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* Department of Medicine, Division of Pulmonary and Critical Care Medicine, and
Will Rogers Institute Pulmonary Research Laboratory, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095; and
Microchemical Facility, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lysozyme has long been presumed to have a significant role in airway host defense due to its in vitro microbicidal activity and ubiquitous, as well as abundant, epithelial expression. Until recently, direct evidence of the biological role of lysozyme has been limited. Akinbi et al. (10) reported that bacterial killing was enhanced in the lungs of mice that transgenically overexpressed rat lysozyme in distal respiratory epithelial cells. The concentration of lysozyme in bronchoalveolar lavage (BAL) from transgenic mice was 2- to 4-fold greater than that of wild-type mice. Mice that overexpressed lysozyme were more efficient than wild-type mice in killing group B streptococci and mucoid Pseudomonas aeruginosa, and had an increased survival rate following infection. In separate studies, slow resolution of hyaline membrane disease-induced bronchopulmonary dysplasia in infants has been found to be associated with reduced production of lysozyme and lactoferrin (11), suggesting a biological role for these molecules in the airways.
The smaller antimicrobial peptides are widely distributed molecular effectors of innate immunity. There are several families of peptides that display similar mechanisms of action against microbes. Many peptides are broad-spectrum microbicides that target Gram-positive and Gram-negative bacteria as well as fungi and some enveloped viruses (1, 12, 13). Antimicrobial peptides are cationic and hydrophobic at physiologic pH, attributes that assist peptide binding and insertion into microbial membranes. Some peptides then aggregate to form pores, and death of the microbe occurs once a threshold number of pores have formed (14, 15).
In the airways, the antimicrobial peptide human 
-defensin (HBD)-1 is
constitutively expressed by epithelia at low levels while the related
peptide HBD-2 is predominantly induced at sites of inflammation
(16, 17). The 
-defensin human neutrophil peptide
(HNP)-1 (18) has also been found at varying concentrations
in BAL (19) and is likely a degranulation product of
transudated neutrophils found in infected airways. Anionic
antimicrobial (poly)peptides may also contribute to the antimicrobial
activity of the airways, and have been detected in ovine and human BAL
fluid (20, 21). The abundance and multiplicity of
antimicrobial peptides and proteins present in airway secretions
suggest that the antimicrobial activity of airway fluid results from
the combined effects of many components.
In contrast to recent studies that have focused on the antibacterial action of individual components of airway fluids (16, 17, 22, 23), we have developed techniques that enable the analysis of whole, minimally manipulated airway fluid (24). These techniques have afforded us the opportunity to assess the combined antimicrobial activity of multiple components under more physiologic conditions than have been heretofore possible. In this study, we used these techniques to provide evidence that cationic antimicrobial polypeptides are responsible for the majority of antibacterial activity of nasal fluid, because the removal of cationic polypeptides from nasal secretions ablates the ex vivo activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and P. aeruginosa. In reconstitution studies, we showed that the quantitatively most abundant antimicrobial polypeptides (lysozyme, lactoferrin, and SLPI) all contribute to the antibacterial activity of nasal fluid.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human neutrophil lysozyme and human milk lactoferrin were purchased from Sigma-Aldrich (St. Louis, MO). Human rSLPI was purchased from R&D Systems (Minneapolis, MN). HNP-1 was purchased from Bachem (Torrance, CA). rHBD-1 and -2 were produced in our laboratory as previously described (17, 25).
Collection and processing of nasal fluid
Nasal secretions were collected from 16 healthy volunteer donors according to a protocol approved by the University of California, Los Angeles, Institutional Review Board. From most donors, nasal secretions were collected by vacuum-aided suction, without chemical stimulation, to avoid introducing foreign substances into the nasal fluids. Gentle manipulation of a narrow rubber-tipped vacuum device inside the nasal passageways mildly stimulated nasal secretions. The range of secretion volumes collected varied between 100 µl and 1.5 ml, depending on the donor. Nasal secretions were pooled and stored at -20°C. Fluid samples were gamma-irradiated at 20 krad and homogenized briefly using a handheld Sonic Dismembranator 100 (Fisher Scientific, Pittsburgh, PA). As previously reported, storage, sonication, and gamma irradiation did not produce discernable changes in microbicidal activity (24).
