The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Caamaño, J.
Right arrow Articles by Hunter, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Caamaño, J.
Right arrow Articles by Hunter, C. A.
The Journal of Immunology, 2000, 165: 5720-5728.
Copyright © 2000 by The American Association of Immunologists

Identification of a Role for NF-{kappa}B2 in the Regulation of Apoptosis and in Maintenance of T Cell-Mediated Immunity to Toxoplasma gondii1

Jorge Caamaño*, Cristina Tato{dagger}, Guifang Cai{dagger}, Eric N. Villegas{dagger}, Kendra Speirs{dagger}, Linden Craig{dagger}, James Alexander{ddagger} and Christopher A. Hunter2,{dagger}

* Medical Research Council Centre for Immune Regulation, School of Medicine, University of Birmingham, Edgbaston, Birmingham, United Kingdom; {dagger} Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104; and {ddagger} Department of Immunology, The Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The NF-{kappa}B family of transcription factors are involved in the regulation of innate and adaptive immune functions associated with resistance to infection. To assess the role of NF-{kappa}B2 in the regulation of cell-mediated immunity, mice deficient in the NF-{kappa}B2 gene (NF-{kappa}B2-/-) were challenged with the intracellular parasite Toxoplasma gondii. Resistance to this opportunistic pathogen is dependent on the production of IL-12, which is required for the development of innate NK cell and adaptive T cell responses dominated by the production of IFN-{gamma} necessary to control replication of this parasite. Although wild-type controls were resistant to T. gondii, NF-{kappa}B2-/- mice developed severe toxoplasmic encephalitis and succumbed to disease between 3 and 10 wk following infection. However, NF-{kappa}B2 was not required for the ability of macrophages to produce IL-12 or to inhibit parasite replication and during the acute stage of infection, NF-{kappa}B2-/- mice had no defect in their ability to produce IL-12 or IFN-{gamma} and infection-induced NK cell responses appeared normal. In contrast, during the chronic phase of the infection, susceptibility of NF-{kappa}B2-/- mice to toxoplasmic encephalitis was associated with a reduced capacity of their splenocytes to produce IFN-{gamma} associated with a loss of CD4+ and CD8+ T cells. This loss of T cells correlated with increased levels of apoptosis and with elevated expression of the pro-apoptotic molecule Fas by T cells from infected NF-{kappa}B2-/- mice. Together, these results suggest a role for NF-{kappa}B2 in the regulation of lymphocyte apoptosis and a unique role for this transcription factor in maintenance of T cell responses required for long-term resistance to T. gondii.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Rel/NF-{kappa}B family of transcription factors, composed of NF-{kappa}B1, NF-{kappa}B2, RelA, RelB, and c-Rel, is induced in response to signals that lead to cell growth, differentiation, inflammatory responses, and apoptosis (1, 2, 3, 4). The types of signals commonly associated with activation of NF-{kappa}B are those which are a consequence of inflammation and infection. Bacteria and their products are some of the best activators of NF-{kappa}B (5, 6, 7, 8) and cytokines such as TNF-{alpha} and IL-1 can also activate NF-{kappa}B (2). The activation and nuclear translocation of NF-{kappa}B leads to increased transcription of a number of different genes including chemokines (IL-8), adhesion molecules (endothelial leukocyte activation molecule, VCAM, ICAM), cytokines (IL-1, TNF-{alpha}, IL-12), and inducible NO synthase (9, 10). Thus, the activation of NF-{kappa}B is strongly associated with the production of cytokines and activation of effector molecules associated with innate immunity to infection. However, the role of NF-{kappa}B in immunity is not restricted to regulation of innate immunity and several NF-{kappa}B members are expressed in T and B cells and are important in the regulation of their responses (11, 12, 13, 14).

The importance of NF-{kappa}B in the development of immune responses and associated effector functions is illustrated by gene deletion studies (15, 16, 17, 18, 19, 20, 21, 22). Thus, mice deficient in different NF-{kappa}B family members are more susceptible to various viral, bacterial, and parasitic infections, but in many cases the basis for the increased susceptibility to these pathogens is uncertain. To understand the role of NF-{kappa}B in the development of innate and adaptive immunity to infection, studies were initiated to determine the role of different NF-{kappa}B family members in the immune response to T. gondii. This parasite is an opportunistic pathogen in patients with acquired and primary deficiencies in cell-mediated immunity (23, 24, 25). Resistance to T. gondii is dependent on the ability of accessory cells to produce IL-12, which stimulates the production of IFN-{gamma} by NK and T cells, which is required to control replication of this parasite (26). Several molecular studies indicate that NF-{kappa}B is likely to be involved in many of these steps. For example, the IL-12 p40 promoter has two NF-{kappa}B binding sites that are involved in the production of IL-12 (27), and NF-{kappa}B is important in the development, differentiation, and function of dendritic cells (20, 21, 28, 29, 30), which can produce IL-12 in response to T. gondii (31). Evidence that NF-{kappa}B is involved in the regulation of the IFN-{gamma} gene is provided by the identification of functional NF-{kappa}B sites in the promoter (32), studies in which inhibition of NF-{kappa}B activation in T cells resulted in reduced production of IFN-{gamma} following primary TCR stimulation (33, 34) and reports that mice deficient in c-Rel or RelB have defects in their ability to produce IFN-{gamma} (13, 35). Furthermore, activation of NF-{kappa}B is involved in the regulation of NO production (9) associated with the control of parasite replication (36, 37). Thus, the association of NF-{kappa}B activation with production of IL-12 and IFN-{gamma} as well as macrophage effector cell functions highlight the possible role that this family of transcription factors plays in resistance to T. gondii.

Direct evidence that NF-{kappa}B is involved in resistance to T. gondii is provided by studies in which mice deficient in NF-{kappa}B2 or RelB were shown to be susceptible to toxoplasmosis (20, 35). However, the mechanism that underlies the increased susceptibility of these mice to toxoplasmosis is unclear and these findings raise several questions about the function of NF-{kappa}B in resistance to infection. The studies presented in this study confirm the important role of NF-{kappa}B2 in resistance to T. gondii and reveal that NF-{kappa}B2 is not required for the induction of the innate or adaptive responses associated with resistance to T. gondii. Rather, these studies identify a unique role for NF-{kappa}B2 in the regulation of lymphocyte apoptosis in chronically infected mice and demonstrate that it is required for the long-term maintenance of adaptive immunity to this parasite.


