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Antigen-antibody mediated desensitization of human lung fragments in vitro
Antigen-anti body mediated desensitization of human lung fragments in vitro Jeffrey M. Drazen, K. Frank Austen,
M.D., Daniel M.D. Bostm,
J. Stechschulte,
M.D.,
and
Mam.
Huma?& lwng fragments incubated with atopio sorwm and specific antigen exhibited marked inhibition. of mediator release upon subsequent antigen challenge. Unrler the oonditioaa reszGlting &a alesonsitizatiolt, mediators were not released into the sensitizing diffmate, the total tissue hist~ine stores were not diminished, ana added histamine was not signijioantly metabolized. Incllbation of lung fragments, atopia serum, an& antigen in a oaloiurfn-free5 miU ethylenediaminetetracetic acid (EDTA) buffer resulted in blockade of desensitization. Thus the presence of antigen rlzvrng passive sensitization does not result in mediator release, yet renders target cells unable to respond to subsequent antigen challenge.
The loss of cellular reactivity to antigen, observed in the leukocytes of selected patients undergoing hyposensitization therapy, has been offered as a possible explanation for amelioration of symptoms in these patients.l Loss of cell reactivity did not appear to result from loss of serum reaginic activity and was attributed to a possible intrinsic change in the way cells respond to antigen.2 It has now been demonstrated that the presence of antigen E during incubation of human lung fragments with atopic serum alters cellular reactivity to subsequent antigen challenge. Antigen-dependent antibody-mediated desensitization is proposed as a possible in vitro model for hyposensitization. MATERIALS
AND
METHODS
Ragweed antigen E was obtained from the Research Resources Branch of the National Institute of Allergy and Infectious Diseases. Serum samples from a ragweed-sensitive patient (R. 8.) (IgE level 250 ng. per milliliter) were used in the present experiments unless otherwise indicated. Tyrode’s solutions was prepared as described. Disodimn and tetrasodium ethylenediaminetetraacetic acid (EDTA) were obtained from Fisher Scientific Corp. (Philadelphia, Pennsylvania). Histamine diphosphate was obtained from Mann Laboratories (Orangeburg, New York). Specimens of macroscopically normal human lung tissue were obtained at the time of surgery for carcinoma of the bronchus and were prepared in 200 mg. replicates as described previously.4 Atopic serum (0.9 ml. per vial) diluted 1:l or 1:3 in Tyrode’s solution was placed in glass vials 2.0 cm. in diameter and 6.0 em. high. One-tenth milliliter of Tyrode’s solution, From the Departments of Medicine, Harvard Medical School, Robert B. Brigham Hospital, and Peter Bent Brigham Hospital. Supported b grants AI-07722, AI-10356, and RR05669 from the National Institutes of Health an cl a grant from the John A. Hartford Foundation, Inc. Dr. Stechschulte is the the recipient of a Research Career Development Award (AI-23405) from the National Institutes of Health. Received for publication Jan. 10, 1973. Re rint requests to: K. Frank Au&en, M.D., Robert B. Brigham Hospital, Boston, Mass. B2120. Vol. 56, No. 9, pp. lS8-166
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Antigen-antibody
TABLE 1. Antigen-antibody
mediated
desensitization
Sensitization Serum ii 8. R.. s.
Dilution
0 1 0
1:3 1:l 1:l 1:3
1 0 1 0 0’
1:3
El. 1%‘.
1:l
El. w. Ii. F. H. F.
1:l 1:l 1:l
of human
lung
Antigen Mg/ml.l
1:l 1:3
I-1. Ii.
desensitization
1 Mediator
1:l
R.. 5. R. S. L. K. L,. K. L. K.
mediated
of lung
fragments*
release
with
antigen
Histamine (% total)
0
challenget SRS-A kt./Gm.)
30
1
159
345
1.5
N.D.:
35 2.1 36 4.1 32 3.0 29 3.0 22
300 45 225
N.D. 300
N.D. 505 N.D. 225 N.D.
1 N.D. ‘Lung fragments were sensitized at 22O C. for 16 hours with serum from ragweed-sensitive patients. t Antigen challenge (1 pg antigen E per milliliter) was at 37” C. for 15 minutes. tLndicates mediator release was not detectable.
containing an appropriate dilution of antigen E, was added to each vial, followed in 2 to 5 seconds by the addition of lung fragments. The vials were incubated at 22’ C. for 16 to 22 hours or at 37” C. for 4 hours unless otherwise indicated in the text. After the incubation Lleriod, the sensitizing diffusate was withdrawn and assayed for histamine or slow-reacting substance of anaphylaxis (SRS-A) activity on the isolated guinea pig ileum in the presence of atropine and mepyramine ma1eate.s The fragments were washed 3 times in Tyrode’s solution, resuspended in 3.0 ml. Tyrode’s solution, and prewarmed at 37” C. for 5 minutes. After challenge with antigen E, the fragments were incubated for 15 minutes at 37” C.; the diffusate was recovered and assayed for histamine and SRS-A activity. The fragments were resuspended in 3.0 ml. Tyrode’s solution and boiled for 8 minutes to extract residual tissue histamine. RESULTS Antigen-dependent fragments
antibody-mediated
desensitization
of human
lung
The effects of introducing antigen E (1.0 pg per milliliter) during the sensitization of human lung fragments was examined with sera from a variety of patients with ragweed sensitivity. There was marked inhibition of mediator release on antigen challenge of fragments sensitized in the presence of antigen (Table I). In another series of experiments a single serum donor was used and the concentration of antigen E present during passive sensitization was varied. The presence of 10.0 or 1.0 pg per milliliter of antigen E during sensitization resulted in a marked desensitization of human lung fragments. to subsequent antigen challenge (Fig. 1). A graded desensitization was observed when smaller doses of antigen E were present during passive sensitization, while passive sensitization in the presence of 1O-4rg per milliliter antigen E resulted in no desensitization to subsequent antigen challenge. In order to determine whether the apparent desensitization to antigen challenge with 1.0 fig per milliliter antigen E was peculiar to this dose, tissues were challenged with antigen E in a dose range from 1O-4 to 10.0 pg per milliliter. A.s shown in Fig. 2, human lung fragments sensitized with atopic
160
Drazen,
Stechschulte,
and
-
FIG. 1. Effects serum [R. S. 1:3) induced (1.0 fig (closed circles).
