Mice deficient in nuclear factor (NF)-kappa B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture - PubMed (original) (raw)

. 1998 Jan 19;187(2):147-59.

doi: 10.1084/jem.187.2.147.

L Carlson, L Poljak, E W Shores, S Epstein, A Leonardi, A Grinberg, T Tran, T Scharton-Kersten, M Anver, P Love, K Brown, U Siebenlist

Affiliations

Mice deficient in nuclear factor (NF)-kappa B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture

G Franzoso et al. J Exp Med. 1998.

Abstract

p52 is a subunit of nuclear factor (NF)-kappa B transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the I kappa B family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.

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Figures

Figure 1

Figure 1

Targeted disruption of the p52/p100 gene. (A) The targeting vector, a partial restriction map of the p52/p100 gene locus, and the mutated allele after homologous recombination of the targeting vector are shown. Probes used and fragments detected in Southern blots are indicated. kb, kilobase pairs; tk, thymidine kinase; B, BamHI; Sm, SmaI; Sp, SpeI; X, XbaI. (B) Southern analyses of ESs (left) and of the offsprings from the resulting mice heterozygous for the mutated allele (PROGENY; right). Correct homologous recombination generated a new Xba I restriction fragment B (7.4 kb) in addition to the wild-type allele-derived fragment A (9.4 kb), and both were detected with probe α (3.4-kb BamHI– XbaI fragment of BS-LC94). Correct insertion of the targeting plasmid was confirmed with SpeI digestion of genomic ES cell DNA and probe β (1.1-kb SmaI fragment of BS-LC60) that detected fragments C and D (wild-type allele [10.0 kb] and mutated allele [7.1 kb], respectively). A neo probe was also used to exclude additional nonhomologous integration of the targeting vector (data not shown). Matings of heterozygous animals then generated p52/p100 (+/−), (+/+), and (−/−) mice that were discerned with probe γ (1.8-kb BamHI fragment of LC60). This probe hybridizes to the BamHI fragments E (wild-type, 1.8 kb) and F (mutant, 3.7 kb) (PROGENY). (C) Western blot analyses of spleens of p52/p100 (+/ +) and (−/−) mice are shown. Antibodies to various NF-κB family members were used as indicated.

Figure 2

Figure 2

Reduction in the B/T cell ratio and reduced levels of total serum antibodies in p52/ p100 (−/−) mice. (A) Representative FCM analysis of splenocytes from 16-wk-old p52/ p100 (−/−) and (+/−) mice. IgM–Red 670 versus IgD-FITC two-color profiles are displayed in the top panels. Single-color profiles depict B220-PE and CD3-FITC staining on total splenocytes (middle and bottom, respectively). Numbers reflect the percentage of positively stained spleen cells. (B) Reduced levels of IgG1, IgG2b, and IgA in sera of p52/p100 (−/−) mice. Titers of total immunoglobulin isotypes from 7–10-wk-old mice of each genotype are shown as indicated. IgG1, IgG2a, and IgA titers differed significantly (P <0.002, P <0.05, and P <0.001) among the three groups (+/+, +/−, and −/−) by one-way analysis of variance. (−/− differed significantly from both other groups.) The reduction in levels of IgG3 observed in p52-deficient mice was not statistically significant (P = 0.079).

Figure 2

Figure 2

Reduction in the B/T cell ratio and reduced levels of total serum antibodies in p52/ p100 (−/−) mice. (A) Representative FCM analysis of splenocytes from 16-wk-old p52/ p100 (−/−) and (+/−) mice. IgM–Red 670 versus IgD-FITC two-color profiles are displayed in the top panels. Single-color profiles depict B220-PE and CD3-FITC staining on total splenocytes (middle and bottom, respectively). Numbers reflect the percentage of positively stained spleen cells. (B) Reduced levels of IgG1, IgG2b, and IgA in sera of p52/p100 (−/−) mice. Titers of total immunoglobulin isotypes from 7–10-wk-old mice of each genotype are shown as indicated. IgG1, IgG2a, and IgA titers differed significantly (P <0.002, P <0.05, and P <0.001) among the three groups (+/+, +/−, and −/−) by one-way analysis of variance. (−/− differed significantly from both other groups.) The reduction in levels of IgG3 observed in p52-deficient mice was not statistically significant (P = 0.079).

