Interleukin-12 (IL-12) is a key cytokine that involved in the development of innate and acquired immunities. It is a potent stimulus for the production of interferon-γ (IFN-γ) in normal lymphocyte, resting and activated T cells and in NK cells (natural killer cells) [1]. The evolvement of cell-mediated immunity is promoted by IL-12. IL-12 has been shown to play roles in activating cytotoxic T cells and NK cells, upregulating the expression of surface receptors of helper T (Th) cells and NK cells, and stimulating IFN-γ secretion from T lymphocytes (Appendix 1). The early decision toward Th1 or Th2 cells is depends on the balance between IL-12, which favors Th1 response, and IL-4, which favors Th2 response [2].
In preclinical studies, IL-12 augments the cytotoxic activity of lymphocytes and the antitumor activity of NK cells from patients with tumors can be augmented the treatment of IL-12 [3-6]. IL-12 is used clinically to cure some inflammatory diseases, such as airway hyper-responsiveness, asthma and allergy [8-10]. However, IL-12 may involve in the pathological process of some autoimmune diseases. The peripheral blood mononuclear cells (PBMCs) from patients with progressive multiple sclerosis were shown to produce more IL-12 [11]. NOD (nonobese diabetic) mice, like type I diabetic patients, develop insulitis, an early infiltration of leukocytes into the pancreas that leads to inflammatory lesions within the islets [12 and 13]. The macrophage depleted NOD mice appear normal without the characteristics of type 1 diabetes, such as death of β cell and the type 1 response of the auto-reactive T-helper cells.
After injecting IL-12, the symptom of diabetes mellitus appeared in the macrophage-depleted NOD mice, suggesting that pathogenic effect of T-cell in type I diabetic patients might be mediated by IL-12 [14].
Active IL-12 is a heterdimeric complex consisting of two subunits, p40 and p35. Recent studies demonstrate that p40 can associate with either p35 to form
the active IL-12 p70 heterodimer, with p19 to form the active IL-23 p59 heterodimer or with itself to form an inhibitory p80 homodimer. The IL-12 p70 can be released by antigen- presenting cells (APCs), such as dendritic cell, macrophage, monocyte, and activated B cell by the stimulation of cytokines, pathogens, nutrients and stresses [15]. The p35 subunit of IL-12 is thought to be expressed constitutively, and regulated post-transcriptionally in most cell types [16 and 17]. Whereas, p40 subunit of IL-12 is inducible upon the stimulation of various stimuli.
Although genes can be regulated through several levels, transcription is the primary control for the gene expression [18]. Upon stimulation, the transcription of IL-12 p40 gene can be upregulated by transcription factors, such as Ets-2, ICSBP (IFN consensus sequence binding protein, also called IRF-8), NF-κB and IRF-1 (interferon regulatory factor-1) (Table 4).
Lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, is a potential activator of macrophages. It has been shown that LPS can induce the expression of IL-12 p40 gene in murine peritoneal macrophages by binding to receptor complexes, consisting of lipopolysaccharide-binding protein (LBP), CD14, Toll like receptor-4 (TLR4) and the accessory protein MD-2 [19 and 20]. Upon the binding its receptor stimulates the activity of mitogenic-activated protein kinase (MAPK) cascades (Appendix 2) [21].
These MAPKs activate different transcription factors, including Elk-1, AP-1, and CREB by phosphorylation. LPS may also activate IKKβ by serially activating MyD88, IRAK,-TRAF-6, and TAK1/MEKK1. Subsequently, IκBs is phosphorylated by IKKβ and targeted for degradation [22 and 23]. The degradation of IκBs permits NF-κB/Rel complex, e.g. p65/RelA, to translocate to nuclear [20]. NF-κB is a primary transcription factor that binds to the DNA sequence between -118 and -107 of the human IL-12 p40 gene [24] or between -132 and -122 of the murine IL-12 p40 gene [25]. Further studies found that
two other sites, Ets and C/EBP residing upstream (-211 to -205) and downstream (-75 to -64) of Rel/NF-κB site, respectively, are important for the trans-activation of Rel/NF-kB [15, 25 and 26]. Interestingly, Ets and C/EBP are conserved in both human and mouse genes.
