- Email: [email protected]
Cromolyn does not modulate human allergic skin reactions in vivo
Cromolyn does not modulate skin reactions in vivo Stanislaus El Paso,
Ting, M.D., Burton
Tex.,
and Philadelphia.
Zweiman, Pa.
Disodium cromoglycate has been used extensively by some physicians for the treatment of asthma, rhinitis, conjunctivitis, and food allergy. ’ The observed beneficial effect of cromolyn in asthma has been thought to be due, at least in part, to its capacity to inhibit mast cell degranulation and subsequent release of mediators.2, 3 There have been scattered reports of a reduction in the itching and skin inflammatory re-sponses in atopic dermatitis after topical application of cromolyn ointment,” but the mechanisms underlying these beneficial effects were not explored. This study describes the results of cromolyn effects on antigen-induced mast cell alterations and release 01’ mediators in the skin of atopic subjects. MATERIALS The Research
design
AND METHODS of the study
was
Committee of William
approved Beaumont
by the Clinical .Army
Medical
Center.
From the Allergy and Immunology Scr\~c. Department ot Medicine, William Beaumont Army Medical Center. El Paso. Tex., and the Allergy/Immunology SectIon, Department ot Medicine, Department of Dermatology, University of Pennsylvania. Philadelphia, Pa. Received for publication April 21. 1982. Accepted for publication July 22, 1982. Reprint requests to: Stanklaus Ting, M.D., Allergy and lmmunoogy Service, Department of Medicine, William Beaumont Army Medical Center. El Paso. TX 79920. The opinions or assertions contained herein arc the private vieus 111 the authors and are not to be construed a5 official or as reflecting the view\ of the Department of the Army or the Department 01 Defense. Vol.
71, No.
1, Part
1, pp.
12-17
human allergic
M.D., and Robert M. Lavker, Ph.D.
VOLUME NUMBER
Cromolyn allergic skin reactions
71 1, Part 1
Abhrr~~iuriom 31
PBS:
II.SC~
Phosphate-buffered salme
intradermal injection of cromolyn would inhibit the expected whealing response to ragweed antigen injected subsequently at the same site. Initially, serial dilution skin testing was carried out in five ragweed-sensitive subjects to determine the concentrations of ragweed extract that would elicit whealing responses of approximately 20 mm’ 20 min later (hereafter called the ED,, for each subject). Then 0.04 ml aliquots of 27r cromolyn solution were injected intradermally in three sites in the outer aspect of the upper arm at least 8 cm apart and at least 8 cm from the previous serial dilution skin tests. These injection sites (noted as A, B, and C) were marked carefully. Aliquots (0.02 ml) of ragweed ED,, were injected into site A 30 min later, into site B 60 set later, and into site C 120 min later. The whealing response was measured 20 min after each injection of ragweed ED2(,, as described.
Measurement chambers
of histamine
release into skin
Four blisters, each with a I cm diameter base, were induced simultaneously (two on each forearm) in four subjects as described previously? each blister was then unroofed aseptically. A specially designed plastic collection chamber with entry and exit ports was placed directly over the denuded base and kept in place by appropriate taping. Each chamber was filled with PBS. Five minutes later, the fluid was removed and the chamber was irrigated and filled with 0.3 ml of one of the following: chamber A, ragweed solution 1000 PNUlml; chamber B. PBS alone (negative control); chamber C, a solution containing an FC of ragweed 1000 PNUiml and cromolyn 2%; chamber D, cromolyn 24. All chamber fluids were removed after 30 min incubation and stored in coded polyethylene tubes at - 20” C until studied. Histamine was analyzed by means of the microenzymatic method of Levy and Widra.”
Measurement in the skin
of eosinophil
responses
Skin testing was carried out in five subjects with (I) ragweed, (2) ragweed plus cromolyn, (3) PBS, and (4) cromolyn alone, as described above. Biopsies were performed on these skin-test sites 2 hr later. The biopsy specimens were coded, embedded in paraffin, and sectioned. The number of eosinophils per square millimeter was determined by an approach described previously.”
