Inhibitory effect of interferon-beta on mouse spleen-derived mast cells

Preparations of murine recombinant interferon (Mu-rIFN)-alpha, -beta and -gamma were assessed for their influence on in vitro growth of mast cells from normal mouse spleen cells (Sp C). Mast cell growth was inhibited by Mu-rIFNs when Sp C were exposed throughout the entire culture period to Mu-rIFNs. The most potent inhibitor of mast cell growth was Mu-rIFN-γ, followed by Mu-rIFN-β; Mu-rlFN-α had little effect. When added to IC-2 cells, clonal mast cell progenitor, both Mu-rlFN-β and −γ), significantly inhibited proliferative response of the target cells. The suppressive effect of Mu-rIFNs on IC-2 cells was selectively abolished by monoclonal antibodies against Mu-rIFN-β and −γ.


Introduction
Interferons (IFNs) are well known to be a family of inducible proteins secreted by several types of cells in response to viruses and other stimuli. It is also generally accepted that IFNs can be divided into three different subclasses, IFN-z,-fl andbased on their structure, cellular origin and biological properties.
The effect of IFNs on cell proliferation was examined extensively by many investigators and IFN-z andhave been shown to suppress the in vitro proliferation and/or differentiation of human pulripotent hemopoietic progenitor cells and of committed progenitor cells. [1][2][3] Recently, Nafzigar et al., 4 using normal mouse bone marrow cells, examined the effect of IFN-y on mast cell growth and reported that IFN-), strongly inhibited their growth when bone marrow cells were exposed throughout the entire culture period to IFN-2 which seemed to exert its inhibitory activity on mast cell progenitors. There is, however, no evidence for a direct effect of IFN-2 on mast cell progenitors.
Furthermore, no information is yet available concerning the effects of IFN-z and -]5 on mast cell growth and proliferation.
The aim of the present study, therefore, is to evaluate the in vitro effects of IFN-0 and-15 on mast cells. In addition, studies were performed to investigate whether an inhibitory activity of IFN-2 on mast cell growth was due to a direct effect on progenitor ceils. Statistical analysis" Data from control and experimental groups were compared by Student's t-test.

Results
Influence of IFNs on mast cell growth" To examine effects of Mu-rIFNs on mast cell induction, 10 x 106 Sp C were cultured in the presence of either IFN-,-/3 or-(100 U/ml each), and the numbers of viable cells and mast cells were counted. As the culture time went on, viable cell numbers decreased gradually in the culture (Fig.  1A). When Sp C were cultured without Mu-rIFNs (Control), the mast cell numbers were gradually increased, peaked on the sixteenth day (Fig. 1B).
In contrast to the results obtained in cultures exposed to Mu-rIFN-0q a statistically significant suppression of mast cell growth was observed when Sp C were exposed throughout the entire culture period to Mu-rIFN-fl and -y (Fig. 1B).
The second experiments were carried out to examine whether mast cell growth inhibition by Mu-rlFN-/3 and -y was associated with concentration of Mu-rIFNs added to cultures. To accomplish this, 5 x 106 Sp C were cultured in the presence of four different doses of Mu-rIFNs and the number of mast cells was counted. As shown in Fig. 2, dose-dependent suppression was observed: 50 and 100 U/ml of Mu-rIFN-/3 failed to suppress mast cell growth but 500 and 1000 U/ml strongly suppressed them. The data in Fig. 2 also indicated that the suppression exerted by Mu-rIFN-y was stronger than that by Mu-rIFN-fl.
The third experiments were designed to examine the effects of Mu-rIFNs in different stages of mast cells. As shown in Fig. 3  growth. However, no such inhibition was observed when Mu-rIFNs were added to the cells after 10 days of culture.
In)quence of Mu-rlFNs on mast cell progenitors: The final experiments were carried out to examine whether Mu-rlFN-fl andexerted their inhibitory effects described above by acting directly on mast cell precursors. For this purpose, IC-2 cells, mast cell progenitors were cultured in the presence of Mu-rlFNs and cell proliferation was examined. As shown in Table 1, addition of Mu-rIFN-fl and -7 significantly inhibited IC-2 cell proliferation in dose   (Table 2).

Discussion
The results of this study demonstrated that: (1) both rIFN-fl and -, but not-, are potent inhibitors of murine mast cell proliferation; (2) rIFN-/ and-2 exert their inhibitory activity on mast cell progenitors, but not on mature mast cells. These conclusions are supported by the following observations: firstly, when Sp C were exposed throughout the entire culture period to rIFN-/ and -, a statistically significant and dose-dependent suppression of mast cell growth was observed, whereas no such inhibition was observed when Mu-rIFN-0 was added to the cultures at seeding of Sp C. Secondly, addition of rIFN-/ andto the culture medium at seeding strongly inhibited mast cell growth. However, addition of rIFNs 10 days after seeding, when differentiation of precursors and proliferation of immature mast cells are progressed, did not affect mast cell growth, as assessed by mast cell number observed on day 16. Finally, proliferation of IC-2 cells, clonal mast cell progenitors, was strongly inhibited when the cells were cultured in vitro with rIFN-/ and -7. This inhibition was completely neutralized when the IFNs were pre-incubated with a monoclonal antibody directed to the appropriate IFN type.
As  15 and on lepromatous leprosy 16 may depend on the activation of macrophages and T-cells, respectively. Mast cells are now considered to play a pivotal role not only in allergic reactions but also in many pathological immune responses, lv'18 It is also well known that infiltration of mast cells and the presence of mast cell-derived soluble mediators in the presence of asthma and atopic allergy are well correlated with the severity of the diseases. From these reports, whatever the mechanisms involved, our in vitro results suggest that IFN-fl and-y could act in vivo as inhibitory factors of mast cell proliferation and that these lymphokines could be of interest in the treatment of asthma and atopic allergy.