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Inhibition of surfactant by lung allograft extracts
Inhibition of surfactant by lung allograft extracts Studies of surfactant following lung reimplantation and transplantation have given contradictory data. Most of these studies have measured surface tension on a modified Wilhelmy balance as an indicator of surfactant but this method has certain limitations. We demonstrated a significant inhibition of surface tension in normal dog lung extracts by transplanted dog lung extracts. We concluded that surfactant may be present in lung extracts but inhibited by various substances, especially in diseased lungs. Therefore, if low surface tension does not develop, only a limited conclusion is warranted. Quantitative data showing increased surfactant turnover in lung allografts as compared to lung autografts or normals have been presented elsewhere.
John A. Drews, M.D., * Donald F. Tierney, M.D., and John R. Benfield, M.D., Torrance and Los Angeles, Calif.
Most measurements of the surface tension of endobronchial lung washings or of lung tissue extracts in recent years have utilized some modification of the method described by Clements' in which surface tension is measured on a modified Wilhelmy balance.> Studies involving canine lung reimplantation, transplantation, or pure vagal denervation have resulted in contradictory data. For example, Trimble and associates" measured a decrease in surface activity of lung extracts following reimplantation whereas Waldhausen and associatesobserved no change. Other reports have supported one or the other of these findings."" The purpose of our study was to find explanations for earlier conflicting results by carefully repeating certain aspects of previFrom the Departments of Surgery and Medicine, Harbor General Hospital, Torrance, and the UCLA School of Medicine, Los Angeles. Supported in part by Research Grants No. HL 13077 and No. HL 14474 from the National Heart and Lung Institute, National Institutes of Health, Bethesda, Md. Received for publication March 18, 1974. Address for reprints: Dr. John R. Benfield, Harbor General Hospital, Box 250, 1000 W. Carson St., Torrance, Calif. 90509. • Present address: Section of Cardio-Thoracic Surgery, Yale-New Haven Hospital, New Haven, Conn.
ously reported surface tension measurements and additionally by studying the surface activity of dipalmitoyl lecithin (DPL), the major surface-active pulmonary phospholipid. Method
Our method was a recent modification of that described by Clements, Nellen, and Trahan." A Teflon trough with inside dimensions of 3.5 by 55 em, was filled with a 0.9 per cent NaCI solution. Weighed samples of lung tissue were homogenized thoroughly with 30 times their weight of normal saline solution and centrifuged at 500 g for 5 minutes. A small sample of the supernatant was dispersed in twice its volume of isopropyl alcohol. The extracts were spread on the 0.9 per cent NaCI solution in increments of 10 A prior to each measurement. Initial surface areas of 192.5 sq. em. were decreased by compression at a constant rate of 4.1 sq. em. per second with a tight-fitting Teflon barrier until a final 7.0 sq. em. area around a platinum plate was achieved. The platinum, rectangular plate was placed at one end of the trough and partially submerged to form a meniscus, and was suspended from a
837
The Journal of
838
Drews, Tierney, Benfield
Thoracic and Cardiovascular Surgery
80 70
. ! E ~
== = = = = = = = = = = = = = = = = = = =~: : : :
HIS CONTROL
60 50
z
Q
Ul
zw
40
I-
PURE
w 30
u
:a:
:>
Ul
DPL
CURVE
20 10 0
0%
100%
Fig. 1. Surface-tension balance tracing of pure DPL solution. Lower portion of tracing represents compression of surface area; upper portion of tracing is normal hysteresis on reexpansion. Note that curve falls well below 12 dynes per centimeter (N/S control curve at 72 dynes per centimeter is shown).
Statham transducer. Changes in surface tension (Y-axis) were plotted against surfacearea changes (X-axis) with an X-Y recorder. * In each instance, the area which the surface film occupied when it was compressed so that surface tension fell to 12 dynes per centimeter or less was used to estimate the amount of surface-active material contained in the sample. We assumed that no other substance but pulmonary surfactant remains in the surface film when the surface tension is 12 dynes per centimeter or lower. By means of the above method, surfacetension-area curves were obtained from the following groups: (l) pure DPL (1.0 mg. in 5 C.c. of isopropyl alcohol) t; (2) pure DPL extracts, plus added contaminants of cholesterol and oleic acid (1.0 mg. in 5 C.c. of isopropyl alcohol); (3 ) normal dog lung extracts (obtained by open biopsy) and endobronchial washings (obtained by lavaging the right accessory lobe of normal dogs with N/S with a balloon catheter inserted under direct bronchoscopic vision); (4) autografted dog lung extracts; 'Houston Instruments, Bellaire, Texas. tCaIbiochem Laboratory, San Diego, Calif.
