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Published in final edited form as: J Invest Dermatol. 2019 Apr 9;139(10):2134–2143.e2. doi: 10.1016/j.jid.2019.03.1145

‘Nephrogenic’ systemic fibrosis is mediated by myeloid C-C chemokine receptor 2

Catherine Do 1,2,*, Viktor Drel 2,*, Chunyan Tan 2, Doug Lee 2, Brent Wagner 3,4,5
PMCID: PMC6756957  NIHMSID: NIHMS1526668  PMID: 30978353

Abstract

Gadolinium-based contrast agents are implicated in several pathologic abnormalities (long-term retention in vital organs such as the skin and brain) and are the cause of a sometimes fatal condition in patients, ‘nephrogenic’ systemic fibrosis (NSF). Bone marrow-derived fibrocytes and the monocyte chemoattractant protein 1 (MCP1) inflammatory pathway have been implicated as mediators of adverse effects induced by gadolinium-based contrast agents. Mechanistic studies are scant. A mouse model of NSF was established. Dermal cellularity was increased in contrast-treated green fluorescent protein (GFP) chimeric mice. Skin GFP and fibrosis were all increased in the contrast-treated chimeric animals. MCP-1 and CCR-2 were increased in the tissues from contrast-treated mice. CCR2-deficient recipients of GFP-expressing marrow had an abrogation of gadolinium-induced pathology and displayed less GFP-positive cells in the skin. Wild-type animals that received CCR2-deficient marrow had a complete abrogation of dermal pathology. That GFP levels and expression increase in an involved organ, the skin, in tandem with a fibrocyte marker supports the blood-borne circulating fibrocyte hypothesis of the disease. Heretofore fibrocyte trafficking has yet to demonstrated. Importantly, our data demonstrate that the monocyte chemoattractant protein 1/C-C chemokine receptor 2 axis plays a critical role in the pathogenesis of NSF.

Keywords: nephrogenic fibrosing dermopathy, skin diseases, chemokines, gadolinium, CCL-2, CCR2, fibrosis, monocyte chemoattractant proteins, bone marrow, renal insufficiency

INTRODUCTION

Gadolinium-based contrast, essential for certain magnetic resonance imaging techniques (Diop et al., 2013), causes ‘nephrogenic’ systemic fibrosis (NSF) in patients with renal insufficiency. This ghastly condition is characterized by a variable onset (sometimes rapid, sometimes years), and symmetric involvement of the bilateral extremities. Skin biopsies exhibit fibrocyte markers such as CD34, procollagen type I, and factor XIIIa; all of this implied that circulating, blood-borne fibrocytes mediate the disease (Cowper and Bucala, 2003). This has been demonstrated experimentally in rats, i.e., that a significant proportion of fibrotic lesions indeed involve myeloid fibrocytes (Wagner et al., 2012). However, there is a paucity of experimental work with mice where there are no studies addressing the role of bone marrow in gadolinium-based contrast-induced disease.

In the dermis, gadolinium-induced fibrotic lesions are characterized by an increased number of CD34-positive spindle-shaped cells (Knopp and Cowper, 2008). Such CD34-positive, bone marrow-derived and circulating cells have been termed fibrocytes (Cowper et al., 2001). These fibrocytes are important in the proliferative stages of wound repair and are unique in that they are peripheral blood cells that have the potential to generate matrix (Quan et al., 2004). It has been postulated that these bone marrow-derived cells are the mediators of NSF (Jimenez et al., 2004, Quan et al., 2004).

We conducted experiments to demonstrate that gadolinium-based contrast agent induces systemic fibrosis in mice. To gauge myeloid involvement in this process, mice with renal insufficiency underwent lethal irradiation followed by salvage bone marrow transplant from green fluorescent (GFP) protein-expressing donors. This permitted tracing the myeloid lineage of the skin cellularity. As the monocyte chemoattractant protein-1 (MCP-1) and its primary receptor, the C-C chemokine receptor 2 (CCR2), have been implicated in mediating the effects of gadolinium-based contrast agent-induced fibrosis (Drel et al., 2016), bone marrow transplantation experiments were performed to identify this system as a primary mechanism of the disease.

RESULTS

Ten percent of patients with NSF have never received hemodialysis (Rosenkranz et al., 2007), therefore the disorder is not exclusive to those afflicted with end-stage renal disease. Wild-type mice (with normal renal function) were randomized to control and gadolinium-based contrast agent treatment. Skin fold thicknesses tended to be increased in the contrast-treated group (Figure 1 a). Similar to what has been described in humans and in our own rat models (Do et al., 2014, Drel et al., 2016, Wagner et al., 2012), there was an increase in dermal cellularity (Figure 1 b, c). The nuclei were dense and spindle-shaped much as we have reported in rats (Do et al., 2014, Drel et al., 2016, Wagner et al., 2012). The dermis from contrast-treated animals demonstrated an increase in fibronectin (Figure 1 d). Skin from contrast-treated animals demonstrated higher levels of the extracellular matrix proteins fibronectin and collagen type I (Figure 1 e).

Figure 1. The effect of gadolinium-based contrast agent treatment in mice.

Figure 1.

