RANTES release contributes to the protective action of PACAP38 against sodium nitroprusside in cortical neurons

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP), a promising neuroprotective peptide, plays an important role during development of the nervous system and in regeneration after injury. PACAP directly promotes survival via multiple signaling systems in neurons. This neuropeptide also has immuno-modulatory properties and can regulate the expression of various inflammatory mediators such as chemokines in nonneuronal cells. Chemokines and their G protein-coupled receptors are widely distributed in the brain, suggesting important functions for these inflammatory proteins in the CNS. The ability of brain endothelial cells and glia to release chemokines has been well documented, whether neurons are also a source for these mediators is unclear. The objective of this study is to determine whether PACAP38 affects expression of regulated on activation normal T expressed and secreted (RANTES) and macrophage inflammatory protein 1-alpha (MIP-1α) in cultured neurons and if these chemokines contribute to the neuroprotective effect of PACAP38. The data show that incubation of neuronal cultures with both PACAP38 and sodium nitroprusside (SNP) reduces the neuronal cell death evoked by SNP alone. PACAP38 dose-dependently increases immunodetectable levels of both RANTES and MIP-1α released in the media by cultured neurons. Co-treatment with a neutralizing antibody to RANTES decreases the PACAP38-mediated protection against SNP. Although RANTES treatment of neurons increased MIP-1α levels in the media and MIP-1α supports neuronal survival in unstressed cultures, MIP-1α does not protect neurons from SNP-induced toxicity. Furthermore, co-treatment with a MIP-1α neutralizing antibody did not affect PACAP38-induced protection against SNP. These results show that the protective effect of PACAP38 on cultured neurons is mediated, in part, by release of RANTES. The ability of PACAP to directly enhance neuronal survival through multiple intracellular signaling pathways as well as via the release of neuroprotective mediators such as RANTES highlights its utility as a potential therapeutic agent for the treatment of neurodegenerative diseases.

Introduction

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) superfamily, and exists in two amidated forms as PACAP38 (38-amino acid residues) and PACAP27 derived from the same precursor (Arimura, 1998; Zhou et al., 2002). The multifunctional PACAP38 demonstrates significant neuroprotection both in vitro and in vivo (Waschek, 2002; Dejda et al., 2005). For example, PACAP38 is able to prevent apoptosis in neuronal cultures exposed to various neurotoxins including amyloid beta (Aβ), hydrogen peroxide and glutamate (Onoue et al., 2002; Vaudry et al., 2002; Shintani et al., 2005). In vivo, PACAP38 administered 1 h after middle cerebral artery occlusion significantly reduces infarct volume (Chen et al., 2006). PACAP exerts its biological action via three different receptors belonging to the family of Gprotein-coupled receptors: PAC1, VPAC1 and VPAC2 (Zhou et al., 2002). All three PACAP receptors are expressed and functional in the rodent brain (Cauvin et al. 1991; D’Agata et al., 1996). Dejda et al. (2008) document that activation of the PAC-1 receptor leads to inhibition of caspase 3 in neurons.

In a recent study we show that multiple intracellular targets contribute to PACAP38-mediated neuroprotection. PACAP38 down-regulates sodium nitroprusside (SNP) -induced cell cycle protein (cyclin E) expression and up-regulates the cyclin-dependent kinase inhibitor p57 as well as the anti-apoptotic protein Bcl-2 in cultured neurons. Also, thrombin-stimulated cell cycle protein (cdk4) expression is decreased by PACAP38 while PACAP38 inhibits thrombin-mediated reduction of p57. Finally, the increase in caspase 3 activity evoked by both SNP and thrombin is decreased by PACAP38 (Sanchez et al., 2009).

Chemokines and their G protein-coupled receptors are widely distributed in the brain, suggesting important functions for this inflammatory superfamily in the CNS (Rostene and Buckingham, 2007). The expression of regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein 1-alpha (MIP-1α) and other chemokines has been linked to pathological changes found in neurodegenerative diseases (Huang et al., 2000; McGeer et al., 2006; Sastre et al., 2006; Heneka and O'Banion, 2007). However, the effects of chemokines in the brain are complex, as both neurotoxic and neuroprotective properties have been documented for these proteins. Exposure of neurons in vitro to pre-aggregated Aβ1–42 results in increased expression of MIP-1α (Lue et al., 2001). An increase in this chemokine is also evoked in vivo by the Parkinson’s disease-producing neurotoxin MPTP (Pattarini et al., 2007). In Down’s syndrome patients with early Alzheimer’s-like dementia, there is a correlation between cognitive dysfunction and MIP-1α level (Carta et al., 2002). In contrast, a study using cDNA microarrays documents a large number of RANTES-responsive genes in cultured neurons that appear to be involved in neuronal survival and differentiation (Valerio et al., 2004). RANTES protects mixed cultures of neurons and astrocytes from HIV-protein or NMDA-induced apoptosis (Eugenin et al., 2003). Also, we recently reported that treatment of primary cortical neuronal cultures with RANTES enhances neuronal survival and that exposure of neuronal cultures to RANTES before treatment with a neurotoxic agent causes a significant reduction in neuronal cell death (Tripathy et al., 2008).

