INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Asthma is a chronic inflammatory disease characterized by eosinophilic airway inflammation and hyperresponsiveness (AHR). Recent studies have emphasized that T lymphocytes and eosinophils are critical effector cells in the production of airway inflammation in asthma (1). In allergic asthma, activated T lymphocytes within airways express a T helper cell type 2 (Th2)-like profile of cytokines, including increased secretion of interleukin-4 (IL-4), IL-5, IL-6, and IL-10. Interleukins-4 and 5 in particular have been strongly implicated in generating and perpetuating the late-phase asthmatic response, including recruitment of activated eosinophils into airways, AHR, and airflow obstruction (4).

Epithelial and submucosal eosinophilia have long been recognized as a pathologic feature in fatal asthma in humans. The degree of peripheral blood, bronchoalveolar lavage (BAL), and tissue eosinophilia has been correlated with the clinical severity of asthma (7). Additionally, improvement in asthma symptoms after steroid treatment is associated with the resolution of airway eosinophilia (8). Although a causal relationship between eosinophilia and AHR has not been conclusively demonstrated, the potential role of eosinophil cells in the pathogenesis of human asthma continues to be the subject of intense investigation (8).

Interleukin-5 is believed to be an especially important mediator in orchestrating the eosinophilic inflammatory response. Indeed, IL-5 enhances proliferation, migration, activation, and survival of eosinophils (13). The importance of IL-5 has been inferred, in part, from studies describing increased IL-5 messenger RNA (mRNA) expression and increased amounts of this cytokine in BAL fluid of asthmatic patients after allergen inhalation challenge (16, 17). A recent study also showed that AHR and histologic changes that are pathognomonic of human asthma develop in transgenic mice that constitutively overexpress IL-5 within lung epithelium (18). Further evidence of the potential importance of IL-5 in asthma comes from animal studies in which treatment with IL-5 induces AHR, and from results of studies using genetically IL-5-deficient mice that fail to develop bronchial eosinophilia or AHR after systemic sensitization and challenge (4). However, additional prior investigations to determine the effects of preventing eosinophilia and AHR with an anti-IL-5 antibody have had conflicting results (10, 19). Nevertheless, these observations have sparked great interest in the potential therapeutic role of an anti-IL-5 antibody as a strategy to prevent eosinophil accumulation and activation in the airways of human asthmatic patients.

Testing the hypothesis that an anti-IL-5 antibody may reduce established pulmonary eosinophilia and AHR has been limited by the lack of an animal model that exhibits these pathologic changes in airway structure and function for a suitably long duration. Therefore, studies using murine asthma models have necessarily initiated treatment during some phase of the antigen sensitization or challenge period, and then evaluated the effects of these interventions on the development of eosinophilia or AHR (10, 19). However, asthma is a chronic inflammatory airway disorder and clinical symptoms generally follow an immediate or late-phase response in the context of chronic inflammatory airway disease. We reasoned that a closer reproduction of the clinical setting, i.e., established inflammation and AHR in an animal model might more closely mimic the human disorder. Additionally, treatment during established disease in this model would also more closely mimic the period of time that is the target for therapeutic intervention in the treatment of human asthma.

We report that TRFK-5 administered to mice after disease is established reversed existing airway eosinophilic inflammation but did not inhibit established AHR. In contrast, dexamethasone (DEX)-treated mice demonstrated a profound reduction in airway eosinophilia and complete reversal of previously established AHR.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Six to 10-wk-old female C57BL/6 mice were purchased from Harlan Sprague-Dawley and housed in a specific pathogen-free facility maintained by the University of Chicago Animal Resources Center. The studies reported here conform to the principles outlined by the Animal Welfare Act and the National Institutes of Health (NIH) guidelines for the care and use of animals in biomedical research.

Schistosoma mansoni Eggs and Antigen

S. mansoni eggs were isolated and purified soluble egg antigen (SEA) was produced as previously described (22). Eggs were stored at 70° C in 1.7% saline before use. To prepare SEA, eggs were homogenized on ice in a Tenbroeck tissue homogenizer. After centrifugation at 106 g for 2 h, the protein content of the recovered aqueous fraction was determined (BioRad, Richmond, CA). SEA was stored at 70° C until used.

