Clemens Gillen

Cloning and characterization of SDF‐1γ, a novel SDF‐1 chemokine transcript with developmentally regulated expression in the nervous system

The cytokines SDF‐1α and ‐1β are two alternatively spliced variants of the CXC (α) chemokines that are highly conserved among species. SDF‐1α was shown to function as a B‐cell maturation factor, a ligand for the CXCR4 (LESTR/fusin) chemokine receptor, thereby inhibiting replication of T cell‐tropic HIV‐1 strains and inducing cell death in human neuronal cell lines. In this report the cloning of the rat SDF‐1β cDNA and a new SDF‐1 isoform, SDF‐1γ, are presented. Using Northern blot analysis, the expression pattern of both isoforms was studied in different tissues and it is shown that during postnatal development of the central and peripheral nervous system SDF‐1β‐ and SDF‐1γ‐mRNA expression is inversely regulated. Whilst SDF‐1β‐mRNA is the predominant isoform in embryonic and early postnatal nerve tissue, SDF‐1γ‐mRNA is expressed at higher levels in adulthood. After peripheral nerve lesion a transient increase in SDF‐1β‐mRNA expression is observed. As revealed by in situ hybridization, neurons and Schwann cells are the main cellular sources of both SDF‐1β and SDF‐1γ mRNAs in the nervous system. Computer‐assisted analysis revealed that both transcripts encode secreted peptides with putative proteolytic cleavage sites which might generate novel neuropeptides.

Sequential expression of mRNA for proinflammatory cytokines and interleukin-10 in the rat peripheral nervous system: comparison between immune-mediated demyelination and Wallerian degeneration.

This study examined the time course of mRNA levels of the proinflammatory cytokines interferon‐γ (IFNγ), interleukin‐1β (IL1β), interleukin‐12 (IL12; p40 subunit), and the immunosuppressant interleukin‐10 (IL10) by semiquantitative reverse transcription polymerase chain reaction (RT‐PCR) in rats with actively induced experimental autoimmune neuritis (EAN) and in distal stumps of crushed sciatic nerves undergoing Wallerian degeneration.

Focal ischaemia of the rat brain elicits an unusual inflammatory response: early appearance of CD8+ macrophages/microglia

Cerebral ischaemia leads to profound glial activation and leukocyte infiltration into the infarct area. In this study, we provide evidence for a dual macrophage response in focal ischaemic lesions of the rat brain. We show that a considerable proportion of macrophages in the ischaemic lesions express the CD8αβ heterodimer to date only described on CD8+ T cells. As known from other lesion paradigms, CD4+ macrophages were also present. Interestingly, CD8‐ and CD4‐expressing macrophages formed two non‐overlapping subpopulations. CD8+ macrophages reached their maximum during the first week with pronounced downregulation thereafter whereas CD4+ cells persisted at high levels into the second week. In contrast to cerebral ischaemia, macrophages in the spleen and in Wallerian degeneration after optic nerve axotomy expressed CD4, but not CD8. In experimental autoimmune encephalomyelitis, CD8 was mainly associated with T cells and very weakly detectable on some ramified cells resembling activated microglia. In conclusion, we show that cerebral ischaemia triggers an unusual inflammatory response characterized by the appearance of CD8+/CD4– macrophages that might exert specific functions in the pathogenesis of ischaemic brain damage.

Differential expression of interleukin‐10 mRNA in Wallerian degeneration and immune‐mediated inflammation of the rat peripheral nervous system

Interleukin‐10 (IL‐10) is a potent immunosuppressant cytokine which downregulates MHC class II antigen expression and inflammatory cytokine production. In this study we localized mRNA for IL‐10 in the rat peripheral nervous system (PNS) by nonradioactive in situ hybridization using a digoxygenin‐labeled riboprobe specific for rat IL‐10. IL‐10 mRNA was expressed by some Schwann cells (SCs) in the normal sciatic nerve. During Wallerian degeneration, SCs strongly expressed IL‐10 mRNA between days 2 and 4 after transection. By day 14 only occasional cells were positive for IL‐10 mRNA. The vast majority of ED1‐positive macrophages were IL‐10 negative after axotomy. Contrastingly, infiltrating macrophages expressed IL‐10 mRNA coincident with beginning clinical recovery in experimental autoimmune neuritis (EAN), the rat model of human Guillain‐Barré syndrome. Our data suggest that SCs provide a constitutive immunosuppressant system in the PNS. In EAN additional macrophage‐derived IL‐10 may be important for the resolution of the T cell‐mediated immune response.

■ REVIEW : Gene Expression in Nerve Regeneration

Injury of peripheral nerve in mammals leads to a complex but stereotypical pattern of histological events that comprise a highly reproducible sequence of degenerative reactions (Wallerian degeneration) succeeded by regenerative responses. These reactions are based on a corresponding sequence of cellular and mo lecular interactions that, in turn, reflect the differential expression of specific genes with functions in nerve degeneration and repair. We report on more than 60 genes and their products that show a specific pattern of regulation following peripheral nerve lesion. The group of regulated genes encoding, e.g., transcription factors, growth factors and their receptors, cytokines, neuropeptides, myelin proteins and lipid carriers, and cytoskeletal proteins as well as extracellular matrix and cell adhesion molecules. We describe and compare the distinct time-courses and cellular origin of expression and further discuss established or putative mo lecular interrelationships and functions with respect to the contribution of these genes/gene products to the molecular regeneration program of the PNS. NEUROSCIENTIST 3:112-122, 1997

Vascular cell adhesion molecule-1 mRNA is expressed in immune-mediated and ischemic injury of the rat nervous system

In this study we used nonradioactive in situ hybridization for the cellular localization of vascular cell adhesion molecule-1 (VCAM-1) mRNA in immune-mediated, ischemic and degenerative diseases of the rat nervous system. In the acute phase of experimental autoimmune encephalomyelitis and neuritis VCAM-1 mRNA was expressed not only on the luminal surface of inflamed vessels but also in perivascular cells suggesting a functional role of VCAM-1 in both endothelial adhesion and local restimulation of autoantigen-specific T cells. Accordingly, perivascular T cell accumulation was most pronounced at sites of local VCAM-1 mRNA expression. In addition, VCAM-1 mRNA was detected in the border zone around photochemically induced cerebral infarcts which is the predeliction site of T cell infiltration and expression of immune activation markers during the first week after ischemia. VCAM-1 mRNA was absent from the center of the infarcts as well as axotomized central and peripheral nerves undergoing Wallerian degeneration. These data indicate that VCAM-1-mediated adhesion processes are involved in immune-mediated and ischemic diseases of the nervous system but not in T cell-independent macrophage recruitment during Wallerian degeneration.

Full‐length Cloning, Expression and Cellular Localization of Rat Plasmolipin mRNA, a Proteolipid of PNS and CNS

We have isolated a 1.476 bp cDNA (NTII11) representing a transcript that is differentially expressed during sciatic nerve development and regeneration in the rat. Nucleotide sequence comparison indicates partial identity with a recently isolated plasmolipin cDNA. However, our clone extends the published sequence by 234 bp at the 5’end and predicts a protein that contains an additional 25 amino acids at the N‐terminus. The open reading frame of the NTII11 transcript encodes a 19.4 kDa protein with four putative transmembrane domains. Northern blot analyses revealed a tissue‐specific expression of the plasmolipin transcript that is restricted to sciatic nerve, brain and kidney. The neural expression was confirmed by in situ hybridization, and cellular localization of plasmolipin mRNA was demonstrated in Schwann cells of the sciatic nerve and in glial cells of myelinated brain structures. The steady‐state levels of plasmolipin mRNA were markedly altered (i) during development of the sciatic nerve and brain, (ii) after sciatic nerve injury, and (iii) in cultured Schwann cells maintained under different conditions of cell growth and arrest. Our data indicate a function of plasmolipin during myelination in the central as well as in the peripheral nervous system.

Cellular localization of the disintegrin CRII‐7/rMDC15 mRNA in rat PNS and CNS and regulated expression in postnatal development and after nerve injury

Disintegrins perform putative functions in cell adhesion, signaling and fusion. We have isolated a 2815‐bp rat cDNA (CRII‐7) representing a transcript that is differentially expressed during sciatic nerve regeneration. Nucleotide sequence comparison indicates that CRII‐7 is the rat homologue to the recently cloned cDNAs MDC15 (ADAM 15) and metargidin (hMDC15) of mouse and human, respectively. The CRII‐7 cDNA (rMDC15) encodes a membrane‐anchored glycoprotein of approximately 85 kDa containing a disintegrin and a metalloprotease domain. Cellular metalloprotease disintegrins are a family of proteins (ADAMs or MDC proteins) with important roles, e.g., in cell–cell interactions during fertilization, muscle and nerve development, or tumor necrosis factor‐α (TNF‐α) cleavage. Northern blot analysis demonstrated a predominant expression of CRII‐7/rMDC15 in the nervous system (PNS and CNS) and lung. Analysis of the CRII‐7/rMDC15 transcript levels following peripheral nerve lesions demonstrated regulated mRNA expression during Wallerian degeneration and nerve regeneration. The steady‐state levels of CRII‐7/rMDC15 transcripts markedly increased within the first day after lesion and then steadily decreased for at least 4 weeks. CRII‐7/rMDC15 mRNA expression was further examined during postnatal development and maturation of rat sciatic nerve and brain, as well as in cultured Schwann cells, meningeal fibroblasts, and astrocytes. In situ hybridization on paraffin sections showed the cellular localization of CRII‐7/rMDC15 mRNA in Schwann cells and endothelial cells of peripheral nerve and in various neuronal populations in brain and spinal cord. GLIA 32:313–327, 2000.

Developmental regulation of decorin expression in postnatal rat brain

Here, we report on the expression of the small chondroitin/dermatan sulfate proteoglycan decorin in the developing postnatal rat brain. Northern analysis of brain RNA demonstrated decorin transcripts with peak expression on postnatal day 3 followed by a slow decline to the lower adult level. In situ hybridization and immunohistochemistry revealed postnatal decorin expression in the grey matter of neocortex, hippocampus and thalamus, in myelinated fibre tracts and in several mesenchymal tissues (blood vessels, pia mater and the choroid plexus). In the neocortex, decorin is expressed in a specific laminar pattern with intense staining of the cortical plate and its derivatives, which differs remarkably from the distributions observed for other proteoglycans [B. Miller, A.M. Sheppard, A.R. Bicknese, A.L. Pearlman, Chondroitin sulfate proteoglycans in the developing cerebral cortex: the distribution of neurocan distinguishes forming afferent and efferent axonal pathways, J. Comp. Neurol. 355 (1995) 615-28]. Thus, decorin seems to serve yet unknown functions in the developing rat brain parenchyma in addition to its well-established role as a constituent of the mesenchymal extracellular matrix.