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. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Stroke. 2014 Jun 10;45(7):2085–2092. doi: 10.1161/STROKEAHA.114.005733

Neuronal production of lipocalin-2 as a help-me signal for glial activation

Changhong Xing 1,*, Xiaoshu Wang 1,2, Chongjie Cheng 1,2, Joan Montaner 3, Emiri Mandeville 1, Wendy Leung 1, Klaus van Leyen 1, Josephine Lok 1, Xiaoying Wang 1, Eng H Lo 1,*
PMCID: PMC4122238  NIHMSID: NIHMS594193  PMID: 24916903

Abstract

Background and purpose

We explored the hypothesis that injured neurons release lipocalin-2 as a help-me signal.

Methods

In vivo lipocalin-2 responses were assessed in rat focal cerebral ischemia and human stroke brain samples using a combination of ELISA and immunostaining. In vitro, microglia and astrocytes were exposed to lipocalin-2 and various markers and assays of glial activation were quantified. Functional relevance of neuron-to-glia lipocalin-2 signaling was examined by transferring conditioned media from lipocalin-2-activated microglia and astrocytes onto neurons to see whether activated glia could protect neurons against oxygen-glucose deprivation and promote neuroplasticity.

Results

In human stroke samples and rat cerebral ischemia, neuronal expression of lipocalin-2 was significantly increased. In primary cell cultures, exposing microglia and astrocytes to lipocalin-2 resulted in glial activation. In microglia, lipocalin-2 converted resting ramified shapes into a long-rod morphology with reduced branching, increased interleukin-10 release, and enhanced phagocytosis. In astrocytes, lipocalin-2 upregulated GFAP, BDNF and thrombospondin-1. Conditioned media from lipocalin-2-treated astrocytes upregulated synaptotagmin, and conditioned media from lipocalin-2-treated microglia upregulated synaptophysin and PSD95 and protected neurons against oxygen-glucose deprivation.

Conclusions

These findings provide proof-of-concept that lipocalin-2 is released by injured neurons as a “help-me” distress signal that activates microglia and astrocytes into potentially pro-recovery phenotypes.

Keywords: lipocalin-2, neuron, microglia, astrocyte

Adaptive signaling within the neurovascular unit is critical for the balance between injury and repair after stroke. 1,2 Damaged neurons can release many factors that activate glia into deleterious forms that worsen neuroinflammation. For example, damaged neurons release glutamate that activate metabotropic receptors on microglia and shift them into neurotoxic phenotypes. 3 After cerebral ischemia, neurons upregulate tumor necrosis factor-alpha (TNF-α) that activates astrocyte and microglia via paracrine and autocrine pathways to amplify neuroinflammation. 4,5 However, what is missing from this formulation is whether injured neurons can also “ask for help” and induce glia to adopt pro-recovery phenotypes. In this study, we assessed the hypothesis that lipocalin-2 (LCN2) is a “help-me” distress signal released from injured neurons.

LCN2, also known as neutrophil-gelatinase-associated-lipocalin or 24p3, is a 25 kDa protein belonging to the lipocalin superfamily. 6 Although initially identified as an antibacterial factor released from neutrophils, 7 LCN2 can be produced by many organs in response to injury. LCN2 is produced by tubular cells during kidney disease. 8 In a mouse model of renal damage, LCN2 protects the kidney against ischemia-reperfusion. 9 In the CNS, LCN2 is associated with Alzheimer’s disease pathology. 10 In rat brain, LCN2 is upregulated after kainate neurotoxicity 11 or after systemic lipopolysaccharide injections. 12 In human stroke, serum levels of LCN2 progressively increase following cerebral ischemia. 13,14

Taken together, these background findings suggest that LCN2 may be an active mediator in tissue inflammation and remodeling. The present study asks whether LCN2 may be a “help-me” signal in stroke-damaged brain. Our data showed that (i) LCN2 is upregulated within neurons in rat focal cerebral ischemia as well as human stroke brain samples, (ii) LCN2 can induce potentially beneficial phenotypes in rat primary microglia and astrocytes, and (iii) LCN2-activated glia may in turn protect neurons against oxygen-glucose deprivation and promote neuroplasticity.

