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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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The development of airway hyperresponsiveness (AHR) is correlated with the infiltration into the lungs of activated eosinophils and T lymphocytes. In large part, influx of eosinophils into the lung is dependent on very late activating antigen-4 (VLA-4) expression. However, the kinetics of eosinophil recruitment and the development of AHR are not fully delineated. Airway function was monitored by changes in lung resistance (RL) and dynamic compliance (Cdyn) to methacholine (MCh) inhalation after anti-VLA-4. After ovalbumin (OVA) sensitization and airway challenge of BALB/c mice, AHR increased as did the number of lung inflammatory cells. Administration of anti-VLA-4 to sensitized mice 2 h before the first (of three) OVA airway challenges significantly prevented changes in RL. Moreover, injection of the antibody from 2 h before the first challenge to 42 h after the last challenge significantly prevented the increases in RL, as well as eosinophil and lymphocyte numbers in the bronchoalveolar lavage fluid (BALF); interleukin-5 (IL-5) and leukotriene concentrations in BALF were also significantly inhibited. Interestingly, treatment with anti-VLA-4 only prevented changes in Cdyn and goblet cell hyperplasia when administered 2 h before the first challenge. These studies demonstrate that the timing of anti-VLA-4 administration can selectively affect pathologic processes that contribute to altered airway function in the central and peripheral airways after allergen challenge.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Bronchial asthma is characterized by allergen-induced airway hyperresponsiveness (AHR) and epithelial damage secondary to chronic airway inflammation. The development of AHR is correlated with the infiltration of eosinophils and activated T lymphocytes (1), thickening of the basement membrane of bronchiolar airways, and goblet cell hyperplasia of the epithelium (5). Eosinophils and T lymphocytes express the very late activating antigen-4 (VLA-4, CD49d/CD29) on their surface, whereas neutrophils do not (8). This integrin binds to the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin and may play an important role in the selective entry of eosinophils and T lymphocytes into inflamed tissues in asthma (12). Moreover, VLA-4 is an important accessory molecule in leukocyte activation and participates in T-cell responses to sensitization as a costimulatory signal (17, 18). Thus, inhibition of VLA-4 could theoretically prevent T-cell responses to antigen. VLA-4 monoclonal antibody (mAb) has been reported to inhibit the migration of eosinophils to inflammatory lesions and antigen-induced airway hyperreactivity in some animal models, including guinea pigs (19), rats (20), sheep (21), and mice (22), but these results have not always been consistent. Laberge and coworkers concluded that anti-VLA-4 inhibited allergen-induced AHR in rats but did not alter eosinophil accumulation in the lung (23). In contrast, Pretolani and coworkers reported that anti-VLA-4 inhibited both antigen-induced bronchial hyperreactivity and the infiltration of eosinophils into guinea-pig airways (19). Recently, Henderson and coworkers reported that intranasal administration of CD49d mAb in mice inhibited all signs of lung inflammation, interleukin-4 (IL-4) and IL-5 release, and hyperresponsiveness to methacholine (MCh) (24). Taken together, these data suggest that VLA-4 plays an essential role in eosinophil accumulation but its role in other aspects of antigen-induced responses is not clear.
In this study, we investigated the role of VLA-4 in allergen-induced lung function and eosinophil and lymphocyte infiltration in the airways and lung tissues in mice sensitized and challenged via the airways with ovalbumin (OVA). We carried out a detailed time-course analysis of the effects of anti-mouse VLA-4 mAb on airway function to inhaled MCh monitored by changes in lung resistance (RL) and dynamic compliance (Cdyn) as well as inflammatory cell recruitment, cytokine production, and leukotriene levels in the bronchoalveolar lavage fluid (BALF), and changes in epithelial cells. These studies demonstrate that VLA-4 is important in the accumulation of eosinophils in the allergic lung but the alteration of selective parameters of lung function was shown to be time-dependent.
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Animals
Female BALB/c mice from 8 to 12 wk of age were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were maintained on OVA-free diets. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center.
Sensitization and Airway Challenge
Mice (6-10 mice/group/experiment) receiving the following treatment were studied: airway challenge after nebulization of OVA alone (N group); intraperitoneal sensitization with OVA and OVA airway challenge (IPN group). Mice were immunized by intraperitoneal injection of 20 µg of OVA (Grade V; Sigma Chemical Co., St. Louis, MO) emulsified in 2.25 mg alum (AlumImuject; Pierce, Rockford, IL) in a total volume of 100 µl on Days 0 and 14. Mice were challenged via the airways by OVA (1% in saline) for 20 min on Days 28, 29, and 30 by ultrasonic nebulization (DeVilbiss, Somerset, PA; particle size 1 to 5 µm). RL and Cdyn were assessed 48 h after the last challenge, and the mice were killed to obtain tissues and cells for further assays.