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

Female CBA/CaJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The NF-{kappa}B2-/- mice originated on a 129/B6 background and had been backcrossed for four to five generations on a C57BL/6 background. Mice were typed using a PCR-based methodology which distinguishes the wild-type (WT)3 NF-{kappa}B2 gene from the targeted NF-{kappa}B2-neo allele as previously described (35). NF-{kappa}B2-/- mice are healthy and do not display any signs of autoimmunity or developmental defects (21). The NF-{kappa}B2-/- and NF-{kappa}B2+/+ mice used in these experiments were littermates generated by crosses of heterozygous mice. As a consequence it was frequently difficult to obtain large numbers of sex-matched controls, thus male and female mice were used for the studies reported here. Direct comparisons within experimental groups used single sexes.

Parasites

Toxoplasma lysate Ags (TLA) were prepared from RH strain tachyzoites as previously described (38). RH strain tachyzoites were routinely maintained in the laboratory in human fibroblasts. Cysts of the ME49 strain of T. gondii were harvested from brains of CBA/CaJ mice infected for 1–2 mo. For experimental infections, mice were given 20 ME49 cysts i.p. in a volume of 0.2 ml.

Histology

At different times postinfection, samples of lung, liver, heart, spleen, and brain were removed from each mouse, fixed in 10% neutral buffered formalin, and embedded in paraffin. Organs were sectioned and stained with hematoxylin and eosin for evaluation of pathological changes. T. gondii parasites and Ags were detected in livers of infected mice by peroxidase-antiperoxidase staining using polyclonal rabbit Abs against T. gondii (39). Cytospin preparations of peritoneal exudate cells were prepared as previously described and used to estimate the percentage of cells infected with T. gondii (40). A value of 0.1% was assigned where the percentage of cells infected was <0.1% but parasites could still be detected. In situ detection of apoptotic cells in histological sections was performed using a TUNEL assay protocol (Boehringer Mannheim, Indianapolis, IN). Briefly, a TdT was used to incorporate fluorescein-dUTP onto the 3' ends of DNA strand breaks. Positively labeled cells were visualized using an anti-fluorescein-peroxidase conjugate in combination with diaminobenzadine. Sections were counterstained with hematoxylin and analyzed using light microscopy.

Reagents

Anti-mouse CD3{epsilon} mAb (145-2C11) was prepared from hybridoma supernatants. IFN-{gamma}, TNF-{alpha}, IL-2, IL-4, IL-6, and IL-10 levels were measured using two-site ELISAs as previously described (41, 42). IL-12 p40 levels were measured using mAb C17.8 and biotinylated mAb C15.6 (grown from hybridomas provided by Giorgio Trinchieri (Wistar Institute, Philadelphia, PA)). Recombinant murine IL-2, IL-4, IL-6, IL-10, TNF-{alpha}, and IFN-{gamma} were purchased from Genzyme (Cambridge, MA). LPS was purchased from Sigma (St. Louis, MO). Levels of reactive nitrogen intermediates were measured using the Greiss assay as previously described (43).

Analysis of T and NK cell responses

Spleens from uninfected or infected animals were harvested and dissociated in complete RPMI 1640 (10% heat-inactivated FCS (Sigma), 1000 U/ml penicillin, 10 mg/ml streptomycin, 0.25 mg/ml fungizone (BioWhittaker, Walkersville, MA)) into single cell suspension as previously described (43). Cells were plated at a cell density of 4 x 105 cells/well in a final volume of 200 µl in 96-well plates and incubated with various stimuli, and supernatants were harvested after 48 h and assayed for the production of IL-2, IL-12, IL-4, and IFN-{gamma}. Cytolysis of 51Cr-labeled YAC-1 cells (American Type Culture Collection, Manassas, VA) was used to measure NK cell cytolytic activity as described previously (44).

Analysis of macrophage functions

Bone marrow-derived macrophages (BMMø) from NF-{kappa}B2-/- and WT littermates were derived from bone marrow cells grown on petri dishes (150 x 15-mm Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) in DMEM containing 20% (v/v) heat-inactivated FCS, 20% L-cell-conditioned medium, 100 U/ml penicillin, and 100 µg/ml streptomycin. After at least 6 days of incubation at 37°C with 5% CO2 in a humidified incubator, adherent cells were harvested using ice-cold buffered saline without calcium or magnesium and washed three times in complete RPMI 1640. For measurement of cytokines and NO production, BMMø were resuspended in complete RPMI 1640 and plated onto 96-well plates, 100 µl/well at 2 x 106 BMMø/ml. Medium alone, 250 ng/ml LPS, or 1000 U rTNF-{alpha} were added to cultures with or without 100 U/ml IFN-{gamma} to a final volume of 200 µl/well. Supernatants were collected at 48 h and used to measure TNF-{alpha}, IL-6, IL-12, and NO production as above. For anti-Toxoplasmaactivity, 4 x 105 BMMø in complete RPMI 1640 were plated on 15-mm glass coverslips in 24-well plates. Cells were then incubated at 37°C with 5% CO2 in medium alone, medium containing 100 U/ml IFN-{gamma}, or medium containing 250 ng/ml LPS or 1000 U/ml TNF-{alpha} with or without the addition of 100 U/ml IFN-{gamma}. After 4 h, cultures were infected with RH tachyzoites at a ratio of one parasite/macrophage, and at 2 and 16 h postinfection, cultures were fixed in formalin and baseline infections and parasite growth was assessed microscopically following staining of coverslip cultures using Diff-Quik (Dade Diagnostics, Aguada, PR).

Cytofluorometric analysis

After dissociation of the spleen and lysis of erythrocytes, splenocytes were resuspended at a final concentration of 1 x 107 cells/ml in FACS buffer composed of 1x PBS (BioWhittaker, Walkersville, MD), 0.2% BSA Fraction V (Sigma), and 4 mM sodium azide. For FACS analysis, 1 x 106 cells were stained with various conjugated mAbs specific for CD4 or CD8 for 20 min on ice in the presence of saturating amounts of Fc Block (PharMingen, San Diego, CA). Cells were then washed and analyzed using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA). For biotinylated mAbs, cells were stained and washed as described above, then incubated with FITC- or PE-conjugated streptavidin (PharMingen) for 20 min on ice. Cells were then washed with FACS buffer and analyzed. Each mAb and streptavidin reagent was used at dilutions empirically determined to give optimal staining for flow cytometric analyses. Results were analyzed using CellQuest software (Becton Dickinson).

RNase protection assay (RPA) analysis

Total RNA was isolated from the spleens of mice by the guanidine isothiocyanate method and was assayed for cytokine mRNA levels using the Riboquant Mutiprobe Protection Assay System (PharMingen). Briefly, 10 µg RNA from each sample was hybridized in solution with the appropriate radiolabeled antisense RNA probe set. mAPO-1, mAPO-2 and mAPO-3 were employed for the detection of mRNA for genes involved in the regulation of apoptosis as recommended by the manufacturers. Following hybridization, free probe and remaining ssRNA were digested with RNases, and the protected probes were purified and resolved on 5% denaturing polyacrylamide gels using Ultra Pure Sequagel reagents (National Diagnostics, Atlanta, GA). Dried gels were then exposed to a phosphorimaging screen (Bio-Rad, Richmond, CA) and visualized using a Bio-Rad Molecular Imager System.

