INTRODUCTION

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Almost all persons with cystic fibrosis (CF) eventually die from progressive lung disease associated with chronic Pseudomonas aeruginosa infection (1). Much of the tissue destruction results from an excessive, predominately neutrophilic, inflammatory response that is more injurious than protective (2). In contrast to bronchoalveolar lavage (BAL) fluid from healthy volunteers, BAL fluid from patients with CF contains high concentrations of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-), interleukin (IL)-1, IL-6, and IL-8, but negligible amounts of the antiinflammatory cytokine IL-10 (3). We have previously demonstrated that airway epithelial cells from healthy volunteers produce IL-10, but that epithelial cells from patients with CF are deficient in IL-10 production (4). Others have reported that T-lymphocyte clones from patients with CF have decreased production of IL-10 (5). Because IL-10 has been shown to inhibit production of TNF-, IL-1, IL-6, and IL-8 (6), it is possible that constitutive production of IL-10, as occurs in the lungs of healthy persons, maintains a balance that inhibits inflammation (7). Conversely, decreased or absent IL-10 may contribute to a regulatory imbalance in which inflammatory responses could become pathologic. Indeed, knockout mice deficient in IL-10 spontaneously develop inflammatory bowel disease (8). Asthma and fatal acute respiratory distress syndrome also have been associated with decreased IL-10, suggesting the importance of this cytokine in human lung diseases (9, 10). Treatment with IL-10 has been shown to reduce leukocyte recruitment, proinflammatory cytokine production, and tissue injury in the airways of sensitized animals after antigen exposure (11, 12), whereas neutralization of endogenous IL-10 was associated with worsening inflammation in this animal model (13). However, in one animal model of acute bacterial infection, IL-10 administration was associated with reduced survival (14). Taken together, these studies suggest that IL-10 plays a physiologic role in preventing excessive activity of the inflammatory cascade. However, the administration of exogenous IL-10 may reduce inflammation, although this may not always be beneficial, should the IL-10 overly suppress normal host defenses.

Successful use of anti-inflammatory therapy provides evidence that the inflammatory response in CF lung disease is excessive relative to the burden of bacterial infection. Clinical trials of alternate-day prednisone in patients with CF demonstrated a beneficial effect on pulmonary function without infectious complications (15, 16). In a rat model of chronic P. aeruginosa infection, ibuprofen significantly reduced the lung inflammation without increasing the burden of bacteria (17). The success with ibuprofen in this model led to a 4-yr clinical trial that demonstrated slower deterioration in pulmonary function in patients with CF treated with high-dose ibuprofen (18). Additionally, P. aeruginosa-infected cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice had increased local concentrations of proinflammatory mediators, more severe weight loss, and increased mortality when compared with their similarly infected wild-type counterparts, but there was no increase in bacterial burden (19). These results also support the notion that the inflammatory response in the CF lung is dysregulated and excessive. Because many effects of IL-10 resemble those of corticosteroids (20) and ibuprofen (21), it seems reasonable to expect that IL-10 might have similar beneficial effects in patients with CF.

We hypothesized that the decrease in IL-10 may be equally important as the increases in proinflammatory cytokines in the pathogenesis of CF lung disease, and that exogenous IL-10 might reduce the excessive inflammatory response. To evaluate these hypotheses, the agar bead model of chronic endobronchial P. aeruginosa infection was used since the histopathology of this model resembles that of the infected CF lung (17, 19, 22). Our results show that IL-10 knockout mice had increased local inflammation and adverse systemic effects as compared with wild-type mice, and that IL-10 treatment of normal mice attenuated local and systemic effects without altering the bacterial burden. These findings suggest that IL-10 deficiency is important in CF lung disease, and that IL-10 treatment might have beneficial effects.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mice

Eight- to 10-wk-old male wild-type C57Bl/10J mice weighing approximately 25 g and IL-10 knockout mice (8) on a C57Bl/10J background matched for age, weight, and sex were obtained from Jackson Laboratories (Bar Harbor, ME) for use in the IL-10 deficiency experiments. This age was selected because at our institution the typical histopathologic changes of inflammatory bowel disease are absent in IL-10 knockout mice younger than 11 wk of age. The genotypes of the mice were confirmed by polymerase chain reaction amplification of tail snip DNA. Eight- to 10-wk-old male CD-1 mice weighing approximately 30 g were obtained from Charles River Breeding Laboratories (Wilmington, MA) for use in the IL-10 treatment experiments. Animals were housed in microisolators and received endotoxin-free water and food.

