Inhibition of NF-κB-Dependent T Cell Activation Abrogates Acute Allograft Rejection

Patricia W. Finn, James R. Stone, Mark R. Boothby and David L. Perkins
J Immunol November 15, 2001, 167 (10) 5994-6001; DOI: https://doi.org/10.4049/jimmunol.167.10.5994

Abstract

Using a heterotopic model of transplantation, we investigated the role of T cell activation in vivo during allograft rejection in I-κB(ΔN)-transgenic mice that express a transdominant inhibitor of NF-κB in T cells. Our results show indefinite prolongation of graft survival in the I-κB(ΔN)-transgenic recipients. Interestingly, at the time of rejection of grafts in wild-type recipients, histology of grafts in the I-κB(ΔN)-transgenic recipients showed moderate rejection; nevertheless, grafts in the I-κB(ΔN) recipients survived >100 days. Analysis of acute phase cytokines, chemokine, chemokine receptors, and immune responses shows that the blockade of NF-κB activation in T cells inhibits up-regulation of many of these parameters. Interestingly, our data also suggest that the T cell component of the immune response exerted positive feedback regulation on the expression of multiple chemokines that are produced predominantly by non-T cells. In conclusion, our studies indicate NF-κB activation in T cells is necessary for acute allograft rejection.

Nuclear factor-κB comprises a family of NF-κB/Rel transcription factors that regulates the transcription of multiple inflammatory and immune genes. NF-κB family members are widely expressed in multiple cell types and include p50, p52, RelA (p65), c-Rel, and RelB. In studies of T cell activation, NF-κB amplifies the expression of multiple chemokine and cytokine genes (1, 2). Thus, NF-κB is a potent proinflammatory signal transduction molecule in T cells. Relevant to clinical transplantation, corticosteroids, which are a common treatment for allograft rejection, inhibit NF-κB activation (3, 4, 5). However, it remains undetermined whether the crucial site of action of corticosteroids involves T cells or other types of inflammatory cells.

In the resting state, NF-κB is retained in the cytoplasm as a complex bound by I-κB. After activation, I-κB is phosphorylated, ubiquinated, and subsequently degraded in the proteosome, thus facilitating NF-κB translocation to the nucleus, where it functions as a regulator of transcription (6, 7, 8). Mutations of the phosphorylation site of I-κBα can create dominant-negative mutants that inhibit NF-κB functions by blocking nuclear translocation (9, 10, 11). For example, I-κB(ΔN), which contains a deletion of the phosphorylation site, functions as a transdominant inhibitor of NF-κB activation. Transgenic mice that express an I-κB(ΔN) transgene regulated by the T cell-specific proximal lck promoter showed decreased cytokine production and defective T cell proliferative responses that are not corrected with the addition of exogenous IL-2 (12). Moreover, the I-κB(ΔN)-transgenic mice exhibit decreased susceptibility to allergic airway hyperresponsiveness and collagen-induced arthritis (13, 14). Our current study analyzes allograft rejection in I-κB(ΔN)-transgenic mice. In addition to multiple parameters of rejection, our study investigated the role of NF-κB-dependent T cell activation on the expression of subsets of chemokines, cytokines, and other immune genes up-regulated during the early vs late immune response after transplantation. Our results demonstrate that the inhibition of NF-κB activation in T cells abrogates up-regulation of chemokines and cytokines during the late phases of rejection, resulting in indefinitely prolonged allograft survival.

Materials and Methods

Vascularized heterotopic cardiac transplantation

Murine hearts were transplanted, as previously described (15). Briefly, hearts were harvested from freshly sacrificed donors and immediately transplanted into 8- to 12-wk-old recipients that were anesthesized via i.p. injection with 60 mg/kg pentobarbital sodium. The donor aorta was attached to the recipient abdominal aorta by end-to-side anastamosis, and the donor pulmonary artery was attached to the recipient vena cava by end-to-side anastamosis. All surgical procedures were completed in less than 60 min from the time that the donor heart was harvested. Donor hearts that did not beat immediately after reperfusion or stopped within 1 day following transplantation were excluded (>95% of all grafts functioned at day 2 following transplantation). Donor grafts were harvested at the indicated times following transplantation and divided into equal sections for preparation of RNA and tissue sections for histology. Clinical rejection was defined as the cessation of a palpable heartbeat and was confirmed by histology using the International Society for Heart and Lung Transplantation criteria (16).