Statistics

Instat software (GraphPad, San Diego, CA) was used for unpaired two-tailed student t test, paired t test evaluations, or Mann-Whitney nonparametric test. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NF-{kappa}B2-/- mice succumb to TE

To address the role of NF-{kappa}B2 in the immune response to T. gondii, NF-{kappa}B2-/- and WT littermate controls were challenged i.p. with 20 cysts of the ME49 strain of T. gondii. In contrast to WT mice, NF-{kappa}B2-/- mice were susceptible to infection and mice succumbed to infection beginning on day 25 with 100% mortality by day 90 of infection (Fig. 1GoA). Histopathological analysis of chronically infected mice revealed that WT mice developed a mild meningoencephalitis (Fig. 1GoB), whereas NF-{kappa}B2-/- mice developed a severe meningoencephalitis characterized by the presence of remarkable numbers of parasites, large numbers of inflammatory cells, and areas of necrosis (Fig. 1GoC). The lungs of infected WT and NF-{kappa}B2-/- mice had a mild to moderate interstitial pneumonia with no organisms readily detected. Similarly, the livers of these mice had a mild mutifocal random and perivascular hepatitis with no obvious differences between WT and NF-{kappa}B2-/- mice (data not shown). These findings confirm previous studies that NF-{kappa}B2-/- mice die earlier than WT mice following infection with T. gondii (20) but demonstrate for the first time that the susceptibility of these mice is associated with increased numbers of parasites and the development of severe TE.



View larger version (122K):
[in this window]
[in a new window]
  		FIGURE 1. NF-{kappa}B2-/- mice are susceptible to TE. A, Groups of WT (n = 14) and NF-{kappa}B2-/- (n = 16) littermates were infected i.p. with 20 cysts of the ME49 strain of T. gondii and survival was monitored. The data presented are the pooled data from three experiments. Histological analysis the brains of chronically infected mice (6 wk) revealed that WT mice had a mild meningoencephalitis (B), whereas NF-{kappa}B2-/- mice developed a severe meningoencephalitis associated with the presence of large numbers of parasites (C). At higher magnifications one of the rare clusters of organisms with only minimal associated inflammation in the WT mice (D) compared with the numerous organisms at the periphery of an inflammatory nodule in the NF-{kappa}B2-/- mice. In the NF-{kappa}B2-/- mice, the inflammation consists of lymphocytes, neutrophils, and macrophages with necrosis characterized by pyknotic nuclei and karyorrhectic debris.

 
Role of NF-{kappa}B2 in macrophage functions

Because activation of NF-{kappa}B is associated with the effector and regulatory functions necessary to control replication of T. gondii, the lack of NF-{kappa}B2 in macrophages could result in increased susceptibility to TE. Therefore, the ability of BMMø from WT and NF-{kappa}B2-/- mice to produce cytokines or control parasite replication was assessed. BMMø from WT and NF-{kappa}B2-/- mice stimulated with IFN-{gamma} and LPS produced similar levels of reactive nitrogen intermediates and were able to control replication of T. gondii (Fig. 2Go, A and B). Analysis of the cytokines produced by BMMø from WT and NF-{kappa}B2-/- mice stimulated with IFN-{gamma} and LPS revealed that they produced similar levels of IL-6 and TNF-{alpha} (data not shown). However, BMMø from NF-{kappa}B2-/- mice produced increased levels of IL-12 but decreased levels of IL-10 (Fig. 2Go, C and D). Because endogenous IL-10 is an inhibitor of macrophage production of IL-12, it is possible that the increased levels of IL-12 are a consequence of a reduced capacity to produce IL-10. Nevertheless, based on the parameters examined, no obvious defects were identified in macrophage function that would provide an explanation for the increased susceptibility of NF-{kappa}B2-/- mice to TE.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. Role of NF-{kappa}B2 in macrophage functions associated with resistance to T. gondii. BMMø were prepared as described in Materials and Methods, and their ability to control parasite replication (A) when unstimulated ({blacksquare}) or stimulated with IFN-{gamma} plus LPS was assessed (). B, The levels of nitrite produced by BMMø stimulated with LPS plus IFN-{gamma} are presented. In addition, BMMø were stimulated with 100 U IFN-{gamma} plus 100 ng/ml LPS for 48 h or LPS alone, and their ability to produce IL-12 (C) and IL-10 (D), respectively, was measured by ELISA. The data presented are pooled from three experiments in which a total of four WT and six NF-{kappa}B2-/- mice were used.

 
NF-{kappa}B2 is not required for infection-induced production of IL-12, IFN-{gamma}, or activation of NK cells

Because NF-{kappa}B is associated with the regulation of innate as well as adaptive immunity to infection, the early events associated with resistance to T. gondii were characterized to determine whether they contributed to the susceptibility of these mice to TE. WT and NF-{kappa}B2-/- mice were infected with T. gondii and the systemic levels of IL-12, IFN-{gamma}, and NK cell activity were used as a measure of the early protective responses to this infection (40, 45). By day 5 postinfection there were elevated serum levels of IL-12 and IFN-{gamma} that were similar in WT and NF-{kappa}B2-/- mice (Fig. 3Go, A and B). Interestingly, similar to the results observed with BMMø, there was a trend to higher levels of IL-12 in the NF-{kappa}B2-/- mice compared with WT mice, but this was not statistically significant. In addition, infection of WT and NF-{kappa}B2-/- mice with T. gondii resulted in a similar increase in NK cell cytolytic activity (Fig. 3GoC). Together, these data indicate that the absence of NF-{kappa}B2 does not inhibit the infection-induced increases in IL-12, IFN-{gamma}, and NK cell activation.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 3. NF-{kappa}B2 is not required for early production of IL-12, IFN-{gamma}, or activation of NK cells. WT and NF-{kappa}B2-/- mice were infected i.p. with 20 cysts of the ME49 strain of T. gondii for 5 days, and serum levels of IL-12 (A) and IFN-{gamma} (B) were measured using ELISA. The data presented are the means ± SD of four experiments, with three to five mice per experimental group. In uninfected mice, serum levels of IL-12 were typically <1 ng/ml and IFN-{gamma} was <19 pg/ml. There was no significant difference in the levels of IFN-{gamma} produced, and IL-12 levels showed a trend toward significance (p = 0.06). C, Splenocytes from uninfected and infected mice were used to measure levels of NK cell cytolytic activity for YAC-1 cells. The data presented are a representative experiment of two performed using two to three mice per experimental group.

