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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Lipocalin 2 (LCN-2) is an innate immune protein that is expressed by a variety of cells and is highly up-regulated during several pathologic conditions, including immune complex (IC)–mediated inflammatory/autoimmune disorders. However, the function of LCN-2 during IC-mediated inflammation is largely unknown. Therefore, this study was undertaken to investigate the role of LCN-2 in IC-mediated diseases.

Methods

The up-regulation of LCN-2 was determined by enzyme-linked immunosorbent assay in 3 different mouse models of IC-mediated autoimmune disease: systemic lupus erythematosus, collagen-induced arthritis, and serum-transfer arthritis. The in vivo role of LCN-2 during IC-mediated inflammation was investigated using LCN-2–knockout mice and their wild-type littermates.

Results

LCN-2 levels were significantly elevated in all 3 of the autoimmune disease models. Further, in an acute skin inflammation model, LCN-2–knockout mice exhibited a 50% reduction in inflammation, with histopathologic analysis revealing notably reduced immune cell infiltration as compared to wild-type mice. Administration of recombinant LCN-2 to LCN-2–knockout mice restored inflammation to levels observed in wild-type mice. Neutralization of LCN-2 using a monoclonal antibody significantly reduced inflammation in wild-type mice. In contrast, LCN-2–knockout mice developed more severe serum-induced arthritis compared to wild-type mice. Histologic analysis revealed extensive tissue and bone destruction, with significantly reduced neutrophil infiltration but considerably more macrophage migration, in LCN-2–knockout mice compared to wild-type mice.

Conclusion

These results demonstrate that LCN-2 may regulate immune cell recruitment to the site of inflammation, a process essential for the controlled initiation, perpetuation, and resolution of inflammatory processes. Thus, LCN-2 may present a promising target in the treatment of IC-mediated inflammatory/autoimmune diseases.

Lipocalin 2 (LCN-2) belongs to a superfamily of small secreted proteins produced by a variety of cells including epithelia and neutrophils (1). LCN-2 and its human ortholog, neutrophil gelatinase–associated LCN, are elevated by several orders of magnitude during inflammation/infection/injury (2, 3). Induction of LCN-2 during sterile inflammation suggests that it may have additional physiologic functions (4). The well-established functions of LCN-2 include antibacterial activity via sequestration of bacterial siderophores, iron homeostasis, and cellular apoptosis (5–7). LCN-2 has been shown to be necessary for neutrophil recruitment in animal models of infection (8, 9), and a recent study confirmed that LCN-2 is indispensable for neutrophil migration, adhesion, and function (10). Accordingly, LCN-2–knockout mice are susceptible to bacterial sepsis (3). Several investigations have shown the up-regulation of LCN-2 in antibody-mediated inflammatory/autoimmune diseases, such as systemic lupus erythematosus (SLE), in humans (11–13).

Autoimmune disease develops from an aberrant immune reaction against the host's own antigens. Pathogenesis of antibody-mediated autoimmune disease involves both cellular and humoral immune responses. Unusual activation of autoantigen-specific T and B cells, viral infections, and changes in the host's cytokine profile are thought to drive development of antibody-mediated autoimmune disease (14). During immune complex (IC)–mediated autoimmune diseases such as arthritis, SLE, and autoimmune vasculitis, autoantibodies bind to target host cells and initiate inflammation, resulting in tissue injury (15, 16). Several in vivo and in vitro studies have shown that the interaction of the Fc domain of the IC with Fcγ receptors (FcγR) expressed on inflammatory cells leads to the destruction of IC-bound target cells/tissues through antibody-dependent cell-mediated cytotoxicity and phagocytosis (17, 18). Therefore, it has been concluded that FcγR plays a major role during the pathogenesis of several IC-mediated autoimmune diseases. Apart from FcγR, ICs also interact with complement components and trigger the release of chemotactic peptide C5a, which also induces degranulation of mast cells (19, 20). Collectively, the interaction of ICs with FcγR and complement components leads to the release of many chemokines and inflammatory mediators followed by destruction of autoantibody-coated target tissues during autoimmune disease.

