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Early effects of corticosteroids on basophils, leukocyte histamine, and tissue histamine
Early effects of corticosteroids on basophils, leukocyte histamine, and tissue histamine Eliot H. Dunsky, M.D., Burton Zweiman, M.D., Elza Fischler, David A. Levy, M.D. Philadelphia, Pa., and Baltimore, Md.
M.D., and
The comparative e#ect in II atopic subjects of a single intravenous injection of methylpredm’solone on sequential studies of blood eosinophils. basophils, leukocyte sensitivity to antigen for histamine release, leukocyte histamine content, and skin histamine was examined. No significant changes occurred in any parameter after placebo treatment. In contrast, 4 hr after intravenous treatment with steroid there were significant decreases in mean eosinophil counts (-95%), basophil counts ( -72%), and histamine content of I x 10’ leukocyte samples ( -62%). Temporal changes in the latter paralleled alterations in circulating basophil levels. No significant changes occurred in the antigen histamine release sensitivity, or the total skin histamine. Studies over a longer period after steroids in 4 subjects showed eosinophil and basophil levels at a nadir at 8 hr, remaining suppressed for 24 hr, and returned to pretreatment levels by 72 hr. Results suggest that corticosteroids induce a prominent decrease in leukocyte histamine due to a depletion of basophils without a decrease in histamine content per basophil, and that skin tissue histamine stores remain unchanged by such treatment.
Despite intensive study, the mechanisms underlying the remarkable efficacy of corticosteroid drugs in many allergic disorders are still not well defined. At least part of the therapeutic activity may be due to effects of steroids on cellular inflammatory responses at the site of immediate hypersensitivity reactions.’ Previous studies in this laboratory have shown that prior steroid administration to atopic humans results in a rapid suppression of the usual eosinophil accumulation in the skin tests site as a response to potent antigen injection. Based upon studies in other experimental models, it is conceivable that various steroid effects on circulating blood elements,2-4 emigration of cells from vessels to the inflammatory sites,5* 6
From the Veterans Administration Hospital and Allergy and Immunology Section, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, and the Department of Biochemistry, The School of Hygiene and Public Health, Johns Hopkins University, Baltimore. Supported in part by National Institutes of Health Grant No. R01 Al 14332 and Veterans Administration Grant MRIS 9967. Received for publication July 31, 1978. Accepted for publication Dec. 5, 1978. Reprint requests to: Eliot H. Dunsky, M.D., 5th Floor Johnson Pavilion, 36th and Hamilton Walk G2, University of Pennsylvania, Philadelphia, PA 19174. Vol. 63, No. 6, pp. 426-432
and mediator release7+’ could be playing a significant role at the tissue level. The present report describes studies of the early effects of intravenous steroids on circulating levels of eosinophils, basophils, and cellular histamine in comparison with tissue histamine content in an attempt to learn more about the antiallergic effects of these important therapeutic agents. MATERIALS Subjects
AND METHODS
Eleven atopic male volunteer subjects (weighing 70 to 90 kg) were studied after their informed consent was obtained. All individuals were clinically sensitive to ragweed and exhibited an immediate type wheal response of at least 10 mm in diameter (and at least 3 times the diameter of a reaction to a negative control injection) 20 min following the injection of 20 protein nitrogen units (PNU) of aqueous ragweed extract (Greer Labs., Lenoir, N. C.). None of thesesubjects was receiving immunotherapy, and all had discontinued medications 2 wk prior to this study. Treatment
program
Eleven subjects were studied on two occasions, at least two weeks apart. On one occasion (in a random order), the subject received an intravenous injection of methylprednisolone sodium succinate (provided as Solu-Medrol by Dr. Sam Stubbs, The Upjohn Co., Kalamazoo, Mich.), in a
VOLUME NUMBER
Effects of corticosteroids
63 6
TABLE I. Mean values
of 8 subjects
Time (hr) Who/r
over a 4-hr period following
0
intravenous
placebo
on histamine
427
injections
1
2
3
4
4,954 2 1,600 210 +- 89 36 -+ 6
7,123 2 2,140 208 + 87 36 + 6.5
6,369 t 2,417 206 + 99 41 +- 5.3
5,838 2 1,833 201 f 85 39 t 3.8
-
356 2 224 62 t 29 52 2 11
blood
WBC/mrti’ Eos/mn? Base/mm”
5,815* * 2,200 207 t 84 36 t 5.8
Sedirnented
l~lood
Eos/mm” Base/mm” Total cellular histamine (ng/tnl) -*Mean z SD. TABLE II. Mean values
320 r 176 64 t 21 61 ? 14
of 8 subjects
Times (hr) Whole
-
over a 4-hr period following 0
intravenous
steroid
injection
1
2
3
4
5,400 2 910 195 k 99 40 ir 9
6,088 k 2,207 95 ? 45 32 i 10
8,042 k 2,140 35 + 30 24 -t 7
9,212 k 2,449 12 k 14 I8 t 10
-
-
-
16 L 19 27 -+ 11 35 5 6
blood
6,260* k 950 232 k 105 47 +- 10
WBC/tnm” Eos/mtn” Baso/mti’ hiood Eos/mtn” Base/mm” Total cellular histamine (@ml) -*Mean + SD.
Sedimented
354 + 217 77 i 30 63 + 15
dose of 1 mg/kg. On the other occasion, an intravenous saline injection was given as a placebo.
Timing
of .studies
At 8:OO A.M. of the study day, immediately prior to the intravenous injections (pretreatment), baseline blood samples were obtained for determination of (1) total leukocyte count (ce11s/mm3); (2) total eosinophil concentration (cells/mm”); (3) total basophil concentration (cells/mm3); and (4) total histamine content in a leukocyte-rich cell suspension. In all 11 subjects, these assays were repeated 4 hr after the intravenous injection of steroids and placebo. In 8 of 11 subjects, similar studies also were carried out hourly for the first 4 hr. In 4 of these 8 subjects, sequential studies were carried out at 4, 8, 24, 48, and 72 hr after the intravenous injection.
