Role and immunomodulatory profile of histamine receptors by H1 and H2 antagonists

The present study was designed to delineate the immunomodulatory role of histamine receptors (H1- and H2-) on induction of antibody response to sheep red blood cells (SRBC), as well as the antibody generation profile, in rabbit system, systemically. The rabbits in two groups received pheniramine (H1-receptor antagonist) and ranitidine (H2-receptor antagonist), respectively, via intramuscular route and were immunized with SRBC intravenously to evaluate suppression or enhancement of antibody responses in sem. A third, control group, received vehicle and were immunized in a similar manner. Histamine released from effector cells (mast cells and basophils) _in vivo_ during inflammatory reactions could influence a detectable antibody response to SRBC as early as day 7-postimmunization (post-I), which lasted until day 28- post-I. Pheniramine-treated rabbits had significantly (*Pa ≤ 0.05 and **Pa ≤ 0.01) more suppressed total serum antibody (IgM + IgG) to SRBC as compared to ranitidine-treated ad cotrol rabbits, while ranitidine-treated rabbits showed different pattern (suppressed or enhanced) during the whole study period. Ranitidine suppressed total antibody level at days 7- and 14- post-I, and enhanced at days 21- and 28- post-I. IgM suppression at day 7- and enhancement at days 14-, 21- and 28- post-I, while IgG suppression during whole study period, as compared to control group was significant (*Pa ≤ 0.05 and **Pa ≤ 0.01) as assessed by direct hemagglutination assay* ad whole SBC-ELISA method**. Here we report that histamine receptor type 2 (H2R)-antagonists have a dominant role on immunosuppression and in immunoregulation of humoral immune responses. Histamine receptor type 2 (H2R)-antagonists are mainly involved in B cell differentiation and proliferation over histamine receptor type 1 (H1R)-antagonists

Role of Protease-Activated Receptors 2 (PAR2) in Ocular Infections and Inflammation

Protease-activated receptors (PARs) belong to a unique family of G protein-coupled receptors (GPCRs) that are cleaved at an activation site within the N-terminal exodomain by a variety of proteinases, essentially of the serine (Ser) proteinase family. After cleavage, the new N-terminal sequence functions as a tethered ligand, which binds intramolecularly to activate the receptor and initiate signaling. Cell signals induced through the activation of PARs appear to play a significant role in innate and adoptive immune responses of the cornea, which is constantly exposed to proteinases under physiological or pathophysiological conditions. Activation of PARs interferes with all aspects of the corneal physiology such as barrier function, transports, innate and adoptive immune responses, and corneal nerves functions. It is not known whether the proteinase released from the microorganism can activate PARs and triggers the inflammatory responses. The role of PAR2 expressed by the corneal epithelial cells and activation by serine protease released from microorganism is discussed here. Recent evidences suggest that activation of PAR2, by the serine proteinases, play an important role in innate and inflammatory responses of the corneal infection

Role of Protease-Activated Receptors 2 (PAR2) in Ocular Infections and Inflammation

Protease-activated receptors (PARs) belong to a unique family of G protein-coupled receptors (GPCRs) that are cleaved at an activation site within the N-terminal exodomain by a variety of proteinases, essentially of the serine (Ser) proteinase family. After cleavage, the new N-terminal sequence functions as a tethered ligand, which binds intramolecularly to activate the receptor and initiate signaling. Cell signals induced through the activation of PARs appear to play a significant role in innate and adoptive immune responses of the cornea, which is constantly exposed to proteinases under physiological or pathophysiological conditions. Activation of PARs interferes with all aspects of the corneal physiology such as barrier function, transports, innate and adoptive immune responses, and corneal nerves functions. It is not known whether the proteinase released from the microorganism can activate PARs and triggers the inflammatory responses. The role of PAR2 expressed by the corneal epithelial cells and activation by serine protease released from microorganism is discussed here. Recent evidences suggest that activation of PAR2, by the serine proteinases, play an important role in innate and inflammatory responses of the corneal infection

CYTOSOLIC PHOSPHOLIPASE A2 ALPHA INHIBITORS ATTENUATE APOPTOSIS OF THE CORNEAL EPITHELIAL CELLS AND MITIGATE ACANTHAMOEBA KERATITIS

