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-pyridyl- ethylamine 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 re- leased 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 alphafluoromethyl- histidine. The plates were incubated at 37°C in a humid atmosphere of 95% air-5% CO2. After 48hr of incu- bation, 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 ex- pressed 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 diphenhyd- ramine (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 prolifer- ation.