More recently, up-regulation of LCN-2, along with other inflammatory cytokines, has been reported in SLE patients (11–13, 21). However, the exact role of elevated LCN-2 levels in autoimmune disease (presumably sterile inflammation/aseptic disease) is largely unknown. Therefore, in this study, we investigated the function of LCN-2 in a model of acute IC-mediated skin inflammation (reverse passive Arthus reaction [RPA]) and a well-established serum-transfer model of arthritis using genetically engineered LCN-2–knockout mice and their wild-type littermates. Since arthritic symptoms in this serum-transfer model of arthritis persist for longer periods of time, it was an excellent model with which to study the role of LCN-2 during chronic inflammatory conditions. Our results demonstrate that LCN-2 levels are significantly elevated in 3 different models of autoimmune disease. Interestingly, in our model of acute IC-mediated skin inflammation, LCN-2 mice exhibited substantially reduced inflammation as compared to wild-type mice, whereas, in the serum-transfer model of arthritis, LCN-2–knockout mice developed severe arthritis, as evidenced by paw swelling and histologic features. Our results demonstrate that LCN-2 is a host-protective factor against systemic autoimmune disease.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Reagents.

Ovalbumin (OVA), Evans blue, and Freund's complete and incomplete adjuvant were purchased from Sigma, rabbit anti-OVA IgG was purchased from Roche Molecular Biochemicals, and horseradish peroxidase substrate and sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels were from Bio-Rad. Iron-, siderophore-, and endotoxin-free mouse recombinant LCN-2, LCN-2–neutralizing monoclonal antibody (mAb; clone 228418), rat IgG2a isotype control antibody (clone 54447), and mouse LCN-2 DuoSet enzyme-linked immunosorbent assay (ELISA) kit were from R&D Systems. A micro–bicinchoninic acid protein assay kit was obtained from Pierce, and bovine type II collagen was obtained from MD Biosciences. Siemens Combistix reagent strips for urinalysis were procured from Emory University's Hospital Pharmacy store. Cell culture reagents were from Life Technologies and 2.4G2 mAb (anti-mouse mAb CD16/32) were from BD Biosciences. Rat anti-mouse neutrophil- specific antibodies (Ly-6G and Ly-6C; clone NIMP-R14) and macrophage-specific antibody (anti-F4/80; clone BM8) were purchased from Abcam. Affinity-purified polyclonal goat anti-rat antibody conjugated with Alexa Fluor 488 was purchased from Jackson ImmunoResearch. K/BxN arthritic serum was a kind gift from Dr. Paul M. Allen (Washington University School of Medicine, St. Louis, MO).

Animal studies.

Arthritis-susceptible female DBA/1J mice and SLE-prone female (NZB × NZW)F1 (NZB/NZW) mice (each 8–10 weeks old) were purchased from The Jackson Laboratory. LCN-2–knockout mice (backcrossed to BL/6 mice for more than 10 generations) were obtained from Dr. Allen Aderem (University of Washington, Seattle, WA) and were originally generated by Dr. Shizuo Akira (Osaka University, Osaka, Japan). Wild-type littermates on a C57BL/6 background were bred and maintained at Emory University's animal facility. Genotyping of LCN-2–knockout mice was performed by Transnetyx. All animal experiments were conducted in accordance with Emory University Institutional Animal Care and Use Committee guidelines.

Reverse passive Arthus reaction.

RPA was carried out as previously described (22, 23). Briefly, a group of LCN-2–knockout mice and wild-type littermates (n = 3 mice per group) were injected intradermally with anti-OVA on the dorsal side (12.5 μg or 25 μg per site). Immediately, 500 μg of OVA in 100 μl of phosphate buffered saline along with 1% Evans blue was injected into the tail vein. Both groups were killed after 4 hours, and the reverse side of the skin at the antibody injection site was collected and evaluated for extravasation of blue dye, since dye intensity corresponds to the severity of inflammation at the site of antibody injection. Photographs of the isolated skin samples were taken, and the intensity of the blue dye was estimated using ImageJ software (National Institutes of Health) and KaleidaGraph (Synergy Software). In some experiments, recombinant LCN-2 (recLcn2; 10 μg/mouse) or LCN-2–neutralizing mAb (100 μg/mouse) was administered intravenously to LCN-2–knockout and wild-type mice, respectively, 1 hour before the initiation of RPA. Skin biopsy samples were subjected to histopathologic analysis using hematoxylin and eosin (H&E) staining.

Collagen-induced arthritis (CIA) and serum-transfer model of arthritis.

