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Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, OH 45229
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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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The spatial expression of pulmonary lysozyme in rodents differs from that in humans in that the enzyme is principally synthesized and secreted by type II alveolar epithelial cells (5, 6). Lysozyme is one of the most abundant proteins in rat bronchoalveolar lavage (BAL)3 fluid, accounting for as much as 6–7% of the total soluble protein (5). Although two lysozyme genes have been identified in the rat, only the type I gene is expressed at detectable levels (7); in contrast, two lysozyme genes are expressed in the mouse, lysozyme M and lysozyme P (8, 9). Lysozyme M is the major form expressed in the lung, and only very low levels of lysozyme P mRNA are detected in mouse lung (8). In the current study rat lysozyme type I, the homologue of mouse lysozyme M, was cloned under control of the human surfactant protein C (SP-C) promoter to direct expression of the transgene to the distal respiratory epithelium (10, 11, 12). Transgenic mice expressing rat lysozyme exhibited significantly enhanced antimicrobial activity, including enhanced bacterial killing, decreased systemic dissemination of pathogen, and increased survival following infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rat lysozyme cDNA was generated from rat type II cell RNA by
RT-PCR using upstream primer 5'-GAA TTC ATG AAG GCT CTC CTA GTT CTG and
downstream primer 5'-GAA TTC TCA GAC TCC GCA GTT CCG AAT AT. The
amplified 400-bp fragment was confirmed to be rat lysozyme by sequence
analysis and was cloned into the EcoRI site of BGI-hSP-C
vector, which contained the 3.7-kb human SP-C promoter, rabbit
-globin intron 2, and bovine growth hormone polyadenylation signal
(13, 14). The transgene DNA construct was excised from the
PUC18 vector backbone by NotI/NdeI digestion, gel
purified using Qiaex resin (Qiagen, Germany), dialyzed for 48 h
against 5 mM Tris (pH 7.4)/1 mM EDTA, and microinjected into fertilized
FVB/N oocytes by the Children’s Hospital Transgenic Core facility.
Founder mice were identified by a transgene-specific PCR with primers
that amplified a 310-bp fragment spanning the junction of the rabbit
-globin intron and the rat lysozyme cDNA (upstream primer, 5'-AAT
TCT GGC TGG CGT GGA AA; downstream primer, 5'-TTG GTA GGG ATC CCC AAG
GCA TT). PCR conditions were 25 cycles at 55°C annealing temperature
with 0.5 µM transgene primers, 0.25 µM dNTPs, 1 U of AmpliTaq DNA
polymerase (Perkin-Elmer/Cetus, Foster City, CA), and 1x AmpliTaq
reaction buffer. PCR results were verified by Southern analyses using a
32P-labeled probe that hybridized to the rabbit
-globin intron. For all studies, transgenic mice were compared with
wild-type (WT) littermate controls. All mice used in this study were
housed in pathogen-free rooms in the animal facility of Children’s
Hospital Medical Center (Cincinnati, OH).
Characterization of transgenic mice
RNA (RT-PCR) analysis.
To identify lines that expressed rat lysozyme mRNA, lung tissues were
collected from 5- to 6-wk-old transgenic mice and control WT
littermates. Total cellular RNA was isolated using TRIzol reagent (Life
Technologies, Grand Island, NY). One microgram of DNase I-treated RNA
was reverse transcribed using the Superscript kit (Life Technologies)
with oligo(dT) as primer. For RT-PCR analyses, rat lysozyme
cDNA-specific primers (upstream, 5'-ACA CAA GCC AGA AAC TAC AACC;
downstream, 5'-GAA TTC TCA GCA TCC GCA GTT CCG AAT) were used with the
PCR conditions described above. -Actin was coamplified in the PCR
reaction as an internal control.
Northern analysis. Ten micrograms of total RNA isolated from lung tissues of 5-wk-old transgenic mice and control WT littermates was fractionated by gel electrophoresis, blotted onto a nylon membrane, probed with biotin-labeled rat or mouse lysozyme cDNA (provided by Dr. Rainer Renkawitz, Max Planck Institut für Biochemie, Martinsried, Germany), and detected by chemiluminescence (Pierce, Rockford, IL).