Mice

Eight- to 12-wk-old male wild-type BALB/cByJ (BALB/c) (H-2d) and C57BL/6J (B6) (H-2b) mice (The Jackson Laboratory, Bar Harbor, ME) were used as donor and recipients, respectively, in the transplant experiments. The I-κB(ΔN)-transgenic mice were also used as recipients and produced by injecting an amino-terminally truncated form of I-κBα, including aa 37–317 linked to the proximal lck promoter plus the locus control region from the human CD2 gene into C57BL/6 × DBA/2 zygotes (12) (see Table I⇓). Founder mice were backcrossed with C57BL/6 mice for four generations, and transgene expression was determined by Southern blot analysis. Mice were maintained in virus Ab-free facility in accordance with federal and state government regulations after Harvard Medical School institutional approval.

View this table:
Table I.

Increased allograft survival in the I-κB(ΔN) recipientsa

‘RNase protection assay

Chemokine, chemokine receptor, and CD marker expression was analyzed by RNase protection assay (RPA),3 as previously described (17). Briefly, total RNA was isolated from hearts using RNAzol and analyzed using the RiboQuant MultiProbe RPA System (BD PharMingen, San Diego, CA). A total of 15 μg of RNA was used per hybridization and RNase reaction with the templates mCK-5 (lymphotactin (Ltn), RANTES, eotaxin, macrophage-inflammatory protein-1β (MIP-1β), MIP-1α, MIP-2, IFN-γ-inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), and macrophage inhibitory factor), mCK2b (IL-1α, IL-1β, IL-1R antagonist (IL-1ra), IL-6), mCK3b (TNF-α), mCR-5 (CCR1, 1β, 4, 5, and 2), a custom template (CXCR2, 3, 4, and CCR6, 8α, 8β, and CXCR5 (BLR-1)), and mCD-1 (TCRδ, TCRα, CD3ε, CD4, CD8α, CD8β, CD19, CD14, CD45) (BD PharMingen). The IP-10 template detects the C57BL/6 allele. The protocol was modified to use 35S-labeled probes to hybridize RNA. After RNase treatment and purification, protected probes were electrophoresed on a denaturing 5% polyacrylamide gel. The gels were exposed in a Molecular Dynamics (Sunnyvale, CA) PhosphorImager. The identity of each protected fragment was established by analyzing its migration distance against a standard curve of the migration distance vs the log nucleotide length for each undigested probe. Samples were normalized to the housekeeping gene, GAPDH. Protected bands were quantitated by densitometry analysis using ImageQuant software (Molecular Dynamics). All results represent a minimum of two independent analyses.

Histology

Recipient native hearts and donor-transplanted hearts were harvested at the indicated times following transplantation and fixed in 10% neutral buffered Formalin. After dehydration and paraffin embedding, 5- to 6-μm-thick sections were routinely stained with H&E. Multiple sections were examined for each heart, and the extent of histological rejection (grade 0–4) and ischemia (grade 0–4, determined by the degree of healing ischemia) was quantified using a modified International Society of Heart and Lung Transplantation grading scale (16). Any samples with an ischemia score >1 were excluded from further analysis.

Mixed lymphocyte culture

A total of 2 × 105 responder spleen cells was stimulated with 4 × 105 stimulator spleen cells that had been irradiated with 2000 rad in 200 μl of RPMI 1640 plus 10% FCS. A total of 1 μCi of [3H]thymidine was added during the last 12 h of culture. After 96 h, cells were harvested and thymidine incorporation was determined, as described elsewhere (18). Cultures were performed in quadruplicate, and SEM were <10%.

