Research reportVIP and d-ala-peptide T-amide release chemokines which prevent HIV-1 GP120-induced neuronal death
Introduction
Cellular interactions which occur between neurons and glia are fundamental to both the function and development of the nervous system [2]. Peptides are among the diffusible neuronal signals that have been shown to interact with receptors on astrocytes. Recent studies have shown that substance P [38] and somatostatin [25] can modulate the release of cytokines, while vasoactive intestinal peptide (VIP) [10], angiotensin and bradykinin [49] have secretagogue activity on astrocytes. In the context of neuronal survival regulation, we have studied the role of VIP as a signal between neurons and glia. The survival-promoting action of VIP [6] has implications both for our understanding of development and the protective mechanisms that are operative during nerve injury or degeneration.
VIP is neuroprotective against clinically significant neurotoxins [22], including the envelope protein, gp120, of the human immunodeficiency virus [11]. Gp120 has been suggested to be a pathogenic factor in neuroAIDS, which features paradoxically low levels of virus in the brain 1, 54. The neurotoxic action of gp120 is potent and requires the presence of glial cells [9] and macrophages [36], which may then secrete neurotoxic products or cytokines 18, 21, 55. However, direct toxic effects of gp120 on cultured hippocampal neurons have been reported [39]. Gp120 was first described to be neurotoxic in hippocampal cultures derived from embryonic mice [11]. Although rodents are not vulnerable to HIV infectivity, the envelope is neurotoxic to neurons from rats 4, 37 and humans 35, 52 through an apoptotic mechanism. Because the envelope protein is key to viral entry and is neurotoxic, search for peptide fragments from gp120 have been investigated for their ability to prevent gp120 binding and/or to prevent gp120-mediated toxicity. Peptides (5–8 amino acids) 41, 46 derived from the gp120 V2 region that are homologous to VIP are also inhibitors of gp120 neurotoxicity 5, 11. A derivative of one of these peptides, DAPTA, has been shown to prevent gp120-induced delayed behavioral milestones, abnormal neuronal dearborization and endocrine abnormalities in rats treated with gp120 30, 40. The protective actions of VIP are mediated through its secretagogue action on astroglia. Among the substances recognized to be secreted after VIP-stimulation of astroglia are interleukin-1α (IL-1α) and β [8], IL-6 [26], protease nexin-1 [19] and activity-dependent neurotrophic factor (ADNF) [7]. ADNF also protects neurons from cell death associated with gp120 [7].
Recent studies have shown that gp120 can use a number of chemokine co-receptors, in conjunction with CD4 3, 13, 15, 34, 50 or alone [43], to allow viral entry of target cells. Chemokine receptors, first characterized on activated immune cells, also have been localized on cerebellar neuronal processes [31], differentiated human neuronal lines [31], and both microglia and astrocytes in human brain cultures [27]. Recent work with the hNT human neuronal cell line indicated that gp120 elicited apoptotic responses through a chemokine receptor (CXCR4) [29]. Orthologs of chemokine receptors (CCR2, CCR3, CCR5 and CXCR4) have been detected in rat spleen, lung, kidney, thymus and macrophages 20, 32. Chemokine receptors have also been reported in primary cultures of rat astrocytes [32]. Recent studies in rat hippocampal cultures have demonstrated numerous chemokine receptors including: CCR1, CCR4, CCR5, CCR9/10, CXCR2, CXCR4 and CX3CR1 [39]. The impetus to test for VIP-mediated regulation of chemokine release resides in observations that VIP prevents neuronal cell death associated with gp120 treatment of murine hippocampal cultures [11]; and chemokines have been shown to block gp120 interactions with chemokine receptors [14]. Importantly, recent studies have shown that several chemokines were able to prevent gp120-induced apoptosis in rat hippocampal cultures [39]. In the present study, VIP is shown to release several β-chemokines and chemokines are shown to act as neuronal survival factors as well as neuroprotectants against gp120-mediated neuronal cell killing.
Section snippets
Materials
Chemokines (stromal derived factor-1 α, eotaxin, RANTES, MIP-1α, MIP-1β) were obtained from PeproTech (Rocky Point, NJ). The gp120 isolates CM, LAV, and IIIB were acquired from the NIH AIDS Research and Reference Reagent Program (Rockville, MD). The SF2 isolate was the gift of Chiron (Emmeryville, CA). The MN isolate was from ABI (Rockville, MD) and RF was a gift from Dr. P. Nara (NCI, NIH). All of the gp120 variants were purified (>95% homogeneous) and previously tested for neurotoxicity with
VIP- and DAPTA-mediated secretion of chemokines
To determine VIP-mediated chemokine secretion, cultures of rat cerebral cortical astrocytes were treated for 90 min with VIP and the release of chemokines was measured in the medium. A concentration-dependent increase in MIP-1α and RANTES was observed (Fig. 1). The secretagogue actions for VIP were potent, with an EC50 of 10 pM observed for the secretion of MIP-1α. The amount of MIP-1α released was attenuated at VIP concentrations≥1 nM. The maximally effective concentration of VIP (0.1 nM)
Discussion
The present study has emphasized five discoveries: (1) that nM concentrations of VIP and DAPTA can cause the release of two beta chemokines: MIP-1α and RANTES; (2) that a MIP-1α-like molecule is necessary for the survival of a subpopulation of neurons in cerebral cortical cultures; (3) that the neuroprotective actions of VIP against the neuronal cell killing effect of gp120 is due, in part, to the release of MIP-1α; (4) some chemokines are potent inhibitors of gp120-mediated neurotoxicity; and
Acknowledgements
This work was supported in part by Advanced Immunity, Stoney Brook, NY.
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