Siglec-E promotes ß2-integrin-dependent NADPH oxidase activation to suppress neutrophil recruitment to the lung.

Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of ß2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b ß2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.

Siglec-F-dependent negative regulation of allergen-induced eosinophilia depends critically on the experimental model.

Siglec-8 and siglec-F are paralogous membrane proteins expressed on human and murine eosinophils respectively. They bind similar sialylated and sulphated glycans and mediate eosinophil apoptosis when cross-linked with antibodies or glycan ligands. In models of allergic eosinophilic airway inflammation, siglec-F was shown previously to be important for negatively regulating eosinophilia. It was proposed that this was due to siglec-F-dependent apoptosis, triggered via engagement with ligands that are upregulated on bronchial epithelium. Our aim was to further investigate the functions of siglec-F by comparing two commonly used models of ovalbumin-induced airway inflammation that differ in the dose and route of administration of ovalbumin. In confirmation of published results, siglec-F-deficient mice had enhanced lung tissue eosinophilia in response to intranasal ovalbumin delivered every other day. However, following aerosolised ovalbumin delivered daily, there was no influence of siglec-F deficiency on lung eosinophilia. Expression of siglec-F ligands in lung tissues was similar in both models of allergen induced inflammation. These data demonstrate that siglec-F-dependent regulation of eosinophilia is subtle and depends critically on the model used. The findings also indicate that mechanisms other than ligand-induced apoptosis may be important in siglec-F-dependent suppression of eosinophilia.

Sialoadhesin deficiency does not influence the severity of lupus nephritis in New Zealand black x New Zealand white F1 mice.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a chronic inflammatory condition with multisystem involvement. One of the key features of the disease is the upregulation of type I interferons, resulting in the so-called "interferon signature". Recent flow cytometric and transcriptomic studies identified Sialoadhesin (Sn, CD169) as an important interferon-induced blood monocyte biomarker in diseased patients. To investigate a potential causative role of Sn in SLE, we generated NZBWF1 (New Zealand Black x New Zealand White F1) mice lacking Sn and compared onset and progression of disease with NZBWF1 expressing normal levels of Sn. METHODS: Sn expression in renal tissues of pre-diseased and diseased NZBWF1 mice was evaluated by Quantitative real time PCR (QPCR) and immunohistochemistry. Sn-/- NZBWF1 mice were generated by speed congenics. Disease severity of Sn+/+ and Sn-/- NZBWF1 mice was assessed by serum immunoassays, flow cytometry, light microscopy and immunohistochemistry. RESULTS: Renal tissues from proteinuric NZBWF1 mice exhibited a significant upregulation of Sn mRNA and protein expression following disease onset. Further immunohistochemical analysis showed that Sn+ macrophages assumed a distinct periglomerular distribution and, unlike CD68+ macrophages, were not present within the glomeruli. Analysis of disease severity in Sn-/- and Sn+/+ NZBWF1 mice revealed no significant differences in the disease progression between the two groups although Sn-deficient mice showed a more rapid onset of proteinuria. CONCLUSIONS: These data confirm a positive correlation of Sn with disease activity. However, Sn deficiency does not have a significant effect on the severity and progression of lupus nephritis in the NZBWF1 mouse model.

Sialoadhesin ligand expression identifies a subset of CD4+Foxp3- T cells with a distinct activation and glycosylation profile.

Sialoadhesin (Sn) is a sialic acid-binding Ig-like lectin expressed selectively on macrophage subsets. In a model of experimental autoimmune encephalomyelitis, Sn interacted with sialylated ligands expressed selectively on CD4(+)Foxp3(+) regulatory T cells (Tregs) and inhibited their proliferation. In this study, we examined the induction of Sn ligands (SnL) on all splenic CD4(+) T cells following in vitro activation. Most CD4(+) Tregs strongly upregulated SnL, whereas only a small subset of ~20% CD4(+)Foxp3(-) T cells (effector T cells [Teffs]) upregulated SnL. SnL(+) Teffs displayed higher levels of activation markers CD25 and CD69, exhibited increased proliferation, and produced higher amounts of IL-2 and IFN-γ than corresponding SnL(-) Teffs. Coculture of activated Teffs with Sn(+) macrophages or Sn(+) Chinese hamster ovary cells resulted in increased cell death, suggesting a regulatory role for Sn-SnL interactions. The key importance of α2,3-sialylation in SnL expression was demonstrated by increased binding of α2,3-linkage-specific Maackia amurensis lectin, increased expression of α2,3-sialyltransferase ST3GalVI, and loss of SnL following treatment with an α2,3-linkage-specific sialidase. The induction of SnL on activated CD4(+) T cells was dependent on N-glycan rather than O-glycan biosynthesis and independent of the mucin-like molecules CD43 and P-selectin glycoprotein ligand-1, previously implicated in Sn interactions. Induction of ligands on CD4(+)Foxp3(-) Teffs was also observed in vivo using the New Zealand Black × New Zealand White F1 murine model of spontaneous lupus and SnL levels on Teffs correlated strongly with the degree of proteinuria. Collectively, these data indicate that SnL is a novel marker of activated CD4(+) Teffs that are implicated in the pathogenesis of autoimmune diseases.

Siglec-E is a negative regulator of acute pulmonary neutrophil inflammation and suppresses CD11b ß2-integrin-dependent signaling.