ELISA

Cytokines TNF-α, IL-6, and IL-1β were evaluated using a Quantikine M immunoassay (R&D Systems, Minneapolis MN) per the manufacturer’s directions. Briefly, the specific Ab for each cytokine was precoated in microtiter wells. A total of 100 μl of serum sample obtained by cardiac puncture or cytokine standard was incubated overnight at 4°C. An enzyme-linked polyconal Ab specific for the particular cytokine was added to the wells and incubated for 2 h. After washing, the substrate tetramethylbenzidine plus hydrogen peroxide was added and incubated 30 min at room temperature. After adding a stop solution of dilute hydrochloric acid, the OD was measured with an Emax microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm wavelength, along with a correction reading at 540 nm wavelength for optical interference by the microtiter plate. Triplicate readings of the control and serum samples and duplicate readings of each standard were averaged after subtracting the background standard OD. Final sample calculations were based on a regression analysis of the log of the final OD vs the log of standard dilutions. The sensitivity of detection is 3, 3.1, and 2 pg/ml for IL-1β, IL-6, and TNF-α, respectively.

Statistics

Graft survival data were calculated as mean, and p values were calculated using Kaplan-Meier/log rank test methods, and differences were considered significant at p < 0.05. Differential expression of mRNA determined by RPA on days 0, 1, 3, 5, and 7 in the I-κB(ΔN) and wild-type recipients was analyzed by two-factor ANOVA. Statistical significance of variances was calculated for p < 0.05 using the F test.

Results

Inhibition of NF-κB activation in T cells prolongs allograft survival

In this study, we investigated the role of NF-κB activation in T cells in a murine model of vascularized heterotopic allogeneic heart transplantation. To inhibit NF-κB activation in T cells, we analyzed I-κB(ΔN)-transgenic mice that express a transdominant inhibitor of NF-κB (I-κB(ΔN)) driven by the the T cell-specific proximal lck promoter. Cardiac transplants were performed across a complete MHC mismatch using BALB/c (H-2d) donors and I-κB(ΔN) (in a C57BL/6 (H-2b) background) recipients. In the wild-type group, BALB/c grafts were transplanted into C57BL/6 (B6) (H-2b) recipients. All grafts transplanted into the I-κB(ΔN) recipients survived >100 days compared with 7.8 days in the wild-type B6 recipients (p < 0.0001) (Table I⇑). Thus, expression of the transdominant inhibitor of NF-κB in T cells provided effective protection against acute allograft rejection in our model.

To characterize acute rejection and the recruitment of inflammatory cells, we analyzed graft histology at days 1 and 7 following transplantation (Fig. 1⇓). On day 1, the I-κB(ΔN) recipients had no evidence of rejection (grade 0) with focal mononuclear cell infiltration. By day 7, when the wild-type control B6 recipients had grade 4 rejection including myocyte necrosis, the I-κB(ΔN) recipients had developed grade 2 rejection with moderate focal infiltrates, but without myocyte necrosis. Interestingly, despite histological evidence of moderate rejection (grade 2) in the I-κB(ΔN) recipients, all of the grafts survived >100 days (Table I⇑).

FIGURE 1.
FIGURE 1.

Histology. Hearts from untransplanted BALB/c (a), and graft hearts from BALB/c→B6 graft heart day 1 (b), BALB→B6 graft heart day 7 (c), BALBc→I-κB(ΔN) day 1 (d), and BALBc→I-κB(ΔN) day 7 (e) are shown. Donor hearts were harvested 1 and 7 days following transplantation and fixed in 10% neutral buffered Formalin. After dehydration and paraffin embedding, 5- to 6-μm-thick sections were routinely stained with H&E. Multiple sections were examined for each heart, and the extent of rejection was quantified on a scale of 0–4 using a modified International Society of Heart and Lung Transplantation grading scale (original magnification, ×400).

