Vasoactive intestinal peptide inhibits IL-8 production in humanmonocytes by downregulating nuclear factor jB-dependent

transcriptional activity

Mario Delgadoa,b,* and Doina Ganeaa

a Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USAb Departamento Biologia Celular, Facultad de Biologia, Universidad Complutense, Madrid 28040, Spain

Received 21 January 2003

Abstract

Although interleukin-8 (IL-8) is a chemokine that plays a beneficial and central role in the inflammatory response, hematopoiesis,

and angiogenesis, excessive IL-8 production can be deleterious to the host, and its selective inhibition represents an important

therapeutic goal. Vasoactive intestinal peptide (VIP) is a neuropeptide that acts as a potent anti-inflammatory agent inhibiting the

function of activated macrophages/monocytes. The present study reports the effect of VIP on IL-8 production by stimulated human

THP1 monocytes. VIP inhibits IL-8 production in a dose- and time-dependent manner at the mRNA level. VIP seems to act by

inhibiting the NF-jB-dependent IL-8 gene activation. The specific VPAC1 receptor mediates the inhibitory effect of VIP. Two

transduction pathways appear to be involved, a major cAMP-independent pathway that preferentially blocks nuclear translocation of

NF-jB and its binding to the jB site of the IL-8 promoter, and a cAMP-dependent pathway that inhibits the activation and binding

to the IL-8 promoter of both CREB-binding protein (CBP) and TATA box-binding protein (TBP), two transcriptional cofactors

strictly required for the transactivating activity of NF-jB. These findings support the proposed role of VIP as a key endogenous anti-

inflammatory agent and describe a novel mechanism, i.e., the inhibition of the production of monocyte-derived IL-8, and are of

obvious physiological significance, because VIP, through the inhibition of IL-8 production, could reduce the monocyte-induced

neutrophil chemotaxis/infiltration, an important event in the pathogenesis of several inflammatory and autoimmune disorders.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Neuroimmunology; Neuropeptides; Monocytes; Chemokines; Inflammation; Chemotaxis; Transcription factors

Systemic administration of lipopolysaccharide (LPS),

an integral outer membrane component of Gram-neg-

ative bacteria, in experimental animals leads to patho-

physiological changes similar to the human septic shock

syndrome, which are systemic responses to severe bac-

terial infections resulting in high mortality. The toxic

effects of endotoxin are exerted through the generation

of endogenous proinflammatory cytokines. Systemicexposure to bacterial endotoxins initiates a rapid, co-

ordinated recruitment of neutrophils, monocytes/mac-

rophages, and T cells into specific host tissues [1–3]. The

infiltration and activation of inflammatory leukocytes,

together with overproduction of proinflammatory me-

diators, initiate the tissue damage that precedes multiple

organ failure. The chemotactic factor IL-8, the best-

characterized member of the a-chemokine or CXC

chemokine family, is a potent activator and chemo-

attractant of neutrophils, but also acts on T cells, ba-

sophils, and eosinophils [1–3]. IL-8 is produced by a

wide variety of cell types, including monocytes/macro-

phages, after exposure to inflammatory stimuli such asthe bacterial endotoxin (LPS), or the proinflammatory

cytokines [1–7], and inhibited by anti-inflammatory

cytokines [8]. Many diverse forms of acute and chronic

inflammatory diseases are characterized by the local

accumulation of inflammatory cells, including neu-

trophils and lymphocytes. IL-8 participates in the

pathogenesis of various inflammatory diseases [3] and

its levels are often correlated with the pathology severity

Biochemical and Biophysical Research Communications 302 (2003) 275–283

www.elsevier.com/locate/ybbrc

BBRC

* Corresponding author. Fax: +34-91-3944981.

E-mail address: mariodm@bio.ucm.es (M. Delgado).

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00149-9

and/or disease outcome [9,10]. Therefore, although IL-8plays a beneficial and central role in the inflammatory

response, hematopoiesis, and angiogenesis [2], excessive

IL-8 production can be deleterious to the host and its

selective inhibition represents an important therapeutic

goal.