Methods

Rat cerebral ischemia

Experiments were randomized, blinded and performed following institutionally-approved protocols. Transient focal ischemia was induced by 90min occlusion of the middle cerebral artery in male Wistar rats (280–300g) under 1.2% isoflurane anesthesia and laser-doppler monitoring. Rectal temperature, blood pressure, pH and gases were all within normal range. Brain samples were obtained from shams at 3 days, and ischemic animals at 1, 3, and 7 days post-reperfusion.

Human stroke brain samples

Proof-of-concept data was collected in one case - a 77 year old female with left middle cerebral artery occlusion, who developed hemorrhagic transformation following thrombolysis, and died 77hours post-stroke (approved by Ethics Committee, Hospital Vall d’Hebron, with informed consent, PR(HG)85/04). Samples were immediately fixed with 4% paraformaldehyde and kept at −80°C. On autopsy and macroscopic examination, morphological features and neuroimaging guided brain tissue sampling from ipsilateral and contralateral cortex. Infarct area was delineated by an experienced neuropathologist and 1cm3 of contiguous tissue was obtained as peri-infarct.

Primary cell cultures

Primary rat microglia, astrocyte, and neurons were prepared following standard methods (details in the online-only Data Supplement). Microglia or astrocyte conditioned media were collected from cultures without or with 1μg/ml of LCN2 treatment for 24hrs. Oxygen-glucose-deprivation experiments were performed using a humidified incubator chamber (90% N2, 5% H2, 5% CO2, 37°C).

Immunohistochemistry

Rats were transcardially perfused with ice-cold PBS. Immunostaining was performed on fresh-frozen 20μm coronal sections at −0.8 and −2.8mm from bregma, representing the maximal infarct area in our models. Brain sections (rat and human) or cultured microglia or astrocytes were probed with primary antibodies against NeuN (Millipore), GFAP (BD for rat brains and cultured astrocytes, and Invitrogen for human brain) or Iba1 (Abcam for rat brains and cultured microglia, and Wako for human brain). Neuronal staining for LCN2 was confirmed with 3 different antibodies (R&D, Abcam, Bioss). Negative controls without primary antibodies showed no immunoreactivity. LCN2 in rat brains was assessed in peri-infarct fields (magnification, ×200) and contralateral homologous areas.

Protein measurements

Western blots were performed with primary antibodies against GFAP (BD), VEGF (Santa Cruz), IGF-1 (Santa Cruz), BDNF (Santa Cruz), TSP-1 (Neomarkers), synaptophysin (Millipore), synaptotagmin (Synaptic Systems), and PSD95 (Abcam). LCN2 levels in equal volume of conditioned media collected from neurons, astrocytes or microglia were detected by Western blot. IL-1β (R&D), IL-10 (eBioscience), and LCN2 (Abcam) in cell culture supernatant or brain tissue lysate were measured by ELISA.

Real-time PCR

Microglial cells were treated with 1 μg/ml of LCN2 (recombinant rat LCN2 from HEK293 cells, Enzo Life Sciences) for 2hrs. For analysis of iNOS, CD86, CD206 and Arg1 mRNA, real-time PCR was performed using TaqMan assays (Applied Biosystems) in triplicates.

Microglia assays

Cell migration assay kit (Chemicon) was used to analyze microglial migration in the presence or absence of 1μg/ml of LCN2 after 4–24hrs incubation. To assess phagocytosis, microglia were seeded onto 96-well plates (1.0×105 cells/well) with vehicle or 1 μg/ml of LCN2, then incubated with fluorescein-labeled Escherichia coli K-12 BioParticles (Invitrogen) for 2hrs at 37°C. Experiments were performed with five replicates per condition and repeated a minimum of three times.

Statistics

3–5 separate experiments were performed for all in vivo and in vitro measurements. Data (mean±SD) were analyzed using t-tests, one-way or two-way ANOVA (SPSS version 16.0). Significance was set at p<0.05.

Results

Upregulation of LCN2 in ischemic neurons

Rats were subjected to 90min of transient focal cerebral ischemia, and then levels of LCN2 in brain tissue were measured with ELISA at 1, 3 and 7 days. LCN2 levels in the brains of normal sham-operated rats were 1.57±0.2ng/mg protein (n=4, measured at 3 days after sham surgery). After focal cerebral ischemia, LCN2 began to increase in the ischemic hemisphere on day 1, and by day 3, was significantly elevated by about 2-fold compared to the contralateral hemisphere (Figure 1A). By day 7, LCN2 slowly decreased back to contralateral levels.

Figure 1.

Figure 1

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LCN2 from neurons. (A) ELISA of rat brain homogenates after focal ischemia showed that LCN2 increased in ipsilateral compared to contralateral hemisphere (p<0.05 between hemispheres, two-way ANOVA; *, p=0.003 at day 3, t-test with Bonferroni correction), n=3–4 per timepoint. (B–C) At 3 days, LCN2 immunoreactivity (green) was stronger in peri-infarct cortex compared to contralateral hemisphere. (B) LCN2-positive cells (green) co-localized with NeuN-postive cells (red). (C) No LCN2 positive signals (green) were found in GFAP-positive cells (red) or Iba-1-positive cells (red). Scale bar = 50μm. (D) In primary neuron cultures, LCN2 increased after oxygen-glucose-deprivation. Representative western blot of LCN2 in conditioned media (neurons, astrocytes, microglia) and bar graph of LCN2 release in neuronal media. **, p<0.01 compared to the control (t-test). n=4.

To explore the cellular localization of LCN2, we performed multi-label immunostaining on rat brain sections at 3 days after ischemic onset. LCN2-positive cells co-localized with NeuN-positive neurons (Figure 1B), and most of the LCN2 signal appeared to be located in cytoplasm (Figure I in the online-only Data Supplement). The intensity of LCN2 immunoreactivity appeared to be higher in peri-infarct cortical areas. However, no LCN2 signals were detected in GFAP-positive astrocytes or Iba1-positive microglia in both contralateral and ipsilateral hemispheres (Figure 1C).

To assess human relevance, we examined LCN2 in post mortem brain sections of a human stroke patient obtained 77hrs after ischemic stroke onset. Cell-specific staining showed co-localization of LCN2 with NeuN-positive neurons in the ipsilateral cortex (Figure 2A). Compared to the contralateral cortex, the numbers of LCN2-positive cells appeared to be increased, and overall fluorescence intensity was stronger in peri-infarct regions (Figure 2A). Consistent with the ischemic animal models, no LCN2 signals were detected in GFAP-positive astrocytes or Iba1-positive microglia in contralateral and peri-infarct cortical regions (Figure 2B).

Figure 2.

Figure 2

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LCN2 expression in neurons from a stroke patient. (A) Compared to the contralateral side, LCN2-positive cells (red) increased in the peri-infarct region. LCN2-positive cells (red) co-localized with NeuN-postive cells (green). (B) No LCN2 signals (red) were found in GFAP-positive cells (green) or Iba-1-positive cells (green). Scale bar = 50μm.

Neuronal release of LCN2 after oxygen-glucose deprivation

To confirm our in vivo finding of LCN2 production by neurons, we measured LCN2 in conditioned media of primary cultured neurons, astrocytes, and microglia before and after oxygen-glucose deprivation. Western blots showed that neuronal release of LCN2 into conditioned media was increased by about 2.5-fold after oxygen-glucose deprivation for 30min and reoxygenation for 24hrs (Figure 1D). In contrast, neither astrocytes nor microglia released LCN2 under normal conditions or after oxygen-glucose deprivation (Figure 1D).

Activation of microglia by LCN2

Our data in rat focal cerebral ischemia and an initial proof-of-concept validation in a human stroke brain sample suggested that ischemia-endangered neurons upregulate LCN2 after stroke. So we next asked whether LCN2 can influence glial behavior as hypothesized. ELISA measurements showed that LCN2 levels ranged between 5 to 10ng/mg protein in ischemic brain tissue from our rat models (Figure 1A). Total protein concentrations in rat brain are known to be approximately 100mg/ml. Hence, LCN2 concentrations should be approximately be 0.5 to 1μg/ml. Of course, it is not possible to directly convert in vivo concentrations to cell cultures, but these levels were tested as a starting point for our cell culture experiments. When primary rat microglia cultures were treated with these concentrations of LCN2 for 24hrs, normally ramified “resting” microglia were converted into a long-rod morphology with reductions in peripheral branching (Figure 3A). The length of the longest axis of sixty cultured microglial cells from three independent experiments were quantified. After 1μg/ml of LCN2 treatment for 24hrs, the length of microglia significantly extended by about 60% compared to the non-treated group (Figure 3A right panel). In addition to these morphological changes, several markers of microglia activation were also examined. Real-time PCR showed that mRNA levels of iNOS, CD86 and CD206 were unchanged (Figure 3B), whereas mRNA level of arginase1 (Arg 1) was significantly increased by LCN2 (Figure 3B).

