Effect of adhesion on inducible nitric oxide synthase (iNOS) production in purified human neutrophils

Abstract

The production of nitric oxide (NO) within neutrophils is an important element of the innate immune response. We have previously shown that cytokines (IL-1alpha, tumour necrosis factor-alpha and interferon-gamma) induce human neutrophils in buffy coat preparations to produce iNOS. In order to define better the exact requirements for iNOS production within human neutrophils, we have studied the conditions needed for the production of iNOS in purified neutrophils. In contrast to buffy coat preparations, purified neutrophils in suspension did not produce an increase in iNOS following addition of cytokines. However, when purified neutrophils were allowed to adhere to glass surfaces either uncoated or coated with fetal calf serum (FCS), plasma, fibronectin or laminin, there was an increase in the percentage of iNOS-positive cells. The addition of cytokines during adhesion of these cells increased this proportion further. This was most marked for glass alone and FCS-coated glass on which the proportion of iNOS-positive cells increased to 22.7% and 35.5%, respectively, a significant increase compared with cytokine-treated neutrophils in suspension. Neither transmigration through activated endothelial monolayers nor the addition of soluble intercellular adhesion molecule-1 to purified neutrophil suspensions increased the percentage of iNOS-positive cells following cytokine stimulation. Adhesion of neutrophils to surfaces coated with IgG or complement also failed to increase cytokine-induced iNOS production. We conclude that iNOS production in human neutrophils requires not only cytokine stimulation, but also additional stimuli from adhesion to a surface.

Fig. 1

iNOS protein in human neutrophils incubated in suspension for 3 h, (A) without cytokine stimulation, (B) with the addition of IFN-γ, IL-1α and tumour necrosis factor-alpha during incubation. Mag. × 1000.

Fig. 2

Effect of adhesion to the indicated surfaces on iNOS production in purified neutrophils. □, Cells in medium alone; ▪, results from cells incubated in the presence of IFN-γ, IL-1α and tumour necrosis factor-alpha. Each bar represents the mean of four separate experiments; error bars are ± s.e.m. At least 200 neutrophils were counted to obtain the percentage of cells that expressed iNOS. *Significant difference from neutrophils in suspension, with cytokines (Mann–Whitney test with Bonferroni correction, P < 0·05).

Fig. 3

iNOS protein in human neutrophils adhered to fetal calf serum-coated coverslips incubated for 3 h, (A) without cytokine stimulation, (B) with the addition of IFN-γ, IL-1α and tumour necrosis factor-alpha during incubation. Mag. × 1000.

Fig. 4

Effect of transmigration through a confluent endothelial cell layer on iNOS production in neutrophils. The treatments are as follows: S, cells in suspension; S + C, cells in suspension + cytokines; S + C + IL-8, cells in suspension + cytokines + IL-8; S + C + N-formyl-methionyl-leucyl-phenylalanine (fMLP), cells in suspension + cytokines + fMLP; T (IL-8), transmigrated neutrophils using IL-8; T, mo (IL-8), transmigrated neutrophils across membrane alone using IL-8; T (fMLP), transmigrated neutrophils using fMLP; T, mo (fMLP), transmigrated neutrophils across membrane alone using fMLP; A (fMLP), neutrophils adherent to endothelial surface with fMLP as chemoattractant. Means of two to seven separate experiments using three different donors are shown; error bars indicate ± s.e.m. Percentages were calculated by counting at least 200 neutrophils.