International Journal of Cell Cloning 6: (1988) Research Laboratory of Blood Physiology, Hunan Medical College, Changsha, Hunan, PRC

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1 Original Paper International Journal of Cell Cloning 6: (1988) The Influence of Histamine at Various Concentrations on the Cell Cycle State of Hematopoietic Stem Cells (CF'U-s) I"i Shounan, XU You-Heng Research Laboratory of Blood Physiology, Hunan Medical College, Changsha, Hunan, PRC Key Words. Histamine Receptors. CFU-s Cell cycle Cimetidine * Pyrilamine Abstract. The influence of histamine at various concentrations on the cell cycle state of hematopoietic stem cells (CFU-s) was investigated. CFU-s were triggered from the Go state into the S phase of the cell cycle by in vitro treatment of mouse bone marrow cells with high concentrations of histamine. This effect could be antagonized by a histamine H2 receptor blocking agent. When bone marrow cells were treated with a histamine H, receptor antagonist prior to histamine treatment, low concentrations of histamine also triggered the entrance of CFU-s into the DNA synthetic phase. Our findings further suggest the existence of histamine H, and H2 receptors on the surface of CFU-s cells and the antagonistic effect of these two histamine receptor subtypes on the cell cycle state of CFU-s. Our results also suggest that histamine may participate in regulating the proliferation of hematopoietic stem cells in vivo during immune or inflammatory responses. Introduction Many types of bone marrow and tissue cells contain histamine. Histamine may play a role in regulating cell proliferation in certain tissues [l, 21 and histaminereleasing agents may meer hematopoietic stem cells (CFU-s) into S phase following in vitro exposure to bone marrow cells [3]. A histamine H2 receptor agonist, 4-methylhistamine (4-MH), may activate the adenylcyclase system, triggering Correspondence: Professor Xu You-Heng, The Research Laboratory of Blood Physiology, Hunan Medical College, Changsha, Hunan, PRC. Received January l3, 1988; provisionally accepted March 7, 1988; accepted for publication April 18, Press

2 Histamine Influence on the Cell Cycle State of CFU-s 291 hematopoietic stem cells (CFU-s) into the S phase of the cell cycle [4]. This effect could be antagonized by the histamine HI receptor agonist, 2-methylhistamine (2-MH)[5]. Our data showed that these two types of histamine receptors might be present on the surface of CFU-s and could affect the cell cycle state of CFU-s. Since histamine is a naturally occurring bioactive substance and is widely distributed in body tissues and cells, including the bone marrow cells, worthwhile information could be obtained by the determination of its effect on the proliferation of CFU-s. Therefore, this study investigates the influence of histamine on the cell cycle state of CFU-s. Materials and Methods Preparation of Bone Marrow Cell Cultures Male and female LACA mice, 8-12 weeks old, weighing g, served as either donors or recipients of bone marrow cells. The bone marrow cell suspensions were prepared by a procedure similar to that reported by Byron [2]. Bone marrow cells from intact femurs were flushed with cold Fischer s medium (GIBCO Laboratories, Grand Island, NY) and allowed to filter through gauze. One ml of prepared cell suspension (6 X lo6 nucleated cells/ml) was pipetted into sterile 12 x 75 mm polystyrene culture tubes (Fischer Scientific Co., Pittsburgh, PA). Fischer s medium, ph 7.4, was supplemented with 10 mmhepes buffer (Sigma Chemical Co., St. Louis, MO). Addition of Drugs to Culture Bone marrow cells were exposed in vitro to histamine (Sigma) alone over a range of concentrations (1 x M to 1 x lo-* M) for 1 h at 37T, or 1 x M cimetidine (Sigma) and 1 X M pyrilamine (Sigma) were added to the cultures 5 min prior to the addition of histamine. Following drug exposure, cultures were treated for an additional hour with 1 x M hydroxyurea (HU; Sigma) at 37 C. The control cultures received a comparable volume of Fischer s medium, in which the drugs were dissolved, bringing the final culture volume to 2 ml. CFU-s Assay CFU-s assay was performed according to the method of fill andmcculloch [6]. After exposure to HU, the cultures were diluted with cold Fischer s medium, and 6 X lo4 bone marrow cells contained in 0.2 ml medium were injected intravenously into total-body irradiated recipients. The radiation dose to recipient mice was 9.5 Gy (60Co gamma ray, 0.66 to 0.80 Gy/min). Spleen colonies were counted nine days later. A comparison was made between numbers of colonies per spleen for control and HU-treated cultures. The percent loss of CFU-s due to HU (suicide rate) was calculated. Statistical Method The unpaired Student s t test was used to compare the mean numbers of spleen colonies between two groups, and the curvilinear regression was used to compare the relationship between the suicide rate of CFU-s and the concentrations of histamine.

