (clone YTS 191.1.2; Cedarlane Laboratories, Hornby, Ontario, Canada) and biotin-conjugated rat anti-mouse CD8 mAb (clone 53-6.7; BD PharMingen, Franklin Lakes, NJ), respectively. Intragraft neutrophil infiltration was detected by staining with a rat anti-mouse Ly-6G mAb (clone RB6-8C5; BD PharMingen), and developed with a secondary biotin-conjugated goat anti-rat Ig (BD PharMingen). Mouse IgG and IgM deposition in grafts was detected using biotin-conjugated goat anti-mouse IgG and goat anti-mouse IgM (Cedarlane). Intragraft IgG1 and IgG2a deposition was detected using a biotin-conjugated rat anti-mouse IgG1 mAb (clone A85-1; BD PharMingen) and rat anti-mouse IgG2a mAb (clone R19-15; PharMingen). Complement 3 (C3) tissue binding was assessed by using a goat anti-mouse C3 Ab (Cappel Research Products, Durham, NC), and developed by using secondary biotin-conjugated rabbit anti-goat IgG mAb (DAKO, Carpenteria, CA). Slides were washed with PBS between steps, and examined under light microscopy. Negative controls were performed by omitting the primary Abs. The sections of immunoperoxidase staining for intragraft IgG, IgG1, IgG2a, IgM, and C3 deposition were graded from 0 to 4+, according to the staining intensity: 0, negative; 1+, equivocal; 2+, weak staining; 3+, moderate staining; and 4+, very intensive staining. The sections of immunoperoxidase staining for intragraft infiltration of CD4+, CD8+ T cells and Ly-6G+ cells (neutrophils) were analyzed by counting the number of all positively stained cells in the entire section and divided by the section area examined (by means of the Empix Northern Eclipse trace application program; Empix Imaging, Mississauga, Ontario, Canada).

Flow cytometry

The circulating anti-donor-specific IgM, IgG, and its IgG1 and IgG2a Abs were evaluated in the recipient serum by FACScan flow cytometry (BD Biosciences, Mountain View, CA) (15, 16). Briefly, C3H mouse splenocytes were isolated and incubated at 37°C for 30 min with serum from various controls and experimental groups: naive BALB/c and C57BL/6 mice; untreated wild-type BALB/c (POD 8), wild-type C57BL/6 (POD 8), IL-4-/- BALB/c (POD 8), IFN-{gamma}-/- C57BL/6 (POD 5), or IL-12p40-/- C57BL/6 (POD 7) recipients; CsA-treated wild-type BALB/c (POD 16), wild-type C57BL/6 (POD 16), wild-type C57BL/6 (POD 100), IL-4-/- BALB/c (POD 88), IFN-{gamma}-/- C57BL/6 (POD 12), IL-12p40-/- C57BL/6 (POD 17) recipients; RAPA-treated wild-type BALB/c (POD 19 and 100) or wild-type C57BL/6 (POD 19) recipients; and CsA-treated C57BL/6 recipients transferred with rejecting sera from either untreated BALB/c or C57BL/6 mice. To stain for total IgG, IgG1, IgG2a, and IgM, the cells were washed and incubated with FITC-conjugated goat Ab specific for the Fc portion of mouse IgG or with PE-conjugated goat Ab specific for the µ-chain of mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA), FITC-conjugated goat anti-mouse IgG1 (Caltag Laboratories, Burlingame, CA), or with FITC-conjugated goat anti-mouse IgG2a (Caltag). After 1 h of staining at 4°C, the cells were washed with PBS, resuspended at 5 x 106/ml, and analyzed by FACS for mean channel fluorescence intensity, which represents the Ab-binding reactivity. Sera from naive BALB/c and C57BL/6 mice were used as controls.

Passive serum transfer

Whole blood (0.6–0.8 ml) was taken from untreated wild-type BALB/c or C57BL/6 mice with rejected C3H mouse heart grafts on POD 8. After 30 min at room temperature, the serum was separated by centrifugation and stored at -80°C until use. Before transfer, the serum was heat inactivated at 56°C for 30 min and injected i.v. into CsA-treated C57BL/6 recipients 8 days after C3H heart transplantation. The CsA (15 mg/kg/day, daily) therapy was continuously given to these C57BL/6 recipients before and after serum transfer (from day 0 to endpoint rejection).

