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(Journal of Leukocyte Biology. 2002;72:1054-1062.)
© 2002 by Society for Leukocyte Biology

Molecular profiling of the role of the NF-{kappa}B family of transcription factors during alloimmunity

Patricia W. Finn*,{dagger}, Hongzhen He*,{dagger}, Chunyan Ma{dagger},{ddagger}, Thomas Mueller{dagger},{ddagger}, James R. Stone§, Hsiou-Chi Liou||, Mark R. Boothby# and David L. Perkins{dagger},{ddagger}

* Pulmonary Division,
{ddagger} Laboratory of Molecular Immunology, and Departments of
{dagger} Medicine and
§ Pathology, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts;
|| Graduate School of Medical Sciences, Weill Medical College, Cornell University Medical Center, New York, New York; and
# Department of Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee

Correspondence: David L. Perkins, Brigham & Women’s Hospital, PBB-170, 75 Francis St., Boston, MA 02115. E-mail: dperkins{at}rics.bwh.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allograft rejection involves a complex network of multiple immune regulators and effector mechanisms. In the current study, we focused on the role of nuclear factor (NF)-{kappa}B/Rel. Previous studies had established that deficiency of the p50 NF-{kappa}B family member prolonged allograft survival only modestly. However, because of its crucial role in signal transduction in inflammatory and immune responses, we hypothesized that other NF-{kappa}B/Rel family members may produce more profound effects on alloimmunity. Therefore, in addition to p50, we analyzed the role of c-Rel, which is expressed predominantly in lymphocytes. Also, to investigate NF-{kappa}B activation in T cells, we examined transgenic mice that express a transdominant inhibitor of NF-{kappa}B [I{kappa}B({Delta}N)] regulated by a T cell-restricted promoter. Allograft survival was prolonged indefinitely in the c-Rel-deficient and I{kappa}B({Delta}N)-transgenic recipients. To determine the molecular basis of NF-{kappa}B modulation of rejection, we analyzed a panel of 58 parameters including effector molecules, chemokines, cytokines, receptors, and cellular markers using hierarchical clustering algorithms and self-organizing maps in p50-/-, c-Rel-/-, and I{kappa}B({Delta}N)-transgenic, experimental groups plus allogeneic-, syngeneic-, and lymphocyte-deficient (alymphoid) control groups. Surprisingly, profiles of gene expression in the c-Rel recipients (which have indefinite graft survival) were similar to the p50-/- and allogeneic recipients (which rapidly reject grafts). As expected, gene expression in the I{kappa}B({Delta}N) recipients (which also have indefinite graft survival) was similar to profiles of nonrejecting syngeneic and alymphoid recipients. Importantly, self-organizing maps identified a small subset of genes including several chemokine receptors and cytokines with expression profiles that correlate with graft survival. Thus, our results demonstrate a crucial role for NF-{kappa}B in acute allograft rejection, identify different molecular mechanisms of rejection by distinct NF-{kappa}B family members, and identify a small subset of inducible genes whose inhibition is linked to graft acceptance.

Key Words: transplantation • chemokines • cytokines • CD8 T cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence from previous studies suggests that the in vivo process of allograft rejection is highly complex [1 , 2 ]. The strongest support of this interpretation is derived from murine studies of knockout or gene-deficient mice. These studies have identified many deficient strains that exhibit delayed but not totally inhibited rejection. To date, only a small subset of strains with targeted gene deletions has indefinitely prolonged allograft survival; these strains include mice deficient in T cells [recombinase-activating gene (RAG)-deficient, T cell receptor (TCR){alpha}-/-, scid, CD4-/-, or nu strains], CD40L-/-, or strains deficient in B7-1 (CD80) and B7-2 (CD86) costimulatory receptors [3 4 5 6 7 8 ]. These observations suggest that many immune-related genes are important but not necessary to produce allograft rejection. Conversely, many different combinations of components of immunity may be sufficient to reject allografts.