CIA was induced by immunizing the DBA/1J mice with bovine type II collagen as previously described (24). Briefly, bovine type II collagen (2 mg/ml) was emulsified thoroughly with an equal volume of Freund's complete adjuvant with heat-killed Mycobacterium (4 mg/ml), and 50 μl of the resulting emulsion (containing 100 μg of collagen) was injected intradermally at the base of the tail. After 21 days, a booster dose was given at the same concentration of collagen with Freund's incomplete adjuvant. Serum prepared from the blood of arthritic K/BxN mice was used to induce the serum-transfer model of arthritis in LCN-2–knockout and wild-type littermates as previously described (25). Briefly, arthritis was induced by injecting 200 μl of K/BxN arthritic serum intraperitoneally.

In both the CIA and serum-transfer models, mice were monitored daily for the development of arthritis. Paw swelling was measured using digital calipers. Blood was collected via the retroorbital plexus, and hemolysis-free serum was collected using serum separator tubes from BD Biosciences and stored at −70°C.

Development of SLE in NZB/NZW mice.

SLE-prone NZB/NZW mice ages 8–10 weeks were purchased from The Jackson Laboratory and maintained at Emory University's animal facility until they developed autoimmune disease. As an index of SLE development, proteinuria was monitored once a week using commercially available Siemens Combistix reagent strips for urinalysis. Protein content was expressed as milligrams of protein per milliliter of urine according to the manufacturer's protocol. Blood samples were collected 8–12 weeks prior to the onset of disease and at 40 weeks, when mice had fully developed disease. Serum samples were prepared and stored at −70°C.

Histologic studies.

Arthritic paws were removed and fixed in 4% buffered formalin, embedded in paraffin, and stained with H&E (Histoserv). The severity of inflammation in the tissue sections was scored as follows: 0 = no symptoms of inflammation (naive paw), 1 = mild infiltration of inflammatory cells, 2 = infiltration of immune cells with muscle destruction, 3 = immune cells with pannus formation, 4 = mild metacarpal bone erosion, and 5 = severe metacarpal and articular bone erosion. Samples were scored by 2 individuals, and the average score was used to plot the graphs. Immunohistochemical analysis of paraffin-embedded tissue was carried out at Emory University's Immunohistochemistry Core Laboratory to detect neutrophils and macrophages using rat anti-mouse Ly-6 (for neutrophils) and anti-mouse F4/80 (for macrophages) as primary antibodies. Bound antibodies were detected using goat anti-rat antibody conjugated with Alexa Fluor 488. Histopathology photographs were taken using an Olympus BX53 microscope (10× objective), and fluorescence images were captured using the 20× and 63× objectives on a Zeiss Axioscope (Carl Zeiss). DAPI staining was performed to detect the nucleus. Polymorphonuclear cells (neutrophils) were identified by nuclear structure followed by anti–Ly-6 antibody.

ELISA.

Hemolysis-free sera were collected via the retroorbital plexus from the animal models described above. These sera were used to quantify circulating LCN-2 using a mouse LCN-2 DuoSet ELISA kit according to the manufacturer's instructions.

Statistical analysis.

One-way analysis of variance and Student's unpaired 2-tailed t-test were performed to compare wild-type and LCN-2–knockout mice. P values less than 0.05 were considered significant. GraphPad Prism 5 software was used to calculate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Up-regulation of systemic LCN-2 in mouse models of autoimmune disease.

To study whether LCN-2 levels are elevated during the development of different IC-mediated autoimmune diseases, we quantified systemic LCN-2 in mouse models of SLE and arthritis. The formation of ICs from autoantibodies against nuclear antigens has been implicated in the development of SLE (26, 27). NZB/NZW mice are susceptible to the development of systemic autoimmune disease symptoms as they age, and most die by 40–50 weeks as a result of multiple organ damage. Urine samples were collected once a week to monitor proteinuria as an index of SLE. Serum samples were collected once NZB/NZW mice began secreting >2 mg/ml of protein in their urine. Serum samples collected before (from 12-week-old mice) and after (from 40-week-old mice) the onset of lupus (Figure 1A, top) were subjected to ELISA to investigate the up-regulation of LCN-2. We observed a 29-fold increase in LCN-2 secretion after the onset of disease (at ∼40 weeks of age) in mice with SLE compared to the level of serum LCN-2 prior to the onset of disease (at 12 weeks of age) (Figure 1A, bottom).