Antigen Sensitization and Challenge

Our protocol extends a sensitization and challenge procedure we have previously reported (23). Mice were immunized intraperitoneally with 5,000 isolated S. mansoni eggs in 0.4 ml saline at Day 0. On Day 7 mice received an intranasal challenge of 10 µg of SEA in 20 µl of phosphate-buffered saline (PBS). Mice (n = 14) were rechallenged intratracheally on Day 14 with 10 µg SEA in 20 µl of PBS. (These mice are referred to as "SCH".) A 1-cm midline incision was made in the ventral neck region, and the tissues overlying the trachea were gently separated. Tracheal injection was performed with a 30-g needle. The incision was closed with a single drop of nexabond glue (superglue). Animals were allowed to recover in a warmed humidified incubator. Anesthesia for this procedure was achieved by intraperitoneal injection of 0.3 to 0.5 ml of a solution of 9.2 ml PBS, 0.65 ml ketamine HCl, and 0.22 ml xylazine HCl. These animals were subsequently sham-treated with saline. Negative control animals (control, n = 8) were sensitized with S. mansoni eggs as described previously, and challenged with saline instead of SEA.

Treatment with TRFK-5 and Dexamethasone

TRFK-5 (n = 12) or DEX-treated mice (n = 12) were sensitized and challenged in an identical manner as the SCH group. However, beginning on Day 4 (see Day +4 in study design below) after tracheal challenge with SEA the experimental groups were injected with either TRFK-5 (4 mg/kg intraperitoneally in 0.4 ml saline) or DEX (2 mg/kg intraperitoneally in 0.4 ml saline) every day until killed at Day 10 (see Day +10 in study design) posttracheal challenge. Additionally, to demonstrate the sensitivity to TRFK-5 in this model to inhibit the development of airway eosinophilia we treated 12 additional mice with TRFK-5 (4 mg/kg intraperitoneally in 0.4 ml saline) every other day beginning at Day 0 until they were killed at Day 24.

Selection of Time Points for DEX/TRFK-5 Treatment

We chose to study Days +4 through +10 (overall Day 18 to 24; see study design above) for the treatment period with either DEX or TRFK-5. We have previously shown that Day +4 mice developed eosinophilic airway inflammation and AHR, and a Th2-like pattern of cytokine secretion (increased IL-4 and IL-5, decreased interferon gamma [IFN-] in BAL and in vitro from cultured lung lymphocytes) (23). In pilot studies we found that these changes persisted for a further 8 to 10 d before declining by Days +12 to +14 (overall Day 26 to 28 after tracheal challenge with SEA). Thus, the interval including Days +4 through +10 consistently produced persistent eosinophilic airway inflammation and AHR. We reasoned that we could treat mice from Day +4 until Day +10 to test the hypothesis that inhibition of IL-5 might reverse established eosinophilic inflammation and also reduce established AHR in this model.

Ventilation and Instrumentation

When killed (Day +4 or Day +10), mice were anesthetized as described previously. The trachea was cannulated with a 20-g, 1-cm metal needle. The jugular vein was isolated and cannulated with P-10 tubing. The mouse was placed into a plexiglass volume plethysmograph and ventilated with 100% oxygen at 140 breaths/min, tidal volume 0.23 to 0.3 ml (0.10 ml/kg body weight; 0.23 to 0.3 ml was much greater than the equipment dead space, which was 40 µl). Vecuronium HCl was administered (0.1 mg/kg, intravenously) to inhibit spontaneous respirations. This paralytic agent was specifically chosen because it does not induce mast cell degranulation.

Measurement of Airway Reactivity

We used a constant volume, variable pressure whole body plethysmograph (230 ml displacement; Penn-Century, Philadelphia, PA) and Honeywell Microswitch solid-state pressure transducers to measure tidal volume excursions and transpulmonary pressure (Ptp = tracheal cannula pressure minus box pressure, with an open-chest animal). Including the PE-10 tracheal cannula, equipment dead space is only 40 µl, and equipment resistance is approximately 2.0 cm H₂O/ml/s. Ptp and volume excursions were recorded digitally (500 Hz each), flow was derived by digital differentiation of the volume signal (Mouse PRC Software; Lakeshore Technologies, Chicago, IL). Lung resistance (RL) was calculated from these signals breath by breath, by the method of Amdur and Mead (24) after subtracting equipment resistance.

