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Nephrogenic Systemic FibrosisImplications for the Pathogenesis ofProinflammatory Phenotype and Macrophages: Establishment of aTLR 4 and TLR 7 in Normal Human Gadolinium Compounds Signaling through

Peter J. Wermuth and Sergio A. Jimenez

ol.1103099http://www.jimmunol.org/content/early/2012/05/30/jimmun

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The Journal of Immunology

Gadolinium Compounds Signaling through TLR 4 and TLR 7in Normal Human Macrophages: Establishment of aProinflammatory Phenotype and Implications for thePathogenesis of Nephrogenic Systemic Fibrosis

Peter J. Wermuth and Sergio A. Jimenez

Nephrogenic systemic sibrosis is a progressive disorder occurring in some renal insufficiency patients exposed to gadolinium-based

contrast agents (GdBCA). Previous studies demonstrated that the GdBCA Omniscan upregulated several innate immunity path-

ways in normal differentiated human macrophages, induced rapid nuclear localization of the transcription factor NF-kB, and

increased the expression and production of numerous profibrotic/proinflammatory cytokines, chemokines, and growth factors. To

further examine GdBCA stimulation of the innate immune system, cultured human embryonic kidney 293 cells expressing one of

seven different human TLRs or one of two human nucleotide-binding oligomerization domain-like receptors were exposed in vitro

for 24 h to various GdBCA. The signaling activity of each compound was evaluated by its ability to activate an NF-kB–inducible

reporter gene. Omniscan and gadodiamide induced strong TLR4- and TLR7-mediated reporter gene activation. The other Gd

compounds examined failed to induce reporter gene activation. TLR pathway inhibition using chloroquine or an inhibitor of IL-

1R–associated kinases 1 and 4 in normal differentiated human macrophages abrogated Omniscan-induced gene expression.

Omniscan and gadodiamide signaling via TLRs 4 and 7 resulted in increased production and expression of numerous

proinflammatory/profibrotic cytokines, chemokines, and growth factors, including CXCL10, CCL2, CCL8, CXCL12, IL-4, IL-

6, TGF-b, and vascular endothelial growth factor. These observations suggest that TLR activation by environmental stimuli may

participate in the pathogenesis of nephrogenic systemic fibrosis and of other fibrotic disorders including systemic sclerosis. The

Journal of Immunology, 2012, 189: 000–000.

Nephrogenic systemic fibrosis (NSF) is a generalized fi-brotic disease occurring in some individuals with renalinsufficiency following exposure to gadolinium-based

contrast agents (GdBCA) used to enhance magnetic resonanceimaging (1–4). Clinically, NSF displays many features in commonwith systemic sclerosis (SSc), including severe and usually pro-gressive skin induration, progressive and eventually incapacitatingjoint flexion contractures, and fibrotic involvement of lungs, heart,and numerous other internal organs (4–6). Histopathologic anal-ysis of affected NSF skin demonstrates marked dermal thickeningwith accumulation of thick collagen bundles extending into thes.c. tissue and fascia (1–6), as well as abundant mucin accumulation(7, 8). Furthermore, several studies have demonstrated Gd depositsin affected tissues (9–12). Although some reports have charac-terized NSF as a noninflammatory disorder, we and others have

demonstrated increased numbers of macrophages, myofibroblasts,and CD34+ fibrocytes (1–6) and described elevation of seruminflammatory markers in NSF patients with recent disease onset(Refs. 3, 4 and unpublished observations) indicative of the in-flammatory nature of the disorder.The mechanisms underlying GdBCA stimulation of tissue fibrosis

remain largely undetermined. GdBCA contain a single Gd3+ ioncomplexed to either a linear or a macrocyclic chelate that increasesGd3+ solubility and decreases Gd3+ toxicity (13–15). According toU.S. Food and Drug Administration data, all NSF cases in which theGdBCA used was identified have been associated with a linearGdBCA (16), most commonly Omniscan (62%) and Magnevist(32%). Other published reports raised the proportion of Omniscan-associated NSF cases to as high as 90% (17). A hypothesis to ex-plain these observations suggested that less thermodynamicallystable linear chelates undergo transmetallation, a process in whichendogenous circulating ions such as Zn2+ displace Gd3+ at greaterrates compared with macrocyclic chelates. It was further suggestedthat transmetallation is exacerbated by the markedly reducedGdBCA clearance rates in patients with renal insufficiency. How-ever, in vitro experiments have demonstrated that exposure to bothlinear and macrocyclic GdBCA induces potent functional effects oncultured human normal dermal fibroblasts (8, 18–26), normal humanPBMCs (27) and differentiated macrophages (28), and fibrocytes(29). One of these studies demonstrated strong activation of the NF-kB pathway in normal human macrophages following exposure toOmniscan and suggested that TLR activation mediated by the che-lated Gd3+ compounds may be responsible for these effects (28).Recently, the insight that TLRs are capable of recognition of

a variety of endogenous damage-associated molecular patternsreleased from injured and inflamed tissues (30) has led to a pro-

Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadel-phia, PA 19107

Received for publication November 1, 2011. Accepted for publication April 24, 2012.

This work was supported in part by National Institutes of Health Grant RO1 AR-019616 (to S.A.J.).

Address correspondence and reprint requests to Dr. Sergio A. Jimenez, JeffersonInstitute of Molecular Medicine, Thomas Jefferson University, Bluemle Life ScienceBuilding Suite 509, 233 South 10th Street, Philadelphia, PA 19107-5541. E-mailaddress: sergio.jimenez@jefferson.edu

