Objectives: Interleukin-6, a pleiotropic cytokine that functions in both innate and adaptive immune responses, has been implicated in allograft rejection. We analyzed the efficacy of anti interleukin-6 receptor monoclonal antibody in delaying allograft rejection in a murine model of a heart.

Materials and Methods: To investigate the role of interleukin-6 receptor signal transduction in acute and chronic allograft rejection, we blocked interleukin-6 receptor signaling to suppress the alloimmune response in C57BL/6 recipients of BALB/c cardiac allografts.

Results: Administration of a high-dose α-interleukin-6 receptor monoclonal antibody prevented the intragraft infiltration of inflammatory cells and lymphocytes and prolonged allograft survival during the peritransplant period. However, all allografts were rejected by 23.5 days after transplant. In contrast, cardiac allograft recipients treated with a cytotoxic T-lymphocyte antigen 4-immunoglobulin plus continued administration of low-dose α-interleukin-6 receptor monoclonal antibody showed long-term graft survival compared with cytotoxic T-lymphocyte antigen 4-immunoglobulin monotherapy. A histologic analysis revealed that graft fibrosis was prevented in cytotoxic T-lymphocyte antigen 4-immunoglobulin plus high-dose α-interleukin-6 receptor monoclonal antibody group, but not in the cytotoxic T-lymphocyte antigen 4-immunoglobulin alone group. This suggests that deterioration of graft function associated with chronic rejection could be prevented by blocking interleukin-6 receptor signaling.

Conclusions: Disruption of interleukin-6 receptor signaling is an effective strategy for modulating proinflammatory immune responses and preventing chronic rejection.

Key words : Proinflammatory immune response, Chronic rejection, Fibrosis, IL-6 receptor signaling, Cardiac allograft.

Introduction

The rejection of transplanted tissues involves interplay between mechanisms that maintain tolerance to the graft and those that promote rejection. While immunologic factors are important for both, the process of rejection is very much an inflammatory one. As a consequence, local production of many proinflammatory cytokines—including interferon (IFN)-γ, interleukin (IL)-2, IL-6, and IL-15, from infiltrating lymphocytes and resident cells (eg, the proximal renal tubular epithelium)—is increased during acute renal graft rejection.^1-3

The pleiotropic cytokine,⁴ IL-6, exerts important biological effects on inflammation, immunity, and stress.^{5, 6} Interleukin-6 production leads to activation of adhesion molecules and leukocyte invasion in all organs. Pretransplant organ dysfunction is a direct consequence of inflammation, and renders many organs unsuitable for transplant. Elevated IL-6 levels in a donor’s heart, kidney, and liver have been shown to worsen ischemia-reperfusion injury in recipients, resulting in deteriorating organ function.^{7, 8}

Although advances in immunosuppressive drugs have greatly decreased the incidence of acute graft rejection and chronic rejection⁹ remain a barrier to long-term graft survival.¹⁰ Chronic rejection of cardiac allografts manifests as interstitial fibrosis, vascular occlusion, and progressive deterioration of graft function.¹¹ Although multiple factors are associated with the onset and progression of chronic rejection, the cause of the disease is poorly understood, and no effective therapy currently exists.

Immune responses against the graft that ultimately results in chronic rejection can be studied in the mouse vascularized heterotopic cardiac transplant model.^12-14 Afanasyeva and associates¹⁵ stated that IL-6 plays a crucial role in cardiac hypertrophy and fibrosis associated with chronic rejection. These results suggest IL-6 as a target for treating graft rejection by protecting the grafted organ from chronic inflammation.

We investigated the effect of disrupting IL-6 receptor signaling on survival of cardiac allografts transplanted cervically in a murine model. Allograft survival was prolonged by blocking IL-6 receptor signaling. Further, we demonstrated the efficacy of a graft infiltrate cell assay and performed histologic analysis, to detect progression of acute graft rejection, chronic rejection, and their association with biological and immunologic changes associated with acute graft and chronic rejection. These histologic and molecular data suggest that IL-6 plays a crucial role in the deterioration of cardiac allograft function during both the peritransplant phase (which is associated with acute graft rejection) and the long-term phase (which is associated with chronic rejection).