Total eosinophil
and basophil
counts
Total eosic,ophil counts were determined in coded specimens using the Unopipette system (Becton, Dickinson & Co., East Rutherford, N. J.). Total basophil counts were determined in coded specimens using the Moore and James technique I5
Determinaltion of leukocyte histamine content A leukocyte-rich supematant suspension was obtained by dextran sedimentations of heparinized venous blood, using
-
a previously described method.“’ The leukocyte-rich suspension was centrifuged, and the cell button washed and resuspended in TRIS-ACM to a final concentration of 1 x 10’ cells/ml. One-milliliter aliquots were boiled for 10 min, centrifuged, and the supematant fluid analyzed for histamine content using the radioenzymatic assay of Levy and Widra. I3 Antigen-induced histamine release was carried out with a leukocyte-rich suspension, as prepared above, concentrated to 1 x 10’ cells/ml. Aliquots of cell suspension were incubated with varying concentrations of ragweed antigen E (provided by The National Institute of Allergy and Infectious Diseases, Catalogue No. A601-901-185). The final concentration of antigen E in the dose response challenge increased progressively from 1 x lo-” pg/ml to 1 x 10-l pg/ml. Incubation was carried out at 37” C for 45 min in TRIS-ACM buffer. The cell suspension was then centrifuged at 1,500 rpm for 10 min and the supematant analyzed for histamine content.
Tissue histamine Three-millimeter dermal punch biopsies were taken from normal-appearing skin sites on the posterolateral aspect of the upper arm in 6 subjects immediately prior to and 4 hr following treatment with either placebo on one occasion or steroid on another occasion. Each biopsy specimen was immediately weighed, minced, and boiled in 1 ml of TRIS-ACM for 10 min. Samples were then centrifuged at
428 Dunsky et al.
J. ALLERGY
TABLE III. Mean levels in 4 subjects Time (hr) Whole
for a 72-hr period following 4
0
intravenous
8
24
placebo
CLIN. IMMUNOL. JUNE 1979
injection
48
72
blood
WBC/mm:’ Eos/mm’l
6,888* + 2,508 181 t 65 39 k 7
Baso/mtij Sedimented
6,163 -+ 2,296 160 ? 90 36 t 4
6,538 2 2,596 6,125 k 1,615 5,350 t 1,075 6,000 r 1,289 178 k 81 156 2 97 151 t 63 168 r 60 36 k 3 37 i 3 36 + 6 43 ? I
blood
Eos/mm” Baso/mm’j
Leukocyte histamine (rig/ml)
386 k 145 70 i 14 53 ?I 3.5
330 2 142 78 k 8 52 k 13
345 5 110 72 + 10 60 2 8
336 k 119 74 k 5 51 k4
363 k 124 78 k 6 63 L 11
272 2 66 74 + I 60 ? 14
*Mean * SD. TABLE IV. Mean values Time (hr) Whole
0
for a 72-hr period following 4
8
intravenous
steroid
injection
24
48
72
9,750 k 3,291 55 k 43 21 k 16
5,963 k 2,750 124 2 42 37 k 4
5,900 k 1,627 165 k 92 37 L 10
79 * 68 39 +- 25 24 k 9
221 ‘-’ 61 79 2 7 46 k 12
300 k 56 69 t 11 565 11
blood
WBC/mm:$ Eos/mm”
5,400* + 1,618 188 + 59 41 k 10
Base/mm:’ Sedimented
7,725 k 1,689 7,181 k 2,026 14 k 18 5k4 17 + 9 10 * 9
blood
EOS /mm”
Base/mm” Histamine (rip/l
of 4 subjects
320 + 121 72 5 25 45 k 13
12.5 2 II 33 k 15 21 k 15
8.5 k 8 16 2 19 16 -+ 12
X 10’
sedimented leukocytes) *Mean 2 SD 1,500 rpm for 10 min. The resulting supematantfluid was frozen and stored at -20” C for subsequent histamine analysis. RESULTS Effects within
4 hr
The sequential hourly changes in blood eosinophil and basophil levels during the first 4 hr after injection of placebo and steroid are shown in Tables I and II. Placebo injections led to no significant changes in mean levels of either eosinophils or basophils (Table I). However, steroid administration was followed by a progressive decrease in the mean level of both cell types (Table II). Eosinophil levels decreased only modestly (- 16%) by one hour, but more prominent decreases were seen by the second (-59%) and third (-85%) hour. And, finally, only a few eosinophils were observed at the fourth hour (-95%). The decreases from the second through the fourth hour, considered in individual subjects, were statistically significant (p = < 0.01). Although basophil levels were reduced to a similar modest degree (- 15%) by 1 hr following steroid
treatment, the subsequent decreases were less prominent and occurred at a somewhat slower rate than those seen with eosinophils. Basophils decreased by -32% at the second hour, -49% at the third hour, and -72% at the fourth hour. In contrast, the total circulating leukocyte levels increased by 49% by the fourth hour after the steroid injection (Table II). This increase reflects a predominant increase in circulating neutrophil levels. There were no significant changes following placebo injection in the histamine contents of 1 X 10’ leukocytes sedimented from peripheral blood (Table I). By the fourth hour after steroid administration, there was a significant (p < 0.001) decrease in the histamine present in 1 x 10’ aliquots of these cells (Table II). Sequential
studies
after 4 hr
Longer term effects of a single intravenous injection were assessed in 4 subjects over a 72-hr period. Again, no significant change in the levels of eosinophils, basophils, or histamine content of sedimented leukocytes were found after placebo injection (Table
VOLUME NUMBER
TABLE
--
Effects of corticosteroids
63 6
V. Ratio
of basophils
levels to cellular
histamine
content
in sedimented
R. Y.
R. z. F. C.
M. M.
R. M. N. S.
l-list (ng120 ~1)
Ratio
84 55 86 78
1.70 0.90 1.15 1.40
0.020 0.017
55
1.10
74
1.00
Baso/mm3
Basolmm3
Hist (ng120 ~1)
Ratio
0.013
0.018 0.020 0.014
41 Y2 YI
1.20 1.00
0.015 0.024
0.95 1.45
0.010
80 78
0.8 I.0
Hist (ng120 ,wl)
Ratio
Basolmm3
Hist (ng/20 ~1)