Abbreviations used in this article: Gd, gadolinium; GdBCA, gadolinium-based con-trast agent; Gd-DTPA, gadolinium diethylenetriaminepentaacetic acid; HEK293,human embryonic kidney 293; IRAK, IL-1R–associated kinase; NLR, nucleotide-binding oligomerization domain-like receptor; NOD, nucleotide-binding oligomeri-zation domain; NSF, nephrogenic systemic fibrosis; SEAP, secreted alkaline phos-phatase; siRNA, small interfering RNA; SSc, systemic sclerosis.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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posed role of innate immunity in the development of various fi-brotic disorders including SSc and pulmonary fibrosis (31–39).Indeed, SSc patients display high levels of expression of IFN-responsive genes, markers of innate immune activation, whichcorrelates with the modified Rodnan skin score, a measure of thedegree of SSc skin involvement, and SSc sera induce IFN-a ex-pression in normal PBMCs (34, 35, 40). Furthermore, the TLR3ligand polyinosinic-polycytidylic acid induces marked activationof IFN-a and increased production of the potent profibrotic cy-tokine TGF-b in normal and SSc fibroblasts, resulting in autocrinestimulation of IFN-a– and TGF-b–responsive gene expressionand dermal inflammation and fibrosis in mice (35). Affected tis-sues from SSc patients often exhibit chronic inflammation, sug-gesting that the release of endogenous TLR ligands during in-flammation and TLR signaling may represent one mechanism thatinitiates and drives this autoimmune fibrotic disease (38). Inter-estingly, NSF occurs almost exclusively in patients with renalinsufficiency (1–4), a clinical condition often accompanied bychronic microinflammation (41).We have previously described changes in the transcriptome of

normal differentiated human macrophages induced by exposure tothe GdBCA Omniscan (28), including the upregulated expressionof IFN-responsive genes accompanied by a rapid and intenseNF-kB activation, as well as NF-kB–dependent expression of nu-merous proinflammatory/profibrotic cytokines, chemokines, andgrowth factors. The results of these studies have led to the hy-pothesis that GdBCA-induced TLR signaling activates expressionof proinflammatory/profibrotic molecules (28). To test this hy-pothesis, we evaluated the effect of Omniscan on NF-kB activa-tion in cells overexpressing a single TLR or nucleotide-bindingoligomerization domain (NOD)-like receptor (NLR). We reportthat cells expressing TLR4 or TLR7 were responsive to Omniscanexposure as measured by the activation of a reporter gene underNF-kB control. We then compared the ability of various GdBCAto signal through TLR4 or TLR7 and examined the effect oftwo TLR pathway signaling inhibitors on Omniscan-mediatedincreases in proinflammatory/profibrotic cytokine, chemokine, andgrowth factor expression in normal human differentiated macro-phages. The data presented in this study strongly indicate a rolefor TLR signaling by Omniscan in normal differentiated humanmacrophages, a pathway that may play a crucial role in the path-ogenesis of NSF. These results also provide support to the partici-pation of TLR in the pathogenesis of SSc and other inflammatoryfibrotic disorders.

Materials and MethodsGd compounds

Dotarem (Guerbet, Bloomington, IL), MultiHance (Bracco Diagnostics,Milan, Italy), ProHance (BraccoDiagnostics), and OptiMark (Mallinckrodt/Covidien, Hazelwood, IN) were supplied as sterile, aqueous solutionscontaining 500 mM Gd chelate. Omniscan and gadodiamide (provided byGE Healthcare, Chalfont St. Giles, U.K.) were supplied as sterile, aqueoussolutions containing 287 mg/ml (500 mM) gadodiamide. The Omniscansolution contained an additional 12 mg/ml (25 mM) caldiamide sodiumin water. Caldiamide (GE Healthcare) was supplied as a sterile aqueoussolution containing 12 mg/ml (25 mM) caldiamide sodium in water.Gd-EDTA (GE Healthcare) was supplied as a sterile aqueous solutioncontaining 250 mM Gd-EDTA in water. Gd-citrate (250 mM) was preparedby mixing 1 ml 250 mM Gd chloride and 1 ml 500 mM sodium citratesolutions at pH 7.4 (42). Gadolinium diethylenetriaminepentaacetic acid(Gd-DTPA; Sigma-Aldrich, St. Louis, MO), the Gd chelate used inMagnevist, was dissolved in sterile PBS solution at 0.5 M concentration.The reagents employed for all of the studies were tested and verified by themanufacturer to be free from endotoxin contamination. The absence ofendotoxin contamination was further confirmed in our laboratories utiliz-ing the E-Toxate assay (Sigma-Aldrich) according to the manufacturer’sinstructions.

TLR screening

Human embryonic kidney 293 (HEK293) cells engineered to express one ofseven different human TLRs (TLRs 2, 3, 4, 5, 7, 8, or 9) or two human NLRs(NOD1 or NOD2) were obtained from InvivoGen (San Diego, CA). Forexperiments the cells were seeded into 96-well plates at a density of 20,000cells per well in DMEM containing 10% FBS (Life Technologies, GrandIsland, NY), 1% vitamins, 2 mM glutamine, antibiotics, and fungizone(DMEM complete medium). These cells also express a reporter constructunder the control of the NF-kB–inducible promoter resulting in expressionof secreted alkaline phosphatase (SEAP) into the culture medium reflectingthe levels of NF-kB activation. Cells in DMEM complete medium wereexposed in duplicate for 20 h to 5 mM of one of the following Gd com-pounds: ProHance, Omniscan, or gadodiamide; or to 2.7 mMGd-EDTA; orto 0.25 mM of the chelate molecule caldiamide in 200 ml medium con-taining Quanti-Blue (InvivoGen) to allow colorimetric detection of SEAP.As positive controls, the cellular response to known specific TLR ligands(InvivoGen) was also measured: heat-killed Listeria monocytogenes at 108

cell/well (TLR2), 1 mg/ml polyinosinic-polycytidylic acid (TLR3), 100 ng/ml Escherichia coli K12 LPS (TLR4), 100 ng/ml Salmonella typhimuriumflagellin (TLR5), 1 mg/ml CL097 (TLR7), 1 mg/ml CL075 (TLR8), 1 mg/ml CpG oligodeoxynucleotide 2006, 100 ng/ml C12-iEDAP (NOD1), or10 ng/ml L18-MDP (NOD2). The absorbance at 650 nm was measured ona Beckman Coulter AD 340C absorbance detector following the 20 h in-cubation.