Increasing doses of methacholine (MCh) (14, 133, 1,200 µg/kg) were infused through the jugular vein catheter at approximately 1-min intervals, at the peak of the response to the preceding dose. This protocol is a modification of the method of Martin and coworkers (25). We have previously shown that this schedule of MCh administration is sufficient to determine differences in airway reactivity between groups, and minimizes the mortality found when higher doses of MCh are used (23).

Bronchoalveolar Lavage

BAL was performed by instilling 0.8 ml of ice-cold PBS through the tracheal cannula, followed by gentle aspiration. This was repeated three additional times. Fluid from all four lavages was pooled. Cells were stained with trypan blue to determine viability, and total nucleated cell counts were established using a Neubauer hemocytometer. Cytocentrifuge preparations were made using a cytocentrifuge (Shandon Southern Instruments, Sewickley, PA) set for 700 × g for 5 min. Cytospin slides were fixed and stained using Diff-Quik (American Scientific Products, McGaw Park, IL). Differential cell counts were determined by counting a minimum of 300 cells/slide, using standard morphologic criteria.

ELISA for Determination of IFN- in BAL Fluid

IFN- in BAL fluid was measured using a commercially available ELISA kit (IFN-; Endogen, Cambridge MA). The lower limit of detection for IFN- was 0.3125 ng/ml.

Histology

Lungs from mice randomly chosen from all groups were removed from the chest cavity and fixed by injection of 10% buffered formalin (1.0 ml) into the tracheal cannula at a pressure of 20 cm H₂O, and immersed in formalin for 24 h. All lobes were sagittally sectioned, embedded in paraffin, cut in 5-µm sections, and stained with hematoxylin and eosin for routine analysis. Additional sections were stained with periodic acid-Schiff (PAS) to identify mucin in epithelial goblet cells and submucosal glands. Goblet cell hyperplasia was graded using a modification of a semiquantitative scoring scheme (0 = none, 1+ = minimal, 2+ = mild, 3+ = moderate, 4+ = marked) that we have previously reported (23, 26).

Statistical Methods

Differences between groups for BAL eosinophils, IFN- content, and for airway reactivity to MCh were determined by analysis of variance (ANOVA). Histologic differences between groups for tissue eosinophilia and numbers of goblet cells in airway epithelium were evaluated with the Mann-Whitney rank sum test. Statistical analyses for all tests were performed using a mean value for each animal. All data are expressed as mean ± SE. Statistical significance was claimed when p  0.05. The number of animals per group was determined by power analysis; the power of each test was established at 0.80 (27).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

BAL Eosinophilia

Although eosinophils were not recovered from BAL fluid of any of the control mice (Figure 1), there were significant numbers of eosinophils (eos) in BAL fluid from SCH mice at Day +4 (583 ± 73 eos/µl BAL fluid, p < 0.0001 versus control). At Day +10 there continued to be a significant BAL eosinophilia (382 ± 60 eos/µl BAL fluid, p < 0.01 versus control). Despite a large degree of variability in the absolute number of eosinophils recovered from BAL of individual mice, eosinophils still represented 73% of all BAL cells recovered in the SCH animals on Day +10. Analysis of the DEX- and TRFK-5-treated groups revealed a much different profile. There was a dramatic reduction in the absolute number of eosinophils recovered in BAL from animals treated with either DEX (120 ± 29 eos/µl, p < 0.01 versus Day +10) or TRFK-5 (86 ± 32 eos/µl, p < 0.01 versus Day +10). Additionally, eosinophils represented only 52 ± 5% of the total cells recovered in DEX-treated mice (p = 0.01 versus Day +10) and 40 ± 3% of the total cells recovered in TRFK-5-treated mice (p < 0.001 versus Day +10). Interestingly, TRFK-5-treated animals had a relative lymphocytosis in BAL fluid that was significantly increased compared with DEX-treated mice (22 ± 3% lymphocytes/µl BAL fluid versus 9 ± 2% lymphocytes/µl BAL fluid, p < 0.01, Table 1). These results demonstrate that treatment with either DEX or TRFK-5 can dramatically reverse established eosinophilic inflammation in S. mansoni sensitized and challenged mice.

View larger version (31K): [in this window] [in a new window]   Figure 1.   Pre-existing BAL eosinophilia is reduced threefold in mice treated with either DEX or TRFK-5. BAL was performed through a previously placed endotracheal tube. Four 0.8-ml aliquots of normal saline were infused, gently aspirated and pooled. No eosinophils were recovered in BAL from the control group. There were 382 ± 60 eos/µl BAL fluid recovered from the Day +10 group representing 72% of all BAL cells recovered. In contrast, eosinophil recovery from mice treated with DEX or TRFK-5 was significantly reduced and represented only 52% and 40% of total BAL cells recovered in these groups, respectively. BAL eosinophilia from TRFK-5- and DEX-treated mice was equivalent. Bars represent the mean and SE of each group. *p < 0.01 for both groups versus Day +10.

TRFK-5 treatment beginning at Day 0 completely ablated the development of BAL eosinophilia seen in nontreated animals (1 ± 1 eos/µl BAL fluid, TRFK-5 treated). These results confirm in this model prior studies demonstrating the effects of TRFK-5 to inhibit the development of airway eosinophilia in other antigen-sensitized and airway-challenged animal models. However, TRFK-5 treatment given beginning at Day 0 was also associated with increased numbers of lymphocytes (13 ± 3%, p < 0.001 versus control or SCH) and neutrophils (26 ± 4%, p < 0.001 versus control or SCH) in BAL fluid (Table 2). Thus, while anti-IL-5 antibody treatment successfully inhibited the development of eosinophilic airway inflammation, it may have unmasked a previously unrecognized mechanism for chemotaxis of additional inflammatory cells into airways. Additionally, these findings suggest a potential mechanism for the induction of AHR in TRFK-5-treated animals beginning on Day 0, in the absence of airway eosinophilia.

Airway Reactivity

Baseline lung resistance of mice in all antigen-sensitized and challenged groups was equivalent (range 0.801 to 0.918 cm H₂O/ml/s). These values were all significantly higher than baseline lung resistance of the control animals (0.593 cm H₂O/ ml/s, p < 0.05 versus each group). As seen in Figure 2, S. mansoni SCH mice demonstrated AHR to intravenously administered MCh at Day +4. These heightened responses were significantly increased at the 1,200 µg/kg dose compared with control (4-fold increase, p < 0.01). At Day +10 airway reactivity to MCh was augmented further compared with Day +4 (p < 0.04). DEX therapy from Day +4 through Day +10 resulted in a profound reversal of airway hyperresponsiveness compared with untreated SCH mice (p < 0.01 versus Day +10). Importantly, for DEX-treated mice, airway reactivity to MCh was equivalent to control animals. However, TRFK-5-treated animals continued to exhibit AHR similar to sensitized and challenged mice (p = ns versus Day +10; Figure 2). Mice treated with TRFK-5 beginning at Day 0 also developed airway responses to MCh (1,200 µg/kg) that were significantly increased compared with control animals and equivalent to SCH mice (13.1 ± 2.0 cm H₂O/ml/s versus 3.8 ± 0.4 cm H₂O/ ml/s for control, p < 0.02, and 11.6 ± 1.2 cm H₂O/ml/s for SCH Day 4, p = ns). These results suggest that factors in addition to IL-5 and/or eosinophilia are sufficient for both the induction and maintenance of AHR observed in this animal model of atopic asthma.

View larger version (38K): [in this window] [in a new window]   Figure 2.   DEX but not TRFK-5 treatment reverses established airway hyperresponsiveness. Anesthetized and paralyzed mice were intubated, ventilated, and placed in a whole body volume plethysmograph. Increasing doses of MCh were infused at 1-min intervals through an indwelling jugular catheter. Mice treated with DEX (2 mg/kg intraperitoneally daily from Day 4-10 after antigen challenge) had airway responses that were equivalent to control animals. These responses were significantly reduced compared with Day +10 mice. Mice treated with TRFK-5 (4 mg/kg intraperitonally daily from Days 4 through 10 after antigen challenge) had responses to MCh that were equivalent to Day +10 animals. RL is the absolute value in response to 1,200 µg/kg MCh. Resistance of the system was subtracted from all measurements. *p < 0.04 versus Day +4, **p < 0.01 versus Day +10.

