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J Appl Physiol 99: 2204-2211, 2005. First published August 4, 2005; doi:10.1152/japplphysiol.00246.2005
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Dose response to rhCC10-augmented surfactant therapy in a lamb model of infant respiratory distress syndrome: physiological, inflammatory, and kinetic profiles

Beth N. Shashikant,1 Thomas L. Miller,1,2 Richard W. Welch,3 Aprile L. Pilon,3 Thomas H. Shaffer,1,2 and Marla R. Wolfson1

1Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania; 2Nemours Research Lung Center, Alfred I. duPont Hospital for Children, Wilmington, Delaware; and 3Claragen, Inc., College Park, Maryland

Submitted 2 March 2005 ; accepted in final form 28 July 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
While surfactant (SF) therapy alone improves respiratory distress syndrome (RDS)-associated gas exchange and lung stability, absence of anti-inflammatory proteins limits efficacy with respect to inflammation. Clara cell secretory protein (CC10), deficient in preterm infants, prevents SF degradation and has anti-inflammatory properties. In this study, intratracheal recombinant human (rh) CC10 (Claragen)-augmented SF (Survanta, Ross) therapy was examined in a premature lamb model of RDS with respect to inflammation and kinetic dose-response profiles. Preterm lambs (n = 24; gestational age: 126 ± 3 days) were delivered via cesarean section, sedated, ventilated, and randomized into groups: 100 mg/kg SF, 100 mg/kg SF followed by 0.5 mg/kg rhCC10, 100 mg/kg SF followed by 1.5 mg/kg rhCC10, and 100 mg/kg SF followed by 5.0 mg/kg rhCC10. Arterial blood chemistry and lung mechanics were monitored; lungs were lavaged and snap-frozen after 4 h. TNF-{alpha}, IL-8 in plasma; TNF-{alpha}, IL-6, IL-8, myeloperoxidase in lung; and rhCC10 in plasma, urine, bronchoalveolar lavage, and lung were analyzed. Improvement in compliance, peak inspiratory pressure, and ventilatory efficiency index were greatest (P < 0.05) with SF + 5.0 mg/kg rhCC10. Plasma, urine, bronchoalveolar lavage, and lung [rhCC10] (where brackets denote concentration) increased (P < 0.01) with dose. Plasma [IL-8] was lower (P < 0.05) with rhCC10 than SF alone. Treatment with at least 1.5 mg/kg rhCC10 resulted in lower (P < 0.05) lung [TNF-{alpha}], [IL-8], and [myeloperoxidase]; SF + 1.5 mg/kg rhCC10 group had lower (P < 0.05) lung [IL-6], compared with all other groups. Compared with SF alone, SF augmented with at least 1.5 mg/kg rhCC10 decreased RDS-induced lung and systemic inflammation. Given that inflammation may lead to functional compromise, these data suggest that early intervention with rhCC10 may enhance SF therapy and warrant longer duration studies to determine its role to decrease long-term complications of ventilator management.

Clara cell secretory protein; mechanical ventilation; cytokines; bronchopulmonary dysplasia

SURFACTANT (SF) REPLACEMENT therapy improves gas exchange and lung stability in respiratory distress syndrome (RDS). However, the ability of SF replacement therapy to reduce inflammation is less clear and may be limited due to the removal of SF proteins A and D, which are believed to play a role in host defense, from commercially available exogenous SFs (23). In addition, exogenous SF therapy has been shown to activate neutrophils (35) and complement (39) in preterm neonates, as well as increase levels of proinflammatory cytokines in models of lung injury (33, 36, 41). Presumably, improvement in both lung function and inflammatory profiles could be achieved by enhancing SF replacement therapy with administration of an anti-inflammatory protein in a model of RDS.

