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.
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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.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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.