Cytokine Content in BAL Fluid

We wished to determine the effect of DEX and TRFK-5 on production of IFN- because (1) IFN- has been implicated in the development of AHR in a murine model of asthma (12), (2) IFN- is critically important in Th1/Th2 differentiation, and (3) IFN- is secreted from T lymphocytes and could be used as a marker to determine if TRFK-5 had effects on T lymphocytes distinct from the single predicted effect of TRFK-5 to neutralize IL-5. We found that at Day +10, IFN- content in SCH mice was 1.36 ± 0.13 ng/ml BAL fluid. This was significantly increased compared with Day +4 (0.87 ± 0.1 ng/ml BAL fluid, p < 0.05) and equivalent to IFN- content of BAL fluid from mice treated with TRFK-5 (1.50 ± 0.24 ng/ml, p = ns). In contrast, the DEX group had only 0.49 ± 0.09 ng IFN-/ml BAL fluid (p < 0.001 versus Day +10 or TRFK-5) (Figure 3). The volume of recovered BAL fluid was equivalent between groups, and ranged from 2.5 to 2.8 ml. Thus, DEX treatment had a significant effect to reduce production of IFN-, whereas TRFK-5 treatment did not affect production of this cytokine. These results strongly suggest that the effect of TRFK-5 to reduce tissue eosinophilia was a specific effect of IL-5 neutralization but not inhibition of cytokine production.

View larger version (40K): [in this window] [in a new window]   Figure 3.   DEX, but not TRFK-5 inhibits IFN- production in BAL fluid. Cell-free BAL fluid from antigen-sensitized mice was collected 4 or 10 d after antigen challenge in trachea. Treatment with TRFK-5 from Days 4 through 10 had no effect on IFN- secretion compared with sham-treated mice (Day +10). In contrast, DEX treatment from Days 4 through 10 resulted in a significant decrease in IFN- content in BAL fluid; this was equivalent to IFN- content in BAL fluid from sham-challenged control animals. *p < 0.001 versus Day +10 or TRFK-5.

Histology

Airways from Day +10 mice had consistent pathologic changes in airway epithelium and submucosa. Specifically, eosinophilic infiltration within epithelium and lamina propria and goblet cell hyperplasia was observed in many airways from all Day +10 mice but was never seen in control airways. Eosinophilic infiltration and mucus cell hyperplasia were significantly reduced in DEX-treated mice, but only the eosinophilic infiltrate was dramatically reversed in the TRFK-5-treated animals. For example, both DEX- and TRFK-5-treated animals had significant reductions in mean scores for eosinophilia within peribronchial spaces compared with Day +10 animals (1.4 ± 0.2 versus 3.3 ± 0.3, p < 0.01 DEX versus Day +10; 1.75 ± 0.3 versus 3.3 ± 0.3, p < 0.05 TRFK-5 versus Day +10; p = ns for DEX versus TRFK-5 animals). However, while DEX-treated mice had a pronounced decrease in goblet cell hyperplasia (1.2 ± 0.3 versus 2.6 ± 0.2 for Day +10, p < 0.01), TRFK-5 treatment did not significantly reduce established goblet cell hyperplasia (2.0 ± 0.3 for TRFK-5 versus 2.6 ± 0.2 for Day +10, p = ns). Thus, treatment with either DEX or   TRFK-5 reduced established airway tissue eosinophilia, whereas DEX alone had a therapeutic effect on established goblet cell hypertrophy in these same animals (Figures 4 and 5).

View larger version (82K): [in this window] [in a new window]   Figure 4.   S. mansoni-sensitized mice develop chronic airway inflammatory changes 10 d after intratracheal antigen challenge. Lungs from mice from all groups were fixed in situ with 10% formalin for 24 h, processed routinely, and stained with PAS to determine differences in goblet cell development. (A) Lung from SCH mouse, 10 d after SEA challenge into the trachea (Day +10). There is a marked, predominantly eosinophilic infiltration with edema around airways, with extension into the adjacent alveolar parenchyma. Goblet cell hyperplasia is extensive. Hematoxylin- eosin (H + E) stain, original magnification: ×100. (B) Higher power magnification of B, original magnification: ×200. (C ) High-power magnification of airway epithelium from A. Plasma cells (arrows) can be seen within the primarily eosinophilic infiltrate, further demonstrating the chronicity of the inflammatory change. H + E stain, original magnification: ×400.

