Simon Y. C. Wong

The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages

We recently identified dectin-1 (βGR) as a major β-glucan receptor on leukocytes and demonstrated that it played a significant role in the non-opsonic recognition of soluble and particulate β-glucans. Using a novel mAb (2A11) raised against βGR, we show here that the receptor is not dendritic cell-restricted as first reported, but is broadly expressed, with highest surface expression on populations of myeloid cells (monocyte/macrophage (Mφ) and neutrophil lineages). Dendritic cells and a subpopulation of T cells also expressed the βGR, but at lower levels. Alveolar Mφ, like inflammatory Mφ, exhibited the highest surface expression of βGR, indicative of a role for this receptor in immune surveillance. In contrast, resident peritoneal Mφ expressed much lower levels of βGR on the cell surface. Characterization of the nonopsonic recognition of zymosan by resident peritoneal Mφ suggested the existence of an additional β-glucan-independent mechanism of zymosan binding that was not observed on elicited or bone marrow-derived Mφ. Although this recognition could be inhibited by mannan, we were able to exclude involvement of the Mφ mannose receptor and complement receptor 3 in this process. These observations imply the existence of an additional mannan-dependent receptor involved in the recognition of zymosan by resident peritoneal Mφ.

The human β-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells

We identified the C‐type‐lectin‐like receptor, Dectin‐1, as the major receptor for fungal β‐glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform‐specific mAb, we show here that human Dectin‐1, the β‐glucan receptor (βGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin‐1 is not myeloid restricted. Both major functional βGR isoforms – βGR‐A and βGR‐B – were expressed by these cell populations in peripheral blood; however, only βGR‐B was significantly expressed on mature monocyte‐derived macrophages and immature DC, suggesting cell‐specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin‐window technique, demonstrated that Dectin‐1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other β‐glucan receptors on mature human macrophages, we have demonstrated that Dectin‐1 acted as the major β‐glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Dectin-1 Expression and Function Are Enhanced on Alternatively Activated and GM-CSF-Treated Macrophages and Are Negatively Regulated by IL-10, Dexamethasone, and Lipopolysaccharide

Dectin-1 is the major macrophage receptor for β-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mφ). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-γ, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-α, although some Mφ-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.

Preferential Induction of CD4+ T Cell Responses through In Vivo Targeting of Antigen to Dendritic Cell-Associated C-Type Lectin-1

Targeting of Ags and therapeutics to dendritic cells (DCs) has immense potential for immunotherapy and vaccination. Because DCs are heterogeneous, optimal targeting strategies will require knowledge about functional specialization among DC subpopulations and identification of molecules for targeting appropriate DCs. We characterized the expression of a fungal recognition receptor, DC-associated C-type lectin-1 (Dectin-1), on mouse DC subpopulations and investigated the ability of an anti-Dectin-1 Ab to deliver Ag for the stimulation of immune responses. Dectin-1 was shown to be expressed on CD8α−CD4−CD11b+ DCs found in spleen and lymph nodes and dermal DCs present in skin and s.c. lymph nodes. Injection of Ag-anti-Dectin-1 conjugates induced CD4+ and CD8+ T cell and Ab responses at low doses where free Ag failed to elicit a response. Notably, qualitatively different immune responses were generated by targeting Ag to Dectin-1 vs CD205, a molecule expressed on CD8α+CD4−CD11b− DCs, dermal DCs, and Langerhans cells. Unlike anti-Dectin-1, anti-CD205 conjugates failed to elicit an Ab response. Moreover, when conjugates were injected i.v., anti-Dectin-1 stimulated a much stronger CD4+ T cell response and a much weaker CD8+ T cell response than anti-CD205. The results reveal Dectin-1 as a potential targeting molecule for immunization and have implications for the specialization of DC subpopulations.

Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor.

The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca2+-dependent and inhibitable withd-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4–7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4–7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.

Expression of the β-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions

Dectin‐1 is a pathogen‐recognition receptor on macrophages (MΦs), neutrophils, and dendritic cells (DCs). On MΦs and bone marrow‐derived DCs, it has been shown to mediate the nonopsonic recognition of and response to soluble and particulate yeast β‐glucans. We have optimized the immunohistochemical detection of Dectin‐1 and demonstrated its expression on neutrophils, subpopulations of MΦs in splenic red and white pulp, alveolar MΦs, Kupffer cells, and MΦs and DCs in the lamina propria of gut villi. This is consistent with its role in pathogen surveillance. A significant proportion of CD11c+ splenic DCs expressed Dectin‐1, but expression was not restricted to any one subset. Dectin‐1 expression was low on resident MΦs and DCs of skin and was not detected on resident MΦs or DCs in kidney, heart, brain, or eye. The proposed, additional role of Dectin‐1 as a coreceptor for T cell activation is supported by its expression on DCs in the T cell areas of the spleen and lymph nodes. Strong expression of Dectin‐1 on subpopulations of MΦs and DCs in the medullary and corticomedullary regions of the thymus suggests a role distinct from pathogen recognition. Tissue localization thus revealed potential roles of Dectin‐1 in leukocyte interactions during innate immune responses and T cell development.

