INTRODUCTION

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Keywords: ventilation; bronchoconstriction; lung transplantation

Long-term survival after lung transplantation is threatened by bronchiolitis obliterans (BO), which is thought to be a form of chronic allograft rejection. With a prevalence exceeding 50% after 5 yr, BO has emerged as the most significant long-term complication and the first cause of late death in lung transplant recipients (1). Although the treatment of established BO is generally unsuccessful, resolution or stabilization may be achieved with augmented immunosuppression if the diagnosis is made before a significant decline in FEV₁ occurs (4). For this reason, there is growing interest in surrogate markers for BO that may help detect and treat the disease in a preclinical stage (3). In this context, two recent studies have shown that having a bronchodilator response at low lung volume or a positive methacholine challenge at 3 mo after transplantation is associated with the development of BO, with a positive predictive value of 81 and 72%, respectively (5, 6).

These observations suggest that posttransplantation nonspecific bronchial hyperreactivity (NSBHR) is a manifestation of a pathologic process in the small airways (6). However, the link between NSBHR and small airways disease is not precisely understood because in nontransplanted hyperreactive subjects, histamine or methacholine-induced bronchoconstriction involves the conductive (proximal) but not the acinar (distal) airways (7). We hypothesized that in transplanted hyperreactive subjects, methacholine-induced bronchoconstriction may involve the small airways. To test this hypothesis, we assessed the alterations in ventilation distribution occurring in the lung periphery in response to methacholine in a group of hyperresponsive lung transplant recipients, and we compared these alterations with those observed in hyperresponsive nontransplanted subjects.

	METHODS

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The population consisted of 15 heart-lung transplant patients who were studied 69-1,636 d after surgery (Table 1), and 17 nonsmoking subjects who had a history of atopy and NSBHR. At the time of testing, all subjects were clinically stable with no respiratory symptoms, and they had normal baseline pulmonary function (Table 2). The transplanted subjects had no clinical sign of rejection or infection, and their mean (± SD) FEV₁ was 100.0 (± 2.7)% of the best postoperative value. Preoperative diagnosis was cystic fibrosis in six patients, primary pulmonary hypertension in six patients, bronchiectasis in one patient, and Eisenmenger syndrome in two patients. No patient had a history of asthma before transplantation, and no donor had a history consistent with asthma. Induction immunosuppressive therapy consisted of antithymocyte globulin, cyclosporine, azathioprine or mycophenolate mofetyl, and methylprednisolone. Maintenance immunosuppression was based on cyclosporine or tacrolimus, azathioprine or mycophenolate mofetil, and methylprednisolone. All subjects gave verbal informed consent to the procedures as approved by the Ethic Committee of the institution.

Baseline measurements of functional residual capacity (FRC), total lung capacity (TLC), residual volume (RV), inspiratory vital capacity (VC), FEV₁, and airway conductance (Gaw) were obtained with the patient seated in a constant volume body plethysmograph (Jaeger, Würzburg, Germany) following the guidelines of the American Thoracic Society (8). The methacholine challenge was performed following a standard protocol (9), using an MEFAR dosimeter and nebulizer (MB3, Bovezzi, Italy). The dose of methacholine required to produce a 20% fall in FEV₁ (PD₂₀) was calculated by linear interpolation between the last two doses on the dose-response curve. Hyperresponsiveness was defined as a PD₂₀ of less than 7.8 µmol (9).