 
To further analyze the role of NF-{kappa}B2 in the development of the early immune response the capacity of splenocytes from uninfected and infected WT and NF-{kappa}B2-/- mice to produce IL-12 and IFN-{gamma} were compared. Stimulation of splenocytes from uninfected mice with TLA resulted in the production of comparable levels of IL-12 by WT and NF-{kappa}B2-/- mice (Fig. 4Go). Stimulation of splenocytes from infected mice with TLA resulted in increased production of IL-12 compared with splenocytes from uninfected mice, but there was no significant difference between WT and NF-{kappa}B2-/- mice (Fig. 4GoA). The ability of splenocytes from uninfected NF-{kappa}B2-/- mice to produce IFN-{gamma} in response to soluble anti-CD3 revealed that equivalent levels of IFN-{gamma} were produced under these conditions and similar results were observed with splenocytes from acutely infected mice (Fig. 4GoB). Splenocytes from uninfected WT and NF-{kappa}B2-/- mice produced minimal levels of IFN-{gamma} in response to TLA, whereas cells from infected WT and NF-{kappa}B2-/- produced increased levels of IFN-{gamma}, but no significant difference was observed between these experimental groups. These results need to be interpreted with care as NF-{kappa}B2-/- mice actually have increased numbers of CD4+ and CD8+ T cells compared with littermate controls (21). Nevertheless, together with the studies which examined NK cell cytolytic activity and serum levels of IFN-{gamma} and IL-12, it appears that in the absence of NF-{kappa}B2, the generation of protective immune responses appear to be intact and these mice are able to survive the acute phase of this infection.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 4. Production of IL-12 and IFN-{gamma} by splenocytes from WT and NF-{kappa}B2-/- mice. Splenocytes from WT or NF-{kappa}B2-/- (KO) mice that were obtained from uninfected mice or mice infected for 5 days were incubated in culture medium alone (control) or in the presence of 1 µg/ml soluble anti-CD3 or 20 µg/ml TLA for 48 h, and the levels of IL-12 (A) and IFN-{gamma} (B) produced were measured by ELISA. The results presented are the pooled data from three experiments using 6 uninfected and 11 infected mice.

 
NF-{kappa}B2 is required for the maintenance of T cell responses

Because NF-{kappa}B2-/- mice succumbed to TE during the chronic stage of the infection the T cell responses of chronically infected mice was analyzed. Interestingly, when chronically infected NF-{kappa}B2-/- mice developed clinical signs of disease, there was a rapid progression and death of mice within a week, whereas other infected NF-{kappa}B2-/- mice in the same group appeared healthy. Therefore, experiments were designed in which groups of WT and NF-{kappa}B2-/- mice were infected, and as NF-{kappa}B2-/- mice became moribund they were sacrificed and their immune responses compared with chronically infected, but healthy, WT and NF-{kappa}B2-/- littermates. These studies revealed that following stimulation of splenocytes with anti-CD3 or TLA, healthy NF-{kappa}B2-/- mice produced lower levels of IFN-{gamma} than chronically infected WT mice and that moribund NF-{kappa}B2-/- mice had a major defect in their ability to produce IFN-{gamma} (Fig. 5GoA). In contrast, IL-12 levels were essentially intact and only reduced in mice that were moribund (Fig. 5GoB). Because endogenous production of IFN-{gamma} enhances TLA-induced IL-12 production (45), the reduced levels of IL-12 detected are likely due to the almost complete lack of IFN-{gamma} in these cultures. Together, these data associate the susceptibility of NF-{kappa}B2-/- mice to TE with a reduced capacity to produce IFN-{gamma} required for resistance to TE.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 5. Production of IL-12, IFN-{gamma}, and IL-2 by splenocytes from chronically infected WT and NF-{kappa}B2-/- mice. Groups of WT and NF-{kappa}B2-/- mice (KO) were infected with T. gondii, and as NF-{kappa}B2-/- mice became moribund between day 40 and 60 postinfection, they were sacrificed, and the ability of their splenocytes to produce IFN-{gamma} (A) or IL-12 (B) was compared with infected WT and healthy NF-{kappa}B2-/- mice. The results presented are the pooled data from three individual experiments using WT (n = 10), NF-{kappa}B2-/- (healthy, n = 12), and NF-{kappa}B2-/- (moribund, n = 6) mice. C, The production of IL-2 by splenocytes from chronically infected WT and healthy NF-{kappa}B2-/- mice stimulated with TLA or anti-CD3 was measured using ELISA. Data presented are from three individual experiments using 10 WT and 12 NF-{kappa}B2-/- mice. D, The production of IL-2 by splenocytes from uninfected WT and NF-{kappa}B2-/- mice stimulated with Con A. The data presented are from a representative experiment of three performed.

 
Analysis of the levels of IL-2 in these experiments revealed that although splenocytes from infected NF-{kappa}B2-/- mice had a major defect in their capacity to produce IFN-{gamma}, they produced higher basal levels of IL-2 than WT mice. However, the levels of IL-2 produced by splenocytes from healthy infected NF-{kappa}B2-/- mice stimulated with TLA were similar to WT mice (Fig. 5GoC) and moribund mice produced negligible levels of IL-2 (data not shown). These findings led us to compare the ability of T cells from uninfected WT and NF-{kappa}B2-/- mice to produce IL-2 in response to polyclonal stimuli. Splenocytes from NF-{kappa}B2-/- mice produced almost 10-fold more IL-2 than splenocytes from WT mice in response to ConA (Fig. 5GoD) and similar difference was also observed with anti-CD3 although the absolute levels of IL-2 produced were much reduced (data not shown). These results demonstrate that in the absence of NF-{kappa}B2 T cells overproduce IL-2.

Previous studies have shown that uninfected NF-{kappa}B2-/- mice have similar numbers of splenocytes compared with WT mice but have an approximate 3-fold increase in the percentage of CD4+ and CD8+ T cells and a reduced number of B cells compared with WT mice (20, 21), and our studies confirmed these findings (data not shown). Analysis of the spleens from chronically infected mice revealed that healthy NF-{kappa}B2-/- mice had similar numbers of CD4+ and CD8+ T cells compared with WT mice (Fig. 6Go) and that moribund NF-{kappa}B2-/- mice displayed a profound loss of CD4+ and CD8+ T cells. In addition, although uninfected NF-{kappa}B2-/- mice have a reduced number of B cells (21), this was further reduced following infection and moribund mice had an almost complete loss of B cells (Fig. 6Go). These data suggest that the reduced levels of IFN-{gamma} produced in recall responses by chronically infected NF-{kappa}B2-/- mice is associated with the loss of T cells from these spleens. The loss of T cells also provides a likely explanation why splenocytes from uninfected NF-{kappa}B2-/- mice produce higher levels of IL-2 than splenocytes from WT mice, but during the chronic stage of infection the levels of IL-2 produced are comparable.