Purpose: The aim of this study was to explore if MIP-133 from Acanthamoeba castellanii trophozoites induces apoptosis of Chinese hamster corneal epithelial cells (HCORN) in vitro via cPLA2ɑ pathway, and to determine the efficacy of cPLA2ɑ inhibitors to alleviate AK in vivo. Methods: HCORN were incubated with or without MIP-133 at doses of 7.5, 15, and 50µg/ml for 6, 12, and 24hrs. Inhibition of cPLA2ɑ was carried out by pre-incubating HCORN for 1hr with cPLA2ɑ inhibitors (10µM MAFP and 20µM AACOCF3) with or without 15µg/ml MIP-133 for 24hrs. Chinese hamsters were injected subconjunctivally with MIP-133 at dosage 40µg/40µl and eyes were removed 3 days after infection. Chinese hamsters were infected with parasite-laden contact lens and treated with cPLA2ɑ inhibitors (AACOCF3 and CAY10650) topically 50µg/5µl three times a day for 14 days post-infection. Expression of cPLA2ɑ at mRNA and enzyme levels was examined by RT-PCR and cPLA2ɑ enzyme assay. Apoptosis was determined by DNA fragmentation assay. CXCL2 expression was examined by RT-PCR and ELISA. In vivo infections were examined by clinical severity of disease scored on a scale of 0 to 5 based on corneal infiltration, corneal neovascularization, and corneal ulceration. Clinical pathology of cornea was examined by histopathology. Results: MIP-133 induces significant increase in cPLA2ɑ and CXCL2 levels from corneal cells. Moreover, cPLA2ɑ inhibitors, MAFP and AACOCF3, significantly reduce cPLA2ɑ and CXCL2 from these cells (P<0.05). Additionally, cPLA2ɑ inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P<0.05). Subconjunctival injection of purified MIP-133 produced cytopathic properties characteristic of MIP-133. Treatments of cPLA2ɑ inhibitors showed significantly less severe keratitis and decreased clinical pathology as compared with control animals. Conclusions: Results suggest that cPLA2ɑ inhibitors therapeutically attenuate apoptosis of the corneal epithelial cells and mitigate AK.EY0975

Identification of Acanthamoeba Membrane Protein That is Recognized by TLR4 on Corneal Epithelial Cells

We have shown that Acanthamoeba spp. activate TLR4 on corneal epithelial cells and induce secretion of chemokines. However, the components of Acanthamoeba trophozoites that induce chemokines production remain unknown. We sought to identify the trophozoite molecules that interact with TLR4 on human corneal epithelial (HCE) cells and trigger IL-8 production. Acanthamoeba membrane protein (AcMP) was isolated by homogenization of trophozoites. The supernatants were collected, solubilized, and membrane fractions were separated by centrifugation using Mem-PERTM plus kit. To examine functional activity of AcMP, HCE and TLR4-expressing HEK293 cells were incubated with or without A. castellanii (1×105 cells/ml) and AcMP (10, 25, and 50 µg/ml) for 24 hours. AcMP was chromatographed by fast protein liquid chromatography (FPLC) and fractions were pooled into four peaks (AcMP-P1 - AcMP-P4). TLR4-ligand in AcMP-P1 - AcMP-P4 was determined by Western blotting. HEK293 and HCE cells were incubated with or without A. castellanii, lipopolysaccharide (LPS, 10 µg/ml), and AcMP-P1 - AcMP-P4 (20 µg/ml) for 24 hours. qRT-PCR and ELISA were used to examine the ability of AcMP-P1 - AcMP-P4 to stimulate IL-8 production in HEK293 and HCE cells. Inhibition of TLR4 involved preincubating HEK293 and HCE cells for 1 hour with neutralizing TLR4-antibody (10 µg/ml) or with the control antibody (10 µg/ml, goat serum) followed by incubation with or without A. castellanii, LPS, and AcMP-P2 for 24 hours. AcMP induced significant IL-8 production at doses of 10, 25, and 50 µg/ml in HEK293 cells while IL-8 mRNA expression and IL-8 secretion were significantly increased in HCE cells at the dose of 50 µg/ml. Treatments of HEK293 with FPLC chromatographed trophozoites’ proteins, AcMP-P1 - AcMP-P4; only AcMP-P2 upregulated significant IL-8 production and mRNA expression. Western blotting of AcMP-P1 - AcMP-P4 showed TLR4-antigen in AcMP-P2 and was recognized an approximate 15-kDa protein band. Anti-TLR4 antibody attenuated IL-8 secretion that is stimulated by AcMP-P2 from HEK293 and HCE cells. These results suggest that A. castellanii trophozoites recognize TLR4 on HCE and HEK293 cells by an approximate 15-kDa molecular mass protein of AcMP and induce IL-8 secretion

PROTEASE-ACTIVATED RECEPTOR 2 (PAR2) IS UPREGULATED BY ACANTHAMOEBA PLASMINOGEN ACTIVATOR (APA) AND INDUCES PROINFLAMMATORY CYTOKINE IN HUMAN CORNEAL EPITHELIAL CELLS

