Background/Purpose: Systemic lupus erythematosus (SLE) is associated with endothelial dysfunction, which can accelerated inflammation. Ingress of neutrophils into tissue requires chemotaxis and adhesion to endothelial cells. The goal of this project was to explore possible mechanisms leading to increased adhesion of neutrophils to glomerular endothelial cells induced by lupus serum.

Methods:

SLE patients in a longitudinal cohort that met four or more ACR criteria had serum collected and stored at -80°C during paired clinic visits with less activity (SLE Disease Activity Index (SLEDAI) score 0-6) and more activity (SLEDAI score 4-14 but always 4 points higher than inactive visit). Primary human renal glomerular endothelial cells (HRGECs) were cultured according to the manufacturer’s protocol and used at passages 3-4 and were seeded at 8 x 10⁴ cells per fibronectin-coated well and grown to 90% confluence. HRGECs were cultured in SLE serum (2.5% in culture medium) for 3 hours in triplicate. Serum was removed and replaced with culture medium. Calcein AM-stained neutrophils isolated from healthy controls were cultured with HRGECs for 20 minutes. Neutrophil adherence to serum-treated HRGECs after five washes was measured as percent of cells (in fluorescence units) adhering relative to a TNFα positive control. Supernatant from “active disease” cultures was collected after neutrophil culture and analyzed for mediators of inflammatory cell vascular adhesion and transmigration (intracellular adhesion molecule-1 (ICAM), vascular CAM-1 (VCAM), neural CAM (NCAM), platelet-derived growth factor, myeloperoxidase, cathepsin D, plasminogen activator inhibitor-1, and interleukin 8 (IL8)) by commercial multiplex bead array. Associations between variables were determined using Spearman correlation and Mann-Whitney U test. Group values were reported as the median (interquartile range).

Results:

32 SLE patients (13 with lupus nephritis) were enrolled with paired active/inactive visits. Patients were 95% female and 73% African-American and had a median age of 40 (18). To explore factors that associated with increased adhesion, supernatant concentrations of mediators were correlated with relative neutrophil adherence and the fold change adherence from inactive to active disease visits. Cathepsin D (r=-0.46, p=0.05) and IL8 (r=0.77, p<0.001) correlated significantly with relative neutrophil adherence. NCAM (r=-0.54, p=002), cathepsin D (r=-0.63, p=0.005), and IL8 (r=0.50, p=0.03) correlated significantly with fold change in adherence with high versus low disease activity.

Conclusion: This study suggests that lupus serum induces expression of mediators that affect neutrophil adhesion. The association of neutrophil adhesion with IL8 is consistent with reports of elevation of IL8 in lupus nephritis and induction by anti-dsDNA antibodies. The negative association with soluble NCAM (not previously reported in HRGECs) may be the result of soluble NCAM competing with the membrane-bound form. Cathepsin D can both induce neutrophil apoptosis and increase the adhesiveness of fibronectin. Neither of these hypotheses was tested in this study.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/lupus-serum-induces-glomerular-endothelial-cell-neutrophil-adhesion-in-association-with-soluble-mediators-of-chemotaxis-and-adhesion/