Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, which causes serious debilitating infections in patients with compromised lung function. The mechanism by which P. aeruginosa is cleared from the lung is not fully defined, although our previous studies have established a role for cellular immunity in protection against P. aeruginosa infections. This study aimed to evaluate the role of P. aeruginosa-specific IgG in protection against P. aeruginosa in a rat model of acute pulmonary infection. Immunoaffinity chromatography was used to purify total rat IgG from rat immune serum (rats immunised with P. aeruginosa) and non-immune serum. Untreated recipient rats were injected intravenously with different concentrations of pure IgG prepared from serum of unimmunised rats (non-immune IgG) or from rats immunised intestinally with killed P. aeruginosa (immune IgG) and infected intratracheally with P. aeruginosa 18 h later. The protective capability of the purified IgG against P. aeruginosa was assessed by measurement of reduction in P. aeruginosa infection in the lung 4 h after instillation of bacteria. Enhanced bacterial clearance induced by IgG was determined to be dose-dependent with a 1 mg dose failing to enhance clearance, whereas 5 mg of immune IgG enhanced clearance from the airways and the lung tissue. Measurement of the IgG1, IgG2a and IgG2b isotypes in serum and the lung lavage following transfer of P. aeruginosa-specific IgG found that all three were present. These results demonstrate that anti-P. aeruginosa IgG can enhance bacterial clearance from the airways in an acute infection and identify an important role for IgG in acute respiratory infections caused by P. aeruginosa.
For over a century, Pseudomonas aeruginosa has been recognised as a bacterial pathogen with the ability to cause serious and destructive infections. Morbidity and mortality have always been disproportionately high for infections with this organism . P. aeruginosa from environmental or endogenous sources may be pathogenic to humans, principally those exposed to invasive surgical implants, such as catheters, or patients who have predisposing factors, including burns, malignant diseases or metabolic disorders. In particular, patients with the genetic disorder cystic fibrosis (CF) are susceptible to chronic P. aeruginosa infections of the airways, causing pulmonary dysfunction that can lead to death . P. aeruginosa is the bacterial species most frequently recovered from the respiratory tracts of chronically infected CF patients and the organism has many activities to evade the immune response . Persistence of P. aeruginosa infection appears to also be related to its ability in favourable circumstances (e.g. copious mucus) to become mucoid (secreting a mucopolysaccharide) and form microcolonies (or biofilms) in which the P. aeruginosa is surrounded by a protective mucopolysaccharide layer. Therapy is therefore most effective against any planktonic bacteria released from the microcolonies or against the initial colonising planktonic form but less effective once bacterial microcolonies develop. Therapy for P. aeruginosa infections with antibiotics is limited due to the extensive antibiotic resistance of the organism and this has led to an interest in immunoprophylaxis by active and passive immunisation [2, 4, 5]. Many patients become at risk of Pseudomonas infection from exposure in the hospital environment, such as in burns or intensive care units, and the delay in the formation of the immune response following active immunisation may not be ideal.
Active and passive immunisation, aimed at elimination of initial infection with P. aeruginosa, might be successful in preventing colonisation and subsequent establishment of a chronic infection. Such therapy may also be useful in elimination of the acute exacerbations of chronic infection. Protection by monoclonal and polyclonal antibodies against P. aeruginosa in an intraperitoneal infection model in the mouse has been demonstrated by others and such antibody-mediated protection may also operate in the lung [6–12]. The majority of these studies have been done in sepsis models in animals that have been made neutropenic. A recent study by Shime et al.  was able to show that IgG therapy aimed at neutralising the type III secretion system showed protective effects in preventing septic shock and death in a mouse model.
We have been particularly interested in studying intestinal immunisation that has the potential to deliver an effective immune response to the mucosal surfaces by way of the common mucosal immune network. An acute respiratory P. aeruginosa infection model, which is well established , was chosen for this study as it mimics the initial episode of infection, allowing us to determine what factors are necessary for elimination of an initial respiratory bacterial infection where bacteria are in the planktonic form. In previous studies we demonstrated a role for cellular immunity in clearance of P. aeruginosa from the lung [13, 14] and transferred immune serum was also shown to enhance bacterial clearance suggesting a role for specific antibody .
