Purification and Characterization of a Novel Chemorepellent Receptor from Tetrahymena thermophila

Chemosensory transduction and adaptation are important aspects of signal transduction mechanisms in many cell types, ranging from prokaryotes to differentiated tissues such as neurons. The eukaryotic ciliated protozoan, Tetrahymena thermophila, is capable of responding to both chemoattractants (O'Neill et al., 1985; Leick, 1992; Kohidai, Karsa & Csaba, 1994, 1995) and chemorepellents (Francis & Hennessey, 1995; Kuruvilla, Kim & Hennessey, 1997). An example of a nontoxic, depolarizing chemorepellent in Tetrahymena is extracellular lysozyme (Francis & Hennessey, 1995; Hennessey, Kim & Satir, 1995). Lysozyme is an effective chemorepellent at micromolar concentrations, binds to a single class of externally facing membrane receptors and prolonged exposure (10 min) produces specific chemosensory adaptation (Kuruvilla et al., 1997). We now show that this lysozyme response is initiated by a depolarizing chemoreceptor potential in Tetrahymena and we have purified the membrane lysozyme receptor by affinity chromatography of solubilized Tetrahymena membrane proteins. The solubilized, purified protein is 42 kD and it exhibits saturable, high affinity lysozyme binding. Polyclonal antibodies raised against this 42 kD receptor block the in vivo lysozyme chemoresponse. This is not only the first time that a chemoreceptor potential has been recorded from Tetrahymena but also the first time that a chemorepellent receptor has been purified from any unicellular eukaryote. Received: 28 July 1997/Revised: 14 November 1997     

Responses of the ciliates tetrahymena and paramecium to vertebrate odorants and tastants

The ciliates Tetrahymena and Paramecium respond to strong depolarizing stimuli with Ca(2+)-based action potentials, ciliary reversals, and consequent bouts of backward and forward swimming called "avoidance reactions" (ARs). We found that several representative tastants and odorants cause repetitive ARs in Tetrahymena and Paramecium at low (nM to microM) concentrations. Tetrahymena responded well to capsaicin, quinine, quinacrine, denatonium benzoate, eugenol, piperine, chloroquine, carvacrol, allyl isothiocyanate (AITC), and menthol. Chemosensory adaptation was seen with carvacrol, eugenol, quinacrine, and capsaicin. Cross-adaptation was seen between some of these compounds, suggesting possible similarities in their chemosensory transduction or adaptation pathways. Paramecium only responded well to AITC, quinacrine, piperine, and eugenol (with the effective concentration for 50% response [EC(50)] values in the microM range) while chemosensory adaptation was only seen to eugenol in Paramecium, suggesting possible species differences. Tetrahymena and Paramecium may have primitive receptors that can recognize these and other compounds or some of these compounds can act independently of specific receptors.     

Chemorepellent signaling through the PACAP/lysozyme receptor is mediated through cAMP and PKC in Tetrahymena thermophila

Pituitary adenylate cyclase-activating peptide and lysozyme are potent chemorepellents which act through the same receptor in Tetrahymena. Using in vivo behavioral studies, we have found that the pituitary adenylate cyclase-activating peptide/lysozyme receptor appears to signal through a G-protein pathway which is mediated through both adenosine 3'5'monophosphate and protein kinase C. Avoidance to pituitary adenylate cyclase-activating peptide and lysozyme is inhibited by the G-protein inhibitor, guanosine 5'-O-(2thiodiphosphate), the adenosine 3'5'monophate analog, Rp-adenosine-3', 5' cyclic monophosphorothioate, and the protein kinase C inhibitors, calphostin C and bisindolylmaleimide IV. A proposed model for signaling through the pituitary adenylate cyclase-activating peptide/lysozyme receptor is briefly outlined.     

GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase

Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.     