RT-PCR

To measure relative differences in cytokine transcript levels between cardiac transplants, we used a semiquantitative RT-PCR technique, as previously described (17). Total RNA was obtained from grafted or normal tissue using TRIzol Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s specifications. First-strand cDNA synthesis was done on total RNA with a first-strand cDNA synthesis kit (Pharmacia, Uppsala, Sweden). For PCR amplification, the reaction was conducted in a volume of 25 µl of PCR Supermix High Fidelity (10790-020; Invitrogen), which was prepared for multiple reactions. Each PCR consisted of 1 µl of cDNA, 100 nM of each of the primers for GAPDH as a reference gene, and 200 nM of each pair of specific primers (IFN-{gamma}, sense, AGC TCT GAG ACA ATG AAC GCT ACA C and antisense, ACC TGT GGG TTG TTG ACC TCA AAC; IL-12, sense, AAA CAG TGA ACC TCA CCT GTG ACA C and antisense, TTC ATC TGC AAG TTC TTG GGC G; IL-2, sense, ACA TTG ACA CTT GTG CTC CGT GTC and antisense, TTG AGG GCT TGT TGA GAT GAT GCT; IL-10, sense, TGC TAT GCT GCC TGC TCT TAC TGA C and antisense, AAT CAC TCT TCA CCT GCT CCA CTG; IL-4, sense, AGC TAG TTG TCA TCC TGC TCT TC and antisense, AGC ATG GTG GCT CAG TAC TACG). The PCR was conducted, as follows: denaturation at 95°C for 3 min, followed by 8 cycles at 95°C for 1 min, annealing of the primers at 60°C, at 72°C for 1 min, followed by 30 cycles at 95°C for 1 min, annealing of primers at 50°C for 1 min, at 72°C for 2 min, and a final extension at 72°C for 5 min. After PCR amplification, 12 µl of the PCR product mixed with loading buffer was subjected to electrophoresis in a 3% agarose gel under 1x Tris base, acetic acid, EDT {Delta} buffer. The PCR product size of 376 bp is for IFN-{gamma}, 451 bp for IL-12, 417 bp for IL-2, 404 bp for IL-10, 423 bp for IL-4, and 249 bp for GAPDH. The cytokine PCR product was compared with the GAPDH PCR product as an internal control for the same cDNA, using the same master mixture at the same time. In addition, to differentially compare cytokine transcription levels between experimental groups, we used densitometry: the ratio of each cytokine mRNA amplification product compared with standardized and titratable levels of GAPDH mRNA amplification product.

Statistical analysis

The data were reported as the mean ± SD. Allograft survival among experimental groups was compared using the rank-log test. Histological and immunohistological findings of intragraft Ab and complement deposition were analyzed using the Mann-Whitney U test. Immunohistological findings of intragraft cellular infiltration, flow cytometric data, and RT-PCR data were analyzed using one-way ANOVA. Differences with p values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BALB/c and C57BL/6 possess distinct patterns of rejection

To understand the impact of strain cytokine predominance on patterns of allograft rejection, we used the prototypic Th1 model, C57BL/6, and Th2 model, BALB/c, as recipients of C3H hearts. In the absence of immunosuppression, distinct patterns of rejection were observed even though graft survival times were similar (BALB/c, 8.2 ± 0.8 days vs C57BL/6, 8.3 ± 0.5 days) (Table I). At endpoint, C3H allografts in wild-type BALB/c recipients showed a typical feature of AVR, characterized by predominant vasculitis (Fig. 1Aa), intravascular thrombosis (Fig. 1Ac), fibrin deposition (Fig. 1Ae), interstitial hemorrhage (Fig. 1A, a and e), and few CD4+ (Fig. 1Ba) and CD8+ (Fig. 1Bc), but massive Ly-6G+ (neutrophil marker) (Fig. 1Be) cell infiltration. In contrast, C3H allografts in wild-type C57BL/6 recipients showed a profile of typical ACR (Fig. 1A, b, d, and f) with heavy CD4+ (Fig. 1Bb) and CD8+ (Fig. 1Bd), but few Ly-6G+ (Fig. 1Bf) cell infiltrates without evidence of AVR. These results were further confirmed by quantitative cell counting. Fig. 1C shows that in C57BL/6 mice, the number of CD4+ and CD8+ cells infiltrating C3H grafts on POD 8 was significantly higher than that in BALB/c mice at the same time point. In contrast, the number of intragraft Ly-6G+ cells in BALB/c mice was significantly higher than that in C57BL/6 mice (Fig. 1C). These results suggest that allografts transplanted into these two strains of mice have different patterns of rejection, and we hypothesized that these differences may contribute to their distinct susceptibility to CsA therapy.


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  		Table I. Survival time and histopathology of cardiac allograftsa

 


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  		FIGURE 1. A, Histology of heart allografts in wild-type BALB/c and C57BL/6 recipient mice. Grafts were harvested at the time of rejection or at 100 days (for CsA-treated C57BL/6 recipients) posttransplantation. H&E-stained or Martius scarlet blue (MSB)-stained paraffin sections of C3H allografts in untreated (a, c, and e) or CsA-treated (g and i) BALB/c recipients, and C3H allografts in untreated (b, d, and f) or CsA-treated (h and j) C57BL/6 recipients. B, Immunoperoxidase staining for cell infiltration in heart allografts of wild-type BALB/c and C57BL/6 recipient mice. Grafts were harvested at the time of rejection or at 100 days (for CsA-treated C57BL/6 recipients) posttransplantation. Immunoperoxidase staining of cryostat sections of CD4+ cells (a, b, g, and h), CD8+ cells (c, d, i, and j), and Ly-6G+ cells (e, f, k, and l) in two strains of mouse recipients treated with or without CsA. The arrows show positive staining when appropriate. C, The infiltrating CD4+, CD8+, and Ly-6G+ cells were determined respectively in the allografts by quantitating all the positively stained cells in the entire section and divided by the section area assessed (cells/mm2). Untreated wild-type C57BL/6 recipients vs untreated wild-type BALB/c recipients at day 8: *, p < 0.01. Untreated wild-type BALB/c recipients vs untreated wild-type C57BL/6 recipients at day 8: *, p < 0.01.