In addition to standard pulmonary function, distribution of ventilation was studied before the challenge in all subjects and after the challenge in the subjects who were hyperresponsive. We used a single-breath washout test, as described in our previous studies (10). The subjects were connected to a double bag-in-box system through a nonrebreathing valve with a 20-ml instrumental dead space. They inhaled a gas mixture containing 5% He, 5% SF₆, and 90% O₂ from FRC to 11 above FRC, and then expired to RV. Both inspiratory and expiratory flows were kept constant at approximately 0.4 L/s. The slope of the alveolar plateau for N₂, SF₆, and He (SN₂, SSF₆, SHe) was obtained from a linear regression analysis performed between 35 and 80% of the expired volume. The downgoing He and SF₆ slopes were treated as positive, as were upgoing N₂ slopes; thus, an increase in SN₂, SSF₆, or SHe indicates a more heterogeneous ventilation. Single-breath washouts were always performed in duplicate by the same investigator and slope values were calculated as the average of two measurements. All signals were sampled at 50 Hz and stored in an Olivetti PC for subsequent analysis.

Predicted values for lung volumes were derived from the ECCS Working Party (13) on the basis of the recipients' anthropometric characteristics. Data are expressed as means ± SD throughout the text and tables. Statistical analysis was made using Student paired and unpaired t tests. A p value < 5% was considered significant.

	RESULTS

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Seven of 15 transplanted subjects (Table 1) and the 17 nontransplanted subjects were hyperresponsive to methacholine. The median PD₂₀ was 1.0 µmol in the hyperresponsive transplanted subjects and 0.51 µmol in the nontransplanted subjects (p = 0.03). All subjects had normal pulmonary function in control conditions; for the seven hyperresponsive transplanted subjects VC, FEV₁, and TLC averaged 85 ± 13, 89 ± 16, and 93 ± 8% of predicted, respectively (Table 2). Similarly, values for SN₂, SSF₆, and SHe were comparable in the two groups and were within the range found in our previous report in 22 healthy subjects (10).

Functional changes in response to methacholine are summarized in Table 3. Subjects in the two groups showed similar average decreases in FEV₁ and specific Gaw, and similar average increases in SN₂ and SSF₆. On the other hand, VC decreased less and FRC increased less after the challenge in the transplanted than in the nontransplanted subjects. In addition, because the increase in SHe in response to methacholine was more than 2.5 times greater in the transplanted than in the nontransplanted subjects, the slope difference between SF₆ and He (SSF₆ SHe) decreased significantly in the former but did not change in the latter; in the transplanted group, SSF₆ SHe became negative after the challenge, whereas it remained positive in the nontransplanted group (Figure 1).

View larger version (10K): [in this window] [in a new window]   Figure 1.   Individual values for the difference in the slope of the alveolar plateau for SF6 and He (SSF6 SHe) in seven hyperresponsive heart-lung transplant recipients (A) and 17 hyperresponsive nontransplanted subjects (B) before and after methacholine. In all transplanted subjects, methacholine produced a decrease in SSF6 SHe, which was positive before and negative after the challenge. This was not observed in the nontransplanted subjects.

	DISCUSSION

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Several previous reports have demonstrated that NSBHR is a frequent finding after heart-lung, double-lung, and single-lung transplantation (6, 14), and in a recent longitudinal study, Stanbrook and Kesten (6) reported that ~ 40% of patients with bilateral lung transplantation had at least one positive methacholine challenge during the course of the first two postoperative years. The incidence of 47% of positive challenges found here in a group of 15 heart-lung transplant subjects studied at 1 yr after surgery is thus consistent with this report.

Before discussing the effects of metacholine on ventilation distribution, it is appropriate to summarize the concepts that support our interpretation of the SF₆ and He single-breath washouts used here. Transport of fresh gas from the entrance of the lung to the alveoli, where it mixes with resident gas, is achieved by convective flow in the conducting airways and by convection and molecular diffusion in the acinus. Theoretical analysis of gas mixing (24) shows that during inspiration the concentration front between inspired and resident gas moves in the acinar zone where it becomes relatively stationary. Inequalities of convective flow at branchpoints located proximal to the diffusion front may contribute to ventilation inhomogeneities, but their contribution to the slope of the alveolar plateau is similar for SF₆ and He. On the other hand, interaction of diffusion and convection at branchpoints subtending intraacinar asymmetrical parallel units creates inhomogeneities of gas concentrations that result in a greater sloping of the alveolar plateau for SF₆ than for He (24). Simulations of intraacinar gas mixing also predict sloping of alveolar plateaus when the airways subtending symmetrical parallel units have different calibers. So, whether physiologic or related to pathologic changes in the airways, diffusion-dependent peripheral inhomogeneities can best be assessed by computing the slope difference between SF₆ and He (SSF₆ SHe). Because the diffusion front for He is located more proximally in the acinus than the diffusion front for SF₆, SHe is expected to be more sensitive to inhomogeneities arising at the level of terminal and respiratory bronchioles, that is, SSF₆  SHe is expected to decrease in disease processes affecting the bronchioles. This theoretical prediction is supported by recent experiments showing a decreased SSF₆ SHe in lung transplant recipients who develop bronchiolar narrowing due to bronchiolitis obliterans (12).