Infiltrating cells in the grafts of I-κB(ΔN) recipients are primarily macrophages

The lack of rejection despite the detection of moderate infiltrates by histology could be due to differential composition of the infiltrating cells. To analyze the types of infiltrating cells, we performed RPA of CD markers, including TCRδ, TCRα, CD3ε, CD4, CD8α, CD8β, F4/80, and CD45 with RNA harvested on days 1, 3, 5, and 7 from graft hearts from I-κB(ΔN) and wild-type B6 recipients (Fig. 2⇓). Quantification of these results by densitometry showed that the infiltrating cells in the graft I-κB(ΔN) recipients from days 1 to 7 predominantly expressed the F4/80 macrophage and CD45 leukocyte markers (Fig. 2⇓, A and B). These results showed that grafts from both the I-κB(ΔN) and wild-type B6 recipients developed macrophage infiltration. However, the level of macrophage infiltration tended to decrease in the I-κB(ΔN) recipients by days 5 and 7, whereas the level increased in the wild-type B6 group. In addition, a low level of T cell markers including TCRα+ and CD8α was detected on days 5 and 7, but the level of expression was less than 5% of control GAPDH. In contrast, in the wild-type B6 group, there were marked increases in TCRα+CD8+ T cells that were detectable by day 5 and exceeded 20% of GAPDH by day 7 (Fig. 2⇓, A and C). To determine the statistical significance of these observations, variances were calculated for each gene analyzed by RPA on days 0, 1, 3, 5, and 7 in the I-κB(ΔN) and wild-type recipients, and significance was calculated as p < 0.05 using the F test (Table II⇓). Based on theseanalyses, the grafts from I-κB(ΔN) recipients had a significant decrease in the level of T cell infiltration compared with the wild-type B6 recipients.

FIGURE 2.
FIGURE 2.

RPA of cell surface markers. RPA analysis of TCRα, CD4, CD8α, F4/80, CD45, and control gene GAPDH in control heart (lane 1), graft hearts in I-κB(ΔN) recipients days 1, 3, 5, and 7 (lanes 2–5), and graft hearts in allogeneic recipients days 1, 3, 5, and 7 (lanes 6–9) (A). Densitometry analysis was performed using ImageQuant software (Molecular Dynamics), and expression of TCRδ, TCRα, CD3ε, CD4, CD8α, CD19, F4/80, and CD45 was quantitated in I-κB(ΔN) (B) and wild-type B6 (C) groups.

View this table:
Table II.

ANOVA of gene expressiona

Diminished chemokine receptor levels in I-κB(ΔN) recipients

Chemokines are important mediators of cell recruitment to inflammatory sites. Therefore, to characterize the cellular infiltrate in the graft hearts, we analyzed the expression of a large panel of chemokine receptors from both the CCR and CXCR families, including CCR1, CCR1β, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8α, and CCR8β, plus CXCR2, CXCR3, CXCR4, and CXCR5 (Fig. 3⇓). CCR2, CCR5, and CXCR4 were moderately increased in the grafts from I-κB(ΔN) recipients (Fig. 3⇓A); however, the level of expression of all of these chemokine receptors remained lower than in the wild-type B6 recipients (Fig. 3⇓B). ANOVA calculations showed that these differences were highly significant for CCR2 (<0.004), CCR5 (<0.001), and CXCR4 (<0.041) (Table II⇑). We also found large increases in chemokine receptor expression in wild-type B6 recipients that includes CCR1, CCR3, and CCR4 in addition to the three receptors up-regulated in the I-κB(ΔN) recipients. Thus, the expression of some chemokine receptors was undetectable in I-κB(ΔN), while detectable in the wild-type controls. Based on the low level of T cell infiltration in the grafts from I-κB(ΔN) recipients, these results suggest that the decreased expression of chemokine receptors in these mice is due to decreased recruitment or decreased cellular activation.

FIGURE 3.
FIGURE 3.

Chemokine receptor expression. RPA analysis of CCR1, 1β, 2, 3, 4, and 5, and CXCR2, 3, 4, and 5 in control, graft hearts in I-κB(ΔN) recipients days 1, 3, 5, and 7, and graft hearts in allogeneic recipients days 1, 3, 5, and 7. Densitometry analysis was performed using ImageQuant software (Molecular Dynamics), and expression was quantitated in I-κB(ΔN) (A) and wild-type B6 (B) allograft recipients.