View larger version (99K):
[in this window]
[in a new window]
  		FIGURE 6. Loss of lymphocytes and increased levels of apoptosis in spleens from chronically infected NF-{kappa}B2-/- mice. Groups of WT and NF-{kappa}B2-/- (KO) mice were infected with T. gondii, and as NF-{kappa}B2-/- mice became moribund between day 40 and 60 postinfection, they were sacrificed, the total numbers of splenocytes was counted, and the total number of CD4+ and CD8+ T cells was estimated based on FACS analysis. The results presented are the pooled data from four separate experimental groups and the figures in parenthesis are the SE. Detection of apoptotic cells using TUNEL staining on the spleen of a WT mouse (B) or an NF-{kappa}B2-/- mouse (C) infected for 6 wk. Note the single TUNEL+ cell (brown) in the left panel and the many TUNEL+ cells in the right panel. In addition, the right panel lacks the many densely staining lymphocytes apparent in the left panel, a sign of lymphoid depletion.

 
Infection of NF-{kappa}B2-/- mice results in increased levels of apoptosis and T cell expression of Fas

Gross pathological analysis of infected NF-{kappa}B2-/- mice that were moribund revealed that their spleens were ~25% of the size of chronically infected WT mice, and histological analysis suggested that although there were no readily detectable areas of parasite replication in the spleen there were high levels of apoptosis compared with chronically infected WT mice. Importantly, based on histological analysis, there were no discernable differences between acutely infected WT and NF-{kappa}B2-/- mice and the use of TUNEL analysis as a measure of the levels of apoptosis in the spleens of WT and NF-{kappa}B2-/- mice revealed that, similar to previous reports (21), uninfected WT and NF-{kappa}B2-/- mice had similar levels of apoptosis (data not shown). However, TUNEL analysis confirmed the preliminary histological analysis and revealed remarkable numbers of apoptotic cells in the spleens of chronically infected NF-{kappa}B2-/- mice, which was not observed in chronically infected WT mice (Fig. 6Go).

The high levels of apoptosis observed in the spleens of chronically infected NF-{kappa}B2-/- mice led us to examine the expression of genes associated with regulation of apoptosis. RPA analysis was used to quantitate the levels of mRNA for Fas ligand (FasL), Fas, Fas-associated death domain (FADD), Fas-associated phosphatase, Fas-associated protein factor, TNF-related apoptosis-inducing ligand, TNFR-assocated death domain protein, receptor interacting protein, Bcl2, bak, bax, bad, and various caspases in the spleens of WT and NF-{kappa}B2-/- mice. This approach did not reveal any difference in the levels of mRNA specific for these genes between uninfected WT and NF-{kappa}B2-/- mice and the only marked difference obtained when infected WT and NF-{kappa}B2-/- mice were compared was in the levels of Fas and FasL. The results presented in Fig. 7Go show the data obtained for Fas, FasL, and FADD using uninfected WT and NF-{kappa}B2-/- mice or WT and NF-{kappa}B2-/- mice infected for 38 days. This analysis revealed that although there were no differences in the levels of Fas, FasL, and FADD between uninfected WT and NF-{kappa}B2-/- mice, infected NF-{kappa}B2-/- mice had increased levels of mRNA for Fas and FasL compared with infected WT mice but there was no significant difference in the levels of FADD (Fig. 7Go). In similar experiments which compared levels of mRNA for Fas and FasL at days 11, 59, and 68 postinfection, similar results were also observed (data not shown). Based on these results, FACS analysis was used to examine T cell expression of Fas. As shown in Fig. 8Go, T cells directly isolated from chronically infected NF-{kappa}B2-/- mice expressed elevated levels of Fas, but this was not observed in uninfected mice or infected WT mice (Fig. 8Go). FACS analysis also revealed a small increase in the expression of FasL (Fig. 8Go). Together, the TUNEL, RPA, and FACS analysis indicate that infection of NF-{kappa}B2-/- mice with T. gondii leads to increased expression of Fas and this correlates with the increased levels of apoptosis observed in these mice.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 7. Analysis of levels of mRNA for Fas, FasL, and FADD in chronically infected WT and NF-{kappa}B2-/- mice. RPA analysis was used to quantify levels of mRNA for Fas, FasL, and FADD in the spleen as described in Materials and Methods. The data presented are based on the ratio from uninfected mice are the means ± range using two WT and two NF-{kappa}B2-/- mice and the data from infected mice is based on the mean ± SD from three WT mice and a single NF-{kappa}B2-/- mouse infected for 38 days. Data are presented as a ratio of the target to the internal L32 ribosomal gene.

 


View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 8. Expression of Fas and FasL by T cells from uninfected and infected WT and NF-{kappa}B2-/- mice. Splenocytes from uninfected (thin line) or mice infected for 60 days (thick line) were analyzed for expression of Fas and FasL using FACS. The data presented are gated on total T cell populations and are representative of three experiments performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies presented here reveal for the first time that NF-{kappa}B2 is not required for the innate NK and adaptive T cell responses that lead to the production of IFN-{gamma} required for resistance to T. gondii, nor is NF-{kappa}B2 required for the macrophage effector functions associated with control of this intracellular pathogen. However, our studies indicate that in chronically infected mice NF-{kappa}B2 has a role in the maintenance of lymphocyte responses necessary to control the chronic stage of this infection. Thus, there is a remarkable loss of T cells in chronically infected NF-{kappa}B2-/- mice which correlates with a reduced capacity to produce IFN-{gamma} and susceptibility to TE. In addition, it is noteworthy that while uninfected NF-{kappa}B2-/- mice have reduced numbers of B cells compared with WT mice (20, 21) there was a further reduction in this population of cells during the chronic stage of infection and this may contribute to the increased susceptibility of these mice to TE (46, 47). Nevertheless, like the patients with AIDS, certain cancers, or who are being treated with immunosuppressive drug regimens (23, 24, 48), the remarkable loss of lymphocytes and/or lymphocyte functions in chronically infected NF-{kappa}B2-/- mice provides a mechanism that underlies the development of TE in these mice.