Chemosensory Adaptation to Lysozyme and GTP Involves Independently Regulated Receptors in Tetrahymena thermophila

ABSTRACT. Chemosensory adaptation is seen in Tetrahymena thermophila following prolonged exposure (ten minutes) to micromolar concentrations of the chemorepellents lysozyme or guanosine triphosphate (GTP). Since these cells initially show repeated backward swimming episodes (avoidance reactions) in these repellents, behavioral adaptation is seen as a decrease in this repellent-induced behavior. The time course of this behavioral adaptation is paralleled by decreases in the extents of surface binding of either [³²P]GTP or [³H]lysozyme in vivo. Scatchard plot analyses of repellent binding in adapted cells suggests the behavioral adaptation is due to a dramatic decrease in the number of surface binding sites, as represented by decreased Bmax values. The estimated K_D values for nonadapted cells are 6.6 μM and 8.4 μM for lysozyme and GTP binding, respectively. Behavioral adaptation and decreased surface receptor binding are specific for each repellent. The GTP adapted cells (20 μM for ten minutes) still respond behaviorally to 50 μM lysozyme and bind [³H]lysozyme normally. Lysozyme adapted cells (50 μM for ten minutes) still bind [³²P]GTP and respond behaviorally to GTP. All the behavioral and binding changes seen are also reversible (deadaptation). Neomycin was shown to be a competitive inhibitor of [³H]lysozyme binding and lysozyme-induced avoidance reactions, but it had no effect on either [³²P]GTP binding or GTP-induced or avoidance reactions. These results are consistent with the hypothesis that there are two separate repellent receptors, one for GTP and the other for lysozyme, that are independently downregulated during adaptation to cause specific receptor desensitization and consequent behavioral adaptation.     

External GTP binding and induction of cell division in starved Tetrahymena thermophila

The extracellular nucleotide, guanosine 5'-triphosphate (GTP) is known to be a chemorepellent for ciliated protozoa such as Paramecium and Tetrahymena. Here, we studied the surface localization of GTP binding sites and also its effects on the cell division of Tetrahymena thermophila. When a ribose-modified and fluorescent analog of GTP, 2'-(or -3')-O-trinitrophenyl (TNP)-GTP was added to the cells starved in non-nutrient buffer, a remarkable fluorescence was observed at the compound cilia of the oral area, while it was weak at other cilia and the somatic membrane. Following transfer of the cells to the starvation medium, the intensity of TNP-GTP fluorescence at the oral area gradually increased and was saturated at 3-4 hours. Addition of GTP to the starved cell induced not only an avoiding reaction in swimming, but also induced a synchronous cell division that occurred 2 hours later. An attempt to search for other stimuli, which induced cell division, revealed that mechanical stimulation by a short period of centrifugation was almost as effective as the addition of GTP. The supernatant after centrifugation had the ability to induce cell division, and such activity was abolished after the supernatant was treated with the phosphatase, apyrase, suggesting the release of GTP by the mechanical stimulation. These results indicate that the released GTP binds mainly to the oral area and this then induces the cell division of starved T. thermophila.     

Chemorepellents in Paramecium and Tetrahymena

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.     

The nucleotide sequences of 5S rRNAs from three ciliated protozoa.

The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined. All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs. The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates.     

Enhancement of Swimming Speed Leads to a More-Efficient Chemotactic Response to Repellent

Negative chemotaxis refers to the motion of microorganisms away from regions with high concentrations of chemorepellents. In this study, we set controlled gradients of NiCl₂, a chemorepellent, in microchannels to quantify the motion of Escherichia coli over a broad range of concentrations. The experimental technique measured the motion of the bacteria in space and time and further related the motion to the local concentration profile of the repellent. Results show that the swimming speed of bacteria increases with an increasing concentration of repellent, which in turn enhances the drift velocity. The contribution of the increased swimming speed to the total drift velocity was in the range of 20 to 40%, with the remaining contribution coming from the modulation of the tumble frequency. A simple model that incorporates receptor dynamics, including adaptation, intracellular signaling, and swimming speed variation, was able to qualitatively capture the observed trend in drift velocity.     

