HIV-infected individuals who remain healthy have a natural CD8 cell non-cytotoxic antiviral response (CNAR) that blocks HIV replication in CD4 lymphocytes without killing the cells . CNAR is associated with secretion of an, as yet unidentified, CD8 cell antiviral factor (CAF)  which functions by blocking HIV transcription [3–7]. CAF lacks identity to a large number of cytokines including interferons, interleukins (IL) 1–16, β-chemokines, and granzymes [8–12].
Recently, it was suggested that CAF represents a mixture of α-defensins . α-Defensins are members of a large family of small antimicrobial proteins (reviewed in ). They are cysteine-rich, cationic polypeptides that function primarily by disrupting the membrane of various microbes including bacteria, yeast, protozoa, and some enveloped viruses. Their primary site of activity is thought to be within the phagolysosome . In humans, α-defensins are found circulating in the plasma, within certain epithelial cells, and within the granules of neutrophils , but until the report by Zhang et al. , they had not been detected in CD8 cells. To address further the potential role of α-defensins in CNAR, we examined the mechanism of the defensin-mediated antiviral activity and evaluated the production of α-defensins by CD8 T cells at the RNA and protein levels. We demonstrated that the α-defensins have anti-HIV activity and are expressed by monocytes, but are not made by CD8 cells. Thus, they do not play a role in the CD8 cell non-cytotoxic anti-HIV immune response.
Materials and methods
Peripheral blood mononuclear cells
Heparinized blood samples were obtained by venipuncture from clinically healthy anti-retroviral-treated or untreated HIV seropositive donors who have been infected for more than 8 years. Buffy coat bags from HIV seronegative donors were provided by the Blood Centers of the Pacific (San Francisco, California, USA). The study received approval from the Committee on Human Research, University of California, San Francisco.
Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood samples or buffy coat bags by Ficoll Hypaque (Sigma Chemicals, St. Louis, Missouri, USA) or FicollPaque Plus (Amersham, Piscataway, New Jersey, USA) density gradient centrifugation according to manufacturer's protocol. These mononuclear cell preparations typically consist of approximately 90–95% lymphocytes and less than 5% each of monocytes and neutrophils. CD4 cells and CD8 cells were subsequently obtained from the PBMC by standard immunomagnetic (IM) bead procedures using Dynal (Great Neck, New York, USA)  or Miltenyi (Auburn, California, USA) IM beads. The purity of these isolated cell subsets was > 95% as verified by standard flow cytometry . Monocyte preparations were generated from HIV seronegative individuals in three ways: (i) OptiPrep density sedimentation (Nycomed Pharma, Silver Spring, Maryland, USA) of buffy coat preparations per the manufacturer's instructions; (ii) additional purification of OptiPrep-enriched monocytes, by adherence to plastic for 24–72 h; and (iii) by elutriation of PBMC as described in Bobak et al. . In all cases, preparations were > 95% pure monocytes with no evidence of polymorphonuclear cell (PMN) contamination. The neutrophil-rich buffy coat cell populations studied (typically > 90% PMN) were obtained by dextran sedimentation of anticoagulated whole blood and subsequent hypotonic lysis of erythrocytes .
Purified α-defensins and α-defensin antibody
A mixture of human neutrophil proteins (HNP) 1–3 was purified from human neutrophils. The purity of these peptides was confirmed by reversed-phase high performance liquid chromatography (HPLC) and acid–urea polyacrylamide gel electrophoresis (PAGE) . This mixture was analyzed for its antimicrobial activities using a microbicidal suspension assay and/or an agar diffusion zone-of-clearance assay . The HNP were shown to be antimicrobial for Staphylococcus aureus 502a (> 4 log kill at 50 μg/ml), Listeria monocytogenes and Cryptococcus neoformans (2 log kill at 40 μg/ml).
Rabbit antibodies to natural HNP1–3 were generated as described [21,22]. Immunoglobulin (Ig) G fractions of pre-immune or immune serum were prepared using DEAE Econo columns (BioRad, Hercules, California, USA) according to the manufacturer's instructions. Purified IgG at a 1 : 10 dilution (44 μg/ml) was used to treat samples for 1 h at room temperature prior to addition to the CAF assay. A 1 : 10 dilution of this antibody can neutralize (by > 95%) the antimicrobial activity of up to 200 ng/ml of the purified HNP 1–3 used in this study (data not shown). The antibody was present continuously during the entire assay.
