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The Journal of Immunology, 2002, 168: 4406-4413.
Copyright © 2002 by The American Association of Immunologists

NF-{kappa}B2 Is Required for Optimal CD40-Induced IL-12 Production but Dispensable for Th1 Cell Differentiation1

Kendra Speirs*, Jorge Caamano{dagger}, Michael H. Goldschmidt*, Christopher A. Hunter* and Phillip Scott2,*

* Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104; and {dagger} Medical Research Council Center for Immune Regulation, School of Medicine, University of Birmingham, Edgbaston, Birmingham, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NF-{kappa}B is a ubiquitously expressed transcription factor involved in the regulation of innate and adaptive immunity. As part of studies to define the role of various NF-{kappa}B family members in Th cell development and maintenance, we infected NF-{kappa}B2-/- and control mice with Leishmania major and followed disease progression. NF-{kappa}B2-/- mice on a normally resistant background develop chronic nonhealing lesions associated with uncontrolled parasite replication and a failure to develop an IFN-{gamma} response. We show that there are no intrinsic defects in Th cell differentiation in the absence of NF-{kappa}B2. Indeed, NF-{kappa}B2-/- T cells are able to develop a Th1 phenotype and protect recombination-activating gene-/- mice from progressive cutaneous leishmaniasis. We demonstrate instead that the susceptibility of NF-{kappa}B2-/- mice to L. major is the result of an IL-12 deficiency, and we provide evidence for a specific impairment in CD40-induced IL-12 production by macrophages lacking this transcription factor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The NF-{kappa}B/Rel eukaryotic transcription factors are crucial regulators of the immune response through their ability to induce the expression of genes for a wide range of cytokines, chemokines, adhesion molecules, and acute phase response proteins (1, 2). The NF-{kappa}B family is composed of five different members, NF-{kappa}B1, NF-{kappa}B2, RelA, RelB, and c-Rel, which are maintained as homo- and heterodimeric complexes within the cytoplasm until the cell is stimulated to induce their nuclear translocation (3). In the cytoplasm, NF-{kappa}B complexes are bound to one of several inhibitory proteins of the I{kappa}B family or the preprocessed NF-{kappa}B precursors, p105 and p100. These serve to sequester the NF-{kappa}B/Rel complexes in the cytoplasm by masking their nuclear localization signal (1, 3). Once cells are stimulated by factors that induce NF-{kappa}B activity (including cytokines such as TNF-{alpha} and IL-1, bacterial and viral products, TCR stimulation, and Ag recognition), a signal transduction cascade is initiated that results in the phosphorylation, ubiquitination, and degradation of I{kappa}B. This allows the translocation of NF-{kappa}B to the nucleus, where it can bind to {kappa}B DNA motifs and induce gene transcription (1, 4).

Until the recent generation of mice deficient in individual NF-{kappa}B components, few studies distinguished between NF-{kappa}B family members. Now, however, infection of various NF-{kappa}B-/- mice with a variety of pathogens has uncovered unique roles for each family member (5, 6, 7, 8). For example, NF-{kappa}B1-deficient mice are resistant to infection with Toxoplasma gondii, whereas mice lacking RelB and NF-{kappa}B2 demonstrate enhanced susceptibility to this parasite (9, 10). Lymphocytic choriomeningitis virus infection of RelB- and NF-{kappa}B2-deficient mice reveals a distinction between these two family members in mediating resistance to viruses. While NF-{kappa}B2 is dispensable for protective immunity to lymphocytic choriomeningitis virus, the absence of RelB results in susceptibility to disease (6, 11). Current research is now focused on defining the mechanisms underlying the distinct phenotypes observed in NF-{kappa}B-/- mice infected with different pathogens.

Resistance to infection often hinges on the appropriate differentiation of CD4+ T cells into Th1 or Th2 subsets. Th1 cells mediate the development of cell-mediated inflammatory responses against intracellular microorganisms, while Th2 cells are required for resistance to extracellular pathogens (12). Molecular studies indicate that NF-{kappa}B can play a potential role at many levels in the development of both types of immune response. There are putative {kappa}B binding sites in the promoters of numerous cytokine genes, including IL-9, IL-12, and IFN-{gamma}, and NF-{kappa}B is important in the activation and proliferation of T lymphocytes (13, 14, 15, 16). A general requirement for the NF-{kappa}B family in Th1-dependent delayed-type hypersensitivity responses was illustrated using transgenic mice whose T cells fail to activate the NF-{kappa}B/Rel signaling pathway (17). Most recently, NF-{kappa}B1 was shown to be essential for Th2 cell development (18). However, the role of the closely related family member, NF-{kappa}B2, in Th cell differentiation has not been investigated. NF-{kappa}B2 is structurally homologous to NF-{kappa}B1 in both its DNA binding and inhibitory domains, but is largely restricted to cells of hemopoietic origin (1, 19). Mice deficient in the nf{kappa}b2 gene lack follicular dendritic cells and have a dramatic reduction in the B cell compartments of all peripheral lymphoid organs (8, 20).