Clara cell secretory protein (CC10), an anti-inflammatory protein native to the lung (7, 28), has immunomodulatory properties (11, 19, 38) and may also prevent SF degradation by PLA2 inhibition (20, 22). Potential benefits of CC10-enhanced SF replacement therapy include inhibition of SF inactivation and regulation of the inflammatory response to both mechanical (high pressures and volumes associated with mechanical ventilation) and biochemical (arachidonic acid, high oxygen tension) stimuli. Ramsay et al. (29) found a direct correlation between low CC10 levels with increased levels of oxidatively modified CC10 in tracheal aspirate of premature infants and the development of bronchopulmonary dysplasia (BPD), suggesting that CC10 may play a role in limiting the inflammatory response that leads to BPD development.

In a preliminary study using a single concentration of CC10 in a rabbit SF washout model to create acute lung injury, we found that markers of inflammation [i.e., IL-8 and myeloperoxidase (MPO)] were lower in the plasma, bronchoalveolar lavage (BAL) fluid, and lung tissue of animals treated with both SF and recombinant human (rh) CC10 compared with SF alone (25). Additionally, using a SF-treated, ventilated, full-term neonatal pig as a ventilator-induced lung injury model, Chandra et al. (6) reported a rhCC10 dose-dependent improvement in compliance and trend toward decreased lung inflammation. Recently in a phase 1, multicentered, placebo-controlled, randomized trial evaluating the safety and pharmacokinetics of rhCC10 in ventilated preterm infants with RDS, Levine et al. (21) reported reductions in total cell count, neutrophil counts, and a trend toward decreased total protein and IL-6 in tracheal aspirate fluid collected over the first 3 days of life. To the degree that other anti-inflammatory proteins have been shown to have a dose-dependent response on inflammation (30) and because developmental deficiencies in the SF and pro/anti-inflammatory profiles predispose the immature neonate to lung injury, in this study we evaluated the dose-response effects of rhCC10 on SF replacement therapy in a SF-deficient preterm lamb model. This model has clinical relevance as it mimics human neonatal RDS and provides the opportunity to clarify the role of CC10 dose in preventing or modifying an inflammatory response in a model with intrinsic SF deficiency. We hypothesized that the use of rhCC10 in conjunction with exogenous SF therapy would promote lung function and minimize the inflammatory response to ventilation in a dose-dependent manner compared with exogenous SF therapy alone.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Animal Protocol

The experimental protocol and procedures for this study were approved by the Institutional Animal Care and Use Committee at Temple University School of Medicine.

Following cesarean section of the ewe, 24 fetal lambs (gestational age: 126 ± 3 days) were anesthetized (intramuscular: 1 µg Fentanyl; subcutaneous ventral neck: ~1 ml –0.5% lidocaine HCl) and instrumented with an endotracheal tube by tracheotomy (3–3.5 mm ID Hi-Lo Jet Tube: Mallincrodkt, Saint Louis, MO) and carotid and venous catheters, paralyzed (intravenous: 0.10 mg/kg pancuronium bromide), and dried. After the umbilical cord was cut, the premature lamb was weighed, and time-cycled, pressure-limited mechanical ventilation (Infant Star Neonatal Ventilator, Infrasonics) was immediately initiated to target arterial PCO2 (PaCO2) within 40–60 Torr, with ventilatory pressures and rate adjusted within the following settings to optimize pressure-volume relationships and to maintain physiological gas exchange and acid-base balance: peak inspiratory pressure (PIP) ≤ 35 cmH2O, positive end-expiratory pressure (PEEP) = 8 cmH2O, flow rate ≤ 10 l/min to provide a tidal volume = 6 ml/kg, respiratory frequency ≤ 70 breaths/min, with inspiratory time between 0.3 and 0.5 s, and fraction of inspired oxygen (FIO2) = 1.0. Airway temperature (35°C) and humidity (100%) were kept constant throughout the 4-h protocol.

Baseline measurements of arterial blood chemistry (Nova Statprofile M, Waltham, MA; Radiometer OSM 3, Copenhagen, Denmark), tidal volume by integrated pneumotachography signals (no. 00, Fleish, Eplinges, Switzerland), airway manometry, calculated respiratory compliance (PeDS-LAB, MAS, Hatfield, PA) (3), mean arterial pressure, and heart rate were performed.

