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Airway mucosal permeability in the Ascaris suum-sensitive rhesus monkey
Airway mucosal permeability in the Ascaris suum-sensitive rhesus monkey R. C. Boucher, M.D., P. D. Pare, M.D., N. J. Gilmore, and J. C. Hogg, M.D. Montreal, Quebec, Canada
M.D., L. A. Moroz,
M.D.,
The permeability of the airways to technetium 99m-labeled albumin was measured in Ascaris suum-sensitive rhesus monkeys. All 8 animals were skin-sensitive to Ascaris suum (AA) antigen, 4 being respiratory responders (R) and 4 nonresponders (NR) to aerosolized antigen. In the absence of antigen challenge there were no differences in the accumulation in the blood of radioactive material from the tracheobronchial tree between the R and NR animals. After a five-minute challenge with aerosolized AA, there was a threefold increase in the rate of accumulation of radioactive material in the blood over control for the R group with no efSect noted in the NR group. Gel filtration data indicated that the radioactivity in the blood most likely represented low molecular weight albumin fragments, resulting from spontaneous degradation of Tc-albumin, that crossed the mucosa and partially bound to circulating albumin. It is concluded that hyperpermeability of the airway mucosa probably is not a factor that contributes to the selective responsiveness of the R group to aerosolized antigen, and that airway permeability is increased consequent to the allergic reaction mediating acute bronchoconstriction.
A defect in the normal protective function of mucosal surfaces may contribute to the atopic state.‘, * Supporting this view is the observation that antigen placed on the nasal mucosa stimulates significantly greater reaginic responses in atopic individuals than in normals,3 while reagin production is similar for both groups when antigen is administered subcutaneously.4 It has been suggested that the increased response to surface immunization in the atopic group results from increased availability of antigen to immunocompetent cells in the submucosa and regional lymph nodes, reflecting either increased mucosal permeability or decreased local antigen elimination.5 Hyperpermeability of the airway mucosa has been offered as an explanation for the increased bronchial reactivity to inhaled antigens in classic extrinsic asthma.6 This mechanism may explain differing
From The Lyman-Duff Laboratory, Department of Pathology, McGill University, and The Harry WebsterThorp Laboratories, Division of Clinical Immunology, McGill University Clinic and Royal Victoria Hospital. Supportedby MRC Grants Mt-4219 and MT-2844. Presentedin part at the Brook Lodge Symposium on Asthma, September, 1976. Received for publication April 2, 1976. Accepted for publication June 3, 1976. Reprint requeststo: Dr. R. C. Boucher, Department of Pathology, McGill University, 3775 University St., Montreal, Quebec, CanadaH3A 2B4. Vol. 60, No. 2, pp. 134-140
bronchial reactivities to a given antigen among individuals with similar cutaneous sensitivities to that antigen. However, it is not known whether observed differences in mucosal permeability are a cause or an effect of the inflammatory state uritier consideration. In the present study, therefore, we attempted to investigate these two possibilities separately. First, are there inherent differences in mucosal permeability which correlate with reactivity to an inhaled antigen in asthma and, second, does the allergic reaction itself induce changes in permeability? To investigate these questions, we used the Ascaris suum-sensitive Rhesus monkey, a model which Patterson and others have shown to share many characteristics with the human asthmatic. ‘* * MATERIALS Animals
AND METHODS
Eight Macaca mulatta monkeys, weighing 3 to 6 kg, were studied. All manifested varying degrees of skin sensitivity to Ascaris suum antigen (AA). Four were bronchial responders (R) and 4 were nonresponders to aerosolized antigen at a dose of 100 yg/ml; the respiratory response of the R animals to antigen challenge was characterized by a 200% to 400% increase in frequency, 100% to 1,OOO% increase in pulmonary resistance, and 40% to 80% fall in dynamic compliance with return to control values within 30 to 60 min, while the NR animals did not vary from control parameters by more than 50%. A detailed description of the skin sensitivity and changes in lung mechanics associated
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with challenge in this rhesus colony has been previously reported. g
mucosal
permeability
in rhesus monkey
135
technetium was also injected intravenously into a monkey, and after 30 min a heparinized blood sample was drawn and plasma was fractionated and counted.
Antigen Crude Axcaris suum extract, described previously,g was supplied by Dr. P. B. Stewart, Pharma Research, Montreal, Quebec, Canada.
Purification
of albumin
Albumin was prepared from normal monkey serum by sequential ammonium sulfate precipitation (fraction precipitating between 2.6 and 3.0 M), diethylaminoethyl (DEAE) cellulose chromatography at pH 8 (Watman Ltd., Chifton, N.Y.),‘O and gel filtration on Bio-Gel A 0.5 (Bio-Rad L,aboratories, Richmond, Calif.). The resulting preparation was homogenous by cellulose acetate electrophoresis. with an estimated purity greater than 99.5%. The concentration of purified albumin was estimated by absorbency at 280 nm, using the extinction coefficient, E;& = 5.8.”
Radiolabeling
of albumin
Purified albumin was prepared for radiolabeling by lyophilization and packaging in glass vials with SnCl, and dextrose under 100% N2 (Chas. Frosst & Co., Montreal, Quebec, Canada). Albumin was radiolabeled with fresh technetium 99m1* immediately prior to each experiment. Because of degradation in the preparative and labeling processes, the freshly labeled albumin was then repurified prior to use by sequential gel filtration on columns of Bio-Gel A 0.5 M (0.9 x 60 cm) and Sephadex G-50 (Pharmacia Fine Chemicals, Montreal, Quebec, Canada) (0.9 x 30 cm) equilibrated with 0.015 M phosphate-buffered 0.15 M NaCl, pH 7.4 (PBS). Aliquots of the freshly labeled albumin were reserved for subsequent dilution and counting in a gamma radiation spectrometer (Packard Instruments, Inc., Downers Grove, Ill.) to estimate the dose delivered (average, 100 &i per animal), and for Sephadex G-50 gel filtration (0.9 x 50 cm column) to assess the extent of fragmentation during the experimental period. The final concentration of the labeled albumin was between 0.05 and 0.1 mg/ml with a specific activity of 1 to 2mCi/mg.