Analysis of protein expression.
To identify transgenic mouse lines that expressed rat lysozyme protein,
Western blotting was performed using a rabbit anti-human lysozyme
Ab (Accurate Chemicals and Scientific Corp, Westbury, NY) that
cross-reacts with both mouse and rat lysozyme
(Mr, 
15K). Lungs were isolated from
5-wk-old offspring of transgenic founders and homogenized in PBS with
1% (v/v) protease inhibitor mixture (Sigma, St. Louis, MO). Protein
concentration was determined by bicinchoninic acid protein assay
(15). One-half microgram of total lung protein was
analyzed by SDS-PAGE and Western blotting, as previously described
(16). To assess the level of secreted lysozyme, BAL fluid
was obtained from six 5-wk-old transgenic mice and five control WT
littermates as described below. One-half microgram of total protein of
BAL fluid was resolved by SDS-PAGE and Western blotted with rabbit
anti-human lysozyme Ab. Levels of lysozyme proteins in lung
homogenates and BAL fluids were quantitated by scanning densitometry
using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
BAL fluid cell count. Five 5-wk-old transgenic mice and five control WT littermates were exsanguinated by transecting the abdominal aorta and the inferior vena cava after a lethal i.p. injection of sodium pentobarbital. The lungs were lavaged three times with 1-ml aliquots of PBS. BAL fluid was centrifuged at 2000 x g for 10 min, and the pellet was resuspended in 0.5 ml of PBS. A 50-µl aliquot was stained with an equal volume of 0.4% trypan blue (Life Technologies) for total cell count on a hemocytometer. Differential cell counts were made on cytospin preparations stained with Diff-Quik (Scientific Products, McGraw Park, IL).
Lysozyme enzyme activity assay. To assess lysozyme enzyme activity, BAL fluid was obtained from 5-wk-old transgenic mice and control WT littermates. BAL fluid containing 1 µg of total protein was incubated with killed Micrococcus lysodeikticus suspended in 0.4 M phosphate buffer, pH 6.7, at an OD (450 nM) of 1 at 37°C. The change in OD was assessed after 1 h of incubation (17). Purified chicken egg lysozyme was used to generate a standard curve (1 U of enzyme activity equals a 0.001 change in OD).
Lung morphology and immunohistochemistry. To assess lung morphology, lungs from four 5-wk-old transgenic mice and four control WT littermates from transgenic line 3.5 were inflation-fixed for immunostaining and light microscopy as previously described (18). Immunostaining for lysozyme was performed using antiserum directed against human lysozyme (Accurate Chemicals and Scientific Corp.). Parallel lung sections were incubated with normal rabbit serum to verify the specificity of immunostaining.
Bacteria killing assays
Group B streptococci (GBS). Stock cultures of GBS used in this study were clinical isolates provided by Dr. J. R. Wright (Duke University, Durham, NC). To minimize variation in virulence, all bacteria used in this study were selected from aliquots of the same passage that had been frozen at -70°C in 20% glycerol/PBS. For each experiment, an aliquot of bacteria was thawed, plated on tryptic soy/5% defibrinated sheep agar plates, and subsequently inoculated in 4 ml of Todd-Hewitt broth (Difco, Detroit, MI). Bacteria were grown for 14–16 h at 37°C with continuous shaking. The bacteria were harvested from the broth by centrifugation at 200 x g for 10 min, washed, and resuspended in sterile PBS at a concentration of 106 CFU/100 µl. The concentration of the inoculum was verified by quantitative culture on sheep blood agar plates.
Pseudomonas aeruginosa. The strain of P. aeruginosa was a mucoid segregant isolated from the sputum of a cystic fibrosis patient (provided by Dr. D. J. Hassett, University of Cincinnati College of Medicine, Cincinnati, OH). P. aeruginosa obtained from a single passage was stored in aliquots at -70°C in 20% glycerol/Luria-Bertoni (LB) broth. For each experiment, an aliquot of bacteria was plated on LB agar followed by inoculation into 4 ml of LB broth. Preparation of the inoculum was conducted as described for GBS; the concentration (CFU) of the inoculum was verified by quantitative culture on LB plates.