PACAP38, in addition to its direct cellular effects, is a neuropeptide with potent immunomodulatory properties including regulation of various inflammatory mediators such as chemokines (Pozo et al., 2002; Abad et al., 2006; Fahrenkrug, 2006; Gomariz et al., 2006). However, whether inflammatory proteins play a role in PACAP38’s neuroprotective effects is unknown. The objective of this study is to determine whether PACAP38 affects expression of RANTES and MIP-1α in cultured neurons and if these chemokines contribute to the neuroprotective effect of PACAP38.

Materials and Methods

Materials

SNP, PACAP38, and 5-fluoro-2’-deoxyuridine were purchased from Sigma (St. Louis, MO, USA). Recombinant rat RANTES was obtained from BioSource (Camarillo, CA) and recombinant MIP-1α was purchased from AbCam (Cambridge, MA). Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). Antibodies for RANTES (AF278-NA, ab9783) and MIP-1α (ab9781) were obtained from R&D Systems and AbCam (Cambridge, MA), respectively. The MTT-based assay kit (Cell Titer 96 One Aqueous solution cell proliferation assay) was obtained from Promega (Madison, WI).

Preparation of cerebral cortical cell cultures

Primary rat cerebral cortical cultures were prepared from cortices isolated from 18-day gestation fetuses, as previously described (Grammas et al., 1999; Reimann-Philipp et al., 2001). Neurons were seeded at a density of 3–5 × 10⁵ cells per ml on 6-well poly-L-lysine coated plates. The medium was changed at day 2 to Neurobasal medium containing B-27 supplement, antibiotic/antimycotic, glutamine (0.5 mM) and 5-fluoro-2’-deoxyuridine (20 µg /ml) to inhibit proliferation of glial cells. On day 5, fresh medium without 5-fluoro-2’-deoxyuridine was added. The primary cerebral cortical cultures utilized were enriched for neurons (>95%). The neuronal identity of the cultures was confirmed using immunofluroescent labeling for beta-3 tubulin, a neuron-specific protein (Promega) and glial fibrillary acidic protein (Promega). Neuronal cultures were used for experiments after 8–9 days in culture.

Treatment of cerebral cortical cell culture

For the neutralizing antibody experiments, neurons were treated with or without 1 mM SNP, PACAP38 (100 nM), 10 µg/ml of neutralizing proteins, or normal IgG (10 µg/ml) for 4 h and cell survival measured. To measure RANTES-induced MIP-1α release, cortical neurons were incubated with RANTES (25, 50, 100, 300 and 500 ng/ml) for 24 h and MIP-1α detected by ELISA.

Measurement of cell survival by MTT assay

Cells were incubated with the MTT reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. This tetrazolium salt is converted to formazan in live cells and quantified by a colorimetric assay. Cells were incubated with the MTT reagent (1:40 dilution) for 10 min at 37°C. The formazan product was read at 490 nm. In each experiment, the number of control cells, i.e. viable cells not exposed to any treatment, was defined as 100%.

Detection of RANTES and MIP-1α by ELISA

Indirect ELISA was used to detect RANTES and MIP-1α released in the supernatant using our previously published protocol (Sanchez et al., 2008; Tripathy et al., 2008). In each experiment, standard curves were generated using 0 – 100 ng of rat recombinant proteins (RANTES or MIP-1α) and levels of RANTES or MIP-1α in samples were determined by extrapolation from standard curves. Supernatants were coated onto 96-well immulon 2HB (Fisher Scientific, Pittsburgh, PA) flat bottom plates with sodium bicarbonate buffer (0.1 M) and incubated overnight at 4°C. The plates were blocked using 1% bovine serum albumin solution and incubated at 37°C for 45 min. After washing two times, 200 µl of primary antibodies (diluted 1:1000 in sodium bicarbonate buffer) were added and incubated overnight at 4°C. Plates were washed extensively to remove unbound antibody and then treated with 200 µl of goat anti-rabbit IgG coupled with horseradish peroxidase (Bio-Rad, 1/1000 dilution) and incubated for 45 min at 37°C, in the dark. The reaction was developed by adding 200 µl/well of ophenylene diamine H₂O₂ (Pierce, Chemicon, CA) for 20 min. Optical density was measured at 450 nm using a microplate ELISA reader (Bio-Rad).

