Bacteria Associated With Obstructive Pulmonary ... - Europe PMC

Bacteria Associated With Obstructive Pulmonary Disease Elaborate Extracellular Products That Stimulate Mucin Secretion by Explants of Guinea Pig Airways

From the Departments of Pathology and Medicine, College of Medicine, University of Vermont, Burlington, Vermont

KENNETH B. ADLER, DANIEL D. HENDLEY, and GERALD S. DAVIS

Certain cell-free filtrates from broth cultures of Pseudomonas aeruginosa, Hemophilus influenzae and Streptococcus pneumoniae stimulate secretion of glycoconjugates by explants of guinea pig trachea. The stimulatory effect is not related to toxicity or damage to the respiratory mucosa, as well as could be determined by ultrastructural examination ofthe explants after exposure. Bacteria isolated from patients with a history of chronic obstructive lung disease (P aeruginosa from cystic fibrosis, Hinfluenzae, and S pneumoniae from chronic bronchitis) do not demonstrate increased frequency of positive strains or greater stimulation of secretion than organisms isolated from other individuals. At least three stimulatory substances are found in cell-free filtrates of P aeruginosa. They appear to be proteins of molecular weight 60,000-100,000 as determined by gel filtration. Within the crude filtrate, they are relatively stable to heat, proteolysis, and storage at 4 C and in liquid nitrogen. The stimulatory activity is not lost upon subculture ofthe bacteria. When isolated from the filtrate by column chro-

matography, they become labile to heat and trypsin. Isolated active fractions show proteolytic activity coinciding with mucin-stimulating capacity, suggesting a relationship with Pseudomonas proteases. Stimulatory substances released by S pneumoniae and H influenzae appear to be diffierent from those elaborated by Pseudomonas. They are extremely labile to heat and storage, and the capacity to stimulate secretion is lost on subculture. Preliminary gel filtration indicates the S pneumoniae stimulatory substance(s) is in a molecular weight range of 100,000-300,000 daltons, while that ofHinfluenzae is between 50,000 and 200,000. The results suggest bacteria which chronically infect or colonize respiratory airways of individuals suffering from obstructive lung disease can elaborate extraceliular product(s) capable of stimulating secretion of mucin. Thus, the bacteria themselves may contribute to local manifestations and, ultimately, to the pathogenesis of obstructive disease. (Am J Pathol 1986, 125:501-514)

BOTH cystic fibrosis (CF) and chronic bronchitis are characterized by increased production and secretion of respiratory mucus, hypertrophy and hyperplasia of mucus-producing cells and glands, and recurrent or chronic respiratory infection and colonization of airways with bacteria. Colonization of the respiratory tract with mucoid strains of Pseudomonas aeruginosa is common in patients with CF1 2; whereas patients with chronic bronchitis suffer recurrent infection, very often with Streptococcus pneumoniae and Hemophilus influenzae.3 4 Despite concurrent bacterial infection and excess mucus during the course of these diseases, relationships between bacterial-derived substances and secretion of respiratory mucin have not been elucidated. In previous reports, we and others have described stimulatory effects of bacterial products on secretion of mucin. Cholera toxin was shown to stimulate secretion of respiratory mucin independent of its action on

adenylate cyclase.5 Cell-free filtrates from strains of P aeruginosa, isolated from individuals with and without cystic fibrosis, stimulate secretion of mucin.6-8 Recently, Klinger et a19 demonstrated that Pseudomonas proteases stimulate mucin release by explants of rabbit airways in vitro. Niles et al10 reported that serine proteases stimulate mucin secretion by explants of hamster trachea. The purpose of the studies reported here was to determine whether or not products elaborated by bacteSupported by Grant 1515 from the Council for Tobacco Research, USA Inc., and Grant Al 18215 from the National Institutes of Health. Accepted for publication July 22, 1986. Address reprint requests to Kenneth B. Adler, PhD, Department of Pathology, College of Medicine, University of Vermont, Burlington, VT 05405.

501

502

ADLER ET AL

ria associated with CF (P aeruginosa) and chronic bronchitis (S pneumoniae, H influenzae) could affect secretion of mucin by explants of guinea pig airways maintained in organ culture. Additional studies were undertaken to characterize stimulatory substances elaborated by these bacterial genera, and to correlate the presence or absence of stimulatory strains of bacteria in the respiratory tract to the disease state and clinical condition of the patients of origin. Materials and Methods Materials Agar plates for bacterial growth (chocolate, sheep blood, and Pseudomonas isolation), Filde's enrichment, and Trypticase soy and casamino acid broth were purchased from Difco (Detroit, Mich). Waymouth's MB 752/1 tissue culture medium, gentamicin, nystatin, and Hanks' balanced salts solution were from GIBCO (Grand Island, NY). Glucosamine HCl (D-6-3H[NJ) was bought from New England Nuclear (Boston, Mass). Centricon microconcentrators were purchased from Amicon Corporation (Danvers, Mass). Trypsin (porcine pancreatic, Type IX); soybean trypsin inhibitor, hyaluronidase (ovine testicular), elastase (porcine pancreatic), pronase (S gresius), phospholipase C (Clostridium perfringens), and azocasein were purchased from Sigma Chemical Co. (St. Louis, Mo). Collagenase was purchased from Worthington Biochemicals, Freehold, NJ. Materials for column chromatography (Sephadex, agarose) were purchased from Pharmacia Corporation (Piscataway, NJ). Instagel was from Packard Corporation (Downers Grove, Ill). Bacterial Isolates: Identification and Characterization Isolates of H influenzae, S pneumoniae, and P aeruginosa recovered from sputum of hospitalized patients were identified by standard methods at the Microbiology Laboratory of the Medical Center Hospital of Vermont. Briefly, H influenzae was characterized by Gram stain, colonial morphology, growth on chocolate agar but not sheep blood agar, and appropriate reactions with S-aminolevulinic acid. S pneumoniae was identified by Gram stain and colonial morphology, by solubility in bile, and by susceptibility to optachin. P aeruginosa identification was based on biochemical reactions as described previously.6 A clinical history of the patient of origin, including major diagnosis at the time of bacterial isolation and presence or absence of chronic lung disease, was obtained. Bacterial colonies were maintained on chocolate agar

