Macrophage recruitment in different models of nerve injury: Lysozyme as a marker for active phagocytosis

Macrophages play critical roles in both degenerative and regenerative processes following peripheral nerve injury. These include phagocytosis of debris, stimulation of Schwann cell dedifferentiation and proliferation, and salvage of myelin lipids for reutilization during regeneration. To better define the role of macrophages, we studied models of primary demyelination (tellurium intoxication) and secondary demyelination (nerve crush and cut). Sections of paraformaldehyde-fixed rat sciatic nerves at various stages of demyelination were stained with monoclonal antibody ED1, a standard macrophage marker, and a polyclonal antiserum specific for lysozyme (LYS). Near the peak of demyelination in all three models, LYS immunoreactivity colocalized with ED1 staining. Macrophages present in nerve after the period of maximal phagocytosis of myelin were much less immunoreactive for LYS. These results suggest LYS is a good marker for macrophages which are active in phagocytosis. Tellurium intoxication, which causes synchronous demyelination and subsequent remyelination of only about 25% of myelin internodes, recruited more macrophages (and induced more lysozyme expression) than either nerve crush or cut, which cause demyelination of all internodes distal to the injury site. This suggests that Schwann cells may recruit macrophages soon after metabolic insult and prior to actual demyelination. The final signal for macrophage recruitment is not directly related to the amount of damaged myelin. In the models listed above, steady state mRNA levels for apolipoprotein E (ApoE; possible mediator of cholesterol salvage), LYS, and Po (major structural protein of PNS myelin), were analyzed by Northern blot analysis. LYS mRNA levels peaked sharply in all models, with a temporal pattern consistent with the expected presence of activated, phagocytic macrophages. The temporal pattern for ApoE mRNA levels differed in the 3 models, but ApoE expression was consistent with its proposed role in salvage of cholesterol during remyelination. © 1995 Wiley-Liss, Inc.     

The chemokine receptor CCR2 is involved in macrophage recruitment to the injured peripheral nervous system

Wallerian degeneration is one of the most elementary reactions of the nervous system after transection of axons, leading to the recruitment of mononuclear cells from the systemic circulation. However, the exact mechanisms regulating this cell invasion have not yet been clarified in detail. Chemokines and their receptors play a central role in leukocyte trafficking, in particular the chemokine MCP-1 has been strongly implicated in macrophage recruitment to the injured nervous system. The present study investigates the course of Wallerian degeneration after transection of the sciatic nerve in mice deficient in two chemokine receptors: CCR2, the main receptor for MCP-1, and CCR5, a marker for Th1 T lymphocytes but also present on macrophages. The number of invading macrophages was determined by immunocytochemistry for three typical macrophage antigens (F4/80, Mac-1, LFA-1). The chemokine receptor CCR2 was expressed by infiltrating cells in the transected nerve stumps. Macrophage invasion was significantly impaired in CCR2-knockout mice when compared with wildtype controls and CCR5-deficient mice. Subsequently, there was a corresponding decrease in myelin phagocytosis due to the reduced invasion of phagocytic macrophages. These data demonstrate the involvement of the chemokine receptor CCR2 in macrophage recruitment to the injured nervous system.     

Monocyte recruitment and myelin removal are delayed following spinal cord injury in mice with CCR2 chemokine receptor deletion

The inflammatory response initiated after spinal cord injury (SCI) is characterized by the accumulation of macrophages at the impact site. Monocyte chemoattractant protein-1 (MCP-1) is a strong candidate for mediating chemotaxis of monocytes to the injured nervous system. To help in defining the role of MCP-1 in inflammation after SCI, we evaluated the time course of macrophage accumulation for 2 weeks following a midthoracic spinal cord contusion injury in mice lacking CCR2, a principal receptor for MCP-1. Mice with a deletion of CCR2 resulted in significantly reduced Mac-1 immunoreactivity restricted to the lesion epicenter at 7 days postinjury. The regions devoid of Mac-1 immunoreactivity corresponded to areas of reduced myelin degradation at this time. By 14 days postinjury, however, there were no differences in Mac-1 staining between CCR2 (+/+) and CCR2 (-/-) mice. Analyses of mRNA levels by RNase protection assay (RPA) revealed increases in MCP-1 as well as MCP-3 and MIP-2 mRNA at 1 day postinjury compared with 7 day postinjury. There were no differences in chemokine expression between CCR2-deficient mice and wild-type littermate controls. The CCR2-deficient mice also exhibited reduced expression of mRNA for chemokine receptors CCR1 and CCR5. Together, these results indicate that chemokines acting through CCR2 contribute to the early recruitment of monocytes to the lesion epicenter following SCI.     