Antibody Treatment
Rat anti-mouse VLA-4 monoclonal antibody, PS/2 (IgG2b) was used in this study (25). The rat mAb was purified from the hybridoma (American Type Culture Collection, Manassas, VA) with the use of a Protein G-Sepharose affinity column (Pharmacia, Uppsala, Sweden) under endotoxin-free conditions, and the mAb was used as purified IgG. Stock mAb was diluted with saline, then filter sterilized through a 0.22-µm filter (Millipore Co., Bedford, MA). Mice received a single intravenous injection (via the tail vein) of anti-VLA-4 or purified rat IgG2b (2 mg/kg) (as control) from 2 h before the first OVA challenge to 2, 12, 24, 42, or 47 h after the last airway challenge. There were no significant differences between OVA-challenged and sensitized mice with or without rat IgG2b treatment in any parameter tested.
Determination of Airway Resistance and Cdyn
Anesthestized, tracheostomized mice were mechanically ventilated and lung function was assessed using a modification of previously described methods (26, 27). Mice were placed in a plethysmograph; a four-way connector was attached to the tracheostomy tube (stainless steel cannula, 18-gauge), with two ports connected to the inspiratory and expiratory sides of two ventilators. Ventilation was achieved at a rate of 160 breaths/min, tidal volume of 150 µl with a positive end- expiratory pressure of 2 to 3 cm H2O by the ventilator (model 683; Harvard Apparatus, South Natick, MA). Transpulmonary pressure was detected by a differential pressure transducer with one side connected to the four-way connector and the other side connected to the plethysmograph. Changes in lung volume were measured by detecting pressure changes in the plethysmographic chamber through a port in the connecting tube with a pressure transducer and then referenced to the second copper gauze-filled 1.0-L glass bottle to stabilize the volume signal for thermal drift and microbarometric pressure changes. Flow was calculated by digital differentiation of the volume signal, and RL and Cdyn were continuously computed by fitting flow, volume, and pressure to an equation of motion (Lab-View; National Instruments, Austin, TX). Aerosolized MCh was administered for 10 breaths at a rate of 60 breaths/min, tidal volume of 500 µl by the ventilator (model SN-480-7-3-2T; Shinano Manufacturing Co., Tokyo, Japan) in increasing concentrations (1.56, 3.125, 6.25, 12.5 mg/ml) (27). After each aerosol MCh challenge, the data were continuously collected for 1 to 5 min and maximum values of RL and minimum values of Cdyn were taken to assess changes in these functional parameters. Baseline (exposure to saline) values as well as the response to MCh between the different groups showed little variation (Table 1).
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Bronchoalveolar Lavage (BAL) and Bone Marrow
After assessment of RL and Cdyn, lungs were lavaged via the tracheal tube with Hanks' balanced salt solution (HBSS, 1 × 1 ml then 1 × 0.4 ml, 37° C). Bone marrow was rinsed out of the right femur, washed, and resuspended in HBSS. The volume of collected BALF was measured in each sample and the number of BAL cells and bone marrow cells were counted by cell counter (Coulter Counter; Coulter Co., Hialeah, FL). Cytospin slides were stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) and differentiated in a blinded fashion by counting at least 300 cells under light microscopy.
Measurement of BALF Cytokines
Cytokine concentrations in the BALF supernatants were measured by ELISA as described (28). Cytokine levels were determined by comparison with the known standards. The limit of detection was 4 pg/ml.
Measurement of Total BALF Leukotrienes
BALF supernatant was loaded onto C-18 Sep-Pak (Varian, Palo Alto, CA); 80% methanol-water was used to rinse out the flask and this was added to Sep-Pak to elute leukotrienes. The samples were evaporated and redissolved in water prior to analysis. These samples were then used for ELISA analysis. Concentrations of leukotrienes were assayed using leukotriene enzyme immunoassay (EIA) kits (Cayman Chemical Co., Ann Arbor, MI) (29). The rabbit antiserum against leukotriene had the following cross reactivities: LTC4 (100%), LTD4 (100%), LTE4 (67%), and N-acetyl-LTE4 (10.5%), but not 5, 12, 15-hydroxyeicosatetraenoic acid (15-HETE), LTB4, 20-hydroxy LTB4, or prostaglandins (< 0.01%). The limit of detection was 12 pg/ml.
Histologic and Immunohistochemistry Studies
Lungs were inflated through the tracheal tube with 2 ml air and fixed in 10% formalin. Blocks of lung tissue were cut around the main bronchus and embedded in paraffin blocks. Tissue sections, 5 µm thick, were affixed to microscope slides, and deparaffinized. The slides were stained with hematoxylin-eosin and periodic acid-Schiff (PAS) for identification of mucus containing cells (28), and examined under light microscopy.