Vasoactive intestinal peptide (VIP) is a neuropeptide

present in the lymphoid microenvironment that elicits a

broad spectrum of biological functions, including ac-tions on natural and acquired immunity [11–15]. Al-

though VIP affects a variety of immune functions, its

primary immunomodulatory function is anti-inflamma-

tory in nature. In agreement with its anti-inflammatory

role, VIP was reported to protect mice from lethal en-

dotoxemia and arthritis, presumably by down-regulat-

ing endogenous proinflammatory macrophage-derived

mediators, including IL-6, TNFa, IL-12, nitric oxide,and several chemokines [16–18]. In a recent report, we

have demonstrated that VIP inhibits endotoxin-induced

production of IL-8 by human monocytes [19]. This

finding is of obvious physiological significance because

VIP, through the inhibition of IL-8 production, reduces

the monocyte-induced neutrophil chemotaxis/infiltra-

tion, an important event in the pathogenesis of several

inflammatory and autoimmune disorders. In this study,we investigate the molecular mechanisms involved in the

inhibitory effect of VIP on IL-8 production by activated

monocytes, including the specific receptors, the intra-

cellular signal pathways, and the nuclear transactivating

factors. This study further clarifies the role played by

VIP in the attenuation of the inflammatory response.

Materials and methods

Reagents. Synthetic VIP was purchased from Calbiochem-Nova-

biochem (Laufelfingen, Switzerland). The PAC1/VPAC2-antagonist

PACAP6–38 was obtained from Peninsula Laboratories (Belmont, CA).

The VPAC1-antagonist [Ac-His1, D-Phe2;K15;R16;L27] VIP [3–7]-

GRF [8–27], the VPAC1-agonist [K15;R16;L27] VIP (1–7)-GRF (8–27),

and the VPAC2-agonist Ro 25–1553 Ac-[Glu8;Lys12;Nle17;Ala19;

Asp25;Leu26;Lys27;28; Gly29;30;Thr31]-VIP cyclo (21–25) were donated

by Dr. Patrick Robberecht (Universite Libre de Bruxelles, Belgium).

The synthetic PAC1 agonist maxadilan was a generous gift from Dr.

Ethan A. Lerner (Massachusetts General Hospital, Charlestown, MA).

Human recombinant TNFa and IL-8, and capture and biotinylated

antibodies against human IL-8 were purchased from Pharmingen (San

Diego, CA). LPS (from Escherchia coli 0111:B4), DEAE-dextran,

calphostin C, and forskolin were purchased from Sigma Chemicals (St.

Louis, MO), and N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quino-

linesulfonamide (H89) was from ICN Pharmaceuticals (Costa Mesa,

CA). Antibodies against p65, p50, cRel, cJun, CBP, TBP, NF-Y (CBF-

A), and HMG-I(Y) were purchased from Santa Cruz Biotechnology

(Santa Cruz, CA).

Cell stimulation. THP-1, a human leukemic monocytic cell line, was

obtained from American Type Culture Collection (Manassas, VA).

THP-1 cells (106 cells/ml, 200ll/well) were cultured in 96-well flat

bottomed tissue culture plate (Costar, Cambridge, MA) in RPMI 1640

medium supplemented with 10% human serum (HS; Gibco-BRL),

containing 10mM HEPES buffer, 1 mM pyruvate, 0.1 M nonessential

amino acids, 2mM glutamine, 50mM of 2-mercaptoethanol, 100U/ml

penicillin, and 10lg/ml streptomycin (complete medium). Cells were

stimulated with different concentrations of LPS (100 ng/ml) or TNFa(10 ng/ml) in the presence or absence of VIP (10�8 M) and/or other

agents as indicated, for various times at 37 �C in a humidified incu-

bator with 5% CO2. Culture supernatants were harvested and stored

at )20 �C until IL-8 determination by ELISA.

Plasmids, transfections, and luciferase assay. 50 deletion constructs

of the IL-8 promoter were produced as described [20] with some

modifications, using the PCR with )420/+44 hIL-8/Luc reporter

plasmid (pGL2-IL-8) [21] as a template and a downstream oligonu-

cleotide hybridizing +86 to +55 of the luciferase cDNA [20]. The

following upstream primers were used to produce 50 deletions (50 nt

indicated by minus; underline indicates BamHI restriction site): )162

hIL-8: 50-AACTTTGGATCCACTCCGTATTTGATAAGG-30; )132

hIL-8: 50-AACAAAGGATCCTGTGATGACTCAGGTTTG-30; )99

hIL-8: 50-TGAAGGGGATCCGCCATCAGTTGCAAATCG-30; and

)54 hIL-8: 50-CATAATGGATCCATGAGGGTGCATAAGTTC-30.

The PCR products were digested with BamHI and HindIII, gel puri-

fied, and subcloned into the pUC vector (Clonthech, Palo Alto, CA).

The plasmid pRc/RSV-p65 containing the entire cDNA of p65 was

kindly provided Drs. G.J. Nabel and J. Stein through the NIH AIDS

Research and Reference Reagent Program. Empty vectors pRc/RSV

and pUC-18 (Invitrogen, Carlsbad, CA) were used to maintain a

constant concentration of total transfected DNA in each experiment.

