In vivo processing of CXCL12α/SDF-1α after intravenous and subcutaneous administration to mice.

CXCL12α has been shown to be selectively processed at the N- and C-termini in blood and plasma in vitro. In order to study the processing in vivo, several versions of CXCL12α were expressed and purified. The protein was administered either iv or sc to mice, and at different time points postadministration plasma was collected and analyzed. To detect modifications of the CXCL12α molecule in crude plasma a SELDI TOF-MS-based method was developed. Anti-CXCL12 antibodies were immobilized on the SELDI chip and CXCL12α binding to the antibodies was detected by SELDI-TOF-MS. The protein was found to be processed both at the C- and N-termini. The same processed CXCL12α forms as detected in vitro were found; however, in addition further processing was detected at the N-terminus, where altogether seven amino acids were removed. At the C-terminus the lysine was removed as has been seen in vitro, and no further processing was detected. The full-length CXCL12α disappeared within minutes after administration, whereas the processed forms of the protein were detectable for up to 6-8 h postadministration. The same processed forms appeared after iv and sc administration, only the kinetics was different. When the shortest processed form detected in plasma, 7ΔN1ΔC-CXCL12α, was administered directly, no further processed forms were detected. Interestingly, a version of CXCL12α containing a N-terminal methionine was protected against N-terminal processing in plasma in vitro; however, in vivo no protection was seen, the protein was processed in the same way as full-length CXCL12α.

Differential effects of chemokines on oligodendrocyte precursor proliferation and myelin formation in vitro.

Chemokines have recently been postulated to have important functions in the central nervous system (CNS) in addition to their principal role of directional migration of leukocytes. In particular, it has been shown that chemokines may play a role in the regulation of oligodendrocyte biology. Here, we have chosen to study the role of certain chemokines in regulating myelination. We have used the murine oligodendrocyte precursor-like cell line, Oli-neu, and primary mixed cortical cultures as experimental systems to assess their activities on oligodendrocyte precursor proliferation and developmental in vitro myelination. GRO-alpha, IL-8, SDF-1alpha and RANTES dose-dependently increased proliferation of this mouse A2B5 precursor-like cell line, while MCP-1 did not. Furthermore, the CXC chemokines GRO-alpha, IL-8 and SDF-1alpha stimulated myelin basic protein synthesis in a dose-dependent manner in primary myelinating cultures and enhanced myelin segment formation in this system, while the CC chemokines MCP-1 and RANTES did not. We also demonstrate that the receptor for SDF-1alpha, CXCR4, is expressed in mixed cortical cultures by PDGFalphaR positive oligodendrocyte precursors (OLPs) as well as by Oli-neu cells. SDF-1alpha induced proliferation in primary mixed cultures and the Oli-neu cell line was mediated through this receptor. We propose, therefore, that CXC chemokines and in particular SDF-1alpha regulates CNS myelination via their effects on cells of the oligodendrocyte lineage, specifically stimulation of OLP proliferation.