Mannose receptor and macrophage galactose-type lectin are involved in Bordetella pertussis mast cell interaction.

Mast cells are crucial in the development of immunity against Bordetella pertussis, and the function of TLRs in this process has been investigated. Here, the interaction between mast cells and B. pertussis with an emphasis on the role of CLRs is examined. In this study, two CLRs, MGL and MR, were detected for the first time on the surface of mast cells. The involvement of MR and MGL in the stimulation of mast cells by heat-inactivated BP was investigated by the use of blocking antibodies and specific carbohydrate ligands. The cell wall LOS of BP was also isolated to explore its role in this interaction. Mast cells stimulated with heat-inactivated BP or BP LOS induced TNF-α, IL-6, and IFN-γ secretion, which was suppressed by blocking MR or MGL. Inhibition of CLRs signaling during BP stimulation affected the ability of mast cells to promote cytokine secretion in T cells but had no effect on the cell-surface expression of ICAM1. Blocking MR or MGL suppressed BP-induced NF-κB expression but not ERK phosphorylation. Syk was involved in the CLR-mediated activation of mast cells by BP. Bacterial recognition by immune cells has been predominantly attributed to TLRs; in this study, the novel role of CLRs in the BP-mast cell interaction is highlighted.

Macrophage receptors for influenza A virus: role of the macrophage galactose-type lectin and mannose receptor in viral entry.

Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca(2+)-dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.

MGL1 promotes adipose tissue inflammation and insulin resistance by regulating 7/4hi monocytes in obesity.

Adipose tissue macrophages (ATMs) play a critical role in obesity-induced inflammation and insulin resistance. Distinct subtypes of ATMs have been identified that differentially express macrophage galactose-type C-type lectin 1 (MGL1/CD301), a marker of alternatively activated macrophages. To evaluate if MGL1 is required for the anti-inflammatory function of resident (type 2) MGL1(+) ATMs, we examined the effects of diet-induced obesity (DIO) on inflammation and metabolism in Mgl1(-/-) mice. We found that Mgl1 is not required for the trafficking of type 2 ATMs to adipose tissue. Surprisingly, obese Mgl1(-/-) mice were protected from glucose intolerance, insulin resistance, and steatosis despite having more visceral fat. This protection was caused by a significant decrease in inflammatory (type 1) CD11c(+) ATMs in the visceral adipose tissue of Mgl1(-/-) mice. MGL1 was expressed specifically in 7/4(hi) inflammatory monocytes in the blood and obese Mgl1(-/-) mice had lower levels of 7/4(hi) monocytes. Mgl1(-/-) monocytes had decreased half-life after adoptive transfer and demonstrated decreased adhesion to adipocytes indicating a role for MGL1 in the regulation of monocyte function. This study identifies MGL1 as a novel regulator of inflammatory monocyte trafficking to adipose tissue in response to DIO.

Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes.

OBJECTIVE: To establish the mechanism of the phenotypic switch of adipose tissue macrophages (ATMs) from an alternatively activated (M2a) to a classically activated (M1) phenotype with obesity. RESEARCH DESIGN AND METHODS: ATMs from lean and obese (high-fat diet-fed) C57Bl/6 mice were analyzed by a combination of flow cytometry, immunofluorescence, and expression analysis for M2a and M1 genes. Pulse labeling of ATMs with PKH26 assessed the recruitment rate of ATMs to spatially distinct regions. RESULTS: Resident ATMs in lean mice express the M2a marker macrophage galactose N-acetyl-galactosamine specific lectin 1 (MGL1) and localize to interstitial spaces between adipocytes independent of CCR2 and CCL2. With diet-induced obesity, MGL1(+) ATMs remain in interstitial spaces, whereas a population of MGL1(-)CCR2(+) ATMs with high M1 and low M2a gene expression is recruited to clusters surrounding necrotic adipocytes. Pulse labeling showed that the rate of recruitment of new macrophages to MGL1(-) ATM clusters is significantly faster than that of interstitial MGL1(+) ATMs. This recruitment is attenuated in Ccr2(-/-) mice. M2a- and M1-polarized macrophages produced different effects on adipogenesis and adipocyte insulin sensitivity in vitro. CONCLUSIONS: The shift in the M2a/M1 ATM balance is generated by spatial and temporal differences in the recruitment of distinct ATM subtypes. The obesity-induced switch in ATM activation state is coupled to the localized recruitment of an inflammatory ATM subtype to macrophage clusters from the circulation and not to the conversion of resident M2a macrophages to M1 ATMs in situ.

Disruption of protein-protein interaction in the Mgl-1 oncoprotein.

Mammalian homologues of the Lethal giant larvae (Lgl) tumor suppressor gene have been identified and these homologues can complement the yeast double mutant of Sop1 and Sop2, the yeast homologue of Lgl, as reported previously. In the absence of these genes in yeast, cellular viability is affected at restrictive temperature and salt environments. Members of this family contain five or more of the WD-40 repeat motifs, which is known to be involved in protein-protein interaction. In order to investigate the biochemical roles for conserved amino acids within the most conserved WD-40 repeat motif amongst these family members, we generated deletion mutants for five conserved amino acids (G450, H451, D453, W459 and D460) in mouse Lgl-1 (Mgl-1), located between 450-460 amino acids. We found that the deletion mutants of Mgl-1, DeltaG450 and DeltaD453, were not capable of complementing yeast mutants of Sop1 and Sop2 at restrictive temperature and high salt environments. These results indicate that the WD-40 repeat motif is important for cellular viability by regulating temperature-sensitivity and salt tolerance in yeast.

Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes.

Development of a reliable method to isolate highly proliferative potential hepatocytes will provide insight into the molecular mechanisms of liver regeneration, as well as proving crucial for the development of a biohybrid artificial liver. The aim of this study is to isolate highly proliferative, e.g., progenitor-like, hepatocytes. To this end, we fractionated hepatocytes expressing low and high levels of the asialoglycoprotein receptor (ASGP-R) based on the difference in their adhesion to poly[N-p-vinylbenzyl-O-beta-d-galactopyranosyl-(1-->4)-d-gluconamide] (PVLA), and examined the proliferative activity and gene expression of these fractionated hepatocytes. The results showed that approximately 0.5 to 1% of the total number of hepatocytes, which showed low adhesion to PVLA, expressed low levels of the ASGP-R, while the rest of hepatocyte population with high adhesion to PVLA expressed high levels of the ASGP-R. Interestingly hepatocytes with low ASGP-R expression levels had much higher DNA synthesizing activity (i.e., are much more proliferative) than those with high ASGP-R expression levels. Moreover, hepatocytes with low ASGP-R expression levels expressed higher levels of epidermal growth factor receptor (EGF-R), CD29 (beta1 integrin) and CD49f (alpha6 integrin) and lower levels of glutamine synthetase than those with high ASGP-R expression. These findings suggested that hepatocytes with low adhesion to PVLA due to their low ASGP-R expression could be potential candidates for progenitor-like hepatocytes due to their high proliferative capacity; hence, the low expression of the ASGP-R could be a unique marker for progenitor hepatocytes. The isolation of hepatocytes with different functional phenotypes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease.

A role of asialoglycoproteins for plasma-membrane-induced inhibition of the switching from alpha 1 to beta subtypes in adrenergic response during primary culture of rat hepatocytes.

Adrenergic responses of rat hepatocytes were studied by measuring Ins(1,4,5)P3(for the response via alpha 1-subtype receptors) and cAMP (for beta-subtype response) generation during brief incubation of cells with respective agonists. Hepatocytes from young rats with an age of 1 week displayed a very high beta response without a significant alpha 1 response. The beta response decreased and the alpha 1 response increased progressively as the age increased; the response was almost exclusively via alpha 1 receptors in hepatocytes of adult rats 9 weeks or more old. The beta response developed, again at the expense of the alpha 1 response, in hepatocytes from adult rats during the primary culture at low cell densities [(1-2.5) x 10(4) cells/cm2]. Such "alpha 1 to beta subtype switching' of adrenergic responses in vitro was totally inhibited by adding plasma membranes prepared from adult rat liver into the low-cell-density culture, but not inhibited at all by membranes from young rat liver. The inhibitory effect of adult rat liver membranes was lost when the membranes had been exposed to endoglycosidase F or beta-galactosidase but was not affected by prior treatment with sialidase. On the contrary, young rat liver membranes became inhibitory to "alpha 1 to beta subtype switching' after prior treatment with sialidase. Thus glycoproteins with unsialylated galactosyl termini on the surface of adult rat hepatocytes are likely to function as a determinant of the relative development of alpha 1/beta subtypes of adrenergic responses; the beta response is predominant in hepatocytes in the juvenile, presumably as a result of sialylation of the galactosyl termini of the functional glycoproteins.

Iron uptake from transferrin and asialotransferrin by hepatocytes from chronically alcohol-fed rats.

Chronic alcohol intake is often associated with alterations to iron homeostasis and an increase in the serum levels of carbohydrate-deficient transferrin. As the liver is a major iron storage site and also synthesizes transferrin, the normal serum iron transport protein, the aim of this study was to test the hypothesis that these disturbances in iron homeostasis were caused by altered hepatocyte iron uptake from the abnormal transferrin. To achieve this, we have investigated iron uptake from both transferrin and asialotransferrin by hepatocytes from male Sprague-Dawley rats fed the De Carli and Lieber alcohol diet. Iron uptake from transferrin by hepatocytes from alcoholic rats was less than 60% that of control values, and in the presence of 50 mM ethanol decreased still further to 35% of the uptake by the corresponding control cells. Iron uptake from rat asialotransferrin was reduced in both groups when compared to that observed from normal transferrin; 13% by control cells and 39% by hepatocytes from alcohol-fed rats. Alcohol, however, had no further effect on asialotransferrin uptake by either hepatocytes from alcohol-fed rats, or their pair-fed controls. Transferrin binding to hepatocytes was also influenced by the alcohol diet. Although there was no difference in binding at 37 degrees C, cells from alcohol-fed rats bound 85% of this total at 4 degrees C, compared to 44% by control hepatocytes. Similar values were also obtained for hepatocyte binding of asialotransferrin; alcohol feeding resulted in an increase in binding at 4 degrees C to 73% from 58% with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease.

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF-induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.