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Age and weight-matched mice were randomized to gadolinium-based contrast agent (gadodiamide, 2.5 mmol/kg intraperitoneally, 20 doses over 4 weeks) or no treatment. (a) Skin fold thickness (n = 4 per group) (b) Dorsal skin sections stained with hematoxylin and eosin (H&E) and periodic acid-Schiff were examined. (c) Dermal cellularity (in triplicate, n = 4 per group). *** P < 0.001 by 2-tailed ‘Student’s’ t test. (d) Fibronectin expression. Immunofluorescence, calibration bar = 0.05 mm. (e) Fibronectin, collagen type I, and the C-C chemokine receptor 2 (CCR2) expression, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as the loading control. Immunoblot. (f) Electron-dense urchin-shaped structures in the gadolinium-based contrast agent-treated mice. Transmission electron microscopy. (g) Energy-dispersive x-ray spectroscopy. Arrows, Mα and Lα emission spectra for gadolinium.

Gadolinium has been detected in nearly every organ tissue from patients with NSF (Sanyal et al., 2011), and there is increasing concern about metal retention in patients with normal renal function exposed to gadolinium-based contrast agents (McDonald et al., 2015). We have found evidence of gadolinium in nearly every organ tissue in contrast-treated rats (Do et al., 2014). Sections of paraffin-embedded skin were processed for transmission electron microscopy without heavy metal staining (e.g., without lead, osmium, or uranium) to assess for electron-densities (Figure 1 f). Several electron-dense materials, upon high magnification, demonstrated mesh-like nanowire and urchin-shaped structures. These same electron-dense crystalline nanostructures peppered the vacuoles of the renal proximal tubules (arrows). Electron-dense deposits demonstrated the presence of gadolinium as assessed by scanning transmission electron microscopy with energy-dispersive x-ray spectroscopy (Figure 1 g). In total, these data demonstrate that gadolinium-based contrast agent treatment induced a hypercellular skin fibrosis with the formation of multinucleated giant cells, the presence of mesh-like nanowire electron densities concomitantly with gadolinium-enriched electron-dense deposits.

Since its discovery, the hypercellularity of NSF was theorized to be from bone marrow-derived ‘fibrocytes’ (Wagner et al., 2016a). This has been experimentally demonstrated in rats (Wagner et al., 2012), but not to date in mice. Lethally-irradiated mice with 5/6th nephrectomies (to model renal insufficiency) were salvaged with tagged bone marrow in order to trace the lineage of infiltrating cells (Figure 2 a). After several weeks for engraftment, recipients were randomized to control and gadolinium-based contrast agent treatment (2.5 mmol/kg intraperitoneally, 20 doses over 4 weeks). At the endpoint, skin fold thickness was greater in the contrast agent-treated group (Figure 2 b). Skin histology demonstrated disorganized collagen bundles and an increase in the number of densely-nucleated cells in the dermis (Figure 2 c). The quantitative increase in dermal cellularity was on the same order as the non-nephrectomized mice treated with gadolinium-based contrast agents (in Figure 1 c), and this is similar to what has been described in patients with NSF (Nazarian et al., 2011). Gadolinium (155Gd) content in the contrast agent-treated skin was 87 ± 5 (mean ± standard error) μg/g tissue as measured in flash-frozen skin by inductively-coupled plasma mass spectroscopy.

Figure 2. The effect of gadolinium-based contrast agent treatment in chimeric ‘tagged’ bone marrow transplanted mice.

Figure 2.

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(a) Mice with 5/6 nephrectomies were lethally-irradiated and salvaged with bone marrow from green fluorescent protein- (GFP-) expressing donors. (b) Skin fold thicknesses were measured in triplicate for each animal. (c) Dermal histology. Dorsal skin from untreated and contrast-treated animals was fixed, embedded, and stained. H&E, calibration bar = 0.05 mm. Accompanying bar chart depicts quantification of dermal nuclei, 3 random high power fields. *** P < 0.001 by two-tailed ‘Student’s’ t-test. (d) Fibronectin and collagen I, immunoblot (Do et al., 2014, Wagner et al., 2012). Fibronectin, collagen type I, immunoblot. (e) Dermal fibronectin, calibration bar = 0.05 mm. (f) Bone marrow marker, GFP, immunoblot. (g) Paraffin-embedded skin, calibration bar = 0.05 mm. * P < 0.05, *** P < 0.001 by two-tailed ‘Student’s’ t-test.

Skin from the contrast agent-treated group demonstrated an accumulation of fibronectin and collagen type I (Figure 2 d). The dermis from the treated group demonstrated widespread and diffuse fibronectin accumulation (Figure 2 e). The myeloid tag, GFP, was much greater in the skin from the contrast-treated animals (Figure 2 f, g).

