- Email: [email protected]
Skin histamine in chronic urticaria
Praphan Phanuphak, and Peter F. Kohler,
M.D., Alan L. Schocket, M.D. Denver, Cola.
M.D., Cartes
M. Arroy~v~,
M.D..
To further study the role of histamine in the pathogenesis oj chronic urticaria, the concentrution of histumine in tissue extracts from skin biopsy samples and in plasma from patients with chronic urticaria was measured by a sensitive radioenzymatic assay. Tissue histamine levels jbom urticarial lesions and uninvolved skin were compared with extracts of biopsy sumples taken ,from normal controls. The average tissue histamine content in 15 biopsy samples,from the chronic urticaria patients was significantly higher than in those of 15 normal controls. Fort> percent of the patients had levels 2 SD greater than the mean of the control group. Elevated histamine levels were also found in biopsy samples of uninvolved skin from some u&curia putients. Circulating histamine levels from chronic urticaria patients were rurely elevated and did not correlate with skin concentration. No correlation was noted between tissue histumine concentration and estimated mast cell concentrution on Giemsa-stained sections of five biops> samples. These results indicate that tissue histamine levels are increased in some patients with c,hronic, urticaria. This suggests that local histamine elevations may be importunt in the pathogenesis of man? patients with this disease. In addition, increased tissue histamine in these patients is not rejlected b! elevated circulating levels.
C wonic ‘ ‘idiopathic ’ ’ urticaria is a common disease, yet its pathogenesis is still not well understood. Vasoactive amines, in particular histamine, have long been considered to be the primary mediators involved in the production of lesions.’ Although histamine, serolonin, SRS-A, and bradykinin all cause vasodilation. only histamine is known to cause pruritus in man.2 Sir Thomas Lewis observed many years ago that an intradermal injection of histamine produces prutitus and a wheal-and-flare reaction identical to spontaneous urticarial lesions.” In addition, this cutaneous response can be suppressed by antihistamine compaunds.4, s These phenomena strongly support the role of histamine as a major, although not necessarily the only, pharmacologic mediator involved in the production of the lesions in urticaria. Direct evi-..-From the Divisions of Clinical Immunology and Rheumatology, Department of Medicine, University of Colorado Medical Cfxlter. Supixxted in part by an Allergic Disease Center Program Grant Al, 12028-06, Allergy and Immunology Training Grant AI0’7i66-01, and a General Clinical Research Program Grant 2.,%IOl-RROOO51-17. Received for publication April 23, 1979. Acc+ed for publication Oct. 8, 1979. Reptint requests to: Alan L. Schocket, M.D., Division of Clinical Immunology, Box B- 164, University of Colorado Medical Center 4200 East Ninth Ave., Denver, CO 80262. 0091-6749/80/050371+05$00.50/0
0 1980
The
C. V. Mosby
dence indicating a pathogenic role for histamine in chronic urticaria, however, ,is scanty. Elevatian of plasma histamine following challenge has been noted in several cases of physical urticaria.R-” On the other hand, in chronic idiopathic u&aria, circetWng histamine levels have been either normallo or only occasionally elevated.” Since histamine is rapidly metabolized or eliminated from the circulation, information about local tissue levels would be more helpful to determine the role of histamine in chronic u&aria. Only a few studies have examined this. K&&m, using negative pressure-induced blisters, has found elevated histamine levels in blister fluid in&d over lesions of chronic idiopathic u&aria patients. I2 Elevated levels were found in 7/13 fluids irwfuced on the clinically normal skin from these patients. Two earlier studies measuring histamine content of skin biopsy samples from lesions in chronic urticaria using less sensitive techniques produced confficting results. Juhlin’” noted increased histamine content in biopsy samples from a small percentage of his patients with “recidivating” urticaria, whereas Zach&ae’* no&d decreased histamine content in some of his m&aria patients. None of the studies examined simultaneous circulating levels. To further study the role of histamine in chronic urticaria, this study was designed t@&@ctly measure histamine content in tissue from skin of chronic urCO.
Vol.
65, No. 5.
pp. 371-375
372
Phanuphak
et
al.
J. ALLERGY
CLIN.
IMMUNOL. MAY 1980
had been established previously in this laboratory. Informed consent was obtained from all patients prior to the study.
Ng Histamine Per Mg Skin
Biopsy
I& v
50
A A
40 38.0 sf 22
30
%
A A0
I 21.8
10 A N=15
N=15
0 URTICARIAL
SKIN
SKIN
FROM NORMAL CONTROLS
FIG. 1. Histamine content of fresh urticarial and normal skin (p < 0.01). (al indicates mean ? 2 SEM. A = upper extremities; o = anterior trunk; q = back; v = lower extremities.