(5) allografted dog lung extracts; (6) allograft extract plus normal lung extract mixtures. Result
Extracts of pure DPL in isopropyl alcohol were used in seven experiments to measure surface tension of a known surfaceactive material. All of these showed a drop in surface tension to well below 12 dynes per centimeter (Fig. 1). However, on six other tracings, where extracts of identical amounts of DPL were mixed with cholesterol or oleic acid extracts, none of the curves fell below 12 dynes per centimeter. Eight normal dogs had lung parenchymal extracts placed on the surface-tension balance. Six of these exhibited drops in surface tension to 12 dynes per centimeter (Fig. 2) and two did not. Eight other normal dogs underwent lobar endobronchial lavage and the fluid return was used in the surface-tension balance. None of these eight exhibited a drop in surface tension below 12 dynes per centimeter. Lung tissue extracts from five animals with reimplanted lungs showed one in which the curve fell just to 12 dynes per centimeter and four in which the curve did not. Of five
Volume 68
Inhibition of surfactant by lung allograft extracts
Number 5 November, 1974
839
80
NIS CONTROL
70
E ~
"~
60
50
~
z
0
40
(ij Z
NORMAL LUNG EXTRACT
LU .... LU
o ~
30
a: :::>
20
10
0
100%
0%
Fig. 2. Surface-tension balance tracing of normal dog lung extract. Curve falls below 12 dynes per centimeter, indicating presence of highly surface-active material, probably surfactant. 80
NIS CONTROL
70
~
60
§
50
., c
z
0
(ij Z
40
....
LU LU
u
TRANSPLANT -EXTRACT
30
~
a: :::>
20
NORMAL EXTRACT
12
(30),)
(30),)
0 100%
0%
Fig. 3. Surface-tension balance tracings of normal lung extract, transplant lung extract, and a mixed extract. (Hysteresis curves have been deleted for clarity.) Note that the transplant extract significantly inhibits the surfactant present in normal lung extract (P < 0.05). extracts of transplanted lungs, none developed surface tensions below 12 dynes per centimeter. Finally, in four separate studies, an amount of normal lung extract which would produce surface tensions below 12 dynes per centimeter (30 A) was mixed with an equal quantity of extract from transplanted lungs. In all of these, the surface tension fell to 12 dynes per centimeter, but at a significantly
more reduced area (mean = 10.5 sq. em.) than when the measurement was made with the normal extract alone (mean = 18.0 sq. em.) (P < 0.05) (see Fig. 3).
Discussion Our understanding of pulmonary surface forces has been advanced greatly by the use of the modified Wilhelmy surface balance as applied initially by Clements. However,
The Journol of
840
Drews, Tierney, Benfield
Thoracic and Cardiovascular Surgery
the method has significant limitations which have not always been considered. The saline extract is obviously a mixture of substances including not only surfactant but other cellular components which may also be surface active. We have used a more recent modification of the method which has been used successfully to estimate the quantity of surface-active material in normal lungs. However, this new method has not previously been reported to be used with lungs thought to have a high surface tension associated with diseases. Many attempts have been made to determine the possible change of surfactant after lung transplantation and the results are conflicting. Most of these reports relied upon an aqueous extract (0.9 per cent NaCl) from minced lung to obtain surfactant. The surfactant was then considered to be present or absent depending upon the minimum surface tension obtained with the Wilhelmy balance. Unfortunately, those methods were not quantitative and the possibility of inhibition or inactivation of the surfactant by substances released during the extraction procedure was not considered. We have tried to make a more nearly quantitative extraction of the surfactant by using Clements' newer method and we have attempted to evaluate the role of inhibition of the surfactant by mixing extracts of normal lungs with extracts from transplanted lungs. Although the surface tension fell to 12 dynes per centimeter when extracts of normal and abnormal lungs were mixed, the surface area at 12 dynes per centimeter was much less than when only the normal extract was used. This result implied a partial loss of surfactant or inhibition of its effect. It indicated that the estimate of quantity of surfactant by this method was not accurate when diseased lungs were used. Presumably the diseased lungs contain a relative excess of blood, plasma, and perhaps cellular debris which could inhibit the surfactant. This inhibition may not occur in situ if the surfactant is physically separated from the inhibiting material. However, if the inhibiting ma-