of
J. ALLERGY
Austen
ANTIGEN
E DURING
geometrically increasing and lung fragments for antigen E per milliliter)
SENSITIZATION
CLIN. IMMUNOL. SEPTEMBER 1973
&g/ml)
amounts of antigen E incubated with atopic 22 hours at 22” C. on the subsequent antigenrelease of histamine [open circles) and SRS-A
serum released histamine and SRS-A in a graded fashion over the range of antigen E challenge doses, When antigen E (1.0 pg per milliliter) was present during sensitization, lung fragments failed to release significant amounts of histamine or SRS-A on antigen challenge with antigen E over the range of doses from 10V4 pg per milliliter to 1.0 ,ug per milliliter. Challenge with 10.0 pg per milliliter antigen E resulted in the release of SRS-A but not histamine. A series of experiments was performed to determine the time course of the antigen-dependent antibody-mediated desensitization. Tissue incubated with allergic serum demonstrated passive sensitization at one hour as evidenced by antigen-induced mediator release (Fig. 3). The degree of sensitization appeared to increase up to 8 to 20 hours. The presence of antigen during the sensitization period permitted no subsequent antigen-induced mediator release up to 4 hours of sensitization, and resulted in a marked inhibition of mediator release at times thereafter. Absence fragments
of mediator and antigen
release
during
incubation
of
atopic
serum
with
lung
In all experiments the diffusate from the sensitization of lung fragments, with or without antigen E, was assayed for the presence of histamine and SRS-A. The histamine present in the diffusate was always less than 2 per cent of the available total histamine and was the same whether or not antigen was present during sensitization. Not only was there no detectable difference in the histamine
VOLUME NUMBER
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52 3
mediated
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200 // SRS-A RELEASE funits / gram I
I50 ,oo
/ P.,
161
/fla
/I
I’
OL c
50 I
5or
HISTAMINE RELEASE
d’
d
20
i ANTIGEN
E CHALLENGE
DOSE
HOURS
OF SENSi/Tf,?ATION
rju/ml~ FIG. 2. on the serum for 22
Effects of geometrically release of histamine (R. S. 1:3) and with hours at 22” C.
FIG. 3. Antigen-induced with (closed circles) at 22” C.
increasing amounts of antigen E at the time of challenge, and SRS-A. All lung fragments were sensitized with atopic antigen E (closed circles) or without antigen E (open circles)
histamine and SRS-A release after sensitization or without (open circles) antigen E (1 pg per milliliter).
for
various Incubation
times was
or SRS-A levels in the sensitizing diffusates, but also the total tissue histamine after sensitization, with or without antigen, was the same. The latter was calculated from the sum of the antigen-induced release and the tissue-extractable histamine as shown in Fig. 4. In another series of experiments, histamine was added at final concentrations ranging from 0 to 400 ng. histamine base per milliliter to vials containing lung and atopic serum with or without antigen; the vials were shaken for 1 minute at 22’ C.; a portion of the fluid was withdrawn from each, and the vials were incubated at 37O C. for 4 hours. The diffusates were withdrawn and both the presensitization and postsensitization diffusates were assayed for histamine (Fig. 5). The amount of histamine detected at 1 minute was within 10 per cent of the calculated final concentration, and the subsequent loss of histamine during the 4 hour incubation, as shown in Fig. 5, was less than 20 per cent and not influenced by the presence of antigen. In other experiments histamine recovery after 22 hours at 22’ C. varied from 60 to 100 per cent of added histamine.
162
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CLIN. IMMUNOL. SEPTEMBER 1973
5
T T
F
SENS.
-RELEASE-
CHALL.
RESID.
TOTAL
o
I!’
2bo
HISTAMINE
-TISSUE-
FIG. 4. Comparison of the available serum and antigen (dark bars) and bars). SENS. = Histamine released released into diffusate at 15 minutes histamine. TOTAL = Sum of SENS. t error of the mean.
Al
360 ADDED
4bo /ng/mlj
histamine from lung fragments sensitized with atopic fragments sensitized with atopic serum alone (light into the sensitizing diffusate. CHALL. = Histamine after antigen challenge. RESID. = Residual tissue CHALL. t RESID. Vertical lines indicate one standard
FIG. 5. Recovery of added histamine after incubation with (closed circles) or without (open circles) antigen E, atopic serum, and lung fragments at 37” C. for 4 hours. Ordinate, Net histamine assayed in the diffusate after 4 hours at 37” C. Abscissa, Histamine assayed in an aliquot of the diffusate after 1 minute mixing period, but before incubation at 37” C. Dashed line through the origin represents 100 per cent recovery. Blockade absence
of antigen-dependent antibody-mediated of calcium and magnesium ions
desensitization
by
the
Human lung fragments were washed 3 times in a modified Tyrode’s solution containing 5 mM EDTA and no calcium or magnesium ions. Fragments were sensitized, with or without antigen, in antiserum diluted in the modified buffer for 4 hours at 37O C. After sensitization the diffusate was withdrawn $nd assayed and the fragments were washed 3 times with normal Tyrode’s solution, suspended in 3.0 ml. normal Tyrode’s solution, and then prewarmed at 37’ C. for 5 minutes before antigen challenge. As shown in Table II, human lung fragments sensitized in the calcium- and magnesium-free buffer yielded amounts of histamine and SRS-A on antigen challenge similar to those obtained from tissue sensitized in normal Tyrode’s solution. The presence of antigen E during sensitization in normal Tyrode’s solution resulted in a greater than 90 per cent inhibition of subsequent antigen-induced mediator release. In contrast, the deletion of calcium and magnesium during sensitization in the presence of antigen blocked the desensitization such that the release of histamine was 50 per cent and that of SRS-A was 88 per cent of that observed with tissue sensitized in the absence of antigen. The release of histamine and SRS-A upon restoration of the cation concentration and challenge with antigen reflected the effects of both
VOLUME NUMBER
Antigen-antibody
52 3
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163
TABLE II. Blockade of antigen-antibody mediated desensitization of human lung fragments by incubation in a calcium- and magnesium-free, 5 mM EDTA buffer* Rekase Sensitization EDTA (mM)
with Calcium (mMlt
atopic
serum Antigen Wglml.)
