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Rapid Publication Rapid Publication Monocyte-Chemotactic Activity of Defensins from Human Neutrophils Mary C. Territo,* Tomas Ganz,** Michael E. Selsted,*1 and Robert Lehrer*II Departments of*Medicine and §Pathology, and * Will Rogers Institute Pulmonary Research Laboratory, Centerfor the Health Sciences, University ofCalifornia, Los Angeles, California 90024; and IlDepartment ofMedicine, Veterans Administration Medical Center, West Los Angeles, California 90073 Abstract Methods We investigated the monocyte-chemotactic activity of frac- Leukocytes for chemotactic studies were obtained from heparinized tionated extracts of human neutrophil granules. Monocyte- peripheral blood by Ficoll-Hypaque density separation to obtain chemotactic activity was found predominantly in the defensin- mononuclear cells, followed by dextran sedimentation to obtain neu- trophils (5). Cells were washed and resuspended at 106 monocytes or containing fraction of the neutrophil granules. Purified prepa- neutrophils/ml in HBSS containing 0.1% BSA (Calbiochem-Behring rations of each of the three human defensins (HNP-1, HNP-2, Corp., La Jolla, CA). HNP-3) were then tested. HNP-1 demonstrated significant Granule-rich fractions were prepared from neutrophils from single chemotactic activity for monocytes: Peak activity was seen at donor leukophoresis packs (Hemacare, Van Nuys, CA) containing 1-3 HNP-1 concentrations of 5 X 10' M and was 49±20% X 10'° cells, of which > 90% were viable PMN. After suspension in (mean±SE, n = 9) of that elicited by 10-8 M FMLP. HNP-2 HBSS (pH 7.4) with 2.5 mM MgCl2, the cell suspension was sealed in a (peak activity at 5 X i0' M) was somewhat less active, yield- nitrogen "bomb" (Parr Instrument Co., Moline, IL) and pressurized to ing 19±10% (n = 11). HNP-3 failed to demonstrate chemotac- 750 psi for 20 min. The suspension was then released drop-wise into tic activity. Checkerboard analysis of monocyte response to HBSS with 5 mM Na2EDTA while stirring. Nuclei and cellular debris HNP-1 and HNP-2 confirmed that their activity was chemo- were removed by centrifugation at low speed (200 g for 10 min). The pellet was examined by phase-contrast microscopy to ensure > 90% tactic rather than chemokinetic. Neutrophils demonstrated a cellular disruption. The granule-rich supernatant was sedimented at low level of response to defensins but this reaction was pri- 27,000 g for 20 min, and the pellets were stored at -70'C until ex- marily chemokinetic. Defensins may play a role in the recruit- tracted. ment of monocytes by neutrophils into inflammatory sites. Granule proteins were extracted and fractionated essentially as de- scribed by Modrzakowski et al. (2). Briefly, the granule pellets (- 1 Introduction X 10°" cell equivalents total) were extracted three times in a volume of 0.2 M sodium acetate buffer with 0.01 M CaCl2 (pH 4.0) at a final Neutrophils, major cellular components of the inflammatory concentration of2 X 108 cell equivalents/ml. Each extraction was done response, are known to contribute to the regulatory control of over 12 h at 40C with gentle stirring, and the residue was separated by the inflammatory cell population by producing low molecular centrifugation at 27,000 g for 20 min. The pooled acetate extracts were weight chemotactic factors such as leukotriene B4, which can concentrated by ultrafiltration (YC-05 filter; Amicon Corp., Danvers, attract more neutrophils and monocytes into the inflamma- MA) and placed on a Sephadex G-100 column (2.5 by 150 cm). - 70 tory site (1). Since many ofthe neutrophil's biologically active mg of crude granule protein extract was eluted with 0.2 M sodium components are contained within its granules and are released acetate buffer (pH 4.0) and collected in 10-ml fractions. The fractions into the extracellular space in response to stimuli, we exam- were pooled into four fractions (A, B, C, and D) defined by their A280 chemotactic activ- pattern (Fig. 1), concentrated by ultrafiltration (Amicon YC-05 filter), ined PMN granules for proteins with direct and dialyzed against PBS in Spectrapore 3 tubing (3,500 mol-wt cutoff; ity for leukocytes. Spectrum Medical Industries, Los Angeles, CA). - 50% ofthe protein In the initial screening we employed gel permeation chro- in the original granule extract was recovered by this technique. matography to fractionate neutrophil granule extracts into Protein concentration was measured by the bicinchoninic acid four principal protein fractions, as described by Modrzakowski protein assay system (Pierce Chemical Co., Rockford, IL) with chicken and colleagues (2). The composition ofthese factions has been egg-white lysozyme as the standard, according to the manufacturer's partially determined: Fraction A contains myeloperoxidase instructions. Electrophoresis of 1-4 gg of crude granule extract, frac- and lactoferrin, fraction B contains proteases (including elas- tions A-D, and purified human lysozyme, neutrophil elastase, and tase and cathepsin G), fraction C contains lysozyme and frac- defensins was performed on 12.5% acid-urea polyacrylamide gels to tion D in rich is defensins (2, 3), a family of peptides with a confirm the identification of each peak. These preparations were di- broad spectrum of microbicidal activity in vitro (4). luted to the indicated concentrations in HBSS containing 0.01% BSA and tested for chemotactic activity. The approximate molar concen- trations of the fractions were estimated using a molecular weight of Address reprint requests to Dr. Mary Territo, Department of Medi- 100,000 for fraction A (myeloperoxidase and lactoferrin), 25,000 for cine, Hematology/Oncology, UCLA School ofMedicine, Los Angeles, fraction B (elastase and cathepsin G), 12,000 for fraction C (lysozyme), CA 90024-1678. and 4,000 for fraction D (defensins). Received for publication 13 April 1989 and in revised form 28 The human defensins HNP-l, HNP-2, and HNP-3 were purified August 1989. from neutrophil granule preparations. The granules were extracted with 10% acetic acid, and the extract was concentrated under vacuum The Journal of Clinical Investigation, Inc. and chromatographed on Biogel P- 10 to yield a crude mixture of the Volume 84, December 1989, 2017-2020 three defensins. These were separated from each other and further Monocyte-Chemotactic Activity ofDefensinsfrom Neutrophils 2017 Figure 1. A representative A chromatogram of neutro- A B C D 0.04 A phil granule extract on a 10( Sephadex G-l00 column. &(0- I B C The pattern of four major 6'0 peaks was highly reproduc- : 4' ible between preparations, ! 2' a x °l IaC ^v ,,^ 0 with only minor variations -201 Till TtT T in the relative size of the -401- aITd absorbance peaks. Arrows -60 m0 - mm0 q0 m0 _ m 0 _ 0 4 designate the cutoff points (M) for 200 1150 fraction pools A, B, C, CONCENTRATION (M) VOLUME OF ELUANT (ml) and D. Figure 2. Chemotactic B activity of neutrophil purified by ion-exchange and reverse phase HPLC as described pre- A B C D fractions A-D viously (6). The purified granule proteins were dissolved in 0.01% acetic acid 10 for monocytes (A) and and diluted to the desired concentrations in HBSS containing 0.01% 0-- for neutrophils (B). BSA (HBSS-BSA). In some assays, 5% autologous human serum (heat 6(0-- Each fraction was tested inactivated at 560C for 30 min) was substituted for BSA in the dilution 42-0 0 !2' at concentrations of buffer (HBSS-serum). iJl,,,_T I 10-6, 10-7, 10-8, 1o-9 Chemotaxis was determined in triplicate samples in multiwell T and 10-10 M. Data chambers (Neuro Cabin -201 rep- Probe, Inc., John, MD) (7) using a 5-,Om pore -40' ! !!TT I TTI TT!T resent mean±SE of four size filter (Nucleopore Corp., Pleasanton, CA) that separated the upper -60- 40 0 - ZD 0 -' s 0 _? QD 0 _ different granule prepa- well which contained the leukocytes from the lower well which con- II each tested in tained the putative chemotactic factors. Negative controls the rations, using CONCENTRATION (M) triplicate. appropriate dilution buffer were run simultaneously. After 1 h incuba- tion at 370C, the filters were removed and stained with Giemsa. With a microgrid, we determined the number ofcells that had migrated to the higher baseline (control) activity of the cells tested (28±7 lower aspect of the filter (cells/hpf) toward the putative chemoattrac- monocytes/hpf in 5% heat-inactivated autologous serum vs. tant, and subtracted from it the number ofcells that migrated toward 6±4 monocytes/hpf in BSA, n = 3), and a lower incremental the diluent control. This activity was then compared with that of 1o-8 above the M FMLP and the results were expressed as percentage of FMLP ac- activity (level baseline) for FMLP (28±13 mono- tivity. cytes/hpf in serum vs. 37±17 monocytes/hpf in BSA, n = 3). Checkerboard analysis (8) was performed to distinguish chemotac- In HBSS-serum, HNP-1 showed peak activity at concentra- tic from chemokinetic activity. Serial dilutions ofthe tested factor were tions of 5 X I0-' M HNP-1, giving 50%±24% (n = 3) of that placed in the lower well ofthe chamber, and similar concentrations of elicited by FMLP. the factor were used to dilute the leukocytes used in the upper well of In HBSS-BSA, HNP-2 demonstrated some chemotactic ac- the chamber. Directional movement of the cells in response to a con- tivity at 5 X 10-8 to 1 X 10-10 M (42 of64 experiments showed centration gradient indicates chemotaxis, whereas increased random activity > 0%, P = 0.006) with peak at 5 X 10-9 M, that was locomotion ofthe cells induced by the factor unrelated to the concen- 19±10% (n = 1 1) of FMLP activity. HNP-3, which differs tration gradient indicates chemokinesis. Results are expressed as from HNP-1 and HNP-2 only in the single amino terminal cells/hpf.
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