Following baseline measurements, animals were randomized to receive either SF alone (100 mg/kg Survanta, Ross Laboratories; 4 ml/kg; n = 6), or SF followed by 0.5, 1.5, or 5 mg/kg of intratracheally administered rhCC10 (Claragen; 5.5 mg/ml adjusted with saline so that all doses of rhCC10 were delivered in a volume of 2 ml/kg; n = 6/group; rhCC10 was administered 15 min after SF). Instillation of the SF and rhCC10 occurred over 5–10 min via the side port of the endotracheal tube, during which time the animals were repositioned to optimize distribution using four equal increments of the total dose with Trendelenberg, reverse Trendelenberg, and left and right lateral decubitus positions.

Arterial pressure, heart rate, pulse oximetry, and rectal temperature were monitored continuously. Arterial blood chemistry and pulmonary mechanics were analyzed every 0.5 h; additional arterial blood samples were collected hourly for subsequent analyses of inflammatory mediators and rhCC10. Fluid and temperature management included 5% dextrose at 5 ml·kg–1·h–1, pancuronium bromide at 0.10 ml·kg–1·h–1 for paralysis to prevent confounding artifact from ineffectual respiratory efforts, buffer to correct pH (bicarbonate/nonbicarbonate based on pH and PCO2), pressors (dopamine ≤20 µg·kg–1·min–1, if mean arterial pressure <40 mmHg), radiant warming, and supplemental anesthesia as needed (Fentanyl: 0.50 µg if 20% increase in blood pressure following deep tissue stimulus).

The oxygenation index {OI = [(MAWP x FIO2)/PaO2 x 100], where MAWP is mean airway pressure, and PaO2 is arterial PO2} and ventilatory efficiency index {VEI = 3,800/[respiratory rate x (PIP – PEEP) x PaCO2 (Torr)]} were computed as previously described (1, 4, 27). OI is accepted as a dimensionless index, since the units cancel when PaO2 and MAWP are expressed in Torr. The VEI is calculated from the equation VEI = (5 ml·kg–1·min–1)/{[(PIP – PEEP) x RF x PaCO2]/760}, where PIP and PEEP are expressed in mmHg, RF is respiratory frequency, and CO2 production is assumed to be 5 ml·kg–1·min–1. When all pressure values are expressed in Torr, VEI would be expressed in ml·Torr–1·kg–1; however, it is conventionally accepted as dimensionless.

Lung Tissue Collection

The animals were deeply anesthetized following the 4-h ventilation period. The trachea was clamped at the final end-expiratory pressure, and a midline thoracostomy with gross inspection was performed. The lungs were perfused through the vasculature with cold Millonig’s phosphate buffer until the perfusate ran clear. The right main stem bronchus was clamped, and a BAL was performed on the left lung by slowly instilling and removing cold phosphate-buffered saline with sodium citrate (9:1 ratio; 3 x 30-ml aliquots) via the endotracheal tube. The total volume of collected BAL fluid was recorded, pooled, and centrifuged. The supernatant was aliquoted and stored at –70°C for subsequent analyses.

Eight consistent regional samples were collected from the left lung, with two samples each from the dependent apex, nondependent apex, dependent base, and nondependent base regions. BAL and tissue samples were snap-frozen in liquid nitrogen and stored at –70°C for analysis of rhCC10 and proinflammatory mediators.

Cytokine Analysis

Cytokine protein concentration in arterial plasma (TNF-{alpha}, IL-8) and lung tissue homogenate (TNF-{alpha}, IL-6, IL-8) were determined by using an ovine-specific sandwich ELISA utilizing mouse anti-ovine capture antibody (IL-6 and IL-8: Serotec; TNF-{alpha}: CSIRO and CAB of University of Melbourne, Australia), rabbit anti-ovine detection antibody (IL-6 and IL-8: Serotec; TNF-{alpha}: CSIRO and CAB of University of Melbourne, Australia), and ovine cytokine protein as standard controls (TNF-{alpha}, IL-6 and IL-8: CSIRO, Australia). All standards and samples were run in duplicate. Standard curves were sensitive at 0.39–200 ng/ml for TNF-{alpha}, 0.39–200 ng/ml for IL-6, and 1.56–200 ng/ml for IL-8 with correlation coefficients >0.95, and interassay and intra-assay coefficients of variance of <10 and <5%, respectively, for all assays. Data are expressed in nanograms per milligram total protein.

