exposure to halothane. The ciliary activity after exposure to increasing concentrations of halothane showed that at 1percent and 2 percent halothane there was no impairment, while 3 percent halothane caused some ciliostasis. Dramatic reduction m the ciliary activity occurred at halothane concentrations of 4 percent and 5 percent. Results of recovery of ciliary activity of tracheal rings exposed to 4 percent halothane concentrations showed that there was immediate and complete recovery for as long as three days aft= the removal of the anesthetic. After four days of exposure, recovery was incomplete, and after one week no recovery of ciliary activity was noted with death of respiratory epithelial cens. Pulmonary alveolar macrophages (PAM)play a key role in the antibacterial defenses of the lung. Their activity is modulated by both humoral and cenular imm ~ n i t y .Phagocytosis ~ and intracellular killing of bacteria by the PAM is an active energy-utilizing process, dependent entirely on aerobic oxidation. Halothane is an agent known to depress oxidative phosphorylation. Also, the microtubular structures of cells are distorted by halothane and their integrity is essential for phagocytosis. Therefore, it could be argued from a theoretic point of view that halothane will depress pulmonary macrophage function. However, a previous study by Goldstem et a14 concluded that halothane failed to depress PAM, whereas methoxyffwane and cyclopropane did depress their activity. Our results are at variance with these data, and suggest halothane does cause significant depression of lung antibacterial activity. This &ding is consistent with the anesthetic action of halothane and rnethoxyllurane at a sub-cellular leveL5 The in uifro organ culture model of tracheal rings has the advantage of assessing the effects of prolonged exposure to halothane on ciliary activity. Our studies showed that halothane concentrations of 4 percent or more impaired ciliary activity and prolonged exposure was cytotoxic after several days. Halothane exposure of less than four days was followed by complete recovery of ciliary activity. Since in clinical practice 4.0 percent halothane concentrations are mly used, and then only briefly for induction, we conclude that at the usual concentrations of halothane there is minimal depression of ciliary activity, which is reversible after termination of the atpomm. RJmmENCPs

1 Modell JH, Moya F: Postoperative camplications, incidence and management. Anesth Analg 45:43!&439,1966 2 Manawadu BR, Mostow SR, LaForce FM: Local anesthetics and tracheal ring ciliary activity. Analg 57:448-

452,1978 3 Kaltmider HB: Expression of immune medmdms of the lung. Am Rev Respir Dis 113:347-370,1976 4 Goldstein E, Munson ES, Eagle C, et a]: The && of anesthetic agents on murine pulmonary bactericidal activity. Anesthesiology 34: 344452,1971 5 SnodgrassPJ,PirasMM:Theeffectofhalothaneonrrrbeof liver mitochondria. Biochemistry (Wpshington ) 5:11401149,1966

CHEST, 75: 2, FEBRUARY, 1979 SUPPLEMENT

Dr. Ryhder: Have you considered that the e!fFect of anesthesia on the incidence of pneumonia may be caused by the inhalation of mucus? There were some papers about 15 years ago which showed that instillation of pure mucus down into the alveolar region very severely depressed bacterial action. Dr. Manuwcrdu: I do not know whether anyone has done any work on the effects of anesthetic agents on the mucus. Dr. Repine: Does halothane or its products remain in the lung and later the multiplication rate of the bacteria independently of host defense mechanisms? Dr. Manatclcrdu: Yes, it might have an effect because halothane per se inhibits bacterial multiplication. Quesfkm: I'd like to make a comment on Dr. Manawadu's paper and then to ask him a question. Dr. Manawadu, you found that 4 percent halothane was required to inhibit the ciliary activity and Dr. Forbes found that lower percentages inhibited mucociliary clearance in dogs. The premise for your study is that ciliary beating is a major determinant of mucociliary clearance. I'd like to put forward an alternative hypothesis that the consistency and the depth of the periciliary fluid could affect the ability of the cilia to sweep the gel up to the mouth At the concentrations used, did the & stop beating or was their activity only impaired? Dr. Manuwadu: The cilia were either beating or not beating. It's a very dramatic change. The difbrence between our system and Dr. Forbes' is in the mucociliary clearance. He is looking at 3 factors: 1) in the ciliary activity, 2) in the mucus, and 3) in the coordination of the ciliary activity. In our study we were only looking at the ciliary activity.