The hematopoietic stem cell marker CD34 is the foundation for linking circulating fibrocytes to systemic fibrosis (Knopp and Cowper, 2008, Wagner et al., 2016b). This marker was increased in the dermis from the contrast-treated animals (Figure 3 a). Activated myofibroblasts will demonstrate α-smooth muscle actin-positive stress fibers (Wagner et al., 2016a), and we have detected this in both gadolinium contrast-treated human fibroblasts and in the skin from contrast-treated rats (Do et al., 2014). Similarly, the dermis from the contrast-treated group was enriched with α-smooth muscle actin-expressing myeloid cells (Figure 3 b). CD45RO is a marker thought to be specific for fibrocytes (Pilling et al., 2009) and proposed to be among the diagnostic criteria for NSF. The dermis from contrast-treated mice demonstrated an increase in the CD45RO-positive myeloid cells (Figure 3 c). We have found that CD163, a marker of alternatively-activated macrophages (Swaminathan et al., 2013), is found in the dermis of contrast-treated rats (Drel et al., 2016). In the dermis from the contrast-treated mice, CD163-positive myeloid cells were similarly abundant (Figure 3 d). In total, these data demonstrate that gadolinium-based contrast agents promote skin infiltration of bone marrow-derived fibrocytes and CD163-positive myeloid cells.

Figure 3. Dermal cellular markers induced by gadolinium-based contrast agent in chimeric ‘tagged’ bone marrow transplanted mice.

Figure 3.

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(a) The myeloid (and fibrocyte) marker CD34 was increased in the dermis of contrast-treated animals. Immunofluorescence, calibration bar = 0.05 mm (b) Expression of α smooth muscle actin (α SMA) by bone marrow-derived cells. Immunofluorescence, calibration bar = 0.03 mm. (c) Co-expression of the fibrocyte marker CD45RO by myeloid cells in the dermis of control and contrast-treated animals. Calibration bar = 0.05 mm. (d) Co-expression of the alternatively-activated macrophage marker CD163 by myeloid cells in the dermis of control and contrast-treated animals. Calibration bar = 0.05 mm.

Monocyte chemoattractant protein 1 and its receptor, the C-C chemokine receptor 2, are elevated in the skin of contrast-treated rats (Drel et al., 2016). When gadolinium-based contrast-treated rats concomitantly receive an antagonist of the C-C chemokine receptor 2, skin fibrosis and dermal cellularity are abrogated (Drel et al., 2016). Therefore, these effectors were examined in the skin of the chimeric mice (Figure 4). Both monocyte chemoattractant 1 (Figure 4 a) and C-C chemokine receptor 2 (Figure 4 b, c) were increased. These data imply that the monocyte chemoattractant protein 1/C-C chemokine receptor 2 are involved in fibrocyte trafficking to the skin.

Figure 4. The monocyte chemoattractant protein 1 (MCP1) and its receptor, the C-C chemokine receptor 2 (CCR2) are increased in the skin from contrast-treated animals.

Figure 4.

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(a) Expression of MCP1 in the dermis of untreated and contrast-treated mice. Calibration bar = 0.05 mm (b) Expression of CCR2 in the dermis of control and contrast-treated mice. Calibration bar = 0.05 mm. Accompanying bar plot depicts the intensity of dermal CCR2 staining in 3 random fields (n = 3 per group). * P < 0.05 by 2-tailed ‘Student’s’ t test. (c) CCR2 was assessed in the skin by immunoblot. The accompanying bar plot depicts the pixel densities of protein expression normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) relative to the control group. ** P < 0.01 by 2-tailed ‘Student’s’ t test.

In order to validate the role of this receptor and the impact on bone marrow-derived fibrocyte trafficking, groups of wild-type (CCR2+/+) and C-C chemokine receptor 2-deficient (CCR2−/−) mice (with normal renal function) underwent lethal irradiation followed by salvage with marrow from GFP-expressing donors (Figure 5 a). After an engraftment period, these groups were sub-randomized to untreated and gadolinium-based contrast agent treatment groups (2.5 mmol/kg intraperitoneally, 20 doses over 4 weeks). The dermis from the contrast-treated CCR2-deficient recipients demonstrated less cellularity with respect to the gadolinium-treated wild-type recipients (Figure 5 b, c). Dermal fibronectin was similarly reduced in the contrast-treated C-C chemokine receptor-2 deficient recipients with respect to the contrast-treated wild-type recipients (Figure 5 d). In parallel, skin fibronectin and the myeloid marker, GFP, were reduced in the contrast-treated CC chemokine receptor 2-deficient recipients with respect to the contrast-treated wild-type recipients (Figure 5 e). Dermal myeloid infiltration and α-smooth muscle actin were both suppressed in the C-C chemokine receptor 2-deficient recipients despite gadolinium-based contrast treatment (Figure 5 f). Similarly, gadolinium-based contrast agent treatment led to less dermal CD34 and CD45RO—the fibrocyte markers—when the recipients were deficient of the C-C chemokine receptor 2 (Figures 5 g and h, respectively). Recipients devoid of CCR had a marked decrease in dermal CD163 expression in response to gadolinium-based contrast agent treatment (Figure 5 i).

Figure 5. The role of host CCR2 in gadolinium-based contrast agent-induced systemic fibrosis.

Figure 5.

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(a) Recipients that expressed CCR2 (CCR2+/+) or were devoid of this receptor (CCR2−/−) were lethally-irradiated and salvaged with marrow from GFP-expressing donors. After an engraftment period, these recipients were randomized to non-treatment or contrast treatment. (b) H&E. (c) Dermal nuclear quantification were counted in three randomly chosen areas from each group (n = 3 each). *** P < 0.001, ** P < 0.01 by analysis-of-variance and Tukey Honest Significant Difference post-hoc test. (d) Fibronectin expression, immunofluorescence. (E) Skin fibronectin, immunoblot. (f-g) Immunofluorescence, GFP (f, g, h, j), and α-smooth muscle actin (α SMA, f), CD34 (g), CD45RO (h, CD163 (i), MCP-1 (j), and CCR2 (k). Calibration bars = 0.05 mm.