terial enters the airspace with pulmonary edema then we would expect the surfactant to be inhibited in situ. The conflicting reports in the literature may also reflect technical differences. As noted in our results, consistent tracings from even normal lung extracts were not obtained. We attributed these results to technical differences. For instance, an optimum extraction procedure would selectively remove surfactant and not inhibitors which may be present in the tissues. Homogenization may result in a thorough extraction of the surfactant but probably includes inhibitors from cells. If a simple saline extraction technique is to be used, then mincing an inflated lung in saline would selectively remove surfactant from the airspaces. In contrast, mincing an atelectatic lung may not give the surfactant on the alveolar surface an opportunity to leave the lung and enter the saline." Another major problem is to spread the surfactant on the surface rather than having it remain in the bulk phase. It is likely that some "inhibitors" of the surfactant are in reality micelles or large particles which essentially dissolve the surfactant. Chylomicrons, for instance;" can inhibit the surfactant with a simple saline extract. To overcome this problem, Clements introduced the use of isopropyl alcohol which dissolved the lipids. When applied directly to an aqueous surface, this provided a more complete spreading of the surfactant at the surface. Although this approach is satisfactory with normal lungs, it apparently is not adequate when diseased lungs are used. We conclude that even recent modifications of the Wilhelmy balance have limited value when used to estimate surfactant quantities in diseased lungs. If lung surface tensions (below 12 dynes per centimeter) develop, it indicates that a highly surface-active substance (not necessarily surfactant) is present. However, if low surface tensions do not develop, the surfactant may be present, but inhibited. We have recently presented quantitative data showing that turnover occurs at a more
Volume 68 Number 5 November, 1974
Inhibition of surfactant by lung allograft extracts
rapid rate in lung allografts than in autografts or normals. We concluded that this greater turnover is probably a direct result of rejection. 11 REFERENCES
2
3
4
5
Clements, 1. A.: Surface Tension of Lung Extracts, Proc. Soc. Exp. BioI. Med. 95: 170, 1957. Clements, J. A., Nellen, B. J., and Trahan, H. J.: Pulmonary Surfactant and Evolution of the Lungs, Science 169: 603, 1970. Trimble, A. S., Kim, J., Bharadwaj, R., Bedard, P., and Wells, C.: Changes in Alveolar Surfactant After Lung Reimplantation, J. THORAc. CARDIOVASC. SURG. 52: 271, 1966. Waldhausen, J. A., Giammona, S. T., Kilman, J. W., and Daly, W. J.: Effect of Transplantation of Canine Lung on Pulmonary Compliance and Surfactant, J. A. M. A. 191: 1002, 1965. Yakeishi, Y., Nozaki, M., Rangel, D. M., Stevens, G. H., Adams, F. H., and Fonkalsrud, E. W. : Effect of Allotransplanation of the
6
7
8
9
10
11
84 1
Canine Lung on Pulmonary Surfactant, Surg. Gynecol, Obstet. 128: 1264, 1969. Lincoln, J. C. R., Gould, B. T., and Reynolds, E. O. R.: Pulmonary Mechanics and Surfactant Measurement in Canine Lungs Following Reimplantation, Thorax 25: 180, 1970. Yeh, T. J., Ellison, L. T., and Ellison, R. G.: Alveolar Surfactant in Lung Homotransplantation and Hilar Stripping, Surg. Forum 15: 191, 1964. Bolande, R. P., and Klaus, M. H.: The Morphologic Demonstration of an Alveolar Lining Layer and Its Relationship to Pulmonary Surfactant, Am. J. Pathol, 45: 449, 1964. Levine, B. E., and Johnson, R. P.: Surface Activity of Saline Extracts From Inflated and Degassed Normal Lungs, J. Appl, Physiol, 19: 333, 1964. Tierney, D. F., and Johnson, R. P.: Altered Surface Tension of Lung Extracts and Lung Mechanics, J. Appl, PhysioI. 20: 1253, 1965. Drews, J. A., Tierney, D. F., and Benfield, J. R.: Effect of Immunosuppression and Lung Transplantation Upon Surfactant, Surgery 76: 80, 1974.
