JOURNAL OF PATHOLOGY, VOL. 153: 257-264 (1 987)

IMMUNOCYTOCHEMICAL DEMONSTRATION OF THE ASSOCIATION BETWEEN LEGIONELLA

PROTEASE, AND PULMONARY LESIONS IN EXPERIMENTAL LEGIONNAIRES’ DISEASE

PNEUMOPHILA, ITS TISSUE-DESTRUCTIVE

ANN WILLIAMS, A. BASKERVILLE, A. B. DOWSETT AND J. W. CONLAN

Experimental Pathology Laboratory, PHLS Centre fttr Applied Microbiology and Research, Porton Down, Salisbury, Wilts. SP4 OJG, U.K.

Received30 April I987 Accepted2 July 1987

SUMMARY Using immunocytochemical techniques at the light and electron microscope levels, Legionella pneumophila and

onc of its extracellular proteases were located in the lungs of guinea pigs with experimental Legionnaires’ disease (LD). L. pneumophila was immunostained by several peroxidase- and gold-labelling methods for light and electron microscopy. The protease was immunolabelled in tissue fixed in Carnoy’s fluid at the light microscopical levcl and on broth-grown organisms at the ultrastructural level. It was not labelled in either formalin- or glutaraldehyde-fixed tissuc.

Using double-labelling tcchniqucs, L. pneumophila and protease were located in the same section and were shown to be intimatcly associated with pulmonary lesions, providing strong evidence for the role of this protease in LD pneumonia.

KEY WORDS-- Legionnaires’ disease, Legionella pneumophila protease, immunolabelling.

INTRODUCTION

The Gram-negative cocco-bacillus Legionella pneumophila is now established as the causative agent of Legionnaires’ disease (LD).’ The disease is an acute fibrinopurulent bronchopneumonia but the mechanisms by which the pulmonary lesions are produced remain uncertain. Recently, indications of the pathogenesis of LD were provided experimentally by Conlan et aL2 and Baskerville et who showed that L. pneumophila in broth culture produced extracellular proteases, one of which, on inoculation intranasally into guinea pigs, causes acute pulmonary lesions similar to those of

-

Addressee for correspondence : Ann Williams, Experimental Pathology Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts. SP4 OJG, U.K.

0 1 9 8 7 by John Wiley & Sons, Ltd. 0022-341 7/87/110257-08$05.00

human LD and experimental LD in this animal.4%5 This tissue destructive protease was detected by an enzyme-linked immunosorbent assay (ELISA) in infected guinea-pig lung homogenates at levels at least equivalent to those which cause death on intranasal inoculation.6 However, in order to con- firm the role of tissue destructive protease in the pathogenesis of LD pneumonia, it is also necessary to demonstrate its presence at the site of tissue damage in the lung. Various peroxidase and gold immunolabelling methods can be used to localize antigens in tissues both at the ultrastructural and at the light microscopical level. Immunoperoxidase and immunofluorescence methods have been used to detect L. pneumophila in human lung t i s s ~ e . ~ - ~ The purpose of this study was to evaluate and modify various immunocytochemical techniques to show, by light and electron microscopy of lung

258 A. WILLIAMS E T A L .

tissue from experimentally infected guinea pigs, the presence of L. pneumophila serogroup specific anti- gen (which delineates the bacteria) and the protease detected by ELISA. The demonstration of a dis- tributional association between organisms, pro- tease, and tissue damage would provide further evidence for a prominent role of the protease in LD.

MATERIALS AND METHODS

Preparation ojantisera

Antisera to L. pneumophila serogroup-specific antigens 1 and 3 were obtained by immunization of rabbits with saline extracts of strains W74/81 and WI 66/81, respectively.’O Antiserum to tissue destructive protease was obtained by immunization with purified protease. Rabbits were injected with 50 yg of purified tissue destructive protease2 in Freund’s incomplete adjuvant (Difco) and boosted with the same dose at 3, 6, and 9 weeks; 3 weeks later the final boost serum was obtained. The IgG fractions of both antisera were obtained by affinity chromatography on a protein A Sepharose column (Pharmacia Fine Chemicals).

