Published ahead of print on May 28, 2003, doi:10.1164/rccm.200210-1148OC
American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 633-639, (2003)
© 2003 American Thoracic Society
Type 1 and Type 2 Cytokine Imbalance in Acute Respiratory Syncytial Virus Bronchiolitis
Julian P. Legg,
Imran R. Hussain,
Jill A. Warner,
Sebastian L. Johnston and
John O. Warner
Infection, Inflammation and Repair Division, University of Southampton, Southampton; and Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College of Science Technology & Medicine, London, United Kingdom
Correspondence and requests for reprints should be addressed to Julian Legg, M.A., M.B., B.Chir, M.R.C.P., Department of Child Health, Infection, Inflammation and Repair Division, University of Southampton, Southampton SO16 6YD, UK. E-mail: julianlegg{at}yahoo.co.uk
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ABSTRACT
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We examined the in vivo immune response of infants to natural respiratory syncytial virus (RSV) infection through analysis of cytokine levels in nasal lavage fluid and stimulated peripheral blood mononuclear cells. Eighty-eight babies with at least one parent with atopy and asthma were prospectively studied through their first winter. Twenty-eight infants had an upper respiratory tract infection where RSV was detected, of whom nine developed signs of acute bronchiolitis. Nasal lavage specimens were assayed for interferon- , interleukin (IL)-4, IL-10, and IL-12 and the RSV load determined by quantitative polymerase chain reaction. Messenger RNA (mRNA) was extracted from stimulated peripheral blood mononuclear cells and interferon-, IL-4, IL-12, and IL-18 mRNA levels determined by polymerase chain reaction. Cytokine profiles were analyzed in relation to clinical outcome. The IL-4/interferon- ratio for infants with acute bronchiolitis was elevated in nasal lavage fluid on both Days 1–2 (p = 0.014) and Days 5–7 (p = 0.001) of the illness compared with infants with upper respiratory tract infection alone. Those with acute bronchiolitis demonstrated a higher IL-10/IL-12 ratio (p = 0.0015) on Days 1–2. IL-18 mRNA levels were reduced (p = 0.019) and the IL-4/interferon- ratio elevated (p = 0.01) in stimulated peripheral blood mononuclear cells from infants with acute bronchiolitis. There was no difference in initial RSV load. These data strongly implicate excess type 2 and/or deficient type 1 immune responses in the pathogenesis of RSV bronchiolitis.
Key Words: respiratory syncytial virus infections • nasal lavage fluid • T helper type 1 cells • T helper type 2 cells • infant
Viral bronchiolitis is the leading cause for hospitalization of infants in the developed world and causes an estimated one million deaths per year worldwide (1, 2). Respiratory syncytial virus (RSV) is associated with the majority of cases (3). Certain underlying conditions including prematurity with or without bronchopulmonary dysplasia, congenital heart disease, immunosuppression, or another underlying respiratory condition increase the risk of contracting and developing severe RSV disease (1). However, although 70–80% of previously healthy infants are infected with RSV in the first year of life, only a small proportion develop bronchiolitis, with the vast majority having upper respiratory tract infection (URTI) alone (1). Airway size (4) and passively acquired maternal IgG antibody (1) are partial explanations for this variation in clinical response to RSV, and consistent with these theories, the peak age of incidence for bronchiolitis is 2–4 months. However, these factors cannot be the whole explanation for RSV bronchiolitis, as the majority of vulnerable infants exposed at the peak age do not develop the disease. Recent interest has therefore focused on the role of the host immune response in the pathogenesis of bronchiolitis (5).
Immune responses can be categorized according to patterns of cytokine production (6). Type 1 cytokines such as interferon- (IFN-) (produced by type 1 effector T cells), IL-12 (from antigen presenting cells), and IL-18 (from activated macrophages) promote cell-mediated immunity and are required for effective responses to intracellular pathogens including viruses. Type 2 cytokines, such as IL-4, IL-5, and IL-10, all produced by type 2 T cells, mediate allergic responses. Cross-regulation occurs between the two responses, and responses deviated toward type 2 or away from type 1 are associated with severe disease in several infectious disease models (7, 8).
