Regulation of Lymphocyte Blastogenesis By

Agric. Biol. Chem., 53 (2), 377-382, 1989 377

Regulation of Lymphocyte Blastogenesis by Histamine Produced in the System per sef Chanho Oh, Hiroshi Okamoto and Kiwao Nakano* Department of Nutritional Regulation, Research Institute for Biochemical Regulation, Nagoya University, Chikusa, Nagoya 464, Japan Received August 9, 1988

The culture of mouse spleen cells with concanavalin A (Con A) or phytohemagglutinin (PHA) increased histidine decarboxylase (HDC)activity as a function of incubation time. The histamine level of the medium increased in parallel with the increase in HDCactivity. Lymphocyte proliferation induced by ConA was enhanced by cimetidine, a histamine H2-antagonist, in the absence of exogenous histamine. Dimaprit, an H2-agonist, blocked lymphocyte blastogenesis at high doses (>10~5 m). Diphenhydramine, an Hl-antagonist, abolished mitogenesis induced by Con A. At low doses (1O~9 ~ 1CT7 m), the I^-agonist 2-pyridylethylamine augumented mitogenesis mediated by Con A. The addition of alphafluoromethylhistidine or histaminase (EC 1.4.3.6), at low doses, to the culture enhanced Con-A-dependent lymphocyte proliferation. At high doses they inhibited the reaction. Histamine itself augmented [3H]thymidine uptake by lymphocytes induced by Con A at the low doses of lO~13 ~ 10~n M;at higher doses ( > 10~5 m), it suppressed this response. These results suggest that mitogen-induced lymphocyte proliferation is regulated by histamine, formed in the system per se through the action of HDC.

Histamine exerts a variety of immunoregu- phocyte proliferation is regulated by histamine latory effects. It inhibits immunoglobulin pro- produced in the system per se. The purpose of duction by B lymphocytes,l) lymphocyte pro- this study was to examine this possibility. The liferation after mitogenic stimulation2'3) or results obtained indicated that lymphocyte antigenic stimulation,40 or in vitro production blastogenesis is regulated by endogenous his- of killer cells.5) The immunosuppressive ef- tamine produced in the system per se. fect of histamine are probably mediated by activation of suppressor T lymphocytes.4^6) Materials and Methods Almost all of these results were obtained from studies on the effects of histamine added exog- Mice. C57BL/6J mice nine to 12 weeks old and of both sexes were obtained from Shizuoka Experimental Animal enously to each immunesystem. The amine is Cooperative, Shizuoka, Japan. released from mast cells or basophils upon activation of the cells with antigen-IgE com- Reagents. Diphenhydramine, histamine, and histam- plex.^ Histamine is synthesized de novo inase (EC 1.4.3.6) were obtained from Sigma Chemical through histidine decarboxylase (HDC*1) in- Co., St. Louis, MO. Cimetidine, dimaprit, and 2-pyridylethylamine dihydrochloride were gifts from Smith duced by the maerophage-T lymphocyte sys- Kline & French Laboratories, Philadelphia, PA. tem, which is activated with concanavalin A Alphafluoromethylhistidine was kindly provided by (Con A) or lipopolysaccharide (LPS).8) It is Merck Sharp & DohmeResearch Laboratories, Rahway, likely, therefore, that mitogen-dependent lym- NJ, respectively. All drugs were dissolved in a serum-free f This work was supported in part by the Naito Foundation and Ishida Foundation. * Address correspondence and reprint requests to: KiwaoNakano, Ph.D., Department ofNutritional Regulation, Research Institute for Biochemical Regulation, Nagoya University, Chikusa, Nagoya 464-01, Japan. *l Abbreviations: HDC, histidine decarboxylase; Con A, concanavalin A; PHA, phytohemagglutinin-/?. 378 C. Oh, H. Okamoto and K. Nakano

syntnetic un meaium(^uaigo rsiutntive e:nemicais to., Osaka). The pH was adjusted, when necessary, before the addition of the solution of the drugs to each culture.

Lymphocyte blastogenesis. Spleens were cut into 2~3 pieces in cold phosphate-buffered saline (PBS, Nissui Pharmaceutical Ltd., Co., Tokyo). The cells were released by gentle pressing of the tissue with forceps, washed three times with PBS, and suspended in GIT medium at the concentration of 2.5 x 106 cells/ml. Then 0.2ml of the cell suspension was placed in each well of a flat-bottomed microtiter plate. Ten microliters each of Con A (0.1 or 0.5/zg/ml,*2 Pharmacia Fine Chemicals, Uppsala, Sweden) or phytohemagglutinin-p (PHA, 5 /zg/ml, Difco Laboratories, Detroit, MI) was added with or without different doses of histamine, its Hr and H2- agonists, antagonists, histaminase, or alphafluoromethylhistidine. The plates were incubated at 37°C in a humid atmosphere of 95% air-5% CO2. After 48hr of incubation, 10,ul of 0.5 /iCi [6-3H]thymidine (specific activity, 25 Ci/mmol, AmershamInternational pic, Buckingham- shire, England) was added for overnight pulsing. The cells were harvested on a automated multiple-sample harvester and the cell-associated radioactivity was count- ed in a liquid scintillation counter. The results were expressed as the means +SEMof three experiments. HDCinduction and histamine biosynthesis. Spleen cells were suspended in GIT medium. The suspension was put Fig. 1. Effects of Con A and PHAon Histidine De- into plastic dishes with 3.5-cm diameters in a volume of carboxylase (HDC) Activity (Upper) and Histamine Lev- 2ml/dish containing 2.5 x 107 cells. Mast cells were not els (Lower) in the Culture of C57BL/6 Mouse Spleen detectable (<0.5%) in the spleen cell suspensions used in Cells as a Function of Incubation Time. this study asjudged by toluidine blue dye staining. Con A Spleen cells (2.5x 107cells/plate) were cultured in a (0.5/^g/ml) or PHA(5/ig/ml) was added to the cultures. The dishes were incubated at 37°C in the CO2incubator serum-free GIT medium in the presence of Con A (O» for 48 hr. After incubation, the cells and the mediumwere 0.5/zg/ml) or PHA (A, 5/ig/ml) or in their absence (#). separated by centrifugation at 250 x g. Cell viability was The results were expressed as picomoles of histamine assayed by trypari blue dye exclusion. The mediumwas produced per hour per plate. Each point represents the assayed for HDCactivity and histamine level as described mean+SEM (bars) of 3 experiments. Statistical differ- previously.8) ences between the control and the stimulated groups: */7 <0.05. Statistic analysis. Duncan'smultiple range test wasused after the analysis of variance or Student's f-test, whichever was a concomitant increase in the histamine was appropriate to each experiment.9) levels of the medium (lower panel). The ad- Results dition of Con A or PHA augmented the spontaneous increase of HDC activity and 1) Effects of Con A or PHAon HDCactivity histamine level of the medium. HDCactivity and histamine content in the culture of mouse increased also within the cells shortly after spleen cells initiation of the culture, reached a peak at 4hr, Culture of mouse spleen cells led to a spon- and declined rapidly (data not shown). These taneous increase in activity of HDC in the results were consistent with those shown culture medium (Fig. 1, upper panel). There previously,8) suggesting that the enzyme was

Optimal concentration of Con A was 0.25 /ig/ml. Regulation of Lymphocyte Blastogenesis by Histamine 379

Fig. 2. Effects of Concentrations of Histamine Hr and H2-Agonists (Left) and Antagonists (Right) on Con- A-induced Proliferation of MouseSplenic Lymphocytes. Results were expressed as the mean± SEMof3 assays. [A, unstimulated control, None; # =Con A plus either cimetidine (CMT), an H2-antagonist, or dimaprit (DM), an H2-agonist; O, Con A plus either diphenhydramine (DH), an Hx-antagonist, or 2-pyridylethylamine (PE), an H1-agonist.] released into the mediumsoon after its syn- 3) Effects of alphafluoromethylhistidine on thesis in the cell. Con-A-dependent lymphocyte transformation We then examined the effects of alpha- 2) Effects of histamine Hr and H2-agonists fluoromethylhistidine, a suicide inhibitor of and antagonists on Con-A-induced lym- HDC, on Con-A-dependent histamine biosyn- phocyte proliferation thesis and lymphocyte blastogenesis. Addition Figure 2 illustrates the effects of cimetidine, of the drug inhibited Con-A-mediated HDC an H2-antagonist, diphenhydramine, an Hr induction and histamine production in the antagonist, dimaprit, an H2-agonist, and 2- culture of spleen cells (Fig. 3b and c). pyridylethylamine, an Hx-agonist on the Con- Lymphocyte proliferation caused by Con A A-induced proliferation of mouse spleen lym- was enhanced by the drug at low doses. At phocytes. Cimetidine enhanced Con-A-me- high doses alphafluoromethylhistidine inhi- diated lymphocyte blastogenesis as a func- bited the reaction (Fig. 3a). tion of the dose, reaching a maximum 1.5-fold increase over the control value at 10~3m. At 4) Effects of histaminase on Con-A-mediat- 10~2 m, it completely suppressed the reaction. ed [3H]thymidine uptake by lymphocyte Diphenhydramine totally abolished Con-A- Next, weexaminedwhether cleavage of en- dependent mitogenesis at concentrations dogenous histamine by histaminase also mod- above 10~4m. 2-Pyridylethylamine enhanced ulates the mitogenesis of lymphocytes. The Con-A-mediated lymphocyte transformation addition of histaminase significantly enhanced at low concentrations, 10~9 to 10~7m, and it the incorporation of [3H]thymidine dependent suppressed the reaction when at 10~3m. on ConA by the spleen cells at concentrations Diamprit inhibited the response at 10~4m or between 0.1 to l.Omg/ml (Fig. 4, upper panel). above. The enzyme abolished the reaction at higher 380 C. Oh, H. Okamoto and K. Nakano

Fig. 4. Effects of Histaminase (EC 1.4.3.6) on Con-A- dependent Proliferation of Lymphocytes (a) and His- tamine Content (b) of Cultures of MouseSpleen Cells. For details, see Materials and Methods. Values are the mean ± SEMof 3 experiments. Significantly different from the culture with added Con A alone: */?<0.05.

concentrations. Histamine in the culture was degraded by histaminase as a function of its dose (Fig. 4, lower panel). 5) Effects of exogenous histamine on Con- A-induced lymphocyte transformation Next, weevaluated the effects of histamine itself on Con-A-induced lymphocyte proliferation. To minimize the effects of endogenously formed histamine, the effects of added histamine were estimated with a subopti- mumdose of Con A (two-fifth optimum). Fig. 3. Effects of Alphafluoromethylhistidine (a-FMH) The results are shown in Fig. 5. Histamine on Con-A-dependent Lymphocyte Blastogenesis (a), HDC Activity (b), and Histamine Levels (c) of Cultures of enhanced the Con-A-mediated uptake of [3H]thymidine at concentrations between Mouse Spleen Cells. For details, see Materials and Methods. Mean of 3 10~13m (151% of the response of the control experiments ± SEM. Significantly different from the cul- culture treated with Con A alone) and 10~n m ture with added Con A alone: */?<0.05. (134%). At higher concentrations, the amine Regulation of Lymphocyte Blastogenesis by Histamine 38 1 via interaction with H2-receptors. Our study was done to settle this question. Wefound that histamine stimulated mitogenesis via interaction with Ht-receptors and suppressed it via H2-receptors. Evidences were 1) that Con A-dependent lymphocyte proliferation was enhanced by a histamine H2- antagonist and suppressed by an Hr antagonist, 2) that 2-pyridylethylamine, an Hi-agonist, enhanced the Con-A-dependent lymphocyte response, but dimaprit, an H2- agonist, inhibited this response. It is note- worthy that the mitogenesis-stimulating actions of the Hj-agonist were exerted at lower doses, 10~9~l(r7M, than doses of >10~4m, at which the H2-agonist exerted its immuno suppressive actions. In addition, the Hr antagonist suppressed lymphocyte prolifer- Fig. 5. Effects of Concentration of Histamine on Con- A-stimulated Mitogenesis of MouseSplenic Lymphocytes. ation at doses as low as 10~4m, while im- Results were expressed as the mean± SEMof three assays. muno-enhancing actions of the ^-antag- Significantly different from the culture with added Con A onist were effective at 10~3m and 10~4m. alone: *p<0.05. These results were consistent with the findings that histamine itself augmented mitogenesis suppressed the response, to 75% and 64% at low doses and inhibited it at high doses. of the control value at 10"5 and 10~4m, It is important to know whether the immu- respectively. no suppressive actions of these histamine- agonists and antagonists are due to their tox- Discussion ic effects on the cells or not. Weevaluated the growth-inhibitory action of these drugs us- Immunomodulatory actions of histamine ing P388D1 macrophage-like cells. Diphenhy- are well documented regarding mitogen- dramine had no growth-inhibitory action on dependent lymphoproliferation.1 ~3) However, the cells at 10~4m, indicating that its inhibi- there are some disagreement about the subtype tory effect on lymphocyte blastogenesis at of the histamine receptor involved. Some in- this dose is not due to any harmful effect on vestigators have reported that histamine- the cells. Cimetidine inhibited the growth of induced suppression of mitogenesis is reversed P388D1 cellsby 36%at 1(T2mandby 31%at by H2- but not by Hrreceptor antagonist,1 ~3) 10~3m, at which the drug inhibited and en- others found that it was not.10'U) An Hr hanced the lymphocyte blastogenesis, respec- agonist also inhibits lymphocyte blastogenesis tively. Therefore, its immuno-modulating ac- in the same way as an H2-agonist does, and tions are not directly dependent on its cyto- these effects are abolished by H2- but not by toxic action. Pyridylethylamine inhibited the Hrantagonists.2) Other findings have been growth ofP388D1 cells by 29% at 1(T3m but that cimetidine, an H2-antagonist, enhances had no noxious effect at 10~7 and at 10~9m. mitogenesis, and that diphenhydramine, an Therefore, the mitogenesis-inhibitory effect of Ht-antagonist, depresses the lymphocyte re- the