Materials and Methods

Mice
Female 5- to 12-week-old BALB/c (H-2d) and C57BL/6 (H-2b) mice purchased from Japan SLC (Hamamatsu, Japan) were housed in a pathogen-free facility. All experiments were performed in accord with protocols set forth by the Animal Care and Use Committee of the Tokyo University of Science.

Vascularized cervical heart transplant
Vascularized heterotopic cervical heart transplant from BALB/c donors to C57BL/6 recipient mice was performed with anastomosis to the vessels of the neck using a nonsuture cuff technique.¹⁶ Graft survival was monitored daily by palpation. Rejection was defined as a loss of palpable cardiac contractions.

Reagents
CTLA4-Ig was generated in our laboratory. Anti-CD44 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). PE-, FITC-, and APC-conjugated anti-CD4 (GK1.5), anti-CD8α (53-6.7.2), anti-Gr1, and anti-CD44 antibodies (Abs) were purchased from BD Biosciences (San Jose, CA, USA). Anti-FcγR (2.4G2) was obtained from the ATCC. Monoclonal antibodies (mAbs) were fluorescein-labeled according to standard techniques.

In vivo monoclonal antibodies therapy
To assess the role of IL-6 receptor signaling in acute and chronic cardiac allograft rejection, we used 2 transplant models. First, we investigated the role of IL-6 receptor signaling during the peritransplant phase. Blocking Abs was administered intraperitoneally (IP), and an untreated control was used for comparison. C57BL/6 mice received 500, 1000, or 2000 µg IP of anti-IL-6R mAbs (MR16-1, rat anti-mouse; Chugai Pharmaceutical Inc., Tokyo, Japan) on days 0, 2, and 4 after transplant. Untreated control mice received no prolonged therapy. On days 7, 14, and 21, each mouse was killed for analyses. Second, we investigated the role of IL-6 during the long-term phase. C57BL/6 mice were treated with 200 µg IP of CTLA4Ig (costimulation blockade) on days 0 and 2 posttransplant for peripheral transplant tolerance induction. Other mice received an additional 500 µg IP of MR16-1 on day 0 for induction and 100 µg every other week. The grafts were recovered at various time points after cardiac transplant.

Graft infiltrate cell analysis and flow cytometry
Graft infiltrate cells were analyzed using the modified method of Afanasyeva and associates.¹⁵ Lymphocytes or spleen cell suspensions from donor heart-transplanted mice were prepared in FACS medium (PBS containing 0.5% calf serum and 0.1% sodium azide). Cells were incubated first with unlabeled anti-FcγR (2.4G2) to block nonspecific binding and then stained with each Ab. A FACSCalibur with CellQuest software (BD Biosciences) was used for 4-color flow cytometric analysis.

Graft histology
Fresh tissues were fixed in 20% neutral buffered formalin until they were processed and embedded in paraffin. Five-micrometer–thick tissue sections were cut on a microtome and stained with hematoxylin and eosin (H&E) and Masson’s trichrome according to standard procedures. We used the modified method of Diaz and associates18 to evaluate the degree of chronic rejection. Briefly, graft fibrosis areas stained with Masson’s trichrome were quantified using iPLab software (Scanalytics Inc., Fairfax, VA, USA). Mean fibrotic area was calculated from 10 to 20 areas per heart section analyzed at ×200 magnification. To evaluate the degree of chronic rejection, the number of vessels (including coronary arteries and intramuscular arterioles affected with obliterative vasculopathy) and the total number of vessels in each section of the histologic specimen, were counted. Histologic specimens were reviewed by a single histologist blinded to the treatment modality. A minimum of 5 individual hearts were analyzed per group for both analytic techniques.

