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J Immunol. Author manuscript; available in PMC Aug 5, 2013.
Published in final edited form as:
PMCID: PMC3733067
EMSID: EMS30427

Sialoadhesin-Positive Macrophages Bind Regulatory T Cells, Negatively Controlling their Expansion and Autoimmune Disease Progression1

Abstract

An important regulatory suppressive function in autoimmune and other inflammatory processes has been ascribed to CD4+Foxp3+ regulatory T cells (Tregs), which requires direct cell-cell communication between Tregs, effector T cells (Teff) and antigen presenting cells. However, the molecular basis for these interactions has not yet been clarified. We show here that sialoadhesin (Sn), the prototype of the siglec family of sialic acid binding transmembrane proteins, expressed by resident and activated tissue-infiltrating macrophages, directly binds to Tregs, negatively regulating their expansion in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In this model, macrophages infiltrate the central nervous system (CNS) exhibiting tissue destructing and demyelinating activity, leading to MS-like symptoms. We show here that severity of EAE symptoms is reduced in Sn knock-out (KO) mice compared to WT littermates, due to an upregulation of CD4+Foxp3+ Treg lymphocytes. Through the use of a Sn fusion protein, Tregs were shown to express substantial amounts of Sn ligand on their cell surface and direct interaction of Sn+ macrophages with Tregs specifically inhibited Treg but not Teff lymphocyte proliferation. Conversely, blocking of Sn on macrophages by Sn-specific antibodies resulted in elevated proliferation of Treg cells. Data indicate that Sn+ macrophages regulate Treg homeostasis which subsequently influences EAE progression. We propose a new direct cell-cell interaction based mechanism regulating the expansion of the Treg cells during the immune response, representing a “dialogue” between Sn+ macrophages and Sn accessible sialic acid residues on Treg lymphocytes.

Introduction

Murine experimental autoimmune encephalomyelitis (EAE), a widely used T cell-mediated animal model for multiple sclerosis, is characterized by self-reactivity directed against several myelin-derived antigens, including myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). During EAE, auto-reactive myelin specific CD4+ T cells penetrate into the central nervous system (CNS) and initiate destruction and demyelination processes executed by activated macrophages (1, 2).

An important role in the regulation of autoimmune and other inflammatory processes has been ascribed to CD4+CD25+ regulatory T cells (Tregs). Abnormalities in the generation and function of these cells and resulting immune dysregulation are considered the primary cause of autoimmune diseases and other immunological disorders (3). Tregs are mainly characterized by expression of the transcription factor, Foxp3, and the IL-2 receptor, CD25, and by a distinct cytokine profile which includes interleukin-10 (IL-10) and TGF-β. Their generation and function, which is the suppression of the activation and proliferation of the CD4+CD25− effector T cells (Teff) during immune responses, is based on their surface markers like CD127, CD62L, CD103, CD122, CTLA-4 and GITR (3). Antibody blocking studies indicate that CTLA-4 (4, 5), GITR (6-8) and IL-2 (9, 10) are required for the generation of Tregs and are thereby involved in the maintenance of the balance between Teff and Treg cells. The suppressive activity of Tregs depends on direct cell-cell communication between Tregs, Teff cells and antigen presenting cells, although the molecular basis for this suppression has not yet been clarified (11). An involvement of carbohydrate structures in Treg functions is evident from recent implications of galactose binding molecules of the galectin family in these activities (12, 13).

Sn is a macrophage restricted prototypic member of the Siglec family of sialic acid binding molecules (14), found on a subpopulation of macrophages within the subcapsular sinus and medulla of lymph nodes, and on metallophilic macrophages in spleen (15, 16). During inflammatory disorders such as rheumatoid arthritis and experimental autoimmune uveoretinitis (EAU), where macrophages are considered to play decisive roles, Sn is expressed by activated macrophages within the inflamed organs (16, 17). In vitro studies have demonstrated that Sn binds several membrane proteins, mainly expressed on leukocytes, via both sialic acid-dependent (CD43, PGSL, MUC1) and independent (MGL1) mechanisms (18-20). However, the biologically relevant interactions of Sn and its ligands are still not clear, and counter-receptors for Sn within macrophage-infiltrated tissues remain to be identified (18). The structural features of Sn and its high conservation on activated macrophages are suggestive of a role in mediating cell-cell interactions.