Neutrophil entry into the lung tissues is a key step in host defense to bacterial and yeast infections, but if uncontrolled can lead to severe tissue damage. Here, we demonstrate for the first time that sialic acid binding Ig-like lectin E (siglec-E) functions to selectively regulate early neutrophil recruitment into the lung. In a model of acute lung inflammation induced by aerosolized lipopolysaccharide, siglec-E-deficient mice exhibited exaggerated neutrophil recruitment that was reversed by blockade of the ß2 integrin, CD11b. Siglec-E suppressed CD11b "outside-in" signaling, because siglec-E-deficient neutrophils plated on the CD11b ligand fibrinogen showed exaggerated phosphorylation of Syk and p38 mitogen-activated protein kinase. Sialidase treatment of fibrinogen reversed the suppressive effect of siglec-E on CD11b signaling, suggesting that sialic acid recognition by siglec-E is required for its inhibitory function. Siglec-E in neutrophils was constitutively associated with the tyrosine phosphatase SHP-1 and may therefore function to constitutively dampen inflammatory responses of neutrophils. These data reveal that siglec-E is an important negative regulator of neutrophil recruitment to the lung and ß2 integrin-dependent signaling. Our findings have implications for the human functional ortholog, siglec-9, and its potential role in regulating inflammatory lung disease.

CD33-related siglecs as potential modulators of inflammatory responses.

The immune system must be tightly regulated to prevent unwanted tissue damage caused by exaggerated immune and inflammatory reactions. Inhibitory and activating immune receptors play a crucial role in this function via phosphotyrosine-dependent signaling pathways. A significant body of evidence has accumulated suggesting that the siglec family of sialic acid binding Ig-like lectins makes an important contribution to this immunoregulation. The CD33-related siglecs are a distinct subset of inhibitory and activating receptors, expressed primarily on leukocytes in a cell type-specific manner. Here, we critically assess the in vitro and in vivo evidence on the functional role for CD33-related siglecs in modulation of inflammatory and immune responses.

Delineating immune-mediated mechanisms underlying hair follicle destruction in the mouse mutant defolliculated.

Defolliculated (Gsdma3(Dfl)/+) mice have a hair loss phenotype that involves an aberrant hair cycle, altered sebaceous gland differentiation with reduced sebum production, chronic inflammation, and ultimately the loss of the hair follicle. Hair loss in these mice is similar to that seen in primary cicatricial, or scarring alopecias in which immune targeting of hair follicle stem cells has been proposed as a key factor resulting in permanent hair follicle destruction. In this study we examine the mechanism of hair loss in GsdmA3(Dfl)/+ mice. Aberrant expression patterns of stem cell markers during the hair cycle, in addition to aberrant behavior of the melanocytes leading to ectopic pigmentation of the hair follicle and epidermis, indicated the stem cell niche was not maintained. An autoimmune mechanism was excluded by crossing the mice with rag1-/- mice. However, large numbers of macrophages and increased expression of ICAM-1 were still present and may be involved either directly or indirectly in the hair loss. Reverse transcriptase-PCR (RT-PCR) and immunohistochemistry of sebaceous gland differentiation markers revealed reduced peroxisome proliferator-activated receptor-γ (PPARγ), a potential cause of reduced sebum production, as well as the potential involvement of the innate immune system in the hair loss. As reduced PPARγ expression has recently been implicated as a cause for lichen planopilaris, these mice may be useful for testing therapies.

Regulating the immune response to tumours.

Naturally occurring regulatory T cells (Tregs) have been shown to suppress immune responses to self-antigens, thereby limiting autoimmunity. In the case of tumours, where immune responses to self-antigens are beneficial and lead to elimination of the tumour, such suppressive activity is actually detrimental to the host. Manipulation of Tregs holds great promise for the immunotherapy of cancer. Several studies performed using rodent models and indicate that Tregs cells inhibit effective anti-tumour immune responses and that their removal promotes tumour rejection. The increasing number of studies of Tregs in patients with cancer also point to a role for these cells in promoting disease progression. This review summarises the findings of these studies and addresses the advantages and potential pitfalls of manipulating Treg activity for the treatment of cancer.

Uncompromised generation of a specific H-2DM-dependent peptide-MHC class II complex from exogenous antigen in Leishmania mexicana-infected dendritic cells.

Leishmania infection inhibits the capacity of macrophages (MPhi) to present antigens to CD4(+) T cells. Relocation of MHC class II and H-2DM to the parasitophorous vacuole (PV) and their subsequent degradation by the parasite may contribute to this defect. Dendritic cells (DC) are critical for initiation of primary T cell responses. DC can process Leishmania antigen and elicit Leishmania-specific T cells, but it is unknown whether exposure to Leishmania impairs this capacity. In particular, it is not clear whether DC containing live parasites efficiently process and present antigens. We investigated the ability of mouse bone marrow-derived DC infected with L. mexicana to generate pigeon cytochrome c (PCC) peptide-MHC class II complexes, using the mAb D4, which recognizes PCC(89-104) H-2E(k), and the PCC-specific T cell hybridoma 2B4. We show that H-2DM-dependent complex generation is not compromised by infection and that complexes are fully recognized by specific T cells. We further show that in contrast to infected MPhi, in infected DC cytoplasmic H-2DM is not down-regulated and not relocated to the parasite-containing vacuole. This observation may explain the continued ability of infected DC to present PCC, and also indicates differences in the habitat of these intracellular parasites in DC compared to MPhi.