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Reversal of histamine-mediated immunosuppression by structurally diverse histamine type II (H2) receptor antagonists
lnt. J. lmmunopharmac., Vol. 6, No. 5, pp. 467-473, 1984. Printed in Great Britain.
0192-0561/84 $3.00+ .00 International Society for lmmunopharmacology.
REVERSAL OF HISTAMINE-MEDIATED IMMUNOSUPPRESSION BY STRUCTURALLY DIVERSE HISTAMINE TYPE II (H2) RECEPTOR ANTAGONISTS ALISON M. BADGER, JEAN YOUNG and GEORGE POSTE Smith Kline & French Laboratories, Philadelphia, PA 19101, U.S.A. (Received 23 September 1983 and in revised form 14 March 1984)
-The effect of a series of structurally-diverse histamine type 1I (H2)-receptor antagonists on histamine-mediated immunosuppression of human peripheral blood lymphocytes (HPBL) has been examined. This analysis of structure- activity relationships was undertaken to examine the validity of the recent proposal arising from clinical studies that H2-receptor antagonists containing a furan ring were devoid of the effects on lymphocyte function reported previously in studies using cimetidine and other H2-receptor antagonists containing an imidazole nucleus. Cimetidine and two furan-containing antagonists, ranitidine and SKF 93479, were found to be devoid of any effect on PHA-induced proliferation of human peripheral blood lymphocytes over a wide concentration range (10 4 to 10-'°M). At high drug concentrations (10 3M) significant suppression of mitogen stimulation was observed but this was accompanied by significant cytotoxicity. All three antagonists were effective in reversing the suppression of PHA-stimulation of HPBL induced by exogenous histamine. Reversal of histamine-induced immunosuppression was obtained at drug concentrations (2.0 x 10-4 to 1.0 x 10-6M) which were non-toxic and did not affect PHA-induced proliferation in the absence of histamine. Ranitidine was the most potent antagonist in reversing histaminemediated immunosuppression. The ability of structurally-diverse H2-receptor antagonists to modify the action of histamine on lymphocyte function lends support to the view that histamine exerts its effects via classical H2-receptors. The ability of ranitidine to alter lymphocyte responsiveness in analogous fashion to cimetidine indicates that the possibility of alterations in immune function should be considered in clinical studies using ranitidine and other furan-containing H2-receptor antagonists. Abstract
Histamine exerts multiple effects on lymphocyte functions, including inhibition of responsiveness to mitogens, antibody formation and lymphocytotoxicity (reviews, Lewis, Nouri, Fan & Gordon, 1982; Plaut & Lichtenstein, 1982). These immunosuppressive effects are believed to be mediated via the interaction of histamine with histamine type II receptors (H2) rather than type I-histamine (H0 receptors (Wang & Zweiman, 1978; Ogden & Hill, 1980; Boot, Hudspith & Brostoff, 1980; Thomas, Huchet & Granjon, 1981; Badger, Young & Poste, 1983) although not all studies report this association (Gordon, Lewis & Nouri, 1981; Vickers, Milliner, Martin & Ganellin, 1982). The identity of the specific subset(s) of lymphocytes that interact with histamine has yet to be identified. H2-receptor antagonists have been reported to abrogate suppressor cell activity in vitro (Osband & McCaffrey, 1980; Palacios &
Alarcon-Segovia, 1981) and to augment a variety of lymphocyte-mediated immune functions in vitro (Gifford, Hatfield & Schmidtke, 1980; Ogden & Hill, 1980; Simon, Salberg & Crane, 1981; Peden, Robertson, Boyd, Brown, Gibbs, Ports, Wormsley & Beck, 1982). The experimental studies published to date on the action of H2-receptor antagonists in reversing histamine-mediated immunosuppression have all used either cimetidine, or its structurally related analogues, burimamide and metiamide. These molecules each contain an imidazole ring. In contrast, two recently described H2-receptor antagonists, ranitidine and SKF 93479, lack this moiety and instead contain a common furan ring structure. Recent reports indicate that, unlike cimetidine, one of these new antagonists, ranitidine, does not affect lymphocyte responsiveness to mitogens (Puppo, Corsini & Mangini, 1982; Peden et al., 1982). It was
Correspondence: Dr. Alison M. Badger, Smith Kline & French Laboratories, 1500 Spring Garden Street, Mail Code L-101, Philadelphia, PA 19101, U.S.A. 467
468
A. M. BADGER,J. YOUNGand G. POSTE
therefore considered of interest to investigate the activity of structurally diverse H2-receptor antagonists on histamine-mediated immunosuppression to determine whether previous reports of the immunological properties of cimetidine and related molecules might be caused by the imidazole ring structure rather than specific antagonism of classical H2-receptors. In this paper we report that H~-receptor antagonists containing furan or imidazole ring structures are both active in reversing histaminemediated immunosuppression in vitro. These data thus support the view that H~-receptors are present on lymphocytes. These findings also indicate that claims that newly developed H~-antagonists such as ranitidine will not affect immune function (Peden et al., 1982) should be treated with caution.
EXPERIMENTAL PROCEDURES
dissolved in RPMI-10% at 10 3M. Log dilutions of the compounds were prepared in the same medium and added at the initiation of the experiment. The final volume in the microtiter wells was adjusted to 200/al with RPMI-10%. Each experimental variable was established in quadruplicate. Cell cultures were incubated for 72 h at 37°C in a 5070 CO2 atmosphere and pulsed with 0.5 Ci 3H-thymidine (specific activity 1.9 taCi/mM; Schwarz/Mann, Orangeburg, NY, U.S.A.) for the last 16 h of culture. The cells were harvested on an automated multiple sample harvester and cellassociated radioactivity measured in a Beckman liquid scintillation counter. The results are expressed as the mean values derived from quadruplicate measurements. Cell viability was determined by measuring the percentage of cells excluding trypan blue after 72 h of incubation. This was carried out by adding 25 tal of trypan blue to the microtiter plate wells, resuspending the cells and counting those that excluded the dye.
