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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 29, 2004 as doi:10.1096/fj.04-1777fje. |
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* Department of Neurology, Heinrich-Heine-University, Düsseldorf, Germany; and
Department of Neurology, Julius-Maximilians-University, Würzburg, Germany
1Correspondence: Department of Neurology, Heinrich-Heine-University, Moorenstr. 5, Düsseldorf D-40225, Germany. E mail: jander{at}uni-duesseldorf.de
SPECIFIC AIMS
Increasing evidence implicates inflammatory processes as modulators of axon regeneration after injury to central and peripheral nervous systems. Osteopontin (OPN) is a macrophage-derived, RGD-containing glycoprotein with cytokine-like, chemotactic, and proadhesive properties. The specific aim of this study was to elucidate a possible role of OPN in crush lesions of the optic and sciatic nerve as models of central and peripheral axotomy, respectively.
PRINCIPAL FINDINGS
1. Lesion-associated macrophages express OPN in crush lesions of the central but not peripheral nervous system
Rat optic and sciatic nerves were subjected to crush injury and analyzed for expression of OPN using immunohistochemistry and quantitative real-time PCR. In optic nerve injury, OPN expression first appeared at day 2, reached its maximum at day 4 (Fig. 1
A, C), and decreased thereafter. OPN was mainly expressed by ED1+ macrophages (Fig. 1B, D
) and remained restricted to the primary lesion site at all time points examined. In contrast to the optic nerve, OPN induction was absent in crushed sciatic nerves (Fig. 1E
) although ED1 immunostaining revealed a similar degree of macrophage activation at the crush site in both paradigms (Fig. 1F
). This pattern of differential OPN expression was confirmed by quantitative PCR analysis of RNA isolated from crushed optic and sciatic nerves. Taken together, these data indicated fundamental differences in the molecular programming of lesion-associated macrophages in the central and peripheral nervous systems.
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2. OPN exerts potent growth inhibitory effects in an in vitro assay of axon outgrowth
Based on the expression data, we hypothesized that lesion-associated expression of OPN might contribute to the nonpermissive environment inhibiting axon regrowth in injured CNS fiber tracts. To test this, we performed an in vitro growth assay using dorsal root ganglia (DRG) from embryonic rats (12.5–14.5 dpc). On dishes coated with laminin as a typical growth-permissive substrate extensive axon outgrowth was observed within 24 h after plating (Fig. 2
A). In contrast, axon outgrowth was strikingly reduced on OPN-coated dishes (Fig. 2B
). On dishes coated with both laminin and OPN (Fig. 2C
) axon outgrowth was slightly better than on OPN alone but still significantly reduced compared with laminin alone. Thus, OPN exerted a dominant inhibitory effect on axon outgrowth from DRG explants in culture. Axons exhibited homogenous radial spreading on laminin-coated dishes (Fig. 2A
) but frequent fasciculation on dishes coated with either OPN alone (Fig. 2B
) or the combination of laminin and OPN (Fig. 2C
).
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We additionally tested vitronectin as an alternative RGD-containing matrix protein sharing receptors such as the 
vß3 and vß5 integrins with OPN. On dishes coated with vitronectin alone (Fig. 2D
), axon outgrowth was as poor as on OPN-coated dishes. However, on dishes coated with the combination of vitronectin and laminin (Fig. 2E
), axon outgrowth was identical to dishes coated with laminin alone. Unlike OPN, vitronectin did not overcome the growth-permissive properties of laminin.
Further studies showed that on laminin-coated dishes axons crossed the border of the underlying Schwann cell corona and extended directly onto the laminin-coated plastic matrix. In contrast, on OPN-coated dishes, axons fasciculated and rarely extended beyond the Schwann cell layer. The few axons extending further onto the OPN-containing matrix displayed small tips of collapsed appearance in contrast to the large growth cones found on laminin-coated surfaces. Thus, our data suggest an active inhibitory effect of matrix-associated OPN on axon growth.
CONCLUSIONS AND SIGNIFICANCE
OPN is a matricellular glycoprotein abundantly expressed by lesion-associated macrophages in a variety of injury models of heart, brain, and skin. Accumulating evidence suggests a critical role of OPN for tissue homeostasis and repair processes after injury. In our present study, we show that lesion-associated macrophages expressed OPN in crush injury of the optic, but not sciatic nerve, indicating fundamental differences in the molecular programming of macrophages between the central and peripheral nervous systems. These findings extend previous observations that macrophages infiltrating in anterograde, Wallerian-type degeneration of sciatic nerves did also not express OPN. Thus, with respect to OPN expression by lesion-associated macrophages, two distinct patterns are discernible: strong OPN induction in heart, skin, brain, and optic nerve injury as opposed to the absence of OPN induction, as a unique finding in peripheral nerve injury.
Our in vitro data revealed inhibition of axon growth as a hitherto unknown effect of OPN. We used DRG explants from embryonic rats as a model to study axon outgrowth on different acellular substrates in vitro. On laminin as a typical growth-promoting substrate we observed extensive radial outgrowth of axons that extended beyond the boundary of the underlying Schwann cells directly onto the laminin-coated surface of the culture dish. In contrast, on surfaces coated with either OPN alone or with a combination of OPN and laminin axons exhibited greatly reduced radial outgrowth suggesting a dominant axon inhibitory effect of extracellular OPN immobilized on the plastic surface. The observation that those axons were growing as fascicles provides further evidence that OPN is a nonpermissive or even repulsive growth substrate that favors an axonal "pulling together." In contrast to OPN, vitronectin as an alternative RGD-containing matrix ligand sharing the vß3/vß5 integrin receptors with OPN did not overcome the growth-promoting properties of laminin. Thus, functional diversity of v-integrin binding matrix ligands with respect to axon reactions is conceivable. In addition to v integrins, OPN binds to several other integrin and non-integrin receptors (36–38) whose expression in CNS and PNS injury remains to be studied.
Apart from direct effects on axons, differential expression of OPN may have additional important consequences for injury responses in the nervous system. OPN exerts proinflammatory effects in the EAE model of immune-mediated CNS injury and provides a chemotactic stimulus for macrophages. Thus, OPN production by macrophages may represent an autocrine signaling mechanism promoting macrophage recruitment and activation in nerve injury. However, with respect to the kinetics of recruitment and the immunophenotype of lesion-associated macrophages at the crush site, no major differences are evident between the optic and sciatic nerve model. OPN exerts chemotactic activity toward astrocytes, suggesting a role for glial scarring in CNS injury. Despite similar inflammatory responses observed in CNS trauma and sciatic nerve crush injury, scar formation only occurs in CNS lesion models where it may represent a key impediment to axonal regeneration.
Our study reveals expression of OPN by lesion-associated macrophages as a novel mechanism that may contribute to the nonpermissive nature of the adult CNS for axon regeneration after injury. Our findings support the view that differential molecular programming of macrophages is a key factor determining injury responses in the CNS and PNS.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1777fje;
This article has been cited by other articles:
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  T. Jang, T. Savarese, H. P. Low, S. Kim, H. Vogel, D. Lapointe, T. Duong, N. S. Litofsky, J. M. Weimann, A. H. Ross, et al. Osteopontin Expression in Intratumoral Astrocytes Marks Tumor Progression in Gliomas Induced by Prenatal Exposure to N-Ethyl-N-Nitrosourea Am. J. Pathol., May 1, 2006; 168(5): 1676 - 1685. [Abstract] [Full Text] [PDF]
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) at the crush site in the optic nerve (CNS), but not sciatic nerve (PNS) and exerts growth inhibitory effects preventing axon regeneration in the CNS paradigm.