Decreased immune responses in I-κB(ΔN) recipients: allospecific proliferation and cytokines

Because expression of the transgene is regulated by the T cell-specific proximal lck promoter, our hypothesis was that the global down-regulation of chemokine receptor expression was due to a defect of T cell regulatory functions. To assess the allospecific response of T cells from I-κB(ΔN) mice, we performed MLR (Fig. 4⇓). These results showed a decreased proliferative response to allogeneic stimulator cells by the I-κB(ΔN) compared with wild-type B6 control responder cells. As expected, the response to control syngeneic B6 stimulator cells was comparable by both strains. We also assessed T cell responses in vivo by analyzing the expression of cytokines, including IL-2, IFN-γ, lymphotoxin (LT)α, and LTβ, which are produced, at least in part, by T cells (Fig. 5⇓). IFN-γ and LTβ expression was markedly increased in the wild-type B6 control grafts, whereas the level of expression remained low (<3% of GAPDH) in the I-κB(ΔN) recipients. Both IL-2 and LTα were expressed at levels barely detectable by RPA at all time points analyzed in both groups. ANOVA calculations showed that the increased expression was significant for IFN-γ (<0.006) and LTβ (<0.042) (Table II⇑). Taken together, these results are consistent with decreased T cell responses both in vitro and in vivo in the I-κB(ΔN) recipients.

FIGURE 4.
FIGURE 4.

Mixed lymphocyte responses. Splenic lymphocytes were harvested from B6 or I-κB(ΔN) mice and stimulated with irradiated syngeneic B6 (syngeneic) or control BALB/c (allogeneic) spleen cells. A total of 1 μCi of [3H]thymidine was added during the last 12 h of culture. After 96 h, cells were harvested and thymidine incorporation was determined. Cultures were performed in quadruplicate, and SEM were <10%.

FIGURE 5.
FIGURE 5.

Expression of Th1 cytokines. RPA analysis of LTα, LTβ, IFN-γ, and IL-2 in control hearts, graft hearts in I-κB(ΔN) recipients days 1, 3, 5, and 7, and graft hearts in allogeneic recipients days 1, 3, 5, and 7. Densitometry analysis was performed using ImageQuant software (Molecular Dynamics), and expression was quantitated in I-κB(ΔN) (A) and wild-type B6 (B) allograft recipients.

Acute phase responses in I-κB(ΔN) recipients: an initial increase is followed by decreased expression

Our analysis of graft histology, the composition of the cellular infiltrates, and chemokine receptor expression suggested decreased positive feedback of the inflammatory response in the transgenic I-κB(ΔN) recipients. Because the defect in the transgenic recipients appears to be limited to the T cells, these observations suggested that the defect was localized within T cell activity. To test this hypothesis, we analyzed the acute phase response, which comprises an aspect of the innate immune response including the cytokines TNF-α, IL-6, IL-1β, and IL-1ra that are produced primarily by non-T cells, but possibly regulated by T cell-dependent mechanisms. Analysis of serum cytokines revealed that on day 1 after transplantation, both IL-6 and IL-1β were increased in both the I-κB(ΔN) and wild-type B6 groups, although to a lesser extent in the I-κB(ΔN) group (Fig. 6⇓A). However, on day 7 after transplantation, IL-6 levels are further increased in the wild-type B6 recipients, whereas expression in the I-κB(ΔN) group has decreased to low levels (Fig. 6⇓B).

FIGURE 6.
FIGURE 6.

Acute phase response cytokine expression in serum and graft heart. Serum was obtained from untransplanted control B6, BALB/c→I-κB(ΔN), and BALB/c→B6 mice at day 1 (A) and day 7 (B) following transplantation, and analyzed by ELISA for the production of cytokines (TNF-α, IL-6, and IL-1β), as described in Materials and Methods. Control serum is from an untransplanted BALB/c mouse.