Bacterial infection. The dose of bacteria selected for these studies was based on the results of previous studies in the FVB/N mouse strain (19). Mice were anesthetized with isofluorane, the trachea was exposed through an anterior midline incision, and 106 CFU (GBS) or 107 CFU (P. aeruginosa), suspended in 100 µl of sterile PBS, was delivered just beneath the cricoid cartilage. The incision was sealed by applying one drop of surgical glue (Nexabrand, Veterinary Products Laboratories, Phoenix, AZ). As a control, 100 µl of nonpyrogenic PBS was similarly instilled into transgenic and WT mice. To assess bacterial load at 6 h (GBS) or 24 h (P. aeruginosa) postinfection, mice were anesthetized with i.p. pentobarbital and exsanguinated by transecting the abdominal aorta, and lung and splenic tissues were harvested, weighed, and subsequently homogenized in sterile PBS. Serial dilutions of homogenates were plated on blood agar (GBS) or LB (Pseudomonas) plates and incubated at 37°C overnight. Viable pathogen counts in the lung and spleen were estimated from the number of colonies after 24 h of quantitative culture and expressed as CFU per gram of tissue.
To determine survival following infection, 20 5- to 6-wk-old transgenic mice from the 3.5 line and 20 WT littermates were infected with 2 x 107 CFU P. aeruginosa intratracheally, as described above. Water and food were provided ad libitum during the period of observation. The number of surviving mice was counted every 12 h for up to 60 h, at which time all surviving mice were sacrificed.
Cytokine levels following infection with P. aeruginosa
Eight 5- to 6-wk-old transgenic mice and eight age-matched WT
littermates were infected intratracheally with
107 CFU of P. aeruginosa suspended in
100 µl of PBS. Mice were sacrificed at either 6 or 24 h, lungs
were lavaged as described above, and lung tissues were homogenized in 1
ml of PBS with 1% (v/v) protease inhibitor mixture; the total volume
after homogenization was adjusted to 2 ml with PBS. Mouse TNF-
,
IL-6, macrophage inflammatory protein-2 (MIP-2), and RANTES were
measured by sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN).
The limits of sensitivity for each cytokine are <5.1, <3.1, 1.5, and
<2 pg/ml, respectively. All samples were assayed in duplicate, and
values were normalized to total protein in the samples.
Statistical analyses
Differences between groups were assessed by one-way ANOVA, and differences between means were assessed by contrast comparisons and the Student-Newman-Keuls test (StatView, Abacus Concepts, Berkeley, CA). Data are expressed as the mean ± SD. Nonparametric survival distributions were estimated to examine differences in survival of transgenic mice and WT littermates. The differences between the two groups were analyzed using Kaplan-Meier curve statistics.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the role of lysozyme in pulmonary host defense, transgenic mouse lines were generated in which rat lysozyme was targeted to the distal respiratory epithelium under the direction of the 3.7-kb human SP-C promoter. Seven of 21 offspring from fertilized oocyte injections were positive for the transgene, as assessed by PCR and confirmed by Southern blot analyses of tail DNA (not shown). Transgenic offspring were indistinct from WT littermates with respect to body weight, lung weight, longevity, and reproductive capability. Rat lysozyme mRNA was detected in the offspring of two of seven transgenic lines (lines 3.5 and 2.6) by RT-PCR, and only these two lines were further characterized.
Analysis of transgene expression
Expression of the lysozyme transgene was assessed by Northern blot
analysis of total RNA isolated from the lungs of 5-wk-old mice. A cDNA
probe specific for the rat lysozyme transgene detected an approximately
1-kb transcript (Fig. 1
A).
When the same blot was probed with a mouse lysozyme cDNA, both the
larger endogenous mouse lysozyme mRNA and rat lysozyme transgene mRNA
were detected.
