The surface presentations of CD18 and β-integrin-like neutrophil antigens were evaluated in six clinically normal horses twice at seven to 30 day intervals. The monoclonal antibodies (MAb) 60.3 and #38 recognize portions of CD18, the β₂-subunit of heterodimeric integrins LFA-1 (i.e. CD11a/CD18), Mac-1 (i.e. CD11b/CD18) and p150,95 (i.e. CD11c/CD18). Monoclonal antibody #25 putatively recognizes a β-integrin-like surface antigen. Neutrophils were isolated from whole blood and incubated with either Hanks’ balanced salt solution or lipopolysaccharide (LPS, No. L7261 from Salmonella typhimurium) and then with one of three primary MAbs (60.3, #38 or #25). Cells were then incubated with the secondary MAb [fluorescein isothiocyanate (FITC)-conjugated affinipure F(ab')₂ fragment-goat antimouse (GAM) IgG], which acted as a label for fluorescence activated cell sorting. Cell viability measurements were performed pre- and post-incubation; and cell type was confirmed.

Results indicate that unstimulated equine neutrophils expressed CD18 cell surface adhesion molecules almost constitutively (p < 0.05) using MAbs 60.3 and #38. Unstimulated cells incubated with MAb #25 had a labeling percentage of 87.67%, indicating that most equine neutrophils express a β-integrin-like antigen on their surface. The labeling percentages, and mean and peak channel numbers (i.e. indicators of fluorescence intensity) were significantly greater (p < 0.05) in neutrophils incubated with MAbs 60.3, #38, and #25 in comparison to control cells (i.e. not incubated with primary MAb). Some autofluorescence was evident in control neutrophils; however, non-selective fluorescence was minimized by use of a secondary MAb composed of F(ab')₂. Monoclonal antibody 60.3 labeled significantly more (p < 0.05) neutrophils than MAb #38 and had greater fluorescence intensity. Conversely, LPS-stimulated cells incubated with MAbs 60.3 and #38 showed significant decreases (p < 0.05) in the percentage of CD18 moieties labeled compared to unstimulated cells. However, there was no significant alteration in percentage labeling with MAb # 25. Mean and peak channel numbers tended to increase after LPS-stimulation in cells incubated with MAbs 60.3 and # 38; however, no significant differences could be ascribed. This data showed that whilst fewer neutrophils were labeled for CD18 after LPS-stimulation, the neutrophils had a higher density of labeling indicative of quantitative up-regulation. Qualitative up-regulation may also have occurred as the number of cells labeled decreased. Viability pre- and post- incubation ranged from 94 to 100% and was not different, indicating that MAb incubation did not adversely effect equine neutrophils. It was -concluded that unstimulated neutrophils from horses almost constitutively express important integrin cell surface antigens, which are crucial to adhesion, interactive communication, and the immune response. Lipopolysaccharide stimulation of neutrophils causes quantitative up-regulation, and may facilitate qualitative alterations in CD18 moieties. Also MAb 60.3 appears superior to MAb #38 in its ability to label CD18 subunit of equine neutrophils. These MAb modalities could be used to manipulate certain diseases, exacerbated by excessive neutrophil numbers and degranulation (eg. ischemia/reperfusion and respiratory distress syndromes).