Analysis of RNA levels of the acute phase cytokines in the graft tissue at day 1 following transplantation indicates that all three of these acute phase reactants, as well as IL-1ra, are modestly increased in both the B6 and I-κB(ΔN) groups (Fig. 7⇓). However, by day 7 following transplantation, IL-6, IL-1β, and IL-1ra are markedly increased in the wild-type B6 group, but not the I-κB(ΔN) recipients. On days 3 and 5 posttransplantation, the levels of acute phase cytokines decreased in the I-κB(ΔN), but increased in the wild-type B6 recipients (not shown). ANOVA calculations showed that the differential expression was significant for IL-1β (<0.008) and IL-1ra (<0.013), but not for TNF-α or IL-6 (Table II⇑). These results indicate that although the acute phase cytokines are up-regulated on day 1 at the level of mRNA in both the I-κB(ΔN) and wild-type B6 groups, a block in NF-κB activation in T cells can prevent further up-regulation of acute phase cytokine levels by day 7.

FIGURE 7.
FIGURE 7.

RPA of acute phase cytokine expression. RPA analysis of TNF-α, IL-1β, IL-6, and IL-1ra in control hearts, graft hearts in I-κB(ΔN) recipients days 1, 3, 5, and 7, and graft hearts in allogeneic recipients days 1, 3, 5, and 7. Densitometry analysis was performed using ImageQuant software (Molecular Dynamics), and expression was quantitated at day 1 (A) and day 7 (B) following transplantation.

Chemokines MIP-1α, MIP-1β, MCP-1, and MIP-2 in I-κB(ΔN) recipients: initial increase, followed by decrease

Chemokines were up-regulated by day 1 posttransplantation in both the I-κB(ΔN) (Fig. 8⇓A) and wild-type B6 recipients (Fig. 8⇓B), although the magnitude of the increase was already greater on day 1 in the control recipients. A kinetic analysis of expression showed that the levels of expression tended to decrease through days 3–7 following transplantation in the I-κB(ΔN) recipients. In contrast, the level of expression markedly increased on days 5 and 7 following transplantation in the control group. ANOVA calculations showed that the differential expression was significant for MIP-1α (<0.001), MIP-1β (0.005), MIP-2 (<0.005), and MCP-1 (<0.030) (Table II⇑). These results suggest that chemokines that are inducible by innate immunity can be further up-regulated by adaptive responses, presumably by T cell-dependent mechanisms. However, inhibition of NF-κB activation in T cells prevents the amplification of the chemokine expression.

FIGURE 8.
FIGURE 8.

Expression of chemokines MIP-1α, MIP-1β, MIP-2, and MCP-1. RPA analysis in control hearts, graft hearts in I-κB(ΔN) recipients days 1, 3, 5, and 7, and graft hearts in allogeneic recipients days 1, 3, 5, and 7. Densitometry analysis was performed using ImageQuant software (Molecular Dynamics), and expression was quantitated in I-κB(ΔN) (A) and wild-type B6 (B) recipients following transplantation.

Chemokines Ltn, RANTES, and IP-10 are increased in wild-type B6 group, but not I-κB(ΔN) recipients

In our analysis of the I-κB(ΔN) recipients, we found minimal increases of Ltn, RANTES, and IP-10 chemokines detected from days 1 through 7 following transplantation in the I-κB(ΔN) recipients (Fig. 9⇓a); however, consistent with our previous results, all four of these chemokines were highly induced by days 5 and 7 in the control B6 recipients (Fig. 9⇓b). ANOVA calculations showed that the differential expression was highly significant for Ltn (<0.003), RANTES (<0.001), and IP-10 (<0.001) (Table II⇑). These results indicate that up-regulation of this subset of chemokines, which is dependent upon the adaptive immune response, is not up-regulated in the I-κB(ΔN) recipients that have deficient NF-κB activation in T cells.

FIGURE 9.
FIGURE 9.

Expression of chemokines Ltn, RANTES, and IP-10. RPA analysis in control heart (lane 1), graft hearts in I-κB(ΔN) recipients days 1, 3, 5, and 7, and graft hearts in allogeneic recipients days 1, 3, 5, and 7. Densitometry analysis was performed using ImageQuant software (Molecular Dynamics), and expression was quantitated in I-κB(ΔN) (A) and wild-type B6 (B) recipients following transplantation.