For TLR ligand dose–response experiments, the above procedure wasperformed using HEK293 cells expressing either human TLR4 or TLR7cultured in DMEM complete medium at a density of 50,000 cells per well.The cells were exposed to 0.5, 1, or 5 mM of one of the following GdBCA:Gd-DTPA, Dotarem, MultiHance, ProHance, OptiMark, gadodiamide, orOmniscan; or to 2.7, 27, or 270 nM Gd-EDTA; or to 0.025, 0.05, or 0.25mM of the chelate molecule caldiamide in 200 ml medium containingQuanti-Blue as described above. As positive controls, the response to 1, 5,or 10 ng/ml E. coli K12 LPS (TLR4) or 1, 5, or 10 mg/ml gardiquimod(TLR7) was measured. No significant effect on cell numbers or cytotox-icity was observed as examined by the WST-1 assay (Roche Diagnostics,Indianapolis, IN).

Macrophage isolation and differentiation

Normal human peripheral blood leukoreduction filters were obtained fromthe Thomas Jefferson University Hospital Blood Bank following Institu-tional Review Board approval. Human PBMCs were isolated from theleukoreduction filters by Ficoll-Hypaque gradient centrifugation (Amer-sham Pharmacia Biotech, Piscataway, NJ) and enriched for monocytes byadherence to plastic culture dishes for 2 h in RPMI 1640 containing 10%FBS (Life Technologies), as described previously (27). To obtain terminallydifferentiated macrophages the monocytes were cultured in RPMI 1640complete medium with 60 ng/ml M-CSF (BioVision, Mountain View, CA)and 25 ng/ml IL-10 (BioVision) for 7 d as described (28). Differentiatedmacrophages (5 3 105 cells/ml) were exposed for 24 h to 1 mM of one ofthe following GdBCA: Gd-DTPA, Dotarem, MultiHance, ProHance,OptiMark, Omniscan, or gadodiamide; or to 0.05 mM caldiamide; or to0.27 mM Gd-EDTA or Gd-citrate. No significant effect on cell numbersor cytotoxicity was observed as examined by the WST-1 assay (RocheDiagnostics).

TLR inhibition studies

To confirm a role of TLRs in GdBCA stimulation of macrophages, normalhuman differentiated macrophages were washed with PBS and exposedto either 50 mM TLR inhibitor chloroquine (InvivoGen) or 300 nMIL-1R–associated kinase (IRAK)1/4 inhibitor 1-(2-(4-morpholinyl)ethyl)-2-(3-nitrobenzoylamino)benzimidazole, N-(2-morpholinylethyl)-2-(3-nitro-benzoylamido)-benzimidazole (Sigma-Aldrich) in RPMI 1640 completemedium for 1 h. This incubation was followed by addition of either PBSor PBS with 1 mM of either Omniscan, gadodiamide, or ProHance; or 0.05mM caldiamide; or 2.7 mM Gd-EDTA for 24 h. As positive controls forTLR-dependent and TLR-independent macrophage stimulation, 100 ng/mlLPS (InvivoGen) or 10 mg/ml TNF-a (Pierce Biotechnology, Woburn,MA), respectively, were used. Macrophage samples cultured with an equalvolume of PBS served as negative controls. No significant effect on cellnumbers or cytotoxicity was observed as examined by the WST-1 assay(Roche Diagnostics). Macrophage culture supernatants were isolated, fil-tered, and maintained frozen for subsequent studies. Cells were washedtwice with PBS and then processed for RNA extraction using the RNeasykit (Qiagen, Valencia, CA) according to the protocol recommended by themanufacturer as described previously (27).

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RNA interference

DharmaFECT 1 small interfering RNA (siRNA) transfection reagent,siGENOME SMARTpool siRNAs specific for human TLR4 and TLR7, andsiGENOME RISC-free control siRNAs were purchased from Dharmacon(Lafayette, CO). Normal human macrophages (5 3 105) differentiated asdescribed above were plated in six-well plates and transfected with 100 nMsiRNA for TLR4 or TLR7 or TLR4 plus TLR7 or with control (scrambled)siRNAs using DharmaFECT 1 (3 ml/well) according to the manufacturer’sinstructions. After 24 h, macrophages were treated with either 1 mMOmniscan, 1 mM gadodiamide, or 1 mM ProHance for 24 h or left untreated.Total RNA was extracted and RNA levels were assessed by real-time PCRas described above. No significant effect on cell numbers or cytotoxicity wasobserved as examined by the WST-1 assay (Roche Diagnostics).

Real-time PCR validation

Expression levels of IL-4, IL-6, IL-13, IFN-g, TGF-b, VEGF, TLR4, TLR7,CCL2 (MCP-1), CCL8 (MCP-2), CXCL9, CXCL10 (IP10), CXCL11(ITAC), and CXCL12 were assayed by real-time quantitative PCR utilizingSYBR Green chemistry (Applied Biosystems, Foster City, CA) followinga standard amplification protocol on an ABI Prism 7900 sequence detec-tion system (Applied Biosystems). The following primers were employed:b-actin, forward, 59-TTGCCGACAGGATGCAGAA-39, reverse, 59-GC-CGATCCACACGGAGTACTT-39; IL-4, forward, 59- TGCTGCCTCCA-AGAACACAA-39, reverse, 59-TGTAGAACTGCCGGAGCACA-39; IL-6,forward, 59-TGAGGAGACTTGCCTGGTGAAA-39, reverse, 39-TGGC-ATTTGTGGTTGGGTCA-39; IL-13, forward, 59-AGCTGGTCAACATC-ACCCAGAA-39, reverse, 59-AGCTGTCAGGTTGATGCTCCAT-39; IFN-g,forward, 59-TTCAGATGTAGCGGATAATGGAAC-39, reverse, 59-TTCT-GTCACTCTCCTCTTTCCA-39; TGF-b, forward, 59-CGAGCCTGAGG-CCGACTA-39, reverse, 59-AGATTTCGTTGTGGGTTTCCA-39; vascularendothelial growth factor (VEGF), forward, 59-AGAAGGAGGAGGGC-AGAATCAT-39, reverse, 59-TAATCTGCATGGTGATGTTGG-39; CCL2,forward, 59-ACCAGCAGCAAGTGTCCCAAA-39, reverse, 59-TTTGCT-TGTCCAGGTGGTCCAT-39; CCL8, forward, 59-TCATGCTGAAGCTCA-CACCCTT-39, reverse, 59-AGAATTGCCATTGCACAACTCTT-39; CXCL10,forward, 59-ACTGCCATTCTGATTTGCTGCC-39, reverse, 59-TGATGC-AGGTACAGC GTACAGT-39; CXCL11, forward, 59-ACTCCTTCCAA-GAAGAGCAGCA-39, reverse, 59-CCATGCCCTTCACACTCATGTT-39;CXCL12, forward, 59-AAAGCCATGTTGCCAGAGCCAA-39, reverse,59-AGCTTCGGGTCAATGCACACTT-39; TLR4, forward, 59-AGAACT-GCAGGTGCTGGATT-39, reverse, 59-AGAGGTGGCTTAGGCTCTGATA-39; TLR7, forward, 59-CTGCTCTCTTCAACCAGACCTCTAC-39, reverse,59-AGAGTGACATCACAGGGCAGAG-39.

