Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Center Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
Received 20 March 2003; returned 4 May 2003; revised 12 June 2003; accepted 12 June 2003
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Abstract |
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Methods: Neutropenic rats with left-sided IPA received either treatment with amphotericin B or remained untreated. At 0, 4, 8, 16, 24, 48, 72 and 120 h after fungal inoculation, the rats were dissected. The size of the macroscopic pulmonary lesions, the number of cfu and amounts of chitin were determined in the infected left lung. Galactomannan concentrations were measured both in the left lung and serum. The cytokines tumour necrosis factor (TNF)-, interleukin (IL)-1ß, IL-6, interferon (IFN)-
, IL-4, IL-10, and the chemokines macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1 were determined quantitatively by ELISA in the infected left lung, uninfected right lung and serum.
Results: Amphotericin B treatment of IPA resulted in changed aspect of pulmonary lesions and significantly reduced levels of left lung chitin (72 and 120 h), left lung galactomannan (72 and 120 h) and serum galactomannan (120 h), but not left lung cfu, compared with untreated infected rats. In addition, amphotericin B treatment resulted in a significant decrease in levels of left lung IL-6 (at 72 and 120 h), MIP-2 (at 120 h) and MCP-1 (at 120 h). No local or systemic increases in TNF-, IL-1ß or IFN-
were observed during infection.
Conclusion: It is concluded that treatment with amphotericin B results in decreased fungal load in the infected lung. This reduction in fungal load probably results in a decreased local inflammatory response, as measured by decreased levels of IL-6, MIP-2 and MCP-1 in the infected lung.
Keywords: chemokines, interleukins, chitin, galactomannan, Aspergillus
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Introduction |
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In the pathogenesis of IPA, both fungal and host factors play a role. In several animal models of IPA, tissue-invasive growth of the fungus was seen with an increase in fungal load over time.57 Antifungal treatment resulted in a reduction in fungal load and tissue damage.810 With respect to host response, cytokines seem to play an important role. Studies showed that resistance to systemic invasive aspergillosis was associated with increased systemic production of Th1 cytokines such as interferon (IFN)-. In contrast, production of interleukin (IL)-4 and IL-10 by interstitial CD4+ Th2 cells was associated with disease progression.1114 In addition, local levels of the pro-inflammatory cytokine tumour necrosis factor (TNF)-
, IL-1ß and IL-6 were elevated in animal models of IPA, and neutralization of TNF-
resulted in increased mortality.15,16 Finally, both the local levels of the C-X-C chemokine macrophage inflammatory protein (MIP)-2 and the C-C chemokine monocyte chemoattractant protein (MCP)-1 were elevated in animal models of IPA, and neutralization of these chemokines or their receptors resulted in decreased fungal clearance.17,18 Few data exist regarding the impact of antifungal treatment on patterns of cytokine release.
In our laboratory, we have developed an inhalation model of unilateral IPA in persistently neutropenic rats.19 In this model, we investigated the effect of treatment with amphotericin B on the kinetics of cytokines and parameters of fungal load in rats with IPA.
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Materials and methods |
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The rat infection model of invasive pulmonary aspergillosis was used, as described previously.20,21 In brief, specified pathogen free female RP strain albino rats (1825 weeks old, 185225 g) were employed. Persistent neutropenia was induced by intraperitoneal (ip) administration of cyclophosphamide 75 mg/kg (Sigma-Aldrich Chemie, Steinheim, Germany) 5 days before fungal inoculation, followed by repeated doses of 60 mg/kg 1 day before and 3 and 7 days after fungal inoculation. This protocol resulted in granulocyte counts of <0.1 x 109/L on the day of fungal inoculation. To prevent bacterial superinfections, animals were given daily doses of amoxicillin 40 mg/kg intramuscularly (im) starting 1 day before inoculation, and a dose of gentamicin 6 mg/kg im on the day of inoculation. In addition, rats received ciprofloxacin (660 mg/L) and polymyxin B (100 mg/L) in their drinking water throughout the experiment. Rats were inoculated with a clinical isolate of A. fumigatus originally isolated from an immunocompromised patient with IPA. Infection was established by intubation of the left main bronchus under general anaesthezia. A cannula was passed through the tube, and the left lung was inoculated with 6 x 104 A. fumigatus conidia suspended in 20 µL of phosphate buffered saline (PBS).
