Respiration 36: 153-159 (1978)

Anaphylaxis in Isolated Rabbit Lungs T. S. Hakim, C. A. Dawson, V. L. Moore and J. J. Darboriak Research Service, Veterans Administration Center, Wood (Milwaukee), and the Departments of Medicine, Pharmacology, Physiology and Pathology, Medical College of Wisconsin, Milwaukee, Wise.

Key Words. Pulmonary vascular resistance • Airway resistance • Lung compliance • Isolated lung

Introduction Anaphylaxis in rabbits is characterized by a marked increase in pulmonary vascular resistance, right heart dilation and failure [5, 15]. Bronchospasm has also been ob­ served [4, 11]; however, the pulmonary vas­ cular response predominates in this species [15], The mechanisms responsible for the increase in pulmonary vascular resistance are not well defined. In part, this is due to the fact that pulmonary vascular responses in intact rabbits are relatively difficult to measure. Therefore, we have developed an isolated rabbit lung model in which the pul-

rnonary response to antigen challenge can be characterized in terms of changes in lung mechanics and vascular resistance. In this preparation, various parameters can be controlled to a degree not possible in the intact animal, thus providing better ac­ cess to the mechanisms involved. We have begun to study these mechanisms by deter­ mining the relative importance of blood and lung tissue factors. This was accomplished by assessing the responses to antigen challenge in lungs from normal animals perfused with blood or plasma from sensitized animals and lungs from sensitized animals perfused with blood or plasma from normal rabbits.

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Abstract. Lungs from rabbits sensitized to ovalbumin or bovine y-globulin were isolated and perfused with autologous blood. The response to antigen challenge via the perfusate was immunologically specific and characterized by a marked increase in perfusion resistance, a moderate increase in airway resistance and a small decrease in lung compliance. The response could also be elicited by specific antigen challenge in lungs from sensitized rabbits perfused with blood from normal rabbits and in lungs from normal rabbits perfused with blood or plasma from sensitized rabbits. The magnitude of the response was greater when blood or plasma from sensitized animals was used as the perfusate. Therefore, blood and/or plasma factors appear to be the major contributors to the response.

Materials and Methods New Zealand rabbits weighing 2.7~4.6 kg were sensitized with 1.25 mg of ovalbumin (OA) or bovine y-globulin (BGG) (Miles Laboratories, Kankakee, 111., 5X crystallized) in complete Freund’s adjuvant (Difco Laboratories, Detroit, Mich.) in a total volume of 2.5 ml in 1 nuchal, 2 axillary and 2 inguinal regions (sc). The animals were evaluated 22-30 days later. Serum antibody concentration was determined in 7 of the OA-sensitizcd and 2 normal rabbits by quantitative pre­ cipitation, as previously described [14]. The technique used to isolate the lung has been described previously [6], The rabbits were preanesthetized with 15 mg chlorpromazine hy­ drochloride (im) (Thorazine® - Smith, Kline & French Lab.) followed 15 min later by 15-20 mg sodium pentobarbital/kg body weight (iv). The an­ imals were heparinized (750 U/kg) through a caro­ tid artery catheter and exsanguinated. 7-10 ml of Rheomacrodex® was infused into the carotid ar­ tery during bleeding. 40-50 ml of blood/kg body weight, with a hematocrit of 30 + 2 (SD) °/o, was obtained. The chest was opened and the pulmon­ ary artery (PA) and left atrium (LA) were cannulated. The lungs were removed and suspended by an endotracheal cannula. The PA and LA cannu­ las were connected to the primed perfusion sys­