Statistical Analysis

Prism version 4.0 software (GraphPad Inc., San Diego CA) was used for graphical presentation and statistical analysis. Statistical analysis used included student’s t-tests and one-way ANOVA followed by Newman-Keuls post hoc multiple comparison tests to compare the different treatment groups. A significant difference was defined as p < 0.05. Data are presented as mean ± SEM of at least 3 independent experiments.

Results

PACAP38 protects neuronal cultures from SNP-induced cell death

Treatment of primary neuronal cultures with 1mM SNP for 4 h caused significant (p<0.001) cell death (Fig. 1). Incubation of neurons with both SNP and PACAP38 (100 nM) significantly (p<0.001) increased neuronal cell survival from 68 to 90% compared to SNP alone. Exposure of neuronal cultures to PACAP38 alone did not affect cell survival (Fig. 1).

Figure 1.

Cortical neurons (day 8) were treated with medium (control), SNP (1 mM), PACAP38 (100 nM), or SNP+PACAP38 for 4 h and cell survival quantitated by MTT assay. The number of control cells was defined as 100%. Data are mean ± SEM of 4 experiments performed in triplicate.

^ap< 0.001 vs. control; ^bp<0.001 vs. SNP.

PACAP38 induces expression of chemokines RANTES and MIP-1α in neuronal cultures

Supernatants from cerebral cortical cultures were assayed by ELISA for the presence of the chemokines RANTES and MIP-1α. There were detectable levels of both chemokines in untreated neuronal cultures (Table 1). Exposure of cultures to increasing concentrations of PACAP38 for 4 h resulted in an increase in RANTES that was significant (p<0.05) at 25 nM and maximal (p<0.01) at 100 nM PACAP. Treatment of with PACAP38 (50–100 nM) also significantly (p<0.01) increased MIP-1α levels (Table 1).

Table 1.

Neurons treated with PACAP38 release RANTES and MIP-1α.

PACAP38 (nM)	RANTES (ng/ml)	MIP1-α (ng/ml)
0	2.66 ± 0.67	1.23 ± 0.12
25	3.99 ± 0.23*	1.24 ± 0.12
50	4.13 ± 0.44*	1.71 ± 0.18*
100	4.15 ± 0.18**	1.98 ± 0.15**

Cortical cultures were incubated with increasing concentrations of PACAP38 for 4 h. RANTES and MIP -1α released into the supernatant were detected by ELISA. Data are mean ± SEM of 2 experiments performed in triplicate.

^*p< 0.05 vs. control

^**p< 0.01vs. control.

Blocking RANTES reduces the neuroprotective effect of PACAP38

Examination of neuronal cultures under basal, unstressed conditions showed that exposure of cells to neutralizing antibody to RANTES significantly (p<0.001) reduced cell survival compared to untreated controls or to cells treated with PACAP (100 nM) (Fig. 2). Exposure of neuronal cultures to PACAP38 plus control IgG did not affect cell survival (data not shown). Treatment of neuronal cultures with a neutralizing antibody to MIP-1α or to antibody plus PACAP38 also caused a significant (p<0.001) reduction in cell survival (Fig. 2).

Figure 2.

Eight day old primary cortical neurons were treated with medium (control), PACAP38 (100 nM), RANTES neutralizing antibody (10 µg/ml), MIP-1α neutralizing antibody (10µg/ml), PACAP38 + RANTES antibody or PACAP38 + MIP-1α antibody for 4 h. Cell survival was determined by MTT assay. Data are mean ± SEM of 3 experiments performed in triplicate.

***p< 0.001 vs. control; ^a p<0.01 vs. RANTES antibody; ^b p<0.01 vs. MIP-1α antibody.

Treatment of neuronal cultures with 1 mM SNP for 4 h significantly (p<0.001) reduced cell survival compared to untreated controls (Fig. 3). This decrease in cell viability was not further affected by addition of a neutralizing antibody to RANTES. In contrast, the ability of PACAP38 to blunt the neurotoxic effect of SNP on neuronal survival was significantly (p<0.001) inhibited when cells were incubated with both PACAP38 and the RANTES antibody (Fig. 3). Addition of a neutralizing antibody to MIP-1α slightly decreased cell death induced by SNP, but showed no significant effect on PACAP38-mediated protection of SNP-treated neurons (Fig. 3).

Figure 3.