Relative quantification was performed by arbitrarily setting the ex-pression level of the PBS-negative control at 100 and by expressing changesin transcript levels of other samples relative to this control sample. Resultsobtained from three experiments with each of the GdBCA alone or in thepresence of each inhibitor examined, utilizing triplicate samples of normalhuman macrophages, were averaged. Relative differences in each PCRsample were corrected using human b-actin mRNA levels as an endoge-nous control.

Multiplex ELISA

SearchLight proteome array analyses (Aushon Biotechnology, Woburn,MA) were conducted to measure the levels of IL-4, IL-6, IL-13, IFN-g,TGF-b, VEGF, CCL2, CCL8, CXCL10, and CXCL11 in macrophageculture supernatants from the TLR inhibition studies described abovefollowing procedures described previously (43). Briefly, culture superna-tant samples were diluted 1:2, 1:50, or 1:1000 and then incubated for 1 hon array plates that had been prespotted with capture Abs specific for eachprotein. Plates were decanted and washed three times with PBS beforeaddition of a mixture of biotinylated detection Abs to each well. Followingincubation with detection Abs for 30 min, plates were washed three timesand incubated for 30 min with streptavidin-HRP. Plates were againwashed, and SuperSignal Femto chemiluminescent substrate (Pierce Bio-technology) was added. The plates were immediately imaged using theSearchLight imaging system and data were analyzed using ArrayVisionsoftware (GE Healthcare).

Statistical analysis

Real-time PCR values reflect themean and SD of three separate experimentseach performed in triplicate with each of the three samples of normal humanmacrophages. The statistical significance of the real-time PCR data wasassessed by a Student two-tailed t test. A p value ,0.05 was consideredstatistically significant.

ResultsIdentification of TLRs involved in Omniscan activation ofNF-kB in HEK293 cells

To explore the ability of GdBCA to induce NF-kB activation viaTLR and NLR signaling, HEK293 cells expressing one of sevenhuman TLRs (TLRs 2, 3, 4, 5, 7, 8, or 9) or one of two humanNLRs (NOD1 or NOD2) were exposed in DMEM complete me-dium for 24 h to one of five compounds: 5 mM linear GdBCAOmniscan; 5 mM gadodiamide, the linear Gd chelate component ofOmniscan; 0.25 mM caldiamide, which is the amount of excesschelate present in the Omniscan formulation; 5 mM macrocyclicGdBCA ProHance; or to 2.7 mM nonchelated Gd compound Gd-EDTA. The effect of these compounds on TLR or NLR signalingwas assessed colorimetrically by measuring production of secretedalkaline phosphatase resulting from the activation of NF-kB in thesecells. Because production of alkaline phosphatase is under thecontrol of a stably integrated NF-kB–inducible promoter, theamount of alkaline phosphatase released by the cells is a directreflection of the level of NF-kB activation. All agents tested wereverified to be endotoxin free by the manufacturer and confirmedusing a Limulus amebocyte lysate gel formation assay. No signif-icant effect on cell numbers or cytotoxicity was observed as ex-amined by the WST-1 assay. The specific response of the HEK293cells expressing one specific TLR or NLR was validated followingactivation with the corresponding TLR- or NLR-specific activator.As portrayed in Fig. 1, 5 mM Omniscan induced significantly

increased NF-kB–dependent SEAP production in HEK293 cellsexpressing either TLR4 or TLR7 compared with control cellsincubated with endotoxin-free PBS. Omniscan did not cause sta-tistically significant stimulation of reporter gene expression in theHEK293 cells expressing any of the other TLRs or NLRs. TLR7expressing cells showed a 5-fold increase in SEAP levels com-pared with control cells in response to Omniscan. These changesin SEAP expression were observed for all replicates for TLR4-and TLR7-expressing cells. Gadodiamide, the Gd chelate inOmniscan, also induced increased NF-kB–dependent SEAP pro-duction in TLR4- or TLR7-expressing HEK293 cells to levelsnearly identical to those induced in response to Omniscan. Incontrast to the linear Gd chelate Omniscan, the macrocyclic Gd

FIGURE 1. Assessment of GdBCA induced NF-kB dependent activa-

tion in HEK293 cells expressing a single TLR or NLR. Measurement of

NF-kB–dependent SEAP levels in HEK293 cells expressing a single TLR

or NLR following exposure to 5 mM Omniscan, gadodiamide, or Pro-

Hance. Values represent the mean (6SD) absorbance at 650 nm of two

replicates of two separate experiments on HEK293 cells in culture. Sta-

tistical significance was calculated by comparing each Gd compound to the

saline control. *p , 0.05, **p , 0.01.

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chelate ProHance did not induce increased NF-kB–dependentSEAP production in HEK293 cells expressing any of the TLRs orNLRs (Fig. 1). Caldiamide and the nonchelated Gd compound Gd-EDTA did not produce significant changes in any TLR or NOD-expressing cell line (data not shown).