Chemokine regulation of macrophage recruitment into the olfactory epithelium following target ablation: involvement of macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1

Target ablation by olfactory bulbectomy synchronizes the degenerative cell death of olfactory receptor neurons (ORNs), infiltration of macrophages, and proliferation of progenitor cells, leading to neurogenesis, ORN replacement, and regeneration of the sensory epithelium. Although macrophages participate in the degenerative and regenerative events, little is known of the molecular and cellular mechanisms associated with their recruitment during the earliest period following target ablation. Macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1), which are members of the CC or beta-chemokine subfamily, are chemoattractants for monocytes/macrophages. Shortly after target ablation, the protein and mRNA levels for MIP-1alpha and MCP-1 were up-regulated, showing peak expression levels from 16 hr to 3 days post-OBX; this coincided with the pattern of infiltration of activated F4/80(+) macrophages. The mRNAs for MIP-1alpha and MCP-1, as well as their cognate receptors CCR1 and CCR2, respectively, were localized in resident and infiltrating macrophages in numbers commensurate with those of F4/80-immunopositive macrophages in adjacent tissue sections. The mRNA(+) macrophages were localized within olfactory epithelial compartments that corresponded with their proposed functions associated with phagocytosis, proliferation, and infiltration. Our data support the hypothesis that MIP-1alpha and MCP-1 are chemoattractant chemokines associated with the recruitment of macrophages into the olfactory epithelium shortly after target ablation.     

Increased expression of chemokines (MCP-1, MIP-1alpha, RANTES) after peripheral nerve transection

After nerve injury, recruitment of circulating macrophages into the endoneurium is essential for degeneration and subsequently for successful regeneration. However, the factors leading to macrophage recruitment are not known in detail. Chemokines are one of many possible factors influencing recruitment. In this study we wanted to examine, immunohistochemically, the expression of MCP-1, MIP-1alpha and RANTES from 6 hours up to 4 weeks after transection of rat sciatic nerve. An increased expression of MCP-1 was noted already 6 hours after transection, mainly in Schwann cells. Later, the MCP-1 positive staining was seen also in macrophages, fibroblast-like cells and endothelial cells. An increased number of MIP-1alpha positive cells could be noticed after 24 hours, the maximum expression in Schwann cells was noted at the 5-day timepoint. Later, part of the positive cells appeared to be macrophages. RANTES was mainly expressed in inflammatory cells. Endothelial cells in the epi- and endoneurium showed positive staining for every chemokine studied after transection. The contralateral non-operated nerves showed an increased number of positive cells for MCP-1 and MIP-1alpha. In the control nerves MCP-1 and MIP-1alpha positive cells were scattered throughout the endoneurium. This study shows that increased expression of chemokines takes place within endoneurium after peripheral nerve transection. Thus, it is probable that chemokines can take part in the recruitment of macrophages. It further shows that there is an increased expression of the studied chemokines in the non-operated contralateral nerves. Even in normal conditions chemokines are needed, probably to keep resident macrophages within endoneurium.     

Monocyte chemoattractant protein (MCP)-1 is rapidly expressed by sympathetic ganglion neurons following axonal injury

EDI-immunoreactive macrophages, absent from the superior cervical ganglia (SCG) of normal rats, appear in these ganglia within 48h after postganglionic axotomy. Further, resident macrophages show changes after axotomy. Since chemokines function as chemoattractants and activators of leukocytes, the effects of axotomy on chemokine expression in the SCG were examined. Within 6 h after nerve transection, increases were seen in mRNA levels for monocyte chemoattractant protein (MCP)-1. MCP-1 mRNA was concentrated in a population of neurons, while MCP-1 protein was localized to endothelial cells. This axotomy-induced neuronal MCP-1 expression may trigger the infiltration and/or activation of macrophages in SCG after injury.     

