Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe
Introduction
The human defense toward microorganisms is largely dependent on the armory of neutrophil granulocytes, cells that are triggered to generate potent bactericidal (and sometimes tissue destructive) metabolites at the sites of infection [1], [2]. Neutrophil triggering may be mediated by a large number of exogenous, proinflammatory mediators [3], [4] or by endogenous agonists such as C5a (a split product from the complement component C5) or IL-8 (a cytokine of the CXC family) [5], [6]. We have recently added two new proteins, galectin-1 and galectin-3, to the list of endogenous proinflammatory mediators that have the ability to activate neutrophils [7], [8].
The galectin family of proteins is defined by their β-galactoside-binding capacity mediated by their conserved sequence elements in their carbohydrate recognition domain (CRD) [9]. At present, fourteen mammalian galectins have been identified and described [10], [11], [12], [13]. The fact that galectins may be present in the cell cytosol as well as in the nucleus or may be secreted extracellularly, suggests that these proteins exert many different functions. This has also been shown in numerous reports over the last decade [14], [15], [16], [17].
Two of the most extensively studied galectins are galectin-1 and galectin-3. Galectin-1 belongs to the prototype galectins, consisting of a carbohydrate recognition domain (CRD) of 14 kDa that may aggregate into homodimers [9]. Galectin-3 is a 31-kDa chimeric galectin, containing one CRD linked by a collagenase-sensitive domain to an N-terminal aggregating domain that enables the molecule to form homodimers or hexamers [9], [18].
The galectins-1 and -3 activate the neutrophil NADPH-oxidase provided that the cells have first been primed [7], [8], [19], [20]. The priming phenomenon has been described for many settings in neutrophil activation processes, prominent examples being extravasation of the cells from the blood stream to the tissue [21], [22] and the effects of bacterial lipopolysaccharides (LPS) [23] and chemoattractants such as formyl-Met-Leu-Phe (fMLF) [24]. Many priming mechanisms have been suggested and it is reasonable to believe that different mechanisms, alone or in concert, may be the cause of different priming events [23], [25], [26]. A prevailing view is that the mechanism for priming involves alterations of intracellular signaling (e.g., changed levels of various second messengers) and/or directs effects on the NADPH-oxidase [27]. With respect to the priming mechanism in relation to the galectins, we have, however, shown that priming induced by LPS and fMLF, as well as during neutrophil extravasation, is due to mobilization of intracellular granules, a process that endows the plasma membrane with new galectin receptors [7], [8], [19].
An alternative route for galectin priming has recently been suggested by Dias-Baruffi et al. [28] who reported that removal of sialic acid residues from the neutrophil cell surface exposes specific galactose-containing binding structures for galectins [28], [29]. It is well known that polylactosamines (potential galectin-3 receptors) can be masked by sialic acid on various cell surface glycoconjugates, and that desialylation by treatment with neuraminidase, a sialic acid-cleaving enzyme, influences the interaction pattern in several cell–cell or cell–agonist binding events. This is illustrated by the facts that desialylation of mononuclear blood cells promotes growth of HIV-1 [30], and that partial removal of sialic acid from the surface of transformed pancreatic cells increases adhesion as well as cellular aggregation [31], [32].
In this study, we have investigated how neutrophil desialylation primes for galectin-3-induced respiratory burst activity. We found that Newcastle disease virus neuraminidase with specificity for α2-3,8-linked sialic acid (but no other neuraminidase) could prime neutrophils not only for galectin-3 but also for the chemoattractant fMLF. The mechanism behind priming is due to exposure of receptor structures, either by unmasking (sialic acid cleavage) of cell surface receptors or by upregulation of granule stored receptors induced by the sialic acid cleavage.
Section snippets
Preparation of galectin-3
Recombinant human galectin-3 was produced in Escherichia coli (E. coli) and purified as previously described [33]. The lectin was stored at 4°C in phosphate-buffered saline (PBS; pH 7.2) containing lactose (150 mM). When used, the lectin preparation was applied to a gel filtration column (PD10; Pharmacia, Uppsala, Sweden) to remove lactose, and diluted to 400 μg/ml in Krebs–Ringer phosphate buffer-containing glucose (10 mM), Ca2+ (1 mM) and Mg2+ (1.5 mM) (KRG, pH 7.3).
Isolation of neutrophils
Blood neutrophils were
Cleavage of sialic acid from the cell surface by Newcastle disease virus neuraminidase primes neutrophils for galectin-3 activation
To investigate whether removal of sialic acid from the glycoconjugates present on the neutrophil cell surface could effect the response of the cell to galectin-3, neutrophils were first treated with neuraminidases (NA) of four different origins: Clostridium perfringens (CP), Salmonella typhimurium (ST), Vibrio cholerae (VC), and Newcastle disease virus (NDV), chosen based on their differences in specificity (Table 1) [35], [36], [37].
The neutrophils were preincubated with neuraminidase for 15
Discussion
In this paper, we show that alteration of the cell surface carbohydrate composition with neuraminidases can alter the neutrophil functionality and responsiveness, for example, to galectin-3 or fMLF. This alteration is accompanied by (and possibly due to) degranulation. The specificity with which NDV-NA can induce such granule mobilization and receptor upregulation is intriguing and the mechanisms driving this effect have to be further studied.
We have previously shown that neutrophils can
Acknowledgements
This work was supported by the Swedish Medical Research Council, the King Gustaf the V 80-Year foundation, the Swedish Society of Medicine, and the Swedish Foundation for Strategic Research Network of Inflammation Research.
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