 
Continuous treatment with CsA achieves indefinite allograft survival in C57BL/6 recipients, but not in BALB/c recipients

To determine whether these two strains have divergent susceptibility to CsA therapy, we treated both C57BL/6 and BALB/c recipients with CsA 15 mg/kg/day, daily, from day 0 to endpoint rejection or to day 100. There was no difference in CsA levels at each time point between these two strains (for example, the CsA trough level in BALB/c mice on POD 16 was 734 ± 46 ng/ml vs 722 ± 48 ng/ml in C57BL/6 mice at the same time point, p > 0.05). Interestingly, we found that continuous treatment with CsA achieved indefinite allograft survival in wild-type C57BL/6 recipients. All the grafts survived over 100 days (Table I). ACR was completely inhibited, and the grafts showed normal histology (Fig. 1A, h and j). There was no cellular infiltration of CD4+ (Fig. 1Bh), CD8+ (Fig. 1Bj), and Ly-6G+ (Fig. 1Bl) cells in these grafts. In contrast, the same therapy failed to prevent AVR, and only marginally prolonged graft survival to 16.5 ± 0.9 days in wild-type BALB/c recipients (Table I). A pattern of mixed AVR and ACR was observed in the grafts of BALB/c recipients at endpoint rejection with moderate infiltrating CD4+ (Fig. 1Bg), CD8+ (Fig. 1Bi), and Ly-6G+ (Fig. 1Bk) cells and a moderate AVR, characterized by thrombosis and interstitial hemorrhage (Fig. 1Ag), vasculitis, and fibrin deposition (Fig. 1Ai). These results were further confirmed by quantitative cell counting (Fig. 1C). These data indicate that C57BL/6 recipients with transplanted C3H allografts, which develop predominant cellular rejection, are sensitive to CsA therapy. Conversely, BALB/c recipients with transplanted C3H hearts, which display predominant humoral rejection, are resistant to CsA treatment.

Opposite cytokine profiles were present in BALB/c and C57BL/6 recipients of C3H allografts

Because C57BL/6 and BALB/c mice have very different cytokine profiles when challenged with intracellular pathogens (7) or transplanted with xenografts (18), we examined the cytokine profiles of transplanted C3H mouse hearts in these two strains of mice. We measured mRNA levels of type 1 cytokines (IL-2, IFN-{gamma}, and IL-12) and type 2 cytokines (IL-4 and IL-10) to determine whether graft rejection or survival, and recipient susceptibility to CsA might correlate with expression of these cytokines in the grafts. Fig. 2, A and B, shows that IL-2, IFN-{gamma}, and IL-12 cytokines were predominantly expressed in C3H allografts of untreated C57BL/6 mice and were significantly higher than those in BALB/c mice at endpoint rejection. In contrast, IL-4 and IL-10 were predominantly expressed in C3H allografts of untreated BALB/c mice and were much higher than those in C57BL/6 recipients at the same day.



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  		FIGURE 2. Intragraft cytokine expression in untreated (A and B) and CsA-treated (C and D) wild-type BALB/c and C57BL/6 mouse recipients. Heart grafts were harvested at the time points indicated. Semiquantitative RT-PCR was performed for intragraft mRNA expression of IL-2, IFN-{gamma}, IL-12, IL-4, and IL-10 (A and C). GAPDH was used as a loading control. The data show one representative of five independent experiments in each group. In addition, mean densitometry measurements for cytokine mRNA to GAPDH mRNA ratios as determined by RT-PCR are shown in bar graph B (untreated wild-type C57BL/6 recipients vs untreated wild-type BALB/c recipients at day 8: *, p < 0.01; untreated wild-type BALB/c recipients vs untreated wild-type C57BL/6 recipients at day 8: **, p < 0.01) and bar graph D (CsA-treated wild-type C57BL/6 recipients vs CsA-treated wild-type BALB/c recipients at day 8: *, p < 0.01; CsA-treated wild-type BALB/c recipients vs CsA-treated wild-type C57BL/6 recipients at day 16: **, p < 0.01).

 
Anti-donor Ab levels correlate with patterns of allograft rejection, and intragraft C3 deposition is associated with AVR

C3H allografts in BALB/c mice showed typical AVR, characterized by vasculitis, thrombosis, fibrin deposition, and interstitial hemorrhage, while C3H allografts in C57BL/6 mice did not. To determine whether the onset of AVR correlated with alloreactive Ab levels, we examined recipient mice for serum Ab levels of anti-donor IgG and IgM, as well as the IgG1 and IgG2a isotypes. Fig. 3A shows that at endpoint rejection, BALB/c mice had significantly higher levels of anti-donor IgG Abs with predominant IgG1 isotype, whereas C57BL/6 recipient mice had very minimal amounts of anti-donor IgG Abs with IgG2a isotype at the same time points. Serum anti-donor IgM levels were increased after heart grafting, but there were no significant differences between these two strains (Fig. 3C). To further investigate the role of Abs and complement in AVR, we determined IgG, IgG1, IgG2a, IgM, and C3 deposition in the grafts using immunostaining techniques. Table II shows that massive deposition of anti-mouse IgG, predominantly IgG1 Abs and C3, was present in C3H allografts in BALB/c mice at endpoint rejection, while only mild deposition of anti-mouse IgG with IgG2a dominant Abs and C3 was found in the allografts of C57BL/6 mice at the same time point. Furthermore, minimal IgM deposition was observed in the grafts of both strains. These results indicate that AVR in BALB/c mice was mediated by anti-donor Ab, primarily IgG1, which is known to be controlled by Th2 cytokines. In contrast, C57BL/6 mice, which have a predominant Th1 cytokine profile, produce low levels of anti-donor IgG, and develop ACR. In addition, C3 deposition in the grafts is associated with the development of AVR in BALB/c recipients in this allotransplant model.