AJP * December 1986

(H influenzae), sheep blood agar (S pneumoniae), or Pseudomonas isolation agar (P aeruginosa). Colonies were subcultured two to three times per week. Preparation of Bacterial Filtrates Ten milliliters Trypticase soy broth (supplemented with Filde's enrichment for H influenzae) or casamino acid broth (for Pseudomonas) was inoculated with each isolate and incubated at 37 C for 24 hours under aerobic, static conditions. The broth cultures then were centrifuged at 2000 rpm for 15 minutes. The supernatants, containing extracellular products of the bacteria, were sterilized by passage through a 0.22-j polymer filter (Millipore, Medford, Mass). Sterilization was confirmed by inoculation of an additional 10 ml broth with 0.1 ml of each filtrate, with no subsequent growth. Colonyforming units (CFUs) were assayed from each broth culture to ascertain similar numbers of bacteria were involved in elaborating the extracellular products to be tested. Cell-free filtrates from 26 isolates of Spneumoniae, 28 isolates of Hinfluenzae, and 18 isolates of P aeruginosa were tested for effects on mucin secretion. Each filtrate was tested at full strength, and at 1:4 and 1:6 dilution in culture medium, as described below.

Tracheal Organ Cultures Explants of trachea from guinea pigs were placed into organ culture as described previously.5""12 Briefly, male Hartley guinea pigs (Charles River of Canada) weighing approximately 250 g, were sacrificed by intraperitoneal injection of sodium pentobarbitol. Trachea were excised aspetically and divided into explants. The entire trachea, from larynx to carina, was removed and immersed in-warm (37 C) Hanks' balance salt solution (HBSS). Adhering connective tissue was dissected away, and the trachea was opened along the dorsal aspect. Explants, approximately 3 x 4 mm in surface area, were placed into 35-mm plastic Petri dishes previously scratched with a scalpel blade to facilitate adherence of the tissue. The organ cultures were incubated in a humid 95% air/5% CO2 environment at 37 C in 0.5 ml of Waymouth's MB 752/1 medium without serum. We added gentamicin (100 gg/ml) and nystatin (25 U/ml) were added to control for microbial contamination. Cultures were rocked at 5 cycles/min. Assay for Mucin Secretion Explants from 6-12 animals (depending on the experiment) were pooled together and incubated for 18 hours in Waymouth's medium containing 10 gCi/ml

Vol. 125 *m No. 3

BACTERIAL PRODUCTS STIMULATE MUCIN SECRETION

glucosamine-HCl (D-6-3H[NJ), specific activity 5-15 mCi/ml. As described previously,5' 1-13 this time period is sufficient for incorporation of tritiated glucosamine into intracellular glycosylated macromolecules subsequently secreted into the culture medium by the tissues (see discussion section). At the end of the 18-hour period, explants were washed with HBSS for removal of unincorporated label and then placed randomly into new 35-mm Petri dishes, 4 explants per dish, and 0.5 ml of fresh, unlabeled Waymouth's medium added. No attempt was made to keep explants from individual animals together; each dish contained explants from several different animals. The dishes were incubated for a 5-hour "baseline" period, at the end of which the medium was removed, the explants washed with 0.1% dithiothreitol in 2 ml HBSS, and the spent medium and washings pooled and saved. At this time, the bacterial filtrates, adjusted to pH 7.4 and 340 mOsm as described previously,6 were added for an additional 2-hour "experimental" period. Sterile broth that had not been exposed to bacteria, but similarly adjusted in pH and osmolarity, was used as a control. At the end of the 2-hour experimental period, the culture media were removed and saved as described above. The media, containing the radiolabeled glycosylated macromolecules, were incubated with 0.107o sheep testicular hyaluronidase for 30 minutes at 37 C. At this time, 10 ml of cold 10%7o trichloracetic acid (TCA) and 1% cold phosphotungstic acid (PTA) were added to each of the medium samples (plus washings) and stored overnight at 4 C. The next day, the precipitates were centrifuged, washed, redissolved in 1 ml of 0.1 N NaOH, and added to 9 ml scintillaton counting fluid (Instagel). Counts per minute of tritium in the precipitates were determined in a Packard scintillation counter after appropriate corrections for quenching. Secretion during the 2-hour "experimental" period was expressed as a percentage of the 5-hour "baseline" period to allow for differences in secretory activity among different dishes of explants. In order to minimize possible effects of interanimal and interexperimental variation on results, we tested paired control samples consisting of sterile broth adjusted in pH and osmolarity to 7.4 and 340 mOsm, respectively, in parallel every time a bacterial product was tested.

Morphologic Studies In studies using the above-described protocol, it is necessary to differentiate true "secretion" from passive release of intracellular labeled macromolecules consequent to toxicity and cell damage (see discussion section). To determine whether or not damage to the explants was associated with exposure to bacterial filtrates,

503

we removed randomly selected explants from culture dishes at the conclusion of the experiments and, after appropriate fixation for light, scanning, or transmission electron microscopy, examined them for morphologic indications of toxicity or cell damage.5'6 11 For light microscopy, tissues were fixed in 1007o phosphate-buffered formalin for 24-48 hours and embedded in paraplast. Sections cut at 6-10 ,u in thickness were examined after staining with hematoxylin and eosin (H&E). Samples for SEM were immersed in Karnovsky's fixative for 24 hours, dehydrated through graded alcohols, and critical point-dried from Freon-13. After vacuum-coating with gold-palladium, specimens were examined in a JEOL JSM 35C scanning electron microscope at 15 kv. Tissues for transmission electron microscopy were fixed in Karnovsky's, dehydrated through graded alcohols and propylene oxide, and embedded in araldite. Sections of 0.5-g thickness were examined after staining with toluidine blue, and selected areas were thin-sectioned (600-800 angstroms), mounted on copper grids, stained with saturated uranyl acetate in methanol and basic lead citrate, and examined at 60 kv in a Zeiss EM-IOC transmission electron microscope.

Stability to Passage Stimulatory strains of bacteria were subcultured two to three times a week on appropriate media for up to 40 passages. Multiple colonies of subcultured strains were picked to assure "population" sampling and inoculated into the appropriate broth medium. They then were tested again (with paired controls) for effects on mucin secretion as described above. Ultrafiltration

Stimulatory strains of P aeruginosa, H influenzae, and S pneumoniae (see Results) were investigated further to elucidate the nature of stimulatory substance(s). The filtrates were concentrated approximately 80 times by ultrafiltration14 through a Centricon-10 microconcentrator, consisting of a low adsoprtion, hydrophilic YM membrane with a 10,000 molecular weight cutoff. The filtrates then were reconstituted in Waymouth's medium. (This procedure did not diminish the potency of the stimulatory strains regarding enhancement of mucin secretion by tracheal explants.) Studies then were undertaken to characterize the stimulatory substances within the reconstituted filtrates. In all experiments described below, paired controls consisting of reconstituted broth that had not supported bacterial growth, but subjected to the identical perturbations, were run in parallel every time a bacterial product was tested.

