We examined the activity of defensins, cysteine-rich cationic peptides that are abundant in the cytoplasmic granules of human and rabbit granulocytes, against various tumor targets. The three human defensins, HNP-1, HNP-2, and HNP-3, lysed human and murine targets in chromium release and dye exclusion assays. Defensin-mediated tumor cell lysis was concentration-dependent, inhibited by serum, and dependent on temperature-sensitive events. Lysis was first detected by three hours of incubation and it reached a plateau between eight and 14 hours. In vitro exposure of murine teratocarcinoma cells to HNP 1–3 abrogated their oncogenicity in vivo. Nonmalignant target cells were also susceptible to defensin-mediated lysis. Four rabbit granulocyte defensins exerted marked (NP-1, NP-2) or moderate (NP-3a, NP-3b) cytotoxic activity, whereas defensin NP-5 was not cytotoxic. When tumor cells were incubated with human defensins in combination with hydrogen peroxide, synergistic cytotoxicity was detected. As defensins are released from granulocytes by various stimuli, their release could contribute to extracellular cytotoxicity which is independent of reactive oxygen intermediates.
Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.
Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.
Myeloperoxidase (MPO), present in the azurophilic granules of polymorphonuclear leukocytes, is a myeloid enzyme whose synthesis is restricted to promyelocytes. Complete hereditary MPO deficiency affects 1 in 2,000 to 4,000 individuals; however, the genetic cause of this defect is unclear. We have determined the molecular basis of MPO deficiency in one individual (SQ). Granulocytes of SQ had no MPO activity, and had complete absence of mature and precursor MPO protein by Western blotting. Scanning MPO gene structure by Southern blotting detected a novel BgI II fragment in SQ; no other alteration in gross gene structure was detected. We hypothesized that a single base pair mutation formed a new BgI II restriction site, and that this occurred in exon 10 of MPO gene. As predicted, exon 10 from SQ was cleaved by BgI II, but DNA from the normal patients and five other MPO-deficient patients was not cleaved by this enzyme. Direct sequencing of the polymerase chain reaction (PCR) product of exon 10 showed a C to T substitution at codon 569 in exon 10, resulting in arginine (CGG) to tryptophan (TGG) substitution and creating a novel BgI II site. The mutation was homozygous, as shown by both sequencing and Southern blotting, and no other alterations in base sequence were detected. To determine the frequency of this mutation, DNA was collected from 400 normal individuals, and the presence of the mutation was examined by digesting with BgI II after amplifying exon 10 by PCR. No other case with the novel BgI II site was detected, suggesting that this is not a restriction fragment length polymorphism. The rest of the coding region of the MPO gene was sequenced in DNA from SQ, as well as from the five other MPO-deficient individuals and one normal person; no other mutations were found. Our results suggest that a point mutation at codon 569 of MPO gene represents one molecular form of MPO deficiency.
Myeloperoxidase (MPO) is a glycosylated hemoprotein contained in the azurophil granules of human polymorphonuclear leukocytes (PMNs). MPO is thought to play a role in the oxidative antimicrobial activity of neutrophils by catalyzing the formation of hypochlorous acid, a potent microbicide, from hydrogen peroxide and chloride anions. Seven unrelated individuals with complete MPO deficiency, a relatively common heritable defect of neutrophils, were identified during routine blood tests. Molecular analyses were conducted to determine the level of the abnormality in these individuals. Western blot analysis showed that 6 of the 7 donors were devoid of immunoreactive MPO protein, while neutrophils from one individual contained only the 55-Kd subunit. Northern analysis of bone marrow RNA from one MPO-deficient donor showed the presence of the normal-sized 3.3-kb transcript indicating that the defect in MPO biosynthesis in this case was posttranscriptional. Southern analysis of four MPO-deficient donors showed a normal Bgl II digestion pattern, whereas an abnormal restriction pattern was observed in a fifth individual. Although the Bgl II pattern was similar to that observed in an unrelated subject described by Nauseef (Blood 73:290, 1989), our study strongly suggests that the point mutation does not reflect a polymorphism. Taken together, these analyses show the existence of diverse abnormalities associated with MPO-deficiency that may be detected at the level of the MPO polypeptide, mRNA, and gene.
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