Major royal jelly protein 3 modulates immune responses in vitro and in vivo

Abstract

We have recently shown that royal jelly has potent antiallergic properties in a mouse model of immediate hypersensitivity. However, it is still unclear which components of royal jelly exhibit antiallergic activity. In this study, we have screened for antiallergic factors in royal jelly based on inhibition of IL-4 production by anti-CD3 stimulated spleen cells derived from OVA/alum-immunized mice. Using a series of column chromatographies, we purified a 70 kDa glycoprotein, major royal jelly protein 3 (MRJP3), that suppresses IL-4 production. In in vitro experiments, MRJP3 suppressed the production of not only IL-4 but also that of IL-2 and IFN-γ by T cells concomitant with inhibition of proliferation. The MRJP3-mediated suppression of IL-4 production was also evident when lymph node cells from OVA/alum-immunized mice were stimulated with OVA plus antigen presenting cells. We next examined the purified suppressive factor on OVA/alum-induced allergic responses in mice. Interestingly, in spite of the antigenicity of MRJP3 itself as an extraneous foreign protein, intraperitoneal administration of MRJP3 inhibited serum anti-OVA IgE and IgG1 levels in immunized mice. In addition, heat-treated soluble MRJP3 treatment reduced its antigenicity while maintaining its inhibitory effects on antibody responses to OVA. These results indicate that MRJP3 can exhibit potent immunoregulatory effects in vitro and in vivo. Furthermore, considering the intriguing immunomodulatory effects of MRJP3, it may be of clinical significance to design MRJP3-derived antiallergic peptides by identifying the associated polypeptide regions.

Introduction

Royal jelly (RJ) is secreted from the cephalic glands of worker honeybees (Apis mellifera L), and directs the development of honeybee larvae into queen bees (Brouwers et al., 1987). RJ is composed of proteins (12–15%), sugars (10–16%), lipids (3–6%), vitamins, and free amino acids, and has been used for medical and nutritional purposes in folk medicine (Howe et al., 1985). Recently, five types of major royal jelly proteins (MRJPs; MRJP1-5) have been characterized by cDNA cloning and sequencing Albert et al., 1999, Klaudiny et al., 1994, Schmitzova et al., 1998. It was found that MRJPs belong to one protein family (60–70% amino acid sequence homology between the proteins) and account for over 80% of the proteins present in RJ.

The biological functions of some components of RJ have been described. The antibacterial activities of the 5.5 kDa protein royalisin, and trans-10-hydroxy-Δ²-decenoic acid found in RJ have been demonstrated against various Gram-positive and Gram-negative bacteria, respectively Fujiwara et al., 1990, Genc and Aslan, 1999. These two antibacterial components are thought to contribute to host defenses in honeybees. One of the MRJPs, MRJP1, which exhibits a molecular mass of 57 kDa as a monomer or 350 kDa as a hexamer after gel-filtration chromatography, promoted the viability and proliferation of primary cultured rat hepatocytes with ED₅₀ of 100 μg/ml Kamakura et al., 2001, Kimura et al., 1995. In addition, peptides derived from MRJP1 as a result of gastrointestinal enzyme hydrolysis, possessed potent angiotensin I-converting enzyme inhibitory activity in the spontaneously hypertensive rat (Matui et al., 2002). Furthermore, others and ourselves have demonstrated that a soluble fraction of RJ shows antiallergic activities, including reduced antigen-specific IgE levels in the sera of allergic mice, although the substance(s) in RJ showing this activity remains unknown Kataoka et al., 2001, Oka et al., 2001.

Th2 cells are immunologically the dominant cell type associated with several notable allergic reactions (Romagnani, 2000). Th2 cells secret interleukin-4 (IL-4), causing IgE class switching in B cells Barner et al., 1998, Fallon et al., 2002, Swain et al., 1990. It has been reported that OVA/alum-sensitized allergic mice harbor a systemic Th2 response characterized by elevated production of Th2 cytokines and antigen-specific IgE synthesis Brewer et al., 1996, Brewer et al., 1999. We have examined here which component in RJ has potent antiallergic activity, and have identified MRJP3 as a factor that inhibits T cell-derived cytokine production in vitro. Furthermore, we have found that MRJP3 shows novel immunomodulatory effects in vivo.