The Janus-like role of neuraminidase isoenzymes in inflammation.

The processes of activation, extravasation, and migration of immune cells to a site are early and essential steps in the induction of an acute inflammatory response. These events are an essential part of the inflammatory cascade, which involves multiple regulatory steps. Using a murine air pouch model of inflammation with LPS as an inflammation inducer, we demonstrate that isoenzymes of the neuraminidase family (NEU1, 3, and 4) play essential roles in these processes by acting as positive or negative regulators of leukocyte infiltration. In genetically knocked-out (KO) mice for different NEU genes (Neu1 KO, Neu3 KO, Neu4 KO, and Neu3/4 double KO mice) with LPS-induced air pouch inflammation, leukocytes at the site of inflammation were counted, and the inflamed tissue was analyzed using immunohistochemistry. Our data show that leukocyte recruitment was decreased in NEU1- and NEU3-deficient mice, while it was increased in NEU4-deficient animals. Consistent with these results, systemic as well as pouch exudate levels of pro-inflammatory cytokines were reduced in Neu1 and increased in Neu4 KO mice. Pharmacological inhibitors specific for NEU1, NEU3, and NEU4 isoforms also affected leukocyte recruitment. Together our data demonstrate that NEU isoenzymes have distinct-and even opposing-effects on leukocyte recruitment, and therefore warrant further investigation to determine their mechanisms and importance as regulators of the inflammatory cascade.

Inhibitors of Human Neuraminidase Enzymes Block Transmigration in vitro.

Cell migration to a site of inflammation is an important step of the immune response. This process is coordinated by cytokines, receptors, and the signal processing machinery of the cell. Many cellular receptors are glycosylated, and their activity can be modulated through changes in glycan structure. Furthermore, glycosylation can be critical to the folding and trafficking of receptors. In this work, we investigated the role of native human neuraminidase enzymes (NEU) in transmigration. We used a cultured T cell line (Jurkat) and a transwell assay with fibronectin (FN) coated wells and cytokines (IL-4 and TNF-α) as chemoattractants in the bottom chamber. We observed that NEU1, NEU3, and NEU4 were positive regulators of transmigration using an siRNA knockdown. Furthermore, we found that pharmacological inhibition of these enzymes inhibited transmigration. We conclude that human NEU isoenzymes NEU1, NEU3, and NEU4 can act as positive regulators of transmigration and should be investigated as targets for anti-inflammatory strategies.

Isoenzyme-Selective Inhibitors of Human Neuraminidases Reveal Distinct Effects on Cell Migration.

The human neuraminidase enzymes (NEU1, NEU2, NEU3, and NEU4) are a class of enzymes implicated in pathologies including cancer and diabetes. Several reports have linked neuraminidase activity to the regulation of cell migration in cancer cells. Using an in vitro cell migration assay on fibronectin (FN) coated surfaces, we have investigated the role of these enzymes in integrin-mediated cell migration. We observed that neuraminidase inhibition caused significant retardation of cell migration in breast cancer (MDA-MB-231) and prostate cancer (PC-3) cell lines when using inhibitors of NEU3 and NEU4. In contrast, inhibition of NEU1 caused a significant increase in cell migration for the same cell lines. We concluded that the blockade of human neuraminidase enzymes with isoenzyme-selective inhibitors can lead to disparate results and has significant potential in the development of anticancer or wound healing therapeutics.

Neuraminidase-3 Is a Negative Regulator of LFA-1 Adhesion.

Within the plasma membrane environment, glycoconjugate-receptor interactions play an important role in the regulation of cell-cell interactions. We have investigated the mechanism and activity of the human neuraminidase (NEU) isoenzyme, NEU3, on T cell adhesion receptors. The enzyme is known to prefer glycolipid substrates, and we confirmed that exogenous enzyme altered the glycolipid composition of cells. NEU3 was able to modify the sialic acid content of purified LFA-1 in vitro. Enzymatic activity of NEU3 resulted in re-organization of LFA-1 into large clusters on the membrane. This change was facilitated by an increase in the lateral mobility of LFA-1 upon NEU3 treatment. Changes to the lateral mobility of LFA-1 were specific for NEU3 activity, and we observed no significant change in diffusion when cells were treated with a bacterial NEU (NanI). Furthermore, we found that NEU3 treatment of cells increased surface expression levels of LFA-1. We observed that NEU3-treated cells had suppressed LFA-1 adhesion to an ICAM-1 coated surface using an in vitro static adhesion assay. These results establish that NEU3 can modulate glycoconjugate composition and contribute to the regulation of integrin activity. We propose that NEU3 should be investigated to determine its role on LFA-1 within the inflammatory cascade.