3 YiIXu 292 Fig. 1. Response of CFU-s to histamine (0) and facilitative effect of 1 x M pyrilamine (0) or antagonistic effect of 1 x M cimetidine (X) on the response of CFU-s to histamine. Results Figure 1 shows the response of CFU-s to histamine and compares the effect on the concentration-response curve for histamine of either 1 x 10-6 M pyrilamine or 1 x M cimetidine. Under the action of histamine alone, the sensitivity of CFU-s to the cytocidal action of HU was obviously increased at concentrations of histamine above 1 X lom4 M. When an H2 receptor blocker, cimetidine (1 X WM), was used prior to reaction of histamine with bone marrow cells, the doseresponse curve shifted to the right, and the suicide rates of CFU-s failed to show a significant increase until the histamine concentration was increased to 1 X 10-2 M. However, 1 x 10-6Mpyrilamine, a histamine HI receptor antagonist, resulted in increased sensitivity of CFU-s to the cytocidal action of HU. Although the concentration of histamine used to treat bone marrow cells was as low as 1 x M, the curve shifted to the left such that a 1,000-fold decrease in the concentration of histamine was effective enough to obtain a maximal increase in sensitivity of CFU-s to the cytocidal effects of HU as that of histamine alone.

4 Histamine Influence on the Cell Cycle State of CFU-s 293 Table I. Response of CFU-s to histamine CFU-s/spleen (mean f SE) Molar conc. of % Loss histamine without HU HU due to HU f 0.81(24) 8.10 f 0.58(23) 5.98 >0.10 lo-' f 1.21(27) f 0.70(28) 9.3b > f 1.46(36) f 0.94(28) 11.8b > f 1.04(30) 8.24 f 0.95(31) 33.1b co f 1.24(30) 7.64 & 0.98(27) 34.3b co.05 'Mean of three experiments bmean of four experiments ( ) = number of spleen counted P Discussion In order to assay the cell cycle state of CFU-s, an S phase-specific agent, HU, was used to inhibit or kill the cells in DNA synthesis phase. Generally, a majority of CFU-s in normal bone marrow are in a resting state (Go), with a relatively small number of them in DNA synthetic phase. Accordingly, they are insensitive to HU (less than 10% of suicide rate) [7]. Any agents that can trigger cells from the Go state into the S phase of the cell cycle are expected to increase the number of CFU-s inhibited by HU. The increased sensitivity (more than 30% of suicide rate) of the LAC4 mouse CFU-s to HU following exposure to high concentrations of histamine (llble I), and the antagonism of this response by the histamine Hz receptor blocking agent, cimetidine (Table 11), suggest that through the H2 receptor high concentrations of histamine may trigger CFU-s into S phase. The result is similar to that of treating bone marrow cells with 4-MH [4]. A histamine HI receptor agonist, 2-MH, has been reported to antagonize the effect of 4-MH on the cell cycle of CFU-s. Pretreatment of bone marrow cells with 4-MH (1 X loq8 M) alone showed a 30%-50% loss of CFU-s due to HU, while bone marrow cells treated with 4-MH (1 x 10-8 M) and 2-MH (1 x 10-6 M) together showed a loss due to HU of only 6%-18% [5]. In this study we demonstrated that when treating bone marrow cells with 1 x Mpyrilamine, a histamine HI receptor blocker, the numbers of CFUs driven into the S phase of the cell cycle were also significantly increased by a much lower concentration (1 x 10-8M) of histamine (Table II). This further suggests that the triggering effect of the Hz receptor on the cell cycle of CFU-s may be antagonized by the H, receptor. Our findings in this study also suggest the ex-