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  		FIGURE 3. Serum levels of anti-donor Abs in wild-type recipients. Mouse sera were harvested at the time of rejection or 16 and 100 days (for CsA-treated wild-type C57BL/6 recipients) after allografting. Serum levels of anti-donor total IgG, as well as IgG1 and IgG2a isotypes in untreated wild-type recipients (A) and CsA-treated wild-type recipients (B) were determined by flow cytometry. Serum levels of anti-donor total IgG and IgM were determined in both untreated and CsA-treated mouse recipients (C). Results are mean ± SD of five experiments. Statistical analyses are described in the text. Serum levels of anti-donor total IgG were compared in different groups. Untreated wild-type C57BL/6 recipients vs untreated wild-type BALB/c recipients at day 8: *, p < 0.01; CsA-treated wild-type C57BL/6 recipients at days 16 and 100 vs CsA-treated wild-type BALB/c recipients at day 16: **, p < 0.01.

 

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  		Table II. Grades for immunoperoxidase staining of heart allograft frozen sectionsa

 
CsA effectively inhibits intragraft expression of Th1 cytokines, Ab production, and C3 deposition in C57BL/6 recipients, but not of Th2 cytokines, Ab production, and C3 deposition in BALB/c recipients

Untreated C3H allografts in C57BL/6 mice expressed predominantly Th1 cytokines, such as IL-2, IFN-{gamma}, and IL-12. CsA treatment (Fig. 2, C and D) markedly decreased intragraft expression of these cytokines on POD 8 and 16 and completely inhibited their expression at day 100 in C57BL/6 recipients. In contrast, CsA failed to effectively inhibit high expression of intragraft IL-4 and IL-10 levels in BALB/c mice. In addition, a mixture of Th1 and Th2 cytokine expression was detected in CsA-treated BALB/c mice at day 16 (endpoint rejection), indicated by more intragraft expressions of IL-2 and IL-12, as well as IL-4 and IL-10, than those in CsA-treated C57BL/6 recipients at the same day. This finding correlates with the histological features of mixed ACR and AVR in CsA-treated BALB/c mice at endpoint rejection.

Fig. 3B shows that CsA effectively inhibited alloreactive Abs on POD 16 and 100 in C57BL/6 mice, while only marginally down-regulated anti-donor total IgG, as well as IgG1 and IgG2a levels in BALB/c recipients on POD 16 (endpoint). Serum anti-donor IgM levels were increased after transplantation, but they remained at similar levels in both strains of untreated and CsA-treated mice (Fig. 3C). Corresponding to circulating Ab levels, high levels of intragraft deposition of IgG with IgG1 Abs predominating and C3 were found in BALB/c mice, but not in C57BL/6 mice when treated with CsA (Table II). These data indicate that CsA effectively inhibits intragraft expression of Th1 cytokines and prevents allograft rejection in C57BL/6 mice, but fails to inhibit Th2 cytokines and humoral responses in BALB/c mice.

Passive transfer of serum from BALB/c, but not from C57BL/6 mice with rejected C3H allografts induces AVR in CsA-treated C57BL/6 recipients

To further define the role of Abs in inducing AVR, we conducted the following passive serum transfer experiments. Sera taken from untreated BALB/c or C57BL/6 recipients with rejected C3H mouse heart grafts on POD 8 were injected i.v. into CsA-treated C57BL/6 mice with C3H mouse heart grafts on POD 8. After transfer of serum from rejecting BALB/c recipients, CsA was no longer effective to prevent rejection in C57BL/6 mice, and the C3H allografts were rejected with typical features of AVR (Fig. 4Aa) with a mean survival of 16.8 ± 1.0 days (Table I). A strong intragraft IgG (IgG1 dominant) and C3 deposition (Table II) was present in these grafts. In contrast, transfer of serum from C57BL/6 mice with rejected C3H allografts did not induce AVR. In fact, these grafts survived for 78.2 ± 3.3 days (Table I) with mild cellular rejection (Fig. 4Ab) and mild intragraft deposition of IgG (IgG2a dominant) and C3 (Table II). To further determine which Ab isotype plays an important role in initiating AVR after transfer, we measured serum Ab subtypes in these recipients using FACS analysis. Fig. 4B shows that serum levels of anti-donor IgG Abs, predominantly IgG1, were markedly elevated after transfer of BALB/c sera. In contrast, transfer from C57BL/6 mice only moderately increased serum IgG levels, and IgG2a was a predominant subtype in these recipients. Adoptive transfer did not significantly affect IgM levels in both strains.



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  		FIGURE 4. A, Histology of heart allografts in CsA-treated wild-type C57BL/6 recipients with passive transfer of rejecting sera from untreated BALB/c (a) and C57BL/6 mice (b). Grafts were harvested at the time of rejection, and the paraffin sections were stained with H&E staining. B, Serum levels of anti-donor IgG, IgG1, IgG2a, and IgM in CsA-treated C57BL/6 recipients transferred with rejecting sera. Mouse sera were harvested at the time of rejection (for serum-transferred groups) or on POD 8 (for nontransferred group, as control). Results are mean ± SD of five experiments. Statistical analyses are described in the text. Serum levels of anti-donor total IgG were compared in two serum-transferred groups, CsA-treated C57BL/6 recipients transferred with rejecting BALB/c sera vs the same recipients transferred with rejecting C57BL/6 sera: *, p < 0.01.