To assess the role of NF-{kappa}B2 in Th cell differentiation and maintenance, we have used the Leishmania major model of infection. Resistance to L. major depends on the production of IL-12, required for CD4+ Th1 cell polarization (21, 22, 23, 24). We found that NF-{kappa}B2-/- mice on a normally resistant background developed chronic nonhealing lesions following infection with L. major. Associated with this increased susceptibility was the failure of NF-{kappa}B2-/- mice to develop a Th1 response and restrict parasite replication. We demonstrate that this nonhealing phenotype was not the result of an intrinsic defect in T cell function, as NF-{kappa}B2-deficient T cells were able to protect recombination-activating gene (RAG)3-/- mice from infection. Instead, NF-{kappa}B2 was shown to play an important role in the production of IL-12 by macrophages. NF-{kappa}B2-/- mice had impaired IL-12 levels following L. major infection, and exogenous IL-12 conferred protection against disease. Importantly, our studies demonstrated a signal-specific requirement for NF-{kappa}B2 in the expression of IL-12. We showed that while LPS signaling was intact in the absence of NF-{kappa}B2, CD40-induced IL-12 production was dramatically impaired. A defect in CD40 signaling may, in part, explain the susceptibility of NF-{kappa}B2-/- mice to L. major.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

NF-{kappa}B2-/- mice were backcrossed to C57BL/6 (five generations) (20). Mice were typed for the absence of the nf{kappa}b2 gene using a PCR-based method, which differentiates between the wild-type (WT) NF-{kappa}B2 gene and the targeted NF-{kappa}B2-neo allele, as previously described (9). Naive NF-{kappa}B2-/- mice are healthy and do not show signs of autoimmunity or developmental abnormalities (20). Controls for our experiments included NF-{kappa}B2+/+ and NF-{kappa}B2+/- littermates of the NF-{kappa}B2-/- mice, as well as C57BL/6 mice. In our studies, no differences were observed between these control groups. Female mice were used between 6 and 8 wk of age.

Infections and in vivo IL-12 and anti-IL-12 treatment

L. major (MHOM/IL/80/Friedlin) parasites were cultured as promastigotes in Grace’s insect cell culture medium (Life Technologies, Grand Island, NY), containing 20% FCS (HyClone Laboratories, Logan, UT), 100 U/ml penicillin 6-potassium, 100 µg/ml streptomycin sulfate, and 2 mM L-glutamine. Stationary phase metacyclics were purified by negative selection with Arachis hypogae agglutinin, as previously described (25). For experimental infections, mice were infected in the hind footpad with 2 x 106 metacyclic parasites. IL-12-treated mice were injected intralesionally with 0.2 µg IL-12 (Genetics Institute, Cambridge, MA) six times over the first 2 wk of infection. Anti-IL-12-treated mice were injected i.p. with 1 mg anti-IL-12 (C17.8) once per week for the first 3 wk of infection. Lesion size was monitored weekly using a dial caliper and expressed as the difference between the infected and uninfected contralateral footpad. Parasite numbers were enumerated by plating homogenates of infected lesions in 10-fold serial dilutions in Grace’s insect culture medium starting with a 1/100 dilution. Each sample was plated in triplicate, and the mean of the negative log parasite titer was calculated after 5 days.

Histology

The footpads were removed from 2-, 4-, and 6-wk infected mice, fixed in 10% neutral buffered formalin, and decalcified in hydrochloric acid. Tissue was embedded in paraffin, and 4- to 6-µm sections were cut. The sections were hydrated and stained with H&E. Histopathologic evaluation of the NF-{kappa}B2-/- and NF-{kappa}B2+/- mice was undertaken.

NO production and parasite killing

Bone marrow macrophages (BMM{phi}) from NF-{kappa}B2-/- and NF-{kappa}B2+/- mice were derived from bone marrow monocytes grown on petri dishes in complete tissue culture medium (CTCM; DMEM containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 25 mM HEPES, and 5 x 10-5 M 2-ME), supplemented with 30% L929 cell-conditioned medium (26, 27). After 7 days of incubation at 37°C, adherent cells were harvested from the plates using disposable cell scrapers, washed twice, and suspended at 1 x 106 cells/ml in 5-ml polypropylene tubes. Cells were primed with 10 U/ml IFN-{gamma} for 4 h and infected in suspension cultures with stationary phase L. major promastigotes at a 2:1 parasite:cell ratio. Infection was allowed to proceed for 2 h, and the cells were washed to remove excess parasites, as previously described (28). Cells were then stimulated with 10 U/ml IFN-{gamma} in the presence or absence of 100 ng/ml LPS (L6143; Sigma-Aldrich, St. Louis, MO) and incubated for 72 h at 34°C. Supernatants were collected and assayed for the presence of reactive nitrogen intermediates using the Greiss assay, as previously described (29). The number of parasites per 100 macrophages was enumerated from cytospins of infected cells.