Monocyte chemoattractant protein-1 was increased in the dermis of the gadolinium-based contrast agent-treated wild-type recipients but not in the C-C chemokine receptor 2-deficient recipients (Figure 5 j). It is expected that the GFP-expressing donor marrow expressed the C-C chemokine receptor 2. In the wild-type recipients, gadolinium-based contrast agent treatment induced an increase in dermal C-C chemokine receptor 2; this effect was not evident in the receptor-deficient animals (Figure 5 k).

To test the degree myeloid C-C chemokine receptor 2 plays in mediating gadolinium-based contrast agent-induced systemic fibrosis, marrow from C-C chemokine receptor 2-deficient mice (with normal renal function) was transplanted into lethally-irradiated wild-type recipients (Supplementary Data 1 a). After an engraftment period, mice were randomized to untreated and contrast-treated groups (as above). Gadolinium content in flash-frozen skin, as assessed by inductively-coupled plasma mass spectroscopy, was 0 in the untreated group, 23 ± 1 μg/g in the contrast-treated group.

There was no difference in dermal cellularity between the gadolinium-based contrast agent-treated and untreated recipients of the C-C chemokine receptor 2-deficient marrow (Supplementary Data 1 b). Gadolinium-based contrast agent treatment failed to increase skin fibronectin content in the recipients of C-C chemokine 2 receptor-deficient marrow (Supplementary Data 1 c). Comparing the untreated and contrast-treated groups, there was no difference in dermal α smooth muscle actin (Supplementary Data 1 d), CD34 (Supplementary Data 1 e), CD163 (Supplementary Data 1 f), or monocyte chemoattractant protein 1 (Supplementary Data 1 g). That the contrast agent-induced increase in dermal cellularity and development of fibrosis were completely abrogated in the recipients of C-C chemokine receptor 2-deficient marrow (when gadolinium was detectable in the tissue) demonstrate that the expression of monocyte chemoattractant protein-1 receptor by myeloid cells is requisite for the disease despite the presence of gadolinium in the tissue.

In order to repeat this using a myeloid marker (analogous to our experimental method to date), lethally-irradiated mice (with normal renal function) were salvaged with bone marrow from red fluorescent protein-expressing and C-C chemokine receptor 2-deficient donors (Figure 6 a). After several weeks for engraftment, these animals were randomized to untreated and gadolinium-based contrast agent treatment (as above). Skin gadolinium, (154, 156, and 157) was 36 ± 7 μg/g in the contrast-treated group. There was no evidence of an increase in dermal cellularity in the contrast-treated group (Figure 6 b). Gadolinium-based contrast agent treatment did not induce fibronectin accumulation in the skin (Figure 6 c, d). There was little increase in dermal CD163 in the contrast-treated animals (Figure 6 e), and no increase in the fibrocyte markers CD34 or CD45RO (Figure 6 f, g). The dermal expression of the myeloid marker, red fluorescent protein, was essentially the same in both groups (Figure 6 h).

Figure 6. The role of myeloid CCR2 in gadolinium-based contrast agent-induced systemic fibrosis.

Figure 6.

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(a) Wild-type mice (n = 20) underwent lethal irradiation followed by salvage bone marrow transplantation from red fluorescent protein-expressing mice with deficiencies in CCR2 (Jackson Laboratory, stock 017586). Weeks after engraftment, mice were randomized to non-treatment or contrast treatment (n = 10 each) (b) Representative dermal histology. H&E, calibration bars = 0.05 mm. Accompanying bar chart depicts quantification of dermal cellularity (n = 3, triplicate randomly-chosen fields). (c) Skin fibronectin. Immunoblot. (d) Dermal fibronectin expression. Immunofluorescence. (e) Dermal CD163 expression. Immunofluorescence, calibration bar = 0.05 mm. (f) Dermal CD34 expression. Immunofluorescence, calibration bar = 0.05 mm. (g) CD45RO expression. Immunofluorescence, calibration bar = 0.05 mm. (h) Red fluorescent protein—the myeloid marker—expression. Immunofluorescence, calibration bar = 0.05 mm.

An in situ co-culture assay (described previously, (Drel et al., 2016)) was used to further demonstrate that the C-C chemokine receptor 2 is requisite for gadolinium-based contrast agent-induced myeloid infiltration of the skin. Bone marrow cells from wild-type and C-C chemokine receptor 2-deficient mice were obtained and stained with 5(6)-carboxyfluorescein diacetate N-hydroxysuccinimidyl ester (CFSE). These stained cells were co-cultured with skin punch biopsies from wild-type animals in cell culture media with or without gadolinium-based contrast agent (Supplementary Data 2). Whereas the contrast stimulated the infiltration of wild-type marrow into the dermis, there was no effect with the C-C chemokine receptor 2-deficient marrow. These data show that myeloid C-C chemokine receptor 2 is requisite for infiltration into the skin.