Ultramicroscopic
studies
of skin-test
sites
In our previous studies we found that antigen-induced histamine release and eosinophil accumulation in human allergic skin reactions were preceded by rapidly developing prominent mast cell alterations.,5 The effects of exogenous
(mm*)
700
-
650
-
600
-
550
-
500
-
450
-
4oo 350
-
300
-
250
-
200
-
150
-
100
-
50
-
WHEALING
O-
13
RESPONSE
*
I-
-
I
FIG. 1. Whealing response (mean 2 SEM) at the sites of injection of ragweed, PBS (control), ragweed plus cromolyn mixture, or cromolyn alone. Ragweed-induced skin whealing response was not inhibited by cromolyn. cromolyn on these events were studied in two of these subjects. Skin biopsy specimens were obtained IO min later at the sites of intradermal injection of (1) ragweed, (2) PBS solution (control), (3) ragweed plus cromolyn, or (4) cromolyn alone. Biopsy specimens were embedded in plastic and coded, and ultrathin sections were prepared for ultramicroscopy. Mast cell alterations were classified based on inspection of at least 20 mast cells in multiple replicate sections as previously described.
RESULTS Cromolyn effects on skin test As shown in Fig. 1, the mean whealing response at the sites of injection of the ragweed plus cromolyn mixture was similar to the mean whealing response at the ragweed injection sites in all subjects (618 +- 27 vs 593 ? 33 mm’, mean ? SEM, p = NS). Very small whealing responses were seen at the skin sites injected with the control PBS solution or with cromolyn solution (9 + 4 vs 10 t 3 mm’). The addition of cromolyn to lower concentrations of ragweed also did not inhibit the whealing responses to these antigen solutions after intradermal injection (Fig. 2). The mean response to injection of ragweed 500 PNU/ml plus cromolyn 2% was similar to the response to ragweed 500 PNU/ml alone (353 * 22 vs 378 +- 22 mm*, p = NS). Likewise, mean responses to ragweed 100 PNU/ml plus cromolyn 2% were similar to those to ragweed 100 PNU/ml alone (12.5 _t 9 vs 118 2 17 mm’, p = NS), and response to ragweed 10 PNU/ml plus cromolyn 2% was similar to that to ragweed 10 PNU/ml alone (93 2 22 vs 80 + 16 mm*, p = NS).
14
J ALLERGY
Ting et al.
WHEALING 400
r
350
-
300
-
250
-
200
-
150
-
100
-
CLIN. IMMUNOL. JANlJARY 1663
RESPONSE
*
(mm2 )
50-
T
Raw500
12-J Rasw 500+ Cromolyn
Rawd 100
RawI 1001 Cromolyn
Ragw
10
Ra6w
10+
CromolYn
FIG. 2. Wheal size (mean t SEM) at skin sites injected with ragweed 500 PNU, 100 PNU, 10 PNU with or without cromolvn. Addition of cromolyn to lower concentrations of ragweed did not inhibit the whealing response.
WHEALING with
EDso (Ragweed)
RESPONSE after
pre-treatment
with
Cromolyn
25 r
20 -
histamine levels in chamber fluids to which ragweed and cromolyn had been added previously (2 1 z 2.6 vs 20 i 2.4 nglml, p = NS). The histamine levels In the chamber where cromolyn alone was added (5 -t 0.4 rig/ml) were similar to the low histamine level> found in the control chamber fluids (4 2 0.85 ng; ml).
Skin biopsy studies 15(mm’) 10 -
5-
Ok 30 lnts.
60 mts.
120 mts.
FIG. 3. Time study of the whealing response (mean -t SEMI to ragweed EDzo when cromolyn was injected at 30, 60, and 120 min before the ragweed. Pretreatment of the skin sites, up to 2 hr with cromolyn, did not modify the ragweed-induced whealing response.
As shown in Fig. 3, injections of cromolyn into skin-test sites up to 2 hr before local injection of ragweed extract did not modify the allergen-induced whealing response.
Cromoiyyn effects on antigen-induced histamine levels Qualitatively a very similar pattern of events was seen in all four subjects (Fig. 4). The histamine levels in skin chamber fluids to which ragweed extract had been added 30 min earlier were similar to the mean
Examination of multiple sections of biopsy specsmens in five of the subjects showed that cosinophils were prominent to similar degrees in skin sites injected 2 hr earlier with ragweed extract alone or ragweed plus cromolyn mixture. These eosinophil responses were significantly greater tllan that seen in sites injected with cromolyn alone or with control PBS solution (Fig. 5). Electron microscopic studies were performed in biopsy tissue obtained in two subjects at sites injected IO min earlier with the test solutions. In sites injected with ragweed alone or ragweed plus cromolyn, there was marked alteration of the mast cells, with swollen and flocculent granules, fusion of granuies into aggregates, and some degranulation (Fig. 6). Ln comparison, in sites injected with cromolyn alone or with PBS. there was little or no mast cell alteratian (Fig. 7).