Ratio
0.016 0.023 0.013 0.019
B. Y.
89
1.35 I .20
0.015 0.021
I9
0.30
58
I.3
0.30
100 120 Il.1
I .25 1.80 I.3
0.013 0.015 0.012
0.55 0.7
0.7
0.010
43 36 59 36
N. S.
70
Mean ratio + SD, 0.014 2 0.004
III). In contrast, the steroid injection was followed by a prominent decrease in levels of both eosinophils and basophils within 4 hr, reaching a nadir at 8 hr (-97% and -78%. respectively). Levels of these cellular elements were still significantly decreased by 24 hr in all subjects: in some subjects suppression was still evident at 48 hr (Table IV). The histamine content of 1 X lo7 sedimented leukocytes obtained prior to treatment ranged between 45 and 63 ng. No specific differences were seen in the histamine content of equal number of cells obtained before and after placebo (Table III). In contrast, a prominent reduction in histamine content was seen in cells obtained (mean - 54%, p = < 0.001) 4 hr after steroids (Table IV). The decrease was maximal (mean - 6!I%) by the eighth hour. Leukocyte histamine cornent was back to pretreatment levels by 72 hr. The ratio of the histamine content to the basophil count in sedimented leukocyte populations was calculated (Table V). The mean ratios were very similar for such populations obtained before and 4 hr after either placebo or steroids. In addition, there was a close correlation between the temporal patterns of changes in basophil levels and histamine content of sedimented 1eu:rocytes obtained at intervals up to 72 hr after intravenous steroids (Fig. 1). Leukocyte sensitivity, as judged by the amount of antigen E needed to induce a release of 50% of the total cellular histamine content, was not significantly
0.010 0.013
4 hr post-steroid
R. z.
F. C. M. M. R. M.
0.016
Mean ratio 2 SD, 0.015 t 0.005
Pre-steroid Basolmma
leukocytes
81
Mean ratio t SD, 0.017 ? 0.003
Subject
429
4 hr post-placebo
Pre-placebo Subject
on histamine
0. I
0.017
0.2
0.006
Mean ratio 5 SD, 0.016 ? 0.006
altered by either steroid or placebo treatment of 5 subjects (Table VI). Steroid
effects on tissue
histamine
stores
Comparison of the total histamine content from skin biopsies before and after intravenous injections showed no significant effect of either the placebo or steroid administration (Fig. 2). Discussion The findings in the present study confirm those of earlier studies and add some new information of potential clinical significance. We observed a rise in total leukocyte level and decreases in the level of basophils and eosinophils within hours after a single intravenous injection of corticosteroid, similar to those reported previously.3* ‘I, i6* l7 Following intravenous steroid administration, a period of 1 to 2 hr passed prior to observing significant changes in the numbers of circulating cellular elements. This interval may represent the time needed for eosinophils and basophils to either marginate locally or to travel to a distant site of sequestration. However, we found that decreases in both eosinophil and basophil levels persisted longer than commonly supposed, with significantly depressed levels of both cell types at 24 hr. Indeed, significant decreases persisted for 72 hr in some subjects. These findings are in concordance with previous observations by our own group that significant eosinopenia was present at 24 hr after sin-
430
Dunsky et al.
J. ALLERGY
MEAN
RESULTS
IN 4 STEROID
FIG. 1. Mean levels of eosinophils, basophils, following intravenous steroids (4 subjects).
SKIN
0
Pre
q
Post
-
TREATED
and histamine
TISSUE
CLIN.
IMMUNOL. JUNE 1979
SUBJECTS
in leukocytes
over a 72-hr period
HISTAMINE
Placebo Placebo
14hd
1
-
FIG. 2. Effect of placebo
or steroid
gle doses of oral steroid given on alternate days.24 Several explanations for this decrease in eosinophils have been postulated on the basis of experimental animal studies.ls There is no direct evidence in humans to support the concept of steroid-induced eosinophil destruction. Destruction of these cells would seem improbable since a return to baseline levels of eosinophils was achieved in a relatively short period of time following the onset of eosinopenia. Rather, our findings suggest a transient redistribution of eosinophils as well as basophils following steroid injection. Recent studies by Dale, Hubert, and Faucilg in hypereosinophilic patients suggest that there may be movement of eosinophils between circulating and marginal blood compartments. Also, animal studies have indicated that eosinophils may migrate to the
on skin histamine
content
(6 subjects).
bone marrow or to various other tissue sites in response to steroid treatment ,25 Another new finding of our study is that a prominent decrease in the histamine content of circulating leukocytes was seen after steroid injection, paralleling the steroid-induced basopenia. Indeed, the leukocyte histamine content correlated closely with the basophil levels in all the leukocyte populations studied, confirming previous observations2’ that much of the circulating leukocyte histamine is present in the basophils. The amount of histamine per basophil was not significantly changed by the steroid administration. The histamine content of the skin from these subjects was unaltered by steroids. This suggests that this group of agents transiently alters circulating levels of basophils but not the tissue mast cells. The histamine
VOLUME NUMBER
Effects of corticosteroids
63 6
TABLE VI. ‘Effect of placebo Subiect M. R. w. N. D.
M!. L. s. S. H.
