Complementation of lymphotoxin alpha knockout mice with tumor necrosis factor-expressing transgenes rectifies defective splenic structure and function

Abstract

Lymphotoxin (LT)alpha knockout mice, as well as double LTalpha/tumor necrosis factor (TNF) knockout mice, show a severe splenic disorganization with nonsegregating T/B cell zones and complete absence of primary B cell follicles, follicular dendritic cell (FDC) networks, and germinal centers. In contrast, as shown previously and confirmed in this study, LTbeta-deficient mice show much more conserved T/B cell areas and a reduced but preserved capacity to form germinal centers and FDC networks. We show here that similar to the splenic phenotype of LTbeta-deficient mice, complementation of LTalpha knockout mice with TNF-expressing transgenes leads to a p55 TNF receptor-dependent restoration of B/T cell zone segregation and a partial preservation of primary B cell follicles, FDC networks, and germinal centers. Notably, upon lipopolysaccharide challenge, LTalpha knockout mice fail to produce physiological levels of TNF both in peritoneal macrophage supernatants and in their serum, indicating a coinciding deficiency in TNF expression. These findings suggest that defective TNF expression contributes to the complex phenotype of the LTalpha knockout mice, and uncover a predominant role for TNF and its p55 TNF receptor in supporting, even in the absence of LTalpha, the development and maintenance of splenic B cell follicles, FDC networks, and germinal centers.

Figure 1

Rescued T/B cell segregation and B cell follicle formation in TgTNF/LTα^−/− mice is p55TNFR dependent. Immunocytochemical analysis was performed on cryostat sections of spleens from wild-type (WT), LTα^−/−, LTβ^−/−, 1278/LTα^−/−, A86/LTα^−/−, and A86/LTα^−/−/p55TNFR^−/− mice. Sections were stained with anti-B220 (blue) for B cells, anti-CD3 (brown) for T cells, anti-IgM (brown) for IgM⁺ B cells, and anti-IgD (blue) for IgD⁺ B cells. Original magnifications: ×40 for B220/CD3 staining, and ×100 for IgM/IgD staining.

Figure 2

Restoration of FDC networks and germinal centers but not marginal zones in TgTNF/LTα^−/− mice. Immunocytochemical analysis was performed on cryostat sections of spleens from LTα^−/−, LTβ^−/−, 1278/LTα^−/−, A86/LTα^−/−, and wild-type control mice. For the detection of FDC networks and germinal centers, spleens were harvested from mice 10 d after immunization with 10⁸ SRBC. Blue, CR1, PNA, and ER-TR9 stainings; brown, B220; red, sialoadhesin (Siaload.). Original magnifications: ×285 for CR1/B220 staining, ×155 for PNA/B220 staining, and ×100 for ER-TR9/ sialoadhesin staining.

Figure 3

Immune response to the TD antigen SRBC. Wild-type (WT), LTα^−/−, and 1278/LTα^−/− mice were immunized intraperitoneally with 10⁸ SRBC in PBS on days 0 and 21. Mice were bled on day 28, at which time serum titers to SRBC-specific IgG1 were determined by ELISA. Data are means ± SE of five mice per group.

Figure 4

ELISA analysis of TNF levels after LPS induction. LTα^−/− (n = 5) mice and wild-type (WT; n = 4) controls were injected intraperitoneally with 100 μg LPS, and serum levels of TNF were determined 90 min later. Thioglycollate-elicited peritoneal macrophages from LTα^−/− (n = 4) mice and wild-type (n = 3) controls were stimulated with 1 μg/ml LPS for 24 h, and supernatants from individual cultures were assayed for the presence of TNF. Data are means ± SE per group. *P <0.03; ⁺⁺ P <0.001 compared with wild-type mice.