Cryptococcal Glucuronoxylomannan Inhibits Adhesion of Neutrophils to Stimulated Endothelium In Vitro by Affecting Both Neutrophils and Endothelial Cells

Abstract

Cryptococcal infections are often characterized by a paucity of leukocytes in the infected tissues. Previous research has shown that the capsular polysaccharide glucuronoxylomannan (GXM) inhibits leukocyte migration. In this study we investigated whether the capsular polysaccharide GXM affects the migration of neutrophils (polymorphonuclear leukocytes [PMN]) through the endothelium by interfering with adhesion in a static adhesion model. Pretreatment of PMN with GXM inhibited PMN adhesion to tumor necrosis factor alpha (TNF-␣)-stimulated endothelium up to 44%. Treatment of TNF-␣-stimulated endothelium with GXM led to a 27% decrease in PMN adhesion. GXM treatment of both PMN and endothelium did not have an additive inhibitory effect. We demonstrated that GXM-induced L-selectin shedding does not play an important role in the detected inhibition of adhesion. L-selectin was still present on PMN in sufficient amounts after GXM treatment, since it could be further inhibited by blocking antibodies. Furthermore, blocking of GXM-related L-selectin shedding did not abolish the GXM-related inhibition of adhesion. GXM most likely exerts its effect on PMN by interfering with E-selectin-mediated binding. The use of blocking monoclonal antibodies against E-selectin, which was shown to decrease adhesion in the absence of GXM, did not cause additive inhibition of PMN adhesion after GXM pretreatment. The use of blocking antibodies also demonstrated that the inhibiting effect found after GXM treatment of endothelium probably involves interference with both intercellular adhesion molecule-1 and E-selectin binding.Cryptococcus neoformans is an encapsulated yeast that can cause life-threatening meningitis in immunocompromised patients. Compared to bacterial meningitis, cryptococcal meningitis is usually characterized by a relative paucity of leukocytes in the cerobrospinal fluid and infected tissues (4, 6), despite adequate stimulation of cytokine production (6,7,12,46,60,65,67). Previous research has shown that the immunomodulatory effects of cryptococcal capsular polysaccharides contribute to the scant leukocyte infiltrates often observed. Cryptococcal culture filtrate, the isolated capsular polysaccharide glucuronoxylomannan (GXM), and mannoprotein 4 (10) all inhibit the influx of leukocytes into inflammation sites (10, 17, 48). Since GXM and mannoprotein 4 have intrinsic chemoattracting properties (10, 15, 16) and high titers of both are found in the bloodstream during infections (10, 13, 25), it has been hypothesized that these compounds, by cross-desensitization, prevent leukocytes from properly responding to chemoattractants, thereby contributing to the scant infiltrate often reported in cryptococcal infections. This has already been demonstrated for GXM in humans as well as in experimental infections in animal models (17,48,49,50). Another mechanism for the diminished leukocyte transendothelial migration, one not yet analyzed in detail in the literature, could be the interference with leukocyte adhesion to the endothelium at ...

O-Acetylation of Cryptococcal Capsular Glucuronoxylomannan Is Essential for Interference with Neutrophil Migration

“…Before the static adherence assay, neutrophils were incubated at 37°C with 0.1-1 g/ml GXM for 45 min, at which maximal inhibition was detected previously (21). Before the rolling experiments, the neutrophils were treated with 10 g/ml GXM (room temperature, 30 min).…”

“…Human neutrophils were isolated from healthy volunteers as previously described (21 Based on the results of previous studies and dose/response curves, the conditions of GXM treatment of neutrophils (i.e., duration, GXM concentration, and temperature) differed among the various assays (20,21). Before the static adherence assay, neutrophils were incubated at 37°C with 0.1-1 g/ml GXM for 45 min, at which maximal inhibition was detected previously (21).…”

“…Static adhesion assays were performed as previously described (21). HUVECs were plated on fibronectin-coated NUNC 72-well plates (Invitrogen Life Technologies) and grown to confluence in 1-2 days.…”

“…Molecular mass distribution of GXM was determined by gel filtration chromatography of 1 mg/ml GXM samples (Superose 6.0 column; Pharmacia, Peapack, NJ) as previously described (21).…”

“…Second, polysaccharides interfere with leukocyte adhesion to the endothelium. GXM has been demonstrated to induce L-selectin shedding (14,18), to bind CD18 (19), and to actually impair both phases of neutrophil adhesion to the endothelium, namely the initial rolling (20) as well as subsequent firm binding to the endothelium (21).…”