Experimental Model

The agarose bead model of chronic endobronchial P. aeruginosa infection was modified for use in mice as described previously (19, 23, 24). Briefly, 5 ml of a suspension of P. aeruginosa strain M57-15, a mucoid clinical isolate grown to late log phase in tryptic soy broth, was added to 50 ml of melted agarose at 50° C. An aliquot of this mixture was added to rapidly stirring mineral oil for 11 min and then gradually cooled in an ice bath over 7 min to form beads. The beads were washed once with 0.5% sodium deoxycholate in phosphate-buffered saline (PBS) (pH, 7.4) and four times with PBS. Bead size, as measured by light microscopy, ranged from 25 to 250 µm (mean, 90 µm). An aliquot of the initial agar bead slurry was diluted and homogenized with a polytron homogenizer, and the number of CFU/ml was determined by plating serial dilutions. The agar bead slurry was then diluted so that each animal received approximately 3 to 4 × 10⁴ CFU in 20 µl.

Mice were anesthetized with 0.015 ml/g of body weight of 2.5% Avertin (2% 2,2,2-tribromoethanol, 2% tert-amyl alcohol, 0.9% NaCl) via intraperitoneal injection and placed in the supine position. A one-inch 22-gauge angiocatheter was inserted into the trachea through a transverse ventral incision and advanced into the right mainstem bronchus. Twenty microliters of agar bead slurry followed by 100 µl of air were rapidly instilled into the right mainstem bronchus. A small number of wild-type, IL-10 knockout, and CD-1 mice were inoculated with sterile agar beads to determine if this resulted in an inflammatory response, and whether this differed in various strains of mice. The experimental protocol was approved by the Case Western Reserve University Institutional Animal Care Committee.

Reagent

Recombinant human IL-10 (rhIL-10) was kindly provided by the Schering-Plough Research Institute (Kenilworth, NJ).

Experimental Protocol

To determine the effects of IL-10 deficiency, chronic endobronchial P. aeruginosa infection was established in 24 wild-type mice and 28 IL-10 knockout mice. The mice were examined and weighed daily until they were killed on Days 3 or 10. The animals were anesthetized with 2.5% Avertin, killed, and exsanguinated via cardiac puncture. The lungs from one third of the animals from each group were preassigned for one of three outcome determinations: quantitative histopathology, quantitative bacteriology, or BAL fluid analysis.

In rhIL-10 treatment experiments, endobronchial P. aeruginosa infection was established in CD-1 mice as described above. The mice then received either 3 µg of rhIL-10 in 200 µl of TRIS HCl (10 mM; pH, 7.4) or 200 µl TRIS HCl alone (placebo) by intraperitoneal injection beginning 12 h after inoculation and continuing every 8 h until the mice were killed on Day 10. This dose of rhIL-10 was extrapolated from pharmacokinetic data provided by the Schering-Plough Research Institute. This treatment regimen was not based on experiments in this model. Thirty-six mice in the rhIL-10 treatment group and 40 mice in the placebo group were evaluated for the same outcome measures described above.

BAL Fluid Analysis

For BAL fluid examination, a 22-gauge bead-tip gavage needle was inserted into the trachea below the larynx and secured with a 4.0 silk suture. Six 0.5-ml aliquots of normal saline were instilled into the trachea, allowed to dwell for 5 s, gently aspirated, and pooled. A cell count and differential was performed on a 250-µl sample. The remainder was centrifuged at 1,000 g for 10 min at 4° C. The supernatants were removed and phenylmethylsulfonyl fluoride and ethylenediaminetetra-acetic acid were added to give final concentrations of 1 × 10⁴ M and 5 × 10³ M, respectively. Supernatants were stored at 70° C until analyzed for murine (m) TNF-, mIL-1, mIL-6, mIL-10, and the chemokines mMIP-2 and mKC/N51 (KC) using commercially available ELISA kits (Endogen Corp., Woburn, MA and R&D Systems, Minneapolis, MN). The results were standardized for dilution of epithelial lining fluid (ELF) using the urea method (25). Cell counts, differentials, and ELISA measurements were performed by investigators unaware of the origin of the samples.