Dose–response analysis of GdBCA signaling through TLR4and TLR7 in HEK293 cells

Following identification of TLR4 and TLR7 as participating inOmniscan-induced activation of NF-kB–dependent SEAP produc-tion in HEK293 cells, dose–response analysis of the effects of sevenGd chelate compounds and two nonchelated Gd compounds wasperformed. HEK293 cells expressing either TLR4 or TLR7 wereexposed in DMEM complete medium to 0.5, 1, and 5 mM con-centrations of the linear GdBCA Gd-DTPA, MultiHance, OptiMark,Omniscan, or gadodiamide; or of the macrocyclic GdBCA Dotaremor ProHance; or to 0.27, 2.7, or 27 mMGd compounds Gd-EDTA orGd-citrate; or to 0.025, 0.05, or 0.25 mM non-Gd chelate moleculecaldiamide. LPS (1 mg/ml) and gardiquimod (10 mg/ml) were usedas positive controls for TLR4 and TLR7 activation, respectively. Theconcentrations of Gd compounds employed in these studies weresimilar to those employed in previously published in vitro studies (8,20, 22–24). Following 24 h exposure to the GdBCA, the levels ofNF-kB–dependent SEAP production were measured. The resultsshowed that some of the Gd compounds induced increased SEAPproduction in a dose-dependent manner. Fig. 2A displays the resultsfor the highest concentration of each agent tested, whereas Fig. 2Bshows the three concentration dose–response for Dotarem, Multi-Hance, Omniscan, and gadodiamide. The linear GdBCA Omniscanand gadodiamide induced a highly significant (p , 0.001) 20-foldincrease in NF-kB–dependent SEAP production in TLR4- ex-pressing HEK293 cells and a 16-fold increase in TLR7-expressingHEK293 cells, levels similar to those induced by the LPS-positive

control. The linear GdBCA MultiHance and the macrocyclic Dot-arem induced much weaker although significant (p , 0.05) 3- to4-fold increases in SEAP production in TLR4- and TLR7-ex-pressing cells, comparable to the 4-fold increase in SEAP pro-duction induced by the nonchelate Gd-EDTA and Gd-citrate. Fig.2B shows that the response to Dotarem, MultiHance, Omniscan,and gadodiamide was dose-dependent. No response was observedat any concentration examined for the linear GdBCA Gd-DTPAor OptiMark or for the macrocyclic ProHance or of the non-Gdchelate molecule caldiamide (Fig. 2B and data not shown).

Stimulation of TLR-dependent cytokine, chemokine, andgrowth factor expression in normal human differentiatedmacrophages

To determine whether TLR signaling was required for Omniscanstimulation of macrophage cytokine, chemokine, and growth factorproduction, normal human macrophages cultured in RPMI 1640complete medium and differentiated in the presence of M-CSF andIL-10 were preincubated with 50 mM chloroquine or with 300 nMIRAK1/4 inhibitor for 1 h followed by addition of either PBS orPBS containing 1 mM either Omniscan, gadodiamide, or Pro-Hance; or 0.05 mM caldiamide; or 2.7 mM Gd-EDTA for 24 h. Aspositive controls for TLR-dependent and TLR-independent mac-rophage stimulation, 100 ng/ml LPS or 10 mg/ml TNF-a, re-spectively, were used. No significant effect on cell numbers orcytotoxicity was observed as examined by the WST-1 assay.Analysis of expression levels of profibrotic/proinflammatory cy-

tokines by real-time PCR demonstrated that Omniscan and ga-dodiamide caused markedly upregulated expression of multiplechemokines (Fig. 3) and cytokines and growth factors (Fig. 4).Incubation of macrophages with LPS also induced increases in che-mokine, cytokine, and growth factor expression, which were abol-ished as expected by both inhibitors. The inhibitors, however, failed

FIGURE 2. Comparison of NF-kB–dependent activation by various GdBCA in HEK293 cells expressing TLR4 or TLR7. Measurement of NF-kB–

dependent SEAP levels in HEK293 cells expressing a single TLR or NLR at 20 h following exposure in DMEM complete medium to GdBCA. Cells were

exposed to 0.5, 1, and 5 mM concentrations of the linear GdBCA Gd-DTPA, MultiHance, OptiMark, Omniscan, or gadodiamide, or of the macrocyclic

GdBCA Dotarem or ProHance; or to 0.27, 2.7, or 27 mM Gd compounds Gd-EDTA or Gd-citrate; or to 0.025, 0.05, or 0.25 mM non-Gd chelate molecule

caldiamide. LPS (1 mg/ml) and gardiquimod (10 mg/ml) were used as positive controls for TLR4 and TLR7 activation, respectively. Values represent the

mean (6SD) absorbance at 650 nm of three replicates of three separate experiments on HEK293 cells in culture. Statistical significance was calculated by

comparing each Gd compound to the saline control. Values for other samples are expressed relative to the saline control. (A) SEAP levels secreted in

response to exposure to the highest concentration of each agent tested. (B) Three concentration dose–response of SEAP levels secreted following exposure

to Dotarem, MultiHance, Omniscan, or gadodiamide. *p , 0.05, **p , 0.01, ***p , 0.001.

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to modify the effects induced by TNF-a (data not shown). Theseresults indicated that the stimulation of cytokines, growth factors, andchemokines induced by the GdBCA was mediated by TLRs.CXCL10 displayed the greatest increase in expression of the

chemokines examined, with maximal 20-fold induction by gado-diamide compared with the saline control (Fig. 3). CCL8 ex-pression was induced 16-fold, CXCL11 expression increased 13-fold, CXCL12 expression increased 12-fold, CXCL9 increased8-fold, and CCL2 showed a 6-fold increase in expression. Gado-diamide induced the strongest levels in expression. A similarpattern was observed for multiple profibrotic/proinflammatorycytokines and growth factors. IL-13 displayed the greatest in-crease in expression, with a maximal 20-fold induction by gado-diamide at 24 h (Fig. 4). Marked increases in the expression of thegrowth factors TGF-b (6-fold) and VEGF (5-fold) and the profi-brotic cytokines IL-4 (2-fold) and IL-6 (6-fold) were also ob-served in response to Gd compounds. ProHance had the smallesteffect on cytokine and growth factor gene expression. Of interestwas the observation that the GdBCA tested induced only minorexpression or did not change the levels of IFN-g, with ProHanceinducing only a 2-fold increase, whereas gadodiamide induceda 1.4-fold increase and Omniscan did not change IFN-g expres-sion levels. Caldiamide did not affect expression levels of any ofthe genes tested (Figs. 3, 4). Preincubation of macrophages witheither chloroquine or IRAK1/4 inhibitor abrogated gadodiamide-and Omniscan-mediated increases in gene expression (Figs. 3, 4).These results demonstrated that Omniscan and gadodiamide elicitstimulation of potent cytokine, chemokine, and growth factor geneexpression in macrophages that is dependent on TLR.

