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(Journal of Leukocyte Biology. 2000;68:693-699.)
© 2000 by Society for Leukocyte Biology

{alpha}-Melanocyte-stimulating hormone peptides inhibit HIV-1 expression in chronically infected promonocytic U1 cells and in acutely infected monocytes

Wilma Barcellini*, Gualtiero Colombo{dagger}, Letteria La Maestra*, Giuliana Clerici*, Letizia Garofalo{dagger}, Anna T. Brini{ddagger}, James M. Lipton§ and Anna Catania{dagger}

Divisions of
* Hematology and
{dagger} Internal Medicine, Ospedale Maggiore di Milano IRCCS, 20122 Milano, Italy;
{ddagger} Department of Pharmacology, Chemotherapy, and Toxicology, University of Milan, 20133 Milano, Italy; and
§ Zengen, Inc., Woodland Hills, California

Correspondence: Anna Catania, III Divisione di Medicina Generale (Pad. Granelli), Ospedale Maggiore di Milano IRCCS, Via F. Sforza 35, 20122 Milano, Italy. E-mail: Anna.Catania{at}unimi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of the present research was to determine if {alpha}-melanocyte-stimulating hormone ({alpha}-MSH) and its C-terminal tripeptide [{alpha}-MSH (11–13), KPV] alter HIV expression in infected cells. The results indicate that chronically HIV-1-infected promonocytic U1 cells produce {alpha}-MSH and that immunoneutralization of the endogenous peptide enhances HIV expression. Because U1 cells express the {alpha}-MSH receptor 1 (MC1R), an autocrine-inhibitory circuit based on the peptide and its receptor likely occurs in these cells. To determine effects of pharmacological concentrations of {alpha}-MSH peptides on HIV expression, we measured p24 antigen release by TNF-{alpha}-stimulated U1 cells exposed to a wide range of concentrations of synthetic {alpha}-MSH and KPV. Viral expression was reduced by both peptides. KPV also effectively reduced HIV replication in acutely infected monocyte-derived macrophages (MDM). The basis of the peptide influence on viral replication is at the transcriptional level; KPV inhibited activation of NF-{kappa}B that is known to enhance viral expression. Endogenous {alpha}-MSH likely contributes to natural defense against HIV. However, greater concentrations of synthetic peptide are much more effective in reducing HIV expression in infected cells.

Key Words: melanocortin peptides • melanocortin receptor 1 (MC1R) • nuclear factor {kappa}B (NF-{kappa}B)


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}-Melanocyte-stimulating hormone [{alpha}-MSH (1–13)] is an endogenous neuroimmunomodulatory, anti-inflammatory peptide [1 , 2 ] derived from pro-opiomelanocortin (POMC) by post-translational processing [1 ]. POMC is expressed widely in peripheral tissues and within the brain, although there are distinct regional differences in expression and post-translational processing [1 ]. Certain cells, including keratinocytes, monocytes, and melanocytes, produce, constitutively or under appropriate stimulation, greater amounts of {alpha}-MSH [2 ]. Influences of {alpha}-MSH are mediated through binding to melanocortin receptors. Five G-protein-linked melanocortin receptors (MC1R–MC5R) have been isolated and cloned [3 ]. They recognize {alpha}-MSH and other melanocortins, such as adrenocorticotropic hormone, with different affinities [3 ]; the MC1R has the greatest affinity for {alpha}-MSH.

The anti-inflammatory effects of {alpha}-MSH are exerted mainly via inhibition of production of inflammatory mediators such as tumor necrosis factor {alpha} (TNF-{alpha}) and nitric oxide [2 ]. Inflammatory activity of macrophages [4 5 6 ] and glial cells [7 ] is modulated via an endogenous circuit that depends on {alpha}-MSH and melanocortin receptors. Macrophages secrete {alpha}-MSH and express the melanocortin receptor MC1R [4 5 6 ]; incubation of macrophages with an antibody to MC1R promotes TNF-{alpha} production [6 ]. Similarly, blockade of endogenous {alpha}-MSH by immunoneutralization increases production of proinflammatory cytokines and nitric oxide in microglia [7 ]. At the cellular level, {alpha}-MSH prevents degradation of I{kappa}-B and, consequently, inhibits activation of nuclear factor-{kappa}B (NF-{kappa}B) and NF-{kappa}B-mediated transcription [8 , 9 ]. The anti-inflammatory "message sequence" of {alpha}-MSH resides in the C-terminal tripeptide [{alpha}-MSH (11–13), KPV] that exerts anti-inflammatory effects in vitro and in animal models of inflammation similar to those of the entire (1–13) sequence [1 , 2 , 10 ]. Although generally, KPV was less potent than {alpha}-MSH (1–13) [1 ], it is promising particularly for therapeutic use in that it is smaller, less expensive, and chemically more stable than the parent molecule.

