Bronchial contractions in transplanted lungs

Bronchial contractions in transplanted lungs Influence of denervation, acute rejection, and the bronchial epithelium The effects of pulmonary denervation and rejection on contractions of bronchial smooth muscle and epithelial modulation of these contractions were studied in dogs after denervation in right lung autotransplantation (n = 6) and acute rejection after right lung aUotransplantation (n = 8). Immunosuppression was withdrawn from the latter group after 5 days; rejection developed after 3 additional days. A significant (p < 0.05) increase in mean peak airway pressure occurred with rejection of aUotransplanted lungs. Rings cut from third-order bronchi of transplanted and contralateral unoperated (native) lungs in each animal were suspended in organ chambers for the measurement of isometric force. In some rings, the epithelium was removed mechanically. Acetylcholine (cholinergic neurotransmitter), serotonin (platelet-product), histamine (mast ceU product), and endothelin-l (endothelium-derived contracting factor) caused concentration-dependent contractions in all rings. In bronchi from native lungs, rings with epithelium contracted less than those without epithelium. This difference was lost after autotransplantation. The smooth muscle and epithelium were affected differendy by autotransplantation. Contractions of rings without epithelium decreased in response to acetylcholine. and endothe6n-l, whereas contractions of rings with epithelium increased in response to histamine and 5-hydroxytryptamine (p < 0.05). During acute rejection, contractions were the same as those after autotransplantation. Bronchial content of endothelin increasedfourfold with rejection. Relaxations to isoproterenol and prostaglandin E2 were similar in both groups. In conclusion, denervation reduced the ability of the smooth muscle to contract. The degree of acute pulmonary rejection seen in this study did not further affect bronchial contractions. Modulation of contractions by the bronchial epithelium was lost with both denervation and rejection. (J THoRAe CARDIOVASC SURG 1993;106:797-804)

Allison J. McLarty, MD,* Virginia M. Miller, Phl)," Henry D. Tazelaar, MD,b and Christopher G. A. McGregor, MB, FRCS, Rochester, Minn.

Lng transplantation is a therapeutic option for patients with end-stage pulmonary disease.l' Limited clinical studies have demonstrated increased bronchial contractions in response to methacholine, a cholinergic agonist, in lung transplant recipients.r'' Denervation Departments of Surgery, Physiology; and Pathology, b Mayo Clinic and Mayo Foundation, Rochester, Minn. Supported by the Mayo Clinic and Mayo Foundation. Received for publication Aug. 25, 1992. Accepted for publication Dec. 21, 1992. Address for reprints: Christopher G. A. McGregor, MB, FRCS, Division of Thoracic and Cardiovascular Surgery, Mayo Clinic, 6-716 Mary Brigh, Saint Mary's Hospital, Rochester, MN 55905. ·Present address: Allison J. McLarty, MD, Department of Surgery, Columbia-Presbyterian Medical Center, New York, NY 10032. Copyright @ 1993 by Mosby-Year Book, Inc. 0022-5223/93 $1.00 +.10

12/1/46209

leading to an up-regulation of muscarinic receptors has been implicated as a cause of this phenomenon. However, exacerbation of bronchoconstriction to histamine, which is not a neurotransmitter, during acute rejection suggests that mechanisms other than denervation may contribute to changes in bronchial constriction.v 7 Bronchial epithelium has a modulatory effect on underlying smooth muscle, such that removal of the epithelium increases the contractile effects of bronchoactive agents such as acetylcholine and histamine.v'" Inflammation of the epithelial layer is associated with airway hyperreactivity in asthma and infection'! I, 12 Similar alteration in epithelial function may contribute to increased bronchial contractions in transplanted lungs in the presence or absence of acute rejection. Therefore, experiments were designed to test the hypotheses that denervation of the lung alters the function of the bronchial epithelium and smooth muscle and that such changes

797

The Journal of Thoracic and Cardiovascular Surgery November 1993

7 9 8 McLarty et al.

are distinct from those occurring during acute pulmonary rejection.

