Effects of low dose methotrexate therapy on the concentration and the glycosylation of alpha 1-acid glycoprotein in the serum of patients with rheumatoid arthritis: a longitudinal study.

OBJECTIVE: The concentration, and the degree of fucosylation and sialylation of human serum alpha 1-acid glycoprotein (AGP) were investigated for changes during 24-week low-dose methotrexate (MTX) or azathioprine treatment (AZA) in rheumatoid arthritis (RA) patients. METHODS: Serum samples from a longitudinal study were analyzed by crossed affinoimmunoelectrophoresis with the fucose specific Aleuria aurantia lectin. RESULTS: In general, the degree of fucosylation of AGP in RA sera was higher than in control sera, but decreased markedly under the influence of successful therapy with MTX. Concomitantly, the degree of sialylation of AGP increased and the concentration decreased. For alpha 1-protease inhibitor and haptoglobin similar results were obtained. In AZA responders less pronounced changes than in MTX responders were observed. In MTX nonresponders no significant trends were found. As in control sera, large interindividual differences in the AGP values were found. CONCLUSION: The heavy fucosylation of AGP in RA sera reflects disease activity rather than an intrinsic characteristic of people genetically predisposed to RA, since it was found to decrease upon disease improvement. The differences in effects on AGP of MTX and AZA suggest either a gradual difference in a similar mechanism of action, or a different mechanism of action of the drugs. Fucosylated and sialylated AGP could be important in the etiopathogenesis of RA, because these molecules potentially can bind to adhesion receptors (selectins), which could prevent the extravasation of leukocytes into inflamed joints.

Inflammation-induced expression of sialyl Lewis X-containing glycan structures on alpha 1-acid glycoprotein (orosomucoid) in human sera.

The glycosylation of the acute phase glycoprotein alpha 1-acid glycoprotein (AGP) in human sera is subject to marked changes during acute inflammation as a result of the cytokine-induced hepatic acute phase reaction. The changes described thus far comprise alterations in the type of branching of the carbohydrate structures as revealed by increased reactivity of AGP with concanavalin A. We now report on acute inflammation-induced increases in alpha 1-->3-fucosylated AGP molecules, as detected by the reactivity of AGP towards the fucose-binding Aleuria aurantia lectin (AAL) in crossed affino-immunoelectrophoresis of human sera. Laparotomy of women, for the removal of benign tumors of the uterus, was used as a model for the development of the hepatic acute phase response. Hugh increases were detected in the amounts of strongly AAL-reactive fractions of AGP, presumably containing three or more fucosylated N-acetyllactosamine units. At least part of these Lewis X-type glycans (Gal beta 1-->[Fuc alpha 1-->3]GlcNAc-R) appeared to be substituted also with an alpha 2-->3-linked sialic acid residue. This was revealed by the laparotomy-induced abundant staining of AGP with an antisialyl Lewis X monoclonal antibody (CSLEX-1) on blots of sodium dodecyl sulfate-polyacrylamide gels containing AGP isolated from the sera of a patient at various days after operation. It is concluded that acute inflammation induces a strong increase in sialyl Lewis X-substituted AGP molecules that persists at a high level throughout the inflammatory period. We postulate that these changes represent a physiological feedback response on the interaction between leukocytes and inflamed endothelium, which is mediated via sialylated Lewis X structures and the selectin endothelial-leukocyte adhesion molecule 1.

Effect of transgenic expression of human alpha 1-acid glycoprotein (AGP) on the glycosylation of human and mouse AGP in various transgenic mouse sera.

The occurrence and the glycosylation of human alpha 1-acid glycoprotein (AGP) was studied in two classes of transgenic mice expressing either the A, B and B' genes (ABB'-mice) or only the A gene of human AGP (A-mice). The glycosylation of the human AGP molecules in the transgenic mouse sera was compared with the glycosylation of mouse AGP in the same animal and with human AGP in normal human serum by studying their heterogeneity in binding to concanavalin A (Con A), using crossed affino immunoelectrophoresis (CAIE) with Con A as the affinocomponent in the first dimension gel. Three to four different glycosylated fractions of human as well as mouse AGP were revealed by this method in all the transgenic mouse sera. A close relationship was apparent between the heterogeneities in Con A binding of human and mouse AGP in the same transgenic mouse. The magnitude of this so-called Con A reactivity was, however, strongly dependent on the transgenic mouse studied. Especially within the group of ABB'-mice dramatic changes in Con A reactivity were found when the human AGP genes were expressed. This indicates in the first place that the oligosaccharide chains of the human AGP molecules expressed also mouse-specific features. Secondly, and more importantly, these findings indicate that the expression of the human AGP genes affected the glycosylation process of the transgenic mouse liver. This organ is the source of the AGP forms occurring in serum. We do not know whether this effect has been caused by the introduction or the expression of the human gene(s) or by the presence of human AGP in the Golgi system or in serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Inflammation-induced changes in expression and glycosylation of genetic variants of alpha 1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

Changes in the serum concentration and the glycosylation of human alpha 1-acid glycoprotein and alpha 1-protease inhibitor in severely burned persons: relation to interleukin-6 levels.