Anticytokine influences of {alpha}-MSH and KPV and their inhibitory effect on NF-{kappa}B activation suggest that the peptides might have anti-HIV properties. Replication of HIV is dependent on the state of activation of infected cells and is regulated by interactions between viral and host factors [11 ]. Among the latter, proinflammatory cytokines have a prominent enhancing effect on HIV replication [12 ]. TNF-{alpha} [13 14 15 ] and other cytokines, such as interleukin 1 (IL-1) [13 ] and IL-6 [16 ], promote HIV replication and have detrimental influences on HIV disease progression [17 ]. Further, the transcription factor NF-{kappa}B is a central mediator in cytokine activation of HIV transcription. TNF-{alpha} stimulates HIV transcription through activation of NF-{kappa}B, which, in turn, binds {kappa}B sequences present in the HIV long terminal repeat (LTR) [13 ].

The purpose of the present research was to learn whether {alpha}-MSH and its C-terminal tripeptide KPV, which inhibited proinflammatory cytokine production in whole blood of HIV-infected patients [18 ], reduce HIV expression likewise in infected cells. To do so, we used two models of HIV infection in vitro: chronically infected monocytic U1 cells and acutely infected monocyte-derived macrophages. U1 cells, which were derived from U937 cells surviving acute HIV-1 infection, are an in vitro model of latent HIV infection in monocytes [19 ]. HIV is present as two integrated proviral copies, and constitutive expression is very low [19 ]. Viral expression can be upregulated by different stimuli including phorbol esters and certain cytokines such as TNF-{alpha}, IL-6, and IL-10 [15 , 16 , 20 ]. Therefore, U1 cells are an appropriate model to investigate influences of agents that modulate HIV expression and replication [21 ]. However, this model does not reproduce natural infection entirely. Primarily, upregulation of HIV does not lead to production of infecting virus [19 ]. Because of differences from naturally infected phagocytes, we investigated the effects of KPV also in acutely HIV-infected monocyte-derived macrophages (MDM) [21 ] that represent more closely the circumstance in HIV infection. The specific aims were to determine 1) whether there is endogenous production of {alpha}-MSH (1–13) and autocrine effects of the peptide on HIV expression in the chronically HIV-1 infected U1 clone; and 2) the influence of treatment with the {alpha}-MSH derivative KPV on HIV expression in chronically and acutely infected monocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides
{alpha}-MSH (1–13) SYSMEHFRWGKPV and (11–13) KPV, N-acetylated and C-amidated, were kindly provided by Dr. Renato Longhi, CNR, Milano, Italy.

Cell cultures and treatments
The chronically HIV-1-infected promonocytic U1 cell line was maintained in complete culture medium RPMI 1640 supplemented with 10 mM HEPES, 2 mM L-glutamine (Sigma-Aldrich, Milwaukee, WI), 10% heat-inactivated fetal calf serum (FCS; HyClone Laboratories, Logan, UT), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco BRL, Grand Island, NY) in log phase of growth. Pilot experiments were performed to determine optimal cell density, stimuli concentration, and kinetics of HIV-1 p24 antigen production in our culture conditions. Before use, cells were washed three times with Hanks’ balanced saline solution (HBSS; Gibco BRL) to remove extracellular virus. Cells were plated onto 24-well flat-bottomed plates at a concentration of 2 x 105/mL (final vol, 1 mL) with medium alone or plus TNF-{alpha} (10 ng/mL; R&D Systems, Oxford, UK) in the presence or absence of {alpha}-MSH (1–13) or KPV in concentrations from 10-13 to 10-4 M. In further experiments, KPV was added in the 10-5 M concentration to U1 cells stimulated with TNF-{alpha} (10 ng/mL), IL-6 (20 ng/mL), IL-10 (20 ng/mL; R&D Systems), or PMA (1 ng/mL; Sigma-Aldrich). Supernatants were collected by centrifugation after 48 h incubation at 37°C in 5% Co2. In crowding experiments, U1 cells were seeded at the density of 2 x 105/mL and maintained in culture at 37°C in 5% CO2 without changing medium for 7 days. KPV (10-5 M), or an equal vol of medium, were added on day 1. All experiments were repeated in at least three independent tests, and each condition was tested in triplicate.