John A. Drews, M.D., * Donald F. Tierney, M.D., and John R. Benfield, M.D., Torrance and Los Angeles, Calif.
Most measurements of the surface tension of endobronchial lung washings or of lung tissue extracts in recent years have utilized some modification of the method described by Clements' in which surface tension is measured on a modified Wilhelmy balance.> Studies involving canine lung reimplantation, transplantation, or pure vagal denervation have resulted in contradictory data. For example, Trimble and associates" measured a decrease in surface activity of lung extracts following reimplantation whereas Waldhausen and associatesobserved no change. Other reports have supported one or the other of these findings."" The purpose of our study was to find explanations for earlier conflicting results by carefully repeating certain aspects of previFrom the Departments of Surgery and Medicine, Harbor General Hospital, Torrance, and the UCLA School of Medicine, Los Angeles. Supported in part by Research Grants No. HL 13077 and No. HL 14474 from the National Heart and Lung Institute, National Institutes of Health, Bethesda, Md. Received for publication March 18, 1974. Address for reprints: Dr. John R. Benfield, Harbor General Hospital, Box 250, 1000 W. Carson St., Torrance, Calif. 90509. • Present address: Section of Cardio-Thoracic Surgery, Yale-New Haven Hospital, New Haven, Conn.
ously reported surface tension measurements and additionally by studying the surface activity of dipalmitoyl lecithin (DPL), the major surface-active pulmonary phospholipid. Method
Our method was a recent modification of that described by Clements, Nellen, and Trahan." A Teflon trough with inside dimensions of 3.5 by 55 em, was filled with a 0.9 per cent NaCI solution. Weighed samples of lung tissue were homogenized thoroughly with 30 times their weight of normal saline solution and centrifuged at 500 g for 5 minutes. A small sample of the supernatant was dispersed in twice its volume of isopropyl alcohol. The extracts were spread on the 0.9 per cent NaCI solution in increments of 10 A prior to each measurement. Initial surface areas of 192.5 sq. em. were decreased by compression at a constant rate of 4.1 sq. em. per second with a tight-fitting Teflon barrier until a final 7.0 sq. em. area around a platinum plate was achieved. The platinum, rectangular plate was placed at one end of the trough and partially submerged to form a meniscus, and was suspended from a
837
The Journal of
838
Drews, Tierney, Benfield
Thoracic and Cardiovascular Surgery
80 70
. ! E ~
== = = = = = = = = = = = = = = = = = = =~: : : :
HIS CONTROL
60 50
z
Q
Ul
zw
40
I-
PURE
w 30
u
:a:
:>
Ul
DPL
CURVE
20 10 0
0%
100%
Fig. 1. Surface-tension balance tracing of pure DPL solution. Lower portion of tracing represents compression of surface area; upper portion of tracing is normal hysteresis on reexpansion. Note that curve falls well below 12 dynes per centimeter (N/S control curve at 72 dynes per centimeter is shown).
Statham transducer. Changes in surface tension (Y-axis) were plotted against surfacearea changes (X-axis) with an X-Y recorder. * In each instance, the area which the surface film occupied when it was compressed so that surface tension fell to 12 dynes per centimeter or less was used to estimate the amount of surface-active material contained in the sample. We assumed that no other substance but pulmonary surfactant remains in the surface film when the surface tension is 12 dynes per centimeter or lower. By means of the above method, surfacetension-area curves were obtained from the following groups: (l) pure DPL (1.0 mg. in 5 C.c. of isopropyl alcohol) t; (2) pure DPL extracts, plus added contaminants of cholesterol and oleic acid (1.0 mg. in 5 C.c. of isopropyl alcohol); (3 ) normal dog lung extracts (obtained by open biopsy) and endobronchial washings (obtained by lavaging the right accessory lobe of normal dogs with N/S with a balloon catheter inserted under direct bronchoscopic vision); (4) autografted dog lung extracts; 'Houston Instruments, Bellaire, Texas. tCaIbiochem Laboratory, San Diego, Calif.