An automated assay for quantifying the swimming behavior of Paramecium and its use to study cation responses

Paramecium tetraurelia is a ciliated protist that alters its swimming behavior in response to various stimuli. Like the sensory responses of many organisms, these responses in Paramecium show adaptation to continued stimulation. For quantitative studies of the initial response to stimulation, and of the time course of adaptation, we have developed a computerized motion analysis assay that can detect deviations from the normal swimming pattern in a population of cells. The motion of an average of ten cells was quantified during periods ranging from 15 to 60 seconds, with a time resolution of 1/15 seconds. During normal forward swimming, the maximum deviation from a straight-line path was less than 17 degrees. Path deviations above this threshold value were defined as changes in swimming direction. The percentage of total path time that cells spent deviating from forward swimming was defined as percent directional changes (PDC). This parameter was used to construct dose-response curves for the behavioral effects of various externally added cations known to induce behavioral changes and also to show the time course of adaptation to a depolarizing K+ stimulus. This assay is a valuable tool for studies of chemoeffectors or mutations that alter the swimming behavior of Paramecium and may also be applicable to other motile organisms.     

Negative Geotactic Behavior of Paramecium Caudatum is Completely Described by the Mechanism of Buoyancy-Oriented Upward Swimming

This paper presents evidence that the negative geotactic behavior of Paramecium caudatum takes place by the mechanism of buoyancy-oriented upward swimming. Photographs of swimming pathways of the organisms were completely described by two dynamic equations for the translational motion of the center of gravity of the organism's body and for the rotational motion of the organism's body about its center of gravity, where the rotational torque is induced by a slight difference in position between the center of gravity and the center of buoyancy. It now seems unlikely that complicated mechanisms such as the statocyst mechanism and the gravity-propulsion mechanism, which have been proposed by many investigators, need be considered for other protozoa since preliminary observation and analysis of other ciliates such as Paramecium multimicronucleatum, Paramecium tetraurelia, and Tetrahymena pyriformis also strongly suggested that their negative geotaxis is due to buoyancy-oriented upward swimming.     

Inhibition of interferon (IFN) gamma-induced Jak-STAT1 activation in microglia by vasoactive intestinal peptide: inhibitory effect on CD40, IFN-induced protein-10, and inducible nitric-oxide synthase expression

Interferon (IFN)-gamma is one of the most important microglia stimulators in vivo participating in inflammation and Th1 activation/differentiation. IFN-gamma-mediated signaling involves the activation of the Jak/STAT1 pathway. The neuropeptides vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP) are two potent microglia-deactivating factors that inhibit the production of proinflammatory mediators in vitro and in vivo. The present study investigated the molecular mechanisms involved in the VIP/PACAP regulation of several IFN-gamma-induced microglia-derived factors, including IFN-gamma-inducible protein-10 (IP-10), inducible nitric-oxide synthase (iNOS), and CD40. The results indicate that VIP/PACAP inhibit Jak1-2 and STAT1 phosphorylation, and the binding of activated STAT1 to the IFN-gamma activated site motif in the IFN regulatory factor-1 and CD40 promoter and to the IFN-stimulated response element motif of the IP-10 promoter. Through its effect in the IFN-gamma-induced Jak/STAT1 pathway, VIP and PACAP are able to control the gene expression of IP-10, CD40, and iNOS, three microglia-derived mediators that play an essential role in several pathologies, i.e. inflammation and autoimmune disorders. The effects of VIP/PACAP are mediated through the specific receptor VPAC1 and the cAMP/protein kinase A transduction pathway. Because IFN-gamma is a major stimulator of innate and adaptive immune responses in vivo, the down-regulation of IFN-gamma-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response in the central nervous system by endogenous neuropeptides.     