Hakim/Dawson/Moore/Barboriak

tem. The ventilation and closed perfusion system is shown in figure 1. The blood was pumped (Masterflex 500) front a reservoir open to atmo­ spheric pressure through a heat exchanger (37 °C) into the PA. The ventilation system consisted of an airtight Plexiglas® box. A piston respirator provided a sinusoidal wave of negative pressure (6 cm H20 peak-to-peak), and a valve and vacuum system maintained the end-expiratory pressure at approxi­ mately -1 cm H20 and compensated for any leaks or temperature changes in the box. The lungs were ventilated with a gas mixture containing 15% O, and 5°/o C 0 2 in nitrogen. This maintained the Po2, Pc02 and pH of the perfusate at 106 + 6.0 (SD) mm Hg, 36 + 5 (SD) mm Hg and 7.42 + 0.05 (SD), respectively. The blood (PA and LA) pressures (measured from side connectors in the perfusion cannulas re­ lative to the LA) and pleural pressure (box pres­ sure) were measured using Statham transducers. The blood flow rate was set at an average of 203 + 30 (SD) ml/min and was held constant throughout each experiment; this provided a mean PA pressure of 8.8 + 3.3 (SD) mm Hg (LA pres­ sure zero). The reservoir volume changes were monitored using a pressure transducer to record the height of the column of blood. Air flow rate was measured using a screen pneumotachometer

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and a Validyne® differential pressure transducer, and volume was obtained by electronically inte­ grating the flow signal. Pulmonary vascular resistance was calculated from the PA-LA pressure difference and the pump flow rate. Since the lung and reservoir were in series, changes in reservoir volume were used to determine changes in lung fluid volume. Pul­ monary compliance and resistance were calculated from the pleural pressure, air flow rate and vol­ ume measurements, as described by Amdur and Mead [2]. To determine the characteristics and the im­ munological specificity of the response to antigen challenge, lungs from 3 groups of rabbits, i.e., normal, OA-sensitized and BGG-sensitized, were challenged by adding either 0.1 mg of OA or 6.0 mg of BGG to the perfusate. Preliminary stud­ ies indicated that these doses were near the opti­ mal quantities required to elicit a consistent vas­ cular response in sensitized lungs. The antigen was added to the reservoir in a physiological saline solution (0.1 or 0.6 ml) within 30 min of the be­ ginning of the perfusion. The need for the larger quantity of BGG cannot be explained completely on the basis of molecular size, and might reflect phylogenetic differences in antigenicity of OA compared to BGG in rabbits.

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To determine the relative importance of blood and lung tissue factors, the following groups were studied: (1) Lungs from normal rabbits perfused with homologous blood from normal animals; (2) lungs from OA-sensitized rabbits perfused with homologous blood from OA-sensitized animals; (3) lungs from OA-sensitized rabbits perfused with blood from normal animals; (4) lungs from OAsensitized rabbits perfused with plasma from nor­ mal animals; (5) lungs from normal rabbits per­ fused with blood from OA-sensitized animals; and (6) lungs from normal rabbits perfused with plas­ ma from OA-sensitized animals. The plasma was prepared by centrifuging the heparinized blood at 20,000 g to remove all cells. The lungs were washed to remove residual autolo­ gous blood remaining following exsanguination by pumping 30 ml of the homologous blood or plas­ ma through the lung and discarding this volume before beginning the closed-circuit perfusion. Lungs in each of these groups were challenged with 2.0 m of OA. The significance of differences between preand postchallenge values of vascular and airway resistance and compliance were evaluated using a paired Student’s t test, and the differences in res­ ponses between groups were evaluated using an unpaired Student’s t test.

Change in Ç l HjO '!

I OA

Time, min

Fig. 2. The mean change in vascular resistance (Rv), pulmo­ nary resistance (Ra) and pulmo­ nary compliance ( C l ) during the 6 min following the addition of OA (arrow) to the perfusion sys­ tem. Lungs from 6 normal, 11 OA-sensitized (OA) and 4 BGGsensitized (BGG) rabbits perfused with autologous blood.

