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The Journal of Immunology, 2007, 178: 1748-1758.
Copyright © 2007 by The American Association of Immunologists, Inc.

Visfatin, an Adipocytokine with Proinflammatory and Immunomodulating Properties1

Alexander R. Moschen*, Arthur Kaser*, Barbara Enrich*, Birgit Mosheimer{dagger}, Milan Theurl{dagger}, Harald Niederegger{ddagger} and Herbert Tilg2,*

* Department of Medicine, Christian Doppler Research Laboratory for Gut Inflammation and Clinical Division of Gastroenterology and Hepatology, {dagger} Department of Medicine, Clinical Division of General Internal Medicine, and {ddagger} Innsbruck Biocentre, Division of Experimental Pathophysiology and Immunology, Innsbruck Medical University, Innsbruck, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adipocytokines are mainly adipocyte-derived cytokines regulating metabolism and as such are key regulators of insulin resistance. Some adipocytokines such as adiponectin and leptin affect immune and inflammatory functions. Visfatin (pre-B cell colony-enhancing factor) has recently been identified as a new adipocytokine affecting insulin resistance by binding to the insulin receptor. In this study, we show that recombinant visfatin activates human leukocytes and induces cytokine production. In CD14+ monocytes, visfatin induces the production of IL-1beta, TNF-{alpha}, and especially IL-6. Moreover, it increases the surface expression of costimulatory molecules CD54, CD40, and CD80. Visfatin-stimulated monocytes show augmented FITC-dextran uptake and an enhanced capacity to induce alloproliferative responses in human lymphocytes. Visfatin-induced effects involve p38 as well as MEK1 pathways as determined by inhibition with MAPK inhibitors and we observed activation of NF-{kappa}B. In vivo, visfatin induces circulating IL-6 in BALB/c mice. In patients with inflammatory bowel disease, plasma levels of visfatin are elevated and its mRNA expression is significantly increased in colonic tissue of Crohn’s and ulcerative colitis patients compared with healthy controls. Macrophages, dendritic cells, and colonic epithelial cells might be additional sources of visfatin as determined by confocal microscopy. Visfatin can be considered a new proinflammatory adipocytokine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adipose tissue has emerged as an important endocrine organ producing a variety of secreted factors including TNF-{alpha} (1), IL-6 and IL-8 (2), plasminogen-activator inhibitor type 1 (3), leptin (4), adiponectin (5, 6), resistin (7), and others. Several of these mediators are predominantly synthesized by adipose tissue and called adipocytokines. Recently, the adipocytokine family has been extended by a novel member—visfatin (8). In search of differentially expressed genes in paired samples of s.c. and visceral fat, Fukuhara et al. (8) detected a transcript that was more abundantly expressed in visceral fat than in s.c. fat. They demonstrated that circulating levels of visfatin correlated strongly with the amount of visceral fat in both humans and mice. Moreover, they reported that recombinant visfatin directly binds to the insulin receptor (IR)3 resulting in its tyrosine phosphorylation as well as phosphorylation of insulin receptor substrate-1 and -2 leading to enhanced glucose uptake in vitro and in vivo (8). This suggests a possible role for visfatin production as a compensatory response in diet- or obesity-induced insulin resistance (9). Notably, Chen and colleagues (10) recently described elevated visfatin plasma levels in patients with type 2 diabetes mellitus.

Visfatin was originally cloned by Samal et al. (11) in search of novel cytokine-like molecules secreted from human PBLs. They described a 52-kDa secreted molecule termed pre-B cell-enhancing factor (PBEF) that was strongly induced by pokeweed mitogen and cycloheximide and enhanced the effect of IL-7 and stem cell factor on pre-B cell colony formation (11). Visfatin (PBEF) is highly conserved in evolution as homologous proteins have been described in bacteria (12), invertebrate sponges (13), and fish (14). Intracellular visfatin (PBEF) acts as a dimeric type II phosphoribosyltransferase (nicotinamide adenine dinucleotide biosynthesis) (12, 15, 16) and growth phase-dependent changes of its subcellular distribution have been reported (17).

Over the last decade, much evidence has emerged that obesity is closely linked to systemic inflammation (18). On the one hand, proinflammatory cytokines such as TNF-{alpha} or IL-6 are overexpressed in adipose tissue of obese patients and contribute to insulin resistance (19, 20). On the other hand, adipocyte-derived cytokines interfere with immune processes. Adiponectin, the most abundant adipocyte protein, has potent anti-inflammatory properties by inhibiting proinflammatory TNF-{alpha} and by inducing anti-inflammatory cytokines like IL-10 and IL-1 receptor antagonist (IL-1Ra) (21, 22). Leptin, the other major product of adipocytes, also affects many aspects of inflammation and immunity (23). We hypothesized that visfatin might share the ambiguity in metabolic and immune functions of other adipocytokines. We therefore set out to study immunological and inflammatory functions of visfatin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials and reagents

Culture medium in all experiments was RPMI 1640 (Biochrom) supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies) and 100 U penicillin/streptomycin (Biochrom). Recombinant human soluble visfatin was purchased from Alexis Biochemicals, and from PeproTech. Both proteins used were >97% pure (SDS-PAGE analysis) and contained <0.01 ng µg–1 LPS as determined by the Limulus amebocyte lysate method. Both proteins showed comparable biological activity with respect to induction of IL-1beta, TNF-{alpha}, and IL-6. Therefore, for all consecutive experiments we used visfatin purchased from Alexis Biochemicals. Recombinant murine soluble visfatin was from Alexis Biochemicals. LPS from Escherichia coli 055:B5, polymyxin B, and insulin were obtained from Sigma-Aldrich. Recombinant human GM-CSF was obtained from Berlex. Human recombinant IL-4 was supplied by Schering-Plough Research Institute. The following Abs were used: PerCP-conjugated CD14, allophycocyanin-conjugated CD40, PE-conjugated CD69, PE-conjugated CD86, FITC-conjugated HLA-DR (BD Biosciences); FITC-conjugated CD54 and allophycocyanin-conjugated CD80 (ImmunoTools).

