Siglec-7 (p70/AIRM) and Siglec-9 are "CD33"-related siglecs expressed on natural killer (NK) cells and subsets of peripheral T cells. Like other inhibitory NK cell receptors, they contain immunoglobulin receptor family tyrosine-based inhibitory motifs in their cytoplasmic domains, and Siglec-7 has been demonstrated to negatively regulate NK cell activation. Based on reports of the presence of these siglecs on T cells, we sought to determine if they are capable of modulating T cell receptor (TCR) signaling using Jurkat T cells stably and transiently transfected with Siglec-7 or Siglec-9. Following either pervanadate stimulation or TCR engagement, both Siglecs exhibited increased tyrosine phosphorylation and recruitment of SHP-1. Effects of Siglec-7 and -9 were also evident in downstream events in the signaling pathway. Both siglecs reduced phosphorylation of Tyr 319 on ZAP-70, known to play a pivotal role in up-regulation of gene transcription following TCR stimulation. There was also a corresponding decreased transcriptional activity of nuclear factor of activated T cells (NFAT) as determined using a luciferase reporter gene. Like all siglecs, Siglec-7 and -9 recognize sialic acid-containing glycans of glycoproteins and glycolipids as ligands. Mutation of the conserved Arg in the ligand binding site of Siglec-7 (Arg 124 ) or Siglec-9 (Arg 120 ) resulted in reduced inhibitory function in the NFAT/luciferase transcription assay, suggesting that ligand binding is required for optimal inhibition of TCR signaling. The combined results demonstrate that both Siglec-7 and Siglec-9 are capable of negative regulation of TCR signaling and that ligand binding is required for optimal activity.The human siglec family of cell receptors is composed of eleven members of the Ig superfamily, which are functionally related by their ability to bind sialic acid-containing carbohydrates of glycoproteins and glycolipids as ligands (1-3). The Siglecs are predominately and differentially expressed on a wide variety of white blood cells (2, 3), the notable exceptions being myelin-associated glycoprotein expressed in glial cells (4, 5) and Siglec-6 expressed in placenta (2, 6). The extracellular region has a variable number of C2-set Ig domains and a single homologous N-terminal "V-set" domain that binds to sialic acids (2). Crystal structure analysis of two Siglecs, sialoadhesin (Siglec-1) and Siglec-7, have revealed a shallow sialic acid binding pocket with a conserved sequence of six amino acids in the tip of the C-CЈ loop that influences the specificity for binding various sialoside sequences found in nature (7-10). Within this sequence, a conserved arginine residue coordinates with the C-1 hydroxyl group of the sialic acid and is required for binding as evidenced by Arg to Ala mutations that abrogate binding to sialic acid-containing ligands (11,12).Another characteristic feature of the siglecs is the presence of consensus immunoglobulin receptor family tyrosine-based inhibitory motifs (ITIM) 1 in the cytoplasmic domains of all but si...

The lymphoid tissue in the omentum, at the so-called milky spots, is known as an initial place for disseminated cancer cells to develop into solid tumors. In the present study, i.p. macrophages significantly took up oligomannose-coated liposomes (OMLs) that were injected into the peritoneal cavity, and then gradually accumulated in the omentum and the other lymphoid tissues within 24 hours of i.p. injection of OMLs. When 5-fluorouracil (5-FU) was encapsulated in the OMLs, >60% of administered 5-FU accumulated in the omentum. Treatment of macrophages at 39°C for 30 minutes led to the release of 5-FU from the macrophages, suggesting that controlled release from macrophages could be achieved by mild hyperthermia. We encased magnetic nanoparticles, which are known to convert electromagnetic energy to heat in the OMLs to achieve in vivo hyperthermia at the site. Using this system in a mouse i.p. metastasis model, we successfully controlled tumor development by coadministration of OML-encased 5-FU and OML-encased magnetic nanoparticles, followed by treatment with an alternating magnetic field. No apparent reduction was seen in tumor growth with the administration of OML-encased magnetic nanoparticles or OML-encased 5-FU alone. Thus, we have established the use of i.p. macrophages as a novel drug delivery system for the control of cancer metastatic to milky spots. (Cancer Res 2006; 66(17): 8740-8)

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