ticaria patients and compare it with simultaneous circulating levels. A sensitive radioenzymatic histamine assayI5 was used to measure histamine in skin extracts, serum, and plasma. MATER1Al.S AND METHODS Patient population Chronic u&aria was defined as persistent or recurrent urticaria lasting 6 wk or longer. Patients entered in this study were referred to the Clinical Immunology Service at the University of Colorado Medical Center from practicing allergists as part of our ongoing vasculitis study. Forty-two patients were evaluated over an 18-mo period. There were 19 men and 23 women in the group, with a mean age of 41 yr (range, 14 to 79 yr). The average duration of urticaria was 4.1 yr (range, 6 wk to 16 yr). All patients had been evaluated previously for a precipitating cause of their urticaria. Work-up included diet manipulation, skin testing for foods and inhalants when indicated, serologic, bacteriologic, and radiologic evaluations including a Venereal Disease Research Laboratory (VDRL) and hepatitis B antigen test. In no patient did this extensive evaluation uncover an etiology for urticaria. Fifteen of the 42 patients underwent a
punch biopsy of a recent lesion. Six of these patients also had biopsy samples taken of uninvolved (normal) skin. All 42 had circulating histamine levels determined. Control skin biopsy samples were obtained from a group of 1.5 healthy volunteers. These included 6 men and 9 women with a mean age of 33 yr (range, 24 to 45 yr). Controls for serum and plasma histamine determinations
procedure
Patients with chronic urticaria undergoing skin biopsy were withdrawn from antihistamines and sympathomimetics for at least 48 hr prior to the procedure. Blood was drawn from another extremity prior to the biopsy, and the plasma or serum was separated and stored at -70” C. A 4-mm diameter, l-mm deep punch biopsy was performed under local anesthesia (0.5 ml of 1% lidocaine without epinephrine) with a disposable trephine punch (Chester A. Baker Labs., Inc., Miami, Fla.). A biopsy sample was taken from either the most recent, well-developed urticarial lesion, uninvolved skin, or both. The biopsy sample was snap-frozen at - 70” C and stored. For consistency, an attempt was made to obtain the biopsy sample from the same area (the forearm) but this was possible in only 50% of patients due to the variation in distribution of the lesions. Additional biopsy specimens were also obtained and mounted for cryostat sectioning in Tissue Tek II (Miles Labs., Inc., Naperville, Ill.), which consists of water-soluble glycol and resins (OCT compound). Skin extraction After thawing each biopsy specimen at room temperature and removal from the mounting media where necessary, subcutaneous fat was cut away from the dermis. The skin was then weighed on an electric balance with a precision of +O. 1 mg. The typical weight ranged between 5 to 6 mg. The skin was vigorously crushed against a No. lOO-mesh stainless steel screen with the plunger of a 2-ml glass syringe in a glass Petri dish with 0.2 ml of 0.1 M phosphate-buffered saline (PBS) (pH 7.9) per milligram of tissue. The final strand of fibrous tissue left on the screen was discarded. The tissue fluid and cell debris were separated by centrifugation at 500 X g for 10 min at mom temperature. Histamine was assayed in duplicate O.l-ml aliquots of the supematant fluid according to the method described below. For the assay of the extract, PBS was substituted for plasma in the standard histamine curve. The mean histamine content was expressed zs nanograms of histamine per milligram of skin extracted. Histamine
deWi$ination
Histamine was measured in edetate (EDTA) plasma, serum, and tissue extracts using an enzymatic isotopic assay as modified by Beaven. I5 Briefly, 100 ~1 of supernatant were incubated with 0.03 +i S-adenosylmethionine (carbon-14-methyl) and 100 ~1 of a preparation of histamineN-methyltransferase derived from guinea pig brain (10 mg protein/ml). Carbon-lCmethyl-histamine was isolated by extraction into chloroform and counted in scintillation fluid (Dilufluor, Mallinckrodt, St. Louis) in a liquid scintillation counter (Packard, Downers Grove, Ill.). All samples were run in duplicate. Absolute histamine concentrations were calculated using standard dilutions of histamine dihydrochloride (Fisher Scientific Co., Los Angeles, Calif.).
Skin
VOL1. ME 65 NUII:3EA 5
Ng
histamine
in chronic
urticaria
373
Histamine W’ Mg
skin
20Histamine
in PfJS
tiirtarninc @CT 1:2
in C?CT in ClsS)
P ‘* 15-
LI
11 N:lO
0
,O
0
i
1
UNEMBEDDED
N-10 EMBEDDED
Comparison of simultaneously embedded (in Tissue Tek II) and unembedded skin histamine. Histamine coW#ent for unembedded skin was 14.4 it 1.8 nglmg and for embedded skin was 11.0 -r- 2.5 nglmg (mean 2 2 SEM, p < 0.05).
TABLE 1. Histamine (nglmg) Patient I
from five patients with chronic urtic&a were sectioned, stained with hematoxylin and eosin, and examined. Thin sections (1 II) were stained with Giemsa stain’” and the number of mast cells on one section (average
3
4 5 6 7* *Patient
histamine
levels
Three of 42 patients (7%) had minimally elevated plasma or serum levels (serum, 1.4 rig/ml; plasma, 1.14 and 3 rig/ml) of histamine. Normal values in our IaMatory are 1 rig/ml or below. Two of these patient:< (1.14 and 3 rig/ml) also had simultaneous skin histamine determinations of 18.4 and 45.8 ng/mg, respectively. Histamine
content
of urtiearial
21)
Determination of histamine standard curve in PBS and in 50% OCT mounting compound fTis$ue Tek 11) end 50% PBS. No histamine is detected in the OCT solution at any added concentration.
2
Cirwlating
is (ng.)
FIG. 3.
