Protocol development for reliable isolation of tear-film neutrophils and in vitro functionality testing
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During prolonged eye closure, such as during sleep, leukocytes are recruited to the ocular surface, with polymorphonuclear neutrophils (PMNs) representing the largest population. PMNs are essential inflammatory cells of the innate immune system, and possess several efficient killing mechanisms, such as phagocytosis and release of antimicrobial substances stored in granules, to protect the host tissues from invading pathogens. Tear-film neutrophils collected from the closed-eye environment have been shown to express high levels of degranulation and activation leukocyte markers (such as CD66b and Mac-1). As these cells may play a role in ocular inflammation and disease, it is important to determine their functionality. However, large variations in response and collection numbers have been observed previously. Limited knowledge also exists on the ability of tear film PMNs to respond to cytokines and mount an oxidative response. Thus, the objectives of this thesis were to develop a standard protocol to process tear-film PMNs to reliably conduct functionality tests in vitro and to assess the production of reactive oxygen species (ROS) in tear film PMNs. Specifically, 1) the sensitivity of tear-film PMNs to experimental procedures (fixation, centrifugation and incubation) was investigated in terms of expression of surface receptors via flow cytometry; 2) two different cell collection methods were evaluated, and changes in the expression of surface receptors of tear-film PMNs when exposed to interleukin-8 (IL-8) and phorbol 12-myristate 13-acetate (PMA) were examined through flow cytometry; and 3) the ability of tear-film PMNs to generate ROS was assessed using luminol-enhanced chemiluminescence. The response of tear-film PMNs was also compared to blood-isolated PMNs. Up to 20 participants were recruited in this research to perform cell collection and donate a small blood sample. A gentle eye wash method was used to collect cells from the closed-eye environment, whereby participants washed their eyes with sterile phosphate buffer saline (PBS) upon awaking at home, and collected the runoff into a sterile polypropylene tube, which was then delivered to the lab within two hours. The patch-OSCCA collection was also tested; participants slept at home and covered one of their eyes with a patch. When they woke up in the morning, they came to the lab directly with one eye covered, and cells were collected using the ocular surface cell collection apparatus (OSCCA), which gently irrigates the ocular surface with warm PBS and collects the solution and cells from a funnel into a centrifuge tube. Very few cells were obtained using the patch-OSCCA collection method and thus this method was not pursued further in this research. When assessing the effect of experimental procedures and measuring cell activation upon stimulation with IL-8 and PMA (a PKC activator), changes in the expression of CD11b (activation and adhesion leukocyte membrane receptor), CD16 (degranulation and phagocytosis marker), CD66b (membrane receptor expressed upon cell degranulation), CD45 (leukocyte common antigen) and CD55 (complement activation marker) were characterized by flow cytometry. ROS production in stimulated (PMA, lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear-film PMNs was measured using luminol-enhanced chemiluminescence (CL). In all experiments, blood-isolated PMNs were also used to allow for comparison in phenotype. Fixation with paraformaldehyde (PFA) is an important step in flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (5-fold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, it was found that an additional centrifugation step prior to antibody incubation and long (4hr) incubation at 37oC significantly altered the expression of membrane receptors with significant reduction in expression of CD11b, CD16 and CD55 when compared to control samples. Therefore, to preserve cell phenotype and cell integrity of tear film PMNs, any additional centrifugation and incubation step should be avoided and post fixation staining is recommended. To gain a further understanding on the phenotype of tear-film PMNs, their ability to respond to IL-8, a cytokine present in the tear film of the closed-eye environment, was characterized via flow cytometry. The expression of surface receptors CD11b, CD16, CD55 and CD66b on tear-film PMNs remained relatively unchanged when exposed to IL-8, whereas some changes in the level of expression of surface receptors were observed in response to PMA but in a lower magnitude compared to blood-isolated PMNs. The respiratory burst is one of the essential killing mechanisms for PMNs and is also related to phagocytosis. PMA stimulation was able to induce ROS production (as measured by chemiluminescence) in tear-film PMNs, and two distinct responder groups were observed, where the high responder group produced significantly more ROS than the low responders. LPS and fMLP failed to induce intracellular ROS production in tear-film PMNs although fMLP-stimulated tear-film PMNs generated ROS extracellularly in the first three minutes. These results suggested that the signalling pathways downstream of PKC as well as the NADPH oxidase were functional but that intracellular signalling pathways upstream the PKC were impaired. This thesis contributes new and important knowledge on tear film PMNs. We proved that the gentle eye wash method is currently the most effective at home collection method. In addition, our study demonstrated that experimental procedures can significantly affect the expression of membrane receptor expression on tear-film PMNs, and if these processing steps are not carefully considered, conclusions on the phenotype of tear-film PMNs can be severely impacted. Our findings also suggest that the lack of response to stimuli may be due to an impairment in the receptor-mediated intracellular signalling pathway and/or insufficient substrates within the cell. Finally, our study on respiratory burst identified two type of responders, which could have significant implications for microbial keratitis and contact lens-induced infiltrates. This thesis highlights the potential role of tear film PMNs in ocular health and inflammation but more work is still needed to gain a better understanding of the phenotype of tear film neutrophils in the closed eye environment and their underlying mechanisms of activation.
Cite this version of the work
Yutong Jin (2019). Protocol development for reliable isolation of tear-film neutrophils and in vitro functionality testing. UWSpace. http://hdl.handle.net/10012/15079