Eight day old primary cortical neurons were treated with medium (control), SNP (1 mM), SNP+ PACAP38 (100 nM), SNP + RANTES antibody (10 µ/ml), SNP + PACAP38 + RANTES antibody, SNP + MIP-1α antibody or SNP + PACAP38 + MIP-1α antibody for 4 h. Cell survival was quantitated by MTT assay. Data are mean ± SEM of 3 experiments performed in triplicate.

#p<0.001 vs. control; ***p< 0.001 vs SNP + PACAP38; **p<0.01 vs. SNP + PACAP38; *p< 0.05 vs. SNP + PACAP38; ^a p<0.01 vs. SNP+RANTES antibody; ^b p<0.05 vs. SNP.

RANTES induces MIP-1α expression in cerebral cortical cultures

Cerebral cortical cultures were incubated with increasing concentrations of RANTES (25 – 500 ng/ml) for 24 h and culture supernatants collected and analyzed by ELISA for MIP-1α. Incubation of neuronal cultures with RANTES resulted in a dose-dependent increase in the release of MIP-1α (Fig. 4). This increase was significant over control (p<0.05) at 50 ng/ml RANTES and highly significant (p<0.001) at 300 ng/ml.

Figure 4.

Cerebral cortical neurons (day 8) were incubated RANTES (25 – 500 ng/ml) for 24 h and MIP-1α released in the media determined by ELISA. Data are mean ± SEM of 3 experiments performed in triplicate.

*p< 0.05 vs. control; **p< 0.01 vs. control; ***p< 0.001 vs. control

Discussion

PACAP, a promising neuroprotective peptide, plays an important role during the development of the nervous system and in regeneration after injury (Fahrenkrug, 2006; Shioda et al., 2006). In vivo, the protective effects of PACAP have been shown in various models of CNS injury and disease, including cerebral ischemia, Parkinson's disease, and trauma (Somogyvari-Vigh and Reglodi, 2004). The upregulation of PACAP following nerve injury suggests that endogenous PACAP plays a regulatory role in the post-traumatic recovery of the nervous system. Despite considerable interest in PACAP38 as a neuroprotective factor, both in vitro and in vivo, the mechanisms that contribute to neuroprotection are poorly understood. In a recent study we documented that multiple intracellular targets in neurons contribute to PACAP38-mediated protection including cell cycle proteins such as cyclin E, cyclin dependent kinase 4, and cyclin kinase inhibitor p57 as well as apoptotic proteins (Bcl-2) and proteases (caspase 3) (Sanchez et al., 2009). PACAP has been demonstrated to decrease neuronal cell death by suppressing cytochrome c release (Ohtaki et al., 2006). Activation of the PACAP specific receptor PAC1R and downstream phosphorylation of ERK and STAT3 also contribute to the neuroprotective properties of PACAP (Ohtaki et al., 2006). A recent study using microarray analysis of PC12 and PACAP knock-out mice indicates that PACAP is an emergency-response peptide inducing a host of immediate early genes including Ier-3, a reported cell survival factor (Eiden et al., 2008). In the current study we extend these observations and document that the protective effect of PACAP38 on cultured neurons is mediated, in part, by release of the chemokine RANTES. This work is consistent with a growing body of literature that implicates PACAP38 as an immunomodulator and a regulator of inflammatory protein expression.

PACAP has been shown to inhibit expression and release of proinflammatory cytokines and chemokines, and enhance the production of the anti-inflammatory factors. In this regard, PACAP38 knock-out mice demonstrate elevated pro-inflammatory markers and a decrease in anti-inflammatory proteins after peripheral crush injury (Armstrong et al., 2008). In an animal model of septic shock systemic administration of PACAP decreases levels of TNF-α (Baranowska-Bik et al., 2006). In the brain, PACAP has been shown to exert neuroprotective effects in Parkinson’s disease models by inhibiting the production of inflammatory mediators (Staines, 2007). However, a direct effect on inflammatory protein release by neurons has not been demonstrated. PACAP attenuates inflammatory activation of microglia under hypoxic conditions, and protects co-cultured PC12 cells from microglial neurotoxicity (Suk et al., 2004). Recent studies have also shown that indirect neuroprotective effects of PACAP38 can be mediated in part by its release of RANTES from astrocytes (Brenneman et al., 2002; Dejda et al., 2005). Here, we demonstrate that PACAP can affect the release of both MIP-1α and RANTES directly from cultured neurons and that RANTES contributes, in part, to the neuroprotective effect of PACAP.

