Role of adhesion molecules in interactions of endothelium with leukocytes in inflammatory processes

This paper reviews recent work regarding the molecules that mediate leukocyte-endothelial cell adhesion, describes the underlying principles of leukocyte migration, and discusses a model of the sequence of events that allows a leukocyte to attach to endothelium and migrate into tissue. Pathophysiological importance of the adhesion molecules in the development of different states of inflammation has been also presented.     

Intercellular adhesion molecule 1 is the major adhesion molecule expressed during schistosome granuloma formation

Endothelial cell adhesion molecules play a key role in inflammation by initiating leukocyte trafficking. One of the most complex inflammatory responses is the formation of a cellular granuloma. Expression of adhesion molecules during granuloma formation was investigated by using the murine host reaction to schistosome parasite eggs deposited in the liver as a model. By both immunohistochemistry and lymphocyte adhesion assays, the predominant interaction identified was between intercellular adhesion molecule 1 (ICAM-1) and its cognate integrin, leukocyte functional antigen 1 (LFA-1). ICAM-1 expression on sinusoidal endothelium was induced when eggs were first deposited in the liver, peaked in parallel with granuloma size, and was downregulated with modulation of the granuloma. Polyacrylamide beads coated with soluble parasite egg antigens could induce ICAM-1 expression on endothelial cells in vitro only in the presence of tumor necrosis factor alpha, a cytokine previously shown to be key to granuloma formation. A role for ICAM-1 in recruiting lymphocytes to the hepatic granuloma was also supported by the observation that lymphocytes preincubated with anti-LFA-1 antibody did not bind to granulomas in tissue sections. While ICAM-1 is the predominant adhesion molecule in schistosome egg granuloma formation in wild-type mice, when the ICAM-1 gene is knocked out, vascular cell adhesion molecule 1 is upregulated and granuloma formation is preserved.     

Neural cell adhesion molecule (N-CAM) domains and intracellular signaling pathways involved in the inhibition of astrocyte proliferation

The neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation in vitro and in vivo, and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other N-CAM-expressing cells also was inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings that RU-486 could partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM was reversed by RU-486. The ability of N-CAM to inhibit astrocyte proliferation was unaffected, however, by agents that block the ability of N-CAM to promote neurite outgrowth. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways.     

Role of neural cell adhesion molecule and polysialic acid in mouse circadian clock function

The suprachiasmatic nuclei (SCN) express the highly polysialylated form of the neural cell adhesion molecule (NCAM) that has been proposed to promote plasticity in the adult brain. To investigate a role for NCAM in SCN circadian clock function, we examined the daily locomotor rhythm of mice homozygous for a mutation, Ncamtm1Cwr, which results in deletion of the NCAM-180 isoform that in brain carries polysialic acid (PSA). Mutant mice entrained well to a 12 hr light/dark cycle but exhibited a significantly shortened free-running period and longer activity duration under constant darkness (DD) than did wild-type mice. By the third week of DD treatment, circadian rhythmicity in the mutant was abolished. Immunocytochemical analyses of the mutant SCN revealed an abnormal number and distribution of vasoactive intestinal polypeptide-producing neurons, suggesting a developmental effect of the mutant phenotype; however, a direct physiological effect of the mutation on clock function was indicated by the fact that removal of PSA from adult wild-type SCN by microinjection of endoneuraminidase shortened the free-running period to a similar extent as in the mutant. Together, these data indicate critical roles for NCAM and PSA in the development and physiology of the mammalian SCN circadian clock.     

The cytoplasmic domains of E- and P-selectin do not constitutively interact with alpha-actinin and are not essential for leukocyte adhesion

The selectins are a family of carbohydrate-binding adhesion molecules involved in the regulation of leukocyte migration. Although there is strong homology between different selectins in their extracellular regions, there is none in the cytoplasmic tails, suggesting selectin-specific functions for these domains. Our previous work showed that the cytoplasmic tail of L-selectin interacts with the actin cytoskeleton via alpha-actinin and vinculin, and that truncation of the cytoplasmic tail of L-selectin blocked both association with alpha-actinin and vinculin and leukocyte adhesion. In the present study, the effects of truncation of the cytoplasmic tails of E- or P-selectin on cell adhesion and cell surface expression were examined, and possible interactions between alpha-actinin and the E- and P-selectin cytoplasmic tails were assessed. In contrast to previous observations demonstrating a requirement for the L-selectin cytoplasmic tail, truncation of the E- or P-selectin cytoplasmic domains had no effect on cell adhesion, or on cell surface expression, when assessed in transiently transfected COS cells. This lack of effect on cell surface expression and adhesion was also observed when transfections were performed with lower amounts of cDNA, which led to submaximal levels of expression. In addition, no interaction between alpha-actinin and the cytoplasmic tails of either E- or P-selectin could be detected under conditions in which binding of alpha-actinin to the L-selectin cytoplasmic tail could be readily demonstrated. Therefore, interactions between the cytoplasmic tail of E- or P-selectin and alpha-actinin or other cytoskeletal proteins are not necessary for leukocyte adhesion per se, but may facilitate downstream biologic events.     