Preparation of PBMCs, monocytes, dendritic cells (DCs), and macrophages

Human peripheral blood from voluntary healthy donors was collected in heparinized tubes and PBMCs were obtained by Lymphoprep gradient centrifugation (Axis Shield) (24). Monocytes were sorted using immunomagnetic anti-CD14 MicroBeads (Miltenyi Biotec).

For preparation of DCs, CD14+ cells were grown at a density of 106/ml in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 1000 U/ml GM-CSF and 800 U/ml IL-4. Culture mediums and cytokines were replenished on days +2 and +5. On day 7, DCs were harvested. For the preparation of macrophages, CD14+ cells seeded at 106/ml in RPMI 1640 supplemented with 5% FCS and 1000 U of GM-CSF, and the medium and GM-CSF were replenished every third day. After 12 days, macrophages were harvested and used for subsequent experiments.

PBMCs, monocytes, DCs, and macrophages were harvested and reseeded in culture medium supplemented with polymyxin B (5 µg/ml) and subsequently stimulated with various concentrations of recombinant human visfatin for 20 h. Additionally, in all experiments, cells were stimulated with 100 ng/ml visfatin in the presence of a specific pharmacologic inhibitor of the indicated protein kinases (p38 kinase (SB 203580), MEK1 (PD 98059), JNK (JNK inhibitor II), PI3K (LY 294002), and Janus protein tyrosine kinase (JAK inhibitor I); all from Calbiochem, EMD Biosciences). Supernatants were harvested and stored at –20°C until measurement of cytokines.

Detection of cytokine production

Concentrations of IL-1beta, IL-1Ra, IL-6, IL-10, and TNF-{alpha} in cell culture supernatants were determined using commercially available Ab pairs and protein standards from R&D Systems (IL-1Ra, IL-6) and BD Pharmingen (IL-1beta, IL-10, and TNF-{alpha}) according to manufacturer’s instructions. Absorption was determined with an ELISA reader (Medgenix Diagnostics) at 450 nm.

Proliferation assays

MLRs were done using CD14+ monocytes as stimulators and adhesion purified allogeneic PBLs as responders. Stimulator monocytes were incubated with indicated concentrations of recombinant visfatin for 20 h. Thereafter, cells were fixed with 0.05% glutaraldehyde in PBS for 30 s and then fixation was stopped by addition of an equal volume of 0.4 M glycine. After washing four times, cells were counted and 0.5 x 105 monocytes were cultured for 5 days in triplicates in round-bottom 96-well plates with the indicated ratios of PBLs.

RNA extraction and quantitative real-time RT-PCR

PBMCs were adjusted to 2 x 106 cells/ml and incubated for 6 h in presence or absence of 100 and 250 ng/ml recombinant visfatin for 6 h. Thereafter, cells were harvested and total RNA extracted using TRIzol reagent (Invitrogen Life Technologies). Reverse transcription was performed with Moloney murine leukemia virus reverse transcriptase (200 U/1 µg of RNA) (Invitrogen Life Technologies) with random hexanucleotide primers (Roche).

Quantitative PCR (qPCR) was performed in a total volume of 25 µl of Brillant QPCR master mix in 40 cycles of 95°C for 30 s and 60°C for 1 min with a Mx4000 quantitative PCR system (Stratagene). Optimal concentrations for forward and reverse primers as well as TaqMan probes were determined before all performed qPCR experiments. Primers used are listed in Table I. For endogenous controls, mRNA expression of GAPDH was determined for human and glucuronidase beta (GUSB) for murine samples using predesigned TaqMan control reagents (Applied Biosciences).


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  		Table I. Primer sets used for quantitative real-time PCR

 
Determination of activated NB-{kappa}B p65 (RelA)-binding activity

For determination of NF-{kappa}B activation, 2 x 106 PBMCs/ml were incubated with or without 250 ng/ml visfatin or 100 ng/ml LPS. Total (cytoplasmatic and nuclear) protein was extracted with M-PER protein extraction reagent (Pierce) in the presence of a protease inhibitor mixture (Sigma-Aldrich) after 1, 3, 6, 9, 12, and 20 h. Protein concentrations were determined by the Bradford protein assay (Bio-Rad). Activated NF-{kappa}B p65 was determined using an EZ-Detect chemiluminescent transcription factor assay (Pierce) that has been described previously by Renard et al. (25). Briefly, 10 µg of total protein was incubated in wells containing biotinylated-consensus DNA duplexes of NF-{kappa}B. The captured active transcription factor was detected by a specific Ab recognizing NF-{kappa}B p65 and then incubated with a secondary HRP-conjugated Ab. A chemiluminescent substrate was added to each well and the resulting signal was detected using an Anthos Lucy 1 luminometer (Anthos Labtec Instruments).

Flow cytometry

For four-color surface flow cytometry, 106/ml visfatin-stimulated monocytes were incubated with indicated FITC-, PE-, PerCP- or allophycocyanin-labeled mAbs or the corresponding isotype controls for 20 min. After washing, cells were acquired with a FACSCalibur and data evaluation was performed by CellQuest Pro software (BD Biosciences). For determination of mannose receptor-mediated Ag uptake, freshly isolated monocytes (1 x 105) were incubated with 0.5 mg/ml FITC-dextran (Sigma-Aldrich) for 60 min at 37°C. Thereafter, cells were washed three times with ice-cold PBS and immediately analyzed by flow cytometry using a FACSCalibur.

