Extracellular acidification decreases the basal motility of cultured mouse microglia via the rearrangement of the actin cytoskeleton

doi:10.1016/S0006-8993(99)02221-0

Brain Research

Volume 853, Issue 1, 17 January 2000, Pages 22-31

https://doi.org/10.1016/S0006-8993(99)02221-0 Get rights and content

Abstract

The present study was undertaken to examine the effect of extracellular pH (pH₀) on the locomotor function of murine microglial cells in vitro. We have found that basal motility of microglia, as measured by a computer-assisted video assay, decreased in an acidic, but not in an alkaline environment. Extracellular acidification affected the architecture of F-actin cytoskeleton, inducing bundling of actin and the formation of stress fibers. The change in intracellular pH (pH_i) resulting from the change in pH₀ seems to be a prerequisite for the motility decrease since other means to decrease pH_i, namely Na⁺-free solution (in the absence of HCO⁻₃) and nigericin-containing solution, mimicked the extracellular acidification. In contrast to its pronounced effect on basal motility of microglial cells, the motility increase, as induced by the chemoattractant complement 5a (C5a), was not affected by the acidic environment. The relationship of pH₀ to the locomotor function was also studied in a long-term microchemotaxis assay where microglia migrated within a pH gradient. Intracellular acidification induced by lowering pH₀ to 6.0 or removal of Na⁺ from the assay medium decreased basal microglial cell migration. The C5a-induced chemotactic migration was moderately decreased by the acidic environment. In conclusion, our results suggest that acidification of the microglial extracellular milieu leads to a decrease in pH_i and thereby reduces the basal microglial motility and C5a-induced chemotaxis via a rearrangement of the cytoskeleton. We would therefore like to speculate that changes in pH_i constitute an important control mechanism in regulating the locomotor function of microglia in culture and probably also in the intact tissue.

Introduction

Microglial cells become activated rapidly in response to any injury 17, 21, 22, 23, 25, 26, 55. Our knowledge on the signal cascades controlling the multi-step activation from the resting to the fully activated form is still rather incomplete. The first stage of activation displays a repertoire in terms of recruitment to the site of lesion, proliferation, increased or de-novo expression of immunomolecules, and functional changes, e.g., the release of reactive oxygen intermediates and inflammatory cytokines 2, 17, 41. In a second stage of activation, if neuronal degeneration occurs, activated microglia further transform into phagocytic cells 31, 42.

Immunohistological in vivo studies in different experimental models present evidence for enhanced motility of microglial cells towards lesions or sites of inflammation. For example, massive migration of intraretinal microglial cells from ganglion cell layer to the photoreceptor cell layer occurred in animals with optic nerve transection 44, 45. In rat central nervous system lesions induced by kainic acid, microglia migrated from remote areas to injured pyramidal neurones [2]. In the leech, damage to the CNS has been shown to be followed by accumulation of microglial cells at the site of injury [20]. Conditions that recruit microglia to the lesion sites are not well characterized. Also not much is known about factors that serve to immobilize microglial cells at the lesioned sites. Much interest was focused on investigating the influence of adhesion molecules, cytokines and other classes of proteins on motility of microglial cells [4]. Recent in vitro studies presented evidence for enhanced motility of microglia after complement 5a (C5a) or EGF receptor stimulation 28, 29, 54. C5a, the marker of central nervous system inflammation [11], not only increased microglial motility but also served as a potent chemoattractant for these cells.

pH could interfere with a motility process two-fold: firstly, the acid–base status of the extracellular space can play a role of the potentially important signaling event. Secondly, intracellular pH changes might be the part of the signal cascade after microglial stimulation. Under many pathological conditions such as ischemia, anoxia, hypoxia, spreading depression or epileptiform activity the pH regulation in the nervous system is impaired leading to an acidosis or alkalosis of the extracellular space in the brain 27, 32, 35, 38, 50. During global ischemia for example, pH₀ can drop from the resting level of 7.4 to as low as 6.2 [38]. Microglia can contribute to the extracellular pH transients in the central nervous system under physiological and pathological conditions. If the resting pH_i of microglial cells follows the changes in pH₀, acidification or alkalization of the extracellular space might influence the organization of the cytoskeleton and hence modulate other cellular processes such as migration to the site of lesion.