To assess variations in transfection efficiencies, cells were transfected

with 2 lg of the control plasmid pCH110 (Amersham Pharmacia

Biotech) that expresses the LacZ gene. Levels of b-galactosidase were

determined using the Galacto-Light assay system (Tropix, Bedfrod,

MA) and exhibited <15% variation between samples.

THP-1 cells were transiently transfected with a total of 10–30lg of

plasmid DNA using DEAE-dextran [22]. Forty-eight hours after

transfection cells were stimulated in complete medium with LPS

(100 ng/ml) in the absence or presence of different concentrations of

VIP. After 6 h incubation, luciferase assays were carried out according

to the instructions of the manufacturer (Promega Biotec). Light

emission was measured in a luminescence microplate counter (Top-

Count; Packard Instrument, Meriden, CT). Luciferase activity, ex-

pressed in arbitrary light units, was corrected for protein concentration

or by normalization to co-expressed b-galactosidase levels.

RNA extraction and Northern blot analysis. Northern blot analysis

was performed according to standard methods. THP-1 cells were

prepared and stimulated as described above. At the various time

points, 1 � 107 cells were harvested and total RNA was extracted by

the acid guanidinium–phenol–chloroform method, electrophoresed on

1.2% agarose–formaldehyde gels, transferred to S&S Nytran mem-

branes (Schleicher and Schuell, Keene, NJ), and cross-linked to the

nylon membrane using UV light.

The probe for human IL-8 (Oncogene, Cambridge, MA) was end-

labeled with [c-32P]ATP (3000Ci/mmol, Amersham, Arlington, IL) by

using T4 polynucleotide kinase. The RNA-containing membranes were

prehybridized for 16 h at 42 �C and then hybridized at 60 �C for 16 h

with the appropriate probes. The membranes were then washed twice

in 2� SSC containing 0.1% SDS at room temperature (20 min each

time), once at 37 �C for 20min, and once in 0:1� SSC containing 0.1%

SDS at 50 �C (20min). The prehybridization and hybridization buffers

were purchased from 50 Prime–30 Prime (Boulder, CO). The mem-

branes were exposed to X-ray films (Kodak, Rochester, NY). Signal

quantitation was performed in a PhosphorImager SI (Molecular Dy-

namics, Sunnyvale, CA). To assure equal loading, the membrane was

stripped and rehybridized with [32P]ATP-labeled b-actin probe

(Stratagene, La Jolla, CA).

Electrophoretic mobility shift assay. Nuclear extracts were prepared

by the mini-extraction procedure of Schreiber et al. with slight

modifications [23]. THP-1 cells were cultured at a density of 107 cells

per well in 6-well plates, stimulated as described above, washed twice

with ice-cold PBS/0.1% BSA, and harvested. The cell pellets were

276 M. Delgado, D. Ganea / Biochemical and Biophysical Research Communications 302 (2003) 275–283

homogenized with 0.4ml buffer A (10mM HEPES, pH 7.9, 10mM

KCl, 0.1mM EDTA, 0.1 mM EGTA, 1mM DTT, 0.5mM PMSF,

10 lg/ml aprotinin, 10lg/ml leupeptin, 10 lg/ml pepstatin, and 1mM

NaN3). After 15min on ice, Nonidet P-40 was added to a final 0.5%

concentration, the tubes were gently vortexed for 15 s, and nuclei were

sedimented and separated from cytosol by centrifugation at 12,000gfor 40 s. Pelleted nuclei were washed once with 0.2 ml of ice-cold buffer

A and the soluble nuclear proteins were released by adding 0.1ml

buffer C (20mM HEPES, pH 7.9, 0.4M NaCl, 1 mM EDTA, 1mM

EGTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 10 lg/ml aprotinin,

10 lg/ml leupeptin, 10lg/ml pepstatin, and 1 mM NaN3). After incu-

bation for 30min on ice, followed by centrifugation for 10min at

14,000 rpm at 4 �C, the supernatants containing the nuclear proteins

were harvested, the protein concentration was determined by the

Bradford method, and aliquots were stored at )80 �C for later use in

EMSAs.

Double-stranded oligonucleotides (50 ng) corresponding to the NF-

jB site of the human IL-8 promoter (50-ATCGTGGAATTTCC

TCTGA-30) [24–26] were end-labeled with [c-32P]ATP by using T4

polynucleotide kinase. For EMSAs with THP-1 nuclear extracts,

20,000–50,000 cpm of double-stranded oligonucleotides, correspond-

ing to approximately 0.5 ng, was used for each reaction. The binding

reaction mixtures (15ll) contain: 0.5–1 ng DNA probe, 5 lg nuclear

extract, 2lg poly(dI-dC) �poly(dI-dC), and binding buffer (50mM

NaCl, 0.2mM EDTA, 0.5 mM DTT, 5% glycerol, and 10mM Tris–

HCl, pH 7.5). The mixtures were incubated on ice for 15min before

adding the probe, followed by another 20min at room temperature.