(5) allografted dog lung extracts; (6) allograft extract plus normal lung extract mixtures. Result
Extracts of pure DPL in isopropyl alcohol were used in seven experiments to measure surface tension of a known surfaceactive material. All of these showed a drop in surface tension to well below 12 dynes per centimeter (Fig. 1). However, on six other tracings, where extracts of identical amounts of DPL were mixed with cholesterol or oleic acid extracts, none of the curves fell below 12 dynes per centimeter. Eight normal dogs had lung parenchymal extracts placed on the surface-tension balance. Six of these exhibited drops in surface tension to 12 dynes per centimeter (Fig. 2) and two did not. Eight other normal dogs underwent lobar endobronchial lavage and the fluid return was used in the surface-tension balance. None of these eight exhibited a drop in surface tension below 12 dynes per centimeter. Lung tissue extracts from five animals with reimplanted lungs showed one in which the curve fell just to 12 dynes per centimeter and four in which the curve did not. Of five
Volume 68
Inhibition of surfactant by lung allograft extracts
Number 5 November, 1974
839
80
NIS CONTROL
70
E ~
"~
60
50
~
z
0
40
(ij Z
NORMAL LUNG EXTRACT
LU .... LU
o ~
30
a: :::>
20
10
0
100%
0%
Fig. 2. Surface-tension balance tracing of normal dog lung extract. Curve falls below 12 dynes per centimeter, indicating presence of highly surface-active material, probably surfactant. 80
NIS CONTROL
70
~
60
§
50
., c
z
0
(ij Z
40
....
LU LU
u
TRANSPLANT -EXTRACT
30
~
a: :::>
20
NORMAL EXTRACT
12
(30),)
(30),)
0 100%
0%
Fig. 3. Surface-tension balance tracings of normal lung extract, transplant lung extract, and a mixed extract. (Hysteresis curves have been deleted for clarity.) Note that the transplant extract significantly inhibits the surfactant present in normal lung extract (P < 0.05). extracts of transplanted lungs, none developed surface tensions below 12 dynes per centimeter. Finally, in four separate studies, an amount of normal lung extract which would produce surface tensions below 12 dynes per centimeter (30 A) was mixed with an equal quantity of extract from transplanted lungs. In all of these, the surface tension fell to 12 dynes per centimeter, but at a significantly
more reduced area (mean = 10.5 sq. em.) than when the measurement was made with the normal extract alone (mean = 18.0 sq. em.) (P < 0.05) (see Fig. 3).
Discussion Our understanding of pulmonary surface forces has been advanced greatly by the use of the modified Wilhelmy surface balance as applied initially by Clements. However,
The Journol of
840
Drews, Tierney, Benfield
Thoracic and Cardiovascular Surgery
the method has significant limitations which have not always been considered. The saline extract is obviously a mixture of substances including not only surfactant but other cellular components which may also be surface active. We have used a more recent modification of the method which has been used successfully to estimate the quantity of surface-active material in normal lungs. However, this new method has not previously been reported to be used with lungs thought to have a high surface tension associated with diseases. Many attempts have been made to determine the possible change of surfactant after lung transplantation and the results are conflicting. Most of these reports relied upon an aqueous extract (0.9 per cent NaCl) from minced lung to obtain surfactant. The surfactant was then considered to be present or absent depending upon the minimum surface tension obtained with the Wilhelmy balance. Unfortunately, those methods were not quantitative and the possibility of inhibition or inactivation of the surfactant by substances released during the extraction procedure was not considered. We have tried to make a more nearly quantitative extraction of the surfactant by using Clements' newer method and we have attempted to evaluate the role of inhibition of the surfactant by mixing extracts of normal lungs with extracts from transplanted lungs. Although the surface tension fell to 12 dynes per centimeter when extracts of normal and abnormal lungs were mixed, the surface area at 12 dynes per centimeter was much less than when only the normal extract was used. This result implied a partial loss of surfactant or inhibition of its effect. It indicated that the estimate of quantity of surfactant by this method was not accurate when diseased lungs were used. Presumably the diseased lungs contain a relative excess of blood, plasma, and perhaps cellular debris which could inhibit the surfactant. This inhibition may not occur in situ if the surfactant is physically separated from the inhibiting material. However, if the inhibiting ma-