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Figure 1. Up-regulation of systemic lipocalin 2 (LCN-2) in animal models of autoimmune disease. A, Total protein content (top) and serum LCN-2 level (bottom) in samples obtained from (NZB × NZW)F1 mice (n = 5) at 12 and 40 weeks of age and subjected to enzyme-linked immunosorbent assay (ELISA). B and C, Paw thickness in DBA/1J mice with collagen-induced arthritis (CIA; n = 5) and C57BL/6J mice with serum-transfer arthritis (SIA; n = 3) (top panels) and serum LCN-2 levels, determined by ELISA, in mice with CIA and mice with serum-transfer arthritis (bottom panels). CIA was induced using bovine type II collagen, and serum-transfer arthritis was induced by injecting mice with K/BxN arthritic serum. Paw thickness was measured as described in Materials and Methods. Values are the mean ± SD of triplicate determinations. Results are representative of 3 individual experiments. WT = wild-type.

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Next, arthritis was induced either by immunizing DBA/1J mice with bovine type II collagen (CIA) or by injecting arthritic K/BxN mouse serum into C57BL/6 mice (serum-transfer model of arthritis). After the onset of arthritis, as determined by swelling of the paw (Figures 1B and C, top), serum samples were collected and subjected to ELISA in order to quantify LCN-2. As shown in the bottom panels of Figures 1B and C, systemic concentrations of LCN-2 were significantly increased in both the CIA model (17-fold) and the serum-transfer model (5-fold) as compared to nonarthritic naive mice. Taken together, these data suggest that LCN-2 levels are substantially elevated, irrespective of the type of autoimmune disease.

Significantly reduced skin inflammation in LCN-2–deficient mice in an RPA model.

To investigate the role of LCN-2 during IC-mediated inflammation, we used an acute antibody-mediated inflammation model known as reverse passive Arthus reaction (28). In this model, inflammation is initiated following antigen–antibody complex formation at the site of antibody injection. We induced inflammation by injecting mice intradermally on the dorsal side with rabbit anti-chicken OVA (anti-OVA). The antigen, chicken OVA, was injected intravenously along with 1% Evans blue dye. Infiltration of inflammatory cells and vascular leakage can be quantified by measuring extravasation of blue dye at the site of antibody injection (22, 23) since the severity of inflammation is directly proportional to the intensity of the blue dye.

As shown in Figure 2A, LCN-2–knockout mice exhibited reduced inflammation as compared to their wild-type littermates. We observed an ∼50% reduction in the intensity of antibody-mediated inflammation in LCN-2–knockout mice as compared to wild-type mice. The intensity of blue dye at the inflamed site was quantitated using KaleidaGraph software (Figure 2B). A group of mice (n = 3) treated intraperitoneally with 40 μg/mouse of an mAb specific for mouse FcγR (2.4G2), such as CD16A and CD32B, showed complete inhibition of IC-mediated inflammation (Figures 2A and B) and were used as a specificity control. Since 2.4G2 does not block FcγRI and FcγRIV, IC-mediated RPA may be mediated solely by FcγRIII. Histopathologic analysis of the inflamed sites (Figure 2C, showing sections labeled site 2 in Figure 2A) showed a notable reduction in immune cell infiltration in LCN-2–knockout mice as compared to wild-type mice.

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Figure 2. Lipocalin 2 (LCN-2) is necessary to potentiate immune complex–mediated inflammation in a reverse passive Arthus reaction (RPA) model. A, Dorsal side of the skin of mice with RPA, killed 3 hours after RPA was initiated as described in Materials and Methods. Photographs are representative of 3 individual mice per group. 1, 2 and 3 indicate injection sites for phosphate buffered saline (PBS), 12.5 μg of antibody, and 25 μg of antibody, respectively. B, Blue dye intensity at sites 2 and 3 in part A, quantified using ImageJ and KaleidaGraph software. Data are expressed as the mean ± SD. C, Top left, Skin biopsy section from the PBS injection site in an untreated wild-type (WT) mouse with RPA (site 1 in part A). No specific pathologic changes were identified. Top right, Skin biopsy section from the anti-ovalbumin antibody (Ab) injection site in an untreated wild-type mouse with RPA (site 3 in part A). The epidermis and dermis were essentially unexceptional. Subdermal fat was edematous and showed infiltration of inflammatory cells at the site of inflammation. Bottom left, Skin biopsy section from the PBS injection site in an untreated LCN-2–knockout (Lcn2KO) mouse with RPA (site 1 in part A). There were no specific pathologic differences between untreated wild-type and LCN-2–knockout mice. Bottom right, Skin biopsy section from the anti-ovalbumin antibody injection site in an LCN-2–knockout mouse (site 3 in part A). Though the pathology in LCN-2–knockout mice was indistinguishable, there was a substantial decrease in the infiltration of inflammatory cells compared to the wild-type mice.