Patients who develop P. aeruginosa infections often develop very good antibody responses and in CF patients antibodies to P. aeruginosa have been associated with poor lung function. This may be related to the ‘hidden colony’ nature once a chronic infection state has been established. Antibody may well play a useful role in the early stages of initial colonisation provided sufficient titres can be established by prior immunisation or passive antibody transfer before development of a chronic infection. Investigation of the role of immunoglobulin subclasses in bacterial clearance from the lung will aid development of such immunotherapy. The present study was undertaken to assess the role of pure immune IgG (purified from serum of rats following intestinal immunisation) in providing protection against P. aeruginosa acute pulmonary infection in an animal model. The main aims of this study were to purify IgG from non-immune and P. aeruginosa-immune rat sera and to examine the role of the IgG antibodies in the clearance of P. aeruginosa from the lungs after intravenous transfer in a rat model of acute pulmonary infection.
Materials and methods
Specific-pathogen-free DA rats aged 6–14 weeks and weighing 135–245 g were used. The rats were obtained from the Central Animal House of the University of Newcastle and were maintained under specific-pathogen-free conditions until the start of the experiment. All procedures were approved by the University of Newcastle Animal Care and Ethics Committee.
Purification of rat IgG
Rat blood was collected from 8–10-week-old male DA rats. Rats were either unimmunised or immunised via the intestinal route by intraluminal injection of 5×109 whole formalin-killed P. aeruginosa in 0.5 ml of phosphate-buffered saline (PBS) 2 weeks prior to blood collection. The blood was allowed to clot and serum was collected and frozen.
Purification of IgG was achieved by immunoaffinity chromatography using goat anti-rat IgG conjugated to agarose (Sigma, St. Louis, MO, USA). A 10 ml (1 cm×10 cm) column (Pharmacia Biotech, Uppsala, Sweden) containing 5 ml (wet volume) of goat anti-rat IgG-agarose was equilibrated with 0.01 M phosphate buffer (0.0075 M disodium hydrogen phosphate, 0.0025 M sodium dihydrogen phosphate, 0.5 M NaCl), pH 7.2, at a flow rate of 0.5 ml min−1. 0.5–1 ml of an ammonium sulfate-precipitated serum fraction resuspended in PBS , or 1 ml of whole rat serum, was applied to the column. Unbound proteins were removed by washing the column with 0.01 M phosphate buffer at a flow rate of 0.5 ml min−1 for 45 min. The bound IgG was then eluted with 0.1 M glycine, 0.15 M NaCl buffer, pH 2.4. 1-ml fractions were neutralised by collection into tubes containing 150 µl of 1 M Tris, pH 8.0. The fractions containing IgG were pooled and analysed. Protein concentration was determined with the Pierce Micro BCA protein assay reagent and the Pierce albumin standard (Laboratory Supplies, Marrickville, NSW, Australia). Total IgG, IgG1, IgG2a, and IgG2b were determined by radial immunodiffusion using BINDARID kits (The Binding Site Ltd, Birmingham, UK). Analysis of the samples was performed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Briefly, samples were diluted in a 1:1 ratio with sample buffer containing SDS and β-mercaptoethanol. High-molecular-mass markers (Pharmacia Biotech) were diluted 1/20 in the sample buffer. Both molecular mass markers and samples were heated at 100°C for 5 min prior to application to the gel. Electrophoresis was performed on a 10–20% gradient gel using the Bio-Rad Mini-Protean II Cell (Bio-Rad Laboratories, Hercules, CA, USA) followed by staining with Coomassie blue R-250. The gel was then scanned on the Imaging Densitometer Model GS-670 (Bio-Rad Laboratories). The pure immune and non-immune IgG preparations were also analysed for the presence of IgG by Western blot. Briefly, samples were separated by SDS–PAGE, transferred to the nitrocellulose membrane in a Bio-Rad Mini Trans Blot Electrophoresis Transfer Cell, washed in 0.5 M Tris-buffered saline (TBS) (20 mM Tris, 0.5 M NaCl, pH 7.5) and incubated for 90 min in TBS containing 5% (w/v) skim milk (Diploma instant skim milk powder, Bonlac Foods Ltd, Australia). The membrane was then washed twice in TBS containing 0.05% (v/v) Tween 20 (polyoxyethylenesorbitan monolaurate) and incubated for 90 min at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Nordic Immunological Laboratories, Tilburg, The Netherlands), diluted 1 in 500 in TBS containing 5% skim milk. The bound conjugated goat anti-rat antibodies were visualised with the HRP-substrate solution (Bio-Rad Laboratories) and the reaction was stopped by washing the nitrocellulose membrane in distilled water.