Interactions between HIV-infected monocyte-derived macrophages and human brain microvascular endothelial cells result in increased expression of CC chemokines

The presence of perivascular monocytic infiltration is a major hallmark of HIV-1-associated dementia. Since CC chemokines are chemoattractant cytokines that are able to attract T cells and monocytes/macrophages to sites of inflammation, and since infiltrating monocytes/macrophages remain in close contact with the brain endothelium, we investigated whether interactions between HIV-1-infected macrophages and brain endothelium result in an altered chemokine production. We found an increased mRNA expression of monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and RANTES by macrophages after HIV-1 infection. Interactions between HIV-infected macrophages and brain microvascular endothelial cells resulted in an additional upregulation of chemokine mRNA expression, during cell-cell contact as well as in a trans-well system. Since IL-1 beta can function as a modulator of chemokine expression we investigated if interleukin-1 beta could be involved in the regulation of chemokine induction. Coculturing of HIV-infected macrophages and endothelial cells resulted in immune-activation as indicated by increased mRNA expression of IL-1 beta. Subsequently, addition of a neutralizing antibody against IL-1 beta resulted in altered chemokine expression by macrophages, but not by endothelial cells. Thus, IL-1 beta appears to play a major role in the regulation of chemokines during cellular interactions in HIV-associated dementia, but other factors may also be involved.     

Interactions between Schwann cells and macrophages in injury and inherited demyelinating disease

In this article we first discuss the factors that regulate macrophage recruitment, activation, and myelin phagocytosis during Wallerian degeneration and some of the factors involved in the termination of inflammation at the end of the period of Wallerian degeneration after peripheral nerve injuries. In particular, we deal with the early events that trigger chemokine and cytokine expression; the role of phospholipase A(2) in initiating the breakdown of compact myelin, and chemokine, cytokine expression; and the role of MCP-1, MIP-1alpha, and IL-1beta in macrophage recruitment and myelin phagocytosis. We also discuss how inflammation may be switched off and the recently identified role of the Nogo receptor on activated macrophages in the clearance of these cells from the injured nerve. In the second half of the article we focus on the role of certain Schwann cell borne cytokines and chemokines, such as M-CSF and MCP-1 as well as intracellular signaling that regulate their expression in animal models of inherited demyelinating disease. Additionally, we present the preservation of sensory nerves fibers from macrophage attack in these animal models as a challenging paradigm for the development of putative treatment approaches. Finally, we also discuss the similarities and differences in these Schwann cell-macrophage responses in injury-induced Wallerian degeneration and inherited demyelinating diseases. Knowledge of the molecular mechanisms underlying Schwann cell-macrophage interaction under pathological conditions is an important prerequisite to develop effective treatment strategies for various peripheral nerve disorders.     

Turn-over of meningeal and perivascular macrophages in the brain of MCP-1-, CCR-2- or double knockout mice

Perivascular and meningeal macrophages are important for immune surveillance in the healthy and the injured brain. Monocyte chemoattractant protein-1 (MCP-1) regulates macrophage migration and permeability of the blood brain barrier. In the present study, we investigated the influence of MCP-1 or/and chemokine receptor 2 (CCR2)-deficiency on macrophage turnover. The results showed no influence of single MCP-1- or CCR-2-deficiency, but double-deficient mice revealed a virtual absence of blood-borne macrophage recruitment. This finding emphasizes that the MCP-1/CCR2 axis is crucially important for macrophage turnover and compensatory mechanisms remain only partially sufficient to sustain regulatory functions.     

“Tissue-repairing” blood-derived macrophages are essential for healing of the injured spinal cord: From skin-activated macrophages to infiltrating blood-derived cells?

Until recently, the local inflammation that occurs in response to spinal cord injury has received a negative reputation; overall, it was assumed to be one of the major causes of a vicious neurotoxic cycle that leads to impaired recovery following injury. This local inflammation involves both the activated tissue-resident microglia and monocyte-derived macrophages infiltrating from the blood. Ten years ago, we proposed that the blood-derived macrophages, reminiscent of “alternatively activated” macrophages (also known as tissue repairing, M2), are not spontaneously recruited in sufficient numbers to sites of injured central nervous system (CNS). We further demonstrated that their exogenous administration to the margins of injured spinal cord improved functional outcome. However, our suggestions evoked criticism, claiming that we were adding macrophages to a site that is already overwhelmed with inflammatory cells. Using experimental paradigms that enabled functional distinction between the resident and infiltrating cells, our most recent studies further corroborated our repair perception, showing that (a) infiltrating monocyte-derived macrophages are recruited following injury and localize to the margins of the lesion, unlike the activated resident microglia that are not compartmentalized, and (b) activated resident microglia and infiltrating monocyte-derived macrophages perform distinct roles; recruited blood-derived macrophages display an (IL-10-dependent) anti-inflammatory phenotype when they become co-localized with the glial scar. We further found that post-injury recruitment of blood monocytes is indeed suboptimal. Augmentation of the levels of naïve blood monocytes leads to their increased recruitment to the same zones that are the targets of the infiltrated endogenous monocytes, and they acquire the same anti-inflammatory activity, leading to improved recovery. Thus, boosting the levels of the relevant blood monocytes reinforces the body’s own repair mechanisms that, for reasons that are currently under investigation, are not optimally triggered within the critical post-injury period.     