Analysis of mannose receptor regulation by IL‐4, IL‐10, and proteolytic processing using novel monoclonal antibodies

The study of the murinemacrophage mannose receptor (MR) has been hampered by the lack of specific reagents. We have generated and characterized novel anti‐MR monoclonal antibodies and used them to analyze MR expression in primary mouse macrophages (MØ). In BioGel‐ and thioglycollate‐elicited MØ, interleukin (IL)‐4 up‐regulated total cell‐associated MR (cMR), correlating with enhanced surface expression. We investigated the influence of IL‐10, a well‐characterized deactivator of MØ function, on MR levels and observed that it had a similar effect to IL‐4. In both cases, enhanced cMR levels translated into increased production of the soluble form of the receptor (sMR). We have demonstrated the presence of sMR in cultures of stable non‐MØ transductants expressing full‐length MR, indicating that the proteolytic activity responsible for cMR cleavage is not MØ‐restricted. These data support a role for the MR in T helper cell type 2 cytokine‐driven, immune responses and suggest a non‐MØ contribution to sMR production in vivo.

Dectin-2 is predominantly myeloid restricted and exhibits unique activation-dependent expression on maturing inflammatory monocytes elicited in vivo

Dectin‐2 is a recently described dendritic‐cell‐associated receptor, suggested to be involved in the initiation and maintenance of UV‐induced tolerance. To understand the physiological relevance of the proposed functions of this C‐type lectin‐like receptor, we have generated monoclonal antibodies against its extracellular domain and performed a detailed study of its expression. In naive mice, Dectin‐2 has a novel distribution pattern compared with other myeloid markers, but is predominantly expressed by a wide variety of tissue macrophages. Its expression was limited on dendritic cells and notably absent from brain microglia and choroid plexus or meningeal macrophages. On peripheral blood monocytes, Dectin‐2 expression was very low on the surface but was transiently and markedly up‐regulated on induction of inflammation in vivo using a variety of stimuli. This change in Dectin‐2 expression occurs on ‘inflammatory’ monocytes after arrival at the inflammatory lesion as demonstrated by adoptive cell‐transfer studies, and is independent of whether the macrophages elicited by the stimuli ultimately expressed Dectin‐2. These observations show Dectin‐2 expression to be characteristic of monocyte activation/maturation at an inflammatory lesion and provide a new perspective on the interpretation of Dectin‐2 function in vivo.

Shaping of adaptive immune responses to soluble proteins by TLR agonists: A role for IFN-α/β

Toll‐like receptors (TLR) are believed to play a major role in the recognition of invading organisms, although their ability to shape immune responses is not completely understood. Our aim was to investigate in vivo the effect of different TLR stimuli on the generation of antibody responses and the induction of CD8+ T‐cell cross‐priming after immunization with soluble protein antigens. While all TLR agonists tested elicited the production of immunomodulatory cytokines, marked differences were observed in their ability to stimulate antigen‐specific immune responses. Zymosan, poly(I:C) and CpG DNA, which signal through TLR2/6, 3 and 9, respectively, were found to strongly induce the production of IgG2a antibodies, whereas R‐848 (TLR7) and LPS (TLR4) did so much more weakly. In contrast, LPS, poly(I:C) and CpG DNA, but not zymosan, induced functional CD8+ T‐cell responses against OVA; peptidoglycan (TLR2/?) and R‐848 were also ineffective in stimulating cross‐priming. Experiments using IFN‐α/βR‐deficient mice showed that the induction of cross‐priming by LPS and poly(I:C) was abrogated in the absence of IFN‐α/β signalling, and induction by CpG DNA was greatly reduced. Overall, our results identify LPS as another TLR agonist that is able to generate functional cross‐priming against a soluble protein antigen. In addition, our results demonstrate that the ability of TLR stimuli to initiate CD8+ T‐cell responses against soluble protein antigens is largely dependent on the IFN‐α/β signalling pathway.

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria.

ABSTRACT Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain,lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxAstrains induced production of tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1α, IL-6, and TNF-α production and induced no detectable IL-12. Addition of exogenous LPS to thelpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.