To investigate the assumed complexity of in vivo alloimmunity, our experimental strategy incorporated two important concepts. First, in addition to graft survival, graft histology, and mixed lymphocyte responses, we analyzed a large panel of 58 genes including effector molecules, chemokines, chemokine receptors, cytokines, and cellular markers previously reported to exert important roles in inflammation and immunity. Our hypothesis was that a subset of these candidate genes would be differentially regulated in the experimental groups. To generate a global interpretation of the integrated functions of these genes, we used hierarchical clustering algorithms to generate dendrograms that visualize dissimilarity between groups based on summed Pearson correlation coefficients of gene expression values. To identify specific subsets of genes with distinct patterns of expression in our experimental groups, we used self-organizing maps (SOM), which are artificial neural network algorithms that have been successfully applied to high-dimensional and nonlinear problems in many areas of science. In combination, this analytical approach would assess global similarity among experimental groups, as well as identify specific genes with distinct patterns of expression.

The second important component of our experimental strategy was the selection of a gene family, nuclear factor-{kappa}B/Rel (NF-{kappa}B), which has been shown to be crucial in the regulation of multiple inflammatory and immune responses [9 , 10 ]. Our hypothesis was that NF-{kappa}B is a major mediator of alloimmunity. Supporting this hypothesis, steroids, which have been shown to inhibit NF-{kappa}B activation [11 ], are important immunosuppressive therapies in clinical transplantation. In animal models, deficiency of the p50 NF-{kappa}B family member modestly prolongs allograft survival to approximately double-graft survival in wild-type controls [12 ]. p50-Deficient mice have normal lymphoid development with modest functional defects, including defective B cell-proliferative responses to lipopolysaccharide and decreased antibody production [13 ]. However, based on studies showing more profound immunodeficiency in mice deficient in other NF-{kappa}B family members, we reasoned that other NF-{kappa}B family members may exert more profound effects on alloimmunity. The NF-{kappa}B family of transcriptional regulators includes p50, p52, p65 (RelA), RelB, and c-Rel. Mice deficient in a single NF-{kappa}B gene develop distinct phenotypes, indicating that the function of each NF-{kappa}B gene is, at least in part, nonoverlapping [14 ]. Based on studies showing that c-Rel is expressed predominantly in lymphocytes [15 ] and that c-Rel deficiency inhibits aspects of T and B cell activation, including cytokine production, proliferation, and antibody production [16 17 18 ], we hypothesized that c-Rel was an important mediator of alloimmunity and included c-Rel-deficient mice in our experimental design.

NF-{kappa}B DNA-binding complexes form homo- and heterodimers. The most abundant NF-{kappa}B transcriptional activator is the p50/RelA heterodimer, which is widely expressed in many cell types and the prototypic NF-{kappa}B transcription complex [10 , 19 ]. However, RelA can also dimerize with other family members. In the p50-deficient strain, the lack of p50/RelA dimers may be compensated, at least in part, by RelA homodimers or complexes with other NF-{kappa}B family members. In our experimental design, we included a transgenic line that expresses a transdominant inhibitor of NF-{kappa}B in T cells [20 ]. In the resting state, NF-{kappa}B is retained in the cytoplasm by the inhibitor protein I{kappa}B. After activation, I{kappa}B is phosphorylated and ultimately degraded in the proteasome, permitting NF-{kappa}B to translocate to the nucleus and function as a transcriptional regulator. I{kappa}B{alpha} has been shown to bind and inhibit NF-{kappa}B complexes containing p50, p52, RelA, and c-Rel [10 , 19 , 21 ]. Thus, the I{kappa}B({Delta}N) strain has constitutive inhibition of multiple NF-{kappa}B complexes in T cells.

Importantly, the c-Rel and I{kappa}B({Delta}N) groups demonstrate indefinite graft survival. In addition, our results indicate that the mechanisms of inhibition of rejection are different in the c-Rel and I{kappa}B({Delta}N) groups. Our results further demonstrate that the three mutant NF-{kappa}B mouse strains [p50-deficient, c-Rel-deficient, and I{kappa}B({Delta}N)-transgenic] modulate different components of alloimmunity. Specifically, each recipient strain generates differential expression of the 58 immune parameters following transplantation. These regulatory differences correlate with different histological scores and different kinetics of rejection. Importantly, the SOM identify a small subset of genes with expression profiles that correlate with graft survival. We propose that these gene products represent critical NF-{kappa}B-regulated mediators of allograft rejection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Vascularized heterotopic cardiac transplantation
Murine hearts were transplanted as previously described [22 ]. Briefly, hearts were harvested from freshly killed donors and were immediately transplanted into 8- to 12-week-old recipients, which were anaesthetized via intraperitoneal 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 inferior 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 2 days after transplantation were excluded (>95% of all grafts functioned at 2 days after transplantation). Donor grafts were harvested at the indicated times after transplantation and divided into equal sections for preparation of RNA and tissue sections for histology. The study endpoint was the time of rejection or 100 days for all recipients with beating allografts.