 
RAPA effectively inhibits AVR and induces indefinite graft survival in BALB/c recipients, but not in C57BL/6 recipients

To further confirm the role of cytokines in regulating the recipient susceptibility to immunosuppressive agents, we treated these two strains of mice with RAPA, another commonly used antirejection drug, which has a distinct immunosuppressive mechanism from CsA (19, 20, 21). Interestingly, these two strain recipients responded to RAPA therapy in opposite ways when compared with CsA therapy. RAPA treatment was not able to effectively prevent ACR and only marginally prolonged the graft survival to 19.8 ± 1.3 days in C57BL/6 recipients (Table I). The grafts were rejected with primarily cellular rejection (Fig. 5Aa), characterized by a large number of CD4+ and CD8+ T cell infiltration (Fig. 5B), but minimal IgG, IgG2a, IgM, and C3 deposition (Table II). In contrast, the same therapy effectively prevented AVR and achieved indefinite allograft survival in BALB/c recipients (Table I) with normal histology on POD 19 and 100 (Fig. 5, A, b and c, and B, and Table II). RAPA treatment markedly inhibited Ab production in BALB/c mice, as reflected by no intragraft Ab deposition and the near absence of anti-donor circulating Ab in BALB/c recipients on POD 100. In contrast, the same therapy only marginally reduced Ab production and did not change the pattern of IgG isoform in C57BL/6 recipients, in which IgG2a was predominant (Fig. 5C). In addition, RAPA treatment was less effective at inhibiting high levels of intragraft Th1 cytokine expression in C57BL/6 recipients. The grafts were rejected with predominant expression of IL-2, IFN-{gamma}, and IL-12 (Fig. 5, D and E). In contrast, RAPA effectively down-regulated intragraft high expression of Th2 cytokines in BALB/c mice. However, in the long-term surviving grafts, we still found low expression of Th2 cytokines (IL-4 and IL-10) at day 100 in BALB/c recipients. These results indicate that C57BL/6 mice with predominant Th1 cytokines are resistant to RAPA, while BALB/c mice with predominant Th2 cytokines are very susceptible to RAPA.



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  		FIGURE 5. A, Histology of heart allografts in BALB/c and C57BL/6 recipients treated with RAPA. Grafts were harvested at the time of rejection or at 19 and 100 days (for RAPA-treated BALB/c recipients) posttransplantation. H&E-stained paraffin sections of C3H allografts in RAPA-treated C57BL/6 recipients at endpoint rejection (POD19) (a), RAPA-treated BALB/c recipients on POD 19 (b) and POD 100 (c). B, The infiltrating CD4+ and CD8+ cells were determined respectively in the allografts by quantitating all the positively stained cells in the entire section and divided by the section area assessed (cells/mm2). RAPA-treated C57BL/6 recipients on POD 19 vs RAPA-treated BALB/c recipients on POD 19 and POD 100: *, p < 0.01; untreated BALB/c recipients vs RAPA-treated BALB/c recipients: **, p < 0.01. C, Serum levels of anti-donor Abs in wild-type recipients. Mouse sera were harvested at the time of rejection or 19 and 100 days (for RAPA-treated BALB/c recipients) after allografting. Serum levels of anti-donor total IgG, as well as IgG1 and IgG2a isotypes in untreated and RAPA-treated recipients were determined by flow cytometry. Results are mean ± SD of five experiments. Statistical analyses are described in the text. Serum levels of anti-donor total IgG were compared in different groups. RAPA-treated BALB/c recipients vs RAPA-treated C57BL/6 recipients at day 19: *, p < 0.01; RAPA-treated BALB/c recipients vs untreated BALB/c recipients: **, p < 0.01. D and E, Intragraft cytokine expression in RAPA-treated BALB/c and C57BL/6 mouse recipients. Heart grafts were harvested at the time points indicated. Semiquantitative RT-PCR was performed for intragraft mRNA expression of IL-2, IFN-{gamma}, IL-12, IL-4, and IL-10 (D). GAPDH was used as a loading control. The data show one representative of five independent experiments in each group. In addition, mean densitometry measurements for cytokine mRNA to GAPDH mRNA ratios as determined by RT-PCR are shown in bar graph E. IL-4 and IL-10 intragraft expression vs IL-2, IFN-{gamma}, and IL-12 intragraft expression in RAPA-treated BALB/c recipients at day 100: *, p < 0.01.

 
IFN-{gamma}- or IL-12-deficient C57BL/6 mice develop accelerated AVR and are resistant to CsA therapy

The data described above indicated that intragraft expression of predominant Th1 cytokines in C57BL/6 mice was associated with cell-mediated rejection, which could be effectively inhibited by CsA, resulting in indefinite graft survival. These data suggest that in this strain, intragraft IFN-{gamma} expression appears to be inversely correlated with AVR. Namely, IFN-{gamma} expression prevents or delays the onset of AVR and renders recipients susceptible to CsA therapy. To determine whether a causal relationship exists between IFN-{gamma} expression and recipients’ susceptibility to CsA therapy, we performed C3H cardiac transplants into C57BL/6 recipient mice lacking IFN-{gamma} expression. Untreated IFN-{gamma}-/- C57BL/6 recipients rejected C3H mouse hearts with a mean survival time of 5.3 ± 0.5 days (Table I) compared with 8.5 ± 0.6 days for untreated wild-type C57BL/6 mice. When compared with the hearts from wild-type C57BL/6 recipients at endpoint rejection, IFN-{gamma}-/- C57BL/6 mice showed accelerated AVR with intravascular thrombosis and interstitial hemorrhage (Fig. 6Aa), yet had very few infiltrating CD4+ or CD8+ cells (Fig. 6, A, b and c, and B), a pattern indistinguishable from wild-type BALB/c recipients. Furthermore, IFN-{gamma}-/- C57BL/6 mice became resistant to CsA treatment, because graft survival time was only prolonged to 12 ± 1.3 days by treatment (Table I). The histology of these grafts also showed typical features of AVR and minimal cell infiltration (Fig. 6, A, j, k, and l, and B).