E
Antigen challenge &g/ml.)
E
of mediators SRS-A
Histamine %
(
S.E.M.
W./Gm.l
S.E.M.
38 280 N.D. N.D. ia: if 18 N.h. 0:o N.D. 5.0 E h*i 3:-i if 360 45 5.0 0:o 1:o 1.0 15:7 2.6 320 21 1.8 170 33 5.0 0.0 1.0 0.0 6.9 *Average of 3 experiments. All lungs were sensitized at 37” C. for 4 hours with serum R.S. at 1:3 dilution of the appropriate buffer. All fragments were challenged, as indicated, with 1.0 +g per milliliter antigen E in normal Tyrode’s buffer. N.D. indicates that mediator release was not detectable. S.E.M. refers to the standard error of the mean. tCa.leium concentration in Tyrode’s buffer. 0.0
1.8
0.0 0.0 1.0
1.0 0.0 1.0 0.0 1.0
31.0 N.D. 1.6
4.8 N.D. 0.6
new antigen and that combined with antibody during the sensitization period. The effects of antigen-antibody interactions during sensitization are revealed by the histamine and SRS-A release after restoration of the cation concentration. DISCUSSION
Human lung fragments sensitized in atopic serum in the presence of ragweed antigen E exhibit a marked inhibition of mediator release upon subsequent antigen challenge. The extent of this antigen-dependent antibody-mediated desensitization was determined by the concentration of antigen present during the sensitization period (Fig. 1). Inhibition of subsequent antigen-induced mediator release was consistently greater than 80 per cent when incubation was carried out with a 1:l or a 1:3 dilution of various atopic sera in the presence of 1.0 pg per milliliter antigen E (Table I). Desensitization was not overcome by varying the antigen challenge dose from 1O-4 to 10.0 pg per milliliter antigen E (Fig. 2). At no time during passive sensitization of tissue in the presence of antigen was there an interval when desensitization was not demonstrable (Fig. 3).
The interpretations of these experiments depend on, the evidence that mediator release does not occur during incubation of human lung fragments in atopic serum and antigen. In all experiments the amount of histamine or SRS-A released into the diffusate during sensitization was the same regardless of whether or not incubation in atopic serum was carried out in the presence of antigen. The possibility that the failure to detect mediator release during sensitization in the presence of antigen was an artefact due to the metabolism of released mediators was excluded by two lines of evidence. First, there was no depletion of tissue histamine stores in desensitized lung fragments (Fig. 4). The maintenance of tissue histamine stores cannot be attributed to the synthesis of new histamine or to the binding of released histamine because antigen challenge of passively sensitized tissue followed by incubation periods from 8 minutes to 4 hours revealed no progressive change in histamine content of the diffusates or tissues I(unpublished observations). Further, Lilja, Lindell, and Saldeene
164
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and
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J. ALLERGY
CLIN. IMMUNOL. SEPTEMBER 1973
demonstrated that 2 to 3 Gm. of normal lung tissue incubated in vitro for 3 hours at 3’7O C. in a buffer containing 40 pg 14C-histidine per milliliter synthesized only 43 ng. of histamine. Our fragments were approximately one-tenth as large and were incubated in a buffer without added histidine, conditions that appear unfavorable for histamine synthesis. Second, there was no unusual metabolism of exogenous histamine added at a concentration comparable to that released during the antigen challenge of passively sensitized tissue (Fig. 5). Eiseman, Bryant, and Waltuch? noted 82 per cent recovery of added histamine after 30 minutes of perfusion through heart-lung preparations from normal dogs. In contrast, Lilja, Lindell, and Saldeen6 and Bouhuys and Lindell* reported slightly greater than 50 per cent recovery of total histamine after incubation with human lung fragments at 37O C. Bennett? noted that recovery of histamine incubated with chopped guinea pig lung fragments varied from 60 to 30 per cent depending on the species and that homogenization rather than chopping increased metabolism. The relatively small amount of histamine metabolism in the present study, compared to previous experiments with human lung fragments, might be accounted for by the introduction of several washes that may possibly have removed intracellular enzymes derived from cells damaged during the fragmentation process. The present study demonstrates that the antigen-induced release of histamine and SRS-A from passively sensitized human lung fragments in vitro is not inhibited if sensitization is carried out in the absence of calcium and magnesium ions and in the presence of a 5 mM EDTA buffer. Similar observations have been made previously in chopped guinea pig lung fragmentslo and in human 1eukocytes.l’ These data suggest that the process of passive sensitization of human lung fragments for the subsequent antigen-induced release of mediators does not require calcium and is not inhibited by the presence of 5 mM EDTA. This phenomenon is in contrast with antigen-induced mediator release that is known to be a calcium-requiring process in human lung tissue12 as well as in other model systems.l3-l5 Desensitization implies an interaction between antigen and antibody at the target cell leading to an activation not productive of mediator release that renders the cell unresponsive to further antigen challenge. It is possible that antigen-antibody interactions in the fluid phase change the characteristics of reagin in such a way. that it cannot bind to target cells. In this case there would be a simple lack of sensitization rather than desensitization. Alternatively, the interaction between human lung fragments, reaginic serum, and antigen could result in true desensitization. In the low calcium incubation experiment (Table II) the absence of calcium during sensitization of lung with atopic serum and antigen prevented the occurence of desensitization. The fact that this tissue could release histamine and SRS-A upon restoration of the cation concentration is evidence that there had been an interaction between antigen and antibody and target cells that had been suspended by the absence of cations. The incremental increase of histamine and SRS-A release when tissues sensitized in low calcium media in.the presence of antigen are subjected to antigen challenge in the presence of cations suggests