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B) and BAL fluid (not shown); total lysozyme protein in
mice from transgenic line 3.5 was increased 4-fold compared with that
in control WT littermates (Fig. 1B). Lysozyme
(Mr, 142) was not detected when lung
homogenates and BAL were blotted with nonimmune rabbit serum.
To determine whether increased lysozyme protein levels were associated
with increased enzyme activity, lysozyme enzyme activity in BAL fluid
from 5-wk-old transgenic mice and wild-type littermate controls was
assessed by a turbidimetric assay using purified chicken egg lysozyme
as a standard. Lysozyme enzyme activity in mice from transgenic line
3.5 was increased 17.7-fold compared with that in wild-type mice (550
U/ng BAL protein vs 31 U/ng BAL protein in WT mice; p =
0.0001; Fig. 2). As predicted from
Western blot analyses, lysozyme enzyme activity in BAL from line 2.6
was increased relative to that in WT controls
(p = 0.02), but was less than that in BAL from
transgenic line 3.5.
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). No staining was detected in the
submucosa or in nonepithelial cells in either WT or transgenic mice or
when parallel lung sections from transgenic mice were incubated with
nonimmune rabbit serum. Lysozyme expression in transgenic mice
therefore conforms to the previously reported expression pattern for
the 3.7-kb human SP-C promoter (10, 11, 12).
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Effect of transgene expression on killing of bacteria in infected mice
Killing of GBS.
To determine whether increased lysozyme levels in the airway enhanced
killing of bacteria in the lungs, quantitative cultures of lung
homogenates from transgenic mice and WT littermate controls were
compared following intratracheal injection of 106
CFU of GBS. All mice survived until sacrifice at 6 h
postinfection. Killing of GBS was enhanced 3.2-fold in mice from
transgenic line 3.5 (2.1 ± 0.1 x 106
CFU/g protein vs 6.8 ± 0.5 x 106 in
WT mice; p = 0.01; Fig. 4). The incidence of systemic
dissemination of infection, as assessed by growth of GBS on plates
inoculated with splenic homogenates, was less in transgenic mice (27 vs
60%; p = 0.04). Killing of GBS was enhanced 1.7-fold
in mice from transgenic line 2.6 (4.2 ± 0.8 x
106 CFU/g protein vs 7.1 ± 0.6 x
106 in WT mice; p = 0.04).
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). Bacterial killing was enhanced
approximately 6.2-fold in mice from transgenic line 2.6 (6.52 ±
0.71 x 106 CFU/g protein; p
= 0.05). Systemic dissemination was not detected in surviving mice at
24 h postinfection. Following intratracheal infection with 2
x 107 CFU of P. aeruginosa, the mean
survival time for transgenic mice (49.0 ± 2.1 h) was
significantly longer than that of WT littermates (36.0 ± 2.6
h; p = 0.0047; Fig. 6).
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). The percentage of neutrophils in BAL
fluid was significantly higher in transgenic mice than in WT
littermates (61.5 ± 5.6 vs 29.9 ± 7.2; p =
0.014) at 6 h postinfection; however, at 24 h postinfection,
the percentage of neutrophils in the BAL fluid was higher in WT mice
(88.9 ± 1.9 vs 63.7 ± 8.0; p = 0.04; Fig. 7B).
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); however, at 24 h postinfection
there was no significant difference between transgenic mice and WT
littermates. There were no significant differences in the levels of
mouse (m) TNF-, mIL-6, and mRANTES between transgenic mice and
control littermates at 6 and 24 h postinfection (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The spectrum of lysozyme antimicrobial activity in vitro appears to be relatively narrow, leading some investigators to conclude that exogenous lysozyme would be of little benefit in controlling bacterial infection (20). However, it is important to note that the results of in vitro studies are not necessarily predictive of lysozyme activity in vivo because of potential synergistic actions with other antimicrobial agents, including lactoferrin, secretory leukocyte antiprotease, cathelicidins, and defensins. For example, the antibacterial activity of lysozyme and LL-37/hCAP-18, a cathelicidin, have been reported to be synergistic in vitro (21). Lysozyme alone is ineffective against Gram-negative bacteria, while lysozyme in combination with lactoferrin is bactericidal for several Gram-negative strains (22). In the current study the disproportional increase in enzyme activity relative to lysozyme transgene protein may also be the result of such synergy. These results underscore the importance of testing the antimicrobial activity of lysozyme in vivo where numerous synergistic interactions are possible.