Chemotaxis

Migration of cells into nitrocellulose to gradients of recombinant visfatin was measured under use of a 48-well Boyden microchemotaxis chamber (Neuroprobe) in which an upper chamber is separated from a lower chamber by a 5-µm pore-size filter (Sartorius) (26).

As indicated, monocytes or B cells were incubated to increasing concentrations of visfatin. After a migration time of 120–240 min, the filters were dehydrated, fixed, and stained with H&E. Migration depth of cells into the filter was quantified by light microscopy, measuring the distance (in micrometers) from the surface to the leading front of cells, before any cells had reached the lower surface (leading front assay). Data are expressed as chemotaxis index, i.e., the ratio of the distance of stimulated and random migration of cells into the nitrocellulose filters.

Mice and in vivo treatment

Pathogen-free 6- to 8-wk-old BALB/c mice were obtained from Harlan Winkelmann and maintained under controlled animal care conditions with free access to standard chow and water. Mice were given two i.p. injections of pyrogen-free saline or 10 µg of recombinant murine visfatin (Alexis Biochemicals) at 0 and after 12 h. Experimental design was as follows: in experimental series 1, blood was collected from the tail vein at 15 h (3 h after the second visfatin injection). The mice were then sacrificed after 20 h. In experimental series 2, the mice were sacrificed at 15 h. The experimental procedure is schematized in Fig. 6A. Blood was centrifuged at 2500 x g for 15 min and serum was stored at –80°C until determination of circulating IL-6 and TNF-{alpha} (OptEIA; BD Biosciences). Liver, spleen, lung, and small intestine from each animal were removed and flash frozen in liquid nitrogen. mRNA expression levels of IL-1beta, IL-6, and TNF-{alpha} were determined by quantitative real-time RT-PCR essentially as described above (for primer and probes see Table I). All animal experiments described were performed in accordance with Austria’s legal requirement.


Figure 6
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  		FIGURE 6. In vivo treatment of mice with murine visfatin leads to increased levels of serum IL-6 and induction of IL-6 gene expression. BALB/c mice (n = 10) were injected i.p. twice at 0 and 12 h with 10 µg of visfatin or saline (A). Blood was taken at 15 (experimental series 1: tail vein (n = 5); experimental series 2: cardiac puncture (n = 5)) and 20 h (experimental series 1: cardiac puncture (n = 5)). Organs were harvested and immediately flash frozen together with cardiac puncture. Serum IL-6 levels were determined by ELISA (B). Total RNA was extracted from individual tissues and IL-1beta (C), IL-6 (D), and TNF-{alpha} (E) mRNAs were quantified by real-time PCR as described in Materials and Methods. Data are normalized to GUSB expression (*, p < 0.05; **, p < 0.001).

 
Human samples

A total of 74 patients with an established diagnosis of inflammatory bowel disease (IBD) (39 Crohn’s disease (CD), 35 ulcerative colitis (UC)) were included in the study. The control group was recruited from 38 age- and sex-matched healthy individuals. Patients’ baseline characteristics are shown in Table II. Informed consent was obtained from each patient involved in the study that has been reviewed and approved by the local ethics committee.


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  		Table II. Patient characteristics

 
For the determination of circulating visfatin, blood was collected into Sarstedt Monovette serum containers, centrifuged at 1200 x g for 15 min, aliquoted into 1-ml portions, and stored at –80°C until assayed. Serum visfatin concentrations were determined using a human visfatin (C-terminal) enzyme immunometric assay (Phoenix Pharmaceuticals).

Visfatin mRNA levels were determined in inflamed and noninvolved colonic biopsy specimens that were collected from nine CD and nine UC patients undergoing diagnostic colonoscopy. The involved character was first identified by gross endoscopic appearance and then further confirmed by histologic evaluation of biopsies taken in parallel. Eight patients undergoing screening colonoscopy served as healthy controls. Biopsy specimens were immediately placed into RNAlater RNA stabilization reagent (Qiagen). RNA extraction, reverse transcription, and qPCR were essentially performed as described above. Sequences of primers and probe are listed in Table I.

Fluorescence microscopy

Colonic tissue specimens were obtained from patients undergoing surgical resection. The tissue samples were immediately embedded in Tissue-Tek OCT compound (Sakura). Six-micrometer sections were prepared on a Leica Cryomicrotom. After fixation in 4°C acetone and rehydration in PBS, nonspecific binding sites were blocked with Image-iT FX signal enhancer (Molecular Probes) and serum-free protein block (DakoCytomation). For detection of visfatin, all sections were stained with two commercially available rabbit anti-visfatin Abs raised either against the entire protein or an N-terminal peptide (both Alexis Biochemicals). For double-labeling, the following mouse Abs were used: anti-human DC-SIGN (CD209) (R&D Systems), anti-human CD3, CD20, CD31, MHC class II, CK18, and smooth muscle actin (all DakoCytomation), and CD163 (BMA Biomedicals). Isotype-matched control Abs were used to exclude nonspecific staining. All primary Abs were diluted in PBS containing 1% BSA and incubated at 4°C overnight. After washing, visfatin was visualized with Alexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes). Subsequently, mouse Abs were detected with Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes). Nuclear counterstaining was performed with 4',6'-diamidino-2-phenylindole (DAPI) 1/7000 in PBS for 4 min. Finally, sections were mounted in Dako fluorescent mounting medium and stored at 4°C until and viewed on a Zeiss Axiovert 100 M microscope with scanning head LSM 510 with the Zeiss Plan-Apochromat objective, x40 oil, numerical aperture of 1.4. Laser lines at 488, 364, and 543 nm were used for excitation. Acquisition was done with the Zeiss LSM Imaging Software version 2.81.