Administration of IgG or serum preparations
The IgG was administered by intravenous infusion. Each rat was lightly anaesthetised by the inhalation of 4% halothane (Zeneca, Macclesfield, UK) in oxygen, the rat tail was slightly warmed with an incandescent lamp, wiped with ethanol, and pure IgG in PBS was injected into the tail vein using a 25-gauge needle 18 h prior to bacterial challenge.
Preparation of the bacteria
A mucoid P. aeruginosa strain Pa 385 was grown on nutrient agar at 37°C for 18 h. The bacteria were then harvested, washed three times by centrifugation and suspension in PBS (Dulbecco's modified phosphate-buffered salts, pH 7.2, Flow Laboratories, UK). The concentration was determined by measuring the optical density at 405 nm and determining the colony-forming units (CFU) ml−1 from a previously prepared regression curve. The concentration of the bacteria was adjusted to give a final concentration of 1.0×1010 CFU ml−1 and checked by plating serial dilutions of the preparation onto nutrient agar plates for enumeration of CFU. A purity check was conducted by examining colonies after overnight incubation.
Acute respiratory infection model
Rats administered IgG 18 h previously were infected intratracheally with live bacteria. Briefly, the rats were sedated with halothane and a cannula (20 gauge by 5 cm, Surflo, Terumo Corporation, Tokyo, Japan) was placed into the trachea. Live P. aeruginosa (5×108 CFU in 50 µl PBS) was administered via the cannula and followed by three 3-ml volumes of air from a 10-ml syringe to allow dispersion of the bacteria into all lobes of the lung. Four hours after infection rats were killed with an overdose of 0.8 ml Nembutal (pentobarbital sodium) (Boehringer Ingelheim, Australia) administered by intraperitoneal injection. The rats were weighed, and blood, bronchoalveolar lavage (BAL) and lung samples were obtained. Blood (4–8 ml) was collected by cardiac puncture. The blood was allowed to clot at room temperature for 1–3 h, after which the clot was removed and the samples were centrifuged at 800×g for 10 min to separate the serum. The serum was stored at −20°C for subsequent analysis of P. aeruginosa-specific antibody. The trachea was exposed, cut, and the lung lavaged five times with 2-ml volumes of PBS. The pooled BAL was serially diluted (10-fold dilutions) in PBS and plated onto nutrient agar plates. After overnight incubation at 37°C, in 5% CO2, the bacterial colonies were counted to determine the number of surviving bacteria. 100 µl of BAL fluid was removed for cytospin preparation and the slides were fixed and stained with Diff Quik fixative and stains (Veterinary Medical and Surgical, Maryville, NSW, Australia) and a differential count performed. The remainder of the BAL was centrifuged for 10 min at 800×g to remove cells and bacteria. The supernatant was removed and frozen for subsequent P. aeruginosa-specific antibody analysis. The pellet was resuspended in 1 ml of PBS and diluted in trypan blue for the enumeration of total viable white blood cells in a Neubauer chamber (Hausser Scientific, Bluebell, PA, USA). The lungs, trachea and the heart were excised intact; the adhering lymph nodes, connective tissues and heart were removed; and the lungs were placed in 10 ml of sterile PBS and homogenised (Heidolph Elektro, Kelheim, Germany). 20-µl portions of serial 10-fold dilutions were plated on nutrient agar to determine the number of surviving bacteria in the lung homogenate. An enzyme-linked immunosorbent assay (ELISA) was performed to measure P. aeruginosa-specific IgG, IgG1, IgG2a and IgG2b concentrations in the serum, BAL and pure IgG preparations, as previously described [5, 17]
Results from three separate experiments were pooled. The data are expressed as the mean±S.E.M. The statistical significance of the differences between the groups was tested by unpaired t-test (Statview).
Purification of rat IgG
Immunoaffinity chromatography, with a specific ligand for rat IgG, successfully separated the contaminants from the IgG. The elution profile produced two major peaks which were resolved from the immunoaffinity column (data not shown). Peak 1 contained the unbound contaminating serum proteins and peak 2, which was eluted with 0.1 M glycine buffer, pH 2.4, contained the pure rat IgG. Both peaks were further analysed by SDS–PAGE (data not shown) to confirm the proteins present in the fractions. Only two distinct bands corresponding to the heavy and the light chains of the IgG molecules were resolved from peak 2 pooled fractions. This demonstrated that the immunoaffinity column was effective for purification of rat IgG. Western blot confirmed the presence of rat IgG in these preparations, by detection with anti-rat IgG antibodies conjugated with HRP. The amount of P. aeruginosa-specific IgG in the pure IgG preparations and starting sera is shown in Table 1. The P. aeruginosa-specific IgG activity was conserved during the purification process. The quantities of total IgG and subclasses were assessed by radial immunodiffusion (for the preparations used in one of the three experiments). It was determined that the absolute quantities of IgG1 were similar in the immune and non-immune IgG preparations but the absolute amount of IgG2a was slightly higher in the immune IgG preparation. The IgG2 subclasses accounted for most of the IgG in the preparations (data not shown).
|Pure IgG||P. aeruginosa-specific IgG in pure IgG preparation (ELISA units)||P. aeruginosa-specific IgG in starting serum (ELISA units)|
|(per mg IgG)||(per ml)||(per mg IgG)|
|Immune||10 227||80 340||11 477|
|Pure IgG||P. aeruginosa-specific IgG in pure IgG preparation (ELISA units)||P. aeruginosa-specific IgG in starting serum (ELISA units)|
|(per mg IgG)||(per ml)||(per mg IgG)|
|Immune||10 227||80 340||11 477|
Assuming approximately 7 mg IgG ml−1 of rat serum.
Effect of transferred IgG on bacterial clearance and immune parameters
The mean recovery of live P. aeruginosa in the BAL fluid and in the lung homogenate samples of the rats immunised with 1 mg and 5 mg of pure IgG was determined for all three experiments (Fig. 1). Significantly enhanced clearance of P. aeruginosa was observed in the BAL of animals administered 5 mg of IgG prepared from serum of immunised animals (immune IgG), when compared with animals receiving 5 mg of IgG prepared from serum of unimmunised animals (non-immune IgG) or with untreated animals (Fig. 1). Administration of 1 mg of immune IgG did not result in enhanced clearance of bacteria from the airways when compared to untreated animals or animals receiving 1 mg non-immune IgG.
Administration of 5 mg of immune or non-immune IgG caused enhanced clearance of P. aeruginosa from the lung tissue compartment when compared to untreated animals (Fig. 1). Analysis of P. aeruginosa-specific IgG in serum and BAL of recipients of immune and non-immune IgG is shown in Fig. 2. Specific antibody was detectable in both serum and BAL of recipients of 5 mg immune IgG, and in serum of recipients of 1 mg of immune IgG. P. aeruginosa-specific IgG1, IgG2a and IgG2b were all found in serum and BAL (Fig. 3) of these rats. The activity for each isotype can be compared between rat groups but the levels of activity between isotypes cannot be directly compared due to the possibility of different affinities of the peroxidase-conjugated anti-isotype antibodies used in the ELISA assays. It is apparent that P. aeruginosa-specific IgG1, IgG2a and IgG2b are all present in the serum following infusion of immune IgG and all three isotypes of specific IgG also appear in the airways. The BAL/serum levels of specific IgG1, IgG2a, and IgG2b are 1/187, 1/97 and 1/61 respectively. Any or all of these isotypes may be responsible for the enhanced bacterial clearance in the airways.
Rats receiving 5 mg of immune IgG had considerably enhanced recruitment of leukocytes to the airways when compared to untreated rats or rats receiving 5 mg of non-immune IgG and this increase was due predominantly to an increased recruitment of neutrophils (Fig. 4). Rats receiving 1 mg of immune or non-immune IgG or 5 mg of non-immune IgG had a much smaller but still significant increase in leukocyte recruitment. There was also a significant increase in numbers of macrophages in recipients of 5 mg of immune IgG compared to untreated rats and an increase in ‘other’ cells in recipients of immune IgG although this was only significantly higher than untreated rats for the 1 mg of immune IgG group.
We have previously established a role for cellular immunity against P. aeruginosa and transfer of immune serum into naive animals was also shown to be protective against P. aeruginosa pulmonary infections . To further define the protective agent in immune serum the present study was undertaken to determine the efficacy of serum-derived IgG in protection against P. aeruginosa acute respiratory infection.
Immunoaffinity chromatography proved reliable in purifying rat IgG with selective binding to rat IgG only resulting in the purification of rat IgG as shown by PAGE and Western blot analysis. A potential drawback of immunoaffinity chromatography is the necessity to use a low-pH buffer to elute bound IgG from the column. While this buffer successfully eluted bound rat IgG from the column, the eluted IgG fractions had to be immediately neutralised to prevent denaturation of the antibodies. This however proved not to be a problem as comparison of P. aeruginosa-specific IgG (as measured by ELISA) in starting serum and pure IgG demonstrated that recovery of antigen-specific antibody was excellent.
Evaluation of the efficacy of the pure immune IgG in enhancement of clearance of P. aeruginosa from the rat lung showed that intravenous administration of 5 mg IgG at a potency of 10,000 ELISA U of P. aeruginosa-specific antibody ml−1 gave significantly enhanced bacterial clearance in the airways but 1 mg (potency of 2000 ELISA U ml−1) did not. This was evidently due to the fact that 5 mg intravenously gave rise to significant levels of immune IgG in the airways whereas 1 mg did not. Both immune and non-immune IgG caused enhanced clearance of P. aeruginosa from the lung tissue compartment suggesting a non-specific mechanism involving IgG was operating at this site. Immunoglobulin therapy has been used to treat certain infectious disease states and it is believed that polyclonal immunoglobulin therapy assists in preventing bacterial tissue attachment . Such an action would be supported by the lower bacterial numbers recovered in the lung tissue, whereas clearance from the BAL (representing bacteria present within the airway spaces) was specific for anti-P. aeruginosa IgG present in the BAL.
Of particular interest is the observation of a significant increase in the BAL total white cell count that was predominantly due to increased neutrophil recruitment. Thus the presence of immune IgG is enhancing recruitment of neutrophils which, together with opsonisation of bacteria by IgG, would lead to increased phagocytosis of bacteria and hence enhanced clearance.
Shryock et al.  have shown that serum from CF patients colonised with P. aeruginosa specifically inhibits phagocytosis of P. aeruginosa by alveolar macrophages. The observed inhibition was a result of increased titres of certain subclasses of IgG directed against P. aeruginosa lipopolysaccharide which contained anti-phagocytic activity. Although the increase in IgG2 and IgG3 specific antibodies is related to poor prognosis, elevated levels of IgG4 have also been reported to be responsible for phagocytic inhibition of P. aeruginosa[19, 20]. In pure IgG preparations, the ratio of opsonising to non-opsonising IgG subclass antibodies may therefore play an important role in determining whether protection against P. aeruginosa infections is or is not achieved. The present study has shown that preparations of P. aeruginosa-specific IgG consisting of IgG1, IgG2a and IgG2b are protective in a model of acute bacterial infection. Whether removal of the IgG2 subclasses would affect the potency of these preparations is yet to be determined. This would aid in a more definitive identification of the importance of the different subclasses of IgG in affecting the clearance of P. aeruginosa from the lungs.
This study has contributed to the understanding of the contribution of antibody in mechanisms involved in the clearance of bacteria from the lung. Intestinal immunisation induces P. aeruginosa-specific IgG in serum and transfer of this immune IgG into recipients resulted in appreciable levels of specific IgG in the recipient serum. The transfused IgG was detected in the airways where clearance of P. aeruginosa occurred which appears to be mediated in part by a significant increase in neutrophil recruitment to the airways. The results have shown serum IgG produced by intestinal immunisation to be an important component of clearance of P. aeruginosa from the airways.