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Similar degrees of IC-induced inflammation in LCN-2–knockout mice treated with recombinant LCN-2 as in wild-type mice.

To further substantiate our results, LCN-2–knockout mice were administered commercially available iron-, siderophore-, and endotoxin-free recombinant LCN-2 (10 μg/mouse) 1 hour prior to RPA. We used a dose of recombinant LCN-2 that produced systemic LCN-2 levels 5-fold higher than basal systemic levels (100 ng/ml of blood) in LCN-2–knockout mice (data not shown). As shown in Figures 3A and B, LCN-2–knockout mice that were treated with recombinant LCN-2 exhibited inflammation similar to that found in wild-type mice in response to ICs, as evidenced by blue dye extravasation. These data suggest that LCN-2 is necessary for immune cell infiltration and, thus, the potentiation of antibody-mediated skin inflammation.

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Figure 3. Administration of recombinant lipocalin 2 (r-Lcn2) to LCN-2–deficient (Lcn2KO) mice induces similar levels of immune complex–induced Evans blue extravasation as in wild-type (WT) mice. A, Dorsal side of the skin of an untreated wild-type control mouse, an untreated LCN-2–knockout control mouse, and a recombinant LCN-2–treated LCN-2–knockout mouse, all with reverse passive Arthus reaction (RPA) (n = 3 mice per group), killed 3 hours after RPA was initiated as described in Materials and Methods. The recombinant LCN-2–treated LCN-2–knockout group consisted of a group of LCN-2–knockout mice (n = 3) injected intravenously with recombinant LCN-2 (100 μg/mouse). After 1 hour, these mice were injected intradermally with phosphate buffered saline (at site 1) or anti-ovalbumin antibody (Ab; 12.5 μg at site 2 and 25 μg at site 3). Results are representative of 3 individual mice per group. B, Intensity of blue dye shown in part A, quantified using ImageJ and KaleidaGraph software for groups with or without recombinant LCN-2 treatment. Data are the mean ± SD from 3 experiments.

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Alleviation of IC-mediated inflammation by neutralization of LCN-2 in wild-type mice.

Next, to confirm the role of LCN-2 in RPA, endogenous LCN-2 in wild-type mice was neutralized using an established mAb against murine LCN-2 (29). LCN-2–specific antibody (10 μg/mouse) was administered 1 hour prior to the initiation of RPA. We observed that neutralization of LCN-2 significantly reduced Evans blue dye leakage in wild-type mice (Figures 4A and B) to levels similar to those exhibited by LCN-2–knockout mice. Collectively, these results suggest that LCN-2 is required for inflammatory cell migration during IC-mediated inflammation. To exclude the possibility that the dose of recombinant LCN-2 we used may exceed biologic levels, leading to difficulty in interpreting our findings, we used an alternate approach involving the development of IC-mediated arthritis to confirm our results.

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Figure 4. Neutralization of lipocalin 2 (LCN-2) in vivo in wild-type (WT) mice reduces the severity of immune complex–induced inflammation. A, Dorsal side of the skin of an untreated wild-type control mouse, a wild-type mouse treated with isotype control monoclonal antibody (mAb), a wild-type mouse treated with LCN-2 mAb, and an untreated LCN-2–knockout (Lcn2KO) control mouse (n = 3 mice per group), all with reverse passive Arthus reaction (RPA), killed 3 hours after RPA was initiated as described in Materials and Methods. The LCN-2 mAb–treated group consisted of a group of wild-type mice (n = 3) injected intravenously with LCN-2 neutralizing mAb (100 μg/mouse). After 1 hour, these mice were injected intradermally with phosphate buffered saline (at site 1) or anti-ovalbumin antibody (12.5 μg at site 2 and 25 μg at site 3). Results are representative of 3 individual mice per group. B, Intensity of blue dye shown in part A, quantified using ImageJ and KaleidaGraph software. Data are the mean ± SD from 3 experiments.

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Aggravation of disease severity in LCN-2–knockout mice in a serum-transfer model of arthritis.