Samples were loaded onto 4% non-denaturing polyacrylamide gels and

electrophoresed in TGE buffer (50mM, Tris–HCl, pH 7.5, 0.38M

glycine, and 2 mM EDTA) at 100V, followed by transfer to Whatman

paper, drying under vacuum at 80 �C, and autoradiography. In some

cases, signal quantitation was performed by phosphoimaging. In

competition and antibody supershift experiments, the nuclear extracts

were incubated for 15min at room temperature with the specific an-

tibody (1lg) or competing cold oligonucleotide (50-fold excess) before

the addition of the labeled probe.

Immunoblotting of proteins bound to the proximal region of the IL-8

promoter. A double-stranded oligonucleotide spanning the proximal

region of the human IL-8 promoter ()162 to +44), generated by PCR

and biotinylated in our molecular biology facility [20,21], was coupled

to Dynabeads M-280 streptavidin (Dynal, Lake Success, NY) ac-

cording to the manufacturer�s recommendations. The IL-8 promoter-

coupled matrix (250 lg) was incubated with 25 ll THP-1 nuclear

extract in binding buffer (50mM NaCl, 5 mM MgCl2, 10mM Tris, pH

7.5, 1 mM DTT, 1mM EDTA, 0.25lg/ml poly(dI-dC), and 5% glyc-

erol) for 20min at room temperature with mixing every 5 min to keep

the Dynabeads in suspension. The non-bound or flow-through frac-

tions were collected and the Dynabeads with bound extract proteins

were washed four times with binding buffer containing 0.5lg/ml

poly(dI-dC). Proteins bound to the Dynabeads were solubilized in 1�Laemmli sample buffer, boiled, and subjected to SDS–PAGE, followed

by transfer to nitrocellulose membranes. The membranes were probed

with antibodies against p65, p50, cRel, TBP, CBP, or NF-Y at dilu-

tions ranging from 1:2,500 to 1:10,000 followed by enhanced chemi-

luminiscent detection.

IL-8 ELISA. IL-8 levels in culture supernatants were determined

using a human IL-8-specific sandwich ELISA (Pharmingen) following

the manufacturer�s instructions.

Results and discussion

Peripheral blood monocytes, as they migrate from the

vascular compartment to a site of inflammation, un-

dergo activation and secrete proinflammatory cytokines

and oxidants, which contribute to pathophysiological

changes associated with several acute and chronic in-flammatory conditions, and inflammatory chemokines

that recruit and activate blood-derived leukocytes. IL-8

is a human chemokine that plays a crucial role in the

pathogenesis of several inflammatory and autoimmune

diseases. The therapeutical action of VIP in several in-

flammatory alterations could be mediated through its

inhibitory effect on chemokine production by activated

macrophages [18,27]. We have recently shown that VIPinhibits IL-8 production by endotoxin-stimulated hu-

man monocytes [19]. Since IL-8 is involved in control-

ling the nature and magnitude of the inflammatory

response, and its selective inhibition represents an im-

portant therapeutic goal, it is crucial to know the mo-

lecular mechanisms through which VIP down-regulates

IL-8 production.

IL-8 synthesis is controlled at several levels, withtranscription as the primary regulatory site. Previous

data indicate that the inhibitory effect of VIP on IL-8

production occurs through the reduction in IL-8 mRNA

levels [19]. The precise molecular mechanisms that ac-

count for the VIP inhibition of IL-8 expression are lar-

gely unknown, and it remains to be established whether

the reduction in steady-state IL-8 mRNA levels results

from a decrease in de novo transcriptional rate, messagestabilization, or both. However, the fact that delayed

treatment with VIP did not affect IL-8 production sug-

gests that VIP inhibits an early event in IL-8 expression

and points to a direct effect of VIP on the novo tran-

scription as the most likely possibility.

Although in other cell types, such as endothelial and

epithelial cells, the regulation of the IL-8 gene tran-

scription is complex and involves multiple cis-acting el-ements, including AP-1, NF-IL-6, and NF-jB sites

[20,26,28–31], transcriptional regulation by LPS and

TNFa of the human IL-8 gene in monocytes has been

shown to solely involve a NF-jB site and transcriptional

factors of the Rel family [21,24–26,32,33]. To investigate

whether VIP affects NF-jB-dependent IL-8 gene tran-

scription, we used the human monocytic cell line THP1.