View larger version (88K): [in this window] [in a new window]   Figure 5.   DEX or TRFK-5 reverses established eosinophilic airway inflammation; only DEX reverses established goblet cell hyperplasia. (A) Lung from Day +10 animal treated with DEX from Days 4 through 10. Note absence of cellular infiltrate surrounding airways or within adjacent lung parenchyma (compare with Figure 4A). H + E stain, original magnification: ×100. (B) Higher power magnification of A. Airway epithelium is normal. H + E stain, original magnification: ×400. (C ) Lung from Day +10 animal treated with TRFK-5 from Days 4 through 10. Note relative absence of cellular inflammatory response. However, airway epithelium appears thickened. H + E stain, original magnification: ×200. (D) Higher power magnification of airway from animal in C. Goblet cells are increased in size and number (compare with B). H + E stain, original magnification: ×400.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study we describe a murine model of atopic asthma in which antigen sensitized mice exhibit airway inflammation, eosinophilia, and AHR for nearly 2 wk after local antigen challenge into the airways. Using this model, we tested the hypothesis that an anti-IL-5 antibody (TRFK-5) given after disease is established may be therapeutic by reducing eosinophilic inflammation and therefore reversing AHR. We studied the changes in cellular response, histology, and airway physiology resulting from treatment with TRFK-5. We compared these changes with the effects of corticosteroid administration. We chose DEX as the pharmacologic "gold standard" for comparison with the results of TRFK-5 treatment because glucocorticoids are the most widely used drugs in the treatment of the late-phase inflammatory response in asthma, and are the therapeutic agents most consistently associated with a decrease in both airway inflammation and AHR. We found that both TRFK-5 and DEX reduced established eosinophilia to a similar degree. However, only DEX but not TRFK-5 reversed established AHR.

We studied S. mansoni-sensitized and airway-challenged C57BL/6 mice because these animals develop airway eosinophilia, AHR, release IL-4 and IL-5 in airways, and secrete large amounts of IL-4 and IL-5 from stimulated lung T lymphocytes (23). Additionally, C57BL/6 mice were chosen because the induction of eosinophilia in this strain is thought to be IL-5-dependent (4, 28). Thus we were able to study the effects of treatment with an anti-IL-5 antibody on previously established eosinophilic airway inflammation in an animal model of asthma for which IL-5 is an important mediator. The pathological changes seen in our model, i.e., increased goblet cell hyperplasia, epithelial and submucosal infiltration with eosinophils and plasma cells, are consistent with chronic changes characteristically seen in human asthma. These are similar to the changes observed by Lee and coworkers (18) in transgenic mice that overexpress IL-5 in lung epithelium. Our observations thus further corroborate that IL-5 is an important mediator in the generation of airway inflammation and that the histologic features in our model parallel some important changes seen in human asthma. Importantly, airway eosinophilia and hyperresponsiveness persists for almost 2 wk after a single antigen challenge in this model. Thus, we chose the Day +4 through Day +10 time interval to evaluate the effects of DEX and TRFK-5 treatment in established disease on the further development of pathologic changes in airway structure and function.

To our knowledge there are only two prior published reports that examined the effect of modulating previously established murine airway eosinophilic airway inflammation. In one prior study, treatment with TRFK-5 at Day 5 after antigen challenge in ovalbumin (OVA)-sensitized mice reduced airway eosinophilia. In this study no additional effects were reported (29). In a second, more recent study, TRFK-5 given 24 h before antigen challenge to previously sensitized and challenged mice inhibited the increased BAL eosinophilia found in mice not treated with TRFK-5. However, the effect of reduced eosinophilia on airway reactivity was not reported (30). Therefore, while there are no equivalent animal studies investigating the relationship between eosinophils and AHR in the setting of established airway inflammation, our data are consistent with previous work in which intraperitoneal administration of alginate encapsulated IL-5-producing cells resulted in the development of AHR prior to the development of pulmonary eosinophilia in mice (10). Lilly and colleagues also reported in mice that airway infiltration with eosinophils could be induced by topical IL-5 application without the development of airway hyperresponsiveness (31).

It has more recently been formally suggested that eosinophilia is a phenomenon that is differentially regulated from AHR, and that perhaps IFN- instead is important in the development of airway hyperresponsiveness (12). We note that IFN- content in BAL fluid from animals in both SCH and TRFK-5 groups was equivalent, increased from Day +4, and significantly higher than the DEX and control groups. Although the present study was not designed to determine the effect of IFN- on AHR, our results are consistent with the report of Hessel and colleagues (12) that IFN- may be important in perpetuating the AHR found in the SCH and TRFK-5 groups in the present study.