We show here that Sn+ macrophages represent a significant proportion of leukocytes infiltrating into the CNS upon EAE induction by immunization with MOG35-55. Sn knockout (KO) mice show significantly reduced EAE severity and incidence compared to wild type (WT) littermates, which is associated with a significant increase in Treg numbers and reduction in Teff numbers within the CNS, but no differences in total macrophages. Through the use of a Sn-Fc fusion protein, a subpopulation of Treg cells expressing Sn ligands was identified in the inflamed CNS, which was elevated in Sn KO mice. In vitro experiments confirmed that the Sn KO macrophages induce higher rates of proliferation of Treg cells than WT macrophages. Thus, Sn-positive macrophages negatively regulate the expansion of Treg cells through Sn-dependent direct cell-cell communication during the immune response. Upon loss of Sn positive macrophages, the proliferation of Treg cells is up-regulated, specifically in the CNS, resulting in a milder disease course.

Materials and Methods

Animals

Sn KO mice (Sn−/−) and WT littermates (Sn+/+) (backcrossed to C57BL/6 at least 10 generations) were employed in active EAE experiments (21). C57BL/6-Ly5.1 (CD45.1) mice were used in passive transfer experiments. Experiments were conducted according to German Animal Welfare guidelines.

Antibodies

Antibodies used in immunofluorescence, FACS and cell sorting included: pan-laminin (455)(22, 23), CD45 (30G.12; BD pharmingen), CD45.2 (104, eBiosciences) and CD45.1 (A20, BD Pharmingen), CD11c (N418, eBiosciences), CD11b/ MAC-1 (M1/70, BD Pharmingen), CD4 (H129.19, BD Pharmingen) and CD8 (53-6.7, eBiosciences), CD25 (7D4, BD pharmingen), CD169 (3D6.112, SeroTec), Foxp3 (ABCAM), F4/80 (BM8, eBioscience), IL-17 (TC11-18H10.1, BD Pharmingen), IFN-γ (XMG1.2, BD Pharmingen), IL-6 (MP5-20F3, eBioscience), IL-10 (JES5-16E3, eBioscience), TNF-α (MP6-XT22, BD Pharmingen), CD16/CD32 (2.4G2, BD Pharmingen), MHCII (M5/114.15.2, eBioscience), CD86 (2331, BD Pharmingen), CD80 (16-10A1,BD Pharmingen), CD40 (3/23, BD Pharmingen), CD69 (H1.2F3, BD Pharmingen). For function blocking experiments, two Sn antibodies were employed, SER-4 and 1C2 (24).

Active EAE

EAE was actively induced in 6-8 week old female C57BL/6 mice by immunization with the 35-55 peptide of myelin/oligodendrocyte glycoprotein (MOG35-55) as described previously (25). The mice were observed daily and scored on a scale of 0-5 for neurological defects: 1 (flaccid tail), 2 (hind limb weakness), 3 (severe hind limb weakness), 4 (hind quarter paralysis) and 5 (forelimb weakness). CNS samples were collected at different EAE stages for FACS analysis or frozen in Tissue-Tek (Sakura Finetek) for immunofluorescence analysis.

Passive EAE

Cells were isolated at day 10 after immunization with MOG35-55 peptide from draining lymph nodes (LN), and stimulated with 20 μg/ml MOG35-55 peptide for 2 days, and then in the presence of 20 ng/ml IL-2 and 10ng/ml IL-1β for further 2 days. On day 4, 10 ng/ml IL-23 was added and the cells cultured for a further 5 days before transfer to recipient mice. 20 ×106 cells/mouse were transferred i.v. and mice injected i.p. with 20 ng Pertussis toxin/mouse on the day of transfer and at day 2 after transfer.

Immunofluorescence

Sn KO or WT mouse brain cryostat sections (5μm) were analysed by immunofluorescence as described previously (23). Primary antibodies employed included anti-Foxp3, anti-Sn (MOMA-1), anti-pan-laminin (405) and anti-CD45, and were visualized using Alexa 488- or Cy3-conjugated goat anti-rat or Cy3- or Cy5-conjugated anti-rabbit secondary antibodies (Jackson Laboratories & Molecular Probes). Negative controls involved incubation of sections with secondary antibodies alone. Sections were examined using a Zeiss AxioImager microscope equipped with epifluorescent optics and documented using a Hamamatsu ORCA ER camera.