Discussion

The NF-κB/Rel family of transcription factors has pleiotropic functions involving the up-regulation of multiple inflammatory and immune genes (19). Previous studies in transplantation investigating NF-κB have focused on the role of p50; these studies showed modest prolongation of graft survival (20). Although NF-κB/Rel proteins are widely expressed in most cell types, in this study we focused on the role of NF-κB activation in T cells in a model of vascularized heterotopic heart transplantation. To inhibit NF-κB activation in T cells, we used I-κB(ΔN)-transgenic mice that express a transdominant inhibitor of NF-κB in T cells (12). Previous studies reported detection of the I-κB(ΔN) transgene in peripheral lymphoid tissue, but not T cell-depleted spleen cells, confirming predominant T cell expression of the transgene (14). As expected, our studies showed decreased T cell responses determined in vitro by mixed lymphocyte reactions and in vivo by decreased cytokines, including reduced levels of IFN-γ and LTβ. These results are consistent with previous studies showing decreased production of IFN-γ in vivo in the I-κB(ΔN)-transgenic mice in a model of allergic pulmonary inflammation (13).

In this study, cardiac transplants in I-κB(ΔN) recipients all survived until the recipients were sacrificed after >100 days compared with a mean survival time of 7.8 days in wild-type B6 recipients. Thus, inhibition of NF-κB in T cells was sufficient to inhibit acute allograft rejection. However, histological analysis of allografts in the I-κB(ΔN) recipients at day 7 following transplantation, a time immediately preceding acute rejection in the wild-type B6 recipients, showed grade 2 rejection with mononuclear cell infiltration compared with grade 0 in syngeneic control grafts. Characterization of the phenotype of the infiltrating cells demonstrated that the composition of the infiltrate consisted predominantly of F4/80+ macrophages and CD45+ leukocytes; however, only extremely low levels of T or B cell markers were detected. Decreased detection of lymphocytes could be due to decreased migration into the graft, decreased proliferation, or increased apoptosis. As expected, the control wild-type B6 recipients had evidence of infiltrating T cells in addition to F4/80+ macrophages and leukocytes. Although F4/80+ macrophages were present, the lack of high levels of TNF-α and IL-1β, cytokines commonly produced by macrophages, suggests that the macrophages may not be fully activated. Also, we analyzed chemokine receptor levels in the graft tissue. These results showed decreased levels of multiple chemokine receptors of both the CCR and CXCR families, including CCR1, CCR1β, CCR2, CCR3, CCR4, CCR5, and CXCR4. Although these receptors can be expressed by multiple cell types, all have been reported to be expressed by T cells (20), which could partially account for the decreased levels in the I-κB(ΔN) recipients following transplantation. Taken together, these studies indicate that T cells expressing the transgene were inefficient at infiltrating allografts.

To investigate mechanisms of rejection that were defective in the I-κB(ΔN) recipients, we focused on kinetic analyses of cytokine and chemokine expression starting at time points 24 h following transplantation. The acute phase response is an important component of innate immunity mediated by cytokines, including IL-1β, IL-6, and TNF-α in response to infection, stress, or injury (21). Interestingly, our analysis of the acute phase reactants, in particular IL-6 and IL-1β, indicates an increase at day 1 after transplantation in both the I-κB(ΔN) and wild-type B6 recipients at the levels of both serum cytokines and graft heart mRNA. In contrast, at day 7 after transplantation, the wild-type B6 recipients expressed increased levels of IL-6 in the serum and IL-6, IL-1β, and IL-1ra mRNA, whereas in the I-κB(ΔN) recipients all of these acute phase reactants were decreased. Both groups express similar levels of TNF-α mRNA at both time points without marked increases in the serum, suggesting that TNF-α may be functioning locally within the graft at these time points. Interestingly, in the wild-type B6 group, increased IL-6 mRNA correlates with the serum cytokine level at both days 1 and 7; however, although IL-1β mRNA is increased at both time points, at day 7 serum IL-1β levels are low. One interpretation is that IL-1β is produced by multiple sites, including locations peripheral to the graft. Alternatively, at day 7 after transplantation, IL-1-converting enzymes could be activated, leading to the degradation of IL-1β in the serum. In contrast, the levels of IL-6 mRNA and serum protein correlate at both time points, suggesting that IL-6 may be produced, at least predominantly, by cells within the graft.