Cells containing major basic protein (MBP) in lung sections were identified by immunohistochemical staining as described using a rabbit anti-mouse MBP (provided by Dr. J. Lee, Mayo Clinic, Scottsdale, AZ) (28). The slides were examined in a blinded fashion with a Zeiss microscope equipped with a fluorescein filter system. Numbers of eosinophils in the peribronchial and perivascular tissue were analyzed using the IPLab2 software (Signal Analytics, Vienna, VA) for the Macintosh counting four different sections per animal (28).
The number of goblet cells in the airway epithelium was counted in at least 20 sections by measuring the length of epithelium defined as basement membrane and the luminal area using an NIH Image analysis system (30). Mucus-containing cells are expressed as the number of goblet cells per 100 µm epithelium.
Statistical Analysis
Values for all measurements are expressed as mean ± SEM. Student's t test was used to determine the levels of difference between two experimental groups. Analysis of variance (ANOVA) followed by Tukey-Kramer honest significant difference (HSD) test was used to compare changes in RL and Cdyn between different groups with the same treatment. ANOVA was used to compare percent changes of RL and Cdyn between different groups with the same treatment. The p values for significance were set at p < 0.05.
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RESULTS |
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DISCUSSION
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Anti-VLA-4 Inhibits Airway Resistance but Does Not Affect Cdyn if Administered Late
Mice received a single intravenous injection of anti-VLA-4 (or control rat IgG) 2 h before the first OVA challenge or from 2 h to 47 h after the last (third) challenge, and airway function was measured 48 h after the last allergen challenge. The increases in RL 48 h after the last allergen challenge were significantly suppressed not only when anti-VLA-4 was injected 2 h before the first OVA challenge, but also when administered 2, 12, 24, and 42 h after the last OVA challenge when compared with control mice (Figure 1A). Further, the reductions in RL were statistically significant between the groups when 12 and 24, 24 and 42, and 42 and 47 h were compared (Figure 1A). The antibody did not significantly affect RL values when injected 47 h after the last challenge. Similarly, treatment with anti-VLA-4 completely inhibited changes in Cdyn when administered 2 h before the first challenge. In contrast, anti-VLA-4 had no significant effect on Cdyn when administered from 2 to 47 h after the last challenge (Figure 1B).
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Anti-VLA-4 Inhibits Eosinophil and Lymphocyte Accumulation in BALF but Does Not Change the Number of Eosinophils in Bone Marrow
The number of inflammatory cells in BALF was determined 48 h after allergen challenge. Administration of anti-VLA-4 (but not control rat IgG) 2 h before the first OVA challenge and 2, 12, 24, and 42 h after the last challenge significantly suppressed the number of eosinophils and lymphocytes in the BALF (eosinophils: 92%, 94%, 90%, 82%, and 55% decrease; lymphocytes: 69%, 42%, 54%, 64%, and 55% decrease compared with control mice at 2 h prechallenge and 2, 12, 24, and 42 h postchallenge, respectively) (Figure 2). The antibody did not significantly suppress either eosinophil or lymphocyte numbers when given 47 h after the last challenge. Sensitization and subsequent challenge with OVA significantly increased the numbers of eosinophils in the bone marrow (from 0.5 ± 0.1 to 1.3 ± 106 cells/femur; p < 0.05) with no significant change in total cell counts. Treatment with anti-VLA-4 when injected 2 h before and from 2 to 47 h after challenge did not affect these increases (data not shown).
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Anti-VLA-4 Decreases IL-5 Concentrations in BALF
IL-5, IL-4, and interferon gamma (IFN-
) concentrations were
measured 48 h after the last OVA challenge by ELISA. After
sensitization and challenge, the increases in IL-5 production in
BALF supernatants were significantly suppressed by anti-VLA-4 when administered from 2 to 42 h after the last challenge (p < 0.05) (Figure 3). Control rat IgG2b had no effect.
Injection of the antibody decreased IL-4 production, but the
effects did not achieve statistical significance; IFN- production in BALF was unchanged by anti-VLA-4 treatment (Figure 3).
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Anti-VLA-4 Antibody Decreases Leukotriene Concentrations in BALF
Leukotriene levels in BALF were determined 48 h after the last challenge by ELISA. Allergen challenge of sensitized mice resulted in a 5-fold increase in the concentration of leukotrienes in BALF compared with nonsensitized mice. Administration of anti-VLA-4 (but not rat IgG2b) from 2 h before the first challenge to 42 h after the last challenge significantly (p < 0.05) decreased leukotriene production (Figure 4).