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m l 'C m

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Results Characteristics of the Responses to Antigen Challenge Figure 2 shows the changes in lung com­ pliance, and airway and vascular resistances in the 3 groups perfused with autologous blood and challenged with OA. The pre­ challenge values were not significantly dif­ ferent among these groups, and the mean values were: vascular resistance, 3.6 ± 1.9 (SD) mm H g s e c -m l_1; pulmonary compli­ ance, 3.7 ± 1.1 (SD) ml • cm H20 _1; and pul­ monary resistance, 48 ± 18 (SD) cm R.O ■sec ■L_1. The vascular resistance in­ creased (p < 0.005) in all 11 OA-sensitized lungs that were challenged with OA, with an average maximum increase of 6.4 ± 1 .3 (SE) mm Hg •sec •ml-1. The pulmonary resist­ ance increased (p < 0.02) 40 ± 12 (SE) cm HoO • sec • L“1. The decrease in lung compli­ ance following specific antigen challenge was small (0.28 ± 0.07 [SE] ml • cm FLO'1), but was consistent and statistically significant (p < 0.005). None of these parameters was detectably changed following OA injection in the 6 normal or 4 BGG-sensitized lungs. The results with the BGG challenged groups were similar to those challenged with OA, i.e., only lungs from BGG-sensitized animals responded. In these BGG-sensitized

lungs, vascular resistance increased 4.0 ± 1.6 (SE) mm Hg • sec • ml-1; pulmonary resist­ ance increased 53 ± 33 (SE) cm FLO • sec ■ I“ 1; and compliance decreased 1.1 ± 0.7 (SE) ml • cm HoO"1. The immunospecificity of the reaction is further exemplified in figure 3, which shows the perfusion pressure response following OA and BGG challenge in 2 lungs. One of the lungs was sensitized with BGG and the other with OA. This procedure was carried out on 4 OA- and 4 BGG-sensitized lungs, and in each case only the sensitizing antigen produced the pressor response. The lung fluid volume changes in the sensitized lungs challenged with specific an­ tigen were variable. The lung fluid volume increased (from 0.5 to 7.0 ml) in 45°/o of the lungs, decreased (from 0.5 to 1.0 ml) in 20% and remained unchanged in 35%. The OA-sensitized animals tested had an average antibody concentration of 1.9 ± 0.7 (SD) mg/ml plasma. No antibodies to OA could be detected in the sera from unsensi­ tized rabbits. Within the sensitized group, no significant correlations between the mag­ nitude of the responses and total antibody concentration were found. Cross Perfusion Studies Figure 4 compares the magnitudes of the vascular responses obtained with the var-

” 1-------- |-------- T~

Time after perfusion w as started, min

46

Fig. 3. Top: a lung from a BGG-sensitized rabbit was chal----------- - lenged with OA and then subse­ quently challenged with BGG. Bottom: a lung from an OA-sen^------------- sitized rabbit was challenged ini­ tially with BGG and then OA.

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either sensitized lung, blood or plasma was sufficient to elicit a response, blood-borne factors were quantitatively more important and included plasma as well as cellular fac­ tors.

Discussion The results of this study indicate that an immunologically specific response could be elicited in isolated rabbit lungs. This re­ sponse was characterized by vascular and airway changes similar to those previously described in intact rabbits, namely, a marked increase in pulmonary vascular resistance [5, 8], increased airway resistance and de­ creased lung compliance [4, 11]. Drinker and Bronfenbrenner [8] ob­ served that the pulmonary arterial pressure increased by more than 100% following an­ tigen challenge in sheep serum sensitized rabbits. Karczewski and Widdicombe [11] and Carrillo and Aviado [4] observed in­ creases in airway resistance of about 70 and Fig. 4. The change in vascular resistance in response to OA chal­ lenge. N. L., N. B. and N. P. are lungs, blood and plasma from normal rabbits, respectively. S. L., S. B. and S. P. are lungs, blood and plasma from sensitized rabbits, respectively. The probability that the difference between any 2 groups was not statistically signi­ ficant is less than the value shown in the inset. N. S. means that p > 0.05. None of the normal lungs perfused with normal homo­ logous blood (group 1) showed any detectable response, and the responses in groups 2-6 were significantly greater than that of group 1 (p < 0.004).

