Both humoral and cellular immune responses are involved in renal allograft rejection. Interleukin (IL)-6 is a regulatory cytokine for both B and Foxp3 (forkhead box P3)-expressing regulatory T (Treg) cells. This study was designed to investigate the impact of donor IL-6 production on renal allograft survival. Donor kidneys from IL-6 knockout (KO) vs. wild-type (WT) C57BL/6 mice (H-2b) were orthotopically transplanted to nephrotomized BALB/c mice (H-2d). Alloantibodies and Treg cells were examined by fluorescence-activated cell sorting analysis. Graft survival was determined by the time to graft failure. Here, we showed that a deficiency in IL-6 expression in donor kidneys significantly prolonged renal allograft survival compared with WT controls. IL-6 protein was upregulated in renal tubules and endothelium of renal allografts following rejection, which correlated with an increase in serum IL-6 compared with that in those receiving KO grafts or naive controls. The absence of graft-producing IL-6 or lower levels of serum IL-6 in the recipients receiving IL-6 KO allografts was associated with decreased circulating anti-graft alloantibodies and increased the percentage of intragraft CD4+CD25+Foxp3+ Treg cells compared with those with WT allografts. In conclusion, the lack of graft-producing IL-6 significantly prolongs renal allograft survival, which is associated with reduced alloantibody production and/or increased intragraft Treg cell population, implying that targeting donor IL-6 may effectively prevent both humoral and cellular rejection of kidney transplants.
- kidney transplant
- humoral rejection
- cellular rejection
- Treg cell
kidney transplantation is the best treatment option for patients with end-stage kidney disease, evidenced by that patients receiving renal transplant live longer and better life over similar patients who are on dialysis (14, 27); however, transplant function can be terminated by the rejection that is mediated by both alloantibodies and cellular immune responses (5, 29). A recent statistical analysis indicates that the 10-yr kidney graft survival rate is only 58% for living donor, 45% for nonexpanded criteria donor, and 28% for expanded criteria donor (21). Thus, development of novel strategies is desperately needed to improve kidney graft survival.
Interleukin (IL)-6 is a key regulatory cytokine for both antibody- and T cell-mediated immune responses; it not only is a growth factor for B cell proliferation and differentiation or an inducer of antibody production, but it also regulates CD4+ T cell differentiation (7, 12). Recently, it has been reported that IL-6 is a negative regulatory factor for the development of CD4+CD25+forkhead box P3 (Foxp3)+ T regulatory (Treg) cells; following antigen stimulation, transforming growth factor (TGF)-β induces the differentiation of nonregulatory CD4+CD25− T cells to CD4+CD25+Foxp3+ Treg cells by upregulation of Foxp3 expression (4, 9), but the Treg cell differentiation can be completely inhibited by the presence of IL-6 (2), resulting in redirecting TGF-β-induced T cell differentiation from Treg cells to IL-17-producing effector Th17 cells (2, 31). Furthermore, IL-6 is found to promote the conversion of Foxp3+ Treg cells to Th17 linage (1). All these studies suggest that IL-6 not only stimulates antibody-mediated immune response, but may also act as a suppressive cytokine for Foxp3+ Treg cell development.
Accumulating evidence in literature suggests that Foxp3+ Treg cells are required for maintaining immune homeostasis and immune tolerance to transplants (3, 16), Our previous study has demonstrated that the population of intragraft Foxp3+ Treg cells declines while renal expression of IL-6 is upregulated following renal allograft rejection in a mouse model (35), and neutralization of IL-6 activity in cocultures of renal tubular epithelial cells (TECs) with allogeneic CD25− splenocytes enhances CD4+Foxp3+ cell expansion (35). In renal allograft biopsies from patients, Foxp3+ cells are present in the tubulitis (11, 32) as well as IL-6 expression in renal resident cells, particularly TECs, is upregulated (26). In heart transplantation, IL-6-deficient grafts survive approximately three times longer than wild-type controls, and graft-producing IL-6 promotes the activation of peripheral CD4+ and CD8+ T cells (17). However, whether renal allograft-derived IL-6 regulates the development of intragraft Foxp3+ Treg cells and alloantibodies and consequently influences the graft survival has not been investigated yet. In this study, we examined whether a deficiency in donor IL-6 expression promoted intragraft Treg cell expansion and reduced alloantibodies, resulting in the prolongation of renal allograft survival in a mouse model.