Inhibition of TLR4 and TLR7 utilizing RNA interference

To confirm that the observed effects of chloroquine and IRAK 1/4inhibitors were due to inhibition of the TLR pathway and were not

the result of possible effects on other inflammatory pathways,TLR4 and TLR7 expression was directly targeted in normal humandifferentiated macrophages employing RNA interference. Normaldifferentiated human macrophages were transfected with controlsiRNA or with siRNA specific for TLR4 or TLR7 alone or incombination. RNA from these experiments was used in validationof gene expression by real-time RT-PCR of TLR4 and TLR7 (Fig.5), chemokines (Fig. 6), and proinflammatory/profibrotic cyto-kines and growth factors (Fig. 7). In control macrophages, theTLR4-specific siRNA induced an 84% decrease in TLR4 mRNAlevels without affecting TLR7 expression levels, whereas theTLR7-specific siRNA induced an 88% decrease in TLR7 ex-pression without affecting the RNA levels of TLR4 (data notshown). Exposure of macrophages to 1 mM Omniscan or 1 mMgadodiamide induced TLR4 (Fig. 5A) and TLR7 (Fig. 5B) ex-pression, and this increased expression was reduced to below thelevels measured in saline control cells by the specific siRNA. Thecontrol siRNA had no appreciable effect on TLR4 or TLR7 ex-pression compared with the saline control. Exposure of cells toeither the TLR4-specific siRNA or to the TLR7-specific siRNAprior to treatment with 1 mM Omniscan or 1 mM gadodiamideresulted in abrogation of the GdBCA-induced overexpression ofchemokines (Fig. 6) and cytokines and growth factors (Fig. 7),reducing their expression to levels equivalent to or lower thanthose measured in the saline controls.

Stimulation of TLR-dependent cytokine, chemokine, andgrowth factor production in normal human differentiatedmacrophages

To confirm that the increased expression observed at the transcriptlevel was reflected at the protein level, SearchLight proteomemultiplex arrays were used to quantitate the amounts of relevantprofibrotic and proinflammatory cytokines, chemokines, and growth

FIGURE 3. Effect of TLR inhibition on GdBCA-induced upregulation of chemokine expression in normal differentiated human macrophages. Dif-

ferentiated normal human macrophages were preincubated for 2 h with TLR inhibitors chloroquine (300 nM) or IRAK1/4 inhibitor (10 mM) followed by 24

h incubation with 1 mM Omniscan (Omni), gadodiamide (Gado), or ProHance (ProHan) or with 0.05 mM caldiamide (Caldia). Expression levels of various

chemokines were assessed by real-time PCR. Values represent the mean (6SD) expression levels of three replicates of three separate experiments with

macrophages differentiated from monocytes obtained from three different normal individuals. LPS (100 ng/ml) and TNF-a (10 mg/ml) were used as

positive controls for TLR4 and TNF-a activation, respectively. Ct values for chemokines were normalized with b-actin. The saline control levels were

arbitrarily set at 100% expression at each time point. Statistical significance of changes in chemokine expression was calculated by comparing each Gd

compound to the saline control. Statistical significance of changes induced by inhibitors was calculated by comparing these levels to those induced by the

GdBCA.*p , 0.05, **p , 0.01, ***p , 0.001.

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factors produced by normal human differentiated macrophagesfollowing GdBCA exposure alone or following preincubation withchloroquine or IRAK 1/4 inhibitor. The results showed that 24 hexposure to some of the Gd-containing compounds resulted in in-creased production and secretion of numerous chemokines, cyto-kines, and growth factors with their significant accumulation in theculture media as shown in Fig. 8. The results, expressed in pico-grams per milliliter, were normalized for the value of b-actintranscripts obtained in the real-time PCR experiments to correct forpossible variance in the number of adherent cells analyzed. All ofthe cytokines/growth factors that exhibited upregulated mRNAexpression following exposure to Omniscan, gadodiamide, Pro-Hance, and Gd-EDTA also demonstrated an increase in the total

amount of the corresponding secreted cytokine/growth factor, withthe greatest increases seen for cells exposed to Omniscan andgadodiamide. Exposure of macrophages to caldiamide did not resultin a detectable increase in the production of any of the cytokines/growth factors analyzed (Fig. 8). As observed for RNA expressionlevels, preincubation of macrophages with either chloroquine (datanot shown) or with IRAK1/4 inhibitor abrogated the Omniscan- andgadodiamide-mediated increased levels of cytokines, chemokines,and growth factors to near baseline levels (Fig. 8).

DiscussionAlthough exposure of patients with renal insufficiency to GdBCAis a primary factor in NSF pathogenesis, the molecular pathways

FIGURE 4. Effect of TLR inhibition on GdBCA-induced upregulation of cytokine/growth factor expression in normal differentiated human macro-

phages. Differentiated normal human macrophages were preincubated for 2 h with the TLR inhibitors chloroquine (300 nM) or IRAK1/4 inhibitor (10 mM)

followed by 24 h incubation with 1 mM Omniscan (Omni), gadodiamide (Gado), or ProHance (ProHan) or with 0.05 mM caldiamide (Caldia). Expression

levels of various cytokines and growth factors were assessed by real-time PCR. Values represent the mean (6SD) expression levels of three replicates of

three separate experiments with macrophages differentiated from monocytes obtained from three different normal individuals. LPS (100 ng/ml) and TNF-a

(10 mg/ml) were used as positive controls for TLR4 and TNF-a activation, respectively. Ct values for cytokines and growth factors were normalized with

b-actin. The saline control levels were arbitrarily set at 100% expression at each time point. Statistical significance of changes in cytokine and growth

factor expression was calculated by comparing each Gd compound to the saline control. Statistical significance of changes induced by inhibitors was

calculated by comparing these levels to those induced by the GdBCA. *p , 0.05, **p , 0.01, ***p , 0.001.