Statistical analysis

Unless otherwise noted, results are expressed as mean ± SEM. The differences among groups were analyzed by Mann-Whitney U test and, where appropriate, by Kruskal-Wallis ANOVA. Significance was assumed for p values <0.05. All data analyses were performed with the SPSS 12.0 software package.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Recombinant visfatin induces the production of cytokines in human PBMCs

To determine the effect of recombinant visfatin on human leukocytes, freshly isolated PBMCs were incubated with visfatin. Stimulation with visfatin resulted in a dose-dependent induction of IL-1beta, IL-1Ra, IL-6, IL-10, and TNF-{alpha} (Fig. 1, A–E). The most pronounced effects were observed for IL-6 production, reaching statistical significance at a concentration as low as 5 ng/ml when compared with untreated controls (Fig. 1C; *, p < 0.05; **, p < 0.01). A total of 50 ng/ml visfatin significantly up-regulated the release of IL-1beta and TNF-{alpha} (Fig. 1, A and E; *, p < 0.05). IL-1Ra and IL-10 induction became significant at visfatin concentrations of 100 and 250 ng/ml, respectively (Fig. 1, B and D; *, p < 0.05; **, p < 0.01).


Figure 1
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  		FIGURE 1. Recombinant visfatin induces cytokine production in human leukocytes. A total of 2 x 106/ml human PBMCs (n = 6) were treated with saline or increasing concentrations of visfatin for 16 h and cytokines were determined by ELISA. Polymyxin B was present at all conditions. A, IL-1beta (*, p < 0.05; **, p < 0.01); B, IL-1Ra (*, p < 0.05); C, IL-6 (*, p < 0.05; **, p < 0.01); D, IL-10 (*, p < 0.05; **, p < 0.01); E, TNF-{alpha} (*, p < 0.05; **, p < 0.01). Preadipocytes from SGBS were differentiated into mature adipocytes and glucose uptakes were performed to test for visfatin’s insulin mimetic effect (F).

 
Recombinant visfatin was derived from E. coli and endotoxin content was below 0.1 EU/µg (~10 pg of endotoxin/µg of protein) as determined by Limulus amebocyte lysate testing. Culture medium was therefore supplemented with 5 µg/ml polymyxin B in all experiments. This concentration efficiently blocked endotoxin-induced cytokine production up to a concentration of 1 ng/ml (data not shown).

Visfatin has been shown to bind to the IR and to mimic insulin action (8). Therefore, PBMCs were incubated with 10 nM insulin alone or in combination with 100 ng/ml recombinant visfatin. Incubation with insulin alone did not induce cytokine production in human PBMCs nor did the presence of insulin alter the visfatin-induced induction of cytokines (n = 3, data not shown).

2-Deoxy-D-glucose transport measurements were performed to confirm visfatin’s biological activity. Preadipocytes from Simpson Golabi Behmel syndrome (SGBS), a gift from Dr. M. Wabitsch (University of Ulm, Ulm, Germany), were grown and differentiated as described previously (27). The SGBS is a rare X-linked recessive disorder characterized by pre- and postnatal overgrowth. The molecular defect causing this syndrome has not yet been exactly characterized although mutations in the glypican 3 gene have been associated with the syndrome in some reported patients (27). As depicted in Fig. 1F, 100 nM insulin significantly up-regulated glucose uptake in SGBS adipocytes. An equimolar concentration of 100 nM (=5.2 ng/ml) visfatin also induced glucose uptake yet to a lesser extent. A high visfatin concentration of 2 µM (=100 ng/ml) could not further enhance glucose uptake in SGBS adipocytes (Fig. 1F). These data indicate that visfatin activates the IR but activation of the IR does not interfere with cytokine production.

Recombinant visfatin modulates cytokine gene expression in human PBMCs

Quantitative real-time PCR was performed to confirm the protein data. Again, visfatin dose-dependently induced IL-6, IL-10, and TNF-{alpha} mRNA expression levels in PBMCs (Fig. 2; *, p < 0.05; **, p < 0.01). As for protein levels, the strongest mRNA up-regulation was seen for IL-6, with an 83.5-fold induction at 100 ng/ml and a 316-fold induction at 250 ng/ml (Fig. 2A, *, p < 0.05; **, p < 0.01). As shown in Fig. 2, B and C, stimulation with 100 and 250 ng/ml visfatin resulted in a 4.4- and 10.5-fold induction of IL-10 and a 3.3- and 6.9-fold induction of TNF-{alpha}, respectively (*, p < 0.05; **, p < 0.01).


Figure 2
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  		FIGURE 2. Increased expression of cytokine mRNA in visfatin-treated leukocytes. Freshly isolated PMBCs were incubated in presence or absence of the indicated concentrations of recombinant human visfatin. Total mRNA was extracted after 5 h, and cytokine expression levels were analyzed by quantitative real-time PCR. A, IL-6 mRNA expression in human PBMCs. B, IL-10 mRNA expression in human PBMCs. C, TNF-{alpha} mRNA expression in human PBMCs. All expression levels are normalized to GAPDH (n = 5; *, p < 0.05; **, p < 0.01).