504

ADLER ET AL

Stability to Heating The reconstituted filtrates were heated at 55 or 90 C for 30 minutes, and effects on mucin secretion were noted in comparison with reconstituted filtrates maintained at room temperature during this ½2-hour period. Each filtrate was tested in triplicate (n = 3).

Sensitivity to Trypsin Reconstituted stimulatory filtrates were treated with porcine pancreatic trypsin (Sigma) at 1-2% of total protein present (as determined by a standard Lowry assay'5) in 0.1 M NaHCO3, pH 7.4, for 15 minutes at 37 C. After 15 minutes, soybean trypsin inhibitor (Sigma) was added at the same concentration to stop the reaction, and the effects of the filtrates on mucin secretion were assayed. An additional control was soybean trypsin inhibitor alone, to preclude the possibility that it could inhibit bacterial enzymes and thus affect the secretory response. Each filtrate was tested in triplicate (n = 3). Stability to Storage/Freezing Reconstituted stimulatory filtrates were stored in cold (4 C), at -25 C, at -70 C, or in liquid nitrogen, in the presence or absence of 0.1-0.2% EDTA, for time periods of 48 hours to 2 weeks. At the end of the storage times, the filtrates were brought to 37 C in a water bath, then tested again, in triplicate, for mucinstimulatory activity.

Characterization of Stimulatory Components: P aeruginosa A stimulatory strain of Pseudomonas (Pa 8) was used. Column chromatography studies were carried out for characterization of the stimulatory components within the culture filtrate.

Molecular Sieve Chromatography Three hundred milliliters of concentrated filtrate was reconstituted in 0.002 M phosphate buffer to a volume of 20 ml, and 2 ml passed through a 38 ml G-200 Sephadex column equilibrated and eluted with 0.002 M phosphate buffer, pH 7.4, at a flow rate of 30 ml/hr. Fractions of 2.0 ml were collected. Each fraction was tested for absorbance at 206 mg, and also for effects on mucin secretion. (Absorbance at 206 mg was measured using a flow cell, path length 2.5 mm, with the buffer baseline set a 0. The UV monitor trace at 206 mg was expressed relative to the buffer baseline, and therefore is expressed as "relative absorbance.") The experiment was repeated three times, and values for mucin secre-

AJP * December 1986

tion were expressed as mean ± standard error (SE) for these three experiments. Ion Exchange Chromatography I The filtrate was passed through a column of DEAE agarose to separate components on the basis of anionic properties. Two milliliters of concentrated filtrate was applied to an 8-ml DEAE agarose column in 0.02 M Tris Cl, pH 8.3. The eluent was a linear gradient of NaCl, 0.0 to 0.2 M, at a flow rate of 30 ml/hr. Again protein content (absorbance at 206 mg) and effects on mucin secretion (n = 3) of each fraction were monitored.

Ion Exchange Chromatography II CM agarose chromatography then was carried out to separate components of the concentrated filtrate on the basis of cationic properties. The concentrated filtrate (10 ml) was run on a Sephadex G-25 column equilibrated with 0.1 M NaAc, pH 4.8, 0.5 mM EDTA, at a flow rate of 30 ml/hr. Active fractions then were passed through an 8-ml CM agarose column (equilibrated with the same buffer) and eluted with a linear gradient of 0.0-0.4 M NaCl, flow rate 25 ml/hr. Relative absorbance at 206 mg and mucin stimulatory activity (n = 3) for each fraction were measured.

Effects of Heat and Trypsin on Isolated Fractions Fractions of the filtrate from the CM Agarose column which showed mucin stimulatory activity (see results) were investigated further. The stimulatory activity of these fractions was tested for heat and trypsin lability (n = 3) as described above. Assay of Isolated Fractions for Protease Activity Since previous studies8-10 have suggested Pseudomonas proteases can stimulate mucin secretion, fractions from the column with mucin-stimulatory activity also were tested for proteolytic activity. A generalized protease assay using azocasein as a substrate, as described by Fischer and Allen,'6 was utilized. Briefly, 50 gl of 2% azocasein (Sigma; final concentration, 0.67%) was mixed with 50 gl each of buffer (Tris HC1:0.0167 M final concentration) in HBSS and test material (or commercial protease [see below] as the positive control), and incubated in stoppered tubes at 37 C for ½ hour. The reaction was stopped by addition of 0.15 ml cold 5% TCA for 5 minutes after chilling the tubes to 0 C. The tubes then were heated to 37 C for 5 minutes for completion of the precipitation. One milliliter of 2.5% TCA then was added, and the mixture was centrifuged at 2000 rpm for 10 minutes. The supernatant was passed through a small glass wool filter made alkaline with 0.1

Vol. 125 * No. 3

BACTERIAL PRODUCTS STIMULATE MUCIN SECRETION

ml of 10 M NaOH. The optical density (OD) at 440 mg then was measured in a Perkin Elmer Lambda 3 spectrophotometer. A unit of protease activity was defined as that amount required to reach a given stage of the reaction (OD of 0.1 in 1 hour at 37 C). Absorbances in the range of 0-0.3 were converted to protease units by means of a plot of enzyme concentration versus absorbance established for trypsin; 1 gl of a 0.1% solution of trypsin (Sigma Type IX) had an activity of 2.5 units. Effects of Commercial Proteases on Mucin Secretion To address further the possibility that bacterial proteases were involved in the mucin stimulatory activity, several additional studies were undertaken. The first question addressed was whether or not "proteases" in general were capable of stimulating secretion. Several commercial preparations of proteases were examined for effects on mucin secretion: Pronase from S gresius, and trypsin, collagenase, and elastase (Sigma, all from porcine pancreas) were adjusted in concentration to an equal activity toward the substrate, azocasein, as the Pseudomonas (Pa 8) filtrate (see above). Effects on mucin secretion at this concentration, and at further dilutions of 1:4 and 1:16, were monitored. Three replicate experiments were carried out at each concentration.