FIG. 2.
biopsy
IO Histamine
skin
Lesions from 15 patients, uninvolved skin from six of these patients, and 15 biopsy samples from normal voltanteers were assayed for histamine content. Histamine content of lesions and normal controls are shown in Fig. 1. Normal control skins had a mean histamine content of 15.7 + 3 (+2 SEM) ng/mg of skin. Urticarial lesion skins had a mean histamine content of 29.9 + 8.1 (&2 SEM) ng/mg of skin, significantly higher than the control skins (p value < 0.01 by Student’s t test). The histamine content of lesions vs uninvolved skin in six patients is shown in Table 1. There is no significant difference between
in lesion
vs uninvolved
Lesion
Unkwo~sltkr
23.8 15.9 14.5 48.6 17.6 10.8 7.6
24.4
skin
10.0
17.2 39.6 x.0 10,4 8.X
with dermatographia
histaminecontent in the lesion and that in uninvolved skin. Six of the 15 patients (40%) with chronic urticaria had skin histamine content greater than 2 SD (12.2 ng/mg) above the mean of the control group. In addition, we have studied one patient with urticaria pigmentosa and one with ~~atog~~~i~. The former had very high levels of histamine(65 ng/mg of lesion skin and 41 ng/mg of normal skin); whereasin the latter, the histamine content of the lesion (7.6 ng/mg) was normal. Both patients’ simultaneous plasmahistamine levels were normal. compsrm of embedded and
in
Preliminary studies showed that placement of urticarial skin in cryostat mounting media (TissueTek II) causeda decreasein histamine content. A direct comparison was therefore made between embedded and unembeddedhalves of the samepiece of skin from 10 normal controls. All the tissueswere similarly kept at - 70” C for 6 to 7 days before histamine assay. Resultsare shown in Fig. 2. Histamine in embeddedskin was lower than the embedded skin in 9 out of 10
374
Phanuphak
et al.
The average reduction in histamine content after cryostat fixation was 28%. To further examine the effect of OCT mounting media (Tissue Tek II) on histamine quantitation, a standard curve for histamine was run with OCT compound 1: 2 in PBS and compared with a curve run, in PBS alone (Fig. 3). The OCT completely eliminated the measurable histamine. It is likely that this binding or inactivation of histamine by OCT compound accounts for the decreased levels of measurable histamine in the OCT-mounted tissue. instances.
Histologic
studies
No significant proliferation of sweat glands, hair bulbs, or blood vessels was seen on hematolylin and eosin section of urticarial lesions. On Giemsa-stained l-pm sections, there was no correlation between the number of mast cells per square millimeter and skin histamine content. DISCUSSION This study is the first to quantitate skin histamine content in patients with chronic urticaria using a sensitive radioenzymatic assay. The results indicate that the mean histamine content in urticarial lesions is significantly higher than that in skin from normal controls. In addition, the histamine content of uninvolved skin closely paralleled that of the lesions in many of the patients. Although there was considerable overlap in skin histamine between the patients and normals, six of 15 patients (40%) had lesional histamine content over 2 SD greater than the mean value from biopsy samples of the normal control group. Previous studies using less sensitive techniques to measure skin histamine in chronic urticaria produced conflicting results. Zachariae,14 using a spectrofluorometric assay, found a lower content of histamine in lesions from urticaria patients than in either normal skin from the same patient or in skin from normal controls. A later study by Juhlin13 seemingly agreed with our results although he omitted any kind of detailed description of his patients, describing their disease only as “recidivating” urticaria. In his group of 18 patients, four had elevated skin histamine content. Although the absolute tissue histamine levels reported in his studies were somewhat lower than we found, he used the less sensitive spectrofluorometric technique. In addition, the lack of patient information provided in Juhlin’s study makes it difficult to compare patient populations. Findings similar to the present study were reported recently by Kaplan et al.‘* using a different technique to determine skin histamine content. He induced suction blisters over urticarial lesions and
J. ALLERGY
CLIN. IMMUNOL. MAY 1980
uninvolved skin and quantitated the histamine content in the blister fluid using the same sensitive radioenzymatic assay used in our study. In 12 of 13 of his patients with chronic urticaria, blister fluid histamine levels taken over urticarial lesions were elevated. In seven of these patients, elevations were also noted in areas of normal-appearing skin. The finding of elevated tissue histamine levels in areas of normalappearing skin in some patients with chronic urticaria was also noted in our study. The source of the elevated histamine content in lesions and uninvolved skin from some of the chronic urticaria patients is unclear. One possibility is diffusion into the skin from the plasma, but as previously noted, circulating histamine levels were normal in almost all but two of our patients with elevated tissue levels. This discordance between tissue and plasma histamine levels in allergic diseases has been observed previously. l7 Possible reasons for this include minimal spillage of histamine from the tissue into the circulation or rapid catabolism of histamine in the blood after release.ls Our findings suggest that local tissue histamine concentration better reflects increased local mediator release in urticaria than does circulating histamine. Another possible explanation for the elevated histamine levels found in lesions and normal skin of some of the chronic urticaria