 
Visfatin activates effector functions of human APCs

Monocytes, DCs, and macrophages are critical regulators of innate as well as adaptive immune responses. Their functions comprise effector tasks with secretion of pro- and anti-inflammatory cytokines, phagocytosis of microorganisms and foreign Ags, Ag presentation, and provision of costimulatory molecules. We therefore studied the effect of visfatin on APCs. As shown in Fig. 3, A–C, visfatin was able to induce the secretion of IL-1beta, IL-6, and TNF-{alpha} from freshly isolated CD14+ monocytes in a dose-dependent manner.


Figure 3
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  		FIGURE 3. Visfatin activates CD14+ monocytes. A–C, CD14+ monocytes (n = 6) were treated with the indicated concentrations of recombinant visfatin for 16 h. Supernatants were harvested and levels of IL-1beta (A), IL-6 (B), and TNF-{alpha} (C) were determined by ELISA (*, p < 0.05). D–F, For the detection of cell surface markers monocytes were incubated with saline or 250 ng/ml visfatin and subsequently analyzed for CD54 (ICAM) (D), CD40 (E), and CD80 (B7-1) (F) by flow cytometry. Representative stainings of three independent experiments are shown (dotted line: isotype control; thin line: solvent-treated cells; thick line: visfatin-treated cells). G–I, Mannose receptor-mediated endocytosis was detected as uptake of FITC-dextran at 37°C, and threshold was set according to baseline uptake of control monocytes that were simultaneously incubated on ice (n = 3). The FITC-dextran uptake of CD14+ monocytes was elevated 4.4-fold at 100 ng/ml (H) and 5.9-fold at 250 ng/ml (I) recombinant visfatin when compared with unstimulated controls (G). J, Visfatin significantly enhanced the alloproliferative response as determined by ANOVA with post hoc Bonferroni (*, p < 0.05). Thymidine incorporation in a MLR using 0.5 x 105 visfatin-preincubated CD14+ monocytes as stimulator cells and MHC-mismatched PBLs in the indicated stimulator:responder ratios (n = 3).

 
Interestingly, visfatin was not able to induce IL-1beta and TNF-{alpha} in GM-CSF-differentiated monocyte-derived macrophages and DCs (data not shown). However, visfatin also induced IL-6 in these cell types (Table III).


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  		Table III. Recombinant visfatin induces IL-6 production in CD14+ monocyte-derived DCs and macrophagesa

 
Expression of cell surface markers in freshly isolated visfatin-stimulated monocytes was studied by flow cytometry. As presented in Fig. 3, D–F, visfatin efficiently up-regulated cell surface expression of CD54 (Fig. 3D), CD40 (Fig. 3E), and CD80 (Fig. 3F). No significant changes were observed for the expression of MHC class II, CD69, and CD86 (data not shown). Mannose receptor-mediated uptake of soluble Ag was measured in visfatin-stimulated as well as control monocytes (Fig. 3, G–I). Treatment with 100 or 250 ng/ml recombinant visfatin significantly enhanced FITC-dextran uptake 4.4- and 5.9-fold, respectively.

To elucidate whether the observed changes in surface expression of costimulatory molecules might alter T cell activation, we performed MLR with CD14+ monocytes as stimulator and PBLs as allogeneic responder cells. To exclude that visfatin might directly affect accessory cells, especially B cells, monocytes were stimulated with visfatin overnight and consequently fixed with glutaraldehyde. Indeed, consistent with their altered immunophenotype, visfatin-stimulated monocytes exhibited a significantly increased allostimulatory capacity (Fig. 3J). As depicted in Fig. 3J, a dose-dependent increase in proliferative response of PBLs was seen more obvious at lower stimulator/responder ratios of 1:1 and 1:3 but was still present at a higher ratio of 1:5. Taken together, these results indicate that visfatin is a potent activator of human monocytes by inducing effector functions and enhancing T cell responses.

Inhibition of p38 MAPK abrogates visfatin-induced cytokine production

Fukuhara et al. (8) demonstrated that visfatin binds to the IR and mimics insulin effects. We speculated that visfatin might activate additional signaling pathways. Thus, CD14+ monocytes were incubated with recombinant visfatin and several specific pharmacologic kinase inhibitors were used to gain insight into possible upstream mechanisms. Inhibition of the p38 MAPK by SB203580 almost completely abrogated all observed changes in visfatin-induced cytokine production indicating a central role for p38 in visfatin signal transduction (Fig. 4, A–E; *, p < 0.05; **, p < 0.01). Inhibition of MEK1 (MAP2K) through PD 98059 also significantly prevented the production of IL-1beta, IL-6, and TNF-{alpha} but not IL-1Ra and IL-10 (Fig. 4, A–E). JNK inhibitor II that selectively blocks JNK activity, which activates AP-1 and related transcription factors like ATF2 (28, 29), significantly inhibited TNF release from visfatin-stimulated monocytes. Inhibition of PI3K by LY294002 significantly down-regulated TNF-{alpha} as well as the anti-inflammatory cytokine IL-10 (Fig. 4, D and E). Because PI3K is critically involved in the control of cell death by activating the survival kinase Akt (30), induction of PI3K could be a possible mechanism involved in visfatin’s antiapoptotic properties as reported in previously published data (31, 32). Blockade of Janus tyrosine protein kinase activity (JAK1–3) did not alter visfatin-induced cytokine production for any of the observed mediators (Fig. 4, A–E).


