Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia - PubMed (original) (raw)

. 1998 Sep 1;95(18):10896-901.

doi: 10.1073/pnas.95.18.10896.

Y Jiang, S Chen, Y Xia, D Maciejewski, R K McNamara, W J Streit, M N Salafranca, S Adhikari, D A Thompson, P Botti, K B Bacon, L Feng

Affiliations

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

J K Harrison et al. Proc Natl Acad Sci U S A. 1998.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Localization of fractalkine mRNA in rat tissues and to neurons in the rat brain. (A) RPA of fractalkine mRNA in various tissues of adult rat. (B_–_I) Localization of fractalkine mRNA in the rat brain by using ISH analysis. Hybridization analysis using “anti-sense” (B, D, F, and H) and “sense” (C, E, G, and I) [35S]-UTP-labeled riboprobes were generated from rat fractalkine cDNA. Hybridized horizontal sections (B and C) were exposed to film for 18 hours (Olf, olfactory bulb; ctx, cortex; cc, corpus collosum; CPu, caudate putamen; Th, thalamus; ca3, ca3 region of the hippocampus; gc, granule cell layer of the hippocampus; CG, central gray; cbm, cerebellum) Hybridization signals (silver grains) reside principally over neurons in the granule cell layer of hippocampus (D), cortex (F), and thalamus (H).

Figure 2

Figure 2

Identification of RBS11 as rat CX3CR1: binding of fractalkine and elevation of intracellular calcium by fractalkine in CHO-RBS11 cells. (A) Whole cell radioligand binding analysis of CHO-CX3CR1-expressing cells. Competition binding of 1 nM labeled peptide with the chemokine domain of rat fractalkine (□), the chemokine domain of human fractalkine (▴), rat monocyte chemoattractant protein 1 (■), rat MIP-1α (♦), rat regulated on activation, normal T-cell expressed and secreted (RANTES) (•), rat stromal cell-derived fator 1 (▾), and murine MIP-2 (▵). Wild-type CHO cells did not display any significant specific binding of 125I-fractalkine (▿). Data were calculated as the fraction of specific binding in the presence of the specified concentration of unlabeled chemokine peptides (Counts per minute bound per 3 million cells: total, 7945 ± 428; nonspecific: 2078 ± 231, n = 7). The results (from triplicate determinations) are presented as the mean ± SEM of three experiments (rat fractalkine) or four experiments (human fractalkine) or the mean of two experiments (all other chemokines). The calculated IC50s for inhibition of labeled peptide binding by the chemokine domains of either rat or human fractalkine are 2.2 nM (95% confidence interval: 0.9–5.3 nM) and 2.9 nM (95% confidence interval: 1.9–4.5 nM), respectively. (B) Rat CX3CR1 protein expression in CHO-CX3CR1-expressing (filled trace) and wild-type CHO (open trace) cells as determined by FACS analysis using anti-RBS11 polyclonal antibody. Neither preimmune rabbit sera nor immune sera preadsorbed with immunogen showed any increased reactivity to the CHO-CX3CR1-expressing cells (data not shown). (C) Representative traces of CHO-CX3CR1 expressing cells in response to application of either 293T cells transiently expressing rat (i, ii) or human fractalkine (iv, v) or the chemokine domain of rat (iii) or human (vi) fractalkine. Wild-type fractalkines (i, iv) or mutant fractalkines (ii, v) in which Arg-Arg residues adjacent to the TM domain were changed to Ser-Ala were expressed transiently in 293T cells. Lower traces depict application of mock transfected 293T cells (vii) to CHO-CX3CR1-expressing cells or addition of the chemokine domain of rat fractalkine (viii) or the wild-type membrane-tethered form of rat fractalkine (ix) to wild-type CHO cells. (D) Concentration-dependent effect of human fractalkine (chemokine domain) on intracellular calcium levels in CHO-CX3CR1-expressing cells. Data are plotted as the fraction of the maximal response seen in the presence of a high dose of fractalkine. Values are the mean of at least two determinations at each concentration. The calculated EC50 from these experiments is 3 nM.