Impairment of Human Alveolar Macrophage Oxygen Consumption, and Superoxide Anion Production by Local Anesthetics Used in ~roncho~o~~* John R Hoidd, M.D.; J a s n e ~G. White, M.D.; and JohnE. Repine, M.D.OO

subsegmental lavage extensively Broncboscopic to obtain human alveolar macrophages for is used

(AM)

*Fmnthe Pulmonary Section, Department of Medidne, and Hematology !kction, of Pediatrics, University of MiMesota Health Sciences Center, Minneapolis.

This work was supported in part by the Minnesota Medical Fadation, ' L hmchtion, Basil O'Conm S m ad of% National Foundation of the March of Dimes, HL171n2. American Cancer Society Institutional Grant PRA-37, and MfnnesoCa Graduate School. **Est&lisbed h m i c a n Heart Association. Reprint r e q m w & , Moyo Box 148¶ Unitwdty of Minnesdo Hoapftcrl, M

W 55455

II(MUWOLO6Y OF THE LUNG 243

study. Two cationic local anesthetics, lidocaine (LDC) or tetracaine (TRC), are commonly used during bronchoscopic procedures to anesthetize the airways. Recent investigations have shown that these drugs alter membrane-dependent responses of a variety of Therefore, it would be important to determine the concentration of these agents in the lavage d u e n t s and their potential effeet on the metabolic activities and morphology of lavaged AM. In the present investigation, we measured the concentration of LDC present in the lavage effluents, and determined the &ect of these agents in uitro on the structure and function of human AM. The results indicate that appreciable amounts of LDC are present in bronchopulmonary lavage efEuents and comparable pharmacologic doses af LDC or TRC in oitro inhibit the oxidative m e t a b o h of human AM and cause striking surface changes in human AM.

Human alveolar macrophages were obtained by subsegmental sterile saline solution lavage of normal volunteers ( e l ) , as well as nonsmokers (9), or d e r s (26) undergoing diagnostic fiberoptic bronchcscopic procedures. Topical anesthesia of the airways was achieved by spraying a maximum of 7 ml of 4 percent LDC into the upper airways and by flushing 2-3 ml boluses of 1percent LDC through the inner channel of the bronchoscape to anesthetize below tbe vocal cords. A marimrrm of 30 ml of 1percent LDC was used for the procedure. Lavage was performed by injecting 80 ml aliquots of sterile saline solution through the bronchoscope which was recovered by gentle suctioa AM wen then separated by centrifugation, resuspended in Hank's bdancd salt solution ( HBSS ) and counted. LDC concentrations in the lavage effiuents were determined by sensitive gas chromrrlographic analysis.4 Oxygen uptake (biologic 0, probe technique) and superoxide anion (0;) release ( superoxide dismutase inhibitable reduction of cytochrome C methodology) were measured for unstimulated AM or AM stimulated by lo0 opsonized heat-killed S u u m (HKB) AM [lo0 opsonized heat-killed bacteria/AM] or the membrane-active chemical, phorbol myristate (PMA).6*6 In some ,stHBSS containing various concentrationrr of LDC or TRC was substituted for HBSS. Morphology of wasbed AM was evaluated in the presence or absemx of LDC or TRC using stpp dard techniques of scanning electron micmcopy (SEM) or transmission electron microscapy (TEM).r &SUL'IS