Although our studies have demonstrated a novel role for NF-{kappa}B2 in the regulation of IL-2, IL-10, and IL-12 production, the most striking observation was the high levels of apoptosis and loss of lymphocytes observed in chronically infected NF-{kappa}B2-/- mice. Importantly, the increased numbers of apoptotic cells was only observed in NF-{kappa}B2-/- mice infected with T. gondii, suggesting that these events are directly related to the regulation of the immune response to this persistent parasite. There are several possible explanations for the loss of T cells during chronic toxoplasmosis. Previous studies have identified a clear role for NF-{kappa}B proteins in the regulation of anti-apoptotic genes, such as TNFR-associated factor, cellular inhibitor of apoptosis, and IEX-1L (49, 50, 51, 52, 53) although, recent studies indicate that NF-{kappa}B can also have a pro-apoptotic role (54). Perhaps the most direct explanation for the increased apoptosis observed in the infected NF-{kappa}B2-/- mice is that NF-{kappa}B2-/- lymphocytes may have reduced expression of anti-apoptotic factors and/or increased levels of proapoptotic proteins such as Fas, rendering these cells more susceptible to apoptosis (55, 56, 57, 58). However, to the best of our knowledge, NF-{kappa}B2 has not been associated with the regulation of apoptosis (20, 21), and further studies will be required to determine whether NF-{kappa}B2 can regulate downstream targets that protect against apoptosis.

Although NF-{kappa}B2 may be directly involved in the regulation of apoptosis, there are alternative explanations for the loss of lymphocytes in the chronically infected NF-{kappa}B2-/- mice. It is possible that the phenotype presented by the NF-{kappa}B2-/- mice during the chronic phase of infection with T. gondii is a result of an immunological environment that promotes apoptosis. For example, NF-{kappa}B2-/- mice lack follicular dendritic cells (21) and NF-{kappa}B is also involved in the generation of metallophilic marginal zone macrophages (59) and the role of these cell types in the maintenance of T cell responses is unknown. In addition, the increased capacity of NF-{kappa}B2-/- T cells to produce IL-2 and elevated levels of expression of Fas by T cells from NF-{kappa}B2-/- mice suggest that activation-induced cell death (AICD) may be involved in the loss of lymphocytes. AICD is a process in which IL-2 primes activated T cells for apoptosis (60) mediated by the interaction of Fas with FasL (61, 62). Of relevance to our studies, the activation of NF-{kappa}B is involved in the expression of Fas, FasL, and IL-2, and several studies have shown that blockade of NF-{kappa}B antagonizes AICD (55, 56, 63, 64, 65, 66). It is not clear why there is only an increase in the number of apoptotic cells and loss of lymphocytes in chronically infected mice, but long-term activated T cells are more sensitive to Fas-induced apoptosis than resting or short-term activated T cells (67, 68). Furthermore, it has been reported that Th1 cells (which dominate during infection with T. gondii) are more susceptible than TH2 cells to AICD (69). Nevertheless, the events that lead to the loss of T cells in infected NF-{kappa}B2-/- mice remain unclear and only by integrating studies which examine how the absence of NF-{kappa}B2 affects susceptibility to apoptosis, production of IL-2 and expression of Fas can we determine the molecular basis for these events in NF-{kappa}B2-/- mice.

An important challenge to understanding the role of NF-{kappa}B in regulation of immunity is to distinguish the role of specific NF-{kappa}B members in the different regulatory and effector functions essential to coordinate the development of protective immunity. The response of NF-{kappa}B2-/- mice to infection with T. gondii contrasts with what is known about the role of other NF-{kappa}B members in resistance to T. gondii. RelB-/- mice are highly susceptible to acute toxoplasmosis associated with a defect in the ability of their NK and T cells to produce IFN-{gamma} (35). In contrast, NF-{kappa}B1-/- mice infected with T. gondii are not more susceptible to toxoplasmosis and can generate and maintain normal T cell responses (J.C. and C.A.H., unpublished observations). Because many of the NF-{kappa}B family members are thought of as being functionally able to compensate for each other, the identification of a role for NF-{kappa}B2 in the regulation of adaptive immunity to infection that is distinct from NF-{kappa}B1 and RelB indicates a unique role for NF-{kappa}B2 in immunity to this infection. It is also important to recognize the parallels between the NF-{kappa}B2-/- mice and the situation in AIDS patients who are chronically infected with T. gondii and who, as their T cell numbers fall, can no longer control this infection and develop TE. T cells from individuals with AIDS have an increased susceptibility to apoptosis and there are high levels of apoptosis of activated T cells in these patients (70, 71, 72, 73), and it has been proposed that AICD contributes to the loss of T cells that allows opportunistic infections to cause disease. Understanding the role of NF-{kappa}B2 in the regulation of T cell survival and maintenance of protective T cell responses may be helpful in the design of strategies to prevent the loss of T cells that renders these patients susceptible to opportunistic pathogens.


   Acknowledgments
 
We thank Dr. Phil Scott for his insightful comments and support during these studies and the preparation of the manuscript, and we acknowledge the support of Rodrigo Bravo and Bristol Myers Squibb for supplying the NF-{kappa}B2-/- mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI 41158-01 and TW00970-02, Center Grant P30 DK50306, and the Commonwealth of Pennsylvania, Department of Agriculture. E.N.V. is supported by a National Institutes of Health predoctoral fellowship (AI 09562); J.A. was on research leave sponsored by the Wellcome Trust; and C.A.H. is a Burroughs Wellcome New Investigator in Molecular Parasitology. Back

2 Address correspondence and reprint requests to Dr. Christopher A. Hunter, Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6008. Back

3 Abbreviations used in this paper: WT, wild type; TLA, Toxoplasma lysate Ag; TE, toxoplasmic encephalitis; BMMø, bone marrow-derived macrophages; RPA, RNase protection assay; FasL, Fas ligand; FADD, Fas-associated death domain; AICD, activation-induced cell death. Back