Figure 4
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  		FIGURE 4. Human CD14+ monocytes were stimulated with 100 ng/ml recombinant visfatin in the presence of either solvent (DMSO) or three times IC50 of the indicated specific kinase inhibitor for p38 kinase (p38) (SB203580), MEK (MEK1) (PD98059), JNK (Inhibitor II), and PI3K (LY204002). Concentrations of IL-1beta (A), IL-1Ra (B), IL-6 (C), IL-10 (D), and TNF-{alpha} (E) in supernatants were determined by ELISA (*, p < 0.05). Visfatin-induced cytokine production in human monocytes (n = 6) is abrogated in the presence of a selective inhibitor of p38 kinase. Inhibition of MEK1 significantly down-regulated the production of proinflammatory cytokines IL-1beta, IL-6, and TNF-{alpha}. Blockade of PI3K significantly suppressed the induction of TNF-{alpha} as well as the anti-inflammatory cytokine IL-10. JNK inhibitor II significantly reduced visfatin-induced TNF release from human monocytes. Visfatin increases NF-{kappa}B p65 (RelA) DNA binding capacity in human leukocytes (F). PBMCs were incubated with visfatin or LPS and p65 DNA-binding capacity was determined by a chemiluminescent transcription factor assay at the indicated time points (n = 3). Data are expressed as relative light units (RLU) (*, p < 0.05).

 
Activation of NF-{kappa}B transcription factors is a central event in the initiation and amplification of inflammatory responses (33). We therefore analyzed the time-dependent activation of p65 (RelA), part of the p50:RelA dimer that is activated by the classical pathway, in freshly isolated leukocytes. As depicted in Fig. 4F, visfatin significantly increased active DNA-binding p65 (RelA) reaching a peak 6 h after stimulation compared with untreated control monocytes (*, p < 0.05).

Recombinant visfatin induces chemotaxis in monocytes and B cells

As depicted in Fig. 5, visfatin dose-dependently induced a migratory response in Boyden chamber microchemotaxis experiments. We found a visfatin-induced migratory response in CD14+ monocytes (Fig. 5A) and in CD19+ B cells (Fig. 5B), but not in CD3+ T cells (data not shown). The observed response was particularly strong reaching levels comparable with fMLP and IL-8 (CXCL8) that were used as positive controls.


Figure 5
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  		FIGURE 5. Effect of visfatin on leukocyte chemotaxis. Freshly isolated CD14+ monocytes and CD19+ B cells were allowed to migrate into nitrocellulose toward various concentrations of visfatin in the lower wells of a Boyden microchemotaxis chamber. Direct chemotaxis of CD14+ monocytes (n = 5) (A), and CD19+ B cells (n = 5) (B). fMLP and IL-8 (CXCL8) served as positive controls. Data are expressed as the chemotaxis index: the ratio of the distance of stimulated and random migration of leukocytes into nitrocellulose filters (*, p < 0.05).

 
Recombinant visfatin induces IL-6 in mice

To understand the in vivo biological relevance of visfatin, we injected recombinant murine visfatin i.p. to BALB/c mice, determined levels of circulating cytokines, and measured cytokine mRNAs in various tissues. Ten mice were treated with either 10 µg of visfatin or saline at 0 and after 12 h. In experimental series 1, blood was collected from the tail vein of five visfatin and five control animals after 15 h and the animals were sacrificed at 20 h. In experimental series 2, five visfatin and five control animals were sacrificed at 15 h (experimental structure is outlined in Fig. 6A). Evaluation of circulating cytokines showed that visfatin-treated animals had significantly elevated serum concentrations of IL-6 after 15 h (3 h after the second visfatin challenge) (Fig. 6B; *, p < 0.05). Elevation of IL-6 serum levels was less pronounced after the first visfatin injection (data not shown). Elevated IL-6 concentrations rapidly declined to control levels and no difference was seen at 20 h (Fig. 6B). Notably, we did not observe any differences in circulating TNF concentrations. To test whether visfatin could induce expression of cytokines in vivo, total tissue RNA was extracted from liver, spleen, lung, and small intestine that were collected from visfatin-treated and control animals of experimental series 2. Consequently, IL-1beta, IL-6, and TNF-{alpha} mRNA transcripts were analyzed by quantitative PCR. mRNA expression of IL-6 was significantly higher in the small intestine of visfatin-treated mice (Fig. 6D; *, p < 0.05). Both IL-1beta and TNF-{alpha} mRNA expression were elevated in the liver of visfatin-treated animals although they did not reach statistical significance (Fig. 6, C and E; p = 0.076).

High circulating visfatin levels are observed in patients with IBD

IBD, in particular CD, is known to express high levels of IL-6 in the gut mucosa, and IL-6 trans-signaling is considered a key factor in apoptosis resistance of lamina propria T cells (34). We therefore investigated the activation state of visfatin in IBD patients.

As shown in Fig. 7A, circulating levels of visfatin were significantly elevated in IBD patients compared with healthy controls. In CD patients, serum visfatin levels were elevated irrespective of disease activity (active disease: CD activity index (CDAI) (35) >150; remission: CDAI <150). In UC, however, visfatin concentrations appeared to be higher in active UC (Rachmilewitz clinical activity score (CAI) (36) >4) compared with UC patients in remission (CAI <4) (Fig. 7A).


Figure 7
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  		FIGURE 7. Circulating visfatin protein and colonic visfatin mRNA expression are elevated in patients with IBD. A, Visfatin serum concentrations were determined in patients with CD and UC and compared with healthy controls. CD and UC patients were divided with respect to disease activity as determined by CDAI for CD and Rachmilewitz CAI for UC patients. The number of patients included is given below the x-axis. B, Total RNA was extracted from IBD (involved and noninvolved) and healthy control colonic biopsy specimen and visfatin mRNA expression was quantified by real-time PCR. Data are expressed as visfatin/GAPDH ratios. The number of patients is indicated below the abscissa.