DISCUSSION

Up to 4.5 million Americans are exposed to gadolinium-based contrast agents annually (in non-federal hospitals) (FDA, 2017). The United States has a high rate of utilization of magnetic resonance imaging compared to other industrialized countries (Papanicolas et al., 2018). As of this writing the United States Food & Drug Administration Adverse Events Reporting System (FAERS) lists 3,094 total cases of ‘nephrogenic systemic fibrosis’; these include 2,962 serious cases and 742 deaths. There are more sequelae than incurable systemic fibrosis that result from gadolinium-based contrast agent administration (Leyba and Wagner, 2019). The top five gadolinium-based contrast agents register 16,517 adverse event cases (57% of these serious). Given the ubiquity and frequency of gadolinium-enhanced magnetic resonance imaging tests, the economic impact is large. Gadolinium-induced disease is a man-made entity. Medical science cannot be indifferent to the patients at risk or to those who are currently suffering from gadolinium-induced symptoms. Heretofore there has yet to be a report of gadolinium-based contrast agents stimulating the recruitment of bone marrow-derived cells to an affected organ, the skin. This was demonstrable in both renal insufficiency and normal renal function. Similar to what has been observed in humans (Kim et al., 2006), multinucleated giant cells were found in the dermis using this model. The morphology of the electron-dense deposit were similar to what has been described for gadolinium oxide in phagolysosmal simulated fluid in vitro (Li et al., 2014). That electron-dense deposits peppered these cells suggests a role in gadolinium-based contrast agent-induced pathology. Hypercellular skin fibrosis and the formation of multinucleated giant cells—each found in our model—are characteristic of NSF. These data demonstrate that the dermal hypercellularity and fibrosis of NSF can be modeled in the mouse. The increase in the myeloid marker shows that gadolinium-based contrast agents promote the infiltration of bone marrow-derived cells—fibrocytes and CD163-positive macrophages—to the dermis. These experiments also suggest that gadolinium-based contrast agents can initiate pathologic responses in healthy individuals.

Gadolinium-based contrast agent-treated human peripheral blood mononuclear cells increase CD163 expression, a marker of alternatively-activated macrophages (Swaminathan et al., 2013); we found that myeloid CD163-positive cells also increases in the dermis of contrast-treated rats (Drel et al., 2016). Partial abrogation of dermal hypercellularity in contrast-treated chimeric GFP-expressing marrow to C-C chemokine receptor 2-deficient recipients and the complete abrogation of dermal cellularity and skin fibronectin in chimeric C-C chemokine receptor 2-deficient marrow to wild-type recipients demonstrate the importance of this C-C chemokine mechanism in gadolinium-induced fibrosis. Myeloid expression of the C-C chemokine receptor 2 is requisite for gadolinium-based contrast induction of systemic fibrosis and dermal hypercellularity even when gadolinium is present in the tissue. These studies now provide a new therapeutic target for gadolinium-induced diseases. Furthermore, that profound systemic effects are induced by gadolinium whether renal insufficiency is present or not should serve to alert patients and clinicians to the biologic activity of these compounds.

The C-C chemokine receptor 2 is important for the recruitment and activation of lung fibrocytes in a mouse model of pulmonary fibrosis (Moore et al., 2005). In a murine ureteral ligation model of renal fibrosis, there is an infiltration of CD45+ fibrocytes, and a large subset of these express the C-C chemokine receptor 2 (Sakai et al., 2006). Antagonism of the monocyte chemoattractant 1/C-C chemokine receptor system did reduce kidney fibrosis in a unilateral ureteral obstruction model (Kitagawa et al., 2004). Similar to NSF, scleroderma is characterized by dermal fibrosis (Boin and Hummers, 2008). Monocyte chemoattractant 1 and the C-C chemokine receptor 2 are involved in bleomycin-induced scleroderma (Yamamoto and Nishioka, 2003) and cutaneous sclerosis (Yamamoto, 2003).

Gadolinium has been found in the brains of patients (on autopsy) with normal renal function several months after exposure to magnetic resonance imaging contrast (Kanda et al., 2015). This appears to be irrespective of the chemical formulation of the gadolinium-binding ligand (McDonald et al., 2017). Sea urchin-shaped mesh-like nanowire structures, similar to what we report in the skin, have now been found in brain tissue of humans exposed to gadolinium-based contrast agents (Rasschaert et al., 2018).

It has yet to be proven that gadolinium (de-chelated or bound with the chaperone) residing in the target organ is what triggers the systemic fibrosis. We know that ‘priming’ bone marrow with prior gadolinium-based contrast exposure is not sufficient to replicate the disease in contrast-naïve recipients (yet such ‘priming’ does increase the severity once recipients are exposed to contrast), (Drel et al., 2016). Gadolinium concentrations are higher in affected skin compared to non-affected skin in patients with NSF (71.4 ± 89.4, mean ± SD compared to 23.1 ± 26.6 μg/g, respectively) (Christensen et al., 2011). These concentrations match what we found in our samples.