Figure 3.

Figure 3

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LCN2-activated microglia. (A) After 1μg/ml of LCN2 treatment for 24hrs, normal ramified “resting” microglia converted into a long-rod morphology with reductions in peripheral branching (left panel), and microglia length were extended (right panel). **, p<0.01 (t-test), scale bar = 50μm. (B) Microglia activation markers were measured with real-time PCR. 1μg/ml of LCN2 treatment for 2hrs rapidly upregulated mRNA levels of Arg1, but did not change iNOS, CD86, or CD206. *, p<0.05 compared to non-treated group (t-test). n=4. (C) LCN2 treatment for 24hrs increased IL-10 release, but did not alter IL-1β levels (ELISA). **, p<0.01 compared to control (one-way ANOVA). n=4. (D) LCN2 increased microglia phagocytosis but did not alter migration. *, p<0.05 compared to control (t-test). n=5.

Next, we asked whether LCN2 could change microglial production of key cytokines. LCN2 treatment for 24hrs significantly increased IL-10 release by microglia, but did not increase IL-1β levels (Figure 3C). Finally, we asked whether LCN2-activated microglia had altered cellular function, as assessed by phagocytosis and migration assays. LCN2-treated microglia showed enhanced phagocytic capacity (Figure 3D). But there were no detectable effects on microglia migration (Figure 3D).

Activation of astrocytes by LCN2

LCN2 treatment did not change the morphology and cellular viability of primary cultured astrocytes (Figure 4A and 4B), but other markers suggestive of more subtle activation were affected. After primary rat astrocyte cultures were treated with LCN2 for 24hrs, Western blots of cell lysates showed that GFAP was significantly increased (Figure 4C and 4D). LCN2 also increased the expression of TSP-1 and BDNF, but did not change the expression of other growth factors such as VEGF or IGF-1 (Figure 4C and 4D).

Figure 4.

Figure 4

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LCN2-activated astrocytes. (A) LCN2 treatment did not change the morphology of cultured astrocytes. (B) Treatment with 1μg/ml of LCN2 for 24hrs did not reduce astrocyte viability. (C) Representative western blots of GFAP, TSP-1, and growth factors (VEGF, IGF-1, and BDNF) in cell lysates. (D) Quantified densitometry of GFAP, TSP-1, VEGF, IGF-1 and BDNF. LCN2 treatment for 24hrs increased the expression of GFAP, TSP-1, and BDNF, but did not change other growth factors. *, p<0.05, **, p<0.01 compared to control (one-way ANOVA). n=4.

Neuronal effects of LCN2-activated glia

Here, we ask whether LCN2-activated glia may be beneficial towards neurons using conditioned media transfer experiments. Microglia or astrocytes were treated with vehicle or 1μg/mL of LCN2, then 24hrs later, conditioned media was collected and added to primary rat neurons, either under normal conditions or after oxygen-glucose deprivation.

Conditioned media from reactive glia or LCN2-treated glia was not directly toxic to normal neurons, and did not induce notable changes in their morphology (Figure II in the online-only Data Supplement). However these media seemed to be neuroprotective against oxygen-glucose deprivation. After 2hrs oxygen-glucose deprivation and 24hrs reoxygenation, neuronal viability significantly decreased by about 30% (Figure 5A and 5B). For microglia, normal conditioned media was not neuroprotective, but after activation with LCN2, microglia conditioned media significantly increased neuron survival after oxygen-glucose deprivation (Figure 5A). For astrocytes, both normal conditioned media as well as conditioned media from LCN2-activated astrocytes significantly increased neuron survival after oxygen-glucose deprivation (Figure 5B). LCN2 alone in control media was not toxic to normal neurons, and had no protective effect on neuronal death induced by oxygen-glucose deprivation (Figure III in the online-only Data Supplement).