Assay for anti-donor monoclonal antibodies
Anti-donor Ab activity was measured by an in vitro killing assay on BALB/c (H-2d) targets.17 To determine anti-donor Ab recognition of the H-2d-restricted target, BALB/c target cells were incubated with a 1:5 dilution of each serum from untreated-rejection, CTLA4-Ig–treated, and CTLA4-Ig + MR16-1–treated mice. The percentage of killing in each target was counted. Target cells were whole spleen cells. The percentage of killing was calculated as follows: The percentage of killing = the percentage of (Ab+rabbit blood complement)/rabbit blood complement.

Statistical Analyses
Each experiment was performed at least 3 times and representative data were collected. The repeated-measures ANOVA, Fisher protected least significant difference test was used. A t test was used to examine the significance of the data obtained by the FACS analysis. Differences in graft survival between the groups were tested with the log-rank sum test. A difference was considered significant at P < .05.

Results

Disruption of IL-6 receptor signaling reduces inflammation and prolongs allograft survival.
To determine if IL-6 has a physiologically important role during graft rejection, we examined mice that received anti-IL-6 receptor mAbs (MR16-1) using our transplant protocol. Under the high-dose MR16-1 administration regimen (2000 µg × 3/mouse), mean survival time was significantly prolonged (mean survival time, 21.5 ± 2.6 d; n=6) compared with untreated grafts (mean survival time, 7.5 ± 0.4 d; n=6) (P < .05) (Figure 1A). Thus, blocking IL-6 receptor signaling significantly prolonged graft survival. To confirm that the increased graft survival observed in mice receiving MR16-1 was MR16-1 dose dependent, we treated wild-type allogeneic transplant recipients (BALB/c→B6) with different dosages of MR16-1 (500 µg × 3 or 1000 µg × 3/mouse). Administering other MR16-1 dosages alone did not prolong graft survival (mean survival time, 8.5 ± 0.6 d for 500 µg × 3; mean survival time, 9.8 ± 1.5 d for 1000 µg × 3; n=6 each [data not shown]). Thus, prolonged survival during the peritransplant phase appeared to be MR16-1 dose dependent, and high-dose MR16-1 alone was sufficient to prolong allograft survival.

Disruption of interleukin-6 receptor signaling enhances survival of cardiac allografts
Recently, it was reported that IL-6 drives development of pathologic hypertrophy and fibrosis during chronic cardiac allograft rejection, suggesting that IL-6 could be a therapeutic target to prevent this disease.¹⁸ To investigate the effect of MR16-1 in the long-term phase after transplant, we next asked whether the combination of CTLA4-Ig and MR16-1 could prevent chronic rejection. As described above, C57BL/6 mice received a BALB/c cardiac allograft together with CTLA4-Ig alone or MR16-1 and CTLA4-Ig. Mice that received 500 µg of MR16-1 on day 0 continued receiving 100 µg IP of MR16-1 every week after transplant. As shown in Figure 1B, a significant difference was observed for long-term graft survival between the CTLA4-Ig alone and CTLA4-Ig + MR16-1 mice. Hence, addition of MR16-1 prolonged graft survival during the long-term phase.

Effects on rejection: Histopathologic analysis
The effects of blocking IL-6 receptor signaling on chronic rejection were analyzed histopathologically (Figure 2 A-C). Mice treated with CTLA4-Ig alone or with MR16-1 and CTLA4-Ig were used for comparison. Despite continued graft survival as assessed by palpation, histologic analysis of the graft in the CTLA4-Ig-alone group on day 200 exhibited evidence of fibrosis. In contrast, grafts that received additional MR16-1 on the same day showed little evidence of chronic rejection (Figure 2B). Cardiac allografts were morphologically normal and remained free of interstitial or vascular pathology, similar to those of naive BALB/c hearts. There were few vessels with obliterative vasculopathy observed in specimens (10 coronary cross-sections/specimens) from recipients in the MR16-1– and CTLA4-Ig–treated groups (Figure 2-c). Collectively, these data indicate that a protocol using additional MR16-1 could support CTLA4-Ig–induced tolerance by preventing morphologic and fibrotic changes in grafts.