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ious perfusion regimes. Introduction of the antigen was always followed by an increase in vascular resistance when the preparation included at least one component: either lung, blood, or plasma from a sensitized rabbit. The mean response was largest when lungs from sensitized rabbits were perfused with blood from sensitized animals. Howev­ er, the response observed when normal lungs were perfused with sensitized blood was not significantly different from that ob­ served when both blood and lungs were from sensitized animals. The responses in sensitized lungs perfused with normal blood or plasma were not significantly different from each other, but both were significantly smaller than any of the groups perfused with sensitized blood or plasma. The re­ sponse in normal lungs perfused with sensi­ tized plasma was significantly smaller than that of normal lungs perfused with sensi­ tized blood. None of the normal lungs per­ fused with normal blood showed any detect­ able response. Thus, the results of these cross perfusion studies indicate that, while

200%, respectively, and decreases in lung compliance of about 30 and 60%, respec­ tively. Thus, the mechanisms which can be called into play within both the lung and blood in the isolated preparation are prob­ ably major contributors to the pulmonary responses seen in the intact animals. Various mechanisms have been implicat­ ed in the increase in pulmonary vascular re­ sistance in rabbits including pulmonary va­ soconstriction [9] and embolization due to aggregation of formed elements [1, 16] and/ or antigen-antibody (Ag-Ab) complexes [13]. The lodging of Ag-Ab complexes in the small pulmonary vessels has been sug­ gested [13]. This appears unlikely in the present study since the response was elicited with an antigen concentration well below that which produced significant precipita­ tion in the quantitative precipitation test. Ag­ gregation of formed elements has been con­ sidered an important contributing factor [1, 7, 16]. However, the observation that a sig­ nificant response could be elicited when no cells were present in the perfusate suggests that cellular aggregation was not the only factor involved. The source(s) and role(s) of mediators which contribute to the response are not clear. Dragstedt et al. [7] concluded that the blood was the important source of histamine which mediated the response. On the other hand, Lecomte [12] concluded that the lung tissue was the important source of media­ to rs) other than histamine because in­ creased vascular resistance, which was not blocked by antihistamines, could be elicited in isolated lungs from sensitized rabbits per­ fused with physiological saline. In the pres­ ent study, either sensitized lung or blood was sufficient to elicit a response. Thus, both may contain factors which can contri­

Hakim/Dawson/Moore/Barboriak

bute to the reaction. Although we attempted to minimize the amount of residual blood left in the sensitized lungs perfused with normal blood or plasma by prewashing the vascular bed, we cannot completely rule out some contribution due to a small amount of residual sensitized blood. Therefore, while the blood-borne factors were certainly most important, the role of the lung tissue is less certain. Formed elements in the blood could play a dual role. As aggregates, they could ob­ struct small vessels [16, 17] or they might release their vasoactive constituents such as histamine and serotonin [3, 10, 16]. The observation that the response could be elic­ ited in normal lungs, even when cells were absent from the perfusate, may indicate a role for the phlogistic activity of soluble AgAb complexes [18] as well. Much of the work which has been done to elucidate the mechanisms involved in the pathophysiologic responses during acute an­ aphylaxis has been directed at the responses of guinea pig airways. Although guinea pig and man share a common shock organ, the lung, there are marked differences in the overall mechanisms involved [15]. The iso­ lated rabbit lung model, in which the phy­ siologic responses can be quantified and the composition of the perfusate altered, should provide a useful tool for developing a more complete description of mechanisms respon­ sible for the pulmonary responses occurring during mammalian anaphylaxis. Acknowledgements This research was supported in part by a grant from the National Heart, Lung and Blood Insti­ tute, H L 15389 (SCOR). Also supported by the Medical Research Service of the Veterans Admin­ istration.