First, we confirmed that VIP affects IL-8 production inTHP1 cells similar to blood isolated monocytes. Indeed,

VIP and a specific VPAC1-receptor agonist inhibit, in a

dose- and time-dependent manner, the IL-8 production

and mRNA expression in LPS-stimulated THP1 cells

(Fig. 1), with very similar kinetics to those observed in

human primary monocytes [19].

Next, we investigated whether VIP inhibits LPS- and

TNFa-induced IL-8 promoter activation. THP1 cellswere transiently transfected with the hIL-8/luciferase

reporter plasmid, containing the )420/+44 hIL-8 pro-

moter region. Forty-eight hours later, the cells were

stimulated with LPS or TNFa in the presence or absence

of VIP and assayed for IL-8 promoter activation 6 h

later. Both LPS and TNFa led to an increase in the IL-8

transcriptional activity (Fig. 2A). Treatment with VIP

M. Delgado, D. Ganea / Biochemical and Biophysical Research Communications 302 (2003) 275–283 277

strongly inhibits LPS- or TNFa-induced IL-8 promoter

activation (Fig. 2A). MG132, a newly described NF-jB

inhibitor, shows a similar inhibitory effect (Fig. 2A),

confirming the involvement of NF-jB on IL-8 tran-

scription. To establish which cis elements are important

in the inhibitory activity of VIP on IL-8 transcriptionalactivity, transient transfections with 50-deletions of the

hIL-8/Luc reporter plasmid were performed. LPS stim-

ulation of THP1 cells transfected with the )162/+44,

)132/+44 or )99/+44 hIL-8/luc plasmids resulted in

similar levels of activation compared to the original hIL-

8 promoter (Figs. 2A and B). However, LPS minimally

increased the luciferase activity in THP1 cells trans-

fected with the )54/+44 hIL-8/Luc vector (Fig. 2B).These results suggest that the essential elements for IL-8

gene regulation by LPS are present in the 50 region

spanning from )99 to )54 bp. This region contains a

jB-like site ()80 to )70 bp) previously reported to be

involved in LPS-induced IL-8 gene activation in

monocytes [21,24–26,32,33]. VIP inhibited IL-8 tran-

scriptional activity in LPS-stimulated THP1 cells

transfected with the )162/+44, )132/+44 or )99/+44,but not )54/+44 hIL-8/Luc vectors (Fig. 2B), suggesting

that VIP could exert its effect through the NF-jB-

binding site.

In mammalian cells, the Rel family includes NF-jB1

(p50), RelA (p65), c-Rel, RelB, and NF-jB2 (p50B, p52)

[34]. NF-jB consists mostly of p50/p65 or p50/c-Rel

heterodimers, which are complexed to the inhibitor IjB

in the cytoplasm of unstimulated cells; stimuli such asLPS or proinflammatory cytokines induce the phos-

phorylation and degradation of IjB, followed by the

release and subsequent nuclear translocation of the p50/

p65 or p50/c-Rel heterodimers, which bind to regulatory

sequences in a variety of target genes [34]. To investigate

whether VIP affects NF-jB binding to the IL-8 pro-moter, we used EMSAs. Stimulation of THP1 cells with

LPS or TNFa led to an increase in NF-jB binding to

the IL-8 promoter; in both cases, treatment with VIP

significantly inhibited NF-jB binding (Fig. 2C, left

panels). The binding specificity is indicated by the

complete displacement of the NF-jB/DNA binding

complexes in the presence of a 50-fold excess of unla-

beled homologous oligonucleotides (Fig. 2C, middlepanels). Addition of anti-p50, anti-p65, or anti-cRel Abs

resulted in a marked reduction in the intensity of the

NF-jB band, and in the appearance of slower migrating

bands, indicating the presence of p50, p65, and cRel in

the NF-jB-binding complex (Fig. 2C, right panel).

The primary level of control for NF-jB is mediated

through its interaction with the inhibitor IjB. We have

previously demonstrated that VIP inhibits NF-jB ac-tivity by blocking LPS-induced IjB degradation and

subsequent NF-jB nuclear translocation in macro-

phages and monocytes [35,36]. If the inhibitory effect of

VIP on NF-jB-dependent IL-8 transcriptional activity

is mediated entirely through the inhibition of NF-jB

nuclear translocation, overexpression of p65 should re-

verse this effect. THP1 cells were transiently transfected

with the hIL-8/Luc reporter plasmid and increasingconcentrations of a vector expressing p65. Increasing

concentrations of p65 only partially reversed the inhib-

itory effect of VIP (Fig. 2D), suggesting that the

neuropeptide affects more than NF-jB nuclear translo-

cation. In contrast, increasing concentrations of p65

completely reversed the inhibitory effect of the NF-jB

inhibitor MG-132 (Fig. 2D).

