Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects

Abstract

The pleiotrophic but overlapping functions of the cytokine family that includes interleukin (IL)-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1 are mediated by the cytokine receptor subunit gp130 as the common signal transducer. Although mice lacking individual members of this family display only mild phenotypes, animals lacking gp130 are not viable. To assess the collective role of this cytokine family, we inducibly inactivated gp130 via Cre-loxP-mediated recombination in vivo. Such conditional mutant mice exhibited neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects, demonstrating the widespread importance of gp130-dependent cytokines.

Figure 1

Gene targeting. (a) Gene targeting vector (bold line) homologously recombined into the gp130 locus (dashed line), thereby generating the targeted allele gp130^NEO3loxP. Exon sequences (rectangles); loxP sites (triangles). The transcriptional orientation of the neomycin resistance gene is shown. Restriction sites used for Southern analysis are given; those marked with an asterisk are not present in the wild-type locus but are introduced via the targeting vector. The probes used for Southern analysis are shown (black). In the gp130^NEO2loxP allele, the loxP site upstream of exon 16 is missing due to lack of cointegration. (b) Southern analysis to identify homologous recombinant clones. The size and position of the bands representing the gp130⁺ (wild-type) and gp130^targeted (gp130^NEO2loxP or gp130^NEO3loxP) alleles are given. (c) Southern analysis to identify clones harboring a cointegrated upstream loxP site. The position and size of the bands representing the gp130^NEO2loxP and gp130⁺ alleles or the gp130^NEO3loxP allele are given. (d) Southern analysis to identify clones which have deleted the neomycin resistance gene but not exon 16 after transient expression of Cre-recombinase. The position and size of the bands representing the gp130⁺, gp130^flox, and gp130^NEO3loxP alleles are given. (e) Southern blot prepared with mouse tail DNA in order to genotype the animals. The position and size of the bands representing the gp130⁺, gp130^flox, and gp130^Δ alleles are shown. Lane 4: bal1, Cre-transgenic (reference 31).

Figure 2

Inactivation of gp130. (a) Southern blot prepared with DNA of various organs of an adult Mx-cre;gp130^flox/flox mouse injected postnatally with IFN to induce expression of Cre-recombinase. The Southern blot was performed as described in Fig.1 e. (b) Concentration of sgp130 in the blood of wild-type, heterozygous, or homozygous gp130^flox animals (shaded circles) and heterozygous gp130^Δ animals (open circles) as measured by ELISA. One symbol represents data obtained for one animal. (c) Cell surface expression of gp130 on peripheral blood CD8⁺ T cells of adult gp130^flox/flox (black line), gp130^Δ/flox (gray line), and Mx-cre;gp130^flox/flox animals which were injected with IFN as newborns (gray histogram).

Figure 3

Degeneration of peripheral nerves in conditional gp130-mutant mice. Electron microscopic analysis of peripheral somatic nerves innervating skeletal muscle (A–D) and vegetative nerves innervating the heart muscle (E and F) or gut (G and H) of control gp130^flox/flox (A and E) and conditional gp130-mutant animals (B–D, F–H). A, Axon; S, Schwann cell; P, cell body. (A) Control, skeletal muscle: the myelinated nerve fibers show a regular myelin and axon structure (A). (B) Conditional gp130-mutant, skeletal muscle: disappearance of laminar myelin structure (arrow) without signs of axon lysis. (C) Conditional gp130-mutant, skeletal muscle: in certain areas the myelin sheath shows a complete lysis (arrow). (D) Conditional gp130-mutant, skeletal muscle: the degeneration of the myelin sheath (arrow) is associated with an axon lysis (A*). (E) Control, heart: the axon bundle has a complete and intact Schwann cell covering (S). (F) Conditional gp130-mutant, heart: the axon bundle (A) shows a partial failure of Schwann cell covering, while the basal lamina completely surrounds it. (G) Conditional gp130-mutant, gut: also in a thicker axon bundle of the gut an incomplete covering of the axon (A) by Schwann cells (S) is recognizable. (H) Conditional gp130-mutant, gut: the glia (S) covering of a myenteric neuron's cell body (P) shows no signs of alteration. For A–D, bar (in D) = 1 μm; for E–G, bar (in G) = 0.25 μm; for H, bar = 1.5 μm.