or steroid
administration
Preolacebo I I I I I
x x x x x
IO-“* 10-a lo-:’ IO-’ IO-’
*Minimum concentration of ragweed antigen E (pg/ml) sensitive donors
on in vitro leukocyte
Post-placebo I I I I I
x x x x x
IO-” lo-:3 lo-:% IO-’ I()~:~
sensitivity
Pre-steroid I I I 1 I
x x x x x
IO-’ 10-a 10-a IO--L IO-’
on histamine
431
to antigen Poet-steroid I I I I I
x x x x x
IO -I IO ~:I IO a IO ’ I()~~
required to induce release of ~50% histamine release from leukocytes of ragweed-
content of antigen-induced immediate hypersensitivity reactions in the skin was not studied in these subjects. It is conceivable that in vivo corticosteroids could affect this response. Others have reported that in vitro steroids inhibited histamine release from sensitized skin slices challenged with antigen. However, we had previously observed22no effect of one week of oral steroid therapy on the wheal-and-flare response to serial dilutnons of antigen injected intradermally in sensitive subjects. In previous studies we have noted that a single intravenous dose of corticosteroids similar to that employed in the present investigation was followed by a rapid inhibition of the cellular inflammatory response in tissue-allergic reaction sites.’ Such findings suggest that an important steroid action would be to diminish the entry of the responding inflammatory cells into the site of mediator release in the allergic reaction, although there is no evidence whether alterations in circulating levels of mediator-rich cells have any influence on allergic reactions in skin sites. These different effects of steroids on mediator-rich cells in the circulating leukocyte and skin should be kept in mind; studies of drug effects in allergic reactivity frequently use the circulating leukocyte for study rather than tissue of the “shock organ” involved because of its ready availability. However, it is possible that steroid effects on circulating basophils may be of therapeutic importance in cases in which allergic reactions occur within the vascular compartment itself. REFERENCES 1. Dunsky E3, Atkins PC, Zweiman B: Histologic responses in human skin tests reactions to ragweed. IV. Effects of a single intravenous injection of steroids, J ALLERGY CLIN IMMLJNOL 5% 142, 1917. 2. Slott RI, Zweiman B: Histologic studies of human skin tests responses to ragweed and compound 48/80. Il. Effects of corticosteroid therapy, J ALLERGY CLIN IMMUNOL 55:232, 1975. 3. Esellier AF, Jeaneret RI,, Marancii L: The mechanism of glucocorticoid eosinopenia, Blood 9:531, 1954. 4. Anderson V, Bro-Rasmussen F, Hourgaard I: Auto-radioraphic studies of eosinophil kinetics: Effects of cortisol, Cell Tissue Kinet 2: 139, 1969.
5. Felarca A, Lowell F: Local effects of cortisol in the time course of eosinophilotaxis with the use of an improved technique, J ALLERGY CLIN IMMUNOL 43: 114, 1969. 6. Weiner SL, Weiner R, Urivetsky M, Shaffer S, Isenberg HD, Janov C, MeiIman E: The mechanism of action of a single dose of methylprednisolone on acute inflammation in vivo, J Clin Invest 56~679, 1975. 7. Borselia AWA: Hormonal influence in blood and tissue basophilia granulocytes, Ann NY Acad Sci 103~394, 1963. 8. Code CF, Mitchell RG, Kennedy JC: The effects of cortisone on the number of circulating basophils and eosinophils, Proc Staff Meet Mayo Clin 29:200, 1954. 9. Greaves MW, Plummer LM: Glucocorticoid inhibition of antigen evoked histamine release from human skin, Immunology 27~359, 1974. 10. Halpern BW, Benaceraf B, Briot M: The role of cortisone, desoxycorticosterone and adrenaline in protecting adrenolectomized animals against hemorrhagic traumatic and histamine shock, Br J Pharmacol 7:287, 1952. 11. Schaver RW, Smiley RL, Davis KJ: Inhibition of cortisone of the binding of new histamine in rat tissues, Proc Sot Exp Biol Med 87:590, 1954. 12. Dunsky E, Atkins P, Zweiman B, Levy D: Early effects of corticosteroids on allergic cellular response, J ALLERGY CLIN IMMUNOL 57: 189, 1976. 13. Levy DA, Widra M: A micro-assay for studying allergic histamine release from human leukocytes using an enzymatic isotopic assay for histamine, J Lab Clin Med 81:29l, 1973. 14. Lichtenstein LM, Osler AG: Studies on the mechanisms of hypersensitivity phenomenom: IX. Histamine release from human leukocytes by ragweed pollen antigen, J Exp Med lXk507, 1964. 15. Moore JE, III, James AW III: Simple direct method for absolute basophil leukocyte count, Proc Sot Exp Biol Med 82~601, 1953. 16. Bishop CR, Athens JW, Boggs DR, Warner HR, Cartwright GE, Wintrobe M: Leukokinetic studies. XIII: A non-steady state kinetic evaluation of the mechanism of cortisone induced granulocytosis, J Clin Invest 47~249, 1968. 17. Chai H, Gilbert A: The effect of alternate day prednisone on the white blood count in children with chronic asthma, J ALLERGYCLIN IMMUNOL 51:65, 1973. 18. Bro-Rasmussen F: Eosinophils in the bone marrow of normal and cortisol-treated rats. Quantitative and autoradiographic studies. Acta Pathol Microbial Stand (A) 81:593. 1973. 19. Dale DC, Hubert RI, Fauci A: Eosinophil kinetics in the hypereosinophilic syndrome, J Lab Clin Med 87:487, 1976. 20. Goetzl EJ, Wasserman SI, Austen F: Eosinophil polymorphonuclear leukocyte function in immediate hypersensitivity, Arch Pathol Lab Med 99: I. 1975.
432
J. ALLERGY
Dunsky et al.
21. Aviado DM, Carillo LR: Anti-asthmatic action of steroids. A review of the literature on their mechanism of action, J Clin Pharmacol lOr3, 1970. 22. Slott RI, Zweiman B: A controlled study of the effect of corticosteroids on immediate skin test reactivity, J ALLERGY CLIN IMMUNOL 54~229, 1974. 23. Buoij-Noord H, Orie NGH, De Vries K: Immediate and late bronchial obstructive reactions to inhalation of house dust and
protective effects of disodium cromglycate and prednisolone, J ALLERGY CLIN IMMUNOL 48:344, 1971. 24. Zweiman B, Scott RJ, Atkins PC: Histologic
study of human skin test response to ragweed and compound 48/80. III. Ef-
Information
CLIN. IMMUNOL. JUNE 1979
fects of alternate day steroid therapy, J ALLERGY CLIN IMMUNOL 58~657, 1976. 25. Ottolenghi A, Barnett
HD: The effect of drugs on the eosinophilic leukocyte population of rat tissue. 1. Dexanithasone, J Pharmacol Exp Ther 189:303, 1974. 26. Parker CW, Huber MG, Baumann ML: Alterations in cyclic AMP metabolism in human bronchial asthma, J Clin Invest 52: 1342, 1973. 27. Ishizaka T, DeBernardo R, Tomioka H, Lichtenstein LM, Ishizaka K: Identification of basophil granulocytes as the site of allergic histamine release, J Immunol IO& 1000, 1972.
