Interaction between a neutrophil and an ICAM-1 coated bead during the adhesion experiment: A. An ICAM-1 coated bead (4.5 μm diameter) is held in the left pipette and the neutrophil is held in the right pipette. B. The contact between the beads and the cell. Adhesion events are scored when there is a small deflection of the cell surface as the cell and bead are separated. The adhesion probability is calculated as the number of adhesive events over the total number of touches.

Effect of NPPB on CBRM1/5 surface expression in Na glucuronate and Na glutamate buffer. Neutrophils (5×10⁶ cells/ml) were incubated in BSS with or without NPPB. Antibody binding capacity (ABC) was obtained using Quantum Simply Cellular Beads as described in Materials and Methods. For Na glucuronate each column shows the mean of four experiments with neutrophils from four different donors (each with duplicate samples). The error bars represent s.e.m. For Na glutamate, each bar represents the mean of two experiments, each with two replicates.

Effect of different anions on the fraction of Mac-1 in the active state. The fraction of active Mac-1 was determined as described in Fig. 2. Na glutamate – filled rhombuses; Na glucuronate – filled circles; Na gluconate – filled stars; BSS – opened squares. Experiments were performed at 21°C. For glutamate, fractions greater than 1.0 reflect measurement uncertainty, either in underestimating the total integrin on the cell surface (obtained from Bear-1 binding) or overestimating activated forms (CBRM1/5 binding).

Effect of chloride substitutes on neutrophil adhesion to ICAM-1. Experiments were performed in BSS, in low Cl^- medium (glucuronate or glutamate), or in low Cl^- medium in the presence of mAb38 (LFA-1 blocking antibody), ICRF44 (Mac-1 blocking antibody), IB4 (β₂ blocking antibody) or 100 μM NPPB as indicated. Each cell-bead pair was contacted 25 times for 2 seconds each time. Adhesion was detected as described in Materials and Methods, and adhesion probability was calculated from the number of adhesive events divided by the number of contacts. Each column represents the mean value from experiments on 4-6 cells from each of 2-4 different donors. Error bars represent s.e.m.

Effect of varying chloride substitute concentration on the expression of Mac-1 activation epitope, recognized by CBRM 1/5 binding. The fit is done using the form of the Michaelis-Menten equation: f = f_max(x/(K_m+x)), where f_max is the fraction of integrins in the high affinity state at saturating concentrations of anion and K_m is the Michaelis-Menten constant, which corresponds to the apparent K_d of the binding reaction. The ratio was calculated based on CBRM1/5 binding measured at each concentration, and an estimate of the total number of integrins on the surface based on the data in Figure 3. A. Chloride was substituted with glucuronate (open circles), glutamate (closed squares) is shown as a reference: f_max=0.55 ± 0.04, K_m = 24.7 ± 7.2 mM, R²=0.894. B. Chloride was substituted with glutamate (closed squares), glucuronate (open circles), is shown as a reference: f_max=1.12 ± 0.04, K_m=25.9 ± 4.3 mM, R²=0.973.

NPPB inhibition of fMLP-induced CBRM1/5 epitope expression and total Mac-1 expression. Neutrophils (5×10⁶ cells/ml) were treated with or without 100 μM NPPB for 5 minutes at 37°C. Cells were then stimulated without (control) or with 10 nM fMLP for 15 minutes at 37°C. 100 μl aliquots were immediately placed in cold microcentrifuge tubes on ice for antibody labeling. Quantum Simply Cellular Beads were used to determine antibody binding capacity. Each column represents the mean of two experiments (with duplicate determinations in each) and the error bars indicate the range for the three experiments.

NPPB inhibition of Cl^- efflux in low Cl^- medium. Net chloride efflux from ³⁶Cl^- loaded cells into Na glucuronate buffer at 37°C was measured as described in Materials and Methods. The cpm of ³⁶Cl^- are plotted as a function of time after resuspension in the glucuronate medium. The plot shows typical data for control efflux (open circles) and efflux into medium with 100 μM NPPB (filled circles). In 3 experiments, with blood from 3 different donors, the average control rate constant was 0.031 (SE 0.005, n=6) and the average with NPPB was 0.0077 (s.e.m. 0.0020, n=5). Thus, on average 100 μM NPPB reduced Cl^- efflux to 25% of the control rate.

Effect of glucuronate on the fraction of Mac-1 in the activated (CBRM1/5 binding) state at A. 37°C and B. 21°C. The fraction of Mac-1 displaying the CBRM1/5 activation epitope was determined by dividing the number of antibody binding sites for CBRM1/5 by the number of binding sites for Bear-1. Mean fluorescence was converted to antibody binding capacity by calibrating with Quantum Simply Cellular Beads. BSS – opened squares; Na glucuronate buffer – filled circles; cells in Na glucuronate returned to BSS after 30 minutes – filled triangles; 10 nM fMLP stimulated cells – filled square. Means from 3 experiments at 37°C (except for the fMLP data, which represents 2 experiments), together with s.e.m. or range (for fMLP data) are shown in A.; B represents a single experiment with duplicate determinations.

Effect of external anions on neutrophil Mac-1 activation epitope expression, recognized by CBRM1/5 antibody binding. Each bar represents the mean of three experiments, each with duplicate determinations, with cells from different donors (except 37.5 mM glutamate where n=1). Experiments were performed at room temperature. The error bars represent s.e.m. Cases shown as +30mM or +60 mM were hypertonic. The 37.5 mM buffers were obtained by replacement of chloride and were isotonic. Applying ANOVA and Duncan's multiple range test (α = 0.05), we find that differences among the four control BSS buffers (leftmost columns) were not statistically significant, and the differences among the three glucuronate buffers (center columns) were also not statistically significant. However, all glucuronate and glutamate buffers were significantly different from controls, the two glutamate concentrations (30 mM and 60 mM) were different from each other and different from all glucuronate-containing buffers.

Effect of glucuronate on Mac 1 expression and activation. Neutrophils (5×10⁶ cells/ml) were either maintained in BSS (opened squares) or resuspended at time zero in glucuronate buffer (filled circles). 100 μl aliquots were taken at the indicated times and placed on ice. At 30 minutes a portion of cells in glucuronate buffer was centrifuged, resuspended in BSS, and returned to the 37°C water bath (filled triangles). Neutrophils were also stimulated with 10 nM fMLP for 30 minutes in BSS (filled square). Antibody binding capacity (ABC) was obtained using Quantum Simply Cellular Beads. Each point represents the mean of three experiments, each with duplicate determinations, with cells from different donors, except for the fMLP data, where the mean of two experiments is shown. Experiments were performed at 37°C. Error bars indicate standard error of the mean (s.e.m.) or, for the fMLP data, range of the two values. A. Antibody binding capacity for the Mac-1 activation epitope, recognized by CBRM1/5 antibody binding. B. Antibody binding capacity for the total Mac-1 surface expression, recognized by Bear-1 antibody binding.