Figure 3

Figure 3

Defective antibody response to T-dependent antigens in p52-deficient mice. 5–30-wk-old mice of both sexes were injected intraperitoneally with (A) 100 μg of TNP (2,4,6 trinitro-phenyl)–KLH (TD), (B) 25 μg of TNP-Ficoll (TI-2) or (C) 50 μg of TNP-LPS (TI-1). Serum levels of anti-TNP specific antibodies were determined by isotype-specific ELISA 14 d after injection of the TNP conjugates. Antibody levels, expressed as OD, are shown for each genotype, as indicated. Anti-TNP antibodies were undetectable in sera of animals before challenge (data not shown). OD values differed significantly among the following groups of animals by one-way analysis of variance. (A) (−/−) versus (+/+) all isotypes tested; (−/−) versus (+/−), IgM, IgG1, IgG2b and IgG3; (+/−) versus (+/+), IgG1, IgG2a, IgG2b. (C) (−/−) versus (+/−) and (+/+), IgM, and IgG1. All other groups were not significantly different.

Figure 4

Figure 4

Germinal centers and B cell follicles are absent in spleens of p52 (−/−) mice, but are present in p50 (−/−) mice. Analyses of spleens taken from an unchallenged, 21-wk-old p52/p10 (−/−) mouse, a (+/−) littermate control (top and middle, respectively), and an unchallenged 15-wk-old p50/ p105 (−/−) mouse (bottom). Bouin-fixed, paraffin-embedded sections were processed with anti-CD3 or anti-B220 antibodies, as indicated.

Figure 5

Figure 5

Absence of germinal centers and dendritic cell networks in spleens of challenged p52/p100 (−/−) mice. 13–15-wk-old p52/p100 (−/−), (+/−), and (+/−) control mice were challenged by intraperitoneal injection of 100 μg of alum-adsorbed TNP-KLH. Frozen sections of spleens were obtained 8 or 17 d after injection and stained (A) with PNA, anti-CD35(CR1), or FDC-M1 antibodies and (B) with PNA ( purple) and anti-B220 antibodies ( pink), as indicated. Stained cryosections from representative pairs of littermates are shown.

Figure 6

Figure 6

Adoptively transferred p52-deficient lymphocytes support formation of germinal centers in RAG–1-deficient mice. Lethally irradiated RAG-1–deficient mice were injected with bone marrow cells isolated from 11-wk-old p52 (−/−), (+/−), and (+/+) donor mice, and 15 wk later challenged intraperitoneally with TNP-KLH (100 μg) adsorbed to alum. Splenic cryosections were obtained 9 d after challenge and stained with PNA ( purple) and anti-B220 antibodies (pink). Stained cryosections from a representative p52/p100 (−/−) marrow transferred animal is shown.

Figure 7

Figure 7

Altered MZ microarchitecture in spleens of p52/p100 (−/−) mice. Splenic cryosections were derived from the same group of p52/p100 (−/ −), (+/−), and (+/+) mice that were used for Fig. 5. Sections were stained with monoclonal antibodies specific to red pulp macrophages (RPM; BM8), MMs (MOMA-1) or MZMs; (ERTR-9), as indicated. Similar results were obtained with spleens from unchallenged animals (data not shown).

Figure 8

Figure 8

p52/p100 (−/−) mice are more susceptible to T. gondii infection. p52/p100 (−/−) mice and (+/−) and (+/+) littermates were injected intraperitoneally with ∼20 cysts of the avirulent T. gondii strain ME49 (initially provided by Dr. J. Remington, Palo Alto Research Foundation, Palo Alto, CA), as detailed previously (62). 10–23-wk-old mice of both sexes were used as follows: 17 (−/−), 15 (+/−), and 4 (+/+) animals. Data are shown as percent survival calculated from two independent experiments.

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