Study On The Inhibitory Effects Of Thymic Immunosuppressive Pentapeptide On Airway Inflammation Of Asthma And Its Action Mechanism

ObjectivesAsthma, a chronic inflammatory disease, is prevalent worldwide. Airway inflammation, resulting from the interactions of various cells and mediators, is fundamental to asthma pathogenesis. All ages of people can suffer from asthma, and asthma has become a major global public health problem for its increasing prevalence. Asthma cannot be cured at present but only controlled with available drugs. Corticosteroids are the most effective and commonly used drugs for asthma treatment. However, severe local or systemic side effects will occur in serious asthma patients for whom high doses of inhaled corticosteroids and long term use are needed. Thus there is a need for the research and development of new antiasthmatic drugs which have high therapeutic effects and low adverse effects.Our laboratory began to study thymic immunosuppressive extract (TISE) in the 1980s. It was found that TISE could inhibit lymphocyte proliferation as well as immune and allergic responses effectively both in vitro and in vivo. TISE effectively suppressed delayed type hypersensitivity reaction on mice and passive cutaneous anaphylaxis on rats, significantly extended the survival time of transplanted skin in rats, and apparently inhibited OVA-induced asthma attacks in guinea pigs. For further investigation of the immunosuppressive activity and mechanism, low molecular weight immunosuppressive factors derived from calf thymus were fractionated with micrococcal nuclease digestion, acid hydrolysis and RP-HPLC separation, through tracking the immunosuppressive activity. After further isolation and purification, a novel pentapeptide with the sequence of Ala-Glu-Trp-Cys-Pro (Mw=604.68) was obtained from TISE, and was named as thymic immunosuppressive pentapeptide (TIPP). In view of the apparent effects of TISE against allergic and inflammatory responses, we speculated that TIPP might have therapeutic actions in allergic inflammation and can be used for the treatment of asthma. In order to confirm our speculation, we first evaluated the in vivo inhibitory effects of TIPP on airway inflammation of asthma with an ovalbumin (OVA)-induced mouse model of allergic asthma, then investigated the anti-allergic activity and action mechanism of TIPP with rat basophilic leukemia cells (RBL-2H3) in vitro. In addition, the HuProt Human Proteome Microarray was used to find the interaction protein of TIPP. As TIPP is a pentapeptide with a novel amino acid sequence, the relationship between the TIPP chemical structure and its immunosuppressive activity was preliminarily tested. The results of this project may find out a new mechanism for asthma treatment and lay foundation for the deep research and development of TIPP.Methods1 Study on the in vitro immunosuppressive activity of TIPP and its structure-activity relationshipThe cytotoxicity of TIPP and its inhibitory activity on lymphocyte proliferation were evaluated with splenocytes stimulated by different mitogens, and the effect on phagocytic activity of macrophages was tested with RAW264.7 cells stimulated by LPS. The structure-activity relationship was investigated by comparing the immunosuppressive activity of TIPP and its three derivatives, including (D-Glu2)-TIPP, [Ala(Ac)1-TIPP and (Ser4)-TIPP.2 Study on the in vivo inhibitory effects of TIPP on airway inflammation of asthmaFive-week-old female BALB/c mice (specific pathogen free) were sensitized with OVA adsorbed in aluminium hydroxide on days 0,7 and 14, and then were challenged with OVA in saline intranasally once daily for 6 consecutive days from days 28 to 33. All mice were analyzed 24 h after the last OVA challenge. Peripheral blood was collected for peripheral white blood cell (WBC) differential counting detection. OVA-specific IgE in serum and IL-4, IL-5, IL-13 and IFN-y in bronchoalveolar lavage fluid (BALF) were determined using ELISA kits. Immune cell subtypes in BALF were analyzed by flow cytometry. Lung tissue sections were stained with haematoxylin-eosin (HE) for inflammation and Periodic acid-Schiff (PAS) for mucus detection. mRNA levels of cytokines in mouse lung tissues were detected by fluorescent quantitative PCR. The expression of MCP-1, VCAM-1 and COX-2, and activation of mitogen-activated protein kinases (MAPKs) and NF-kB were determined by Western blot. In addition, guinea-pig trachea ring experiments were used to evaluate the effect of TIPP on airway smooth muscle contraction induced by histamine and methacholine.3 Study on the in vitro anti-allergic activity and action mechanism of TIPPFor further investigation on the in vitro anti-allergic activity and action mechanism of TIPP, RBL-2H3 cells were sensitized with anti-DNP-IgE and then stimulated with DNP-HSA. Release of β-hexosaminidase and histamine were determined as degranulation biomarkers. The intracellular calcium levels were detected with Fluo 3-AM, a calcium-specific fluorescent probe. The mRNA levels of pro-inflammatory cytokines were assessed by fluorescent quantitative PCR. F-actin was labeled with rhodamin-phalloidin to observe membrane ruffling. Besides, Western blot analysis was used for the detection of COX-2 expression and signaling pathway activation.4 Search for the target of TIPPConfocal microscope observation was used to detect the binding character of TIPP and RBL-2H3 cells. Then flow cytometry analysis was used to