Methods Animal care was in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). Adult mongrel dogs of either sex matched for size and weight (25 kg) were used. Right lung autotransplantation was performed in six dogs; right single lung allotransplantation was performed in eight dogs as described previously.P except that University of Wisconsin solution (60 ml/kg) was used for pulmoplegia instead of Euro-Collins solution. The mean ischemic time of the transplanted lungs was 55 minutes. Animals that underwent allotransplantation also underwent immunosuppression with intravenous cyclosporine (I to 3 rug/kg per day) to maintain plasma concentrations of 250 to 500 ng/rnl, radioimmunoassay for parent compound plus metabolites, and administration of azathioprine (2.5 mg/kg per day) beginning after induction of anesthesia. Methylprednisolone (50 mg intravenously) was given at 8-hour intervals for 24 hours, reduced to 0.5 rug/kg per day by day 3, and maintained at this dose until day 5. In the postoperative period, all dogs received gentamicin (40 mg) and clindamycin (300 mg) intravenously and benzylpenicillin (1.2 million U) intramuscularly before the operation. The gentamicin and clindamycin were repeated every 12 hours after the operation for 3 days. Animals subsequently received Bactrim DS (160 mg trimethoprim and 800 mg sulfamethoxazole) orally twice a day until they were killed. All intravenous drugs were administered through a catheter placed in the jugular vein the day of operation. After opacification was absent on chest radiography for 5 days, immunosuppression was discontinued, allowing the allotransplant to be rejected, When rejection was suspected from clinical signs, radiographic confirmation was obtained, at which point the dogs were used for experiments, generally after 8 days. Dogs that underwent autotransplantation were likewise studied after 8 days. Animals were anesthetized with pentobarbital sodium (30 rng/kg intravenously). Airway pressures were measured with a double-lumen endotracheal tube in both transplanted and native lungs immediately after the operation and when the dogs were killed. Transplanted and native lungs were ventilated in turn with a tidal volume of 10 ml/kg during single lung ventilation and 15 ml/kg during two lung ventilation. Inspiratory flow of oxygen was 40%, respiratory rate was set at 20 breaths/min, and positive end-expiratory pressure was 4 em H 20. The mean peak inspiratory airway pressure in each bronchial tree was measured. Animals were then exsanguinated. The heart-lung block was excised, and the third-order bronchi from the lower lobes were dissected and placed in modified Krebs-Ringer bicarbonate solution (control solution, in millimoles per liter: NaCI, 118.3; KCI, 4.7; CaCI 2, 2.5; MgS04, 1.2; KH2P04, 1.2; NaHC0 3 , 25.0; calcium disodium edetate, 0.26; and glucose, 11.1) for studies in organ chambers. The remaining lobes oflung were prepared for either biochemical assays of endothelin content or histologic analysis. Organ chamber studies. After removal of connective tissue, third-order bronchi of the lower lobes, which were used exclu-

sively for the heterogeneity of response to bronchoactive agents along the bronchial tree, 14 were cut into rings of 5 to 6 mm in length. In some rings the epithelial layer was deliberately removed by gently rubbing the luminal surface with the tip of a pair of watchmaker's forceps." The rings were suspended between a fixed point and the transducer for the measurement of isometric force in organ chambers filled with control solution (25 ml) at 37° C and bubbled with 95% oxygen and 5% carbon dioxide. Bronchial rings were equilibrated at a passive tension of less than 0.5 gm for 30 minutes. The tissue was placed at the optimal point on length-tension curves by progressively stretching the rings and determining contraction in response to 20 mmol/L potassium chloride at each level of stretch. Maximal contraction in response to potassium chloride (60 mmol/L) was measured at optimal length. After an equilibration period of I hour, cumulative concentration-response curves were obtained for response to acetylcholine (10- 9 to 10"4 mol/L), histamine (10- 8 to 10-4 mol/L), 5-hydroxytryptamine (10- 8 to 10- 5 mol/L), or endothelin-I (10- 11 to 10- 7 mol/L), Rings of bronchi from each transplanted lung, with and without epithelium, were studied in parallel with rings from the corresponding native lung. A total of three agonists was tested in each group of four rings in identical order throughout the study. The sequence of drug administration was kept constant to reduce variability caused by drugs interacting. In some rings, relaxations to isoproterenol (10- 9 to 10- 6 mol/L) or prostaglandin E 2 (10- 13 to 10-9 mol/L) were measured in bronchial rings contracted with 5-hydroxytryp-