In vivo and in vitro behavior labeled albumin fragments
in plasma of
Low molecular weight (LMW) fragments of labeled albumin, obtained by Sephadex G-50 gel filtration as described in “Results” (Fig. 4), were incubated with heparinized monkey blood at 37” C for 10 mm, centrifuged, and the plasma obtained and fractionated on Sephadex G-50 (0.9 x 50 cm column) or Bio-Gel A 0.5 m (0.9 x 50 cm column). Low molecular weight fragments were also injected intravenously and instilled intratracheahy into monkeys, and plasma samples fractionated on Sephadex G-50 (0.9 :< 50 cm column). Free technetium (NaTcO,) was incubated with heparinized whole monkey blood in vitro for 24 hr at 22” C and plasma prepared and fractionated as described above. Free
Procedure The animals were lightly anesthetized with pentobarbital sodium (20 mg/kg) and phencyclidine hydrochloride (2 to 3 mg/kg), intubated with a cuffed endotracheal tube, and studied in the upright position with a Fleisch pneumotachograph (No. 00) and arterial line in place. Each study was initiated by instilling 0.9 ml of the albumin solution into the trachea via a catheter. One-ml blood samples were obtained for counting at 30 set, I, 3, 5, 10, 20, 30, 40, 60, and 90 min after instillation. At 30 min and 60 min, additional 2.5-ml blood samples (anticoagulated with 50 U heparin) were obtained, centrifuged, and l-ml plasma samples fractionated on Sephadex G-50 (0.9 X 50 cm) and fractions counted. Each animal was studied at least twice on different days. One occasion served as a control study for a second when a 5-min aerosolized challenge of antigen (Hudson disposable nebulizer, Wadsworth, Ohio) was administered 30 min after the instillation of the radiolabeled albumin. Respiratory frequency, a sensitive indicator of AA response, was used as an index of bronchial response.g At the end of two of the control studies, rapid shallow breathing was induced manually with a syringe, and blood samples were obtained subsequently at 5-min intervals for counting.
Lung scanning The intrathoracic distribution of instilled Tc-albumin in 2 animals was assessed with an external gamma scintillation camera (Picker Canada, Montreal). After initial threedimensional scans were obtained, the animals were manually hyperventilated with a syringe and the distribution of labeled material assessed with repeat scans.
Effects of antigen challenge filtration rate (GFR)
on glomerular
The steady-state inulin infusion methodI was used to assess the effect of antigen-induced bronchospasm on GFR. The animals were given a priming dose of inulin (Nutritional Biochem. Co., Cleveland, Ohio) dissolved in normal saline calculated to give an inulin concentration of 0.25 mg/ml in extracellular water. An intravenous inulin infusion was maintained with an LKB Variopex peristaltic pump (Uppsala, Sweden) at a rate calculated to replace filtered losses (GFR estimated at 25 ml/min with urinary inulin concentration estimated to be 0.25 mg/ml). Inulin therefore was delivered at a concentration of 12.5 mg/ml and at a rate of 0.5 ml/min. Blood samples (2 ml with 40 U heparin) were obtained at 35, 40, and 45 mm after the priming dose and initiation of the infusion. A standard 5min antigen challenge was delivered to 4 sensitive monkeys beginning at the 45-min point, and blood samples were obtained 5, 10 and 30 min later (50, 55, and 75 min after the priming dose). Plasma inulin levels were measured calorimetrically.”
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TIME
FIG. 1. Appearance tracheally instilled
CLIN. IMMUNOL. AUGUST 1977
lmml
in blood of radioactivity from intraTc-albumin for R and NR monkeys.
TlME
kiln1
FIG. 3. Appearance in blood of radioactivity in the blood for a responding monkey. Two challenge runs (one from preliminary study) and one control run shown.
Appearance
0
10
20
30
40
50 TIME
60
70
80
90
~IlWl)
FIG. 2. Appearance in blood of radioactivity for a single nonresponding monkey. Two control runs (one from a preliminary study) and one challenge run shown.
Analysis
of data and statistical
methods
Preliminary studies indicated the rapid accumulation of radioactive material in the blood in the 0- to IO-min period with a relatively constantrate of accumulationfrom 10 to 90 min. Therefore, slopes of accumulation of radioactivity in the blood for the 0- to IO-min period, the IO- to 30min (prechallenge) interval, and the 40- to 90-min (postchallenge) interval were computed using the leastsquaresmethod. The ratio of the slope of the postchallenge to prechallenge interval for each animal was used as an index of the effect of antigen challenge on permeability, thus taking into account small differences in rates for the prechallengeinterval. The differences in theseratios (challenge vs control run) for animals within each group were comparedby a standard t test. RESULTS Respiratory
response
The responsive animals responded to antigen challenge with a mean increase in respiratory frequency of 304% (range, 200% to 379%), while the NR group showed no change in frequency with challenge (100% k 10%).
of radioactivity
in the blood
Fig. 1 shows the percentage of the delivered dose of radioactive material present in the blood vs time for the R and NR groups. The points for each group up to 30 min represent the average for the control and challenge runs. There are no evident differences between the two groups in the amount of radioactivity present in the blood at any given time. Fig. 2 shows the results of two control and one challenge run for the same nonresponding monkey. The radiolabeled material accumulates in the blood at a similar rate in the three studies. The aerosolized antigen challenge does not appear to alter this rate. In contrast, Fig. 3, which shows the results of one control and two challenge runs for a responding monkey, demonstrates that in this animal AA challenge produces a marked and reproducible increase in the rate of appearance of counts in blood. This increase was sustained at a similar rate for the 60-min postchallenge period. Manual hyperventilation in two control studies did not alter the baseline rates. Table I presents the average rates of accumulation of counts in the blood over the three time intervals for the control and challenge runs of the R and NR animals. The ratios of the rate of accumulation of counts for the 40 to 90 min interval to the one corresponding to the 10 to 30 min interval are presented in the last column. It can be seen that for both the R and NR groups the ratios do not differ appreciably from one in the two sets of control runs. Antigen challenge in the R group increases this ratio threefold, while in the NR group the ratio increases by less than 50%. Comparison of the differences between the challenge vs control runs in these ratios computed for each animal individually showed a significant increase for the R group (p = 0.05). with no difference for the NR group.
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TABLE I. Rates of accumulation
mucosal
permeability
in rhesus monkey
137
in blood (mean 2 SD)
of radioactivity
Time periods (min) 40-90 O-10
Responder Nonresponder
Control Challenge
0.011 rt 0.004 0.015 k 0.004
Control Challenge
0.015 + 0.009
Nature of the radiolabeled appearing in the blood
0.007
-c
0.003
material
Fig. 4 shows the results of Sephadex G-50 gel. filtration analysis of an aliquot of the radiolabeled albumin which had been stored for 6 hr at room temperature in PBS, i.e., the time period corresponding to end of experimental use. Two peaks are seen with similar radioactive and absorbency (280 nm) profiles, suggesting degradation of albumin into LMW fragments predominantly less than 3,500 daltons. Despite initial purification, spontaneous degradation of albumin occurred during a time corresponding to the experimental period. The mean degradation for all labeled preparations was 5% (range 4 to 6%) when analyzed at 6 hr. The importance of this fragmentation is indicated by the data depicted in Fig. 5. When labeled albumin fragments obtained by gel filtration on Sephadex G-50 (Fig. 4) were incubated with monkey blood in vitro for 10 min, centrifuged, and the plasma obtained fractionated again on Sephadex G-50, approximately 30% of the radioactivity now appears in the void volume (Fig. 5 upper left), indicating binding of the labeled albumin fragments to higher molecular weight proteins in plasma. Upon similar fractionation of the mixture of plasma and labeled fragments on Bio-Gel A 0.5 m, radioactivity due to the labeled albumin fragments eluted at a volume similar to that of pure albumin alone, indicating that labeled fragments were bound either to albumin or to some plasma protein of similar molecular weight. Identical results were obtained by gel filtration analysis of plasma samples obtained from monkeys after intravenous injection or intratracheal instillation (Fig. 5, lower left) of labeled albumin fragments. Free technetium did not appear to contribute to this phenomenon, since the labeled albumin fragments eluted from Sephadex G-50 ahead of and distinct from the elution volume of free technetium (Fig. 4), and less than 0.1% of free technetium bound to albumin when mixed with monkey blood in vitro, or after intravenous administration of technetium to monkeys. The Sephadex G-50 gel filtration profiles from the experimental studies (employing Tc-labeled albumin) of responsive and nonresponsive monkeys, pre- and
10-30
40-90
10-30 1.20 3.14
0.005
2
It
_f
0.002 0.002
0.006
0.007
0.022
-e 0.012
0.008 0.004
2
0.003
0.007
c
0.005
k
0.001
0.005
k
0.004
postchallenge, files obtained ments alone. shown in Fig.