Blocking interleukin-6 receptor signaling reduces T-lymphocyte graft infiltration
The rejection of transplanted tissues involves the interplay between mechanisms that maintain graft tolerance and factors that promote rejection. While immunologic factors are important for both, inflammation is commonly involved. It has been shown that the local production of many proinflammatory cytokines, such as IFN-γ, IL-2, IL-6, and IL-15 from infiltrating lymphocytes and resident cells such as the proximal renal tubular epithelium increases during acute renal graft rejection.^1-3 Thus, we analyzed and counted graft-infiltrating leukocytes with a fluorescence-activated cell sorter.

First, high-dose MR16-1 mice and untreated rejection mice were used for comparison. In the high-dose MR16-1 versus untreated control groups (n=6 each), graft infiltrate cells on the CD3 gate revealed that the median values (range) for myocardial staining positive for CD4+ cells were 49.6% (range, 37.3%-60.2%) versus 39.2% (range, 29.2%-43.7%) (NS); for CD8+ cells, the values were 16.8% (range, 12.3%-18.4%) versus 50.8% (range, 47.6%-52.4%) (P < .001) (Figure 3A).

The total number of graft-infiltrating CD4+, CD8+ T cells, macrophages, and neutrophils was determined (Figure 3B). Treatment with high-dose MR16-1 decreased these cells’ infiltration into allografts to low levels compared with untreated control allografts. In addition, the number of CD8+ T cells infiltrating the allograft was statistically lower than the number of CD4+ T cells (P < .001).

Thus, high-dose MR16-1 administration reduced leukocyte infiltration into the allografts during the peritransplant phase, particularly for CD8+ T cells. Similarly, additional administration of low-dose MR16-1 reduced leukocyte infiltration into allografts compared with CTLA4-Ig alone. The median values (range) for myocardial staining positive for CD4+ T cells were 29.1% (range, 20.5%-34.3%) versus 45.6% (range, 40.8%-48.4%) (P < .05); for CD8+ T cells, it was 43.1% (range, 39.7%-45.5%) versus 40.5% (range, 28.5%-50.4%) (P < .05) (Figure 3C). The total number of graft-infiltrating CD4, CD8 T cells, and macrophages (except neutrophils) was significantly lower than CTLA4-Ig alone (Figure 3D). Thus, in the CTLA4-Ig mice and in the model that added MR16-1 to the regimen, which is a long-term phase model, graft-infiltrating leukocyte numbers also were reduced, particularly CD4+ T cells. Collectively, these data indicate that, during the peritransplant acute phase, high-dose MR16-1 prevents leukocyte recruitment, especially CD8+ cells, to allografts. In contrast, during the long-term phase, the additional use of low-dose MR16-1 supported CTLA4-Ig–induced tolerance by preventing leukocyte recruitment—especially CD4+ cells—to allografts.

T-cell activation decreased in high-dose MR16-1 mice compared with untreated mice
It has been demonstrated previously that blockage of the IL-6 receptor signaling reduces T-cell activation; that is, blocking IL-6 down-regulates T-cell activation as evidenced by decreased expression of effector markers.¹⁹ To confirm this in our model and to determine the extent to which this occurs, we looked at CD44 expression using CD4+ and CD8+ T-cell markers by flow cytometry.

As shown in Figure 4A, T-cell expression of CD44 in CD4+ T cells was decreased in high-dose MR16-1–treated mice compared with untreated rejection mice. Similarly, CD44 expression in CD8+ T cells was decreased in high-dose MR16-1–treated mice compared with untreated mice. Collectively, these data indicate that the blockade of IL-6 receptor signaling by high-dose MR16-1 reduces T-cell activation during the peritransplant phase and supports the avoidance of acute rejection.

In contrast, in the combined CTLA4-Ig and MR16-1 regimen, there was a significant difference in CD44 expression in CD8+ cells. No significant difference was observed in CD4+ cells (Figure 4B). However, CD44 expression in CD4 and CD8 T cells in the CTLA4-Ig + MR16-1 mice appeared to be lower than that of CTLA4-Ig–alone mice.

