Human Neutrophil Granule Extracts Research Articles

The Capsule Sensitizes Streptococcus pneumoniae to α-Defensins Human Neutrophil Proteins 1 to 3

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Its polysaccharide capsule causes resistance to phagocytosis and interferes with the innate immune system's ability to clear infections at an early stage. Nevertheless, we found that encapsulated pneumococci are sensitive to killing by a human neutrophil granule extract. We fractionated the extract by high-performance liquid chromatography and identified alpha-defensins by mass spectrometry as the proteins responsible for killing pneumococci. Analysis of sensitivity to the commercial alpha-defensins human neutrophil proteins 1 to 3 (HNP1-3) confirmed these findings. We analyzed the sensitivities of different pneumococcal strains to HNP1-3 and found that encapsulated strains are efficiently killed at physiological concentrations (7.5 microg/ml). Surprisingly, nonencapsulated, nonvirulent pneumococci were significantly less sensitive to alpha-defensins. The proposed mechanisms of alpha-defensin resistance in nonencapsulated pneumococci is surface charge modification, e.g., by introduction of positive charge by D-alanylation of surface-exposed lipoteichoic acids. These mechanisms are surmounted by the presence of the capsule, which we hypothesize is masking these charge modifications. Hence, alpha-defensins in the phagolysosome of neutrophils possibly contribute to intracellular killing after antibody-mediated opsonophagocytosis of encapsulated pneumococci.

Human Neutrophils Kill Bacillus anthracis

Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified α-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that α-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

Cleavage of gonococcal pilin and alteration of common pilin epitopes by human neutrophil lysosomal elastase

Incubation of purified gonococcal pili with human neutrophil granule extracts and highly purified neutrophil elastase, but not cathepsin G, resulted in digestion of pilin (the protomeric subunit of pili). Elastase cleaved pilin (18.6 kDa) in a fashion such that either two, three or four products (14.5, 10.5, 9.0, 4.0 kDa) were generated. With both neutrophil extracts and elastase, digestion of pilin was dependent on enzymatic activity. The 10.5 and 9.0 kDa proteolytic fragments of pifin reacted with antisera to conserved epitopes of the cyanogen bromide 2 fragment of gonococcaln pilin. However, the 14.5 kDa fragment was poorly reactive and the 4.0 kDa fragment was nonreactive. Our results indicate that elastase and possibly other serine proteases of human neutrophils have the capacity to degrade gonococcal pili.

Cleavage of gonococcal pilin and alteration of common pilin epitopes by human neutrophil lysosomal elastase

Incubation of purified gonococcal pili with human neutrophil granule extracts and highly purified neutrophil elastase, but not cathepsin G, resulted in digestion of pilin (the protomeric subunit of pili). Elastase cleaved pilin (18.6 kDa) in a fashion such that either two, three or four products (14.5, 10.5, 9.0, 4.0 kDa) were generated. With both neutrophil extracts and elastase, digestion of pilin was dependent on enzymatic activity. The 10.5 and 9.0 kDa proteolytic fragments of pifin reacted with antisera to conserved epitopes of the cyanogen bromide 2 fragment of gonococcaln pilin. However, the 14.5 kDa fragment was poorly reactive and the 4.0 kDa fragment was nonreactive. Our results indicate that elastase and possibly other serine proteases of human neutrophils have the capacity to degrade gonococcal pili.

Rapid damage to membranes of Neisseria gonorrhoeae caused by human neutrophil granule extracts.

Neisseria gonorrhoeae were exposed to extracts of human neutrophil granules and effects on gonococcal growth and membranes were determined. Enumeration of gonococci by phase-contrast microscopy at 0 and 60 min revealed that they underwent very limited cell division after exposure to granule extract. At 60 min, treated gonococci tended to clump, and some lost their refractivity under phase-contrast optics, indicating membrane damage. Treated and untreated gonococci utilized oxygen at similar rates at time 0; treated gonococci utilized oxygen at a relatively constant rate for 60 min, even though colony-forming ability (i.e. viability) decreased by 90%, whereas untreated gonococci showed a steadily increasing rate of oxygen consumption over the same period, which essentially paralleled increase in colony-forming ability. Membrane ultrastructure of untreated and treated gonococci was compared in thin section by transmission electron microscopy. Extract treatment resulted in a time-related increase in disruption of the bacterial outer membrane, which became apparent almost immediately after treatment. This was accompanied by increasingly aberrant septum structure. Extract treatment also increased the resolution of peptidoglycan by electron microscopy, as early as 10 min after treatment. These data suggest that extract treatment of gonococci caused a rapid loss of the ability to form colonies on agar concomitant with alteration of gonococcal peptidoglycan and outer-membrane structure, but with little alteration of inner-membrane function.

Defensins mediate the microbicidal activity of human neutrophil granule extract against Acinetobacter calcoaceticus.