Received for publication June 14, 2000. Accepted for publication August 23, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Siebenlist, U., G. Franzoso, K. Brown. 1994. Structure, regulation and function of NF-{kappa}B. Annu. Rev. Cell Biol. 10:405.
  2. Ghosh, S., M. J. May, E. B. Kopp. 1998. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. W. E. Paul, and C. G. Fathman, and L. E. Glimcher, eds. In Annual Review of Immunology Vol. 16:225. Annual Reviews, Palo Alto. [Medline]
  3. Gerondakis, S., R. Grumont, I. Rourke, M. Grossmann. 1998. The regulation and roles of Rel/NF-{kappa}B transcription factors during lymphocyte activation. Curr. Opin. Immunol. 10:353.[Medline]
  4. May, M. J., S. Ghosh. 1998. Signal transduction through NF-{kappa}B. Immunol. Today 19:80.[Medline]
  5. Geng, Y., B. Zhang, M. Lotz. 1993. Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes. J. Immunol. 151:6692.[Abstract]
  6. Lousie, C. B., M. C. Tran, T. G. Obrig. 1997. Sensitization of human umbilical vein endothelial cells to shiga toxin: involvement of protein kinase C and NF-{kappa}B. Infect. Immun. 65:3337.[Abstract]
  7. Toosi, Z., B. D. Hamilton, M. H. Phillips, L. E. Averill, J. J. Ellner, A. Salvekar. 1997. Regulation of nuclear factor-{kappa}B and its inhibitor I{kappa}B-{alpha}/MAD-3 in monocytes by Mycobacterium tuberculosis and during human tuberculosis. J. Immunol. 159:4109.[Abstract]
  8. Ebnet, K., K. D. Brown, U. K. Siebenlist, M. M. Simon, S. Shaw. 1997. Borrelia burgdorferi activates nuclear factor-{kappa}B and is a potent inducer of chemokine and adhesion molecule gene expression in endothelial cells and fibroblasts. J. Immunol. 158:3285.[Abstract]
  9. Xie, Q. W., Y. Kashiwabara, C. Nathan. 1994. Role of transcription factor NF-{kappa}B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269:4705.[Abstract/Free Full Text]
  10. Liu, S. F., X. Ye, A. B. Malik. 1997. In vivo inhibition of nuclear factor-{kappa}B activation prevents inducible nitric oxide synthase expression and systemic hypotension in a rat model of septic shock. J. Immunol. 159:3976.[Abstract]
  11. Lernbecher, T., U. Muller, T. Wirth. 1994. Distinct NF-{kappa}B/Rel transcription factors are responsible for tissue-specific and inducible gene activation. Nature 365:767.
  12. Weih, F., D. Carrasco, R. Bravo. 1994. Constitutive and inducible Rel/NF-{kappa}B activities in mouse thymus and spleen. Oncogene 9:3289.[Medline]
  13. Gerondakis, S., A. Strasser, D. Metcalf, G. Grigoriadis, J. Y. Scheerlinck, R. J. Grumont. 1996. Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor. Proc. Nat. Acad. Sci. USA 93:3405.[Abstract/Free Full Text]
  14. Ishikawa, H., D. Carrasco, E. Claudio, R.-P. Ryseck, R. Bravo. 1997. Gastric hyperplasia and increased proliferative responses of lymphocytes in mice lacking the COOH-terminal ankyrin domain of NF-{kappa}B. J. Exp. Med. 186:999.[Abstract/Free Full Text]
  15. Sha, W. C., H. C. Liou, E. I. Tuomanen, D. Baltimore. 1995. Targeted disruption of the p50 subunit of NF-{kappa}B leads to multifocal defects in immune responses. Cell 80:321.[Medline]
  16. Grigoriadis, G., Y. Zhan, R. Grumont, D. Metcalf, E. Handman, C. Cheers, S. Gerondakis. 1996. The Rel subunit of NF-{kappa}B-like transcription factors is a positive and negative regulator of macrophage gene expression: distinct roles for Rel in different macrophage populations. EMBO J. 15:7099.[Medline]
  17. Weih, F., G. Warr, H. Yang, R. Bravo. 1997. Multifocal defects in immune responses in RelB-deficient mice. J. Immunol. 158:5211.[Abstract]
  18. Schwarz, E. M., P. Krimpenfort, A. Berns, I. M. Verma. 1997. Immunological defects in mice with a targeted disruption in Bcl-3. Genes Dev. 11:187.[Abstract/Free Full Text]
  19. Franzoso, G., L. Carlson, T. Scharton-Kersten, E. W. Shores, S. Epstein, A. Grinberg, T. Tran, E. Shacter, A. Leonardi, M. Anver, et al 1997. Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic architecture, and germinal center reactions. Immunity 6:479.[Medline]
  20. Franzoso, G., L. Carlson, L. Poljak, E. W. Shores, S. Epstein, A. Leonardi, A. Grinberg, T. Tran, T. Scharton-Kersten, M. Anver, et al 1998. Mice deficient in nuclear factor (NF)-{kappa}B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J. Exp. Med. 187:147.[Abstract/Free Full Text]
  21. Caamano, J. H., C. A. Rizzo, S. K. Durham, D. S. Barton, C. Raventos-Suarez, C. M. Snapper, R. Bravo. 1998. NF-{kappa}B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses. J. Exp. Med. 187:185.[Abstract/Free Full Text]
  22. Harling-McNabb, L., G. Deliyannis, D. C. Jackson, S. Gerondakis, G. Grigoriadis, L. E. Brown. 1999. Mice lacking the transcription factor subunit rel can clear an influenza infection and have functional anti-viral cytotoxic T cells but do not develop an optimal antibody response. Int. Immunol. 11:1431.[Abstract/Free Full Text]
  23. Luft, B. J., R. Hafner, A. H. Korzun, C. Leport, D. Antoniskis, E. M. Bosler, D. D. Bourland, R. Uttamchandani, J. Fuhrer, J. Jacobson, et al 1993. Toxoplasmic encephalitis in patients with the acquired immunodeficiency syndrome. N. Engl. J. Med. 329:995.[Abstract/Free Full Text]
  24. Israelski, D. M., J. S. Remington. 1993. Toxoplasmosis in patients with cancer. Clin. Infect. Dis. 17:(Suppl. 2):S423.
  25. Tsuge, I., H. Matsuoka, A. Nakagawa, Y. Kamachi, K. Aso, T. Negoro, M. Ito, S. Torii, K. Watanabe. 1998. Necrotizing toxoplasmic encephalitis in a child with the X-linked hyper-IgM syndrome. Eur. J. Pediatr. 157:735.[Medline]
  26. Denkers, E. Y., R. T. Gazzinelli. 1998. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin. Microbiol. Rev. 11:569.[Abstract/Free Full Text]
  27. Yoshimoto, T., H. Nagase, T. Ishida, J. Inoue, H. Nariuchi. 1997. Induction of interleukin-12 p40 transcript by CD40 ligation via activation of nuclear factor-{kappa}B. Eur. J. Immunol. 27:3461.[Medline]
  28. Burkly, L., C. Hesslon, L. Ogata, C. Reilly, L. A. Marconi, D. Olson, R. Tizard, R. Cate, D. Lo. 1995. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature 373:531.[Medline]