It has been shown that C57BL/6 mice are resistant to developing CIA (30, 31). Therefore, we used a well-established serum-transfer model of arthritis to investigate whether LCN-2 deficiency confers a similar degree of protection during the development of arthritis. We induced the serum-transfer model of arthritis in age- and sex-matched LCN-2–knockout mice and their wild-type littermates by injecting K/BxN arthritic serum (0.2 ml) intraperitoneally, and we monitored disease severity over a period of 3 weeks. The serum-transfer model of arthritis developed in wild-type and LCN-2–knockout mice in a time-dependent manner and reached disease severity by day 12 (Figure 5A). We observed that there was significantly more severe disease (P < 0.05) in LCN-2–knockout mice as compared to wild-type mice, as indicated by paw thickness (Figure 5A). By day 12, LCN-2–knockout mice had more severe disease than their wild-type littermates and were rendered nearly immobile as a result of paw swelling (Figure 5B). Consistent with clinical severity, synovial inflammation was significantly elevated (P < 0.05) in LCN-2–knockout mice (Figure 5G), as evidenced by histopathologic features 12 days after K/BxN serum transfer.

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Figure 5. LCN-2–deficient (Lcn2KO) mice with serum-transfer arthritis (SIA) develop severe arthritis as compared to wild-type (WT) mice with serum-transfer arthritis. Serum-transfer arthritis was induced as described in Materials and Methods. A, Paw thickness, indicating the development of arthritis. Values are the mean ± SEM. B, Photographs of mouse limbs obtained on day 12 after induction of serum-transfer arthritis. C–F, Histologic sections of ankle joints from a naive wild-type mouse (C), a naive LCN-2–knockout mouse (D), a wild-type mouse with serum-transfer arthritis (E), and an LCN-2–knockout mouse with serum-transfer arthritis (F) stained with hematoxylin and eosin. Tissue sections of hind limbs from naive wild-type and LCN-2–knockout mice showed notably intact bone structure. Tissue sections of arthritic hind limbs exhibited greater infiltration of immune cells, bone erosion, and cartilage destruction in LCN-2–knockout mice compared to wild-type mice. Broken lines differentiate the metacarpal (MC) bone from the articular cartilage (AC). T = tendon muscle; P = pannus formation. Results are representative of 3 individual mice per group. Original magnification × 10. G, Histologic scores for bone erosion. Assessment of bone erosion was carried out as described in Materials and Methods. A group of naive wild-type mice treated with phosphate buffered saline (n = 3) was used as a specificity control. Each data point represents a single mouse; horizontal lines and error bars show the mean ± SEM.

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As a specificity control, nonarthritic paws from naive LCN-2–knockout and wild-type mice were compared. The paws of nonarthritic mice showed intact metacarpal bone, articular cartilage structure, and tendon muscle without any detectable immune cells (Figures 5C and D). As shown in Figure 5F, there was marked destruction of soft tissue, metacarpal bone, and articular cartilage, with extensive pannus formation, in LCN-2–knockout mice as compared to their wild-type littermates (Figure 5E). Taken together, these results suggest that LCN-2 plays a protective role during chronic IC-mediated inflammation and may also play an indispensable role during tissue remodeling and in preserving bone homeostasis.

LCN-2 is essential for neutrophil extravasation but may not be required for macrophage migration to the site of inflammation.

It has been shown that neutrophils and macrophages are necessary for the initiation and progression of inflammation and play major roles during the development of the serum-transfer model of arthritis (25, 32). Therefore, to confirm the type of immune cell responsible for causing the severe arthritic damage observed in LCN-2–knockout mice, immunohistochemical analysis was performed. Consistent with a recent report (10), we also observed a significant reduction (∼50–60%) in neutrophil infiltration (Figure 6A) in LCN-2–knockout mice (Figure 6E) compared to wild-type mice (Figure 6D), whereas neutrophils were undetectable in tissue sections from naive mice (Figures 6B and C). The presence of neutrophils was confirmed based on polynuclear structures (DAPI staining) and Ly-6 surface marker (Figure 6D, inset). These results suggest that LCN-2 is required for the migration of neutrophils to the site of inflammation.