RANTES and its receptor CCR5 have been implicated in a wide array of pathological conditions in the brain and neurodegenerative diseases (Mines et al., 2007). Despite these associations, the role of RANTES in the diseased CNS is unclear because in addition to its established role in leukocyte recruitment and activation, RANTES has been shown to protect mixed cultures of human neurons and astrocytes from HIV- or NMDA-induced apoptosis (Brenneman et al., 2002). Also RANTES can suppress LPS-induced expression of IL-1β, IL-6, and TNFα and inducible nitric oxide synthase (Gamo et al., 2008). We recently documented that RANTES exerts direct neuroprotective effect on neurons in culture (Tripathy et al., 2008). In that study exposure of neuronal cultures to RANTES results in an increase in cell survival and a protective effect against the toxicity of thrombin and SNP. RANTES may be an atypical chemokine because it can signal via the orphan G protein-coupled receptor (GPR75) (Pease, 2006). Activation of this receptor has been shown to protect a hippocampal neuronal cell line following Aβ-induced injury by stimulation of the PLC/PI3K/Akt signaling pathway and mitogen-activated protein kinase (Ignatov et al., 2006; Pease, 2006). RANTES has also been shown to affect subcellular localization and phosphorylation of the cell cycle protein pRb in human mixed fetal neuronal and glial cultures (Jordan-Sciutto et al., 2001). Taken together, these data suggest that RANTES has significant neuroprotective properties.

In the current study we show that RANTES is capable of increasing neuronal survival both under basal, unstressed conditions and can help protect neurons from the toxic effects of SNP. Although PACAP stimulates the release of both RANTES and MIP-1α from neurons, the actions of MIP-1α on neuronal survival are limited. While MIP-1α supports neuronal survival in control, unstressed cultures, it does not protect neurons from the neurotoxic effects of SNP. Paradoxically, it appears that cell survival in cultures exposed to both PACAP plus an antibody to MIP-1α is slightly less than in neuronal cultures exposed to the MIP-1α antibody alone. In vivo the pleiotropic PACAP elicits release of numerous cytokines and chemokines that ultimately act in concert to produce the potent neuroprotective effects of the protein. It is possible that in vitro introduction of a blocking antibody to one of these mediators disrupts this balance unmasking an untoward effect of a PACAP-induced protein. For example, IL-6, which has been shown to increase in response to PACAP, has both neuroprotective and neurotoxic effects (Conroy et al., 2004; Peng et al., 2005; Wang et al., 2009). Because both PACAP and RANTES cause release of MIP-1α this chemokine is likely to have other important functions in the brain. In this regard, in both Alzheimer’s disease and in multiple sclerosis, MIP-1α is strongly associated with microglia/macrophages (Balashov et al., 1999; Lue et al., 2001). In a model of transient forebrain ischemia in rats, MIP-1α expression in CA1 pyramidal neurons contributes to microglial recruitment, suggesting that MIP-1α released from neurons contributes to activation of other cell types and propagation of inflammatory cascades in the brain (Cartier et al., 2005; Wang et al., 2008).

As we document in the current study, neurons long recognized as a target for cytokines and chemokines can also serve a source of these bioactive proteins. These results are supported by in vitro data showing that IL-1 induces IL-6 expression in cultured neurons and in vivo studies that indicate spinal cord injury causes upregulation of cytokines in neurons (Tsakiri et al., 2008). Also in a recent paper we show that neuronal cultures that are oxidatively stressed release TNFα, IL-1, MIP-1α and RANTES (Tripathy and Grammas, 2009). These data support the idea that inflammatory proteins can be induced in both neuronal and non-neuronal cells and likely contribute importantly to intercellular communication in the brain.

It has been suggested that autoimmune disorders of endogenous vasocative neuropeptides such as PACAP and vasoactive intestinal peptide (VIP) contribute to the pathogenesis of diseases such as Parkinson’s disease, multiple sclerosis and ALS (Staines, 2007; 2008). Indeed, both PACAP and VIP have neuroprotective effects in Parkinson’s disease models by inhibiting the production of inflammatory mediators, and PACAP specifically protects against neurotoxicty induced by rotenone (Wang et al., 2005). The distribution of PACAP and its receptors further supports the pleiotropic function of this neuropeptide in the central and peripheral nervous systems (Zhou et al., 2002; Shioda et al., 2006). Endogenous PACAP38 as well as exogenously administered PACAP38 contribute to neuroprotection (Chen et al., 2006; Ohtaki et al., 2008). The ability of PACAP to directly enhance neuronal survival through multiple intracellular signaling pathways as well as via the release of neuroprotective mediators such as RANTES highlights its utility as a potential therapeutic agent for the treatment of neurodegenerative diseases.