Light microscopy

Tissue preparation-Three groups of six female Dunkin-Hartley guinea pigs were given a high dose of L. pneumophila administered as a small particle aerosol as described by Baskerville et ~ 1 . 4 , The animals developed a widespread bron- chopneumonia and died at approximately 3 days post infection. The lungs were removed and 2 -3 mm thick slices were taken from areas of consolidation. These were either snap-fro7en in liquid nitrogen or immersed in either 4 or 10 per cent phosphate- buffered neutral formalin for various times up to 2 days, or Carnoy’s fixative for 1 h. Frozen tissue sections were cut at -20”C, picked up on glass slides, allowed to dry, and then stored over silica gel at -20°C until required. Formalin- or Carnoy’s-fixed tissue was processed by standard procedures, embedded in paraftin wax, and 5pm thick sections were cut. Unstained paraffin sections of human lung from two bacteriologically con- firmed cases of LD and two non-Legionella pneu- monias were also used in the study. Lung tissue from an adult Vervet monkey experimentally infec- ted with L. pneumophila by aerosol and killed 5 days later was also examined.

Immunolahelling methods

Prior to labelling, frozen sections were fixed in dry acetone at -20°C for 20min. Paraffin- embedded sections were dewaxed and rehydrated through an alcohol series to water.

Immunoperoxiduse

Endogenous peroxidase activity was blocked with 3 per cent H,O, in methanol for 5-15 min (depending on the type of section and method used). Sections were washed thoroughly in tap water, then in Tris-buffered saline (TBS) at pH 7.6, and incubated with newborn calf serum (NBCS) for 30- 60 min to reduce non-specific staining. All washes between steps consisted of three changes of TBS, pH 7.6. In all methods, peroxidase was visualized using 3,3-diaminobenzidine tetrahydrochloride (DAB), 25 mg/l00 ml TBS, with 50 pl of 30 per cent H202. The counterstain used was Mayer’s haematoxylin, after which tissue was dehydrated through an alcohol series, cleared in Histosol (Shandon Ltd.), and mounted in DPX. A direct immunoperoxidase method involving a single over- night incubation with horseradish peroxidase (HRP)-conjugated primary antibodies and an indirect method using a HRP-conjugated anti- rabbit IgG were used, but principally the peroxidase anti-peroxidase (PAP) and avidin-biotin complex (ABC) methods were used.

The PAP procedure was basically that of Sternberger et al.” with the following modifica- tions ; incubation with primary antibodies was over- night at 4OC, the secondary antibody was swine anti-rabbit IgG (Nordic) diluted 1 :50, and rabbit PAP (Miles) was diluted 1 :loo. The ABC method was basically as follows: primary antibodies were highly diluted (all dilutions were determined by optimum titrations) and incubation was overnight at 4°C. After the TBS wash, sections were immersed in 10 per cent NBCS for 30min. A biotinylated swine anti-rabbit IgG (Dakopatts) diluted I :500 was applied for 30 min followed by an avidin and biotinylated HRP complex (Dakopatts).

lmmunogold

Primary antibodies (IgG) were adsorbed to col- loidal gold particles (see section on electron microscopy) and used as direct labels. Sections were washed in TRS, pH 8.2, and non-specific labelling blocked with NBCS for I h before incubation with

IMMUNOCYTOCHEMICAL DEMONSTRATION OF L. PNEUMOPHILA PROTEASE 259

the gold labels overnight at 4°C. After washing in buffer and water, they were counterstained with haematoxylin, dehydrated, cleared, and mounted.

An indirect immunogold silver staining (TGSS) method was also used. This was essentially that of Springall et a1.,12 but a commercial silver develop- ment solution was used (Intense, Janssen Pharmaceutica).

Douhle-labelling

The ABC and direct immunogold techniques were used to label both the serogroup specific anti- gen and tissue destructive protease in the same sec- tion. The ABC method as described was used to label tissue destructive protease. The section was thoroughly washed in TBS (pH 8.2), NBCS was applied for 1 h, and then bacterial serogroup speci- fic antigen was labelled with direct immunogold. Double-labelling of these two antigens was also per- formed using direct peroxidase conjugates and direct immunogold. Tn all methods, negative con- trols were included in which either primary anti- bodies were replaced by normal rabbit serum or secondary conjugates were omitted.

Electron microscopy

Immunogold labelling methods were used to demonstrate the presence of serogroup specific anti- gen and protease on both whole mounted and sec- tioned, broth-grown organisms. Subsequently, these methods were applied to sections of macrophages and lung tissue from infected guinea pigs.

Preparation of cells and tissue

Whole organisms-Broth cultures of L. pneumophila serogroups I and 3 were grown in yeast extract broth (YEB),13 incubated aerobically at 37°C. Organisms were harvested by centrifugation at 7500 g for 1 h and the pellet was resuspended in 0.1 per cent formalin for 10 min. The formalin was removed after centrifugation and the pellet gently resuspended in a small volume of distilled water. Approximately 20 pl of this thick suspension was placed on formvar/carbon-coated, 400 mesh, cop- per specimen grids. After approximately 30 sec, the excess fluid was removed with moist filter paper and the grids were allowed to dry.