FIGURE 5. Effect of RNA interference of TLR expression on GdBCA-induced upregulation of TLR4 and TLR7 expression in normal differentiated

human macrophages. Differentiated normal human macrophages were transfecte with 100 nM siGENOME SMARTpool siRNA specific for TLR4 or TLR7

or both TLR4 plus TLR7 with DharmaFECT 4 (3 ml/well) followed by 24 h incubation with 1 mM Omniscan (Omni), gadodiamide (Gado), or ProHance

(ProHan). Expression levels of TLR4 and TLR7 were assessed by real-time PCR. Values represent the mean (6SD) expression levels of three replicates of

three separate experiments with macrophages differentiated from monocytes obtained from three different normal individuals. An siGENOME RISC-free

control siRNA was used as a negative control. Ct values for TLRs were normalized with b-actin. The saline control levels were arbitrarily set at 100%

expression at each time point. Statistical significance of changes in TLR expression was calculated by comparing each Gd compound to the saline control.

***p , 0.001.

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stimulated following exposure to GdBCA have not been identified.The data presented in this study demonstrating that Omniscansignals via TLR4 and TLR7 provide a possible mechanism for theinitiation of GdBCA-induced inflammation and fibrosis. Omniscanand gadodiamide, the linear Gd chelate present in Omniscan, in-duce potent stimulation of NF-kBmediated expression of an SEAPreporter protein in HEK293 cells expressing either TLR4 or TLR7

but not in cells expressing one of the other TLRs or the NODreceptors (Figs. 1, 2). The stimulatory effects were comparable to thelevels of stimulation induced by the TLR4 ligand LPS or the TLR7ligand gardiquimod, whereas the chelate molecule caldiamide usedin the Omniscan formulation did not stimulate SEAP expression(Fig. 2). One linear GdBCA, MultiHance, showed a much weakerinduction of SEAP expression (Fig. 3), as did one of the macrocylic

FIGURE 6. Effect of RNA interference of TLR expression on GdBCA-induced upregulation of chemokine expression in normal differentiated human

macrophages. Differentiated normal human macrophages were transfected with 100 nM siGENOME SMARTpool siRNA specific for TLR4, TLR7, or

TLR4 plus TLR7 with DharmaFECT 4 (3 ml/well) followed by 24 h incubation with 1 mM Omniscan (Omni), gadodiamide (Gado), or ProHance (ProHan).

Expression levels of various chemokines were assessed by real-time PCR. Values represent the mean (6SD) expression levels of three replicates of three

separate experiments with macrophages differentiated from monocytes obtained from three different normal individuals. An siGENOME RISC-free control

siRNA was used as a negative control. Ct values for chemokines were normalized with b-actin. The saline control levels were arbitrarily set at 100%

expression at each time point. Statistical significance of changes in chemokine expression was calculated by comparing each Gd compound to the saline

control. *p , 0.05, **p , 0.01, ***p , 0.001.

FIGURE 7. Effect of RNA interference of TLR expression on GdBCA-induced upregulation of cytokine/growth factor expression in normal differen-

tiated human macrophages. Differentiated normal human macrophages were transfected with 100 nM siGENOME SMARTpool siRNA specific for TLR4,

TLR7, or TLR4 plus TLR7 with DharmaFECT 4 (3 ml/well) followed by 24 h incubation with 1 mM Omniscan (Omni), gadodiamide (Gado), or ProHance

(ProHan). Expression levels of various cytokines and growth factors were assessed by real-time PCR. Values represent the mean (6SD) expression levels of

three replicates of three separate experiments with macrophages differentiated from monocytes obtained from three different normal individuals. LPS (100

ng/ml) and TNF-a (10 mg/ml) were used as positive controls for TLR4 and TNF-a activation, respectively. Ct values for cytokines and growth factors were

normalized with b-actin. The saline control levels were arbitrarily set at 100% expression at each time point. Values for other samples are expressed relative

to the saline control. Statistical significance of changes in cytokine and growth factor expression was calculated by comparing each Gd compound to the

saline control. **p , 0.01, ***p , 0.001.

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GdBCA, ProHance, in HEK293 cells. Two nonchelated Gd com-pounds, Gd-EDTA and Gd-Citrate, stimulated SEAP expression inTLR4- or TLR7-expressing HEK293 cells at a level intermediatebetween the high levels induced by Omniscan or gadodiamide andthe low levels induced by MultiHance or ProHance.In normal human differentiated macrophages, both Omniscan

and gadodiamide strongly induced multiple profibrotic and proin-flammatory cytokines, chemokines, and growth factors as mea-sured by real-time PCR of total RNA (Figs. 3, 4) and ELISA ofculture supernatants (Fig. 8), whereas ProHance and Gd-EDTAinduced a less pronounced increase in expression of these genes.Preincubation of differentiated macrophages with chloroquine orwith the IRAK1/4 inhibitor for 1 h abrogated the Omniscan- orgadodiamide-induced expression and production of these proin-flammatory/profibrotic molecules (Figs. 3, 4, 8), indicating thecrucial role of TLR4 and TLR7 in these effects. These observa-tions were confirmed in experiments targeting TLR4 and TLR7 byRNA interference (Figs. 6, 7), thus conclusively demonstratingthat the increased expression levels observed are TLR-dependent.Increased expression induced by TNF-a, which acts downstreamof the TLRs, was unaffected by these inhibitors.Exposure of patients with renal insufficiency to GdBCA is a pri-

mary factor in the pathogenesis of NSF (1–4), with the majority ofNSF cases attributed to exposure to Omniscan (16, 17). Although theexact mechanisms responsible for the development of NSF followingGdBCA exposure in patients with renal insufficiency are not known,there are two hypotheses that have been proposed, as illustrated inFig. 9. In the transmetallation hypothesis, Gd-containing compoundsescape into the extravascular space and transmetallation is induced byendogenous ions allowing free Gd3+ to escape. The transmetallationprocess is dependent on the thermodynamic stability of the GdBCA,

and therefore it is more pronounced for the less stable linear chelates(44). The released Gd3+ can then escape from the chelate and interactwith tissue macrophages, resulting in the production and secretion ofproinflammatory/profibrotic cytokines, chemokines, and growth fac-tors by these cells. These secreted macrophage products act on res-ident tissue fibroblasts, inducing their differentiation into a-smoothmuscle actin-expressing myofibroblasts with the consequent increasein production and secretion of a variety of molecules involved in thefibrotic process including the interstitial collagens. The exact mech-anisms involved in the escape of Gd-containing compounds into theextravascular space and subsequent transmetallation are not entirelyknown; however, it is likely that factors such as the higher GdBCAconcentrations and their increased retention in the circulation owingto renal insufficiency, associated with alterations in endothelial per-meability and tissue edema resulting from inflammatory or throm-botic events, may all contribute to this process (44–46).In disagreement with the transmetallation hypothesis, however,