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Antigen-antibody
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165
all cell-bound IgE antibody did not react with antigen during the sensitization period. The failure to appreciate a comparable increment of histamine and SRS-A release in the absence of blockade by cation depletion is consistent with the view that not all of the cell-bound reagin needs to have reacted with antigen to fully desensitize the tissue. In addition an IgE-rich fraction prepared by DE-52 and G-200 column chromatographylG possessedboth sensitizing and desensitizing capabilities; other fractions could not be studied for antigendependent antibody-mediated desensitization since they did not sensitize human lung fragments for the release of mediators. Previous investigators have observed phenomena similar to antigen-dependent antibody-mediated desensitization in vivo. I7 Passively sensitized guinea pigs challenged with a dose of antigen that did not result in clinical manifestations were sacrificed 48 hours later, and their lungs were examined for total and antigen-releasable histamine. Although the total lung histamine of the group receiving antigen challenge in vivo was the same as that of a control group, the lung fragments contained only one sixth of the antigen-releasable histamine obtained from the control group. Kisil and associates18studied the effects of pretreatment of human skin sites with antigen-reagin mixtures that did not lead to wheal and flare reactions. After 24 hours the skin sites were injected with reaginic serum alone, followed after another 24 hour interval by injection of specific antigen. The skin sites pretreated with antigen-reagin mixtures did not give wheal and flare reactions. The findings of impaired leukocyte reactivity to grass antigen in selected patients undergoing immunotherapy for ragweed pollinosis’ might be another example of antigen-dependent antibody-mediated desensitization, The half-life of IgEl is such that sensitization in vivo might be an ongoing process, and thus antigen from immunotherapy would be available to mediate d.esensitization. that
REFERENCES 1 Lichtenstein, L. M., and Levy, D. A.: Is “desensitization” for ragweed hay fever immunologically specific.cS Int. Arch. Allergy Appl. Immunol. 42: 615,1972. 2 Levy, D. A., Lichtenstein, L. M., Goldstein, E. O., and Ishizaka, K.: Immunologic and cellular changes accompanying the therapy of pollen allergy, J. Clin. Invest. 59: 360, 1971. 3 Tyrode, M. V.: The mode of action of some purgative salts, Arch. Int. Pharmacol. 20: 205, 1910. 4 Orange, R. P., Austen, W. C., and Austen, K. F.: Immunological release of histamine and slow-reacting substance of anaphylaxis from human lung. I. Modulation by agents influencing cellular levels of cyclic 3’,5’-adenosine monophosphate, J. Exp. Med. 184: 136s, 1971. 5 Stechschulte, D. J., Au&en, K. F., and Bloch, K. J.: Antibodies involved in antigen-induced release of slow-reacting substance of anaphylaxis (SRS-A) in the guinea pig and rat, J. Exp. Med. 125: 127, 1967. 6 Lilja, B., Lindell, S. E., and Saldeen, T.: Formation and destruction of 04 histamine in human lung tissue in vitro, J. ALLERGY 31: 492, 1960. 7 Eiseman, B., Bryant, L., and Waltuch, T.: Metabolism of vasomotor agents by the isolated perfused lung, J. Thorac. Cardiovasc. Surg. 48: 798, 1964. 8 Bouhuys, A., and Lindell, S. E.: Release of histamine by cotton dust extracts from human lung tissue in vitro, Experientia 17: 211, 1961. 9 Bennett, A.: The metabolism of histamine by guinea-pig and rat lung in vitro, Br. J. Pharmacol. 24: 147, 1965. 10 Mongar, J. L., and Schild, H. 0.: A study of the mechanism of passive sensitization, J. Physiol. 150: 546, 1960.
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11 Levy, D. A., and Osler, A. G.: Studies on the mechanisms of hypersensitivity phenomena. XIV. Passive sensitization in vitro of human leukocytes to ragweed pollen antigen, J. Immunol. 97: 203, 1966. 12 Orange, R. P., Kaliner, M. A., and Au&en, K. F.: The immunological release of histamine and slow reacting substance of anaphylaxis from human lung. III. Biochemical control mechanisms involved in the immunologic release of chemical mediators, in Austen, K. F., and Becker, E. L., editors: Second international symposium on the biochemistry of the acute allergic reactions, Oxford, 1971, Blackwell Scientific Publications, p. 189. 13 Mongar, J. L., and Schild, H. 0.: The effect of calcium and pH on the anaphylactie reaetion, J. Physiol. 140: 272, 1958. 14 Austen, K. F., and Humphrey, J. IF.: In vitro studies of the mechanisms of anaphylaxis, Adv. Immunol. 3: 1, 1963. 16 Lichtenstein, L. M., and Osler, A. G.: Studies on the mechanisms of hypersensitivity phenomena. IX. Histamine release from human leukocytes by ragweed pollen antigen, J. Exp. Med. 120: 507, 1964. of E-antibodies as a carrier of reaginic 16 Ishizaka, K., and Ishizaka, T.: Identification activity, J. Immunol. 99: 1187, 1967. effect of desensitization on release of 17 Burdon, K. L., and Schultz, G. A.: Inhibiting histamine from guinea pig lung by specific antigen, 5. ALLERGY 38: 100, 1966. 18 Kisil, F. T., Centeno, E. R., Attallah, N. A., and Sehon, A. H.: Demonstration of reaginallergen complexes formed on elution of reagins from immunosorbent with allergens, Int. Arch. Allergy Appl. Immunol. 42: 40, 1972. 19 Waldman, T. A.: Disorders of immunoglobulin metabolism, N. Engl. J. Med. 281: 1170, 1969.
M.D., Daniel M.D. Bostm,
J. Stechschulte,
M.D.,
and
Mam.
Huma?& lwng fragments incubated with atopio sorwm and specific antigen exhibited marked inhibition. of mediator release upon subsequent antigen challenge. Unrler the oonditioaa reszGlting &a alesonsitizatiolt, mediators were not released into the sensitizing diffmate, the total tissue hist~ine stores were not diminished, ana added histamine was not signijioantly metabolized. Incllbation of lung fragments, atopia serum, an& antigen in a oaloiurfn-free5 miU ethylenediaminetetracetic acid (EDTA) buffer resulted in blockade of desensitization. Thus the presence of antigen rlzvrng passive sensitization does not result in mediator release, yet renders target cells unable to respond to subsequent antigen challenge.
The loss of cellular reactivity to antigen, observed in the leukocytes of selected patients undergoing hyposensitization therapy, has been offered as a possible explanation for amelioration of symptoms in these patients.l Loss of cell reactivity did not appear to result from loss of serum reaginic activity and was attributed to a possible intrinsic change in the way cells respond to antigen.2 It has now been demonstrated that the presence of antigen E during incubation of human lung fragments with atopic serum alters cellular reactivity to subsequent antigen challenge. Antigen-dependent antibody-mediated desensitization is proposed as a possible in vitro model for hyposensitization. MATERIALS
AND
METHODS
Ragweed antigen E was obtained from the Research Resources Branch of the National Institute of Allergy and Infectious Diseases. Serum samples from a ragweed-sensitive patient (R. 8.) (IgE level 250 ng. per milliliter) were used in the present experiments unless otherwise indicated. Tyrode’s solutions was prepared as described. Disodimn and tetrasodium ethylenediaminetetraacetic acid (EDTA) were obtained from Fisher Scientific Corp. (Philadelphia, Pennsylvania). Histamine diphosphate was obtained from Mann Laboratories (Orangeburg, New York). Specimens of macroscopically normal human lung tissue were obtained at the time of surgery for carcinoma of the bronchus and were prepared in 200 mg. replicates as described previously.4 Atopic serum (0.9 ml. per vial) diluted 1:l or 1:3 in Tyrode’s solution was placed in glass vials 2.0 cm. in diameter and 6.0 em. high. One-tenth milliliter of Tyrode’s solution, From the Departments of Medicine, Harvard Medical School, Robert B. Brigham Hospital, and Peter Bent Brigham Hospital. Supported b grants AI-07722, AI-10356, and RR05669 from the National Institutes of Health an cl a grant from the John A. Hartford Foundation, Inc. Dr. Stechschulte is the the recipient of a Research Career Development Award (AI-23405) from the National Institutes of Health. Received for publication Jan. 10, 1973. Re rint requests to: K. Frank Au&en, M.D., Robert B. Brigham Hospital, Boston, Mass. B2120. Vol. 56, No. 9, pp. lS8-166
VOLUME NIJMBER
52 3
Antigen-antibody
TABLE 1. Antigen-antibody
mediated
desensitization
Sensitization Serum ii 8. R.. s.
Dilution
0 1 0
1:3 1:l 1:l 1:3
1 0 1 0 0’
1:3
El. 1%‘.
1:l
El. w. Ii. F. H. F.
1:l 1:l 1:l
of human
lung
Antigen Mg/ml.l
1:l 1:3
I-1. Ii.
desensitization
1 Mediator
1:l
R.. 5. R. S. L. K. L,. K. L. K.
mediated
of lung
fragments*
release
with
antigen
Histamine (% total)
0
challenget SRS-A kt./Gm.)
30
1
159
345
1.5
N.D.:
35 2.1 36 4.1 32 3.0 29 3.0 22
300 45 225
N.D. 300
N.D. 505 N.D. 225 N.D.
1 N.D. ‘Lung fragments were sensitized at 22O C. for 16 hours with serum from ragweed-sensitive patients. t Antigen challenge (1 pg antigen E per milliliter) was at 37” C. for 15 minutes. tLndicates mediator release was not detectable.
containing an appropriate dilution of antigen E, was added to each vial, followed in 2 to 5 seconds by the addition of lung fragments. The vials were incubated at 22’ C. for 16 to 22 hours or at 37” C. for 4 hours unless otherwise indicated in the text. After the incubation Lleriod, the sensitizing diffusate was withdrawn and assayed for histamine or slow-reacting substance of anaphylaxis (SRS-A) activity on the isolated guinea pig ileum in the presence of atropine and mepyramine ma1eate.s The fragments were washed 3 times in Tyrode’s solution, resuspended in 3.0 ml. Tyrode’s solution, and prewarmed at 37” C. for 5 minutes. After challenge with antigen E, the fragments were incubated for 15 minutes at 37” C.; the diffusate was recovered and assayed for histamine and SRS-A activity. The fragments were resuspended in 3.0 ml. Tyrode’s solution and boiled for 8 minutes to extract residual tissue histamine. RESULTS Antigen-dependent fragments
antibody-mediated
desensitization
of human
lung
The effects of introducing antigen E (1.0 pg per milliliter) during the sensitization of human lung fragments was examined with sera from a variety of patients with ragweed sensitivity. There was marked inhibition of mediator release on antigen challenge of fragments sensitized in the presence of antigen (Table I). In another series of experiments a single serum donor was used and the concentration of antigen E present during passive sensitization was varied. The presence of 10.0 or 1.0 pg per milliliter of antigen E during sensitization resulted in a marked desensitization of human lung fragments. to subsequent antigen challenge (Fig. 1). A graded desensitization was observed when smaller doses of antigen E were present during passive sensitization, while passive sensitization in the presence of 1O-4rg per milliliter antigen E resulted in no desensitization to subsequent antigen challenge. In order to determine whether the apparent desensitization to antigen challenge with 1.0 fig per milliliter antigen E was peculiar to this dose, tissues were challenged with antigen E in a dose range from 1O-4 to 10.0 pg per milliliter. A.s shown in Fig. 2, human lung fragments sensitized with atopic
160
Drazen,
Stechschulte,
and
-
FIG. 1. Effects serum [R. S. 1:3) induced (1.0 fig (closed circles).
of
J. ALLERGY
Austen
ANTIGEN
E DURING
geometrically increasing and lung fragments for antigen E per milliliter)
SENSITIZATION
CLIN. IMMUNOL. SEPTEMBER 1973
&g/ml)
amounts of antigen E incubated with atopic 22 hours at 22” C. on the subsequent antigenrelease of histamine [open circles) and SRS-A
serum released histamine and SRS-A in a graded fashion over the range of antigen E challenge doses, When antigen E (1.0 pg per milliliter) was present during sensitization, lung fragments failed to release significant amounts of histamine or SRS-A on antigen challenge with antigen E over the range of doses from 10V4 pg per milliliter to 1.0 ,ug per milliliter. Challenge with 10.0 pg per milliliter antigen E resulted in the release of SRS-A but not histamine. A series of experiments was performed to determine the time course of the antigen-dependent antibody-mediated desensitization. Tissue incubated with allergic serum demonstrated passive sensitization at one hour as evidenced by antigen-induced mediator release (Fig. 3). The degree of sensitization appeared to increase up to 8 to 20 hours. The presence of antigen during the sensitization period permitted no subsequent antigen-induced mediator release up to 4 hours of sensitization, and resulted in a marked inhibition of mediator release at times thereafter. Absence fragments
of mediator and antigen
release
during
incubation
of
atopic
serum
with
lung
In all experiments the diffusate from the sensitization of lung fragments, with or without antigen E, was assayed for the presence of histamine and SRS-A. The histamine present in the diffusate was always less than 2 per cent of the available total histamine and was the same whether or not antigen was present during sensitization. Not only was there no detectable difference in the histamine
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200 // SRS-A RELEASE funits / gram I
I50 ,oo
/ P.,
161
/fla
/I
I’
OL c
50 I
5or
HISTAMINE RELEASE
d’
d
20
i ANTIGEN
E CHALLENGE
DOSE
HOURS
OF SENSi/Tf,?ATION
rju/ml~ FIG. 2. on the serum for 22
Effects of geometrically release of histamine (R. S. 1:3) and with hours at 22” C.
FIG. 3. Antigen-induced with (closed circles) at 22” C.
increasing amounts of antigen E at the time of challenge, and SRS-A. All lung fragments were sensitized with atopic antigen E (closed circles) or without antigen E (open circles)
histamine and SRS-A release after sensitization or without (open circles) antigen E (1 pg per milliliter).
for
various Incubation
times was
or SRS-A levels in the sensitizing diffusates, but also the total tissue histamine after sensitization, with or without antigen, was the same. The latter was calculated from the sum of the antigen-induced release and the tissue-extractable histamine as shown in Fig. 4. In another series of experiments, histamine was added at final concentrations ranging from 0 to 400 ng. histamine base per milliliter to vials containing lung and atopic serum with or without antigen; the vials were shaken for 1 minute at 22’ C.; a portion of the fluid was withdrawn from each, and the vials were incubated at 37O C. for 4 hours. The diffusates were withdrawn and both the presensitization and postsensitization diffusates were assayed for histamine (Fig. 5). The amount of histamine detected at 1 minute was within 10 per cent of the calculated final concentration, and the subsequent loss of histamine during the 4 hour incubation, as shown in Fig. 5, was less than 20 per cent and not influenced by the presence of antigen. In other experiments histamine recovery after 22 hours at 22’ C. varied from 60 to 100 per cent of added histamine.
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5
T T
F
SENS.
-RELEASE-
CHALL.
RESID.
TOTAL
o
I!’
2bo
HISTAMINE
-TISSUE-
FIG. 4. Comparison of the available serum and antigen (dark bars) and bars). SENS. = Histamine released released into diffusate at 15 minutes histamine. TOTAL = Sum of SENS. t error of the mean.
Al
360 ADDED
4bo /ng/mlj
histamine from lung fragments sensitized with atopic fragments sensitized with atopic serum alone (light into the sensitizing diffusate. CHALL. = Histamine after antigen challenge. RESID. = Residual tissue CHALL. t RESID. Vertical lines indicate one standard
FIG. 5. Recovery of added histamine after incubation with (closed circles) or without (open circles) antigen E, atopic serum, and lung fragments at 37” C. for 4 hours. Ordinate, Net histamine assayed in the diffusate after 4 hours at 37” C. Abscissa, Histamine assayed in an aliquot of the diffusate after 1 minute mixing period, but before incubation at 37” C. Dashed line through the origin represents 100 per cent recovery. Blockade absence
of antigen-dependent antibody-mediated of calcium and magnesium ions
desensitization
by
the
Human lung fragments were washed 3 times in a modified Tyrode’s solution containing 5 mM EDTA and no calcium or magnesium ions. Fragments were sensitized, with or without antigen, in antiserum diluted in the modified buffer for 4 hours at 37O C. After sensitization the diffusate was withdrawn $nd assayed and the fragments were washed 3 times with normal Tyrode’s solution, suspended in 3.0 ml. normal Tyrode’s solution, and then prewarmed at 37’ C. for 5 minutes before antigen challenge. As shown in Table II, human lung fragments sensitized in the calcium- and magnesium-free buffer yielded amounts of histamine and SRS-A on antigen challenge similar to those obtained from tissue sensitized in normal Tyrode’s solution. The presence of antigen E during sensitization in normal Tyrode’s solution resulted in a greater than 90 per cent inhibition of subsequent antigen-induced mediator release. In contrast, the deletion of calcium and magnesium during sensitization in the presence of antigen blocked the desensitization such that the release of histamine was 50 per cent and that of SRS-A was 88 per cent of that observed with tissue sensitized in the absence of antigen. The release of histamine and SRS-A upon restoration of the cation concentration and challenge with antigen reflected the effects of both
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TABLE II. Blockade of antigen-antibody mediated desensitization of human lung fragments by incubation in a calcium- and magnesium-free, 5 mM EDTA buffer* Rekase Sensitization EDTA (mM)
with Calcium (mMlt
atopic
serum Antigen Wglml.)
