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Phagocytosis by Alveolar Macrophages: Pharmacologic Features
208
15TH ANNUAL ASPEN CONFERENCE
adverse reaction? Dr. Farr: It would be arduous to do the testing on enough "normals" to find the occasional adverse reaction. Dr. Gary Gross, Denver: Regarding the propellant, in a patient at National Jewish Hospital, the pulmonary function deteriorated in response to the inhaled medication but not in response to the propellant. Dr. Godfrey: There may be a cultural difference. Children in Great Britain stay at home longer, whereas children in the United States see the doctor sooner. Thus, because they could treat themselves at home in Britain this may have caused an even further delay in obtaining the doctors. Dr. Stolley: I think that the pharmacologic effect
was more important than the cultural effect. This is suggested by the fact that Norway had a higher death rate from asthma than Denmark or Sweden during the 1960's. Norway marketed the concentrated isoproterenol and Sweden and Denmark didn't. Dr. Nadel: What is the acute effect of these aerosols? If the pH of the solution is acid, this in itself could be a factor. Also, regarding the death rate, did the type of nebulizer or the cleaning procedure make a difference? Dr. Reisman: What kind of nebulizers were used? Dr. Stolley: This information can only be obtained by history and with great difficulty from the survivors of those who died. The highly concentrated preparation was over-represented in the death rate.
SESSION IV: PULMONARY DEFENSES
Phagocytosis by Alveolar Macrophages: Pharmacologic Features· J. B. L. Gee, M.D .; F. L. Sacs, M.D .; P. McKeever, M.D .; J. S. Douglas, M.D.; and S. E. Malawista, M.D. The alveolar macrophage (AM) plays an essential clearance and antimicrobial role in the lung by virtue of its phagocytic capacity. We have studied the process of phagocytosis in the AM employing a number of pharmacologic agents in order to elucidate some of the basic mechanisms. Phagocytosis may be considered as three interrelated processes: 1) particle:cell membrane reaction; 2) particle entry or ingestion; 3) secondary metabolic events which include the triad of increases on Q02, glucose oxidation and H 202 formation together with release of lysosomal enzymes. The AM were isolated from rabbits and studied in vitro employing staphylococci as test particles. The effects of certain drugs on the following features of phagocytosis were observed: 1) particle entry by light microscopy of Gram stained preparations and by clearance of live bacteria from the supernatant from shaking bacteria-AM suspensions; 2) 02 consumption and 14(:02 production from 14C-Iabeled substrates, namely 14C-l-glucose, 14C-6-glucose, 14C-formate, 14C-l-pyruvate, 14C-lacetate and 14C-I-4-succinate; 3) lysosomal activation by release of lysozyme (muramidase) into the incubation medium. Two key enzymes of the pen-From the Department of Internal Medicine and Yale University Lung Research Center, Yale University, New Haven.
tose shunt were measured, namely, glucose-B-phcsphate and 6-phosphogluconis acid dehydrogenases. Phospholipase C (PLC)
This enzyme (phosphatidylcholine-choline-phosphohydrolase EC 3.1.4.3) cleaves L-a-Iecithin to form diglycerides and phosphoryl choline. Exposure of AM to PLC (0.25 units/ml final concentration) in the presence of ImEq Ca'" evokes changes quantitatively very similar to those observed in phagocytosis. 1 Q02 increased by 35 percent. 14C02 production from the following substrates also increased: 1) 14C-l-glucose by 400 percent; 2) 14C_6_ glucose by 342 percent; 3) HC-l-pyruvate by 262 percent. PLC did not change the 14C02 production from two substrates whose conversion to CO 2 depends on mitochondrial activity, namely 14(:-1acetate and 14C-1-4-succinate. However, increased H 202 formation in the presence of PLC may be inferred from the increase (60 percent) in 14C02 production from 14C-formate since the latter has previously been shown to be a catalase: H 202 dependent reaction.1 Finally, PLC evokes release of intracellular lysozyme into the incubation medium. These features demonstrate the similar metabolic patterns between phagocytosis and PLC . It is suggested that both PLC and phagocytosis evoke