0.003
,118 1.25
were indistinguishable from the proafter administration of albumin fragResults of a typical experiment are 5 (right).
Lung scans Lung scans from two monkeys revealed that the 0.9 ml of instilled labeled albumin was deposited centrally in the trachea and main stem bronchi. Two minutes of manual hyperventilation did not alter this distribution. lnulin
data
Table II presents the steady-state and postchallenge plasma inulin levels for 4 responsive monkeys. It can be seen that in three monkeys, A, W, and E, there are clearly no changes in inulin concentrations, and hence GFR, postchallenge. In monkey I there is a late 6% increase in plasma inulin which would correspond to a similar decline in GFR. Such a small change is probably within the limits of experimental error and cannot account for the observed increases in rates of accumulation of radioactive material in the blood postchallenge (threefold increase). DISCUSSION The data summarized in Table 1 suggest that in the absence of antigen challenge there are no differences in the rates of accumulation of radiolabeled material between the responding and nonresponding monkeys. Following antigen change this rate is increased threefold for the responding group with no change in the nonresponding group. However, before interpreting these results an evaluation of the possible sources of error introduced by the fragmentation of the radiolabeled albumin and the physiologic changes accompanying bronchoconstriction is necessary. In this study, although no evidence was found for significant release of free technetium from Tcalbumin, there was evidence for the spontaneous degradation of Tc-albumin into LMW fragments that, though not rigorously sized, largely were eluted later than glucagon (mw = 3,500 dabons) on Sephadex G-50 (Fig. 4). As shown in Fig. 5, left these LMW fragments, when mixed with blood in in vitro and in
138
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I5 -
et al.
J. ALLERGY
Albumm VO
,4 -
,3 -
2
I
0
20
30
40 FRACTION
50 NUMBER
60
70
FIG. 4. Sephadex G-50 elution profile of Tc-albumin 6 hr after preparation and purification. Radioactive profile plotted semilogarithmically.
vivo experiments, tended to bind to albumin (or a protein of similar molecular weight) in a relatively fixed percentage. Consequently, gel filtration data from actual experimental studies (Fig. 5, right) that apparently indicate transport of intact albumin across the mucosa may be misleading. A more satisfactory explanation is that the radioactivity in the blood reflects the accumulation of fragments, considerably smaller than intact albumin or the active fraction of Ascuris antigen (mw = 18,000 daltons),15 that crossed the mucosa and partially bound to circulating albumin. There are alternatives to increased permeability as explanations for the increased accumulation of labeled albumin fragments in blood after antigen challenge in responsive animals. The possibility that decreased renal clearance of these LMW fragments secondary to allergic bronchoconstriction might account for their increased blood levels is rendered unlikely by the absence of the necessary changes in glomerular filtration rate (Table II). Alternatively, increases in proteolytic activities in the airways or pulmonary interstitium following allergic bronchoconstriction might result in a greater pool of labeled fragments available for absorption in the responsive animals. Although direct data on proteolytic activity are not available in the rhesus model of asthma, it is known that proteolytic activity in the blood of human asthmatics and of Bordetella pertussis-immunized mice is decreased16 and proteinase inhibitor activity of trypsin in human asthmatics is increased when compared with patients with other lung diseases,” making such an explanation unlikely. Finally, it is conceiv-
CLIN. IMMUNOL. AUGUST 1977
able that the rapid shallow breathing associated with allergic bronchoconstriction could have redistributed the radiolabeled albumin in the airways, leading to greater absorption of LMW fragments by virtue of this larger surface area. However, we were unable to detect redistribution of labeled albumin during rapid shallow breathing produced passively as assessed by lung scanning, nor did we detect an increase in the rate of accumulation of counts in the blood following this maneuver. In view of these considerations, we interpret the increased rate of accumulation of counts in blood following antigen challenge in the responsive monkeys to indicate a change in airway mucosal permeability rather than other factors. Returning then to the first question examined, our data suggest there are no inherent differences in mucosal permeability between responding and nonresponding animals in the absence of antigen challenge. Since both groups are sensitive by skin test criteria to Ascaris antigen, it therefore seems unlikely that differences in bronchial mucosal permeability provide an explanation for differences in bronchial reactivity to inhaled antigen. However, with respect to the second question, our data show that bronchial mucosal permeability is not a constant parameter. The increase in the rate of appearance of counts in the blood following allergic bronchoconstriction suggests that airway permeability is acutely increased and remains altered for at least one hour after challenge, well after the change in respiratory mechanics induced by challenge had returned to normal. These findings are of interest with reference to the data of Buckle and Cohen,6 who reported a patient with symptomatic allergic rhinitis with greatly increased nasal permeability when compared to quiescent atopic patients with similar histories. Such observations are consistent with our data and suggest that increases in mucosal permeability clearly can be an effect of allergic reactions. Reported data concerning relative mucosal permeabilities of quiescent atopic and normal individuals are conflicting.6* l* The fact that antigen challenge of a sensitive, but asymptomatic, animal can increase mucosal permeability may help to reconcile such inconsistencies. It is conceivable that exposure to antigen insufficient to produce clinical symptoms might induce significant permeability changes in an apparently asymptomatic patient, and unless appropriately sensitive techniques are employed to monitor such changes in permeability, the allocation of the individual for clinical research purposes may be faulty. From a pathogenetic viewpoint, the observation that mucosal permeability can be altered by antigen challenge has several implications. First, increased bronchial mucosal permeability following antigen
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mucosal
permeability
139
in rhesus monkey
FIG. 5. Sephadex G-50 elution profiles of 4 monkey plasma samples. Upper left, in vitro incubation of LMW fragments with whole monkey blood. Lowerleft, plasma profile 30 min after intratracheal instillation of LMW fragments. Upper right, plasma sample from a responsive monkey 30 min after instillation of Tc-albumin. Lower right, plasma sample from the same animal 30 min after antigen challenge. (II, represents void; F, fragment peak).
challenge would provide a mechanism for amplification of the initial allergic reactions by permitting increased access of antigen to the mast cell-rich submucosal tissues, a mechanism that would be operative whether the initial reaction between antigen and target cell occurs in the submucosa, or, as others have suggested, in the lumen of the airway.lg In addition, increased mucosal permeability following antigen challenge may offer an explanation for the increased sensitivity to aerosolized carbachol and histamine postchallenge reported for the monkeyzO and guinea pk2’ respectively. It is tempting to speculate that similar induced increases in mucosal permeability might contribute to the increased bronchial reactivity of asthmatics to a variety of low molecular weight agonists, including histamine and methacholine,22, 23 and the increased bronchial response to histamine following tracheobronchial infection.24 Finally, increases in mucosal permeability are likely bidirectional and an accelerated flux from the interstitium to airway lumen may contribute to the increased amounts of albumin reported in the sputum of asthmatic patients.25 The site and mechanisms underlying the alterations in mucosal permeability are unknown and may be related to the microvasculature, epithelium, or both. The microvasculature likely becomes more permeable as a result of local mediator release from mast cells, but changes in the epithelium may contribute as well.