Quantitative Histopathology

A 22-gauge bead-tip needle was inserted into the trachea and fixed in place with a suture. The heart-lung block was removed and rinsed in heparinized saline. The lungs were inflated with a 2% paraformaldehyde-agarose mixture in PBS solution and fixed for 5 d. The lungs were cut midsagittally and embedded in paraffin. Tissue sections were then cut sagittally every 5 µm and stained with hemotoxylin-eosin. One section from the center of each lobe and one section halfway lateral through each lobe were examined for histopathologic changes using a grid point counting method as was used in the rat study of ibuprofen (17). The stained sections were viewed on a monitor at 145-fold magnification. Each section was scored by sequentially moving the tissue section 0.88 mm stepwise on a mechanical stage, recording the presence or absence of inflammation beneath four equally spaced points in each 0.88-mm square. Overall, approximately 1,200 points were counted in the lungs from each animal. Because of the small but variable spillover from the right lung into the left lung, both lungs were examined. The points from all lobes of both lungs were added together and the percent inflammation was determined by dividing the number of points falling on loci of inflammation by the number of total points counted. The grid point counting was performed by an experienced observer who was unaware of the origin of the tissue sections.

Quantitative Bacteriology

The lungs were isolated and removed using sterile technique, then placed in 50 ml of sterile PBS and homogenized together. Serial 10-fold dilutions were spread on replicate MacConkey agar plates. CFU were counted at 24 and 48 h. The spleens were removed from all nonsurviving mice and from mice whose lungs were designated for quantitative bacteriology, homogenized in sterile PBS, and plated at a dilution of 1:100 to determine if bacteremia was present.

Statistical Analysis

The data were analyzed using the SAS statistical package (SAS Inc., Cary, NC). Differences in survival were determined by the log-rank procedure. In experiments comparing wild-type mice with IL-10 knockout mice, the log rank statistic was 0.05 with one degree of freedom. In experiments comparing rhIL-10 with placebo, the log rank statistic was 4.43 with one degree of freedom. Groups were compared for summary descriptors of change in body weight using a nonparametric approach for longitudinal data, which is particularly useful for longitudinal data with modest sample sizes (26, 27). Because sample sizes were limited and to avoid making invalid distributional assumptions, Wilcoxon's nonparametric rank sum procedure was used to compare experimental groups for all outcome measures, including summary measures of longitudinal weight change (26). Significance probabilities were obtained by exact tables for small sample sizes (28). Data are expressed as mean ± standard error of the mean (SEM).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Endobronchial P. aeruginosa Inoculation in Wild-Type Mice and IL-10 Knockout Mice

Because our major goal was to determine if IL-10 deficiency would result in increased local inflammation and increased systemic morbidity because of chronic P. aeruginosa infection, we inoculated wild-type mice and their IL-10 knockout counterparts with P. aeruginosa embedded in agar beads. This model has been shown to resemble the chronic endobronchial infection in CF in many ways (17, 19, 22). In preliminary studies, we used intratracheal instillation to establish bilateral infection. Although mice sufficient in IL-10 had better survival than did mice deficient in IL-10, the overall mortality precluded completion of all the analyses we wished to perform. Therefore in subsequent experiments, we performed unilateral inoculation into the right mainstem bronchus.

Survival

There was no difference in overall survival at the end of the 10-d period of observation between wild-type and IL-10 knockout mice. Both groups had a survival of 71% (p = 0.82 by the log rank procedure) (Figure 1). However, IL-10 knockout mice did have a tendency towards earlier death. On Day 5, wild-type mice had a survival of 96%, whereas IL-10 knockout mice had a survival of 71%. On necropsy, all nonsurvivors had marked pulmonary involvement. Further, we examined the small and large intestines of all mice and found no evidence of inflammatory bowel disease. No bacteria grew in cultures of spleen homogenates from any nonsurvivors. There was no difference in overall survival between wild-type (n = 8) and IL-10 knockout (n = 10) mice inoculated with sterile agar beads (p = 0.44). One wild-type mouse and no IL-10 knockout mice died.

