Although Galβ1–4GlcNAc (LacNAc) moieties are the most common constituents of N-linked glycans on vertebrate proteins, GalNAcβ1–4GlcNAc (LacdiNAc, LDN)-containing glycans are widespread in invertebrates, such as helminths. We postulated that LDN might be a molecular pattern for recognition of helminth parasites by the immune system. Using LDN-based affinity chromatography and mass spectrometry, we have identified galectin-3 as the major LDN-binding protein in macrophages. By contrast, LDN binding was not observed with galectin-1. Surface plasmon resonance (SPR) analysis and a solid phase binding assay demonstrated that galectin-3 binds directly to neoglycoconjugates carrying LDN glycans. In addition, galectin-3 bound to Schistosoma mansoni soluble egg Ags and a mAb against the LDN glycan inhibited this binding, suggesting that LDN glycans within S. mansoni soluble egg Ags contribute to galectin-3 binding. Immunocytochemistry demonstrated high levels of galectin-3 in liver granulomas of S. mansoni-infected hamsters, and a colocalization of galectin-3 and LDN glycans was observed on the parasite eggshells. Finally, we demonstrate that galectin-3 can mediate recognition and phagocytosis of LDN-coated particles by macrophages. These findings provide evidence that LDN-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.

Monocyte infiltration into inflamed tissue requires their initial arrest onto the endothelial cells (ECs), followed by firm adhesion and subsequent transmigration. Although several pairs of adhesion molecules have been shown to play a role in the initial adhesion of monocytes to ECs, the mechanism of transendothelial migration is poorly defined. In this study, we have investigated the role of signal-regulatory protein (SIRP)α-CD47 interactions in monocyte transmigration across brain ECs. CD47 expression was observed in vivo on cerebral endothelium of both control animals and animals suffering from experimental allergic encephalomyelitis. To investigate whether SIRPα-CD47 interactions are instrumental in the trafficking of monocytes across cerebral EC monolayers, in vitro assays were conducted in which the migration of monocytes, but not adhesion, was found to be effectively diminished by blocking SIRPα and CD47 on monocytes and ECs, respectively. In this process, SIRPα was found to interact solely with its counterligand CD47 on ECs. Overexpression of the CD47 molecule on brain ECs significantly enhanced monocytic transmigration, but did not affect adhesion. SIRPα-CD47-mediated transendothelial migration involved Gi protein activity, a known signaling component of CD47. Finally, cross-linking of CD47 on brain ECs induced cytoskeletal reorganization of the endothelium, a process that was Gi protein independent. These data provide the first evidence that the interaction of CD47 with its monocytic counterligand SIRPα is of importance in the final step of monocyte trafficking into the brain, a critical event in the development of neuroinflammatory diseases.

Monocyte infiltration into inflamed tissue requires the initial arrest of the cells on the endothelium followed by firm adhesion and their subsequent migration. Migration of monocytes and other leukocytes is believed to involve a coordinated remodeling of the actin cytoskeleton. The small GTPases RhoA, Rac1, and Cdc42 are critical regulators of actin reorganization. In this study, we have investigated the role of Rho-like GTPases RhoA, Rac1, and Cdc42 in the adhesion and migration of monocytes across brain endothelial cells by expressing their constitutively active or dominant-negative constructs in NR8383 rat monocytic cells. Monocytes expressing the active form of Cdc42 show a reduced migration, whereas Rac1 expression did not affect adhesion or migration. In contrast, expression of the active form of RhoA in monocytes leads to a dramatic increase in their adhesion and migration across endothelial cells. The effect of RhoA was found to be mediated by its down-stream effector Rho kinase (ROCK), as pretreatment with the selective ROCK inhibitor Y-27632 prevented this enhanced adhesion and migration. These results demonstrate that RhoA activation in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells.

Signal regulatory protein α (SIRPα) is a glycoprotein receptor that recruits and signals via the tyrosine phosphatases SHP-1 and SHP-2. In macrophages SIRPα can negatively regulate the phagocytosis of host cells and the production of tumor necrosis factor alpha. Here we provide evidence that SIRPα can also stimulate macrophage activities, in particular the production of nitric oxide (NO) and reactive oxygen species. Ligation of SIRPα by antibodies or soluble CD47 triggers inducible nitric oxide synthase expression and production of NO. This was not caused by blocking negative-regulatory SIRPα-CD47 interactions. SIRPα-induced NO production was prevented by inhibition of the tyrosine kinase JAK2. JAK2 was found to associate with SIRPα in macrophages, particularly after SIRPα ligation, and SIRPα stimulation resulted in JAK2 and STAT1 tyrosine phosphorylation. Furthermore, SIRPα-induced NO production required the generation of hydrogen peroxide (H2O2) by a NADPH oxidase (NOX) and the phosphatidylinositol 3-kinase (PI3-K)-dependent activation of Rac1, an intrinsic NOX component. Finally, SIRPα ligation promoted SHP-1 and SHP-2 recruitment, which was both JAK2 and PI3-K dependent. These findings demonstrate that SIRPα ligation induces macrophage NO production through the cooperative action of JAK/STAT and PI3-K/Rac1/NOX/H2O2 signaling pathways. Therefore, we propose that SIRPα is able to function as an activating receptor.