An acid extract of human neutrophil granules was fractionated on a Sephadex G-100 column and tested for microbicidal activity against Acinetobacter calcoaceticus HO-1 as described previously (M.C. Modrzakowski and C. M. Paranavitana, Infect. Immun. 32:668-674, 1981). The low-molecular-weight protein fraction, peak D, accounted for about 30% of the protein and nearly all of the activity of the crude extract against strain HO-1. Peak D protein proved to be a mixture of the three human defensin peptides HNP1, HNP2, and HNP3. Purified defensins reproduced the microbicidal activity of peak D. The data suggest that defensins could play a major role in the killing of A. calcoaceticus by human neutrophils.

A new elastase inhibitor from Streptococcus pneumoniae protects against acute lung injury induced by neutrophil granules.

A neutrophil elastase-inhibitor isolated from lysed pneumococcal cells, as well as trypsin-digest peptides derived from this factor, were tested for their ability to suppress acute lung injury in mice treated with human neutrophil granule extracts. Injury was assessed by measuring pulmonary sequestration of circulating 125I-labeled albumin, lung water, and lung hemoglobin. Both the native inhibitor and the tryptic-peptides gave good protection when preincubated with granule extract for brief periods before intrapulmonary instillation. Lesser, but still significant, protection was observed in the absence of preincubation. Protection was not simply due to addition of exogenous proteins to the granule extract because substitution of goat immunoglobulin for pneumococcal fraction was ineffective. These results suggest that pneumococcal elastase-inhibitors, recently described by us, may play a role in minimizing lung injury during pneumococcal pneumonia.

Effects of human neutrophil granule extracts on macromolecular synthesis in Neisseria gonorrhoeae.

Neisseria gonorrhoeae were exponentially killed for 120 min (i.e., they were prevented from forming colonies on agar) by extracts of human neutrophil granules; however, macromolecular synthesis, indicated by incorporation of radiolabeled precursors in trichloroacetic acid-precipitable material, continued at or above zero time control values for 45 min. Protein, deoxyribonucleic acid, and ribonucleic acid synthesis appeared to decrease simultaneously after 45 min. Little or no lysis gonococci occurred during the first 60 min of incubation. The ions K+, Na+, Ca2+, Cl1-, SO4(2-) and PO4(3-) at concentrations of less than or equal to 100 mM did not affect granule extract bactericidal activity. On the other hand, 20 mM Mg2+ completely inhibited killing when initially present along with granule extract or when added within 2 to 5 min after granule extract was added to a suspension of gonococci. Gonococci treated with granule extract, washed, and then incubated in gonococci. Gonococci treated with granule extract, washed, and then incubated in the absence of extract died as if extract were still present. The ability of subinhibitory concentrations of actinomycin D or erythromycin to inhibit growth and protein and nucleic acid synthesis was synergistically increased in the presence of granule extract. The above information suggests that a bactericidal component(s) of human neutrophil granules sticks to gonococci, altering their outer membrane permeability and their ability to divide.

Killing of Neisseria gonorrhoeae by human polymorphonuclear neutrophil granule extracts.

Neisseria gonorrhoeae was grown in vitro (on agar and in broth) and in vivo (in 10-day chicken embryos) and tested for its sensitivity to the bactericidal action of human neutrophil granule extracts. Under all conditions studied, type 1 and type 4 N. gonorrhoeae were killed equally well by dialyzed extracts of neutrophil granules (containing both azurophil and specific granule contents) and by the myeloperoxidase-Cl- - H2O2 bactericidal system. However, sensitivity to the bactericidal activity of granule extracts depended upon growth conditions and growth phase. Log-phase, egg-grown gonococci were the most sensitive; they were killed 100% by 250 to 300 micrograms of granule extract (60 min, 37 degrees C) per ml. N. gonorrhoeae grown on agar for 20 h (to stationary phase) were the least sensitive, being killed only 80 to 90% with 500 micrograms of granule extract per ml. Thus, susceptibility to granule extract of gonococci grown under the four conditions studied in this report decreased in the order: log phase, egg grown; log phase, broth grown; stationary phase, egg grown; and stationary phase, agar grown. Killing was time and temperature dependent; little killing occurred when incubations were done at 10 degrees C. Boiled granule extract had only minimal effects on N. gonorrhoeae viability. Addition of catalase (500 U/ml) to the granule extract bactericidal system did not protect; however, the same concentration of catalase completely inhibited the bactericidal activity of the myeloperoxidase-Cl- - H2O2 system.

Myeloperoxidase-Cl--H2O2 bactericidal system: effect of bacterial membrane structure and growth conditions

The human-myeloperoxidase-Cl--H2O2 bactericidal system killed (i) smooth Enterobacteria spp. greater than or equal to rough and (ii) static-grown, stationary-phase bacteria greater than or equal to aerated-grown, log-phase. This is in contrast to human neutrophil granule extracts (involved in nonoxidative bactericidal mechanisms) that kill rough and aerated log-phase Enterobacteria spp. much more efficiently.