Acanthamoeba keratitis (AK) is a vision-threatening disease caused by pathogenic strains of Acanthamoeba. The main risk factors for AK are wearing eye contact lenses, corneal injuries, and contact with contaminated water. Diagnosis of AK is not straightforward and treatment is very demanding because of Acanthamoeba cysts resist to most antimicrobial agent. Our project is based on the observations that the innate immune system plays an important role in AK, and exploring the sequential pathogenic cascades of AK and targeting the therapeutic approaches to determine new remedies to prevent and treat AK. We have shown that Acanthamoeba trophozoites secrete cytopathic serine proteases, MIP-133 and aPA. Role of MIP-133 in the pathogenesis of AK via cPLA2α pathway has been explored; however, aPA interaction to corneal epithelial cells in disease progression is still unknown. In the present study, we have shown that aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK. Purpose (a): Acanthamoeba plasminogen activator (aPA), is a serine protease elaborated by Acanthamoeba trophozoites, facilitates invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA induces proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptor PAR2 pathway. Methods (b): A. castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system and aPA activity was determined by zymography assays. HCE cells were incubated with or without aPA (100µg/ml), PAR1-agonists (Thrombin, 10µM; TRAP-6, 10µM), and PAR2-agonists (SLIGRL-NH2, 100µM; AC55541, 10µM) for 24 hours. Inhibition of PAR1 and PAR2 involved pre-incubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH79797, 60μM) and PAR2 (FSLLRY-NH2, 100μM) and then incubated with or without aPA, Thrombin, TRAP-6, SLIGRL-NH2, and AC55541 for 24 hours. Expression of PAR1 and PAR2 was examined by qRT-PCR, flow cytometry, and immunocytochemistry. IL-8 expression was quantified by qRT-PCR and by ELISA. Results (c): PAR1 and PAR2 surface protein were expressed in HCE cells. aPA and PAR2-agonists significantly upregulated PAR2 expression (~1-2 times) (P<0.05). PAR2-antagonist significantly inhibited aPA and PAR2-agonists-induced PAR2 expression (~2-5 times) (P<0.5) in HCE cells. PAR1-agonists, but not aPA, significantly upregulated PAR1 expression, which was significantly inhibited by PAR1-antagonist (~30-45 times) in HCE cells. aPA and PAR2-agonists, but not PAR1-agonists, stimulated IL-8 production, which is significantly diminished by PAR2-antagonist (~2-10 times) (P<0.5). PAR1-antagonist did not diminish aPA-induced IL-8 production in HCE cells. Conclusions (d): aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK

Role and immunomodulatory profile of histamine receptors by H1 and H2 antagonists

AbstractThe present study was designed to delineate the immunomodulatory role of histamine receptors (H1- and H2-) on induction of antibody response to sheep red blood cells (SRBC), as well as the antibody generation profile, in rabbit system, systemically. The rabbits in two groups received pheniramine (H1-receptor antagonist) and ranitidine (H2-receptor antagonist), respectively, via intramuscular route and were immunized with SRBC intravenously to evaluate suppression or enhancement of antibody responses in sem. A third, control group, received vehicle and were immunized in a similar manner. Histamine released from effector cells (mast cells and basophils) in vivo during inflammatory reactions could influence a detectable antibody response to SRBC as early as day 7-postimmunization (post-I), which lasted until day 28- post-I. Pheniramine-treated rabbits had significantly (&amp;42;Pa &amp;le; 0.05 and &amp;42;&amp;42;Pa &amp;le; 0.01) more suppressed total serum antibody (IgM + IgG) to SRBC as compared to ranitidine-treated ad cotrol rabbits, while ranitidine-treated rabbits showed different pattern (suppressed or enhanced) during the whole study period. Ranitidine suppressed total antibody level at days 7- and 14- post-I, and enhanced at days 21- and 28- post-I. IgM suppression at day 7- and enhancement at days 14-, 21- and 28- post-I, while IgG suppression during whole study period, as compared to control group was significant (&amp;42;Pa &amp;le; 0.05 and &amp;42;&amp;42;Pa &amp;le; 0.01) as assessed by direct hemagglutination assay&amp;42; ad whole SBC-ELISA method&amp;42;&amp;42;. Here we report that histamine receptor type 2 (H2R)-antagonists have a dominant role on immunosuppression and in immunoregulation of humoral immune responses. Histamine receptor type 2 (H2R)-antagonists are mainly involved in B cell differentiation and proliferation over histamine receptor type 1 (H1R)-antagonists.</jats:p