FACS

Mice were perfused with PBS before spleens, LNs, thymus and brains were harvested. Spleens and brains were pretreated with 2 mg/ml collagenase D (Roche Diagnostics) and 1 mg/ml DNAse I (Roche Diagnostics), and total cells isolated using cell strainers (BD Bioscience); 70μm for the spleens and 100μm for the brains. Brain homogenates were separated into neuronal and leukocyte populations by discontinuous density gradient centrifugation using isotonic Percoll (Amersham) (26). For intercellular staining, isolated leukocytes were stimulated with PMA (10 ng/ml)/ ionomycin (1 μg/ml) (Sigma) in the presence of 10 μg/ml BrefA (Sigma) 37°C for 5h. An intracellular staining kit (eBioscience) was employed to permeabilize and fixed the cells. Primary antibodies employed are listed above and stained cells were analysed using a FACS Calibur (Becton Dickinson). In all cases, isotypes control antibodies were employed to determine background staining.

Cytokine measurements

Cytokine activity in culture supernatants of Treg cells, Teff cells and macrophages from immunized Sn KO and WT mice was tested using a cytometric bead array kit (Bender MedSystems). Intracellular staining and FACS analyses were performed as described above.

T cell proliferation assays

To test Treg function, T cells were isolated from draining LNs, and CD4+CD25− and CD4+CD25+ cells were sorted by MACS (Miltenyi) and placed in RPMI 1640 plus 5% FCS and β-mercaptoethanol. These Teff and Treg cells were mixed in different ratios and 2 ×104 of the mixed T cells were cultured at 37°C for 3d together with irradiated splenic DCs (from a non-immunized mouse) in the presence of different concentrations of MOG35–55. Cell proliferation was determined by 3H-thymidine incorporation.

To test antigen presentation function of WT versus Sn KO macrophages, 2 ×104 CD4+ T lymphocytes from LNs of immunized mice were cultured at 37°C for 3d together with 1 ×104 macrophages (from the CNS of immunized mice) and different concentrations of MOG35–55. Cell proliferation was determined by 3H-thymidine incorporation.

To measure proliferation of Teff versus Treg lymphocytes, 2 ×106 Teff or Treg lymphocytes isolated from immunized mice (by MACS sorting as described above) were incubated with 1μM carboxy-fluorescein diacetate succinimidyl ester (CFSE) and cocultured with 106 macrophages (from EAE) mice plus 20μg/ml MOG35–55 for 4 days before FACS analysis.

Sialoadhesin binding assay

Sn-Fc protein (15μg/ml) (24) was preincubated on ice for 1h with 10 ng/ml Cy5-conjugated anti-human-IgG Fc fragment antibody. Cells were then stained using the Sn-Fc protein-Cy5-anti-human-IgG complex or the Cy5-conjugated anti-human-IgG Fc antibody alone, in combination with other cell marker antibodies and analyzed by FACS. Sialidase treatment involved adding 50μl (60 mU) neuraminidase from Vibrio cholerae (Calbiochem) in 1ml DMEM to 5 ×106 cells, and subsequent 2h incubation at 37°C followed by extensive washing with PBS.

Bromodeoxyuridine (BrdU) incorporation

5 × 106 donor Ly5.1 naive CD4+CD25+ T cells were transferred i.v. to Ly5.2 WT and KO recipient mice, and 3 days after transfer the recipients were immunized with MOG35-55. On days 7, 9 and 11 after immunization the mice were injected i.p. with 1.5mg BrdU. Brains and spinal cords were removed 12h after the last injection. Lymphocytes were isolated as described above, and analyzed by FACS using the BrdU kit (BD Biosciences) and cell markers listed above.

Statistical analysis

Quantitative data is expressed as means ± s.e.m. Statistical significance of EAE results was analysed with a unpaired Student’s T test and by Mann Whitney test, whereas proliferation rates, cytokine production, and cell numbers were assessed by Student’s t-test. P values of less than 0.05 were considered significant.