Correlation of Mucin-Stimulatory and Proteolytic Activity The next question addressed was whether or not the protease activity of the active fractions correlated with mucin stimulating activity. The comparative effects of freezing/thawing (- 25 C or - 70 C for 48 hours), heating to 55 C and 90 C for 30 minutes, and trypsin digestion (1-2% total protein present for 15 minutes) on the respective mucin-stimulatory and proteolytic activities of active column fractions were examined. Each determination was carried out in triplicate (n = 3). Effects of Phospholipase C on Mucin Secretion The possibility was investigated that phospholipase, another product often associated with Pseudomonas, could be involved in the mucin-stimulating activity. Phospholipase C from Clostridium perfringens (Sigma) was tested for effects on mucin secretion over a concentration range of 5-100 gl/ml (n = 9; see Table 7). Concentrations of phospholipase that stimulated secretion were compared to the phospholipase activity of stimulatory filtrates with the use of the lipase assay described by Rodball (17).

505

Characterization of Stimulatory Components: H influenzae and S pneumoniae Most of these biochemical studies were carried out on filtrates of Pseudomonas. This is because the stimulatory strains of both S pneumoniae and H influenzae proved to be highly unstable to storage at any temperature, losing their stimulatory effect quite rapidly, even at liquid nitrogen temperatures, and upon subculture. Therefore, only preliminary chromatography of these filtrates on a Sephadex G-200 column, separating on the basis of molecular weight (as described above), was possible. Statistical Analysis In studies involved with mucin secretion, experimentals were compared with controls with the use of the Schefe procedure for multiple comparisons."' A confidence level of co0

.

-i

t-

a

;-

-

,

;

4 AL.. A.

4

A

a

a

%.:'i

BACTERIAL PRODUCTS STIMULATE MUCI

Vol. 125 * No. 3

SECRETION

509

tein [see Materials and Methods]) had a far greater inhibitory effect on filtrates of H influenzae and S pneu-

E

-

0

0

0

C) 40

CM c0

.0 D

o

C

0

.0

4

.2 0

0 m

V 0 C,)

0 .5

C

.) 8

4

0

12

16

20

24

28

Fraction # (2.Oml) Figure 2-Gel filtration through Sephadex G-200 of culture filtrate from stimulatory strain of P aeruginosa (Pa 8). The 38 ml column was equilibrated and eluted with 0.02 M phosphate buffer, pH 7.4. Two milliliters of reconstituted filtrate was added to the column at a flow rate of 30 ml/hr. Mucinstimulatory activity (-0 -) was confined to Fractions 11-21 (peak at 0.08 M phosphate buffer) corresponding to a protein peak --- -) of molecular weight approximately 60,000-100,000 daltons. n = 3 at each point; mean + 1 SEM.

(Hi) 11 and 13) to enhance mucin secretion. Reconstituted filtrates from each of the Pseudomonas strains appeared slightly but not significantly affected by heating to 55 C for 30 minutes. Heating to 90 C had more of an inhibitory effect, but significant stimulation of secretion still was observed. In marked contrast, reconstituted strains of both S pneumoniae and H influenzae lost much of their stimulatory effect after heating at 55 C for ½ hour and essentially all of their capacity to stimulate mucin secretion after heating to 90 C for this time. Similarly, exposure to trypsin (1-207 total pro-

moniae.

This overall schema held true for other forms of pertubation. The stimulatory effect of the Pseudomonas strains was preserved after freezing of filtrates in liquid nitrogen, whereas that of other bacterial filtrates was lost after storage at every temperature and time tested. The presence of 0.1-0.2% EDTA stabilized the Pseudomonas, but not the Streptococcus or Hemophilus product(s), to freezing at -25 and -70 C. Isolation and Characterization of Stimulatory Substance(s): Pseudomonas The stimulatory Pseudomonas strain Pa 8 was examined further for characterization of the active components within the filtrate. As illustrated in Figure 2, gel filtration through a Sephadex G-200 column gave a single peak of mucin-stimulatory activity in fractions corresponding to a molecular weight range of 60,000-100,000. Passage of the stimulatory filtrate through a DEAE agarose column (Figure 3) resulted in a peak of stimulatory activity in fractions eluting after the bulk of the 206 mu absorbing material, suggesting an acidic protein. CM agarose chromatography, however, (Figure 4) revealed a large peak of stimulatory activity in fractions corresponding to a major protein peak late in the gradient, and thus a basic protein

E

A 0

I

0

/I I

E D 0

C) C

I

1.0

200o

C4

0.

z

c.0

1n

I I I I-

0

/

0

150 .o

0

C.) b-

0

0 N.

0

0

.0

0 c

._

C

0

.0.

4 0

C%

0

0

(n

4.5

100

0

2

:~~~~~~~~~~~. *. 6 4 8 10 12

14

16

18

8

2

n

Fraction # (1.5 ml) Figure 3- DEAE-agarose chromatography of culture filtrate from stimulatory strain of Pseudomonas aeruginosa (Pa8). Twenty milliliters of culture filtrate was concentrated by ultrafiltration and applied to an 8-mI DEAEagarose column in 0.02 M Tris-CI, pH 8.3. Two milliliters was added to the column at a flow rate of 30 ml/hr. The eluant was a linear gradient of NaCI, 0.0 to 0.2 M. A single peak of mucin-stimulatory activity (-*-) appears in fractions eluting at 0.13 M NaCI, after the bulk of the 206 mtt absorbing material --- -), suggesting an acidic protein. n = 3 at each point; mean + 1 SEM.

Fraction # ( 2.0 ml) Figure 4-CM-agarose chromatography of culture filtrate from a stimulatory strain of P aeruginosa (Pa 8). Thirty milliliters of filtrate was concentrated to 2 ml by ultrafiltration and gel-filtered on Sephadex G-25 equilibrated with 0.01 M NaAc, pH 4.8, with 0.5 mM EDTA (flow rate, 25 ml/hr). Fractions corresponding to protein peaks then were run on an 8-mi CM-agarose column (equilibrated with the same buffer) and eluted with a linear gradient of 0.0 to 0.4 M NaCI. A peak of stimulatory activity (-0-) was observed concentrated in Fractions 16-20, corresponding to a major protein peak (--- ) late in the gradient (0.36 M NaCI) and thus a basic protein. Stimulatory activity in early fractions not sticking to the column might correspond to the acidic stimulatory material observed in the DEAE-agarose column (see Figure 3), suggesting the existence of more than one stimulatory protein. n = 3 at each point; mean + 1 SEM.