It is well known that CD44 expression in both CD4+ and CD8+ T cells is inhibited by CTLA4-Ig.^{20, 21} Our study produced similar results. Nevertheless, for CD44 expression in CD8+ cells, additional use of MR16-1 provided more-effective inhibition than CTLA4-Ig monotherapy. Thus, in this long-term phase model, blockage of IL-6 receptor signaling by low-dose MR16-1 also appeared to retain low T-cell activation, especially in CD8+ cells, and support transplant tolerance induced by CTLA4-Ig.

Blockage of interleukin-6 receptor signaling reduces B-cell recruitment to lymph nodes and decreases anti-donor Ab formation
Hirano and associates reported that IL-6 induces the differentiation of B cells into Ab-forming cells.²² Therefore, we investigated the effect of blocking IL-6 receptor signaling on B-cell function. As shown in Figure 5A, the proportion of B cells in the lymph nodes of CTLA4-Ig + MR16-1 mice was lower than that in CTLA4-Ig–alone mice. This proportion was roughly equivalent to that in the naive control. Thus, recruitment of B cells to lymph nodes was not inhibited by CTLA4-Ig monotherapy.

The proportion of B cells in the spleens of the CTLA4-Ig + MR16-1 mice was roughly equivalent to that in mice treated with only CTLA4-Ig (data not shown). Thus, blocking IL-6 receptor signaling reduced recruitment of B cells to regional lymph nodes. Based on this result, we conducted an investigation of anti-donor Ab function using an in vitro killing assay. Our data suggest that the killing activity of the serum of the CTLA4-Ig + MR16-1 mice was lower not only than that of the naive control, but also than that of the CTLA4-Ig–alone mice. Over 90% of the BALB/c cells were not killed by CTLA4-Ig + MR16-1 mouse serum (Figure 5B). This suggests that differentiation of B cells into Ab-forming cells was inhibited by blocking IL-6 receptor signaling. Collectively, these data suggest that blocking IL-6 receptor signaling not only reduces recruitment of B cells to regional lymph nodes, but also decreases their production of anti-donor Abs.

Discussion

Our results, obtained using a murine model of vascularized cardiac transplant, demonstrate that blocking IL-6 receptor signaling moderates the proinflammatory state during the peritransplant phase. Blocking cytokine activity (eg, using mAbs or soluble receptors) is a rational approach for immunosuppressive therapy of allograft rejection. We found that anti-IL-6 receptor mAbs mitigated tissue inflammation in donor hearts and delayed acute allograft rejection in a mouse model. Therefore, in clinical use, a suitable clinical dose should be determined in combination with other immuno­suppressive therapy.

Although advances in immunosuppressive drugs have greatly decreased the incidence of acute graft rejection in clinical settings, chronic rejection remains a barrier to long-term graft survival.¹⁰ Chronic rejection in cardiac allografts manifests as interstitial fibrosis, vascular occlusion, and progressive deterioration of graft function.¹¹

In naive rodent models, it is easy to inhibit immune responses by blocking T-cell costimulation, but difficult to inhibit immune responses against the graft that ultimately results in chronic rejection.^{23, 24} Immune responses against grafts that ultimately result in chronic rejection can be studied in a mouse vascularized heterotopic cardiac transplant model.^12-14

Our investigation indicates that IL-6 receptor signaling blockade down-regulated activation of peripheral T cells. In the high-dose MR16-1–alone regimen, activation level of CD4+ and CD8+ T cells was down-regulated 7 days after transplant; however, activation levels became equivalent to those of the untreated controls 14 days after transplant (data not shown). Based on these results, we investigated whether this down-regulated activation in the peritransplant phase influenced T-cell recruitment to the allograft using a graft infiltrate cell assay. Our data revealed that blocking IL-6 receptor signaling resulted in not only a decreased number of T cells, but also macrophages and neutrophils in the allograft during the peritransplant phase. Furthermore, the number of CD8+ T cells infiltrating the allograft was significantly lower than the number of CD4+ T cells. This is consistent with previous reports that neutrophils are initially recruited into cardiac allografts through innate immune mechanisms that are independent of the CD8 T cells, and that activation of the CD8 T cells amplifies infiltration and activation of neutrophils to mediate tissue injury within the allograft.^{25, 26} In our experiment using MHC-disparate donor and recipient mice, we demonstrated that blocking IL-6 receptor signaling during the peritransplant phase prolonged cardiac allograft survival and inhibited leukocyte recruitment to the allograft; however, this effect did not continue without additional treatment such as costimulatory blockade.

