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. 1998 Oct 5;188(7):1343-52.
doi: 10.1084/jem.188.7.1343.

A critical role of the p75 tumor necrosis factor receptor (p75TNF-R) in organ inflammation independent of TNF, lymphotoxin alpha, or the p55TNF-R

E Douni  1 , G Kollias
Affiliations

A critical role of the p75 tumor necrosis factor receptor (p75TNF-R) in organ inflammation independent of TNF, lymphotoxin alpha, or the p55TNF-R

E Douni et al. J Exp Med. .

Abstract

Despite overwhelming evidence that enhanced production of the p75 tumor necrosis factor receptor (p75TNF-R) accompanies development of specific human inflammatory pathologies such as multi-organ failure during sepsis, inflammatory liver disease, pancreatitis, respiratory distress syndrome, or AIDS, the function of this receptor remains poorly defined in vivo. We show here that at levels relevant to human disease, production of the human p75TNF-R in transgenic mice results in a severe inflammatory syndrome involving mainly the pancreas, liver, kidney, and lung, and characterized by constitutively increased NF-kappaB activity in the peripheral blood mononuclear cell compartment. This process is shown to evolve independently of the presence of TNF, lymphotoxin alpha, or the p55TNF-R, although coexpression of a human TNF transgene accelerated pathology. These results establish an independent role for enhanced p75TNF-R production in the pathogenesis of inflammatory disease and implicate the direct involvement of this receptor in a wide range of human inflammatory pathologies.