assess the effects of concentration, temperature, and incubation time on the interaction between TIPP and RBL-2H3 cells. The HuProtTM Human Proteome Microarray was used to find the interaction protein of TIPP and the possible target was speculated with prior results. In addition, the inhibitory activity of TIPP on the proliferation of K562 (Bcr-Abl positive) and HL60 (Bcr-Abl negative) was determined.Results1 In vitro immunosuppressive activity of TIPP was related to its structureThe proliferation assay showed that TIPP apparently inhibited splenocyte proliferation stimulated by Con A in a dose-dependent manner, while only had weak inhibitory effect on LPS stimulated splenocyte proliferation at high doses (200 and 800μg/mL). TIPP also significantly inhibited the up-regulation of the phagocytic activity of RAW264.7 cells stimulated by LPS. The cytotoxicity assay indicated that TIPP did not show cytotoxic effect on splenocytes and RAW264.7 cells. The results suggested that the inhibitory activity of TIPP was not relevant to cytotoxicity.The immunosuppressive activity of TIPP was relevant to its chemical structure and the -SH group in Cys4 could strengthen the immunosuppressive activity of TIPP. There was a significant decrease in TIPP activity when its-SH group is substituted by-OH group.2 TIPP shows inhibitory effects on lung allergic inflammationTIPP showed an apparent inhibitory activity on lung allergic inflammation in asthmatic mice, and had lower toxicity in vivo. TIPP significantly inhibited the increase in total BALF cell numbers and effectively resisted the balance changes of cells in BALF. Histological analysis showed that TIPP decreased the inflammation cell infiltration and goblet cell hyperplasia at peribronchial areas. Meanwhile, TIPP could inhibit the increase in Th2 cytokines and OVA-specific IgE production, mRNA levels of IL-4, TNF-a and eotaxin-1 and the expression of MCP-1, VCAM-1 and COX-2 in lung tissues, and could attenuate the activation of MAP kinases and NF-κB in the lung tissues of the allergic mice. Besides, the results of guinea-pig trachea ring experiments showed that TIPP had no effect on airway smooth muscle contraction induced by histamine and methacholine.3 TIPP effectively blocked Raf/MEK/ERK and NF-κB signaling pathwaysTIPP effectively suppressed IgE-mediated activation of RBL-2H3 cells during antigen stimulation process. TIPP significantly inhibited the degranulation in IgE-antigen complex-stimulated RBL-2H3 cells without cytotoxicity, suppressed the increase of intracellular calcium and the rearrangement of F-actin, attenuated the transcription of pro-inflammatory cytokines (IL-3,-4,-6,-13, TNF-a, MCP-1), and decreased the expression of COX-2. Western blot analysis showed that TIPP had an inhibitory activity on the activation of Raf/MEK/ERK signaling pathway and the nuclear translocation of NF-κB, but showed no effect on the activation of JNK, p38 and AKT.4 Grb2 is the possible action target of TIPPConfocal microscopic results showed that TIPP bound with the cell membrane and could get into cells. Flow cytometry analysis indicated the effect of concentration-, time-and temperature-dependence in the binding between TIPP and RBL-2H3 cells. The HuProtTM Human Proteome Microarray assay showed that TIPP had a specific interaction with Grb2 (grow factor receptor bound protein-2, FI=1187.500±27.577, SNR=26.159±0.998). As Raf/MEK/ERK is the main downstream pathway of Ras and Grb2 is an important adaptor protein for Ras activation, we supposed that TIPP is a Grb2 blocking agent and TIPP shows an anti-allergic activity through inhibiting Ras activation via blocking the interaction between Grb2 and SOS or pY in LAT. Besides, TIPP showed a higher inhibitory activity on the proliferation of K562 cells (Bcr-Abl positive) than on that of HL60 cells (Bcr-Abl negative), and could markedly arrested K562 cell cycle in G2 phase with a dose-dependent manner. The higher inhibitory activity of TIPP on the proliferation of K562 cells supported the hypothesis that TIPP is a Grb2 blocking agent.Conclusions and significances(1) The immunosuppressive activity of TIPP was studied for the first time. TIPP could inhibit splenocyte proliferation stimulated by Con A/LPS and inhibited the up-regulation of phagocytic activity of RAW264.7 cells stimulated by LPS without cytotoxicity.(2) The immunosuppressive activity of TIPP is relevant to its structure and the-SH group in Cys4 is crucial for the activity of TIPP.(3) The in vivo inhibitory effects of TIPP on airway inflammation of asthma were studied for the first time. TIPP effectively inhibited the lung allergic inflammation in asthmatic mice and had lower toxicity in vivo.(4) The in vitro anti-allergic activity of TIPP was evaluated for the first time. TIPP could inhibit the IgE-antigen mediated RBL-2H3 cell activation.(5) The action mechanism of TIPP was investigated for the first time. TIPP could block Raf/MEK/ERK signaling pathway and suppress the nuclear translocation of NF-κB. TIPP may be a Grb2 blocking agent and TIPP may show an anti-allergic activity through blocking Ras activation via binding with Grb2.(6) The action mechanism of TIPP as a Grb2 blocking agent was primarily verified. TIPP showed a higher inhibitory activity on the proliferation of K562 cells (Bcr-Abl positive) than on that of HL60 cells (Bcr-Abl negative), and could markedly arrested K562 cell cycle in G2 phase, which supported the hypothesis that TIPP is a Grb2 blocking agent.