View larger version (10K): [in this window] [in a new window]   Figure 1.   Survival of wild-type (n = 24) and IL-10 knockout (n = 28) mice with chronic endobronchial P. aeruginosa infection. There was no difference in overall survival during the 10-d period of observation as determined by the log rank test (p = 0.82). One wild-type (n = 8) mouse inoculated with sterile agar beads died on Day 7 and no IL-10 knockout (n = 10) mice inoculated with sterile agar beads died (p = 0.44).

Change in Body Weight

Wild-type mice had less severe weight loss than did IL-10 knockout mice (p = 0.04) (Figure 2). Wild type mice lost weight for the first 3 d after inoculation but steadily gained thereafter. IL-10 knockout mice lost weight for 4 d after inoculation before beginning to regain weight. Interestingly, the timing of the most severe weight loss (Days 4 to 5) corresponds with the most dramatic difference in mortality. At 10 d, wild-type mice had a mean net change in body weight of 0.02 ± 0.4 g, whereas IL-10 knockout mice had a mean net change in body weight of 1.6 ± 0.5 g. There was no overall difference in weight loss between wild-type (n = 7) and IL-10 knockout (n = 10) mice inoculated with sterile agar beads (p > 0.1). The maximum weight loss in animals given sterile agar beads was less than 1.9 g.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Change in cumulative body weight of wild-type (n = 17) and IL-10 knockout (n = 20) mice with chronic endobronchial P. aeruginosa infection. Values represent the mean change in daily body weight ± SEM in mice surviving 10 d. The net change in body weight of P. aeruginosa-infected wild-type and IL-10 knockout mice was significantly different as determined by Wilcoxon's rank sum procedure (p = 0.04). In wild-type (n = 7) and IL-10 knockout (n = 10) mice inoculated with sterile agar beads, the maximal weight loss occurred on Day 4 and the mean weight loss in both groups was less than 1.9 g. Overall, there was no significant difference in change in cumulative body weight in wild-type and IL-10 knockout mice inoculated with sterile agar beads (p > 0.1).

BAL Fluid Analysis

BAL was performed 10 d after pulmonary inoculation. Although wild-type mice tended to have a lower mean absolute neutrophil (PMN) count (8.7 × 10⁵/ml ELF, n = 4) than did IL-10 knockout mice (2.0 × 10⁶/ml ELF, n = 5), the difference was not statistically significant (p > 0.2) primarily because of the variability within each group. However, the percent PMNs recovered on bilateral BAL was significantly less in the wild-type mice (13 ± 7%) than in the IL-10 knockout mice (44 ± 9%) (p < 0.05). There was no statistically significant difference in BAL fluid TNF-, IL-1, IL-6, MIP-2, or KC, measured 10 d after inoculation, although most of the measured concentrations were at the lower limits of detection in the ELISA assays. Wild-type mice (n = 3) inoculated with sterile agar beads had a mean absolute PMN count of 4.5 × 10⁴/ml ELF and 1.3 ± 0.4% PMNs recovered on BAL, whereas IL-10 knockout mice (n = 4) given sterile beads had a mean absolute PMN count of 3.4 × 10⁵/ml ELF (p > 0.1) and 8 ± 2% PMNs (p > 0.1) recovered on BAL.

In order to determine if there were differences in cytokines during the acute phase of P. aeruginosa infection that might contribute to the findings seen during the chronic phase, subsequent experiments were undertaken in which BAL was performed 3 d after inoculation. All proinflammatory cytokines were present in lesser concentrations in BAL fluid from wild-type mice (n = 8) than from IL-10 knockout mice (n = 9). Statistically significant differences in IL-6 (19.9 ± 4.1 ng/ml ELF versus 99.2 ± 15.4 ng/ml ELF, p < 0.02) and KC (3.5 ± 0.6 ng/ ml ELF versus 7.2 ± 1.3 ng/ml, p < 0.05) were seen between wild-type mice and IL-10 knockout mice, respectively.