Several mechanisms have been proposed for the microbicidal activity of lysozyme in vitro. Lysozyme is a muramidase whose enzymatic activity is typically assessed by hydrolysis of the glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine in the cell wall of Micrococcus lysodeikticus. Muramidase activity in BAL of transgenic mice expressing rat lysozyme was increased 17.7-fold relative to that in WT mice, suggesting that elevated lysozyme enzyme activity may account for much of the enhanced clearance of P. aeruginosa. However, there is also evidence that lysozyme possesses antibacterial activity that is independent of muramidase activity. Enzymatically inactive lysozyme was shown to retain bactericidal activity (23, 24), and During et al. (25) recently demonstrated that small amphipathic helical peptides derived from T4 lysozyme were both bacteriostatic and fungistatic. In the present study, clearance of GBS and Escherichia coli (data not shown) in transgenic mice was enhanced only 3.2-fold, similar to the 4-fold increase in lysozyme protein levels but much less than the increase in lysozyme enzyme activity. These results are consistent with a nonenzymatically mediated bactericidal action of lysozyme against some bacteria.
Enhanced bacterial killing in transgenic mice may be partly due to increased recruitment of neutrophils early in the course of infection. Neutrophil influx into the airspaces was slightly, but significantly, higher in transgenic mice at 6 h postinfection; further, the level of mMIP-2, a neutrophil chemotactic factor, was significantly elevated in transgenic mice at this time point. In contrast, neutrophil numbers and mMIP-2 levels in unchallenged transgenic mice were not different from those in WT littermates. These data suggest that constitutive overexpression of lysozyme in the airways may enhance neutrophil recruitment following infection. Although it is reasonable to speculate that increased neutrophil influx may contribute to enhanced bacterial killing in these transgenic mice, this hypothesis has not been directly tested.
Lysozyme was previously identified in rat alveolar type II epithelial cells by immunohistochemistry (5, 6). In the present study lysozyme was amplified by PCR using cDNA generated from highly purified isolated rat type II epithelial cells; the deduced amino acid sequence of the amplified product was identical with the previously reported sequence for rat lysozyme (26) with the single exception of a substitution of glycine for tryptophan at position 46. Immunogold labeling and subcellular fraction of type II epithelial cells localized lysozyme to lamellar bodies, secretory granules in which pulmonary surfactant is stored (27, 28, 29). Following secretion, lysozyme was detected in association with tubular myelin, a lattice-like network of surfactant membranes (27, 28). Interestingly SP-A, another lamellar body protein involved in airway host defense (30, 31), was also associated with tubular myelin (28, 32), raising the possibility that this structure may serve as a scaffold for host defense proteins involved in alveolar surveillance.
Although the present study has not defined the precise mechanism by which lysozyme promotes bacterial killing in vivo, these results have important implications for enhancing airway host defense. Lysozyme levels in transgenic mice were constitutively elevated without affecting lung structure, suggesting that lysozyme could be safely administered for extended therapy. Most importantly, elevated levels of lysozyme significantly enhanced killing of P. aeruginosa, the most common pathogen associated with chronic colonization of the airway in cystic fibrosis patients. Lysozyme administration alone or in combination with other agents, such as elastase inhibitors (33), may provide an important adjunct to treatment of chronic lung infections with antibiotics.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Henry T. Akinbi, Division of Pulmonary Biology, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039.
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; SP-C, surfactant protein C; GBS, group B streptococci; LB, Luria-Bertoni; WT, wild type; MIP-2, macrophage-inflammatory protein-2; m, murine.
Received for publication February 7, 2000. Accepted for publication August 17, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, WT mice; •, transgenic
mice.
,
transgenic mice.
, transgenic mice.