Results of our graft infiltrate cell assay revealed that blocking IL-6 receptor signaling by continually administering low-dose MR16-1 also resulted in a decreased number of leukocytes in the allograft during the long-term phase (Figure 3D). In particular, the number of CD4+ T cells infiltrating the allograft was significantly lower than the number of CD8+ T cells. This result is opposite to that of the peritransplant phase model. Additionally, the results of our in vitro killing assay showed that the killing activity in the serum of the CTLA4-Ig + MR16-1 mice was significantly reduced compared with that in mice treated with CTLA4-Ig alone. Hirano and associates²² reported that blocking IL-6 receptor signaling inhibited differentiation of B cells into Ab-forming plasma cells. Our data also show that blocking IL-6 receptor signaling markedly reduced anti-donor Ab levels. In this long-term phase model, CD4+ T-cell infiltration to the graft and anti-donor Ab production was reduced by low-dose MR16-1, and so, such a regimen may prevent chronic tissue damage.

Interleukin-6 is primarily induced by antigen-independent mechanisms after transplant.⁸ However, our results show that anti-donor Ab formation is reduced by blocking IL-6 receptor signaling. Thus, multiple factors (including adaptive immunity) may be responsible for inducing allograft-produced and recipient-produced IL-6 after clinical transplant. Additional studies are required to see whether physiological stimuli are involved in this process.

Interleukin-6 has been suggested as a therapeutic target for fibrosis^{18, 27}; therefore, we blocked IL-6 receptor signaling to assess its role in the fibrosis associated with chronic rejection. Blocking IL-6 receptor signaling reduced the fibrotic area indicative of cardiac fibrosis in chronic rejection grafts (Figures 2A and B). These results implicate IL-6 receptor signaling in the induction of fibrosis in chronic rejection. Further, targeting IL-6 receptor signaling may ameliorate fibrosis while stabilizing functional parameters in cardiac allografts undergoing chronic rejection. These findings also are consistent with those of previous in vitro studies in which blocking IL-6 receptor signaling decreased cardiac fibroblast proliferation.⁹

Beside its roles in fibrosis, IL-6 is also a potent modulator of immune responses in multiple cell types of both the innate and adaptive systems.^28-30 Hence, it is possible that blocking IL-6 receptor signaling ameliorates chronic rejection in this model through immunomodulatory effects. The anti-IL-6 receptor mAbs may prolong graft survival by impairing transition of graft-reactive immunity from innate responses to adaptive responses.³⁰

Additionally, as described above, B-cell trafficking to lymph nodes also was reduced in anti-IL-6 mAb–treated recipients in our study. Collectively, these data suggest that blocking IL-6 signaling inhibits B-cell recruitment to lymph nodes and differentiation into Ab-producing cells. Similar to our investigation, many researchers have found that anti-IL-6 mAbs may alter lymphocyte trafficking to the graft,^31-33 lymphocyte survival,³⁴ activation, and differentiation,³⁵ as well as the immunologic events initiating fibrosis.

In conclusion, we reported the effect of disruption of IL-6 receptor signaling on survival of cardiac allografts during acute and chronic rejection. Our results led to identifying development of cardiac allograft rejection during the peritransplant and long-term phases. These histologic and molecular findings suggest a crucial role for IL-6 in cardiac injury during the peritransplant phase associated with acute graft rejection and long-term phase associated with chronic rejection. Together, these observations indicate that blocking IL-6 receptor signaling represents an effective approach to ameliorate the proinflammatory state associated with acute graft rejection and fibrosis associated with chronic rejection, while stabilizing anatomic and functional parameters of the graft. Further, identifying the underlying mechanisms of the disease process should allow for design of specific therapies aimed at preventing cardiac allografts from entering the proinflammatory state associated with acute graft rejection and so halting the progression of chronic rejection.

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