Mice
Eight- to 12-week-old male mice including BALB/cByJ (BALB/c; H-2d), C57BL/6J (B6; H-2b), C57BL/6J-Rag-1tm1Mom (B6-Rag-deficient; H-2b), B6,129-PNfkb1tm1Ba1 (p50-/-; JAX, Bar Harbor, ME), and BALB/c-AnNTac-Rag2tm1N12 (BALB/c-Rag-deficient; H-2d; Taconic, Petersburgh, NY) were used as donor and recipients in the transplant experiments (Table 1 ). Mice are maintained in vented racks with constant temperature and humidity in our animal facility under virus antibody-free conditions. The I{kappa}B({Delta}N) transgenic mice were also used as recipients and produced by injecting an amino-terminally truncated form of I{kappa}B({Delta}N), including amino acids 37–317 linked to the proximal lck promoter plus the locus control region from the human CD2 gene into C57BL/6 x DBA/2 zygotes [20 ]. Founder mice were back-crossed with C57BL/6 mice for four generations, and transgene expression was determined by Southern blot analysis. The I{kappa}B({Delta}N) transgene is regulated by the proximal lck promoter, which generates predominantly T cell-restricted expression [20 ]. Mice were maintained in a virus antibody-free facility in accordance with federal and state government regulations after Harvard Medical School (Boston, MA) institutional approval. As previously reported, c-Rel-deficient mice were produced by gene targeting to produce mice deficient in exons 1, 2, 3, and 5 of the c-Rel gene [23 ].


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  		Table 1. Allograft Survival

 
Mixed lymphocyte culture
Responder spleen cells (2x105) were stimulated with 4 x 105 stimulator spleen cells, which had been irradiated with 2000 R in 200 µl RPMI plus 10% fetal calf serum. 3H-Thymidine (1 µCi) was added during the last 12 h of culture. After 96 h, cells were harvested, and thymidine incorporation was determined as described [24 ]. Cultures were performed in quadruplicate, and SEM were <10%.

Ribonuclease protection assay (RPA)
Chemokine, chemokine receptor, and CD marker expression was analyzed by RPA. Briefly, total RNA was isolated from hearts using RNAzol and analyzed using the RiboQuant multiprobe RPA system (PharMingen, San Diego, CA). RNA (15 µg) was used per hybridization and RNase reaction with the templates mCK-5 [lymphotactin (Ltn), migration inhibitory factor, regulated on activation, normal T expressed and secreted (RANTES), eotaxin, macrophage inflammatory protein (MIP)-1ß, MIP-1{alpha}, MIP-2, interferon (IFN)-inducible protein 10 (IP-10), and monocyte chemoattractant protein (MCP)-1], mCR-5 [chemokine receptor (CCR)1, 1ß, 4, 5, and 2], a custom template [CXC chemokine receptor (CXCR)2, 3, 4, and 5 and CCR6, 8{alpha}, and 8ß], mCK2b [interleukin (IL)-1{alpha}, IL-1ß, IL-1RA, IL-6], mCK3b [tumor necrosis factor {alpha} (TNF-{alpha})], mCR-5 (CCR1, 1{alpha}, 4, 5, and 2), and mCD-1 (TCR{gamma}, TCR{alpha}, CD3{varepsilon}, CD4, CD8{alpha}, CD8ß, CD19, F4/80, CD45; PharMingen). The IP-10 template detects the C57BL/6 allele [25 ]. The protocol was modified by labeling probes with 35S. After hybridization with the 35S-labeled probes, the samples were treated with RNase and purified according to the manufacturer’s protocol. The protected probes were electrophoresed on a denaturing 5% polyacrylamide gel. The gels were exposed in a Molecular Dynamics PhosphorImager. The identity of each protected fragment was established by analyzing its migration distance against a standard curve of the migration distance versus the log nucleotide length for each undigested probe. Samples were normalized to the housekeeping gene, glyceraldehyde 3-phospahte dehydrogenase. Protected bands were quantitated by densitometry analysis using ImageQuant software (Molecular Dynamics).