multiple experimental studies have demonstrated that both linearand macrocyclic GdBCA induce potent metabolic changes in cells,including increased hyaluronan and collagen production by normalhuman fibroblasts (8, 18–26), increased expression of proin-flammatory and profibrotic cytokines, chemokines, and growthfactors by normal human PBMCs (27), and differentiated humanmacrophages (28), and increased differentiation of normal humanPBMCs into fibrocytes (29). These experimental results havesuggested an alternative hypothesis that posits that intact chelatedGdBCA are responsible for GdBCA-mediated stimulation (47). Inthe chelate hypothesis (Fig. 9), the metabolic and molecular eventsdescribed above are initiated by intact chelated GdBCA ratherthan by free Gd3+. The cellular pathways exploited by eitherchelated GdBCA or transmetallated free Gd3+ to initiate these

FIGURE 8. Effect of TLR inhibition on amounts of Gd compound-stimulated cytokines, chemokines, and growth factors in normal differentiated human

macrophages. Quantitative measurement of cytokines, chemokines, and growth factors present in the culture media of Gd compound-exposed cells was

performed employing multiplex ELISA proteome array analysis. Differentiated normal human macrophages were preincubated with either 300 nM

chloroquine or 10 mM IRAK1/4 inhibitor. Following preincubation, cells were incubated with either saline, 1 mM Omniscan (Omni), 1 mM gadodiamide

(Gado), or 0.05 mM caldiamide (Caldia). Values are expressed in picograms per milliliter and represent the mean value of values obtained from duplicate

results at three dilutions: 1:2, 1:50, and 1:1000. Statistical significance of changes in chemokine, cytokine, and growth factor production was calculated by

comparing each Gd compound to the saline control. Statistical significance of changes induced by inhibitors was calculated by comparing these levels to

those induced by the GdBCA. **p , 0.01, ***p , 0.001.

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cellular changes is largely unknown. The data presented in thisstudy suggest an important role of TLR signaling in NSF patho-genesis in particular and for the development of fibrosis in general.First, the data suggest that the ability to trigger TLR signaling ismore potent following exposure to Omniscan and gadodiamide.The most obvious explanation is that these molecules possess aunique, specific molecular shape or pattern that renders them ca-pable of signaling via TLR4 and TLR7. Caldiamide, the chelatemolecule contained in Omniscan, failed to activate NF-kB, andGd-EDTA activated only weakly NF-kB, suggesting that the great-est effect on TLR signaling is mediated by an intact Omniscan Gdchelate molecule. Although the molecular pattern responsible forTLR activation is not known, it is possible that the formation ofcoordination bonds with Gd3+ apparently alters the nonstimulatorymolecular pattern of caldiamide sufficiently to render the chelatecomplex capable of activating TLR signaling.Omniscan and gadodiamide signaling via both TLR4 and TLR7

allows these compounds to exert their effects at the cell surface andin the endosome. Endosomal TLR signaling is particularly relevantfor macrophages owing to the stimulatory effect of TLR signalingon phagocytosis by macrophages (48). Initial engagement of TLR4by Omniscan at the macrophage cell surface could induce andincrease the rates of Omniscan phagocytosis. Once phagocytosed,Omniscan would be able to amplify its TLR signaling capacity byengaging TLR7 within the endosome. Several reports of Gddeposits found in affected tissues from NSF patients described thepresence of macrophages in close proximity (9–12), suggestingthat the highly acidic environment of the endosome could facili-tate deposition of Gd salts, such as Gd phosphate and Gd car-bonate (49). These precipitated Gd salts could then maintain aconstant state of TLR activation in both macrophages and fibro-blasts, producing a chronic proinflammatory/profibrotic phenotyperesponsible for disease persistence and progression. Gd salts couldact as a secondary trigger for the innate immune system, pre-venting the normal resolution of the TLR response followingGdBCA clearance, inducing the release of profibrotic cytokines,chemokines, and growth factors.Fibroblasts express TLR4 at the cell surface, and it is possible

that some of the direct effects of Gd compounds on fibroblastssuch as inducing their differentiation into a-smooth mucle actin-

expressing myofibroblasts and the increased production and se-cretion of molecules involved in the fibrotic process such as col-lagens and hyaluronan could be due to engagement of fibroblastTLRs. We are currently investigating whether Gd compounds arecapable of engaging normal fibroblast TLR signaling. It is alsopossible that GdBCA-induced hyaluronan could also signal viaTLR4 expressed on macrophages and fibroblasts, contributing toa chronic proinflammatory/profibrotic environment (39).Taken together, the results described in this study demonstrate

that Omniscan and gadodiamide signal through TLR4 and TLR7,resulting in the increased expression of genes encoding severalwell-characterized profibrotic and proinflammatory cytokines,chemokines, and growth factors in normal human differentiatedmacrophages. The role of TLR4 and TLR7 in this stimulation wasconfirmed by the abrogation of these effects by specific TLR4and TLR7 inhibitors as well as by RNA interference studies. Thepresent study provides strong evidence and plausible pathophys-iological mechanisms for initiation and maintenance of a proin-flammatory/profibrotic phenotype by Omniscan and gadodiamidein the development of NSF. The marked ability of Omniscan andgadodiamide to signal through TLR4 and TLR7 compared withnonchelated Gd compounds and the inability of the chelate back-bone, caldiamide, to induce this response suggest that the intact Gdchelate complex is capable of initiating innate immunity activationand that the specificity of the stimulation may be dependent onan intrinsic molecular pattern present in this chelate molecule.Further study and characterization of the cellular effects of thesecompounds and of the mechanisms of these effects may providevaluable information regarding the early events in the pathogenesisof NSF and other fibrosing diseases such as SSc.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 9. Diagramatic representation of two pos-

sible pathways that may participate in the induction of

NSF by GdBCA. This schematic diagram represents

two possible mechanisms by which GdBCA result in

activation of target cells. In the transmetallation model,

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