Murine studies indicate that prior sensitization to individual RSV surface proteins followed 3 weeks later by RSV infection can induce polarized cytokine responses that follow broad type 1 and type 2 repertoires (9). Mice with type 2 cytokine responses developed enhanced disease with pulmonary hemorrhage and eosinophilia, whereas those with type 1 responses had reduced immunopathology and enhanced viral clearance (10). Similar immune mechanisms have been suggested in human studies of RSV bronchiolitis (11–14), whereas other similar studies reach directly conflicting conclusions (15–18). These studies are conflicting and inconclusive as a result of problems in their design. Although two studies examined children with proven RSV bronchiolitis and the appropriate control group (infants also infected with RSV but with mild illness only), none have either investigated cytokine responses in the airway or prospectively sampled bronchiolitis and control subjects at the same time relative to the onset of symptoms and none have analyzed responses while controlling for age (cytokine responses are known to change rapidly in the first year of life) or assessed viral load.
We have therefore performed a carefully designed prospective birth cohort study to control for all the above confounding factors to investigate whether RSV bronchiolitis is associated with excess type 2/deficient type 1 cytokine responses. We examined the in vivo immune responses of infants to their first natural proven RSV infection through analysis of cytokine production in both nasal lavage fluid and stimulated blood mononuclear cells taken at identical times from onset of symptoms of infection. Subjects were prospectively monitored to determine if infection resulted in bronchiolitis or upper respiratory signs and symptoms alone. Cytokine responses were then analyzed in relation to age and disease outcome as well as virus load and virus clearance.
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METHODS
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Subjects
Ninety-one babies with at least one parent with atopy and asthma were recruited antenatally. Eighty-eight infants were subsequently monitored through their first winter (see expanded METHODS in the online supplement).
Study Design
Whenever a baby developed respiratory symptoms, one of the research team (J.P.L.) visited the home (Days 1 and 2), where a respiratory questionnaire was completed, clinical examination performed, and nasal lavage specimen collected. If this specimen tested positive for RSV (RSV Testpack; Abbott Diagnostics, Maidenhead, UK), another home visit was arranged. A further questionnaire, examination, and nasal lavage were performed and venous blood collected (Days 5–7). Acute URTI was defined as new-onset rhinorrhea with or without fever or cough but without bronchiolitis. Acute bronchiolitis (AB) was diagnosed if the infant had all three of tachypnoea (> 60 breaths/minute), subcostal recession at rest, and inspiratory crackles on auscultation.
Nasal Lavage—Collection and Cytokine Analysis
Nasopharyngeal lavage was performed supine with 2.5 ml of phosphate buffered saline in each nostril and collection into a standard mucus extractor. The specimen was vortexed and a 500 µl aliquot mixed with 2 ml of virus transport medium for storage at -70°C. The remaining specimen was filtered through a 100-µm cell strainer and centrifuged at 400 x g for 10 minutes. Supernatants were separated, aliquoted, and stored at -70°C until analysis.
Cytokine levels in nasal lavage supernatants were measured using matched antibody pairs according to the manufacturers' instructions. IFN- (lower detection limit 5 pg/ml), IL-10 (6 pg/ml), and IL-12 (6 pg/ml) antibody pairs were obtained from Pharmingen (San Diego, CA) and IL-4 (0.32 pg/ml) high sensitivity ELISA kit from CLB (Amsterdam, The Netherlands).
Quantitative Polymerase Chain Reaction for RSV F protein
The viral load in the samples was analyzed using real-time quantitative fluorescent polymerase chain reaction (PCR) (TAQMAN; Applied Biosystems, Foster City, CA) with the ABI 7700 sequence detector. Dilutions of a plasmid containing the F protein of RSV A2 were used to generate a standard curve. The PCR detected both A and B strains of RSV and the common A strain subtypes to a lower limit of detection of five gene copies.
Venous Blood—Processing and Analysis
Peripheral blood mononuclear cells (PBMC) were suspended at 1 x 106 cells/ml in culture medium supplemented with 10% filtered autologous plasma and stimulated with phytohemagglutinin (1 µg/ml) or LPS (5 µg/ml) for 24 hours at 37°C with 5% CO2. Cultures were centrifuged at 400 x g for 5 minutes and total cellular RNA extracted and analyzed by reverse transcription–PCR using specific primers for IFN-, IL-4, IL-12 (p35 subunit), and IL-18 messenger RNA (mRNA) and internal control glyceraldehyde phosphate dehydrogenase mRNA.