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  		FIGURE 6. A, Histology and immunohistology for cellular infiltrates in heart allografts of IFN-{gamma}-/- or IL-12p40-/- C57BL/6 and IL-4-/- BALB/c mice. Grafts were harvested at the time of rejection. H&E-stained paraffin sections of C3H allografts in untreated (a, d, and g) or CsA-treated (j, m, and p) cytokine-deficient mouse recipients. Immunoperoxidase staining of cryostat sections of CD4+ cells (b, e, h, k, n, and q) and CD8+ cells (c, f, i, l, o, and r) in cytokine-deficient mice treated with or without CsA. The arrow shows positive staining when appropriate. B, The infiltrating CD4+ and CD8+ cells were determined respectively in the allografts by quantitating all the positively stained cells in the entire section and divided by the section area assessed (cells/mm2). Untreated IFN-{gamma}-/- or IL-12p40-/- C57BL/6 recipients vs untreated wild-type C57BL/6 recipients at endpoint rejection: *, p < 0.01. Untreated IL-4-/- BALB/c recipients vs untreated wild-type BALB/c recipients at endpoint rejection: **, p < 0.01.

 
Because IL-12 and IFN-{gamma} up-regulate each other (22), one possibility was that IL-12, in addition to IFN-{gamma}, regulated the pattern of allograft rejection and recipient susceptibility to CsA therapy. Consistent with this, C3H hearts transplanted into untreated IL-12p40-/- C57BL/6 recipients were rejected as quickly as untreated wild-type C57BL/6 mice (mean survival time of 7.3 ± 0.5 days vs 8.3 ± 0.5 days, Table I), and the grafts displayed a very aggressive AVR (Fig. 6Ad). Furthermore, very few CD4+ and CD8+ T cells infiltrated the transplanted hearts (Fig. 6, A, e and f, and B). Similar to those of IFN-{gamma}-/- C57BL/6 mice, CsA failed to prolong graft survival past day 20 in IL-12p40-/- C57BL/6 recipients because the grafts were rejected with severe AVR (Fig. 6Am) and few infiltrating cells (Fig. 6, A, n and o, and B) at 17.8 ± 3.3 days (Table I).

Disruption of IFN-{gamma} or IL-12 genes in C57BL/6 mice significantly increased intragraft IL-4 expression, and IL-12 disruption decreased IFN-{gamma} expression when compared with wild-type controls, whereas the levels of other measured cytokines were similar between gene mutant and wild-type mice (Fig. 7, A and B). Interestingly, CsA therapy marginally inhibited IL-2 and IFN-{gamma} levels in IL-12p40-/- C57BL/6 mice, as well as IL-2 and IL-12 levels in IFN-{gamma}-/- mice, but was not able to effectively inhibit IL-4 and IL-10 levels in heart grafts of these mice. The intragraft expression of Th2 cytokines was still significantly higher than that of Th1 cytokines in these gene mutant mice after CsA treatment (Fig. 7, A and B).



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  		FIGURE 7. Intragraft cytokine expression in IFN-{gamma}-/- or IL-12p40-/- C57BL/6 (A and B) and IL-4-/- BALB/c mice (C and D) with or without CsA treatment. Heart grafts were harvested at the time of rejection. Semiquantitative RT-PCR was performed for intragraft mRNA expression of IL-2, IFN-{gamma}, IL-12, IL-4, and IL-10 (A and C). GAPDH was used as a loading control. The data show one representative of five independent experiments in each group. In addition, mean densitometry measurements for cytokine mRNA to GAPDH mRNA ratios as determined by RT-PCR are shown in bar graph B (untreated IFN-{gamma}-/- or IL-12p40-/- C57BL/6 recipients vs untreated wild-type C57BL/6 recipients at endpoint rejection: *, p < 0.01; untreated IL-12p40-/- C57BL/6 recipients vs untreated wild-type C57BL/6 recipients at endpoint rejection: **, p < 0.01) and bar graph D (untreated IL-4-/- BALB/c recipients vs untreated wild-type BALB/c recipients at endpoint rejection: *, p < 0.01).

 
Furthermore, untreated IFN-{gamma}-/- or IL-12p40-/- C57BL/6 mice (Fig. 8A) had higher levels of anti-donor IgG Abs, with predominant IgG1 levels, when compared with wild-type C57BL/6 mice. Unlike wild-type mice, CsA therapy was no longer effective at inhibiting anti-donor Ab production in mice lacking IFN-{gamma} or IL-12 (Fig. 8A). Although serum levels of anti-donor IgM were elevated after allografting, they had similar levels in wild-type and IFN-{gamma}-/- or IL-12p40-/- C57BL/6 recipients with or without CsA therapy (Fig. 8C). In addition, strong intragraft deposition of IgG (IgG1 dominant) and C3, but not IgM, was found in both untreated and CsA-treated IFN-{gamma}-/- or IL-12p40-/- C57BL/6 mice (Table II). These results suggest that IL-12 or IFN-{gamma}, which facilitates cellular immunity, negatively regulates AVR and makes recipients susceptible to CsA therapy.