Histology
Recipient native hearts and donor-transplanted hearts were harvested 1 day after transplantation and fixed in 10% neutral-buffered formalin. After dehydration and paraffin embedding, 5–6 mc-thick sections were routinely stained with hematoxylin and eosin. Multiple sections were examined for each heart, and the extent of rejection (grades 0–4) and ischemia (grades 0–4) were quantified using a modified International Society of Heart and Lung Transplantation grading scale [26 ]. Any graft hearts with an ischemia score >1 were excluded from analysis.

Statistics and algorithms
Serum cytokines were calculated as the mean + SD of quadruplicate assays. Differential expression of mRNA was determined by RPA 24 h following transplantation in the native and graft hearts of the syngeneic, alymphoid, and allogeneic recipients by two-factor ANOVA. Statistical significance of variances was calculated for P < 0.05 by F-test. Differential expression of mRNA determined by RPA on days 1, 3, 5, and 7 in syngeneic, alymphoid, and allogeneic samples and untransplanted control heart RNA was analyzed by two-factor ANOVA. Statistical significance of variances was calculated for P < 0.05 by F-test. Cluster analysis was performed using Cluster and TreeView software [27 ] (courtesy of Michael Eisen, Lawrence Livermore Radiation Laboratory, Berkeley, CA). RPA values were analyzed by Cluster using the hierarchical clustering algorithm with complete linkage clustering. Briefly, dissimilarity is determined by calculation of the Pearson correlation coefficient between each series of values from each experimental group. After processing, the dendrogram was visualized by TreeView [27 ]. Self-organizing maps were generated by GeneCluster [28 ]. RPA values were normalized to a mean of 0 and variance of 1 using a 2 x 2 geometry of four seed maps. Data were filtered with a threshold of a minimum of a threefold change for each parameter. Four maps were selected empirically to eliminate clusters with few genes or large standard deviations. The centroids and standard deviations of the groupings were analyzed using 100 epochs. Additional epochs did not alter the gene clusters of the maps.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
3c-Rel but not p50 is essential for cardiac allograft acceptance
To investigate the role of NF-{kappa}B in allograft rejection, we analyzed three NF-{kappa}B groups [p50-/-, c-Rel-/-, and I{kappa}B({Delta}N) transgenic strains] and three control groups (allogeneic, syngeneic, and alymphoid) in a murine heterotopic heart transplant model (Table 1) . Our results show indefinitely prolonged allograft survival (>100 days) in c-Rel-/- and I{kappa}B({Delta}N) recipients. Consistent with previous reports, the p50-/- recipients have modestly prolonged survival with a median survival time of 15 days [12 ]. As expected, the wild-type allogeneic group rejected allografts at a median of 8 days, whereas the syngeneic and alymphoid (deficient in T and B lymphocytes as a result of deficiency of the recombinase-activating gene) groups did not reject grafts. To explore the basis for NF-{kappa}B modulation of rejection, we analyzed alloreactivity in mixed lymphocyte reactions in vitro (Fig. 1 ). These results showed that the proliferative response was reduced by the c-Rel-/- (16% of wild-type), I{kappa}B({Delta}N; 19% of wild-type), and to a modest extent, in the p50-/- (79% of wild-type) responder cells. Together, these results indicate decreased alloreactivity and prolonged allograft survival in strains with impaired NF-{kappa}B function. Importantly, different graft survival times in the three NF-{kappa}B mutant strains support a model in which the various NF-{kappa}B family members perform different functions in alloimmunity.



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  		Figure 1. Mixed lymphocyte reactions. Splenic lymphocytes were harvested from wild-type B6, p50-/-, c-Rel-/-, and I{kappa}B({Delta}N) mice and were stimulated with irradiated syngeneic B6 (hatched bars) or allogeneic BALB/c (solid bars) spleen cells. 3H-Thymidine (1 µCi) 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. Data represent means ± SD.