tamine.P

Drugs and chemicals. All drugs were obtained from Sigma Chemical Company, St. Louis, Missouri, except for endothelinI, which was obtained from Peptides International, Inc., Louisville, Kentucky. Acetylcholine, a cholinergic neurotransmitter, and isoproterenol, a ,a-adrenergic agonist, were used because they are released from autonomic nerves that innervate the lung.!" Histamine is released from activated mast cells that participate in the inflammatory response.!? and 5-hydroxytryptamine is released from platelets'"; these drugs were administered to determine their effect on bronchial contractions in the early postoperative period and during rejection. Endothelin-I, a vascular endothelium-derived contracting factor.l? was used because modifications of its release might occur during vascular events of pulmonary rejection. Prostaglandin E2, a metabolite of arachidonic acid, which is a source of many mediators of the inflammatory response.i" was also used. Drugs, with the exception of prostaglandin E2, were dissolved in distilled water and added to the organ chamber in volumes of less than 0.5 ml. The prostaglandin E 2 was dissolved in ethanol; preliminary studies (not described) indicated no effect of the alcohol on response ofthe tissue. Concentrations of drugs are expressed as final molar concentrations in the organ bath. Assay for endothelin. Rings cut from pulmonary arteries, third-order bronchi, and segments of lung parenchyma were blotted dry, weighed, and frozen in liquid nitrogen. Tissue content of endothelin was measured as described previously." Histologic analysis. Lobes of lung were infused with 10% buffered formalin and fixed for at least 24 hours before histologic sectioning and staining with hematoxylin and eosin. Severity of rejection was classified by means of light microscopy from grade 0 to grade 4 according to the standard Working Formulation for the Standardization of Nomenclature in the Diagnosis of Heart and Lung Rejection developed by the Lung

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McLarty et al.

Table I. Mean peak inspiratory airway pressure Day 0

Day 8

Tx

32.0 ± 2.8 28.8 ± 1.5

31.6 ± 2.3 30.8 ± 2.7

Tx

1.9 30.2 ± 1.4

30.4 ± 1.7 49.2 ± 1.0*

Autotransplant Native

Rejected allotransplant Native 31.2 ±

Data are presented as mean ± standard error of the mean in millimeters of mercury; n; 5 in all groups. Native, Unoperated contralateral lung; Tx, transplanted lung. 'Statistically different from native lung (p < 0.05).

799

Table II. Mean maximal tension of bronchial rings with and without epithelium to Kel (60 mmol/L)

Autotransplant Native Tx

With epithelium

Without epithelium

18.! ± 3.3 10.3 ± 1.7

12.0 ± 1.0 10.3 ± 2.5

Rejected allotransplant Native 15.5 ± Tx

2.4 6.2 ± 1.0*

12.3 ± 2.0 7.4 ± 1.1

Data are presented as mean ± standard error of the mean in grams tension; n ; 5 in all groups. Native, Unoperated contralateral lung; Tx, transplanted lung. 'Statistically significant difference between rings with epithelium in the rejected allotransplant group (p < 0.05).

