The MHP36 line of murine neural stem cells expresses functional CXCR1 chemokine receptors that initiate chemotaxis in vitro
Introduction
The central nervous systems of adult rodents and humans contain neural stem cells that are capable of differentiating into neurons and glia (Eriksson et al., 1998, Gage, 2000). The properties and therapeutic potential of neural stem cells are currently the focus of tremendous scientific endeavor, as cell loss within the CNS often results in severe disability or death. Transplanted embryonic tissue has already been used to treat patients suffering from Huntington's disease (Rosser et al., 2002) and Parkinson's disease (Freed et al., 2001, Olanow et al., 2003); some see these trials as the forerunners of formal stem cell transplantation programs (Bjorklund et al., 2003). Disappointments and setbacks in the development of effective therapeutic neuroprotectants for stroke and traumatic brain injury have only served to increase the interest in stem cell therapies for cerebral ischemia (del Zoppo, 2004, Turnpenny et al., 2005).
In 2001, Veizovic et al. showed that murine neural stem cells can effect brain repair in a rat model of middle cerebral artery occlusion (MCAO). They transplanted murine neural stem cells from a temperature sensitive conditionally immortalised line (known as MHP36 cells (Gray et al., 1999)) into the contralateral cerebral hemisphere of rats that had undergone 60 min of middle cerebral artery ischemia–reperfusion 2 to 3 weeks earlier, and compared functional outcome and post mortem histology with appropriate controls after 42 weeks. Animals that had received MHP36 cell transplants made a significantly better functional recovery and had significantly smaller infarct volumes; strikingly some MHP36 cells had migrated from the transplant site across the corpus callosum to the lesion. Other investigators have since published similar findings, showing transhemispheric migration of murine, rat and human neural stem cells in other rodent models of hypoxic–ischemic cerebral injury (Imitola et al., 2004, Kelly et al., 2004), furthermore serial magnetic resonance imaging has been used to map migratory pathways (Hoehn et al., 2002, Modo et al., 2004). MHP36 cell transplants have also proved to be beneficial in other rodent models, such as lesions of the forebrain cholinergic projections, and old age (Hodges et al., 2000, Grigoryan et al., 2000).
Current understanding of the mechanisms and molecules that are essential for neural stem cell migration centres on adhesion molecules. For example, the interaction of the migrating neurons with their environment through expression of the polysialated glycoprotein neural cell adhesion molecule (PSA-NCAM) is necessary for proper migration, as null mutation for NCAM or the deletion of the polysialic acid moiety results in migratory defects (Bonfanti and Theodosis, 1994, Ono et al., 1994, Rousselot et al., 1995). Migration through the rostral pathway is influenced by members of the ephrin-B family (Conover et al., 2000), Slit integrin family members (Jacques et al., 1998, Wu et al., 1999), unknown astrocyte-derived factors (Mason et al., 2001), and the direction of flow of cerebrospinal fluid (CSF) (Sawamoto et al., 2006). The extracellular matrix molecule tenascin-R also appears to provide a molecular cue that induces neuroblasts to begin their migration towards the olfactory bulb (Saghatelyan et al., 2004).
Chemokines are also important chemotactic factors in brain development and disease. The stromal-cell derived factor-1α (SDF-1α)-CXCR4 system is involved in development of the cerebellum (Reiss et al., 2002). SDF-1α is secreted by the meninges and acts as a chemoattractant and mitogen to embryonic stem cells, maintaining the integrity of the external granular layer and later orchestrating the development of the mature cerebellum. Expression of the gene encoding SDF-1α is upregulated in penumbral vessels after cerebral ischemia in mice and is associated with a flux of CXCR4-expressing leukocytes into the brain (Welsh et al., 1987). In cerebral ischemia, neural stem cells migrate towards areas rich in SDF-1α (Imitola et al., 2004), which can also elicit migration of neural stem cells in vitro (Imitola et al., 2004, Tran et al., 2004, Robin et al., 2006). Neural stem cells also seem to transcribe a broad range of other chemokine receptor genes including CXCR1 and CXCR2 (Tran et al., 2004).
