Normal but not altered mucins activate neutrophils

Abstract

Interactions between leucocytes and their surroundings are mediated through oligosaccharide epitopes, some of which are also expressed on ocular mucins. Neutrophils represent the majority of immune cells in the proinflammatory environment of the ocular surface during sleep. We have tested whether changes in mucin glycosylation, as occur in dry eyes, influence the phenotype and activation of neutrophils. Peripheral blood leucocytes were circulated over equal concentration mats of ocular surface mucins purified from normal volunteers and dry-eye patients, and in sequence over normal and pathological mucins in all combinations. Non-adherent cells were tagged with monoclonal fluorescent antibodies to leucocyte determinants and analysed by flow cytometry. Oxidative burst, assessed with dihydrorhodamine, was followed in cells and supernatant. At a speed similar to that of leucocyte traffic in the retina, normal mucins caused a decrease in neutrophil cathepsin G fluorescence, a decrease that was not observed with mucins from patients with Meibomian gland disease or Sjogren syndrome. No effect was detected at a higher flow. Supernatant and cells collected after circulation over normal mucin showed increased rhodamine fluorescence, indicative of oxidative burst. Fluorescence could also be observed in intact cells adherent to dry-eye mucins. Non-adherent cells could be activated with phorbol 12-myristate 13-acetate after flow over any mucin or combination of mucins. Differences in neutrophil activation after exposure to normal and pathological mucins highlight reciprocal influences at the interface between local and systemic immunity.

Interactions between human neutrophils and mucin-coated surfaces

“…Since the electrostatic repulsion between the adsorbed mucins and the neutrophils should be largely screened at the high ionic strength used (150 mM), we suggest surfaceshielding through steric hindrance to be the most critical factor determining mucin coating performance. This finding is well in line with previous observations [14,15].…”

“…Our results showed that all mucins could be used to effectively suppress the adhesion of neutrophils to the material surface. Furthermore, in good agreement with previous findings [14,15], it was indicated that dense mucin surface packing is a prerequisite for good coating performance.…”

“…Of more direct relevance to the application of mucins as biomaterial coatings, a previous study suggested the commercial mucin preparation from which we purified the present more albumin-and aggregate-free BSM, to inhibit lymphocyte surface function through steric hindrance [14]. Another study reported that ocular mucins from dry eye patients, which are underglycosylated and adopt compact surface conformations, activate neutrophils to a lower degree than their normal, more diffusely bound counterparts [15].…”

“…Correlations were made between the neutrophil activation and the individual mucin uptakes on the substrates. In complement to previous studies on mucin-leukocyte interaction [14,15], we employed the quartz crystal microbalance technique supplemented with dissipation monitoring (QCM-D) to evaluate possible differences in organization between different mucin coatings. Finally, to predict the behavior of mucin-coated materials in vivo, the neutrophil response towards mucin-coated substrates contacting human serum was studied.…”

Mucin Coatings Suppress Neutrophil Adhesion to a Polymeric Model Biomaterial

“…Presumably, neutrophil-substrate contact is reduced due to the improved surface-shielding associated with higher mucin surface densities. Studies by Berry and coworkers on the interactions between adsorbed human ocular mucins and neutrophils support this hypothesis (Aknin et al, 2004). Thus, they showed that normal mucins activate neutrophils to a higher extent than pathological mucins from dry eye patients.…”