CHEST, VOL 63, NO.4, APRIL 1973 SUPPLEMENT
215
15TH ANNUAL ASPEN CONFERENCE
these changes as a result of effects on cell membrane phospholipids. Cytochalasin B
This agent (2 y I ml) disrupts microfilaments in many cell types including the AM.2 It does not impair microtubular morphology. Quantitative bacteriologic methods demonstrate diminished uptake of live Staphylococcus aureus from shaking bacteriaAM suspensions in the presence of cytochalasin B. These effects are associated with striking diminution of "C02 production from 14C-I-glucose and 14C-6-glucose (77 percent and 42 percent respectively in AM exposed to killed Staphylococcus epidermidis) . Cytochalasin B did not affect 14C02 production from 14C-I-pyruvate, 14C-I-acetate or 14C_I_4 succinate, nor impair the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase. Further, the effects are reversible by washing out the cytochalasin B. The association of diminished bacterial 'ingestion and disruption of the microfilaments suggests that the latter serve as actin-like contractile proteins in the ingestion process. The diminution of C02 formation from labelled glucose may possibly arise from a combination of diminished ingestion and impaired glucose transport. The latter possibility is suggested by preliminary evidence indicating that the uptake by AM of the transportable but nonmetabolized glucose analogue, 2-deoxyglucose is inhibited by 2 ylml of cytochalasin B. Drugs Mediated by Cyclic AMP
Agents presumed to elevate intracellular cyclic
AMP, such as theophylline (10"3 M), prostaglandin E I ( I0"4M ), the cyclic AMP analogue, dibutyryl cyclic AMP (10"3 M) and also dibutyryl cyclic 3', 5' guanosine-monophosphate all produce concentration dependent diminution of 14C02 production from both I4C-I-glucose and 14C-6-glucose in both resting and phagocytosing AM. These agents did not impair bacterial uptake. Isoproteronol (up to 10"3 M), epinephrine (10"3 M) and sublethal concentrations of propranolol had no effects on either glucose conversion to CO 2 or -bacterial uptake. In the absence of measurements of cyclic AMP, it is not possible to make definite statements, but it appears likely that cyclic AMP regulates either glucose transport or glucose metabolism without effects on particle entry. CoNCLUSION
These studies are examples of the use of pharmacologic agents to analyze some mechanisms of phagocytosis in an important pulmonary defense cell. They suggest that: I) the metabolic features of phagocytosis may result from modification of cell membrane phospholipids, 2) AM phagocytosis depends on contractile microfilaments, and 3) cyclic AMP regulates glucose metabolism in the AM. REFERENCES
1 Gee JBL, Vassallo CL, Kaskin J, et al: Catalase dependent peroxidative metabolism in alveolar macrophages during phagocytosis. J Clin Invest 49 :1280-87, 1970 2 Malawista SE, Gee JBL, Bensch KG: Cytochalasin B reversibly inhibits phagocytosis . Functional, metabolic and ultrastructural effects in human blood leukocytes and rabbit alveolar macrophages, Yale Bioi Med 44:286-300, 1971
Retarded Clearance of Macroaggregated Albumin from the Lung of Asthmatic Patients" W . Busse, M.D. ; C. E. Reed, M.D.; I. Tyson, M.D.; and M. Birnbaum, M.D.
During the course of study of an asthmatic patient who had more than ten episodes of atelectasis of the left lower lobe from mucous plugs, a lung scan with macroaggregated albumin lSI I (MAAlSI I) was performed. The scan (not the injection of isotope) was repeated daily . Radioactivity appeared normally in the lung, but unexpectedly remained there for five days. Radioactivity is normally cleared from the lung with a half-time of 4.5 to 10 hours.1.2 Lung scans were performed in 14 asthmatic °From the University of Wisconsin Medical School, Madison.
