Co-expression of different types of Fc receptors on murine peritoneal macrophages

Simultaneous expression of particular immunoglobulin Fc receptors (FcR) was studied on the plasma membranes of murine peritoneal macrophages. This was facilitated by the use of sheep red blood cells (SRBC) and/or synthetic microspheres coated with monoclonal antibodies of different isotypes. It was concluded that a majority of macrophages bear more than one type of FcR; macrophages bearing at least three types of FcR were present in the peritoneal cavity; macrophages bearing Fc mu R did not bind IgE, IgA or IgG; all macrophages bearing Fc alpha R also expressed Fc gamma 2bR, Fc gamma 3R and Fc epsilon R; all macrophages bearing Fc epsilon R also expressed Fc gamma 2bR and Fc alpha R. Except for Fc alpha R, essentially equivalent numbers of FcR-bearing macrophages were detected when antibody-coated SRBC or polymeric microspheres were used. Simultaneous applications of these reagents permitted the most detailed and direct investigations yet performed of multiple FcR expression on individual cells.     

Tumor immunology and cytokines

Abstracts of the 4th International Congress of the Metastasis Research Society: Science and Medicine in Cancer MetastasisSymposium 3

Tumor immunology and cytokines     

Antimetastatic activities of modified heparins: selectin inhibition by heparin attenuates metastasis

Heparin, which is traditionally used as an anticoagulant but has a variety of additional biological activities, was shown in several retrospective and prospective clinical trials to have an effect on cancer survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. To clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. Cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. Heparin is also a potent inhibitor of selectin-mediated interactions. P- and L-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet-tumor cell thrombi formation. To delineate the biological activities of heparin contributing to metastasis inhibition, modified heparins with specific activities were evaluated. Low anticoagulant heparin preparations still inhibited metastasis efficiently, indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. Modified heparins characterized for heparanase inhibitory activity are also potential inhibitors of selectins. Selectin inhibition is a clear component of heparin inhibition of metastasis. The contribution of selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity.     

Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase

Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs) endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation.     

P-selectin mediates the adhesion of sickle erythrocytes to the endothelium

The adherence of sickle red blood cells (RBCs) to the vascular endothelium may contribute to painful vaso-occlusion in sickle cell disease. Sickle cell adherence involves several receptor-mediated processes and may be potentiated by the up-regulated expression of adhesion molecules on activated endothelial cells. Recent results showed that thrombin rapidly increases the adhesivity of endothelial cells for sickle erythrocytes. The current report presents the first evidence for the novel adhesion of normal and, to a greater extent, sickle RBCs to endothelial P-selectin. Studies of the possible interaction of erythrocytes with P-selectin revealed that either P-selectin blocking monoclonal antibodies or sialyl Lewis tetrasaccharide inhibits the enhanced adherence of normal and sickle cells to thrombin-treated endothelial cells. Both RBC types also adhere to immobilized recombinant P-selectin. Pretreating erythrocytes with sialidase reduces their adherence to activated endothelial cells and to immobilized recombinant P-selectin. Herein the first evidence is presented for the binding of normal or sickle erythrocytes to P-selectin. This novel finding suggests that P-selectin inhibition be considered as a potential approach to therapy for the treatment of painful vaso-occlusion in sickle cell disease.     

Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs

Puga Yung GL, Li Y, Borsig L, Millard A‐L, Karpova MB, Zhou D, Seebach JD. Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock‐out pigs. Xenotransplantation 2012; 19: 196–206. © 2012 John Wiley & Sons A/S. Abstract: Background: Anti‐Galα1,3Galβ‐R natural antibodies are responsible for hyperacute rejection in pig‐to‐primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody‐mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb₃) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. Methods: The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb₃ synthase mRNA was tested by RT‐PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. Results: Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb₃ on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H‐type sugar structures present in GalT knockout cells as compared to wild‐type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb₃ was also totally absent, whereas a fucosylated form of iGb₃ was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild‐type or GalT knockout animals. Conclusions: These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non‐Galα1,3Gal antibodies and cellular responses.     

Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c+ cells through TLR4

Breast milk oligosaccharides shape the intestinal environment by affecting mucosal immunity and bacterial colonization. To clarify the role of milk oligosaccharide sialyl(α2,3)lactose (3SL) in intestinal physiology and disease, we investigated colitis development in Il10^−/− mice exposed to normal or 3SL-deficient milk during lactation. Onset and progression of intestinal inflammation were delayed in Il10^−/− mice deficient for the α2,3 sialyltransferase 4 (ST3GAL4) responsible for 3SL biosynthesis. The proinflammatory role of 3SL was confirmed by showing that oral supplementation of newborn Il10^−/−;St3gal4^−/− mice with 3SL increased colitis severity. Conversely, fostering of newborn Il10^−/− mice to lactating St3gal4^−/− mothers reduced colitis severity. 3SL directly stimulated mesenteric lymph node CD11c⁺ dendritic cells and induced production of cytokines required for expansion of T_H1 and T_H17 T cells. The stimulatory effect of 3SL was attenuated in Tlr4-deficient CD11c⁺ cells, demonstrating that 3SL induces inflammation through Toll-like receptor 4 (TLR4) signaling. Thus, 3SL directly modulates mucosal immunity, which increases susceptibility to colitis.Inflammatory bowel disease (IBD) affects up to 0.8% of the Western population and this number is constantly growing worldwide (1). The etiology of IBD is not fully understood, although a number of genetic and environmental factors leading to aberrant mucosal immune responses have been identified (2). Nutrition and especially breastfeeding affects the risk for IBD (3). Breastfeeding for at least 3 mo, accordingly, contributes to a lower incidence for IBD (4); however, these data still are controversial. Oligosaccharides are major constituents of breast milk fulfilling various functions, such as promoting growth of beneficial bacteria, acting as soluble receptors preventing attachment of pathogens in the gastrointestinal tract, and reducing adhesion of leukocytes (4, 5). The exact functions of individual oligosaccharides remain however largely unknown.The limited structural diversity of mouse milk oligosaccharides, including only sialyl(α2,3)lactose (3SL) and sialyl(α2,6)lactose (6SL), enables assessing the specific functional contribution of these oligosaccharides to intestinal homeostasis (6). A recent study provided unique evidence that milk-derived 3SL, but not 6SL, increased susceptibility to dextran sulfate sodium (DSS)-induced acute colitis (6). These findings indicate that individual milk oligosaccharides may not only mediate protective effects, but may promote inflammation as well.Mucosal innate immunity has a pivotal role in regulating inflammatory responses (2). Dendritic cells (DCs) and macrophages sense luminal antigens and provide signals for induction of tolerance to ingested antigens and commensal bacteria or for initiation of inflammatory immune responses, facilitating activation of adaptive immunity (7, 8). Intestinal DCs consist of two functionally distinct subsets based on expression of CD103 and chemokine (C-X3-C motif) receptor 1 (CX3CR1) (9). CD103⁺CX3CR1⁻ cells are the main population of migratory intestinal DCs influencing regulatory T cells in a TGF-β and retinoic-acid–dependent manner (10, 11). In contrast, CD103⁻CX3CR1⁺ DCs are resident cells sampling luminal antigens, thereby initiating local immune responses (12).Mucosal immunity is a key to the maintenance of gut homeostasis. Several classes of pattern recognition receptors mediate innate immune responses of intestinal epithelial cells and lamina propria-resident leukocytes. Toll-like receptors (TLR) and Nod-like receptors facilitate the recognition of bacterial fragments such as peptidoglycan (TLR2), lipopolysaccharides (TLR4), flaggelin (TLR5), unmethylated CpG DNA sequences (TLR9), and muramyl di- and tripeptides (Nod1 and Nod2) (13). In particular, glycans are recognized by different families of receptors, such as C-type lectins, galectins, and siglecs, which are expressed both by antigen-presenting cells and intestinal epithelial cells (14). Although, beneficial effects of milk oligosaccharides are well known, the mechanisms mediating the “sensing” of these oligosaccharides remain to be determined.The present study addresses the proinflammatory effect of milk oligosaccharide 3SL in contributing to spontaneous colitis in Il10^−/− mice. The 3SL supplementation of adult mice demonstrated the ability of milk oligosaccharides to directly affect mucosal immunity.