Cytokine analysis

At 2, 4, and 8 wk postinfection, single cell suspensions were prepared using splenocytes from infected mice. Cells were resuspended in CTCM and cultured in 24-well tissue culture plates at 4 x 106 cells/ml. Cells were stimulated with 50 µg/ml soluble leishmanial Ag (SLA) alone or in the presence of 10 ng/ml IL-12 (Genetics Institute), and cultured with 2.5 µg/ml anti-IL-4R mAb (M1; generously provided by F. Finkleman, University of Cincinnati, Cincinnati, OH, and Immunex, Seattle, WA). SLA was prepared as previously described (30). Supernatants were harvested following a 72-h incubation at 37°C, and IFN-{gamma} and IL-4 levels were measured using two-site ELISAs, as previously described (31). IL-12 p40 levels were measured at 4 and 6 wk postinfection, using mAb C17.8 and biotinylated mAb C15.6 (prepared from hypbridoma supernatants originally provided by G. Trinchieri, Wistar Institute, Philadelphia, PA).

To assess in vitro T cell differentiation, NF-{kappa}B2-/- and NF-{kappa}B2+/+ splenocytes from naive mice were stimulated with 1 µg/ml plate-bound anti-CD3 (145-2C11) and anti-CD28 (BD PharMingen, San Diego, CA) under Th1 (10 µg/ml anti-IL-4 (11B11) and 10 ng/ml IL-12), Th2 (40 ng/ml IL-4 and 50 µg/ml anti-IFN-{gamma} (XMG6)), or neutral conditions (without cytokines or anti-cytokine mAbs). After 7 days, the cells were harvested, washed, and restimulated under neutral conditions for an additional 3 days. Supernatants were harvested, and IFN-{gamma} and IL-4 levels were measured by ELISA.

For the in vitro detection of IL-12 p40, BMM{phi} were cultured in flat-bottom 96-well plates at 2 x 105 cells/well. Cells were primed for 4 h with 10 U/ml murine rIFN-{gamma} and stimulated with 1, 5, and 10 g/ml anti-CD40 (clone IC10; R&D Systems, Minneapolis, MN), or 100 ng/ml LPS. Supernatants were harvested after 72 h at 37°C, and IL-12 p40 levels were measured by ELISA. For the in vivo detection of IL-12, each mouse was treated i.p. with 0.3 mg LPS (L4641; Sigma-Aldrich) or 0.5 mg anti-CD40. Mice were tail bled at 3 and 24 h to measure serum IL-12 p40 levels in response to LPS and anti-CD40, respectively.

Cytofluorometric analysis

Splenocytes were harvested from naive mice, and single cell suspensions were prepared. To assess cell division, splenocytes were stained with the dye CFSE (Molecular Probes, Eugene, OR), as previously described (32). Briefly, cells were washed and suspended in PBS at a concentration of 4 x 106 cells/ml and incubated with an equal volume of CFSE for 5 min at room temperature. Labeling was quenched by adding 10 ml FCS and washing twice in CTCM. Splenocytes were cultured in 96-well U-bottom plates at a concentration of 2 x 105 cells/well, with 1 µg/ml anti-CD3 under neutral or Th1 conditions.

After 4 days, the cells were harvested and intracellular staining was performed, as previously described (33). Briefly, the splenocytes were stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin, and 10 µg/ml brefeldin A for 4 h. The cells were then washed, incubated with 10 µg anti-FcR{gamma}III/IIR Ab (2.4G2) and 10 µg rat IgG (Sigma-Aldrich) for 5 min to inhibit nonspecific binding of Abs, surface stained with anti-CD4-CyChrome (BD PharMingen), and fixed overnight with 1% paraformaldehyde. Cells were then permeabilized with 0.1% saponin in FACS buffer (PBS containing 0.1% BSA and 0.1% sodium azide) for intracellular staining with anti-IFN-{gamma}-PE or an isotype-matched control Ab (BD PharMingen). Cells were washed and analyzed using a FACSCalibur cytometer (BD Biosciences, San Jose, CA). Numbers of proliferating cells producing IFN-{gamma} were quantitated using CellQuest software (BD Biosciences).

Adoptive transfer

Spleens from naive NF-{kappa}B2-/-, NF-{kappa}B2+/-, and WT B/6 mice were harvested, and single cell suspensions were prepared. CD3+ T cells were purified by negative selection (cell purification columns; R&D Systems) and adoptively transferred by i.v. injection (5 x 106 cells/mouse) into RAG-/- mice. One day later, the mice were infected with L. major, and the course of infection was monitored, as described above. Mice were sacrificed at 14 wk postinfection, and single splenocyte suspensions were prepared, as described above.