Selective depletion of cationic polypeptides from nasal fluid
A cation-exchange resin was used to remove cationic (poly)peptides from minimally manipulated nasal fluid without significantly affecting the concentrations of other major ions. Carboxymethyl weak cation-exchange (CM) resin (Bio-Rad, Hercules, CA) was equilibrated with a buffer, 1x nasal salt buffer (pH 7.4; described in Ref. 24), which approximates the electrolyte composition of nasal fluid. In preparation for the experiment, the equilibrated CM resin was adjusted to pH 6.0, sedimented for 15 min at 10,000 x g, and removed of all overlying 1x NSB. An equal volume of minimally manipulated nasal fluid supernatant was added to the bed volume of 1x NSB-equilibrated CM resin, vortexed briefly, and incubated end-over-end for 48 h at 4°C. Following incubation, the pH of the sample was measured (ranged from 6.8 to 7.0), and the resin was removed by centrifugation (10,000 x g, 10 min). The conductivities of whole (20.8 mS/ml) and CM-depleted (18.5 mS/ml) nasal fluids were measured using a conductivity meter (Oakton, Singapore) in two batches of nasal fluid and found to be quite similar. Cationic peptides bound to the CM resin were extracted by incubating end-over-end with two resin volumes of 5% acetic acid at 4°C for subsequent 2 and 24-h extractions. The peptide extract was vacuum-dried and resuspended to the original volume of nasal fluid. The volume of CM-depleted nasal fluid was equivalent to the starting volume of whole nasal fluid. All samples were frozen at -20°C in aliquots for subsequent experiments.
One-dimensional gel electrophoreses and semiquantitative Western analysis
Nasal secretions were separated on duplicate gels by acid urea (AU)-PAGE (25) and Tricine-SDS-PAGE (26). Following AU-PAGE or SDS-PAGE, gels were immunoblotted to Immobilon-PSQ polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) for 30 min (HBD-1, HBD-2, and HNP-1 to -3), 1 h (lysozyme), 2 h (SLPI), to 4 h (lactoferrin) in 0.7% acetic acid/10% methanol as described previously (25). The membranes were then incubated with either 1/250 dilution of rabbit polyclonal anti-human lysozyme (DAKO, Carpinteria, CA), 1/5000 dilution of anti-human lactoferrin (DAKO), 1/1000 dilution of anti-HBD-1 (25), 1/1000 dilution of anti-HBD-2 (27), or 1/1000 dilution of goat anti-human SLPI (R&D Systems, Minneapolis, MN) overnight at room temperature. After several washings in 1x Blotto TBS (TBS plus 1% dried milk and 0.1% BSA), membranes were incubated with 1/2000 dilution of alkaline phosphatase-conjugated polyclonal goat anti-rabbit (lysozyme, lactoferrin, HBD-1, and HBD-2) secondary Ab (Pierce, Rockford, IL) or rabbit anti-goat (SLPI) secondary Ab (Organon Teknika, West Chester, PA) for 1 h at room temperature. The membranes were washed and then developed using nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate as a chromogenic substrate. Serial dilutions of standard human neutrophil lysozyme (Sigma-Aldrich), human milk lactoferrin (Sigma-Aldrich), SLPI (R&D Systems), HBD-1, and HBD-2 (25, 27) were used as positive controls and for semiquantitative studies.
Lysoplate assay for muramidase activity of lysozyme
Briefly, 0.5 mg/ml lyophilized Micrococcus lysodeiktikus was suspended in 66 mM sodium phosphate buffer (Na2HPO4/NaH2PO4, pH 7.4), combined with 1% low EEO-agarose in 66 mM sodium phosphate, and poured in a level 9 x 9 cm square petri dish (28). Sixteen or 25 evenly spaced 3.2-mm wells were punched in the solidified agar, and 5 µl of sample was introduced into each well. Lysozyme enzymatic activity was determined by measuring the diameters of zones of clearance relative to standards of human neutrophil lysozyme.
Bacterial strains and culture conditions
The P. aeruginosa strain, designated "CF," is an isolate (29) from a cystic fibrosis patient and was generously donated by Dr. M. J. Welsh (University of Iowa, Iowa City, IA). P. aeruginosa CF and the laboratory strains E. coli ML-35p and L. monocytogenes EGD were cultured 18 h at 37°C in 50 ml of 3% trypticase soy broth (TSB; strains described in Ref. 30). Each strain was snap-frozen by rapidly freezing aliquots in liquid nitrogen and stored at -80°C for up to 1 year. Aliquots (50 µl for E. coli, 500 µl for all other bacteria) were thawed and subcultured 2.5 h immediately before use in antibacterial assays in 50 ml of 3% TSB to obtain mid-logarithmic growth phase. Subcultures were then centrifuged at 1400 x g for 10 min and washed once in 1x HBSS or 1x NSB. The bacteria were collected at 1400 x g for 10 min, and resuspended in 1–5 ml of HBSS or 1x NSB. An OD625 of 0.16 to 0.18 approximated 2.5 x 107 bacterial CFU/ml. E. coli and L. monocytogenes were selected because of their high sensitivity in screening assays (radial diffusion assays (RDAs)). P. aeruginosa was used in the most biologically relevant model (CFU assay) of ex vivo colonization of nasal fluid.
Radial diffusion assay
RDAs were performed as described previously (31). Briefly, the underlay consisted of 1% agarose and 1/100 dilution of TSB in 10 mM sodium phosphate (pH 7.4), either alone or supplemented with 50 or 100 mM NaCl. The overlay consisted of 6% TSB and 1% agarose in dH2O for all assays. Bacteria or fungi (4 x 106) were mixed with 10 ml of underlay gel solutions kept molten at 48°C and poured into 100-cm2 square petri dishes. A series of 3.2-mm diameter wells were punched after the agarose solidified, and 5 µl of sample was added into designated wells. Plates were incubated at 37°C for 3 h to allow for peptide diffusion. The microbe-laden underlay was then covered with 10 ml of molten overlay, and the plates were allowed to harden. Antibacterial activity was identified as a clear zone around the well absent of microbial growth after 18-h incubation at 37°C. The activity was represented in radial diffusion units (RDU) defined as: (diameter of clear zone (in millimeters) - 3.2 mm) x 10. Assays were repeated at least twice.
Gel-overlay assay
The gel-overlay assay measures the activity of peptides and proteins that diffuse from an electrophoresed PAGE gel into an agarose gel embedded with microcolonies of bacteria and was performed as described previously (31). Briefly, nasal fluid was extracted with 5% acetic acid, vacuum-dried, resuspended in 3x loading dye, and separated by AU-PAGE. The gel was washed 20 min in 10 mM sodium phosphate pH 7.4 then placed on a premade 1% agarose plate containing 10 mM sodium phosphate with 100 mM NaCl (pH 7.4), 0.03% (w/v) of TSB powder, and 4 x 106 L. monocytogenes or E. coli. The plate was then incubated at 37°C for 3 h to allow the proteins and peptides in the polyacrylamide gel to diffuse into the underlying bacterial layer. The polyacrylamide gel was then removed, and the bacterial layer was overlaid with a nutrient layer that contained 6% TSB in 1% agarose. Clear zones without bacterial growth represented antibacterial activity. Duplicate AU gels were electrophoresed and Coomassie-stained to correlate antibacterial activity with protein bands.
CFU microassays
Bacteria (OD625 = 0.2) were diluted 100- to 300-fold in HBSS for use in the CFU microassay (modification of a method described in Ref. 32). Samples consisted of 6 µl of either bacterial or yeast dilution in HBSS/0.2% TSB plus 24 µl of nasal fluid for each condition, which allowed quadruplicates of three time points (0-, 3-, and 24-h incubation). Separate tubes with 6 µl of bacteria or yeast and 24 µl of 1x NSB/0.2% TSB were used as controls for microbial growth. Each well of a 72-well Terasaki microtiter plate (Nalge Nunc International, Roskilde, Denmark) was loaded with 2 µl of liquid wax (MJ Research, Watertown, MA) to prevent evaporation. Two microliters of the sample (or buffer-only control) was loaded into each of 12 wells by pipetting directly underneath the liquid wax. The entire plate was incubated at 37°C/5% CO2. To recover the incubated fluid at the specified time points, wells were washed thoroughly with 46 µl of HBSS and the wash was placed on ice in a microcentrifuge tube. The fluid was then plated on tryptic soy agar plates, and CFUs were counted following overnight incubation at 37°C.
Two-dimensional gel electrophoresis
Nasal secretions were separated on a 25-cm AU-PAGE gel (12.5% acrylamide/0.33% bis-acrylamide) as the first dimension and transferred to a reducing SDS-PAGE as the second dimension. Nasal fluid (20 µl) was extracted with 200 µl of 5% acetic acid, concentrated to near dryness under vacuum, and resuspended in 20 µl of AU-PAGE loading dye (9 M urea in 5% acetic acid with a few grains of methyl green). The AU-PAGE gel was electrophoresed 16 h under pulsed field (160–170 V forward pulse for 0.1–18 s, reverse pulse for 0.03–6 s; pulses incremented by 2-fold for 10 replications and then reset to shortest time; Programmable Pulse Inverter-200; MJ Research). The gel was stained using 0.1 x amido black (0.04% napthol blue-black, 2.5% isopropanol, and 1% acetic acid) for 10–20 s to visualize protein bands. Strips of AU-PAGE gel, consisting of an entire lane of an AU gel with electrophoresed nasal secretion proteins, was excised and incubated at room temperature in dH2O for 3 min. Subsequent incubations in 50 mM Tris (pH 8.8) were performed for three rinses (15 min per rinse). The gel strip was trimmed and placed atop the cathodal side of a precast 8–18% SDS-PAGE gel (Amersham Pharmacia Biotech, Piscataway, NJ) and electrophoresed for 45 min at 20 mA in a MultiPhor II electrophoresis system (Amersham Pharmacia Biotech) using ExcelGel SDS buffer strips (Amersham Pharmacia Biotech) as ion reservoirs. Once the proteins migrated from the AU-PAGE slice into the SDS-PAGE gel, the buffer strips were repositioned, and the gel was electrophoresed for an additional 145 min at 40 mA. Protein spots were visualized in the gel with either silver stain or SYPRO Ruby Gel (Bio-Rad), or on Immobilon-PSQ PVDF membranes with SYPRO Ruby Blot or Colloidal Gold total protein stain (Bio-Rad) according to the manufacturer’s instructions.
Polypeptide identification
N-terminal sequencing was done by automated Edman degradation,
in a model Procise-cLC protein sequencer (Applied Biosystems, Foster
City, CA), of protein spots excised from large-format (two-dimensional
gel) SDS-PAGE transferred to PVDF membranes as described in Ref.
33 . For mass-matching techniques, two-dimensional gels
were stained as described above, and each protein spot of interest was
excised using a clean razor blade and immersed in 1% acetic acid for
storage. Silver-stained cored gel pieces were washed three times with
HPLC grade water (Sigma-Aldrich), three times with Farmer’s reducer
(30 mM potassium ferricyanide/65 mM sodium thiosulfate) in HPLC grade
water, and five to 10 times with HPLC grade water. Cored SYPRO Ruby gel
pieces were washed three times with 50% acetonitrile/0.01 M
NH4HCO3 and vacuum-dried
for 30 min. Sequencing grade trypsin (200–500 ng; Promega, Madison,
WI) was mixed with 0.01 M
NH4HCO3 (5 µl); the pH
was adjusted to 8.5, and in-gel digestion continued for 15 h at
37°C (34). The peptides were extracted with 60%
acetonitrile/0.01 M
NH4HCO3. The extracted
peptides were microdesalted using C18 ZipTip (Millipore), mixed with
-cyano-4-hydroxycinnamic acid (Agilent Technologies, Wilmington,
DE), and analyzed by delayed-extraction reflectron matrix-assisted
laser desorption ionization-time of flight mass spectrometry
using a model ReflexIII mass spectrometer (Bruker Daltonics,
Billarica, MA) as described in Ref. 35 . The proteins
were tentatively identified by matching the monoisotopic masses of
tryptic peptides against the All Taxa proteome using either the
ProFound (http://129.85.19.192/profound_bin/WebProFound.exe) or the
Mascot
(http://www.matrixscience.com/cgi/index.pl?page=/search_form_select.html)
algorithms.