Drugs Histamine dihydrochloride was obtained from Calbiochem-Behring, La Jolla, California, USA. Cimetidine, SKF 93479 and SKF 91581 were provided by Smith KIine & French Laboratories, Welwyn Garden City, U.K. and ranitidine (Glaxo, Greenford, U.K. Lot # 1HP001) was obtained from a commercial source. Mononuclear Cell Preparation Mononuclear cells were isolated from heparinized blood from normal healthy volunteers on FicollHypaque (Pharmacia Fine Chemicals, Inc., Piscataway, N J, U.S.A.) density gradients (Badger et al., 1983). The cells were washed three times in Hank's balanced salt solution (HBSS) and resuspended at the desired concentration in RPM! 1640 (Flow Laboratories, Rockville, Maryland, U.S.A.) containing 10°70 heat-inactivated (56°C, 30 rain) fetal calf serum (FCS) (Flow), 2mM glutamine, penicillin (100 units/ml) streptomycin (100 /ag/ml). This medium will be referred to as RPMI-10%. Cell Cultures Fifty microliter aliquots of the HPBL cell suspension (1 × 105 ceils) were placed in 96 well round bottomed microtiter plates. Phytohaemagglutinin M (Difco Laboratories, Detroit) was added to the cultures in RPMI-10% at dilutions ranging from 1:256 to 1:1,024. Histamine, SKF 91581 and the H2-receptor antagonists cimetidine, SKF 93479 and ranitidine were
RESULTS
The chemical structures of a series of H2-receptor antagonists containing either an imidazole ring (burimamide, metiamide, cimetidine) or a furan ring (ranitidine and SKF 93479) are shown in Table 1. SKF 91581 is structurally similar to cimetidine but has no activity at H~-receptors. Since several studies have shown that the imidazole-containing antagonists produce similar effects on a variety of immune functions in vitro and in vivo (Plaut & Lichtenstein, 1982) only cimetidine was used in the following comparison with the newer furan-containing antagonists. The effect of cimetidine, ranitidine and SKF 93479 on PHA-induced mitogenesis in HPBL is shown in Table 2. The results indicate that neither imidazole nor furan-containing antagonists have any effect over a wide concentration range (10 -4 and 10 ~°M). Limited but significant suppression was observed with ranitidine at 10 ~M but at this high concentration about 1 0 - 15°70 cytotoxicity was detected. SKF 93479 produced significant cytotoxicity at both 10-3M (50070 cytotoxicity) and 10 4M (25070 cytotoxicity) and this probably accounts for the immunosuppression obtained with this agent at these physiologically irrelevant concentrations. In contrast to their lack of effect on lymphocyte responsiveness to PHA, all three antagonists reversed the suppression of the PHA response induced by exogenous histamine (Table 3). The possibility that
469
Reversal of Histamine-Mediated I m m u n o s u p p r e s s i o n i
Table 1. Structure of H2-receptor antagonists and analogs
Compound
Blockade of Gastric Acid Secretion ID5o (#mol/kg)~11"
Structure
Ranitidine
@HNO2 CH2SCH2CH2NHCNHCH 3
(CH3)2NCH2
SK&F 93479
o
(CH~)~NC.~ Cimetidine
0.13
CH~SCH~CH ..~H N~cH~")-"
CH3
0.14
c.~
CH2SCH2CH~NHCNHCH 3
Metiamide CH3~ "
1.4
L,4c.
HN,~N
CH2SCH2CH2NH~NHCH 3 S
1.6
HN, ~ N
Burimamide
CH2CH2CH2CH2NHCNHCH3
6.1
HN,~N
SK&F91581
r~ o,~c,.c,.,,~,,o..
250
S
HN~N
the antagonists themselves might have a stimulatory effect on HPBL proliferation was excluded by p a r a l l e l c o n t r o l e x p e r i m e n t s c o n d u c t e d at t h e s a m e t i m e u s i n g r e p l i c a t e cell s a m p l e s ( T a b l e 4). S K F 91581, a c o m p o u n d w h i c h is s t r u c t u r a l l y s i m i l a r t o c i m e t i d i n e ( T a b l e 1), b u t w h i c h h a s n o a c t i v i t y at
H 2 - r e c e p t o r s w a s n o t a b l e to r e v e r s e h i s t a m i n e induced suppression (data not shown). T h e ability o f r a n i t i d i n e to r e v e r s e h i s t a m i n e m e d i a t e d s u p p r e s s i o n in H P B L is s h o w n in Fig. 1. D o s e r e l a t e d e f f e c t s w e r e o b t a i n e d w h e n 10-SM h i s t a m i n e (or h i g h e r ) , w h i c h gives 3 0 - 5 0 % sup-
Table 2. Effect of H2-antagonists on PHA-stimulated h u m a n peripheral blood lymphocytes
Concentration (M) 10 -3 10 -4 10 5 10 6 10 7 10 8 10 -9 10 ,o
Cimetidine c p m _+ SD ( × 10 3)
% change
Ranitidine c p m ± SD ( × 10 3)
45,297 ___2565 -6al" 43,610 47,038 _+ 2298 -3 47,820 51,873 _+ 4961 +7 48,032 45,461 +_ 9746 -6 49,832 52,873 _+ 3557 +9 49,046 47,661 _+ 1939 -2 48,312 47,925 _+ 3491 - 1 50,733 47,404 _+ 2073 -2 50,425 Unstimulated control 593 _+ 328 P H A stimulated control 48,423 -4- 2380
_+ 2001 _+ 1997 _+ 1384 + 2369 +__ 1268 + 3327 _+ 2614 _ 1216
% change + + + +
10" 1 1 3 1 1 5 4
SKF 93479 cpm +_ SD ( × 10 3) 383 22,495 45,385 49,082 48,107 47,880 55,245 49,585
_+ 206:1: _+ 1590~ _+ 3376 +_ 1707 _+ 6575 _+ 4260 _+ 6629 _+ 1860
*P < 0.05. ***P < 0.001. I-All values were compared to P H A control, n = 4 for all samples. ~ T h e percentage of trypan-blue stained cells with SKF 93479 at 10-~M was 50 and 25°70 at 10-4M.
% change -99*** -53*** - 6 + 1 + 1 - 1 + 14" + 3
470
A. M. BADGER, J. YOUNG and G. POSTE Table 3. Reversal of histamine suppression by the H2-antagonists cimetidine ranitidine and SKF 93479 3 HT-lncorporation into D N A (cpm ± SD x 10 3). 1-256 1-512 1 - 1024
P H A Dilution Control
75,774 ± 14,547
68,840 ± 13,762
44,678 ± 10,960
Histamine 2.5 × 10 5M
53,708 ± 12,984 -29%P = <0.001t
40,525 ± 12,932 -41°/0 P < 0.001
22,405 _+ 10,497 - 5 0 % P = < 0.001
Histamine 2.5 x 10-SM + Cimetidine* 2.5 × 10 4M
67,768 ± 13,566 +36%:[: P = < 0.01§
56,499 ± 16,132 + 44°70 P = < 0.01
34,490 ___ 12,816 + 46°7o P = < 0.01
Histamine 2.5 x 1 0 ~ M + Ranitidine 2.0 × lO-4M
68,724 ± 13,366 +32°7o P = <0.01
57,775 ± 13,497 +39°7o P = < 0.01
34,050 ± 10,697 +48°70 P = < 0.05
Histamine 2.5 x I0 ~ + SKF 93479 2.0 x 10 ~M
64,881 ± 13,530 +49% P = <0.05
56,550 ± 15,103 +43°70 P = < 0 . 0 1
33,521 ± 11,760 +50°7o P = < 0 . 0 5
*The results are presented as m e a n _+ SD of 16 values from 4 different experiments. -[-The histamine treated groups are compared to the control group by Student's t-test. t T h e 07o increase = 100 - [ - c p m (Histamine + a n t a g o n i s t ) - c p m (Histamine) "1 x 100.