Lung tissue samples for cytokine and rhCC10 (see below) analysis were homogenized by using the following procedure: 200 mg tissue were thawed slightly on ice, washed twice with PBS, and homogenized rapidly in 1 ml RIPA solution (50 mM Tris·HCl, 150 mM NaCl, 1% Igepal, 0.5% NaDOC, 0.1% SDS) with Complete protease (i.e., serine, cysteine, and metalloproteases) inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), before centrifugation at 12,000 g for 10 min at 4°C to obtain supernatant.

MPO Assay

MPO concentration in lung tissue was determined by using a colorimetric bioassay. Tissue samples (200 mg) were homogenized in 0.05 M potassium phosphate buffer (1:10 weight to volume) and centrifuged at 1,700 g for 30 min at 4°C. The supernatant was collected, incubated for 2 h in a 60°C water bath, and again centrifuged for 5 min at 10,000 g and 4°C to obtain supernatant for assay (31).

The assay was performed by using the following procedure: 10 µl of standard (human leukocyte MPO, ICN Biomedicals, Aurora, OH) or sample were incubated with 100 µl of substrate buffer (0.1 M sodium citrate, 0.1% o-dianisidine, 1 mM hydrogen peroxide, pH = 5.5), in duplicate, for 1 min in a 96-well plate. The plate was read immediately at 560 nm in an automated plate reader (MRX Revelation, Thermo Labsystems, Franklin, MA). Linear standard curves were obtained with sensitivity from 0.0625 to 1.44 U/ml; interassay and intra-assay coefficients of variance were <10 and <6%, respectively. Data are expressed as units per milligram total protein.

rhCC10 Assay

rhCC10 concentration in lung tissue, plasma, BAL fluid, and urine was determined by using a human-specific competitive ELISA assay, developed by Claragen, which utilizes human-specific CC10 antibodies and rhCC10 standard (Claragen, College Park, MD). The limit of detection in this assay was 10 ng/ml, and four parameter logistic standard curves were obtained that ranged from 10 to 250 ng/ml. All standards and samples were run in duplicate with interassay and intra-assay coefficients of variance of <12 and <7%, respectively. Data are expressed as nanograms per milliliter (plasma and urine) or nanograms per milligram total protein (BAL fluid and lung tissue).

Total Protein Concentration

TNF-{alpha}, IL-6, IL-8, MPO, and rhCC10 concentration in lung tissue and rhCC10 concentration in BAL fluid were normalized to total protein concentration of lung homogenate by using the method described by Bradford (5).

Statistical Analysis

Data analysis was performed by using Microsoft EXCEL Groups and Graph Pad PRISM. Values are expressed as means ± SE. For data that were normally distributed, the parametric ANOVA was used to establish a significant overall difference among the groups. If not normally distributed, the Kruskal-Wallis test was used for an overall comparison of group differences. Significance was accepted with a P < 0.05, and, when omnibus significance was found, Dunnett’s post hoc comparison to SF alone as the control was applied. Multiple pairwise comparisons among all groups were made with Dunn’s test.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Physiological Profile