MATERIALS AND METHODS
All strains of mice, wild-type C57BL/6J (B6, WT), B6.129S2-Il6tm1Kopf/J (IL-6 KO), and BALB/cJ, were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facility of the Jack Bell Research Centre of the University of British Columbia (Vancouver, British Columbia, Canada) or The University of Western Ontario (London, Ontario, Canada). Animal experiments were performed in accordance with the Canadian Council on Animal Care guidelines under protocols approved by the Animal Use Subcommittee at our institution.
Kidney donors from male WT or IL-6 KO mice (10–12 wk old, H-2b) were orthotopically transplanted to bilaterally nephrectomized male BALB/cJ (10–12 wk old, H-2d) recipient mice as described previously (8). Briefly, animals were anesthetized with isoflurane, and the donor kidney, ureter, and bladder were recovered en bloc, including the renal artery and the renal vein. After bilateral recipient nephrectomies, the harvested donor graft was revascularized with end-to-side anastomoses between the donor renal artery and recipient abdominal aorta, as well as the donor renal vein and recipient inferior vena cava. Total ischemic time was limited to 25 min. Donor and recipient bladders were attached dome to dome. Both native kidneys were removed at the time of transplantation. The life-supporting kidney allograft survival was followed by daily examinations of overall animal health.
Protein expression of IL-6 and Foxp3 in sections of renal allograft was assessed by the immunohistochemical method as described in our previous study (35). In brief, after deparaffin and rehydration, tissue sections were incubated with goat anti-IL-6 antibody (clone AF-406-NA, R&D System, Minneapolis, MN) or with rat anti-mouse/rat Foxp3 antibody (clone FJK-16s, eBioscience, San Diego, CA) overnight at 4°C. The immune complex of IL-6/anti-IL-6 antibody or Foxp3/anti-Foxp3 antibody on the tissue section was detected using anti-goat Ig (for IL-6) or anti-rat Ig antibody (for Foxp3) conjugated with biotin, which was visualized with the 3,3′-diaminobenzidine peroxidase substrate kit (Vector Labs, Burlington, ON). Negative controls for staining were the sections stained with normal rat IgG as the first antibody or without this antibody. Foxp3+ cells were semiquantitatively counted under ×400 magnification [high-power field (hpf)] in a blinded fashion. The average at least 20 nonoverlapping views were obtained for each graft.
Isolation and determination of Treg cells.
The infiltrates in kidney graft were isolated as described in our previous study (35). In brief, after perfusion with PBS, kidney grafts were minced and digested with collagenases D (Worthington Biochemicals, Lakewood, NJ). The resultant cell suspensions were filtered through 30-μm nylon mesh, and leukocytes from the cell suspension were recovered from interphase by a density centrifugation using 1.083 g/ml Histopaque (Sigma Canada, Oakville, ON).
CD4+CD25+Foxp3+ Treg cell population in cell suspensions (graft infiltrates and splenocytes) was determined by fluorescence-activated cell sorting (FACS) analysis using Mouse Regulatory T cell Staining kit following manufacturer's protocol (eBioscience). Briefly, cells were probed with anti-CD4-FITC (Fluorescein isothiocyanate) and anti-CD25-PE (phycoerythrin) for 30 min at 4°C. After fixation, cells were probed for intracellular Foxp3 using APC (allophycocyanin)-labeled anti-Foxp3 antibody or isotype control antibody (PharMingen) for 30 min at room temperature (RT) or 4°C overnight. Finally, cells were washed with permeabilization buffer and counted using a calibur flow cytometer (BD Biosciences, Mississauga, ON). Data were analyzed using either CellQuest (BD Biosciences) or FlowJo software.
Measurement of serum IL-6 and anti-graft alloantibodies.
Serum levels of IL-6 in recipient mice vs. naive BALB/c mice were measured by ELISA using Mouse IL-6 ELISA Ready-SET-GO kit (eBioscience) following manufacturer's protocol.
Anti-graft alloantibodies in sera were measured by FACS analysis using the calibur flow cytometer as described previously (15). Briefly, splenocytes (0.5 × 106 cells in 0.1 ml of RPMI 1640 medium containing 2% bovine calf serum) were isolated from B6 mice as target cells and were incubated with sera from recipient mice vs. naive BALB/c mice (background control) at a serial dilution (1:100, 1:300, and 1:600) for 30 min at RT. After being washed twice with ice-cold PBS, cell suspension was incubated with goat anti-mouse IgG-FITC (BD Biosciences) at 4°C. The fluorescence intensity for alloantibodies against allogeneic lymphocytes in each serum-incubated sample was determined using FACS analysis as described in results. Briefly, the lymphocytes were gated in the scatter plot of forward scatter (SSC) vs. side scatter (SSC), followed by visualizing the fluorescence of these cells in a dot plot against SSC. The mean fluorescence intensity (MFI) of antigraft alloantibody-bound cells was measured in a histogram.