Statistics

Significance was determined by Student’s paired t test, with a p value < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NF-{kappa}B2-/- mice are susceptible to L. major infection

To assess whether NF-{kappa}B2 is required for resistance to L. major, NF-{kappa}B2-/-, NF-{kappa}B2+/-, and WT B/6 mice were infected in the hind footpad with 2 x 106 metacyclic promastigotes, and lesion size was measured over time. NF-{kappa}B2+/- and WT BL/6 mice exhibited a resistant phenotype, characterized by small lesions that healed by 12 wk. In contrast, NF-{kappa}B2-/- mice developed chronic nonhealing lesions (Fig. 1GoA). Histopathologic evaluation of lesions from 4-wk infected NF-{kappa}B2+/- mice showed extensive dermal fibroblast proliferation interspersed with numerous lymphocytes, and very few infected macrophages (Fig. 1GoB). In contrast, NF-{kappa}B2-/- lesions showed a loss of dermal collagen, large numbers of infected macrophages, and few lymphocytes (Fig. 1GoC). Evaluation of lesions from 2- and 6-wk infected mice revealed a similar discrepancy between NF-{kappa}B2-/- and NF-{kappa}B2+/- footpads (data not shown). Parasite quantitation at 2, 8, and 12 wk postinfection correlated well with these observations (Table IGo). At later time points, there was evidence for a systemic spread of the infection. Viable amastigotes could also be recovered from the spleen and liver of L. major-infected NF-{kappa}B2-/- mice (data not shown).



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  		FIGURE 1. NF-{kappa}B2-/- mice fail to resolve L. major infection. NF-{kappa}B2-/-, NF-{kappa}B2+/-, and WT BL/6 mice were infected in the right hind footpad with 2 x 106 metacyclic promastigotes. A, The course of infection was monitored by measuring footpad size. Results are expressed as the mean ± SEM of five mice per group. One representative experiment of five is shown. Histopathologic analysis of footpads from 4-wk infected mice revealed that NF-{kappa}B+/- mice had very few parasites and a large lymphocytic infiltrate (B), whereas NF-{kappa}B2-/- mice had few lymphocytes and could not contain infection (C).

 

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  		Table I. Parasite quantitation from footpads of L. major-infected NF-{kappa}B2-/-, NF-{kappa}B2+/-, and C57BL/6 mice

 
Effector cell function is intact in NF-{kappa}B2-/- mice

NF-{kappa}B2 is expressed at high levels in myeloid cells, and the NF-{kappa}B family of transcription factors is implicated in a large number of effector functions, including the production of NO and reactive oxygen intermediates (34, 35). Therefore, we asked whether the susceptibility of NF-{kappa}B2-/- mice results from an inability of L. major-infected macrophages to respond to IFN-{gamma} and produce sufficient levels of NO necessary to restrict parasite replication. Macrophages from NF-{kappa}B2-/- and NF-{kappa}B2+/- mice were infected with L. major in vitro and stimulated with IFN-{gamma} alone, or IFN-{gamma} and LPS for 2 or 72 h. It should be noted that the percentage of infected macrophages ranged from 50 to 60% after the initial 2 h and did not reveal a discrepancy in the infectivity of NF-{kappa}B2-/- and NF-{kappa}B2+/- cells. NF-{kappa}B2-/- and NF-{kappa}B2+/- macrophages produced equivalent levels of nitrate after 72 h and were equally capable of controlling parasite replication (Fig. 2Go). Based on these data, we concluded that the immune response downstream of IFN-{gamma} production is functionally intact in NF-{kappa}B2-/- mice.



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  		FIGURE 2. Macrophage activation is normal in the absence of NF-{kappa}B2. BMM{phi} were infected with L. major promastigotes, as described in Materials and Methods, and their ability to control parasite replication (A) and produce nitrate (B) in response to IFN-{gamma} and/or LPS was assessed. Shown are the means ± SEM of duplicates for each condition. This experiment was performed five times with similar results.

 
NF-{kappa}B2-/- mice exhibit defective IFN-{gamma} expression following L. major infection

To assess the Th effector cell phenotype in L. major-infected NF-{kappa}B2-/- mice, we stimulated splenocytes from infected mice with SLA and measured IFN-{gamma} and IL-4 levels in the culture supernatants. NF-{kappa}B2-/- splenocytes produced substantially less IFN-{gamma} than NF-{kappa}B2+/- cells even when IL-12 was added to the cultures (Fig. 3Go). However, NF-{kappa}B2-/- and NF-{kappa}B2+/- mice produced comparable amounts of IL-4, demonstrating that the observed defect in IFN-{gamma} production was not the result of an overwhelming Th2 response in the absence of NF-{kappa}B2 (Fig. 3Go).



