Objective　To investigate the function and characteristics of histamine receptors on the hemopoietic progenitor cells.
　　Methods　BDF1 mice (both male and female), inbred at our university, aged 8-12 weeks, weighing 20-24 g, were used in this study. Bone marrow cells were incubated for 1 hour at 37℃ with 2-AT (H1 receptor agonist) or impromidine (H2 receptor agonist) alone, or in combination with the antagonists pyrilamine or cimetidine respectively. Control experiment was performed in Dulbecco's modified Eagle's Medium (DMEM) alone. Cells treated with different drugs were performed by colony forming unit-granulocyte-macrophage (CFU-GM) and colony forming unit-megakaryocyte (CFU-Meg) assay.
　　Results　When bone marrow cells were treated with 10-8 mol/L to 10-5 mol/L of 2-thiazolylethylamine (2-AT) which had no influence on CFU-GM and CFU-Meg proliferation, 10-8 mol/L to 10-5 mol/L of impromidine could increase the number of CFU-GM and CFU-Meg colonies. The effects of H2 receptor agonists on CFU-GM, CFU-Meg could be antagonized by H1 receptor agonist.
　　Conclusions　Our findings suggest the existence of histamine H1 and H2 receptors on the hemopoietic progenitor cells and the antagonism between two different histamine receptor subtypes on the proliferation of CFU-GM and CFU-Meg.

Chin Med J 1998; 111(2):132-135

　　Histamine is a bioactive amine found extensively in various tissues of mamals. Byron1,2　first reported the involvement of histamine in hemopoiesis using 4-methylhistamine (4-MH, an agonist of histamine H2 receptor) to trigger CFU-S into cell cycle and cimetidine (a histamine H2 receptor blocker) to antagonize this effect. Xu YH 3　found that histamine H1 receptor existed on murine CFU-S. The effect of stimulating histamine H2 receptors to trigger CFU-S into cell cycle could be antagonized by 2-methylhistamine (2-MH, a histamine H1 receptor agonist). Recently, Yi SN and Xu YH 4　 further proved the existance of H1 and H2 receptors on CFU-S of mouse bone marrow cells and the antagonistic effect of these two histamine receptor subtypes on the cell cycle state of CFU-S.

METHODS

Chemicals
　　Dulbecco's modified Eagle's medium (DMEM), agar, methylcellulose, phytohemagglutinin (PHA), cimetidine, 2-mercaptoethanol (2-ME) and L-glutamine were all purchased from Sigma Chemical Co. (U.S.A); Erythropoietin (EPO) from AMGEN Thousand Oaks (U.S.A.). Pyrilamine, 2-thiazolylethylamine (2-AT), impromidine, dimaprit and 4-methylhistamine (4-MH) were kindly presented by Smith Kline & French Research Ltd.

Preparation of bone marrow cells
　　BDF1 mice (both male and female) inbred at our university, aged 8-12 weeks, weighing 20-24 g were used in this study. Bone marrow cells from mouse femurs were flushed with cold DMEM and allowed to pass 27-gauge needles and were kept in ice until use.

Cells treatment with different drugs
　　Bone marrow cells were incubated for 1 hour at 37℃ with 2-AT (H1 receptor agonist) or impromidine (H2 receptor agonist 10-8-10-5 mol/L) alone, or in combination with the antagonists pyrilamine (10-5 mol/L) or cimetidine (10-7-10-5 mol/L) respectively. The antagonists were added 15 minutes prior to the histamine agonists. Control experiments were performed in DMEM alone.

CFU-GM assay
　　The CFU-GM assay was performed by the method of Metcalf.5　Bone marrow cells were suspended in DMEM containing 26% horse serum, 10% conditioned medium from mouse lung, 0.3% agar, at a final conentration of bone marrow cells (10×104 /ml). The cell suspensions of 1 ml volume were planted into 35-mm culture dish. All cultures were done in triplicate and were incubated at 37℃ in a fully humidified atmosphere of 5% CO2. The colonies (≥50 cells) were counted after 7 days of culture. Different types of CFU-GM colony were identified by Giemsa stain in situ, and the percentage of each type was calculated.

CFU-Meg assay
　　Cultures were established in micro-methylcellulose by the method previously reported by Pan.6　Briefly, marrow mononuclear cells 1×105/0.2 ml were suspended in DMEM containing 26% horse serum, 10-4 mol/L 2-ME, 1 U/ml Epo, 5% phytohemagglutinin stimulated mononuclear cell conditioned medium (PHA-MCM), 0.3% L-glutamine and the culture was then made semisolid with 0.8% methylcellulose. After mixing, the cell suspensions of 0.2 ml in volume were dispensed into each well of the microplate and incubated at 37℃ in fully humidified atmosphere of 5% CO2 for 7 days. An aggregate of 3 or more cells were scored as a colony with an inverted microscope. The cells of CFU-Meg were positive stained by cholinesterase.

Statistical analyses
　　The standard student's t test was used to establish statistical significance.