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Figures

Figure 1
Figure 1
S1 nuclease protection analysis of total RNA from tissues of normal, TgE1334 and TgE1335 mice shows correct patterns of expression for the transgenic p75TNF-R mRNA. β-actin acts as an internal control for sample loading.
Figure 2
Figure 2
Production of a functional hup75TNF-R protein on the surface of transgenic thymocytes. (A) Freshly isolated or ConA-stimulated thymocytes from normal, TgE1335, and mup75TNF-R knockout mice were analyzed by flow cytometry for the expression of murine and human p75TNF-R protein. Approximately 20 and 22% of freshly isolated normal and transgenic thymocytes were found to express endogenous or transgenic p75TNF-R, respectively. ConA stimulation resulted in the induction of both the murine p75TNF-R in normal mice (60% of cells positive) and the human p75TNF-R in transgenic mice (57% of cells positive). Thymocytes from normal or p75TNF-R–deficient mice are used as negative controls. (B) Proliferative response of ConA-treated TgE1335 (•) and normal (○) thymocytes to human rTNF reveals a functional human p75TNF-R protein. The amount of 3H incorporation in either normal or transgenic thymocytes treated with ConA alone is indicated by a dashed line. Results are representative of three independent experiments.
Figure 2
Figure 2
Production of a functional hup75TNF-R protein on the surface of transgenic thymocytes. (A) Freshly isolated or ConA-stimulated thymocytes from normal, TgE1335, and mup75TNF-R knockout mice were analyzed by flow cytometry for the expression of murine and human p75TNF-R protein. Approximately 20 and 22% of freshly isolated normal and transgenic thymocytes were found to express endogenous or transgenic p75TNF-R, respectively. ConA stimulation resulted in the induction of both the murine p75TNF-R in normal mice (60% of cells positive) and the human p75TNF-R in transgenic mice (57% of cells positive). Thymocytes from normal or p75TNF-R–deficient mice are used as negative controls. (B) Proliferative response of ConA-treated TgE1335 (•) and normal (○) thymocytes to human rTNF reveals a functional human p75TNF-R protein. The amount of 3H incorporation in either normal or transgenic thymocytes treated with ConA alone is indicated by a dashed line. Results are representative of three independent experiments.
Figure 3
Figure 3
Production of soluble and cell surface hup75TNF-R protein in transgenic sera and on peripheral blood leukocytes. (A) Serum levels of soluble mup75 and hup75TNF-Rs were measured by specific ELISAs, before or after LPS-administration in normal (n = 4), and TgE1335 heterozygous (n = 4) mice. Total (murine and human) sp75TNF-R production in heterozygous TgE1335 mice is increased approximately fivefold in comparison to the production of endogenous sp75TNF-R protein in normal mice (49 ± 4.5 ng/ml total p75TNF-R protein in transgenic mice versus 10 ± 2.5 ng/ml of endogenous p75TNF-R protein in normal mice). Transgenic p75TNF-R production in LPS-treated TgE1335 mice is regulated similarly to normal LPS-treated mice. TgE1335 homozygous mice (n = 4) spontaneously produce highly elevated levels of both soluble murine and human p75TNF-Rs. (B) Flow cytometric analysis of peripheral blood leukocytes from 3-wk-old TgE1335 heterozygous or homozygous mice shows the enhanced surface expression of hup75TNF-R on leukocytes taken from homozygous transgenic animals. Data are representative of three independent experiments.
Figure 3
Figure 3
Production of soluble and cell surface hup75TNF-R protein in transgenic sera and on peripheral blood leukocytes. (A) Serum levels of soluble mup75 and hup75TNF-Rs were measured by specific ELISAs, before or after LPS-administration in normal (n = 4), and TgE1335 heterozygous (n = 4) mice. Total (murine and human) sp75TNF-R production in heterozygous TgE1335 mice is increased approximately fivefold in comparison to the production of endogenous sp75TNF-R protein in normal mice (49 ± 4.5 ng/ml total p75TNF-R protein in transgenic mice versus 10 ± 2.5 ng/ml of endogenous p75TNF-R protein in normal mice). Transgenic p75TNF-R production in LPS-treated TgE1335 mice is regulated similarly to normal LPS-treated mice. TgE1335 homozygous mice (n = 4) spontaneously produce highly elevated levels of both soluble murine and human p75TNF-Rs. (B) Flow cytometric analysis of peripheral blood leukocytes from 3-wk-old TgE1335 heterozygous or homozygous mice shows the enhanced surface expression of hup75TNF-R on leukocytes taken from homozygous transgenic animals. Data are representative of three independent experiments.
Figure 4
Figure 4
p75TNF-R-triggered multi-organ inflammation and hematopoietic abnormalities develop even in the absence of TNF. Histopathological analysis (H/E) in liver and pancreas of 4-mo-old heterozygous TgE1335 mice (TgE1335het) and 3-wk-old TgE1335 homozygous (TgE1335hom) or homozygous TgE1335 × TNF−/− mice (TgE1335hom/TNF −/−). Lesions in heterozygous Tg1335 mice involve mainly the liver and pancreas where inflammatory infiltrates develop and persist chronically from 2–3 mo of age. In homozygous TgE1335 mice a more severe pathology develops, characterized by extensive periportal inflammation and tissue necrosis (asterisk) in the liver, and in a severely hypoplastic and inflamed pancreas. A similar histopathology evolves in homozygous TgE1335 transgenic mice bred into a null TNF background. Original magnification ×108. Spleen sections from homozygous TgE1335 mice are characterized by markedly reduced numbers of IgM+ B cells (brown) whereas CD3+ T cell localization (blue) seems unaffected. A similar phenotype occurs even in the absence of endogenous TNF. Original magnification ×114.
Figure 5
Figure 5
Survival of hup75TNF-R transgenic mice in the absence of TNF or the p55TNF-R. All TgE1335 heterozygous mice (Tghet, n = 10, ○) survive usually past 10 mo of age whereas TgE1335 homozygous mice (Tghom, n = 10, •) succumb to the disease during their first month of age even in the absence of the p55TNF-R (Tghom/p55 −/−, n = 6, ▵). The presence of TNF contributes positively but is not essential for the development of the lethal pathology (Tghom/TNF −/−, n = 10, ▪). Survival is measured as a percentage of the initial number of animals in each genotype group.
Figure 6
Figure 6
Enhanced NF-κB binding activity in PBMC from hup75 transgenic mice. EMSA of nuclear extracts from PBMC isolated from heterozygous TgE1335 mice or normal control littermates (n = 5 per group) at 1, 2, and 6 mo of age. Specific NF-κB complexes are indicated by arrows, whereas OCT-1 probe acts as an internal control for sample loading. Cold NF-κB or OCT-1 probe was used to indicate specific NF-κB or OCT-1 binding, respectively.
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