Results

Reduced EAE Severity and Incidence in Sn KO mice

Active EAE induction in Sn KO mice and WT littermates revealed reduced disease severity throughout the disease course in KO mice but no significant alteration in the day of onset of disease symptoms (Fig. 1A). While 92% of WT mice developed disease symptoms, first appearing on average between days 10-12 after immunization, approximately 80% of the KO mice developed symptoms throughout the experimental period (Fig. 1A), indicating reduced disease susceptibility.

FIGURE 1
Reduced EAE susceptibility and disease severity in Sn KO mice. (A) Active EAE induction in Sn KO mice and WT littermates showing reduced mean clinical score, disease incidence and maximal disease severity in KO mice. Data shown are means ± s.e.m. ...

Despite reduced disease scores in KO animals, extensive leukocyte infiltration of the CNS parenchyma was apparent by immunofluorescence staining at peak disease severity (day 17) in both KO and WT brain sections (Fig. 1B). However, quantitative microscopic examination of CNS sections revealed slightly fewer inflammatory cuffs/ cm2 in Sn KO mice compared with WT mice (Fig.1B). FACS analysis of perfused CNS (brain and spinal cord) at peak disease severity confirmed the absence of the Sn+ CD11b+ macrophage population in KO mice, which represented 2% of total leukocytes in WT CNS (Fig. 1C) and 20% of total macrophages, but no significant difference in total CD11b+ cells, or in CD8+ T cells (Fig. 2A) and CD11c DCs (not shown) in the CNS of WT and KO mice. However, there was a moderate reduction in CD4+ T cells in KO CNS (Fig. 2A). No differences were observed in total CD45+ cells, CD4+ T cells, CD8+ T cells, CD11b+ (Fig. 2A) and CD11c+ DC cells (not shown) in draining lymph nodes, or in the circulation (not shown) of KO mice versus WT littermates.

FIGURE 2
Analysis of CD4+ T lymphocytes in Sn KO and WT mice. A) FACS of Sn KO mice and WT littermates at peak disease severity (day 17) for total infiltrating CD45+CD4+ or CD8+ T lymphocytes, or CD45+CD11b+ macrophages recovered from perfused brain (CNS) and ...

To define whether the reduced numbers of CD4+ T cells in Sn KO brains were due to defects in T cell migration into the CNS, passive transfer experiments were performed using CD45.2+ WT or KO MOG35-55-specific T cell blasts transferred to CD45.1+ WT recipients, or CD45.1+ WT MOG35-55-specific T cell blasts transferred to CD45.2+ WT or KO recipients, and analysis of brains at day 3 after transfer, a time point when T cell proliferation in the brain has not yet occurred (27). FACS revealed no differences in the number of donor WT or KO T cell blasts in the brains of WT or KO recipient mice (Fig. 2B), indicating the absence of significant T cell migration defects in KO mice.

Upregulation of Treg cells in Sn KO CNS during EAE

Since Sn KO mice showed reduced EAE severity and incidence without major deficiencies in total leukocyte infiltration, and as published data suggests that the balance between Teff and Tregs is important in development of EAE symptoms (28, 29), analyses were extended to T cell subpopulations: Th1 and Th17 cells were analysed in the CNS of KO and WT mice at peak disease severity (day 17), revealing no differences in the proportion of Th17 cells but a reduced proportion of Th1 cells in Sn KO mice. Absolute numbers of both Th1 and Th17 cells, however, were lower in the CNS of KO mice compared to WT littermates (Fig. 2C). Tregs as defined by CD4+CD25+Foxp3+ were also elevated in various organs of Sn KO mice as compared to WT littermates, in particular the CNS (Fig. 2D). FACS revealed that naïve WT and KO mice showed identical numbers of Treg cells in multiple organs, indicating that there is no defect in Treg development (Supplementary Fig. 1). The elevated Treg and reduced Th1 and Th17 cell numbers in the CNS of KO mice suggests increased T cell suppressor function, implicating Sn in the regulation of the balance between Treg and Teff cells during the immune response. Other regulatory immune cell populations were also examined during EAE, including NKT cells, γ/δ T cells and B cells, revealing no significant differences between Sn KO mice and WT littermates (not show)

Sn ligands are detectable on the Treg in multiple organs during EAE

To determine how Sn+ macrophages upregulate Treg numbers, expression of Sn ligands was investigated on activated immune cells during EAE by flow cytometry using a fusion protein that combines a human IgG-Fc fragment with the first 3 Ig-domains of Sn (Sn-Fc). This chimeric protein specifically detects sialic acid-containing structures recognized by the Sn molecule and, therefore, recognizes potential Sn counterreceptors (24). In combination with specific cell markers, this Sn-Fc therefore permits identification of the cell population/s that potentially interact with Sn expressed naturally by macrophages. Furthermore, it permitted quantification of Sn ligands on different cells during the immune response.