Figure 3

Figure 3

Expression of CX3CR1 in primary cultures of rat microglia; fractalkine dependent responses mediated by CX3CR1. (A) RPA of CX3CR1 in peritoneal macrophage, cultured microglia, and brain by using RBS11 and L32 riboprobes. (B) Cell surface expression of CX3CR1 in cultured microglia assessed by FACS analysis by using anti-CX3CR1 antibody and reaction of preimmune (open trace) and immune sera (filled trace) to cultured microglia. Immune sera preadsorbed with immunogen did not react with the microglia (data not shown). (C) Chemotaxis of microglia in response to recombinant chemokine domain of fractalkine (left). Each point represents the mean ± SEM cell number in five high-power (×1,000) fields (hpf) from three experiments performed in quadruplicate. Inhibition of fractalkine-mediated chemotaxis by anti-RBS11 antibody is shown on the right. Unshaded histogram represents unstimulated migration in the presence of anti-RBS11 antibody (1:100 dilution). Black histograms represent dilution-related inhibition of fractalkine-induced microglial cell migration by using anti-RBS11 antibody. Hatched histograms represent MIP-1α-mediated microglial cell migration in the absence (0) or presence (1:100) of anti-RBS11 antibody. Each histogram represents the mean ± SEM cell number per five hpfs from three experiments performed in quadruplicate. (D) Effect of fractalkine forms on intracellular calcium levels in primary cultures of rat microglia. i–iii Show dose-dependent increases in intracellular calcium mediated by rat fractalkine expressed as a membrane-bound form in 293T cells. The lower trace of iii shows the effect of anti-RBS11 antibody (1:100). iv shows 293T-membrane-bound human fractalkine ± anti-RBS11 antibody (1:100). v shows wild-type 293T, and vi shows soluble rat fractalkine (50 nM). Representative traces from n = 3 experiments are depicted.

Figure 4

Figure 4

ISH analysis of CX3CR1 expression in the rat facial nucleus after motor neuron axotomy. ISH was performed on rat brainstem sections from animals killed at 1 (A), 4 (B), 7 (C), 14 (D), and 21 (E) days after nerve transection by using [35S]UTP-labeled riboprobe. F depicts a quantitative analysis of the hybridization signal on the operated (•) and contralateral (○) sides. Results are presented as the mean ± SEM of data derived from at least three sections from three different animals. The hybridization signal was specific for CX3CR1 because no increase in signal was observed by using a sense riboprobe (H) when compared with a corresponding anti-sense riboprobe hybridized-section (G). Hybridization signals (silver grains) from anti-sense [33P]UTP-labeled riboprobe hybridized-sections are found principally over lectin (GSA-I-B4)-staining microglia in the neuropil of the unoperated facial motor nucleus (J) or lesioned facial motor nucleus (arrowhead in I) whereas several CX3CR1 mRNA-containing microglial cells are found perineuronally (arrows in I) surrounding axotomized motor neurons (N, motor neuron cell body). (K) Sense [33P]UTP-labeled riboprobe-hybridized section showing only a few scattered silver grains.

Figure 5

Figure 5

Fractalkine expression in the rat facial nucleus after motor neuron axotomy. ISH was performed on rat brainstem sections from animals killed 4 days after nerve transection. Shown is hybridization analysis of the contralateral (A and B) and ipsilateral (C and D) facial motor nuclei by using anti-sense (A and C) and sense (B and D) [33P]UTP-labeled riboprobes generated from rat fractalkine cDNA. Hybridization signals are found principally over the motor neuron cell bodies. (E) Western blot analysis of recombinant (bacterial- and mammalian cell-expressed) fractalkines, as well as protein extracts from the facial motor nucleus from the control and lesioned sides after peripheral nerve transection. Fractalkine forms were subject to SDS/PAGE and include bacterial-expressed fusion protein (10 ng) containing the chemokine module of rat fractalkine (lane 1), and cell lysates prepared from COS cells transfected with either antisense (lane 2) or sense (lanes 3–5) constructs containing membrane-anchored forms of rat fractalkine. In addition, protein extracts (10 μg/lane) from the facial motor nucleus from the control (lanes 6, 8, 10, 12, and 14) and lesioned (lanes 7, 9, 11, 13, and 15) sides 1 (lanes 6 and 7), 4 (lanes 8 and 9), 7 (lanes 10 and 11), 14 (lanes 12 and 13), and 21 (lanes 14 and 15) days after axotomy were subject to SDS/PAGE. Immunoblot analysis was performed by using anti-rat fractalkine rabbit sera (lanes 1–3, 6–15), preimmune sera (lane 4), or immune sera preadsorbed with recombinant rat fractalkine, chemokine domain (lane 5).

References

    1. Baggiolini M, Dewald B, Moser B. Adv Immunol. 1994;55:97–179. - PubMed
    1. Baggiolini M, Dewald B, Moser B. Annu Rev Immunol. 1997;15:675–705. - PubMed
    1. Bazan J F, Bacon K B, Hardiman G, Wang W, Soo K, Rossi D, Greaves D R, Zlotnik A, Schall T J. Nature (London) 1997;385:640–644. - PubMed
    1. Pan Y, Lloyd C, Zhou H, Dolich S, Deeds J, Gonzalo J A, Vath J, Gosselin M, Ma J, Dussault B, et al. Nature (London) 1997;387:611–617. - PubMed
    1. He J, Chen Y, Farzan M, Choe H, Ohagen A, Gartner S, Busciglio J, Yang X, Hofmann W, Newman W, et al. Nature (London) 1997;385:645–649. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources