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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 2003, p. 3901-3906, Vol. 47, No. 12
0066-4804/03/$08.00+0 DOI: 10.1128/AAC.47.12.3901-3906.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
Paul B. McCray Jr.,2 and Peter S. Thorne1*
Department of Occupational and Environmental Health,1 Department of Pediatrics, The University of Iowa, Iowa City, Iowa 522422
Received 5 June 2003/ Returned for modification 19 August 2003/ Accepted 9 September 2003
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ABSTRACT |
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METHODSResultsDiscussionReferences |
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INTRODUCTION |
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The members of one class of promising antimicrobial peptides resemble the 18 amino acids of the N terminus of SMAP-29, derived from sheep neutrophils (16, 19). While showing a broad spectrum of antimicrobial activity and low toxicity in ovine models, the parent compound, SMAP-29, exhibits unacceptably high hemolytic and cytotoxic activity in human cells (1, 16). A series of substituted peptides has been assembled to circumvent the toxic potential while retaining antimicrobial activity. These substituted peptides are termed ovispirins; the structures and functional three-dimensional properties of these peptides are given by Sawai et al. (16) and Tack et al. (23). One peptide in this series, novispirin G10 (in which glycine is substituted at position 10 for isoleucine), has been shown to be significantly less hemolytic for human cells while retaining high antimicrobial activity against gram-negative and gram-positive bacteria (16, 21, 22).
Few in vivo studies have been reported for the ovispirins and novispirins. Brogden and colleagues (1) tested the efficacy of SMAP-29 in an ovine model of pulmonary infection by using Mannheimia haemolytica. Steinstraesser and colleagues (21) examined the activity of novispirin G10 in a burned skin model by using Pseudomonas aeruginosa. We wished to establish a mouse model of subacute bacterial lung infection in order to examine novispirin G10 alone and in combination with an established pulmonary therapeutic agent in the absence of foreign substances to prolong bacterial seeding (e.g., agarose beads). Our hypothesis was that the novispirin G10 would reduce lung toxicity by binding lipopolysaccharide (LPS) released by gram-negative cells during treatment of the infection. For our model, we chose Klebsiella pneumoniae, an organism known to elicit a strong cytokine response in mouse lung (8). The efficacy of the test antimicrobial peptide was compared to that of tobramycin sulfate, a standard antibiotic for the treatment of pulmonary infections.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and
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Endotoxin binding assay. Endotoxin was measured by the kinetic chromogenic Limulus amebocyte lysate (LAL) assay (Kinetic-QCL; BioWhittaker, Inc., Walkersville, Md.) as previously described (24). Cathelicidins bind to lipopolysaccharide (LPS) at the lipid A moiety and extinguish endotoxic activity but do not affect the LAL assay itself (11). Accordingly, dilutions of novispirin G10 were added to pyrogen-free water (BioWhittaker, Inc.) containing 175 EU of endotoxin (Escherichia coli O55:B5; BioWhittaker, Inc.) per ml. The concentration of endotoxin was confirmed by activity in the LAL assay. The 50% endotoxin binding capacity was calculated from a regression of the recovered endotoxic activity against the spiked activity.
MICs and
MBCs.
K.
pneumoniae (ATCC43816) was obtained from the American Type Culture
Collection (Manassas, Va.). The MIC of the peptide was measured by
using a modification of the protocol of Steinberg and Lehrer
(20). Briefly, the
bacterial culture was inoculated into tryptic soy broth (Difco,
Franklin Lakes, N.J.) and grown overnight at 37°C. Aliquots of
the overnight culture were inoculated into fresh tryptic soy broth and
grown to log phase in a shaking water bath (2 to 3 h). The
bacteria were centrifuged at 3,000 x g for 10 min,
washed, and resuspended in 10 mM phosphate-buffered saline (pH 7.4).
Dilutions of novispirin G10 were made in 0.01% acetic
acid-0.1% BSA, and 50-µl aliquots were dispensed
in duplicate into wells of a 96-well cell culture plate (Costar 3595).
Washed bacteria were added to the peptide dilutions or buffers (final
concentrations, 2 x 105 CFU/ml) and preincubated for
3 h at 37°C. At the end of the preincubation period,
100 µl of 2x concentrated Mueller-Hinton broth (MHB)
(BBL; Becton Dickinson and Co., Cockeysville, Md.) was added to each
well, and the wells were incubated for a further 16 h. After
incubation, the optical densities of the wells were determined at 650
nm (OD650) by using a SPECTRAmax 340 microplate
spectrophotometer (Molecular Devices, Sunnyvale, Calif.). The
positive-control wells contained MHB inoculated with bacteria, and the
negative-control wells contained MHB without bacteria. The lowest
concentration of antimicrobial agent showing 
50%
growth control was considered to be the MIC. Aliquots from wells with
no growth were plated onto tryptic soy agar (TSA; Difco) and incubated
overnight. The minimal bactericidal concentration (MBC) was determined
to be the lowest dilution concentration that showed no growth on TSA.
The MIC and MBC of tobramycin sulfate (Eli Lilly & Co.,
Indianapolis, Ind.) were determined for K. pneumoniae by using
both a standard broth microdilution assay
(13) and by the method
described above for antimicrobial peptides.
A checkerboard assay was used to test for synergistic interaction between G10 and tobramycin sulfate (3). The fractional inhibitory concentration (FIC) of the combined antibiotics was determined by using the following relationship: FIC = (lowest concentration of antibiotic A/MIC of A) + (lowest concentration of antibiotic B/MIC of B).
Pulmonary toxicity models. The University of Iowa Institutional Animal Care and Use Committee approved all experimental protocols. Eight-week-old male C57BL/6 mice were quarantined for 2 weeks in the Inhalation Toxicology Facility vivarium before treatment.
The in vivo pulmonary toxicity of G10 and tobramycin was tested by intratracheal instillation. The mice were anaesthetized with inhaled ether and restrained, ventral side up, on a reclined support. A Jelco 19-mm catheter (Johnson & Johnson, Arlington, Tex.) was guided into the trachea of the recumbent mouse. One hundred microliters of antibiotic or saline control was transferred to a 1-ml syringe by micropipette. The syringe was then attached to the catheter hub, and the fluid was gently expelled into the catheter, where it was aspirated by the mouse. The mouse was placed in a recovery jar before being returned to its cage. Food (Purina mouse chow) and water were available ad libitum.
Before necropsy, the mice were anaesthetized with ether and euthanized by cervical dislocation. A total of 4 ml of pyrogen-free saline (1 ml per wash) was introduced through a cannula inserted into the trachea. The saline and associated bronchoalveolar fluid (BAL) were collected by gravity drain and then centrifuged, after which the supernatant was divided into aliquots for biochemical characterization. The cell pellet was prepared for cell count and differential staining as previously described (25).
Pulmonary infection model. Single colonies of K. pneumoniae grown for 16 h on TSA were emulsified into pyrogen-free saline, and the turbidity of the suspensions was measured at OD360. Mouse lungs were instilled with Klebsiella as described above as part of an established infection model (8). The concentrations of instilled Klebsiella organisms were checked by spread plating the inocula on TSA and ranged from 8.27 to 8.72 log10. Five hours after the introduction of the Klebsiella suspension, the mice were instilled with one of five treatment regimens: saline (control), G10 (0.4 mg/kg), tobramycin (1 mg/kg), tobramycin (1 mg/kg) plus G10 (0.4 mg/kg), or tobramycin (10 mg/kg). Necropsies were performed, and the BAL was collected at 24 h postinstillation. At the inocula concentration chosen, Klebsiella was recovered from the lungs of instilled mice for up to 72 h without mortality.
Pulmonary endotoxin stimulation model. In order to examine the role of the gram-negative cell membrane in a noninfectious model, a suspension of Klebsiella was prepared as above and pasteurized at 70°C for 1 h. One hundred microliters of the pasteurized culture was plated on TSA to ensure that 99.9% of the cells were nonviable. The cell suspension was adjusted to the same optical density and instilled as described for the infection model. At 5 h postinstillation, the mice were instilled intratracheally with saline, G10 (0.4 mg/kg), or tobramycin (1 mg/kg) plus G10 (0.4 mg/kg). Necropsies were performed at 24 h, and BAL was collected. Comparison groups of mice were instilled with commercial endotoxin prepared from E. coli O111:B4 (BioWhittaker, Inc.) at two concentrations, 10 mg/kg and 40 mg/kg. Necropsies were performed at 5 and 24 h postinstillation.
Measures of
response.
Inflammation of
the lung was assessed by leukocytosis and neutrophil recruitment and by
the release of the proinflammatory cytokines interleukin-6 (IL-6) and
tumor necrosis factor alpha (TNF-
). Increased permeability of
the lung was assessed by measurement of hemoglobin and albumin
concentrations in the BAL. Colonization of the lung was assessed by the
continuing presence of Klebsiella, indicative of a failure to
clear the infection.
The cell pellet of the centrifuged BAL was resuspended in 150 µl of Hanks' buffered salt solution (Cellgro Mediatech, Inc., Herndon, Va.). Twenty-five microliters of the suspension was diluted 1:20 and dispensed into 3% acetic acid to lyse the erythrocytes. The leukocyte suspension was counted by using a hemocytometer at 400x magnification. Fifty microliters of the remaining cell suspension was centrifuged onto microscope slides by using a cytofuge (StatSpin, Norwood, Mass.) and stained with Diff Quick (Dade Behring AG, VWR) for differential cell counting.
Cytokine concentrations in BAL fluid were determined by
using commercial enzyme-linked immunosorbent assay kits (IL-6 and
TNF- DuoSet; R&D Systems, Minneapolis, Minn.). The assays
were measured at OD450 with a correction wavelength of
OD540. BAL albumin was measured by using a colorimetric
assay against a bovine serum albumin (BSA) standard curve
(Microprotein-PR; Sigma, St. Louis, Mo.). The concentration of
hemoglobin was determined by a spectrophotometric scan at
OD380, OD415, and OD450
(26) by using a BSA
standard curve.
Aliquots of uncentrifuged BAL were plated onto TSA to determine the concentration of surviving Klebsiella. To test the effect of the BAL fluid itself on the antibiotics, the MICs of tobramycin and G10 were determined by using the protocol outlined above with the following modification: BAL from mice instilled with saline, pasteurized Klebsiella, G10 alone, or pasteurized Klebsiella plus G10 was used as the diluent for the antibiotics. A control MIC using the normal diluent, 0.01% acetic acid-0.1% BSA, was determined on the same microtiter plate.
Plasma cytokines. At the time of necropsy, blood was collected by cardiac puncture into a 1-ml syringe containing 75 µl of heparin. The blood from all mice in the same treatment group was pooled and measured, and an equal volume of pyrogen-free saline was added. The blood was divided into 900-µl aliquots and incubated for 5 h at 37°C in 5% CO2. The samples were gently centrifuged in a microfuge to pellet the cells. The supernatant was stored at -85°C until analyzed for cytokine concentration as described for the BAL fluid.
Statistical
analyses.
The data were
analyzed using SPSS (Chicago, Ill.) version 10 software for Windows.
The distributions of the data were examined. The distribution of the
cytokine concentrations (IL-6 and TNF-) was approximately log
normal and was transformed to the base 10 logarithm for use in
parametric statistics. Tests of significance for multiple comparisons
were performed by using the post hoc procedure of Scheffé
(4).
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RESULTS |
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TopAbstractIntroductionMaterials and
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When
administered to uninfected mice, neither the peptide diluent
(0.01% acetic acid-0.1% BSA) G10 nor tobramycin
was inflammatory as measured by neutrophil recruitment to the lung or
production of the proinflammatory cytokines IL-6 and TNF-
(Table
2). There was no evidence of toxicity with these treatments, as assessed
by the concentration of hemoglobin or protein in the BAL fluid,
compared to that for the naïve controls or the controls
instilled with saline (Table
2). The combination of G10
and tobramycin was not toxic or inflammatory.
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. In
these mice, the instilled Klebsiella had not been killed and
yielded 7 x 103 CFU/ml (3.84 log units). Infected
mice treated with tobramycin at 1 mg/kg had comparable levels of
infection, whereas treatment at the higher dose of 10 mg/kg reduced the
bacterial recovery by nearly two log units. When infected mice were
treated with G10 alone or in combination with low-dose tobramycin,
higher concentrations of Klebsiella were recovered and strong
evidence of inflammation and lung toxicity emerged. Treatment with G10
resulted in increased permeability of the lung, as evidenced by
increased hemoglobin and protein in the BAL. This effect was
significantly greater than for the naïve mice and the infected
mice treated with saline. The instillation of Klebsiella
itself resulted in inflammation, but this effect was exacerbated by the
administration of G10 (Table
3). In order to determine
if infection was necessary for this effect, mice were instilled with
pasteurized Klebsiella, followed by the treatments described
above. This experiment showed that killed bacteria could induce
inflammation and some toxicity, which novispirin G10 failed to
alleviate (Table
4). High doses of bacterial LPS with or without added G10 did not produce
the high degree of inflammation and toxicity seen with lung infection
and G10.
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concentrations in blood
are reported in Table
5. In this series, the blood of mice infected intratracheally with
Klebsiella had significantly elevated levels of both IL-6 and
TNF- (Table 5).
However, there was a profound increase in the concentration of the
cytokine IL-6 in blood drawn from infected mice treated with G10 alone
or in combination with tobramycin, an effect which was not seen in mice
instilled only with LPS, a noninvasive control stimulant (P
< 0.001). This finding suggests that in the infection model the
cathelicidin, G10, produced a strong lung response and profound
systemic inflammation that was not observed with 10 mg/kg of
tobramycin, with LPS, or with infection and saline
treatment.
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DISCUSSION |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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However, when this peptide was used alone or in combination with standard treatment for gram-negative bacterial lung infection, the resulting toxicity was unexpected and profound. Interestingly, the antecedents of this reaction were noted but downplayed in previous work. For example, several studies have shown that novispirin G10 is inhibited by calcium or magnesium ions in test broth (9, 21; Lehrer, personal communication), but because the peptide is active in a dose-dependent fashion, the reduction in efficacy did not extinguish its antimicrobial properties beyond therapeutic ranges. In addition, other studies have shown novispirin G10 to be sensitive to inhibition by the presence of serum. Steinstraesser and colleagues (21) found that the addition of serum in the gel underlay of the plate inhibition test increased the apparent MIC by 10-fold for P. aeruginosa. In a previous study, we confirmed the reduction in effect for P. aeruginosa but also found that the addition of (bovine) serum completely inhibited the bactericidal action of novispirin G10 for K. pneumoniae.
In in vivo models, related compounds, CAP18 (rabbit derived peptide), for example, instilled alone (5 µg) resulted in significant lung edema. When CAP18 was mixed with P. aeruginosa prior to intratracheal instillation in mice, the bacterial counts in the lung homogenates were not significantly decreased compared to those for the lungs instilled with bacteria alone (15). In Steinstraesser's burned skin model, peripheral blood samples showed that the numbers of white blood cells and neutrophils were significantly increased in the novispirin G10-treated group compared to those for the controls, supporting our findings with BAL and circulating blood. However, this cytotoxicity may be limited to interspecies differences between peptide and host, as Brogden and colleagues (1) showed reduced inflammation in lambs infected with M. haemolytica treated with SMAP-29 compared to that for lambs with infection but no treatment.
Previous studies have shown that cathelicidin mixed with LPS or bacteria prior to instillation reduced lung damage (15). However, these are not sufficient models to use if the intent is to reduce the number of bacteria in a fulminant lung infection. In the model presented here, a gram-negative bacterial pathogen that would resist clearance for 48 to 72 h in mouse lung was used to approximate a chronic infection. Two treatment concentrations of tobramycin were used in building our pulmonary model. Tobramycin at 1 mg/kg was chosen to be suboptimal to allow observations of synergy between the aminoglycoside and the peptide. Tobramycin at 10 mg/kg was a therapeutic dose. The concentration of G10 was chosen based on the synergistic MIC and on previous work of Travis et al. (27). The treatment outcomes between the regimens was significant (3.9 log10 bacteria recovered at necropsy for tobramycin at 1 mg/kg versus 1.9 log10 for tobramycin at 10 mg/kg; P = 0.08). The unique contribution of G10 in combination therapy was to bind to LPS, thereby reducing the endotoxic potential of the gram-negative bacteria for the host.
Contrary to expectations,
the addition of G10 to the treatment regimen increased both the
inflammatory and toxic responses of the host. IL-6 and TNF-
are proinflammatory cytokines that are sensitive to endotoxin
stimulation, and if unregulated, they contribute to pulmonary disease
in gram-negative infections. These cytokines were clearly elevated in
the BAL of mice treated with either G10 alone or G10 in combination
with tobramycin. In addition, the lung epithelium was more permeable in
the infection model when G10 was added as part of the treatment. This
effect was also seen when killed Klebsiella were present in
the lung. The mechanism for this action and whether it is an effect
residing with the neutrophils are not known at present but deserve
further study. One intriguing observation was that the BAL of mice
instilled with killed Klebsiella in combination with G10 and
tobramycin appeared to have elevated MICs of G10, consistent with
observations for cation and serum inhibition of the peptide. The
products of the profound inflammatory response in the mouse lung
similarly blocked the action of G10 in vitro. The IL-6 concentration in
the lungs was higher when killed Klebsiella was used as the
stimulant (218 pg/ml) than when endotoxin was used (62.5 pg/ml), but
the addition of a sham G10 treatment to either killed
Klebsiella or LPS stimulation did not elevate the levels of
cytokine further.
The response for circulating cytokines was very similar to that for pulmonary cytokines, but the difference in response to the two stimulants was much greater, particularly for IL-6. The concentration of circulating IL-6 was elevated after challenge of mouse lung with purified endotoxin, but the concentration was 10,000-fold lower than that after challenge with the killed Klebsiella.
In summary, a mouse pulmonary infection model was developed that can be used as an early in vivo test of the efficacy and toxicity of antimicrobial peptides and should be included in the work of developing these compounds. The study was limited to the testing of one peptide in a mouse model; however, this peptide underwent screening in vitro with other SMAP- and CAP-derived compounds, and at no stage of the in vitro investigation was there any indication that G10 would prove to be toxic in vivo. In addition, G10 was chosen for the model based on its apparent lack of action on erythrocytes in preliminary screening of its hemolytic index. Further work is planned to examine the mechanism for the effects observed in this study.
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ACKNOWLEDGMENTS |
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This study was supported in part by Environmental Health Sciences Research Center grant NIEHS P30 ES05605.K.H.B. was the recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research.
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FOOTNOTES |
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Present
address: School of Occupational and Environmental Hygiene, University
of British Columbia, Vancouver, BC V6T 1Z3,
Canada.
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REFERENCES |
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