Our second goal was to determine the effeet of LDC or TRC on 0,consumption and 0 , 'release by unstimulated or stimulated AM. LDC (216mM)or TRC (0.5 mM) in a dosedependent manner rapidly reduced 0, uptake and 0, release by unstimulated or stimulated human AM (Fig 1). The effects were produced by concentrations of LDC which were present in the lavage &uents, but were more marked at higher concentrations. LDC (16mM) decreased 0, consumption by unstirnulated or stimulated AM by 67 percent or 74 percent respectively (P < .001). Similarly, LDC inhibited 0; release by unstimulated or HKB-stimulated AM by approximately 80 percent ( P < .001). In a camparable pattern, TRC in a dose-dependent manner progressively reduced 0,uptake and 0,release by human AM. The degree of inhibition of metabolism by 2rnM TRC was similar to that by 16m LDC. Additional experiments showed that in concentrations used LDC or TRC were not cytotoxic to human AM since their inhibitory &ect on 0, consumption was reversed by washing AM in HBSS. Our third goal was to assess the influence of cationic anesthetics on the ultrastructure of human AM. Examination of control AM and samples of AM which had been incubated with TRC (0.5-2mM) revealed striking differences in surface morphology. Human AM exposed

I

WTUULATED

24

1

13

16t

STIHOL ATE0

Bottera

T

UIT*KATED

PMA

T

STIMULATED &/wM

PM

AND DISCU%ION

Our first goal was to determine the range of concentrations of LDC to which AM were potentially exposed in the lavage fluid M-Me levels d LDC were uniformly present in lavage returns, but the concentrations varied widely. The mean amcentration of LDC present in the flnid recovered fnrm the initial 60 ml aliquot from eight subjects was 5.5 mM; (range 0.9mM to 11.5mM) Signihntly (P < .01) bwer concentrations of LDC were present in subsequent lavage ef€Iuents. The mean concentration present in the second lavage a u e n t was 0.8 m M and dm mean concentration present in the remainder of the saline solution recovered was 0.4mM. 244 21st ASPEN LUNQ COHFEREHCE

RCURE 1. T 0 p - 0 ~amsumption by washed human AM. LDC ( l&nM) or TRC (2mM ) caused significant ( P< .001) reduction in O2 consumption by unstimulated or stimulated human AM. B o t t o m - * release by washed human AM. The results are expressed as n moles of superoxide dismutase (lo0 &mI) inhibitable cyto&ane C reduced. LJX ( l6mM) or TRC (2mM) caused significant inhibition of 0; release by unstimulated or stimulated human AM.

CHEST, 75: 2, fXBRUARY, 1979 SUPPLEMENT

shown that the cationic local anesthetics, LDC or TRC, are potent inhibitors of d a t i v e metabolism of AM and cause marked alterations of their surface membranes with rounding of the cells. The potential influence of these agents must be carefully considered in design and interpretation of all studies evaluating human cells obtained by bronchopuhnonary lavage. While this effect must be considered, these cationic anesthetics may be valuable agents for evaluating membrane related events of human AM.

Scanning electron micrograph of human alveolar macrophage before and after exposure to tetracaine. The cells on the left are washed control human AM. Pleats or &dike folds are the dominant features of the surface architecture. 'I'he cells on the right were incubated with 2mM tetracaine. They appear more spherical than cells on the left and the sheetlike folds are largely replaced by tightly padred villous projection (original magnification x 3,000). ~ G U R E2.

to TRC were uniformly covered by villous projections, but lacked the sheet-like protuberance seen on all control AM (Fig 2). Thin sections of control AM and AM incubated with TRC revealed no sigdcant differences in internal structure or organization. In summary, the findings show that anesthetic agents routinely present in lavage efauents have the capacity to alter the function and structure of AM. The results of the present experiments suggest that the site of action of these anesthetic agents on the human AM is in part the cell membrane, and that they alter the response of these cells to surface stimuli. This is sup ported by three lines of evidence. First, PMA, whose effect was inhibited by LDC or TRC is a surface active agent that binds to the cell membrane.8 the generation of Oiwhich was inhibited by LDC or TRC has been recently demonstrated to be a membrane dependent respon~e.~ Third, our ultrastructural findings demonstrated striking differences in surface morphology between control and TRGtreated AM, but no sig&cant differences in internal structure. In conclusion, the findings of this investigation have