patients, ranging in age from 18 to 49 years old, and in severity ranging from mild to very severe. Half the patients required either daily or alternate day prednisone. There was prolonged retention of radioactivity in the lungs of the asthmatics with median radioactivity half-time of 17.5 hours. Normal values at our institution are 8-10 hours (Fig I) . The degree of retention of radioactivity was not dependent upon the severity of the asthma or the use of corticosteroids. Swiss-Webster mice were given 109 Bordetella pertussis organisms intraperitoneally. The intraperitoneal or intravenous injection of B pertussis can
CHEST, VOL. 63, NO.4, APRIL 1973 SUPPLEMENT
15TH ANNUAL ASPEN CONFERENCE
adverse reaction? Dr. Farr: It would be arduous to do the testing on enough "normals" to find the occasional adverse reaction. Dr. Gary Gross, Denver: Regarding the propellant, in a patient at National Jewish Hospital, the pulmonary function deteriorated in response to the inhaled medication but not in response to the propellant. Dr. Godfrey: There may be a cultural difference. Children in Great Britain stay at home longer, whereas children in the United States see the doctor sooner. Thus, because they could treat themselves at home in Britain this may have caused an even further delay in obtaining the doctors. Dr. Stolley: I think that the pharmacologic effect
was more important than the cultural effect. This is suggested by the fact that Norway had a higher death rate from asthma than Denmark or Sweden during the 1960's. Norway marketed the concentrated isoproterenol and Sweden and Denmark didn't. Dr. Nadel: What is the acute effect of these aerosols? If the pH of the solution is acid, this in itself could be a factor. Also, regarding the death rate, did the type of nebulizer or the cleaning procedure make a difference? Dr. Reisman: What kind of nebulizers were used? Dr. Stolley: This information can only be obtained by history and with great difficulty from the survivors of those who died. The highly concentrated preparation was over-represented in the death rate.
SESSION IV: PULMONARY DEFENSES
Phagocytosis by Alveolar Macrophages: Pharmacologic Features· J. B. L. Gee, M.D .; F. L. Sacs, M.D .; P. McKeever, M.D .; J. S. Douglas, M.D.; and S. E. Malawista, M.D. The alveolar macrophage (AM) plays an essential clearance and antimicrobial role in the lung by virtue of its phagocytic capacity. We have studied the process of phagocytosis in the AM employing a number of pharmacologic agents in order to elucidate some of the basic mechanisms. Phagocytosis may be considered as three interrelated processes: 1) particle:cell membrane reaction; 2) particle entry or ingestion; 3) secondary metabolic events which include the triad of increases on Q02, glucose oxidation and H 202 formation together with release of lysosomal enzymes. The AM were isolated from rabbits and studied in vitro employing staphylococci as test particles. The effects of certain drugs on the following features of phagocytosis were observed: 1) particle entry by light microscopy of Gram stained preparations and by clearance of live bacteria from the supernatant from shaking bacteria-AM suspensions; 2) 02 consumption and 14(:02 production from 14C-Iabeled substrates, namely 14C-l-glucose, 14C-6-glucose, 14C-formate, 14C-l-pyruvate, 14C-lacetate and 14C-I-4-succinate; 3) lysosomal activation by release of lysozyme (muramidase) into the incubation medium. Two key enzymes of the pen-From the Department of Internal Medicine and Yale University Lung Research Center, Yale University, New Haven.
tose shunt were measured, namely, glucose-B-phcsphate and 6-phosphogluconis acid dehydrogenases. Phospholipase C (PLC)
This enzyme (phosphatidylcholine-choline-phosphohydrolase EC 3.1.4.3) cleaves L-a-Iecithin to form diglycerides and phosphoryl choline. Exposure of AM to PLC (0.25 units/ml final concentration) in the presence of ImEq Ca'" evokes changes quantitatively very similar to those observed in phagocytosis. 1 Q02 increased by 35 percent. 14C02 production from the following substrates also increased: 1) 14C-l-glucose by 400 percent; 2) 14C_6_ glucose by 342 percent; 3) HC-l-pyruvate by 262 percent. PLC did not change the 14C02 production from two substrates whose conversion to CO 2 depends on mitochondrial activity, namely 14(:-1acetate and 14C-1-4-succinate. However, increased H 202 formation in the presence of PLC may be inferred from the increase (60 percent) in 14C02 production from 14C-formate since the latter has previously been shown to be a catalase: H 202 dependent reaction.1 Finally, PLC evokes release of intracellular lysozyme into the incubation medium. These features demonstrate the similar metabolic patterns between phagocytosis and PLC . It is suggested that both PLC and phagocytosis evoke