Statistics
Bacterial colony counts (CFU assay) and RDAs were performed in at least triplicate in each independent experiment. Statistical analyses were performed on log10 values to enhance normal distribution. Sets of independent experiments were compared with a Tukey pairwise comparison (Sigma-Stat; SPSS, Chicago, IL). Error bars represent SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Some of our initial studies, which defined the antimicrobial
activity of minimally manipulated nasal secretions, used boiling to
heat inactivate nasal secretions (24). Subsequent addition
of lysozyme and lactoferrin could not fully restore the activity of the
boiled fluid in CFU assays against a mucoid strain of P.
aeruginosa, thus implicating other antimicrobials that potentially
act in concert with lysozyme and lactoferrin (24).
However, the boiling procedure aggregates much of the soluble protein
(Fig. 1
) and unpredictably alters the
macromolecular composition of nasal fluid. Therefore, we sought to
develop gentler methods to selectively deplete airway secretions of
specific cationic polypeptides, while retaining physiologic
concentrations of the remaining polypeptides and electrolytes.
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The cationic peptides and proteins were extracted from nasal
secretions by incubation with the cation-exchange resin, CM-Prep
(Bio-Rad), which had been pre-equilibrated with 1x NSB, a buffer that
has the electrolyte and divalent cation composition of nasal fluid.
Several studies were performed to test the effects of CM depletion.
AU-PAGE, which separates polypeptides based on their cationic charge
density, was performed on nondepleted and CM-depleted nasal fluid as
well as the (poly)peptides recovered from the resin. Lysozyme, SLPI,
and small amounts of several other cationic polypeptides were depleted
from the nasal fluid, while most of the other proteins and peptides
remained unchanged (Fig. 2A).
As predicted, overlaying an unstained duplicate of this AU-PAGE gel
onto a lawn of a test organism, L. monocytogenes, in 10 mM
sodium phosphate (pH 7.4) plus 100 mM NaCl revealed an absence of
lysozyme-mediated killing only for the CM-depleted nasal fluid (data
not shown). Semiquantitative Western analysis revealed that lysozyme
was nearly absent from the CM-depleted fluid (Fig. 2B) and
that concentrations of other known antimicrobial (poly)peptides were
also significantly reduced (Table I). The
concentration of lactoferrin was not appreciably diminished in the
CM-depleted nasal fluid, which may be related to the low cationic
charge density of lactoferrin and reduced binding to CM resin in the
physiologic concentration of electrolytes in nasal fluid.
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C).
Likewise, the corresponding bactericidal activity of CM-depleted nasal
fluid was significantly reduced as compared with intact nasal fluid
under RDA analysis against our test strains L. monocytogenes
(Figs. 2C and 3) and E. coli (data not shown) in
low electrolyte conditions (10 mM sodium phosphate, pH 7.4). Similar
activities were also apparent at higher electrolyte conditions (10 mM
sodium phosphate (pH 7.4) supplemented with either 50 mM NaCl or 100 mM
NaCl; Fig. 3). Most importantly, for each
test condition the cationic polypeptide extract was as active as the
whole nasal fluid. Taken together, these data indicate that the
antibacterial activity of nasal fluid is contained in the cationic
fraction. Whole nasal fluid, CM-depleted fluid, and the extracted
cationic polypeptides were used in subsequent electrophoretic and
reconstitution assays.
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We used a novel proteomic technique to identify cationic
antimicrobial polypeptides in nasal fluid. By using a combination of
AU-PAGE and gel overlays against E. coli and L.
monocytogenes, we identified a region that contained a number of
antimicrobial polypeptides (Fig. 4A). A slice of AU-PAGE gel
from the antimicrobial region (the first dimension of a two-dimensional
gel) was electrophoresed into an SDS-PAGE gel (the second dimension).
The resulting silver-stained two-dimensional electrophoretogram of the
antimicrobial region is shown in Fig. 4B with most of the
major polypeptide spots in this region identified. Table II indicates how each polypeptide spot
was characterized in this study and summarizes the previously reported
biological functions for each identified polypeptide. Note that every
polypeptide identified in this region was either previously reported to
be antimicrobial or has other purported roles in host defense.
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A). Lysozyme, SLPI, -defensins, and
-microseminoprotein localized in the region of the gel that was
active against L. monocytogenes, while palate lung uvula
nasal epithelial clone, lipocalin-1, calgranulins A and B,
lipophilin C, and 2-microglobulin localized in
the region of the gel that was active against E. coli.
Specifically, lysozyme was more active against L.
monocytogenes than E. coli in gel overlays (Fig. 4A) and in RDAs (data not shown), which is likely attributed
to the ability of lysozyme to digest peptidoglycan that is exposed in
Gram-positive, but not Gram-negative, bacteria. Two-dimensional gel analysis of CM-depleted fluid
Two-dimensional gel electrophoresis was next used to characterize
the cationic polypeptides that remained in the nasal fluid after CM
depletion, as well as those that were extracted with the CM resin.
Representative two-dimensional gel electrophoretograms are given in
Fig. 5, revealing the polypeptide spot
profile of whole nasal fluid (Fig. 5A), CM-depleted nasal
fluid (Fig. 5B), and cationic polypeptide extract (Fig. 5C). The antimicrobial region designated in Fig. 4 is
underlined in each panel. Although the concentration of several
peptides was slightly diminished in the CM-depleted nasal fluid (Fig. 5B, arrows), there was a complete depletion of lysozyme
and SLPI. Two-dimensional gels of the cationic polypeptide
extract (Fig. 5C) indicated that only lysozyme, SLPI, and
lactoferrin were recovered. Taken together with Figs. 2 and 3, this
implies that lysozyme, SLPI, and lactoferrin contribute most of the
antibacterial activity of nasal fluid. The next set of experiments
explores reconstitution of CM-depleted fluid with these three cationic
polypeptides.
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The ex vivo antibacterial activity of CM-depleted fluid was tested
in the presence or absence of physiologic concentrations of lysozyme,
SLPI, and lactoferrin (either alone or in combination) in CFU assays
against a mucoid strain of P. aeruginosa isolated from a
patient with cystic fibrosis. To determine the "physiologic" amount
of polypeptide for these restoration assays, the concentration of
lysozyme, SLPI, and lactoferrin was measured by Western blot for whole
nasal fluid and CM-depleted fluid, and the difference in concentration
was added back to the CM-depleted fluid. Fig. 6 reveals that, although whole nasal
fluid was able to reduce the CFU per milliliter by nearly 10-fold after
3-h incubation, the number of CFU per milliliter within CM-depleted
fluid increased 
5-fold over inoculum and was significantly different
from whole nasal fluid (n = 6; p <
0.002). The CFU per milliliter of whole nasal fluid and the cationic
polypeptide extract at 3 h were not significantly different
(n = 6; p = 0.245).
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CM-depleted nasal fluid supplemented with lysozyme alone (n = 4, p < 0.001), lysozyme plus lactoferrin (n = 4, p = 0.021), or lysozyme plus lactoferrin plus SLPI (n = 4, p = 0.002) could significantly reduce CFU per milliliter as compared with CM-depleted nasal fluid. RDAs against E. coli and L. monocytogenes confirmed that lysozyme alone or in combination with SLPI and/or lactoferrin restored the activity of CM-depleted nasal fluid (data not shown). These data suggest that lysozyme, either alone or in combination with lactoferrin and SLPI, is responsible for a majority of the antibacterial potential of nasal fluid.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although one-dimensional electrophoretic techniques revealed the major
antimicrobial components within nasal fluid, such as lysozyme and SLPI,
the resolving power of these gels is not sufficient for the
identification of other putative antimicrobial polypeptides. To
overcome these limitations, we turned to two-dimensional
electrophoresis to help characterize the proteome of nasal fluid,
specifically focusing on cationic polypeptides. Standard techniques use
isoelectric focusing of protein samples in the first dimension,
followed by SDS-PAGE in the second dimension. The resulting protein
spots can be visualized by a variety of staining techniques, excised
from the gel, and sequenced by mass-matching techniques or N-terminal
Edman degradation. However, these standard techniques proved
problematic in our system because many antimicrobial peptides and
proteins are extremely cationic and are not resolved by conventional
isoelectric focusing methods, resulting in the loss of all polypeptides
whose isolelectric point is >9 (data not shown). This includes such
common antimicrobial substances as lysozyme, lactoferrin, defensins,
and many other cationic antimicrobial (poly)peptides. For this reason,
we adopted a two-dimensional gel system that resolves proteins by
cationic charge to mass ratio in the first dimension using AU-PAGE and
molecular mass in the second dimension using SDS-PAGE. Although
the first-dimension AU-PAGE revealed putative proteins other than
lysozyme, lactoferrin, and SLPI in the cationic polypeptide extract
(Fig. 2A), these were not noted in the two-dimensional gels
(Fig. 5C) and may represent proteins with posttranslational
modifications (e.g., glycosylation) that are not easily resolved by
SDS-PAGE. We also noted that protein bands, which appear as a single
bands by AU-PAGE, may separate into two or more protein spots in the
second-dimension SDS-PAGE. We are currently purifying several of these
polypeptides from large volumes of whole nasal fluid, and will use the
purified polypeptides in reconstitution assays against a wide range of
target microbes.
Our studies (Table I and Ref. 24), as well as those from
other groups (2, 4, 37, 38), indicate that the predominant
antimicrobial polypeptides within nasal fluid are lysozyme,
lactoferrin, and SLPI. However, we have also identified a number of
additional antimicrobial (and putatively antimicrobial) polypeptides
within nasal fluid. One might question why humans have retained such
high concentrations of several antimicrobial polypeptides, or what the
necessity is for the production of numerous, yet quantitatively less
abundant, microbicidal peptides and proteins. One conjecture is that
antimicrobial polypeptides have evolved different but partially
overlapping spectra of activity. If this is correct, microbes that are
resistant to one type of peptide or protein might still preserve
sensitivity to another. Likewise, microbes encountering a variety of
antimicrobials would be less likely to develop resistance or to
otherwise subvert the host’s defenses. A convincing example of
differential antimicrobial activity has been reported for
-defensins. Staphylococcus aureus is markedly insensitive
to HBD-1 and HBD-2, yet even multidrug-resistant strains are
susceptible to a similar molecule, HBD-3 (39). Although it
is too early to speculate that the combination of lysozyme,
lactoferrin, and SLPI is responsible for innate airway host defense
against all potential pathogens, our studies serve as a template for
future investigation as to the role of quantitatively less abundant
antimicrobial peptides, such as defensins, in the presence and absence
of the major microbicidal components of airway fluid.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alexander M. Cole, Department of Medicine, Division of Pulmonary and Critical Care, University of California, Los Angeles, School of Medicine, 10833 Le Conte Avenue, Room CHS 37-055, Los Angeles, CA 90095-1690. E-mail address: acole{at}mednet.ucla.edu
3 Abbreviations used in this paper: SLPI, secretory leukoprotease inhibitor; BAL, bronchoalveolar lavage; HBD, human -defensin; HNP, human neutrophil peptide; AU, acid urea; PVDF, polyvinylidene difluoride; RDA, radial diffusion assay; RDU, radial diffusion unit; CM, carboxymethyl weak cation exchange.
Received for publication July 16, 2002. Accepted for publication October 9, 2002.
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  J. A. Martellini, A. L. Cole, N. Venkataraman, G. A. Quinn, P. Svoboda, B. K. Gangrade, J. Pohl, O. E. Sorensen, and A. M. Cole Cationic polypeptides contribute to the anti-HIV-1 activity of human seminal plasma FASEB J, October 1, 2009; 23(10): 3609 - 3618. [Abstract] [Full Text] [PDF]
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 G. Diamond, N. Beckloff, and L.K. Ryan Host Defense Peptides in the Oral Cavity and the Lung: Similarities and Differences Journal of Dental Research, October 1, 2008; 87(10): 915 - 927. [Abstract] [Full Text] [PDF]
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 T. Q. Do, S. Moshkani, P. Castillo, S. Anunta, A. Pogosyan, A. Cheung, B. Marbois, K. F. Faull, W. Ernst, S. M. Chiang, et al. Lipids Including Cholesteryl Linoleate and Cholesteryl Arachidonate Contribute to the Inherent Antibacterial Activity of Human Nasal Fluid J. Immunol., September 15, 2008; 181(6): 4177 - 4187. [Abstract] [Full Text] [PDF]
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 C. G. Clement, S. E. Evans, C. M. Evans, D. Hawke, R. Kobayashi, P. R. Reynolds, S. J. Moghaddam, B. L. Scott, E. Melicoff, R. Adachi, et al. Stimulation of Lung Innate Immunity Protects against Lethal Pneumococcal Pneumonia in Mice Am. J. Respir. Crit. Care Med., June 15, 2008; 177(12): 1322 - 1330. [Abstract] [Full Text] [PDF]
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 S. R. Clarke and S. J. Foster IsdA Protects Staphylococcus aureus against the Bactericidal Protease Activity of Apolactoferrin Infect. Immun., April 1, 2008; 76(4): 1518 - 1526. [Abstract] [Full Text] [PDF]
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J. A. Nash, T. N. S. Ballard, T. E. Weaver, and H. T. Akinbi The Peptidoglycan-Degrading Property of Lysozyme Is Not Required for Bactericidal Activity In Vivo J. Immunol., July 1, 2006; 177(1): 519 - 526. [Abstract] [Full Text] [PDF]
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N. Venkataraman, A. L. Cole, P. Svoboda, J. Pohl, and A. M. Cole Cationic Polypeptides Are Required for Anti-HIV-1 Activity of Human Vaginal Fluid J. Immunol., December 1, 2005; 175(11): 7560 - 7567. [Abstract] [Full Text] [PDF]
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 A. M. Cole, D. R. Thapa, V. Gabayan, H.-I Liao, L. Liu, and T. Ganz Decreased clearance of Pseudomonas aeruginosa from airways of mice deficient in lysozyme M J. Leukoc. Biol., November 1, 2005; 78(5): 1081 - 1085. [Abstract] [Full Text] [PDF]
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 R. Dajani, Y. Zhang, P. J. Taft, S. M. Travis, T. D. Starner, A. Olsen, J. Zabner, M. J. Welsh, and J. F. Engelhardt Lysozyme Secretion by Submucosal Glands Protects the Airway from Bacterial Infection Am. J. Respir. Cell Mol. Biol., June 1, 2005; 32(6): 548 - 552. [Abstract] [Full Text] [PDF]
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C. Geetha, S.G. Venkatesh, L. Bingle, C.D. Bingle, and S.-U. Gorr Design and Validation of Anti-inflammatory Peptides from Human Parotid Secretory Protein Journal of Dental Research, February 1, 2005; 84(2): 149 - 153. [Abstract] [Full Text] [PDF]
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D. Proud, S. P. Sanders, and S. Wiehler Human Rhinovirus Infection Induces Airway Epithelial Cell Production of Human {beta}-Defensin 2 Both In Vitro and In Vivo J. Immunol., April 1, 2004; 172(7): 4637 - 4645. [Abstract] [Full Text] [PDF]
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T. Ganz Antimicrobial polypeptides J. Leukoc. Biol., January 1, 2004; 75(1): 34 - 38. [Abstract] [Full Text] [PDF]
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R. L. Gibson, J. L. Burns, and B. W. Ramsey Pathophysiology and Management of Pulmonary Infections in Cystic Fibrosis Am. J. Respir. Crit. Care Med., October 15, 2003; 168(8): 918 - 951. [Abstract] [Full Text] [PDF]
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 F. Hubalek, J. Pohl, and D. E. Edmondson Structural Comparison of Human Monoamine Oxidases A and B: MASS SPECTROMETRY MONITORING OF CYSTEINE REACTIVITIES J. Biol. Chem., August 1, 2003; 278(31): 28612 - 28618. [Abstract] [Full Text] [PDF]
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