Biochemical evidence for a P2Y-like receptor in Tetrahymena thermophila

Extracellular nucleotides are ubiquitous signaling molecules. ATP signals through two receptor types: the ionotropic P2X receptors, and the metabotropic P2Y receptors. ATP acts as a chemorepellent in Tetrahymena thermophila, where it causes a distinct avoidance response. The intracellular mechanisms by which ATP causes avoidance in this organism, however, are unknown. In this study, we use in vivo pharmacological assays along with enzyme immuno-assays to obtain information about the ATP chemorepellent pathway and its associated second messenger systems. Our data show strong similarities between the presumed ATP receptor of T. thermophila and members of the P2Y family of receptors. The ATP response of T. thermophila appears to be coupled to phospholipase C, a defining characteristic of the P2Y receptor family. In addition, the ATP chemoresponse appears to be linked to a G_i/o protein, nitric oxide synthase, and adenylyl cyclase, all of which are characteristic of some P2Y receptors. This is an important first step in describing the pathways involved in ATP chemoresponse of this organism.Abbreviations cAMP adenosine 3'5'-monophosphate - ATP--S adenosine-5'-O-(3-thiotriphosphate) - EIA enzyme immunoassay - GDP--S guanosine 5'-O-(2-thiodiphosphate) - cGMP guanosine 3'5'-monophosphate - IMP 2-imino-4-methylpiperidine - IP₃ inositol 1,4,5-trisphosphate - NO nitric oxide - iNOS inducible nitric oxide synthase - PACAP pituitary adenylate cyclase activating polypeptide - PKA cAMP-dependent protein kinase - PKC protein kinase C - PKG cGMP-dependent protein kinase - Rp-cAMPs Rp-adenosine-3',5' cyclic monophosphorothioate     

The role of cortical orientation in the control of the direction of ciliary beat in Paramecium

The swimming behavior of many ciliate protozoans depends on graded changes in the direction of the ciliary effective stroke in response to depolarizing stimuli (i.e., the avoiding reaction of Paramecium). We investigated the problem of whether the directional response of cilia with a variable plane of beat is related to the polarity of the cell as a whole or to the orientation of the cortical structures themselves. To do this, we used a stock of Paramecium aurelia with part of the cortex reversed 180 degrees. We determined the relation of the orientation of the kineties (ciliary rows) to the direction of beat in these mosaic paramecia by cinemicrography of particle movements near living cells and by scanning electron microscopy of instantaneously fixed material. We found that the cilia of the inverted rows always beat in the direction opposite to that of normally oriented cilia during both forward and backward swimming. In addition, metachronal waves of ciliary coordination were present on the inverted patch, travelling in the direction opposite to those on the normal cortex. The reference point for the directional response of Paramecium cilia to stimuli thus resides within the cilia or their immediate cortical surroundings.     

Orientation of paramecium swimming in a DC magnetic field

We found that a ciliated protozoan, Paramecium, swam perpendicular to a static (DC) magnetic field (0.68 T). The swimming orientation was similar even when the ionic current through the cell membrane disappeared after saponin treatment. To determine the diamagnetic anisotropy of intracellular organs, macronuclei, cilia, and secretory vesicles, trichocysts, were selectively isolated. Both cilia and trichocysts tended to align their long axis parallel to the magnetic field (0.78 T). Paramecium mutants that lack trichocysts also swam perpendicular to the magnetic field, although the proportion fraction was smaller than the normal population. Since large numbers of cilia and trichocysts are arranged at right angles to the long axis of the cell, the diamagnetic anisotropies of cilia and trichocysts cause the long axis of the cell to align perpendicular to the magnetic field. In contrast to the DC magnetic field, an alternative (AC) magnetic field (60 Hz, 0.65 T) had almost no effect on the swimming orientation of Paramecium.     

PACAP and VIP increase the expression of myelin-related proteins in rat schwannoma cells: Involvement of PAC1/VPAC2 receptor-mediated activation of PI3K/Akt signaling pathways

PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24 h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 μM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 μM) but not the MAPKK inhibitor (PD98059, 50 μM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating diseases.     

Temperature-dependent changes in the swimming behaviour of Tetrahymena pyriformis-NT1 and their interrelationships with electrophysiology and the state of membrane lipids

The swimming velocity and the amplitude of the helical swimming path of T. pyriformis-NT1 cells grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C) were monitored between 0 and 40 degrees C in the presence and absence of electric fields. Within physiological limits the swimming velocity increased and the amplitude decreased as temperature was raised. The temperature profiles of these properties were not linear, and showed discontinuities at different temperatures for the different cultures. The break points in Arrhenius plots of the resting potential, regenerative spike magnitude, repolarization time, swimming velocity and swimming amplitude are tabulated and compared. The initial breakpoints upon cooling were clustered about the breakpoints in fluorescence polarization of D.P.H. in extracted phospholipids, and around the transition temperatures estimated from the literature for the pellicular membrane of these cells. The average of the initial breakpoints on cooling was 22.9 degrees C for Tg 38 degrees C cells and 13.7 degrees C for Tg 20 degrees C cells, a shift of 9.2 degrees C. Unlike Paramecium there is no depolarizing receptor potential in Tetrahymena upon warming. It is suggested that this may be the basis of a behavioural difference between Tetrahymena and Paramecium--namely that in Tetrahymena maximum swimming velocity occurs above growth temperature whereas in Paramecium the two points coincide. Swimming velocity and resting potential were correlated with membrane fluidity within physiological limits, but for other parameters the relationship with fluidity was more complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotection: a comparative view of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide

The neuroprotective properties of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) place these peptides in a special category of ligands that have implications for our understanding of pathological conditions as well as a potential basis for therapeutic intervention. It is remarkable that these peptides have a protective impact against such a wide variety of clinical relevant toxic substances. This protective diversity is consistent with the multiple pathways that are activated or inhibited by the action of these peptides. Although knowledge is emerging on the neuroprotective mechanisms of VIP and PACAP, it is already evident that these two peptides are not identical in their action and each peptide has multiple mechanisms that allow for neuroprotective diversity. The multiple intracellular signaling pathways and differing extracellular mediators of neuroprotection contribute to this diversity of action. In this review, examples of neuroprotective actions will be presented that serve to demonstrate the remarkable breadth of neuroprotective processes produced by VIP and PACAP.     

Anorexigenic effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide in the chick brain are mediated by corticotrophin-releasing factor

Intracerebroventricular (ICV) injection of pituitary adenylate cyclase-activating polypeptide-38 (PACAP) or vasoactive intestinal peptide (VIP) inhibits feeding in chicks. However, the underlying anorexigenic mechanism(s) has not yet been investigated. The present study investigated whether these peptides influence the activity of corticotrophin-releasing factor (CRF) neural pathways in the brain of chicks. Firstly, we found that ICV injections of PACAP and VIP increased plasma corticosterone concentrations. The corticosterone-releasing effect of PACAP was completely attenuated by co-injection of astressin, a CRF receptor antagonist, but this effect was only partial for VIP. These results demonstrated that CRF neurons mediate the actions of PACAP and, to a lesser extent, VIP, and suggest that the signaling mechanisms differ between the two peptides. This difference may arise from the two peptides interacting with different receptors because the corticosterone-releasing effect of PACAP, but not VIP, was completely attenuated by co-injection of PACAP (6–38), a PACAP receptor antagonist. Finally, we examined the effect of ICV co-injection of astressin on the anorexigenic effects of PACAP and VIP and found that the effects of both peptides were attenuated by astressin. Overall, the present study suggests that the anorexigenic effects of PACAP and VIP are mediated by the activation of CRF neurons.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit LPS-stimulated MIP-1alpha production and mRNA expression

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides with immunomodulatory properties, including the regulation of several proinflammatory mediators. Such mediators, for example chemokines, influence trafficking of inflammatory cells and contribute to shaping the immune response. In the present work, we studied the effect of VIP and PACAP on the CC chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) production in LPS-stimulated RAW 264.7 macrophage cell line. VIP and PACAP inhibited the production of MIP-1alpha in a dose-dependent manner and over a broad spectrum of LPS concentrations. The use of selective agonists and antagonists of VIP/PACAP receptors showed that type 1 VIP receptor (VPAC1) is the major receptor involved, but the type 2 VIP receptor (VPAC2) may be also implicated. By using selective PKA and PKC inhibitors and cAMP mimicked agents, we demonstrated a cAMP-dependent signalling pathway for the inhibitory effect of VIP/PACAP on MIP-1alpha production, although a minor non-mediated cAMP pathway was also involved. mRNA expression studies showed a down-regulation of MIP-1alpha gene expression by VIP and PACAP. Taken together, the present work strongly supports an anti-inflammatory role of VIP and PACAP by a new mechanism associated with impairment of a key component of the chemokine network.