Figure 4

Hematopoietic defects in the absence of functional gp130. Conditional gp130-mutant (shaded symbols); gp130^flox/flox (filled symbols). (a) Basal hematology profile. The number of leukocytes, erythrocytes, and thrombocytes was determined in blood collected from the tail vein. (b) Recovery after 5-FU–induced hematopoietic ablation. The kinetics of recovery of leukocytes, erythrocytes, and thrombocytes in the peripheral blood after a single intraperitoneal administration of 150 mg/kg 5-FU at day 0 was determined over a period of 30 d (n = 7 for each genotype). (c) Survival after immune thrombocytopenia. Thrombocytopenia was induced by intraperitoneal injection of 3 μl adsorbed rabbit anti–mouse platelet serum (Inter-Cell Technologies, Inc., Hopewell, NJ) diluted in PBS at day 0, and survival of animals was subsequently monitored.

Figure 5

Antibody production in conditional gp130-mutant animals. Levels of different Ig isotypes in 12–15-wk-old conditional gp130-mutant (open circles) and gp130^flox/flox animals (filled circles) as quantitated by ELISA. One symbol represents data obtained for one animal.

Figure 6

Increased susceptibility to viral and bacterial infections of conditional gp130-mutant animals. (a and b) The anti-VSV IgG (a) and IgM (b) response after infection was determined as described in Materials and Methods in conditional gp130-mutant (open circles), IL-6–deficient (shaded circles), and gp130^flox/flox (filled circles) control animals (at least three per group and time point). (c) The number of vaccinia virus recovered from lung at day 5 after infection (see Materials and Methods) was determined in conditional gp130-mutant (open circles), IL-6–deficient (shaded circles), and gp130^flox/flox (filled circles) control animals. One symbol represents data obtained from one mouse. (d) The number of viable L. monocytogenes in the spleen 5 d after infection (see Materials and Methods) was determined in conditional gp130-mutant (open circles), IL-6–deficient (shaded circles), and gp130^flox/flox (filled circles) control animals. One symbol represents data obtained from one mouse.

Figure 7

Hepatic abnormalities in the conditional mutants. Light (A–D) and electron (E and F) microscopic analysis of control (A, C, and E) and gp130-mutant (B, D, and F) liver. N, Nucleus; S, liver sinus. After 6 wk, gp130-mutant liver (B) has a high amount of lipid droplets (arrowheads), widening of the Disse and intercellular space (arrows), a reduced content of binucleated hepatocytes, and an increase in Kupffer cells compared with controls (A). In 12-mo-old gp130-mutant liver, a part of the liver parenchyma is replaced by fibrotic material (D, arrows), a phenomenon not observed in the gp130^flox/flox control animals (C). The ultrastructure of gp130-mutant liver (F) gives evidence for a rarification of smooth and rough endoplasmic reticulum (ER), a dense clustering of mitochondrial (Mi) and monoparticulate (β) glycogen granules (triangle), a decrease of microvilli (*), and an enlargement of Disse and intercellular spaces (arrows) compared with the gp130^flox/flox control (E). For A and B, bar = 5 μm; for C and D, bar = 20 μm; for E and F, bar = 0.5 μm.

Figure 8

No increased synthesis of APP in the absence of functional gp130. (a) Conditional gp130-mutant (gray bars, n = 5) and gp130^flox/flox control mice (black bars, n = 5) were injected intraperitoneally with 1 mg/kg LPS, and the concentration of the APP SAP and C3 in the serum was measured 24 h later by ELISA. (b) The hematic glucose and serum SAP concentrations were monitored over a period of 80 h after challenge with 1 mg/kg LPS in gp130-mutant (shaded symbols) and control (filled symbols) animals.

Figure 9

Emphysema development in conditional gp130-mutant animals. Representative histological appearance of lungs from different ages of gp130^flox/flox and conditional gp130-mutant mice. (A and B) Orcein staining. After 5 mo of age, conditional gp130-mutants (B) showed a distinct reduction of elastic fibers (red) compared with controls (A). (C and D) Methylene blue staining. At the age of 12 mo, a distinct rarification of alveolar walls was detectable in the absence of functional gp130 (D) compared with gp130^flox/flox controls (C). Bar = 60 μm.