l
cpm ( C o n t r o l ) - c p m (Histamine) §All values are compared to the histamine control by Student's t-test. pression of the PHA response, was used. However, at l o w e r h i s t a m i n e c o n c e n t r a t i o n s ( 1 0 - 6 M ) , w h i c h gives o n l y 2 0 - 2 5 % suppression, ranitidine consistently reversed the suppression though dose related e f f e c t s were m o r e d i f f i c u l t to d e m o n s t r a t e . E v a l u a t i o n o f t h e relative p o t e n c y o f c i m e t i d i n e a n d ranitidine by titrating different concentrations of t h e s e m o l e c u l e s a g a i n s t a single c o n c e n t r a t i o n o f h i s t a m i n e (10 5M) r e v e a l e d t h a t r a n i t i d i n e is t h e m o r e p o t e n t a n t a g o n i s t (Fig. 2). S t u d i e s o n t h e reversal of histamine suppression revealed that
]
r a n i t i d i n e w a s 100 f o l d m o r e p o t e n t t h a n c i m e t i d i n e . I n c o m p a r i s o n t h e I D 5 0 o f r a n i t i d i n e in i n h i b i t i n g g a s t r i c acid s e c r e t i o n w a s o n l y 10 t i m e s l o w e r t h a n t h a t o f c i m e t i d i n e ( T a b l e 1).
DISCUSSION The present study has examined the ability of s t r u c t u r a l l y d i v e r s e H 2 - r e c e p t o r a n t a g o n i s t s to reverse histamine-mediated immunosuppression.
Table 4. Effect of cimetidine, ranitidine and SKF 93479 on H P B L stimulated with 3 different doses of P H A
P H A Dilution
3 HT-lncorporation into D N A (cpm ± SD x 10 -3) 1 - 256 1 - 512 I - 1024
Control
75.774 ± 14,547"
68,840 _+ 13,762
44,678 ± 10,960
Cimetidine (2.5 x 10 4M)
80,214 ± 16,673 +6°/o N.S.'["
72,465 + 18,012 + 5°7o NS
43,989 _ 14,476 - 2% NS
Ranitidine (2.0 x 10-4M)
73,959 ± 17,988 +2o7o NS
65,563 ± 16,790 -5o70 NS
41,069 ± 9474 -80/0 NS
SKF 93479 (2.0 × 10-~M)
73,892 ± 13,091 + 2 % NS
66,629 ± 15,176 - 3 % NS
44,353 _+ 13,441 - 1 % NS
*The results are presented as mean _+ SD o f 16 values from 4 different experiments. ~All values are compared to the P H A control by Student's t-test. NS = Not significant.
Reversal of Histamine-Mediated Immunosuppression 40
response of HPBL to mitogens, bacterial antigens and all•antigens (Gifford et al., 1980; Ogden & Hill,
1980). Similarly, oral administration of cimetidine to patients for treatment of gastro-intestinal ulceration
30
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471
2O
[]
10-
10 6 J x
/
10" 5 Concentration (M)
-10
Fig. 1. Reversal of histamine-induced suppression by O, Histamine (10 6 and 10 ~M); ranitidine. • [] r-q, Histamine (10 6 and 10 ~M) + Ranitidine 10-6M; (3 - O, 10 5M; X X, 10 ~M. All ranitidine treated groups were significantly different from the histamine controls when compared by student's ttest (P = 0.01).
These experiments were prompted by two recent reports suggesting that a new series of H~-receptor
antagonists containing a furan ring structure might differ from antagonists containing an imidazole ring in their ability to affect lymphocyte function (Peden et al., 1982; Puppo et al., 1982).
Several studies have shown that the imidazolecontaining antagonist, cimetidine, and its congeners, metiamide and burimamide, can augment the in vitro
has been reported to augment the responsiveness of their lymphocytes to both mitogens and all•antigens (Gifford, Schmidtke & Ferguson, 1981; Simon et al., 1981; Peden et al., 1982). In contrast, Puppo et al. (1982) found that ranitidine did not alter the in vitro response of HPBL to mitogens, though cimetidine was not examined in this study. However, Peden et al. (1982) reported that lymphocytes obtained from patients treated with cimetidine showed enhanced responsiveness to P H A stimulation while patients in the same trial receiving ranitidine did not show augmented lymphocyte reactivity. They interpreted these data as evidence that HPBL lack classical H2-receptors and suggested that the previously reported effects of cimetidine and other H2-
antagonists containing an imidazole nucleus on immune function resulted from actions unrelated to antagonism of H~ receptors.
In these studies however, the H2 antagonists were not examined for their ability to antagonize histamine
mediated
suppression
of
mitogen
responsiveness. These experiments are, of course difficult to interpret if cimetidine alone enhances lymphocyte proliferation to mitogens. The present experiments and those of several others however (Brostoff, Pack & Lydyard, 1980; Brown, Badzer & Poste, 1982; Festen, DePauw, Smeulders & Wagener, 1981; Merety, Room & Maini, 1981) have failed to shown any stimulatory effect of cimetidine
on lymphocyte-proliferation and this has enabled us
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Fig. 2. Comparison of cimetidine and ranitidine for their ability to reverse histamine induced suppression. [] PHA control (dilution 1-512); [] Histamine (10~SM). Histamine (10 ~M) + ranitidine 1-7, or cimetidine Q, at 10 6, 5 x 10 -6, 10 5, 5 × 10 5, 10-aM. All cimetidine and ranitidine treated samples were compared to the histamine control by Student's t-test. Vertical bars represent mean + SD *P < 0.05; **P < 0.01; ***P < 0.001.
