Soluble polysialylated NCAM: a novel player of the innate immune system in the lung

Abstract

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1β. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1β or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing "neutrophil extracellular traps", which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.

Fig. 1

Model of polySia-NCAM-140. Complex-type N-glycan chains were computationally added to N-glycosylation sites N222, N315, N347, N423, N449, and N478 of a homology model of NCAM-140. The glycan chain at position N449 (cyan) contains two polySia chains, one consisting of 38 and one of 20 sialic acid residues. Three polySia chains are present in the N-glycan at position N478 (yellow), consisting of 38, 30, and 20 sialic acid residues. The figure was created with Yasara [59]

Fig. 2

Detection of polySia-NCAM-140 in protein lysates of healthy human lung donors. a Lung homogenates of human tissue were separated by SDS-PAGE for Western blotting using 10 μg protein per lane with or without endoN pretreatment. Immunostaining was performed with an anti-polySia mAb. Apparent molecular weights of standard proteins are indicated in kDa. b Polysialylated NCAM was isolated using inactive endoN coupled to magnetic beads and visualized by Western blotting using a mAb against NCAM (mAb 123C3). De-polysialylated NCAM was additionally examined after endoN treatment. c PolySia chains were released from purified polySia-NCAM and directly labeled with DMB in situ. Resulting fluorescently tagged sialic acid polymers were separated on an anion exchange column according to polySia chain length. Respective chain length of polySia residues is given for selected peaks on top of the profiles

Fig. 3

Immunohistological localization of polySia-NCAM-140 in lung tissue of healthy human donors. Paraffin embedded serial human lung sections were stained with a mAb against NCAM (mAb 123C3) and polySia (arrows). For negative control of polySia-staining lung sections were exposed to endoN. Scale bars 50 μm

Fig. 4

Expression and distribution of polySia-NCAM in lungs of COPD patients. a PolySia was analyzed in lung lysates of healthy donors and COPD patients by Western blotting. As specificity control, lysates were exposed to endoN prior to analysis. As loading control actin was used. Apparent molecular masses of standard proteins are indicated in kDa. Protein bands representing polySia were quantified by densitometry, and values are means of eight healthy donors and ten COPD patients each (100 % was set for healthy donors). b mRNA expression levels of NCAM, ST8SiaII and ST8SiaIV in lungs of donor and COPD patients were determined by quantitative real-time PCR; GAPDH was used as standard housekeeping gene. Values represent means of three donors and 11 COPD patients, respectively. The statistical evaluation was performed by Student’s t test (unequal variances, two-tailed). Significance levels are indicated by n.s. (not significant), p > 5 %, *p < 0.05, **p < 0.01, ***p < 0.001. c, d The distribution of polySia was visualized with anti-polySia mAb on paraffin embedded lung section from healthy donors and COPD patients; scale bar equals c 50 μm and d 25 μm. e Polysialylated NCAM were purified from lung lysates of donor and COPD patients and visualized by Western blotting using a mAb against polySia and an anti-NCAM (mAb 123C3) before and after polySia degradation by endoN

Fig. 5

Activation of the inflammasome leads to increased levels of polySia-NCAM-110 in murine lung tissue. Western-blot analyses of lysated lung tissue of bleomycin-treated and control mice were performed using a mAb against a polySia and b NCAM (mAb H28) before and after degradation of polySia by endoN. To this end, polysialylated NCAM was isolated using inactive endoN coupled to magnetic beads. a Protein bands representing polySia were quantified by densitometry, and values are means of three NaCl-treated and three bleomycin-treated mice (100 % was set for three untreated mice). The statistical evaluation was performed by Student’s t test (unequal variances, two-tailed). Significance levels are indicated by n.s. (not significant), p > 5 %, *p < 0.05, **p < 0.01, ***p < 0.001. Serial murine lung sections of c untreated mice and d mice treated with bleomycin were stained with a mAb against polySia. Arrows indicate immunostaining. Scale bars 10 μm

Fig. 6

The secretion of intracellularly localized polySia-NCAM of human lung epithelial cell line A549 is induced by IL-1β and LPS. a Western-blot analyses of cell lysates of human A549 cells were performed using a mAb against polySia and NCAM (123C3) before and after endoN treatment. b, c Cultured A549 cells were fixed with methanol and stained with fluorescently labeled inactive endoN and b anti-NCAM mAb 123C3 or c polyclonal antibodies to visualize the trans-Golgi apparatus (anti-TGN38). For nuclear staining DAPI was used. d In addition, polySia was visualized in cells after stimulation with IL-1β (200 ng/ml) or LPS (100 ng/ml). Scale bars for c and d = 25 μm. e, f Polysialylated proteins were purified from supernatants of differently treated A549 cells and visualized by Western blotting using an anti-polySia mAb and after depolysialylation with a mAb against NCAM (123C3). g In an analogous cell lysates of IL-1β-treated cells were analyzed. h Western blots against polySia and NCAM (after depolysialylation) were performed using supernatants of untreated A549 cells as well LPS-stimulated cells in the absence or presence of TAPI-2. Apparent molecular masses of standard proteins are indicated in kDa

Fig. 7

Soluble polySia-NCAM decreases histone- and NET-mediated cytotoxicity and stimulation of polysialylation increases the protective capability of soluble polySia-NCAM. a, b A549 cells were exposed to histones (10 μg/ml) and the cytotoxicity was determined. In parallel, A549 cells were stimulated by a IL-1β (200 ng/ml) and b LPS (2 μg/ml). To calculate the impact of polySia, endoN was added to the cell culture medium. c Moreover, cells were exposed to NET with or without LPS (2 μg/ml) stimulation and/or additional supplemental degradation of polySia by endoN. d A549 cells were exposed to increasing amount of histones and the cytotoxicity was determined (diamonds). In parallel, A549 cells were stimulated by IL-1β (200 ng/ml) (squares). To examine a possible effect of Neu5Ac precursors on the protective capability of soluble polySia-NCAM, A549 cells were fed with ManNAc prior to IL-1β stimulation (circles). The impact of polySia after ManNAc supplemental was evaluated by endoN treatment during IL-1β stimulation (triangles). e The influence of ManNAc treatment on polySia biosynthesis was analyzed by Western blotting using antibodies against polySia. In addition, depolysialylated NCAM was visualized with mAb 123C3. All values ± SD in Fig. 7 are means of three independent experiments. The statistical evaluation was performed by Student’s t test (unequal variances, two-tailed). Significance levels are indicated by ns (not significant), p > 5 %, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Proposed model for the secretion of polySia-NCAM-110 and its subsequent cytoprotective function. Stimulation of lung epithelial cells by IL-1β or LPS induced ectodomain shedding of polySia-NCAM-140 by metalloproteinases resulting in the release of polySia-NCAM-110. PolySia chains of soluble NCAM-110 bind nucleosomes, thereby inhibiting their cytotoxic activity