Lipocalin‐2 (NGAL/LCN2), a “help‐me” signal in organ inflammation

The authors received funding from the German Research Foundation (SFB/TRR 57) and the Interdisciplinary Center for Clinical Research (IZKF) Aachen. Potential conflict of interest: Nothing to report. Author names in bold designate shared co‐first authorship. To the Editor: There are numerous experimental and clinical studies available showing that lipocalin 2 (LCN2), also known as neutrophil gelatinase‐associated lipocalin (NGAL), is up‐regulated during inflammation and in response to cellular stress, promoting protective effects during acute and chronic injury.1 In a recent report, Xu et al. demonstrated that hepatocyte‐derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration.2 The presented data corroborate our previous findings that injury‐induced up‐regulation of hepatic LCN2 has a significant hepatoprotective effect in acute liver injury and that hepatocytes are the major source for hepatic LCN2.3 Furthermore, these data suggest that LCN2 might act as an intrinsic “help‐me” sensor that, upon injury, develops an activity necessary to recruit inflammatory cells. In line with this assumption, we observed that lcn2‐deficient mice showed a significant lower recruitment of neutrophils and leukocytes, compared to wild‐type (WT) animals, when fed with a methionine‐choline–deficient (MCD) diet that induces hepatic inflammation and injury (Fig. 1). The staining and activity measurement of myeloperoxidase (MPO), representing an enzyme that is most abundantly expressed in neutrophilic granulocytes, further confirmed the assumption that LCN2 is an essential factor promoting hepatic neutrophil recruitment (Fig. 2). In line with this, after MCD feeding, hepatic MPO expression analyzed by quantitative real‐time polymerase chain reaction (qRT‐PCR) was higher in WT animals than in mice lacking LCN2 (not shown). Moreover, the important role of LCN2 for neutrophil recruitment was also documented in mice that were injected with lipopolysaccharide (LPS). When MPO‐positive neutrophil infiltrates were comparatively analyzed in WT and Lcn2 null mice that received LPS for 6 hours, we found a statistically significant higher number of respective cells (P = 0.0139 when counting each 10 representative high power fields) in WT animals (not shown).Figure 1: Leukocyte/neutrophil recruitment in hepatic inflammation. WT or Lcn2‐deficient mice were fed with a standard chow or MCD diet for 4 weeks. (A) Liver samples were stained with Oil Red, hematoxylin and eosin (H & E), or antibodies directed against the leukocyte common antigen CD45 or neutrophil marker MPO. (B) RNA was prepared and subjected to qRT‐PCR analysis for CD45 expression. (C) Protein extracts from respective livers were subjected to western blotting analysis for analysis of CD45 expression. Abbreviations: GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; mRNA, messenger RNA.Figure 2: Quantitative analysis of neutrophil recruitment. (A) MPO was stained in liver cryosections obtained from WT or Lcn2‐deficient mice fed with either a control or an MCD diet for 4 weeks (magnification, ×40). As a negative control, livers were stained with a nonimmune immunoglobulin. As a positive control, tonsillitis tissue was stained with the antibody directed against MPO. (B) Cells positive for MPO were counted in 10 representative high power fields each using the public domain Java image processing and analysis program (ImageJ, version IJ 1.46r). Statistically significant differences are indicated. (C) MPO activity was quantified in liver tissue samples using the MPO activity quantification kit (ab105136; Abcam, Cambridge, MA), according to the manufacturer's instructions. Statistically significant differences are indicated. (D) Liver cryosections taken from WT or Lcn2‐deficient animals that received a single injection of LPS or a standard saline solution (NSS) for 6 hours were stained for LCN2 and MPO. Magnification, ×200. Abbreviation: DAPI, 4’,6‐diamidino‐2‐phenylindole.It is most likely that this biological activity of LCN2 is organ independent given that LCN2 was already reported to promote production of neutrophil‐attracting chemokines and neutrophil recruitment during mycobacterial pulmonary infection by inducing alveolar macrophage‐produced granulocyte colony‐stimulating factor, a key regulator of neutrophil production and keratinocyte chemoattractant (KC/CXCL1) that is involved in chemotaxis and cell activation of neutrophils through chemokine receptor CXCR2.5 Of note, another recent report has demonstrated that inflammatory cell‐derived LCN2 at the sites of inflammation plays important roles in central sensitization and the subsequent nociceptive behavior in the complete Freund's adjuvant‐induced chronic inflammatory pain mouse model.6 All these attributes render this lipocalin as a reliable biomarker for hepatic inflammation and as a novel drug target for lesions associated with overshooting inflammatory reactions in liver and other organs.