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  		FIGURE 8. Serum levels of anti-donor Abs in cytokine-deficient mouse recipients. Mouse sera were harvested at the time of graft rejection after allografting. Serum levels of anti-donor total IgG, as well as IgG1 and IgG2a isotypes in IFN-{gamma}-/- or IL-12p40-/- C57BL/6 recipients with or without CsA therapy (A), and IL-4-/- BALB/c recipients with or without CsA treatment (B). Serum levels of anti-donor total IgG and IgM in both untreated and CsA-treated gene mutant mouse recipients (C). Results are mean ± SD of five experiments. Statistical analyses are described in the text. Serum levels of anti-donor total IgG were compared in different groups, untreated wild-type C57BL/6 recipients vs C57BL/6 recipients with IFN-{gamma} or IL-12 deficiency treated with or without CsA: *, p < 0.01; CsA-treated IL-4-/- BALB/c recipients vs untreated wild-type and IL-4-/- BALB/c recipients: **, p < 0.01.

 
Disruption of IL-4 gene attenuates AVR and renders BALB/c mice CsA sensitive

To further determine the relationship between Th2 cytokines such as IL-4 and AVR, we transplanted C3H allografts into IL-4-/- BALB/c mice. Untreated IL-4-/- BALB/c recipients rejected C3H mouse hearts with a mean survival of 8.5 ± 0.6 days (Table I), which was at the same rate as that of untreated wild-type BALB/c mice (8.2 ± 0.8 days). However, the histopathological features were shifted from typical AVR to a mixed pattern of AVR and ACR, characterized by moderate vasculitis, hemorrhage, and mild thrombosis (Fig. 6Ag), but massive intragraft-infiltrating CD4+ and CD8+ T cells (Fig. 6, A, h and i, and B). Furthermore, IL-4-/- BALB/c mice became susceptible to CsA treatment. The graft survival time of CsA-treated recipients was prolonged to 88.8 ± 9.3 days (Table I). Significant attenuation of both humoral and cellular rejection with minimal vasculitis and interstitial hemorrhage (Fig. 6Ap), as well as mild infiltration of CD4+ (Fig. 6, Aq and B) and CD8+ (Fig. 6, Ar and B) T cells were shown in the grafts at endpoint rejection.

Disruption of IL-4 enhanced intragraft expression of IL-2, IFN-{gamma}, IL-12, and IL-10 mRNA (Fig. 7, C and D) as compared with wild-type BALB/c recipients. In this case, the up-regulation of IL-10 mRNA level may compensate for the deficiency of IL-4. Notably, CsA effectively inhibited IL-2, IFN-{gamma}, and IL-12 gene expression in IL-4-/- BALB/c mice, as compared with wild type BALB/c mice (Fig. 7, C and D). Untreated IL-4-/- BALB/c mice displayed decreased anti-donor total IgG and IgG1 Abs, but increased levels of IgG2a in their sera (Fig. 8B) and decreased IgG (predominantly IgG1) and C3 deposition in the grafts (Table II) when compared with wild-type BALB/c mice. CsA therapy strongly inhibited anti-donor Abs of total IgG, IgG1, and IgG2a in the sera (Fig. 8B) and the deposition of IgG and its isotypes, and C3 in the grafts (Table II) in IL-4-/- BALB/c mice as compared with wild-type mice. In addition, serum anti-donor IgM levels were increased after transplantation, but there were no significant differences between wild-type and IL-4-/- BALB/c mice with or without CsA treatment (Fig. 8C). Intragraft IgM deposition remained at minimal level in these mice (Table II). These results indicate that IL-4, which promotes humoral immunity, facilitates AVR and renders recipients resistant to CsA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The discovery of CsA in the 1980s has remarkably improved the short-term success rates of transplantation (23). However, some individuals are known to be resistant to CsA therapy. Kabelitz et al. (3) indicated that in vitro, human alloreactive CTL precursors from 2 of 21 normal individuals were CsA resistant. In addition, Hess and Colombani (4) suggested that lymphocytes or their functions can be arbitrarily divided into CsA sensitive and CsA resistant. Understanding the mechanisms underlying these phenomena is important to improve the outcome of clinical transplantation. CsA is a potent noncytotoxic, immunosuppressive agent (24). The ability of CsA to prevent IL-2 production and CTL activation, and thereby inhibiting T cell-mediated cellular immunity is currently considered to be the primary mechanism for its immunosuppressive efficacy (25, 26, 27). Furthermore, secretion of other lymphokines/monokines such as IFN-{gamma} is also inhibited in the presence of CsA (27, 28). As cytokine gene polymorphism in humans does exist, we hypothesized that the pattern of allograft rejection and recipient susceptibility to CsA would be regulated by each individual’s cytokine profile. To test this hypothesis, we used two genetically well-defined mouse strains: C57BL/6 and BALB/c. These two strains display opposite patterns of cytokine profiles when challenged by pathogens, such as Leishmania major (7, 29), and autoantigens (30, 31, 32), or are exposed to injury (8) or to xenografts (18).