Physiological responses are shown in Fig. 1. There were no statistically significant group differences in baseline (15-min postdelivery, pre-SF) values of PaO2, PaCO2, compliance, PIP, OI, or VEI. Following treatment with SF or SF + rhCC10, PaO2 (Fig. 1A) increased significantly (P < 0.0001) compared with baseline values, independent of group. Following the initial increase, PaO2 decreased significantly (P < 0.0001) over time, independent of group. PaCO2 (Fig. 1B) decreased significantly (P < 0.0001) following treatment for all groups compared with baseline values and remained lower than baseline over time (P < 0.005), independent of group. Following treatment, respiratory compliance (Fig. 1C) increased significantly (P < 0.0001) compared with baseline values, independent of group. Compliance continued to increase significantly (P < 0.005) over time for all groups. Improvement in compliance was group dependent, with the greatest improvement (P < 0.0001) over time occurring with 5.0 mg/kg rhCC10. PIP (Fig. 1D) was not significantly different initially following treatment compared with baseline values, independent of group. However, PIP decreased significantly in all groups (P < 0.0001) throughout the treatment protocol and was significantly lower (P < 0.05) with SF + 5.0 mg/kg rhCC10 over time compared with SF alone. Following treatment with SF or SF + rhCC10, the OI (Fig. 1E) initially decreased significantly (P < 0.0001) compared with baseline values, then increased significantly (P < 0.0001) over time in all groups. The VEI (Fig. 1F) initially increased significantly (P < 0.05) following treatment with either SF or SF + rhCC10 compared with baseline values. The VEI in animals treated with 5.0 mg/kg rhCC10 continued to improve (P < 0.05) following the initial increase and was significantly greater (P < 0.0001) compared with all other groups, over time.



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  		Fig. 1. Lung function parameters during treatment in a premature lamb model of respiratory distress syndrome (RDS). A: arterial PO2 (PaO2) increased significantly (*P < 0.0001) initially following treatment with surfactant (SF) or SF + recombinant human Clara cell secretory protein (rhCC10) compared with baseline values (BL) and then decreased significantly (**P < 0.0001) over time, independent of group. B: arterial PCO2 (PaCO2) decreased significantly (*P < 0.0001) following treatment with SF or SF + rhCC10 compared with BL and remained lower than BL, which continued over time (**P < 0.005), independent of group. C: respiratory compliance increased significantly (*P < 0.0001) following treatment with SF or SF + rhCC10 compared with BL and continued to increase significantly (**P < 0.005) over time in all groups, with the greatest improvement ({dagger}P < 0.0001 vs. all other groups) over time occurring with 5.0 mg/kg rhCC10. D: peak inspiratory pressure (PIP) was not significantly different than BL initially following treatment with SF or SF + rhCC10, independent of group; however, it decreased significantly (**P < 0.0001) in all groups over time and was significantly lower ({dagger}P < 0.05) with SF + 5.0 mg/kg rhCC10 over time compared with SF alone. E: oxygenation index decreased significantly (*P < 0.0001) following treatment with SF or SF + rhCC10 compared with BL values and then increased significantly over time (**P < 0.0001) in all groups. F: ventilatory efficiency index increased significantly (*P < 0.05) from BL following treatment with either SF or SF + rhCC10 and continued to improve over time (**P < 0.05) in animals treated with SF + 5.0 mg/kg rhCC10 ({dagger}P < 0.0001 vs. all other groups). Values are means ± SE. Symbol legends are the same for all graphs. All doses of rhCC10 are in mg/kg (i.e., SF + 0.5 mg/kg rhCC10).

 
rhCC10 Kinetic Profile

Plasma rhCC10 concentration increased significantly (P < 0.001) over time in all rhCC10-treated groups (Fig. 2A) and was significantly different (P < 0.0001) as a function of dose at all time points with 5.0 mg/kg rhCC10 group significantly greater (P < 0.001) than all other rhCC10-treated groups. Urinary excretion of rhCC10 was significantly greater (P < 0.05) with 5.0 mg/kg rhCC10 than with 0.5 or 1.5 mg/kg rhCC10 at all time points (Fig. 2B). rhCC10 concentration in BAL fluid increased significantly (P < 0.0001) as a function of rhCC10 dose and was significantly greater (P < 0.05) in lambs treated with 5.0 mg/kg rhCC10 compared with all other rhCC10-treated groups (Fig. 2C). Lung rhCC10 concentration increased significantly (P < 0.0001) as a function of rhCC10 dose (Fig. 2D) and was significantly greater (P < 0.05) in the animals treated with 1.5 and 5.0 mg/kg rhCC10 compared with 0.5 mg/kg rhCC10.