Several studies have shown that, in addition to DNAbinding, the transactivating activity of NF-jB requires

interaction with coactivators that bridge various tran-

scriptional activators and components of the basal

transcriptional machinery, such as the CREB-binding

protein (CBP) and the TATA-binding protein (TBP)

[37–42]. CBP is a ubiquitously expressed nuclear coac-

tivator present in limiting amounts [37]. A diverse and

increasing number of transcription factors and someelements of the basal transcriptional machinery are able

to form stable physical complexes with, and respond to,

CBP [43]. CBP functions as an integrator linking various

transcription factors to the basal transcriptional appa-

ratus, by binding to the basal transcription factor

TFIIB, which in turn contacts the TBP of the TFIID

complex in the basal apparatus [38–40]. The interaction

of p65 with CBP is essential for NF-jB transcriptionalactivity [41,42,44], and this interaction can be strength-

ened by p65 phosphorylation [38,45] or impeded by

competition from other CBP-binding factors such as

CREB, c-Jun, c-Fos, p53, steroid receptors, c-Myb, and

Myo-D [38–40,43]. In a previous study, we have dem-

onstrated that VIP inhibits NF-jB-dependent gene ac-

tivation at multiple levels in THP-1 cells, by affecting

Fig. 1. VIP inhibits IL-8 production and mRNA expression by acti-

vated THP-1 monocytes. THP-1 cells (106 cells/ml) were stimulated

with LPS (100 ng/ml) in the absence or presence of VIP or the VPAC1-,

VPAC2-, and PAC1-agonists (10�8 M). At different times, IL-8 con-

tents were determined by ELISA and IL-8 mRNA expression was

determined by Northern blot. Results are means�SD of three ex-

periments performed in duplicate.

278 M. Delgado, D. Ganea / Biochemical and Biophysical Research Communications 302 (2003) 275–283

NF-jB nuclear translocation and DNA binding, inhib-

iting the interaction of CBP with p65, and reducing TBP

activation and binding [35]. In order to investigate

whether similar mechanisms participate in the inhibitory

effect of VIP on IL-8 gene activation, we evaluated the

effects of VIP on the LPS-induced transcriptional acti-

vators binding to the human IL-8 promoter. To identify

the IL-8 promoter binding factors, a biotinylated affinity

matrix spanning the proximal hIL-8 regulatory region

()162 to +44 bp) was generated and coupled to strep-

tavidin-coated magnetic beads. This biotinylated probe

was incubated with nuclear extracts from unstimulated

or LPS-stimulated THP1 cells treated with or without

VIP. The factors bound to the IL-8 probe were released

from the magnetic beads by boiling in SDS sample

buffer and identified by immunoblotting.

Fig. 2. VIP inhibits NF-jB dependent transcription of IL-8 in human monocytes. (A) VIP inhibits both LPS- and TNFa-induced IL-8 promoter

activation. THP-1 cells were transiently transfected with the hIL-8/Luc construct (10lg). Forty-eight hours after transfection cells were incubated

with medium alone or stimulated with LPS (100 ng/ml) or TNFa (10 ng/ml) in the absence or presence of VIP (10�8 M) or the NF-jB inhibitor

MG132 (100lM) for 6 h. Cells were then lysed and cytosolic extracts (100 lg) were used in luciferase assays. Fold induction is relative to luciferase

activity in unstimulated cells. Data are expressed as means�SD of three independent experiments performed in duplicate. (B) Involvement of kB site

in the inhibitory effect of VIP on IL-8 gene activation. THP-1 cells transiently transfected with selected 50 deletions of the hIL-8/Luc construct were

stimulated with LPS (100 ng/ml) in the absence or presence of VIP (10�8 M) for 6 h prior to reporter assay. Fold induction is relative to luciferase

activity in unstimulated cells. Data are expressed as means� SD of three independent experiments performed in duplicate. (C) VIP inhibits binding to

the jB site of the hIL-8 promoter. Nuclear extracts were prepared from THP-1 cells incubated for 2 h with LPS (100 ng/ml) or TNFa (10 ng/ml) in the

presence or absence of VIP (10�8 M). NF-jB binding was assessed by EMSA using a radiolabeled oligonucleotide containing the jB site of the

human IL-8 promoter. Specificity was determined by the addition of 50-fold excess of unlabeled non-homologous (AP-1) or homologous (jB) ol-

igonucleotides to the nuclear extracts (Competition). Identification of the proteins bound to the jB site by supershift analysis: nuclear extracts (2 h

incubation) were incubated with polyclonal antibodies against p65, cRel, p50, or cJun for 20min before the addition of the jB probe. Similar results