TABLE ulin
II. Effect
(mg1100 Control
Mon-
of antigen
ml) levels (steady
challenge
on PlSSmS
in 4 responding
in-
monkeys
Postchallenge
state)
35 min
40 min
45 min
50 min
55 min
75 min
I
30.9 24.8 28.3
21.4 25.4 27.6
E
ND*
ND
29.9 25.1 21.9 25.3
31.8 25.0 27.9 24.0
31.1 26.0 31.4 24.1
32.4 24.7 30.2 25.0
key
W A
*Not done.
Recent evidence suggests that both histamine and methacholine can alter the protective function of the epithelial tight junctions and allow increased permeability to horseradish peroxidase, a macromolecule with a molecular weight of 40,000 daltons. Such observations suggest a potential role for these mediators in the changes in permeability seen with allergic bronchoconstriction.26 We wish to thank Dr. Morton Levy and Ms. C. Cox for the inulin determinations; Mr. H. Ghezzo for assistance in the statistical analysis; and Ms. E. Hervas, Ms. M. Kim, and Mr. M. Mann for technical assistance. REFERENCES
I. Cohen, M. B., Ecker, E. E., Briebart, J. R., and Rudolph, J. A.: The rate of absorptionof ragweedpollen material from the nose, J. Immunol. l&419, 1929.
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2. Walzer, W.: Absorption of antigens, J. ALLERGY 13554, 1942. 3. Salvaggio, J. E., Cavanaugh, J. J. A., Lowell, F. C., and Leskowitz, S.: A comparison’of the immunologic responses of normal and atopic individuals to intranasally administered antigen, J. ALLERGY 3562, 1964. 4. Salvaggio, J. E., and Leskowitz, S.: A comparison of the immunologic responses of normal and atopic individuals to parenterally, alum precipitated protein antigen, Int. Arch. Allergy Appl. Immunol. 26264, 1965. 5. Leskowitz, S., Salvaggio, J. E., and Schwartz, H. J.: A hypothesis for the development of atopic allergy in man, Clin. Allergy 2:237, 1972. 6. Buckle, F. G., and Cohen, A. B.: Nasal mucosal hyperpermeability to macromolecules in atopic rhinitis and extrinsic asthma, J. ALLERGY CLIN. IMMUNOL. 55213, 1975. 7. Patterson, R., and Kelley, J. F.: Animal models of the asthmatic state, Ann. Rev. Med. 25:53, 1974. 8. Patterson, R., Harris, K. E., Suszko, I. M., and Roberts, M.: Reagin-mediated asthma in rhesus monkeys and relation to bronchial cell histamine release and airway reactivity to carbocholine, J. Clin. Invest. 57:586, 1976. 9. Pare, P. D., Michoud, M. C., and Hogg, J. C.: Lung mechanics following antigen challenge of Ascaris SUU~ sensitive rhesus monkeys, J. Appl. Physiol. 41:668, 1976. 10. Fahey, J. L., McCoy, P. F., and Goulian, M.: Chromatography of serum proteins in normal and pathologic sera: Distribution of protein-bound carbohydrate and cholesterol, siderophilin, thyroxin-binding protein, B-12 binding protein, alkaline and acid phosphatases, radioiodinated albumin and myeloma proteins, J. Clin. Invest. 37:272, 1958. 11. Schoenberger, M. Z.: Streulichtmessungen an plasma-protein, Z. Naturforsch. 105:474, 1955. 12. Lin, M. S., Winchell, H. S., and Shipley, B. A.: Use of Fe (11) or Sn (11) alone for labelling of albumin, J. Nucl. Med. 12:204, 1971. 13. Smith, H. W.: The kidney; structure and function in health and disease, New York, .1951, Oxford University Press, p. 62. 14. Fuhr, J., Kacmarczyk, J., and Kruttgen, C. D.: Eine einfache colorimetrische methode zur inulinbestimmung fiir niegren-
J. ALLERGY
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clearance-untersuchungen bei stoffwechelgesunden und diabetikern, Klin. Wochenschr. 33:729, 1955. 1.5. Hussain, R. Bradbury, S., and Stregan, Cl.: Hypersensitivity to ascaris antigens. VIII. Characterization of a highly purified allergen, J. Immunol. 111:260, 1973. 16. Busse, W. W., and Reed, C. E.: Abnormal degradation of macroaggregated albumin particles in patients with asthma, J. ALLERGY CLIN. IMMUNOL. 53271, 1974. 17. Smith, J. M.: Interference with tryptic digestion by sputum from asthmatic patients, Am. Rev. Respir. Dis. g&858, 1963. 18. Kontu-Karakitsos, K., Salvaggio, J. E., and Mathews, K. P.: Comparative nasal absorption of allergens in atopic and nonatopic subjects, J. ALLERGY CLIN. IMMUNOL. 55241, 1975. 19. Richardson, J. B., Hogg, J. C., Bouchard, T., and Hall, D. L.: Localization of antigen in experimental bronchoconstriction in guinea pigs, J. ALLERGY CLIN. IMMUNOL. 52~172, 1973. 20. Patterson, R. W., and Harris, K. E.: The effect of cholinergic and anticholinergic agents on the primate model of allergic asthma, J. Lab. Clin. Med. 87:65, 1976. 21. Popa, V., Douglas, J. S., and Bouhuys, A.: Airway responses to histamine, acetylcholine, and propranalol in anaphylactic hypersensitivity in guinea pigs, J. ALLERGY CLIN. IMMUNOL. 51:344, 1973. 22. Curry, J. J.: The action of histamine on the respiratory tract in normal and asthmatic subjects, J. Clin. Invest. 25:785, 1947. 23. Herxheimer, H.: Bronchial obstruction induced by allergens, histamine, and acetyl-beta-methyl-choline-chloride, Int. Arch. Allergy Appl. Immunol. 2:27, 1951. 24. Empey, D. W., Laitinen, L. A., Jacobs, L., Gold, W. M., and Nadel, J. A.: Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection, Am. Rev. Respir. Dis. 113:131, 1976. 25. Dunhill, M. S.: The morphology of airways in bronchial asthma, in‘Stein, M., editor: New directions in asthma, Parkridge, Ill., 1975, American College of Physicians, 213. 26. Boucher, R. C., Ranga, V., Pare, P. D., Inoue, S., Moroz, L., and Hogg, J. C.: Effect of histamine and methacholine on respiratory mucosal permeability, Am. Rev. Respir. Dis., vol. 115, April, 1977. (Abst.)