PCR products were electrophoresed on 2% agarose gels containing VISTRAGreen and analyzed by direct fluorescence on a Storm 850 imager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
Statistical Analysis
Nasal lavage and PBMC cytokine and the derived cytokine ratios were logarithmically transformed before analysis. To account for age differences between groups, transformed cytokine levels and ratios were compared using analysis of covariance with age as a covariate. The magnitude of difference between groups for these data is summarized using the geometric mean ratio (GMR) together with a 95% confidence interval. If a particular cytokine was not detected in a specimen by ELISA, the value of the lower limit of detection of that assay was used for statistical analysis.
Comparison of viral load between AB and URTI groups was made using the Mann–Whitney U test. Other continuous variables were analyzed using the unpaired Student's t test. Categoric data were analyzed by  2 or Fisher's exact test. A p value of less than 0.05 was considered significant.
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RESULTS
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Patient Characteristics and Outcome
Three of the recruited infants were withdrawn from the study before the start of the study period. Two infants developed symptoms and signs consistent with asthma, and one infant was withdrawn for personal reasons. A total of 123 episodes of respiratory tract infection were reported to the study team in the 88 babies monitored. RSV was detected by enzyme immunoassay (RSV Testpack) in 28 (32%) of the studied infants. Nine of the 28 developed symptoms and signs of acute bronchiolitis, of whom three required hospitalization. The remaining 19 infants had signs of an URTI alone (Figure 1)
. No significant differences in demographic data were observed between the two groups of patients (Table 1) . Of the 28 infants with detectable RSV, all had nasal lavage specimens collected at the time of infection. Due to technical problems with venesection in four infants, venous blood was obtained from only 24 (15 URTI, 9 AB)—this subgroup did not differ demographically from the larger group (data not shown). Six infants had two or more separate RSV infections during the study period but only data from the first infection were included in the analysis.
Cytokine Levels in Nasal Lavage Fluid
Using the described technique, nasal lavage proved a reliable and reproducible means of collecting nasal secretions during an acute URTI. Over 60% ( 3 ml) of the instilled lavage fluid was recovered in the majority of cases (> 89%), with a median collection of 3.85 ml (range, 1.9–4.9 ml). There were no significant complications associated with the technique.
To investigate whether the nature of the host immune response differed between those who developed AB and those with URTI alone, nasal lavage fluid from both groups of patients was analyzed by ELISA for cytokine production. In those RSV-positive infants who developed AB, IFN- levels were significantly lower on Days 1–2 than in those RSV-positive infants with signs of an URTI alone (p = 0.029; URTI:AB GMR 3.61 [1.15, 11.35], Table 2) . Conversely, IL-4 was significantly higher on Days 5–7 in the bronchiolitis group (p = 0.011; AB:URTI GMR 2.03 [1.19,3.44]), with a trend toward higher levels on Days 1–2 (Table 2). There were no significant differences in IL-10 and IL-12 levels observed between the two groups (Table 2). To examine the balance between type 1 and type 2 cytokine responses and to correct for dilutional variation between samples, the results were expressed as ratios (IL-4/IFN- and IL-10/IL-12). The ratio of IL-4/IFN- from infants with AB was markedly higher on both Days 1 and 2 (p = 0.014; AB:URTI GMR 6.00 [1.48,24.32]) and Days 5–7 (p = 0.001; AB:URTI GMR 4.94 [2.06,11.89]) than from infants with an URTI. In addition, those with AB demonstrated a significantly higher IL-10/IL-12 ratio (p = 0.015; AB:URTI GMR 10.23 [1.64,63.68]) on Days 1 and 2 of the illness (Figure 2)
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Figure 2. Impaired type 1 and augmented type 2 airway cytokine production in infants with respiratory syncytial virus (RSV) bronchiolitis. Nasal lavage specimens were collected from infants with proven RSV-induced upper respiratory tract infection (URTI, open circles, n = 19) or bronchiolitis (filled circles, n = 9) on Days 1–2 and Days 5–7 of illness. Geometric means of type 1 and type 2 cytokine ratios are shown on Days 1–2 and Days 5–7 of illness. IL-4/IFN- ratios (A) were increased five- to sixfold at both time points and the IL-10/IL-12 ratio (B) was increased 10-fold on Days 1–2 in the bronchiolitis group compared with those with URTI alone.