 
Visfatin mRNA expression is increased in CD and UC

Real-time PCR analysis was performed to quantitate visfatin mRNA expression in colonic biopsy specimens of patients with IBD and healthy controls. Data were normalized to human GAPDH. A significant up-regulation of visfatin mRNA expression was observed in inflammatory colonic biopsy specimens of both CD and UC patients compared with control subjects (Fig. 7B; *, p < 0.05). Visfatin mRNA expression in noninvolved CD and UC biopsy specimens was still elevated when compared with healthy control specimens, but this difference did not reach statistical significance (Fig. 7B).

Cellular sources of visfatin in inflammatory colonic tissue

To identify cellular sources of human visfatin, we performed confocal microscopy with double-immunofluorescence staining of visfatin with several specific cellular markers. As depicted in Fig. 8A, we detected visfatin in adipocytes of mesenteric tissue adjacent to the colonic wall (white arrows). Furthermore, Fig. 8A shows CD163+ double-positive tissue macrophages that reside between adipocytes (arrowheads). Fig. 8B also displays double-positive CD163+ tissue macrophages within the submucosa. Moreover, visfatin colocalized with DCs (Fig. 8D), detected by an Ab directed against CD209 (DC-SIGN), and cytokeratin 18-positive epithelial cells (Fig. 8C). Although visfatin expression has been described in PBLs, we did not colocalize visfatin within mucosa-infiltrating CD3+ T cells (Fig. 8E), nor in secondary follicle-associated CD20+ B cells (Fig. 8F). No colocalization was found in CD31+ endothelial cells (Fig. 8G). Smooth muscle cells as identified by staining for smooth muscle actin were slightly positive for visfatin (data not shown).


Figure 8
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  		FIGURE 8. Double-immunofluorescence microscopy on frozen sections of visceral adipose tissue and colonic wall from CD resections. Cellular localization of visfatin was identified by indirect staining (red) to identify visfatin+ cell types. Cell nuclei are shown in blue (DAPI). Costainings for identification of specific cell types are stained in green. A, Visfatin+ adipocytes (white arrows) of visceral fat from CD patients. CD163 double-positive tissue macrophages (green to yellow; red + green = yellow; white arrowheads) reside within the adipose tissue next to the adipocytes. B, CD163 double-positive tissue macrophages in the submucosa of the inflamed colonic wall. C, Cytokeratin 18 (CK18) double-positive colonic epithelial cells from colonic crypts. D, CD209 (DC-SIGN) double-positive DCs in the submucosa. E, CD3+ T cells (green) adjacent to visfatin single-positive cells (red). Some T cell are penetrating into colonic crypts consisting of visfatin+ epithelial cells. F, CD19 slightly double-positive B cells of a submucosal secondary follicle. G, CD31+ (PECAM) endothelial cells. H, MHC class II (MHC II) single- and double-positive cells. Specificity of staining was confirmed by omitting the first Ab (for visfatin) and isotype-matched irrelevant monoclonal control Abs for all other Abs used (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have reported proinflammatory activities exerted by the recently characterized adipocytokine visfatin. We have demonstrated that visfatin, initially described as PBEF, dose-dependently up-regulated the production of the pro- and anti-inflammatory cytokines IL-1beta, IL-1Ra, IL-6, IL-10, and TNF-{alpha} in human monocytes. These cytokines play a substantial role in a wide range of infectious and inflammatory diseases (37, 38, 39).

APCs such as monocytes, DCs, and macrophages execute one of the central processes inducing and regulating immune functions by establishing cell-cell contacts with T cells. Besides the interaction of the peptide-Ag-MHC complex with the TCR, additional signals delivered by costimulatory cell surface molecules are crucial for effective lymphocyte activation. On the one hand, APC-derived CD80 (B7-1) and CD86 (B7-2) provide important costimulatory signals to augment and sustain T cell response via ligation with CD28 (40). On the other hand, ligation of T cell-derived CD154 (CD40L) with CD40 activates APCs and induces their persistence (41). We demonstrate that visfatin induces expression of the costimulatory molecules CD80 (B7-1) and CD40 in human monocytes. Moreover, we observed a significant induction of ICAM-1 (CD54), another costimulatory ligand that binds to LFA-1, thereby promoting the activation of T cells (42). Evidence that visfatin affects primary lymphocyte responses was demonstrated by an increased dose-dependent proliferative response after preincubating monocytes with visfatin. Notably, visfatin was able to increase significantly mannose receptor-mediated phagocytosis by human monocytes. Altogether, we provide evidence that visfatin activates APCs, up-regulates the expression of costimulatory molecules and provokes an enhanced proliferative response in the MLR therefore regulating and affecting these central immune functions. Finally, in accordance with previous data (31), APCs might be a major source of visfatin themselves as identified by immunofluorescence double staining with macrophage and DC markers (CD163, DC-SIGN, MHC class IIhigh) in colonic tissue samples of IBD patients. Trafficking of cells to the sites of inflammation is another critical function of the immune system and largely orchestrated by chemokines (43). We provide evidence that visfatin is a potent chemotactic factor particularly for CD14+ monocytes and CD19+ B cells.