Quantitative Histopathology

The typical histopathology is seen in Figure 3. Both wild-type (Figures 3A and 3B) and IL-10 knockout (Figures 3C and 3D) mice had qualitatively similar infiltrates dominated by PMNs. However, the extent of inflammation differed markedly. At 10 d after inoculation (Figure 4), wild-type mice (n = 8) had a significantly decreased area of lung inflammation (10 ± 2%) compared with IL-10 knockout mice (28 ± 4%, n = 8) (p < 0.002). Wild-type mice (n = 3) inoculated with sterile agar beads had 2.3 ± 0.4% inflammation and similarly inoculated IL-10 knockout mice (n = 4) had 3.2 ± 0.6% inflammation (p > 0.2) on histopathologic examination at 10 d.

View larger version (165K): [in this window] [in a new window]   Figure 3.   Lung histopathology of wild-type and IL-10 knockout mice 10 d after inoculation with agarose beads containing P. aeruginosa. Tissue sections were stained with hemotoxylin-eosin. A and B reveal the typical histopathology of wild-type mice at original magnifications ×120 and ×600, respectively. C and D reveal the typical histopathology of IL-10 knockout mice at original magnifications ×120 and ×600, respectively. Notice the large peribronchial neutrophilic infiltrates in both animals and the absence of granulomas. The extension of leukocytes into the surrounding lung parenchyma was less in the wild-type mice than in the IL-10 knockout mice.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Area of lung inflammation in wild-type (n = 8) and IL-10 knockout mice (n = 8) inoculated with P. aeruginosa containing agar beads, and of wild-type (n = 3) and IL-10 knockout mice (n = 4) inoculated with sterile agar beads. Values represent the mean ± SEM of the area percentage of inflammation from lung sections taken from mice 10 d after inoculation. Compared with IL-10 knockout mice inoculated with Pseudomonas agar beads, wild-type mice had significantly less area of the lung involved in inflammation as determined by Wilcoxon's rank sum procedure (p < 0.002). There was no difference in the area of inflammation between wild-type and IL-10 knockout mice inoculated with sterile agar beads (p > 0.2).

Quantitative Bacteriology

In order to determine the effect of the absence of IL-10 on the bacterial burden, quantitative bacteriology was performed on lung homogenates from mice killed 10 d after inoculation. There was no difference in the amount of P. aeruginosa recovered from the lungs of wild-type mice (mean log CFU/mouse lung = 3.4 ± 0.6, n = 5) and IL-10 knockout mice (mean log CFU/mouse lung = 3.2 ± 0.7, n = 7) (p > 0.4). No bacteria grew in cultures of spleen homogenates from either wild-type or IL-10 knockout mice killed 10 d after inoculation. No bacteria were isolated on cultures of spleen or lung homogenates from any mouse inoculated with sterile agar beads.

Because the greatest difference in mortality during the chronic infection occurred 4 d postinoculation, quantitative bacteriology was performed on lung homogenates from wild-type and IL-10 knockout mice killed 3 d after endobronchial inoculation. Although there was a trend for decreased bacterial counts in wild-type (mean log CFU/mouse lung = 5.7 ± 1, n = 8) versus IL-10 knockout mice (mean log CFU/mouse lung = 6.7 ± 1.3, n = 9), it was not statistically significant (p > 0.2).

Summary of Results for IL-10 Wild-type Knockout Mice

Thus, IL-10 deficiency was associated with increased inflammation and increased systemic morbidity, as indicated by weight loss, without an increased bacterial burden. These results are consistent with those found in CFTR knockout versus wild-type mice with a similar infection (19). We therefore speculate that IL-10 deficiency may be responsible for the excessive inflammation in CFTR knockout mice and in the airways of humans with CF as well.

IL-10 Treatment in CD-1 Mice with Endobronchial P. aeruginosa Infection

Having shown that IL-10 deficiency was associated with increased local inflammation and systemic morbidity, we sought to determine if exogenous IL-10 administration could safely ameliorate inflammation in this type of infection in normal mice. To demonstrate the effect of IL-10 deficiency on inflammation in the previous experiments, a wild-type strain of mice was chosen that developed only mild inflammation at 10 d. To demonstrate the effect of rhIL-10 treatment on inflammation, multiple strains of mice were evaluated for their ability to develop large amounts of inflammation in our model without resulting in overwhelming mortality. Of all strains studied (Balb/C, C57Bl/6J, C57Bl/10J, C3H/FEJ, and CD-1), CD-1 mice best met these criteria (data not shown). We therefore inoculated CD-1 mice with P. aeruginosa containing agar bead and treated them with either rhIL-10 or placebo. Our goal was not to compare the inflammatory response between different strains of mice, but to determine the effect of IL-10 administration on inflammatory response caused by endobronchial P. aeruginosa infection in a single strain. However, similar responses were seen in both strains of mice that we used. Mice with more IL-10 had less inflammation than did mice with less IL-10 in both C57Bl/10J and CD-1 mice.