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  		FIGURE 3. NF-{kappa}B2-/- mice fail to generate a Th1 response during L. major infection. Splenocytes from NF-{kappa}B2-/- or NF-{kappa}B2+/- mice were harvested at designated time points following infection, and incubated in culture medium alone or in the presence of SLA ± IL-12. The levels of IFN-{gamma} and IL-4 produced after 3 days were measured by ELISA. The data presented are the means ± SEM of individual mice at each time point. This experiment was performed three times with similar results (*, p < 0.05 compared with NF-{kappa}B2-/- mice).

 
NF-{kappa}B2-/- T cells can mediate resistance to L. major

One possible explanation for the susceptibility of NF-{kappa}B2-/- mice to L. major is an inability of T lymphocytes to differentiate into Th1 effectors. To address this, we cultured splenocytes from NF-{kappa}B2-/- and NF-{kappa}B2+/+ mice under neutral or Th1 (IL-12, anti-IL-4) conditions in the presence of anti-CD3. After 4 days, the cells were harvested and stained for intracellular IFN-{gamma} expression. NF-{kappa}B2-/- T cells proliferated normally and produced similar levels of IFN-{gamma} as NF-{kappa}B2+/+ lymphocytes under both culture conditions (Fig. 4GoA). We also examined whether polarized NF-{kappa}B2-/- splenocytes could reveal a defect in Th cytokine production upon restimulation. To do this, we cultured NF-{kappa}B2-/- and NF-{kappa}B2+/+ splenocytes under neutral, Th1, or Th2 (anti-IFN-{gamma}, IL-4) conditions for 7 days. The cells were then harvested and stimulated under neutral conditions for an additional 3 days. IFN-{gamma} and IL-4 levels were comparable in supernatants from NF-{kappa}B2-/- and NF-{kappa}B2+/+ cultures, demonstrating that T lymphocytes cannot only become Th1 effectors but also effectively differentiate into Th2 cells in the absence of NF-{kappa}B2 (Fig. 4GoB). This observation contrasts with NF-{kappa}B1-/- T cells, which are deficient in their ability to undergo Th2 cell polarization (18).



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  		FIGURE 4. Naive NF-{kappa}B2-/- T lymphocytes can proliferate and polarize to become Th1 or Th2 effectors. A, Splenocytes from naive NF-{kappa}B2-/- and NF-{kappa}B2+/+ mice were labeled with CFSE and cultured under Th0 or Th1 conditions (IL-12 and anti-IL-4) in the presence of anti-CD3. After 3 days, the cells were harvested and stained for CD4 and intracellular IFN-{gamma}. Shown are the percentages of CD4+ T cells that produced IFN-{gamma} and divided at least once. Data shown are representative of three experiments. B, NF-{kappa}B2-/- and NF-{kappa}B2+/+ splenocytes were cultured under Th0, Th1, or Th2 (IL-4 and anti-IFN-{gamma}) conditions in the presence of anti-CD3 and anti-CD28. After 7 days, the cells were harvested and restimulated with anti-CD3 for an additional 3 days. The levels of IFN-{gamma} and IL-4 produced were measured by ELISA.

 
The experiments described above suggest that the T cell arm of the immune response is sufficiently intact in the absence of NF-{kappa}B2 to mediate resistance to L. major. To definitively address this issue, we asked whether reconstitution of RAG-/- mice with NF-{kappa}B2-/- T cells could confer protection to L. major. As expected, B/6 T cells were able to control lesion development in RAG-/- mice, whereas unreconstituted RAG-/- mice remained susceptible and developed severe cutaneous leishmaniasis (Fig. 5GoA). More importantly, the adoptive transfer of NF-{kappa}B2-/- T cells also conferred a healing phenotype to RAG-/- mice. The healing observed in these mice correlated with the development of a Th1 response. Thus, when RAG-/- recipients were sacrificed at 14 wk of infection, splenocytes were stimulated with SLA or anti-CD3, and the number of IFN-{gamma}-producing cells was assessed. As seen in Fig. 5GoB, similar numbers of cells from RAG-/- mice that received either NF-{kappa}B2-/- or control splenocytes produced IFN-{gamma} in response to both SLA and anti-CD3. These results demonstrate that NF-{kappa}B2-/- T cells can differentiate into Th1 effector cells to promote healing in vivo and provide compelling evidence that the susceptibility of NF-{kappa}B2-/- mice to L. major is not explained by an intrinsic defect in T cell function.



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  		FIGURE 5. Adoptive transfer of T cells from naive NF-{kappa}B2-/- mice protects RAG 1-/- mice from L. major infection. A, CD3+ T lymphocytes were purified from NF-{kappa}B2-/- and WT BL/6 mice and adoptively transferred into RAG 1-/- mice by i.v. injection. One day later, the mice were infected with 2 x 106 metacyclic promastigotes. Results are expressed as the mean ± SEM of five mice per group. Data are representative of two independent experiments. B, Splenocytes were harvested at 14 wk following infection and incubated in the presence of SLA or anti-CD3. After 6 days, the cells were stained for CD4 and intracellular IFN-{gamma} and analyzed by flow cytometry. Shown are the percentages of CD4+ and non-CD4+ cells that produced IFN-{gamma}. Less than 2% of unstimulated cells from both groups were found to be positive for IFN-{gamma}.