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Cytokine mRNA Production by Stimulated PBMC
PHA-stimulated PBMC from infants with acute bronchiolitis expressed significantly less IFN- mRNA than PBMC from infants with signs of an URTI alone (p = 0.002; URTI:AB GMR 24.04 [3.81,151.71]). There was no significant difference in IL-4 mRNA levels observed between the two groups. The ratio of IL-4/IFN- mRNA production was significantly higher (p = 0.010; AB:URTI GMR 67.92 [3.13,1472.31]) in the AB group when compared with the URTI group (Figure 3)
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LPS-stimulated PBMC from infants with acute bronchiolitis also expressed significantly less IL-18 mRNA (p = 0.019; URTI:AB GMR 14.45 [1.61,129.71], Figure 4)
than PBMC from infants with signs of an URTI. IL-12 mRNA expression in LPS-stimulated PBMC was not significantly different (p = 0.215; URTI:AB GMR 2.7 [0.54,13.58]) between the two groups.
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Figure 4. Initial viral load in nasal lavage was similar between groups, but there was a trend toward impaired virus clearance in bronchiolitis. Nasal lavage specimens were collected from infants with proven respiratory syncytial virus (RSV)-induced upper respiratory tract infection (URTI, open circles, n = 19) or bronchiolitis (filled circles, n = 9) on Days 1–2 and Days 5–7 of illness. RSV F protein expression was determined by quantitative reverse transcription–polymerase chain reaction. Individual values at both time points are shown and connected by lines to demonstrate a change within each subject. There was no difference in initial viral load between groups (p = 0.308) but there was a trend toward impaired viral clearance in the bronchiolitis group (p = 0.064).
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RSV F Protein in Nasal Lavage
The balance between type 1 and type 2 cytokine responses is known to be determined, at least in part, by antigen load (19) and imbalance in type 1/type 2 responses is known to affect virus clearance (20). Therefore, to determine initial viral load and to investigate relative rates of viral clearance between the two groups, RSV F protein mRNA was quantified in nasal lavage fluid using quantitative PCR. Initial viral load was similar—F protein gene copies in the nasal lavage fluid obtained on Days 1 and 2 of the illness did not differ significantly between the two groups (p = 0.308). However, there was a trend toward impaired viral clearance in the AB group, in that four of the nine infants with AB had a rise in F protein gene copies between Days 1 and 2 and Days 5–7 compared with only 2 of the 19 infants with URTI (p = 0.064, Figure 4).
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DISCUSSION
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We report the first prospective study to investigate type 1 and type 2 cytokine responses in infants proven to have RSV bronchiolitis and in infants also with RSV infection but with mild clinical illness without bronchiolitis. The study design also permitted controlling for age and dilutional factors in nasal lavage, sampling of both airway and peripheral blood, sampling of the two groups concurrently in relation to onset of signs of infection, and analysis of initial virus load and virus clearance. We have demonstrated that RSV bronchiolitis is associated with a profound imbalance in type 1/type 2 cytokines with deficient type 1 and excess type 2 responses. This imbalance was manifest in both inducing (IL-12 and IL-18) and effector (IFN- and IL-4) cytokines and was present in both airway secretions and in peripheral blood. We have further demonstrated that this imbalance cannot be attributed to differences in age, dilution, or initial viral load and that it is associated with impaired virus clearance in the bronchiolitic group.
IFN- and IL-4 represent the archetypal cytokines of the type 1/type 2 paradigm and are the most substantiated example of a pair of mutually counter-regulatory cytokines. Whereas IFN- inhibits the development of a type 2 immune response and promotes type 1 responses, IL-4 induces type 2 cytokine production and suppresses type 1 responses (21, 22). Our data demonstrate reduced IFN- and increased IL-4 production (Table 2) and a highly significantly increased IL-4/IFN- ratio in RSV bronchiolitis in both the respiratory tract (Figure 2) and systemically (Figure 3). This provides strong evidence for an excess type 2 and/or a deficient type 1 immune response in infants with bronchiolitis.