Survival

Mice treated with rhIL-10 had improved survival compared with placebo-treated mice (p = 0.035 by the log rank procedure) (Figure 5). There were no deaths in either group during the first 3 d. Twenty-four of 36 IL-10-treated mice (67%) survived over the 10-d period of observation as compared with 18 of 40 placebo-treated mice (45%). On necropsy, all nonsurvivors in both groups had marked lung pathology. No bacteria grew in cultures of spleen homogenates from nonsurvivors in either rhIL-10-treated or placebo-treated groups, thus confirming the absence of systemic infection.

View larger version (9K): [in this window] [in a new window]   Figure 5.   Survival in CD-1 mice with chronic endobronchial P. aeruginosa infection and treated with either rhIL-10 (n = 36) or placebo (n = 40). Survival was significantly greater in rhIL-10-treated mice as determined by the log rank test (p = 0.035).

Change in Body Weight

Both groups of mice lost weight during the first 5 d after inoculation, but they slowly regained weight thereafter (Figure 6). Mice treated with rhIL-10 (n = 24) had less severe weight loss than did mice treated with placebo (n = 18) (p = 0.005). By Day 10, mice treated with rhIL-10 had a mean net change in body weight of 0.7 ± 0.4 g, whereas mice treated with placebo had a mean net change in body weight of 2.5 ± 0.6 g.

View larger version (17K): [in this window] [in a new window]   Figure 6.   Effect of rhIL-10 treatment on change in cumulative body weight in mice with chronic endobronchial P. aeruginosa infection. Values represent the mean change in daily body weight ± SEM of rhIL-10-treated (n = 24) and placebo-treated (n = 18) mice surviving 10 d. The difference in net change in body weight between rhIL-10 treatment and placebo was significant as determined by Wilcoxon's rank sum procedure (p = 0.005).

BAL Fluid Analysis

Bronchoalveolar lavage was performed on eight mice treated with rhIL-10 and on five mice treated with placebo. In animals surviving through Day 10, rhIL-10-treated mice had 4 ± 2% PMNs and a mean absolute PMN count of 3 × 10⁵/ml ELF (mean log PMNs/ml ELF = 4.9 ± 0.2), whereas placebo-treated mice had 32 ± 17% PMNs and a mean absolute PMN count of 5 × 10⁶/ml ELF (mean log PMNs/ml ELF = 6.0 ± 0.4). The differences in percent PMNs and absolute PMN counts were statistically significant (p < 0.02 and p < 0.05, respectively) (Figure 7). CD-1 mice (n = 8) inoculated with sterile agar beads had a mean absolute PMN count of 2.5 × 10⁴/ml ELF and 1 ± 0.3% PMNs recovered from BAL 10 d after inoculation. Although there were no statistically significant differences in BAL fluid TNF-, IL-1, IL-6, MIP-2, or KC, 10 d after pulmonary inoculation with Pseudomonas agar beads, mean KC concentrations were decreased in the rhIL-10 treatment group compared with the placebo group (378 ± 81 pg/ml ELF versus 662 ± 239 pg/ml ELF), but the range of individual variation was too wide for statistical significance.

View larger version (8K): [in this window] [in a new window]   Figure 7.   BAL fluid PMNs in CD-1 mice with chronic endobronchial P. aeruginosa infection and treated with rhIL-10 (n = 8) or placebo (n = 5). The values represent the number of PMNs in BAL fluid, corrected for the dilution of ELF, 10 d after inoculation. The horizontal lines indicate the group means. The rhIL-10-treated mice had significantly fewer PMNs as determined by Wilcoxon's rank sum procedure (p < 0.02).

Quantitative Histopathology

The histopathology in both groups was similar to that shown in Figure 3, with no qualitative differences. However, there was a significant difference in the extent of inflammation (Figure 8). IL-10-treated mice (n = 8) had a significantly reduced area of inflammation (11 ± 2%) compared with placebo-treated mice (35 ± 7%, n = 7) (p < 0.01). Ten days after inoculation with sterile agar beads, CD-1 mice (n = 9) had 4 ± 0.7% inflammation.

