TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation.

The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.

Phenotypic and functional characterization of mice that lack the type I receptor for IL-1.

IL-1 alpha and IL-1 beta bind to receptors termed the type I and type II IL-1 receptors. The type I IL-1 receptor is responsible for specific signaling, while the type II IL-1 receptor functions as a nonsignaling decoy receptor. To determine the effect of a defect in IL-1-mediated signaling, mice have been produced with a genetically disrupted type I IL-1 receptor gene. Mice lacking type I IL-1 receptors are of normal vigor and exhibit no overt phenotype. B cells from type I IL-1R-/- mice activated in vitro with anti-IgM do not proliferate in response to IL-1, but do so in response to IL-4. Injection of murine IL-1 alpha does not induce detectable serum IL-6 levels in type I IL-1R-/- mice, but equivalent levels are produced in response to LPS. Type I IL-1R-/- mice have normal serum Ig levels and generate equivalent primary and secondary Ab responses as wild-type mice. In response to LPS, acute phase protein mRNA induction are equivalent in type I IL-1R-/- and wild-type mice. Type I IL-1R-/- mice do not differ from control mice in susceptibility to either a lethal challenge with D-galactosamine plus LPS or high dose LPS. Interestingly, ICE-/-/type I IL-1R-/- double mutant mice are resistant to high dose LPS. Type I IL-1R-/- mice backcrossed to the C57BL/6 background were as equally resistant as wild-type mice to Listeria monocytogenes.

Chromosomal assignment and genomic structure of Il15.

Interleukin-15 (IL-15) is a novel cytokine whose effects on T-cell activation and proliferation are similar to those of interleukin-2 (IL-2), presumably because IL-15 utilizes the beta and gamma chains of the IL-2 receptor. Murine IL-15 cDNA and genomic clones were isolated and characterized. The murine Il15 gene was found to consist of eight exons spanning at least 34 kb and was localized to the central region of mouse chromosome 8 by interspecific backcross analysis. Intron positions in a partial human IL15 genomic clone were identical with positions of corresponding introns in the murine gene. The human IL15 gene was mapped to human chromosome 4q31 by fluorescence in situ hybridization.

Isolation and mapping of human T-cell protein tyrosine phosphatase sequences: localization of genes and pseudogenes discriminated using fluorescence hybridization with genomic versus cDNA probes.

This work reports the isolation, partial characterization, and chromosomal mapping of several human T-cell protein tyrosine phosphatase (PTPase) sequences and provides a direct comparison of the specificity of cDNA versus genomic probes in discriminating the location of genes versus pseudogenes by fluorescence in situ hybridization. In initial attempts to map the T-cell (TC) PTP gene using a 2-kb cDNA, several labeled sites were noted, raising the possibility of multiple related sequences within the genome. To address this, four genomic clones were obtained with homology to the TC PTP cDNA and characterized for their primary structure and their position within the human genome. Based on the presence or absence of an open reading frame and the intron/exon structure, two of these clones were found to be overlapping sequences encoding the true TC PTP gene and two were highly related but distinct processed pseudogenes. The TC PTP gene (gene symbol PTPN2) encoded by clones L17-2 and L5-1 localized to chromosome 18p11.2-p11.3, whereas pseudogenes encoded by clone L17-1, entitled TCPS1 (gene symbol PTPN2P1), and clone L18, entitled TCPS13 (gene symbol PTPN2P2), mapped to chromosomes 1q22-q24 and 13q12-q13, respectively. A direct comparison of the specificity of genomic and cDNA probes demonstrated that under identical conditions the genomic probes (containing both exon and intron sequences) readily identified a single specific site of hybridization, whereas the cDNA identified sites of both the gene and its pseudogenes. While providing mapping and sequencing information on the TC PTPase sequences, this work illustrates a strategy for addressing a recurrent problem in gene mapping studies where highly related sequences exist within the genome.

Naltrindole, an opioid delta receptor antagonist, blocks cocaine-induced facilitation of responding for rewarding brain stimulation.