 
Molecular profiling of the allograft during graft acceptance and rejection
Analysis of graft survival and intragraft cytokine mRNA clearly indicated differential regulation of gene expression by p50, c-Rel, and NF-{kappa}B-dependent T cell activation. To extend these observations to a global analysis of NF-{kappa}B-modulated alloimmunity, we analyzed a panel of 58 parameters, which includes effector molecules, cytokines, chemokines, chemokine receptors, and cellular markers previously reported to play important roles in immunity (Fig. 2 and Table 2 ). We elected to monitor mRNA because of the technical feasibility of analyzing a large subset of genes. Although mRNA levels do not always correlate with protein expression, a recent study combining genomics and proteomics in yeast demonstrated that 95% of changes in mRNA levels were associated with changes at the protein level [29 ]. To compare all parameters in a global analysis, we used hierarchical clustering algorithms to generate dendrograms from data at three time points: day 1, day 7, and the study endpoint (the time of graft rejection or survival >100 days; Fig. 3 ).



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  		Figure 2. Serum cytokines. The levels of TNF-{alpha} (open bars), IL-1ß (left cross-hatched bars), IL-6 (solid bars), and IFN-{gamma} (right cross-hatched bars; pg/ml) were determined by ELISA on days 1, 3, 5, and 7 following transplantation in the alymphoid (a), syngeneic (b), allogeneic (c), p50-/- (d), c-Rel-/- (e), and I{kappa}B({Delta}N; f) groups. Control serum (Day 0) is from untransplanted B6 mice.

 

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  		Table 2. ANOVA of Gene Expression

 


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  		Figure 3. Hierarchical clustering analysis. The level of expression of a panel of 54 candidate genes including effector molecules, cytokines, chemokines, chemokine receptors, and cellular markers was determined at day 1 (top), day 7 (middle), and the time of rejection or 100 days for nonrejecting grafts (bottom) by RPA of graft hearts and was analyzed by Cluster and Tree software [27 ]. Controls are untransplanted BALB/c hearts. The degree of dissimilarity is proportional to the total length of the horizontal distance in the dendrogram between groups.

 
At day 1, gene expression profiles in all six transplanted groups were relatively similar (proportional to the x-axis distance separating groups; Fig. 3a ). This dendrogram is consistent with our previous report that a major component of the early response at day 1 after cardiac transplantation is composed of an inflammatory response that is independent of adaptive immunity [30 ] causing the allogeneic, syngeneic, alymphoid, and mutant recipient groups to be closely linked in dendrograms. In contrast, at day 7, when the allogeneic group has developed a robust, adaptive, alloimmune response, the groups showed a high level of dissimilarity (Fig. 3b) . As expected, at day 7, the untransplanted control, syngeneic, and alymphoid groups were most similar and were most closely linked to the I{kappa}B({Delta}N) group, which has indefinite graft survival. Surprisingly, the c-Rel group, which also has indefinite graft survival, was most similar with the allogeneic and p50 groups, which reject grafts in 8 and 15 days, respectively. The day 7 dendrogram is consistent with examination of mRNA expression patterns by ANOVA. Compared with the allogeneic group, only five genes were not significantly up-regulated in the c-Rel group. In sharp contrast, 16 and 23 genes were not up-regulated in the p50 and I{kappa}B({Delta}N) groups, respectively (Table 2) . At the study endpoint, the allogeneic and p50 groups are most similar, whereas the untransplanted, alymphoid, syngeneic, and I{kappa}B({Delta}N) groups, which do not reject grafts, form a second cluster (Fig. 3c) , and the c-Rel group remained dissimilar from the other nonrejecting groups at the study endpoint but became less similar to the allogeneic and p50 groups. Thus, the p50 and I{kappa}B({Delta}N) groups show predicted clustering patterns; however, the c-Rel group, despite the fact that grafts were not rejected, showed an unexpected high level of similarity with the rejecting groups.

Graft mononuclear cell infiltration without tissue damage in c-Rel-/- recipients
To investigate the discordance between graft survival and clustering patterns in the c-Rel group, we analyzed histological evidence of rejection (Fig. 4 ). At day 7, the syngeneic, alymphoid, and I{kappa}B({Delta}N) groups showed grades 0–+1 rejection, whereas the allogeneic, p50, and c-Rel groups all showed +2 or +3 mononuclear cell infiltration; in addition, at day 100, the c-Rel group showed extensive +2 mononuclear cell infiltration. However, at days 7 and 100, the c-Rel group (and the other nonrejecting groups) did not show significant myocyte necrosis, whereas the allogeneic and p50 groups develop myocyte necrosis at days 7 and 15, respectively. These observations indicate that the deficiency of c-Rel does not block mononuclear cell infiltration but does inhibit crucial effector functions required to promote acute rejection.