In accordance with previously published observations (15, 16), CD45+CD11b+ cells were identified as the major population of Sn ligand expressing cells in non-immunized animals (Supplementary Fig. 2). Especially in the bone marrow, more than 40% of the CD45+CD11b+ cells expressed high levels of Sn accessible sialic acid residues, which were removed by sialidase treatment (not shown). These Sn ligand expressing cells were both Sn+ and Sn−. In the spleen and lymph nodes, approximately 35% and 10% of the CD45+CD11b+ cells were Sn ligand positive, respectively, while CD4+ and CD8+ T cells and B220+ B cells showed little or no expression (Supplementary Fig. 3A).

During EAE, Sn ligand levels on CD45+CD11b+ cells were reduced (Supplementary Fig. 2), and an additional CD4+Foxp3+ Treg population was detected that expressed Sn ligand and was particularly enriched in the CNS of both WT and KO mice (Fig.3A). By contrast, naive Tregs from spleen and LN show low levels of Sn-ligand (Supplementary Fig. 3B), while CD4+Foxp3− Teff cells do not express the Sn ligand (Fig. 3A).

FIGURE 3
Expression of Sn ligands on immune cell populations. A) FACS analysis of Sn-Fc protein binding to CD4+Foxp3+ Tregs and CD4+Fox3p− Teff cells isolated from CNS, spleen and LN of WT mice at peak EAE symptoms (day 17), before and after sialidase ...

In Sn KO mice, which showed elevated numbers of CD4+Foxp3+ Tregs, the proportion of Sn ligand+ Tregs was also significantly higher (32.6%) than in WT littermates (19.3%) (Fig. 3B). CD4+Foxp3− Teff cells, in contrast, did not show any up-regulation of Sn ligand in KO mice (Fig. 3B). The elevated numbers of Sn ligand+ Tregs in KO mice during EAE suggests that direct interaction of Tregs with Sn+ macrophages down regulates Treg numbers, in particular in the CNS.

Activated Sn+ macrophages inhibit Treg expansion in vitro

In view of the elevated Treg numbers and increased levels of Sn ligand on Treg surfaces in Sn KO mice, in vitro proliferation assays were performed to exclude the possibility that high levels of Sn ligand on Tregs alters their suppressor effects on Teff cell proliferation: CD4+CD25− T lymphocytes isolated from MOG35-55 immunized WT mice were cultivated together with WT splenic DC as antigen presenting cells (APC) plus MOG35-55, in the presence of increasing ratios of CD4+CD25+ Tregs isolated from immunized WT or KO mice. Antigen-specific CD4+CD25− Teff lymphocyte proliferation was inhibited by increasing ratios of Treg cells regardless of whether they were isolated from WT or KO mice (Fig. 4A). Levels of IL-10 as an indicator of Treg activity (30) was also comparable in KO and WT Tregs (Fig. 4B), indicating that Treg function per se is not altered by the higher than usual Sn ligand levels on KO cells.

FIGURE 4
Normal Treg function in Sn KO mice. (A) CD4+CD25− Teff cells isolated from MOG35-55 immunized WT mice were cultivated together with WT irradiated splenic DC as APC plus MOG35-55, in the presence of increasing ratios of Tregs isolated from immunized ...