Data were collected from individual experiment or mouse in each study for statistical analysis. Kaplan-Meier survival analysis with log-rank test was used for comparison of graft survival between groups, and one-way or two-way ANOVA or t-tests (1-tailed distribution) were used as appropriate for other data analyses. A P value of ≤0.05 was considered significant.
Prolonged survival of renal allografts by genetic knockout of IL-6 from donor kidneys.
It has demonstrated that upregulation of IL-6 expression in renal TECs is seen in renal allografts from patients as well as in a murine model following graft rejection (26, 35). To investigate the impact of donor-derived IL-6 on renal allograft survival, graft survival was first examined in BALB/c recipient mice receiving of B6 donors (WT vs. IL-6 KO). In this kidney transplantation model (B6 to BALB/c recipients, major histocompatibility complex fully mis-matched), accumulative data indicate that ∼80% of renal allografts lose their function from 30 to 60 days after transplantation (35). Similar to this observation, the median survival of recipient BALB/c mice receiving WT B6 renal allografts was 38.5 days (n = 6), and none of them survived more than 100 days in this group, while four out of seven recipient mice transplanted with IL-6 KO renal allografts survived over 100 days (Fig. 1A). The survival analysis using the Mantel-Cox/logrank test indicated that IL-6 KO grafts significantly survived longer than WT controls (P = 0.0018), implying the beneficial effect of donor IL-6 deficiency on renal allograft survival.
To further confirm whether the clinical deterioration of recipient mice, including those with IL-6 KO grafts, was really due to the loss of life-supporting graft function, blood urea nitrogen (BUN) in these recipients was measured compared with that in those with functional grafts. As shown in Fig. 1B, none of the recipients that showed clinical deterioration had a serum level of BUN below 100 mg/dl (108.56 ± 8.72 mg/dl in whole group), while the serum levels of BUN in the recipients with functional IL-6 KO grafts at the end of experiment were 41.25 ± 10.25 mg/dl. Furthermore, histological examination of rejected grafts confirmed that these rejected grafts had much more severity of cellular and vascular rejection, characterized by interstitial hemorrhage and dilation, tubular necrosis or atrophy, and massive cellular infiltration than functional grafts (Fig. 1C).
Reduction of serum IL-6 level in recipients by IL-6 deficiency in kidney graft cells.
In addition to activated leukocytes, renal cells can produce IL-6 in response to stimulation with many inflammatory mediators and cell-cell contact with allogeneic lymphocytes (24, 35), and serum IL-6 levels in patients elevate following kidney transplantation (6, 18, 33). To differentiate the contribution of kidney graft cells vs. recipient's immune cells to serum IL-6, donor kidneys from WT vs. IL-6 KO mice were transplanted to allogeneic recipient mice, and IL-6 expression in graft tissues and recipients' sera were examined on postoperative day 14. At this particular time point, histological examination with hematoxylin and eosin staining indicated that WT and IL-6 KO allografts shared similar cellular infiltration and tubulitis, acute cellular rejection, that were involved almost the whole graft section (Fig. 2). Despite that, graft function in both groups was normal, evidenced by the normal levels of serum creatinine and BUN (data not shown).
Immunohistochemical stain of IL-6 protein in graft sections showed that IL-6 expression was clearly upregulated in both tubular epithelial cells and endothelial cells of WT kidney grafts but not in IL-6 KO grafts following acute rejection, whereas the staining of IL-6 in infiltrates was relatively weak and was the same between these two groups (Fig. 3, A–D). Concurrently, serum IL-6 was measured in kidney transplant recipients vs. naive mice of the same age. As shown in Fig. 3E, the serum IL-6 levels in recipients receiving WT allografts were significantly higher than that in those with KO grafts (mean 7.38 pg/ml in WT group vs. 1.16 pg/ml in KO group, P = 0.0319, n = 4) and in naive mice (mean 0.94 pg/ml; WT group vs. naive group, P = 0.0207). Compared with naive mice, serum IL-6 levels in IL-6 KO transplant recipients were only a slight increase but that was not statistical difference (mean 1.16 pg/ml in KO group vs. 0.94 pg/ml in naive group, P = 0.1242) in this limited number of animals. Taken together, these data show that a deficiency in IL-6 expression in donor kidneys significantly reduces serum IL-6, suggesting that graft cell-producing IL-6 may largely contribute to the increase of serum IL-6 in kidney transplant recipients.
