Peripheral nerve induces macrophage neurotrophic activities: regulation of neuronal process outgrowth, intracellular signaling and synaptic function

Abstract

Rat cortical neurons cultured in conditioned media from human monocyte-derived macrophages (MDM) show increased neuronal protein synthesis, neurite outgrowth, mitogen-activating protein kinase activity, and synaptic function. Neurotrophic properties of human MDM-conditioned media are significantly enhanced by human peripheral nerve and to a more limited extent by CD40 ligand pre-stimulation. Such positive effects of MDM secretions on neuronal function parallel the secretion of brain-derived neurotrophic factor (BDNF). MDM activation cues may serve to balance toxic activities produced during neurodegenerative diseases and thus, under certain circumstances, mitigate neuronal degeneration.

Introduction

Central nervous system (CNS) innate immune responses play a fundamental role in health and disease. Brain macrophages and T-cell lymphocytes mediate inflammatory responses that facilitate both neurotoxic and neurotrophic processes in the CNS. In the environment of CNS degenerative disease and traumatic injury, macrophages secrete inflammatory cytokines, alpha and beta chemokines, quinolinic acid, arachidonic acid and its metabolites, nitric oxide and free radicals Gendelman, 2002, Kiefer et al., 2001 as well as growth inhibiting proteins such as chondroitin sulfate proteoglycans Al-Omaishi et al., 1999, Cotter et al., 1999, Epstein and Gelbard, 1999, Fitch and Silver, 1997, Gendelman, 2002, Jansen and Reinhard, 1999, McGeer and McGeer, 1999. Secretion of inflammatory cytokines and growth-inhibiting proteins by activated immune cells has been shown to cause secondary degeneration following cell death caused by disease or trauma Popovich et al., 2002, Popovich and Jones, 2003.

Specific environmental activation cues present during neurodegenerative disease and CNS injury may also be responsible for neurotrophic responses. A growing body of evidence suggests that CNS innate inflammatory responses promote neuronal process outgrowth and functional recovery following injury (Schwartz, 2002). As observed in the peripheral nervous system, macrophages, once recruited to the site of CNS injury, promote phagocytosis of myelin debris and recycling of lipid degradation products required for the repair and regeneration of damaged neurons and their processes (Lotan and Schwartz, 1994). In addition, macrophages secrete a wide range of cytokines and growth factors with neurotrophic properties including nerve growth factor (NGF) Caroleo et al., 2001, Elkabes et al., 1996, brain-derived neurotrophic factor (BDNF) Batchelor et al., 1999, Batchelor et al., 2002b, Dougherty et al., 2000, glial-derived neurotrophic factor (GDNF) Batchelor et al., 1999, Batchelor et al., 2002b, Satake et al., 2000, Wei et al., 2000, Yamamoto et al., 1998, growth/differentiation factor-15/macrophage inhibitory cytokine (GDF-15/MIC) (Strelau et al., 2000) and interleukin-6 (IL-6) (Kiefer et al., 2001). Moon et al. (2002) recently showed that macrophages support the synthesis of neuronal growth promoting heparan sulfate proteoglycans while microglia and astrocytes synthesize growth inhibiting chondroitin sulfate proteoglycans. In toto, macrophage secretory and substrate bound proteins can increase neurite output and branching (Patrick et al., 1996), activate mitogen-activated protein kinase (MAPK) pathways and increase choline acetyltransferase (ChAT) activity (Jonakait et al., 2000). Upregulation of cellular processes such as these is necessary for neuronal differentiation or recovery from injury (Jonakait et al., 2000).

Further, in vivo studies have shown that enhanced sprouting of dopaminergic neurons following striatal injury directly corresponds to the recruitment of activated brain macrophages (Batchelor et al., 2002a). Implantation of peripheral macrophages into regions of damaged spinal cord results in degradation of growth inhibiting myelin proteins and production of regenerative extracellular matrix molecules (Franzen et al., 1998). Partial restoration of motor function has been shown to occur when macrophages and T-cell lymphocytes are activated with peripheral nerve (PN) and subsequently injected into areas of spinal cord injury Prewitt et al., 1997, Rapalino et al., 1998, Schwartz, 2000, Schwartz et al., 1999a, Schwartz and Moalem, 2001, Zeev-Brann et al., 1998. Recent work has begun to characterize the role of T cell lymphocytes in the neurotrophic immune response. T cell mediated protective responses induced by active or passive immunization of CNS-injured rats with myelin-associated peptides or Nogo A Hauben et al., 2000, Hauben et al., 2001 protect injured axons from secondary degeneration by inducing neurotrophin production by surrounding astrocytes and macrophages (Barouch and Schwartz, 2002). Clearly, T lymphocytes play a major role activating neuroprotective and neuroregenerative processes in the damaged CNS, however, the role of macrophages has not been defined.

Given that environmental cues can influence the balance of neurotoxic and neurotrophic properties of macrophages, we developed in vitro cellular assays to assay the pathways for neuroregeneration. In this regard, we hypothesized that CD40 ligand (CD40L) and human peripheral nerve explants differentially influence macrophage neurotrophic function. CD40 receptor is expressed by macrophages van Kooten, 2000, van Kooten and Banchereau, 1997 and plays a major role in the regulation of the immune response. The expression of CD40L by infiltrating T-cells during autoimmune disorders, endothelial cells lining cerebral microvessels (Mach et al., 1997) and astrocytes (Calingasan et al., 2002) suggest a broad range of physiologically relevant functions for CD40L in the CNS. Explants of human peripheral nerve and myelin proteins have been shown to augment immune cell mediated neurotrophic activities Hauben et al., 2000, Schwartz and Moalem, 2001.

We now show that monocyte-derived macrophage (MDM)-conditioned media and MDM-conditioned media from CD40L- and, significantly, from PN-stimulated macrophages increase neuronal survival, synthesis of proteins associated with neurite outgrowth, activity of mitogen-activating protein kinases, ERK1 and ERK2, and synaptic transmission. Production of BDNF and other neurotrophins was associated with these neuronal responses. All together, enhancement of human macrophage neurotrophic and regenerative function by specific environmental cues may play an important role in recovery from CNS injury. Such recovery may serve, in part, to affect the tempo of neurodegenerative diseases.