M.D., L. A. Moroz,
M.D.,
The permeability of the airways to technetium 99m-labeled albumin was measured in Ascaris suum-sensitive rhesus monkeys. All 8 animals were skin-sensitive to Ascaris suum (AA) antigen, 4 being respiratory responders (R) and 4 nonresponders (NR) to aerosolized antigen. In the absence of antigen challenge there were no differences in the accumulation in the blood of radioactive material from the tracheobronchial tree between the R and NR animals. After a five-minute challenge with aerosolized AA, there was a threefold increase in the rate of accumulation of radioactive material in the blood over control for the R group with no efSect noted in the NR group. Gel filtration data indicated that the radioactivity in the blood most likely represented low molecular weight albumin fragments, resulting from spontaneous degradation of Tc-albumin, that crossed the mucosa and partially bound to circulating albumin. It is concluded that hyperpermeability of the airway mucosa probably is not a factor that contributes to the selective responsiveness of the R group to aerosolized antigen, and that airway permeability is increased consequent to the allergic reaction mediating acute bronchoconstriction.
A defect in the normal protective function of mucosal surfaces may contribute to the atopic state.‘, * Supporting this view is the observation that antigen placed on the nasal mucosa stimulates significantly greater reaginic responses in atopic individuals than in normals,3 while reagin production is similar for both groups when antigen is administered subcutaneously.4 It has been suggested that the increased response to surface immunization in the atopic group results from increased availability of antigen to immunocompetent cells in the submucosa and regional lymph nodes, reflecting either increased mucosal permeability or decreased local antigen elimination.5 Hyperpermeability of the airway mucosa has been offered as an explanation for the increased bronchial reactivity to inhaled antigens in classic extrinsic asthma.6 This mechanism may explain differing
From The Lyman-Duff Laboratory, Department of Pathology, McGill University, and The Harry WebsterThorp Laboratories, Division of Clinical Immunology, McGill University Clinic and Royal Victoria Hospital. Supportedby MRC Grants Mt-4219 and MT-2844. Presentedin part at the Brook Lodge Symposium on Asthma, September, 1976. Received for publication April 2, 1976. Accepted for publication June 3, 1976. Reprint requeststo: Dr. R. C. Boucher, Department of Pathology, McGill University, 3775 University St., Montreal, Quebec, CanadaH3A 2B4. Vol. 60, No. 2, pp. 134-140
bronchial reactivities to a given antigen among individuals with similar cutaneous sensitivities to that antigen. However, it is not known whether observed differences in mucosal permeability are a cause or an effect of the inflammatory state uritier consideration. In the present study, therefore, we attempted to investigate these two possibilities separately. First, are there inherent differences in mucosal permeability which correlate with reactivity to an inhaled antigen in asthma and, second, does the allergic reaction itself induce changes in permeability? To investigate these questions, we used the Ascaris suum-sensitive Rhesus monkey, a model which Patterson and others have shown to share many characteristics with the human asthmatic. ‘* * MATERIALS Animals
AND METHODS
Eight Macaca mulatta monkeys, weighing 3 to 6 kg, were studied. All manifested varying degrees of skin sensitivity to Ascaris suum antigen (AA). Four were bronchial responders (R) and 4 were nonresponders to aerosolized antigen at a dose of 100 yg/ml; the respiratory response of the R animals to antigen challenge was characterized by a 200% to 400% increase in frequency, 100% to 1,OOO% increase in pulmonary resistance, and 40% to 80% fall in dynamic compliance with return to control values within 30 to 60 min, while the NR animals did not vary from control parameters by more than 50%. A detailed description of the skin sensitivity and changes in lung mechanics associated
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with challenge in this rhesus colony has been previously reported. g
mucosal
permeability
in rhesus monkey
135
technetium was also injected intravenously into a monkey, and after 30 min a heparinized blood sample was drawn and plasma was fractionated and counted.
Antigen Crude Axcaris suum extract, described previously,g was supplied by Dr. P. B. Stewart, Pharma Research, Montreal, Quebec, Canada.
Purification
of albumin
Albumin was prepared from normal monkey serum by sequential ammonium sulfate precipitation (fraction precipitating between 2.6 and 3.0 M), diethylaminoethyl (DEAE) cellulose chromatography at pH 8 (Watman Ltd., Chifton, N.Y.),‘O and gel filtration on Bio-Gel A 0.5 (Bio-Rad L,aboratories, Richmond, Calif.). The resulting preparation was homogenous by cellulose acetate electrophoresis. with an estimated purity greater than 99.5%. The concentration of purified albumin was estimated by absorbency at 280 nm, using the extinction coefficient, E;& = 5.8.”
Radiolabeling
of albumin
Purified albumin was prepared for radiolabeling by lyophilization and packaging in glass vials with SnCl, and dextrose under 100% N2 (Chas. Frosst & Co., Montreal, Quebec, Canada). Albumin was radiolabeled with fresh technetium 99m1* immediately prior to each experiment. Because of degradation in the preparative and labeling processes, the freshly labeled albumin was then repurified prior to use by sequential gel filtration on columns of Bio-Gel A 0.5 M (0.9 x 60 cm) and Sephadex G-50 (Pharmacia Fine Chemicals, Montreal, Quebec, Canada) (0.9 x 30 cm) equilibrated with 0.015 M phosphate-buffered 0.15 M NaCl, pH 7.4 (PBS). Aliquots of the freshly labeled albumin were reserved for subsequent dilution and counting in a gamma radiation spectrometer (Packard Instruments, Inc., Downers Grove, Ill.) to estimate the dose delivered (average, 100 &i per animal), and for Sephadex G-50 gel filtration (0.9 x 50 cm column) to assess the extent of fragmentation during the experimental period. The final concentration of the labeled albumin was between 0.05 and 0.1 mg/ml with a specific activity of 1 to 2mCi/mg.
In vivo and in vitro behavior labeled albumin fragments
in plasma of
Low molecular weight (LMW) fragments of labeled albumin, obtained by Sephadex G-50 gel filtration as described in “Results” (Fig. 4), were incubated with heparinized monkey blood at 37” C for 10 mm, centrifuged, and the plasma obtained and fractionated on Sephadex G-50 (0.9 x 50 cm column) or Bio-Gel A 0.5 m (0.9 x 50 cm column). Low molecular weight fragments were also injected intravenously and instilled intratracheahy into monkeys, and plasma samples fractionated on Sephadex G-50 (0.9 :< 50 cm column). Free technetium (NaTcO,) was incubated with heparinized whole monkey blood in vitro for 24 hr at 22” C and plasma prepared and fractionated as described above. Free
Procedure The animals were lightly anesthetized with pentobarbital sodium (20 mg/kg) and phencyclidine hydrochloride (2 to 3 mg/kg), intubated with a cuffed endotracheal tube, and studied in the upright position with a Fleisch pneumotachograph (No. 00) and arterial line in place. Each study was initiated by instilling 0.9 ml of the albumin solution into the trachea via a catheter. One-ml blood samples were obtained for counting at 30 set, I, 3, 5, 10, 20, 30, 40, 60, and 90 min after instillation. At 30 min and 60 min, additional 2.5-ml blood samples (anticoagulated with 50 U heparin) were obtained, centrifuged, and l-ml plasma samples fractionated on Sephadex G-50 (0.9 X 50 cm) and fractions counted. Each animal was studied at least twice on different days. One occasion served as a control study for a second when a 5-min aerosolized challenge of antigen (Hudson disposable nebulizer, Wadsworth, Ohio) was administered 30 min after the instillation of the radiolabeled albumin. Respiratory frequency, a sensitive indicator of AA response, was used as an index of bronchial response.g At the end of two of the control studies, rapid shallow breathing was induced manually with a syringe, and blood samples were obtained subsequently at 5-min intervals for counting.