Generation of CAF fluids from CD8 cells
Purified CD8 cells were stimulated with anti-CD3 coupled IM beads  at a bead : cell ratio of approximately 2 : 1 in RPMI 1640 complete medium containing 10% heat-inactivated (56°C, 30 min) fetal calf serum (FCS), 2 mM L-glutamine, and 1% antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin) supplemented with 200 U/ml of recombinant IL-2 (rIL-2; generously provided by Glaxo-Wellcome, Research Triangle Park, North Carolina, USA). After 3 days, the IM beads were removed and the cells were passaged into serum-free AIM-V medium (Gibco-BRL, Gaithersberg, Maryland, USA) containing 200 U/ml rIL-2. Culture fluids were collected every 2 days and the cells passed at 2 × 106 cells/ml in fresh AIM-V medium. The collected culture fluids were filtered (0.45 μm) and stored (−70°C) until tested.
The CAF content in the CD8 cell fluids was measured by a standardized assay . In brief, phytohemagglutinin (PHA)-stimulated CD4 cells obtained from HIV seronegative subjects were acutely infected for 1 h with 4000 50% tissue culture infectious does (TCID50) of HIV-1SF2c/107 cells . This HIV isolate is syncytium inducing (SI) and chemokine-insensitive . In some cases, HIV-1SF162, an NSI chemokine-sensitive isolate [24,9] was used. After washing, 1 × 105 infected CD4 cells/well were plated in triplicate in 96-well culture plates in the presence of a 50% dilution of a CD8 cell culture fluid (or control medium). The cultures were passed every 2 days, replenishing with fresh medium and CD8 cell supernatant, and monitored for viral reverse transcriptase (RT) activity . A reduction in CAF activity was determined by dividing the level of RT activity in culture fluid-treated wells by that found in infected CD4 cells receiving control medium . An inhibition of HIV replication by ≥ 50% was considered CAF-positive; inhibition by < 10% was considered CAF-negative.
Measurement of α-defensin levels in CD8 cell culture fluids
A 100 μl aliquot of CD8 cell culture fluids (stored at −80°C) was removed, acidified to 0.1% trifluoroacetic acid (TFA), and injected onto a 0.21 × 15 cm Vydac C4 reversed-phase HPLC column equilibrated in water containing 0.1% TFA at a flow rate of 0.2 ml/minute. The chromatograph was developed by applying a linear 0–70% acetonitrile : 0.1% TFA gradient at 1% per min. To minimize protein loss, 0.4-ml fractions were collected in low retention, siliconized 1.5-ml conical tubes, containing 200 ng bovine serum albumin dissolved in 20 μl 1% acetic acid. Fractions corresponding to the retention time of HNP1–3 (52–55.6 min) were concentrated 20-fold by roto-evaporation. One μl of the concentrated sample was mixed with an equal volume of 10 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in water : acetonitrile (1 : 1) containing 0.1% TFA. Samples were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) using a Voyager DE-STR mass spectrometer (PerSeptive Biosystems, Framingham, Massachusetts, USA) set at 2600–3000 relative laser intensity.
Treatment of HIV-1 virions with α-defensins
Two hundred and 1000 TCID50 of HIV-1SF33  (diluted in RPMI 1640, or with RPMI containing 10% FCS when noted) were separately incubated with 200 μg/ml purified HNP1–3 (or buffer control) for 1 h in a 37°C water bath. The treated virus was then diluted 1 : 5 and 1 : 20 in RPMI 1640 complete medium and used to inoculate 3 × 106 PHA-stimulated PBMC (previously treated with polybrene ). After 2 h, the cells were washed to remove free virus. Then, 1 × 106 cells were plated in a 48-well plate in quadruplicate in 1 ml RPMI 1640 complete medium containing 100 U/ml rIL-2. Virus replication was monitored by RT activity .
Pretreatment of CD4 cells with α-defensins
PHA-stimulated CD4 cells  were incubated with 100 μg/ml of the defensin mixture (diluted in RPMI 1640), or buffer control, for 1 h (in duplicate for each concentration) in a 37°C incubator. After washing, each treated population was inoculated separately with 200 and 1000 TCID50 of HIV-1SF33, and 100 TCID50 of HIV-1KP1 for 1 h in a 37°C incubator. HIV-1KP1 is a chemokine-sensitive nonsyncitium inducing (NSI) primary isolate. The cells were washed to remove free virus and 0.5 × 106 cells were plated in 700 μl of the RPMI 1640 complete medium in a 48-well plate (in quadruplicate). Virus replication was monitored by RT assay .
Quantification of α-defensin mRNA expression
Total RNA was extracted from purified CD8 cells using TriZol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer's protocol. Total RNA was quantified by measuring the optical density at 260 nm using the SmartSpec 3000 (BioRad, Hercules, California, USA). For quantitative PCR, 500 ng (unless otherwise indicated) of total RNA was reverse transcribed using 250 U murine leukemia virus RT (Invitrogen) in 1 × Amplitaq Buffer (Applied Biosystems, Foster City, California, USA) supplemented with 7.5 mM MgCl2 with 5 μM random hexamers (Invitrogen) 1 mM each of dNTPs (Invitrogen) and 40 U RNase inhibitor (Promega, Madison, Wisconsin, USA). The reaction mixture was incubated at 25°C for 10 min, 48°C for 40 min, and 95°C for 5 min. Five μl of a 1 : 10 dilution of the RT reaction was subjected to PCR using the GeneAmp 5700 Sequence Detection System, SYBR Green PCR Master Mix (Applied Biosystems) and 4 μM primer mix in a 25 μl reaction. Cycling conditions were as follows: initial denaturation at 95°C for 10 min followed by 50 cycles at 95°C for 15 sec and 60°C for 1 min. The forward and reverse α-defensin specific primers were 5'–GTCTGCCCTC TCTGGTCAC–3’ and 5'–AAGCTCAGCAGCAGA ATGC–3', respectively.
To determine the relative number of α-defensin transcripts in each sample, a standard PCR amplification product was made by reverse transcription and amplification of the Reference Human RNA pool (Stratagene, Carlsbad, California, USA) using the α-defensin specific primers described above. The resulting 340 base-pair band was quantified and serially diluted to cover a range of 10–100 000 copies of PCR standard. The standard curve was constructed by plotting the copy number versus the PCR cycle at which SYBR green fluorescence is first detected (Ct).
For analyzing HNP mRNA in purified human monocytes, RT–PCR was performed essentially as described above, except that amplification was carried out for 35 cycles. The amplified product was of the correct size (340 bp) and hybridized to an HNP-1 cDNA in Southern blots (data not shown).
Cell samples were washed in phosphate-buffered saline (PBS), and about 1 × 105 cells were then placed into a chamber of a Shandon cytofunnel (Shandon Lipshaw, Pittsburgh, Pennsylvania, USA) and centrifuged in a Shandon Southern cytospin for 10 min at 1200 rpm. The adhered cells were air-dried onto the glass slide (Fisherbrand Superfrost) at room temperature. Immunocytochemistry was performed essentially as described previously . Briefly, cytospin slides were fixed in 4% paraformaldehyde in phosphate buffer, treated with Fc blocker (Innovex, Litchfield, Minnesota, USA) and Avidin/Biotin (Vector Laboratories, Burlingame, California, USA) according to the manufacturers’ protocols. Slides were then incubated with 1 : 20 rabbit anti-HNP 1–3 IgG or pre-immune IgG. Immunoreactivity was visualized by ABC-Glucose Oxidase and NBT detection kits (both from Vector Laboratories) and counter-stained with Nuclear Fast Red.
Effect of α-defensins on HIV replication in CD4 cells
A standard CAF assay was conducted to evaluate the effect of α-defensins on HIV replication in acutely infected purified CD4 cells . Culturing the infected CD4 cells in the continued presence of various concentrations of a purified α-defensin mixture (HNP1–3), added immediately after infection, did not affect the extent of the ensuing viral replication. This observation was made using NSI or SI isolates of HIV (Fig. 1), and in the case of the NSI isolate, with high and low virus inputs (data not shown).
Effect of α-defensins on HIV virions
Virions, diluted in RPMI 1640, were treated with various concentrations of the HNP1–3 mixture. Serum-free medium was used for dilutions because α-defensin activity is reduced by serum proteins  (see below). The treated virus was further diluted (to reduce the defensins concentration below cytotoxic levels, see below) and used to infect PBMC. A dose-dependent reduction in the subsequent replication of the virus resulted from this treatment (Fig. 2) that was nearly completely abrogated by the presence of 5% FCS (data not shown). In multiple experiments, treatment of 200 TCID50 of HIV with 200 μg/ml of α-defensins (the highest concentration tested) led to a 70–98% inhibition of virus replication, whereas similar treatment of 1000 TCID50 of HIV resulted in only 42–60% inhibition.
To rule out the possibility that the inhibitory activity on the virions reflected an effect of residual α-defensins on the target cells, HIV particles were pelleted by centrifugation (10 000 g for 1 h) immediately after incubating with the α-defensins and resuspended to the original volume. This procedure showed that virions exposed to 200 μg/ml of defensins underwent a similar reduction in infectious virus (70%).
Effect of α-defensins on CD4 cells
Purified CD4 cells were pretreated with various concentrations of the HNP1–3 mixture (ranging from 0 to 100 μg/ml) in the absence of FCS. After washing, the cells were infected with a low input of HIV. Equal numbers of viable defensin-treated or untreated infected CD4 cells were then cultured to measure virus replication. A dose-dependent reduction of virus replication was observed with the HNP-treated cells (Fig. 3). However, pretreatment with the α-defensins did not affect the extent of HIV replication when a fivefold higher input dose of virus was used (data not shown). Enumeration of the CD4 cells, either immediately following pretreatment or after the approximately 1-h infection period, indicated that exposure to the α-defensins caused a dose-dependent reduction in cell number (data not shown). At the highest concentration of defensins tested, an approximately 40% reduction was observed. Viability analyses at these time periods using Trypan blue dye exclusion did not reveal a corresponding decrease in viability suggesting that the reduction in cell number possibly resulted from defensin-mediated lysis of a portion of the cells. At completion of the experiments, a reduction in virus production was observed without evidence of further loss of CD4 cells (data not shown). These latter results most likely reflect an antiviral mechanism that is independent of cytotoxicity. Performing the pretreatment procedure in the presence of 5% FCS reduced the antiviral effect by > 80% and the cytotoxic effect by > 95%.
Lack of evidence for α-defensin production by purified CD8 cells
Studies aimed at defining the role of α-defensins in the mediation of CAF activity did not reveal any evidence for their involvement. In these studies, quantitative RT–PCR was used to measure the level of α-defensin mRNA expression in unstimulated (freshly-isolated) and PHA-stimulated CD8 cells isolated from two HIV-infected individuals (showing strong CNAR activity) and three uninfected individuals. During a time course of 0, 6, 48, and 96 h, CD8 cells were not found to express detectable levels of mRNA coding for α-defensins (< 10 copies/500 ng RNA). However, in these same experiments, freshly isolated PBMC were shown to produce appreciable levels of message for the α-defensins (4500 copies/500 ng RNA). In a separate experiment, we detected α-defensin message from as low as 63 ng of total RNA isolated from unstimulated PBMC from an uninfected individual. However, the levels of message were undetectable in purified CD8 cells even when high amounts of total RNA were analyzed (1000 ng). The detected α-defensin message in PBMC was most likely from monocytes present in the PBMC. Purified human monocytes were found to contain substantial levels of mRNA for HNP1–3 that were readily detected by RT–PCR (Fig. 4).
Intracellular expression of α-defensin proteins was analyzed in CD8 cells purified from the PHA-activated PBMC of two HIV-infected individuals who showed strong CNAR activity, and buffy coat leukocytes (mostly PMN) and PBMC from HIV seronegative donors. Strong HNP immunoreactivity was observed in neutrophils in buffy coat and PBMC preparations (Fig. 5c, e, and g), but was absent in purified activated CD8 cells (Fig. 5a and b) and lymphocytes in PBMC preparations (Fig. 5f and g)). HNP peptides were also detected in human monocytes (Fig. 5g) confirming our mRNA expression data (Fig. 4) and demonstrating that cells of the monocyte lineage possess these important antimicrobial molecules. Unexpectedly, we found that lymphocytes in cytospin preparations of unfractionated buffy coat cells, consisting mostly of neutrophils (data not shown), or a 50 : 50 mixture of buffy coat cells with PBMC were immunopositive for defensins (Fig. 5e). In repeated experiments, lymphocytes in buffy coat preparations were immunopositive for HNP1–3, but these cells were uniformly defensin-negative when first separated from buffy coat granulocytes by Percoll or Ficoll-Hypaque gradient centrifugation prior to the preparation of cytospins.
Quantification of α-defensin peptides by biochemical/MS methods in five CAF-positive and five CAF-negative culture fluids from CD8 cells  also revealed undetectable levels of α-defensin peptides. Analysis of control fluids, to which α-defensin was added exogenously, showed the method used could easily detect levels as low as 50 ng/ml (Fig. 6). This amount is more than an order of magnitude below the amount of these peptides required to demonstrate antimicrobial activity . Finally, treatment of CAF-containing culture fluids with an amount of anti-HNP1–3 antibodies capable of completely neutralizing more than 100 ng/ml of these peptides did not reduce the extent of CAF activity in a standard CAF assay  (Fig. 7).
Human α-defensins are small, antimicrobial peptides produced primarily by neutrophils and epithelial cells . They have been recently reported to have anti-HIV activity and were proposed to constitute the CD8 cell anti-HIV factor, CAF . In the present study we show that a mixture of purified HNP1–3 can directly render HIV particles noninfectious and can also affect CD4 cells so as to reduce their ability to replicate HIV, but only under low protein conditions (Figs 2 and 3). We also demonstrate that, in addition to expression by neutrophils, the α-defensins are produced by monocytes of uninfected individuals. A previous study had suggested the presence of α-defensins in monocytes/macrophages , but in that study there was no evidence of HNP mRNA expression. Importantly, these peptides are not produced by CD8 cells of uninfected or HIV-infected subjects neither at the RNA or protein levels. Thus, they do not play a role in the CD8 cell mediated noncytotoxic inhibition of HIV replication.
The direct antiviral effect of α-defensins on virions may result from disruption of the virus particle, as proposed for other defensin-like molecules [28,30], or possibly prevention of binding/entry into the target cells. In either case, this direct action on HIV particles distinguishes the defensins from CAF which, upon treatment of HIV virions does not alter HIV infectivity . The finding that brief pretreatment of CD4 cells with the α-defensins inhibits subsequent HIV infection/replication could reflect a competitive interference with virion binding to (or uptake by) the cell. Alternatively, the defensins may signal the cell in a receptor-dependent manner [31–35] resulting in an intracellular antiviral state that affects a post-entry step(s) in the virus life cycle. We found that the antiviral effect on both virions and cells to be sensitive to the presence of serum proteins (i.e., FCS), as has been observed upon treating other enveloped viruses with these proteins . This finding questions the potential relevance of this anti-HIV activity in the serum of peripheral blood. Other small defensin-like peptides have been shown to exhibit anti-HIV effects at concentrations ranging from 2 to 100 μg/ml [36–40]. Some of these peptides appear to block HIV at either entry or fusion [39,40] which, like the activity of the α-defensins, is different from CAF activity [5,8].
Our studies (in contrast to the results of Zhang et al. ) demonstrate that the α-defensins are not produced by CD8 T cells and thus are not CD8 cell antiviral factors. α-Defensin RNA was not found in purified CD8 cells from HIV-infected and uninfected subjects, regardless of whether these cells were stimulated or not with the mitogen PHA. At the protein expression level, purified CD8 cells were devoid of detectable α-defensin polypeptides in both intracellular compartments and extracellular fluids (Figs 5 and 6). Moreover, CAF activity in CD8 cell supernatants could not be reduced by anti-HNP1–3 neutralizing antibodies (Fig. 7).
It is noteworthy that we found α-defensin expression by human monocytes, both at the mRNA and protein levels (Fig. 5). Besides their antimicrobial properties, α-defensins have also been shown to have immune modulating activities implicated in bridging innate and acquired immune responses , and can induce the proliferation of various cell types [32,33]. Thus, local release of α-defensins by monocytes may have other physiologic importance.
The finding of α-defensin expression in monocytes (Fig. 5) may explain the discrepancy between our results and those of Zhang et al.  regarding α-defensin expression by CD8 cells. We showed that in vitro manipulation of lymphocyte–granulocyte mixtures can result in immunocytochemical staining of lymphocytes, which only occurs if substantial numbers of neutrophils are present (Fig. 5). The studies of Zhang et al.  showed α-defensin immunopositivity in CD8 T cells grown on irradiated feeder cell layers which typically include human monocytes and contaminating neutrophils. Therefore, their results could reflect α-defensin transfer during in vitro manipulations, or the uptake of defensins from the cells of the feeder layer by the activated CD8 cells during the co-culture period. The latter possibility is supported by the observation that small amphipathic cationic peptides can be taken up by T cell lines .
In summary, our studies demonstrate that the α-defensins can exhibit modest antiviral activity against HIV in vitro and are produced by monocytes. However, they are not expressed by CD8 cells and thus do not play a role in the CD8 cell noncytotoxic anti-HIV response.
After this paper was submitted, an article by Chang et al. (J Virol 2003, 77:6777–6784) was published indicating that alpha-defensins can affect HIV replication following viral entry but are not the product of CD8 cell lines.
The authors thank H. Foster and B. Ashlock for technical assistance and A. Murai and K. Peter for help in the preparation of the manuscript.
Sponsorship: Supported by NIH grants (RO1-AI-30350 and R37-AI22931).
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