The chemokines MIP-1α, MIP-1β, MIP-2, and MCP-1 were increased 1 day following transplantation in both the I-κB(ΔN) and wild-type B6 groups, although the level expressed was lower in the I-κB(ΔN) mice. Using lymphocyte-deficient RAG knockout mice, we have found that innate immune responses were sufficient to induce these same four chemokines in the absence of lymphocytes 1 day following transplantation (data not shown). These observations suggest the possibility that the lower chemokine levels observed in this study were due to the lack of positive feedback regulation from activated T cells in the I-κB(ΔN)-transgenic mice. This hypothesis is supported by our kinetic analysis indicating decreased chemokine levels in the I-κB(ΔN) recipients at days 3 through 7, whereas chemokine levels were markedly increased by days 5–7 in the wild-type B6 recipients. This conclusion is further supported by previous reports indicating that the major producers of these four chemokines are non-T cells (22, 23). In contrast, increased production of TGF-β1 and TGF-β3 is not inhibited in the I-κB(ΔN) recipients.

A common paradigm of immunity is that activation of innate immunity is important, or even a prerequisite, for the initiation of an adaptive immune response (24). In this paradigm, innate responses produce chemokines and cytokines and up-regulate costimulatory ligands and adhesion molecules, creating a milieu that promotes and regulates the activation of adaptive immunity. Interestingly, our results suggest an additional reciprocal pattern of regulation in which adaptive immunity produces positive feedback regulation of the innate response. Furthermore, our studies indicate that NF-κB-dependent activation of the T cell component of adaptive immunity is required for the positive feedback.

In our current experiments, levels of the chemokines RANTES, Ltn, and IP-10 were also markedly decreased in the I-κB(ΔN) recipients. Previous studies have shown that RANTES and Ltn, although produced by multiple cell types, are highly up-regulated in T cells. Thus, these results suggest that inhibition of NF-κB in T cells may directly decrease production of RANTES and Ltn. In contrast, IP-10 is produced primarily by monocytes, endothelial cells, keratinocytes, and fibroblasts, but not T cells. However, IP-10 is induced by IFN-γ and TNF-α, two cytokines that can be produced by Th1 T cells. Thus, decreased production of IP-10 in the I-κB(ΔN) recipients may be due to reduced secretion of IFN-γ or TNF-α by T cells. Previous studies have shown that transcription of TNF-α is regulated, at least in part, by NF-κB (25, 26). These observations indicate that the T cell component of adaptive immunity is crucial for the up-regulation of these chemokines during days 5 to 7 of allograft rejection. Thus, in the I-κB(ΔN) recipients of allografts, the expression of RANTES, Ltn, and IP-10 is blocked.

In this study, we have analyzed allograft survival, T cell functions, and levels of genes, including cell surface markers, chemokine receptors, chemokines, and cytokines. Using this approach, we have assessed the NF-κB-dependent components of an immune response in a model of transplantation. Future studies will be required to analyze the function of individual genes in the process of rejection. Our results demonstrate that NF-κB activation in T cells is necessary for multiple parameters of in vitro and in vivo T cell activation and for acute rejection of vascularized cardiac allografts. In addition, our results indicate that the T cell component of the adaptive immune response exerts positive feedback regulation on the early inflammatory response.

Acknowledgments

We are grateful to Thomas Mueller for critical review of this manuscript. We thank Min Xu for technical support.

Footnotes

  • ↵1 This work was supported by grants from the American Heart Association, Arthritis Foundation, and National Institutes of Health (AI44085) to D.L.P.

  • ↵2 Address correspondence and reprint requests to Dr. David L. Perkins, Brigham and Women’s Hospital, PBB-170, 75 Francis Street, Boston, MA 02115. E-mail address: dperkins{at}rics.bwh.harvard.edu

  • ↵3 Abbreviations used in this paper: RPA, RNase protection assay; IP-10, IFN-γ-inducible protein-10; LT, lymphotoxin; Ltn, lymphotactin; MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein; IL-1ra, IL-1R antagonist.

  • Received June 18, 2001.
  • Accepted August 31, 2001.

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