Defensins impair phagocytic killing by neutrophils in biomaterial-related infection

Abstract

The implantation of foreign material carries a risk of infection which frequently is resistant to all treatment short of removing the implant. We have previously shown that these materials activate neutrophils by contact, leading to production of oxygen free radicals accompanied by release of granule products. Such activation further results in depletion of local host defenses, including the capacity of biomaterial-activated neutrophils to kill bacteria. Among the granule products released from neutrophils are small cationic antibacterial peptides (human neutrophil peptides [HNP]) known as defensins. Here we tested the hypothesis that defensins, released from activated neutrophils onto the surface of biomaterials, might play a role in the deactivation of subsequent neutrophil populations. Incubation of neutrophils with purified HNP resulted in a dose-related impairment of stimulus-induced oxygen radical production and of phagocytic killing. Furthermore, fresh neutrophils added to biomaterial-associated neutrophils exhibited impaired phagocytic killing. This impairment could be abrogated by antibody to HNP but not by an irrelevant antibody. Taken together, these observations support the idea that neutrophils activated at a material surface can create, by means of HNP release, an environment hostile to their microbicidal function and that of their infiltrating brethren.

FIG. 1

Effect of polystyrene preincubation on staphylocidal activity. The percent viable staphylococci during 24 h of incubation with neutrophils is shown. Control neutrophils were inoculated with S. aureus at the same time the cells were placed in the polystyrene wells (□). The decrease in the number of viable staphylococci occurred rapidly during the first 2 h of incubation and more slowly after that. These cells killed 65% ± 3% of the inoculum after 2 h and 88% ± 2% of the inoculum after 24 h. The figure also shows the effect on the staphylocidal behavior of fresh neutrophils added to neutrophils that had been preincubated for 1 h in polystyrene wells (●). These cells killed only 38% ± 4% of the inoculum at 2 h and 41% ± 8% of the inoculum at 24 h (P ≤ 0.001 compared to the control at both of these times). The error bars indicate standard errors.

FIG. 2

Extractable defensin of control neutrophils (A), cells which had been incubated on polystyrene for 30 min (B) and for 2 h (C), and neutrophils incubated for 2 h on woven Dacron (D) or silastic (E). Exposure to all three materials was associated with significantly reduced defensin content (P ≤ 0.01 under all conditions). The error bars indicate standard errors.

FIG. 3

(A) Effect of HNP1 on staphylocidal activity. The effect of preincubating neutrophils for 1 h with several concentrations of HNP1 (or buffer control) is shown. The cells were washed and resuspended in KRPG with 10% plasma and then inoculated with staphylococci. The control cells (□) killed 91% ± 1% of the inoculum after 2 h and 88% ± 2% of the inoculum after 24 h. HNP1 at 10 μg/ml (■) did not affect staphylocidal activity. HNP1 at 25 μg/ml (▴) reduced staphylocidal activity to 75% ± 7% killed at 24 h (P ≤ 0.03 compared to the control or HNP1 at 10 μg/ml). HNP1 at 35 μg/ml (○) reduced staphylocidal activity to 31% ± 13% killed at 2 h and 37% ± 13% killed at 24 h (P ≤ 0.01 compared to the control). (B) Effect of HNP2 on staphylocidal activity. The effect of HNP2 on the staphylocidal activity of human PMN is more potent than the effect of HNP1 shown in panel A. Although HNP2 at 10 μg/ml (■) did not significantly affect killing, HNP2 at 15 μg/ml (●) partially inhibited staphylocidal activity: 41% ± 20% of the inoculum was killed at 2 h, and 40% ± 8% was killed at 24 h compared to 89% ± 5% and 85% ± 2% killed at 2 and 24 h by control cells (□) (P ≤ 0.01 at 2 and 24 h). HNP2 at 25 μg/ml (▵) almost completely abrogated staphylocidal activity: 5% ± 15% killed at 2 h and 8% ± 20% killed at 24 h (P ≤ 0.01 at 2 and 24 h compared to the control; P ≤ 0.05 at 2 and 24 h compared to HNP2 at 10 μg/ml).

FIG. 4

Effect of HNP1 on superoxide release. Incubation of neutrophils with 35 μg of HNP1/ml (▨) significantly reduced the superoxide release of unstimulated (unstim) cells and of cells stimulated with PMA, fMLP, and OZ (P ≤ 0.01) compared to unexposed (control) cells (■). Incubation with 17.5 () and 25 (░⃞) μg of HNP1/ml had little effect (mean of two experiments).

FIG. 5

Effect of antibody to HNP on polystyrene-associated inhibition of staphylocidal activity. Compared to control cells, in which staphylococci were added without a prior 1-h incubation of PMN on polystyrene (■), staphylocidal activity was impaired when fresh neutrophils were added to the polystyrene-exposed neutrophils (), but this is overcome by addition of monoclonal antibody specific for HNP (░⃞) but not by irrelevant antibody to VCAM (▨) prior to the addition of fresh neutrophils. This effect was highly significant at both 2 and 24 h after the inoculation of neutrophils with staphylococci (P = 0.00008).