As macrophages are potential APC that can regulate T cell proliferation (31, 32), the T cell proliferative response induced by Sn+ versus Sn− macrophages was investigated. Since Sn could not be used to isolate macrophages from the KO mice, the CD11b and F4/80 double positive macrophage population was isolated from immunized KO and WT mice, which in the WT mice contained the Sn+ population (Fig. 5A). This macrophage population showed identical levels of surface markers, including MHC-II, B7-1, B7-2 and CD40 (33), in KO and WT animals (not shown) but significant differences in cytokine profiles, with reduced intracellular levels of TNFα, IFNγ, IL-6 and increased IL-10 in KO cells (Fig. 5B). Similarly, supernatants from cultured Sn KO macrophages also showed reduced levels of TNFα, IFNγ, IL-6, IL-1α and increased IL-10 in comparison to WT macrophages (Fig. 5C). This altered cytokine profile in the Sn KO mice provides evidence for altered macrophage function.

FIGURE 5
Sn KO macrophages stimulate Treg proliferation. (A) Expression of Sn on CD11b+F4/80+ sorted from the CNS of WT immunized mice; open histogram is Sn staining on the CD11b+F4/80+ cells, gray histogram is the isotype control. (B) Intracellular cytokine profile ...

In vitro proliferation of MOG35-55-specific CD4+ T cells using the KO or WT macrophages as APC, revealed reduced T cell proliferation in the presence of Sn KO as compared to WT macrophages, suggesting that Sn KO macrophages are compromised in their ability to induce an antigen specific T cell proliferative response (Fig. 5D). In view of the elevated Treg numbers in KO mice, this inhibitory effect was further investigated using isolated WT Teff or Treg cells: Due to the low numbers of Tregs that can be isolated even from inflamed brains and the limited sensitivity of the 3H-thymidine incorporation method, it was necessary to use enriched Teff and Treg lymphocyte samples isolated from immunized mice, which were CFSE-labeled, cultured together with antigen-loaded CD11b+F4/80+ WT or KO macrophages, and subsequently analysed by FACS to determine the proportion of proliferating cells. Teff cells exhibited comparable proliferation in the presence of either WT or KO macrophages. By contrast, Treg lymphocyte expansion was upregulated by coculture with KO macrophages (Fig. 5E), indicating that Sn KO macrophages specifically induce proliferation of Treg cells but have no impact on the CD4+Foxp3− Teff cells. Hence, reduced proliferation of the CD4+ T cell population in the presence of the Sn KO macrophages is likely to be due to an expansion of Treg lymphocytes, which in turn reduced proliferation of Teff cells.

Blocking Macrophage Sn Upregulates Tregs

To determine whether regulation of Treg proliferation requires direct interaction between the Sn molecule on the macrophage cell surface and Tregs, WT macrophage Sn was blocked by incubation with the Sn-specific antibodies, SER-4 and 1C2, and proliferation of antigen specific CD4+ T lymphocytes was measured in the presence of increasing proportions of anti-Sn-treated or untreated macrophages. Anti-Sn-treated WT macrophages induced the same inhibition of CD4+ T lymphocyte proliferation as Sn KO macrophages (Fig. 6A). Similar experiments performed with CFSE-labeled isolated Teff or Tregs revealed enhanced proliferation of Treg by anti-Sn-treated WT macrophages and no effect on Teff cell proliferation (Fig. 6B).

FIGURE 6
Direct interaction between Sn+ macrophages and Tregs. (A) Proliferation of total MOG35-55 specific CD4+ T lymphocytes cocultured in the presence of increasing numbers of WT or Sn KO CD11b+F4/80+ macrophages isolated from CNS of the immunized mice, in ...

Sn+ Macrophages Interact with Tregs in the CNS

The above data suggest a direct interaction between Sn+ macrophages and Treg cells. To investigate whether Treg lymphocytes and Sn+ macrophages colocalize in vivo, spleens and inflamed WT brains were immunofluorescently stained for Sn+ macrophages, Foxp3+ Tregs, and pan-laminin to define the perivascular space and the marginal sinus. Sn+ macrophages occurred in close contact to Foxp3+ Treg in the splenic marginal sinus (Supplementary Fig. 4) and, within the brain, both in the perivascular cuff and in the CNS parenchyma, with the majority of the interactions occurring in the perivascular space (Fig. 7A).

FIGURE 7
In vivo localization of Tregs and Sn+ macrophages and proliferation of Tregs in Sn KO CNS. (A) Triple immunofluorescence staining of an inflamed WT brain for Foxp3, Sn (as defined by the MOMA-1 antibody) and pan-laminin, to define the perivascular space, ...