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Figure 6. Lipocalin 2 (LCN-2) is a prerequisite for neutrophil, but not macrophage, migration to the site of inflammation during the pathogenesis of serum-transfer arthritis. A, Green fluorescence intensity of sections from wild-type (WT) mice and LCN-2–knockout (Lcn2KO) mice examined for neutrophils, calculated using ImageJ software and graphically represented using GraphPad Prism 5. B–E, Histologic sections from LCN-2–knockout and wild-type mice, stained to detect neutrophils as described in Materials and Methods. Nonarthritic paws of wild-type mice (B) and LCN-2–knockout mice (C) did not exhibit neutrophils, whereas neutrophil infiltration (arrows) was significantly reduced in arthritic LCN-2–knockout mice (E) as compared to wild-type mice (D). Inset, Neutrophils were identified based on nuclear structure (DAPI) followed by surface marker (Ly-6) staining. F, Green fluorescence intensity of sections from wild-type mice and LCN-2–knockout mice examined for macrophages, calculated using ImageJ software and graphically represented using GraphPad Prism 5. G–J, Histologic sections from LCN-2–knockout and wild-type mice, stained to detect macrophages as described in Materials and Methods. Nonarthritic paws of wild-type mice (G) and LCN-2–knockout mice (H) did not exhibit macrophage accumulation near metacarpal bone or tendon muscle, whereas considerably more macrophages (arrows) accumulated in arthritic LCN-2–knockout mice (J) as compared to wild-type mice (I). Original magnification × 20 in B–E and G–J; × 63 in inset. Results are representative of 3 individual mice per group. Values in A and F are the mean ± SD.

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To investigate whether macrophages are associated with severe pathogenesis in the serum-transfer model of arthritis in LCN-2–knockout mice, we performed macrophage staining using F4/80 antibody. Interestingly, we observed considerably more macrophage accumulation in arthritic LCN-2–knockout mice than in wild-type mice (Figure 6F), with macrophages concentrated in the pannus region near the metacarpal bone (Figures 6I and J). Macrophages were not detected in nonarthritic LCN-2–knockout and wild-type mice near the metacarpal bone or tendon muscle regions (Figures 6G and H). At present, the cause of such elevated macrophage accumulation in LCN-2–knockout mice is unclear (Figure 6F), although we have made similar observations in a Dextran sulfate sodium–induced inflammatory bowel disease (colitis) model (Vijay-Kumar M, et al: unpublished observations). Therefore, it is possible that macrophages may be responsible for causing severe serum-transfer model pathology in LCN-2–knockout mice.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

In humans, systemic up-regulation of LCN-2 correlates significantly with the development of many inflammatory disorders, such as peritonitis, atherosclerosis (33), vasculitis/Kawasaki disease (34), inflammatory bowel diseases (35), and SLE (11–13, 21). Apart from these studies, the up-regulation of LCN-2 has also been observed in sputum from patients with asthma and those with chronic obstructive pulmonary disease and in synovial fluid from rheumatoid arthritis patients (36). LCN-2 was recently identified as an inducer of chemoattractants that mediate cell migration during CNS injury (37). Though the bactericidal activity of LCN-2 has been well characterized (3, 8, 38) and is known to be up-regulated during various inflammatory conditions (4, 39), its biologic role during IC-mediated inflammation is still obscure. In this report, we have shown that LCN-2 plays a protective role in the development of IC-mediated inflammatory diseases such as arthritis and SLE. We observed a several-fold increase in circulating LCN-2 during IC-mediated autoimmune disease such as SLE and arthritis.

To delineate the function of LCN-2 during IC-mediated inflammation, we used a model of acute inflammation known as RPA. We found that extravasation of inflammatory cells at the site of inflammation was reduced in LCN-2–knockout mice as compared to wild-type mice. Treating LCN-2–knockout mice with recombinant LCN-2 facilitated inflammation, such that treated LCN-2–knockout mice were indistinguishable from wild-type mice, whereas neutralizing circulating endogenous LCN-2 using mAb in wild-type mice reduced inflammation to levels comparable to those in LCN-2–knockout mice. These data support earlier studies (8, 37) indicating that LCN-2 is required for the migration of inflammatory cells to the site of inflammation in RPA.

Studies with Fcγ- and FcγR-knockout mice have shown that RPA is mediated largely by FcγR (40–42). To investigate whether the interaction of FcγR with ICs leads to the secretion of LCN-2 from inflammatory cells such as macrophages, we incubated ICs with P388D1 cells (mouse macrophage cell line) for 24 hours at 37°C and measured their secretion of LCN-2 in culture supernatants. Our results demonstrated a 5–6–fold increase in LCN-2 secretion as compared to untreated cells (data not shown). Hence, it is possible that the interaction of ICs with FcγR expressed on inflammatory cells residing at the site of inflammation causes up-regulation of LCN-2, which, in turn, may act as an inducer of chemoattractants. Therefore, we hypothesize that there might be cross-talk between FcγR and LCN-2 up-regulation during IC-mediated inflammation. It has been shown that the complement pathway also plays a vital role during IC-mediated inflammation and, while it is possible this may also be a contributing factor, we have previously shown that the complement pathway plays little or no role during RPA in C57BL/6 mice (22).