Rejection Study Group of the International Society for Heart

Transplantation.P Further grading ofspecific bronchial (carti-

laginous) and bronchiolar involvement with rejection was performed by using an arbitrary semiquantitative scale of 0 to 4 (0 = no involvement, I = minimal, 2 = mild, 3 = moderate, 4 = severe involvement). Sections of bronchial rings used in organ chamber studies were also examined with light microscopy to confirm the presence or absence of epithelium. Calculations andstatisticalanalysis. Results areexpressed as the mean ± the standard error of the mean, and n is the number of animals from which bronchi were taken in each group. Student's t testwas used forpaired results of rings with and without epithelium or unpaired results between rings from autotransplants and allotransplants. A p value of <0.05 was considered to bestatistically significant. Results Airway pressure. Mean peak inspiratory airway pressures of native and transplanted lungs of either group were not different immediately after the operation (day 0,Table I). In dogs that underwent autotransplantation, peak airwaypressuresin nativeand transplanted lungsat the time the animals were killedwere not different from each other or from those measured before the operation. Fordogs in whichtransplants were rejected,airway pressurein the native lungs remained unchanged at the time theanimalswere killed; however, there was a significant (p < 0.05) mean increase of 19 mm Hg in resistance to airflow in the rejected lungs (Table I). Organ chamber experiments. Contraction of bronchial. rings from native lungs of dogs that underwent autotransplantation was in all casesthe same as that from dogs that did not undergo operation (unpublished data) and therefore served as controls in the present experiments. Contraction Potassium chloride. Potassium chloride caused concentration-dependent increases in tension in all rings (Table 11). No statistically significant differences were observed in maximal tensions between rings with and without epithelium in any groups. Autotransplantation

did not significantly alter maximal contractions. In the presence of acute pulmonary rejection, maximal tension of transplanted rings with epithelium was significantly less than those from native lungs (Table II). Because of this difference, contractile responses to all other agonists are expressed as a percentageof the maximal contraction to potassium chloride. Acetylcholine and endothelin-I, Acetylcholine and endothelin-l caused concentration-dependent increases in tensionin all rings. In the native lungs of the dogs that underwent autotransplantation (control), the maximal contractionof ringswith epithelium was significantly less than for those without epithelium. Maximal contraction of rings without epithelium were decreased significantly in autotransplanted bronchi. The difference in contraction betweenrings with and without epithelium was lost. Acute rejectiondid not significantly change the contractionof the allotransplantedbronchi from that observedin autotransplanted bronchi (Figs. 1 and 2). Histamine and 5-hydroxytryptamine. Histamine and 5-hydroxytryptamine caused concentration-dependent increasesin tensionin all bronchial rings. In native lungs of dogs that underwent autotransplantation, rings without epitheliumcontracted more than those with epithelium (Figs. 3 and 4). In autotransplanted lungs, the difference in contraction between rings with and without epithelium was lost. This was due to significantly increased maximal contraction of rings with epithelium. In native lungs of dogs in which transplants were rejected, no significant differences occurred in contractions between rings with and without epithelium. Rings from rejected lungs contracted the same as those from autotransplanted lungs (Figs. 2 and 3). Relaxation. Relaxation in response to isoproterenol and prostaglandin E2 were obtained in rings contracted with a submaximal concentration of 5-hydroxytryptamine. The contraction in response to 5-hydrox-

800

The Journal of Thoracic and Cardiovascular Surgery November 1993

McLarty et al.

Native auto

120 100 80 60 'i!. 40 C 20 o o iii c

-

o without epithelium

Gl

9

·5

8

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_.L----''----'-_....I.

7

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Rejecting

1:~~ n=7

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Acetylcholine, -log M

Fig. 1. Cumulative concentration-response curves of third-order canine bronchi in response to acetylcholine. Data are expressed as mean ± standard error of mean and represent percentage of increase in tension of a maximal contraction to KCl (60 mmoljL); n equals number of animals from which rings were taken. From native, unoperated, control lung of dogs that underwent autotransplantation, maximal contraction of rings without epithelium was greater than that of those with epithelium (Native auto). This difference was lost with denervation (Autotransplant), with decreased contraction of rings without epithelium compared with control (p < 0.05). Rejection did not significantly alter responses in native (Native rejecting) or transplanted (Rejecting) lungs.