Decrease of serum alloantibodies in recipients receiving IL-6 KO renal allografts.
IL-6 is a key inducer of B cell differentiation to antibody-producing plasma cells and IgG production in vivo (13, 28). The effect of graft cell-producing IL-6 on donor-induced alloantibody production was examined in kidney transplant recipients receiving WT grafts (WT group) vs. IL-6 KO grafts (KO group). Sera were harvested from recipients on postoperative day 14 (n = 4) as mentioned above and naive mice as controls (n = 2), and the detailed procedure of anti-graft alloantibody assay was diagrammed in Fig. 4A. In the scatter plot of forward scatter (FSC) vs. side scatter (SSC), the lymphocytes were gated, and the intensity of FITC was analyzed. Two groups of FITC-bound cells were identified: “higher” intensity and “lower” intensity. The difference of these FITC-bound cells between naive (without graft) and recipient (including response to the graft) mice was found in the intensity of lower group, suggesting that the change of lower group was anti-graft alloantibody response. The MFI of this group of cells was measured using a histogram (Fig. 4A). As shown in Fig. 4, B and C, the levels of alloantibodies in the sera from KO group were significantly lower than those from WT group, evidenced by that the MFI of FITC in KO group was 1,084 ± 14 at a 1:600 dilution, 1,917 ± 25 at a 1:300 dilution, and 4,140 ± 18 at a 1:100 dilution, compared with in WT group 3,886 ± 493 at a 1:600 dilution, 6,832 ± 932 at a 1:300 dilution, and 12,702 ± 1,683 at a 1:100 dilution (P = 0.0007, n = 4), while in control sera from naive mice the MFI was minimal at all of different dilutions (from 253 ± 4 to 293 ± 10). These data suggest that a lack of graft cell-producing IL-6 or lower levels of serum IL-6 (Fig. 3E) result in significantly decreasing serum alloantibodies in kidney transplant recipients.
Increase in intragraft Foxp3+ Treg cell population in IL-6 KO renal allografts.
IL-6 inhibits TGF-β-induced Foxp3+ Treg cell differentiation in vitro (2) and positively regulates the conversion of natural Treg cells to Th17 cells (1); however, the evidence for the negative regulation of Foxp3+ Treg cells by IL-6 in vivo is limited. In the mouse model of kidney transplantation, we demonstrated that the population of intragraft Foxp3+ Treg cells declines following the graft rejection and IL-6 upregulation (35). Here, we examined whether the lack of IL-6 expression in graft resident cells resulted in retaining or increasing intragraft Foxp3+ infiltrates. Both WT and IL-6 KO kidney grafts had a similar interstitial infiltration of mononuclear cells on postoperative day 14 as described above (Fig. 2), but immunohistochemical stain of Foxp3 showed that more Foxp3+ infiltrates were found in the sections of IL-6 KO kidney grafts than those in WT grafts (Fig. 5A), that was also demonstrated by a semiquantitative analysis, showing 75.87 ± 7.65/hpf in IL-6 KO group compared with 40.55 ± 7.03/hpf in WT group (P = 0.0243, n = 4; Fig. 5B).
To further confirm this result and to answer whether the increase in Treg cell population in IL-6 KO group specifically occurred within the grafts, the CD4+CD25+Foxp3+ cells in grafts vs. spleens were analyzed by FACS analysis. As shown in Fig. 6, the percentages of intragraft CD4+CD25+Foxp3+ cells in IL-6 KO renal allografts were higher than those in WT grafts, as indicated by that 43.1 ± 3.4% of gated CD4+CD25+ infiltrates were Foxp3+ cells in IL-6 KO allografts compared with 23.1 ± 4.4% in WT grafts (P = 0.004, n = 4), while this phenotype of Treg cell population in the spleens between these two groups was not significantly different (6.67 ± 1.37% in KO group vs. 5.12 ± 1.78 in WT group). These data suggest that IL-6 produced from graft resident cells may mostly affect intragraft Treg cell population.
In our previous study, we demonstrated a decrease of intragraft Foxp3+ Treg cell population following upregulation of Th17-related cytokines including IL-6 during the graft rejection in a mouse renal allograft model (35), and in vitro neutralization of IL-6 activity favored Foxp3-expressing Treg cell development in cocultures of TECs with allogeneic lymphocytes (35). In this report, we present the data showing that a deficiency in IL-6 expression in donor kidneys significantly prolongs renal allograft survival that correlates with a decrease in both serum IL-6 and alloantibodies, and an increase in intragraft Foxp3+ Treg cell population.
In both human and mouse renal allografts, IL-6 is predominantly produced by renal TECs following graft rejection (26, 35) and largely contributes to the increase in serum IL-6 in kidney transplant recipients as the serum levels of IL-6 in recipients receiving IL-6 KO renal allografts are close to the basal levels in naive mice (Fig. 3E). The beneficial outcome of IL-6-deficient kidney allografts in their survival (Fig. 1) is consistent with the genetic studies of patients with GG vs. CC genotypes at −174 of IL-6 gene promoter polymorphism; −174 C/C of IL-6 gene polymorphism may be prone to producing more IL-6 than those with −174 G/G polymorphism (23), and −174C/C kidney donors are rejected with higher incidence and severity compared with those with −174 G/G or −174G/C polymorphism (20). All these studies indicate that a decrease in graft cell-producing IL-6 could benefit kidney transplant survival.
In organ transplantation, Foxp3+ Treg cells are very potent in the suppression of both alloantigen-reactive CD8+ and CD4+ effector T cells (30) and can specifically induce graft tolerance under appropriate conditions (36). Recent studies demonstrate that an increase in Foxp3+ Treg cells within the grafts is associated with spontaneous acceptance of renal allografts in mice (22, 34) and provide immune privilege to protect allogeneic hematopoietic stem/progenitor cell niche from rejection in the bone marrow (10). Many studies have demonstrated that IL-6 is a negative regulator factor for Foxp3+ Treg cell development (1, 2, 19, 31). Thus, if naive T cells are primed at the site of renal allografts (24), and/or in regional draining lymph nodes during rejection, a lack of IL-6 production from the resident graft cells or lower levels of circulating IL-6 in the lymph nodes may favor Foxp3+ Treg cell differentiation or expansion in vivo (Figs. 5 and 6) while effector Th17 cell development is suppressed. Indeed, our previous study demonstrates the association of disappearing intragraft Foxp3+ Treg cells with upregulation of Th17-related cytokines or Th17 cell development (35), indicating the possibility of infiltrating Foxp3+ Treg cells converting to Th17 cells following the graft rejection. Thus, a high level of IL-6 within renal allograft tissue may downregulate intragraft Foxp3+ Treg cell development in two ways: one is to inhibit Foxp3 expression during T cell differentiation, and the other is to promote the conversion of intragraft Foxp3+ Treg cells to Th17 cells. Therefore, IL-6 deficiency in graft cells may result in an increase in the percentages of intragraft Foxp3+ Treg cells and prolongs graft survival.
It has also been documented that alloantibodies mediate humoral rejecton to kidney transplant and are associated with glomerulopathy, multilamination of peritubular capillary basemembranes, and C4d deposition in the promotion of chronic rejection (5, 25). IL-6 has originally been identified as a B cell growth factor for B cell proliferation and differentiation (12), and an early study showed that IL-6 deficiency in mice results in less antigen-specific IgG production (13). Our present study shows that the lack of IL-6 expression in donor kidneys significantly results in decreasing systemic IL-6 in the recipients in response to renal allografts (Fig. 3E), which may attenuate alloantibody production (Fig. 4) that may consequently benefit to long-term survival of kidney transplants.
In conclusion, current immunosuppressive drugs can improve the short-term survival of organ transplant in patients; however, achieving long-term graft survival still remains a clinically unsolved problem. Better understanding of the role of graft resident cells in graft rejection may provide a solution for inducing long-term graft acceptance. Results from the current experimental study demonstrate that a deficiency of IL-6 expression in renal donors reduces circulating levels of IL-6 and alloantibodies and increases intragraft Foxp3+ Treg cell population that are associated with prolongation of renal allograft survival. These results suggest that targeting IL-6 particularly in the donors could benefit kidney transplant survival in patients.
This work was supported by a grant funded by the Kidney Foundation of Canada (C. Du, H. Chen, and C. Y. C. Nguan; 2008–2010). S. Li received a training award from the Canadian Institutes of Health Research for Health Research Strategic Training Program in Transplantation Research, Vancouver Coastal Health Research Institute.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: H.W., Q.G., Z.L., S.L., W.G., and C.D. analyzed data; H.W., C.Y.N., and C.D. interpreted results of experiments; H.W., Q.G., Z.L., and C.D. prepared figures; H.W., H.C., C.Y.N., and C.D. edited and revised manuscript; H.W., Q.G., Z.L., S.L., W.G., H.C., C.Y.N., and C.D. approved final version of manuscript; Q.G., Z.L., S.L., and W.G. performed experiments; H.C., C.Y.N., and C.D. conception and design of research; C.D. drafted manuscript.
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