DISCUSSIUN Cromolyn pretreatment has been found beneficial in some individuals with asthma.‘. B Prior inhalation of this agent will often inhibit or reduce the degree ot bronchospasm after antigen inhalation challenge in sensitive subjects.“, If’ Such in vivo effects have been thought to result, at least in part, from the inhibitory effect of cromolyn on antigen-induced release of mediators from lung mast cells.” Since such mast
VOLUME NUMBER
71 1. Part 1
Cromolyn
allergic
skin reactions
15
HISTAMINE 25
20
f
*
15 (rig/ml
1 10
5
0 Ragweed
rl
Control
rl
Cromolyn
Ragweed + Crom0lyn
FIG. 4. Histamine levels (mean & SEM) in skin chamber fluids after 30 min incubation with ragweed, PBS (control), ragweed plus cromolyn, and cromolyn. Ragweed induced significant release of histamine, which was not inhibited by cromolyn. PBS and cromolyn induced small amounts of histamine release.
SKIN
BIOPSY
25
20
*
% 2 A i n. 0 f v) 0 w
15
10
5
I
0 Ragweed
Ragweed Cromolyn
+
I
I
Cromolyn
FIG. 5. Tissue eosinopl hil response (cells/mm2) at skin sites injected 2 hr earlier with ragweed, PBS, ragweed plus cromolyn, and cromolyn alone. Ragweed and ragweed plus cromolyn mixture induced prominent eosinophil response, significantly greater than that induced by PBS (control) and cromolyn alone.
cell-derived mediators are thought to cause some of the manifestations of atopic dermatitis-like itching and inflammatory cell accumulation, a trial of locally applied cromolyn in this skin disorder would be reasonable. Results of such clinical trials have varied with impressive improvement after use of 10%
cromolyn ointment.4 However, there has been no systematic investigation in which such locally applied cromolyn alters antigen-induced mast cell alterations and mediator release in human skin. In this study we used a human skin-test model in which we have previously shown consistent striking
16
J ALLERGY
Ting et al.
FW. 6. Electron plus cromolyn,
micrograph of a typical mast cell IO min after injection of ragweed showing evidence of marked degranulation. (x 10,000.)
CLIN. IMMUNOL. JANUARY 1983
and ragweed
FIG. 7. Electron micrograph of typical mast cell 10 min after injection with cromolyn or PBS (control), showing large amounts of intact electron-dense granules with no evidence of degrenuletion. (x14,000.)
VOLUME NUMBER
71 1. Part 1
mast cell alterations starting within 15 set after intradermal antigen injection.” This is followed over the next 30 min by increasing levels of histamine released into a skin chamber appended to such test sites and by ingress of eosinophils into the area starting 1 to 2 hr later. 3 Using this model we found no inhibitory effects of cromolyn on the mast cell ultramicroscopic alterations, mediator release, or inflammatory cell accumulation when cromolyn was added to antigen in the skin chambers at the challenge site. Our results, showing that cromolyn did not inhibit in vivo the anaphylactic histamine release from human skin mast cells, are consistent with the in vitro findings of Pearce et al.12 It is conceivable but unlikely that no inhibition was seen in this because the cromolyn in solution did not penetrate the base of the chamber at the dermalepidetmal junction; addition of cromolyn to the antigen solution did not inhibit the whealing reaction after intradermal injection. It is conceivable that higher concentrations of cromolyn in solution could inhibit such reactions. In some clinical reports, up to 10% cromolyn in petrolatum had been employed for treatment of skin disorders.4 Our attempts to use cromolyn solution in concentrations greater than 2% led to nonspecific irritative effects. A more likely explanation for the results observed here is that mast cells in the skin are less affected than those in the human respiratory tract. The heterogeneity of mast cells from different organ systems has recently been stressed.13,I4 It is not clear whether lack of cromolyn effects in the skin reflects decreased binding to uptake of this agent into skin mast cells or other events involved in mediator release during allergic reactions. Further studies that are ethically and logistically feasible will be needed to help our understanding of these fascinating questions.