The experimental protocols adhered to the rules laid down in The Dutch Animal Experimentation Act (1977) and the published Guidelines on the Protection of Experimental Animals by the Council of the EC (1986). The present protocols were approved by the Institutional Animal Care and Use Committee of the Erasmus Medical Center Rotterdam.
Antifungal treatment
Amphotericin B (Fungizone) was obtained from Bristol-Meyers BV (Woerden, The Netherlands), and diluted in 5% dextrose. Fungizone was administered intravenously (iv) via the lateral tail vein at a dose of 1 mg/kg/day. Treatment was started at 16 h after fungal inoculation, a time point at which hyphal growth was established. Treatment was continued for 10 days.
Survival rate
A separate experiment was performed to observe the death rate in amphotericin B-treated infected rats, and untreated infected rats (n = 2528 rats per group). The survival rate was monitored twice daily until day 11 after fungal inoculation.
Kinetics of parameters of fungal load and cytokines
Parameters of fungal load were compared in amphotericin B-treated infected rats and untreated infected rats. Cytokines were determined in these groups, and also in uninfected rats. At designated time points (n = 56 rats per group per time point), rats were anaesthetized with pentobarbital 50 mg/kg ip (Ceva Sante Animale, Maassluis, The Netherlands). The chest cavity was opened aseptically and the right ventricle punctured to obtain blood. Subsequently, through the same needle the lungs were perfused with 5 mL of PBS. The infected left lung and uninfected right lung were removed, kept on ice and homogenized separately in 10 mL of PBS with 1 x complete protease inhibitor (Roche Diagnostics, Mannheim, Germany) using a tissue homogenizer (The Virtis Co. Inc., Gardiner, NY, USA). The homogenates were centrifuged (10 min, 4000g, 4°C), and supernatants were passed through a 0.45 µm pore size filter (Schleicher and Schuell, Dassel, Germany) and stored at 20°C for cytokine and galactomannan assays. The pellet was resuspended in 10 mL of PBS and used for cfu and chitin assays.
Pulmonary macroscopic lesion and histopathology
Separate groups of rats were used for macroscopic observation and histopathology. The left and right lung were fixed with formalin and embedded in paraffin. Every lung was cut at three levels ±1 mm apart. At every level, two adjacent sections were obtained, of which one was stained with haematoxylin and eosin (H&E) and the other with Grocotts methenamine silver.22
Pulmonary macroscopic lesions were classified and measured as described previously.23 In brief, angio-invasive lesions, seen as macroscopic dark-red lesions, were characterized histologically by extensive hyphal broncho- and angio-invasion and haemorrhagic infarction. Responsive lesions, seen as macroscopic light-red lesions, were characterized histologically by the presence of relatively short hyphae, little angio- or broncho-invasion, with resulting less haemorrhagic infarction.23 The pulmonary lesion was measured from photographs of the anterior of the lungs taken immediately after dissection. The sizes of the two types of pulmonary lesion were expressed as percentages of the total left lung surface.
Fungal cultures of organs
cfu in lungs were counted in 1:10 and 1:100 dilutions of left lung homogenate, and in 1:10 dilutions of right lung homogenate. Dilutions of homogenates were spread onto Sabouraud agar plates. The cfu were counted after incubation at 37°C for 36 h. The remaining homogenate was used for the chitin assay and the galactomannan assay.
