Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury

Abstract

Diverse functions have been reported for lipocalin 2. To investigate these functions in vivo, we generated gene-targeted lipocalin 2-deficient mice (Lcn2-/- mice). In vitro studies have suggested that lipocalin 2 is important for cellular apoptosis induced by IL-3 withdrawal, and for the induction of kidney differentiation during embryogenesis. Analysis of Lcn2-/- mice showed normal cell death upon IL-3 withdrawal and normal kidney development. However, we found that Lcn2-/- mice exhibited an increased susceptibility to bacterial infections, in keeping with the proposed function of lipocalin 2 in iron sequestration. Neutrophils isolated from Lcn2-/- mice showed significantly less bacteriostatic activity compared with WT controls. The bacteriostatic property of the WT neutrophils was abolished by the addition of exogenous iron, indicating that the main function of lipocalin 2 in the antibacterial innate immune response is to limit this essential element. Another important function ascribed to lipocalin 2 has been its protective role against kidney ischemia-reperfusion injury. We analyzed Lcn2-/- mice using a mouse model for severe renal failure and could not detect any significant differences compared with their WT littermates.

Fig. 1.

Lipocalin 2-deficient cells respond normally to IL-3 withdrawal and a variety of apoptotic stimuli. (A) Cultured bone marrow cells from WT and Lcn2^−/− littermates were subjected to IL-3 withdrawal, and the percentage of viable cells remaining was measured by annexin–propidium iodide staining at the indicated time points. Results shown are the mean percentage of viable cells (±SD) of three independent experiments involving at least two mice for each genotype. (B) Thymocytes from WT and Lcn2^−/− littermate mice were treated with the indicated chemical and physical stimuli, and cell viability was assessed by annexin–propidium iodide staining after 24 h.

Fig. 2.

Antibacterial effects of lipocalin 2 in vivo and in vitro. (A) Lipocalin 2 protects against bacterial infection in vivo. Shown are survival curves of Lcn2^−/− and WT mice that were i.p. injected with 1.5 × 10⁸ cfu/30 g of E. coli (ATCC 25922). Results shown are the percentage survival from three independent experiments of a total of 19 Lcn2^−/− mice and 24 WT mice (P > 0.005; log-rank test). (B) Lcn2^−/− neutrophils exhibit impaired bacteriostatic activity in vitro. Neutrophils isolated from Lcn2^−/− mice (black bars) and WT littermates (white bars) were incubated with E. coli at a multiplicity of infection of 1. Gray bars indicate cultures of bacteria and WT neutrophils to which 50 μM iron was added. Bacterial growth was determined at the indicated time points as described in Materials and Methods. Data shown are the mean cfu of bacteria (±SD) of triplicate measurements from three independent experiments. Significant differences in bacterial growth between WT and Lcn2^−/− cultures were found at 30 min (∗, P < 0.001) and at 60 and 120 min (∗∗, P < 0.0002).

Fig. 3.

Lipocalin 2 deficiency does not increase kidney damage after ischemia-reperfusion injury. In a murine model for acute tubular necrosis (ATN), the renal pedicle was clamped for 30 min and the contralateral kidney was removed. (A) After 24 h of reperfusion, the serum creatinine level rose from 0.22 ± 0.05 mg/dl (n = 5) in sham-operated mice to 2.26 ± 0.34 mg/dl in WT mice and to 2.36 ± 0.27 mg/dl in Lcn2^−/− mice (n = 7; P > 0.5, for WT vs. Lcn2^−/−), demonstrating no difference. (B) Similarly, blood urea nitrogen levels showed no significant difference between Lcn2^−/− mice and WT mice, rising from 26 ± 8 mg/dl in sham-operated mice (n = 5) to 167 ± 25 mg/dl in WT mice and 202 ± 31 mg/dl in Lcn2^−/− mice (n = 7; P > 0.05, for WT mice vs. Lcn2^−/− mice). (C) Ischemic kidneys in both Lcn2^−/− and WT mice showed similar loss of tubular nuclei (ATN, Bottom) and an equal presence of cortical and medullary intratubular casts (ATN, Middle). (D) The area of the proximal convoluted tubular necrosis was evaluated by the Jablonski scale, which also demonstrated no significant difference (0, no necrosis; 1, isolated necrosis; 2, focal necrosis in inner cortex; 3, diffuse necrosis in inner cortex; 4, necrosis involving whole cortex). (E) Percentage of apoptotic tubules containing at least one apoptotic nucleus. Ischemia-reperfusion injury resulted in a similar increase of apoptotic tubules in Lcn2^−/− and WT mice.