Our mouse model provides a method of studying the mechanism of gadolinium-based contrast-induced disease and demonstrates the centrality of the C-C chemokine receptor 2 to this disease process. Gadolinium-based contrast treatment induces the myeloid cellular infiltration of a target organ, the skin, in a systemic manner. Furthermore, our experiments pinpoint the C-C chemokine receptor 2 as a critical mediator of fibrocyte trafficking. This work is limited by the concentrations of gadolinium-based contrast agent and the repetitive dosing. Nonetheless, elucidation of this pathway may permit sharply focusing on the multitude of undiscovered risk factors that permeate in patients with gadolinium-induced afflictions. There is a thread that consists of gadolinium-based contrast agents, the C-C chemokine receptor 2, and myeloid cell-induced fibrosis. This study provides a rational target for therapy in gadolinium-based contrast agent-induced diseases.

MATERIALS & METHODS

Animals.

All experimental procedures and protocols were in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH and approved by the Institutional Animal Care and Use Committee (IACUC). Endpoints were 1) weight loss of 10%, 2) dermatologic findings previously described (Wagner et al., 2012), or 3) completion of 4 weeks of contrast treatment (unless otherwise noted). Animals were examined daily for any signs of systemic fibrosis.

NSF model.

Analogous to the gadolinium-based contrast agent-induced systemic fibrosis model established in rats (Do et al., 2014, Drel et al., 2016, Wagner et al., 2016a, Wagner et al., 2012), _weight-matched female C57 black mice 33–34 weeks of age were randomized to control and gadolinium-based contrast agent (Omniscan—gadodiamide/caldiamide—2.5 mmol/kg intraperitoneally, 5 doses per week aiming for 40 doses over 8 weeks or gross evidence of systemic effect).

GFP chimeric mice.

For all bone marrow transplant experiments, fully-acclimated (either C57 black or on a C57 black background) mice underwent lethal irradiation (950 rads divided into two sessions spaced 4 hours apart, GammaCell 40, Atomic Energy of Canada Limited, Mississauga, ON, Canada) followed by bone marrow cells (1 × 107) harvested from donors. Recipients were 1) wild-type mice with 5/6 nephrectomies at 10–12 weeks salvaged with GFP-expressing marrow (C57BL/6-Tg(CAG-EGFP)1310sb/LeySopJ [stock # 006567], The Jackson Laboratory, Bar Harbor, ME), 2) CCR2-deficient mice salvaged with GFP-expressing marrow, 3) wild-type mice salvaged with CCR-deficient marrow (C57BL/6–129S4-Ccr2tmIfc/J [004999], and C57BL/6-B6.129(Cg)-Ccr2tm2.1Ifc/J [017586]). For the first group, partial nephrectomies were performed at 10–12 weeks followed by 2 weeks acclimation. For all groups, 4 weeks were used for engraftment, then animals were randomized into control or gadolinium contrast-treated groups (Omniscan, General Electric HealthCare, Little Chalfont, UK; 2.5 mmol/kg intraperitoneally, aiming for 20 doses over 4 weeks).

Tissue fixation, sectioning, and histology.

Sections of dorsal skin were dissected off the underlying fascia and sliced into equal pieces. These were processes and the dermal nuclei quantified as previously described (Do et al., 2014, Wagner et al., 2012). A portion was fixed in 10% neutral-buffered formalin, and the cortex of the remnant was flash-frozen (for immunoblot or immunofluorescence). Unless otherwise noted, all photomicrographs were obtained with a Nikon Eclipse 55i with a 40× objective.

Immunofluorescence.

Paraffin-embedded tissue was sectioned on glass slides, deparaffinized in xylene, and re-hydrated. Tissues were incubated with citric acid-based unmasking solution (Vector Laboratories, Burlingame, CA), blocked with bovine serum albumin (1%), goat serum (10%) in phosphate-buffered saline (pH 7.4) for 2 hours. Antibodies are listed in Table 1. For mouse antibodies, blocking was with a mouse on mouse immunodetection kit (Vector Laboratories, Burlingame, CA) per the manufacturer’s instructions. Goat secondary antibodies to mouse and rabbit immunoglobulins were AlexaFluor594 and AlexaFluor488 (Thermo Fisher Scientific Life Sciences, Waltham, MA).

Table 1.

Immunofluorescence stain synopsis

Marker Dilution Company City, State
Green fluorescent protein (ab290) 1:100 Abcam Cambridge, MA
CD34 (ab81289) 1:200 Abcam Cambridge, MA
CD163 (bs-2527R) 1:100 Bioss Woburn, MA
Monocyte chemoattractant protein 1 1:100 Novus Biologicals Littleton, CO
Fibronectin (F3648) 1:100 Sigma-Aldrich St. Louis, MO
α smooth muscle actin (A5228) 1:100 Sigma-Aldrich St. Louis, MO
C-C chemokine receptor 2 (PA523037) 1:100 Thermo Fisher Scientific Life Sciences Waltham, MA
CD45RO (MA5-11532) 1:25 Thermo Fisher Scientific Life Sciences
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Immunoblot.

Tissue was homogenized in radioimmunoprecipitation assay buffer as previously described (Do et al., 2014, Wagner et al., 2012). Antibodies for fibronectin (F3648) from Sigma-Aldrich (St. Louis, MO), C-C chemokine receptor 2 (3415R) from Biovision, Inc. (Milpitas, CA), GFP (ab290) and collagen type I (ab34710) from Abcam (Cambridge, MA), and glyceraldehyde 3-phosphate dehydrogenase from Santa Cruz Biotechnology (Dallas, TX).