Interferon-γ (IFN-γ), a cytokine mainly produced by T and NK cells, regulates the inflammatory and the antimicrobial/tumoricidal activity of macrophages by binding to its specific receptor [27]. The IFN-γ receptor, in turn, enhances the production of mediators, such as TNF-α, IL-1, NO, and IL-12, from macrophage [28]. The IFN-γ receptor consists of α and β chains, forming a heterodimer upon the binding of IFN-γ. Dimerization triggers receptor trans- phosphorylation and activates the associated JAK1 (Janus kinase) and JAK2 tyrosine kinases [29 and 30]. Subsequently, the activated JAK1 and JAK2 phosphorylate and activate downstream γ-activated factor (GAF), consisting of STAT1 (signal transduction ans activator of transcription) α dimmer and possibly another DNA binding protein. The activated GAF then upregulates transcription of IFN-γ-responsive genes by binding to their GAS (γ-activated site) [31-34]. IRF-1 and ICSBP, two GAF-activated proteins, have been shown to modulate important cellular events in response to IFN-γ [31 and 35]. ICSBP and IRF-1 can form a complex and binds to the Ets site in the promoter region from -224 to -214 of human IL-12 p40 gene [35].
In murine IL-12 p40 gene, ICSBP can associate with NF-AT (nuclear factor of activated T cell) and binds to the NF-AT site (-68 to -63) [17]. On the other hand, IRF-1 was found to activate the transcription by binding at site (-63 to -56) downstream the Rel/NF-κB. The binding of IRF-1 at this site could either up or down regulate the transcription of IL-12 p40 in response to different stimuli [31]. Mice with deficient of the IRF-1, IRF-2, or ICSBP exhibit a defect in IL-12 production [17, 36 and 37], suggesting the essential role of these transcription factors in the expression of IL-12. A synergistic
effect was observed on promoting the human IL-12 p40 gene in RAW264.7 cells with pretreatment of IFN-γ 8 h prior to LPS stimulation [2]. The promoter region responsible for the synergistic response of LPS and IFN-γ was located within -222 and -212 relative to the transcription start site of human IL-12 p40 gene [2]. These results suggest that multiple transcription factors probably work cooperatively to enhance the transcription of IL-12 p40 gene.
However, the cooperation between LPS and IFN-γ signal pathway is still unknown. Earlier works have shown that most known functional transcription binding site were located in the region between -250 and -50 of human IL-12 p40 gene (Fig. 1 and Table 4). Since the total length of reported promoter region of p40 gene is about 2500 basepairs, the promoter region upstream of -250 remain to be explored.
Owning to the importance of IL-12 on regulating cellular immune response and acting as bridge between innate and adaptive immunities, we have constructed a cell-based assay system for the screening of functional lead compounds from Chinese herbs that can modulate the transcription of IL-12 p40 gene. Various traditional Chinese herbs (Table 3) were chosen to screen for the potential lead components.
II. Materials and Methods
I. Materials
1. Cell Line
Murine macrophage cell line RAW264.7 and human embryonic kidney cell line HEK 293 cell lines were purchased from Food Industrial Research and Development Institute, Hsinchu.
2. Reagents
The crude extracts of various Chinese herb including coptis chinensis, Lian E Gu Jing, and Xiao Gu Jing, were provides by Ho,Jiau-Ching of Ta-Hwa Institute of Technology. Lipopolysaccharide (LPS) of Salmonella typhimurium and Escherichia coli G418, inhibitors or antagonists were from Calbiochem.
Restriction enzymes were from New England BioLabs. Prestained protein standard marker (broad range) and Bradford’s reagent were obtained from BioRad. BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit was purchased from ABI. Biomax film was from Kodak. Hyperfilm, PVDF and ECL were from Amersham. Nylon membrance was from Schleicher &Schaell.
The pfuTurbo DNA polymerase was from STRATAGENE. Taq DNA polymerase was from Viogene. Agarose, gel DNA extraction kit and BSA were from Boehringer Mannheim. T4-DNA ligase was from MBI. Fetal bovine serum (FBS) and Dulbecco’s modified egale medium (DMEM) were purchased from Hyclone. Penicillin, streptomycin, fetal calf serum (FCS), agarose, 0.25% trypsin-EDTA and ethidium bromide (EtBr) were purchased from GIBCO BRL. 3’end Biotin labeling kit, electropherotic mobility shift assay (EMSA) kit and Nonidat P-40 (NP-40) was purchased from PIERCE.
All other reagents and chemicals used in the experiments were reagent grade.
3. Primers
All the primers used in the thesis are listed in Table. 1.