472
A. M. BADGER, J. YOUNG and G. POSTE
to d e m o n s t r a t e the H2-antagonist reversal of suppression. The present results indicate t h a t b o t h cimetidine a n d ranitidine are effective in reversing h i s t a m i n e - i n d u c e d suppression o f H P B L responses to mitogens. Studies with the HI a n t a g o n i s t d i p h e n d y d r a m e i n revealed t h a t it not only failed to reverse h i s t a m i n e induced suppression b u t increased the suppressive activity in c o m b i n a t i o n with h i s t a m i n e (Badger et al., 1983). In all experimens the a n t a g o n i s t s were a d d e d to the cultures at the same time or slightly before the histamine. This was done so t h a t our results can be c o m p a r e d with those in the literature. The greater p o t e n c y o f ranitidine in these experiments is consistent with its greater potency
w h e n c o m p a r e d with cimetidine in inhibiting gastric acid secretion in m a n via H~-receptor b l o c k a d e ( B o h m a n , M y r e n & Larsen, 1980; Walt, Male, Rawlings, H u n t , M i l t o n - T h o m p s o n & Misiewicz, 1981; K o n t u r c k , Obtulowica, Kwiecien, Sito, Mikos & Oleksy, 1980). In a d d i t i o n to providing evidence to support previous findings showing t h a t histamine affects lymphocyte f u n c t i o n via an action at classical H2-receptors, o u r results suggest t h a t claims that f u r a n - c o n t a i n i n g H2-receptor antagonists such as ranitidine do not affect i m m u n e f u n c t i o n (Peden et al., 1982) are p r e m a t u r e a n d should be interpreted with caution.
REFERENCES
BADGER, A. M., YOUNG, J. & POSTE, G. (1983). Inhibition of phytohaemagglutinin-induced proliferation of human peripheral blood lymphocytes by histamine and histamine H1 and H~ agonists. Clin. Exp. Immunol., 51, 178 - 184. BOHMAN, T., MYREN, J. & LARSEN, S. (1980). Inhibition of the histamine-stimulated gastric secretion in healthy subjects by the H2-receptor antagonist ranitidine. Scand. J. Gastroent., 15, 183- 185. BOOT, J. R., HUDSPITH, B. N. & BROSTOFF, J. (1980). The effect of the major mediators of type 1 hypersensitivity on lymphocytes from normal and atopic subjects. Int. J. Immunopharm., 2, 7 - 1 I. BROSTOFF, J., PACK, S. & LYDYARD, P. M. (1980) Histamine suppression of lymphocyte activation. Clin. Exp. Immunol., 39, 7 3 9 - 745. BROWN, A. E., BADGER,A. M. & POSTE, G. (1982). The effect of cimetidine on immune cell function and host response to tumors. In: Developments in Immunology Vol. 17. Current Concepts in Human Immunology and Cancer Immunomodulation (Edited by Serrou, B., Rosenfeld, C., Daniels, J. C. & Saunders, J. P.) pp. 5 1 3 - 519. Elsevier Biomedical Press, Amsterdam. FESTEN, H. P. M., DEPAUW, B. E., SMEULDERS, J. J. & WAGENER, D. J. T. (1981). Cimetidine does not influence immunological parameters in man. Clin. lmmunol. & Immunopathol., 21, 33 - 38. GIFFORD, R. P. M., HATFIEED, S. M. & SCHMIDTKE, J. R. (1980). Cimetidine-induced augmentation of human lymphocyte blastogenesis by mitogen, bacterial antigen and alloantigen. Transplantation, 29, 143 - 148. GIFFORD, R. P. M., SCHMIDTKE, J. R., & FERGUSON, R. M. (1981). Cimetidine modulation of lymphocytes from renal allograft recipients. Transplantation Proc., XIII, 663 - 667. GORDON, D., LEWIS, G. P. & NOURI, A. M. E. (1981). Inhibition of lymphocyte proliferation by histamine and related compounds not mediated via H1- or H~-receptors. Br. J. Pharmac., 74, 137-141. KONTURCK, S. J., OBTUEOWICA,W., KWIECIEN, N., SITO, E., MIKOS, E. & OLEKSY, J. (1980) Comparison of ranitidine and cimetidine on the inhibition of histamine, strain feeding and meal-induced gastric secretion in duodenal ulcer patients. Gut., 21, 181- 186. LEWIS, G. P., NOURI, A. M. E., FAY, D. T. P. & GORDON, D. (1982). Effect of histamine and related compounds on the immune response. Adv. Pharm. Ther. 11, 159- 169. MERETY, K., ROOM, G. & MAINI, R. N. (1981) Effect of histamine on the mitogenic response of human lymphocytes and its modification by cimetidine and levamisole. Agents Actions. 11, 8 4 - 88. OGDEN, B. E. & HILL, H. R. (1980) Histamine regulated lymphocyte mitogenic responses through activation of specific H~ and H2 histamine receptors. Immunology, 41, 107- 114. OSBAND, M. & MCCAEFREV, R. (1980) Concanavalin A activation of suppressor cells is blocked by cimetidine. Blood, 54, 89A. PALAC1OS, R. & ALARCON-SEGOVIA,D. (1981) Cimetidine abrogates suppressor T cell function in vitro. ImmunoL Letters, 3, 33 - 37. PEDEN, N. R., ROBERTSON,A. J., BOYD, E. J. S., BROWN, R. A., GIBBS,J. H., POTTS, R. C., WORMSLEY,K. G. & BECK, J. S. (1982). Mitogen stimulation of peripheral blood lymphocytes of duodenal ulcer patients during treatment with cimetidine or ranitidine. Gut, 23, 3 9 8 - 403. PLAUT, M. & LICHTENSTEIN, L. M. (1982). Pharmacology of histamine receptors. (Edited by C. R. Ganellin & M. E. Parsons) pp. 392-435. John Wright & Son Ltd., Bristol, U.K.
Reversal of Histamine-Mediated Immunosuppression
473
PupPo, F., CORSINI,G. • MANGINI, P. (1982). Ranitidine does not modify lymphocyte in vitro reactivity to the mitogens PHA, Con A and PWM. IRCS Med. Sci, 10, 538. SIMON, M. R., SALBERG, O. J. t~£ CRANE, S. (1981). In vivo cimetidine augmentation of phytohemagglutinin-induced human lymphocyte thymidine uptake. Transplantation, 31, 4 0 0 - 402. THOMAS, Y., HUCHET, R. & GRANJON, D. (1981). Histamine-induced suppressor cells of lymphocyte mitogenic response. Cell. Immunol., S9, 2 6 8 - 275. VICKERS, M. R., MILLINER, K., MARTIN, D. & GANELLIN, C. R., (1982). Histamine-induced inhibition of lymphocyte proliferation and lysosomalenzyme release from polymorphs may not be mediated via H,- or H2-receptors. Agents Actions, 12, 6 3 0 - 634. WALT, R. P., MALE,P. J., RAWLINGS,J., HUNT, R. H., MILTON-THOMPSON, G. J. & MISIEWlCZ, J. J. (1981). Comparison of the effects of ranitidine, cimetidine and placebo on the 24 hour intragastric acidity, and nocturnal acid secretion in patients with duodenal ulcer. Gut, 22, 4 9 - 5 4 . WANG,S. R., & ZWEIMAN,B. (1978). Histamine suppression of human lymphocyte responses to mitogens. Cell. Immunol., 36, 28 - 36.