One of the most conspicuous findings of the present study has been that alterations in ventilation distribution induced by methacholine were different in the transplanted versus the nontransplanted hyperresponsive subjects, though both groups had similar average changes in FEV₁ and SGaw (Table 3). In all hyperresponsive transplanted subjects, methacholine-induced bronchoconstriction increased SHe more than SSF₆, such that SSF₆ SHe invariably decreased and became negative (Figure 1); this suggests that ventilation inhomogeneities developed in the zone of membranous and respiratory bronchioles. On the other hand, SSF₆ SHe was not affected significantly by methacholine in the nontransplanted subjects (Figure 1). This observation is fully consistent with a recent work by Verbanck and colleagues (7) who showed that inhalation of histamine in nontransplanted subjects with NSBHR makes ventilation more heterogeneous in the conductive but not in the acinar airways.

The reason for the different pattern of response observed in the transplanted and the nontransplanted subjects is unknown, but denervation of the airways may play a role. Baseline lung function (Table 2) indicates that values of FEV₁/VC were higher in the transplanted than in the nontransplanted subjects (p < 0.001), which is likely to reflect denervation of the airways with loss of resting vagal bronchomotor tone and increased airway caliber (25). This alteration, and the fact that denervation may reduce bronchoconstriction of proximal airways that contain most cholinergic receptors (26), might promote a more peripheral distribution of methacholine in the transplanted subjects. As a result, bronchoconstriction and inhomogeneities of ventilation distribution may involve smaller airways.

Denervation might also affect the development of dynamic pulmonary hyperinflation in response to methacholine. A striking difference between the two groups of subjects studied here was that methacholine produced a much greater increase in FRC in the nontransplanted than in the transplanted subjects; in fact, the increase in FRC in the latter did not reach statistical significance (p = 0.059). Bronchoconstriction induced by methacholine in nontransplanted subjects is generally accompanied by an increase in FRC, but the mechanism of this response is still controversial. Pellegrino and Brusasco (27) have suggested that expiratory flow limitation may be the triggering factor to generate dynamic hyperinflation, that is, when expiratory flow limitation is achieved, stimulation of afferent mechanoreceptors in the airway wall would initiate inspiration by reflexly activating the inspiratory muscles before the resting volume of the system is reached. This mechanism is expected to be absent after transplantation due to denervation of the airways; as a result, the increase in FRC during bronchoconstriction would be reduced or even completely abolished. Whether or not this is the correct explanation for the different response seen in the transplanted versus the nontransplanted subjects, the smaller increase in FRC found in the former may enhance narrowing of the distal airways produced by bronchoconstriction because their caliber depends in part on the elastic recoil of the lung, and hence on lung volume.

In conclusion, the present studies have shown that the response of transplanted subjects to methacholine involves the small airways, which is not seen in nontransplanted subjects with NSBHR. This observation suggests that assessing bronchial reactivity after lung transplantation may be regarded as a useful tool to explore the small airways, and it helps in understanding why the response to methacholine may provide information on the presence of a pathologic process affecting the bronchioles, and hence on the risk of progression to BO (6).