Figure 5.

Figure 5

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Effects of LCN2-activated glia on neuronal oxygen-glucose deprivation. (A) Conditioned media from normal (MCM) or LCN2-activated microglia (L-MCM) was non-toxic to normal neurons. L-MCM increased neuronal survival after oxygen-glucose deprivation. #, p<0.05 compared to normoxia group; *, p<0.05 compared to the control (one-way ANOVA). n=4. (B) Conditioned media from normal (ACM) or LCN2-activated astrocytes (L-ACM) was non-toxic to normal neurons, but increased neuronal survival after oxygen-glucose-deprivation. #, p<0.05 compared to normoxia group; *, p<0.05 compared to control (one-way ANOVA). n=4.

Besides neuroprotection, promotion of neuroplasticity may also be a critical part of neurorecovery. Microglia or astrocyte cultures were treated with vehicle or 1μg/mL of LCN2, then 24hrs later, conditioned media was collected and added to primary rat neurons, and after another 48hrs, neuronal lysates were probed for presynaptic and postsynaptic proteins. Conditioned media from normal microglia did not change synaptic proteins. But conditioned media from LCN2-activated microglia significantly upregulated synaptophysin (Figure 6A) and PSD95 (Figure 6C). Both normal unactivated astrocytes and LCN2-activated astrocytes upregulated synaptophysin (Figure 6A) and PSD95 (Figure 6C), but only LCN2-activated astrocytes significantly upregulated synaptotagmin (Figure 6B).

Figure 6.

Figure 6

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Effects of LCN2-activated glia on synaptic proteins. (A) Conditioned media from normal astrocytes and LCN2-activated astrocytes and microglia upregulated synaptophysin. *, p<0.05, **, p<0.01 compared to the control (one-way ANOVA). (B) Conditioned media from LCN2-activated astrocytes upregulated synaptotagmin. **, p<0.01 compared to the control (one-way ANOVA). (C) Conditioned media from normal astrocytes and LCN2-activated astrocytes and microglia upregulated PSD95. *, p<0.05, **, p<0.01 compared to control (one-way ANOVA). n=4.

Discussion

We assessed the idea that LCN2 is released by injured neurons as a “help-me” signal for glial activation. In human stroke brain samples, rat focal cerebral ischemia and primary neuron cultures, injured neurons upregulated and released LCN2. Directly adding LCN2 to primary microglia and astrocytes activated them. LCN2-activated microglia showed enhanced phagocytosis and released IL-10, whereas LCN2-activated astrocytes released BDNF and TSP-1. Conditioned media from LCN2-activated microglia and astrocytes protected neurons against oxygen-glucose deprivation and upregulated synaptic proteins. Taken together, this combination of human stroke samples, rat models, and cell culture approaches suggest that LCN2 may be a “help-me” signal that is released by ischemically-endangered neurons to guide astrocytes and microglia into pro-recovery phenotypes (Figure IV in the online-only Data Supplement).

Reactive responses in glia play a central role in stroke recovery. Traditionally, reactive glia were thought to be deleterious. Reactive astrocytes may form inhibitory glial scars that block neural remodeling. 15 Activated microglia may release neurotoxic factors and free radicals that amplified secondary neuronal death. 16 Recently, however, this relatively simplistic idea has been mostly overturned. Reactive glia are now known to play more nuanced roles, and under some conditions, may be beneficial. 17,18 During acute stroke, astrocytes can support glutamate homeostasis and blood-brain barrier function. 19,20 During delayed phases post-stroke, astrocytes may release neurotrophic and angiogenic factors. 21,22 Astrocytic release of HMGB1 may promote neurovascular remodeling. 23 Production of TSP-1 or tissue plasminogen activator by reactive astrocytes may enhance synaptic and dendritic plasticity. 24–26 Our results are consistent with this concept. LCN2-activated astrocytes increased their production of BDNF and TSP-1, both of which are beneficial after stroke 22,25 and conditioned media from LCN2-activated astrocytes upregulated pre and post-synaptic proteins on recipient neurons.