patients might be an increased concentration of mast cells or hair follicles. Histamine in the normal skin has been localized by fluorescent technique in the walls of small blood vessels in the mast cells and around the eccrine sweat glands and hair bulbs. l3 Routine histologic studies failed to reveal any increase of the sweat glands, hair bulbs, or blood vessels in the urticarial lesions. Estimation of mast cell number in Giemsa-stained 1-p thick section of skin did not show significant differences in the number of mast cells between normal and urticarial skin regardless of cellular infiltration or histamine content. In addition, no apparent qualitative or quantitative difference in mast cell granules was found. To better evaluate these possibilities, however, more detailed grid counting of mast cells in tissue sections will have to be done. Other explanations which we did not explore for the increased skin histamine in the urticaria patients include either a higher histamine content per mast cell or an increased histamine in other cells or tissues such as vessels. In addition, an impaired.catabolism of the tissue histamine may also contribute to increased local levels. This possibility is under investigation in our laboratory. Several other variables in addition to techniques of histamine assay must be considered in interpreting
Skin
VOLL ME 65 NUFI3ER 5
tissue histamine levels. These include site and technique of biopsy and the method of tissue processing and extraction. A variation of skin histamine content according to biopsy site in normal humans was reported hy Johnson.‘g Our results, however, showed no tiifferences related to the site of biopsy (Fig. 1). Skir histamine contents have been reported both per dry weight and wet weight of the skin. We feel that the processes of lyophilization and defatting of the skir, are unnecessary and cumbersome procedures as ion!! as the homogenization of skin is standardized. To our surprise, as noted earlier, we also found that fixation of the skin in the cryostat mounting media (Ti>iue Tek II) significantly lowers skin histamine conent (Fig. 2), resulting in the exclusion of several of cur original biopsy samples from the study. This was directly related to the effect of the mounting media, because we were unable to detect any histamine when the standard curve was run in 50% Tissue Tek II. This indicates that the resin either inactivates or binds the free histamine or the methyltransferase. Cocsequently, tissue prepared in this way cannot be usea to assay histamine content. Ir summary, this study indicates that tissue levels of histamine are elevated in many patients with chronic urticaria. In general, elevations were noted in biopsy samples from lesions; in some patients, elevat4 levels were also noted in normal-appearing skia. Simultaneous determinations of circulating histamine levels were normal in almost all patients, indicatilg that circulating levels poorly reflect local mediator release. Finally, it is important for tissue determinations that biopsy samples are not mounted in 6XT resin hecause this compound interferes with histiimine determinations. The authors Ciiernsa-stained
thank Dr. skin section.
E.
G.
Thorne
for
examining
REPMENCES I. \Varin RP. Champion RH: IV. B. Saunders Co.. p. 15.
Urticaria.
Philadelphia,
1974,
histamine
in chronic
tirticaria
375
2. Juhhn L, Michaelson G: Cutaneous reactions to kallikrein, bradykinin and histamine in healthy subjects and in patients with urticaria. Acta Derm Venereal 4%26, 1969. 3. Lewis T: Blood vessels of the human skin and their response. London, 1927, Shaw & Sons, Ltd. 4. Galant SP, Bullock J, Won& D. Maihach HI: Inhibitory effect of antiallergic drugs on allergenand histamine-induced wheal-and-flare response. J Al.1 ERGY GIN IMM~WJL 49: 119. 1972. 5. Rhoades RB, Leifer KN. Cohan R, Witting HJ: Suppression of histamine-induced pnuitus hy three antihistaminic drugs. J ALLERCV CLIN IMMUNOL 55: 180, 1975. 6. Kaplan AP, Gray L. Shaff RE, Horakova Z, Beaven MA: In vivo studies of mediator release in cold urticaria and chofinergic urticaria. J ALLERGY CLIN IMMUNOI. 55~394. 1975. 7. Kaplan AP, Beaven MA: In vivo studies of the pathogenesis of cold urticaria. cholinergic urticaria and variation induced swelling. J Invest Derm 67:327, 1976. 8. Soter NA, Wasserman SI, Austen KF: Cold unicaria: Release into the circulation of histamine and eosinophil chemotactic factor of anaphylaxis during cold challenge. N Engl J Med 294:687, 1976. 9. Kaplan AP: Mediators of urticaria and an&edema. J At.LERGY CLIN 1~~~~01.60:324, 1977. 10. Beah GM: Plasma histamine concentrations in allergic diseases. J ALLERGY 34:8, 1963. 11. Mathison DA, Arroyave CM, Bhat KN, Hurewitz DS, Marnell DJ: Hypocomplementemia in chronic idiopathic urticaria. Ann Intern Med 86:534, 1977. 12. Kaplan AP, Horakova 2, Katz SI: Assessment of tissue fluid histamine levels in patients with urticaria. J AI.I.I:RGy CL-IN IMMUNOI. 61:350, 1978. 13. Juhlin L: Localization and content of histamine in normal and diseased skin. Acta Derm Venereol 47:383, 1967. 14. Zachariae H: Skin histamine in urticaria: A specirofluorometric assay. Acta Derm Venereol 47:383, 1963. 1.5. Beaven MA: Radiochemical assay procedures for drugs and transmitters. in Iversen LL, Snyder SH, editors: Handbook of psychopharmacology. Biochemical principles and techniques in neuropharmacology. vol. 1. New York, 1975. Plenum Press, pp. 253-290. 16. Dvorak HF, Mihm MC Jr: Basophilic leukocytes in allergic contact dermatitis. J Exp Med 135:2X, 1972. 17. Porter JF, Mitchell RG: Distribution of histamine in human blood. Physiology Rev 52~361, 1972. 18. Beaven MA: Histamine. N Engl J Med 294~30. 1976. 19. Johnson HH: Histamine levels in human skin. Arch Dermatol 76~726. 1957.
M.D., Alan L. Schocket, M.D. Denver, Cola.
M.D., Cartes
M. Arroy~v~,
M.D..
To further study the role of histamine in the pathogenesis oj chronic urticaria, the concentrution of histumine in tissue extracts from skin biopsy samples and in plasma from patients with chronic urticaria was measured by a sensitive radioenzymatic assay. Tissue histamine levels jbom urticarial lesions and uninvolved skin were compared with extracts of biopsy sumples taken ,from normal controls. The average tissue histamine content in 15 biopsy samples,from the chronic urticaria patients was significantly higher than in those of 15 normal controls. Fort> percent of the patients had levels 2 SD greater than the mean of the control group. Elevated histamine levels were also found in biopsy samples of uninvolved skin from some u&curia putients. Circulating histamine levels from chronic urticaria patients were rurely elevated and did not correlate with skin concentration. No correlation was noted between tissue histumine concentration and estimated mast cell concentrution on Giemsa-stained sections of five biops> samples. These results indicate that tissue histamine levels are increased in some patients with c,hronic, urticaria. This suggests that local histamine elevations may be importunt in the pathogenesis of man? patients with this disease. In addition, increased tissue histamine in these patients is not rejlected b! elevated circulating levels.
C wonic ‘ ‘idiopathic ’ ’ urticaria is a common disease, yet its pathogenesis is still not well understood. Vasoactive amines, in particular histamine, have long been considered to be the primary mediators involved in the production of lesions.’ Although histamine, serolonin, SRS-A, and bradykinin all cause vasodilation. only histamine is known to cause pruritus in man.2 Sir Thomas Lewis observed many years ago that an intradermal injection of histamine produces prutitus and a wheal-and-flare reaction identical to spontaneous urticarial lesions.” In addition, this cutaneous response can be suppressed by antihistamine compaunds.4, s These phenomena strongly support the role of histamine as a major, although not necessarily the only, pharmacologic mediator involved in the production of the lesions in urticaria. Direct evi-..-From the Divisions of Clinical Immunology and Rheumatology, Department of Medicine, University of Colorado Medical Cfxlter. Supixxted in part by an Allergic Disease Center Program Grant Al, 12028-06, Allergy and Immunology Training Grant AI0’7i66-01, and a General Clinical Research Program Grant 2.,%IOl-RROOO51-17. Received for publication April 23, 1979. Acc+ed for publication Oct. 8, 1979. Reptint requests to: Alan L. Schocket, M.D., Division of Clinical Immunology, Box B- 164, University of Colorado Medical Center 4200 East Ninth Ave., Denver, CO 80262. 0091-6749/80/050371+05$00.50/0
0 1980
The
C. V. Mosby
dence indicating a pathogenic role for histamine in chronic urticaria, however, ,is scanty. Elevatian of plasma histamine following challenge has been noted in several cases of physical urticaria.R-” On the other hand, in chronic idiopathic u&aria, circetWng histamine levels have been either normallo or only occasionally elevated.” Since histamine is rapidly metabolized or eliminated from the circulation, information about local tissue levels would be more helpful to determine the role of histamine in chronic u&aria. Only a few studies have examined this. K&&m, using negative pressure-induced blisters, has found elevated histamine levels in blister fluid in&d over lesions of chronic idiopathic u&aria patients. I2 Elevated levels were found in 7/13 fluids irwfuced on the clinically normal skin from these patients. Two earlier studies measuring histamine content of skin biopsy samples from lesions in chronic urticaria using less sensitive techniques produced confficting results. Juhlin’” noted increased histamine content in biopsy samples from a small percentage of his patients with “recidivating” urticaria, whereas Zach&ae’* no&d decreased histamine content in some of his m&aria patients. None of the studies examined simultaneous circulating levels. To further study the role of histamine in chronic urticaria, this study was designed t@&@ctly measure histamine content in tissue from skin of chronic urCO.
Vol.
65, No. 5.
pp. 371-375
372
Phanuphak
et
al.
J. ALLERGY
CLIN.
IMMUNOL. MAY 1980
had been established previously in this laboratory. Informed consent was obtained from all patients prior to the study.
Ng Histamine Per Mg Skin
Biopsy
I& v
50
A A
40 38.0 sf 22
30
%
A A0
I 21.8
10 A N=15
N=15
0 URTICARIAL
SKIN
SKIN
FROM NORMAL CONTROLS
FIG. 1. Histamine content of fresh urticarial and normal skin (p < 0.01). (al indicates mean ? 2 SEM. A = upper extremities; o = anterior trunk; q = back; v = lower extremities.