In this study, we have demonstrated that C57BL/6 mice with transplanted C3H allografts displayed typical ACR and low levels of circulating and intragraft anti-donor IgG with predominant IgG2a Abs, whereas BALB/c mice with C3H allografts developed typical AVR and high levels of circulating and intragraft anti-donor IgG with predominant IgG1 Abs. This rejection pattern was associated with a predominance of Th1 cytokines (IL-2, IFN-{gamma}, and IL-12) in C57BL/6 recipients, and a Th2 cytokine (IL-4 and IL-10) predominance in BALB/c recipients. In C57BL/6 mice, CsA therapy effectively inhibited ACR, resulting in indefinite graft survival, while in BALB/c mice, the same therapy failed to prevent AVR, and only marginally prolonged allograft survival. Disruption of Th1 cytokine genes such as IFN-{gamma} or IL-12 in C57BL/6 mice promoted AVR, resulting in resistance to CsA therapy; whereas in BALB/c mice, disruption of Th2 cytokine genes such as IL-4 attenuated AVR, resulting in susceptibility to CsA therapy. These data indicate that cytokines regulate both the patterns of allograft rejection and recipient response to CsA therapy.

To further define the role of Ab in inducting AVR, we conducted passive serum transfer from either BALB/c mice or C57BL/6 mice with rejected allografts into CsA-treated C57BL/6 mice. We found that serum transfer from BALB/c mice undergoing rejection induced AVR in CsA-treated C57BL/6 mice and resulted in concomitant recipient resistance to CsA therapy. We further demonstrated that transfer of serum from these BALB/c mice significantly increased the circulating level of IgG, predominantly IgG1. These results demonstrate the important role of alloantibodies, especially IgG, in mediating acute allograft rejection and on the outcome of CsA therapy. For these experiments, we transferred sera to CsA-treated C57BL/6 recipients 8 days after heart transplantation. This time was selected because it coincides with vigorous alloantibody production and heart allograft rejection in both strains of wild-type mouse recipients. In addition, our immunohistological data showed that intragraft C3 deposition correlated with Ab production and the development of AVR in this model. This result indicates that Abs initiate complement activition in allograft rejection. Abs can activate circulating complement components (33), but also can increase the secretion of complement components by macrophages at the site of inflammation (34, 35).

Interestingly, these two strains responded to RAPA in opposite ways compared with CsA therapy. BALB/c mice were very sensitive to RAPA therapy. Two weeks of this therapy effectively prevented rejection and induced long-term graft survival. In contrast, C57BL/6 mice were resistant to RAPA and the grafts were rejected in 19 days. Although the precise mechanism of these differences is unknown, in this study, RAPA seems more effective to inhibit Th2 cytokines than Th1 cytokines. These findings are supported by previous studies, which indicate that RAPA is very potent to inhibit host alloantibody responses in a rat-accelerated graft rejection model (36).

In this study, we have clearly demonstrated that the two different mouse strains when confronted with the same stimuli, C3H heart allografts, display different production of Th1- or Th2-dominant cytokines. Less is understood about the molecular basis of Th1-Th2 development after Ag challenge. It is possible that the observed differences in cytokine gene expression are the results of more fundamental differences in immune responses between the two strains (i.e., differences in signaling pathways, Ag processing and presentation, distinct role of CD4 vs CD8 T cells, etc.). It is well known that T lymphocytes require signals normally provided by macrophages or dendritic cells to respond to Ags (37, 38, 39, 40). Some researchers (41, 42) indicated that macrophages can be an important factor in determining the direction of Th1/Th2 differentiation and of other immune responses because they are typically the first cells to receive signals. Also, it has been suggested that dendritic cell subsets play an important role in regulating the Th1/Th2 balance (43, 44, 45).

It is well documented that cytokine gene polymorphism in humans plays an important role in graft rejection. The studies by Hutchinson et al. (46) have demonstrated that there is a great variation of cytokine expression in humans. We would predict that these individuals would have different rejection patterns and thus variable susceptibility to certain immunosuppressive drugs. These data point to the importance of genomic studies in future clinical transplantation such as genetic prescreening to determine specific cytokine profiles in individual recipients, as a predictive tool in assessing the outcome of transplantation and antirejection therapy. Our data also suggest that modulation of the cytokine responses among individuals may provide a therapeutic approach for inhibition of graft rejection or for augmentation of the efficacy of immunosuppressive drugs.


   Acknowledgments
 
We acknowledge Drs. Gill Strejan, Jacqueline Arp, and Xian-Chang Li for reviewing the manuscript; Dr. Xuyan Huang for excellent technical support; and Sharon Mutch for secretarial assistance.


   Footnotes
 
1 This study was supported by Canadian Institutes of Health Research, National Institutes of Health (5U19 AI15173-2), and Multi-Organ Transplant Program, London Health Sciences Centre. Back

2 Address correspondence and reprint requests to Dr. Robert Zhong, Department of Surgery, London Health Sciences Centre-University Campus, 339 Windermere Road, P.O. Box 5339, London, Ontario, Canada N6A 5A5. E-mail address: zzhong{at}uwo.ca Back

3 Abbreviations used in this paper: CsA, cyclosporine; ACR, acute cellular rejection; AVR, acute vascular rejection; POD, postoperative day; RAPA, rapamycin. Back

Received for publication July 18, 2002. Accepted for publication July 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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H. Wang, J. Arp, W. Liu, S. J. Faas, J. Jiang, D. R. Gies, S. Ramcharran, B. Garcia, R. Zhong, and R. P. Rother
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