View larger version (9K): [in this window] [in a new window]   Figure 8.   Area of inflammation in CD-1 mice with chronic endobronchial P. aeruginosa infection, and treated with rhIL-10 (n = 8) or placebo (n = 7). Values represent the mean ± SEM of the area percentage of inflammation from lung sections taken from mice 10 d after inoculation with P. aeruginosa. The rhIL-10-treated mice had significantly less area of lung involved in inflammation as determined by Wilcoxon's rank sum procedure (p < 0.01).

Quantitative Bacteriology

To determine if treatment with rhIL-10 resulted in any change in the bacterial burden, quantitative bacteriology was performed on lung homogenates from mice killed on Day 10 after inoculation. There was no difference in the amount of P. aeruginosa recovered from the lungs of rhIL-10-treated mice (mean log CFU/mouse lung = 3.8 ± 0.7, n = 8) versus placebo-treated mice (mean log CFU/mouse lung = 4.6 ± 0.7, n = 6) (p > 0.5). No bacteria grew in cultures of spleen homogenates in rhIL-10-treated or placebo treated mice killed 10 d after inoculation.

Summary of Results for IL-10 Treatment

The results of these experiments comparing IL-10 treatment with placebo treatment were similar to those results found in experiments comparing wild-type mice with IL-10 knockout mice. In both instances, the mice with greater IL-10 had decreased inflammation, milder systemic morbidity, and decreased mortality, but no increase in the burden of bacteria.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Several lines of evidence suggest that in CF lung disease, inflammation is excessive relative to the bacterial burden (7). The inflammatory response is characterized by a predominately neutrophilic infiltrate. The PMNs release mediators that damage tissues, result in deleterious functional alterations, and interfere with local defense mechanisms (2, 7). As the lung disease progresses, severe bronchiectasis results that ultimately leads to death. Reports that local inflammation in CF may occur in the absence of bacterial infection (29) suggest that dysregulation of the inflammatory response may be directly linked to defects in CFTR function. Compared with non-CF infants, infants with CF have large quantities of IL-8, PMNs, and PMN elastase in their BAL fluid. In some cases, this may occur even in the absence of detectable bacterial infection (29). In patients undergoing BAL for clinical indications, CF children with H. influenzae as their only pathogen had higher BAL PMN counts and IL-8 levels than did non-CF children with similar burdens of H. influenzae (30). Comparable differences were seen in CF versus wild-type mice with experimental endobronchial P. aeruginosa infection (19). Furthermore, treatment of patients with CF with anti-inflammatory drugs slows the progression of lung disease without resulting in exacerbation of infection (15, 16, 18). Taken together, these studies suggest that the inflammatory response in the CF lung may be dysregulated and disproportionately severe as compared with the burden of infection. We have demonstrated that the anti-inflammatory cytokine IL-10 is deficient in the airways of patients with CF (3). This prompted us to determine whether IL-10 deficiency contributes to the excessive inflammatory response, and to determine if exogenous IL-10 might moderate inflammation without exacerbating infection.

