Differential IL-6 mRNA expression by stimulated peripheral macrophages of Staggerer and Lurcher cerebellar mutant mice.

We have recently reported about a hyperexpression of interleukin-1 mRNA by stimulated peripheral macrophages of several cerebellar mutant mice exhibiting complex patterns of neuronal degeneration in the cerebellum. Interestingly, studying the staggerer mutant mice, our data showed a hyperexpression of IL-1 beta mRNA and a hyperproduction of the cytokine. The hyperexpression of IL-1 beta mRNA was observed whatever the conditions of stimulation and the stimulating agent used, a hyperexpression of IL-1 alpha and TNF alpha mRNAs was also detected. This set of data suggests a hyperexcitability state of (sg/sg) macrophages. In the present study, we examined the IL-6 mRNA expression by stimulated peripheral macrophages of two cerebellar mutant mice, the staggerer and the lurcher mutants. Our results show that IL-6 mRNA is hyperexpressed in stimulated macrophages of staggerer mutant mice. On the contrary, no hyperexpression is observed in stimulated macrophages of lurcher mutant mice. These results suggest that the neuronal degeneration affecting the cerebellum in the two mutant mice do not lead to the same immunological defect at the peripheral level.

Evidence for a hyperexcitability state of staggerer mutant mice macrophages.

We recently reported an abnormal production of interleukin-1 (IL-1) in peripheral macrophages of several neurological mutant mice that exhibit patterns of neuronal degeneration, especially in the cerebellum. After in vitro activation by lipopolysaccharide acid (LPS), these macrophages hyperexpress IL-1 beta mRNA and hyperproduce IL-1 protein in comparison with +/+ controls. In the present study, focused on the staggerer mutant mice, we investigate if this genetic dysregulation is specific for IL-1 beta or if it reflects a generalized hyperexcitability of these macrophages. The hyperexpression of IL-1 beta mRNA in sg/sg macrophages is present whatever the duration of LPS stimulation, even for periods as short as 15 min, although it reaches a maximum after 4 h of stimulation. The hyperinducibility of sg/sg macrophages is observed even when very low doses of LPS are used (0.01 microgram/ml) and reaches its maximum for 5 micrograms/ml LPS. Synthetic molecules (muramyl dipeptides), such as N-acetylmuramyl-L-alanyl-D-isoglutamine or murabutide, known as macrophage activators, are also efficient in revealing the cytokine hyperexpression in sg/sg macrophages. In addition, hyperexpression of two other cytokines, i.e., tumor necrosis factor-alpha and IL-1 alpha mRNAs, is also detected in LPS-stimulated macrophages of mutant mice. Finally, the effect of an inhibitor of protein synthesis, cycloheximide, is similar in +/+ and sg/sg macrophages. As a whole, these data lead us to conclude that the sg/sg macrophages are in a state of general hyperexcitability when compared with +/+ ones.

Interleukin-1 hyperproduction by in vitro activated peripheral macrophages from cerebellar mutant mice.

Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin-1 (IL-1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL-1 cDNA probes, revealed a clear-cut hyperexpression of IL-1 mRNA in staggerer macrophages. An IL-1 bioassay using the IL-1-responsive D10.G4 cell line also revealed a sixfold increase of IL-1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3-week to 1-year-old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL-1 production by peripheral macrophages.

Peripheral macrophage abnormalities in mutant mice with spinocerebellar degeneration.

We recently reported hyperproduction of interleukin-1 (IL1) and hyperexpression of IL1 beta mRNA, after in vitro activation by lipopolysaccharide (LPS) in peripheral macrophages of several neurological mutant mice, i.e. staggerer, lurcher, pcd and reeler, that exhibit patterns of neuronal degeneration in the cerebellum; in the present study, we investigated the expression of several cytokine mRNA in peripheral macrophages of other mutants with neuronal degeneration in the cerebellum or in the spinal cord to determine whether this genetic dysregulation is specific for IL1 beta or whether it reflects a generalized hyperexcitability of these macrophages. Hyperexpression of IL1 beta mRNA was present in the cerebellar mutants nodding and nervous, but not in weaver. A similar phenomenon was found, but to a lesser extent, in the spinal mutants dystonia musculorum, wobbler and motor neuron degeneration. On the contrary, no hyperexpression of IL1 beta mRNA was found in non-genetic models of neuronal degeneration (Wistar rats treated with X irradiation or with 3-acetyl-pyridine). In the heterozygote staggerer +/sg, which exhibits a late onset of cerebellar neuronal loss, hyperexpression was found not only in 12-month old animals but also in 2-month old ones, i.e. when the number of cerebellar neurons is still normal. Synthetic molecules (muramyl dipeptides) like MDP or murabutide (Mu), known as macrophage activators, were also efficient in inducing IL1 hyperexpression in sg/sg macrophages. Hyperexpression of two other cytokine mRNA, i.e. IL1 alpha and tumour necrosis factor alpha mRNA, was also detected in LPS-stimulated macrophages of staggerer and lurcher mutant mice. These data led us to conclude that the macrophages of spinal and cerebellar mutants are in a state of general hyperexcitability. Work is in progress to establish whether the cytokine abnormalities result from a defect intrinsic to the macrophages of the mutant mice or are secondary to the degenerative process ultimately leading to neuronal loss.