E
Antigen challenge &g/ml.)
E
of mediators SRS-A
Histamine %
(
S.E.M.
W./Gm.l
S.E.M.
38 280 N.D. N.D. ia: if 18 N.h. 0:o N.D. 5.0 E h*i 3:-i if 360 45 5.0 0:o 1:o 1.0 15:7 2.6 320 21 1.8 170 33 5.0 0.0 1.0 0.0 6.9 *Average of 3 experiments. All lungs were sensitized at 37” C. for 4 hours with serum R.S. at 1:3 dilution of the appropriate buffer. All fragments were challenged, as indicated, with 1.0 +g per milliliter antigen E in normal Tyrode’s buffer. N.D. indicates that mediator release was not detectable. S.E.M. refers to the standard error of the mean. tCa.leium concentration in Tyrode’s buffer. 0.0
1.8
0.0 0.0 1.0
1.0 0.0 1.0 0.0 1.0
31.0 N.D. 1.6
4.8 N.D. 0.6
new antigen and that combined with antibody during the sensitization period. The effects of antigen-antibody interactions during sensitization are revealed by the histamine and SRS-A release after restoration of the cation concentration. DISCUSSION
Human lung fragments sensitized in atopic serum in the presence of ragweed antigen E exhibit a marked inhibition of mediator release upon subsequent antigen challenge. The extent of this antigen-dependent antibody-mediated desensitization was determined by the concentration of antigen present during the sensitization period (Fig. 1). Inhibition of subsequent antigen-induced mediator release was consistently greater than 80 per cent when incubation was carried out with a 1:l or a 1:3 dilution of various atopic sera in the presence of 1.0 pg per milliliter antigen E (Table I). Desensitization was not overcome by varying the antigen challenge dose from 1O-4 to 10.0 pg per milliliter antigen E (Fig. 2). At no time during passive sensitization of tissue in the presence of antigen was there an interval when desensitization was not demonstrable (Fig. 3).
The interpretations of these experiments depend on, the evidence that mediator release does not occur during incubation of human lung fragments in atopic serum and antigen. In all experiments the amount of histamine or SRS-A released into the diffusate during sensitization was the same regardless of whether or not incubation in atopic serum was carried out in the presence of antigen. The possibility that the failure to detect mediator release during sensitization in the presence of antigen was an artefact due to the metabolism of released mediators was excluded by two lines of evidence. First, there was no depletion of tissue histamine stores in desensitized lung fragments (Fig. 4). The maintenance of tissue histamine stores cannot be attributed to the synthesis of new histamine or to the binding of released histamine because antigen challenge of passively sensitized tissue followed by incubation periods from 8 minutes to 4 hours revealed no progressive change in histamine content of the diffusates or tissues I(unpublished observations). Further, Lilja, Lindell, and Saldeene
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demonstrated that 2 to 3 Gm. of normal lung tissue incubated in vitro for 3 hours at 3’7O C. in a buffer containing 40 pg 14C-histidine per milliliter synthesized only 43 ng. of histamine. Our fragments were approximately one-tenth as large and were incubated in a buffer without added histidine, conditions that appear unfavorable for histamine synthesis. Second, there was no unusual metabolism of exogenous histamine added at a concentration comparable to that released during the antigen challenge of passively sensitized tissue (Fig. 5). Eiseman, Bryant, and Waltuch? noted 82 per cent recovery of added histamine after 30 minutes of perfusion through heart-lung preparations from normal dogs. In contrast, Lilja, Lindell, and Saldeen6 and Bouhuys and Lindell* reported slightly greater than 50 per cent recovery of total histamine after incubation with human lung fragments at 37O C. Bennett? noted that recovery of histamine incubated with chopped guinea pig lung fragments varied from 60 to 30 per cent depending on the species and that homogenization rather than chopping increased metabolism. The relatively small amount of histamine metabolism in the present study, compared to previous experiments with human lung fragments, might be accounted for by the introduction of several washes that may possibly have removed intracellular enzymes derived from cells damaged during the fragmentation process. The present study demonstrates that the antigen-induced release of histamine and SRS-A from passively sensitized human lung fragments in vitro is not inhibited if sensitization is carried out in the absence of calcium and magnesium ions and in the presence of a 5 mM EDTA buffer. Similar observations have been made previously in chopped guinea pig lung fragmentslo and in human 1eukocytes.l’ These data suggest that the process of passive sensitization of human lung fragments for the subsequent antigen-induced release of mediators does not require calcium and is not inhibited by the presence of 5 mM EDTA. This phenomenon is in contrast with antigen-induced mediator release that is known to be a calcium-requiring process in human lung tissue12 as well as in other model systems.l3-l5 Desensitization implies an interaction between antigen and antibody at the target cell leading to an activation not productive of mediator release that renders the cell unresponsive to further antigen challenge. It is possible that antigen-antibody interactions in the fluid phase change the characteristics of reagin in such a way. that it cannot bind to target cells. In this case there would be a simple lack of sensitization rather than desensitization. Alternatively, the interaction between human lung fragments, reaginic serum, and antigen could result in true desensitization. In the low calcium incubation experiment (Table II) the absence of calcium during sensitization of lung with atopic serum and antigen prevented the occurence of desensitization. The fact that this tissue could release histamine and SRS-A upon restoration of the cation concentration is evidence that there had been an interaction between antigen and antibody and target cells that had been suspended by the absence of cations. The incremental increase of histamine and SRS-A release when tissues sensitized in low calcium media in.the presence of antigen are subjected to antigen challenge in the presence of cations suggests