Similarly, simplistic profiles cannot be applied to microglia. After stroke, prolonged microglia accumulation can last for weeks, thus potentially contributing to both injury and repair. 27,28 Selective ablation of proliferating resident microglia worsens neuronal apoptosis and infarction. 29 In contrast, microglial CX3CR1 deletion in mice promoted a protective inflammatory milieu and protected against ischemia at early time points. 30 More recently, it was found that microglial P2Y12 deficiency protected against global cerebral ischemia in mice. 31 These biphasic effects have led to analogies with peripheral macrophages, where damaging M1 phenotypes are contrasted against beneficial M2 phenotypes. 32 In the CNS, the situation may be even more complex, and microglia do not just comprise either classically activated M1 or alternatively activated M2, but instead, span the range from deleterious to regulatory to remodeling modes. 32,33 Our results support this idea. LCN2 activates microglia but their shape (long-rod morphology with reduced branching) is different from typical morphologies after lipopolysaccharide activation. 34 In LCN2-activated microglia, M1-like markers such as iNOS and CD86 are not altered, whereas for M2-like markers, CD206 was unchanged and Arg1 is elevated. In the end, this LCN2-induced phenotype may still be beneficial for stroke recovery. LCN2-activated microglia showed increased phagocytosis which may help with debris clean-up and remodeling 28, produced IL-10 which has been suggested to be pro-recovery 35, and their conditioned media protected neurons against oxygen-glucose deprivation and upregulated pre- and post-synaptic proteins which may help with neuroplasticity.

Taken together, our findings suggest that LCN2 is released by injured neurons as a “help me” signal to promote beneficial phenotypes in astrocytes and microglia. However, a few caveats should be kept in mind. First, it is likely that injured neurons produce many other factors that can act as distress signals. For example, in zebrafish brains, damaged neurons release leukotriene C4 that shifts inflammatory cells into a pro-neurogenic phenotype. 36 In mouse neurons, excitotoxic glutamate triggers the release of soluble fractalkine that promotes microglial phagocytosis of neuronal debris. 37 How LCN2 interacts with other “help-me” signals should be explored. Second, although neurons produce LCN2 in our models of cerebral ischemia, other cell types may produce LCN2 in other models and diseases. For example, reactive astrocytes can produce LCN2 after inflammatory stimulation 38, in spinal cord injury 39 and multiple sclerosis 40, or in response to neurodegeneration. 41 A third caveat may be related to the effects of LCN2 per se. In our models that attempt to mimic stroke, LCN2 may serve as a beneficial “help me” signal. However, under other disease conditions, LCN2 may have other effects. For example, under iron-mediated stress conditions or exposure to higher concentrations for prolonged periods of time, LCN2 may increase cell death in neurons and astrocytes. 38,41,42 In macrophages, LCN2 may downregulate proinflammatory cytokines and neutrophil infiltration after S. pneumoniae infections in mice. 43 Furthermore, neutrophils can also release LCN2 when they invade inflammed tissue, with both good and bad effects. 44 How LCN2 ultimately affects the balance between survival and death in multiple cell types warrants deeper analysis. A fourth limitation involves the question of signaling. LCN2 has two receptors, megalin and 24p3R. How these receptors operate should be carefully studied in the future. Finally, it must be acknowledged that the present study only provides proof of principle for this concept of “help-me” signaling. Rigorously understanding how LCN2 or other mediators contribute to the balance between injury and repair in vivo will require rigorous knock-out and knock-in experiments in the future.

In conclusion, we used a combination of human stroke samples, in vivo rat models and cell culture approaches to define a mechanism whereby damaged neurons release LCN2 as a “help-me” distress signal that guides microglia and astrocytes into potentially beneficial pro-recovery phenotypes. Further studies are warranted to dissect this pathway and explore new therapeutic opportunities for augmenting LCN2 or other help-me mediators in the remodeling neurovascular unit after stroke.

Supplementary Material

Supplemental Material

Acknowledgments

Funding: Supported in part by NIH and the Rappaport Foundation.

Footnotes

Disclosures: None.

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