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Localization of Eosinophils and Lymphocytes in Lung Tissue
Evidence of inflammatory cell infiltration and the effect of anti-VLA-4 were further investigated through histologic examination of hematoxylin-eosin, anti-MBP, and PAS-stained lung sections. Intraperitoneal sensitization and subsequent challenge with OVA via the airways increased the number of eosinophils and lymphocytes in the peribronchial and perivascular tissue. In mice challenged alone, very few eosinophils or lymphocytes were detected in these sites. Examination of tissue sections showed that treatment with anti-VLA-4 (but not rat IgG2b) abolished eosinophil and lymphocyte infiltration in the peribronchial and perivascular area and the effects of the antibody could be seen even when injected for up to 42 h after the last challenge (Figure 5). Further, staining with anti-MBP revealed that the marked increase in eosinophils in the peribronchial and perivascular tissue after sensitization and challenge was suppressed by anti-VLA-4 when injected from 2 h before the first OVA challenge to 42 h after the last challenge (Figure 6).
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In addition, lung sections were stained with PAS to identify mucus-containing cells in the airway epithelium. A large number of cells staining positive for mucus were found in sensitized and challenged mice compared with mice that were challenged alone. Surprisingly, treatment with anti-VLA-4 only inhibited PAS staining when administered 2 h before the first challenge. Administration of the mAb after OVA challenge did not have any effect on goblet cell mucus production (Figures 7 and 8).
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DISCUSSION
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VLA-4 is expressed on the surface of eosinophils and T lymphocytes (8, 9) and plays an important role in the selective recruitment of these cells into and around the airways, resulting in chronic inflammation (12). However, the importance, particularly the kinetics, of VLA-4 blockade on eosinophil recruitment, inflammation, and components of AHR is unknown. To further delineate these relationships, we performed a time-course analysis of the effects of anti-VLA-4 on airway function monitored by changes in pulmonary resistance and dynamic compliance. In parallel, we examined inflammatory cell recruitment, cytokine production, and leukotriene concentrations in BALF, and changes in goblet cells. Airway challenge of sensitized BALB/c mice triggered marked increases in RL and a reduction in Cdyn to inhaled MCh in a dose-dependent manner. Airway eosinophilia in BALF and lung tissue, significant increases of eosinophils in the bone marrow, and increased concentrations of IL-4, IL-5, and leukotrienes in BALF were also demonstrated. Allergen-induced AHR and the accompanying inflammatory changes reached maximum levels 48 h after the last challenge.
Intravenous administration of anti-VLA-4 but not control rat IgG2b 2 h before the initial airway challenge significantly affected RL and Cdyn to inhaled MCh, as well as eosinophil accumulation in BALF. The early increase in lymphocyte numbers was also significantly reduced. Moreover, injection of the antibody from 2 h before the first challenge to 42 h after the last challenge significantly prevented, in a progressively decreasing manner, the increases in RL and eosinophil and lymphocyte infiltration in the airways without altering the number of eosinophils in the bone marrow 48 h after the last challenge. Similarly, IL-5 and leukotriene concentrations in BALF were also significantly inhibited at 48 h depending on the time of VLA-4 antibody administration. In contrast to these effects of anti-VLA-4, treatment with the antibody only prevented changes in Cdyn and mucus production when administered 2 h before the first challenge. Thus, despite the inhibition of eosinophil numbers in the BALF following treatment with anti-VLA-4 after the OVA challenges, changes in Cdyn or mucus production could not be prevented unless the VLA-4 antibody was administered before airway challenges were initiated. These studies reveal critical temporal differences in the control of allergen-induced changes in RL and changes in Cdyn. Such changes could reflect differences in airflow obstruction in the central and peripheral airways, as discussed subsequently, indicating that the timing of anti-VLA-4 administration selectively affects distinct pathologic processes in this model.
The mechanism underlying the effects of the VLA-4 antibody appears linked to the suppression of the recruitment of
eosinophils or lymphocytes, or both, into the airways (14). T
lymphocytes are one of the major populations of cells that accumulate at sites of allergic inflammation and express VLA-4
on their surface (11, 12). Pacheco and colleagues demonstrated that allergen stimulation upregulated CD49d expression on T cells derived from BAL of asthmatic patients in a
time-dependent manner, and was coordinated with expression
of human leukocyte-associated antigen-DR (HLA-DR), a
marker of T-cell activation (31). In the present study, the number of lymphocytes recruited into the airways and IL-5 production in BALF were significantly suppressed by administration of anti-VLA-4. Suppression of IL-4 production was also
observed but did not achieve statistical significance whereas
IFN- production was unaffected. In the present study, a major consequence of anti-VLA-4 in sensitized and challenged
mice was the profound reduction in eosinophil recruitment into the lung without affecting eosinophil expansion in the
bone marrow. Cumulatively, the findings suggest that VLA-4
takes part in allergen-induced pathophysiologic inflammatory
responses, likely by recruiting both eosinophils and T lymphocytes that express this adhesion molecule. Because both eosinophils and lymphocyte numbers were reduced by anti-VLA-4, it
is difficult to distinguish their relative roles in the development and suppression of AHR at the present time.