 
The susceptibility of NF-{kappa}B2-/- mice to L. major is associated with a defect in IL-12 production

IL-12 stimulates the production of IFN-{gamma} from lymphoid cells, and NF-{kappa}B has been implicated in the regulation of IL-12 synthesis (36, 37). To address a potential defect in IL-12 production during L. major infection of NF-{kappa}B2-/- mice, we measured IL-12 p40 production by SLA-stimulated splenocytes from 4- and 6-wk infected animals. NF-{kappa}B2-/- splenocytes produced significantly less IL-12 p40 than NF-{kappa}B2+/- cells at both time points (Fig. 6Go). To determine whether the susceptibility of NF-{kappa}B2-/- mice is linked to this defect in IL-12 production, we administered exogenous IL-12 to NF-{kappa}B2-/- mice and measured lesion size over time. As reported previously, IL-12-treated BALB/c mice resisted L. major and controlled infection (23, 24). IL-12 treatment of NF-{kappa}B2-/- mice also provided protection against disease, resulting in the development of small lesions that had healed by 6 wk postinfection (Fig. 7GoA). Viable promastigotes were recovered at >10-8 dilutions for untreated NF-{kappa}B2-/- mice. In contrast, NF-{kappa}B2-/- mice treated with IL-12 exhibited 3- to 4-log fewer parasites (data not shown).



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  		FIGURE 6. IL-12 production is impaired following L. major infection of NF-{kappa}B2-/- mice. Splenocytes from NF-{kappa}B2-/- and NF-{kappa}B2+/- mice were harvested at 4 and 6 wk postinfection and incubated in culture medium alone or in the presence of SLA for 3 days. IL-12 p40 levels were quantitated by ELISA. The data presented are the means ± SEM of individual mice at each time point. This experiment was performed three times with similar results (*, p < 0.05 compared with NF-{kappa}B2-/- mice).

 


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  		FIGURE 7. Exogenous IL-12 provides protection to NF-{kappa}B2-/- mice, and anti-IL-12 results in the development of progressive cutaneous leishmaniasis. A, NF-{kappa}B2-/- and NF-{kappa}B2+/- mice were infected in the right hind footpad with 2 x 106 metacyclic promastigotes. IL-12-treated mice were administered IL-12 six times over the first 2 wk of infection. This experiment was performed four times with similar results. B, NF-{kappa}B2-/- and NF-{kappa}B2+/- mice were infected in the right hind footpad with 2 x 106 metacyclic promastigotes. Some mice received anti-IL-12 once per week for the first 3 wk of infection. Results are expressed as the mean ± SEM of five mice per group.

 
Infection of IL-12-deficient mice with L. major results in uncontrolled lesion growth, similar to that of a susceptible BALB/c mouse (38, 39). We suggest that the nonhealing phenotype of NF-{kappa}B2-/- mice is explained by a deficiency in IL-12 production, and yet these animals prevent progressive lesion development. As shown in Fig. 6Go, NF-{kappa}B2-/- mice still produce low levels of IL-12 during infection with L. major. To address whether the NF-{kappa}B2-independent production of IL-12 may account for the chronic nature of this infection, we treated NF-{kappa}B2-/- mice with anti-IL-12 and measured lesion size over time. In the complete absence of this cytokine, NF-{kappa}B2-/- mice were unable to contain lesion growth and developed a course of infection similar to that observed in IL-12 or CD40-/- mice (Fig. 7GoB). Viable promastigotes were recovered at >10-17 dilutions for anti-IL-12-treated NF-{kappa}B2-/- and NF-{kappa}B2+/- mice, at 10-10–10-11 dilutions for untreated NF-{kappa}B2-/- mice, and at 10-1 for untreated NF-{kappa}B2+/- mice (data not shown).