ytryptamine averaged 56% of the maximal tension in response to 60 mmoljL potassium chloride. Isoproterenol and prostaglandin E2 caused concentration-dependent relaxations in all rings. No statistically significant differences were seen between rings with or without epithelium or between native and transplanted lungs (data not shown, n = 5 to 7 in all groups). Endothelin levels. Bronchial endothelin-I content did not differ statistically between native and transplanted bronchi in animals that underwent either autotransplantation or allotransplantation (Table III). In the bronchi of the rejected allotransplanted lungs, content of endothelin ranged from 172.5 to 1113.9 pgjml per gram of tissue, and the mean was fourfold that of the autotransplanted bronchi (Table III). Native bronchi from animals with rejected allotransplanted lungs also showed large variability in content of endothelin-l; the mean was about two times that of native lungs from animals that underwent autotransplantation. Content of endothelin-l in pulmonary arteries was elevated about twofold after autotransplantation relative to the native arteries and was not affected further by rejection. Endothelin-l in the lung parenchyma also was variable (67.3 to 268.7 pgjml per cubic centimeter) but was not elevated significantly by rejection (Table III).

Histologic analysis. Gross and histologic examination of the transplanted lungs, bacterial and fungal cultures of the blood, and tracheal secretion were negative for significant infection. In the animals that underwent allotransplantation, the grade of pulmonary rejection was either 2 or 3.22 Further specific grading of the airways showed no bronchial involvement in any rejecting animal and grade I bronchiolar changes in one animal, grade 2 in three animals, and grade 3 in three animals. Histologic analysis of bronchi used in organ chamber studies confirmed the absence or presence of epithelium. Infiltration of lymphocytes into the bronchial muscle layer was absent or minimal in rings from rejected lungs. Discussion Previous studies have demonstrated differential effects of denervation and acute rejection on the function of pulmonary arteries after single lung transplantation in the canine model.P The present study examines changes in bronchial contractility with the same animal model. Denervation. In the present study, denervation altered bronchial contractions by two mechanisms: one affecting the bronchial smooth muscle and the other the bronchial epithelium. These effects were demonstrated with different agonists. Denervation did not result in atrophy of the

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 5

McLarty et al.

Native auto

100 80

~

C 0

ii

• .5 • as

..• III

u

.5

Autotransplant

100

• with epithelium o without epithelium

80

60

60

40

40

20

20 10

9

8

7

Native rejecting

100

80

60

60

40

40

20

20

0

0 9

8

I

9

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8

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100

80

10

, • • •, • '~6

0 , 10

0

C

80 1

• • • •

.~,

I

I

,

!

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Fig. 2. Cumulativeconcentration-response curvesto endothelin-l. Data are expressed as mean ± standard error of mean and representpercentageof increasein tensionof a maximalcontractionin response to KCI (60 mmoljL); n equals number of animals from which rings were taken. Rings from control lung without epitheliumcontracted more than those with epithelium (Native auto). Denervation eliminated this difference in contraction (p < 0.05, Autotransplant). Rejection did not significantly change responses in native (Native rejecting) or transplanted (Rejecting) lungs from those contractions seen after autotransplantation.

Table III. Content of endothelin-l in pulmonary tissue after lung transplantation Autotransplant

Allotransplant

Tissue

Native

Transplanted

Native

Rejecting transplanted

Bronchi (pg/rnl per gram) Intralobar pulmonary arteries (pg/rnl per gram) Lung parenchyma (pg/rnl per cubic centimeter)

172.9 ± 38.6 161.9 ± 42.8 85.1 ± 17.3

169.4 ± 25.0 350.6 ± 100.6 115.7 ± 31.9

438.4 ± 109.1 137.6 ± 26.7 111.9 ± 35.9

681.3 ± 240.4 438.1 ± 128.7 130.4 ± 29.1

Ratio of rejecting allotransplant/ autotransplant 4.0 1.2 1.12

Data are presented as mean ± standard error of mean. Native, Unoperated contralateral lung.

smooth muscle because contractions in response to potassium chloride, which directly depolarizes the smooth muscle, were not decreased, and histologic evidence of atrophy was not observed. However, maximal contractions of the bronchial smooth muscle (rings without epithelium) in response to acetylcholine and endothelin-I were decreased after autotransplantation. This is probably not a nonspecific decrease in the ability of the smooth muscle to contract because responses to 5-hydroxytryptamine and histamine were not affected in the same way. Rather, denervation probably affects selective intracellular mechanisms shared by both acetylcholine and endo-

thelin-I, which couple their receptors to activation of contractile proteins. Decreased contractions to acetylcholine are not consistent with clinical observations in human heart-lung transplant recipients in whom methacholine, a cholinergic agonist delivered by means of aerosol, caused bronchospasm with a fall in forced expiratory volume." The reasons for these differences in contraction of bronchial smooth muscle to cholinergic activation in vitro and in vivoare not clear. This difference may be due to the length of time between transplantation and the studies. Alternatively, it could represent differences between isometric (organ

The Journal of Thoracic and Cardiovascular Surgery November 1993

8 0 2 McLarty et at.