Endogenous production and immunoneutralization of {alpha}-MSH
{alpha}-MSH production of U1 cells was determined by measuring concentrations of the peptide in supernatants from cells seeded as described above for 48 h in the presence of medium alone or with phorbol 12-myristate 13-acetate (PMA; 1 ng/ml). In immunoneutralization experiments, an affinity-purified rabbit anti-{alpha}-MSH antibody (Euro-Diagnostica, Malmö, Sweden), diluted 1:250 with medium, was used for blocking the influence of {alpha}-MSH produced by U1 cells. Rabbit immunoglobulin G (IgG) at the same dilution was used as control. Antibody-treated cells were coincubated with medium or PMA (1 ng/ml). After 48 h incubation, supernatants were separated and tested for p24 antigen release. In crowding experiments, the anti-{alpha}-MSH antibody or the control IgG was added on day 1, and the supernatants were harvested on day 7. {alpha}-MSH was measured with a competitive radioimmunoassay kit (Euro-Diagnostica). The detection limit was 0.9 pmol/mL.

Northern blot analysis of HIV-1 expression
U1 cells (20x106, at a density of 2x105/mL in complete medium) were incubated with medium alone or stimulated with PMA (1 ng/mL) for 24 h, in the presence or absence of KPV 10-5 M. Total RNA was prepared using an extraction kit based on the guanidine isothiocyanate phenol method (Tripure, Boehringer Mannheim, Indianapolis, IN), according to the manufacturer’s instructions. Total RNA (10 µg) was resolved on a denaturing 0.8% agarose/formaldehyde gel, transferred onto a nylon membrane, and hybridized for 18 h at 65°C to a [{alpha}-32P]dCTP-labeled, HIV, full-length probe (kind gift of L. Turchetto and E. Vicenzi, S. Raffaele Hospital, Milan, Italy). Probe was labeled by random priming using the Ready-To-Go labeling kit (Amersham Pharmacia Biotech, Sunnyvale, CA). Following hybridization, filters were washed twice in 1 x saline sodium citrate (SSC), 0.1% (w/v) sodium dodecyl sulfate (SDS) at room temperature for 10 min, twice in 0.1 x SSC, 0.1% (w/v) SDS at 65°C for 10 min, and then exposed to X-ray film for 5 days. After removal of the HIV full-length probe, filters were rehybridized with [{alpha}-32P]dCTP-labeled, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe for quantity normalization. Densitometric analysis was performed using ImageMaster VDS 3.0 software (Amersham Pharmacia Biotech).

MC1R gene expression
Total RNA extracts from U1 cells, unstimulated or stimulated with PMA, were used for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of {alpha}-MSH receptor (MC1R) mRNA expression also. To overcome possible misinterpretation as a result of genomic contamination (MC1R gene lacks introns), total RNA was treated with RNase-free DNase I (Boehringer Mannheim) for 30 min at 37°C. DNase I was then inactivated by phenol-chloroform extraction. First-strand cDNA synthesis was performed, using 1 µg of each RNA sample, 0.2 µM random primers, and 200 U Avian murine virus RT (Boehringer Mannheim) in a 20-µl reaction vol. PCR amplification was performed on a portion (10%) of each cDNA mixture in a 50-µl reaction vol containing 0.8 µM upstream and downstream primer, 2 U AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems, Norwalk, CT), 0.2 mM each deoxynucleoside triphosphate, 1.5 mM MgCl2. The MC1R primer pair, forward 5'-GCCACCATCGCCAAGAACC-3' and reverse 5'-ATAGCCAGGAAGAAGACCA-3', generated a 416-bp product. To minimize nonspecific amplification, we used a hot-start procedure by adding the Taq DNA polymerase to PCR mixtures prewarmed to 80°C. The PCR temperature profile consisted of 35 cycles of 45-sec denaturation at 94°C, 45-sec annealing at 57°C, and 1-min extension at 72°C, followed by a 7-min final elongation at 72°C. All PCR products were analyzed by 2% agarose gel electrophoresis. A GAPDH primer pair, forward 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and reverse 5'-CATGTGGGCCATGAGGTCCACCAC-3', generating a 980-bp product, was used for normalization.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from 20 x 106 U1 cells, seeded at 2 x 105/mL in complete medium and stimulated for 4 h with TNF-{alpha} (20 ng/mL) in the presence or absence of 10-5 M KPV. Briefly, cells were washed once with cold phosphate-buffered saline (PBS) and twice with buffer A [10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM dithiothreitol (DTT)] and were incubated for 10 min on ice in buffer A plus 0.1% Nonidet-P40 (Sigma-Aldrich). Tubes were then mixed vigorously on a vortex machine for 10 s, and the homogenates were centrifuged at 4°C for 10 min at maximum speed in a microfuge. Supernatants were removed, and nuclear pellets were resuspended in 15 µl of buffer C [20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.42 M KCl, 0.2 mM ethylenediaminetetraacetate (EDTA), 25% glycerol, 0.5 mM PMSF, and 0.5 mM DTT], incubated for 15 min on ice, mixed, and centrifuged as above. Then, supernatants were diluted with 75 µl buffer D (20 mM HEPES, pH 7.9, 0.05 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM PMSF, and 0.5 mM DTT) and stored at -80°C for gel-retardation assay.

The binding reaction was carried out by incubating 10 µg of nuclear protein extract and 0.5 ng of 32P end-labeled (30,000 cpm/µl), 35-mer, double-stranded, NF-{kappa}B consensus oligonucleotide for 15 min at room temperature in a binding buffer (12 mM Tris-HCl, pH 7.8, 60 mM KCl, 0.2 mM EDTA, 0.3 mM DTT, plus 5% glycerol, and 2 µg/mL bovine serum albumin), including 1 µg/mL ssDNA. In this study, we used a sense oligonucleotide for NF-{kappa}B, 5'-GATCCAAGGGGACTTTCCGCTGGGGACTTTCCATG-3' (the NF-{kappa}B consensus binding sites are underlined), and an antisense one, 5'-GATCCATGGAAAGTCCCCAGCGGAAAGTCCCCTTG-3', to examine the specificity of binding of NF-{kappa}B to the DNA. Each oligonucleotide was annealed to its complementary strand and end-labeled with [{gamma}-32P]dATP (Amersham Pharmacia Biotech) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Specificity of the binding reaction was determined in a competition assay also: Nuclear extracts were first incubated with 100-fold molar excess of unlabeled NF-{kappa}B or activator protein 1 consensus probe for 5 min and then with the labeled probe. Reaction products were separated on a 5% (30:1) native polyacrylamide gel in 1 x Tris-borate-EDTA buffer. Gels were dried and exposed to film for autoradiography (3 days).

p24 and RT determinations
p24 antigen release (Cellular Products, Inc., Buffalo, NY) and RT activity (RetroSys RT assay, Innovagen, Lund, Sweden) were determined using commercial ELISA kits. Measure of RT activity in the sample is based on immunoenzymatic quantification of bromo-deoxyuridine triphosphate (BrdUTP) incorporated in newly synthesized DNA.

Acute infection of MDM
Human peripheral blood mononuclear cells (PBMC) were isolated from normal donors by Ficoll-Hypaque density-gradient centrifugation. Monocytes were isolated by Percoll gradient separation and allowed to differentiate into macrophages by seeding them in complete RPMI medium with 20% FCS in 24-well tissue-culture plates at 106 cells/mL density for 7 days. MDM were infected with a monocytotropic HIV-1Ba-L strain (1:10). The undiluted viral stock contained 107 infectious U/mL. After 24 h, MDM were washed and maintained in complete medium for 3 weeks; medium was replaced three times a week. RT activity was measured weekly postinfection. KPV (10-5 M) was added at the time of HIV infection and daily until harvest.

Statistical analysis
All values are given as mean ± SE. Comparison of group means that it was performed using analysis of variance (ANOVA) of ranks followed by Dunn’s test for specific comparisons. Two sample comparisons were performed using Mann-Whitney rank-sum test. Probability values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Influence of endogenous {alpha}-MSH on HIV expression in chronically infected U1 cells
Supernatants of resting and stimulated U1 cells were analyzed for production of {alpha}-MSH. There was a small but consistent production of peptide after 48 h culture in unstimulated conditions (5.2±0.3 pmol/mL). Further, when cells were coincubated with PMA, {alpha}-MSH in the supernatants was increased two-fold (to 12.90±0.42 pmol/mL). To determine effects of blockade of endogenous {alpha}-MSH on HIV replication, the peptide was immunoneutralized with a specific anti-{alpha}-MSH antibody. p24 antigen was measured in the supernatants from resting cells and from those exposed to PMA or in crowding conditions. In cells incubated with the anti-{alpha}-MSH antibody, there was a substantial increase in p24 release under unstimulated and crowding conditions and after stimulation with PMA (Fig. 1 ). Immunoneutralization of endogenous peptide causes a 100% increase in p24 release by resting U1 cells and a 25–30% increase in stimulated cells. The irrelevant IgG did not alter p24 release in any condition. {alpha}-MSH receptor MC1R gene expression was determined in resting and PMA-stimulated U1 cells. In both conditions, a PCR product specific for MC1R with the expected length of 416 bp was detected (Fig. 2 ).



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  		Figure 1. Effect of immunoneutralization of endogenous {alpha}-MSH on p24 release by U1 cells. Immunoneutralization of endogenous {alpha}-MSH increased p24 release by U1 cells in resting and crowding conditions and after stimulation with PMA. In this and the following figures, bars represent the mean ± SE. *p < 0.05; **p < 0.01.

 


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  		Figure 2. MC1R expression in U1 cells. In resting and PMA-stimulated cells, a PCR product specific for MC1R with the expected length of 416 bp was detected.

 
Influence of {alpha}-MSH peptides on HIV expression in chronically infected U1 cells and in acutely infected MDM
{alpha}-MSH (1–13) and the tripeptide KPV significantly inhibited p24 release from TNF-{alpha}-stimulated U1 cells (Fig. 3 ). Inhibitory effects of {alpha}-MSH occurred over a broad range of peptide concentrations including picomolar concentrations that occur naturally in human plasma [22 ]. These peptide concentrations inhibited p24 release significantly (34–36%), suggesting that the small amounts of endogenous {alpha}-MSH present in the circulation normally inhibit HIV expression. Low concentrations of KPV were less effective relative to the parent molecule. However, greater concentrations caused pronounced HIV inhibition, with the most effective concentration for both peptides being 10-5 M. In this concentration, {alpha}-MSH (1–13) and KPV caused 52.7% and 56.0% inhibition of p24 release, respectively.



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  		Figure 3. Effect of treatment with synthetic {alpha}-MSH (1–13) or KPV on p24 release by TNF-{alpha}-stimulated U1 cells. Both {alpha}-MSH peptides inhibited p24 release over a broad spectrum of concentrations.

 
On the basis of these parallel effects, and because KPV possesses advantages over {alpha}-MSH (1–13) in terms of cost and absorption, we elected to use the tripeptide in the highly effective 10-5 M concentration in further tests on HIV replication. These tests were designed to determine the scope of inhibitory influences of KPV on HIV expression. Therefore, HIV expression was induced with different stimuli already known to upregulate the virus in promonocytic U1 cells such as IL-6, IL-10, PMA, and crowding condition [12 ]. KPV inhibited significantly p24 and RT release from U1 cells induced by all these stimuli (Fig. 4 ).



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  		Figure 4. Effect of KPV on RT and p24 release by stimulated U1 cells. Treatment with KPV (10-5 M) inhibited RT and p24 release from U1 cells exposed to different stimuli.

 
The inhibitory activity of KPV on HIV expression was confirmed by Northern blot analysis of HIV-RNA in PMA-stimulated U1 cells (Fig. 5 ). PMA was used because it determined the highest stimulation of p24 release in the present and in previous experiments on HIV expression in U1 cells [19 ]. Addition of KPV reduced by approximately 50% spliced and unspliced HIV-1 RNA in PMA-stimulated cells.



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  		Figure 5. Effect of treatment with KPV on HIV RNA in resting and PMA-stimulated U1 cells. Addition of KPV (10-5 M) reduced spliced and unspliced HIV-1 RNA by ~50% in PMA-stimulated U1 cells.

 
The U1 cell clone provides an in vitro model of latent HIV infection, in which induction of viral replication does not lead to production of infecting virus [19 ]. Therefore, we investigated effects of KPV also in acutely infected MDM, which are a more realistic model of productive HIV infection [21 ]. Treatment with the tripeptide inhibited RT release significantly in acutely infected MDM (Fig. 6 ). This inhibitory effect was more pronounced on day 6 but was still statistically significant on day 21.



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  		Figure 6. Effect of treatment with KPV on HIV replication in acutely infected MDM. Treatment with the tripeptide (10-5 M) inhibited RT release significantly from acutely HIV-infected MDM. Inhibitory effect was more pronounced on day 6 (p<0.01) but was still statistically significant on days 13 and 21 (p<0.05).

 
NF-{kappa}B DNA-binding activity in U1 cells
The transcription factor NF-{kappa}B is a central mediator in cytokine activation of HIV transcription. TNF-{alpha} stimulates HIV transcription through activation of NF-{kappa}B, which, in turn, binds {kappa}B sequences present in the HIV LTR [13 ]. Therefore, we determined the effect of KPV on NF-{kappa}B DNA binding in U1 cells. TNF-{alpha} treatment greatly enhanced NF-{kappa}B DNA-binding activity, and coincubation of cells with 10-5 M peptide significantly reduced NF-{kappa}B activation (Fig. 7 ). The tripeptide did not alter NF-{kappa}B activation in resting cells.



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  		Figure 7. Effect of treatment with KPV on NF-{kappa}B activation. KPV (10-5 M) markedly reduced NF-{kappa}B activation induced by TNF-{alpha} in U1 cells. There was no change in NF-{kappa}B activation in resting cells treated with the tripeptide.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results indicate that endogenous {alpha}-MSH has anti-HIV effects in chronically infected promonocytic U1 cells. Indeed, U1 cells produced {alpha}-MSH and expressed the gene for the {alpha}-MSH receptor MC1R, and immunoneutralization of the autocrine peptide enhanced HIV expression. Such antiviral influences based on endogenous {alpha}-MSH could be significant to host protection. That is, in phagocytes, which are the main reservoir of the virus [23 , 24 ], production and action of {alpha}-MSH could reduce viral burden. Further, low concentrations of synthetic {alpha}-MSH (1–13), comparable with those found in human plasma [22 ], caused substantial inhibition of HIV expression in U1 cells. Therefore, in addition to the autocrine peptide, circulating {alpha}-MSH, could contribute to reduce HIV replication. That circulating peptide may exert beneficial effects is suggested by our previous data showing that greater concentrations of {alpha}-MSH are associated with reduced disease progression or death in HIV-infected patients [25 ].

{alpha}-MSH is part of the host response to infection and inflammation. The peptide increases in the circulation of rabbits [26 ] and human subjects after endotoxin administration [27 ]. Immunoneutralization of central {alpha}-MSH augments the duration of fever in rabbits injected with endogenous pyrogen [28 ] and increases circulating TNF-{alpha} and nitric oxide (NO) in mice with endotoxin shock [29 ]. Our recent observation that the peptide has antimicrobial activity against two representative pathogens, the yeast Candida albicans and the Gram-positive bacterium Staphylococcus aureus [30 ], further supports the idea that this ancient peptide contributes to host defense and natural immunity. {alpha}-MSH, which occurs in high concentrations in barrier organs, such as the gut and the skin [1 ], appears to have broad antimicrobial effects like other natural antimicrobial peptides [31 ]. The present research indicates that {alpha}-MSH can contribute to host defense against HIV infection as well.

The hallmark of AIDS immunopathogenesis is a clear link between cellular activation and HIV production. Activated cells are preferential targets for viral infection and production [11 ]. HIV expression is increased significantly by agents such as TNF-{alpha} and other cytokines, and augmentation of viral expression in these circumstances maps to sequences included in the viral LTR that bind NF-{kappa}B [13 ]. NF-{kappa}B is retained in an inactive form in cytoplasm [32 ]; its prototypic form consists of a heterodimer of p50 and p65 that is normally bound by members of the I{kappa}B family, including I{kappa}B{alpha}. Activation of NF-{kappa}B requires degradation of the cytoplasmic inhibitor I{kappa}B{alpha} [32 ]. Phosphorylation of I{kappa}B by various agents, such as drugs, cytokines, bacterial products, and viruses, leads to I{kappa}B degradation and translocation of NF-{kappa}B to the nucleus [32 ]. Through LTR binding to NF-{kappa}B, HIV is thus linked to the state of activation of infected cells. Stimuli that activate NF-{kappa}B enhance HIV production [33 ]. In these circumstances, viral RNA increases, and the pattern of expression changes to include the singly spliced and unspliced messenger RNA transcripts encoding virion constituents. Spliced and unspliced HIV RNA was substantially reduced by treatment of PMA-stimulated U1 cells with KPV.

{alpha}-MSH reduces inflammation via three mechanisms of action [1 , 2 ]: direct action of the peptide on its receptors in peripheral inflammatory cells; modulation of brain inflammation via local influences of the peptide on its receptors in glial cells; and indirect effects on peripheral inflammation through descending anti-inflammatory neural pathways induced by stimulation of {alpha}-MSH receptors within the brain. All these effects of the peptide are partly exerted through inhibition of NF-{kappa}B. In the monocytic cell line U937, {alpha}-MSH downregulated NF-{kappa}B activation induced by a variety of inflammatory stimuli, including TNF, endotoxin, ceramide, and okadaic acid [8 ]. Suppression of NF-{kappa}B was mediated through generation of cAMP and activation of protein kinase A [8 ]. Consistent with previous results in noninfected cells, the present data show that {alpha}-MSH (11–13) inhibits NF-{kappa}B DNA binding also in U1 cells. Further, {alpha}-MSH and KPV modulated central nervous system (CNS) inflammation by inhibiting NF-{kappa}B activation in experiments on human glioma cells and whole mouse brains stimulated with lipopolysaccharide [9 ]. In both models of CNS inflammation, the evidence was consistent with {alpha}-MSH-induced modulation of NF-{kappa}B activation by limiting I{kappa}B{alpha} degradation. Finally, recent research showed that centrally administered {alpha}-MSH inhibits peripheral NF-{kappa}B activation by central action [34 ]. All these observations suggest that {alpha}-MSH could be a candidate for treatment of pathologic conditions in which activation of NF-{kappa}B is involved [8 , 32 ]. HIV infection is one such condition clearly. Indeed, the peptide, through inhibition of NF-{kappa}B, can reduce viral replication directly in peripheral and central phagocytes and in glial cells, which are the main reservoir of the virus. A third possible mechanism, perhaps the most exciting with regard to the host defense, is the inhibition of NF-{kappa}B activation and viral replication in peripheral cells through an action within CNS. Such a neuroimmunomodulatory circuit involving neural pathways could be significant in the host response to HIV infection.

Discovery of effective antiviral molecules has improved treatment of patients with HIV infection [35 ]. However, elevated costs of antiviral drugs, emergence of resistant viral strains, and disease relapse after treatment withdrawal remain unsolved problems [35 ]. Persistence of HIV transcription in PBMC of patients receiving antiretroviral therapy indicates that HIV infection is not eradicated with current treatments [36 ]. Therefore, therapies that reinforce specific anti-HIV treatments are actively sought; they could be beneficial in association with antiviral molecules targeting HIV genes. The present data show that the tripeptide KPV has anti-HIV properties in infected cells. If such anti-HIV properties are confirmed in vivo, the peptide could be used as an adjunctive therapy for HIV infection. The moderately scarce magnitude of KPV effects relative to more specific anti-HIV drugs and the biphasic pattern in the peptide influence should not discourage its use. {alpha}-MSH is a natural modulatory peptide, and its activity is likely regulated to avoid excessive inhibition. Therefore, it is reasonable to believe that a regulatory mechanism, perhaps receptor downregulation, preserves cells from excessive peptide effects. Such a regulatory mechanism might account for the biphasic curve observed in the present and in previous research on anti-inflammatory influences of {alpha}-MSH peptides [1 , 2 ]. Indeed, 30–50% inhibition of viral replication is not negligible, also considering that such inhibition would occur together with other potential beneficial effects, such as antimicrobial and anti-TNF activity.

Received March 19, 2000; revised May 30, 2000; accepted June 2, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Catania, A., Lipton, J. M. (1993) {alpha}-Melanocyte stimulating hormone in the modulation of host reactions Endocr. Rev. 14,564-576[Medline]
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