Objective To investigate the effect of calcitonin gene-related peptide (CGRP) on the regulation of group 2 innate lymphoid cells (ILC2) in the peripheral blood of patients with allergic rhinitis (AR). Methods Peripheral blood mononuclear cells (PBMCs) were extracted from normal healthy individuals and AR patients, then stimulated with CGRP, interleukin 33 (IL-33) and CGRP combined with IL-33 for 3 days, with blank stimulus as control. The percentage of ILC2 in the four groups was measured by flow cytometry. After being sorted, ILC2 was given to CGRP, IL-33 and CGRP combined with IL-33 stimulation for 3 days, with blank stimulus as control. The percentage of IL-5 and IL-13 positive cells in ILC2 was detected by flow cytometry, and the levels of IL-5 and IL-13 in ILC2 supernatant were measured by ELISA. Results The percentage of ILC2 in the peripheral blood of AR patients was significantly higher than that of the control group. The levels of IL-5⁺ILC2 and IL-13⁺ILC2 were significantly increased by IL-33 single stimulation after culturing PBMCs. After adding IL-33 combined with CGRP stimulation, the levels of IL-5⁺ILC2 and IL-13⁺ILC2 in PBMCs were significantly reduced; after CGRP single stimulation, the levels of IL-5⁺ILC2 and IL-13⁺ILC2 in PBMCs were further decreased. After ILC2 was sorted and cultured, the levels of IL-5⁺ILC2 and IL-13⁺ILC2 showed significant increase after IL-33 single stimulation. The levels of IL-5⁺ILC2 and IL-13⁺ILC2 were decreased by IL-33 and CGRP co-stimulation, and they were further reduced after CGRP single stimulation. Compared to IL-33 single stimulation, IL-5 and IL-13 levels dropped significantly due to the IL-33 and CGRP co-stimulation. The levels of IL-5 and IL-13 were further reduced by CGRP single stimulation. Conclusion CGRP inhibits the proliferation and activation of peripheral blood ILC2 in AR and exert anti-inflammatory effects in AR.
Humans ; Calcitonin Gene-Related Peptide/pharmacology* ; Leukocytes, Mononuclear ; Immunity, Innate ; Interleukin-33/pharmacology* ; Interleukin-13 ; Lymphocytes ; Interleukin-5/pharmacology* ; Rhinitis, Allergic ; Cell Proliferation

OBJECTIVETo evaluate the effect of intranasal liposome-mediated IL-12 gene therapy on the eosinophils and IL-5 in the murine model of allergic rhinitis.

METHODSThirty-six BALB/C mice were randomly divided into allergic rhinitis (AR) group, gene therapy group and control group. Allergic rhinitis group were sensitized and stimulated by ovalbumin (OVA), and gene therapy group were administered with liposome-mediated pGEG. mIL-12 transnasally before stimulated. The eosinophils in bone marrow were counted by Wright's staining, and the eosinophils in nasal mucosa were counted by HE staining. The eosinophils of peripheral blood were detected by flow cytometry. The expression of IL-5 in bone marrow and nasal mucosa was examined by immunohistochemistry. The IL-5 in serum was detected by ELISA.

RESULTSAmong the three groups, the difference of all data was statistically significant (P<0.01). Multiple Comparison showed that the ratio of eosinophils to white cells and the mount of IL-5 positive cells in nasal mucosa and bone marrow of gene therapy group was significantly lower than that of AR group (P<0.05). The ratio of eosinophils to granulocyte (0.124 +/- 0.031) and the expression level of IL-5 [(29.51 +/- 6.68) pg/ml] in peripheral blood [ 0.184 +/- 0.079 and (56.58 +/- 16.80) pg/ml] were significantly lower in gene therapy group than in AR group (P<0.05).

CONCLUSIONSTransnasal administration of liposome- mediated pGEG. mIL-12 could depress the expression of IL-5 in bone marrow, peripheral blood, and nasal mucosa, to influence the proliferation and differentiation of eosinophils and decrease the delivery and transference of eosinophils to peripheral blood and nasal mucosa. It may be a new treatment for respiratory tract allergic inflammation.

Animals ; Eosinophils ; metabolism ; Genetic Therapy ; Interleukin-12 ; genetics ; pharmacology ; Interleukin-5 ; metabolism ; Liposomes ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; metabolism ; Rhinitis, Allergic, Perennial ; metabolism ; therapy

OBJECTIVETo study the regulattory effect of Astragalus membranaceus on immune disturbance of the rats with IgA nephropathy.