for authors
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M.D., and
The comparative e#ect in II atopic subjects of a single intravenous injection of methylpredm’solone on sequential studies of blood eosinophils. basophils, leukocyte sensitivity to antigen for histamine release, leukocyte histamine content, and skin histamine was examined. No significant changes occurred in any parameter after placebo treatment. In contrast, 4 hr after intravenous treatment with steroid there were significant decreases in mean eosinophil counts (-95%), basophil counts ( -72%), and histamine content of I x 10’ leukocyte samples ( -62%). Temporal changes in the latter paralleled alterations in circulating basophil levels. No significant changes occurred in the antigen histamine release sensitivity, or the total skin histamine. Studies over a longer period after steroids in 4 subjects showed eosinophil and basophil levels at a nadir at 8 hr, remaining suppressed for 24 hr, and returned to pretreatment levels by 72 hr. Results suggest that corticosteroids induce a prominent decrease in leukocyte histamine due to a depletion of basophils without a decrease in histamine content per basophil, and that skin tissue histamine stores remain unchanged by such treatment.
Despite intensive study, the mechanisms underlying the remarkable efficacy of corticosteroid drugs in many allergic disorders are still not well defined. At least part of the therapeutic activity may be due to effects of steroids on cellular inflammatory responses at the site of immediate hypersensitivity reactions.’ Previous studies in this laboratory have shown that prior steroid administration to atopic humans results in a rapid suppression of the usual eosinophil accumulation in the skin tests site as a response to potent antigen injection. Based upon studies in other experimental models, it is conceivable that various steroid effects on circulating blood elements,2-4 emigration of cells from vessels to the inflammatory sites,5* 6
From the Veterans Administration Hospital and Allergy and Immunology Section, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, and the Department of Biochemistry, The School of Hygiene and Public Health, Johns Hopkins University, Baltimore. Supported in part by National Institutes of Health Grant No. R01 Al 14332 and Veterans Administration Grant MRIS 9967. Received for publication July 31, 1978. Accepted for publication Dec. 5, 1978. Reprint requests to: Eliot H. Dunsky, M.D., 5th Floor Johnson Pavilion, 36th and Hamilton Walk G2, University of Pennsylvania, Philadelphia, PA 19174. Vol. 63, No. 6, pp. 426-432
and mediator release7+’ could be playing a significant role at the tissue level. The present report describes studies of the early effects of intravenous steroids on circulating levels of eosinophils, basophils, and cellular histamine in comparison with tissue histamine content in an attempt to learn more about the antiallergic effects of these important therapeutic agents. MATERIALS Subjects
AND METHODS
Eleven atopic male volunteer subjects (weighing 70 to 90 kg) were studied after their informed consent was obtained. All individuals were clinically sensitive to ragweed and exhibited an immediate type wheal response of at least 10 mm in diameter (and at least 3 times the diameter of a reaction to a negative control injection) 20 min following the injection of 20 protein nitrogen units (PNU) of aqueous ragweed extract (Greer Labs., Lenoir, N. C.). None of thesesubjects was receiving immunotherapy, and all had discontinued medications 2 wk prior to this study. Treatment
program
Eleven subjects were studied on two occasions, at least two weeks apart. On one occasion (in a random order), the subject received an intravenous injection of methylprednisolone sodium succinate (provided as Solu-Medrol by Dr. Sam Stubbs, The Upjohn Co., Kalamazoo, Mich.), in a
VOLUME NUMBER
Effects of corticosteroids
63 6
TABLE I. Mean values
of 8 subjects
Time (hr) Who/r
over a 4-hr period following
0
intravenous
placebo
on histamine
427
injections
1
2
3
4
4,954 2 1,600 210 +- 89 36 -+ 6
7,123 2 2,140 208 + 87 36 + 6.5
6,369 t 2,417 206 + 99 41 +- 5.3
5,838 2 1,833 201 f 85 39 t 3.8
-
356 2 224 62 t 29 52 2 11
blood
WBC/mrti’ Eos/mn? Base/mm”
5,815* * 2,200 207 t 84 36 t 5.8
Sedirnented
l~lood
Eos/mm” Base/mm” Total cellular histamine (ng/tnl) -*Mean z SD. TABLE II. Mean values
320 r 176 64 t 21 61 ? 14
of 8 subjects
Times (hr) Whole
-
over a 4-hr period following 0
intravenous
steroid
injection
1
2
3
4
5,400 2 910 195 k 99 40 ir 9
6,088 k 2,207 95 ? 45 32 i 10
8,042 k 2,140 35 + 30 24 -t 7
9,212 k 2,449 12 k 14 I8 t 10
-
-
-
16 L 19 27 -+ 11 35 5 6
blood
6,260* k 950 232 k 105 47 +- 10
WBC/tnm” Eos/mtn” Baso/mti’ hiood Eos/mtn” Base/mm” Total cellular histamine (@ml) -*Mean + SD.
Sedimented
354 + 217 77 i 30 63 + 15
dose of 1 mg/kg. On the other occasion, an intravenous saline injection was given as a placebo.
Timing
of .studies
At 8:OO A.M. of the study day, immediately prior to the intravenous injections (pretreatment), baseline blood samples were obtained for determination of (1) total leukocyte count (ce11s/mm3); (2) total eosinophil concentration (cells/mm”); (3) total basophil concentration (cells/mm3); and (4) total histamine content in a leukocyte-rich cell suspension. In all 11 subjects, these assays were repeated 4 hr after the intravenous injection of steroids and placebo. In 8 of 11 subjects, similar studies also were carried out hourly for the first 4 hr. In 4 of these 8 subjects, sequential studies were carried out at 4, 8, 24, 48, and 72 hr after the intravenous injection.
Total eosinophil
and basophil
counts
Total eosic,ophil counts were determined in coded specimens using the Unopipette system (Becton, Dickinson & Co., East Rutherford, N. J.). Total basophil counts were determined in coded specimens using the Moore and James technique I5
Determinaltion of leukocyte histamine content A leukocyte-rich supematant suspension was obtained by dextran sedimentations of heparinized venous blood, using
-
a previously described method.“’ The leukocyte-rich suspension was centrifuged, and the cell button washed and resuspended in TRIS-ACM to a final concentration of 1 x 10’ cells/ml. One-milliliter aliquots were boiled for 10 min, centrifuged, and the supematant fluid analyzed for histamine content using the radioenzymatic assay of Levy and Widra. I3 Antigen-induced histamine release was carried out with a leukocyte-rich suspension, as prepared above, concentrated to 1 x 10’ cells/ml. Aliquots of cell suspension were incubated with varying concentrations of ragweed antigen E (provided by The National Institute of Allergy and Infectious Diseases, Catalogue No. A601-901-185). The final concentration of antigen E in the dose response challenge increased progressively from 1 x lo-” pg/ml to 1 x 10-l pg/ml. Incubation was carried out at 37” C for 45 min in TRIS-ACM buffer. The cell suspension was then centrifuged at 1,500 rpm for 10 min and the supematant analyzed for histamine content.
Tissue histamine Three-millimeter dermal punch biopsies were taken from normal-appearing skin sites on the posterolateral aspect of the upper arm in 6 subjects immediately prior to and 4 hr following treatment with either placebo on one occasion or steroid on another occasion. Each biopsy specimen was immediately weighed, minced, and boiled in 1 ml of TRIS-ACM for 10 min. Samples were then centrifuged at
428 Dunsky et al.
J. ALLERGY
TABLE III. Mean levels in 4 subjects Time (hr) Whole
for a 72-hr period following 4
0
intravenous
8
24
placebo
CLIN. IMMUNOL. JUNE 1979
injection
48
72
blood
WBC/mm:’ Eos/mm’l
6,888* + 2,508 181 t 65 39 k 7
Baso/mtij Sedimented
6,163 -+ 2,296 160 ? 90 36 t 4
6,538 2 2,596 6,125 k 1,615 5,350 t 1,075 6,000 r 1,289 178 k 81 156 2 97 151 t 63 168 r 60 36 k 3 37 i 3 36 + 6 43 ? I
blood
Eos/mm” Baso/mm’j
Leukocyte histamine (rig/ml)
386 k 145 70 i 14 53 ?I 3.5
330 2 142 78 k 8 52 k 13
345 5 110 72 + 10 60 2 8
336 k 119 74 k 5 51 k4
363 k 124 78 k 6 63 L 11
272 2 66 74 + I 60 ? 14
*Mean * SD. TABLE IV. Mean values Time (hr) Whole
0
for a 72-hr period following 4
8
intravenous
steroid
injection
24
48
72
9,750 k 3,291 55 k 43 21 k 16
5,963 k 2,750 124 2 42 37 k 4
5,900 k 1,627 165 k 92 37 L 10
79 * 68 39 +- 25 24 k 9
221 ‘-’ 61 79 2 7 46 k 12
300 k 56 69 t 11 565 11
blood
WBC/mm:$ Eos/mm”
5,400* + 1,618 188 + 59 41 k 10
Base/mm:’ Sedimented
7,725 k 1,689 7,181 k 2,026 14 k 18 5k4 17 + 9 10 * 9
blood
EOS /mm”
Base/mm” Histamine (rip/l
of 4 subjects
320 + 121 72 5 25 45 k 13
12.5 2 II 33 k 15 21 k 15
8.5 k 8 16 2 19 16 -+ 12
X 10’
sedimented leukocytes) *Mean 2 SD 1,500 rpm for 10 min. The resulting supematantfluid was frozen and stored at -20” C for subsequent histamine analysis. RESULTS Effects within
4 hr
The sequential hourly changes in blood eosinophil and basophil levels during the first 4 hr after injection of placebo and steroid are shown in Tables I and II. Placebo injections led to no significant changes in mean levels of either eosinophils or basophils (Table I). However, steroid administration was followed by a progressive decrease in the mean level of both cell types (Table II). Eosinophil levels decreased only modestly (- 16%) by one hour, but more prominent decreases were seen by the second (-59%) and third (-85%) hour. And, finally, only a few eosinophils were observed at the fourth hour (-95%). The decreases from the second through the fourth hour, considered in individual subjects, were statistically significant (p = < 0.01). Although basophil levels were reduced to a similar modest degree (- 15%) by 1 hr following steroid
treatment, the subsequent decreases were less prominent and occurred at a somewhat slower rate than those seen with eosinophils. Basophils decreased by -32% at the second hour, -49% at the third hour, and -72% at the fourth hour. In contrast, the total circulating leukocyte levels increased by 49% by the fourth hour after the steroid injection (Table II). This increase reflects a predominant increase in circulating neutrophil levels. There were no significant changes following placebo injection in the histamine contents of 1 X 10’ leukocytes sedimented from peripheral blood (Table I). By the fourth hour after steroid administration, there was a significant (p < 0.001) decrease in the histamine present in 1 x 10’ aliquots of these cells (Table II). Sequential
studies
after 4 hr
Longer term effects of a single intravenous injection were assessed in 4 subjects over a 72-hr period. Again, no significant change in the levels of eosinophils, basophils, or histamine content of sedimented leukocytes were found after placebo injection (Table
VOLUME NUMBER
TABLE
--
Effects of corticosteroids
63 6
V. Ratio
of basophils
levels to cellular
histamine
content
in sedimented
R. Y.
R. z. F. C.
M. M.
R. M. N. S.
l-list (ng120 ~1)
Ratio
84 55 86 78
1.70 0.90 1.15 1.40
0.020 0.017
55
1.10
74
1.00
Baso/mm3
Basolmm3
Hist (ng120 ~1)
Ratio
0.013
0.018 0.020 0.014
41 Y2 YI
1.20 1.00
0.015 0.024
0.95 1.45
0.010
80 78
0.8 I.0
Hist (ng120 ,wl)
Ratio
Basolmm3
Hist (ng/20 ~1)
Ratio
0.016 0.023 0.013 0.019
B. Y.
89
1.35 I .20
0.015 0.021
I9
0.30
58
I.3
0.30
100 120 Il.1
I .25 1.80 I.3
0.013 0.015 0.012
0.55 0.7
0.7
0.010
43 36 59 36
N. S.
70
Mean ratio + SD, 0.014 2 0.004
III). In contrast, the steroid injection was followed by a prominent decrease in levels of both eosinophils and basophils within 4 hr, reaching a nadir at 8 hr (-97% and -78%. respectively). Levels of these cellular elements were still significantly decreased by 24 hr in all subjects: in some subjects suppression was still evident at 48 hr (Table IV). The histamine content of 1 X lo7 sedimented leukocytes obtained prior to treatment ranged between 45 and 63 ng. No specific differences were seen in the histamine content of equal number of cells obtained before and after placebo (Table III). In contrast, a prominent reduction in histamine content was seen in cells obtained (mean - 54%, p = < 0.001) 4 hr after steroids (Table IV). The decrease was maximal (mean - 6!I%) by the eighth hour. Leukocyte histamine cornent was back to pretreatment levels by 72 hr. The ratio of the histamine content to the basophil count in sedimented leukocyte populations was calculated (Table V). The mean ratios were very similar for such populations obtained before and 4 hr after either placebo or steroids. In addition, there was a close correlation between the temporal patterns of changes in basophil levels and histamine content of sedimented 1eu:rocytes obtained at intervals up to 72 hr after intravenous steroids (Fig. 1). Leukocyte sensitivity, as judged by the amount of antigen E needed to induce a release of 50% of the total cellular histamine content, was not significantly
0.010 0.013
4 hr post-steroid
R. z.
F. C. M. M. R. M.
0.016
Mean ratio 2 SD, 0.015 t 0.005
Pre-steroid Basolmma
leukocytes
81
Mean ratio t SD, 0.017 ? 0.003
Subject
429
4 hr post-placebo
Pre-placebo Subject
on histamine
0. I
0.017
0.2
0.006
Mean ratio 5 SD, 0.016 ? 0.006
altered by either steroid or placebo treatment of 5 subjects (Table VI). Steroid
effects on tissue
histamine
stores
Comparison of the total histamine content from skin biopsies before and after intravenous injections showed no significant effect of either the placebo or steroid administration (Fig. 2). Discussion The findings in the present study confirm those of earlier studies and add some new information of potential clinical significance. We observed a rise in total leukocyte level and decreases in the level of basophils and eosinophils within hours after a single intravenous injection of corticosteroid, similar to those reported previously.3* ‘I, i6* l7 Following intravenous steroid administration, a period of 1 to 2 hr passed prior to observing significant changes in the numbers of circulating cellular elements. This interval may represent the time needed for eosinophils and basophils to either marginate locally or to travel to a distant site of sequestration. However, we found that decreases in both eosinophil and basophil levels persisted longer than commonly supposed, with significantly depressed levels of both cell types at 24 hr. Indeed, significant decreases persisted for 72 hr in some subjects. These findings are in concordance with previous observations by our own group that significant eosinopenia was present at 24 hr after sin-
430
Dunsky et al.
J. ALLERGY
MEAN
RESULTS
IN 4 STEROID
FIG. 1. Mean levels of eosinophils, basophils, following intravenous steroids (4 subjects).
SKIN
0
Pre
q
Post
-
TREATED
and histamine
TISSUE
CLIN.
IMMUNOL. JUNE 1979
SUBJECTS
in leukocytes
over a 72-hr period
HISTAMINE
Placebo Placebo
14hd
1
-
FIG. 2. Effect of placebo
or steroid
gle doses of oral steroid given on alternate days.24 Several explanations for this decrease in eosinophils have been postulated on the basis of experimental animal studies.ls There is no direct evidence in humans to support the concept of steroid-induced eosinophil destruction. Destruction of these cells would seem improbable since a return to baseline levels of eosinophils was achieved in a relatively short period of time following the onset of eosinopenia. Rather, our findings suggest a transient redistribution of eosinophils as well as basophils following steroid injection. Recent studies by Dale, Hubert, and Faucilg in hypereosinophilic patients suggest that there may be movement of eosinophils between circulating and marginal blood compartments. Also, animal studies have indicated that eosinophils may migrate to the
on skin histamine
content
(6 subjects).
bone marrow or to various other tissue sites in response to steroid treatment ,25 Another new finding of our study is that a prominent decrease in the histamine content of circulating leukocytes was seen after steroid injection, paralleling the steroid-induced basopenia. Indeed, the leukocyte histamine content correlated closely with the basophil levels in all the leukocyte populations studied, confirming previous observations2’ that much of the circulating leukocyte histamine is present in the basophils. The amount of histamine per basophil was not significantly changed by the steroid administration. The histamine content of the skin from these subjects was unaltered by steroids. This suggests that this group of agents transiently alters circulating levels of basophils but not the tissue mast cells. The histamine
VOLUME NUMBER
Effects of corticosteroids
63 6
TABLE VI. ‘Effect of placebo Subiect M. R. w. N. D.
M!. L. s. S. H.