510

ADLER ET AL

AJP * December 1986

g

E 0

It

0.2

strate.17 As illustrated in Figure 5, three peaks of mucinstimulating activity (fractions 15-20 [ncot retained by column]; 31-37 and 40-44) also containeed potent pro-

teolytic activity.

I200 0

U. >

20

3

40/5

(A

0

2.

00

o--

2

0.~~~~~~~~~~~ 20

14

40

30

50

Fraction # (2.Oml) Figure 5-Correlation between mucin-stimulatory and protease acti fractions from Pa 8 eluting from a 20 ml CM agarose column (2.0 r ded; flow rate, 30 ml/hr). Three peaks of mucin-stimulatory activity (are observed: Fractions 15-20 (not retained on column, peak at 0 NaCI), fractions 31-37 (peak at 0.32 M NaCI), and Fractions 40-44 at 0.38 M NaCI). Proteolytic activity (- -) also is confined only to fractions. n = 4 at each point; mean 1 SEM.

(stimulatory activity in early fractions not adherin the column could correspond to the acidic stimula material observed with the DEAE column seen in ure 3). This suggested the existence of more than stimulatory substance within the bacterial filtrat4

Effects of Phospholipase C on Mucin Secretion As illustrated in Table 5, commercial phospholipase C stimulated secretion of mucin in a dose-dependent Table 4-Effects of Heat, Trypsin, and Freeze/Thawing on Mucin-Stimulatory and Proteolytic Activity of Active Fractions of Pseudomonas Filtrate (DEAE Cellulose Chromotography) Mucin secretion Strain (% control)t Proteolytic activityt

Assay of Isolated Fractions for Protease Activit) The possibility that the stimulatory substances ir active filtrates were related to proteases was investiga Active fractions (stimulating mucin secretion) elu from a 20-ml CM agarose column were tested general proteolytic activity with azocasein as a Table 3-Proteolytic Activity of Bacterial Filtrates

Organism

Protease (units/ml)*

Pseudomonas Pa 3 Pa 4 Pa 5 Pa 6 Pa 8 Pa 10 Pa 16 Streptococcus Sp 12 Sp 22 Sp 26

0.051 3.332 0.028 0.097 14.622 0.023 0.033

Mucin secretion (% of control)t

92 102

9 13

0.000 0.002 0.001 0.003 0.001

112 77 196

2 10

0.000 0.000 0.000

144 136 80

136 143 96 130 145

Pa 14 Heat (C)* 37 55 90 Freezing (C)11 -25 -70 Trypsin1

1

2t

8t

38t

Hemophilus

Hi 11 Hi 13 Hi 14

14t 8t 8

*Protease activity against substrate, azocasein, expressed units tivity/ml for triplicate experiments; mean 1 SEM. t Means 1 SEM; Values for mucin secretion are those from Table 1. t P < 0.05, significantly different from control.'9 as

±

Pa 6 Heat (C)* 37 55 90 Freezing (C)II -25 -70

Trypsinl

5t 5t

0.012 0.277 0.003 0.009 0.778 0.002 0.001

Correlation of Mucin-Stimulatory and Proteolytic Activities The above data suggested that Pseudomonas proteases were related to the components responsible for stimulation of mucin secretion by the cell-free filtrates. To test this theory further, we tested filtrates from other stimulatory strains of Pseudomonas for proteolytic activity with the same azocasein procedure. As illustrated in Table 3, several stimulatory filtrates also had potent proteolytic activity; but, surprisingly, others did not. Results of additional studies testing the effects of heating, trypsinization, and freezing/thawing on concurrent mucin-stimulatory and proteolytic capacity are illustrated in Table 4.

ac-

207 + 33§ 151 + 14§ 103 ± 7 107 119 86

6 5 8

100 (Control) 97 2 2 1§

98 92 4

7 5 5§

177 ± 11§ 142 + 9§ 124 ± 15

100 (Control) 99 ± 5 24 + 13§

109 ± 8 129 + 4§ 101 ± 11

107 ± 11 100 4 0 ±§

* Exposed to these temperatures for 10 minutes. t All values are means ± 1 SEM. n = 3 at each point. Paired control samples were tested in parallel for each experiment. t See Table 3. Expressed as percentage of proteolytic activity of fraction kept at 37 C. n = 3 at each point. § P < 0.05, significantly different from control.1'9 I Stored for 48 hours at the indicated temperature. 1 Trypsin adjusted to 1-2% of total protein present, incubated at 37 C, for 15 minutes.

Table 5-Effect of Proteolytic and Lipolytic Enzymes on Secretion of Mucin by Cavine Tracheal Explants Mucin secretion (% of control)* 1:4 Fullt n Conc: Enzyme Trypsin Elastase Collagenase Pronase

261 278 300 311

9 9 9 9

*

9

± 44t ± 32t

39t 16t 100411/ml

Conc: Phospholipase C§

511

BACTERIAL PRODUCTS STIMULATE MUCIN SECRETION

Vol. 125 * No. 3

± ±

247 ± llt

187 ± 23t 169 + 12t 188 ± 21t 200 ± 4t 50 gI/ml 229 ± 14t

1:16 144 129 155 139

± 11t ± 7t ± 131 ± llt

5 tll/ml 154 ± 3t

All values are means ± 1 SEM. Paired control samples were tested in parallel for each experiment.

t Proteases were adjusted to 2-3 units of protease activity toward the substrate, azocasein/ml; 0.1% solution in Tris buffer, pH 8.0 (see text). They were tested at this concentration and at 1:4 and 1:16. Controls were this solution without addition of proteases.

t P < 0.05, significantly different from control."9 § Phospholipase C from Clostridium perfringens was tested at concentrations of 100, 50 and 5 Ill/ml.

manner. However, none of the stimulatory strains of Pseudomonas demonstrated phospholipase activity (as measured by the general egg yolk turbidity assay of Rodball17). (Other assays more specific for Pseudomonas phospholipase, such as the p-nitrophenylphosphorycholine assay of Berka et a120 were not carried out; so it is still possible that the Pseudomonas isolates may have had phospholipase activity, and it would be premature at this point to conclude that Pseudomonas phospholipases were not contributing to stimulation of mucin secretion provoked by the cell-free filtrates.)