Indeed, pH_i plays an important role in the regulation of cytoskeletal organization and various types of cell motility, including ameboid movement, shape changes, chemotaxis and phagocytosis (for review, see [43]). For example, cell morphotype and motile activity of the rat fibrosarcoma cells (A870N) are significantly influenced by the pH of the medium [30]. Motile function of human neutrophils can also be regulated via their pH_i[39]. In agreement with this hypothesis, certain steps in the actin polymerization sequence and the binding of actin filaments to membrane-anchoring proteins are pH-dependent events [8].

The possible role of pH₀ in the locomotor function of microglia has not been studied. The purpose of this study was therefore (i) to investigate whether the changes in pH₀ may serve as a signal for microglial motility, migration and cytoskeletal reorganization, and (ii) to test whether pH₀ can interfere with a migratory response of microglia under chemotactic conditions after C5a receptor activation.

Section snippets

Microglial cell culture

Microglial cells were prepared from cortex of newborn mice (strain NMRI; Naval Medical Research Institute) essentially as described previously 10, 12. In brief, cortical tissue was carefully freed of blood vessels and meninges. Tissue was trypsinized for 2 min, carefully dissociated with a fire-polished Pasteur pipette and washed twice. The cortical cells were cultured for 2 weeks in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum with change of medium every

Basal motility of microglia: effect of intracellular acidification and alkalinization

Time-lapse videomicroscopy of cultured microglial cells revealed that these cells exhibited a high resting motility. They did not show a distinct polarity and they rarely translocated, but protruded and retracted their processes within minutes.

In order to study the effect of pH_i on microglial motility, we changed the pH of the perfusing buffer over the range 5.8–8.3. The pH_i of microglial cells followed the changes in pH₀. Similarly to our previous observation [9], decreasing pH of the

Extracellular acidification as a possible regulator of microglial motility

The cultured microglia is characterized by its ameboid shape, dynamic stationary movement and complex shape changes, the latter properties being the prerequisite for migration. In this study we determined a possible relationship between the extracellular pH and the locomotor function of microglial cells. Most interestingly, basal motility of microglial cells, observed by time-lapse video microscopy, rapidly decreased in an acidic environment while overall cell viability remained unaffected.

Acknowledgements

This research was supported by Alexander von Humbold fellowship (to L.F.). The authors thank G. Muller and R. Krauss for the excellent technical assistance.

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Research reportExtracellular acidification decreases the basal motility of cultured mouse microglia via the rearrangement of the actin cytoskeleton

Abstract

Introduction

Section snippets

Microglial cell culture

Basal motility of microglia: effect of intracellular acidification and alkalinization

Extracellular acidification as a possible regulator of microglial motility

Acknowledgements

Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Trends Neurosci.

Brain Res.

Neuroscience

Cell Immunol.

Neurosci. Lett.

Biochem. Biophys. Res. Commun.

Int. Rev. Cytol.

Brain Res.

Int. J. Dev. Neurosci.

Eur. J. Cancer Clin. Oncol.

Radiother. Oncol.

Eur. J. Cancer Clin. Oncol.

Effect of lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures

J. Neurosci. Res.

NMR spectroscopic investigation of the recovery of energy and acid–base homeostasis in the cat brain after prolonged ischemia

J. Cereb. Blood Flow Metab.

Inflammatory cytokines within the central nervous system: Sources, function and mechanism of action

Cell Physiol.

Global cerebral ischemia and intracellular pH during hyperglycemia and hypoglycemia in cats

Stroke

Protein kinase C-induced redistribution of the cytoskeleton and phosphorylation of vimentin in cultured brain macrophages

J. Neurosci. Res.

A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments

J. Cell. Biol.

Intracellular pH regulation in cultured microglial cells from mouse brain

J. Neurosci. Res.

Antigen presentation and tumor cytotoxicity by interferon-gamma-treated microglial cells

Eur. J. Immunol.

Expression of the receptor for complement C5a (CD88) is up-regulated on reactive astrocytes, microglia, and endothelial cells in the inflamed human central nervous system

Am. J. Pathol.

Characterization of ameboid microglia isolated from developing mammalian brain

J. Neurosci.

Extra- and intracellular pH during near-complete forebrain ischemia in the rat

J. Neurochem.

Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments

Biochemistry

Research report
Extracellular acidification decreases the basal motility of cultured mouse microglia via the rearrangement of the actin cytoskeleton