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  		Figure 4. Histology and immunohistochemistry. Analysis of control of an untransplanted BALB/c heart (a), allogeneic graft d 7 (b), p50-/- graft day 7 (c), c-Rel-/- graft day 7 (d), I{kappa}B({Delta}N) graft day 7 (e), p50-/- graft day 15 (f), c-Rel-/- graft day 100 (g), and I{kappa}B({Delta}N) graft day 100 (h). Graft hearts were harvested at 7 days after transplantation and at the study endpoint and stained with hematoxylin and eosin. 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 Heart Transplantation grading scale.

 
Self-organizing maps identify subsets of NF-{kappa}B-modulated genes correlated with graft rejection
To determine the molecular basis of c-Rel deficiency in blocking rejection, we used SOM to identify specific subsets of genes that were differentially regulated in 10 experimental samples, including untransplanted control heart, the allogeneic, syngeneic, and alymphoid groups at day 7, and the p50, c-Rel, and I{kappa}B({Delta}N) groups at day 7 and the study endpoint (Fig. 5 and Table 3 ). Several distinct profiles of gene expression emerged from this analysis. Maps 1, 2, and 3 all include genes that are induced in the allogeneic group; however, the genes are differentially regulated in the three NF-{kappa}B groups. For example, Map 1 includes genes that are markedly up-regulated in the p50 group but not increased in the I{kappa}B({Delta}N) group at the experiment endpoint. Map 1 includes several chemokine receptors and cytokines; supporting the validity of the analysis, IL-1ß and IL-6, cytokines up-regulated in Map 1, are increased at the level of serum protein and graft mRNA. Map 2 includes genes not increased in the p50 and I{kappa}B({Delta}N) groups at day 7 and study endpoint but induced in the c-Rel group, particularly at day 100. Last, Map 3 includes 13 genes that are not increased in the p50, c-Rel, and I{kappa}B({Delta}N) groups but induced in the allogeneic group. Of note, the level of gene expression in Map 1 [high in allogeneic and p50 and low in c-Rel and I{kappa}B({Delta}N)] correlates with graft survival, suggesting the genes in this subset contribute to the vigorous alloimmune response in the p50-/- and wild-type allogeneic recipients. Conversely, the failure to induce these genes may contribute to the prolonged graft survival in the c-Rel-/- and I{kappa}B({Delta}N) recipients.



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  		Figure 5. Self-organizing maps of gene expression. For each map, the diamonds from left to right represent mean expression for each map of genes from control untransplanted BALB/c, syngeneic day 7, alymphoid day 7, allogeneic day 7, p50-/- day 7, c-Rel-/- day 7, I{kappa}B({Delta}N) day 7, p50-/- day 15, c-Rel-/- day 100, and I{kappa}B({Delta}N) day 100. The y-axis represents relative expression calculated for each self-organizing map. Individual maps from 0 through 3 include a total of 7, 8, 4, and 13 genes, respectively (see Table 3 ). Data were edited with a threshold of threefold change in value. Values of each gene for the 10 experimental groups were normalized to mean = 0 and variance = 1. Maps were generated with a 2 x 2 geometry with 100 epochs. Heuristic analysis showed that increased number of nodes produced multiple, similar maps, and decreased number of nodes increased the SD; also, more epochs did not modify the grouping or decrease standard deviations.

 

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  		Table 3. Gene Clusters Selected by Self-Organizing Maps

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results analyzing allograft rejection in p50-/-, c-Rel-/-, and I{kappa}B({Delta}N) transgenic recipients in a vascularized allogeneic cardiac transplant model indicate that some but not all NF-{kappa}B genes are crucial modulators of acute rejection. A striking result in our study was that graft survival was indefinitely prolonged in the c-Rel-/- and I{kappa}B({Delta}N) strains. In contrast, p50-/- recipients had only modestly prolonged survival, which was consistent with previous reports [12 ]. Together, these results indicate that c-Rel and NF-{kappa}B activation in T cells produce more robust effects on alloimmunity than p50. In this heart transplant model, many different strains with gene deficiencies have been analyzed as graft recipients. These studies have shown that the most common result is that these mutant recipients have delayed rejection; however, graft survival is indefinitely prolonged only in a small subset of deficient strains. Specifically, indefinite survival has been observed only in mice deficient in T cells (RAG-/-, TCR{alpha}-/-, scid, CD4-/-, or nu strains), CD40 ligand-/-, or the B7-1 and B7-2 costimulatory molecules. All of the other gene-deficient strains studied do reject grafts, although often with delayed kinetics. Thus, our results demonstrate that some NF-{kappa}B family members including c-Rel have an absolutely essential role in allograft rejection shared with only a few previously identified immune mediators.