Isolation and Characterization of Stimulatory Substances: Hemophilus influenzae and Streptococcus pneumoniae In general, the stimulatory activity of the S pneumoniae and Hinfluenzae appeared far more labile than that of the Pseudomonas filtrates. This inhibited severely further efforts to isolate active components in filtrates of H influenzae and S pneumoniae. For this reason, only preliminary estimations of molecular weight range for the active components in cell-free filtrates from these two bacterial types were possible. Column chromatography studies carried out with a stimulatory strain of S pneumoniae (concentrated 80 times by ultrafiltration and reconstituted in buffer, 2 ml applied to the column at a flow rate of 30 ml/hr) revealed stimulatory component to elute from a Sephadex G-200 column (equilibrated and eluted with 0.02 M phosphate buffer, pH 7.4, as described above for Pseudomonas) in a region corresponding to a molecular weight range of 100,000-300,000 daltons. The stimulatory component from H influenzae, in preliminary studies, appears to be of similar molecular weight. Filtrates from isolates from both of these bacteria contained no measurable proteinase activity with the use of the azocasein substrate method (Table 3).

Discussion The results of this study demonstrate that certain strains of bacteria that colonize and infect respiratory tracts of individuals suffering from obstructive lung disease are capable of elaborating extracellular substances that can stimulate secretion of respiratory mucin by explants of guinea pig trachea. This is true for P aeruginosa, an organism that colonizes airways of patients with CF, and, to a lesser extent, with both S pneumoniae and H influenzae, organisms associated with respiratory tract infections in individuals suffering from chronic bronchitis. The stimulatory effect on secretion is not secondary to toxicity, damage to secretory cells, or proliferative changes in the mucosa, as well as could be determined by ultrastructural examination of tissues and cells after exposure to cell-free filtrates from these organisms. Throughout this report, the term "mucin" is used to describe the glucosamine-labeled, hyaluronidase-resistant macromolecules recovered by precipitation with TCA/PTA. Although we did not carry out extensive biochemical analysis of the recovered material in these specific studies, this methodology, or others quite similar, have been described extensively in the literature as appropriate for estimating release of mucous glycoprotein from explants of respiratory airways, 21-5 gut,26 and uterine cervix." These investigations involved biochemical and chromatographic assay of the radiolabeled precipitated macromolecules, showing them to be mostly mucous glycoprotein. In other studies utilizing column chromatography of TCA/PTA-precipitable, glucosamine-labeled macromolecules, we have found, after treatment with hyaluronidase, that the overwhelming majority of recovered material is mucous glycoprotein. However, since extensive biochemical analysis of the recovered glycoconjugates was not carried out in these studies, what we refer to as "mucin" in this report

512

ADLER ET AL

technically is high-molecular-weight, glycosylated macromolecules resistant to hyaluronidase. As reviewed by Boat,28 many exogenous substances, when applied to in vitro systems, can elicit release of radiolabeled macromolecules by generating cell damage or necrosis. Therefore, it is necessary to in some way ascertain that the secretagogue action of an experimental agent is not the result of a toxic response in secretory cells. There are several different ways to approach this problem. The most sensitive assays for toxic injury or cell damage are biochemical; assays such as release of lactate dehydrogenase (LDH), labeled chromium, or selenium are well suited to address this question. However, in an organ culture system, as opposed to a pure cell culture system, there are several different cell types within an explant. We would not be able to identify the cellular source of, for example, released LDH. In these studies we specifically were interested in epithelial cells, and whether or not release of macromolecules was related to active "secretion," rather than a result of membrane perturbation or a generalized toxic response. Since, to our knowledge, there are no reliable markers specific for epithelial cell injury, it was believed that ultrastructural examination of the epithelium after exposure to the filtrates or other agents of interest was the best overall way to assess damage or toxicity, with the realization that subtle biochemical alterations not yet observable in the electron microscope could have occurred. The stimulatory products from Pseudomonas appear related to proteases of molecular weight 60,000-100,000 daltons. There appear to be several different substances capable of stimulating secretion of mucin within a single bacterial filtrate (see Figure 5). Several commercial preparations of proteases: pronase, trypsin, collagenase, and elastase, adjusted in concentration to an equal activity toward the substrate azocasein as a stimulatory Pseudomonas filtrate (Pa 8), elicited roughly comparable stimulation of mucin secretion (see Table 5), supporting the concept that Pseudomonas proteases could be related to the mucin-stimulatory activity. This conclusion would fit in well with preliminary reports from this laboratory8 implicating Pseudomonas proteases in this action. In addition, Klinger et al9 have demonstrated that purified elastase and alkaline proteinase from P aeruginosa filtrates increase secretion of mucin by rabbit tracheal explants. Although proteases in the Pseudomonas filtrates could be related to the mucin-stimulatory components, the correlation is far from exact. Whereas significant protease activity was absent from nonstimulatory strains, several stimulatory filtrates of Pseudomonas, specifically Pa 6 and Pa 4, had only 0.2% and 0.7%,

AJP

*

December 1986

respectively, of the protease activity of Pa 8, yet stimulated secretion as strongly (see Table 3). In addition, the mucin-stimulatory activity seemed more stable to freeze/thawing than the proteolytic activity. This is difficult to interpret, but other substances besides proteases in the filtrates could be contributing to stimulation of secretion, or effects of these enzymes other than proteolysis could be involved in the mucin-stimulatory action. In addition, it should be pointed out that failure to degrade azocasein does not prove conclusively that protease activity is absent. The stimulatory action of crude Pseudomonas filtrates appeared relatively stable to both heat and trypsin. In contrast, when the material is recovered from an agarose column in a more purified form, it becomes labile to heat and trypsin (see Table 4). In its native form, the stimulatory substances within the filtrate could be protected, possibly via binding to another protein or other compound, against proteolytic digestion and heat. In this regard, it is of interest that EDTA stabilized substances in the Pseudomonas filtrates frozen at - 25 and - 70 C. It is known that EDTA inhibits at least two of the extracellular proteases produced by Pseudomonas9 and a Pseudomonas collagenase.30 Thus, one may speculate that the lability of certain filtrates could be due to degradation of the stimulatory substance(s) by intrinsic proteolytic action. However, since the stimulatory substances in the Pseudomonas filtrates appear related to proteases, and filtrates from Hemophilus and Streptococcus failed to demonstrate potent proteolytic activity, ascribing the lability of certain filtrates to the presence of active proteolytic enzymes is tenuous at best. The extreme lability of the stimulatory substance(s) in the cell-free filtrates from both S pneumoniae and H influenzae precluded detailed studies as carried out for the more stable Pseudomonas products. A stimulatory effect of a cell-free filtrate from Hemophilus influenzae on secretion of mucin by rabbit tracheal explants has been reported, supporting our own findings.9 It appears unlikely that the stimulatory substances in these filtrates are related to proteases, because none of the filtrates from either H influenzae or S pneumoniae contained measurable proteolytic activity against azocasein. Preliminary Sephadex gel filtration of stimulatory strains suggested both the Hinfluenzae and Spneumoniae substance(s) to be in a molecular weight range of 50,000-300,000 daltons. Future studies should be directed toward finding means of increasing the stability of these substances to enable more detailed analysis, similar to that described above for Pseudomonas. The concept of products produced by bacteria within the respiratory tract having a direct stimulatory effect

