Similarities and differences in RANTES- and (AOP)-RANTES-triggered signals: implications for chemotaxis

Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.     

Molecular cloning and functional characterization of Cynomolgus monkey (Macaca fascicularis) CC chemokine receptor,CCR3

We have cloned and performed the first functional characterization of the chemokine receptor, CCR3, of Cynomolgus monkey (Macaca fascicularis). The deduced amino acid sequence of the cloned Cynomolgus CCR3 was found to be more similar to that of a previously-reported Rhesus (Macaca mulatta) CCR3 (99.4%) than that of a reported Cynomolgus CCR3 (98.0%). Stably-transfected Cynomolgus CCR3 bound human eotaxin (CCL11) with similar kinetics (Kd 240 pM) and was responsive to human CCR3 ligands (eotaxin [CCL11], eotaxin-2 [CCL24], and MCP4 [CCL13]) in Ca(2+) mobilization and chemotaxis assays, thus provides a useful alternative species model system for the analysis of modulators of eotaxin--CCR3 induced signaling and activation.     

Cytokine-Induced Inflammation in the Central Nervous System Revisited

Cytokines play an essential role as mediators of the immune response. They usually function as part of a network of interactive signals that either activate, enhance, or inhibit the ensuing reaction. An important contribution of this cytokine cascade is the induction of an inflammatory response that recruits and activates subsets of leukocytes that function as effector cells in the response to the sensitizing antigen. Proinflammatory cytokines activate endothelial cells (EC) to express adhesion molecules and induce the release of members of the chemokine family, thus focusing and directing the inflammatory response to sites of antigen recognition. However, the vasculature of the central nervous system (CNS) is highly specialized and restricts the access of components of the immune system to the CNS compartment. In this review, we address the question as to whether endothelial cells in the CNS respond differently to specific cytokines known to induce either a proinflammatory effect or a regulatory effect in systemic vascular beds.     

Contribution of cytokines on the suppression of lung metastasis

Weekly injection of a protein-bound polysaccharide PSK in mice with Lewis Lung Cancer (LLC) significantly decreased the number of lung metastatic foci concomitant with enhancement of cytostatic activity in the bronchoalveolar lavage (BAL) cells. These effects were more marked when the agent was given intratracheally, inducing a larger number of pulmonary macrophages, lymphocytes and neutrophils concomitant with increases in BAL tumor necrosis factor- (TNF-), mouse inflammatory protein- (MIP-1), mouse inflammatory protein- (MIP-1), interleukin-1 (IL-1) and interleukin-6 (IL-6), but not interleukin-2(IL-2) and interleukin-4 (IL-4). Pre-treatment with anti TNF- antibody reduced these effects. The time course and production of PSK-induced cytokines were similar between the tumor-bearing mice and control mice. BAL neutrophils in mice with LLC showed a tendency toward acceleration of O2- production compared with circulating neutrophils. Pulmonary macrophage phagocytosis was also significantly higher in the LLC mice.These results suggest that enhancement of cytostasis appears to be induced by activation and/or improvement of function in inflammatory and immune cells through cytokines under immunomodulator treatment in lung metastasis, possibly via a TNF--dependent mechanism.     

Analysis of hydrophobicity in the alpha and beta chemokine families and its relevance to dimerization.

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.     

Specific induction of macrophage inflammatory protein 1-alpha in glial cells of Sandhoff disease model mice associated with accumulation of N-acetylhexosaminyl glycoconjugates

Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), due to a primary defect of the beta-subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylhexosamine residues at their non-reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). We demonstrated that chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 (monocyte chemotactic protein-1), SLC (secondary lymphoid-tissue chemokine), fractalkine and SDF-1 (stromal derived factor-1), were detected. Significant up-regulation of MIP-1alpha mRNA and protein in the above-mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP-1alpha-immunoreactivity (IR) in the above-mentioned brain regions of SD mice was co-localized in Iba1-IR-positive microglial cells and partly in glial fibrillary acidic protein (GFAP)-IR-positive astrocytes, in which marked accumulation of N-acetylglucosaminyl (GlcNAc)-oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP-1alpha-IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP-1alpha might coincide with the accumulation of GlcNAc-oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.     

Enhanced expression and shedding of the transmembrane chemokine CXCL16 by reactive astrocytes and glioma cells

The transmembrane chemokine CXCL16 is expressed by dendritic and vascular cells and mediates chemotaxis and adhesion of activated T cells via the chemokine receptor CXCR6/Bonzo. Here we describe the expression and shedding of this chemokine by glioma cells in situ and in vitro. By quantitative RT-PCR and immunohistochemistry, we show that CXCL16 is highly expressed in human gliomas, while expression in normal brain is low and mainly restricted to brain vascular endothelial cells. In cultivated human glioma cells as well as in activated mouse astroglial cells, CXCL16 mRNA and protein is constitutively expressed and further up-regulated by tumour necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma). CXCL16 is continuously released from glial cells by proteolytic cleavage which is rapidly enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). As shown by inhibitor studies, two distinct members of the disintegrin-like metalloproteinase family ADAM10 and 17 are involved in the constitutive and PMA-induced shedding of glial CXCL16. In addition to the chemokine, its receptor CXCR6 could be detected by quantitative RT-PCR in human glioma tissue, cultivated murine astrocytes and at a lower level in microglial cells. Functionally, recombinant soluble CXCL16 enhanced proliferation of CXCR6-positive murine astroglial and microglial cells. Thus, the transmembrane chemokine CXCL16 is expressed in the brain by malignant and inflamed astroglial cells, shed to a soluble form and targets not only activated T cells but also glial cells themselves.     

Constitutive expression of CCR2 chemokine receptor and inhibition by MCP-1/CCL2 of GABA-induced currents in spinal cord neurones

In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT-PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABA(A)-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.     

Sensing plasma membrane pore formation induces chemokine production in survivors of regulated necrosis

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