The aims of the current study also did not include elucidation of potential mechanisms underlying the inhibitory effect of DEX on AHR. Importantly, baseline lung resistance at Day +10 in TRFK-5, DEX, and SCH groups was equivalent, thus differences in baseline airway caliber could not account for the reduction to baseline in airway responses to MCh in the DEX group. We speculate therefore that DEX was able to reduce AHR due to a multitude of anti-inflammatory effects, including the reduction in IFN- secretion noted above. Glucocorticoids inhibit eosinophil chemotaxis and adhesion and reduce eosinophil influx at the site of inflammation. They also increase eosinophil apoptosis (32). These effects are in part mediated by the inhibition of eosinophil-active cytokines such as IL-5 from T lymphocytes or eosinophils themselves (33). DEX can also affect cytokine production by Th2-type CD4+ cells both in in vitro cell cultures and in asthmatic patients (34, 35). Glucocorticoids also inhibit transcription factors such as NF-kappa B which regulates many of the genes that have an increased expression in patients with asthma, including chemokines (IL-8, RANTES, macrophage inhibitory protein-1 [MIP-1], monocyte chemoattractant protein-1 [MCP-1]), tumor necrosis factor- (TNF-), IL-1, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), inducible nitric oxide synthase, and adhesion molecules (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule [VCAM-1], E-selectin) (36). Other explanations for a reduction in both eosinophilia and AHR in DEX-treated animals besides a direct effect on cytokine production may be inhibition of phospholipase A2 activity in lung tissue and decrease in synthesis of arachidonic acid metabolites including the leukotrienes (37). Lastly, DEX-treated mice but not TRFK-5-treated animals had a significant reduction in goblet cell hyperplasia. Although the exact stimulus for mucus cell hyperplasia is not known, it is likely that a number of mediators found in asthmatic BAL fluid are potent secretogogues. Many of these same stimuli for mucus secretion are also inhibited by DEX, thus the effects of DEX on both eosinophilia and goblet cell hypertrophy in our model are consistent with known effects of DEX in humans with asthma. Importantly, mucus hypersecretion may cause airflow obstruction and in this manner increase reactivity to bronchoreactive agents including MCh. In contrast, TRFK-5 reduced eosinophilia but would not predictably have had an effect on other synergistic pathways that may play a role in development and/or maintenance of AHR. Our data therefore suggest that intense tissue eosinophilia may not be necessary to maintain established AHR, however increased mucus secretion may play a role in perpetuating already established AHR.

We note that TRFK-5 treatment resulted in increased numbers of BAL lymphocytes and neutrophils in mice treated from Day 0 compared with control or SCH mice. Mice treated with TRFK-5 from Day 4 through 10 also had a significant increase in lymphocytes recovered in BAL fluid compared with DEX-treated mice (Table 1). Airway neutrophilia and lymphocytosis is commonly found in horses with naturally occurring AHR (heaves) (38) and in experimental animal models of asthma (39). The potential role of T cells and products of T cells, including cytokines, in generating AHR has also recently been reviewed (40). Thus, increased number of inflammatory cells including lymphocytes in TRFK-5-treated mice compared with DEX-treated mice may partially explain the continued AHR in the TRFK-5, but not the DEX-treated group.

We recognize that one potential limitation to the interpretation of our data is our use of saline as a "sham" treatment control (instead of the relevant human Ig) for the TRFK-5-treated group. We and others have previously reported in murine studies of asthma, transplantation, and autoimmunity that the more "relevant" control, ie, Ig never had a significant effect by itself (19, 23, 41), and was equivalent to the (lack of) effect of saline as a negative control. Therefore, we feel confident that our data using saline instead of L6 or another irrelevant Ig in the SCH group can be reasonably interpreted compared with the TRFK-5-treated group.

In conclusion, we have described a reproducible murine model with chronic airway inflammation and AHR that mimics some of the defining features of human asthma. Using this model, we have demonstrated that once airway inflammation is established, antagonizing the effects of IL-5 and/or reducing eosinophilia alone are not successful in reducing existing AHR. These findings imply that targeting a reduction of eosinophils as a therapeutic goal in asthmatic patients may not result in diminished symptoms of asthma, and suggest that additional strategies to reduce structural inflammatory airway pathology including mucus cell hyperplasia and smooth muscle thickening may be more effective.