CD40-induced IL-12 production is impaired in the absence of NF-{kappa}B2

The development of a protective cell-mediated immune response to leishmaniasis is dependent on CD40-induced IL-12 production by macrophages and dendritic cells (39, 40, 41). In an effort to explain why IL-12 levels are reduced in L. major-infected NF-{kappa}B2-/- mice, we asked whether CD40 signaling is impaired in the absence of this transcription factor. Several labs have shown that NF-{kappa}B is activated in response to CD40, and one study provides evidence for its specific involvement in anti-CD40-induced IL-12 transcription (13). To address whether NF-{kappa}B2 is required in the CD40 signaling pathway to produce IL-12, we stimulated NF-{kappa}B2-/- and NF-{kappa}B2+/- macrophages with anti-CD40 or LPS. Importantly, CD40 expression was comparable between both cell types (data not shown). IL-12 levels were not impaired in response to LPS; however, anti-CD40-induced IL-12 production was defective in the absence of NF-{kappa}B2 (Fig. 8GoA). We also addressed CD40 signaling defects in NF-{kappa}B2-/- dendritic cells but failed to see a reproducible deficiency in IL-12 (data not shown). This is consistent with data suggesting that NF-{kappa}B-inducing kinase-deficient dendritic cells, which have very little NF-{kappa}B2 activation, produce normal levels of IL-12 in response to anti-CD40 (42, 43). To confirm our macrophage results in vivo, we administered anti-CD40 or LPS to NF-{kappa}B2-/- and NF-{kappa}B2+/- mice and quantitated serum IL-12 p40 levels at 3 and 24 h posttreatment. NF-{kappa}B2-/- mice produced higher levels of IL-12 than NF-{kappa}B2+/- mice in response to LPS (Fig. 8GoB). Importantly, however, CD40-induced IL-12 levels were reduced in NF-{kappa}B2-/- mice. These results suggest that NF-{kappa}B2 is required for optimal anti-CD40-induced IL-12 production.



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  		FIGURE 8. Anti-CD40-induced IL-12 production is defective in the absence of NF-{kappa}B2. IL-12 p40 levels were quantitated in the supernatants of macrophages primed with IFN-{gamma} and stimulated with anti-CD40 or LPS for 3 days (A), or in the serum of mice treated with LPS for 3 h or anti-CD40 for 24 h (B). The data presented are the means ± SEM of three individual mice and are representative of three experiments (*, p < 0.05 compared with NF-{kappa}B2-/- mice).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that NF-{kappa}B2 is dispensable for T cell proliferation and polarization into Th subsets. We use the L. major model of infection to show in vivo that NF-{kappa}B2-/- T cells can promote resistance to a disease that requires Th1 cell development for effective healing. This is intriguing considering that numerous studies have shown NF-{kappa}B activation in Th cells and a role for this transcription factor in normal T cell function (44). Because NF-{kappa}B2 appears to be of minimal importance to T lymphocytes during L. major infection, the susceptibility of NF-{kappa}B2-/- mice must be explained by a defect in another cell lineage. We show that macrophages require NF-{kappa}B2 for optimal CD40-stimulated IL-12 production, and provide evidence that the failure of NF-{kappa}B2-/- mice to resist cutaneous leishmaniasis results from a deficiency in IL-12 during infection. Importantly, NF-{kappa}B2 is not essential for all aspects of macrophage function. Parasite killing and NO production are intact in the absence of this transcription factor.

We were interested in addressing a role for NF-{kappa}B2 in T cells because gene deletion studies have defined roles for other NF-{kappa}B family members in modulating T cell function. For example, T cells lacking c-Rel cannot effectively proliferate in response to mitogenic stimuli (45). In addition, the susceptibility of RelB-/- mice to T. gondii is explained by an intrinsic defect in T cell-induced IFN-{gamma} production (9). Most recently, Th2 cell development was shown to require NF-{kappa}B1, potentially through its role in inducing GATA-3 expression (18). It is surprising that T cells are not more affected by the absence of NF-{kappa}B2, considering the structural similarity between this and other NF-{kappa}B family members. One explanation is that compensation by other NF-{kappa}B components masks a potential role for NF-{kappa}B2. Indeed, evidence for partial redundancy of NF-{kappa}B1 and NF-{kappa}B2 functions has already been shown using mice deficient in both family members (11). It is also possible that NF-{kappa}B2 is not essential in T lymphocytes because its activation patterns are tightly regulated. Research demonstrates that NF-{kappa}B1 is ubiquitously expressed in mouse tissues, while NF-{kappa}B2 activation is largely restricted to myeloid cells of the immune system (46, 47).

To determine why NF-{kappa}B2-/- mice fail to resist L. major, we addressed potential defects in two myeloid cell functions known to involve NF-{kappa}B, inducible NO synthase expression, and IL-12 production. The susceptibility of c-Rel-/- mice to L. major is correlated with impaired NO induction and parasite killing (48). However, despite an association between c-Rel and NF-{kappa}B2 in some tissues (15), NF-{kappa}B2 is not essential for macrophage effector cell function. Our results indicate that a more likely explanation for the susceptibility of NF-{kappa}B2-/- mice is a deficiency in IL-12 production during infection. Given that CD40-CD40 ligand (CD40L) ligation provides the primary stimulus of IL-12 during leishmaniasis, we investigated potential defects in CD40 signaling to produce IL-12 in the absence of NF-{kappa}B2 (39, 40, 41, 49). Although this signaling pathway is involved in a number of immunological processes, including the maturation and survival of dendritic cells and the production of Ab, IL-12 production appears to be its only required function for resistance to L. major (50). Administration of exogenous IL-12 to CD40L knockout mice confers protection against disease (41).

