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Updated: Aug 4, 2026

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Published on: August 23, 2013
Beta 2-integrin (CD11b/CD18) is the primary adhesive glycoprotein complex involved in neutrophil-mediated immune
1Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Abstract:
The pathogenesis of antisperm antibody (ASA)-mediated infertility is postulated to be related in part to complement (C)-dependent neutrophil-mediated injury to sperm in the female genital tract. We have reported that sperm-bound IgG activated human C and deposited C3 fragments on motile sperm. We also demonstrated that IgG and C3-bound motile sperm adhered to human neutrophils in vitro, and that this adhesion potentiated the localized release of oxygen radicals at the site of neutrophil/sperm membrane contact. The goal of the present study was to identify the neutrophil surface receptor(s) involved in neutrophil/sperm adhesion and to evaluate their relevance to the pathogenesis of neutrophil-mediated immune injury to sperm. Neutrophils were coincubated with motile sperm in the presence of C-fixing ASA+ sera or control sera. After defined incubation periods, the following neutrophil variables were evaluated: 1) surface expression of Fc (Fc gamma RII and Fc gamma RIII) and C receptors (CR1[CD35], CR3 [CD11b/CD18], and CR4 [CD11c/CD18]) by flow cytometry, 2) neutrophil aggregation by flow cytometry, 3) tyrosine phosphorylation of neutrophil proteins by flow cytometry, and 4) the immune adherence and ingestion of sperm by light and scanning electron microscopy (SEM). The functionality of adhesion receptors was studied by use of a panel of anti-leukocyte monoclonal antibodies (mAbs) for their ability to block neutrophil/sperm adhesion and neutrophil aggregation. Only the incubation of neutrophils and sperm in the presence of C-fixing ASA+ sera resulted in marked (> 70%) sperm binding to neutrophils. Consistent with this pattern, a significant (p < 0.0001) increase in surface expression of neutrophil CD11b and neutrophil aggregation was evident. According to SEM, most of the sperm were linked to the neutrophil by the acrosomal region of sperm head. Maximum expression of CD11b antigen was obtained when neutrophils were coincubated with sperm in the presence of C-fixing ASA+ sera. CD11b up-regulation correlated with a significant (p < 0.05) increase in tyrosine phosphorylation of neutrophil proteins during sperm phagocytosis. Only the combination of mAbs directed to the beta 2-integrin, CD11b (D12/SHCL-3), or CD18 (MHM23) subunits maximally inhibited ASA- and C-mediated sperm adhesion to neutrophils (by 70%) or sperm phagocytosis (by 75%), as well as neutrophil aggregation (by 96%). These findings strongly implicate the CD11b/CD18 glycoprotein complex (CR3) in the adhesive events involved in ASA- and C-mediated immune destruction of motile sperm by neutrophils.
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