Geneviève Durand

Prevalence of tri- and tetraantennary glycans of human alpha 1-acid glycoprotein in release of macrophage inhibitor of interleukin-1 activity.

Based on the affinity for concanavalin A (Con A), human α1-acid glycoprotein (AGP) can be separated by chromatography on Con A-Sepharose gel into three variants: Con A unreactive AGP, Con A weakly reactive AGP, and Con A strongly reactive AGP. When exposed to native AGP or to its glycan variants, murine peritoneal macrophages released a factor that inhibited the interleukin-1 (IL-1) proliferative activity as measured in terms of the thymocyte comitogenic assay. Con A unreactive AGP, which contains tri- and tetraantennary glycans and no biantennae, proved to be more effective than Con A weakly and Con A strongly reactive variants, which contain one and two diantennary glycans, respectively. The inhibitory effect was not a function of the negative charge related to the sialyl residues and was not mediated by the maanosyl-fucosyl receptor.

Asialoglycoprotein receptor in human isolated hepatocytes from normal liver and its apparent increase in liver with histological alterations.

We have characterized a binding site for galactosyl terminal glycoproteins in hepatocytes isolated from human biopsies. The binding of asialoorosomucoid on hepatocytes previously treated by Triton X-100 was saturable, calcium-dependent and highly affine (Ka = 1.11 +/- 0.87.10(9) M-1) thus corresponding to a ligand-receptor binding. The total number of receptors in the normal human liver was 140,000 +/- 65,000 sites per cell. This corresponded to the value obtained in the human hepatoma cell line HepG2, but was significantly lower than for isolated rat hepatocytes. Furthermore, in hepatocytes isolated from livers with histological features of either fibrosis, cirrhosis, hepatocarcinoma with cirrhosis or nodular regenerative hyperplasia, the number of asialoglycoprotein receptors per cell was increased, while the binding affinity was unchanged.

Human α 1-acid glycoprotein-exposed macrophages release interleukin 1 inhibitory activity

The concanavalin A-unreactive variants of α 1-acid glycoprotein introduced in culture medium of monocytes/macrophages induces the inhibition of thymocyte proliferative activity of interleukin 1. LPS or LPS receptors were not involved in the activity of α 1-acid glycoprotein on macrophages. α 1- acid glycoprotein did not show any direct effect on thymocytes whereas the monocyte/macrophage-supernatant inhibited the interleukin 1 proliferative effect. The activity of tumor necrosis factor and interleukin 2 was not altered by the α 1- acid glycoprotein-macrophage supernatant.

An immunochemical procedure to evaluate the degree of desialylation of α1-acid glycoprotein in rat serum

When radial immunodiffusion (RID) and electroimmunodiffusion (EID) were used for the determination of rat alpha 1-acid glycoprotein (alpha 1-AGP) a significant discrepancy in the results was encountered depending on the degree of sialylation. When alpha 1-AGP was desialylated, the amounts estimated by EID were much lower than those actually present as assayed by the RID method. The relationship between the percentage of desialylation of alpha 1-AGP and the percentage of its underestimation by EID relative to RID was determined and a calibration curve was plotted to evaluate the degree of desialylation of rat alpha 1-AGP. When compared to other procedures (rat membrane inhibition assay and isoelectrofocusing), the proposed method was easier to perform and allowed the specific evaluation of the degree of undersialylation of the glycoprotein.

Diabetes-induced variation in hepatic binding protein

The total capacity of hepatocytes to bind asialoorosomucoid was measured on normal and streptozotocin diabetic rats. 4 days after the streptozotocin injection, a slight decrease of total receptor concentration was observed while a more marked reduction of cell surface receptor occurred. In animals sacrificed 11 days after the streptozotocin injection, the total capacity of hepatocytes to bind asialoorosomucoid was about 70% of the normal level.

A macrophage-derived factor induced by α1-acid glycoprotein that inhibits IL-1 comitogenic activity

After exposure to a concanavalin A (Con A)-unreactive variant of alpha 1-acid glycoprotein (AGP), macrophages released an inhibitor of interleukin-1 (IL-1) proliferative activity in the thymocyte comitogenic assay. This effect was observed with AGP concentrations above 100 micrograms/ml in the macrophage supernatant and would appear to be mediated by the macrophages, since native AGP had no activity on thymocyte proliferation. Preliminary physicochemical characterization showed that the factor was partially resistant to heating, undialyzable, and eluted with an apparent molecular mass of 50-100 kDa when subjected to Sephacryl S-200 chromatography. Murine IL-1 and human (h) recombinant (r) IL-1 were affected by this factor to the same extent. IL-1 and IL-2 co-induced thymocyte proliferation, which is mitogen-independent, was also inhibited, whereas hrIL-2 activity was not suppressed when assayed in thymocytes with PHA at a submitogenic concentration or in CTLL cells. The factor did not interfere with TNF alpha or hrIL-6 activity when tested against their specific cell line. These data indicate that the inhibitor may act specifically against IL-1 activity and further elucidate the possible role of AGP in the modulation of IL-1 activity via the secretion of an inhibitor.

Direct evidence for sialic acid oxidation in periodate-induced lymphocyte proliferation

Abstract Normal mouse spleen cells treated with periodate were stimulated to undergo blastogenesis. In contrast spleen cells from nude mice did not respond to periodate. Such a treatment of normal and nude spleen cells led to the oxidation of the cell surface sialic acid residues with formation of N-AN8. Prior treatment with neuraminidase of normal spleen cells greatly impaired their capacity to respond to periodate activation with a decrease in the amount of N-AN8 formed.

In vivo quantification of removal of asialo-orosomucoid from the circulation in anaesthetized streptozotocin-diabetic rats

SummaryThe in vivo kinetic of removal of3H asialo-orosomucoid from plasma was investigated in control and streptozotocin-diabetic rats after intravenous injection of 1 mg of asialo-orosomucoid/100 g body wt. Michaelis-Menten kinetics of disappearance were observed. In diabetic rats the maximal rate (Vmax) of disappearance of3H asialo-orosomucoid was decreased by 30% with no modification of Michaelis constant. Since no accumulation of desialylated orosomucoid in the circulation was observed, the slower rate of removal of3H asialo-orosomucoid was attributed to a decrease in the number of hepatic asialoglycoprotein receptors which are largely involved in the catabolism of asialoglycoproteins. Our estimate on in vivo maximal rates was 10- to 20-fold greater than our previous in vitro estimate of the maximal rate of endocytosis. In contrast, the values of the Michaelis constant obtained in vivo and in vitro were very similar.

Kinetic constanis of asialoorosomucoid endocytosis comparison between hepatocytes from normal and streptozotocin-diabetic rats

Abstract The initial velocity of asialoorosomucoid internalization is determined for normal and diabetic rat hepatocytes. The analysis of results according to Woolf-Hofstees method, showed no modification of the endocytosis constant. In contrast, the maximum velocity of asialoorosomucoid internalization is decreased by threefold in diabetic rat hepatocytes as compared to normal rat hepatocytes. No modification of internalization constant is observed between the two groups of rats. This suggests that the decrease of asialoorosomucoid total uptake, previously reported for diabetic rat hepatocytes is directly related to a decrease of total surface receptor number.

Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rat

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.