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all cell-bound IgE antibody did not react with antigen during the sensitization period. The failure to appreciate a comparable increment of histamine and SRS-A release in the absence of blockade by cation depletion is consistent with the view that not all of the cell-bound reagin needs to have reacted with antigen to fully desensitize the tissue. In addition an IgE-rich fraction prepared by DE-52 and G-200 column chromatographylG possessedboth sensitizing and desensitizing capabilities; other fractions could not be studied for antigendependent antibody-mediated desensitization since they did not sensitize human lung fragments for the release of mediators. Previous investigators have observed phenomena similar to antigen-dependent antibody-mediated desensitization in vivo. I7 Passively sensitized guinea pigs challenged with a dose of antigen that did not result in clinical manifestations were sacrificed 48 hours later, and their lungs were examined for total and antigen-releasable histamine. Although the total lung histamine of the group receiving antigen challenge in vivo was the same as that of a control group, the lung fragments contained only one sixth of the antigen-releasable histamine obtained from the control group. Kisil and associates18studied the effects of pretreatment of human skin sites with antigen-reagin mixtures that did not lead to wheal and flare reactions. After 24 hours the skin sites were injected with reaginic serum alone, followed after another 24 hour interval by injection of specific antigen. The skin sites pretreated with antigen-reagin mixtures did not give wheal and flare reactions. The findings of impaired leukocyte reactivity to grass antigen in selected patients undergoing immunotherapy for ragweed pollinosis’ might be another example of antigen-dependent antibody-mediated desensitization, The half-life of IgEl is such that sensitization in vivo might be an ongoing process, and thus antigen from immunotherapy would be available to mediate d.esensitization. that
REFERENCES 1 Lichtenstein, L. M., and Levy, D. A.: Is “desensitization” for ragweed hay fever immunologically specific.cS Int. Arch. Allergy Appl. Immunol. 42: 615,1972. 2 Levy, D. A., Lichtenstein, L. M., Goldstein, E. O., and Ishizaka, K.: Immunologic and cellular changes accompanying the therapy of pollen allergy, J. Clin. Invest. 59: 360, 1971. 3 Tyrode, M. V.: The mode of action of some purgative salts, Arch. Int. Pharmacol. 20: 205, 1910. 4 Orange, R. P., Austen, W. C., and Austen, K. F.: Immunological release of histamine and slow-reacting substance of anaphylaxis from human lung. I. Modulation by agents influencing cellular levels of cyclic 3’,5’-adenosine monophosphate, J. Exp. Med. 184: 136s, 1971. 5 Stechschulte, D. J., Au&en, K. F., and Bloch, K. J.: Antibodies involved in antigen-induced release of slow-reacting substance of anaphylaxis (SRS-A) in the guinea pig and rat, J. Exp. Med. 125: 127, 1967. 6 Lilja, B., Lindell, S. E., and Saldeen, T.: Formation and destruction of 04 histamine in human lung tissue in vitro, J. ALLERGY 31: 492, 1960. 7 Eiseman, B., Bryant, L., and Waltuch, T.: Metabolism of vasomotor agents by the isolated perfused lung, J. Thorac. Cardiovasc. Surg. 48: 798, 1964. 8 Bouhuys, A., and Lindell, S. E.: Release of histamine by cotton dust extracts from human lung tissue in vitro, Experientia 17: 211, 1961. 9 Bennett, A.: The metabolism of histamine by guinea-pig and rat lung in vitro, Br. J. Pharmacol. 24: 147, 1965. 10 Mongar, J. L., and Schild, H. 0.: A study of the mechanism of passive sensitization, J. Physiol. 150: 546, 1960.
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11 Levy, D. A., and Osler, A. G.: Studies on the mechanisms of hypersensitivity phenomena. XIV. Passive sensitization in vitro of human leukocytes to ragweed pollen antigen, J. Immunol. 97: 203, 1966. 12 Orange, R. P., Kaliner, M. A., and Au&en, K. F.: The immunological release of histamine and slow reacting substance of anaphylaxis from human lung. III. Biochemical control mechanisms involved in the immunologic release of chemical mediators, in Austen, K. F., and Becker, E. L., editors: Second international symposium on the biochemistry of the acute allergic reactions, Oxford, 1971, Blackwell Scientific Publications, p. 189. 13 Mongar, J. L., and Schild, H. 0.: The effect of calcium and pH on the anaphylactie reaetion, J. Physiol. 140: 272, 1958. 14 Austen, K. F., and Humphrey, J. IF.: In vitro studies of the mechanisms of anaphylaxis, Adv. Immunol. 3: 1, 1963. 16 Lichtenstein, L. M., and Osler, A. G.: Studies on the mechanisms of hypersensitivity phenomena. IX. Histamine release from human leukocytes by ragweed pollen antigen, J. Exp. Med. 120: 507, 1964. of E-antibodies as a carrier of reaginic 16 Ishizaka, K., and Ishizaka, T.: Identification activity, J. Immunol. 99: 1187, 1967. effect of desensitization on release of 17 Burdon, K. L., and Schultz, G. A.: Inhibiting histamine from guinea pig lung by specific antigen, 5. ALLERGY 38: 100, 1966. 18 Kisil, F. T., Centeno, E. R., Attallah, N. A., and Sehon, A. H.: Demonstration of reaginallergen complexes formed on elution of reagins from immunosorbent with allergens, Int. Arch. Allergy Appl. Immunol. 42: 40, 1972. 19 Waldman, T. A.: Disorders of immunoglobulin metabolism, N. Engl. J. Med. 281: 1170, 1969.