Chitin assay
The chitin assay was performed as described by Lehmann & White.24 In brief, the lung homogenate was centrifuged (1800g, 15 min), resuspended in 4 mL of 3% sodium lauryl sulfate (SDS; Sigma) and heated at 100°C for 15 min. After cooling, the pellet was washed once with distilled water, resuspended in 3 mL of 120% KOH solution and heated to 130°C for 1 h. Subsequently, 8 mL of ice-cold 75% ethanol was added, tubes were kept at 4°C for 15 min and 0.3 mL of Celite suspension (Celite 545; Sigma) was added. After centrifugation (1800g, 5 min, 4°C), the pellet was washed with cold ethanol (40%) and cold distilled water successively, and suspended in 0.5 mL of NaNO2 (5%) and 0.5 mL of KHSO4 (95%). After centrifugation (1800g, 15 min), volumes of the supernatant were mixed with 12.5% NH4SO3NH2, followed by MBTH (3-methyl-benzo-2-thiazolone hydrazone HCl monohydrate; Sigma). After heating for 3 min, the supernatants were cooled, and allowed to stand for 30 min, following the addition of FeCl36H2O (0.83%). The optical density at 650 nm was read in a spectrophotometer. The chitin content was expressed as micrograms of glucosamine per left lung. Final measurements of chitin were corrected for the loss of volume of homogenate.
Galactomannan assay
Concentrations of galactomannan in organs and serum were measured, as described previously.20, 23 Briefly, 300 µL of each sample of supernatant of left or right lung homogenate, or serum, were used in a sandwich ELISA (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Belgium). Each plate contained a calibration curve derived from rat serum or lung homogenate samples containing 0, 1, 1.5, 2, 3, 4, 6, 8 and 12 ng/mL galactomannan. The concentration of galactomannan in positive test samples was expressed as nanograms of galactomannan per mL of serum or lung homogenate.
Cytokine ELISA
Rat TNF-, IFN-
, IL-1ß, IL-4, IL-6, IL-10, MIP-2 and MCP-1 were quantified in supernatants of left and right lung homogenates and serum using ELISA kits (Cytoscreen; Biosource International, Camarillo, USA). Each kit contained a calibration curve with different concentrations of the respective cytokines.
Statistical analysis
Differences in rat survival rate were assessed by log rank test. Differences in parameters of fungal infection and cytokines were assessed by Students t-test.
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Results |
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The survival rate of neutropenic rats with IPA was compared in amphotericin B-treated infected rats and untreated infected rats. Treatment was started 16 h after fungal inoculation, by which time hyphal growth was established (data not shown). Per group, 2528 rats were used. Untreated infected rats died from day 5 after fungal inoculation. Death of all rats had occurred by day 11 (Figure 1). Treatment with amphotericin B resulted in a significant increase in survival rate (P < 0.0001), with the first rats dying at day 6 after fungal inoculation, and with 52% surviving at day 11.
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Parameters of fungal load and levels of cytokines were compared in amphotericin B-treated infected rats and untreated infected rats. Cytokines were also determined in uninfected rats. To avoid selection bias, time points were chosen at which all the rats in the three groups were still alive, i.e. at 0, 4, 8, 16, 24, 48, 72 and 120 h after fungal inoculation. In each group, 56 rats per time point were used.
Pulmonary macroscopic lesions and histopathology
In untreated infected rats, dark-red, haemorrhagic pulmonary lesions were seen at 48 h and later, increasing in size over time. At 120 h after fungal inoculation, the lesions comprised, on average, 49 ± 4% of the total left lung surface. Histologically, these angio-invasive lesions showed extensive fungal broncho- and angio-invasion and tissue haemorrhagia. In amphotericin B-treated infected rats, pulmonary lesions also increased over time, but the aspect of these lesions was different. A shift was seen from dark-red, angio-invasive lesions in untreated infected animals to responsive lesions in amphotericin B-treated infected rats. Histologically, these responsive lesions had shorter hyphae, reduced fungal broncho- and angio-invasion and less tissue haemorrhagia than untreated infected rats. At 120 h after fungal inoculation, these responsive lesions in amphotericin B-treated rats comprised 46 ± 15% of the total left lung surface.