IL-10 is a product of various cell types including T cells and monocytes and possesses a wide range of activities including suppression of type 1 cytokine secretion (23). IL-12 is rapidly released by antigen presenting cells in response to infection and is essential for the development of a type 1 immune response (24). IL-10 and IL-12 represent a further counter-regulatory balance between type 1 and type 2 responses (25, 26). The significantly increased IL-10 to IL-12 ratio in nasal lavage fluid on Days 1 and 2 in those with bronchiolitis also demonstrates the development of an excessive type 2 or deficient type 1 immune response in these infants (Figure 2).
IL-18 (previously known as IFN-–inducing factor) is a cytokine synthesized by activated macrophages that, acting in concert with IL-12, plays an important role in promoting type 1 immune responses (27). Our data for the first time demonstrate deficient IL-18 expression by stimulated PBMC in infants with bronchiolitis (Figure 4), suggesting that deficiency of this cytokine may be important in regulating the deficiency of IFN- in these individuals.
During RSV infection, viral replication occurs in the respiratory epithelium and inflammatory processes in the nasal air passages have been shown to reflect those in the lower airways (28, 29). Nasal lavage therefore represents a minimally invasive, reproducible method of obtaining specimens with immunologic relevance to the respiratory tract as a whole. No reliable method exists for calculating the volume of actual epithelial lining fluid collected during nasal lavage. This raises the possibility that the differences in nasal lavage cytokine levels (Table 2) between groups may merely represent differences in dilution of nasal secretions. By expressing results as ratios (Figure 2), we were not only able to examine the balance between type 1 and type 2 cytokines but also to eliminate the need to control for dilutional differences between specimens. The fact that the results were similar whether expressed as cytokine levels or as a ratio confirms that dilutional factors were not important.
The type of immune response induced by exposure to a foreign antigen is influenced by many factors including antigen dose (19). To address the possible contribution of antigen dose to the observed cytokine differences, we used quantitative real-time reverse transcription–PCR to determine the quantity of RSV F protein mRNA present in the Days 1 and 2 nasal specimens. No difference in initial viral load was observed between those with and without bronchiolitis, thus making differences in antigen dose an unlikely explanation for our findings. Impaired RSV clearance has previously been shown to correlate with disease severity using quantitative viral cultures (30). Animal studies of RSV infection indicate that high levels of IL-4 delay virus clearance (31). We have observed impaired type 1/augmented type 2 immunity and a trend toward impaired virus clearance in the bronchiolitis group, confirming that the observed imbalanced immune response is associated with not only clinical but also, possibly, with virologic outcomes.
Cytokine responses to acute respiratory viral infections are known to rise and fall during the first days of infection, with varying time courses depending on the cytokines measured (32). Infants admitted to hospital with acute bronchiolitis can present several days after the onset of infection, whereas uncomplicated URTI is a short-lived condition. For this reason, cytokine responses measured in cross-sectional studies of infants admitted to hospital with bronchiolitis cannot be reliably compared with control infants with mild URTI, as the timing of sampling relative to the onset of infection is certain to be different between groups and can never be reliably established. This confounding influence can only be removed by employing a prospective study design in which sampling of the index and control groups is simultaneous relative to the onset of signs/symptoms of infection. This study is the first to employ such a design and therefore remove confounding by timing of sampling.
The current study was designed to enable a sensitive analysis of the systemic immune response during RSV infection through polyclonal stimulation of PBMC. Polyclonal stimulation, with PHA (a T cell mitogen) and LPS (a B cell mitogen), was used in preference to specific T cell cloning to avoid the acute selective pressures observed during the cloning process and to enable a more global overview of the systemic immune response rather than having to extrapolate findings from a small component of the T cell repertoire. Moreover, live virus stimulation was not employed so as to avoid the effects of cell death on cytokine production. A relatively short period of stimulation (24 hours) minimized the potential for deviation of the intrinsic cytokine response by polyclonal activation.
The ontogeny of the human immune response has great relevance to studies of early childhood. Prescott and coworkers demonstrated a rapid suppression of type 2 responses in children without atopy but a persistence of these responses in children with atopy over the first 6–12 months of life (33). To take account of the small difference in age between the groups (Table 1), all comparisons of cytokine values were adjusted for age using analysis of covariance. The imbalanced cytokine responses we have observed therefore cannot be accounted for by differences in age between the two groups.