Since RPA is a model of acute IC-mediated inflammation, we extended our studies to investigate the function of LCN-2 in the K/BxN arthritis model. In contrast to our acute RPA model, we observed that LCN-2–knockout mice developed severe arthritis as compared to wild-type mice. The reason for contrasting results between the RPA and serum-transfer models is not clear; however, it may be due to the fact that RPA is an acute model featuring only transient tissue damage that may not require all possible tissue repair mechanisms for recovery of the inflamed region. It is interesting to note that LCN-2 is highly elevated during tissue involution/remodeling (43). Therefore, it is possible that, since serum-transfer arthritis persists for a longer period of time, the absence of LCN-2–mediated tissue repair may result in the increased severity of arthritis observed in LCN-2–knockout mice as compared to wild-type mice.

Histopathologic analysis of LCN-2–knockout and wild-type arthritic paws revealed significant metacarpal bone erosion and articular cartilage damage with extensive pannus formation in LCN-2–knockout, but not wild-type, mice, suggesting that LCN-2 may play a vital role in preserving bone architecture. It has been shown that neutrophils and macrophages are predominantly responsible for the initiation and progression of serum-transfer arthritis (25, 32). Similar to Schroll et al (10), we observed that neutrophil infiltration is severely reduced in LCN-2–knockout mice during the development of serum-transfer arthritis, suggesting that LCN-2 is a prerequisite for neutrophil infiltration and that, perhaps, serum-transfer arthritis is mediated by cells other than neutrophils in LCN-2–deficient mice.

Consistent with that reasoning, we observed greater macrophage accumulation in LCN-2–knockout mice during the development of serum-transfer arthritis. While the mechanism of such increased macrophage accumulation is unknown, it is possible that the production of macrophage-specific chemokines may have increased over time in LCN-2–knockout mice as a compensatory mechanism to mediate inflammation in the absence of a timely neutrophil response. Therefore, we hypothesize that macrophages may be responsible for causing the severe arthritic pathology observed in LCN-2–knockout mice. Various in vivo studies have shown that the inflammation and tissue repair mechanisms occur in parallel during inflammatory conditions in order to limit host tissue damage, and that LCN-2 plays a significant role in tissue remodeling (44–46). Therefore, in continuation of this view, our data suggest that LCN-2–knockout mice with serum-transfer arthritis experience continuous inflammation on a background of impaired tissue remodeling capacity and, thereby, have exacerbated arthritis. Such damage in LCN-2–knockout mice may be primarily mediated by macrophages as compared to wild-type mice. Nonetheless, other cell types including mast cells and platelets may also play a role, as has been observed during the pathogenesis of serum-transfer arthritis (47, 48).

Further, apart from the innate immune cellular contribution to the pathogenesis of serum-transfer arthritis, it is possible that LCN-2–deficient mice exhibit elevated intracellular labile iron that may participate in the Fenton reaction, generating reactive oxygen species and thus increasing oxidative stress. Up-regulation of LCN-2 during inflammatory conditions may aid in scavenging reactive oxygen species indirectly by chelating iron. We have also found that LCN-2 offers significant protection against endotoxin-induced sepsis in mice whereby LCN-2–knockout mice exhibit significantly elevated oxidative stress markers and lipopolysaccharide-induced toxicity (49).

In conclusion, the data presented in this report suggest that engagement of FcγR expressed on local inflammatory cells by ICs leads to the induction of LCN-2. Such IC-induced LCN-2 expression may be necessary for the migration of inflammatory cells to the site of not only infection (8, 9, 50), but also inflammation during acute IC-mediated inflammatory events, whereas in chronic IC-mediated disorders, LCN-2 may play a protective role and prevent severe tissue damage by facilitating tissue remodeling. Therefore, our results show that LCN-2 may play a dual role (initiation and resolution) during the pathogenesis of IC-mediated inflammation. Taken together, these studies suggest that LCN-2 plays a vital role during the pathogenesis of IC-mediated inflammatory/autoimmune disease conditions by modulating the inflammatory response of immune cells.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Shashidharamurthy and Vijay-Kumar had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Shashidharamurthy, Vijay-Kumar.

Acquisition of data. Shashidharamurthy, Machiah, Aitken, Putty, Srinivasan.

Analysis and interpretation of data. Shashidharamurthy, Machiah, Aitken, Chassaing, Parkos, Selvaraj.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES