Sialoadhesin-positive host macrophages play an essential role in graft-versus-leukemia reactivity in mice.
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We recently established an effective immune T-cell-mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell-mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell-mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen beta-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous beta-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages. (+info)
Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis.
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The molecular interactions between sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of sialoadhesin for several sialosides. The results indicate that alpha2, 3-sialyl-lactose and alpha2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does alpha-methyl-N-acetylneuraminic acid (alpha-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with alpha-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins. (+info)
Cell-specific glycoforms of sialoadhesin and CD45 are counter-receptors for the cysteine-rich domain of the mannose receptor.
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We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domain of the mannose receptor, binds to marginal zone metallophilic macrophages (Mo) and B cell areas in the spleen and to subcapsular sinus Mo in lymph nodes of naive mice (CR-Fc(+) cells). Several CR-Fc ligands were found in spleen and lymph node tissue lysates using ligand blots. In this paper we report the identification of two of these ligands as sialoadhesin (Sn), an Mo-specific membrane molecule, and the leukocyte common antigen, CD45. CR-Fc bound selectively to Sn purified from spleen and lymph nodes and to two low molecular weight isoforms of CD45 in a sugar-dependent manner. CR-Fc binding and non-binding forms of Sn, probably derived from CR-Fc(+) and CR-Fc(-) cells respectively, were selected from spleen lysates. Analysis of the glycan pool associated with the CR-Fc-binding form revealed the presence of charged structures resistant to sialidase, absent in the non-binding form, that could correspond to sulfated structures. These results confirm the identification of the CR region of the mannose receptor as a lectin. We also demonstrate that the same glycoprotein expressed in different cells of the same organ can display distinct sugar epitopes that determine its binding properties. (+info)
Sialic acid binding receptors (siglecs) expressed by macrophages.
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Sialic acids are structurally and topographically well-suited to function as ligands in cellular recognition events. Sialoadhesin (Sn) is a sialic acid binding receptor uniquely expressed by macrophage subsets. It is a member of the immunoglobulin (Ig) superfamily with 17 extracellular domains. Sn is a prototypical member of the siglec family of sialic acid binding proteins, which includes CD22, myelin-associated glycoprotein, CD33, and siglec-5. These membrane proteins are involved in discrete functions in the hemopoietic, immune, and nervous systems. The sialic acid binding region of siglecs is localized within the membrane-distal, amino-terminal domain and in the case of Sn, it has been characterized in atomic detail by X-ray crystallography, nuclear magnetic resonance, and site-directed mutagenesis. Our studies on Sn indicate that this receptor is likely to function as a macrophage accessory molecule in a variety of cell-cell and cell-extracellular matrix interactions. CD33 and siglec-5 are also expressed on macrophage subsets as well as other myeloid cells. However, unlike Sn, the properties of these molecules indicate a predominant role in signaling functions rather than in cell-cell interactions. (+info)
Macrophage-tumour cell interactions: identification of MUC1 on breast cancer cells as a potential counter-receptor for the macrophage-restricted receptor, sialoadhesin.
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In many carcinomas, infiltrating macrophages are commonly found closely associated with tumour cells but little is known concerning the nature or significance of adhesion molecules involved in these cellular interactions. Here we demonstrate in primary human breast cancers that sialoadhesin (Sn), a macrophage-restricted adhesion molecule, is frequently expressed on infiltrating cells that often make close contact with breast carcinoma cells. To determine whether Sn could act as a specific receptor for ligands on breast cancer cell lines, binding assays were performed with a recombinant form of the protein fused to the Fc portion of human immunoglobulin G1 (IgG1) (Sn-Fc). Sn-Fc was found to bind specifically and in a sialic acid-dependent manner to the breast cancer cell lines MCF-7, T47.D and BT-20 both in solid- and solution-phase binding assays. To investigate the nature of the sialoglycoproteins recognized by Sn on breast cancer cells, MCF-7 cells were labelled with [6-3H]glucosamine. Following precipitation with Sn-Fc, a major band of approximately 240000 MW was revealed, which was shown in reprecipitation and Western blotting experiments to be the epithelial mucin, MUC1. (+info)
Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs.
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The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5. (+info)
Functional and in situ evidence for nitric oxide production driven by CD40-CD40L interactions in graft-versus-leukemia reactivity.
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In a murine tumor model, complete tumor remission is achievable at even advanced metastasized stages by transfer of immune T cells from donor B10.D2 (H-2d, Mls(b)) into tumor-bearing DBA/2 (H-2d, Mls(a)) mice. We showed previously that this graft-versus-leukemia (GvL) effect is dependent on synergistic interactions of transferred CD4+ and CD8+ T cells with host sialoadhesin (SER)-positive macrophages. We now show that the CD40-CD40L (CD154) interaction is involved in the induction of inducible nitric oxide synthase (iNOS) expression during adoptive immunotherapy (ADI). We demonstrate that during ADI, the level of CD40 expression in the liver becomes significantly augmented in comparison to livers of tumor-bearing, untreated animals. CD40 expression is found mostly on SER+ macrophages and to a lesser extent on dendritic cells (DCs). In GvL animals, more SER+ macrophages express iNOS than untreated animals. iNOS expressing cells are found in close proximity to apoptotic cells, at early time points of the therapy in areas of metastasis, and at late stages around portal veins, where CD4+ and CD8+ T lymphocytes form clusters with SER+ macrophages. Blocking of CD40L in vivo at days 5 and 20, when all iNOS+ cells express CD40, leads to significantly reduced CD40 and iNOS expression as well as to a marked inhibition of the therapeutic effect. These data provide functional and in situ evidence that the increased CD40 and iNOS expression observed during ADI contribute to the eradication of liver metastases and to the clearance of donor lymphocytes from the liver. (+info)
Characterization of human sialoadhesin, a sialic acid binding receptor expressed by resident and inflammatory macrophage populations.
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Sialoadhesin is a macrophage-restricted cellular interaction molecule and a prototypic member of the Siglec family of sialic acid binding immunoglobulin (Ig)-like lectins. So far, it has only been characterized in rodents. Here, we report the molecular cloning, binding properties, and expression pattern of human sialoadhesin. The predicted protein sequences of human and mouse sialoadhesin are about 72% identical, with the greatest similarity in the extracellular region, which comprises 17 Ig domains in both species. A recombinant protein consisting of the first 4 N-terminal domains of human sialoadhesin fused to the Fc region of human IgG1 mediated sialic acid-dependent binding with a specificity similar to its mouse counterpart, preferring sialic acid in the alpha2,3 glycosidic linkage over the alpha2,6 linkage. By flow cytometry with peripheral blood leukocytes, recombinant sialoadhesin bound strongly to granulocytes with intermediate binding to monocytes, natural killer cells, B cells, and a subset of CD8 T cells. Using antibodies raised to the recombinant protein, sialoadhesin was immunoprecipitated from the THP-1 human monocytic cell line as an approximate 200-kd glycoprotein. The expression pattern of human sialoadhesin was found to be similar to that of the mouse receptor, being absent from monocytes and other peripheral blood leukocytes, but expressed strongly by tissue macrophages in the spleen, lymph node, bone marrow, liver, colon, and lungs. High expression was also found on inflammatory macrophages present in affected tissues from patients with rheumatoid arthritis. (+info)