0192-0561/84 $3.00+ .00 International Society for lmmunopharmacology.
REVERSAL OF HISTAMINE-MEDIATED IMMUNOSUPPRESSION BY STRUCTURALLY DIVERSE HISTAMINE TYPE II (H2) RECEPTOR ANTAGONISTS ALISON M. BADGER, JEAN YOUNG and GEORGE POSTE Smith Kline & French Laboratories, Philadelphia, PA 19101, U.S.A. (Received 23 September 1983 and in revised form 14 March 1984)
-The effect of a series of structurally-diverse histamine type 1I (H2)-receptor antagonists on histamine-mediated immunosuppression of human peripheral blood lymphocytes (HPBL) has been examined. This analysis of structure- activity relationships was undertaken to examine the validity of the recent proposal arising from clinical studies that H2-receptor antagonists containing a furan ring were devoid of the effects on lymphocyte function reported previously in studies using cimetidine and other H2-receptor antagonists containing an imidazole nucleus. Cimetidine and two furan-containing antagonists, ranitidine and SKF 93479, were found to be devoid of any effect on PHA-induced proliferation of human peripheral blood lymphocytes over a wide concentration range (10 4 to 10-'°M). At high drug concentrations (10 3M) significant suppression of mitogen stimulation was observed but this was accompanied by significant cytotoxicity. All three antagonists were effective in reversing the suppression of PHA-stimulation of HPBL induced by exogenous histamine. Reversal of histamine-induced immunosuppression was obtained at drug concentrations (2.0 x 10-4 to 1.0 x 10-6M) which were non-toxic and did not affect PHA-induced proliferation in the absence of histamine. Ranitidine was the most potent antagonist in reversing histaminemediated immunosuppression. The ability of structurally-diverse H2-receptor antagonists to modify the action of histamine on lymphocyte function lends support to the view that histamine exerts its effects via classical H2-receptors. The ability of ranitidine to alter lymphocyte responsiveness in analogous fashion to cimetidine indicates that the possibility of alterations in immune function should be considered in clinical studies using ranitidine and other furan-containing H2-receptor antagonists. Abstract
Histamine exerts multiple effects on lymphocyte functions, including inhibition of responsiveness to mitogens, antibody formation and lymphocytotoxicity (reviews, Lewis, Nouri, Fan & Gordon, 1982; Plaut & Lichtenstein, 1982). These immunosuppressive effects are believed to be mediated via the interaction of histamine with histamine type II receptors (H2) rather than type I-histamine (H0 receptors (Wang & Zweiman, 1978; Ogden & Hill, 1980; Boot, Hudspith & Brostoff, 1980; Thomas, Huchet & Granjon, 1981; Badger, Young & Poste, 1983) although not all studies report this association (Gordon, Lewis & Nouri, 1981; Vickers, Milliner, Martin & Ganellin, 1982). The identity of the specific subset(s) of lymphocytes that interact with histamine has yet to be identified. H2-receptor antagonists have been reported to abrogate suppressor cell activity in vitro (Osband & McCaffrey, 1980; Palacios &
Alarcon-Segovia, 1981) and to augment a variety of lymphocyte-mediated immune functions in vitro (Gifford, Hatfield & Schmidtke, 1980; Ogden & Hill, 1980; Simon, Salberg & Crane, 1981; Peden, Robertson, Boyd, Brown, Gibbs, Ports, Wormsley & Beck, 1982). The experimental studies published to date on the action of H2-receptor antagonists in reversing histamine-mediated immunosuppression have all used either cimetidine, or its structurally related analogues, burimamide and metiamide. These molecules each contain an imidazole ring. In contrast, two recently described H2-receptor antagonists, ranitidine and SKF 93479, lack this moiety and instead contain a common furan ring structure. Recent reports indicate that, unlike cimetidine, one of these new antagonists, ranitidine, does not affect lymphocyte responsiveness to mitogens (Puppo, Corsini & Mangini, 1982; Peden et al., 1982). It was
Correspondence: Dr. Alison M. Badger, Smith Kline & French Laboratories, 1500 Spring Garden Street, Mail Code L-101, Philadelphia, PA 19101, U.S.A. 467
468
A. M. BADGER,J. YOUNGand G. POSTE
therefore considered of interest to investigate the activity of structurally diverse H2-receptor antagonists on histamine-mediated immunosuppression to determine whether previous reports of the immunological properties of cimetidine and related molecules might be caused by the imidazole ring structure rather than specific antagonism of classical H2-receptors. In this paper we report that H~-receptor antagonists containing furan or imidazole ring structures are both active in reversing histaminemediated immunosuppression in vitro. These data thus support the view that H~-receptors are present on lymphocytes. These findings also indicate that claims that newly developed H~-antagonists such as ranitidine will not affect immune function (Peden et al., 1982) should be treated with caution.
EXPERIMENTAL PROCEDURES
dissolved in RPMI-10% at 10 3M. Log dilutions of the compounds were prepared in the same medium and added at the initiation of the experiment. The final volume in the microtiter wells was adjusted to 200/al with RPMI-10%. Each experimental variable was established in quadruplicate. Cell cultures were incubated for 72 h at 37°C in a 5070 CO2 atmosphere and pulsed with 0.5 Ci 3H-thymidine (specific activity 1.9 taCi/mM; Schwarz/Mann, Orangeburg, NY, U.S.A.) for the last 16 h of culture. The cells were harvested on an automated multiple sample harvester and cellassociated radioactivity measured in a Beckman liquid scintillation counter. The results are expressed as the mean values derived from quadruplicate measurements. Cell viability was determined by measuring the percentage of cells excluding trypan blue after 72 h of incubation. This was carried out by adding 25 tal of trypan blue to the microtiter plate wells, resuspending the cells and counting those that excluded the dye.