To confirm that the association between Sn KO macrophage and Tregs in the CNS has the same inhibitory effect on Treg expansion as observed in vitro, in vivo expansion of Treg was measured in the CNS of WT and Sn KO mice during EAE: WT CD45.1+CD4+ encephalitogenic T cells were passively transferred to MOG35-55 immunized CD45.2+ Sn KO mice and WT littermates, and their proliferation in the CNS was measured by BrdU incorporation. At day 12 after transfer CD45.1+CD4+ donor T cells were recovered from the CNS and analyzed by flow cytometry for the expression of Foxp3 and the incorporation of BrdU. Since passively transferred autoreactive T cells directly migrate to the CNS without extra activation and proliferation in the periphery, and the majority of T cell proliferation observed in the periphery is dominated by host cells, specific measurement of BrdU incorporation into donor CD4+CD25+Foxp3+ Tregs in the CNS reflects mainly cells proliferating within that organ. Approximately 13% of the donor CD4+CD25+Foxp3+ Treg cells recovered from the CNS of KO mice represented proliferating cells, while only 6% of donor CD4+CD25+Foxp3+ Treg, had proliferated in WT recipient mice (Fig. 7B), supporting an in vivo immunosuppressive role for Sn KO macrophages.

Discussion

Sn positive macrophages have been previously shown to represent a population of macrophages that stem from the circulation and are present in the CNS early after the induction of EAE (34, 35). Injection of chlodronate-loaded liposomes and subsequent transfer of encephalitogenic T lymphocytes results in resistance to EAE, even in the presence of infiltrating encephalitogenic T lymphocytes and a Th1 proinflammatory cytokine millieu, and correlates with reduction in F4/80+ and Mac-1+ macrophages and complete absence of Sn+ macrophages. The data suggest a significant role for macrophages within the CNS at early stages of EAE that is independent of later demyelinating events. Data presented here indicate that one of these functions is regulation of Treg numbers and thereby Teff lymphocyte function by a subpopulation of Sn+ macrophages.

The reduced EAE incidence and severity observed in Sn KO mice, and associated upregulation of Treg and reduction in Th1 and Th17 cells, suggests that Sn+ macrophages regulate the balance between Treg and Teff lymphocytes in EAE. Data shown here clearly indicate that Sn+ macrophages represent a functionally distinct macrophage population that directly interacts with Sn ligand on Tregs to inhibit Treg proliferation but not their function. Absence of defects in Treg function in the Sn KO mice was demonstrated by the normal cytokine profiles of Tregs isolated from Sn KO mice, and in particular their ability to inhibit proliferation of MOG35-55-specific Teff lymphocytes to the same extent as Tregs from WT littermates. In vitro assays suggest that direct interaction between Sn+ macrophages and Tregs is required for these proliferation effects as antibody blocking of the Sn molecule on the macrophage surface was sufficient to demonstrate the same enhanced proliferation of Tregs as observed in the presence Sn negative macrophages. The direct interaction between Sn+ macrophages and Tregs was supported by the in vivo localization studies which showed that these two cell populations occur in close proximity both in the periphery, in the LN and spleen, and during EAE also in the CNS. During EAE, Treg numbers were particularly elevated in the brain where Foxp3+ Treg and Sn+ macrophages colocalized in both the parenchyma and in the perivascular cuffs, bordered by the endothelial and parenchymal basement membranes, which represent sites of high cell density and therefore high potential for cell-cell interactions.

In addition to direct interaction between Sn+ macrophages and Tregs, the altered cytokine profile of the macrophages, with downregulation of proinflammatory cytokines such as TNF-α, IL-6, IFN-γ and upregulation of the anti-inflammatory cytokine IL-10, may enhance the immunosuppressive phenotype as documented in other inflammatory situations (36-38). It is not possible to determine whether this altered cytokine profile results from the loss of the Sn molecule from the macrophage subpopulation, or changes in macrophage phenotype due to the altered Teff to Treg ratios. However, in view of the proproliferative effect of activated macrophages treated with Sn antibodies on Tregs in vitro, the altered cytokine profile is unlikely to account for the upregulation of Tregs observed in the Sn KO brain. Rather, the data suggest that this requires direct interaction between Sn+ macrophages and Tregs, the nature of which remains to be defined. Sn can potentially bind a wide range of glycoconjugates carrying sialic acid ligands in α2,3, α2,6 and α2,8 linkages, and although previous studies identified CD43 as a potential counterreceptor on T cells, it is not clear whether upregulation of sialoadhesin binding to Tregs reflects an overall increased sialylation or the induction of a high avidity counterreceptor(s). While investigations are underway to identify potential Treg Sn counterreceptors, they are hampered by the fact that they are expressed only on activated Tregs, which are present in very low numbers, and by the very low affinity of Sn for sialic acids.