Statistics.

Unless otherwise indicated, multi-group comparisons were analyzed using analysis of variance with the Tukey honest significant difference post-hoc test using the statistical software R version 3.4.3.

Data availability statement.

Datasets related to this article can be found at https://digitalrepository.unm.edu/hsc_data/KINM/1/ hosted at the University of New Mexico (Wagner, 2019).

Supplementary Material

Supplemental Figure 1
Supplemental Figure 2

ACKNOWLEDGEMENTS

This is dedicated to Dr. Yves Gorin, Ph.D. The research was funded by the Veterans Administration Merit Award (I01 BX001958, BW); the National Institutes of Health through Grants R01 DK 102085 (BW), UL1 TR001120 (YG), R01 DK-079996 (YG), R01 DK 78971 (YG); the Qatar National Research Fund (YG); and Dialysis Clinic, Inc. (BW).

Abbreviations used:

CCR2

C-C chemokine receptor 2

CFSE

5(6)-carboxyfluorescein diacetate N-hydroxysuccinimidyl ester

DAPI

4′,6-diamidino-2-phenylindole

FAERS

Food & Drug Administration Adverse Events Reporting System

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GFP

green fluorescent protein

IACUC

Institutional Animal Care and Use Committee

NSF

‘nephrogenic’ systemic fibrosis

MCP-1

monocyte chemoattractant protein-1

NIH

National Institutes of Health

Footnotes

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CONFLICT OF INTEREST

BW serves as an expert witness for a firm representing patients with adverse events from gadolinium-based contrast agents. The remaining authors do not have any relationships that pose conflicts of interest.