CHEST, 75: 2, FEBRUARY, 1979 SUPPLEMENT

1 Rabinovitch M, DeStefano MJ: Cell shape changes induced by cationic anesthetics. J Exp Med 143:290304, 1976 2 Nicokon G 4 Smith JR, Pate G: Effect of local anesthetics on cell morphology and m e m b r a n e - d cytmkeletal organizPtion in BALB/3T3 cells. J Cell Biol 68:39540e. 1976 3 Coldstein IM,Lind S, Haffstein S, et al: Iduence of local anesthetics upon human polymorphonuclear leukocyte function in uitm. Reduction of lysosomal enzyme release and superoxide anion production. J Exp Med 146:483494, 1977 4 Keenaghan JB: The determination of lidocaim or prilocaine in whole blood by gas chromatography. Anesthesiology 29: 110-112,1968 5 Hoidal JFt, Repine JE, Beall CD, et al: The effect of phorbol myristrate acetate on the metabolism and ultrastructure of human alveolar macrophages. Am J Path 6 Hoidal JR, Beall GD,Rasp FL Jr, et al: Comparison of the metabolism of alveolar macrophages from hnmans, rats

and rabbits; response to heat-killed bacteria or phorbol myristate acetate. J Lab Clin Med 92:787-794, 1978 7 White JC: Giant organelles containing tubules in ChedialcHigashi lymphocytes. Am J Path 69:s-238,1972 8 Sivak A, van Dnunen BL: Cellular interactioos of phorbol myristate acetate in tumor promotion. Chcm Biol Interact

3:401-411, 1971 9 Coldstein IM,Cerqueiro M, Lind S, et al: Evidence that the superoxide generating system of human leukocytes is associated with the cell surface. J Clin Invest 59:249-254, 1977

Dr. Davis: I congratulate you on a very complete and multi-faceted study. Just to give you the reassurance that it gave me, your values for oxygen uptake by human alveolar macrophages, both stimulated and unstimulated, virtually match ours to the decimal point. I wonder if you would comment on the fact that oxygen uptake and superoxide generation appear to be matched quite closely when you inhibit them with lidocaine. Since oxygen uptake is much easier to do and requires fewer cells, is it necessary to look at both in order to say anything about killing potential?

IMMUNOLOGY OF THE LUNG 245

Dr. Hoidal: To take the last part first, I think it's necessary to look at both. Using a very special inhibitor of mitochondria1 respiration antimycin A, which inhibits the electron transfer from cytochrame B to C,, we were able to inhibit approximately two-thirds of alveolar macrophage oxygen uptake, but not superoxide release. Subsequently, we have been interested in comparing alveolar macrophages from smokers and nonsmokers. Our initial studies compared a population of older patients undergoing diagnostic fiberoptic bronchoscopy. The 0,uptake by smokers was approximately 50 percent greater than the 0, uptake by nonsmokers. We then evaluated alveolar macrophages from young smoking volunteers and age-matched nonsmokers. The 0,uptake was approximately the same in these two groups, but the pathways of oxygen utilization appeared different. Specifically, the smokers' alveolar macrophages when stimulated had a greater superoxide release than the nonsmokers. So I think that 0,uptake above doesn't tell you how the oxygen is utilized by the cell. Dr. Brody: Where do you think that the lidocaine that you applied to the vocal cords ends up? Do you think that the effect you are seeing on the macrophage occurs in the small airspaces or subsequent to the removal of the cells? Dr. Hoidol: I think that a good part of the anesthetic which produced an &ect on the cells came from the smaller airways. In the study all tubing was changed after anesthetization of lower airways and prim to recovering any fluid during the lavage. Dr. Lynn: Did you happen to do any of your studies m the absence of protein or serum? Dr. Hoidal: Yes, we sedimented the cek and washed with balanced salt solution. Dr. Reiss: Do you wish to speculate as to the mechanism af action of lidocaine in the light of the fact that it's highly soluble in lipid. Dr. Hoidal: We've tried to determine the mechanism and action of the lidocaine on these cells. L i d h e has been reported to have numerous actions. It removes the adsorbed calcium from the cell membrane. Secondly, it probably blocks the sodium and potassium channels in the cell membrane. And thirdly, it fluidizes the phospholipid bylayers of the cell membrane. To date, the study has been directed at determining whether removal of adsorbed calcium from the cell membrane was the mechanism of lidocaine action. We have not been able to establish this. We've made two types of preliminary studies. First we've replaced the calcium and tried to override the block, without success. Secondly we've tried to utilize ionophores to transport calcium across the membrane. Again this did not prevent the effect of lidocaine. Dr. M u s o n : Dr. Hoidal, were you looking at production of superoxide and the consumption of oxygen with adherent cell cultures or were those dtures in suspension?