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1 Abell, R. C. and Schenck, H. I\: Microscopic observations on the behavior of living blood vessels of the rabbit during the reacion of an­ aphylaxis. J. Immun. 34: 195 (1938). 2 Amdur, M. P. and Mead, J.: Mechanics of re­ spiration in unanesthetized guinea pigs. Am. J. Physiol. 192: 364 (1958). 3 Benveniste, J.: Platelet activating factor, a new mediator of anaphylaxis and immune complex deposition from rabbit and human basophils. Nature 249: 581 (1974). 4 Carrillo, L. R. and Aviado, D. M.: Mecha­ nisms for the bronchodilator effects of corti­ costeroids in the sensitized rabbit. J. Pharmac. exp. Ther. 164: 302 (1968). 5 Cohen, S. G.; Franke, F. R., and Karlson, E. L.: Studies on the mechanism of fatal anaphy­ laxis in the rabbit. J. Allergy 22: 160 (1951). 6 Dawson, C. A.; Jones, R. L., and Hamilton, L. H.: Hemodynamic responses of isolated cat lungs during forward and retrograde perfusion. J. appl. Physiol. 35: 95 (1973). 7 Dragstedt, C. A.; Ramirez de Arellano, M.; Lawton, A. H., and Youmas, G. P.: Passive sensitization of rabbits’ blood. J. Immun. 39: 537 (1940). 8 Drinker, C. K. and Bronfenbrenner, J.: The pulmonary circulation in anaphylactic shock. J. Immun. 9: 387 (1924). 9 Grove, E. F.: Studies in anaphylaxis in the rabbit. V. On the role of unstripped muscle in acute anaphylaxis in the rabbit. J. Immun. 23: 147 (1932). 10 Humphrey, J. H. and Jaques, R.: The release of histamine and 5-hydroxytryptamine (sero­ tonin) from platelets by antigen-antibody reac­ tion (in vitro). J. Physiol. 128: 9 (1955). 11 Karczewski, W. and Widdicombe, J. G.: The role of the vagus nerves in the respiratory and

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circulatory reactions to anaphylaxis in rabbits. J. Physiol. 201: 293 (1969). Lecomte, J.: Reactions anaphylactiques in vi­ tro des artères pulmonaires du lapin. Int. Archs Allergy appl. Immun. 12: 339 (1958). McKinnon, G. E.; Andrews, E. C.; Heptinstall, R. H., and Gcrmuth, F. G.: An immunohistologic study on the occurrence of intravascular antigen-antibody precipitation and its role in anaphylaxis in the rabbit. Bull. Johns Hopkins Hosp. 101: 258 (1957). Moore, V. L. and Fink, J. N.: Immunologic studies in hypersensitivity pneumonitis - Quan­ titative precipitins and complement-fixing anti­ bodies in symptomatic and asymptomatic pi­ geon breeders. J. Lab. clin. Med. 85: 540 (1975). Stechschulte, D. J. and Austen, K. F.: Anaphy­ laxis, pp. 237-276 (Academic Press, New York 1974). Waalkes, T. P.; Wcissbach, H.; Bozicevich, J., and Udenfriend, S.: Serotonin and histamine release during anaphylaxis in the rabbit. J. clin. Invest. 36: 1115 (1957). Waalkes, T. P. and Doburn, H.: The role of platelets and the release of serotonin and his­ tamine during anaphylaxis in the rabbit. J. Al­ lergy 30: 394 (1959). Weigle, W. O.; Cochrane, C. G., and Dixon, F. J.: Anaphylactic properties of soluble antigenantibody complexes in the guinea pig and rab­ bit. J. Immun. 85: 469 (1960).

Received: May 31, 1977 Accepted: August 23, 1977 Dr. C. A. Dawson, Research Service/151, Veterans Administration Center, Wood, WI 53193 (USA)

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References

Anaphylaxis in isolated rabbit lungs.

Respiration 36: 153-159 (1978) Anaphylaxis in Isolated Rabbit Lungs T. S. Hakim, C. A. Dawson, V. L. Moore and J. J. Darboriak Research Service, Vete...
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