In contrast to these results, the effects of anti-VLA-4 on eosinophil recruitment and airway hyperreactivity in animal models have not always been consistent (19). Henderson and coworkers recently reported that intraperitoneal treatment with the same antibody used in this study prevented eosinophil recruitment into BALF but did not suppress allergen-induced airway hyperreactivity. Only intranasal administration of the antibody abrogated airway hyperreactivity as well as eosinophil and lymphocyte accumulation in BALF in their model, prompting the conclusion that CD49d-positive intrapulmonary leukocytes other than eosinophils are the critical effectors of allergen-induced airway hyperreactivity and lung inflammation (24). It is not presently obvious why such inconsistencies exist.
The reduction in eosinophil numbers was accompanied by a marked reduction in leukotriene concentrations in BALF. Anti-VLA-4 treatment from 2 h before the first challenge to 42 h after the last challenge significantly suppressed leukotriene levels in BALF. Moreover, examination of cells containing eosinophilic MBP identified by immunohistochemistry, indicated that the marked increase in eosinophils in the peribronchial and perivascular tissue after sensitization and challenge was also suppressed by anti-VLA-4, paralleling the findings for eosinophil and leukotriene concentrations in BALF. It is possible that allergen challenge of sensitized mice likely resulted in the recruitment and activation of eosinophils and that the eosinophils recruited into the airways released the leukotrienes, which could affect airway smooth muscle function, epithelial cell functions, and vascular permeability (29, 32). Furthermore, because leukotrienes in turn can influence eosinophil recruitment (32), recruited eosinophils may be triggered to release additional leukotriene, MBP, and IL-5 in an autocrine manner, thus further enhancing allergic lung inflammation. Thus, although the correlation between development of AHR and eosinophilic inflammation is not always linear, these findings confirm that both IL-5-dependent eosinophilic inflammation and the production of leukotrienes can play critical roles in the development of AHR. In support of this cascade is the study of Irvin and coworkers (35) which demonstrated marked reduction in lung eosinophilia and AHR in mice in which 5-lipoxygenase was genetically deleted.
A provocative finding in the present study was the dissociation of changes in RL and Cdyn after administration of anti-VLA-4. It has been proposed that changes in Cdyn reflect narrowing of peripheral airways; in contrast, changes in RL represent airflow obstruction of central airways (26, 36, 37). Anti-VLA-4 given from 2 h before the first challenge to 42 h after the last challenge significantly prevented increases in RL, but normalization of Cdyn was only observed when anti-VLA-4 was administered 2 h before the initial challenge. Furthermore, changes in Cdyn did not appear correlated with the number of eosinophils in BALF or the concentrations of IL-5 and leukotrienes in BALF. If these differences in Cdyn and RL truly reflect differences in peripheral and central airways, the data suggest different pathogenetic mechanisms contributing to airway dysfunction. Bronchial asthma is thought to be caused by epithelial damage arising from airway inflammation, which is characterized by the accumulation of activated eosinophils and T lymphocytes in the airways (1). Goblet cell hyperplasia has been described as one of the important findings in the airway epithelium of newly diagnosed asthmatics (38). The excessive production of mucus resulting from goblet cell hyperplasia leads to airway occlusion with plugs of mucus, exudation, and cell debris. Blyth and coworkers demonstrated that in a murine model of atopic asthma, extensive hyperplasia of airway goblet cells is induced by repeated OVA challenge of sensitized mice (39). In their extended protocol, goblet cell hyperplasia appeared before eosinophils and lymphocytes migrated across the airway epithelial layer (39). Cohn and coworkers (40) demonstrated that goblet cell hyperplasia could be induced by T cells in the absence of eosinophils. Using PAS staining to monitor mucus production, we saw similar increases in goblet cell staining after sensitization and challenge. As observed for Cdyn, treatment with anti-VLA-4 inhibited mucus production only when administered 2 h before the first challenge, whereas intravenous injection of anti-VLA-4 after allergen challenge was initiated had no effect on mucus production. How goblet cell hyperplasia/mucus production may be linked to Cdyn and not to RL is unclear at present but airway epithelial function may be a critical factor in the development of altered dynamic compliance.
Recently, Hojo and coworkers showed that anti-VLA-4
prevented early responses in the lung through inhibition of mast
cell activation (41). It is possible that resident (or recruited) lung inflammatory cells, which release tumor necrosis factor-alpha (TNF-
), IL-4 (or IL-13), and other mediators, play an
important role in initiating goblet cell hyperplasia and mucus
hypersecretion, even before eosinophilic infiltration. IL-4 (or
IL-13) may be essential for mucus production in sensitized and
challenged mice (28, 40). Increases in IL-4 production may
precede increases in IL-5 production (unpublished observations).