ticaria patients and compare it with simultaneous circulating levels. A sensitive radioenzymatic histamine assayI5 was used to measure histamine in skin extracts, serum, and plasma. MATER1Al.S AND METHODS Patient population Chronic u&aria was defined as persistent or recurrent urticaria lasting 6 wk or longer. Patients entered in this study were referred to the Clinical Immunology Service at the University of Colorado Medical Center from practicing allergists as part of our ongoing vasculitis study. Forty-two patients were evaluated over an 18-mo period. There were 19 men and 23 women in the group, with a mean age of 41 yr (range, 14 to 79 yr). The average duration of urticaria was 4.1 yr (range, 6 wk to 16 yr). All patients had been evaluated previously for a precipitating cause of their urticaria. Work-up included diet manipulation, skin testing for foods and inhalants when indicated, serologic, bacteriologic, and radiologic evaluations including a Venereal Disease Research Laboratory (VDRL) and hepatitis B antigen test. In no patient did this extensive evaluation uncover an etiology for urticaria. Fifteen of the 42 patients underwent a
punch biopsy of a recent lesion. Six of these patients also had biopsy samples taken of uninvolved (normal) skin. All 42 had circulating histamine levels determined. Control skin biopsy samples were obtained from a group of 1.5 healthy volunteers. These included 6 men and 9 women with a mean age of 33 yr (range, 24 to 45 yr). Controls for serum and plasma histamine determinations
procedure
Patients with chronic urticaria undergoing skin biopsy were withdrawn from antihistamines and sympathomimetics for at least 48 hr prior to the procedure. Blood was drawn from another extremity prior to the biopsy, and the plasma or serum was separated and stored at -70” C. A 4-mm diameter, l-mm deep punch biopsy was performed under local anesthesia (0.5 ml of 1% lidocaine without epinephrine) with a disposable trephine punch (Chester A. Baker Labs., Inc., Miami, Fla.). A biopsy sample was taken from either the most recent, well-developed urticarial lesion, uninvolved skin, or both. The biopsy sample was snap-frozen at - 70” C and stored. For consistency, an attempt was made to obtain the biopsy sample from the same area (the forearm) but this was possible in only 50% of patients due to the variation in distribution of the lesions. Additional biopsy specimens were also obtained and mounted for cryostat sectioning in Tissue Tek II (Miles Labs., Inc., Naperville, Ill.), which consists of water-soluble glycol and resins (OCT compound). Skin extraction After thawing each biopsy specimen at room temperature and removal from the mounting media where necessary, subcutaneous fat was cut away from the dermis. The skin was then weighed on an electric balance with a precision of +O. 1 mg. The typical weight ranged between 5 to 6 mg. The skin was vigorously crushed against a No. lOO-mesh stainless steel screen with the plunger of a 2-ml glass syringe in a glass Petri dish with 0.2 ml of 0.1 M phosphate-buffered saline (PBS) (pH 7.9) per milligram of tissue. The final strand of fibrous tissue left on the screen was discarded. The tissue fluid and cell debris were separated by centrifugation at 500 X g for 10 min at mom temperature. Histamine was assayed in duplicate O.l-ml aliquots of the supematant fluid according to the method described below. For the assay of the extract, PBS was substituted for plasma in the standard histamine curve. The mean histamine content was expressed zs nanograms of histamine per milligram of skin extracted. Histamine
deWi$ination
Histamine was measured in edetate (EDTA) plasma, serum, and tissue extracts using an enzymatic isotopic assay as modified by Beaven. I5 Briefly, 100 ~1 of supernatant were incubated with 0.03 +i S-adenosylmethionine (carbon-14-methyl) and 100 ~1 of a preparation of histamineN-methyltransferase derived from guinea pig brain (10 mg protein/ml). Carbon-lCmethyl-histamine was isolated by extraction into chloroform and counted in scintillation fluid (Dilufluor, Mallinckrodt, St. Louis) in a liquid scintillation counter (Packard, Downers Grove, Ill.). All samples were run in duplicate. Absolute histamine concentrations were calculated using standard dilutions of histamine dihydrochloride (Fisher Scientific Co., Los Angeles, Calif.).
Skin
VOL1. ME 65 NUII:3EA 5
Ng
histamine
in chronic
urticaria
373
Histamine W’ Mg
skin
20Histamine
in PfJS
tiirtarninc @CT 1:2
in C?CT in ClsS)
P ‘* 15-
LI
11 N:lO
0
,O
0
i
1
UNEMBEDDED
N-10 EMBEDDED
Comparison of simultaneously embedded (in Tissue Tek II) and unembedded skin histamine. Histamine coW#ent for unembedded skin was 14.4 it 1.8 nglmg and for embedded skin was 11.0 -r- 2.5 nglmg (mean 2 2 SEM, p < 0.05).
TABLE 1. Histamine (nglmg) Patient I
from five patients with chronic urtic&a were sectioned, stained with hematoxylin and eosin, and examined. Thin sections (1 II) were stained with Giemsa stain’” and the number of mast cells on one section (average
3
4 5 6 7* *Patient
histamine
levels
Three of 42 patients (7%) had minimally elevated plasma or serum levels (serum, 1.4 rig/ml; plasma, 1.14 and 3 rig/ml) of histamine. Normal values in our IaMatory are 1 rig/ml or below. Two of these patient:< (1.14 and 3 rig/ml) also had simultaneous skin histamine determinations of 18.4 and 45.8 ng/mg, respectively. Histamine
content
of urtiearial
21)
Determination of histamine standard curve in PBS and in 50% OCT mounting compound fTis$ue Tek 11) end 50% PBS. No histamine is detected in the OCT solution at any added concentration.
2
Cirwlating
is (ng.)
FIG. 3.
FIG. 2.
biopsy
IO Histamine
skin
Lesions from 15 patients, uninvolved skin from six of these patients, and 15 biopsy samples from normal voltanteers were assayed for histamine content. Histamine content of lesions and normal controls are shown in Fig. 1. Normal control skins had a mean histamine content of 15.7 + 3 (+2 SEM) ng/mg of skin. Urticarial lesion skins had a mean histamine content of 29.9 + 8.1 (&2 SEM) ng/mg of skin, significantly higher than the control skins (p value < 0.01 by Student’s t test). The histamine content of lesions vs uninvolved skin in six patients is shown in Table 1. There is no significant difference between
in lesion
vs uninvolved
Lesion
Unkwo~sltkr
23.8 15.9 14.5 48.6 17.6 10.8 7.6
24.4
skin
10.0
17.2 39.6 x.0 10,4 8.X
with dermatographia
histaminecontent in the lesion and that in uninvolved skin. Six of the 15 patients (40%) with chronic urticaria had skin histamine content greater than 2 SD (12.2 ng/mg) above the mean of the control group. In addition, we have studied one patient with urticaria pigmentosa and one with ~~atog~~~i~. The former had very high levels of histamine(65 ng/mg of lesion skin and 41 ng/mg of normal skin); whereasin the latter, the histamine content of the lesion (7.6 ng/mg) was normal. Both patients’ simultaneous plasmahistamine levels were normal. compsrm of embedded and
in
Preliminary studies showed that placement of urticarial skin in cryostat mounting media (TissueTek II) causeda decreasein histamine content. A direct comparison was therefore made between embedded and unembeddedhalves of the samepiece of skin from 10 normal controls. All the tissueswere similarly kept at - 70” C for 6 to 7 days before histamine assay. Resultsare shown in Fig. 2. Histamine in embeddedskin was lower than the embedded skin in 9 out of 10
374
Phanuphak
et al.
The average reduction in histamine content after cryostat fixation was 28%. To further examine the effect of OCT mounting media (Tissue Tek II) on histamine quantitation, a standard curve for histamine was run with OCT compound 1: 2 in PBS and compared with a curve run, in PBS alone (Fig. 3). The OCT completely eliminated the measurable histamine. It is likely that this binding or inactivation of histamine by OCT compound accounts for the decreased levels of measurable histamine in the OCT-mounted tissue. instances.
Histologic
studies
No significant proliferation of sweat glands, hair bulbs, or blood vessels was seen on hematolylin and eosin section of urticarial lesions. On Giemsa-stained l-pm sections, there was no correlation between the number of mast cells per square millimeter and skin histamine content. DISCUSSION This study is the first to quantitate skin histamine content in patients with chronic urticaria using a sensitive radioenzymatic assay. The results indicate that the mean histamine content in urticarial lesions is significantly higher than that in skin from normal controls. In addition, the histamine content of uninvolved skin closely paralleled that of the lesions in many of the patients. Although there was considerable overlap in skin histamine between the patients and normals, six of 15 patients (40%) had lesional histamine content over 2 SD greater than the mean value from biopsy samples of the normal control group. Previous studies using less sensitive techniques to measure skin histamine in chronic urticaria produced conflicting results. Zachariae,14 using a spectrofluorometric assay, found a lower content of histamine in lesions from urticaria patients than in either normal skin from the same patient or in skin from normal controls. A later study by Juhlin13 seemingly agreed with our results although he omitted any kind of detailed description of his patients, describing their disease only as “recidivating” urticaria. In his group of 18 patients, four had elevated skin histamine content. Although the absolute tissue histamine levels reported in his studies were somewhat lower than we found, he used the less sensitive spectrofluorometric technique. In addition, the lack of patient information provided in Juhlin’s study makes it difficult to compare patient populations. Findings similar to the present study were reported recently by Kaplan et al.‘* using a different technique to determine skin histamine content. He induced suction blisters over urticarial lesions and
J. ALLERGY
CLIN. IMMUNOL. MAY 1980
uninvolved skin and quantitated the histamine content in the blister fluid using the same sensitive radioenzymatic assay used in our study. In 12 of 13 of his patients with chronic urticaria, blister fluid histamine levels taken over urticarial lesions were elevated. In seven of these patients, elevations were also noted in areas of normal-appearing skin. The finding of elevated tissue histamine levels in areas of normalappearing skin in some patients with chronic urticaria was also noted in our study. The source of the elevated histamine content in lesions and uninvolved skin from some of the chronic urticaria patients is unclear. One possibility is diffusion into the skin from the plasma, but as previously noted, circulating histamine levels were normal in almost all but two of our patients with elevated tissue levels. This discordance between tissue and plasma histamine levels in allergic diseases has been observed previously. l7 Possible reasons for this include minimal spillage of histamine from the tissue into the circulation or rapid catabolism of histamine in the blood after release.ls Our findings suggest that local tissue histamine concentration better reflects increased local mediator release in urticaria than does circulating histamine. Another possible explanation for the elevated histamine levels found in lesions and normal skin of some of the chronic urticaria