Our experimental approach was based on the hypothesis that the role of NF-{kappa}B in alloimmunity is a complex process differentially regulated by different NF-{kappa}B family members. To produce a comparative overview of rejection mechanisms, we generated dendrograms using hierarchical clustering algorithms that calculated dissimilarity based on the sum of Pearson correlation coeffecients of all 58 parameters (Fig. 1) . As expected, the I{kappa}B({Delta}N), syngeneic, and alymphoid groups, which do not reject grafts, were closely linked. In addition, the dendrograms closely linked the p50 and allogeneic groups, which rapidly reject grafts. Surprisingly, the c-Rel group, which accepts grafts indefinitely, was most similar to the allogeneic and p50 groups but highly dissimilar from the other nonrejecting groups [including the I{kappa}B({Delta}N), syngeneic, and alymphoid groups]. These observations indicate that although the I{kappa}B({Delta}N) and c-Rel-/- recipients have indefinite graft survival, the mechanisms preventing rejection can be differentiated at a molecular level.

The similarity between the c-Rel and allogeneic groups in the dendrograms is consistent with the ANOVA results showing that in comparison to the allogeneic group, the c-Rel group up-regulated fewer parameters (five genes) than the I{kappa}B({Delta}N) group (23 genes). Thus, the profile of gene expression in the c-Rel grafts, which survive >100 days, was highly similar to the profile in allogeneic grafts, which reject in 8 days. Histological and immunohistochemical analyses also showed the highest levels of mononuclear cell infiltration including CD8+ cells in the c-Rel and allogeneic groups. The simplest interpretation of these results is that c-Rel deficiency blocks effector components of rejection but does not inhibit mononuclear and T cell infiltration. This interpretation is further supported by the observation that despite extensive CD8 T cell infiltration, the grafts in the c-Rel recipients lack myocyte necrosis, a hallmark of rejection.

In contrast to the c-Rel group, dendrograms of the I{kappa}B({Delta}N) recipients showed strong similarity with the syngeneic, alymphoid, and untransplanted groups, none of which reject allografts. This finding suggests that there are distinct mechanisms of graft acceptance in the c-Rel-/- and I{kappa}B({Delta}N) recipients. Consistent with this observation, the I{kappa}B({Delta}N) recipients did not significantly increase 23 parameters based on ANOVA. As the NF-{kappa}B inhibitor is expressed predominantly in T cells, these results indicate that NF-{kappa}B-dependent T cell activation is crucial for the up-regulation of these genes in wild-type mice during an alloimmune response. The most striking conclusion from these observations is that the c-Rel and I{kappa}B({Delta}N) groups produce indefinite graft survival but with markedly different patterns of cellular infiltration and profiles of gene expression.

Graft survival in the p50 group was only moderately prolonged. Consistent with the survival data, the day 7 dendrogram showed strong similarity between the p50 and allogeneic groups. It is interesting that the analysis of expression profiles of specific genes using ANOVA showed differential gene expression between the allogeneic and p50-/- groups. Specifically, a subset of 15 genes was not up-regulated in the p50-/- group, suggesting that they may contribute to the modest delay in graft rejection. In addition, a second subset of genes was super-induced in the p50-/- group to levels greater than the wild-type allogeneic group. The greatest induction was observed for IL-1ß, which was increased in mRNA and serum protein determinations. Previous studies have shown that p50/RelA heterodimers are activators of transcription, whereas p50 homodimers can repress the transcription of some genes [31 ]. Thus, the loss of transcriptional repression as a result of deficiency of p50 is consistent with increased expression of IL-1ß. Thus, not only does the p50 group have different profiles of expression compared with the c-Rel and I{kappa}B({Delta}N) groups, it also differs from the allogeneic group.