Drugs Histamine dihydrochloride was obtained from Calbiochem-Behring, La Jolla, California, USA. Cimetidine, SKF 93479 and SKF 91581 were provided by Smith KIine & French Laboratories, Welwyn Garden City, U.K. and ranitidine (Glaxo, Greenford, U.K. Lot # 1HP001) was obtained from a commercial source. Mononuclear Cell Preparation Mononuclear cells were isolated from heparinized blood from normal healthy volunteers on FicollHypaque (Pharmacia Fine Chemicals, Inc., Piscataway, N J, U.S.A.) density gradients (Badger et al., 1983). The cells were washed three times in Hank's balanced salt solution (HBSS) and resuspended at the desired concentration in RPM! 1640 (Flow Laboratories, Rockville, Maryland, U.S.A.) containing 10°70 heat-inactivated (56°C, 30 rain) fetal calf serum (FCS) (Flow), 2mM glutamine, penicillin (100 units/ml) streptomycin (100 /ag/ml). This medium will be referred to as RPMI-10%. Cell Cultures Fifty microliter aliquots of the HPBL cell suspension (1 × 105 ceils) were placed in 96 well round bottomed microtiter plates. Phytohaemagglutinin M (Difco Laboratories, Detroit) was added to the cultures in RPMI-10% at dilutions ranging from 1:256 to 1:1,024. Histamine, SKF 91581 and the H2-receptor antagonists cimetidine, SKF 93479 and ranitidine were
RESULTS
The chemical structures of a series of H2-receptor antagonists containing either an imidazole ring (burimamide, metiamide, cimetidine) or a furan ring (ranitidine and SKF 93479) are shown in Table 1. SKF 91581 is structurally similar to cimetidine but has no activity at H~-receptors. Since several studies have shown that the imidazole-containing antagonists produce similar effects on a variety of immune functions in vitro and in vivo (Plaut & Lichtenstein, 1982) only cimetidine was used in the following comparison with the newer furan-containing antagonists. The effect of cimetidine, ranitidine and SKF 93479 on PHA-induced mitogenesis in HPBL is shown in Table 2. The results indicate that neither imidazole nor furan-containing antagonists have any effect over a wide concentration range (10 -4 and 10 ~°M). Limited but significant suppression was observed with ranitidine at 10 ~M but at this high concentration about 1 0 - 15°70 cytotoxicity was detected. SKF 93479 produced significant cytotoxicity at both 10-3M (50070 cytotoxicity) and 10 4M (25070 cytotoxicity) and this probably accounts for the immunosuppression obtained with this agent at these physiologically irrelevant concentrations. In contrast to their lack of effect on lymphocyte responsiveness to PHA, all three antagonists reversed the suppression of the PHA response induced by exogenous histamine (Table 3). The possibility that
469
Reversal of Histamine-Mediated I m m u n o s u p p r e s s i o n i
Table 1. Structure of H2-receptor antagonists and analogs
Compound
Blockade of Gastric Acid Secretion ID5o (#mol/kg)~11"
Structure
Ranitidine
@HNO2 CH2SCH2CH2NHCNHCH 3
(CH3)2NCH2
SK&F 93479
o
(CH~)~NC.~ Cimetidine
0.13
CH~SCH~CH ..~H N~cH~")-"
CH3
0.14
c.~
CH2SCH2CH~NHCNHCH 3
Metiamide CH3~ "
1.4
L,4c.
HN,~N
CH2SCH2CH2NH~NHCH 3 S
1.6
HN, ~ N
Burimamide
CH2CH2CH2CH2NHCNHCH3
6.1
HN,~N
SK&F91581
r~ o,~c,.c,.,,~,,o..
250
S
HN~N
the antagonists themselves might have a stimulatory effect on HPBL proliferation was excluded by p a r a l l e l c o n t r o l e x p e r i m e n t s c o n d u c t e d at t h e s a m e t i m e u s i n g r e p l i c a t e cell s a m p l e s ( T a b l e 4). S K F 91581, a c o m p o u n d w h i c h is s t r u c t u r a l l y s i m i l a r t o c i m e t i d i n e ( T a b l e 1), b u t w h i c h h a s n o a c t i v i t y at
H 2 - r e c e p t o r s w a s n o t a b l e to r e v e r s e h i s t a m i n e induced suppression (data not shown). T h e ability o f r a n i t i d i n e to r e v e r s e h i s t a m i n e m e d i a t e d s u p p r e s s i o n in H P B L is s h o w n in Fig. 1. D o s e r e l a t e d e f f e c t s w e r e o b t a i n e d w h e n 10-SM h i s t a m i n e (or h i g h e r ) , w h i c h gives 3 0 - 5 0 % sup-
Table 2. Effect of H2-antagonists on PHA-stimulated h u m a n peripheral blood lymphocytes
Concentration (M) 10 -3 10 -4 10 5 10 6 10 7 10 8 10 -9 10 ,o
Cimetidine c p m _+ SD ( × 10 3)
% change
Ranitidine c p m ± SD ( × 10 3)
45,297 ___2565 -6al" 43,610 47,038 _+ 2298 -3 47,820 51,873 _+ 4961 +7 48,032 45,461 +_ 9746 -6 49,832 52,873 _+ 3557 +9 49,046 47,661 _+ 1939 -2 48,312 47,925 _+ 3491 - 1 50,733 47,404 _+ 2073 -2 50,425 Unstimulated control 593 _+ 328 P H A stimulated control 48,423 -4- 2380
_+ 2001 _+ 1997 _+ 1384 + 2369 +__ 1268 + 3327 _+ 2614 _ 1216
% change + + + +
10" 1 1 3 1 1 5 4
SKF 93479 cpm +_ SD ( × 10 3) 383 22,495 45,385 49,082 48,107 47,880 55,245 49,585
_+ 206:1: _+ 1590~ _+ 3376 +_ 1707 _+ 6575 _+ 4260 _+ 6629 _+ 1860
*P < 0.05. ***P < 0.001. I-All values were compared to P H A control, n = 4 for all samples. ~ T h e percentage of trypan-blue stained cells with SKF 93479 at 10-~M was 50 and 25°70 at 10-4M.
% change -99*** -53*** - 6 + 1 + 1 - 1 + 14" + 3
470
A. M. BADGER, J. YOUNG and G. POSTE Table 3. Reversal of histamine suppression by the H2-antagonists cimetidine ranitidine and SKF 93479 3 HT-lncorporation into D N A (cpm ± SD x 10 3). 1-256 1-512 1 - 1024
P H A Dilution Control
75,774 ± 14,547
68,840 ± 13,762
44,678 ± 10,960
Histamine 2.5 × 10 5M
53,708 ± 12,984 -29%P = <0.001t
40,525 ± 12,932 -41°/0 P < 0.001
22,405 _+ 10,497 - 5 0 % P = < 0.001
Histamine 2.5 x 10-SM + Cimetidine* 2.5 × 10 4M
67,768 ± 13,566 +36%:[: P = < 0.01§
56,499 ± 16,132 + 44°70 P = < 0.01
34,490 ___ 12,816 + 46°7o P = < 0.01
Histamine 2.5 x 1 0 ~ M + Ranitidine 2.0 × lO-4M
68,724 ± 13,366 +32°7o P = <0.01
57,775 ± 13,497 +39°7o P = < 0.01
34,050 ± 10,697 +48°70 P = < 0.05
Histamine 2.5 x I0 ~ + SKF 93479 2.0 x 10 ~M
64,881 ± 13,530 +49% P = <0.05
56,550 ± 15,103 +43°70 P = < 0 . 0 1
33,521 ± 11,760 +50°7o P = < 0 . 0 5
*The results are presented as m e a n _+ SD of 16 values from 4 different experiments. -[-The histamine treated groups are compared to the control group by Student's t-test. t T h e 07o increase = 100 - [ - c p m (Histamine + a n t a g o n i s t ) - c p m (Histamine) "1 x 100.