Signaling via CD40 is mediated in part by TNFR-associated factor family members and results in the subsequent activation of mitogen-activated protein kinases, I-{kappa}B kinases, and NF-{kappa}B (51, 52, 53). The defect in CD40-induced IL-12 production by NF-{kappa}B2-/- macrophages is similar to that observed in human PBMCs lacking NF-{kappa}B essential modulator (or I-{kappa}B kinases) activation (54). Importantly, LPS signaling is intact in the absence of either NF-{kappa}B2 or NF-{kappa}B essential modulator, highlighting a signal-specific requirement for these molecules in mediating IL-12 production. However, recent work in these laboratories demonstrates that, in contrast to NF-{kappa}B2, NF-{kappa}B1 is dispensable for normal IL-12 production via CD40 ligation (D. Artis, manuscript in preparation).

The distinct requirement for NF-{kappa}B2 in CD40-stimulated IL-12 production may also explain why IL-12 levels are normal during acute T. gondii infection of NF-{kappa}B2-/- mice (10). In contrast to L. major infections, the CD40-CD40L interaction is not essential for early IL-12 production and innate immunity to toxoplasmosis (55). However, a requirement for this pathway has been defined recently in the control of parasite replication during the chronic phase of infection (55). CD40L-/- mice are increasingly defective in IL-12 as the disease progresses (55). A similar reduction in IL-12 observed during chronic T. gondii infection of NF-{kappa}B2-/- mice was previously associated with the reduced capacity of these mice to produce IFN-{gamma} (10). Thus, this diminution in IL-12 may instead be explained by a defect in CD40-induced IL-12 production in the absence of NF-{kappa}B2. Interestingly, infection of NF-{kappa}B2-/- mice with T. gondii was accompanied by a massive increase in lymphocyte apoptosis (10). Although we found no evidence of enhanced cell death in the spleens of L. major-infected NF-{kappa}B2-/- mice (data not shown), an increase in apoptosis may provide an explanation for the reduced lymphocytic infiltrate in the footpad lesions of these animals.

Another possibility for the low numbers of lymphocytes observed in NF-{kappa}B2-/- lesions is a defect in T lymphocyte recruitment by cells at the site of infection. While many adhesion molecules and chemokines require NF-{kappa}B family members for their induction and activity, a specific role for NF-{kappa}B2 has not been defined (56, 57). Endothelial cells lacking NF-{kappa}B2 may reveal a defect in E-selectin expression, for example, which could impair the ability of T cells to home to the infected footpad. Alternatively, if NF-{kappa}B2 is involved in cutaneous T cell-attracting chemokine production, its absence might prevent efficient trafficking of lymphocytes to skin lesions (58). Further studies will be required to determine how enhanced lymphocyte apoptosis or impaired recruitment may influence the susceptibility of NF-{kappa}B2-/- mice to L. major.

We propose a role for NF-{kappa}B2 in the production of the IL-12 required to heal L. major infection and suggest that a defect in CD40 signaling may explain the in vivo deficiency in this cytokine. However, it is important to note that anti-CD40-induced IL-12 production is not completely abrogated in the absence of NF-{kappa}B2. This may explain why NF-{kappa}B2-/- mice still produce low levels of IL-12 and prevent uncontrolled parasite replication. Indeed, treatment of NF-{kappa}B2-/- mice with anti-IL-12 results in progressive lesion development, a manifestation of disease similar to that observed in IL-12- or CD40-deficient mice (38, 39). It is possible that NF-{kappa}B2 acts in concert with other transcription factors to stimulate maximal IL-12 promoter activity. Alternatively, NF-{kappa}B2 may be indirectly required for the synthesis of elements, such as IFN regulatory factor 1, which regulate IL-12 gene transcription (59). Defining the role of NF-{kappa}B2 in mediating IL-12 production aids us in viewing the NF-{kappa}B family as a set of individual transcription factors, each with unique roles in modulating immune function. The selectivity of NF-{kappa}B2 in CD40-induced IL-12 production highlights this NF-{kappa}B component as a potential drug target in the treatment of diseases resulting from a hyperactive Th1 response.


   Acknowledgments
 
We thank Dr. David Artis for his insightful comments and critical reading of the manuscript, and we acknowledge the support of Bristol-Myers Squibb (New York, NY) for supplying the NF-{kappa}B2-/- mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI-35914, AI-41880, AI-41158, and AI-0752, and the Burroughs Wellcome Fund for New Initiatives in Malaria Research. Back

2 Address correspondence and reprint requests to Dr. Phillip Scott, Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6008. E-mail address: pscott{at}vet.upenn.edu Back

3 Abbreviations used in this paper: RAG, recombination-activating gene; BMM{phi}, bone marrow macrophage; CD40L, CD40 ligand; CTCM, complete tissue culture medium; SLA, soluble leishmanial Ag; WT, wild type. Back

Received for publication October 24, 2001. Accepted for publication February 22, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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