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  		Fig. 2. rhCC10 concentration during and after treatment in a premature lamb model of RDS. A: plasma rhCC10 concentration over time was significantly greater (*P < 0.05 vs. SF + 0.5 mg/kg rhCC10 and SF + 1.5 mg/kg rhCC10) in animals treated with 5.0 mg/kg rhCC10 compared with all other rhCC10-treated groups. B: animals treated with 5.0 mg/kg rhCC10 had significantly greater (*P < 0.05 vs. SF + 0.5 mg/kg rhCC10 and SF + 1.5 mg/kg rhCC10) urinary concentration of rhCC10 during treatment compared with all other rhCC10-treated groups (group symbols are the same as for plasma rhCC10). C: end bronchoalveolar lavage (BAL) rhCC10 was significantly greater (*P < 0.05 vs. SF + 0.5 mg/kg rhCC10 and SF + 1.5 mg/kg rhCC10) in the 5.0 mg/kg rhCC10 group compared with all other rhCC10-treated groups. D: lung rhCC10 concentration increased as a function of rhCC10 dose and was significantly greater (**P < 0.05 vs. SF + 0.50 mg/kg rhCC10) in animals treated with SF + 1.5 or SF + 5.0 mg/kg rhCC10 compared with SF + 0.50 mg/kg rhCC10. Values are means ± SE. All doses of rhCC10 are in mg/kg (i.e., SF + 0.5 mg/kg rhCC10).

 
Proinflammatory Mediator Profile

Plasma.   As shown in Fig. 3A, arterial plasma TNF-{alpha} concentration increased within the first 2 h of ventilation and then decreased over time. These changes were not significantly different as a function of treatment group (Fig. 3A). Plasma IL-8 (Fig. 3B) increased significantly from baseline (P < 0.0001) in animals treated with SF alone and increased significantly (P < 0.05) over time in all groups. Treatment with SF + rhCC10 resulted in significantly lower plasma IL-8 compared with SF alone (P < 0.001). Plasma IL-8 concentration was not significantly different within the rhCC10-treated groups.



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  		Fig. 3. Arterial plasma cytokine concentration over time in a premature lamb model of RDS. A: plasma TNF-{alpha} was not significantly different as a function of treatment group. B: plasma IL-8 increased significantly from BL (*P < 0.0001) in animals treated with SF alone and increased significantly (**P < 0.05) over time in all groups; treatment with SF + rhCC10 resulted in significantly lower plasma IL-8 compared with SF alone ({dagger}P < 0.001, all rhCC10-treated groups vs. SF alone at all time points following BL). Values are means ± SE. All doses of rhCC10 are in mg/kg (i.e., SF + 0.5 mg/kg rhCC10).

 
Lung.   rhCC10 decreased proinflammatory cytokines in a dose-dependent manner. Animals treated with SF + 1.5 or 5.0 mg/kg rhCC10 had significantly less (P < 0.05) TNF-{alpha} in the lung compared with animals treated with SF alone and SF + 0.5 mg/kg rhCC10 (Fig. 4A). Animals treated with SF + 1.5 mg/kg rhCC10 had significantly lower (P < 0.05) IL-6 concentration in the lung tissue compared with all other groups (Fig. 4B). Treatment with either SF + 1.5 or 5.0 mg/kg rhCC10 resulted in significantly lower (P < 0.05) lung tissue IL-8 concentration compared with SF alone or SF + 0.5 mg/kg rhCC10 (Fig. 4C). Lung tissue MPO concentration was significantly lower (P < 0.05) in lambs treated with either SF + 1.5 mg/kg or SF + 5.0 mg/kg rhCC10 compared with lambs treated with both SF alone and SF + 0.5 mg/kg rhCC10 (Fig. 4D).