Various extracellular signals are integrated and processed by MAPK cascades (44). p38 MAPK, ERK, and JNK are three distinct MAPK pathways. Our results indicate a central role for p38 and MEK-1 for visfatin-induced signal transduction. Visfatin has originally been defined as cytokine which acts on pre-B cell formation together with IL-7 (11). Notably, IL-7 is a key cytokine for early B and T cell development (45) and recently Wan and coworkers (46) demonstrated that p38 activation can be found upon IL-7 stimulation. Further studies are required to exactly characterize a possible role of IL-7- and visfatin-induced p38 activation in pre-B cell formation. NF-{kappa}B plays an important role in triggering and coordinating immune responses including regulation of cytokines like IL-1, IL-6, and TNF (47). Activators of NF-{kappa}B induce rapid, I{kappa}B kinases dependent, phosphorylation, polyubiquitination, and finally proteasomal degradation of I{kappa}B (48). Visfatin up-regulated NF-{kappa}B p65 (RelA) DNA-binding activity in human leukocytes. However, it remains to be determined whether the observed NF-{kappa}B activation is a direct effect or caused secondary due to induction of other cytokines. Visfatin binds to and activates the insulin receptor but insulin does not interact with its cytokine-inducing effects (data not shown). Our observations support the hypothesis that cytokine induction by visfatin might be induced by engagement of another so far unidentified receptor (49). Treatment of human monocytes with recombinant visfatin leads to p38- and MEK-1-dependent induction of IL-1beta, IL-6, and TNF-{alpha} and identifies visfatin as a new upstream activator of these stress-activated kinases.

When administered to mice, murine visfatin significantly increased the level of circulating IL-6. We did not detect elevated levels of TNF-{alpha} or IL-1beta after visfatin administration. Fukuhara et al. (8) demonstrated that acute administration of recombinant visfatin resulted in a significant fall of plasma glucose levels within 30 min that quickly returned to control levels after 60 min. Their results suggest a short plasma half-life for visfatin whose biological activity might be regulated by enzymatic inactivation or potential natural occurring antagonists that might be a rationale for the comparably weak in vivo effects. The increase in IL-6 levels was paralleled by an up-regulation of IL-6 mRNA levels in the intestine that seemed to be the major source because no differences were observed in liver, spleen, or lung. This result fits well with our in vitro data in human leukocytes where IL-6 was the cytokine most prominently up-regulated. Moreover, it is notable that IL-6 was the only cytokine found to be up-regulated in human macrophages and DCs after visfatin stimulation (Table III). IL-6 is known to be a pleiotropic cytokine that is critically involved in a variety of immunological processes, such as activation of acute phase responses (50), hemopoiesis (51), final B cell maturation, T cell activation and proliferation (52), induction of chemokines and leukocyte recruitment (53), and liver and neuronal regeneration (54, 55). Moreover, visfatin-induced IL-6 expression might be involved in the pathogenesis of insulin resistance associated with visceral obesity (56). IL-6 has been demonstrated to promote insulin resistance via induction of suppressor of cytokine signaling proteins (57). Our results raise the possibility that obesity-related enhanced visfatin expression (8, 56) induces IL-6 production which is likely to promote insulin resistance.

The proinflammatory cytokine IL-6 is also highly expressed in patients with IBD (58). By binding to its soluble receptor IL-6 can stimulate cells lacking the IL-6R. This IL-6 trans-signaling activates STAT3, bcl-2, and bcl-xL and mediates resistance of T cells to apoptosis (34). Thus, we investigated the activation state of visfatin in CD and UC. We observed significantly increased visfatin serum levels in IBD patients compared with control subjects. This is in accordance with recent reports that demonstrated high circulating visfatin levels in rheumatoid arthritis and acute lung injury (59, 60). Significantly higher visfatin mRNA expression in inflamed IBD colonic biopsies suggests that the colonic mucosa is a potential source of elevated visfatin plasma levels. By histological examination, we identified potential cellular sources of visfatin in inflamed colonic tissue that included APCs, like DCs and macrophages, as well as epithelial cells. There are several reports demonstrating enhanced tissue expression of visfatin in inflammatory conditions including acute lung injury (60), clinical sepsis (31), and severe generalized psoriasis (61). However, with serum concentrations between 1 and 3 ng/ml circulating visfatin levels are low, even in patients with active IBD when compared with the effective concentrations required for in vitro cytokine induction. It remains to be established whether the enhanced tissue-specific visfatin expression might be sufficient to propose a role for visfatin as an autocrine/paracrine inflammatory cytokine. Visfatin was shown to be more abundantly expressed in visceral compared with s.c. adipose tissue (8). As expected, visfatin could be detected in adipocytes of the mesenteric adipose tissue. Notably, adipose tissue-infiltrating macrophages also stained positive for visfatin and should be considered to contribute to the overall visfatin expression level at this location.

The functional profile of visfatin reported in this study would suggest a potential role of this adipocytokine in the pathogenesis of inflammatory disorders. Further studies focusing on the identification of a potential cellular receptor apart from the insulin receptor and its pharmacological manipulation in experimental and human disease will further illuminate the role of this novel proinflammatory adipocytokine.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Austrian Science Foundation (P17447). Back

2 Address correspondence and reprint requests to Dr. Herbert Tilg, Department of Medicine, Clinical Division of Gastroenterology and Hepatology, Innsbruck Medical University and Christian Doppler Research Laboratory for Gut Inflammation, Anichstrasse 35, 6020 Innsbruck, Austria. E-mail address: Herbert.Tilg{at}uibk.ac.at Back

3 Abbreviations used in this paper: IR, insulin receptor; PBEF, pre-B cell-enhancing factor; IL-1Ra, IL-1 receptor antagonist; DC, dendritic cell; GUSB, glucuronidase beta; qPCR, quantitative PCR; IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; SGBS, Simpson Golabi Behmel syndrome; CDAI, CD activity index; CAI, clinical activity score. Back

Received for publication August 3, 2006. Accepted for publication November 2, 2006.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosures
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