or steroid
administration
Preolacebo I I I I I
x x x x x
IO-“* 10-a lo-:’ IO-’ IO-’
*Minimum concentration of ragweed antigen E (pg/ml) sensitive donors
on in vitro leukocyte
Post-placebo I I I I I
x x x x x
IO-” lo-:3 lo-:% IO-’ I()~:~
sensitivity
Pre-steroid I I I 1 I
x x x x x
IO-’ 10-a 10-a IO--L IO-’
on histamine
431
to antigen Poet-steroid I I I I I
x x x x x
IO -I IO ~:I IO a IO ’ I()~~
required to induce release of ~50% histamine release from leukocytes of ragweed-
content of antigen-induced immediate hypersensitivity reactions in the skin was not studied in these subjects. It is conceivable that in vivo corticosteroids could affect this response. Others have reported that in vitro steroids inhibited histamine release from sensitized skin slices challenged with antigen. However, we had previously observed22no effect of one week of oral steroid therapy on the wheal-and-flare response to serial dilutnons of antigen injected intradermally in sensitive subjects. In previous studies we have noted that a single intravenous dose of corticosteroids similar to that employed in the present investigation was followed by a rapid inhibition of the cellular inflammatory response in tissue-allergic reaction sites.’ Such findings suggest that an important steroid action would be to diminish the entry of the responding inflammatory cells into the site of mediator release in the allergic reaction, although there is no evidence whether alterations in circulating levels of mediator-rich cells have any influence on allergic reactions in skin sites. These different effects of steroids on mediator-rich cells in the circulating leukocyte and skin should be kept in mind; studies of drug effects in allergic reactivity frequently use the circulating leukocyte for study rather than tissue of the “shock organ” involved because of its ready availability. However, it is possible that steroid effects on circulating basophils may be of therapeutic importance in cases in which allergic reactions occur within the vascular compartment itself. REFERENCES 1. Dunsky E3, Atkins PC, Zweiman B: Histologic responses in human skin tests reactions to ragweed. IV. Effects of a single intravenous injection of steroids, J ALLERGY CLIN IMMLJNOL 5% 142, 1917. 2. Slott RI, Zweiman B: Histologic studies of human skin tests responses to ragweed and compound 48/80. Il. Effects of corticosteroid therapy, J ALLERGY CLIN IMMUNOL 55:232, 1975. 3. Esellier AF, Jeaneret RI,, Marancii L: The mechanism of glucocorticoid eosinopenia, Blood 9:531, 1954. 4. Anderson V, Bro-Rasmussen F, Hourgaard I: Auto-radioraphic studies of eosinophil kinetics: Effects of cortisol, Cell Tissue Kinet 2: 139, 1969.
5. Felarca A, Lowell F: Local effects of cortisol in the time course of eosinophilotaxis with the use of an improved technique, J ALLERGY CLIN IMMUNOL 43: 114, 1969. 6. Weiner SL, Weiner R, Urivetsky M, Shaffer S, Isenberg HD, Janov C, MeiIman E: The mechanism of action of a single dose of methylprednisolone on acute inflammation in vivo, J Clin Invest 56~679, 1975. 7. Borselia AWA: Hormonal influence in blood and tissue basophilia granulocytes, Ann NY Acad Sci 103~394, 1963. 8. Code CF, Mitchell RG, Kennedy JC: The effects of cortisone on the number of circulating basophils and eosinophils, Proc Staff Meet Mayo Clin 29:200, 1954. 9. Greaves MW, Plummer LM: Glucocorticoid inhibition of antigen evoked histamine release from human skin, Immunology 27~359, 1974. 10. Halpern BW, Benaceraf B, Briot M: The role of cortisone, desoxycorticosterone and adrenaline in protecting adrenolectomized animals against hemorrhagic traumatic and histamine shock, Br J Pharmacol 7:287, 1952. 11. Schaver RW, Smiley RL, Davis KJ: Inhibition of cortisone of the binding of new histamine in rat tissues, Proc Sot Exp Biol Med 87:590, 1954. 12. Dunsky E, Atkins P, Zweiman B, Levy D: Early effects of corticosteroids on allergic cellular response, J ALLERGY CLIN IMMUNOL 57: 189, 1976. 13. Levy DA, Widra M: A micro-assay for studying allergic histamine release from human leukocytes using an enzymatic isotopic assay for histamine, J Lab Clin Med 81:29l, 1973. 14. Lichtenstein LM, Osler AG: Studies on the mechanisms of hypersensitivity phenomenom: IX. Histamine release from human leukocytes by ragweed pollen antigen, J Exp Med lXk507, 1964. 15. Moore JE, III, James AW III: Simple direct method for absolute basophil leukocyte count, Proc Sot Exp Biol Med 82~601, 1953. 16. Bishop CR, Athens JW, Boggs DR, Warner HR, Cartwright GE, Wintrobe M: Leukokinetic studies. XIII: A non-steady state kinetic evaluation of the mechanism of cortisone induced granulocytosis, J Clin Invest 47~249, 1968. 17. Chai H, Gilbert A: The effect of alternate day prednisone on the white blood count in children with chronic asthma, J ALLERGYCLIN IMMUNOL 51:65, 1973. 18. Bro-Rasmussen F: Eosinophils in the bone marrow of normal and cortisol-treated rats. Quantitative and autoradiographic studies. Acta Pathol Microbial Stand (A) 81:593. 1973. 19. Dale DC, Hubert RI, Fauci A: Eosinophil kinetics in the hypereosinophilic syndrome, J Lab Clin Med 87:487, 1976. 20. Goetzl EJ, Wasserman SI, Austen F: Eosinophil polymorphonuclear leukocyte function in immediate hypersensitivity, Arch Pathol Lab Med 99: I. 1975.
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21. Aviado DM, Carillo LR: Anti-asthmatic action of steroids. A review of the literature on their mechanism of action, J Clin Pharmacol lOr3, 1970. 22. Slott RI, Zweiman B: A controlled study of the effect of corticosteroids on immediate skin test reactivity, J ALLERGY CLIN IMMUNOL 54~229, 1974. 23. Buoij-Noord H, Orie NGH, De Vries K: Immediate and late bronchial obstructive reactions to inhalation of house dust and
protective effects of disodium cromglycate and prednisolone, J ALLERGY CLIN IMMUNOL 48:344, 1971. 24. Zweiman B, Scott RJ, Atkins PC: Histologic
study of human skin test response to ragweed and compound 48/80. III. Ef-
Information
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fects of alternate day steroid therapy, J ALLERGY CLIN IMMUNOL 58~657, 1976. 25. Ottolenghi A, Barnett
HD: The effect of drugs on the eosinophilic leukocyte population of rat tissue. 1. Dexanithasone, J Pharmacol Exp Ther 189:303, 1974. 26. Parker CW, Huber MG, Baumann ML: Alterations in cyclic AMP metabolism in human bronchial asthma, J Clin Invest 52: 1342, 1973. 27. Ishizaka T, DeBernardo R, Tomioka H, Lichtenstein LM, Ishizaka K: Identification of basophil granulocytes as the site of allergic histamine release, J Immunol IO& 1000, 1972.
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