Taken together, the data may support the contention that AHR
is the result of more than one mechanism with IL-5-dependent
eosinophilic inflammation and leukotriene production causing
changes in the central airways whereas changes in the epithelium
of peripheral airways, including goblet cell hyperplasia, may
relate to changes in Cdyn. Peripheral, small airway function
has been underevaluated in asthma owing to the difficulty of
sampling and lack of specificity of physiologic measurements for
this parameter. In this regard, the study of Kaminsky and coworkers (42) is of note because significant changes in peripheral
lung function were present in asthmatics even when spirometry
was within normal limits. Another possibility relates to the timing
of cell trafficking to different inflammatory sites: an early parenchymal response is followed by a later airway response, potentially reflecting differences in parenchymal versus bronchial circulation. Additional studies are needed to evaluate the precise mechanism of specific aspects of lung function in animal models of asthma, as AHR measured solely as changes in RL may not
totally define airway function.
In summary, our findings demonstrate that, similar to human asthma, eosinophils and T lymphocytes accumulate in the bronchial mucosa, accompanied by AHR in allergen-sensitized and challenged mice. VLA-4 appears important in the selective accumulation and activation of eosinophils in the allergic lung. VLA-4 also appears to play a role in the triggering of goblet cell hyperplasia and mucus production. Because goblet cell hyperplasia can develop in the absence of eosinophils (40), this effect of VLA-4 antibody may be on the accumulation of lymphocytes. Furthermore, the timing of anti-VLA-4 administration can selectively affect pathologic processes that contribute to altered airway function in the central and peripheral airways. This investigation suggests that IL-5-dependent eosinophilic inflammation plays an essential role in the development of changes in RL, especially in the central airways, whereas rapid induction of goblet cell hyperplasia in the epithelium might be an important factor or marker in the development of altered dynamic compliance in this model. The further identification of the requirements for alteration of specific components of lung function has important implications for the treatment of the complex syndrome of asthma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Erwin W. Gelfand, M.D., National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: gelfande{at}njc.org
(Received in original form October 25, 1999 and in revised form February 23, 2000).
Acknowledgments: The authors are grateful to Dr. Z.-H. Cui for help in quantitating goblet cell numbers, Ms. Diana Nabighian for preparation of the manuscript, and Ms. Lynn Cunningham for her help in preparing the tissue slides.
Supported in part by a grant from the National Institutes of Health (HL-36577 to E.W.G. and HL-56638 to C.G.I.).
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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1. Bradly, B. L., M. Azzawi, M. Jacobson, B. Assoufi, J. V. Collins, A. M. A. Irani, L. B. Schwartz, S. R. Durham, P. K. Jeffery, and A. B. Kay. 1991. Eosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with relationship to bronchial hyperresponsiveness. J. Allergy Clin. Immunol. 88: 661-674 [Medline].
2. Azzawi, M., B. Bradley, P. K. Jeffery, A. J. Frew, A. J. Wardlaw, B. Assoufi, J. V. Collins, S. R. Durham, G. K. Knowles, and A. B. Kay. 1990. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am. Rev. Respir. Dis. 142: 1407-1413 [Medline].
3. De Monchy, J. G., H. F. Kauffman, P. Venge, G. H. Koeter, H. M. Jansen, H. J. Sluiter, and K. De Vries. 1985. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am. Rev. Respir. Dis. 131: 373-376 [Medline].
4. Weller, P. F.. 1994. Eosinophils: structure and functions. Curr. Opin. Immunol. 6: 85-90 [Medline].
5. Busse, W. W., W. F. Calhoun, and J. D. Sedgewick. 1993. Mechanism of airway inflammation in asthma. Am. Rev. Respir. Dis. 147: S20-S24 [Medline].
6. Roche, W. R., R. Beasley, J. H. Williams, and S. T. Holgate. 1989. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1: 520-524 [Medline].
7. Blyth, D. I., M. S. Pedrick, T. J. Savage, E. M. Hessel, and D. Fattah. 1996. Lung inflammation and epithelial changes in a murine model of atopic asthma. Am J. Respir. Cell Mol. Biol. 14: 425-438 [Abstract].
8. Smith, C. H., J. N. Barker, and T. H. Lee. 1993. Adhesion molecules in allergic inflammation. Am. Rev. Respir. Dis. 148: S75-S78 [Medline].
9.
Weller, P. F.,
T. H. Rand,
S. H. Goelz,
G. Chi-Rosso, and
R. J. Lobb.
1991.