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  		Fig. 4. Lung tissue cytokine concentration following treatment in a premature lamb model of RDS. A: animals treated with SF and either 1.5 or 5.0 mg/kg rhCC10 had significantly less (*P < 0.05 vs. SF alone and SF + 0.5 mg/kg rhCC10) TNF-{alpha} in the lung compared with SF alone and SF + 0.5 mg/kg rhCC10. B: treatment with SF + 1.5 mg/kg rhCC10 resulted in significantly lower (#P < 0.05 vs. all other groups) lung IL-6 concentration compared with all other treatment groups. C: treatment with at least 1.5 mg/kg rhCC10 resulted in significantly lower (**P < 0.01 vs. SF alone; ***P < 0.05 vs. SF + 0.5) lung IL-8 compared with SF alone or SF + 0.5 mg/kg rhCC10. D: animals treated with SF + 1.5 mg/kg or SF + 5.0 mg/kg rhCC10 had significantly lower (*P < 0.05 vs. SF alone and SF + 0.5 mg/kg rhCC10) lung myeloperoxidase (MPO) compared with SF alone and SF + 0.5 mg/kg rhCC10. Values are means ± SE. All doses of rhCC10 are in mg/kg (i.e., SF + 0.5 mg/kg rhCC10).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
This study finds that rhCC10-augmented SF treatment in the preterm lamb results in 1) dose-dependent improvement in lung mechanics, improvement in ventilation efficiency, and a reduction in lung inflammation; and 2) reductions in the systemic inflammatory profile compared with treatment with exogenous SF alone.

Although new treatments have improved the clinical course and survival, the incidence of BPD in preterm infants has not been substantially affected (16). Acute inflammatory changes, secondary to toxic reactive oxygen species, and mechanical stress shortly after initiation of supplemental oxygen and mechanical ventilation have been implicated in the evolution of BPD (16, 17, 26). Developmental deficiencies in SF composition and content, and antioxidant and anti-inflammatory profiles make the preterm lung vulnerable to injury. Thus augmentation of anti-inflammatory capabilities in the lung is a logical strategy to prevent BPD. Intratracheal delivery of therapeutic agents has proven effective in attenuating lung injury in various animal preparations (15, 24, 25, 42). Whereas initial studies suggested that early postnatal steroids (dexamethasone, hydrocortisone) could reduce the incidence and severity of BPD, more recent reports are inconclusive (12, 32, 37), reporting significant adverse effects, including increased mortality, cerebral palsy, bowel perforation, and infection (2, 34, 40). In addition, although widespread use of exogenous SFs has contributed to improvement in lung stability (18), their effect on lung inflammation is inconclusive and may be limited by the lack of anti-inflammatory SF proteins A and D in commercial SF preparations. Within this context, new therapies are urgently needed that specifically target the acute inflammatory responses. Recent studies in mechanically ventilated neonatal pigs, saline-lavaged juvenile rabbits, and human infants suggest that intratracheally administered rhCC10 may decrease lung inflammation in acute lung injury and RDS (6, 21, 25). In this study, we questioned whether this approach was effective in an immature animal model of RDS, with a native SF and endogenous CC10 deficiency, and whether this effect was dose dependent.

SF replacement therapy results in improved oxygenation through various mechanoprotective mechanisms, including reduction in surface tension and pressure requirements to inflate the lung, thus improving lung stability (8–10, 24, 25). We found that treatment with SF and rhCC10 resulted in a rhCC10 dose-dependent improvement in compliance, reduction in ventilatory pressure, and improvement in the VEI, with the greatest effects noted following treatment with SF + 5.0 mg/kg rhCC10 compared with SF alone. These findings suggest that rhCC10 imparts additional protection to the lung compared with the primarily mechanoprotective attributes of SF replacement therapy alone. There are several possible explanations for the rhCC10 dose-dependent improvements. Consistent with previous reports of the direct effect of decreased chemotactic activity of defense cells and anti-inflammatory properties of CC10 (13, 38), rhCC10 decreased the proinflammatory cytokine and MPO profile in the SF-treated preterm lung. This effect could mitigate alveolar-capillary membrane leak and subsequent mechanical instability. Additionally, as rhCC10 inhibits secreted PLA2, which is upregulated and released during an inflammatory process and hydrolyzes a major component of SF (dipalmitoyl phosphatidylcholine), the dose-dependent improvement in compliance is consistent with a more functional and/or greater SF pool size, either of which would be supported by the effect of rhCC10 to inhibit inflammatory-related secreted PLA2 release/activation and subsequently preventing SF degradation (20, 22). Whether by directly attenuating local inflammation or indirectly by preventing SF degradation, the rhCC10 dose-dependent improvement in lung stability and reduction in ventilatory requirements is consistent with a lung protective strategy to foster development of the immature lung.