Sections of organisms-Pellets of 0. I per cent glutaraldehyde-fixed L. pneumophila were prepared

as above, resuspended in phosphate buffer, and pel- leted again. Approximately 50pl of molten agar (Oxoid No. 1) was added and gently mixed. When set, the agar was cut into 1- 2 mm cubes, which were dehydrated through a graded alcohol series and taken through propylene oxide to Araldite embcd- ding. Silver sections (80 nm) were cut on a Reichert OMU2 ultramicrotome fitted with a diamond knife and picked up on uncoated nickel 400 mesh grids.

Macrophage sections-Alveolar macrophages were infected in vivo by aerosol exposure of guinea pigs to L. ~neum~phi la and obtained by pulmonary 1a~age . I~ They were separated by centrifugation from the lavage, fixed in 0.1 per cent glutaralde- hyde, and processed to Araldite, as above.

Tissue sections-Legionella-infected guinea pig lung was obtained at necropsy immediately after death and cut into 1-2 mm cubes. Fixation was for 2 h in 0.1 per cent glutaraldehyde; blocks were transferred to phosphate buffer containing 0.2 M sucrose before dehydration and embedded in Araldite. One micrometre thick sections were cut and stained with toluidine blue to locate suitable areas for ultrathin sectioning. Eighty nanometre sections were cut and picked up on uncoated nickel grids.

Preparation of gold reagents

A colloidal gold solution of particles approx- imately 15 nm in diameter was prepared by the reduction of chloroauric acid with tri-sodium citrate.Is Four millimetres of a 1 per cent tri-sodium citrate solution was added to 100ml of a boiling 0.1 per cent chloroauric acid solution. Boiling was continued until the colour became red-orange. After cooling, the pH of the solution was adjusted using 0.2 M K,CO,. Since the binding of a protein to gold particles is optimal at a pH close to or slightly above its isoelectric point, the pH of the gold sol was adjusted accordingly. Using indicators, an aliquot of the solution was adjusted to the desired pH and then an equivalent volume of K,CO, was added to the bulk of the gold. Phenol red indi- cator was used to estimate a neutral pH and phenolphthalein to estimate pH 8 -9.

Staphylococcal protein A, anti-serogroup 1 TgG, anti-serogroup 3 IgG, and anti protease IgG were adsorbed to 15 nm gold sols. The amount of protein required to stabilize the sol was determined in each case by finding the quantity which renders the sol resistant to electrolyte flocculation. A dilution of protein was made to which volumes of gold were

260 A. WILLIAMS ETAL.

added. The stabilizing amount was the lowest con- centration which prevents a colour change from red to blue on addition of KCl. A stabilizing amount of protein was added to a larger volume of gold with constant stirring. This was further stabilized with bovine serum albumin and then purified by centrifugation.'

Labelling

All incubations were carried out by floating grids on drops of reagents. Washes were with TBS drop- ped from a syringe through a 0.22 pm (Millipore) filter. TBS was at pH 8.2 for all gold reagents and at pH 7.6 for any primary antibodies. Prior to label- ling, grids were incubated with NBCS for 1 h. An initial etching step was performed on araldite- embedded sections using 10 per cent H202 for 10 min.

A direct method was used, involving incubation overnight at 4°C with appropriate reagents. After washing with buffer and distilled water, grids were allowed to dry. All araldite-embedded sections were then stained with uranyl acetate and lead citrate.

The indirect method used was the protein-A gold (pAAu) technique, employing either commercial (Bioclin, 20 nm) or laboratory prepared (1 5 nm) pAAu. Grids were incubated with primary anti- bodies at optimal dilutions in TBS, pH 7.6, over- night at 4°C. After washing in TBS, pH 7.6, and then twice in TBS, pH 8.2, they were incubated with NBCS for 30 min. Excess NBCS was drained off and they were incubated with pAAu at optimal dilu- tion for 1-2 h at room temperature.

Negative controls were, for the direct method, inappropriate antibody-gold reagents and, for the indirect method, incubation with normal rabbit serum or PAAu only.