Human eosinophil adherence to vascular endothelium mediated by binding to VCAM-1 and ELAM-1.
Proc. Natl. Acad. Sci. U.S.A.
88:
7430-7433
10. Walsh, G. M., J. J. Mermod, A. Hartnell, A. B. Kay, and A. J. Wardlaw. 1991. Human eosinophil, but not neutrophil, adherence to IL-1 stimulated human umbilical vascular endothelial cells in alpha 4 beta 1 (very late antigen-4) dependent. J. Immunol. 146: 3419-3423 [Abstract].
11.
Carlos, T. M.,
B. R. Schwatz,
N. L. Kovach,
E. Yee,
M. Rosso,
L. Osborn,
G. Chi-Rosso,
B. Newman,
R. Lobb, and
J. M. Harlan.
1990.
Vascular cell adhesion molecule-1 mediates lymphocyte adherence to
cytokine-activated cultured human endothelial cells.
Blood
76:
965-970
12. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301-314 [Medline].
13.
Carlos, T. M., and
J. M. Harlan.
1994.
Leukocyte-endothelial adhesion
molecules.
Blood
84:
2068-2101
14.
Lobb, R. R., and
M. E. Hemler.
1994.
The pathophysiologic role of 4
integrins in vivo.
J. Clin. Invest.
94:
1722-1728
.
15.
Masumoto, A., and M. E. Hemler. 1993. Mutation of putative divalent cation
sites in the 4 subunit of the integrin VLA-4: distinct effects on adhesion
to CS1/fibronectin, VCAM-1, and invasin. J. Cell Biol. 123L:245-253.
16. Elices, M. J., L. Osborn, Y. Takada, C. Crouse, S. Luhowskyj, M. E. Hemler, and R. R. Lobb. 1990. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 60: 577-584 [Medline].
17.
Damle, N. K., and
A. Aruffo.
1991.
Vascular cell adhesion molecule 1 induces T cell antigen receptor-dependent activation of CD4+ T lymphocytes.
Proc. Natl. Acad. Sci. U.S.A.
88:
6403-6407
18. Hynes, R. O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11-25 [Medline].
19.
Pretolani, M.,
C. Ruffie,
J. R. Lapa e Silva,
D. Joseph,
R. R. Lobb, and
B. B. Vargaftig.
1994.
Antibody to very late activation antigen 4 prevents antigen-induced bronchial hyperreactivity and cellular infiltration in the guinea pig airways.
J. Exp. Med.
180:
795-805
20. Richards, I. M., K. P. Kolbasa, C. A. Hatfield, G. E. Winterrowd, S. L. Vonderfecht, S. F. Fidler, R. L. Griffin, J. R. Brashler, R. F. Krzesicki, L. M. Sly, K. A. Ready, N. D. Staite, and J. E. Chin. 1996. Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized Brown Norway rats. Am. J. Respir. Cell Mol. Biol. 15: 172-183 [Abstract].
21.
Abraham, W. M.,
M. W. Sielczak,
A. Ahmed,
A. Cortes,
I. T. Lauredo,
J. Kim,
B. Pepinsky,
C. D. Benjamin,
D. R. Leone,
R. R. Lobb, and
P. F. Weller.
1994.
4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.
J.
Clin. Invest.
93:
776-787
.
22.
Nakajima, H.,
H. Sano,
T. Nishimura,
S. Yoshida, and
I. Iwamoto.
1994.
Role of vascular cell adhesion molecule 1/very late activation antigen
4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue.
J. Exp. Med.
179:
1145-1154
23. Laberge, S., H. Rabb, T. B. Issekutz, and J. G. Martin. 1995. Role of VLA-4 and LFA-1 in allergen-induced airway hyperresponsiveness and lung inflammation in the rat. Am. J. Respir. Crit. Care Med. 151: 822-829 [Abstract].
24.
Henderson, W. R.,
E. Y. Chi,
R. K. Albert,
S. Chu,
W. J. E. Lamm,
Y. Rochon,
M. Jonas,
P. E. Christie, and
J. M. Harlan.
1997.
Blockade of
CD49d (4 integrin) on intrapulmonary but not circulating leukocytes
inhibits airway inflammation and hyperresponsiveness in a mouse
model of asthma.
J. Clin. Invest.
100:
3083-3092
[Medline].
25.
Miyake, K.,
I. L. Weissman,
J. S. Greenberger, and
P. W. Kincade.
1991.
Evidence for a role of the integrin VLA-4 in lymphohemopoiesis.
J.
Exp. Med.
173:
599-607
26.
Martin, T. R.,
N. P. Gerard,
S. J. Galli, and
J. M. Drazen.
1988.
Pulmonary responses to bronchoconstrictor agonists in the mouse.
J. Appl.
Physiol.
64:
2318-2323
27.