To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, FcαRI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcαRI tail is important for FcαRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in FcαRI activation. PP2A binds to the intracellular tail of FcαRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, FcαRI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcαRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis of Neisseria meningitidis. The present findings demonstrate PP2A involvement as a molecular mechanism for FcαRI ligand binding regulation, a key step in initiating an immune response.

Activation of granulocyte effector functions, such as induction of the respiratory burst and migration, are regulated by a variety of relatively ill-defined signaling pathways. Recently, we identified a novel Ca 2؉ /calmodulin-dependent kinase I-like kinase, CKLiK, which exhibits restricted mRNA expression to human granulocytes. Using a novel antibody generated against the C-terminus of CKLiK, CKLiK was detected in CD34 ؉ -derived neutrophils and eosinophils, as well as in mature peripheral blood granulocytes. Activation of human granulocytes by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF), but not the phorbol ester PMA (phorbol 12-myristate-13-acetate), resulted in induction of CKLiK activity, in parallel with a rise of intracellular Ca 2؉ [Ca 2؉ ] i . To study the functionality of CKLiK in human granulocytes, a cell-permeable CKLiK peptide inhibitor (CKLiK 297-321 ) was generated which was able to inhibit kinase activity in a dose-dependent manner. The effect of this peptide was studied on specific granulocyte effector functions such as phagocytosis, respiratory burst, migration, and adhesion. Phagocytosis of Aspergillus fumigatus particles was reduced in the presence of CKLiK 297-321 and fMLP-induced reactive oxygen species (ROS) production was potently inhibited by CKLiK 297-321 in a dosedependent manner. Furthermore, fMLPinduced neutrophil migration on albumin-coated surfaces was perturbed, as well as ␤2-integrin-mediated adhesion. IntroductionHuman granulocytes, which include neutrophils and eosinophils, play an important role in host defense against invading pathogens. 1 During induction of inflammatory reactions, granulocytes in the peripheral blood become primed or preactivated, leave the bloodstream by diapedesis through the endothelium, and migrate toward the site of inflammation. 2 At the inflammatory site, granulocytes become highly activated, secrete proteolytic enzymes by degranulation, and form cytotoxic reactive oxygen species (ROS) to eliminate invading microorganisms. 3 These granulocyte effector functions are initiated by inflammatory mediators, such as chemoattractants, which activate the G-protein-coupled receptors (GPCRs). One of the signaling events that occurs after ligand binding to GPCRs, is the elevation of intracellular Ca 2ϩ ([Ca 2ϩ ] i ). Ligand binding to GPCRs initially activates trimeric G-proteins and subsequent dissociation of the G␣ and G␤␥ subunits. The dissociation of the G␤␥ subunit results in activation of phosphatidylinosytol-3-OH kinase (PI3K) and phospholipase C␤ (PLC␤). PLC␤ activation in turn leads to the hydrolysis of phosphatidylinositol4,5phosphate (PIP 2 ), producing diacylglycerol (DAG) and inositol-3 phosphate (IP 3 ). 4 Subsequently, IP 3 binds to its receptor on the Ca 2ϩ stores, resulting in Ca 2ϩ release into the cytoplasm. Elevation of [Ca 2ϩ ] i in granulocytes has been observed during activation of several granulocyte effector functions, for example in N-formyl-methionylleucyl-phenylalanine (fMLP)-induced respira...

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

Sections of human atherosclerotic plaques, obtained from 21 autopsy cases with various degrees of atherosclerosis, were stained with the indirect immunoperoxidase technique using specific monoclonal antibodies against macrophages and smooth muscle cells. Distinctive results were found in differing stages: Single blood monocytes were observed in diffuse intimal thickening and the foam cells seen in fatty streaks were mostly identified as mature tissue macrophages, while only very few blood monocytes were present. The spindle cells observed in fibroelastic plaques showed positive reactions to antibodies against desmin, which points to their derivation from smooth muscle cells, whereas only a few macrophage-derived foam cells were seen in these lesions. In the complicated lesions the majority of foam cells were macrophage-derived, but there was also a small number of foam cells positive to antibodies against desmin, suggesting a smooth muscle cell derivation. Our results confirm that in human atherosclerotic plaques the majority of the foam cells are obviously macrophage-derived, which emphasizes the important role of macrophages in the morphogenesis of these lesions.