Neutrophil defensins (or human neutrophil peptides-HNP) are major constituents of the azurophilic granules of human neutrophils and have been shown to display broad-spectrum antimicrobial activity. Other activities of these defensins, which are released from stimulated neutrophils, include cytotoxic, stimulatory, and chemotactic activities toward a variety of target cells. We studied the potential use of HNP-1 for antibacterial therapy of experimental bacterial infections in mice. In experimental peritoneal Klebsiella pneumoniae infections in mice, HNP-1 injection was shown to markedly reduce bacterial numbers in the infected peritoneal cavity 24 h after infection. This antibacterial effect was found to be associated with an increased influx of macrophages, granulocytes, and lymphocytes into the peritoneal cavity. These leukocytes appeared to be a requirement for the antibacterial effect, since in leukocytopenic mice administration of HNP-1 did not display antibacterial activity. HNP-1 treatment also reduced bacterial numbers in experimental K. pneumoniae or Staphylococcus aureus thigh muscle infections. In this model, radiolabeled HNP-1 was found to accumulate at the site of infection, whereas most of the injected HNP-1 was rapidly removed from the circulation via renal excretion. These results demonstrate that neutrophil defensins display marked in vivo antibacterial activity in experimental infections in mice and that this activity appears to be mediated, at least in part, by local leukocyte accumulation. ( J. Clin. Invest. 1998. 102:1583-1590.)

Previously we have identified and characterized three murine microbicidal proteins purified from the granule fraction of cells from the murine macrophage cell line RAW264.7. During these studies evidence was obtained for the presence of an additional antimicrobial protein in the cytosolic fraction of RAW264.7 cells that had been activated with interferon-␥ (IFN-␥). In this study we have purified this protein, designated ubiquicidin, to apparent homogeneity and demonstrated that it is a cationic, small (M r 6654) protein. Ubiquicidin displayed marked antimicrobial activity against Listeria monocytogenes and Salmonella typhimurium. Using a gel overlay procedure evidence was obtained that the protein also displays activity against Escherichia coli, Staphylococcus aureus, and an avirulent strain of Yersinia enterocolitica. Aminoterminal amino acid sequencing and mass spectrometry analysis of purified ubiquicidin indicated that it is most likely identical to the ribosomal protein S30. This protein is produced by posttranslational processing of the Fau protein, a 133-amino-acid fusion protein consisting of S30 linked to an unusual peptide with significant homology to ubiquitin. The fau gene has been reported to be expressed in a variety of tissues in humans and various animal species. The presence of ubiquicidin in the cytosol of macrophages may serve to restrict the intracellular growth of microorganisms. In addition, because macrophage disintegration will likely lead to release of ubiquicidin into the extracellular environment, it may contribute to host defense after macrophage death. J. Leukoc. Biol. 66: 423-428; 1999.

The intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes was investigated independently of the ingestion of these bacteria. Granulocytes were allowed to phagocytose preopsonized bacteria for only 3 min, after which the noningested bacteria were removed by differential centrifugation and 2 washes. With this technique, the number of viable cell-associated bacteria at the start of the assay, determined after lysis of the granulocytes, includes about 80% intracellular bacteria. Intracellular killing depends on (a) the temperature (no killing occurring at 4 "C, maximal killing at 33-39"C, and a decrease in the capacity of the granulocytes to kill ingested bacteria at temperatures above 42"C), and (b) the number of bacteria ingested (after phagocytosis at bacteria-to-cell ratios of 100 : 1 and 1000 : 1, not all of the ingested bacteria are killed, whereas after phagocytosis at lower bacteria-to-cell ratios, almost all ingested bacteria are killed). To determine the maximum number of bacteria that can be killed by granulocytes, intracellular killing was measured after phagocytosis of bacteria at various bacteria-to-granulocyte ratios in the presence of phenylbutazone, a drug which inhibits killing during the ingestion period. Phenylbutazone proved to be a useful tool in the study of intracellular killing, since this drug provides a reversible inhibition of the killing when granulocytes are incubated in its presence for up to 3 min, whereas after longer incubation, the inhibitory effect is irreversible. Calculation based on the data obtained in this study gave maximum rates of intracellular killing amounting to 3.7 x lo5 bacteriai5 x lo6 granulocytesimin for Staph. aureus and 8.5 x lo5 bacteria/5 x 10' granulocytesimin for E. coli.Using the rate of intracellular killing after phagocytosis at a bacteria-to-granulocyte ratio of 1 : 1 and the rate of ingestion obtained in an earlier study, we were able to compute the theoretical numbers of viable extracellular not (yet) ingested, viable intracellular, killed intracellular and total intracellular bacteria. The theoretical curves fit well with the experimental data.

A B S T R A C T The role of serum factors in the intracellular killing of bacteria by monocytes was studied on the basis of an assay independent of phagocytosis. After 3 min of phagocytosis of preopsonized bacteria and removal of noningested bacteria, the monocytes containing bacteria are reincubated for various periods and the number of unkilled bacteria is determined by a microbiological method after lysis of the cells.Evidence that this assay measures the killing of ingested bacteria was provided by scanning electron microscopy, lysostaphin treatment, and the effect on the rate of intracellular killing of inactivated serum lacking specific opsonic activity.

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 ;g/ml resulted in full restoration of the activity of the alternative pathway. Using an enzymelinked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.