METHODSRats IgA nephropathy (IgAN) model was duplicated by oral feeding of bovine serum albumin (BSA), subcutaneous injection of carbon tetrachloride (CCl4) and injection of lipopolysaccharide (LSP) into vena caudalis. The rats were divided into three groups randomly for the normal, IgAN model group and the group treated with Astragalus membranaceus (treatment group). The treatment group was given the Astragalus membranaceus granules via intragastric administratsion, the normal group and the IgAN model group were given the equal amount of aqua destillata by gastric perfusion. The rats were examined for albuminuria, hematuria and pathological changes of renal tissue and the distribution of TGF-beta and interleukin-5 in renal tissue was determined by immunohistochemistry and the IFN-gamma and IL-4 of cytokine of Th1 and Th2 types were detected in rats IgA nephropathy model by sandwich enzyme linked immunosorbent assay (ELISA).

RESULTS(1) The hematuria in rats with IgA nephropathy significantly increased compared with normal control group and Astragalus treatment group (P < 0.05). There was significant increase in albuminuria in rats with IgA nephropathy, compared with normal control group and astragalus treatment group (P < 0.01). (2) The pathological change of glomerular mesangium, renal tubules and renal interstitia became serious in rats IgA nephropathy model when compared with normal control group and astragalus treatment group. Immumofluorescence showed renal IgA density in rats IgA nephropathy model was significantly higher than that in the normal control group (P < 0.001) and astragalus treatment group (P < 0.001). (3) The result of immuno histochemistry showed that there was only weak expression of TGF-beta and interleukin 5 in normal renal tissue. The expression of TGF-beta and interleukin 5 in IgA nephropathy model was significantly stronger than those in normal control group (P < 0.05) and astragalus treatment group (P < 0.05). (4) The serum IL-4 levels were (33.74 +/- 7.52) pg/ml in rats IgA nephropathy model, significantly higher than that in normal control group (2.36 +/- 0.85) pg/ml and astragalus treatment group (3.24 +/- 1.13) pg/ml. The IFN-gamma level in serum of rats IgA nephropathy model was (18.79 +/- 3.80) pg/ml, which was significantly higher than that in normal control group (46.53 +/- 5.56) pg/ml and astragalus treatment group (41.28 +/- 2.95) pg/ml.

CONCLUSIONSThe astragalus could lower the level of hematuria and 24 hours-albuminuria of the IgAN model, and amelioratse the change of the renal pathology and reduce the deposit of IgA in glomerular mesangium. The possible mechanism of the effect is that astragalus could regulate the derangement of Th1, Th2, accordingly could improve the level of IL-4 and IFN-gamma in the serum and diminish the expression of cytokine Th2 TGF-beta1 and IL-5 of the renal tissue, and thereby could postpone the development of IgAN.

Animals ; Astragalus membranaceus ; chemistry ; immunology ; Cattle ; Drugs, Chinese Herbal ; pharmacology ; Glomerulonephritis, IGA ; immunology ; Interleukin-4 ; pharmacology ; Interleukin-5 ; pharmacology ; Kidney Tubules ; drug effects ; Rats ; Transforming Growth Factor beta ; immunology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo observe the inhibitory effect of mizolastine on substance P(SP)-induced production of leukotriene B(4) (LTB(4)) and interleukin 5 (IL-5) in mouse skin.

METHODSMice were fed with different doses of mizolastine or other control drugs, 30 min after administration animals were injected intradermally with SP on the back. The treated skin samples were taken and competitive enzyme-link immunoassay (ELISA) method was applied to detect LTB (4) and IL-5 in the skin samples.

RESULTThe LTB(4) and IL-5 levels in 10 mg/kg mizolastine group were (1.23 +/-0.29)pg/ml and (34.28 +/-11.00)pg/ml, respectively, which were lower than those in saline control group [(5.52+/-1.88)pg/ml and (179.62 +/-46.25)pg/ml respectively] or loratadine group [(3.89+/-1.27)pg/ml and (127.74 +/-43.27)pg/ml respectively]. No significant difference was found between 10 mg/kg mizolastine group and dexamethasone group (P=0.161 and P=0.508).

CONCLUSIONMizolastine might inhibit the production of LTB(4) and IL-5 induced by substance P in mouse skin, suggesting that anti-inflammatory effect and the blockade of histamine H1 receptors might be involved in its anti-pruritic mechanisms.

Animals ; