Histamine and serotonin suppression of lymphocyte response to phytohemagglutinin and allogeneic cells

CELLULAR

IMMUNOLOGY

83,280-29 1 ( 1984)

Histamine and Serotonin Suppression of Lymphocyte Response to Phytohemagglutinin and Allogeneic Cells MIREILLE BONNET,* GENEV&VE LESPINATS,*AND CLAUDE BURTIN? *Institut de RecherchesScientifques sur le Cancer, Villejuif; France, and f U. 203 INSERM, Faculte’ de Midecine Necker-E&ants Malades, Paris, France Received July 14. 1983; accepted September 15, 1983 in vitro proliferation of lymphocytes induced Histamine added to murine spleencells suppressed by PHA or allogeneic spleen cells. Another vasoactive amine, serotonin (Shydroxytryptamine), exerted a similar inhibitory activity on PHA- or allogeneic cell-induced lymphocyte proliferation. Anti-H2 histamine antagonists, cimetidine, metiamide, and ranitidine, blocked the histamine and serotonin suppressive effect. Cyproheptadine, an anti-H 1 histamine and anti-serotonin antagonist, and methysergide, an anti-serotonin antagonist, also blocked histamine and serotonin inhibitory activities. These data suggestthe presence,on lymphocytes, of receptors for serotonin which might be related to histamine receptors.

INTRODUCTION Histamine modulates a great number of physiological reactions which are mediated by two classes of receptors called, respectively, Hl and H2 histamine receptors (1, 2). Recent works suggestthat this vasoactive amine may be implicated in the host’s defenseagainst a tumor, and that tumor growth is probably associatedwith a decrease in histamine availability (3, 4). In a previous paper, we demonstrated that histamine injected ip into fibrosarcomabearing mice induced an inhibition of tumor growth and an increase in survival time (5). This effect was even greater when animals were similarly injected with 5-hydroxytryptamine (serotonin), an important vasoactive amine in mice, thus indicating the likelihood of increased vascular permeability (6). However, other mechanisms could be involved, especially at the level of the immune system,and we were interested in studying the immunomodulatory action of histamine and serotonin. The action of histamine on stimulated lymphocytes has been determined mainly on human and guinea pig lymphocytes: when added to mitogen-stimulated lymphocytes, histamine induces a decreasein cellular proliferation and in lymphokine production (7,8). Few studies have been performed on mice where histamine also inhibits cellular proliferation by activation of a T-suppressive subpopulation (9, 10). We have confirmed that histamine inhibits the mitogen-induced lymphocyte proliferation of murine spleen cells, and specified experimental conditions for preventing histamine-induced inhibition by antagonists. We have shown that histamine also inhibits lymphocyte proliferation induced in mixed lymphocyte culture. 280 OOOS-8749184 $3.00 Cbwigbt0 1984 by Academic AU rights of reproduction

Press, Inc. in any form rrsmd

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The action of serotonin on lymphocyte proliferation has not yet been described. We have demonstrated here that serotonin has an activity similar to histamine activity on lymphocyte stimulation induced by mitogens. The sameactivity is found in mixed lymphocyte culture. MATERIAL

AND METHODS

Animals. Male and female CH3/He, C57BL/6, and DBA/2 mice, 6-20 weeks old, with defined intestinal flora, were obtained from the production colonies of the Institut de Recherches Scientifiques sur le Cancer. Spleen cell suspensions. Spleens were aseptically removed, minced with scissors, filtered through gauze, and washed twice in Eagle’s minimum essential medium. Viable cells were counted and adjusted at the desired concentration. Culture medium. Cultures of lymphocytes with PHA’ (Wellcome Laboratory) were prepared in RPM1 1640 medium (Gibco) supplemented with 2 mM glutamine (G&co), antibiotics (100 U/ml penicillin and 100 &ml streptomycin), and 5% calf serum (Gibco). For MLC, 5-mercaptoethanol (Sigma) 5-10m5M was added to this medium. Culture of spleen cells with PHA. PHA was used at a final concentration of 3 pg/ ml. Preliminary experiments determined that this concentration was suboptimal, and it was used in order to eventually detect an increase in stimulation. C3H/He spleen cells, 3-lo5 cells per well, were placed in Falcon 3040 microplates. PHA and the different drugs and reagentswere added, at the desired concentration; the final volume in each well was 200 ~1. The plates were incubated for 48 hr in an atmosphere of 5% CO* plus 95% air. Mixed lymphocyte culture. Spleen cells from C57BL/6 mice, 5-lo5 cells per well, and mitomycin-treated DBA/2 spleen cells, 5 X lo5 cells per well, were placed in microplates, with the drugs and reagents at the desired concentration, in a total volume of 200 ~1. Cultures were made in triplicate and incubated for 72 hr in CO* 5% Terminution of cultures. Five hours before the end of the culture, 1 &i of [3H]dThd (TMM79B; CEA, Gif sur Yvette, France; sp. act. 25 Ci/mmol) was added to each well. Cultures were harvested on glassfiber filters with a multiple sample collector (Skatron, Flow Laboratories). The glass fiber discs were placed in scintillation fluid (Lipoluma, Lumac) and the radioactivity was counted in an SL 30 Intertechnique scintillation spectrometer. Expression of results. The percentageof stimulation was determined by the following formula: cpm in presence of reagent x 100. cpm in absenceof reagents The Student’s t test was used to compute the statistical significance of differences between groups. Drugs and reagents. All reagents were stored in dry form at room temperature, dissolved immediately before use in the culture medium, and filtrated for sterilization. Histamine dihydrochloride, serotonin creatinin bisulfate, and creatinin bisulfate were purchased from Prolabo. ’ Abbreviations used: PHA, phytohemagglutinin; MLC, mixed lymphocyte culture.

282

BONNET,

LESPINATS,

AND

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Dimaprit, SKF 9 1449 A2 (histamine H2 agonist), Nordimaprit SKF 9 1487 A2 (the inactive homolog of dimaprit), cimetidine, and metiamide (H2 antagonists) were kindly provided by Dr. Ganellin (Smith, Kline and French Laboratories, England). Ranitidine (H2 antagonist) was a gift from Dr. Alexandre (Glaxo Laboratory). Cyproheptadine (anti-H2 and anti-serotonin antagonist) was purchased from Merck Sharp. Methysergide bimaleate (serotonin antagonist) was obtained from Sandoz. Toxicity of drugs and reagents. A number of agonists and antagonists of histamine have been described. Before adding them to lymphocyte cultures we controlled their toxicity on lymphoid cells. The cytotoxicity was measured by chromium releasefrom labeled C3H/He lymphocytes after 4 hr incubation (11). Nordimaprit (an inactive homolog of dimaprit) could not be used, because it had a direct cytotoxic effect on lymphoid cells. For each antagonist, we employed the highest concentration among the noncytotoxic ones: most of them were used at 10V5and lop4 M, except for methysergide at 10m6and 10m5M and cyproheptadine at lop5 M.

I ,o-'2

I ,o-"

I ,o-lo

I

I

,o-9

,o-8

HISTAMINE

I

I

I

I

I

10-7

10-e

10-5

1o-4

10-S

(M)

FIG. 1. Effect of histamine on the lymphocyte response to PHA. Values are expressed as mean percentage of control f SE for 16 experiments.

HISTAMINE

283

AND SEROTONIN SUPPRESSION

RESULTS

Action of Histamine on PHA-Stimulated Lymphocytes At high concentrations, histamine exerted an inhibitory effect on cellular cultures, whether the cells were PI-IA stimulated or not. We chose to more extensively study the stimulated cultures as this allowed us to determine the effect of antagonists and also because the nonstimulated cells were too poorly proliferating. A wide range of concentrations from lo-” to lop3 M was tested. Figure 1 shows the effect of histamine on PHA-stimulated C3H splenic cells. Histamine at 10V4and 10e3M regularly induced a significant inhibition of the response to PHA, while 10V5 A4 concentration induced inhibition in about half of the experiments. At low concentrations, from lo-‘* to lo-” M, a slight enhancement of the response was occasionally encountered, but was not significant. In order to verify that histamine induced a true inhibition and not a kinetics displacement of the lymphocyte response to the mitogen, we incubated spleen cells with histamine and PHA for various periods of time, between 6 and 72 hr. Results reported in Fig. 2 show that during a 6-hr incubation, lop3 M histamine already induced a significant inhibition. This time-related effect achieved a maximum of 77% inhibition, for the 72-hr incubation. For the lop4 M histamine concentration, a significant inhibition was observed from 24 hr on, increasing progressively afterward. In similar way, we tested the action of dimaprit, an H2 agonist. Dimaprit strongly inhibited the lymphocyte response to PHA at lOeMand 1O-3M. Table 1 compares

6

24

46 INCUBATION ( hours

72 TIME

)

FIG. 2. Kinetics of the lymphocyte response to PHA in the presence of histamine, 10m3M (0 and lo-‘M(O---0).

0)

284

BONNET, LESPINATS, AND BURTIN TABLE 1 Comparison of Histamine and Dimaprit Percentage inhibition PHA”

Concentration (W

Histamine

1O-5 1O-4 10-r

MLCb Dimaprit

Histamine

Dimaprit

21

0

0

0

38 59

70

14 54

51 82

100

a PHA-stimulated C3H/He spleen cells. b MLC:C57BL/6 spleen cells stimulated by DBA/Z mitomycin-treated spleen cells.

the inhibitions induced by histamine and dimaprit for the same molar concentrations. As can be seen the dimaprit effect was more pronounced than the histamine effect. To make the influence of histamine on lymphocytes more precise, we studied the action of type H2 antagonists (anti-H2): cimetidine, metiamide, and ranitidine. We also employed cyproheptadine (anti-H1 and anti-serotonin) and methysergide, a serotonin antagonist (also called UML). When these antagonists were added to the culture at high concentrations, they inhibited the lymphocyte response. These results, combined with the results of toxicity (see Materials and Methods) show that anti-H2 antagonists could be used at 10e4A&, while cyproheptadine and methysergide could not be employed at concentrations higher than lop5 M. In order to determine whether they could prevent the inhibitory effect of histamine, antagonists were added at the same time as histamine to PHA-stimulated cultures. The results obtained were variable: in some experiments inhibition was able to be reversed, while in others it was not. In contrast, when antagonists were added to PHA-stimulated cultures 1 hr before histamine, they were regularly able to induce a 2 100 0

a + 5 0

80

: 80 + z “, 40 PC w n

20

H3

+c4

fM4

+Fi4

+cY5

+UMLG

FIG. 3. Effectof histamine and serotonin receptor antagonistson histaminsinduced inhibition of lymphocyte responseto PHA. Antagonists and PHA were added to lymphocytes at the initiation of the culture, histamine 1 hr later. *, P < 0.05; **, P < 0.02; *+*, P < 0.01.

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SUPPRESSION

reversal of the histamine effect: cimetidine, metiamide, ranitidine, cyproheptadine, and methysergide were able to prevent the inhibitory effect of histamine. Figure 3 shows a representative example of this type of experiment. In other experiments, antagonists were added to spleen cells at initiation of the culture; histamine was then added 1 hr later, and PHA 24 hr later. Similar results were obtained: cimetidine, metiamide, ranitidine, cyproheptadine, and methysergide prevented the histamine-induced inhibition. The same antagonists were tested on dimaprit-induced inhibition. In this case, inhibition was prevented only by the antiH2 antagonists, cimetidine, metiamide, and ranitidine. In the experiment shown in Fig. 4, the inhibition exerted by 10e4 M dimaprit could be prevented by anti-H2

120

100

80

60

A

40

0

[L 5

20

0

0 1

lL 0

H4

+c4 l

= 120w 0

LT : loo-

+M4

+R4

+cy5

+UMLFj

+M4

+F34

+cys

+UMLG

**

B

ao60-

40 -

20

tl D4

+c4

FIG. 4. Effect of histamine and serotonin receptor antagonists on histamine and dimaprit-induced inhibition of lymphocyte response to PHA. Antagonists were added to lymphocytes at initiation of the culture, histamine or dimaprit 1 hr later, and PHA 24 hr later. *, P -z 0.05.

286

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antagonists 1O-4AI. The inhibition induced by 1Oe3A4 dimaprit could not be reversed by antagonists lop4 AI, probably becauseit was too high. Action of Histamine on Mixed Lymphocyte Cultures Figure 5 shows the effect of histamine on C57BL/6 splenic cells stimulated by DBA/2 mitomycin-treated splenic cells. Histamine inhibited MLC at IO-’ M concentration in all experiments, and at 1Oe4and low5M in about half of the experiments. 14c

120

1OC

4c

x

,o-l2

10-11

,o-‘0

10-g

10-E

HISTAMINE

10-7

10-S

10-5 1(y4

1o-3

(M)

FOG.5. Effect of histamine on MLC C57BL/6 spleen cells stimulated by DBA/Z mitomycin-treated spleen cells. Mean percentage of control + SE for 12 experiments.

HISTAMINE

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In some cases,we observed a slight but significant enhancement for the low concentration 1O-*‘2to 10-i’ it4 The ‘H2 receptor agonist dimaprit inhibited the reaction at 1OP4and 1Om3M concentration. As reported in Table 1, it induced a greater inhibition than histamine. It is noteworthy that for each molecule, histamine and dimaprit, the inhibition induced in both culture systemshad the same order of magnitude, with the effect of dimaprit being more pronounced than that of histamine in both cases.

,o-12

,o-11

,(p

10-S

10-a SEROTO

10-7 NIN

10-6

10-5

1o-4

lo-3

( M)

FIG. 6. Effect of serotonin on PHA stimulated lymphocytes. Mean percentage of control f SE for 7 experiments.

288

BONNET,

LESPINATS,

AND

BURTIN

Action of Serotonin on PHA-Stimulated Lymphocytes Figure 6 shows the effect of serotonin on PHA-stimulated C3H splenic cells. At 1Om3M concentration, serotonin regularly induced a strong inhibition on PHA-stimulated lymphocytes. At lop4 M concentration, the inhibition was slight and was not observed in all experiments. This inhibitory effect was due to the 5-hydroxytryptamine part of the molecule: bisulfate creatinin crystallized together with 5-HT had no significant effecton PHA-stimulated cultures when added alone at the sameconcentration as serotonin. 141

121

41

I ,o-'2

I ,o-ll

I ,(-p

I ,&II

I 10-8

SEROTONIN

I 10-7

I 10-G

I

10-5

I

1o-4

I

1o-3

IM)

FIG. 7. Effect of serotonin on MLC. Mean percentage of control k SE for 7 experiments.

HISTAMINE

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AND SEROTONIN SUPPRESSION

Kinetics showed that after 6-hr incubation, inhibition was already detected, and increased during the course of the incubation time. The action of antagonists on this serotonin-induced inhibition was tested. Due to their own inhibitory effect on cultures, they were used at the same concentrations as previously described. When added simultaneously or 1 hr before serotonin, they were unable to prevent the suppression exerted by serotonin. When antagonists were added to spleen cells at initiation of the culture, serotonin 1 hr later, and PHA 24 hr later, cyproheptadine, methysergide, and anti-H2 receptor antagonists were able to prevent serotonin-induced inhibition. Action of Serotonin on Mixed Lymphocyte Culture As shown in Fig. 7, serotonin strongly inhibited MLC at lo-’ M concentration, and was inactive at 10e4M. The effects of histamine and serotonin were compared on both culture systems, and are summarized in Table 2. It can be noted.that, at 10V3M, in both systems, histamine and serotonin induced a similar percentage of inhibition. DISCUSSION Histamine at high concentrations induced an inhibition of PHA-induced lymphocyte stimulation, as described by others (9, 10). This effect is a true inhibition, occurring from the first hours of the culture, in agreement with the findings of Ogden and Hill (12). This inhibition is mediated mainly by histamine H2 receptors, as demonstrated by the similar activity of dimaprit, an H2 receptor agonist, and the ability of antiH2 receptor antagonists to prevent the inhibition induced by histamine and dimaprit. It must be noted that cimetidine and ram&line prevented histamine-induced inhibition in a similar manner. Cimetidine and ranitidine are both anti-H2 antireceptor antagonists, but their chemical structures are entirely different (13). As already described by others (14, 15) we observed that nordimaprit (an analog of dimaprit without H2 agonist activity) decreasedthymidine incorporation of mitogenTABLE 2 Comparative Effects of Histamine and Serotonin Percentage inhibition Histamine

Serotonin

Concentration wf)

PHA”

MLC”

PHA

MLC

1o-5 IO--’ lo-’

21 38 59

0 14 46

0 12 55

0 1 55

’ PHA-stimulated C3H/He spleen cells. ’ C57BL/6 spleen cells stimulated by mitomycin-treated DBA/Z spleen cells.

290

BONNET, LESPINATS, AND BURTIN

stimulated lymphocytes. However, under our experimental conditions, nordimaprit proved to be toxic. The decreasein thymidine incorporation by lymphocytes in the presenceof nordimaprit was not due to inhibition of lymphocyte stimulation by this product, but rather to cell death. Therefore, results obtained with nordimaprit were not interpretable. Cyproheptadine, an anti-H 1 and anti-serotonin receptor antagonist, prevented the inhibition induced by histamine. Thus, this could be an argument for the participation of Hl receptor in this inhibition. However, mepyramine (an anti-H1 receptor antagonist) did not prevent histamine-induced inhibition (data not shown) and it seems likely that prevention exerted by cyproheptadine could be due to steric hindrance. The results of blockage of histamine activity by antagonists were highly dependent upon experimental conditions, and the respective time of addition of histamine, antagonists, and PHA to lymphocytes. This could explain the discrepancies found in the literature where some authors find blocking of the reaction only by histamine anti-H2 receptor antagonists (9-16) and others find no blocking either by anti-H 1 or by anti-H2 receptor antagonists (14-17). Even under the most favorable experimental conditions, we sometimes failed to obtain a reversal of histamine-induced inhibition by antagonists. All antagonists, at 1O-3M concentration proved to be toxic for lymphocytes. Therefore, they could not be used at concentrations higher than the histamine concentration necessaryto induce inhibition of lymphocyte stimulation by PHA. Indeed, many investigators use 5- to lo-fold higher antagonist concentrations in reversal studies, which were not possible in our culture systemsbecauseof toxicity. It may be that these difficulties in reversal studies are due to the impossibility of increasing the antagonist/agonist concentration ratio. This was also true for serotonin. We showed that histamine also induced an inhibitory activity on mixed lymphocyte culture, and our results support the hypothesis of H2 receptor involvement, since dimaprit had a similar inhibiting activity. We demonstrated that serotonin, at similar molar concentrations, had the same activity as histamine on PHA or allogeneic spleencell-induced lymphocyte stimulation. Lymphocyte receptors for serotonin have not yet been described. In our experiments, histamine anti-H2 receptors were able to block the action of both histamine and serotonin. Anti-serotonin antagonists were also able to block both activities. Many anti-serotonin antagonists are also anti-histamine antagonists in the central nervous system (18). The question of whether there are common receptors for histamine and serotonin on lymphocytes remains to be demonstrated. Further studies of receptors on lymphocytes for histamine and for serotonin now appear to be necessary. REFERENCES I. Ash, A. S. F., and Schild, H. O., Brit. J. Pharmacol. 27, 427, 1966. 2. Black, J. W., Duncan, W. A. M., Durant, E. J., Ganellin, C. R., and Parsons, E. M., Nature (London) 236, 385, 1972. 3. Lynch, N. R., and Salomon, J. C., Immunology 32, 645, 1977. 4. But-tin, C., Scheinmann, P., Salomon, J. C., Lespinats, G., Frays&et, C., Lebel, B., and Cant, P., Brit. J. Cancer 43, 684, 1981. 5. Burtin, C., Scheinmann, P., Salomon, J. C., Lespinats, G., Loisillier, F., and Canu, P., Cancer Lett. 12, 195, 1981.

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6. Burtin, C., Scheinmann, P., Salomon, J. C., Lespinats, G., and Canu, P., Brit. J. Cancer 45, 54, 1982. 7. Beer, D. J., Rosenwasser,L. J., Dinarello, C. A., and Rocklin, R. E., Cell Immunol. 69, 101, 1982. 8. Rocklin, R. E., Greineder, D., Littman, B. H., and Melmon, K. L., Ceil. Immunol. 37, 162, 1978. 9. Suzuki, S., and Huchet, R., Cell. Immunol. 62, 396, 1981. 10. Suzuki, S., and Huchet, R., Cell. Immunol. 68, 349, 1982. 11. Wang, S. R., and Zweiman, B., Cell Immunol. 36, 28, 1978. 12. Ogden, B. E., and Hill, H. R., Immunology 41, 107, 1980. 13. Plind, A. C., and Rowley-Jones, D., Lancet 8272,601, 1982. 14. Gordon, D., Lewis, G. P., and Now-i, A. M. E., Brit. J. Pharmacol. 74, 137, 1981. 15. Vickers, M. R., Milliner, K., Martin, D., and Ganellin, C. R., Agents Actions 12, 5, 1982. 16. Rocklin, R. E., J. Clin. Invest. 57, 1051, 1976. 17. Brostoff, J., Pack, S., and Lydyard, P. M., Clin. Exp. Immunol. 39, 739, 1980. 18. Peroutka, S. J., and Snyder, S. H., Fed. Proc. 42, 2 13, 1983.