Number of cfu
In untreated infected rats, no increase was seen in the number of cfu in the infected left lung over time, despite progression of the fungal infection (Figure 2). Treatment with amphotericin B did not reduce the number of left lung cfu significantly compared with untreated infected rats. Right lungs were positive for cfu in two untreated infected rats at 120 h after fungal inoculation (mean: 1.15 log10 cfu). In amphotericin B-treated infected rats, all right lungs were negative. The difference was not significant (data not shown).
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An increase was seen in the amount of chitin in the infected left lung in untreated infected rats over time, especially between 48 and 120 h after fungal inoculation (Figure 3). Amphotericin B treatment resulted in significant suppression of the amount of chitin at 72 (P = 0.02) and 120 h (P = 0.03) after fungal inoculation. All right lungs were negative for chitin in both groups (data not shown)
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Concentrations of galactomannan in the infected left lung increased over time, both in untreated infected rats and amphotericin B-treated infected rats (Figure 4). Treatment with amphotericin B suppressed the increase in galactomannan concentrations, with significant differences at 72 (P = 0.03) and 120 h (P = 0.006). Similar data were seen for galactomannan concentrations in serum, with a significant difference between untreated infected rats and amphotericin B-treated infected rats at 120 h (P = 0.04). Galactomannan concentrations in the right lungs of both groups of rats were not significantly elevated compared with uninfected controls (data not shown).
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Concentrations of the cytokines TNF-, IFN-
, IL-1ß, IL-4, IL-6, IL-10, MIP-2 and MCP-1 were measured in the left and right lungs and serum. The concentrations of TNF-
, IFN-
, IL-1ß, IL-4 and IL-10 did not differ significantly among the three groups of rats. In addition, differences in concentrations between the infected left lung and the uninfected right lung were not significant in all groups (data not shown). In contrast, IL-6 in the left lung of untreated infected rats increased over time up to six-fold compared with uninfected controls (Figure 5a). Amphotericin B treatment resulted in suppression of the increase in left lung IL-6 levels, with significant differences compared with untreated infected rats at 72 (P = 0.04) and 120 h (P = 0.02). Concentrations of IL-6 in the right lung and serum were not significantly different among the three groups of rats (data not shown). Concentrations of the chemokine MCP-1 in the left lung also increased over time (Figure 5b). Amphotericin B treatment resulted in lower levels of this chemokine, with a significant difference compared with untreated infected rats at 120 h (P = 0.01). There were no significant differences in MCP-1 levels in the right lung and serum among the three groups of rats (data not shown). Similar to IL-6 and MCP-1, levels of left lung MIP-2 increased over time, and were suppressed in infected rats receiving amphotericin B treatment (Figure 5c). The difference between untreated infected rats and amphotericin B-treated infected rats was significant at 120 h (P = 0.02). There were no significant differences in concentrations of this chemokine in right lung and serum among the three groups of rats (data not shown).