RESULTS

The effects of histamine H1 and H2 receptor agonists on hemopoietic progenitor cells
　　2-AT alone at concentration of 10-8 to 10-5 mol/L had no significant effect on the colony number of CFU-GM and CFU-Meg, but impromidine at concentration of 10-8 to 10-5 mol/L could strikingly increase the number of CFU-GM and CFU-Meg colonies. The colony numbers reached peak at impromidine 10-6-10-5 mol/L (compared with control group P<0.01). When bone marrow cells were pretreated by cimetidine (a blocker of H2 receptor), the enhancing effect of impromidine or dimaprit on the production of CFU-GM and CFU-Meg colonies was abolished (Fig.1).

Fig. 1. Influence of histamine H1 and H2 receptor agonists on the colony number of CFU-GM and CFU-Meg. Results are expressed as ±s of four separate experiments.

The antagonistic effect of histamine H1 and H2 receptor on the production of CFU-GM and CFU-Meg
　　2-AT (10-5 mol/L) could antagonize the enhancing action of impromidine (10-5 mol/L) on the colony number of CFU-GM and CFU-Meg. This antagonizing effect was abolished by pretreatment of bone marrow cells with pyrilamine (10-5 mol/L, an antagonist of histamine H1 receptor, Fig.2).

The effect of H2 receptor agonist on the differentiation of CFU-GM
　　The percentage of granulocytic colonies among the total CFU-GM of murine bone marrow cells with impromidine treatment was significantly higher than the control group (P<0.01, Table).

DISCUSSION

　　Bone marrow and many tissues contain histamine. Under conditions such as irradiation, inflamation, allergic reactions and hypoxia, the concentration of histamine in bone marrow and blood was increased.7,8 The enhancement of erythropoiesis resulting from hypoxia could be attenuated by cimetidine treatment. Cimetidine might affect bone marrow

Fig. 2. The antagonistic effect of 2-AT (10-5 mol/L) and impromidine (10-5 mol/L) on the production of CFU-Meg and CFU-GM colonies. Results are expressed as the mean percentage of colonies ±S of four separete experiments. C: control; I: impromidine; A+I: 2-AT+impromidine; P+A+I: pyrilamine+2-AT+impromidine; * P<0.01, compared with control.

Table. Effect of impromidine on the differentiation of CFU-GM

Group Percentage of different types of colony
G M GM
Control 15.0±4.5 57.2±9.6 27.6±9.3
Impromidine (10-5 mol/L) 23.4*±5.5 53.0±11 23.5±9.3
　　Data are ±s of 3 seperate experiments. * P<0.01, compared with control. G: granulocytic colony; M: macrophagic colony; GM: granulo-macrophagic colony.

hemopoietic reconstruction of the mice after irradiation.9 The combination of cimetidine and recombinant alpha interferon had a synergistic inhibitory effect on the growth of CFU-GM.10 As cimetidine has been widely used clinically, many cases of agranulocytosis, thrombocytopenia and pancytopenia have been reported.11,12　The observation suggests that histamine partcipated in the regulation of hemopoiesis. The presence of histamine H1 and H2 receptors on hemopoietic stem cells of murine bone marrow has been evidenced.1-3 Anagnostou13 et al found that stimulating H1 receptor could inhibit proliferation of CFU-E and BFU-E. Recently, Schneider 14　 et al found that histamine modulates IL-3 induced 8-day CFU-S cell cycling via its H2-receptor. These show that histamine possesses an important regulating function in hemopoiesis, which prompts us to investigate the function of histamine receptors on hemopoietic progenitors. In this work, our data indicated that 2-AT (H1 receptor agonist) alone had no effects on the colony number of CFU-GM and CFU-Meg whereas impromidine (H2 receptor agonist) could strikingly increase the numbner of CFU-GM and CFU-Meg colony. The histamine H2 receptor antagonist, cimetidine could completely abrogate the effect of impromidine on CFU-GM and CFU-Meg proliferation, indicating the specificity of this effect. In addition, we found that 2-AT could antagonize the enhancing action of impromidine on the production of CFU-GM and CFU-Meg colonies. When bone marrow cells were pretreated by pyrilamine (an antagonist of histamine H1 receptor) prior to the addition of 2-AT, the effect of 2-AT antagonizing impromidine on CFU-GM proliferation was abolished. These results proved the existence of histamine H1 and H2 receptors on hemopoietic progenitors and the antagonism of these two histamine receptor subtypes on the proliferation of CFU-GM and CFU-Meg. We observed the differentiation of CFU-GM by morphological identification of colony cells. The percentage of granulocytic colonies after treatment of the bone marrow cells with impromidine was significantly increased as compared with that of the control group. This finding indicates that stimulation of the histamine H2 receptor may promote the differentiation of CFU-GM toward granulocytic cell line. This result is similar to the report by Nakaya.15

　　In conclusion, histamine has an effect on the proliferation and differentiation of hemopoietic progenitor (CFU-GM, CFU-Meg). Clinical administration of H2 receptor antagonists possibly causes granulocytopenia or pancytopenia. The intracellular and molecular mechanisms underlying the effect of histamine receptor agonists on the proliferation and differentiation of hemopoietic progenitors need to be further investigated.

Laboratory of Blood Physiology, Hunan Medical University, Changsha 410078, China (Tan MQ, Pan ZJ, Wang QR and Xu YH)
　　This work was supported by the National Natural Sciences Foundation of China (No. 39170323).

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