Native auto

Autotransplant

.DD! ' .", ..,...,. . 80

80 .DD! 60

0 without epithelium

~

C Ui 0

...

C Gl

60 40 20 0 9

.=

Gl

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.=

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40 20 0 7

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80 lDD! 60

8

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Rejecting

80 .DD! 60 40 20

40 20

o

o

I

9

8

7

6

4

5

I

9

8

7

Histamine, -log M Fig. 3. Cumulative concentration-response curves of third-order canine bronchi in response to histamine. Data are expressed as mean ± standard error of mean and represent percentage of increase in tension of a maximal contraction in response to KCI (60 mmol jL); n equals number of animals from which rings were taken. In control lung (Native auto), rings with epithelium contracted less than those without epithelium. Denervation caused an increase in contraction in rings with epithelium (Autotransplant, p < 0.05). Rejection did not significantly alter responses in native (Native rejecting) or transplanted (Rejecting) lungs. Native auto

Autotransplant

lDI' .", ......... 80

80 lDD~ 60

0 without epithelium

~

C 0 Ui

... C

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60 40 20

o

I

9

.=

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8

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40 20 0 7

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'D80 D[ 60

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40 20

o

8

n=7

I

9

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8

7

40 20

o ,

6 9 5 7 8 5-Hydroxytryptamine, -log M

n=7

6

5

Fig. 4. Cumulative concentration-response curves in response to 5-hydroxytryptamine. Data are expressed as mean ± standard error of mean and represent percentage of increase in tension of a maximal contraction in response to KCI (60 mmol/L): n equals number of animals from which rings were taken. Rings from control lung (Native auto) with epithelium contracted less than those without. This difference was lost in denervation, with an increase in contraction (p < 0.05) of rings with epithelium (Autotransplant). Rejection did not significantly alter responses in native (Native rejecting) or transplanted (Rejecting) lungs.

chamber) and isotonic (in vivo) contractions of the smooth muscle. In the latter, shortening of the smooth muscle would reduce airway diameter and therefore increase resistance to airflow. Another possible explana-

tion for the differences in study findings is the initial stimulation of only the epithelium by methacholine, with the release of an epithelium-derived contracting factor in vivo. In addition, in in vivo studies, there is a shear stress

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 5

of air movement across the epithelium that may affect release of epithelium-derived factors. The present study confirms that the bronchial epithelium modifies the tone of the underlying smooth muscle. Both basal and receptor-mediated release of an epithelium-derived relaxing factor(s) have been proposed.P This study suggests that denervation results in decreased release of epithelium-derived relaxing factor and subsequent increased contraction of the underlying smooth muscle. The absence of a difference in sensitivity between rings with and without epithelium to four different agonists favors the interpretation that basal release of epithelium-derived relaxing factor rather than receptor-operated mechanisms is affected by denervation. Therefore, it may be suggested that neurogenic input has trophic effects on basal release of epithelium-derived relaxing factor(s). The epithelium may modify the tone of larger (second-order) bronchi by basal release of relaxing factor and of smaller (fourth-order) airways by receptor-mediated release.P It is possible that in the third-order bronchi studied in the present experiments, basal release of these factors is also of greater importance. Decrease in basal release of an epithelium-derived relaxing factor may, in part, explain the clinical observation of hyperactive response to provocative airway tests in lung transplant recipients. Acute rejection. The degree of acute pulmonary rejection present in these experiments did not significantly alter bronchial contractions from that observed after denervation alone. This contrasts with findings in the pulmonary artery, where a clear distinction exists between receptor-mediated responses to agonists in rejected lungs and those responses in denervated lungs.l ' These differences may be related to the nature of pulmonary rejection. Rejection of the lung histologically begins as a perivascular phenomenon that later spreads to the airways. Bronchiolar involvement with rejection mayor may not be commensurate with blood vessel involvement. 22 The relative frequency of rejection affecting the cartilaginous bronchi remains unknown, but this rejection was not present in the animals in this study. The absence of a lymphocytic or mononuclear infiltrate around the cartilaginous bronchi, which were the subject of the organ chamber studies, may accountfor the absence of any further effects on bronchial contractions caused by acute rejection. The findings of this study do not exclude the possibility that bronchial contractions may be altered with more severe or prolonged rejection affecting the cartilaginous bronchi. The rejection process, however, seems to affect contraction of bronchi of the native lung because differences in contraction between rings with and without epithelium in response to acetylcholine and histamine were reduced