RESULTS Light microscopy

L. pneumophila bacteria were demonstrated by both peroxidase- and gold-labelling of serogroup specific antigen in frozen and in paraffin-embedded tissue (fixed in formalin or Carnoy's fluid). Tissue destructive protease was demonstrated in frozen sections and in paraffin-embedded tissue which had been fixed in Carnoy's fluid, but could not be detec- ted in formalin-fixed tissue. Figures 1 and 2 show immunoperoxidase labelling of the protease in guinea pig lung tissue while Figs. 4 and 5 dem- onstrate the immunogold techniques used. Organ-

isms were localized principally in the foci of tissue damage, a large proportion being intracellular in macrophages and polymorphs. Some diffuse extra- cellular specific staining was seen in the same areas which may be due to the presence of soluble antigen. Protease was observed only at the sites of lung damage as a diffuse intra- and extracellular staining, although whole organisms were delineated occasionally.

Figure 6 shows the simultaneous localization of organisms by immunogold (red) and protease by immunoperoxidase (brown). In these double- labelling experiments, staining for organisms demonstrated them mostly as discrete cocco-bacilli with diffuse staining for protease in the surrounding area. The protease was found at sites with large numbers of bacteria and both were associated only with pulmonary lesions, particularly those showing necrosis.

Of the labelling techniques used, the direct immunogold gave the lowest background staining and greatest specificity (Fig. 5 ) while the ABC was the most sensitive with minimal background (Figs 1-3). Alveolar structure was poorly preserved in frozen tissue compared with that in paraffin- embedded material. Figure 7 shows organisms labelled by immunoperoxidase in a bacteriologi- cally known positive case of human LD. A further known case was also positive by this technique but lung tissue from two other patients with non-LD lobar pneumonia was negative. Sections from the lung of a Vervet monkey, 5 days after experimental infection, were stained positively by immunoperox- idase for L. pneumophila (Fig. 8). Negative controls (Fig. 3) showed no specific staining but a light back- ground staining was observed due to the presence of unblocked, endogenous peroxidase.

Electron microscopy

L. pneumophila serogroups 1 and 3 were labelled for serogroup specific antigen by the direct immunogold and/or indirect pAAu methods. Figures 9 and 10 demonstrate the surface labelling of both whole and sectioned bacteria. Whole organ- isms showed intense surface labelling, while in sec- tion, bacteria were peripherally delineated with gold particles. In sections of macrophages obtained by lavage, organisms showing similar peripheral label- ling were found in vacuoles (Fig. 11). The lung tissue examined contained very few extracellular organisms and all labelling was observed within macrophage phagosomes (Fig. 12). Figures 13 and

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IMMUNOCYTOCHEMICAL DEMONSTRATION OF L. PNEUMOPHILA PROTEASE 26 1

Fig. 9-Direct immunogold labelling of whole, broth-grown L. pneumuphila. x 14 500

Fig. 11-Direct immunogold labelling of L. pneumophila in alveolar macrophages obtained by lavage from infected guinea pigs. x20400

Fig. 10-Direct immunogold labelling for serogroup-specific antigen in sections of broth-grown L. pneumuphila. x 42 000

Fig. 12-Indirect PAAu labelling of L. pneumuphilu in an alveolar macrophage in infected guinea pig lung. x 18 200

262 A. WILLIAMS ETAL.

Fig. 13 - Indirect, protein-A gold (PAAu) labelling for protease on whole, broth-grown L. pneurnophila. x 24 600

Fig. 15-Negative control. Serogroup 3 L. pneumophila sections incubated with gold-coupled antiserum to serogroup 1. x 47 600

14 show a similar but less intense pattern of label- ling for protease to that of the serogroup-specific antigen. The negative control showed minimal sec- tion staining (Fig. 15). Protease could not be label- led in macrophage and lung sections which had been fixed in glutaraldehyde. Labelling of organ- isms and protease in tissue fixed in Carnoy’s fluid was not possible due to extremely poor tissue preservation.

DISCUSSION

Legionella pneumophila extracellular proteases have been suggested as possible pathogenic factors in Legionnaires’ disease and evidence for this has been provided by in vivo studies in a guinea pig r n ~ d e l . ~ ~ ~ In this study, the tissue destructive pro- tease known to cause pulmonary lesions similar to those of experimental LD in the guinea pig was demonstrated in infected lung by various immuno- cytochemical labelling techniques. At the light microscopal level, the protease was shown only to

double-labelling experiments, in which organisms Fig. 14-Direct immunogold labelling for protease in sections be present at the sites of pulmonary lesions, and of broth-grown L. pneumophila. x 50 400

IMMUNOCYTOCHEMICAL DEMONSTRATION OF L. PNEUMOPHILA PROTEASE 263

and protease were labelled in the same section, showed their intimate association. There also appeared to be a quantitative correlation between protease, organisms, and tissue damage in that the greatest quantities of protease were present in large lesions containing masses of bacteria.

Although all the mechanisms operating in LD pneumonia have not yet been elucidated, this paper provides further evidence that the L. pneumophila protease directly causes the pulmonary damage. When the protease is administered intranasally to guinea pigs, acute pulmonary lesions which are sim- ilar to those of human and experimental LD result in a few hours.3 The time course of these lesions is rapid and occurs before the recruitment of macrophages and polymorphs (PMN). It has been suggested that at least some of the damage in LD could be caused by polymorph enzymes or their oxygen-dependent antimicrobial systems. This is probably not the case, since a recent studyI7 has shown that in guinea pigs depleted of circulating PMN, LD lung lesions were identical to those of intact, infected controls.

The choice of fixative for paraffin-embedded material was found to be critical in the labelling of protease. The routine fixative, formalin, immobilizes peptides and proteins by cross-linking, which can lead to severe antigen denaturation due to reactions with primary amino groups and sub- sequent conformational changes. Protease could not be demonstrated in formalin-fixed material, even after minimal exposure to low concentrations (4 per cent) of the fixative.

recently reported similar prob- lems with formalin when attempting to immunostain a mast cell protease and found that Carnoy’s fixative, an alcohol, chloroform, and acetic acid mixture, enabled that protease to be labelled. When Carnoy’s-fixed lung was used in this study, the Legionella protease was readily stained. Alcoholic fixatives immobilize proteins by precipi- tation due to disruption of hydrogen bonds result- ing in denaturation of their s t ruc t~re . ’~ It would appear that this denaturation has a less severe effect on the antigenic properties of proteins than the cross-linking induced by aldehydes. Protease was not found in the human lung sections but only formalin-fixed tissue was available so that the results are probably falsely negative.

The use of frozen tissue avoids such difficulties with fixatives and protease was readily demon- strable in frozen sections for light microscopy. However, the conditions for freezing, storage, and

Garrett et

sectioning of the tissue are difficult to optimize and often result in sections of poorer definition and structural morphology than is the case with paraffin-embedded material.

Ultrastructurally, protease was weakly labelled on organisms grown in broth, but could not be demonstrated in infected lung tissue or macro- phages. This could be due to denaturation of the enzyme by the glutaraldehyde fixative or because the quantities found in the ultrathin tissue section are below the sensitivity limit of the labelling methods used. Immunoelectron microscopy was attempted on tissue fixed in Carnoy’s fluid but was unsuccessful since this fixative rendered tissue archi- tecture and cell cytoplasm and membranes barely recognizable at the ultrastructural level. The use of cryoultramicrotomy may enable this difficulty to be circumvented.

Several immunolabelling techniques were cvalu- ated for the demonstration of Legionella antigens by light microscopy, the most successful being the direct immunogold and the avidin-biotin peroxi- dase (ABC). The direct immunogold gave clear, specific labelling of organisms with minimal non- specific staining. However, as with all direct label- ling, sensitivity was reduced by comparison with indirect methods. The ABC method showed high sensitivity with low background staining and was particularly useful for detecting low numbers of organisms and protease. The main difficulty with this and other immunoperoxidase methods was that of endogenous peroxidase staining. Macrophages and red blood cells contain significant amounts of peroxidase and both types of cells are abundant in Legionella-infected lung. Pretreatment of tissue sec- tions with hydrogen peroxide quenched most of the endogenous activity but this was not always com- plete, resulting in a low level of false labelling of macrophages. It was noted in some human lung specimens that haemosiderin-containing macrophages were particularly rich in endogenous peroxidase which could not be readily blocked.

When used in succession on the same tissue sec- tion, these two methods produced clear double- labelling of protease and organisms. Using the direct immunogold after the ABC eliminated the problem of cross-reactions between primary and bridging antibodies. However, a blocking of anti- genic sites may occur with the avidin-biotin com- plex preventing some binding of antibodies coupled to gold. The contrast between the red (gold) and brown (DAB) staining in these double-labelled sec- tions was readily apparent and illustrated the

264 A. WILLIAMS ETAL.

intimate relationship between L. pneumophila, its tissue destructive protease, and pulmonary lesions.

ACKNOWLEDGEMENTS

This work was funded by an MRC Project grant to A. W. and J. W. C. We wish to thank Mrs Emily Elphick and Mrs Iris Francis for skilled assistance.

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