A further unique feature of this birth cohort study was the fact that all infants in both URTI and AB groups were at a similar risk of developing skewed cytokine responses as a result of having at least one parent with atopy. Furthermore, both groups had similar proportions of infants with two parents with atopy (Table 1). We have previously shown that as a group such infants have a reduced ability to produce IFN- from cord blood mononuclear cells, compared with infants with no family history of atopy (34), suggesting an innate skew toward a type 2 cytokine response. To test the hypothesis that excess type 2/deficient type 1 immune response is associated with bronchiolitis, we selected such infants to optimize the potential of detecting such responses during RSV infections. We now show that within the group, those who develop bronchiolitis also have impaired type 1 immunity relative to those who did not. To substantiate our findings for the general population, similar (but necessarily much larger) studies are now required using randomly selected families.
Type 2 T cells are involved with the switching of B cells to IgE production through both cell contact and IL-4 production, whereas a type 1 cytokine response can inhibit this switch (35). Increased levels of RSV-specific IgE have previously been demonstrated in nasopharyngeal secretions of infants with RSV bronchiolitis, providing indirect evidence of a type 2 response in human RSV infection (36). Further recent human studies have implicated an association between a type 2 immunologic response and RSV bronchiolitis. Roman and colleagues found a significantly increased IL-4/IFN- production ratio on mitogen exposure of PBMC taken from infants while hospitalized with RSV bronchiolitis when compared with healthy control subjects identified retrospectively (12). Similarly, Aberle and coworkers identified lower levels of IFN- mRNA in PBMC from infants with severe RSV bronchiolitis than in those with a milder clinical course (11). Indirect evidence for a type 2 immune response has also emerged from the analysis of cell surface markers in RSV-positive bronchiolitis when compared retrospectively with control infants (13). Our study supports the findings of these studies and is the first to investigate both respiratory tract and systemic cytokine responses, to control for age, to ensure simultaneous timing of sampling relative to onset of infection, to investigate initial virus load, and to monitor virus clearance. It is also unique in using prospectively identified control subjects from the same birth cohort and geographic area who are infected in the same season with the same virus but who do not develop bronchiolitis. It is likely that the absence of one or more of these design features together with the atopic family history of the recruited cohort explains the conflicting results reported in other previous studies (15–18).
RSV bronchiolitis is an important risk factor for the development of asthma (37) and atopic sensitization (38), both of which are characterized by an excess of type 2 cytokines (39). However it is debated whether RSV bronchiolitis causes asthma by deviating subsequent immune responses toward a type 2 or away from a type 1 phenotype, or whether asthma and bronchiolitis simply have common risk factors, with bronchiolitis being the first presenting illness in a child already at risk of developing asthma as a result of impaired type 1 immunity that pre-dated RSV bronchiolitis (40). The present data clearly demonstrate an excess type 2 and deficient type 1 response in RSV bronchiolitis and are very consistent with the increased risk of subsequent asthma in these patients. Further studies of this nature, and perhaps intervention studies, will be required to indicate whether RSV infection also acts as a cause of the increased risk of asthma observed in RSV bronchiolitis.
Each winter, outbreaks of RSV infection result in over 2% of all infants under 1 year of age requiring hospitalization (2). The health-care costs of these annual epidemics are substantial, amounting to almost $700 million per annum in the United States alone (41). Despite the obvious magnitude of the problem, advances in both prevention and therapy have been slow. Thus far, efforts to develop a vaccine have failed (42) and treatment is controversial, varying worldwide (43) and often including potentially toxic and expensive medication. As our understanding of disease immunopathogenesis increases, so the potential benefits of appropriate immunotherapy become apparent. We have demonstrated the association of an excessive type 2/deficient type 1 cytokine response to RSV infection in the respiratory tract with a more severe clinical course. This raises the possibility that treatments that restore the type 1–type 2 cytokine balance to the relative type 1 predominance of URTI will ameliorate RSV disease. Further studies are required to confirm these findings in other patient groups.
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Acknowledgments
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The authors thank Dr. J. Lorraine Low for help with statistical analyses, Brenda Colwell for secretarial support, and Norma Diaper for organizational assistance.
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FOOTNOTES
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Supported by grants from the National Asthma Campaign (UK), the British Lung Foundation, and the British Medical Association.
This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org
Received in original form October 8, 2002;
accepted in final form May 27, 2003
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