Polysialic acid, a glycan with highly restricted expression, is found on human and murine leukocytes and modulates immune responses

Abstract

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.

Figure 1. NCAM/CD56+ human NK cells modulate polySia and NCAM expression with activation

Flow cytometric analyses of total PBMCs after 24 h in culture with (right) or without (left) the polySia-specific neuraminidase Endo-N (A), and of total PBMCs after 48 h culture in the presence (heavy line, no fill) or absence (light line, grey fill) of IL-2 (B).

Figure 2. Human NK cell activation results in an increased production of short to medium-length polySia chains

Purified primary NK cells from five individual donors were cultured separately for 48 h with or without IL-2, combined and analyzed for DP. Fractions of increasing DP were collected (x-axis), and analyzed for total sialic acid (as NANA) content (y-axis).

Figure 3. ST8Sia IV produces polySia on subsets of mouse bone marrow and peripheral myeloid cells

Flow cytometric analyses of total wild-type (WT) bone marrow comparing polySia and cKit expression (A). We studied four populations: PSA^neg/Kit^hi (1), PSA^lo/Kit^hi (2), PSA^hi/Kit^hi (3), and PSA^lo/Kit^lo (4). These subsets were present in ST8Sia II−/− mice, but absent from ST8Sia IV−/− animals. In the periphery (B), wild-type Gr-1+ myeloid cells expressed polySia, while the ST8Sia IV−/− cells were displayed fluorescence similar to that of the isotype control. In contrast to the human, mouse NK cells did not express polySia (C). DX5+ splenocytes were labeled with isotype control (light line, grey fill) or anti-polySia (heavy line, no fill) antibodies and analyzed by flow cytometry.

Figure 4. The PSA^neg/Kit^hi and PSA^lo/Kit^hi subsets contain erythroid and myeloid progenitors

PolySia (PSA)/cKit-defined subsets were sorted from wild-type bone marrow and plated in culture using conditions that promote erythroid (A) or myeloid (B) development. (A) Cells were maintained for 7 d in methyl-cellulose with erythropoietin, stem cell factor and IL-3, then analyzed microscopically for burst-forming units erythroid (BFU-E), and colony-forming units granulocyte-macrophage (CFU-GM). BFU-E and CFU-GM counts were combined to determine the total number of colonies formed. (B) Bone marrow cells were maintained in culture for 7 d, then scored microscopically for colonies (>50 cells) and clusters (10–49 cells). The results of two separate experiments are shown.

Figure 5. The PSA^neg/Kit^hi and PSA^lo/Kit^hi subsets contain lymphoid progenitors

PolySia (PSA)/cKit-defined subsets were sorted from wild-type bone marrow and placed in co-culture with OP9 stromal cells expressing either GFP and DL-1, or GFP alone. OP9-DL1 promotes the differentiation of lymphoid progenitors into T cells and NK cells, while OP9-GFP supports the development of B cells and NK cells. Differentiation was monitored by flow cytometry; results after 18 d in culture are shown. B cells were identified by B220 expression, and NK cells by cell surface DX5. T cell development was monitored by appearance of double negative stage 2 and 3 cells, identified by expression of CD44 and CD25. Representative data from one experiment are shown. The experiment was repeated three times with similar results.

Figure 6. Development of the polySia (PSA)/cKit-defined subsets is temporally linked

Wild-type mice were injected with a single dose of 5-FU to deplete cycling cells, and then sacrificed at various intervals over a 12-day period. The loss and restoration of the PSA/cKit-defined subsets was monitored by flow cytometry.

Figure 7. G-CSF increases the production of the PSA^lo/Kit^hi, PSA^hi/Kit^hi and PSA^lo/Kit^lo subsets

Wild-type mice were injected every 24 h with either vehicle alone or vehicle containing 2 µg of G-CSF. Animals were sacrificed after 5 d and polySia/cKit-defined bone marrow subsets were monitored by flow cytometry. The change in percent of total bone marrow was calculated for each population. The results of two separate experiments were combined and plotted in this graph. * p ≤ 0.007

Figure 8. NCAM on the surface of mouse bone marrow cells is the scaffold for polySia

Polysialylated proteins were immunoprecipitated from lysates of wild-type (WT) or ST8Sia IV−/− (IV−/−) bone marrow (BM) cells and Endo-N-treated (A). Recovered proteins were silver stained and two bands were visible in wild-type, but not ST8Sia IV−/− samples. The bands were isolated and both were identified as NCAM, on the basis of two peptides each, by mass spectrometry. Flow cytometry using anti-NCAM and anti-cKit on wild-type mouse bone marrow cells showed NCAM was expressed on populations that were analogous to those observed by staining with anti-polySia and anti-cKit (B). Immunoblotting with anti-polySia and anti-NCAM mAbs showed a polySia signal in WT and ST8Sia II−/− BM, but not ST8Sia IV−/− BM. In contrast, an NCAM signal was observed in association with all the samples (C). Adult WT brain lysate was used as a positive control for both polySia and NCAM signals, and Endo-N-treated (+EN) wild-type bone marrow was used as a negative control for polySia expression. Three major isoforms of NCAM were visible in brain lysates, while two (of ∼140 and ∼120 kDa) were present in bone marrow samples. Note that the polysialyated bands migrated with a lower mobility than those recognized by anti-NCAM, as only a fraction of this molecule carried these specialized glycans.

Figure 9. The contact hypersensitivity response is excessive in ST8Sia IV−/− mice

Wild-type and ST8Sia IV−/− mice were sensitized to DNFB. One week later the left ear was treated with DNFB and the right ear with vehicle alone as a negative control. We assessed the inflammatory response by measuring ear thickness with digital calipers at 24, 48 and 72 h (A). Each ear measurement was taken three times. The data represent the average ear thickness ± SD for the DNFB-treated ears of all mice (n = 3 WT, n = 3 ST8Sia IV−/−). At each timepoint, ST8Sia IV−/− mice had significantly more ear swelling than did wild-type animals (* p = 0.01; ** p ≤ 0.001). Histological analyses showed excessive tissue damage observed in ST8Sia IV−/− mice as compared to wild-type mice (B). Vehicle- (B i and ii) and DNFB-treated (B iii and iv) ears were removed, fixed, sectioned and stained with hematoxylin and eosin for histological analysis. Asterisks denote areas of edema, which appeared as empty spaces. Bar = 60 um.

Figure 10. RMA tumors grow faster in ST8Sia IV−/− mice than in wild-type animals

Wild-type, ST8Sia IV−/−, and TCRβ−/− mice (negative controls for immunocompetence) were injected subcutaneously with 10⁴−10⁵ RMA cells. When tumors became ulcerated, or grew larger than 1.5 cm, or mice lost >20% of their original body weight, animals were sacrificed. Animals surviving more than 60 d were considered tumor-free; all deaths are plotted on the graph. There was a statistically significant difference in survival rates between wild-type and ST8Sia IV−/− animals (p = 0.02, two-tailed Mann-Whitney test).