were observed in other three independent experiments. (D) Overexpression of p65 partially reverses the inhibitory effect of VIP on IL-8 promoter

activation. THP-1 cells were transiently cotransfected with the )162 hIL-8/Luc construct (10lg) and increasing concentrations (0, 2.5, 5, 10, or 15 lg)

of the pRSV-p65 vector (p65). The total DNA amount in each transfection was brought up to 25 lg with the empty vector pUC DNA. After 48 h, the

cells were stimulated with LPS (100 ng/ml) in the absence or presence of 10�8 M VIP or MG132 (100lM) and incubated for an additional 6 h before

determining the luciferase activity. Fold induction is relative to luciferase activity in unstimulated cells. Results represent means�SD of three in-

dependent experiments performed in duplicate.

M. Delgado, D. Ganea / Biochemical and Biophysical Research Communications 302 (2003) 275–283 279

Both cRel and p65 are present in the extracts fromLPS-treated samples, but not from unstimulated or

LPS-stimulated cells treated with VIP (Fig. 3, p65 and

cRel, input). The p65 and cRel present in the extract

from LPS-treated cells bind to the IL-8 promoter region

(Fig. 3, p65 and cRel, bound). In contrast, p50 is con-

stitutively expressed in THP1 cells and binds partially to

the IL-8 promoter (Fig. 3, p50, input, bound, and flow-

thru). The p50 binding is not affected by LPS or VIP.Both TBP and CBP are constitutively present in the

nucleus and neither LPS nor VIP affects their levels (Fig.

3, CBP and TBP, input). However, whereas LPS induced

the binding of both CBP and TBP to the IL-8 promoter

region, VIP drastically inhibited it (Fig. 3, CBP and

TBP, bound). As a control, we used the nuclear factor-Y

(NF-Y), a transcription factor present in the nucleus

which binds constitutively to various promoters, in-cluding IL-8. None of the treatments affected NF-Y

binding (Fig. 3, NF-Y). Finally, this experimental design

allowed us to investigate an additional possible regula-

tory element of the NF-jB transactivation, i.e., the

nonhistone chromosomal proteins of the high mobility

group (HMG)-I(Y) family, two chromatin architectural

proteins that play a role in the transcriptional regulation

of certain mammalian genes, by enhancing the DNAbinding of several transcription factors, including NF-

jB [46,47]. HMG-I(Y) was present in the nucleus from

unstimulated, LPS-stimulated, and VIP-treated THP1

cells, and none of the treatments affected its binding to

the IL-8 promoter (Fig. 3). Almost identical results were

previously obtained when human TNFa promoter wasstudied [35], but some differences have been found. For

example, CBP is constitutively bound to the TNFapromoter and VIP does not affect CBP binding [35].

However, CBP is almost absent in the IL-8 promoter of

unstimulated THP1 cells and VIP inhibits LPS-induced

CBP binding (Fig. 3). The explanation for these differ-

ences is that, in the TNFa promoter, CBP is bound to

CREB in the CRE site in unstimulated cells, and to p65and cJun in the jB and CRE sites, respectively, in LPS-

stimulated cells. VIP restores the basal state with CBP

bound to CREB [35]. In contrast, CREB is not involved

in the regulation of IL-8 gene activation and the pres-

ence of CBP in the IL-8 promoter is dependent only on

p65/cRel binding.

The immunological actions of VIP are exerted

through a family of receptors consisting of VPAC1,VPAC2, and PAC1 [11–15,17]. Human monocytes ex-

press both high and low affinity VIP binding sites [48,49]

and THP1 cells constitutively express VPAC1 and PAC1

mRNA and VPAC2 mRNA following LPS-stimulation

[35]. The VPAC1 is coupled primarily to the adenylate

cyclase system and VIP increases intracellular cAMP

levels in human monocytes [50]. The inhibitory effect of

VIP on IL-8 production is mediated primarily throughVPAC1 and only slightly through cAMP [19]. This has

been partially confirmed in the present study, because

the VPAC1 agonist, but not the VPAC2 or PAC1 ag-

onists, mimicked the inhibitory effect of VIP on IL-8

production (Fig. 1). Therefore, we determined the effect

of a specific VPAC1 antagonist and of the PKA inhib-

itor H89 on the changes induced by VIP on NF-kB-

dependent IL-8 gene activation. The VIP inhibition ofLPS-induced IL-8 promoter activation is completely

reversed by the VPAC1 antagonist and only slightly

reversed by H89 (Fig. 4A). In contrast, the PAC1/

VPAC2-antagonist PACAP6–38 and the PKC inhibitor

calphostin C did not affect the VIP effect (IL-8 promoter

activation for LPS+VIP treated monocytes in the pres-

ence of 10�6 M PAC1/VPAC2-antagonist or 100 ng/ml

calphostin C was 11 � 3 or 13 � 2 fold induced, re-spectively). In addition, forskolin (a cAMP-inducing

agent) affects only slightly IL-8 gene activation (Fig.