The data presented here are consistent with recent studies of experimental autoimmune uveoretinitis (EAU) in Sn KO mice (39). Like EAE, EAU is an organ specific (retina) CD4+ T cell-mediated autoimmune disease, which when induced in the Sn KO mouse shows the same reduced disease severity and incidence as reported here. Reduced EAU symptoms were shown to be associated with reduced Teff proliferation, and while a molecular mechanism for this effect was not proposed, they could be explained by elevated numbers of Tregs as described here. Similarly, in two genetic models of peripheral and central nervous system demyelination, in which not only macrophages but also CD8+ T lymphocytes play an important pathogenic role, disease symptoms are ameliorated in Sn KO mice and are associated with reduced CD8+ T cell and macrophage numbers (40, 41). These results are also consistent with an upregulation of the CD4+CD25+Foxp3+ Treg population in the Sn KO mice. However, since glycosylation pathways in CD4 and CD8 pathways are known to be differentially regulated (42) direct interactions between Sn and activated CD8 T cells may also be important (43).

A large body of data documents the suppressive effects of CD4+CD25+Foxp3+ Treg cells on Teff cell proliferation and pro-inflammatory cytokine production (3, 11, 44), and development and differentiation of Treg cells has been elucidated by different research groups (45-47). However, the expansion of this subpopulation of T cells during the immune response is not well understood. Existing data on the regulation of Treg cells focuses on dendritic cells (DCs), the long-known classic APCs. There are several examples of specific DC populations being required for Treg expansion, for example, epidermal RANKL expression alters DC function to maintain the number of the peripheral regulatory T cells (48) and, in EAE, SOCS3 deficient DCs have been shown to selectively induce expansion of Foxp3+ Treg cells in a TGF-β-dependent manner (49). We show here that in addition to DCs, Sn+ macrophages also regulate Treg expansion, however, in a negative manner. During EAE development, engagement of macrophage Sn with its ligand on the cell surface of Tregs inhibits Treg proliferation, permitting the expansion of the Teff cell population and induction of disease symptoms. These events are early in the EAE development and the in vivo proliferation study together with the absence of defects in T cell migration to the CNS in the Sn KO mice, suggest that passively transferred Treg lymphocytes hyperproliferate both in the periphery and in the CNS of the Sn KO mice compared with WT mice, which correlated with the milder phenotype of the disease. Sn KO macrophages recovered from the CNS of EAE mice also showed a significantly higher capacity of inducing Treg proliferation compared with those recovered from the spleen (not shown), indicating that after penetration into the CNS macrophages acquire additional functions that favour Treg suppression.

In conclusion, we show here that during EAE Sn+ macrophages represent a distinct subpopulation of macrophages that interact directly with Treg lymphocytes, principally at the site of inflammation, to inhibit their proliferation and, thereby, regulate Teff lymphocyte function. Targeting Sn may, therefore, represent a novel means of regulating autoimmune disease progression.

Supplementary Material

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Acknowledgements

The authors thank Rupert Hallmann for advice throughout the project and for critical reading of the manuscript.

Abbreviations

Sn
sialoadhesin

Footnotes

1This work was supported by the German (SFB293 A14) and Swedish Research Councils (621-2001-2142 and K2004-33X-15076-01A), the Alfred Österlund stiftelse, Greta och Johan Kocks stiftelse, Crafoordaska stiftelsen, Kungliga fysiografiska sällskapet i Lund, the Interdisciplinary Clinical Research Center (IZKF; Lo2/017/07) Münster, Germany, and a Wellcome Trust Senior Research Fellowship WT081882MA (PRC).

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