Vol. 125 * No. 3

BACTERLAL PRODUCTS STIMULATE MUCIN SECRETION

on mucin secretion is of extreme interest with regard to the pathogenesis of obstructive pulmonary disease. For example, patients with CF are colonized recurrently with mucoid strains of Paeruginosa.1 2,31,32 Excess mucus in the CF airways may precede33 or occur consequent to34'35 Pseudomonas infection. Products produced by Pseudomonas may be important in the manifestations of CF in the airways. A number of proteases produced by these organisms2'36'37 as well as exotoxin A,38 exoenzyme S,39 and hydrocyanic acid39 can cause infllammation and injury and lead to compromised pulmonary defense.39'40 Phenazine pigments produced by P aeruginosa may predispose to local immunoincompetency.41 Pseudomonas alkaline protease and elastase have been shown to inhibit antibacterial function (eg, oxygen consumption, superoxide production) of human neutrophils in vitro.42 These bacteria also elaborate an extracellular substance that can discoordinate and inhibit beating of respiratory cilia in vitro43'" as well as a 25,100-dalton exotoxin (leukocidin), which can affect function of granulocytes and lymphocytes.454' Interestingly enough, this exotoxin has been shown to activate metabolism of arachidonic acid within a variety of cell types48'49 an action which could lead to the production of a medley of bioactive metabolites (such as prostaglandins and leukotrienes) which have been shown to affect secretion of respiratory mucin. 1221,23 Collectively, these actions support the concept that these organisms may be acting within the respiratory system to enhance an environment (excess mucus, compromised pulmonary defense) which would favor their own survival and growth.

The role of H influenzae and S pneumoniae in the pathogenesis of COPD also is not clear. Patients with chronic bronchitis usually suffer from recurrent bouts of infection with these two organisms during times of acute exacerbation of the disease, whereas they remain in a stable, uninfected phase at other times.34 As reported here, a percentage of these organisms elaborate extracellular products stimulatory to mucin secretion within the airways. Combined with compromised pulmonary defense mechanisms associated with the disease process, further accumulations of mucus in the airways favor increased colonization by more bacteria, possibly leading to a "symbiotic relationship" of sorts whereby bacterial infection leads to increased mucus favoring, in turn, increased bacterial infection, etc., in these patients. Thus, in airways of patients with chronic bronchitis, as in those of cystic fibrotics, certain bacteria could be releasing products to aid in development of an environment favoring their own growth, and in so doing contributing to the pathogenesis of obstructive lung disease in the host.

513

References 1. Kulczycki LL, Murphy TM, Bellanti JA: Pseudomonas colonization in cystic fibrosis. JAMA 1978, 240:30-36 2. Hoiby N: Pseudomonas aeruginosa infection in cystic fibrosis. Acta Pathol Microbiol Scand 1977, C77(Suppl 262):1-96 3. Tager I, Speizer FE: Role of infection in chronic bronchitis. N Engl J Med 1975, 292:563-571 4. Gump DW, Phillips CA, Forsyth BR, McIntosh K, Lamborn KR, Stouch WH: Role of infection in chronic bronchitis. Am Rev Respir Dis 1976, 113:465-474 5. Adler KB, Hardwick DH, Craighead JE: Effect of cholera toxin on secretion of mucin by explants of guinea pig trachea. Lab Invest 1981, 45:372-377 6. Adler KB, Winn WC Jr, Alberghini TV, Craighead JE: Stimulatory effect of Pseudomonas aeruginosa on mucin secretion by the respiratory epithelium. JAMA 1983, 249:1615-1617 7. Adler KB, Winn WC Jr, Hardwick DH, Craighead JE: Effects of bacterial products on secretion of mucin by rodent trachea in vitro. Chest 1982, 81(suppl):37-39 8. Adler KB, Hendley DD: Stimulation of airway mucin secretion by an exoproduct elaborated by Pseudomonas aeruginosa. Preliminary isolation (Abstr). CF Club Abstracts 1984, 25:121 9. Klinger JD, Tandler B, Liedtke CM, Boat TF: Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium. J Clin Invest 1984, 74:1669-1678 10. Niles RM, Stone PJ, Christensen TG, Snider GL: Serine proteases stimulate release of mucus glycoproteins from hamster trachea in organ culture (Abstr). J Cell Biol 1983, 97:437a 11. Adler KB, Mossman BT, Butler GB, Jean LM, Craighead JE: Interaction of Mt. St. Helens' volcanic ash with cells of the respiratory epithelium. Environ Res 1984, 35:346-361 12. Adler KB: Mucin secretion by respiratory tract tissue in vitro. In Vitro Models of Respiratory Epithelium. Edited by LJ Schiff. Boca Raton, CRC Press 1986, pp 23-47 13. Adler KB, Brody AR, Craighead JE: Studies on the mechanism of mucin secretion by cells of the porcine tracheal epithelium. Proc Soc Exp Biol Med 1981, 166:96-106 14. Blatt WF, Robinson SM, Bixler HJ: Membrane ultrafiltration: The diafiltration technique and its application to microsolute exchange and binding phenomena. Anal Biochem 1968, 26:151-160 15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 1951, 193:265-275 16. Fischer E Jr, Allen JH: Corneal ulcers produced by cellfree extracts of Pseudomonas aeruginosa. Am J Opthamol 1958, 46:21-27 17. Rodball M: Metabolism of isolated fat cells: II. The similar effects of phospholipase C (Clostridium perfringens a toxin) and of insulin and glucose and amino acid metabolism. J Biol Chem 1966, 241:130-139 18. Berka RM, Gray GL, Vasil ML: Studies of Phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa. Infect Immun 1981, 34:1071-1073 19. Schefe H: A method for judging all contrasts in the analysis of variance. Biometrika 1953, 40:87-104 20. Snedecer GW, Cochran WG: Statistical Methods. 6th edition. Ames: Iowa State University Press, 1967 21. Coles SJ, Neill KH, Reid LM, Austen KF, Nii Y, Corey EJ, Lewis RA: Effects of leukotrienes C4 and D4 on glycoprotein and lysozyme secretion by human bronchial mucosa. Prostaglandins 1983, 25:155-170 22. Coles SJ, Reid LM: Inhibition of glycoconjugate secretion by cochicine and cytochalasin B: An In-vitro study of human airway. Cell Tissue Res 1981, 214:107-116

514

ADLER ET AL

23. Marom Z, Shelhamer JH, Bach MK, Morton DR, Kaliner M: Slow reacting substances, leukotrienes C4 and D4, increase the release of mucus from human airways in vitro. Am Rev Respir Dis 1982, 126:449-451 24. Shelhamer JH, Marom Z, Kaliner M: Human respiratory mucous glycoproteins. Exp Lung Res 1984, 7:149-162 25. Jennings M, Cross CE, Last JE: Glycoprotein synthesis by tracheal explants from various mammalian species. Comp Biochem Physiol 1977, 57A:317-320 26. Lamont JT, Turner BS, Dibenedetto D, Handin R, Schafer Al: Arachidonic acid stimulates mucin secretion in prairie dog gallbladder. Am J Physiol 1983, 245:G92-G98 27. Adler KB, Alberghini TV, Counts DF, Auletta F: Cellular mechanisms of mucus secretion by rabbit and human cervical explants in-vitro. Biol Reprod 1983, 29:751-765 28. Boat TF: Quantitation of mucous glycoprotein secretion by airways epithelium. Chest 1982, 81::29S-31S 29. Morihara K, Tzuzuki H: Pseudomonas aeruginosa peptide peptidohydrolase: III. Some characters as a Ca2+metalloenzyme. Biochim Biophys Acta 1964, 92:351-360 30. Carrick L, Berk S: Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa. Biochim Biophys Acta 1975, 391:422-434 31. Doggett RG, Harrisson GM, Stillwell RN, Wallis ES: An atypical Pseudomonas aeruginosa associated with cystic fibrosis of the pancreas. J Pediatr 1966, 68:215-221 32. Hoiby N: Prevalence of mucoid strains of Pseudomonas aeruginosa in bacteriological specimens from patients with cystic fibrosis and patients with other diseases. Acta Pathol Microbiol Scand 1975, 83:549-552 33. Evans LR, Linker A: Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol 1973, 116:915-924 34. Oppenheimer EH: Similarity of the tracheobronchial mucous glands and epithelium in infants with and without cystic fibrosis. Hum Pathol 1981, 12:36-48 35. Sturgess J, Imrie J: Quantitative evaluation of the development of tracheal submucosal glands in infants with cystic fibrosis and control infants. Am J Pathol 1982, 106:303-311 36. Hastie AT, Hingley ST, Kueppers F, Higgins ML, Tannenbaum GS, Weinbaum G: Protease production by Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Inf Immun 1983, 40:506-513 37. Doring G, Obernessen HJ, Botzenhart K, Flehmig B, Hoiby N, Hofmann A: Proteases of Pseudomonas aeruginosa in patients with cystic fibrosis. J Infect Dis 1983, 147:744-750 38. Pollack M: Pseudomonas aeruginosa exotoxin A. N Engl J Med 1980, 302:1360-1362

AJP * December 1986

39. Liu PV: Extracellular toxins of Pseudomonas aeruginosa. J Infect Dis 1974, 130:594-599 40. Gray L, Kruger A: Microscopic characterization of rabbit lung damage produced by Pseudomonas aeruginosa proteases. Infect Immun 1979, 23:150-159 41. Sorenson U, Klinger JD, Cash HA, Chase PA, Dearborn DG: In vitro inhibition of lymphocyte proliferation by Pseudomonas aeruginosa phenazine pigments. Infect Immun 1983, 41:321-330 42. Kharazami A, Doring G, Hoiby N, Valerius NH: Interaction of Pseudomonas aeruginosa alkaline protease and elastase with human polymorphonuclear leukocytes in vitro. Infect Immun 1984, 43:161-165 43. Wilson R, Roberts D, Cole P: Effect of bacterial products on human ciliary function in vitro. Thorax 1985, 59:297-306 44. Reimer A, Klementsson K, Ursing J, Wretland B: The mucociliary activity of the respiratory tract: I. Inhibitory effects of products of Pseudomonas aeruginosa on rabbit trachea in vitro. Acta Otolarynggol (Stockholm) 1980, 90:462-469 45. Scharmann W: Purification and characterization of leucocidin from Pseudomonas aeruginosa. J Gen Microbiol 1976, 93:292-302 46. Lutz F: Purification of a cytotoxic protein from Pseudomonas aeruginosa. Toxicon 1979, 17:467-475 47. Hirayama T, Kato I: Mode of cytotoxic action of pseudomonal leukocidin on phosphatidylinositol metabolism and activation of lysosomal enzyme in rabbit leukocytes. Infect Immun 1984, 43:21-27 48. Suttorp N, Seeger W, Uhl J, Lutz F, Roka L: Pseudomonas aeruginosa cytotoxin stimulates prostacyclin production in cultured pulmonary artery endothelial cells: membrane attack and calcium influx. J Cell Physiol 1985, 123:64-72 49. Bremm KD, Brom J, Konig W, Spur B, Crea A, Bhakdi S, Lutz F, Fehrenbach FJ: Generation of leukotrienes and lipoxygenase factors from human polymorphonuclear

granulocytes during bacterial phagocytosis and interaction with bacterial exotoxins. Zentralbl Bakteriol Mikrobiol Hyg [A] 1983, 254:500-514

Acknowledgments The writers thank Janet E. Schwarz, Todd V. Alberghini, Bettie Clements, Robert A. Highland, Elaine Mohrbach, Britt Murphy, Mary Navin, Jane Rivers, and Maria Salvaggio for excellent technical and editorial assistance.