SDF-1α is a potent chemoattractant for resting lymphocytes and monocytes (Kim and Broxmeyer, 1998). However, neutrophils are the effectors of early inflammation, and we chose to examine whether neutrophil chemoattractants might influence the migration of MHP36 murine neural stem cells. Interleukin-8 (IL-8; CXCL8) is produced during reperfused cerebral ischemia in rabbits (Matsumoto et al., 1997) and monkeys (Popivanova et al., 2003), and its levels are raised in the CSF of patients with ischemic stroke (Matsumoto et al., 1997, Tarkowski et al., 1997, Kostulas et al., 1999, Losy et al., 2005). It acts mainly via the chemokine receptor CXCR1. The levels of GROα (CXCL1; CINC), which acts mainly via CXCR2, increase rapidly in ischemic brain areas after permanent MCAO (Liu et al., 1993) and transient ischemia (Yamasaki et al., 1995, Yamagami et al., 1999) in rodents, and stroke patients (Losy et al., 2005). The early influx of neutrophils into the brain after cerebral ischemia has been observed in rodents (Davies et al., 1998), dogs (Hallenbeck et al., 1986), primates (Garcia and Kamijyo, 1974) and humans (Price et al., 2004).
We sought evidence that IL-8 influences the migration of neural stem cells in the same way as neutrophils, namely via CXCR1 and/or CXCR2 (Murphy, 1997). We sought chemokine receptor messenger RNA (mRNA) and protein, and evidence of receptor function using calcium fluorophores and in vitro migration assays. We also examined whether this neural stem cell line shares any other characteristics with neutrophils, for example the expression of the CD15 antigen (Crocker and Burnett, 1986). We also assessed whether IL-8 upregulated the expression of doublecortin (Dcx), a protein expressed by migrating neuroblasts in several species (Gleeson et al., 1998, Francis et al., 1999). Dcx is expressed in health in the rostral migratory stream (Yang et al., 2004), and also after experimental focal cerebral ischemia by proliferating neural progenitor cells in the subventricular zone of adult rodents and in grafted human CNS stem cells migrating towards ischemic cerebral cortex (Jiang et al., 2001, Jin et al., 2001, Kelly et al., 2004).
Section snippets
Materials
All reagents were purchased from Sigma (Poole, United Kingdom) unless otherwise stated.
Maintenance of MHP36 cells in culture
MHP36 cells were grown in adherent culture in serum-free medium at their permissive temperature of 33 °C in an atmosphere of 5% CO2 and humidified air as previously described (Kershaw et al., 1994, Sinden et al., 1997). Ambient temperature was increased to 37 °C (non-permissive temperature) 24 h before all experiments.
Reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA was extracted using guanidium thiocyanate and glass fiber filters, and purified by
RT-PCR for CXCR1 and CXCR2
Having subjected MHP36 cell lysates to RT-PCR reactions, an amplimer was identified demonstrating the presence of mRNA transcribed from the CXCR1 gene (Fig. 1a). There was no evidence that MHP36 cells transcribe mRNA from CXCR2 (Fig. 1b). The specificity of primer pairs and suitability of reaction conditions were confirmed by the presence of amplimers in positive controls (in this case murine PBMCs), reactions examining the expression of GAPDH, and by sequencing. The relative brightness of the
Discussion
Previous studies of the influence of chemokines on neural stem cell migration have concentrated on the SDF-1α-CXCR4 ligand–receptor system. However, as Tran et al. (2004) demonstrated, neural stem cells transcribe a wide range of genes encoding chemokine receptors. We have shown that MHP36 cells, a conditionally immortalised murine neural stem cell line, express functional CXCR1 receptors. When the cells are stimulated by IL-8, alterations in intracellular Ca++ flux are seen, and Dcx is
Acknowledgements
We wish to thank Drs N Menager of the Department of Clinical Veterinary Medicine, University of Cambridge, for assistance in isolating murine PBMCs, and Mr R Hicks for assistance with flow cytometry. This work was supported by a grant from the Medical Research Council of the UK to DWW.
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