Few studies have examined the effects of SF replacement therapy on inflammation. In a study of commercial SFs, Ikegami and Jobe (14) found higher proinflammatory mRNA levels in the lung with SF treatment compared with nonventilated controls, but they found no difference between the different SFs. SF treatment in preterm humans has been shown to increase neutrophil (35) and complement activation (39) and, in animal models of lung injury, has been shown to result in increased inflammatory cytokine release (33, 36, 41). In contrast, in the present study, treatment with SF + rhCC10 resulted in lower arterial plasma IL-8 concentration compared with SF replacement therapy alone. Additionally, compared with preterm lambs treated with SF alone, SF augmented with at least 1.5 mg/kg rhCC10 resulted in lower inflammatory cytokine (TNF-{alpha}, IL-6, IL-8, and MPO) levels in the lung tissue. The anti-inflammatory properties observed for rhCC10 in this study are consistent with those recently reported from tracheal aspirants collected over the first 3 days of life from human SF-treated preterm neonates treated with a single, 5 mg/kg dose of rhCC10 (21). Taken together, these studies suggest that early intervention with rhCC10 may mitigate inflammatory processes associated with ventilation of the immature lung. Furthermore, as the results of this study show, with the presence of rhCC10 as well as reduced levels of inflammatory cytokines in the systemic circulation, the effects of intratracheally administered rhCC10 extend beyond the lung. Mechanistically, we speculate that intratracheal delivery of rhCC10 allows the anti-inflammatory protein to pass from the alveolar space into the interstitium for local effects, as well as through the alveolar-capillary membrane into the pulmonary and systemic blood vessels, where it can modulate inflammation.

Arterial plasma, BAL, urine, and lung rhCC10 concentration increased with rhCC10 dose. Additionally, lambs treated with 5.0 mg/kg rhCC10 had higher concentrations of rhCC10 in the plasma, BAL, and lung. While comparable reduction in inflammation was noted at doses of 1.5 and 5.0 mg/kg, coupled with the significant improvements in the physiology and higher lung capacitance of rhCC10, the data suggest that the higher dose may afford more prolonged anti-inflammatory protection. This dose allows for a significant amount of the agent to enter into the circulation to reduce systemic inflammation while providing a reservoir of rhCC10 in the lung to control pulmonary inflammation, prevent SF degradation, and replenish the circulating rhCC10 lost through urinary excretion. As such, intratracheal delivery of rhCC10 incorporates dual mechanisms to downregulate inflammation: 1) by direct delivery to the target organ of ventilator-induced inflammation; and 2) secondarily, to the systemic circulation. Further studies are warranted to assess the impact of exogenous rhCC10 on the endogenous protein and gene expression.

Compared with SF therapy alone, intratracheal rhCC10-augmented SF therapy decreased RDS-induced lung and systemic inflammation by combining the benefits of mechanoprotection and cytoprotection. To the degree that inflammation may lead to functional compromise and that anti-inflammatory defense mechanisms are deficient in prematurity, these data suggest that early intervention with rhCC10 may augment SF therapy and warrant studies of longer duration to clarify its role to decrease long-term complications associated with ventilator management of RDS.


   GRANTS
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The authors gratefully acknowledge Claragen, Inc. (College Park, MD), Ross Laboratories (Columbus, OH), Sigma Xi Society, American Heart Association, and National Institutes of Health Grants HL-67612 and 1 P20 RR-020173–01 for support of this study.


   DISCLOSURES
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REFERENCES
 
R. W. Welch and A. L. Pilon are consultants to Claragen, Inc., have Claragen stock options, and have patents pending.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. R. Wolfson, Dept. of Physiology, Temple Univ. School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140 (e-mail: Marla.wolfson{at}temple.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 RESULTS
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