Lung scanning The intrathoracic distribution of instilled Tc-albumin in 2 animals was assessed with an external gamma scintillation camera (Picker Canada, Montreal). After initial threedimensional scans were obtained, the animals were manually hyperventilated with a syringe and the distribution of labeled material assessed with repeat scans.
Effects of antigen challenge filtration rate (GFR)
on glomerular
The steady-state inulin infusion methodI was used to assess the effect of antigen-induced bronchospasm on GFR. The animals were given a priming dose of inulin (Nutritional Biochem. Co., Cleveland, Ohio) dissolved in normal saline calculated to give an inulin concentration of 0.25 mg/ml in extracellular water. An intravenous inulin infusion was maintained with an LKB Variopex peristaltic pump (Uppsala, Sweden) at a rate calculated to replace filtered losses (GFR estimated at 25 ml/min with urinary inulin concentration estimated to be 0.25 mg/ml). Inulin therefore was delivered at a concentration of 12.5 mg/ml and at a rate of 0.5 ml/min. Blood samples (2 ml with 40 U heparin) were obtained at 35, 40, and 45 mm after the priming dose and initiation of the infusion. A standard 5min antigen challenge was delivered to 4 sensitive monkeys beginning at the 45-min point, and blood samples were obtained 5, 10 and 30 min later (50, 55, and 75 min after the priming dose). Plasma inulin levels were measured calorimetrically.”
136
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et al.
J. ALLERGY
TIME
FIG. 1. Appearance tracheally instilled
CLIN. IMMUNOL. AUGUST 1977
lmml
in blood of radioactivity from intraTc-albumin for R and NR monkeys.
TlME
kiln1
FIG. 3. Appearance in blood of radioactivity in the blood for a responding monkey. Two challenge runs (one from preliminary study) and one control run shown.
Appearance
0
10
20
30
40
50 TIME
60
70
80
90
~IlWl)
FIG. 2. Appearance in blood of radioactivity for a single nonresponding monkey. Two control runs (one from a preliminary study) and one challenge run shown.
Analysis
of data and statistical
methods
Preliminary studies indicated the rapid accumulation of radioactive material in the blood in the 0- to IO-min period with a relatively constantrate of accumulationfrom 10 to 90 min. Therefore, slopes of accumulation of radioactivity in the blood for the 0- to IO-min period, the IO- to 30min (prechallenge) interval, and the 40- to 90-min (postchallenge) interval were computed using the leastsquaresmethod. The ratio of the slope of the postchallenge to prechallenge interval for each animal was used as an index of the effect of antigen challenge on permeability, thus taking into account small differences in rates for the prechallengeinterval. The differences in theseratios (challenge vs control run) for animals within each group were comparedby a standard t test. RESULTS Respiratory
response
The responsive animals responded to antigen challenge with a mean increase in respiratory frequency of 304% (range, 200% to 379%), while the NR group showed no change in frequency with challenge (100% k 10%).
of radioactivity
in the blood
Fig. 1 shows the percentage of the delivered dose of radioactive material present in the blood vs time for the R and NR groups. The points for each group up to 30 min represent the average for the control and challenge runs. There are no evident differences between the two groups in the amount of radioactivity present in the blood at any given time. Fig. 2 shows the results of two control and one challenge run for the same nonresponding monkey. The radiolabeled material accumulates in the blood at a similar rate in the three studies. The aerosolized antigen challenge does not appear to alter this rate. In contrast, Fig. 3, which shows the results of one control and two challenge runs for a responding monkey, demonstrates that in this animal AA challenge produces a marked and reproducible increase in the rate of appearance of counts in blood. This increase was sustained at a similar rate for the 60-min postchallenge period. Manual hyperventilation in two control studies did not alter the baseline rates. Table I presents the average rates of accumulation of counts in the blood over the three time intervals for the control and challenge runs of the R and NR animals. The ratios of the rate of accumulation of counts for the 40 to 90 min interval to the one corresponding to the 10 to 30 min interval are presented in the last column. It can be seen that for both the R and NR groups the ratios do not differ appreciably from one in the two sets of control runs. Antigen challenge in the R group increases this ratio threefold, while in the NR group the ratio increases by less than 50%. Comparison of the differences between the challenge vs control runs in these ratios computed for each animal individually showed a significant increase for the R group (p = 0.05). with no difference for the NR group.
VOLUME NUMBER
Airway
60 2
TABLE I. Rates of accumulation
mucosal
permeability
in rhesus monkey
137
in blood (mean 2 SD)
of radioactivity
Time periods (min) 40-90 O-10
Responder Nonresponder
Control Challenge
0.011 rt 0.004 0.015 k 0.004
Control Challenge
0.015 + 0.009
Nature of the radiolabeled appearing in the blood
0.007
-c
0.003
material
Fig. 4 shows the results of Sephadex G-50 gel. filtration analysis of an aliquot of the radiolabeled albumin which had been stored for 6 hr at room temperature in PBS, i.e., the time period corresponding to end of experimental use. Two peaks are seen with similar radioactive and absorbency (280 nm) profiles, suggesting degradation of albumin into LMW fragments predominantly less than 3,500 daltons. Despite initial purification, spontaneous degradation of albumin occurred during a time corresponding to the experimental period. The mean degradation for all labeled preparations was 5% (range 4 to 6%) when analyzed at 6 hr. The importance of this fragmentation is indicated by the data depicted in Fig. 5. When labeled albumin fragments obtained by gel filtration on Sephadex G-50 (Fig. 4) were incubated with monkey blood in vitro for 10 min, centrifuged, and the plasma obtained fractionated again on Sephadex G-50, approximately 30% of the radioactivity now appears in the void volume (Fig. 5 upper left), indicating binding of the labeled albumin fragments to higher molecular weight proteins in plasma. Upon similar fractionation of the mixture of plasma and labeled fragments on Bio-Gel A 0.5 m, radioactivity due to the labeled albumin fragments eluted at a volume similar to that of pure albumin alone, indicating that labeled fragments were bound either to albumin or to some plasma protein of similar molecular weight. Identical results were obtained by gel filtration analysis of plasma samples obtained from monkeys after intravenous injection or intratracheal instillation (Fig. 5, lower left) of labeled albumin fragments. Free technetium did not appear to contribute to this phenomenon, since the labeled albumin fragments eluted from Sephadex G-50 ahead of and distinct from the elution volume of free technetium (Fig. 4), and less than 0.1% of free technetium bound to albumin when mixed with monkey blood in vitro, or after intravenous administration of technetium to monkeys. The Sephadex G-50 gel filtration profiles from the experimental studies (employing Tc-labeled albumin) of responsive and nonresponsive monkeys, pre- and
10-30
40-90
10-30 1.20 3.14
0.005
2
It
_f
0.002 0.002
0.006
0.007
0.022
-e 0.012
0.008 0.004
2
0.003
0.007
c
0.005
k
0.001
0.005
k
0.004
postchallenge, files obtained ments alone. shown in Fig.
0.003
,118 1.25
were indistinguishable from the proafter administration of albumin fragResults of a typical experiment are 5 (right).
Lung scans Lung scans from two monkeys revealed that the 0.9 ml of instilled labeled albumin was deposited centrally in the trachea and main stem bronchi. Two minutes of manual hyperventilation did not alter this distribution. lnulin
data
Table II presents the steady-state and postchallenge plasma inulin levels for 4 responsive monkeys. It can be seen that in three monkeys, A, W, and E, there are clearly no changes in inulin concentrations, and hence GFR, postchallenge. In monkey I there is a late 6% increase in plasma inulin which would correspond to a similar decline in GFR. Such a small change is probably within the limits of experimental error and cannot account for the observed increases in rates of accumulation of radioactive material in the blood postchallenge (threefold increase). DISCUSSION The data summarized in Table 1 suggest that in the absence of antigen challenge there are no differences in the rates of accumulation of radiolabeled material between the responding and nonresponding monkeys. Following antigen change this rate is increased threefold for the responding group with no change in the nonresponding group. However, before interpreting these results an evaluation of the possible sources of error introduced by the fragmentation of the radiolabeled albumin and the physiologic changes accompanying bronchoconstriction is necessary. In this study, although no evidence was found for significant release of free technetium from Tcalbumin, there was evidence for the spontaneous degradation of Tc-albumin into LMW fragments that, though not rigorously sized, largely were eluted later than glucagon (mw = 3,500 dabons) on Sephadex G-50 (Fig. 4). As shown in Fig. 5, left these LMW fragments, when mixed with blood in in vitro and in
138
Boucher
I5 -
et al.
J. ALLERGY
Albumm VO
,4 -
,3 -
2
I
0
20
30
40 FRACTION
50 NUMBER
60
70
FIG. 4. Sephadex G-50 elution profile of Tc-albumin 6 hr after preparation and purification. Radioactive profile plotted semilogarithmically.
vivo experiments, tended to bind to albumin (or a protein of similar molecular weight) in a relatively fixed percentage. Consequently, gel filtration data from actual experimental studies (Fig. 5, right) that apparently indicate transport of intact albumin across the mucosa may be misleading. A more satisfactory explanation is that the radioactivity in the blood reflects the accumulation of fragments, considerably smaller than intact albumin or the active fraction of Ascuris antigen (mw = 18,000 daltons),15 that crossed the mucosa and partially bound to circulating albumin. There are alternatives to increased permeability as explanations for the increased accumulation of labeled albumin fragments in blood after antigen challenge in responsive animals. The possibility that decreased renal clearance of these LMW fragments secondary to allergic bronchoconstriction might account for their increased blood levels is rendered unlikely by the absence of the necessary changes in glomerular filtration rate (Table II). Alternatively, increases in proteolytic activities in the airways or pulmonary interstitium following allergic bronchoconstriction might result in a greater pool of labeled fragments available for absorption in the responsive animals. Although direct data on proteolytic activity are not available in the rhesus model of asthma, it is known that proteolytic activity in the blood of human asthmatics and of Bordetella pertussis-immunized mice is decreased16 and proteinase inhibitor activity of trypsin in human asthmatics is increased when compared with patients with other lung diseases,” making such an explanation unlikely. Finally, it is conceiv-
CLIN. IMMUNOL. AUGUST 1977
able that the rapid shallow breathing associated with allergic bronchoconstriction could have redistributed the radiolabeled albumin in the airways, leading to greater absorption of LMW fragments by virtue of this larger surface area. However, we were unable to detect redistribution of labeled albumin during rapid shallow breathing produced passively as assessed by lung scanning, nor did we detect an increase in the rate of accumulation of counts in the blood following this maneuver. In view of these considerations, we interpret the increased rate of accumulation of counts in blood following antigen challenge in the responsive monkeys to indicate a change in airway mucosal permeability rather than other factors. Returning then to the first question examined, our data suggest there are no inherent differences in mucosal permeability between responding and nonresponding animals in the absence of antigen challenge. Since both groups are sensitive by skin test criteria to Ascaris antigen, it therefore seems unlikely that differences in bronchial mucosal permeability provide an explanation for differences in bronchial reactivity to inhaled antigen. However, with respect to the second question, our data show that bronchial mucosal permeability is not a constant parameter. The increase in the rate of appearance of counts in the blood following allergic bronchoconstriction suggests that airway permeability is acutely increased and remains altered for at least one hour after challenge, well after the change in respiratory mechanics induced by challenge had returned to normal. These findings are of interest with reference to the data of Buckle and Cohen,6 who reported a patient with symptomatic allergic rhinitis with greatly increased nasal permeability when compared to quiescent atopic patients with similar histories. Such observations are consistent with our data and suggest that increases in mucosal permeability clearly can be an effect of allergic reactions. Reported data concerning relative mucosal permeabilities of quiescent atopic and normal individuals are conflicting.6* l* The fact that antigen challenge of a sensitive, but asymptomatic, animal can increase mucosal permeability may help to reconcile such inconsistencies. It is conceivable that exposure to antigen insufficient to produce clinical symptoms might induce significant permeability changes in an apparently asymptomatic patient, and unless appropriately sensitive techniques are employed to monitor such changes in permeability, the allocation of the individual for clinical research purposes may be faulty. From a pathogenetic viewpoint, the observation that mucosal permeability can be altered by antigen challenge has several implications. First, increased bronchial mucosal permeability following antigen
VOLUME NUMBER
Airway
60 2
mucosal
permeability
139
in rhesus monkey
FIG. 5. Sephadex G-50 elution profiles of 4 monkey plasma samples. Upper left, in vitro incubation of LMW fragments with whole monkey blood. Lowerleft, plasma profile 30 min after intratracheal instillation of LMW fragments. Upper right, plasma sample from a responsive monkey 30 min after instillation of Tc-albumin. Lower right, plasma sample from the same animal 30 min after antigen challenge. (II, represents void; F, fragment peak).
challenge would provide a mechanism for amplification of the initial allergic reactions by permitting increased access of antigen to the mast cell-rich submucosal tissues, a mechanism that would be operative whether the initial reaction between antigen and target cell occurs in the submucosa, or, as others have suggested, in the lumen of the airway.lg In addition, increased mucosal permeability following antigen challenge may offer an explanation for the increased sensitivity to aerosolized carbachol and histamine postchallenge reported for the monkeyzO and guinea pk2’ respectively. It is tempting to speculate that similar induced increases in mucosal permeability might contribute to the increased bronchial reactivity of asthmatics to a variety of low molecular weight agonists, including histamine and methacholine,22, 23 and the increased bronchial response to histamine following tracheobronchial infection.24 Finally, increases in mucosal permeability are likely bidirectional and an accelerated flux from the interstitium to airway lumen may contribute to the increased amounts of albumin reported in the sputum of asthmatic patients.25 The site and mechanisms underlying the alterations in mucosal permeability are unknown and may be related to the microvasculature, epithelium, or both. The microvasculature likely becomes more permeable as a result of local mediator release from mast cells, but changes in the epithelium may contribute as well.