drug at 10~3m may be a nonspecific in- sponse even whenused without any exogenous jurious effect on the cells, but its immuno- histamine.1'12) Thus, it has been difficult to enhancing action at <10"7m is due to its decide whether histamine inhibits mitogenesis specific effect. Growth of P388D1 cells was 382 C. Oh, H. Okamoto and K. Nakano decreased by 34% by dimaprit at 10 4m. In contrast to the H2-receptor-mediated ac- Therefore, the immuno-suppressive action of tions of histamine, little information is avail- the drug can be ascribed to its toxic effect on able concerning Hi-receptor-dependent ac- the cells. tions of the amine. This study showed that We also showed here that these immuno- histamine enhanced lymphocyte blastogenesis regulatory actions of histamine were due to via the H1-receptor. The results seem to be those of histamine produced endogenously in consistent with the observation that a his- the system per se. The evidence was: 1) that tamine H1-receptor agonist, 2-pyridylethyl- culture of mouse spleen cells with ConA or amine, activates contrasuppression, although PHAincreased HDCactivity with a concom- the H2-receptor agonist dimaprit activates sup- itant accumulation of histamine in the culture, pressor cells.5) Those results, together with the 2) that histamine Hr and H2-antagonists results of this study, indicate that histamine regulated mitogenesis even in the absence of may have opposing actions. Endogenous his- any exogenous histamine, and 3) that partial tamine may stimulate lymphocyte blasto- degradation of endogenous histamine by low genesis via H1-receptors of target cells in the doses of histaminase or partial inhibition of early stages, when production of the amine is histamine biosynthesis by low doses of alpha- low. In later stages the large amount of his- nuoromethylhistidine, a suicide inhibitor of tamine that has accumulated in the culture HDC, enhanced Con-A-dependent mitogen- now may inhibit the response via H2-receptors. esis, although complete decomposition of the In this way, homeostasis is achieved. amine by high doses of histaminase or total inhibition of histamine synthesis by high doses References of alphafluoromethylhistidine abrogated Con- 1) B. E. Ogden and R. Hill, Immunology, 41, 104(1980). A-dependent lymphocyte blastogenesis. Our 2) A. Badger, J. Young and G. Poste, Clin. Exp. suggestion may explain the reason of previ- Immunol, 51, 178 (1983). ous findings which have not been fully under- 3) Y. Thomas, R. Huchet and D. Grandjon, Cell stood that antihistaminics have actions of Immunol, 59, 268 (1981). their own.1'4'6'12'13* For example cimetidine 4) I. S. Barsoum, H. S. S. Dahawi, F. M. Gamil, M. Habib, M. A. El Alamy and D. G. Colley, J. alone, in the absence of any exogenous hista- Immunol, 133, 1576 (1984). mine, enhances lymphocyte blastogenesis.M2) 5) J. N. Siegel, A. Schwartz, P. W. Askenase and R. K. Schnaper et al.6) have recently demonstrated Gershon, Proc. Natl Acad. Scl, U.S.A., 79, 5052 (1982). that addition of ConAto the culture of human 6) H. W. Schnaper, T. M. Aune and R. K. Roby, J. PBL activates suppressor T lymphocytes, lead- Immunol, 139, 1185 (1987). ing to production of soluble immuneresponse 7) A. Leutosakos and F. L. Pearce, Biochem. J., 36, suppressor (SIRS) and that histamine-type 2 1373 (1986). antagonists, cimetidine and ranitidine, ab- 8) C. Oh, S. Suzuki, I. Nakashima, K. Yamashita and rogates the response. However,no persuading K. Nakano, Immunology, in press. explanation has been given of these immuno- 9) S. Dowdyand S. Wearden, "Statistics for Research," modulating actions of histamine-type 2 antag- John Wiley and Sons, New York, 1983, pp. onists per se. Theresults of this study provide 10) J. Brostoff,262S. Pack and P.-272.M. Lydyard, Clin. Exp. a good explanation; histamine, which is pro- Immunol 39, 739 (1980). duced in the culture of immune cells per se, ll) D. Gorden, G. P. LewisandA. M. E. Nouri, Brit. J. would modulate mitogen-dependent lym- Pharmacol, 74, 137 (1981). phocyte blastogenesis or production of SIRS 12) R. R. M. Gifford, S. M. HatfieldandJ. R. Schmidke, Transplantation, 29, 143 (1981). by activated suppressor T cells. Cimetidine 13) D. M. Sahasrabuhde, C. S. Mccune, R. W. may block the actions of endogenous hista- O'Donnell and E. C. Henshaw, J. Immunol, 138, mine. 1185 (1987).