CHEST, VOL 63, NO.4, APRIL 1973 SUPPLEMENT
215
15TH ANNUAL ASPEN CONFERENCE
these changes as a result of effects on cell membrane phospholipids. Cytochalasin B
This agent (2 y I ml) disrupts microfilaments in many cell types including the AM.2 It does not impair microtubular morphology. Quantitative bacteriologic methods demonstrate diminished uptake of live Staphylococcus aureus from shaking bacteriaAM suspensions in the presence of cytochalasin B. These effects are associated with striking diminution of "C02 production from 14C-I-glucose and 14C-6-glucose (77 percent and 42 percent respectively in AM exposed to killed Staphylococcus epidermidis) . Cytochalasin B did not affect 14C02 production from 14C-I-pyruvate, 14C-I-acetate or 14C_I_4 succinate, nor impair the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase. Further, the effects are reversible by washing out the cytochalasin B. The association of diminished bacterial 'ingestion and disruption of the microfilaments suggests that the latter serve as actin-like contractile proteins in the ingestion process. The diminution of C02 formation from labelled glucose may possibly arise from a combination of diminished ingestion and impaired glucose transport. The latter possibility is suggested by preliminary evidence indicating that the uptake by AM of the transportable but nonmetabolized glucose analogue, 2-deoxyglucose is inhibited by 2 ylml of cytochalasin B. Drugs Mediated by Cyclic AMP
Agents presumed to elevate intracellular cyclic
AMP, such as theophylline (10"3 M), prostaglandin E I ( I0"4M ), the cyclic AMP analogue, dibutyryl cyclic AMP (10"3 M) and also dibutyryl cyclic 3', 5' guanosine-monophosphate all produce concentration dependent diminution of 14C02 production from both I4C-I-glucose and 14C-6-glucose in both resting and phagocytosing AM. These agents did not impair bacterial uptake. Isoproteronol (up to 10"3 M), epinephrine (10"3 M) and sublethal concentrations of propranolol had no effects on either glucose conversion to CO 2 or -bacterial uptake. In the absence of measurements of cyclic AMP, it is not possible to make definite statements, but it appears likely that cyclic AMP regulates either glucose transport or glucose metabolism without effects on particle entry. CoNCLUSION
These studies are examples of the use of pharmacologic agents to analyze some mechanisms of phagocytosis in an important pulmonary defense cell. They suggest that: I) the metabolic features of phagocytosis may result from modification of cell membrane phospholipids, 2) AM phagocytosis depends on contractile microfilaments, and 3) cyclic AMP regulates glucose metabolism in the AM. REFERENCES
1 Gee JBL, Vassallo CL, Kaskin J, et al: Catalase dependent peroxidative metabolism in alveolar macrophages during phagocytosis. J Clin Invest 49 :1280-87, 1970 2 Malawista SE, Gee JBL, Bensch KG: Cytochalasin B reversibly inhibits phagocytosis . Functional, metabolic and ultrastructural effects in human blood leukocytes and rabbit alveolar macrophages, Yale Bioi Med 44:286-300, 1971
Retarded Clearance of Macroaggregated Albumin from the Lung of Asthmatic Patients" W . Busse, M.D. ; C. E. Reed, M.D.; I. Tyson, M.D.; and M. Birnbaum, M.D.
During the course of study of an asthmatic patient who had more than ten episodes of atelectasis of the left lower lobe from mucous plugs, a lung scan with macroaggregated albumin lSI I (MAAlSI I) was performed. The scan (not the injection of isotope) was repeated daily . Radioactivity appeared normally in the lung, but unexpectedly remained there for five days. Radioactivity is normally cleared from the lung with a half-time of 4.5 to 10 hours.1.2 Lung scans were performed in 14 asthmatic °From the University of Wisconsin Medical School, Madison.
patients, ranging in age from 18 to 49 years old, and in severity ranging from mild to very severe. Half the patients required either daily or alternate day prednisone. There was prolonged retention of radioactivity in the lungs of the asthmatics with median radioactivity half-time of 17.5 hours. Normal values at our institution are 8-10 hours (Fig I) . The degree of retention of radioactivity was not dependent upon the severity of the asthma or the use of corticosteroids. Swiss-Webster mice were given 109 Bordetella pertussis organisms intraperitoneally. The intraperitoneal or intravenous injection of B pertussis can
CHEST, VOL. 63, NO.4, APRIL 1973 SUPPLEMENT