patients might be an increased concentration of mast cells or hair follicles. Histamine in the normal skin has been localized by fluorescent technique in the walls of small blood vessels in the mast cells and around the eccrine sweat glands and hair bulbs. l3 Routine histologic studies failed to reveal any increase of the sweat glands, hair bulbs, or blood vessels in the urticarial lesions. Estimation of mast cell number in Giemsa-stained 1-p thick section of skin did not show significant differences in the number of mast cells between normal and urticarial skin regardless of cellular infiltration or histamine content. In addition, no apparent qualitative or quantitative difference in mast cell granules was found. To better evaluate these possibilities, however, more detailed grid counting of mast cells in tissue sections will have to be done. Other explanations which we did not explore for the increased skin histamine in the urticaria patients include either a higher histamine content per mast cell or an increased histamine in other cells or tissues such as vessels. In addition, an impaired.catabolism of the tissue histamine may also contribute to increased local levels. This possibility is under investigation in our laboratory. Several other variables in addition to techniques of histamine assay must be considered in interpreting
Skin
VOLL ME 65 NUFI3ER 5
tissue histamine levels. These include site and technique of biopsy and the method of tissue processing and extraction. A variation of skin histamine content according to biopsy site in normal humans was reported hy Johnson.‘g Our results, however, showed no tiifferences related to the site of biopsy (Fig. 1). Skir histamine contents have been reported both per dry weight and wet weight of the skin. We feel that the processes of lyophilization and defatting of the skir, are unnecessary and cumbersome procedures as ion!! as the homogenization of skin is standardized. To our surprise, as noted earlier, we also found that fixation of the skin in the cryostat mounting media (Ti>iue Tek II) significantly lowers skin histamine conent (Fig. 2), resulting in the exclusion of several of cur original biopsy samples from the study. This was directly related to the effect of the mounting media, because we were unable to detect any histamine when the standard curve was run in 50% Tissue Tek II. This indicates that the resin either inactivates or binds the free histamine or the methyltransferase. Cocsequently, tissue prepared in this way cannot be usea to assay histamine content. Ir summary, this study indicates that tissue levels of histamine are elevated in many patients with chronic urticaria. In general, elevations were noted in biopsy samples from lesions; in some patients, elevat4 levels were also noted in normal-appearing skia. Simultaneous determinations of circulating histamine levels were normal in almost all patients, indicatilg that circulating levels poorly reflect local mediator release. Finally, it is important for tissue determinations that biopsy samples are not mounted in 6XT resin hecause this compound interferes with histiimine determinations. The authors Ciiernsa-stained
thank Dr. skin section.
E.
G.
Thorne
for
examining
REPMENCES I. \Varin RP. Champion RH: IV. B. Saunders Co.. p. 15.
Urticaria.
Philadelphia,
1974,
histamine
in chronic
tirticaria
375
2. Juhhn L, Michaelson G: Cutaneous reactions to kallikrein, bradykinin and histamine in healthy subjects and in patients with urticaria. Acta Derm Venereal 4%26, 1969. 3. Lewis T: Blood vessels of the human skin and their response. London, 1927, Shaw & Sons, Ltd. 4. Galant SP, Bullock J, Won& D. Maihach HI: Inhibitory effect of antiallergic drugs on allergenand histamine-induced wheal-and-flare response. J Al.1 ERGY GIN IMM~WJL 49: 119. 1972. 5. Rhoades RB, Leifer KN. Cohan R, Witting HJ: Suppression of histamine-induced pnuitus hy three antihistaminic drugs. J ALLERCV CLIN IMMUNOL 55: 180, 1975. 6. Kaplan AP, Gray L. Shaff RE, Horakova Z, Beaven MA: In vivo studies of mediator release in cold urticaria and chofinergic urticaria. J ALLERGY CLIN IMMUNOI. 55~394. 1975. 7. Kaplan AP, Beaven MA: In vivo studies of the pathogenesis of cold urticaria. cholinergic urticaria and variation induced swelling. J Invest Derm 67:327, 1976. 8. Soter NA, Wasserman SI, Austen KF: Cold unicaria: Release into the circulation of histamine and eosinophil chemotactic factor of anaphylaxis during cold challenge. N Engl J Med 294:687, 1976. 9. Kaplan AP: Mediators of urticaria and an&edema. J At.LERGY CLIN 1~~~~01.60:324, 1977. 10. Beah GM: Plasma histamine concentrations in allergic diseases. J ALLERGY 34:8, 1963. 11. Mathison DA, Arroyave CM, Bhat KN, Hurewitz DS, Marnell DJ: Hypocomplementemia in chronic idiopathic urticaria. Ann Intern Med 86:534, 1977. 12. Kaplan AP, Horakova 2, Katz SI: Assessment of tissue fluid histamine levels in patients with urticaria. J AI.I.I:RGy CL-IN IMMUNOI. 61:350, 1978. 13. Juhlin L: Localization and content of histamine in normal and diseased skin. Acta Derm Venereol 47:383, 1967. 14. Zachariae H: Skin histamine in urticaria: A specirofluorometric assay. Acta Derm Venereol 47:383, 1963. 1.5. Beaven MA: Radiochemical assay procedures for drugs and transmitters. in Iversen LL, Snyder SH, editors: Handbook of psychopharmacology. Biochemical principles and techniques in neuropharmacology. vol. 1. New York, 1975. Plenum Press, pp. 253-290. 16. Dvorak HF, Mihm MC Jr: Basophilic leukocytes in allergic contact dermatitis. J Exp Med 135:2X, 1972. 17. Porter JF, Mitchell RG: Distribution of histamine in human blood. Physiology Rev 52~361, 1972. 18. Beaven MA: Histamine. N Engl J Med 294~30. 1976. 19. Johnson HH: Histamine levels in human skin. Arch Dermatol 76~726. 1957.