Cromolyn
allergic
skin reactions
17
REFERENCES 1 Pepys J, Edwards AM, editors: The mast cell, its role in health and disease. Marshfield, Mass., 1979, Pitman Medical, pp. 199, 447, 506. 2 Ankier SI: Disodium cromoglycate and inhibition of passive cutaneous anaphylaxis. Int Arch Allergy 41: 163, 197 I. 3. Goose J, Blair AMJN: Passive cutaneous anaphylaxis in the rat induced with two hemologous reagin-like antibody sera, and its specific inhibition with disodium cromoglycate. Immunology 16:749, 1969. 4. Haider SA: Treatment of atopic eczema in children: clinical trial of 10% sodium cromoglycate ointment. Br Med J I: 1570. 1977. 5. Ting S, Dunsky EH, Lavker RM, Zweiman B: Pattern of mast cell alterations and in viva mediator release in human allergic skin reaction. J ALLERGY CLIN IMMUNOL 66:417, 1980. 6. Levy DA, Widra M: A micro-assay for studying allergic histamine release from human leukocytes using an enzymaticisotopic assay for histamine. J Lab Clin Med 81:291, 1973. 7. Altounyan REC: Inhibition of experimental asthma by a new compound-disodium cromoglycate. Acta Allergol 22:487, 1967. 8. Altounyan REC, Howell JBL: Treatment of asthma with disodium cromoglycate. Respiration 26: 131, 1969. 9. Anand SC. Goodman DH: Protective effect of cromolyn sodium on bronchial challenge tests in pollen asthma. Ann Allergy 30:64, 1972. 10. Pepy J, Hargreave FE, Chan M, McCarthy DS: Inhibitory effects of disodium cromoglycate on allergen-inhalation tests. Lancet 2: 134, 1968. 11. Atkins PC, Norman MD, Zweiman B: Antigen induced neutrophil chemotactic activity in man. Correlation with bronchospasm and inhibition by disodium cromoglycate. J ALLERGY CLIN IMMUNOL 62: 149, 1978. 12. Pearce CA, Greaves MW, Plummer VM, Yamamoto ES: Effect of disodium cromoglycate on antigen evoked histamine release from human skin. Clin Exp Immunol 17:437, 1974. 13. Kaliner MA: Is a mast cell a mast cell a mast cell? J ALLERGY CLIN IMMUNOL 66: 1, 1980. 14. Bierenstock J, Bejus AD: Mucosal immunology. Immunology 41:249. 1980.
Ting, M.D., Burton
Tex.,
and Philadelphia.
Zweiman, Pa.
Disodium cromoglycate has been used extensively by some physicians for the treatment of asthma, rhinitis, conjunctivitis, and food allergy. ’ The observed beneficial effect of cromolyn in asthma has been thought to be due, at least in part, to its capacity to inhibit mast cell degranulation and subsequent release of mediators.2, 3 There have been scattered reports of a reduction in the itching and skin inflammatory re-sponses in atopic dermatitis after topical application of cromolyn ointment,” but the mechanisms underlying these beneficial effects were not explored. This study describes the results of cromolyn effects on antigen-induced mast cell alterations and release 01’ mediators in the skin of atopic subjects. MATERIALS The Research
design
AND METHODS of the study
was
Committee of William
approved Beaumont
by the Clinical .Army
Medical
Center.
From the Allergy and Immunology Scr\~c. Department ot Medicine, William Beaumont Army Medical Center. El Paso. Tex., and the Allergy/Immunology SectIon, Department ot Medicine, Department of Dermatology, University of Pennsylvania. Philadelphia, Pa. Received for publication April 21. 1982. Accepted for publication July 22, 1982. Reprint requests to: Stanklaus Ting, M.D., Allergy and lmmunoogy Service, Department of Medicine, William Beaumont Army Medical Center. El Paso. TX 79920. The opinions or assertions contained herein arc the private vieus 111 the authors and are not to be construed a5 official or as reflecting the view\ of the Department of the Army or the Department 01 Defense. Vol.
71, No.
1, Part
1, pp.
12-17
human allergic
M.D., and Robert M. Lavker, Ph.D.
VOLUME NUMBER
Cromolyn allergic skin reactions
71 1, Part 1
Abhrr~~iuriom 31
PBS:
II.SC~
Phosphate-buffered salme
intradermal injection of cromolyn would inhibit the expected whealing response to ragweed antigen injected subsequently at the same site. Initially, serial dilution skin testing was carried out in five ragweed-sensitive subjects to determine the concentrations of ragweed extract that would elicit whealing responses of approximately 20 mm’ 20 min later (hereafter called the ED,, for each subject). Then 0.04 ml aliquots of 27r cromolyn solution were injected intradermally in three sites in the outer aspect of the upper arm at least 8 cm apart and at least 8 cm from the previous serial dilution skin tests. These injection sites (noted as A, B, and C) were marked carefully. Aliquots (0.02 ml) of ragweed ED,, were injected into site A 30 min later, into site B 60 set later, and into site C 120 min later. The whealing response was measured 20 min after each injection of ragweed ED2(,, as described.
Measurement chambers
of histamine
release into skin
Four blisters, each with a I cm diameter base, were induced simultaneously (two on each forearm) in four subjects as described previously? each blister was then unroofed aseptically. A specially designed plastic collection chamber with entry and exit ports was placed directly over the denuded base and kept in place by appropriate taping. Each chamber was filled with PBS. Five minutes later, the fluid was removed and the chamber was irrigated and filled with 0.3 ml of one of the following: chamber A, ragweed solution 1000 PNUlml; chamber B. PBS alone (negative control); chamber C, a solution containing an FC of ragweed 1000 PNUiml and cromolyn 2%; chamber D, cromolyn 24. All chamber fluids were removed after 30 min incubation and stored in coded polyethylene tubes at - 20” C until studied. Histamine was analyzed by means of the microenzymatic method of Levy and Widra.”
Measurement in the skin
of eosinophil
responses
Skin testing was carried out in five subjects with (I) ragweed, (2) ragweed plus cromolyn, (3) PBS, and (4) cromolyn alone, as described above. Biopsies were performed on these skin-test sites 2 hr later. The biopsy specimens were coded, embedded in paraffin, and sectioned. The number of eosinophils per square millimeter was determined by an approach described previously.”
Ultramicroscopic
studies
of skin-test
sites
In our previous studies we found that antigen-induced histamine release and eosinophil accumulation in human allergic skin reactions were preceded by rapidly developing prominent mast cell alterations.,5 The effects of exogenous
(mm*)
700
-
650
-
600
-
550
-
500
-
450
-
4oo 350
-
300
-
250
-
200
-
150
-
100
-
50
-
WHEALING
O-
13
RESPONSE
*
I-
-
I
FIG. 1. Whealing response (mean 2 SEM) at the sites of injection of ragweed, PBS (control), ragweed plus cromolyn mixture, or cromolyn alone. Ragweed-induced skin whealing response was not inhibited by cromolyn. cromolyn on these events were studied in two of these subjects. Skin biopsy specimens were obtained IO min later at the sites of intradermal injection of (1) ragweed, (2) PBS solution (control), (3) ragweed plus cromolyn, or (4) cromolyn alone. Biopsy specimens were embedded in plastic and coded, and ultrathin sections were prepared for ultramicroscopy. Mast cell alterations were classified based on inspection of at least 20 mast cells in multiple replicate sections as previously described.
RESULTS Cromolyn effects on skin test As shown in Fig. 1, the mean whealing response at the sites of injection of the ragweed plus cromolyn mixture was similar to the mean whealing response at the ragweed injection sites in all subjects (618 +- 27 vs 593 ? 33 mm’, mean ? SEM, p = NS). Very small whealing responses were seen at the skin sites injected with the control PBS solution or with cromolyn solution (9 + 4 vs 10 t 3 mm’). The addition of cromolyn to lower concentrations of ragweed also did not inhibit the whealing responses to these antigen solutions after intradermal injection (Fig. 2). The mean response to injection of ragweed 500 PNU/ml plus cromolyn 2% was similar to the response to ragweed 500 PNU/ml alone (353 * 22 vs 378 +- 22 mm*, p = NS). Likewise, mean responses to ragweed 100 PNU/ml plus cromolyn 2% were similar to those to ragweed 100 PNU/ml alone (12.5 _t 9 vs 118 2 17 mm’, p = NS), and response to ragweed 10 PNU/ml plus cromolyn 2% was similar to that to ragweed 10 PNU/ml alone (93 2 22 vs 80 + 16 mm*, p = NS).
14
J ALLERGY
Ting et al.
