Differential role of lipocalin 2 during immune complex-mediated acute and chronic inflammation in mice

Abstract

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.

Figure 1. Upregulation of systemic Lcn2 in animal models of autoimmune diseases

(A) Serum samples from NZB/WF1 mice (n=5) at 12 and 40 weeks old (upper panel) were collected and subjected for ELISA (lower panel). (B upper panel) CIA was induced in DBA/J1 mice (n=5) using bovine collagen type-II. (C upper panel) SIA in C57BL/6J mice (n=3) by injecting K/BxN arthritic serum and paw thickness was measured as described in Materials and Methods. Serum samples were collected and assayed for Lcn2 by ELISA (B and C lower panel) as described under Materials and Methods. Figures are representative of three individual experiments. Data are mean ± SD of triplicates.

Figure 2. Lcn2 is required to potentiate IC-mediated inflammation in RPA model

(A) RPA was initiated as described under Materials and Methods. After 3h mice were euthanized and the dorsal side of the skin photographed for analysis. The photographs are representative of three individual mice. (B) Blue dye intensity was quantified using ImageJ and KaleidaGraph software. Data are expressed as ± SD. (C) Panel-1: Skin biopsy from PBS injection site of WT untreated mouse with RPA (from Figure 2A, site 1). No specific pathologic changes are identified. Panel-2: Skin biopsy from anti-Ova antibody injection site of WT untreated mouse with RPA (from Figure 2A, site 3). The epidermis and dermis are essentially unexceptional. Subdermal fat is edematous and shows infiltration of inflammatory cells at the site of inflammation. Panel-3: Skin biopsy from PBS injection site of Lcn2KO untreated mouse with RPA (from Figure 2A, site 2). No specific pathologic difference between untreated WT and Lcn2KO mice. Panel-4: Skin biopsy from anti-Ova antibody injection site of Lcn2KO mouse (from Figure 2A, site 3). Though the pathology of Lcn2KO is indistinguishable, there is a substantial decrease in the infiltration of inflammatory cells than the WT mice.

Figure 3. Rec-Lcn2 administration to Lcn2 deficient mice induces similar levels of IC-induced Evan’s blue extravasation in WT mice

(A) A group of Lcn2KO mice (n=3) were injected with rec-Lcn2 intravenously (100 µg/mouse). After 1h, these mice were injected intradermally with PBS (site 1) or anti-Ova (12.5 µg at site 2 and 25 µg at site 3). RPA was initiated as described above in three separate groups of mice (n=3): WT untreated control, Lcn2KO untreated control and rec-Lcn2 treated Lcn2KO. After 3h the mice were euthanized and the dorsal side of the skin was photographed for analysis. The figure shows three representative mice. (B) The blue dye seen in the photographs was quantified using ImageJ and KaleidaGraph softwares for groups with or without rec-Lcn2 treatment. Data are presented as the mean ± SD from three experiments.

Figure 4. Neutralization of Lcn2 in vivo in WT mice reduces severity of IC-induced inflammation

(A) A group of WT mice (n=3) were injected with Lcn2 neutralizing mAb intravenously (100 µg/mouse). After 1h, these mice were injected intradermally with PBS (site 1) or anti-Ova (12.5 µg in site 2 and 25 µg in site 3). RPA was initiated as described above in four separate groups of mice (n=3): WT untreated control, WT treated with Lcn2 mAb, WT treated with isotype control mAb and Lcn2KO untreated control. After 3h the mice were euthanized and the dorsal side of the skin was photographed for analysis. The figure shows three representative mice. (B) Intensity of dermal lesions, seen blue in the photographs, was quantified using ImageJ and KaleidaGraph software. Data are presented as the mean ± SD from three experiments.

Figure 5. Lcn2 deficient mice develop severe arthritis when compared to WT in SIA model

SIA was induced as described under Materials and Methods. The mice were monitored for the development of arthritis by ankle thickness (A) and photographs were taken on day 12 (B). Histological section of ankle joints stained with haematoxylin and eosin (original magnification 10x). Tissue sections of hind limbs from naïve WT (C) and Lcn2KO mice (D) show remarkably intact bone structure. Tissue sections of arthritic hind limbs exhibit greater infiltration of immune cells, bone erosion and cartilage destruction in Lcn2KO (F) compared to WT mice (E). Histological scoring and assessment of bone erosion in arthritic paws of Lcn2KO and WT mice were carried out as described under Materials and Methods and represented graphically (G). The blue dotted line differentiates the metacarpal (MC) bone from articular cartilage (AR). P and T stand for panus formation and tendon muscle, respectively. A group of mice (n=3) treated with PBS (naïve) served as specificity control. Photographs are representative of three individual mice. Data are expressed as ± SEM.

Figure 6. Lcn2 is a prerequisite for neutrophil, but not macrophage, migration to the site of inflammation during the pathogenesis of SIA

Histological sections of Lcn2KO and WT mice were stained to detect neutrophils/macrophages as described under Materials and Methods. Unarthritic paws of WT (B) and Lcn2KO (C) mice did not exhibit neutrophils whereas neutrophil infiltration was significantly reduced in arthritic Lcn2KO (E) when compared to WT (D) mice. Inset: Neutrophils are identified based on nuclear structure (DAPI) followed by surface marker (Ly-6) staining. These pictures were taken at 63x original magnification. Unarthritic paws of WT (G) and Lcn2KO (H) mice did not exhibit macrophage accumulation near metacarpal bone or tendon muscle whereas considerably more macrophages accumulated in arthritic Lcn2KO (J) when compared to WT (I) mice. Green fluorescence intensity of each section was calculated using Image J software and graphically represented using GraphPad Prism 5 software (A and F). Pictures were taken at 20x original magnification and the regions of neutrophil/macrophage accumulation are presented in the figure for the purpose of clarity. Arrows indicates the presence of neutrophils/macrophages. Data are representative of three individual mice (n=3) and are expressed as ± SD.