Takeda, K.,
E. Hamelmann,
A. Joetham,
L. D. Shultz,
G. L. Larsen,
C. G. Irvin, and
E. W. Gelfand.
1997.
Development of eosinophilic airway
inflammation and airway hyperresponsiveness in mast cell-deficient
mice.
J. Exp. Med.
186:
449-454
28.
Tomkinson, A.,
A. Kanehiro,
N. Rabinovitch,
A. Joetham,
G. Cieslewicz, and
E. W. Gelfand.
1999.
The failure of STAT6 deficient mice to
develop airway eosinophilia and airway hyperresponsiveness is overcome by IL-5.
Am. J. Respir. Crit. Care Med.
160:
1283-1291
29. Szczeklik, A., K. Sladek, R. Dworski, E. Nizankowska, J. Soja, J. R. Sheller, and J. Oates. 1996. Bronchial aspirin challenge causes specific eicosanoid response in aspirin-sensitive asthmatics. Am. J. Respir. Crit. Care Med. 154: 1608-1614 [Abstract].
30. Cui, Z.-H., B.-E. Skoogh, T. Pullertis, and J. Lotvall. 1999. Bronchial hyperresponsiveness and airway wall remodeling induced by exposure to allergen for nine weeks. Allergy 54: 1074-1082 [Medline].
31.
Pacheco, K. A.,
M. Tarkowski,
J. Klemm, and
L. J. Rosenwasser.
1998.
CD49d expression and function on allergen-stimulated T cells from
blood and airway.
Am. J. Respir. Cell Mol. Biol.
18:
286-293
32. Munoz, N. M., I. Douglas, D. Mayer, A. Hernreiter, X. Zhu, and A. R. Leff. 1997. Eosinophil chemotaxis inhibited by 5-lipoxygenase blockade and leukotriene receptor antagonism. Am. J. Respir. Crit. Care Med. 155: 1398-1403 [Abstract].
33. Underwood, D. C., R. R. Osborn, S. J. Newsholme, T. J. Torphy, and D. W. P. Hay. 1996. Persistent airway eosinophilia after leukotriene (LT) D4 administration in the guinea pig. Am. J. Respir. Crit. Care Med. 154: 850-857 [Abstract].
34.
Weller, P. F.,
C. W. Lee,
D. W. Foster,
E. J. Corey,
K. F. Austin, and
R. A. Lewis.
1983.
Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of
leukotriene C4.
Proc. Natl. Acad. Sci. U.S.A.
80:
7626-7630
35.
Irvin, C. G.,
Y.-P. Tu,
J. R. Sholler, and
C. D. Funk.
1997.
5-lipoxygenase
products are necessary for ovalbumin-induced airway responsiveness
in mice.
Am. J. Physiol.
272:
L1053-L1058
36. Woolcock, A. J., N. J. Vincent, and P. T. Macklem. 1969. Frequency dependence of compliance as a test for obstruction in the small airways. J. Clin. Invest. 48: 1097-1106 .
37. Moy, M. L., and S. H. Loring. 1998. Compliance. Semin. Respir. Crit. Care Med. 19: 349-359 .
38. Jeffery, P. K., A. J. Wardlaw, F. C. Nelson, J. V. Collins, and A. B. Kay. 1989. Bronchial biopsies in asthma: an ultrastructural, quantitative study and correlation with hyperreactivity. Am. Rev. Respir. Dis. 140: 1745-1753 [Medline].
39.
Blyth, D. I.,
M. S. Pedrick,
T. J. Savage,
H. Bright,
J. E. Beesley, and
S. Sanjar.
1998.
Induction, duration, and resolution of airway goblet cell
hyperplasia in a murine model of atopic asthma: effect of concurrent
infection with respiratory syncytial virus and response to dexamethasone.
Am. J. Respir. Cell Mol. Biol.
19:
38-54
40.
Cohn, L.,
R. J. Homer,
H. MacLeod,
M. Mohrs,
F. Brombacher, and
K. Bottomly.
1999.
Th2-induced airway mucus production is dependent
on IL-4R, but not on eosinophils.
J. Immunol.
162:
6178-6183
41.
Hojo, M.,
K. Maghni,
T. B. Issekutz, and
J. G. Martin.
1998.
Involvement
of 4 integrins in allergic airway responses and mast cell degranulation in vivo.
Am. J. Respir. Crit. Care Med.
158:
1127-1133
42. Kaminsky, D. A., C. G. Irvin, D. A. Gurka, D. C. Feldsien, E. M. Wagner, M. C. Liu, and S. E. Wenzel. 1995. Peripheral airways responsiveness to cool, dry air in normal and asthmatic individuals. Am. J. Respir. Crit. Care Med. 152: 1784-1790 [Abstract].
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