4A). These results suggest that whereas the effect of VIP

on IL-8 transcriptional activity is entirely VPAC1-de-

pendent, the effects are mediated primarily through a

cAMP-independent pathway. This is in agreement with

previous reports describing that, in peripheral blood

monocytes, cAMP-inducing agents do not significantlyaffect IL-8 production [24,51–53]. When NF-jB and

p65/cRel DNA binding were analyzed, we found again

that the VPAC1 antagonist completely reversed the in-

hibitory effect of VIP and that H89, even at the highest

concentration, reversed these effects only slightly (Figs.

4B and C). This correlates with the fact that forskolin

inhibits only weakly NF-jB binding to the IL-8

Fig. 3. VIP changes the composition of the nuclear factors bound to

the IL-8 promoter. Nuclear extracts prepared from THP-1 cells incu-

bated for 2 h with LPS (100 ng/ml) in the presence or absence of VIP

(10�8 M) were added to a biotinylated oligonucleotide spanning the

proximal region of the human IL-8 promoter ()162 to +44). The

bound proteins were identified by Western blotting using the indicated

antibodies. One representative experiment from three is shown.

280 M. Delgado, D. Ganea / Biochemical and Biophysical Research Communications 302 (2003) 275–283

promoter (Fig. 4B). Therefore, the major pathway for

the inhibition of p65–cRel DNA binding by VIP is

mainly non-cAMP mediated. In contrast, the VPAC1antagonist and the PKA inhibitor completely reversed

the inhibitory effect of VIP on the binding of TBP and

CBP to the IL-8 promoter, and forskolin mimicked the

effect of VIP (Fig. 4C), suggesting the involvement of the

cAMP/PKA pathway. Therefore, in contrast to the VIP

effect on p65 and cRel, which is cAMP-independent, the

reduction in CBP and TBP bound to the IL-8 promoter

is mediated by increases in intracellular cAMP. This is inagreement with a previous report [35], in which we

demonstrated that VIP inhibits NF-jB transactivationin THP1 cells through three molecular mechanisms.

First, VIP inhibits the nuclear translocation of p65,

primarily through the cAMP-independent stabilization

of IkB. Second, increases in intracellular cAMP led to

the phosphorylation of CREB, and subsequent seques-

tration of CBP, resulting in a decrease in CBP-p65

complexes. Third, VIP reduces TBP phosphorylation

(required for the recruitment of RNA polymerase II),through a cAMP-dependent inhibition of the MEKK1/

MEK6/p38 MAPK pathway. The MAPK p38 pathway

has been indeed shown to play a regulatory role in IL-8

production [54,55]. Similar to VIP, b-adrenergic agon-

ists inhibit IL-8 production by LPS-stimulated THP1

cells, involving increases in cAMP levels [56]. In addi-

tion, adenosine that increases intracellular cAMP

through stimulation of adenylate cyclase inhibits IL-8production in monocytes [57], although the exact

molecular mechanisms remain to be elucidated. The

involvement of both cAMP-dependent and cAMP-

independent pathways was shown previously for the

inhibitory effect of VIP on TNFa, IL-12, chemokine,

and nitric oxide production in macrophages (reviewed in

[11–15]), on TNFa production in monocytes [35], and

on IL-2 and IL-10 production in lymphocytes [58].However, the relative involvement of these pathways in

the later cases and in the IL-8 production is different.

Whereas the effect of VIP on TNFa, IL-12, chemokines

and nitric oxide production is mediated primarily

through the cAMP-dependent pathway, the major me-

diator in the inhibition of IL-8 production is cAMP-

independent. The nature of the cAMP-independent

transduction pathway remains to be determined.

Acknowledgments

We thank Dr. Patrick Robberecht (Universite Libre de Bruxelles,

Brussels, Belgium) for the VPAC1 agonist and antagonist, Drs. David

Bolin and Ann Welton (Hoffmann-LaRoche, Nutley, NJ) for the

VPAC2 agonist Ro 25-1553, and Dr. Ethan Lerner (Massachusetts

General Hospital, Charlestown, MA) for the PAC1 agonist maxadilan.

This work was supported by grants PHS AI 041786-03 (D.G.), by

Grant PM98-0081 (M.D.), and by the postdoctoral fellowships from

the Spanish Department of Education and Science and Johnson &

Johnson (M.D.).

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