5 ~ ~- YiIXu 294 Table 11. Effect of cimetidine or pyrilamine on the response of CFU-s to histamine Treatment CFU-s/spleen (mean f SE) % Loss P Molar conc. of histamine without HU HU due to HU I x 10-5 M f 0.66(27) f 0.85(24) 7.3' >0.25 Cimetidine f 1.26(24) 9.25 f 1.02(23) 9.8' > f 0.86(26) 7.56 f 0.95(25) 17.7' >0.10 Histamine f 1.20(22) 5.76 * 0.75(25) 33.1' co.01 lo-' 9.52 f 0.63(21) 6.19 f 0.41(23) 35.0a co.01 1 x 10-6 M f 1.19(26) 9.88 * 1.16(25) 12.0' >0.10 Pyrilamine f 1.15(30) 8.08 i-0.81(32) 23.4b ~ f 1.06(31) 7.63 f 0.74(33) 23.3b C0.025 Histamine f 0.79(32) 6.28 f 0.93(32) 33.1b C0.025 'Mean of three experiments bmean of four experiments ( ) = number of spleen counted f 1.07(31) 8.15 f 0.78(33) 34.1b ~0.01 istence of HI and H2 receptors on the marrow CFU-s surface of the LACA mouse, and these two histamine receptor subtypes have been shown to interact in modulating the proliferation of CFU-s. Histamine plays an important role in the process of immune reaction or inflammation of the body. Normally, the concentration of histamine in blood plasma is in the range of 1 X M to 1 x 10-7 M [ 81. In an allergic situation, local concentrations of histamine in certain tissues may reach as high as 1 x M [9]. Some researchers reported that the number of receptors is dynamically regulated both by hormones and drugs that ordinarily combine with the receptors. Such regulation of receptor numbers in turn often control cellular sensitivity to the hormone or drug action [lo]. Our findings suggest that in normal conditions, because of low concentrations of histamine in bone marrow, the HI receptor may be more sensitive than the H2 receptor to the stimulation of histamine. Hence, additional CFU-s are not triggered into the S phase of the cell cycle. During the process of inflammation or allergic reaction, the concentration of histamine in bone marrow may increase, primarily stimulating the H2 receptor. The triggering effect of histamine on DNA synthesis in CFU-s is then released. Accordingly, the modulating effect of histamine on the proliferation of CFU-s may be the result of its own synthetic action on these two subtypes of histamine receptors.

6 Histamine Influence on the Cell Cycle State of CFU-s 295 Acknowledgments This work was supported by the National Natural Science Foundation of China. We gratefully acknowledge Professor Zhou Yunjiao and Dr. Jim Dnhao for their comments and suggestions. References Kahlson G, Rosengren E. New approaches to the physiology of histamine. Physiol Rev 1968;48: Byron JW. Mechanism for histamine H,-receptor induced cell-cycle changes in the bone marrow stem cell. Agent Actions 1977;7: Byron JW. Histamine H,-receptor and the hematopoietic stem cell (CFU-S). In: Clarkson B, Marks PA, Till JE, eds. Differentiation of Normal and Neoplastic Hematopoietic Cells. Cold Spring Harbor Conference on Cell Proliferation. New York: Cold Spring Harbor Laboratory, 1978: Byron JW. Pharmacodynamic basis for the interaction of cimetidine with the bone marrow stem cells (CFUs). Exp Hematol 1980;8: Xu YH. The antagonistic effect of histamine H2-receptor agonist and HI-receptor agonist on the cell-cycle response of hematopoietic cells. Bulletin of Hunan Medical College 1984;9:1-5. Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 1961;14: Becker AJ, McCulloch EA, Siminovitch L, Till JE. The differing demands for blood cell production on DNA synthesis by hemopoietic colony-forming cells of mice. Blood 1965 ;26: Beer DJ, Matloff SM, Rocklin RE. The influence of histamine on immune and inflammatory responses. Adv Immunol 1984;35: Adams M, Lichtenstein LM. In vitro studies of antigen-induced broncho-spasm: effect of antihistamine and SRS-A antagonist on response of sensitized guinea pig and human airways to antigen. J Immunol 1979;122: Leftkowitz RJ, Michel T. Plasma membrane receptor. J Clin Invest 1983;72: