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*Vasculitis
Endocrinology Vol. 144, No. 1 360-370
Copyright © 2003 by The Endocrine Society

ARTICLE

{alpha}-Melanocyte Stimulating Hormone Prevents Lipopolysaccharide-Induced Vasculitis by Down-Regulating Endothelial Cell Adhesion Molecule Expression

T. E. Scholzen, C. Sunderkötter, D.-H. Kalden, T. Brzoska, M. Fastrich, T. Fisbeck, C. A. Armstrong, J. C. Ansel and T. A. Luger

Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin (T.E.S., D.-H.K., T.B., M.F., T.F., T.A.L.), Institute of Experimental Dermatology (C.S.), Department of Dermatology (C.S., T.A.L.), University of Münster, 48149 Münster, Germany; and Department of Dermatology (C.A.A., J.C.A.), Northwestern University, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Dr. Thomas Scholzen, Ph.D., University of Münster, Department of Dermatology, Ludwig Boltzmann Institute for Cell and Immunobiology of the Skin, Von-Esmarch-Strasse 58, 48149 Münster, Germany. E-mail: thoscho{at}uni-muenster.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neuroendocrine hormone {alpha}-melanocyte stimulating hormone (MSH) has profound antiinflammatory and immunomodulating properties. Here we have examined the possibility that {alpha}-MSH may interfere with the expression and function of cell adhesion molecules (CAMs) expressed by human dermal microvascular endothelial cells (HDMECs) in response to lipopolysaccharide (LPS) or TNF{alpha} in vitro and in vivo. In HDMEC, {alpha}-MSH (10-8/10-12 M) profoundly reduced the mRNA and protein expression of E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 induced by LPS or TNF{alpha} as determined by semiquantitative RT-PCR, ELISA, and fluorescence-activated cell sorter analysis. In addition, {alpha}-MSH significantly impaired the LPS-induced ICAM-1 and VCAM-1-mediated adhesion of lymphocytes to HDMEC monolayer in a functional adhesion assay. Likewise, {alpha}-MSH effectively inhibited the transcription factor nuclear factor-{kappa}B activation in HDMEC, which is required for CAM gene expression. Importantly in vivo, in murine LPS-induced cutaneous vasculitis (local Shwartzman reaction), a single ip injection of {alpha}-MSH significantly suppressed the deleterious vascular damage and hemorrhage by inhibiting the sustained expression of vascular E-selectin and VCAM-1. This persistent expression has been implicated in the dysregulation of diapedesis and activation of leukocytes, which subsequently leads to hemorrhagic vascular damage. Our findings indicate that {alpha}-MSH may have an important therapeutical potential for the treatment of vasculitis, sepsis, and inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROOPIOMELANOCORTIN (POMC)-derived tridecapeptide {alpha}-MSH is a neuropeptide involved in a variety of biological processes, such as pigmentation (1), body weight regulation (2), and inflammation (3, 4, 5). It is now well established that {alpha}-MSH participates in the regulation of immune responses by impairing important functions of both antigen-presenting cells as well as T cells (5), and several lines of evidence indicate that {alpha}-MSH has a variety of antiinflammatory activities (6, 7, 8, 9, 10).

POMC peptides such as {alpha}-MSH exert their biological activities through the activation of a family of five different G protein-coupled melanocortin receptors (MC-Rs) (11). The MC-1R, which is capable of binding {alpha}-MSH with high affinity in humans and rodents, is expressed on immune cells such as monocytes, macrophage cell lines, dendritic cells, and neutrophils. Many of the antiinflammatory and immunomodulatory activities of {alpha}-MSH such as suppression of fever induced by IL-1 or IL-6, induction of the antiinflammatory mediator IL-10, or inhibition of macrophage functions and leukocyte migration are mediated by the activation of this MC-R (9, 12, 13, 14).

Leukocyte-endothelial cell interaction is a multistep process involving rolling, firm adhesion, and finally transmigration into the target tissue. These events are tightly regulated by the consecutive expression of a set of cell adhesion molecules (CAMs) such as E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 as well as integrin counterparts expressed by leukocytes (15, 16). Endothelial cells (ECs) also participate in inflammatory events by synthesizing a repertoire of growth factors, cytokines, and chemokines. Increased endothelial CAM and cytokine or chemokine expression can be observed after exposure of cells to a variety of agents that include certain cytokines (17, 18, 19), endotoxin [lipopolysaccharide (LPS)], UV irradiation (20, 21), or neuropeptides (22, 23).

Recruitment of the inflammatory infiltrate is the main function of endothelial CAMs, and several CAMs such as E-selectin or P-selectin can substitute for each other in case one is neutralized by antibodies or genetically deleted (24). However, in leukocytoclastic vasculitis (LcV), i.e. in leukocyte-induced destruction of postcapillary venules, E-selectin and VCAM-1 have been shown to have additional exclusive roles in mediating vessel damage (25). LcV occurs as isolated primary cutaneous disorder or as a symptom of some autoimmune and infectious diseases. Despite the different causes such as vascular deposition of immune complexes or activation by infection the common pathophysiological feature is a deleterious interaction between activated leukocytes and ECs of postcapillary venules during infiltration of leukocytes (24). The Shwartzman reaction (Shw-r), for example, has been shown to depend on a strong and sustained expression of E-selectin and VCAM-1, a condition not encountered in nonvasculitic inflammation (25, 26). Neutralization of these CAMs did not prevent recruitment of leukocytes but markedly suppressed the cellular events leading to vascular damage, which indicates their special role in this process.

We have previously demonstrated that human dermal microvascular endothelial cells (HDMECs) express the MC-1R (27). In addition, HDMECs are capable of producing POMC and releasing POMC peptides such as ACTH and {alpha}-MSH (28). To further elucidate the biologic role of HDMEC MC-1R and {alpha}-MSH in acute inflammation, we used an Shw-r animal model system of cutaneous vasculitis. Our studies indicate that {alpha}-MSH effectively prevented the deleterious vascular damage in this model of cutaneous inflammatory vasculitis. This therapeutic effect of {alpha}-MSH appears to be mediated in part by the down-regulation of CAM on dermal microvascular endothelial cells and the inhibition of HDMEC nuclear factor-{kappa}B (NF{kappa}B) activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
BALB/c mice were obtained from Charles River (Sulzfeld, Germany). Mice were housed in a special pathogen-free barrier facility with free access to water and food according to state and federal regulations. Males and females were studied at 10–14 wk with 18 mice per experimental condition. All experiments were approved by the state review board (Münster, G24/96, G46/93, G38/90).

Cell culture
HDMECs were obtained from PromoCell (Heidelberg, Germany). Cells were grown in endothelial cell basal medium, microvascular (EBM-MV kit system, PromoCell) supplemented with 5% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 0.4% (vol/vol) endothelial cell growth supplement/heparin, 1 µg/ml hydrocortisone with the antibiotics 50 µg/ml gentamicin, and 50 ng/ml amphotericin-B (growth media) in a humidified atmosphere at 37 C and 5% CO2. Experiments were conducted with cells in passage 3–6. HDMEC cultures were characterized by their typical cobblestone morphology using light microscopy and analysis for their capacity to express factor VIII-like antigen or CD31 (platelet endothelial CAM-1). The cell line HMEC-1 was cultured and maintained in endothelial basal media (SFM, Life Technologies, Inc., Grand Island, NY) supplemented with 10% FBS and antibiotics. Before stimulation, HDMEC or HMEC-1 were deprived from growth factors and FBS by culturing in stimulation medium (HDMEC: EBM-MV, no supplements except 0.5% FBS, and antibiotics; HMEC-1: SFM with 2% FBS plus antibiotics) overnight. Likewise, these media were used for stimulation. Human B-lymphoblastoid JY-1 cells and T-lymphoblastoid Molt-4 cells (generously provided by Dr. Jack Strominger, Dana Faber Cancer Institute, Boston, MA) were cultured in RPMI supplemented with 10% FBS, 100 U/ml penicillin, 0.25 mg/ml Amphotericin B, and 10 µg/ml streptomycin (Life Technologies, Inc.).

RNA isolation
Total RNA was isolated 3 h after stimulation using TRIzol reagent (Life Technologies, Inc.). To avoid genomic DNA contamination that may interfere with PCR amplification, total RNA was treated with 10 U ribonuclease-free deoxyribonuclease I (Roche Diagnostics, Mannheim, Germany) as described (28).

RT-PCR
Semiquantitative RT-PCR was conducted as described (28). One microgram RNA was subjected to reverse transcription using the Reverse Transcription System (Promega Corp., Madison, WI). PCR amplification was run in 50-µl reactions containing appropriate volumes of diluted cDNA, deoxynucleotide triphosphates (0.2 mM each), 1.5 mM MgCl2, 20 pM of each primer and the standard buffer supplemented with RedTaq polymerase (2.5 µl/reaction, Sigma, St. Louis, MO). Nucleotide sequences of PCR primers for human E-selectin, VCAM-1, and ICAM-1 were generated as previously published (29). The following amplification programs were used: 1) E-selectin (473-bp PCR fragment): 1 cycle of 94 C/5 min, 58 C/2 min, 72 C/3 min, 28 cycles of 94 C/1 min, 58 C/2 min, 72 C/3 min, and 1 cycle of 94 C/1 min, 58 C/2 min, and 72 C/7 min; 2) VCAM-1 (451-bp PCR product): 1 cycle of 94 C/5 min, 57 C/2 min, 72 C/3 min, 28 cycles of 94 C/1 min, 57 C/2 min, 72 C/3 min, and 1 cycle of 94 C/1 min, 57 C/2 min, and 72 C/7 min; and 3) ICAM-1 (446-bp PCR product): 1 cycle of 94 C/5 min, 60 C/2 min, 72 C/3 min, 28 cycles of 94 C/1 min, 60 C/2 min, 72 C/3 min, and a final cycle of 94 C/1 min, 60 C/2 min, and 72 C/7 min. ß-Actin was amplified using primers and an amplification program as described (28). Aliquots of reaction products were run on 1.5% agarose gels and analyzed by product size, compared with a coamplified control template.

Quantification of PCR products
E-selectin, VCAM-1, or ICAM-1 mRNA expression relative to ß-actin was quantified as previously described (28). Amplification fragments were separated on 1.5% agarose gels, and the signal intensity of the target PCR product was compared with that of a ß-actin PCR product amplified from the same cDNA in a separate PCR. Densitometrical evaluation was performed using an image analysis and photo documentation system (transilluminator hood/Pieper FK75121Q-IR CCD video camera connected to a PC equipped with a frame grabber video card; Biostep GmbH, Jahnsdorf, Germany) with Phoretix Grabber 3.01 and Phoretix Totallab 1.00 image processing and analyzing software (Nonlinear Dynamics, Newcastle upon Tyne, UK). Densitometer readings of target-specific PCR products were normalized to the ß-actin product density in the respective sample. Subsequently, the density of the target product amplified from cDNA prepared from stimulated cells was related to that of unstimulated control cells at any given time point. Unless stated otherwise, results from three different experiments were taken and expressed in percent of control as mean ± SEM with the density of unstimulated controls set to 100% for each time point analyzed.

Determination of HDMEC or HMEC-1 adhesion molecule expression
Cells grown in 96-well plates were either left untreated or stimulated for 4–16 h with LPS (100 ng/ml) alone or in combination with {alpha}-MSH (10-8/10-12 M). HDMEC or HMEC-1 expression of E-selectin and VCAM-1 was assessed by a two-step whole-cell ELISA as described (22) using mouse antihuman E-selectin or VCAM-1 monoclonal antibodies (mAbs) (BD Biosciences Europe, Heidelberg, Germany) followed by a peroxidase-conjugated goat-antimouse IgG and detection by 3,3',5,5'-tetramethylbenzidine colorimetric reaction. Results are expressed as relative OD read at 450 nm ± SEM with unstimulated cells set to 1. Alternatively, HDMECs were stimulated as above, detached from culture dishes using accutase (PAA Laboratories, Linz, Austria), stained with mouse antihuman VCAM-1 mAb (clone B-K9, Diaclone Research, Besançon, France) followed by fluorescein isothiocyanate-conjugated antimouse IgG1 Ab (clone A85-1; BD Biosciences) or a fluorescein isothiocyanate-conjugated antihuman ICAM-1 mAb (Immunotech, Marseille, France) and subjected to fluorescence-activated cell sorter (FACS) analysis (EPICS XL, Coulter, Miami, FL). FACS data were calculated from positively gated cells in percent as change in mean fluorescence intensity (MFI) = [MFI (TNF or LPS+MSH)] - [MFI (control)]/[MFI (TNF or LPS)] - [MFI (control)] ± SEM, n = 3.

EMSAs
EMSAs were performed as described (30). Briefly, HDMECs were plated at a density of 25,000 cells/cm2 and either left untreated or stimulated with LPS alone (100 ng/ml) or in combination with {alpha}-MSH (10-8/10-12 M). After stimulation, nuclear proteins were extracted, the binding reactions were carried out by adding 2 µg poly(deoxyinosine-deoxycytidine) and 104 cpm of 32P-labeled, ds NF{kappa}B oligonucleotide (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 5'-AGT TGA GGG GAC TTT CCC AGG C-3') to 7.5 µg of the extracted nuclear proteins followed by incubation at 22 C for 30 min. Reaction samples were separated electrophoretically on native high ionic gels at 150 V for 1.5 h and subjected to autoradiography. Competition analysis was performed by adding a 40-M excess of unlabeled {kappa}B oligonucleotide. Supershift analysis was performed by adding anti-p75/cRel, anti-p68/RelB, anti-p65/Rel A, or anti-p50 Ab (Santa Cruz Biotechnology, Inc.) to the binding reaction.

Western blotting
Cells were lysed in Margolis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton-X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM disodium pyrophosphate, 0.01% NaN3, and Complete protease inhibitor cocktail for 15 min on ice. After centrifugation, supernatants were collected, and protein content measured by a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). The protein samples were subjected to 10% SDS-PAGE, blotted on nitrocellulose membranes, and incubated with antibody directed against I{kappa}B{alpha} (Upstate Biotechnology, Inc., Lake Placid, NY). Equal loading was monitored by reprobing membranes with an antibody directed against {alpha}-tubulin (Calbiochem, San Diego, CA). Signals were detected with an ECL-kit (Amersham, Buckinghamshire, UK). Membranes were exposed to X-OMAT film (Eastman Kodak Co., Rochester, NY) and subjected to densitometrical analysis.

Adherence of Molt-4 or JY-1 lymphoblastoid cells to HDMECs
Binding assays measuring cellular adherence to HDMECs were performed as described (22) using human T-lymphoblastoid Molt-4 or B-lymphoblastoid JY-1 cells. HDMECs (35,000 cells/cm2) were plated in 24-well plates, deprived overnight, and then either left untreated or treated for 16 h in quadruplicate with {alpha}-MSH (10-8/10-12 M) in combination with 100 ng/ml LPS (Sigma) or TNF{alpha} (10 ng/ml), respectively. In selected control and treated wells, mouse antihuman ICAM-1 or antihuman VCAM-1 mAbs (Beckton Dickinson PharMingen) were added in a final concentration of 10 µg/ml and allowed to incubate for 10 min at 37 C/5% CO2 before the addition of leukocytes. Leukocytes were labeled with 350 µCi Na251CrO4 for 1 h at 37 C/5% CO2, washed with Earl’s balanced salt solution supplemented with 5% FBS, added to the control and various treated HDMECs at a concentration of 70,000 cells/well, and incubated for 30 min at 37 C/5% CO2. Nonadherent cells were removed by repeated washing with Earl’s balanced salt solution/5% FBS. Cells were lyzed with 1% SDS and endothelial cell-bound radioactivity was counted in a {gamma} counter. Adherence was calculated as relative leukocyte binding = [cpm/well (unstimulated control)]/[cpm/well (stimulated)] ± SEM with unstimulated controls set to 1.0.

Local Shw-r
Preparation and induction of a Shw-r was performed as described (25): In each experiment, 18 mice were injected with 7.5 µg LPS (Sigma) sc into the left ear (preparatory dose). After 6 h and 24 h, ears of six mice were harvested for histologic and immunohistochemical analysis. The remaining mice were challenged with 150 µg LPS ip. For {alpha}-MSH treatment, mice were injected with 25 µg {alpha}-MSH (Bachem, Heidelberg, Germany) in 100 µl PBS ip 3 h after sc LPS preparation. This time point was selected after a small pilot study (injection of {alpha}-MSH 1, 3, and 6 h after sc LPS priming) demonstrating the highest efficacy of {alpha}-MSH at this time point.

Control mice were injected with 100 µl PBS. Macroscopically, the Shw-r response elicited by LPS challenge in the presence or absence of {alpha}-MSH was determined by a semiquantitative score from 1 to 4 for the vascular hemorrhage (size and number of small hemorrhages) as described (25). In addition, the infiltrate was subjected to histologic examination at 2, 3.5, and 6 h after challenge (different experiments). Expression of E-selectin, VCAM-1, or ICAM-1 was examined at the end of the preparatory phase after 24 h.

Immunohistochemistry
Immunohistochemical staining of mouse ears was performed as described (25) using mAb 10E9.6 (rat IgG2a) against murine E-selectin (kindly provided by D. Vestweber, Münster) and mAb M/K-1.9 (rat IgG1) against murine VCAM-1 (ATCC, Manassas, VA) or murine ICAM-1 (kindly provided by F. Takei, Vancouver, Canada) followed by incubation with a gt(ab')2 antirat IgG conjugated to peroxidase (Dianova, Hamburg, Germany) as secondary antibody.

Microscopic evaluation
To identify and count positive vessels, sections were examined using an ocular endowed with an eyepiece graticule (x160). To compare areas of equal size, 10 graticule fields were evaluated for each section. Results were presented in terms of the absolute number of positive ECs (E-selection, VCAM-1) in the defined areas.

Statistical analysis
Results are expressed as arithmetic mean ± SE. The unpaired t test was used to calculate the statistical significance. Differences between multiple groups were examined using an ANOVA (Bonferroni t test) or the U test according to Mann and Whitney (local Shw-r). Mean differences with a P value less than 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
{alpha}-MSH antagonizes the LPS-induced mRNA expression of E-selectin, VCAM-1, and ICAM-1
The expression of adhesion molecules such as ICAM-1, VCAM-1, and E-selectin in cytokine-activated ECs is regulated at the transcriptional level. Thus, we first examined the effect of {alpha}-MSH on the LPS-induced endothelial cell E-selectin, VCAM-1, and ICAM-1 mRNA expression by RT-PCR analysis and subsequent densitometrical evaluation (Fig. 1Go). Because {alpha}-MSH at a concentration of 10-8 to 10-12 M was previously described to be effective in modulating HDMEC functions (31), these concentrations were used in the experiments in this study. LPS stimulation strongly up-regulated mRNA expression of all three HDMEC adhesion molecules after 3 h (Fig. 1Go). However, when HDMECs were simultaneously treated with LPS and {alpha}-MSH, a significant, concentration-dependent reduction of the LPS-induced mRNA expression of E-selectin (Fig. 1BGo), VCAM-1 (Fig. 1CGo), and ICAM-1 (Fig. 1DGo) was detected. Likewise, {alpha}-MSH antagonized the LPS-induced mRNA expression of these adhesion molecules in the microvascular endothelial cell line HMEC-1 (data not shown). In addition, RT-PCR analysis of HDMECs and HMEC-1 treated with TNF{alpha} in combination with {alpha}-MSH revealed that {alpha}-MSH also antagonized the TNF{alpha}-induced adhesion molecule mRNA expression at 3 and 6 h (data not shown).



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  		Figure 1. Regulation of HDMEC E-selectin, VCAM-1, and ICAM-1 adhesion molecule mRNA expression by LPS and {alpha}-MSH. HDMECs (2 x 106 cells) were stimulated with LPS (100 ng/ml), LPS + {alpha}-MSH (10-8 or 10-12 M), or left untreated (-). Total RNA was harvested after 3 h and subjected to RT-PCR analysis using primer specific for ß-actin, E-selectin, VCAM-1, or ICAM-1, respectively (A–D). The resulting PCR fragments separated on a 1.5% agarose gel were photographed, and the digitized images were processed for densitometrical evaluation as described in Materials and Methods (A–D). RT-PCR data (photographs) are representative results from one of three similar experiments, densitometry (bar graphs) are expressed in percent as mean relative OD ± SEM with n = 3. ***, P < 0.001 vs. unstimulated control (-). #, P < 0.05; ##, P < 0.01 vs. LPS.

 
{alpha}-MSH down-regulates the LPS- and TNF{alpha}-induced expression of E-selectin, VCAM-1, and ICAM-1 on HDMECs
We next examined the effect of {alpha}-MSH on LPS-induced VCAM-1 and E-selectin expression on HDMECs using specific whole-cell ELISAs (22). LPS alone induced the expression of E-selectin and VCAM-1 peaking at 4 h (Fig. 2AGo) and 16 h (Fig. 2BGo), respectively. The addition of {alpha}-MSH (10-8/10-12 M) significantly inhibited the LPS-induced expression of both adhesion molecules at all time points with 10-12 M {alpha}-MSH being more effective (Fig. 2Go). Likewise, as demonstrated by FACS analysis, {alpha}-MSH (10-12 M) was also capable of significantly antagonizing the TNF{alpha}-induced expression of ICAM-1 (-42.8% ± 4.3% MFI reduction, P < 0.05; Fig. 3AGo) or VCAM-1 (-36.1% ± 6.4% MFI reduction, P < 0.05, Fig. 3CGo), compared with cells stimulated with TNF{alpha} alone. In addition, {alpha}-MSH inhibited the LPS-induced HDMEC VCAM-1 expression (-65.2% ± 10.4% MFI reduction, P < 0.05; Fig. 3DGo) but only moderately reduced the LPS-induced expression of ICAM-1 (-22.7% ± 3.3% MFI reduction; Fig. 3BGo). These data demonstrate that {alpha}-MSH is capable of reducing the TNF{alpha}- or endotoxin-induced cell surface expression of E-selectin, VCAM-1, and ICAM-1 on HDMECs.



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  		Figure 2. {alpha}-MSH antagonizes the LPS-induced cell surface expression of E-selectin and VCAM-1 on microvascular endothelial cells. HDMECs were cultured in 96-well plates and left untreated or stimulated in quadruplicate for the indicated time points at 37 C with LPS alone or in the presence of {alpha}-MSH (10-8/10-12 M). E-selectin (A) and VCAM-1 (B) cell surface expression was determined by ELISA as described (22 74 ). Results are expressed as relative OD ± SEM with n = 3. ***, P < 0.001 vs. unstimulated control (-). #, P < 0.05; ##, P < 0.01 vs. LPS.

 


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  		Figure 3. {alpha}-MSH antagonizes the TNF{alpha}- or LPS-induced cell surface expression of ICAM-1 and VCAM-1 on HDMECs. HDMECs (25,000 cells/cm2) were left untreated (control) or stimulated with either 10 ng/ml TNF{alpha} (A and C) or 100 ng/ml LPS (B and D) alone or in the presence of {alpha}-MSH (10-12 M) for 16 h. Subsequently cells were harvested, stained with an antihuman ICAM-1 (A and B) or antihuman VCAM-1 mAb (C and D), and subjected to FACS analysis. Treatment with {alpha}-MSH reduced the TNF{alpha}/LPS-induced expression of HDMEC ICAM-1 and VCAM-1, compared with cells stimulated with TNF{alpha} or LPS alone. Changes in the MFI of positively gated cells were calculated as described in Materials and Methods.

 
{alpha}-MSH prevents the activation of NF{kappa}B in endothelial cells
During inflammation, the transcriptional activation of many genes including nitric oxide synthase, adhesion molecules, and cytokines is mediated by the transcription factor NF{kappa}B (32). Because bacterial endotoxin has been reported to induce gene expression by NF{kappa}B activation (33), we subsequently examined the effect of {alpha}-MSH on the activation of this transcription factor. EMSA analysis of HDMEC nuclear extracts revealed the formation of two DNA-protein complexes (I and II) that could be competed with nonradioactive {kappa}B oligonucleotide (Fig. 4AGo, bold arrows I and II). LPS stimulation resulted in the activation of complex I within 30 min. The addition of {alpha}-MSH immediately before LPS stimulation resulted in a partially abrogated activation and nuclear translocation of this NF{kappa}B transactivator complex (Fig. 4AGo). Supershift analysis using antibodies against the NF{kappa}B proteins p50, p65/Rel A, p68/Rel B, and p75/c-Rel revealed that the LPS-inducible complex I was heterodimeric and consisted of the NF{kappa}B subunits p50 and p65 (Fig. 4AGo, dotted arrows), whereas incubation with anti-c-Rel (p75) or anti-Rel B (p68) did not result in a remarkable supershift of complex I. Because phosphorylation and subsequent degradation of I{kappa}B{alpha} is a prerequisition for p65/p50 translocation into the nucleus, we next examined the effect of LPS/{alpha}-MSH on cytosolic I{kappa}B{alpha} by Western blotting. Compared with untreated controls, stimulation of HDMECs with LPS (100 ng/ml) resulted in a 50% and 70% reduction of cytosolic I{kappa}B{alpha} protein after 15 min and 30 min, respectively (Fig. 4Go, B and C). In particular, after 30 min, addition of {alpha}-MSH completely prevented the LPS-induced degradation of I{kappa}B{alpha} (Fig. 4Go, B and C). Similar data were obtained when cells were treated with TNF{alpha} alone and in combination with 10-12 M {alpha}-MSH (Scholzen, T. E., and T. Brzoska, unpublished observation). These results indicate that in HDMECs {alpha}-MSH is capable of inhibiting the NF{kappa}B activation initialized by bacterial endotoxin presumably by preventing I{kappa}B{alpha} degradation, which may account for the reduced transactivation of adhesion molecule genes.



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  		Figure 4. {alpha}-MSH interferes with the LPS-mediated HDMEC NF{kappa}B nuclear translocation. HDMECs plated at a density of 25,000 cells/cm2 were left untreated (-) or treated with (LPS 100 ng/ml) alone or in the presence of {alpha}-MSH (10-8/10-12 M) for 30 min. Subsequently, cells were harvested, nuclear and cytosolic proteins were isolated, and EMSA was performed as described (30 ). Bold arrows indicate positions of HDMEC NF{kappa}B complexes that could be competed with unlabeled {kappa}B oligonucleotide (comp). Determination of NF{kappa}B complex composition in LPS-stimulated HDMEC was performed by adding antibodies against the {kappa}B proteins c-Rel, Rel B, p50, or p65 to the binding reaction (LPS + Ab). Dotted arrows indicate positions of supershifted bands. Treatment with {alpha}-MSH reduced the LPS-induced formation of the p65/p50 transactivator complex (bold arrow I), compared with cells stimulated with LPS alone (A). Cytosolic proteins (15 µg per lane) from HDMECs stimulated for 15 and 30 min with LPS/{alpha}-MSH were separated on a 10% SDS PAGE and blotted on nitrocellulose membrane. Membranes were enhanced chemiluminescence stained using a polyclonal anti-I{kappa}B{alpha} antibody and an antibody specific for {alpha}-tubulin to verify equal gel loading (B). The resulting digitized film was subjected to densitometrical analysis (C). {alpha}-MSH prevented the degradation of I{kappa}B{alpha} following LPS incubation. Data are representatives of three similar experiments.

 
{alpha}-MSH inhibits the LPS- and TNF{alpha}-induced adhesion of leukocytes to HDMECs in culture
To further investigate the impact of {alpha}-MSH on the LPS-induced leukocyte-HDMEC interactions, functional in vitro adhesion assays were performed. 51Cr-labeled, CD49d (VLA-4)-expressing Molt-4 lymphoblastoid T cells, which preferentially adhere to HDMECs by binding to VCAM-1 (22), or CD11a/CD18 (LFA-1)-expressing JY-1 lymphoblastoid B-cells, which preferentially adhere via ICAM-1 (34), were added to cultured HDMEC monolayers that had been stimulated for 16 h with LPS alone or in combination with {alpha}-MSH (Fig. 5Go, A and B). Our studies indicated that LPS alone strongly up-regulated the adhesion of both Molt-4 (Fig. 5AGo) and JY-1 (Fig. 5BGo) to HDMECs. This LPS-mediated adhesion of Molt-4 or JY-1 could be significantly inhibited by the addition of anti-VCAM-1 (Molt-4; Fig. 5AGo) or anti-ICAM-1 antibodies (JY-1; Fig. 5BGo), demonstrating that these adhesion molecules are involved in Molt-4 and JY-1 binding, respectively. In addition, pretreatment of Molt-4 with VLA-4 or JY-1 with LFA-1 antibodies prevented adhesion of these lymphocytes to the HDMEC monolayer (data not shown). Most importantly, HDMECs stimulated with 100 ng/ml LPS in combination with {alpha}-MSH (10-8/10-12 M) demonstrated dose-dependent reduction in the LPS-induced Molt-4 adhesion to HDMECs (Fig. 5AGo). Likewise, a significant dose-dependent decrease in JY-1 cell adhesion was observed when HDMECs were exposed to both LPS and {alpha}-MSH (10-8/10-12 M; Fig. 5BGo). These results indicated that {alpha}-MSH is capable of directly inhibiting the LPS-induced ICAM-1- or VCAM-1-mediated adhesion of leukocytes to HDMECs. In a similar manner, {alpha}-MSH antagonized the TNF{alpha}-induced adhesion of Molt-4 to HDMEC monolayer with {alpha}-MSH at a concentration of 10-12 M being most effective (Fig. 5CGo).



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  		Figure 5. {alpha}-MSH antagonizes the LPS- or TNF{alpha}-induced adhesion of 51Cr-labeled Molt-4 and JY-1 lymphocytes to HDMECs. The {alpha}-MSH-dependent modulation of functional binding of T-lymphoblastoid Molt-4 or B-lymphoblastoid JY-1 cells to LPS-activated (A and B) or TNF{alpha}-activated HDMEC (C) was determined in a quantitative cell adhesion assay. HDMECs were cultured in 24-well plates and left untreated (-) or stimulated in quadruplicate for 16 h with LPS (100 ng/ml), LPS + {alpha}-MSH (10-8/10-12 M), or TNF{alpha} (10 ng/ml) alone or in combination with {alpha}-MSH. Then 70,000 T-lymphoblastoid Molt-4 cells (A and C) or B-lymphoblastoid JY cells (B) per well labeled with 350 µCi 51Cr were added to the HDMEC monolayer. The radioactivity resulting from adherent 51Cr-labeled lymphocytes was counted in a {gamma}-counter as described in Materials and Methods. Specificity of preferentially VCAM-1 mediated Molt-4 or ICAM-1 mediated JY-1 binding was determined by pretreatment of LPS-treated HDMECs for 10 min with antihuman VCAM-1 (Molt-4; a, c) or antihuman ICAM-1 (JY-1; b) mAb, respectively (LPS + Ab; TNF + Ab). Results are expressed as relative leukocyte binding ± SEM with unstimulated controls set to 1.0. n = 3. ns, not significant. *, P < 0.05; **, P < 0.01 compared with untreated controls (-). #, P < 0.05, ##, P < 0.01, compared with cells stimulated with LPS or TNF{alpha} only.

 
{alpha}-MSH decreases the severity of LPS-induced leukocytoclastic vasculitis (local Shw-r)
To test the physiological relevance of the in vitro observations in an in vivo model, we subsequently analyzed the effect of {alpha}-MSH in the local Shw-r, a mouse model for cutaneous LcV in which the sustained expression of VCAM-1 and E-selectin in particular has been shown to be one of the pathophysiological steps in vessel damage. The local Shw-r in mice is initiated by the sc injection of low-dose LPS into one ear followed by systemic (ip) LPS challenge 24 h later (25). The clinical and histopathologic changes that occur in a Shw-r include edema, hemorrhages, and microthrombi consistent with the histopathologic alterations found in LcV. Our in vitro studies demonstrated that {alpha}-MSH interferes with the induction of adhesion molecule expression. Because we hypothesized that this may also be of relevance in vivo, {alpha}-MSH was applied 3 h after the sc preparatory dose of LPS.

Two hours after triggering an Shw-r by ip LPS application (26 h after sc LPS priming), the macroscopic examination of mouse ears subjected to sc LPS priming demonstrated several small hemorrhages (Fig. 6AGo). In contrast, when mice were pretreated with systemic {alpha}-MSH before the initiation of the Shw-r, a significant reduction of vascular hemorrhage could be detected (Fig. 6BGo). Quantification of the extent of cutaneous hemorrhages revealed that number and size of hemorrhage lesions at 2, 3.5, and 6 h after LPS challenge was significantly reduced by ip pretreatment with {alpha}-MSH 3 h after sc LPS priming (Table 1Go). In the initial phase of the Shw-r, a sustained expression of E-selectin was detected, unlike other models of local skin inflammation such as contact dermatitis in which E-selectin is rapidly down-regulated. In accordance with previous observations (25, 35), we detected long-lasting expression of vascular E-selectin for more than 24 h in ears of mice not treated with {alpha}-MSH (Fig. 7AGo and Table 2Go). In contrast, ears of {alpha}-MSH-treated mice revealed significantly less E-selectin-positive vessels at the end of the preparatory phase (Fig. 7BGo and Table 2Go). VCAM-1 was also suppressed after treatment with {alpha}-MSH, but its reduction was not continuously significant, whereas the expression of ICAM-1 as with HDMECs after LPS treatment in vitro was not significantly affected (data not shown). We also noted that {alpha}-MSH treatment slightly but not significantly inhibited edema formation and the size of the cutaneous infiltrate as determined by ear swelling measurement and histologic examination (data not shown). Thus, these data indicate that the prevention of the LPS-induced hemorrhagic response in {alpha}-MSH-treated mice correlated with the inhibition of vascular expression of E-selectin and VCAM-1 in the preparatory phase of the local Shw-r.



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  		Figure 6. {alpha}-MSH reduces the number and size of hemorrhagic lesions during the local Shw-r. BALB/c mice (n = 6 per group) were treated by sc injection of LPS (A) or sc LPS into one ear + ip {alpha}-MSH (B) during the preparatory phase of the local Shw-r. The macroscopic appearance of the ears was assessed 2 h after systemic challenge of mice with LPS. Mice injected with {alpha}-MSH during the preparatory phase had significantly fewer and smaller hemorrhagic lesions (B; blue arrows), compared with control mice not treated with {alpha}-MSH (A).

 

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  		Table 1. Effect of {alpha}-MSH on hemorrhage during LPS-induced local Shw-r

 


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  		Figure 7. Expression of E-selectin during the preparatory phase of the local Shw-r. BALB/c mice were treated with sc LPS (A) or sc LPS + ip {alpha}-MSH (B) in the preparatory phase of the local Shw-r. Ears were harvested after 24 h and stained for E-selectin. Mice injected with {alpha}-MSH revealed significantly fewer E-selectin-positive vessels (arrows), compared with control mice treated with LPS only. No staining was observed in mice incubated with an irrelevant antibody (IgG control, C). Scale bars, 50 µm.

 

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  		Table 2. Effect of {alpha}-MSH on the LPS-induced expression of E-selectin in the preparatory phase of the Shw-r

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Dermal microvascular endothelial cells play a pivotal role for skin homeostasis and inflammation. The tightly regulated expression of CAMs by HDMECs are crucial events, which mediate adhesion and diapedesis of leukocytes into the extravascular tissue during cutaneous inflammation (36) and in some cases additional effects such as in vasculitic destruction of vessels. E-selectin is an inducible glycoprotein exclusively expressed by cytokine-activated ECs, which mediates rolling of neutrophils, monocytes, and some memory T cells on the vascular endothelium (37, 38, 39). ICAM-1 and VCAM-1 mediate firm adherence of leukocytes to blood vessels and are two of the most important cell surface molecules for endothelial-mononuclear cell interactions (40, 41, 42). TNF{alpha} and LPS have been previously demonstrated to induce VCAM-1, which is only minimally expressed by resting HDMECs, and increase the constitutive ICAM-1 cell surface expression (43). In this study, we have demonstrated that {alpha}-MSH is capable of antagonizing the LPS- or TNF{alpha}-induced activation of HDMEC in vitro by suppression of E-selectin, VCAM-1, and ICAM-1 mRNA and protein expression in a time- and concentration-dependent manner.

Proinflammatory stimuli such as LPS, TNF{alpha}, and IL-1 play a critical role in immune and inflammatory responses as they activate NF{kappa}B as a key transcription factor required for the synthesis of proinflammatory cytokines, chemokines, adhesion molecules, and other mediators of inflammation (44). Cytosolic NF{kappa}B is associated with inhibitory proteins (I{kappa}B), and to be translocated into the nucleus, the NF{kappa}B:I{kappa}B complex has to be disrupted through the activation of an inhibitor of {kappa}B kinase (IKK) complex containing the two kinases IKK{alpha} and IKKß and the regulatory subunit IKK{gamma} (45, 46). The latter results in I{kappa}B phosphorylation, its subsequent proteosomal degradation, and the release of NF{kappa}B (32). Although a relative small set of adhesion molecule-specific transcription factors is required for the individual transcriptional activation of E-selectin, VCAM-1, and ICAM-1, NF{kappa}B is the key transcription factor for all three adhesion molecules (47). In accordance with this notion, incubation of HDMECs with the cell-permeable proteasome and NF{kappa}B inhibitor MG132 resulted in an almost complete abrogation of the LPS-induced cell surface expression of E-selectin, VCAM-1, and ICAM-1 (Scholzen, T. E., unpublished observation). {alpha}-MSH has been shown to inhibit NF{kappa}B activation in cell lines such as melanoma, glioma, and U937 (48, 49, 50). Our findings demonstrates that {alpha}-MSH, possibly by maintaining the cytosolic steady-state levels of I{kappa}B{alpha} via a not-yet-identified mechanism, acts as potent inhibitor of LPS- or TNF{alpha}-activated NF{kappa}B in primary dermal ECs. This may involve cAMP/protein kinase A-dependent signal transduction because reduction of the TNF{alpha}-induced ICAM-1 expression by {alpha}-MSH could be mimicked by the protein kinase A activator forskolin (Scholzen, T. E., unpublished observation). Therefore, NF{kappa}B inhibition may account at least in part for the down-regulation of endothelial CAM expression and thus for the antiinflammatory activity of {alpha}-MSH in the local Shw-r.

The functional relevance of the above findings was supported by an in vitro adhesion assay demonstrating an {alpha}-MSH-mediated down-regulation of the adhesion of the T- and B-lymphoblastoid cell lines Molt-4 and JY-1 induced by LPS and TNF{alpha}. This binding was mediated by ICAM-1 and/or VCAM-1 because incubation of activated HDMECs with antibodies against these adhesion molecules significantly reduced lymphocyte binding. Thus, by interfering with the induced expression of these important cell surface molecules, {alpha}-MSH constitutes a powerful inhibitor of endothelium-mediated inflammatory responses.

Most importantly, our data provide strong evidence that the administration of the neuroendocrine hormone {alpha}-MSH effectively prevented the endotoxin-induced vasculitis in a murine model of the local Shw-r. This inflammatory process is characterized by segmental vascular inflammation encompassing infiltration of neutrophils, necrosis of the vascular wall, and subsequently extravasation of erythrocytes (26, 51). These multiple sequential steps are regulated by the expression of cytokines, chemokines, and their receptors as well as adhesion molecules (24). As demonstrated previously, the development of LcV in the Shw-r strongly correlated with the sustained expression of E-selectin and VCAM-1, which is in contrast to other types of acute inflammation such as irritant contact dermatitis (25). In vitro, the expression of E-selectin on vascular ECs is transient peaking at 4–8 h post induction, with a more persistent expression on microvascular EC, particularly after repetitive stimulation with TNF{alpha} or LPS, compared with ECs of macrovascular origin such as human umbilical vein endothelial cells (52). The prolonged expression of E-selectin apparently depends on the turnover rate of cell surface E-selectin, which is more slowly internalized and degraded in HDMECs than in human umbilical vein endothelial cells (53). This long-lasting expression of E-selectin that is also found in some other types of chronic cutaneous and noncutaneous inflammation (54, 55, 56) may interfere with the sequence of events necessary for undisturbed diapedesis of leukocytes in vasculitic inflammations. Because these adhesion molecules may not only mediate rolling or adherence of polymorphonuclear cells (PMNs) in LcV, but also signaling for subsequent steps of diapedesis (57, 58) or for release of toxic cell products (59), their sustained expression may result in a prolonged adherence of PMN cells and an increased release of toxic compounds, which ultimately mediates damage of the vascular endothelium.