Recent experimental results have led to the suggestion that opioid antagonists can modulate the reinforcing properties of cocaine. In this experiment, rats were fixed with chronically indwelling bipolar electrodes for stimulation of the medial forebrain bundle (MFB) as it courses through the hypothalamus. Rats were taught to press a lever for brief trains of electrical stimulation of the MFB. Subsequently, they were allowed to press for varying intensities of stimulation daily until their response rates were stable. Cocaine (5 mg/kg, s.c.) enhanced the rate of pressing for lower intensities of brain stimulation. Naltrindole (3 mg/kg, i.p.) had no effect on response rate alone but blocked the cocaine-induced facilitation of pressing for rewarding brain stimulation. An implication that can be drawn from these data is that naltrindole, or other delta-selective opioid antagonists, might be effective as medicines for use in treating cocaine abuse.

Isolation of the cDNA encoding rat skeletal muscle myosin light chain kinase. Sequence and tissue distribution.

A cDNA clone encoding skeletal muscle myosin light chain kinase (MLCK) was isolated from a rat skeletal muscle library using oligonucleotide probes. The total length of the rat skeletal muscle MLCK cDNA was 2823 base pairs with an open reading frame of 1830 base pairs. The deduced sequence of the 610-amino acid protein exhibited 96% amino acid identity to rabbit skeletal muscle MLCK in the carboxyl-terminal portion of the molecule, which contains the catalytic and the calmodulin-binding domains, and 58% identity in the amino-terminal region. Analysis of total rat mRNA revealed a single mRNA species of 3.4 kilobases that was unique to skeletal muscle. Further analysis of skeletal muscle tissue using fast-twitch glycolytic, fast-twitch oxidative glycolytic, and slow-twitch oxidative fibers isolated from rat leg revealed that the mRNA level for MLCK varied among the three fiber types. The results of kinase assays performed on the fibers showed that MLCK activity levels paralleled the MLCK mRNA levels found in each of the three types of skeletal muscle fibers studied. Fast-twitch oxidative glycolytic (gastrocnemius red) and slow-twitch oxidative (soleus) exhibited 60 and 13%, respectively, of the enzymatic activity present in fast-twitch glycolytic (gastrocnemius white) fibers.

The molecular cloning of a type II regulatory subunit of the cAMP-dependent protein kinase from rat skeletal muscle and mouse brain.

A cDNA clone for a type II regulatory (R) subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from a rat skeletal muscle library using a specific 47-base oligonucleotide probe. The rat cDNA was 1.2 kilobases (kb) in length and contained an open reading frame of 1.113 kb representing 92% of the coding region of the molecule. Nick-translated rat cDNA was then used to isolate a mouse RII cDNA clone from a brain library that contained an open reading frame of 1.143 kb. Because both cDNAs lacked complete coding sequences, the remainder of the RII coding region was obtained from a 15-kb mouse genomic clone. The mouse RII coding region contains 1.2 kb corresponding to a 400-amino acid protein of 51.141 kDa. The mouse cDNA hybridizes to two mRNA species, a 2.4-kb form that was only observed in testis and a 6.0-kb form found in a wide range of tissues, including testis.

Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase.

The amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) was determined recently [Scott, J. D., Fischer, E. H., Takio, K., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5732-5736]. An earlier report [Scott, J. D., Fischer, E.H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383] showed that at least part of the inhibitory domain of PKI is located in a 20-residue segment extending from residue 11 to residue 30: Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile-Leu-Val-Ser- Ser-Ala . In the present study, we further mapped the inhibitory region of PKI by addition or deletion of residues at both ends of this peptide and by substitutions for specific amino acids. The results show that (i) deletion of residues 25-30 did not change inhibitory activity but addition of residues toward the amino terminus increased the inhibitory potency up to 150-fold (Ki 4.8 nM), to a level approaching that of PKI; (ii) replacement of alanine-21 by serine converted the inhibitor into a substrate having a relatively low affinity (Km 280 microM) for the enzyme; (iii) replacement of alanine-21 by phosphoserine or alpha-aminobutyric acid decreased inhibitory activity by a factor of 120 and 20, respectively; (iv) replacement of serine-13 had essentially no effect, whereas substitution of threonine-16 decreased inhibitory activity. The greatest decreases of inhibitory potency occurred with replacements of the arginines in positions 18 and 19.