  29. Weih, F., D. Carrasco, S. K. Durham, D. S. Barton, C. A. Rizzo, R.-P. Ryseck, S. A. Lira, R. Bravo. 1995. Mutiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-{kappa}B/Rel family. Cell 80:331.[Medline]
  30. Wu, L., A. D’Amico, K. D. Winkel, M. Suter, D. Lo, K. Shortman. 1998. RelB is essential for the development of myeloid-related CD8{alpha}- dendritic cells but not of lymphoid-related CD8{alpha}+ dendritic cells. Immunity 9:839.[Medline]
  31. Reis e Sousa, B. C., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charset, R. N. Germain, A. Sher. 1997. In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J. Exp. Med. 186:1819.[Abstract/Free Full Text]
  32. Sica, A., L. Dorman, V. Viggiano, M. Cippitelli, P. Ghosh, N. Rice, H. A. Young. 1997. Interaction of NF-{kappa}B and NFAT with the interferon-promoter. J. Biol. Chem. 272:30412.[Abstract/Free Full Text]
  33. Aune, T. M., A. L. Mora, S. Kim, M. Boothby, A. H. Lichtman. 1999. Costimulation reverses the defect in IL-2 but not effector cytokine production by T cells with impaired I{kappa}B{alpha} degradation. J. Immunol. 162:5805.[Abstract/Free Full Text]
  34. Ferreira, V., N. Sidenius, N. Tarantino, P. Hubert, L. Chatenoud, F. Blasi, M. Korner. 1999. In vivo inhibition of NF-{kappa}B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis. J. Immunol. 162:6442.[Abstract/Free Full Text]
  35. Caamaño, J., J. Alexander, L. Craig, R. Bravo, C. A. Hunter. 1999. The NF-{kappa}B family member RelB is required for innate and adaptive immunity to Toxoplasma gondii. J. Immunol. 163:4453.[Abstract/Free Full Text]
  36. Adams, L. B., J. B. Hibbs, R. R. Taintor, J. L. Krahenbuhl. 1990. Microbiostatic effect of murine activated macrophages for Toxoplasma gondii: role for synthesis of inorganic nitrogen oxides from L-arginine. J. Immunol. 144:2725.[Abstract]
  37. Scharton-Kersten, T. M., G. Yap, J. Magram, A. Sher. 1997. Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplasma gondii. J. Exp. Med. 185:1261.[Abstract/Free Full Text]
  38. Sharma, S. D., J. Mullenax, F. G. Araujo, A. A. Erlich, J. S. Remington. 1983. Western blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies. J. Immunol. 131:977.[Abstract]
  39. Conley, F. K., K. A. Jenkins. 1981. Immunohistological study of the anatomic relationship of Toxoplasma antigens to the inflammatory response in the brains of mice chronically infected with Toxoplasma gondii. Infect. Immun. 31:1184.[Abstract/Free Full Text]
  40. Hunter, C. A., L. Ellis-Neyer, K. Gabriel, M. Kennedy, P. Linsley, J. S. Remington. 1997. The role of the CD28/B7 interaction in the regulation of NK cell responses during infection with Toxoplasma gondii. J. Immunol. 158:2285.[Abstract]
  41. Abrams, J. S., M. G. Roncarolo, H. Yssel, G. J. Andersson, J. E. Silver. 1992. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol. Rev. 127:5.[Medline]
  42. Sander, B., I. Hoiden, U. Andersson, E. Moller, J. S. Abrams. 1993. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. J. Immunol. Meth. 166:201.[Medline]
  43. Candolfi, E., C. A. Hunter, J. S. Remington. 1995. Roles of {gamma} interferon and other cytokines in suppression of the spleen cell proliferative response to concanavlin A and Toxoplasma antigen during acute toxoplasmosis. Infect. Immun. 63:751.[Abstract]
  44. Hunter, C. A., C. S. Subauste, V. H. Van Cleave, J. S. Remington. 1994. Production of {gamma} interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor {alpha}. Infect. Immun. 62:2818.[Abstract/Free Full Text]
  45. Scharton-Kersten, T. M., T. A. Wynn, E. Y. Denkers, S. Bala, E. Grunvald, S. Hieny, R. T. Gazzinelli, A. Sher. 1996. In the absence of endogenous IFN-{gamma}, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157:4045.[Abstract]
  46. Sayles, P. C., G. W. Gibson, L. L. Johnson. 2000. B cells are essential for vaccination-induced resistance to virulent Toxoplasma gondii. Infect. Immun. 68:1026.[Abstract/Free Full Text]
  47. Kang, H., J. S. Remington, Y. Suzuki. 2000. Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-{gamma}, TNF-{alpha}, and inducible nitric oxide synthase. J. Immunol. 164:2629.[Abstract/Free Full Text]
  48. Slavin, M. A., J. D. Meyers, J. S. Remington, R. C. Hackman. 1994. Toxoplasma gondii infection in marrow transplant recipients: a 20 year experience. Bone Marrow Transplant. 13:549.[Medline]
  49. Wu, M. X., Z. Ao, K. V. S. Prasad, R. Wu, S. F. Schlossman. 1998. IEX-1L, an apoptosis inhibitor involved in NF-{kappa}B-mediated cell survival. Science 281:998.[Abstract/Free Full Text]
  50. Lee, S. Y., D. R. Kaufman, A. L. Mora, A. Santana, M. Boothby, Y. Choi. 1998. Stimulus-dependent synergism of the antiapoptotic tumor necrosis factor receptor-associated factor 2 (TRAF2) and nuclear factor {kappa}B pathways. J. Exp. Med. 188:1381.[Abstract/Free Full Text]
  51. Wang, C.-Y., M. W. Mayo, R. G. Korneluk, D. V. Goeddel, A. S. Baldwin. 1998. NF-{kappa}B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281:1680.[Abstract/Free Full Text]
  52. Zong, W. X., L. C. Edelstein, C. Chen, J. Bash, C. Gelinas. 1999. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-{kappa}B that blocks TNF{alpha}-induced apoptosis. Genes Dev. 13:382.[Abstract/Free Full Text]
  53. Grumont, R. J., I. J. Rourke, S. Gerondakis. 1999. Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis. Genes Dev. 13:400.[Abstract/Free Full Text]
  54. Ryan, K. M., M. K. Ernst, N. R. Rice, K. H. Vousden. 2000. Role of NF-{kappa}B in p53-mediated programmed cell death. Nature 404:892.[Medline]
  55. Ouaaz, F., M. Li, A. A. Beg. 1999. A critical role for the RelA subunit of nuclear factor {kappa}B in regulation of multiple immune-response genes and in Fas-induced cell death. J. Exp. Med. 189:999.[Abstract/Free Full Text]
  56. Teixeiro, E., A. Garcia-Sahuquillo, B. Alarcon, R. Bragado. 1999. Apoptosis-resistant T cells have a deficiency in NF-{kappa}B-mediated induction of Fas ligand transcription. Eur. J. Immunol. 29:745.[Medline]
  57. Hsu, S. C., M. A. Gavrilin, H. H. Lee, C. C. Wu, S. H. Han, M. Z. Lai. 1999. NF-{kappa}B-dependent Fas ligand expression. Eur. J. Immunol. 29:2948.[Medline]