Monocyte chemoattractant protein-1 is a pathogenic component in a model for a hereditary peripheral neuropathy

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in models for inherited demyelinating neuropathies. It is presently unknown which factors link the Schwann cell-based myelin mutation to the activation of endoneurial macrophages. Here we identified the chemokine monocyte chemoattractant protein-1 (MCP-1) as a first and crucial factor upregulated in Schwann cells of mice heterozygously deficient for the myelin protein zero. The chemokine could be identified as an important mediator of macrophage immigration into peripheral nerves. Furthermore, a 50% reduction of chemokine expression by crossbreeding with MCP-1-deficient mice reduced the increase in macrophage numbers in the mutant nerves and lead to a robust amelioration of pathology. Surprisingly, the complete absence of MCP-1 aggravated the disease. Our findings show that reducing but not eliminating chemokine expression can rescue genetically caused demyelination that may be an interesting target in treating demyelinating diseases of the peripheral nervous system.     

A role for CXCL12 (SDF-1alpha) in the pathogenesis of multiple sclerosis: regulation of CXCL12 expression in astrocytes by soluble myelin basic protein

The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1alpha), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1beta and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1beta. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS.     

Selective chemokine mRNA expression following brain injury

Injury in non-neuronal tissues stimulates chemokine expression leading to recruitment of inflammatory cells responsible for orchestration of repair processes. The signals involved in directing repair of damage to the brain are less well understood. We hypothesized that following brain injury, chemokines are expressed and regulate the rate and pattern of inflammatory cell accumulation. The two chemokine subfamilies are alpha(α)-chemokines, which primarily function as neutrophil chemoattractants, and the beta(β)-chemokines, which function primarily as monocyte chemoattractants. We assessed α and β chemokine mRNA expression patterns and leukocyte accumulation following a cerebral cortical lesion. Cortical lesions were produced with and without addition of endotoxin, Escherichia coli lipopolysaccharide (LPS), which stimulates cytokine expression. We studied the expression of the β-chemokines: monocyte chemoattractant protein (gene product JE; MCP-1/JE), macrophage inflammatory protein-1 alpha and beta (MIP-1α and MIP-1β), and the regulated upon activation normal T expressed and secreted chemokine (RANTES) as well as the α-chemokines: interferon-γ-inducible protein (IP-10) and N51/KC (KC; a murine homologue of MIP-2). Changes in gene expression were analyzed by northern analysis at different time points following injury. Leukocyte and macrophage densities were analyzed by immunohistochemistry at the same time intervals. All chemokines were elevated following cortical injury/endotoxin. MCP-1 and MIP-1α were elevated at 2 h and peaked 6 h, MIP-1β peaked at 6 h, but declined more rapidly than MCP-1 or MIP-1α, and IP-10 peaked at 6 h and showed the most rapid decline. KC was elevated at 1 h, and peaked at 6 h following LPS. RANTES was elevated at 1 h and achieved a plateau level between 6 and 18 h, then declined. In contrast, sterile injuries produced in the absence of endotoxin only induced the mRNA of the β-chemokine MCP-1, and its expression was delayed compared to the cortical injury/endotoxin group. The presence of chemokine message as early as 1 h indicates that expression of this class of molecules is an early response in the repair process following traumatic brain injury. Macrophage/microglia accumulation occurred more rapidly, activated microglia further from the lesion border, and more cells accumulated in cortical injury/endotoxin than in cortical lesions produced under sterile conditions. Thus, there was a positive correlation between β-chemokine expression and the number of β-chemokine responsive cells (i.e. microglia) accumulating in injury sites. This is the first comprehensive study using a panel of chemokine probes and specific marcophage/microglial markers to study in vivo activation of the brain following injury. Our data show that the brain is capable of expression of multiple chemokine genes upon appropriate stimulation (e.g. LPS-treatment). The gradient of microglial activation is consistent with physical damage stimulating release of chemokines that diffuse from the injury site. These data strongly suggest that chemokines are instrumental in the initiation of repair processes following brain injury.     