terial enters the airspace with pulmonary edema then we would expect the surfactant to be inhibited in situ. The conflicting reports in the literature may also reflect technical differences. As noted in our results, consistent tracings from even normal lung extracts were not obtained. We attributed these results to technical differences. For instance, an optimum extraction procedure would selectively remove surfactant and not inhibitors which may be present in the tissues. Homogenization may result in a thorough extraction of the surfactant but probably includes inhibitors from cells. If a simple saline extraction technique is to be used, then mincing an inflated lung in saline would selectively remove surfactant from the airspaces. In contrast, mincing an atelectatic lung may not give the surfactant on the alveolar surface an opportunity to leave the lung and enter the saline." Another major problem is to spread the surfactant on the surface rather than having it remain in the bulk phase. It is likely that some "inhibitors" of the surfactant are in reality micelles or large particles which essentially dissolve the surfactant. Chylomicrons, for instance;" can inhibit the surfactant with a simple saline extract. To overcome this problem, Clements introduced the use of isopropyl alcohol which dissolved the lipids. When applied directly to an aqueous surface, this provided a more complete spreading of the surfactant at the surface. Although this approach is satisfactory with normal lungs, it apparently is not adequate when diseased lungs are used. We conclude that even recent modifications of the Wilhelmy balance have limited value when used to estimate surfactant quantities in diseased lungs. If lung surface tensions (below 12 dynes per centimeter) develop, it indicates that a highly surface-active substance (not necessarily surfactant) is present. However, if low surface tensions do not develop, the surfactant may be present, but inhibited. We have recently presented quantitative data showing that turnover occurs at a more
Volume 68 Number 5 November, 1974
Inhibition of surfactant by lung allograft extracts
rapid rate in lung allografts than in autografts or normals. We concluded that this greater turnover is probably a direct result of rejection. 11 REFERENCES
2
3
4
5
Clements, 1. A.: Surface Tension of Lung Extracts, Proc. Soc. Exp. BioI. Med. 95: 170, 1957. Clements, J. A., Nellen, B. J., and Trahan, H. J.: Pulmonary Surfactant and Evolution of the Lungs, Science 169: 603, 1970. Trimble, A. S., Kim, J., Bharadwaj, R., Bedard, P., and Wells, C.: Changes in Alveolar Surfactant After Lung Reimplantation, J. THORAc. CARDIOVASC. SURG. 52: 271, 1966. Waldhausen, J. A., Giammona, S. T., Kilman, J. W., and Daly, W. J.: Effect of Transplantation of Canine Lung on Pulmonary Compliance and Surfactant, J. A. M. A. 191: 1002, 1965. Yakeishi, Y., Nozaki, M., Rangel, D. M., Stevens, G. H., Adams, F. H., and Fonkalsrud, E. W. : Effect of Allotransplanation of the
6
7
8
9
10
11
84 1
Canine Lung on Pulmonary Surfactant, Surg. Gynecol, Obstet. 128: 1264, 1969. Lincoln, J. C. R., Gould, B. T., and Reynolds, E. O. R.: Pulmonary Mechanics and Surfactant Measurement in Canine Lungs Following Reimplantation, Thorax 25: 180, 1970. Yeh, T. J., Ellison, L. T., and Ellison, R. G.: Alveolar Surfactant in Lung Homotransplantation and Hilar Stripping, Surg. Forum 15: 191, 1964. Bolande, R. P., and Klaus, M. H.: The Morphologic Demonstration of an Alveolar Lining Layer and Its Relationship to Pulmonary Surfactant, Am. J. Pathol, 45: 449, 1964. Levine, B. E., and Johnson, R. P.: Surface Activity of Saline Extracts From Inflated and Degassed Normal Lungs, J. Appl, Physiol, 19: 333, 1964. Tierney, D. F., and Johnson, R. P.: Altered Surface Tension of Lung Extracts and Lung Mechanics, J. Appl, PhysioI. 20: 1253, 1965. Drews, J. A., Tierney, D. F., and Benfield, J. R.: Effect of Immunosuppression and Lung Transplantation Upon Surfactant, Surgery 76: 80, 1974.