To identify specific subsets of genes modulated during the late- or adaptive-phase of rejection, we used SOM (Figs. 2 and 3) , which are artificial neural network algorithms that have been successfully applied to high-dimensional and nonlinear problems in many areas of science. The SOM identified specific subsets of genes with distinct patterns of expression. For example, Map 3 contains a subset of genes highly up-regulated in the allogeneic group at the time of rejection but markedly inhibited in the p50, c-Rel, and I{kappa}B({Delta}N) groups. This subset includes markers for CD8 T cells and chemokines, such as RANTES and Ltn, previously shown to be important in promoting rejection. Thus, decreased expression of these genes correlates with increased graft survival. However, as these genes are expressed at low levels in the p50-/- group, which has only modest prolongation of graft survival, inhibition of expression of this subset is not sufficient to explain the indefinite graft survival in the c-Rel-/- and I{kappa}B({Delta}N) groups. Map 2 contains genes up-regulated in the allogeneic group, not increased in the p50 and I{kappa}B({Delta}N) groups, but interestingly up-regulated in the c-Rel group. Based on the observation that the c-Rel group has indefinite graft survival, this subset, which includes MCP-1, IP-10, and TCA-3, would be predicted to be important in the activation of immunity but not sufficient to promote the effector functions of rejection.

Importantly, Map 1 contains a subset with low levels of expression in the c-Rel-/- and I{kappa}B({Delta}N) recipients but increased expression in the wild-type and p50-/- recipients at the end point of rejection. Thus, Map 1 identifies a subset of genes with expression levels that inversely correlate with graft survival, suggesting a shared molecular basis among the distinct mechanisms of rejection in these groups. Three genes in this subset (IL-1ß, IL-6, and TNF-{alpha}) are components of the acute-phase response, which may not be stimulated in the absence of acute rejection. Also, three chemokine receptors in this subset correlate with rejection. CCR1, which has multiple ligands including RANTES, MIP-1{alpha}, MCP-2, and MCP-3, is expressed on activated T cells and monocytes. Deficiency of CCR1 delays acute and chronic cardiac allograft rejection in mice [32 ]. Also, in biopsies of renal allografts, increased numbers of CXCR4+ leukocytes were detected in rejecting grafts [33 ].

The nearly ubiquitous and most abundant NF-{kappa}B DNA-binding complex is the p50/RelA heterodimer. The modestly prolonged graft survival in the p50-/- recipients could be a result of disruption of the p50/RelA complex; however, our results showing indefinite graft survival in the c-Rel-/- and I{kappa}B({Delta}N) recipients indicate that non-p50/RelA complexes that include c-Rel have major effects on alloimmunity. Specifically, results from the c-Rel group indicate that NF-{kappa}B complexes containing c-Rel have a necessary function in promoting alloimmunity. Although c-Rel can form complexes with p50, our results suggest that the crucial complexes can be formed with other NF-{kappa}B family members or be mediated by c-Rel homodimers. In addition, our analysis of the I{kappa}B({Delta}N) group indicates that NF-{kappa}B complexes not containing c-Rel are also important in alloimmunity. This conclusion is based on several observations including different histology, different serum cytokine production, and different profiles of gene expression in the c-Rel group compared with the I{kappa}B({Delta}N) group. I{kappa}B{alpha} has been shown to inhibit not only p50 and RelA but also complexes containing c-Rel and p52. Thus, the profound blockade of gene expression in the I{kappa}B({Delta}N) group must be, at least in part, a result of inhibition of complexes not containing c-Rel. Although we have no direct evidence, deficiency of p50 and c-Rel could contribute to attenuated innate-immune responses, whereas the I{kappa}B({Delta}N) results are obviously a result of diminished T cell adaptive-immune responses. Taken together, these observations indicate that NF-{kappa}B has a crucial but complex role in alloimmunity. Common therapeutic agents in clinical transplantation are steroids, which are known inhibitors of NF-{kappa}B. In our studies, p50, c-Rel, and NF-{kappa}B-dependent T cell activation all produced unique profiles of gene expression and distinct effects of graft rejection, suggesting that optimal therapeutic modalities should target specific NF{kappa}B complexes. Additional understanding of the mechanisms of NF-{kappa}B modulation of rejection could be important to develop a molecular definition of rejection and develop improved therapeutic strategies.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the American Heart Association, Arthritis Foundation, and National Institutes of Health (AI44085) to D. L. P. and HL61752 to M. R. B. We thank C. McKee for critical review of the manuscript.

Received March 29, 2002; revised May 16, 2002; accepted June 27, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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