In vivo nerve-macrophage interactions following peripheral nerve injury

In vertebrates, the peripheral nervous system has retained its regenerative capacity, enabling severed axons to reconnect with their original synaptic targets. While it is well documented that a favorable environment is critical for nerve regeneration, the complex cellular interactions between injured nerves with cells in their environment, as well as the functional significance of these interactions, have not been determined in vivo and in real time. Here we provide the first minute-by-minute account of cellular interactions between laser transected motor nerves and macrophages in live intact zebrafish. We show that macrophages arrive at the lesion site long before axon fragmentation, much earlier than previously thought. Moreover, we find that axon fragmentation triggers macrophage invasion into the nerve to engulf axonal debris, and that delaying nerve fragmentation in a Wld(s) model does not alter macrophage recruitment but induces a previously unknown 'nerve scanning' behavior, suggesting that macrophage recruitment and subsequent nerve invasion are controlled by separate mechanisms. Finally, we demonstrate that macrophage recruitment, thought to be dependent on Schwann cell-derived signals, occurs independently of Schwann cells. Thus, live cell imaging defines novel cellular and functional interactions between injured nerves and immune cells.     

Tellurium-induced neuropathy: a model for reversible reductions in myelin protein gene expression.

Inclusion of 1.1% tellurium in the diet of developing rats causes a highly synchronous primary demyelination of peripheral nerves, which is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis and accumulation of squalene. We now report that the demyelination resulting from this limiting of the supply of an intrinsic component of myelin (cholesterol) leads to repression of the expression of mRNA for myelin-specific proteins. Tellurium exposure resulted in an increase in total RNA (largely rRNA) in sciatic nerve, which could not be accounted for by cellular proliferation; these increased levels of rRNA may be a reactive response of Schwann cells to toxic insult and may relate to the higher levels of protein synthesis required during remyelination. In contrast, steady-state levels of mRNA, determined by Northern blot analysis, for P0 and myelin basic protein were markedly decreased (levels after 5 days of tellurium exposure were only 10-15% of control levels as a fraction of total RNA and 25-35% of control levels when the increased levels of total RNA were taken into account). Message levels increased during the subsequent period of remyelination and reached near-normal levels 30 days after beginning tellurium exposure. Although message levels for the myelin-associated glycoprotein showed a similar temporal pattern, levels did not decrease as greatly and subsequently increased sooner than did levels for P0 and myelin basic protein. The coordinate alterations in message levels for myelin proteins indicate that Schwann cells can down-regulate and then up-regulate the synthesis of myelin in response to alterations in the supply of membrane components.     

Interaction between Schwann cells and other cells during repair of peripheral nerve injury

Peripheral nerve injury(PNI) is common and, unlike damage to the central nervous system injured nerves can effectively regenerate depending on the location and severity of injury. Peripheral myelinating glia, Schwann cells(SCs), interact with various cells in and around the injury site and are important for debris elimination, repair, and nerve regeneration. Following PNI, Wallerian degeneration of the distal stump is rapidly initiated by degeneration of damaged axons followed by morphologic changes in SCs and the recruitment of circulating macrophages. Interaction with fibroblasts from the injured nerve microenvironment also plays a role in nerve repair. The replication and migration of injury-induced dedifferentiated SCs are also important in repairing the nerve. In particular, SC migration stimulates axonal regeneration and subsequent myelination of regenerated nerve fibers. This mobility increases SC interactions with other cells in the nerve and the exogenous environment, which influence SC behavior post-injury. Following PNI, SCs directly and indirectly interact with other SCs, fibroblasts, and macrophages. In addition, the inter-and intracellular mechanisms that underlie morphological and functional changes in SCs following PNI still require further research to explain known phenomena and less understood cell-specific roles in the repair of the injured peripheral nerve. This review provides a basic assessment of SC function post-PNI, as well as a more comprehensive evaluation of the literature concerning the SC interactions with macrophages and fibroblasts that can influence SC behavior and, ultimately, repair of the injured nerve.