Dr. Hoido2: The biochemical tests were done on mcemtly harvested cells in suspension.

246 2 1 ~ ASPEN t LUNG CONFERENCE

Local and Systemic Immunity following Localized Deposition of Antigen in the l.ung* David E. Bice, Ph.D.; Dorothy L. Harris, B.S.; Joseph0.H a , Ph.D.; and Bruce A. Muggenburg, D.V.M.,Ph.D.

he results of previous studies using dogs indicated Tthat intrapulmonary immunization resulted in

an increase in antigen-specific antibody-forming cells in the population of cells removed by lung lavage.' However, it remains unclear whether these cells were produced by antigen deposition and stimulation of cells in airwayassociated lymphoid tissues, or in regional and distant lymphoid tissues followed by a migration and accumulation of antibody-forming cells in the lung. To obtain additional data concerning the role of local and systemic immune responses following instillation of antigen into the lung, dogs were immunized in specific airways and the resulting immune response was measured in immunized as well as control airways, in regional lymphoid tissues and in blood and spleen. Beagle dogs two and three years of age were immunized by instillation of 1010 sheep red blood cells (SRBC) in 1.0 ml saline solution into airways of individual lung lobes. A fiberoptic bronchoscope was used to locate specific airways and the SRBC suspension was delivered through 1.6 mm diameter polyethylene tubing into airways approximately 2 mm in diameter. The SRBC were instilled into the airways of the right or left apical and right or left diaphragmatic lung lobes of 16 dogs. These dogs were sacrificed five days after immunization and cell suspensions were prepared from individual lung-associated lymph nodes and the spleen. The Jerne plaque assay as modified by Cunningham2 was used to determine the number of anti-SRBC plaque-forming cells (PFC). To evaluate the local response in immunized and control airways, nine additional dogs were immunized in airways of either the left or right apical lung lobes. The immunized airway and a control airway in the opposite a p i d lobe were lavaged with five saline solution washes of 10 ml each using the fiberoptic bronchoscope on days 3,5,7,10, 12, 14 and 21 after SRBC exposure. An average volume of 42.2 ml lavage fluid was recovered. The cells recovered were counted and the percentage of macrophages, lymphocytes and granulocytes was determined. Blood samples were also taken and the number of direct ( IgM ) and indirect ( I&) PFC in the blood, and in the cells from immunized and control airways was determined.

The number of plaque-forming cells in lung-associated .From the Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute. Albuquerque. Research performed under U.S.De artrnent of Ener Contract No. EY-76-04-1013 in facieties fully accAd by the American Association for ~dvancementof Laboratory Animal Care. Reprint requests: Dr. Bice, Looelace Research Institute, BIdg 9217, Areo Y, Kirtland AFB East, Albuquerque 87115

CHEST, 75: 2, FEBRUARY, 1979 SUPPLEMENT