Galectin-3 alters the lateral mobility and clustering of ß1-integrin receptors.

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5ß1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased ß1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins.

Integrin-mediated cell migration is blocked by inhibitors of human neuraminidase.

Integrins are critical receptors in cell migration and adhesion. A number of mechanisms are known to regulate the function of integrins, including phosphorylation, conformational change, and cytoskeletal anchoring. We investigated whether native neuraminidase (Neu, or sialidase) enzymes which modify glycolipids could play a role in regulating integrin-mediated cell migration. Using a scratch assay, we found that exogenously added Neu3 and Neu4 activity altered rates of cell migration. We observed that Neu4 increased the rate of migration in two cell lines (HeLa, A549); while Neu3 only increased migration in HeLa cells. A bacterial neuraminidase was able to increase the rate of migration in HeLa, but not in A549 cells. Treatment of cells with complex gangliosides (GM1, GD1a, GD1b, and GT1b) resulted in decreased cell migration rates, while LacCer was able to increase rates of migration in both lines. Importantly, our results show that treatment of cells with inhibitors of native Neu enzymes had a dramatic effect on the rates of cell migration. The most potent compound tested targeted the human Neu4 isoenzyme, and was able to substantially reduce the rate of cell migration. We found that the lateral mobility of integrins was reduced by treatment of cells with Neu3, suggesting that Neu3 enzyme activity resulted in changes to integrin-co-receptor or integrin-cytoskeleton interactions. Finally, our results support the hypothesis that inhibitors of human Neu can be used to investigate mechanisms of cell migration and for the development of anti-adhesive therapies.

Phytochemical screening and in vitro bioactivities of the extracts of aerial part of Boerhavia diffusa Linn.

OBJECTIVE: To investigate the bioactivities of crude n-hexane, ethyl acetate and methanol extracts of aerial part of Boerhavia diffusa Linn. (B. diffusa) and its phytochemical analysis. METHODS: The identification of phytoconstituents and assay of antioxidant, thrombolytic, cytotoxic, antimicrobial activities were conducted using specific standard in vitro procedures. RESULTS: The results showed that the plant extracts were a rich source of phytoconstituents. Methanol extract showed higher antioxidant, thrombolytic activity and less cytotoxic activity than those of n-hexane and ethyl acetate extracts of B. diffusa. Among the bioactivities, antioxidant activity was the most notable compared to the positive control and thus could be a potential rich source of natural antioxidant. In case of antimicrobial screening, crude extracts of the plant showed remarkable antibacterial activity against tested microorganisms. All the extracts showed significant inhibitory activity against Candida albicuns, at a concentration of 1000 µg/disc. CONCLUSIONS: The present findings suggest that, the plant widely available in Bangladesh, could be a prominent source of medicinally important natural compounds.

Antinociceptive and anti-inflammatory activity of the ethanolic extract of Cymbidium aloifolium (L.).

The ethanol leaf extract of Cymbidium aloifolium (L.) was evaluated for its analgesic and antiinflammatory activities. The extract, at the dose of 200 and 400 mg kg(-1) body weight, exerted the analgesic activity by observing the number of abdominal contractions and anti-inflammatory activity against Carrageenin induced paw edema in mice by measuring the paw volume. The ethanolic extract of Cymbidium aloifolium (L.) showed statistically significant (p < 0.05) reduction of percentage of writhing of 33.57 and 61.31% at 200 and 400 mg kg(-1) oral dose, respectively, when compared to negative control. The Ethanolic plant extract also showed significant (p < 0.05) dose dependent reduction of mean increase of formation of paw edema. The results of the experiment and its statistical analysis showed that the ethanolic plant extract had shown significant (p < 0.05) dose dependent analgesic and anti-inflammatory activities when compared to the control.