Glial cell interactions with tenascin-C: adhesion and repulsion to different tenascin-C domains is cell type related

The multimodular glycoprotein tenascin-C is transiently expressed, predominantly by glial cells, during the development of the central and peripheral nervous systems. This extracellular matrix glycoprotein is involved in the control of cell adhesion, neuron migration and neurite outgrowth. Distinct functional properties for neuronal cell types have been attributed to separate tenascin-C domains using antibody perturbation studies and in vitro experiments on tenascin-C fragments. In order to study potential roles of tenascin-C for glial cell biology, a library of recombinant tenascin-C domains was used in a bioassay in vitro. Embryonic day 14 astrocytes, various astroglial-derived cell lines (C6, A7 and Neu7) and oligodendroglial-derived cell types (Oli-neu and G26-20) were examined in an adhesion assay and compared to the neuroblastoma cell line N2A. A binding site for most cell types, except for A7 and N2A, could be assigned to the first three fibronectin type III domains. Repulsive properties could be mapped to three different sites the epidermal growth factor-like repeats, fibronectin type III repeats 4 and 5 and to the alternatively spliced region of the molecule. The responses to these repulsive sites varied according to the cell type. These data are consistent with the interpretation that different cell types express distinct sets of tenascin-C receptors which might regulate cellular responses via distinct second messenger pathways.     

Role of neural cell adhesion molecule and polysialic acid on the neuronal development and regeneration

The results of the study of Enterovirus as viral meningoencephalitis producing agents, carried out from 1990 to 1994, are described, 546 feces samples, 95 cerebrospinal fluids and 1,058 matched sera were studied and obtained from 1,388 patients clinically diagnosed with this disease. Samples for viral isolation were inoculated into two different cellular systems. The highest number of isolation was found in diploid cells from human fibroblast. Antibody determinations were carried out by a neutralization test (micromethod) with 11 Enterovirus antigens (Echo 4, 6, 9, 11 and 30; and Coxsackie B1, 2, 3, 4, 5 and 6) and in epidemic periods with the isolated virus. During the years under study, 2 epidemic outbreaks took place: on caused by Coxsackie A9 (1990-1991) and the other one by Echo 30 (1994). A greater positivity to Echo 6 and 11 was found among the matched sera.     

Beta2 integrin/ICAM-1 adhesion molecule interactions in cutaneous inflammation and tumor promotion

Leptin is a protein that is produced primarily in fat tissue and is thought to be a lipostatic feedback signal for the regulation of body fat stores. The purpose of this study was to determine the behavioral specificity of i.c.v.-administered mouse leptin in rats and to assess the effects on meal patterns. Using a modified two-bottle paradigm we examined the putative aversive response to i.c.v. doses of 1, 5, 7, 10, and 30 microg of mouse leptin. Artificial CSF and intraperitoneal lithium chloride served as negative and positive controls, respectively. Saccharin consumption in all leptin treatments was not significantly different from the negative control. Following a recovery period, rats from the same group were used to assess the effects of a 30-microg i.c.v. dose on cumulative food intake and meal patterns using a computer-based system for acquisition of feeding data. Leptin (i.c.v.) significantly increased intermeal interval and decreased meal size. We, therefore, conclude that mouse leptin, at doses up to 30 microg i.c.v., is not aversive in the rat, and that leptin has a multiphasic effect on meal patterns.     

Carbohydrate-carbohydrate interactions in adhesion

Cell-cell interactions play an important role in the development, maintenance, and pathogenesis of tissues. They are highly dynamic processes which include migration, recognition, signaling, adhesion, and finally attachment. Cells on their pathway to a final location have to pass and interact with their substratum formed of matrix and cell layers. Testing and recognition are important keys for the proper result of tissue formation. They can, however, also lead to diseases when they are misused in pathological situations, by microorganisms or malignant cells, for instance. Carbohydrates, which are the most prominent surface-exposed structures, must play an important role as recognition molecules in such processes. The rich variability of carbohydrate sequences which cell surfaces can present to lectins, adhesion molecules, and other ligands creates a refined pattern of potential attachment sites. The subtle control of the surface presentation density can provide variations in attachment strength. Not only the carbohydrate sequences but also the fact that carbohydrates can be branched while proteins cannot and that the oligosaccharide chains can be attached to the protein backbone in different densities and patterns will create yet more interaction possibilities. Maximal use of the combinatorial richness of carbohydrate molecules would be made when carbohydrate sequences could interact with other carbohydrate sequences. Such interactions have only very rarely been considered for biochemically and biologically relevant situations since they are difficult to measure. A few are known and will be summarized here with the hope that this wealth of possible chemical interactions may be considered more and more by surface cell biochemists when analyzing fine tuning in cellular interactions.     

Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow

We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.     

Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins

The contribution of noncadherin-type, Ca2+-independent cell-cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM-positive transfectants behave like cells with a decreased strength of cell-cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM-cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of alpha- and beta-catenins decreased in cells overexpressing Ep-CAM. While the total beta-catenin content remains unchanged, a reduction in total cellular alpha-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell-cell adhesions diminish, Ep-CAM-mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell-cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell-cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.     

Role of the microtubule cytoskeleton in gravisensing Chara rhizoids

Docosahexaenoic acid and arachidonic acid are deposited in large amounts in the developing central nervous system, and concentrations are particularly high in synaptic plasma membrane and retina ethanolamine phospholipids. Arachidonic acid and docosahexaenoic acid are present in human milk. The precursors linoleic [18:2(n-6)] and alpha-linolenic [18:3(n-3)] acid, but not arachidonic acid or docosahexaenoic acid, are present in formulas. Desaturation and elongation of 18:2(n-6) and 18:3(n-3) to arachidonic acid and docosahexaenoic acid, respectively, depend on the dietary content and ratio of 18:2(n-6) and 18:3(n-3), but appropriate levels and ratios of 18:2(n-6) and 18:2(n-3) for formula are not well defined. The effect of formula with 1 or 4% fatty acids 18:3(n-3) and 16, 30 or 35% fatty acids 18:2(n-6) on synaptic plasma membrane and retina ethanolamine phospholipid fatty acids was therefore studied in piglets, with reference to piglets fed milk. Piglets fed 4% fatty acids 18:3(n-3), but not those fed 1% fatty acids 18:3(n-3), had similar central nervous system docosahexaenoic acid levels but had significantly lower brain weights than piglets fed sow milk. Synaptic plasma membrane and retina arachidonic acid were lower in piglets fed the formulas with 4% rather than 1% fatty acids 18:3(n-3). The dietary 18:3(n-3) content, rather than the 18:2(n-6) to 18: 3(n-3) ratio, seemed more important for deposition of docosahexaenoic acid in brain. However, synaptic plasma membrane and retina docosahexaenoic acid levels were further reduced in piglets fed 1% fatty acids 18:3(n-3) (0.4% energy) with 30% rather than with 16% fatty acids 18:2(n-6). The need for further study of upper limits of dietary 18:3(n-3) during development is suggested.     

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis.     

Increased neural cell adhesion molecule in the CSF of patients with mood disorder

Neural cell adhesion molecule (N-CAM) is involved in cell-cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N-CAM in the CSF. Soluble human N-CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N-CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N-CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N-CAM immunoreactive proteins, primarily the 120-kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N-CAM concentrations. It is possible that the 120-kDa N-CAM band present in the CSF is derived from CNS cells as a secreted soluble N-CAM isoform. Our results suggest the possibility of latent state-related disturbances in N-CAM cellular function, i.e., residue from a previous episode, or abnormal N-CAM turnover in the CNS of patients with mood disorder.     

Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.     

Role of the endothelial cytoskeleton in blood-brain-barrier permeability to protein

The role of the cytoskeletal elements, microfilaments and microtubules in cerebral endothelial permeability to protein during steady states was investigated by studies of cerebrovascular permeability to horseradish peroxidase (HRP) in rats pretreated with cytochalasin B or colchicine, agents known to disrupt microfilaments and microtubules, respectively. In addition, the effect of colchicine pretreatment on the alterations in cerebrovascular permeability that occur in acute hypertension were studied. Rats infused with cytochalasin B showed increased cerebrovascular permeability to HRP in multifocal areas of the ipsilateral hemisphere. Most of the permeable vessels were arterioles; however, capillaries and venules also showed increased permeability. Ultrastructural studies of permeable vessels showed HRP in all layers of vessel walls and in endothelial and smooth muscle cell pinocytotic vesicles, which were increased in number. Although segments of interendothelial spaces were labeled by tracer, continuous labeling of interendothelial spaces from the luminal to the abluminal end was not seen and tight junctions were not disrupted. Normotensive rats pretreated with colchicine showed no alteration in cerebrovascular permeability to HRP. Colchicine pretreatment attenuated the permeability alterations that were observed in acutely hypertensive rats. This study demonstrates that integrity of endothelial actin filaments is important for maintenance of the blood-brain barrier to protein during steady states since increased permeability occurred in the presence of an actin disrupting agent. The microtubular network had no demonstrable role during steady states; however, disruption of the microtubular network had a protective effect and prevented the development of alterations in permeability to protein in acute hypertension.     

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.