The relation between interleukin-6 (IL-6) levels and changes in serum concentrations and glycosylation (concanavalin A affinity) of two human acute-phase glycoproteins, alpha 1-acid glycoprotein (AGP) and alpha 1-protease inhibitor (PI), was studied in sequential serum samples of burn patients. The level of IL-6 was already increased at the first day following injury, and after a dip at day 2 or 3 rapidly reached a second maximal value at day 4 or 5. The serum concentrations of AGP and PI reached their maximal values after day 5 and remained at a high level throughout the total period studied (7 weeks). The concanavalin A reactivities of both acute-phase glycoproteins were found to be elevated only during the first 2-2.5 weeks. Maximal values were observed on day 2 and from day 7 to 16, following closely the rise and fall of the IL-6 serum level. After day 16, the concanavalin A affinity rapidly declined long before a decrease was observed in the serum concentrations of AGP and PI. Our previous in vitro studies have indicated an involvement of IL-6 in the induction of both secretion and increased concanavalin A affinity. This study indicates that IL-6 could play a causal role in the induction of both phenomena in vivo.

Isolation and characterization of BanLec-I, a mannoside-binding lectin from Musa paradisiac (banana).

A lectin (BanLec-I) from banana (Musa paradisiac) with a binding specificity for oligomannosidic glycans of size classes higher than (Man)6GlcNAc was isolated and purified by affinity chromatography on a Sephadex G-75 column. It did not agglutinate untreated human or sheep erythrocytes, but it did agglutinate rabbit erythrocytes. BanLec-I stimulated T-cell proliferation. On size-exclusion chromatography, BanLec-I has a molecular mass of approx. 27 kDa, and on SDS/PAGE the molecular mass is approx. 13 kDa. The isoelectric point is 7.2-7.5. BanLec-I was found to be very effective as a probe in detecting glycoproteins, e.g. on nitrocellulose blots.

Con A-nonreactive human alpha 1-acid glycoprotein (AGP) is more effective in modulation of lymphocyte proliferation than Con A-reactive AGP serum variants.

Human alpha 1-acid glycoprotein (AGP) has been shown to modulate various cellular and humoral immune reactions in vitro. Using glycosidase-modified derivatives of AGP, the importance of its carbohydrate moiety with regard to these effects has been noted. In normal serum, three molecular AGP forms interacting differently with concanavalin A (Con A) are present. The ratio of these forms is often changed during various physiopathological conditions. In this study, we could show that differences exist between the three AGP forms with regard to their immunomodulatory effectiveness. At physiological concentrations, the Con A-nonreactive variant AGP-A induced a stronger inhibition of the anti-CD3 stimulated lymphocyte proliferation than the other forms. Interestingly, AGP-A was also found to be responsible for the stimulation of lymphocyte proliferation induced by low AGP concentrations in vitro. Both immunomodulatory effects of AGP were abrogated by desialylation of the glycoprotein. These results support an immunomodulatory role of AGP in conditions characterized by a changed ratio of the differently glycosylated AGP forms.

Con A affinity of rat alpha 1-acid glycoprotein (rAGP): changes during inflammation, dexamethasone or phenobarbital treatment as detected by crossed affino immunoelectrophoresis (CAIE) are not only a reflection of biantennary glycan content.

Using crossed affino immunoelectrophoresis (CAIE), the secretion of the Con A most reactive form (CAIE-3) of rat alpha 1-acid glycoprotein (rAGP) has been shown to be increased in sera of Wistar and Sprague Dawley rats during inflammation and treatment with dexamethasone or phenobarbital. Primary hepatocyte cultures prepared from experimentally treated Wistar rats reflect these in vivo findings, since rAGP as present in corresponding secretion media shows similar changes in Con A reactivity. In this study, the relation of this increase towards the amount of biantennary glycans was investigated for both differently treated rat strains. For this purpose, metabolically labelled rAGP, secreted by isolated hepatocytes under the various conditions, was separated on Con A-Sepharose into four fractions. For each fraction of rAGP its behaviour in CAIE was established, revealing a positive correlation for Con A reactivity between the two methods. However, the enormous increase in Con A reactivity of rAGP in CAIE during inflammation and other conditions (increase in CAIE-3), could not be shown using Con A-Sepharose chromatography. Glycopeptides of each fraction were prepared and the amount of biantennary glycans was assessed. Contrary to expectations, an increase of the total amount of biantennary glycans of rAGP, secreted during conditions associated with an increase in CAIE-3 was not found. The independency of the results with regard to rat strain and procedures used underlined the generality of these findings. Consequently, not only the biantennary glycan content is responsible for the separation of rAGP in CAIE. The importance of other differences in glycosylation, e.g. sialylation, for the increase of rAGP CAIE-3 during various experimental conditions is discussed.