l
cpm ( C o n t r o l ) - c p m (Histamine) §All values are compared to the histamine control by Student's t-test. pression of the PHA response, was used. However, at l o w e r h i s t a m i n e c o n c e n t r a t i o n s ( 1 0 - 6 M ) , w h i c h gives o n l y 2 0 - 2 5 % suppression, ranitidine consistently reversed the suppression though dose related e f f e c t s were m o r e d i f f i c u l t to d e m o n s t r a t e . E v a l u a t i o n o f t h e relative p o t e n c y o f c i m e t i d i n e a n d ranitidine by titrating different concentrations of t h e s e m o l e c u l e s a g a i n s t a single c o n c e n t r a t i o n o f h i s t a m i n e (10 5M) r e v e a l e d t h a t r a n i t i d i n e is t h e m o r e p o t e n t a n t a g o n i s t (Fig. 2). S t u d i e s o n t h e reversal of histamine suppression revealed that
]
r a n i t i d i n e w a s 100 f o l d m o r e p o t e n t t h a n c i m e t i d i n e . I n c o m p a r i s o n t h e I D 5 0 o f r a n i t i d i n e in i n h i b i t i n g g a s t r i c acid s e c r e t i o n w a s o n l y 10 t i m e s l o w e r t h a n t h a t o f c i m e t i d i n e ( T a b l e 1).
DISCUSSION The present study has examined the ability of s t r u c t u r a l l y d i v e r s e H 2 - r e c e p t o r a n t a g o n i s t s to reverse histamine-mediated immunosuppression.
Table 4. Effect of cimetidine, ranitidine and SKF 93479 on H P B L stimulated with 3 different doses of P H A
P H A Dilution
3 HT-lncorporation into D N A (cpm ± SD x 10 -3) 1 - 256 1 - 512 I - 1024
Control
75.774 ± 14,547"
68,840 _+ 13,762
44,678 ± 10,960
Cimetidine (2.5 x 10 4M)
80,214 ± 16,673 +6°/o N.S.'["
72,465 + 18,012 + 5°7o NS
43,989 _ 14,476 - 2% NS
Ranitidine (2.0 x 10-4M)
73,959 ± 17,988 +2o7o NS
65,563 ± 16,790 -5o70 NS
41,069 ± 9474 -80/0 NS
SKF 93479 (2.0 × 10-~M)
73,892 ± 13,091 + 2 % NS
66,629 ± 15,176 - 3 % NS
44,353 _+ 13,441 - 1 % NS
*The results are presented as mean _+ SD o f 16 values from 4 different experiments. ~All values are compared to the P H A control by Student's t-test. NS = Not significant.
Reversal of Histamine-Mediated Immunosuppression 40
response of HPBL to mitogens, bacterial antigens and all•antigens (Gifford et al., 1980; Ogden & Hill,
1980). Similarly, oral administration of cimetidine to patients for treatment of gastro-intestinal ulceration
30
¢~
471
2O
[]
10-
10 6 J x
/
10" 5 Concentration (M)
-10
Fig. 1. Reversal of histamine-induced suppression by O, Histamine (10 6 and 10 ~M); ranitidine. • [] r-q, Histamine (10 6 and 10 ~M) + Ranitidine 10-6M; (3 - O, 10 5M; X X, 10 ~M. All ranitidine treated groups were significantly different from the histamine controls when compared by student's ttest (P = 0.01).
These experiments were prompted by two recent reports suggesting that a new series of H~-receptor
antagonists containing a furan ring structure might differ from antagonists containing an imidazole ring in their ability to affect lymphocyte function (Peden et al., 1982; Puppo et al., 1982).
Several studies have shown that the imidazolecontaining antagonist, cimetidine, and its congeners, metiamide and burimamide, can augment the in vitro
has been reported to augment the responsiveness of their lymphocytes to both mitogens and all•antigens (Gifford, Schmidtke & Ferguson, 1981; Simon et al., 1981; Peden et al., 1982). In contrast, Puppo et al. (1982) found that ranitidine did not alter the in vitro response of HPBL to mitogens, though cimetidine was not examined in this study. However, Peden et al. (1982) reported that lymphocytes obtained from patients treated with cimetidine showed enhanced responsiveness to P H A stimulation while patients in the same trial receiving ranitidine did not show augmented lymphocyte reactivity. They interpreted these data as evidence that HPBL lack classical H2-receptors and suggested that the previously reported effects of cimetidine and other H2-
antagonists containing an imidazole nucleus on immune function resulted from actions unrelated to antagonism of H~ receptors.
In these studies however, the H2 antagonists were not examined for their ability to antagonize histamine
mediated
suppression
of
mitogen
responsiveness. These experiments are, of course difficult to interpret if cimetidine alone enhances lymphocyte proliferation to mitogens. The present experiments and those of several others however (Brostoff, Pack & Lydyard, 1980; Brown, Badzer & Poste, 1982; Festen, DePauw, Smeulders & Wagener, 1981; Merety, Room & Maini, 1981) have failed to shown any stimulatory effect of cimetidine
on lymphocyte-proliferation and this has enabled us
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Fig. 2. Comparison of cimetidine and ranitidine for their ability to reverse histamine induced suppression. [] PHA control (dilution 1-512); [] Histamine (10~SM). Histamine (10 ~M) + ranitidine 1-7, or cimetidine Q, at 10 6, 5 x 10 -6, 10 5, 5 × 10 5, 10-aM. All cimetidine and ranitidine treated samples were compared to the histamine control by Student's t-test. Vertical bars represent mean + SD *P < 0.05; **P < 0.01; ***P < 0.001.