In this study, {alpha}-MSH application in the early priming phase of the LPS-induced local Shw-r significantly reduced the detrimental effects elicited by systemic LPS challenge, resulting in fewer and smaller petechial hemorrhages. Interestingly, this attenuation of the local Shw-r did not result from a reduction of leukocyte infiltrate because we observed only a minor reduction of PMN influx after {alpha}-MSH administration. Strikingly as in vitro, {alpha}-MSH reduced the sustained expression of endothelial adhesion molecules, particularly of E-selectin and, to a lesser degree, VCAM-1, thus interfering with an important step in the pathogenesis of this disorder. However, in contrast to our in vitro studies, in which {alpha}-MSH clearly antagonized the expression of VCAM-1 induced by LPS and also by TNF{alpha}, suppression of VCAM-1 in vivo was not statistically significant in our evaluation, which may be related to the fact that this CAM, like ICAM-1, is also expressed by other cells in the tissue. This may explain why inhibition of VCAM-1 by {alpha}-MSH was not as prominent as that of E-selectin, although injection of neutralizing antibodies against both E-selectin and VCAM-1 reduced the hemorrhage in the local Shw-r more effectively than application of each antibody alone (25). This provides direct evidence that the expression level and function of E-selectin and potentially of VCAM-1 are causally related to the severity of cutaneous vasculitis in the local Shw-r.

Nonetheless, the local reduction of vascular adhesion molecule expression may not exclusively account for the {alpha}-MSH-mediated inhibition of LcV in the local Shw-r. Proinflammatory cytokines such as IL-12, interferon {gamma}, and in particular TNF{alpha} that are induced by LPS have been implicated as powerful mediators of vasculitis and endotoxic shock (60, 61). {alpha}-MSH is capable of reducing an inflammatory response by down-regulating the production of such mediators in vivo or in vitro in cells such as monocytes, macrophages, or neutrophils, which have been shown to play a role in the pathogenesis of LcV (62, 63, 64, 65). On the other hand, {alpha}-MSH also increases the production of the antiinflammatory cytokine IL-10 in monocytes (66). This is of particular importance because IL-10 is capable of reducing cytokine synthesis in murine monocytes and macrophages (67, 68). {alpha}-MSH has been demonstrated to increase IL-10 in the bronchoalveolar fluid in a mouse model of airway inflammation (69), and the {alpha}-MSH-mediated inhibition of murine allergic contact dermatitis could be abrogated by the injection of anti-IL10 antibodies (10). With respect to endotoxic shock and vasculitis, it was proposed that IL-10 may prevent activation of immune cells such as macrophages by IL-12 and interferon {gamma} that are induced by small amounts of LPS during the preparatory phase of the Shw-r (60, 70). In accordance with these observations, administration of endotoxin induced IL-10 production in mice and humans (71, 72, 73). In this scenario, by inducing additional IL-10 during the preparatory phase of the local Shw-r, {alpha}-MSH could prevent activation of mononuclear cells and PMN cells, resulting in a dampened inflammatory response to ip LPS challenge and a decreased susceptibility to systemic shock.

In conclusion, by directly targeting leukocyte-endothelial interactions, the neuroendocrine hormone {alpha}-MSH had a strong therapeutic effect on the LPS-induced LcV. The importance of our findings presented in this study is further supported by recent studies demonstrating a strong antiinflammatory effect of {alpha}-MSH in animal models for endotoxin-mediated liver inflammation or contact hypersensitivity (8, 10). Preliminary studies in humans also suggest an {alpha}-MSH antiinflammatory potential when applied locally to treat contact allergy-induced eczematous lesions (3). Whereas therapeutic strategies currently used to treat different forms of vasculitis such as corticosteroids or immunosuppressive drugs are associated with adverse side effects and often lack specificity for the underlying pathomechanisms, we did not observe any adverse effects of {alpha}-MSH in this as well as in our previous contact hypersensitivity studies (10), and in addition, only very low pharmacological doses of {alpha}-MSH were required. In summary, these findings strongly suggest that the neuroendocrine POMC peptide {alpha}-MSH may effectively prevent onset or progression of certain states of acute or chronic inflammation. Thus, {alpha}-MSH and its analogs may have an important therapeutical potential for the treatment of inflammatory, autoimmune, and allergic diseases.


   Acknowledgments
 
The authors gratefully acknowledge the technical assistance of S. Merfeld, K. Fischer, and E. Nottkämper.


   Footnotes
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB293 B2) and the Volkswagenstiftung (I 74582; to T.A.L.); an Innovative Medical Research (IMF) grant and a grant from the Interdisciplinary Center for Clinical Research (IKF D15; to C.S.); NIH (HD-33024 and AI-41493; to J.C.A.); NIH R03AR44969 (to C.A.A.); and Centre de Recherché et Investigations Epidermiques et Sensorielles, (C.E.R.I.E.S.), Paris, France.

T.E.S. was supported by a fellowship grant from the Deutsche Forschungsgemeinschaft (SCHO629 1/1), and H.K. was supported by the Dermatology Foundation.

Abbreviations: CAM, Cell adhesion molecule; EC, endothelial cell; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; HDMEC, human dermal microvascular endothelial cell; ICAM, intercellular CAM; IKK, inhibitor of {kappa}B kinase; LcV, leukocytoclastic vasculitis; LPS, lipopolysaccharide; mAb, monoclonal antibody; MC-R, melanocortin receptor; MFI, mean fluorescence intensity; MSH, melanocyte stimulating hormone; NF{kappa}B, nuclear factor-{kappa}B; PMN, polymorphonuclear; POMC, proopiomelanocortin; Shw-r, Shwartzman reaction; VCAM, vascular CAM.

Received June 30, 2002.

Accepted for publication September 25, 2002.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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