REFERENCES

  1. Boin F, Hummers LK. Scleroderma-like fibrosing disorders. Rheum Dis Clin North Am 2008;34(1):199–220; ix. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Christensen KN, Lee CU, Hanley MM, Leung N, Moyer TP, Pittelkow MR. Quantification of gadolinium in fresh skin and serum samples from patients with nephrogenic systemic fibrosis. J Am Acad Dermatol 2011;64(1):91–6. [DOI] [PubMed] [Google Scholar]
  3. Cowper SE, Bucala R. Nephrogenic fibrosing dermopathy: suspect identified, motive unclear. Am J Dermatopathol 2003;25(4):358. [DOI] [PubMed] [Google Scholar]
  4. Cowper SE, Su LD, Bhawan J, Robin HS, LeBoit PE. Nephrogenic fibrosing dermopathy. Am J Dermatopathol 2001;23(5):383–93. [DOI] [PubMed] [Google Scholar]
  5. Diop AD, Braidy C, Habouchi A, Niang K, Gageanu C, Boyer L, et al. Unenhanced 3D turbo spin-echo MR angiography of lower limbs in peripheral arterial disease: a comparative study with gadolinium-enhanced MR angiography. AJR Am J Roentgenol 2013;200(5):1145–50. [DOI] [PubMed] [Google Scholar]
  6. Do C, Barnes JL, Tan C, Wagner B. Type of MRI contrast, tissue gadolinium, and fibrosis. Am J Physiol Renal Physiol 2014;307(7):F844–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drel VR, Tan C, Barnes JL, Gorin Y, Lee DY, Wagner B. Centrality of bone marrow in the severity of gadolinium-based contrast-induced systemic fibrosis. FASEB J 2016;30(9):3026–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FDA. Medical Imaging Drugs Advisory Committee Meeting. Gadolinium retention after gadolinium based contrast magnetic resonance imaging in patients with normal renal function. Briefing document., https://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/MedicalImagingDrugsAdvisoryCommittee/UCM572848.pdf; 2017. [accessed.
  9. Jimenez SA, Artlett CM, Sandorfi N, Derk C, Latinis K, Sawaya H, et al. Dialysis-associated systemic fibrosis (nephrogenic fibrosing dermopathy). Arthritis Rheum 2004;50(8):2660–6. [DOI] [PubMed] [Google Scholar]
  10. Kanda T, Fukusato T, Matsuda M, Toyoda K, Oba H, Kotoku J, et al. Gadolinium-based Contrast Agent Accumulates in the Brain Even in Subjects without Severe Renal Dysfunction: Evaluation of Autopsy Brain Specimens with Inductively Coupled Plasma Mass Spectroscopy. Radiology 2015;276(1):228–32. [DOI] [PubMed] [Google Scholar]
  11. Kim RH, Ma L, Hayat SQ, Ahmed MM. Nephrogenic fibrosing dermopathy/nephrogenic systemic fibrosis in 2 patients with end-stage renal disease on hemodialysis. J Clin Rheumatol 2006;12(3):134–6. [DOI] [PubMed] [Google Scholar]
  12. Kitagawa K, Wada T, Furuichi K, Hashimoto H, Ishiwata Y, Asano M, et al. Blockade of CCR2 ameliorates progressive fibrosis in kidney. Am J Pathol 2004;165(1):237–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Knopp EA, Cowper SE. Nephrogenic systemic fibrosis: early recognition and treatment. Semin Dial 2008;21(2):123–8. [DOI] [PubMed] [Google Scholar]
  14. Leyba K, Wagner B. Gadolinium-based contrast agents: why nephrologists need to be concerned. Curr Opin Nephrol Hypertens 2019;28(2):154–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Li R, Ji Z, Chang CH, Dunphy DR, Cai X, Meng H, et al. Surface interactions with compartmentalized cellular phosphates explain rare earth oxide nanoparticle hazard and provide opportunities for safer design. ACS nano 2014;8(2):1771–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McDonald RJ, McDonald JS, Dai D, Schroeder D, Jentoft ME, Murray DL, et al. Comparison of Gadolinium Concentrations within Multiple Rat Organs after Intravenous Administration of Linear versus Macrocyclic Gadolinium Chelates. Radiology 2017;285(2):536–45. [DOI] [PubMed] [Google Scholar]
  17. McDonald RJ, McDonald JS, Kallmes DF, Jentoft ME, Murray DL, Thielen KR, et al. Intracranial Gadolinium Deposition after Contrast-enhanced MR Imaging. Radiology 2015;275(3):772–82. [DOI] [PubMed] [Google Scholar]
  18. Moore BB, Kolodsick JE, Thannickal VJ, Cooke K, Moore TA, Hogaboam C, et al. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol 2005;166(3):675–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nazarian RM, Mandal RV, Kagan A, Kay J, Duncan LM. Quantitative assessment of dermal cellularity in nephrogenic systemic fibrosis: a diagnostic aid. J Am Acad Dermatol 2011;64(4):741–7. [DOI] [PubMed] [Google Scholar]
  20. Papanicolas I, Woskie LR, Jha AK. Health Care Spending in the United States and Other High-Income Countries. JAMA 2018;319(10):1024–39. [DOI] [PubMed] [Google Scholar]
  21. Pilling D, Fan T, Huang D, Kaul B, Gomer RH. Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS One 2009;4(10):e7475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Quan TE, Cowper S, Wu SP, Bockenstedt LK, Bucala R. Circulating fibrocytes: collagen-secreting cells of the peripheral blood. Int J Biochem Cell Biol 2004;36(4):598–606. [DOI] [PubMed] [Google Scholar]
  23. Rasschaert M, Schroeder JA, Wu TD, Marco S, Emerit A, Siegmund H, et al. Multimodal Imaging Study of Gadolinium Presence in Rat Cerebellum: Differences Between Gd Chelates, Presence in the Virchow-Robin Space, Association With Lipofuscin, and Hypotheses About Distribution Pathway. Invest Radiol 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rosenkranz AR, Grobner T, Mayer GJ. Conventional or Gadolinium containing contrast media: the choice between acute renal failure or Nephrogenic Systemic Fibrosis? Wien Klin Wochenschr 2007;119(9–10):271–5. [DOI] [PubMed] [Google Scholar]
  25. Sakai N, Wada T, Yokoyama H, Lipp M, Ueha S, Matsushima K, et al. Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis. Proc Natl Acad Sci U S A 2006;103(38):14098–103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanyal S, Marckmann P, Scherer S, Abraham JL. Multiorgan gadolinium (Gd) deposition and fibrosis in a patient with nephrogenic systemic fibrosis--an autopsy-based review. Nephrol Dial Transplant 2011;26(11):3616–26. [DOI] [PubMed] [Google Scholar]
  27. Swaminathan S, Bose C, Shah SV, Hall KA, Hiatt KM. Gadolinium contrast agent-induced CD163+ ferroportin+ osteogenic cells in nephrogenic systemic fibrosis. Am J Pathol 2013;183(3):796–807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wagner B ‘Nephrogenic’ systemic fibrosis is mediated by myeloid C-C chemokine receptor 2: Datasets. Journal of Investigative Dermatology: University of New Mexico Libraries; 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wagner B, Drel V, Gorin Y. Pathophysiology of gadolinium-associated systemic fibrosis. Am J Physiol Renal Physiol 2016a;311(1):F1–F11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wagner B, Drel VR, Gorin Y. Pathophysiology of gadolinium-associated systemic fibrosis. Am J Physiol Renal Physiol 2016b;311(1):F1–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wagner B, Tan C, Barnes JL, Ahuja SS, Davis TL, Gorin Y, et al. Nephrogenic systemic fibrosis: Evidence for bone marrow-derived fibrocytes in skin, liver, and heart lesions using a 5/6 nephrectomy rodent model. Am J Pathol 2012;181(6):1941–52. [DOI] [PubMed] [Google Scholar]
  32. Yamamoto T Potential roles of CCL2/monocyte chemoattractant protein-1 in the pathogenesis of cutaneous sclerosis. Clinical and experimental rheumatology 2003;21(3):369–75. [PubMed] [Google Scholar]
  33. Yamamoto T, Nishioka K. Role of monocyte chemoattractant protein-1 and its receptor,CCR-2, in the pathogenesis of bleomycin-induced scleroderma. J Invest Dermatol 2003;121(3):510–6. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure 1
NIHMS1526668-supplement-Supplemental_Figure_1.jpg (1.1MB, jpg)
Supplemental Figure 2
NIHMS1526668-supplement-Supplemental_Figure_2.jpg (329.2KB, jpg)

Data Availability Statement

Datasets related to this article can be found at https://digitalrepository.unm.edu/hsc_data/KINM/1/ hosted at the University of New Mexico (Wagner, 2019).

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