472
A. M. BADGER, J. YOUNG and G. POSTE
to d e m o n s t r a t e the H2-antagonist reversal of suppression. The present results indicate t h a t b o t h cimetidine a n d ranitidine are effective in reversing h i s t a m i n e - i n d u c e d suppression o f H P B L responses to mitogens. Studies with the HI a n t a g o n i s t d i p h e n d y d r a m e i n revealed t h a t it not only failed to reverse h i s t a m i n e induced suppression b u t increased the suppressive activity in c o m b i n a t i o n with h i s t a m i n e (Badger et al., 1983). In all experimens the a n t a g o n i s t s were a d d e d to the cultures at the same time or slightly before the histamine. This was done so t h a t our results can be c o m p a r e d with those in the literature. The greater p o t e n c y o f ranitidine in these experiments is consistent with its greater potency
w h e n c o m p a r e d with cimetidine in inhibiting gastric acid secretion in m a n via H~-receptor b l o c k a d e ( B o h m a n , M y r e n & Larsen, 1980; Walt, Male, Rawlings, H u n t , M i l t o n - T h o m p s o n & Misiewicz, 1981; K o n t u r c k , Obtulowica, Kwiecien, Sito, Mikos & Oleksy, 1980). In a d d i t i o n to providing evidence to support previous findings showing t h a t histamine affects lymphocyte f u n c t i o n via an action at classical H2-receptors, o u r results suggest t h a t claims that f u r a n - c o n t a i n i n g H2-receptor antagonists such as ranitidine do not affect i m m u n e f u n c t i o n (Peden et al., 1982) are p r e m a t u r e a n d should be interpreted with caution.
REFERENCES
BADGER, A. M., YOUNG, J. & POSTE, G. (1983). Inhibition of phytohaemagglutinin-induced proliferation of human peripheral blood lymphocytes by histamine and histamine H1 and H~ agonists. Clin. Exp. Immunol., 51, 178 - 184. BOHMAN, T., MYREN, J. & LARSEN, S. (1980). Inhibition of the histamine-stimulated gastric secretion in healthy subjects by the H2-receptor antagonist ranitidine. Scand. J. Gastroent., 15, 183- 185. BOOT, J. R., HUDSPITH, B. N. & BROSTOFF, J. (1980). The effect of the major mediators of type 1 hypersensitivity on lymphocytes from normal and atopic subjects. Int. J. Immunopharm., 2, 7 - 1 I. BROSTOFF, J., PACK, S. & LYDYARD, P. M. (1980) Histamine suppression of lymphocyte activation. Clin. Exp. Immunol., 39, 7 3 9 - 745. BROWN, A. E., BADGER,A. M. & POSTE, G. (1982). The effect of cimetidine on immune cell function and host response to tumors. In: Developments in Immunology Vol. 17. Current Concepts in Human Immunology and Cancer Immunomodulation (Edited by Serrou, B., Rosenfeld, C., Daniels, J. C. & Saunders, J. P.) pp. 5 1 3 - 519. Elsevier Biomedical Press, Amsterdam. FESTEN, H. P. M., DEPAUW, B. E., SMEULDERS, J. J. & WAGENER, D. J. T. (1981). Cimetidine does not influence immunological parameters in man. Clin. lmmunol. & Immunopathol., 21, 33 - 38. GIFFORD, R. P. M., HATFIEED, S. M. & SCHMIDTKE, J. R. (1980). Cimetidine-induced augmentation of human lymphocyte blastogenesis by mitogen, bacterial antigen and alloantigen. Transplantation, 29, 143 - 148. GIFFORD, R. P. M., SCHMIDTKE, J. R., & FERGUSON, R. M. (1981). Cimetidine modulation of lymphocytes from renal allograft recipients. Transplantation Proc., XIII, 663 - 667. GORDON, D., LEWIS, G. P. & NOURI, A. M. E. (1981). Inhibition of lymphocyte proliferation by histamine and related compounds not mediated via H1- or H~-receptors. Br. J. Pharmac., 74, 137-141. KONTURCK, S. J., OBTUEOWICA,W., KWIECIEN, N., SITO, E., MIKOS, E. & OLEKSY, J. (1980) Comparison of ranitidine and cimetidine on the inhibition of histamine, strain feeding and meal-induced gastric secretion in duodenal ulcer patients. Gut., 21, 181- 186. LEWIS, G. P., NOURI, A. M. E., FAY, D. T. P. & GORDON, D. (1982). Effect of histamine and related compounds on the immune response. Adv. Pharm. Ther. 11, 159- 169. MERETY, K., ROOM, G. & MAINI, R. N. (1981) Effect of histamine on the mitogenic response of human lymphocytes and its modification by cimetidine and levamisole. Agents Actions. 11, 8 4 - 88. OGDEN, B. E. & HILL, H. R. (1980) Histamine regulated lymphocyte mitogenic responses through activation of specific H~ and H2 histamine receptors. Immunology, 41, 107- 114. OSBAND, M. & MCCAEFREV, R. (1980) Concanavalin A activation of suppressor cells is blocked by cimetidine. Blood, 54, 89A. PALAC1OS, R. & ALARCON-SEGOVIA,D. (1981) Cimetidine abrogates suppressor T cell function in vitro. ImmunoL Letters, 3, 33 - 37. PEDEN, N. R., ROBERTSON,A. J., BOYD, E. J. S., BROWN, R. A., GIBBS,J. H., POTTS, R. C., WORMSLEY,K. G. & BECK, J. S. (1982). Mitogen stimulation of peripheral blood lymphocytes of duodenal ulcer patients during treatment with cimetidine or ranitidine. Gut, 23, 3 9 8 - 403. PLAUT, M. & LICHTENSTEIN, L. M. (1982). Pharmacology of histamine receptors. (Edited by C. R. Ganellin & M. E. Parsons) pp. 392-435. John Wright & Son Ltd., Bristol, U.K.
Reversal of Histamine-Mediated Immunosuppression
473
PupPo, F., CORSINI,G. • MANGINI, P. (1982). Ranitidine does not modify lymphocyte in vitro reactivity to the mitogens PHA, Con A and PWM. IRCS Med. Sci, 10, 538. SIMON, M. R., SALBERG, O. J. t~£ CRANE, S. (1981). In vivo cimetidine augmentation of phytohemagglutinin-induced human lymphocyte thymidine uptake. Transplantation, 31, 4 0 0 - 402. THOMAS, Y., HUCHET, R. & GRANJON, D. (1981). Histamine-induced suppressor cells of lymphocyte mitogenic response. Cell. Immunol., S9, 2 6 8 - 275. VICKERS, M. R., MILLINER, K., MARTIN, D. & GANELLIN, C. R., (1982). Histamine-induced inhibition of lymphocyte proliferation and lysosomalenzyme release from polymorphs may not be mediated via H,- or H2-receptors. Agents Actions, 12, 6 3 0 - 634. WALT, R. P., MALE,P. J., RAWLINGS,J., HUNT, R. H., MILTON-THOMPSON, G. J. & MISIEWlCZ, J. J. (1981). Comparison of the effects of ranitidine, cimetidine and placebo on the 24 hour intragastric acidity, and nocturnal acid secretion in patients with duodenal ulcer. Gut, 22, 4 9 - 5 4 . WANG,S. R., & ZWEIMAN,B. (1978). Histamine suppression of human lymphocyte responses to mitogens. Cell. Immunol., 36, 28 - 36.