In order to ascertain if endogenous IL-10 deficiency was associated with an increased inflammatory response to P. aeruginosa, we established chronic endobronchial infection in wild-type mice and IL-10 knockout mice using the agarose bead model. The P. aeruginosa containing agar beads lodge in the small airways of mice and establish a chronic infection. This, in turn, incites a luminal exudate and an intense peribonchial infiltrate that contains a large proportion of neutrophils, distinctly resembling the inflammation found in the lungs of patients with CF. As the infection persists and the inflammatory response progresses, the cellular infiltrate may spread to involve the local lung parenchyma, but there is little sepsis or spread to distal sites (19). All animals that died before the tenth day had some degree of lung disease on necropsy. None had inflammatory bowel disease or apparent bacteremia. Outcome measures included survival, change in body weight, BAL fluid PMN counts, BAL fluid cytokine concentrations, percent area of lung inflammation, and quantitative bacteriology. The overall survival was identical in wild-type and IL-10 knockout mice. However, when compared with wild-type mice, IL-10-deficient mice had more severe weight loss, increased percent PMNs in BAL, and increased area of lung inflammation; without alterations in the bacterial burden. Although a large percentage of PMNs was seen on histopathologic examination (Figure 3), the mean percent PMNs in BAL fluid was less than 50% in all groups. This may be explained by two facts. First, although the inoculation was predominately unilateral, the BAL procedure was performed on both lungs together. Thus, alveolar macrophages from the uninvolved lung may be diluting out the percentage of neutrophils from infected lung segments. Second, neutrophils may be trapped behind agar beads that are occluding the airways in the most involved areas and therefore may be inaccessible by BAL. Similar phenomena may account for the fact that no significant difference was demonstrated in BAL fluid cytokines between wild-type and IL-10 knockout mice at Day 10. The dilutional effects of large volume BAL of both lungs may obscure local differences in the involved lung segments. Furthermore, the concentrations of all cytokines in both groups were near or below the sensitivity of the assays. Therefore, in order to determine if IL-10 deficiency altered the cytokine profile during the acute phase of the infection, we killed a set of animals during the height of the acute inflammatory response on Day 3. Compared with wild-type mice, IL-6 and the chemokine KC were significantly increased in IL-10 knockout mice. Overall, despite the fact that the absence of endogenous IL-10 was associated with increased inflammation and more severe weight loss, these changes were not associated with more efficient host defense mechanisms since the bacterial burden did not differ between the two groups. Hence, the absence of IL-10 mimicked the situation in CF in which local and systemic responses to chronic infection appear to be excessive, and injurious rather than protective.

We also found that treatment with exogenous rhIL-10 was associated with reduced inflammation in this model of chronic endobronchial P. aeruginosa infection. Again all animals that died before the tenth day had some degree of lung disease on necropsy, but a small percentage of mice in both groups also had an associated ileus, which may have been related to the multiple intraperitoneal injections. Mice receiving exogenous rhIL-10 had improved survival, less severe weight loss, reduced absolute PMN counts in BAL, reduced percentage of PMNs in BAL, and reduced percent area of inflammation, but no alterations in the bacterial burden of the lung. This last point is important because of concerns regarding the administration of an anti-inflammatory agent in the face of ongoing bacterial infection. Like ibuprofen and corticosteroids in patients with CF (15, 16, 18), rhIL-10 did not result in sepsis or overwhelming bacterial infection in this animal model. Although some studies have not demonstrated that IL-10 is associated with improved survival (14, 31), ours did. This is most likely due to differences in the models. We studied chronic endobronchial P. aeruginosa infection, whereas other studies used models of acute or systemic infection.

Our data agree with the findings of others who used related animal models. IL-10 deficiency was associated with increased lung inflammation in mice repeatedly exposed to aerosolized P. aeruginosa (32). Exogenous mIL-10 was associated with reduced lung injury and mortality in mice with acute invasive P. aeruginosa pneumonia (33). The effects of IL-10 are likely due to inhibition of NF-B activation through increased production and stabilization of the mRNA transcript for NF-B's inhibitor, IB (34, 35). Other investigators have recently speculated that defective CFTR function may result in increased NF-B activation, which in turn may result in increased proinflammatory cytokine secretion by airway epithelial cells (36).

Our results support the hypothesis that the reductions in IL-10 production may be as important as the increases in proinflammatory cytokines in the pathogenesis of the excessive inflammatory response to the chronic endobronchial P. aeruginosa infection in patients with CF. We have also shown that exogenous rhIL-10 reduced the local inflammation as well as the systemic morbidity without increasing the bacterial burden. This suggests that administration of this anti-inflammatory cytokine will not likely result in deleterious inhibition of local host defense mechanisms.

Although survival in CF has greatly improved during the past 30 yr, the beneficial effects of antibiotics, airway clearance, and improved nutrition appear to have arrived at a plateau (1). Despite the hope for treatments aimed at correcting the basic defect, most successful current therapies still address only secondary pathology. Because treatments directed at modifying the basic defect may not benefit patients with CF with established chronic infection and inflammatory lung disease, it is important to be able to interrupt the inflammatory cascade and slow the disease progression. Our observations strongly suggest that further investigation of IL-10 as a potential therapy for CF is warranted.