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Discussion |
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Evidence is accumulating that cytokines play a key role in the host response against bacterial as well as fungal infections.2932 TNF- is one of the first cytokines detectable following bacterial or endotoxic challenge, and has been implicated in the cardiopulmonary dysfunction, increased microvascular permeability and metabolic derangements that typify bacteraemic septic shock.3335 Increases in TNF-
concentrations are also seen in infections with non-bacterial pathogens including rickettsiae, viruses and protozoa. These findings have been taken as evidence of a common host-response mechanism involving TNF-
, irrespective of the taxonomic class of the organism.36,37 In contrast to this hypothesis, in our model, levels of TNF-
in the serum and infected lungs of rats with IPA were not elevated compared with uninfected controls. Other investigators also found that experimental fungal infection did not result in increased levels of TNF-
. In a systemic rat model of lethal invasive candidiasis, serum levels of TNF-
and IL-1
remained low, even at death. These results were the same for immunocompetent and neutropenic rats. In contrast, when rats were infected with Escherichia coli or Staphylococcus aureus, large increases in TNF-
and IL-1
were seen.38,39 These findings suggest that TNF-
is not an essential mediator in lethal fungal infections. The same may be true for IL-1ß, since in our model this cytokine also showed no increase in rats with IPA. In contrast, IL-6, another pro-inflammatory cytokine, increased over time in the left lung of rats with IPA in our model. IL-6 can suppress levels of TNF-
, both in human monocytic cell lines and in intact mice,40 and has a function that limits the inflammatory sequelae of TNF and other inflammatory mediators.41 It is therefore also possible that rapid development of high levels of IL-6 in our model may have down-regulated levels of TNF, and possibly also of IL-1. IL-6 has been shown to play a role in IPA, since IL-6-deficient mice were more susceptible to IPA than wild-type mice,42 and IL-6 was increased in the lungs of immunocompromised mice with IPA compared with uninfected controls.15 A possible role for IL-6 in IPA might be the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, which is a molecule known to activate macrophages,43 which in turn are known to be able to kill Aspergillus.44
A number of studies in mice from one group of investigators suggest that survival in IPA was associated with a Th1 response, and succumbing to the infection with a Th2 response.1114,45 In the present study, the Th1-associated cytokine IFN- was not elevated in rats with IPA that survived, and the Th2-associated cytokines IL-4 and IL-10 were not elevated in rats that succumbed to the infection. IL-6, which is considered to be a Th2-associated cytokine in murine models,46 was elevated in both treated and untreated rats in our study. Therefore, it could be concluded that, in our model, infection with Aspergillus resulted in a predominantly Th2 response, which was significantly decreased, but not reversed into a Th1 response, as a consequence of amphotericin B treatment. The difference in cytokine profiles between our study and the above-mentioned studies may be explained by the use of a lower inoculum and persistently neutropenic animals in our study, factors that have been shown to have an effect on cytokine concentrations.16,29,47
In our model, we found an increase over time of the left lung levels of the C-X-C chemokine MIP-2, as well as the C-C chemokine MCP-1. These chemokines are produced by a number of cells including macrophages, lymphocytes, and endothelial and epithelial cells.43 Possibly, the production of these chemokines during IPA is a result of endothelial damage during vasal invasion of the fungus and/or direct stimulation by fungal toxins. Elevated levels of these chemokines in animal models of IPA have also been described by others.16,48 In addition, antibody-mediated neutralization of the C-X-C receptor resulted in an invasive aspergillosis infection, and MCP-1 neutralization decreased conidial clearance in otherwise immunocompetent mice.17,18 This indicates that these chemokines may play a significant role in the host defence against A. fumigatus.
Antifungal treatment of infected rats with amphotericin B in the present model resulted in decreased levels of IL-6, MIP-2 and MCP-1 compared with untreated infected rats. This may be a direct effect of amphotericin B on the immune system, since this antifungal agent is known to have immunomodulatory characteristics.49 However, this is not likely since amphotericin B was shown to induce, not suppress, gene expression for TNF-, IL-1ß, MCP-1 and the MIP-2 homologue IL-8.5052 In addition, experiments in our laboratory showed that amphotericin B treatment in uninfected rats did not influence cytokine levels in lungs and serum compared with untreated uninfected rats (data not shown). It is therefore more likely that the decreased cytokine response is a result of the reduction of the fungal load caused by amphotericin B treatment.
From the present study it can be concluded that treatment with amphotericin B reduces the increase in fungal load over time. This reduction in fungal load probably results in a decreased local inflammatory response and hence decreased levels of IL-6, MIP-2 and MCP-1 in the infected lung. TNF- does not play a role in IPA in neutropenic rats. These insights may be useful in the development of new immunomodulatory strategies for the treatment of IPA.
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Footnotes |
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