WHEALING 400
r
350
-
300
-
250
-
200
-
150
-
100
-
CLIN. IMMUNOL. JANlJARY 1663
RESPONSE
*
(mm2 )
50-
T
Raw500
12-J Rasw 500+ Cromolyn
Rawd 100
RawI 1001 Cromolyn
Ragw
10
Ra6w
10+
CromolYn
FIG. 2. Wheal size (mean t SEM) at skin sites injected with ragweed 500 PNU, 100 PNU, 10 PNU with or without cromolvn. Addition of cromolyn to lower concentrations of ragweed did not inhibit the whealing response.
WHEALING with
EDso (Ragweed)
RESPONSE after
pre-treatment
with
Cromolyn
25 r
20 -
histamine levels in chamber fluids to which ragweed and cromolyn had been added previously (2 1 z 2.6 vs 20 i 2.4 nglml, p = NS). The histamine levels In the chamber where cromolyn alone was added (5 -t 0.4 rig/ml) were similar to the low histamine level> found in the control chamber fluids (4 2 0.85 ng; ml).
Skin biopsy studies 15(mm’) 10 -
5-
Ok 30 lnts.
60 mts.
120 mts.
FIG. 3. Time study of the whealing response (mean -t SEMI to ragweed EDzo when cromolyn was injected at 30, 60, and 120 min before the ragweed. Pretreatment of the skin sites, up to 2 hr with cromolyn, did not modify the ragweed-induced whealing response.
As shown in Fig. 3, injections of cromolyn into skin-test sites up to 2 hr before local injection of ragweed extract did not modify the allergen-induced whealing response.
Cromoiyyn effects on antigen-induced histamine levels Qualitatively a very similar pattern of events was seen in all four subjects (Fig. 4). The histamine levels in skin chamber fluids to which ragweed extract had been added 30 min earlier were similar to the mean
Examination of multiple sections of biopsy specsmens in five of the subjects showed that cosinophils were prominent to similar degrees in skin sites injected 2 hr earlier with ragweed extract alone or ragweed plus cromolyn mixture. These eosinophil responses were significantly greater tllan that seen in sites injected with cromolyn alone or with control PBS solution (Fig. 5). Electron microscopic studies were performed in biopsy tissue obtained in two subjects at sites injected IO min earlier with the test solutions. In sites injected with ragweed alone or ragweed plus cromolyn, there was marked alteration of the mast cells, with swollen and flocculent granules, fusion of granuies into aggregates, and some degranulation (Fig. 6). Ln comparison, in sites injected with cromolyn alone or with PBS. there was little or no mast cell alteratian (Fig. 7).
DISCUSSIUN Cromolyn pretreatment has been found beneficial in some individuals with asthma.‘. B Prior inhalation of this agent will often inhibit or reduce the degree ot bronchospasm after antigen inhalation challenge in sensitive subjects.“, If’ Such in vivo effects have been thought to result, at least in part, from the inhibitory effect of cromolyn on antigen-induced release of mediators from lung mast cells.” Since such mast
VOLUME NUMBER
71 1. Part 1
Cromolyn
allergic
skin reactions
15
HISTAMINE 25
20
f
*
15 (rig/ml
1 10
5
0 Ragweed
rl
Control
rl
Cromolyn
Ragweed + Crom0lyn
FIG. 4. Histamine levels (mean & SEM) in skin chamber fluids after 30 min incubation with ragweed, PBS (control), ragweed plus cromolyn, and cromolyn. Ragweed induced significant release of histamine, which was not inhibited by cromolyn. PBS and cromolyn induced small amounts of histamine release.
SKIN
BIOPSY
25
20
*
% 2 A i n. 0 f v) 0 w
15
10
5
I
0 Ragweed
Ragweed Cromolyn
+
I
I
Cromolyn
FIG. 5. Tissue eosinopl hil response (cells/mm2) at skin sites injected 2 hr earlier with ragweed, PBS, ragweed plus cromolyn, and cromolyn alone. Ragweed and ragweed plus cromolyn mixture induced prominent eosinophil response, significantly greater than that induced by PBS (control) and cromolyn alone.
cell-derived mediators are thought to cause some of the manifestations of atopic dermatitis-like itching and inflammatory cell accumulation, a trial of locally applied cromolyn in this skin disorder would be reasonable. Results of such clinical trials have varied with impressive improvement after use of 10%
cromolyn ointment.4 However, there has been no systematic investigation in which such locally applied cromolyn alters antigen-induced mast cell alterations and mediator release in human skin. In this study we used a human skin-test model in which we have previously shown consistent striking
16
J ALLERGY
Ting et al.
FW. 6. Electron plus cromolyn,
micrograph of a typical mast cell IO min after injection of ragweed showing evidence of marked degranulation. (x 10,000.)
CLIN. IMMUNOL. JANUARY 1983
and ragweed
FIG. 7. Electron micrograph of typical mast cell 10 min after injection with cromolyn or PBS (control), showing large amounts of intact electron-dense granules with no evidence of degrenuletion. (x14,000.)
VOLUME NUMBER
71 1. Part 1
mast cell alterations starting within 15 set after intradermal antigen injection.” This is followed over the next 30 min by increasing levels of histamine released into a skin chamber appended to such test sites and by ingress of eosinophils into the area starting 1 to 2 hr later. 3 Using this model we found no inhibitory effects of cromolyn on the mast cell ultramicroscopic alterations, mediator release, or inflammatory cell accumulation when cromolyn was added to antigen in the skin chambers at the challenge site. Our results, showing that cromolyn did not inhibit in vivo the anaphylactic histamine release from human skin mast cells, are consistent with the in vitro findings of Pearce et al.12 It is conceivable but unlikely that no inhibition was seen in this because the cromolyn in solution did not penetrate the base of the chamber at the dermalepidetmal junction; addition of cromolyn to the antigen solution did not inhibit the whealing reaction after intradermal injection. It is conceivable that higher concentrations of cromolyn in solution could inhibit such reactions. In some clinical reports, up to 10% cromolyn in petrolatum had been employed for treatment of skin disorders.4 Our attempts to use cromolyn solution in concentrations greater than 2% led to nonspecific irritative effects. A more likely explanation for the results observed here is that mast cells in the skin are less affected than those in the human respiratory tract. The heterogeneity of mast cells from different organ systems has recently been stressed.13,I4 It is not clear whether lack of cromolyn effects in the skin reflects decreased binding to uptake of this agent into skin mast cells or other events involved in mediator release during allergic reactions. Further studies that are ethically and logistically feasible will be needed to help our understanding of these fascinating questions.
Cromolyn
allergic
skin reactions
17
REFERENCES 1 Pepys J, Edwards AM, editors: The mast cell, its role in health and disease. Marshfield, Mass., 1979, Pitman Medical, pp. 199, 447, 506. 2 Ankier SI: Disodium cromoglycate and inhibition of passive cutaneous anaphylaxis. Int Arch Allergy 41: 163, 197 I. 3. Goose J, Blair AMJN: Passive cutaneous anaphylaxis in the rat induced with two hemologous reagin-like antibody sera, and its specific inhibition with disodium cromoglycate. Immunology 16:749, 1969. 4. Haider SA: Treatment of atopic eczema in children: clinical trial of 10% sodium cromoglycate ointment. Br Med J I: 1570. 1977. 5. Ting S, Dunsky EH, Lavker RM, Zweiman B: Pattern of mast cell alterations and in viva mediator release in human allergic skin reaction. J ALLERGY CLIN IMMUNOL 66:417, 1980. 6. Levy DA, Widra M: A micro-assay for studying allergic histamine release from human leukocytes using an enzymaticisotopic assay for histamine. J Lab Clin Med 81:291, 1973. 7. Altounyan REC: Inhibition of experimental asthma by a new compound-disodium cromoglycate. Acta Allergol 22:487, 1967. 8. Altounyan REC, Howell JBL: Treatment of asthma with disodium cromoglycate. Respiration 26: 131, 1969. 9. Anand SC. Goodman DH: Protective effect of cromolyn sodium on bronchial challenge tests in pollen asthma. Ann Allergy 30:64, 1972. 10. Pepy J, Hargreave FE, Chan M, McCarthy DS: Inhibitory effects of disodium cromoglycate on allergen-inhalation tests. Lancet 2: 134, 1968. 11. Atkins PC, Norman MD, Zweiman B: Antigen induced neutrophil chemotactic activity in man. Correlation with bronchospasm and inhibition by disodium cromoglycate. J ALLERGY CLIN IMMUNOL 62: 149, 1978. 12. Pearce CA, Greaves MW, Plummer VM, Yamamoto ES: Effect of disodium cromoglycate on antigen evoked histamine release from human skin. Clin Exp Immunol 17:437, 1974. 13. Kaliner MA: Is a mast cell a mast cell a mast cell? J ALLERGY CLIN IMMUNOL 66: 1, 1980. 14. Bierenstock J, Bejus AD: Mucosal immunology. Immunology 41:249. 1980.