Assessment of inhibition and epileptiform activity in the septal dentate gyrus of freely behaving rats during the first week after kainate treatment.

Mossy fiber reorganization has been hypothesized to restore inhibition months after kainate-induced status epilepticus. The time course of recovery of inhibition after kainate treatment, however, is not well established. We tested the hypothesis that if inhibition is decreased after kainate treatment, it is restored within the first week when little or no mossy fiber reorganization has occurred. Chronic in vivo recordings of the septal dentate gyrus were performed in rats before and 1, 4, and 7-8 d after kainate (multiple injections of 5 mg/kg, i.p.; n = 17) or saline (n = 11) treatment. Single and paired-pulse stimuli were used to assess synaptic inhibition. The first day after kainate treatment, only a fraction of rats showed multiple population spikes (35%), prolonged field postsynaptic potentials (76%), and loss of paired-pulse inhibition (29%) to perforant path stimulation. Thus, inhibition was reduced in only some of the kainate-treated rats. By 7-8 d after treatment, nearly all kainate-treated rats showed partial or full recovery in these response characteristics. Histological analysis indicated that kainate-treated rats had a significant decrease in the number of hilar neurons compared to controls, but Timm staining showed little to no mossy fiber reorganization. These results suggest that a decrease in synaptic inhibition in the septal dentate gyrus is not a prerequisite for epileptogenesis and that most of the recovery of inhibition occurs before robust Timm staining in the inner molecular layer.

The conformation of Ld induced by beta 2-microglobulin is fixed during de novo synthesis and irreversible by exchange or dissociation.

Data are presented that support the hypothesis that beta 2m controls the folding of the ligand binding site of newly synthesized class I molecules. This conclusion was indicated by comparisons of two antigenic forms of the Ld molecule separated by sequential immunoprecipitation. Whereas, mAb 30-5-7+ Ld molecules were found to exist either as free H chains or associated with beta 2m, 30-5-7- Ld molecules showed no beta 2m association. Chemical comparisons showed 30-5-7- Ld molecules to be highly sensitive to proteolysis relative to 30-5-7+ Ld molecules. Experiments employing a construct with the Ld gene juxtaposed to the inducible metallothionein promoter indicated that the ratio of the antigenic forms of Ld was determined by the relative synthesis of beta 2m vs class I proteins. Pulse-chase experiments demonstrated that the two antigenic forms of Ld do not share a precursor-product relationship, but do display disparate rates of intracellular transport. beta 2m dissociation or exchange at the cell surface was found not to affect the ratio of the two antigenic forms of Ld. In contrast to these findings with Ld, the Dd and Ddml molecules were not detected in alternative conformations, thus mapping this property to the N-terminus of the class I molecule. These findings support the notion that beta 2m induces conformation on the alpha 1/alpha 2 domains of Ld molecules during de novo synthesis and once beta 2m-conformed, the class I structure is fixed and irreversible.

Interleukin 1 is processed and released during apoptosis.

Interleukin (IL-) 1 alpha and 1 beta are synthesized as 31- to 34-kDa pro molecules. They are released from monocytes and macrophages as proteolytically processed 17-kDa mature molecules that bind with high affinity to specific receptors on target cells. IL-1 is not released via the classic secretory pathway. The pro molecules are synthesized as cytosolic proteins without signal peptides. Although the proteases that convert the pro molecules to the mature forms are cytosolic enzymes, processed IL-1 is not detected associated with the cell but is found only in culture supernatants. We demonstrate here that release of IL-1 is efficiently induced by cell injury. When the injury causes cellular necrosis, IL-1 alpha is released as a mixture of unprocessed and processed molecules but IL-1 beta is released exclusively as the biologically inactive pro form. In contrast, when cells undergo apoptosis, maturation of both IL-1 alpha and IL-1 beta is efficient. When apoptosis is rapid, as in macrophages that are targets for allospecific cytotoxic T lymphocytes, processing is observed to occur intracellularly. These findings suggest that cell injury is an important physiologic stimulus for release of IL-1. The nature of the injury profoundly affects the forms of IL-1 that are released.

Generation of monoclonal antibodies to murine IL-1 beta and demonstration of IL-1 in vivo.

The role of murine IL-1 beta in vitro and in vivo has not been defined. We describe here the production of neutralizing and immunoprecipitating mAb and polyclonal antibodies specific for murine IL-1 beta and their application to a characterization of the murine IL-1 beta protein. Immunization of either hamsters or rabbits with the recombinant mature form of murine IL-1 beta emulsified in CFA elicited antisera and hamster mAb that only recognized denatured IL-1 beta. In contrast, immunization with rIL-1 beta adsorbed to alum resulted in the generation of neutralizing and immunoprecipitating rabbit and hamster antisera and hamster mAb. All of the mAb recognize both the pro-form of IL-1 beta and the mature bioactive form produced by cultures of murine peritoneal macrophages. Using these antibodies, we demonstrate that approximately half of the IL-1 activity present in supernatants of LPS-treated cultured mouse macrophages is composed of IL-1 beta. Additionally, IL-1 beta as well as IL-1 alpha can be detected in the plasma of LPS-treated mice. These studies, therefore, demonstrate the production of IL-1 beta both in vitro and in vivo.

Molecular cloning of the murine IL-1 beta converting enzyme cDNA.

IL-1 beta is a potent modulator of immune and inflammatory responses. Murine IL-1 beta is initially synthesized as an inactive 33-kDa pro-molecule that is activated by proteolytic cleavage between Asp-117 and Val-118 to generate the 17-kDa mature IL-1 beta protein. This cleavage is catalyzed by a specific protease that has been designated the IL-1 beta converting enzyme (or IL-1 beta convertase). We have used a human IL-1 beta convertase cDNA to isolate murine convertase cDNA from a WEHI-3 library. These cDNA predicted that the murine convertase is a 402-residue protein. Overall, the murine convertase showed 71% nucleotide and 62% predicted amino acid sequence identity with the human convertase. Southern blot analysis of interspecific backcross mice indicated that the murine IL-1 beta convertase is encoded by a single copy gene located on murine chromosome 9. The murine convertase showed broad constitutive expression, being detected in mononuclear phagocyte and T lymphocyte cell lines as well as in spleen, heart, brain, and adrenal glands. The expression of the murine convertase in mononuclear phagocytes was up-regulated by treatment with LPS or rIFN-gamma. These studies establish that the IL-1 beta convertase is an evolutionarily conserved, widely expressed enzyme that can be regulated at a pretranslational level.