McLarty et al.

80 3

in this group. Histologic analysis of bronchi from native lungs did not show lymphocytic infiltrate or injury to the smooth muscle. Therefore, in the absence of an anatomic change such as denervation, these observations suggest a role of blood-borne factors, which are either released or not cleared from the rejecting allograft, in the altering of bronchial contractions. This idea is further supported by the observations that serum levels of angiotensin-converting enzyme decreased with acute rejection 13 and that tissue content of endothelin increased in native unoperated bronchi of the rejected allograft. Airway pressures. In vivo measurements of bronchial reactivity differed qualitatively from in vitro results. Peak inspiratory _airway pressure increased with rejection; however, in organ chamber studies, maximal bronchial contractions were reduced. Because airway pressures were unchanged after autotransplantation and in the contralateral lung of rejecting allografts, the increase in airway pressure in the rejecting lung may reflect tissue edema and decreased compliance of the pulmonary tissue rather than change in smooth muscle tone. Alternatively, humoral factors in vivo may affect bronchial tone, and, as discussed previously, shortening of the bronchial smooth muscle may participate in the increased resistance to airflow in the bronchi of rejecting lungs in vivo. We thank Drs. Amita Rastogi,Giovanni Spezialli, and Pertti Aarnio and Ronald Lee for assistancein surgicalpreparation of the animals, Denise Heublein from Dr. John Burnett's laboratory forperformingthe assaysforendothelin, Robert Lorenz forpreparationofthe illustrations, and EllenGladwelland Amy Pelot for secretarial assistance.

I.

2.

3.

4.

5. 6.

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7. Higenbottam T, Jackson M, Rashdi T, Stewart S, Coutts C, Wallwork J. Lung rejection and bronchial hyperresponsiveness to methacholine and ultrasonically nebulized distilled water in heart-lung transplantation patients. Am Rev Respir Dis 1989;140:52-7. 8. Barnes PJ, Cuss FM, Palmer JB. The effect of airway epithelium on smooth muscle contractility in bovine trachea. Br J Pharmacol 1985;86:685-92. 9. Flavahan NA, Aarhus LL, Rimele TJ, Vanhoutte PM. Respiratory epithelium inhibits bronchial smooth muscle tone. J Appl Physiol 1985;58:834-8. 10. Flavahan NA, Vanhoutte PM. The respiratory epithelium releases a smooth muscle relaxing factor. Chest 1985;87: I89S-90S. II. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985;131:599606. 12. Boushey HA, Holtzman MJ, Sheller JR, Nadel JA. Bronchial hyperreactivity. Am Rev Respir Dis 1980;121:389413. 13. Nilsson FN, Miller VM, McGregor CGA. Pulmonary arterial reactivity after transplantation: differential effects of denervation and rejection. J THORAC CARDIOVASC SURG 1992;103:751-62. 14. Stuart-Smith K, Vanhoutte PM. Heterogeneity in the effects of epithelium removal in the canine bronchial tree. J Appl PhysiolI987;63:2510-5. 15. Stuart-Smith K, Vanhoutte PM. Epithelium, contractile

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