TABLE ulin
II. Effect
(mg1100 Control
Mon-
of antigen
ml) levels (steady
challenge
on PlSSmS
in 4 responding
in-
monkeys
Postchallenge
state)
35 min
40 min
45 min
50 min
55 min
75 min
I
30.9 24.8 28.3
21.4 25.4 27.6
E
ND*
ND
29.9 25.1 21.9 25.3
31.8 25.0 27.9 24.0
31.1 26.0 31.4 24.1
32.4 24.7 30.2 25.0
key
W A
*Not done.
Recent evidence suggests that both histamine and methacholine can alter the protective function of the epithelial tight junctions and allow increased permeability to horseradish peroxidase, a macromolecule with a molecular weight of 40,000 daltons. Such observations suggest a potential role for these mediators in the changes in permeability seen with allergic bronchoconstriction.26 We wish to thank Dr. Morton Levy and Ms. C. Cox for the inulin determinations; Mr. H. Ghezzo for assistance in the statistical analysis; and Ms. E. Hervas, Ms. M. Kim, and Mr. M. Mann for technical assistance. REFERENCES
I. Cohen, M. B., Ecker, E. E., Briebart, J. R., and Rudolph, J. A.: The rate of absorptionof ragweedpollen material from the nose, J. Immunol. l&419, 1929.
140
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et al.
2. Walzer, W.: Absorption of antigens, J. ALLERGY 13554, 1942. 3. Salvaggio, J. E., Cavanaugh, J. J. A., Lowell, F. C., and Leskowitz, S.: A comparison’of the immunologic responses of normal and atopic individuals to intranasally administered antigen, J. ALLERGY 3562, 1964. 4. Salvaggio, J. E., and Leskowitz, S.: A comparison of the immunologic responses of normal and atopic individuals to parenterally, alum precipitated protein antigen, Int. Arch. Allergy Appl. Immunol. 26264, 1965. 5. Leskowitz, S., Salvaggio, J. E., and Schwartz, H. J.: A hypothesis for the development of atopic allergy in man, Clin. Allergy 2:237, 1972. 6. Buckle, F. G., and Cohen, A. B.: Nasal mucosal hyperpermeability to macromolecules in atopic rhinitis and extrinsic asthma, J. ALLERGY CLIN. IMMUNOL. 55213, 1975. 7. Patterson, R., and Kelley, J. F.: Animal models of the asthmatic state, Ann. Rev. Med. 25:53, 1974. 8. Patterson, R., Harris, K. E., Suszko, I. M., and Roberts, M.: Reagin-mediated asthma in rhesus monkeys and relation to bronchial cell histamine release and airway reactivity to carbocholine, J. Clin. Invest. 57:586, 1976. 9. Pare, P. D., Michoud, M. C., and Hogg, J. C.: Lung mechanics following antigen challenge of Ascaris SUU~ sensitive rhesus monkeys, J. Appl. Physiol. 41:668, 1976. 10. Fahey, J. L., McCoy, P. F., and Goulian, M.: Chromatography of serum proteins in normal and pathologic sera: Distribution of protein-bound carbohydrate and cholesterol, siderophilin, thyroxin-binding protein, B-12 binding protein, alkaline and acid phosphatases, radioiodinated albumin and myeloma proteins, J. Clin. Invest. 37:272, 1958. 11. Schoenberger, M. Z.: Streulichtmessungen an plasma-protein, Z. Naturforsch. 105:474, 1955. 12. Lin, M. S., Winchell, H. S., and Shipley, B. A.: Use of Fe (11) or Sn (11) alone for labelling of albumin, J. Nucl. Med. 12:204, 1971. 13. Smith, H. W.: The kidney; structure and function in health and disease, New York, .1951, Oxford University Press, p. 62. 14. Fuhr, J., Kacmarczyk, J., and Kruttgen, C. D.: Eine einfache colorimetrische methode zur inulinbestimmung fiir niegren-
J. ALLERGY
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clearance-untersuchungen bei stoffwechelgesunden und diabetikern, Klin. Wochenschr. 33:729, 1955. 1.5. Hussain, R. Bradbury, S., and Stregan, Cl.: Hypersensitivity to ascaris antigens. VIII. Characterization of a highly purified allergen, J. Immunol. 111:260, 1973. 16. Busse, W. W., and Reed, C. E.: Abnormal degradation of macroaggregated albumin particles in patients with asthma, J. ALLERGY CLIN. IMMUNOL. 53271, 1974. 17. Smith, J. M.: Interference with tryptic digestion by sputum from asthmatic patients, Am. Rev. Respir. Dis. g&858, 1963. 18. Kontu-Karakitsos, K., Salvaggio, J. E., and Mathews, K. P.: Comparative nasal absorption of allergens in atopic and nonatopic subjects, J. ALLERGY CLIN. IMMUNOL. 55241, 1975. 19. Richardson, J. B., Hogg, J. C., Bouchard, T., and Hall, D. L.: Localization of antigen in experimental bronchoconstriction in guinea pigs, J. ALLERGY CLIN. IMMUNOL. 52~172, 1973. 20. Patterson, R. W., and Harris, K. E.: The effect of cholinergic and anticholinergic agents on the primate model of allergic asthma, J. Lab. Clin. Med. 87:65, 1976. 21. Popa, V., Douglas, J. S., and Bouhuys, A.: Airway responses to histamine, acetylcholine, and propranalol in anaphylactic hypersensitivity in guinea pigs, J. ALLERGY CLIN. IMMUNOL. 51:344, 1973. 22. Curry, J. J.: The action of histamine on the respiratory tract in normal and asthmatic subjects, J. Clin. Invest. 25:785, 1947. 23. Herxheimer, H.: Bronchial obstruction induced by allergens, histamine, and acetyl-beta-methyl-choline-chloride, Int. Arch. Allergy Appl. Immunol. 2:27, 1951. 24. Empey, D. W., Laitinen, L. A., Jacobs, L., Gold, W. M., and Nadel, J. A.: Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection, Am. Rev. Respir. Dis. 113:131, 1976. 25. Dunhill, M. S.: The morphology of airways in bronchial asthma, in‘Stein, M., editor: New directions in asthma, Parkridge, Ill., 1975, American College of Physicians, 213. 26. Boucher, R. C., Ranga, V., Pare, P. D., Inoue, S., Moroz, L., and Hogg, J. C.: Effect of histamine and methacholine on respiratory mucosal permeability, Am. Rev. Respir. Dis., vol. 115, April, 1977. (Abst.)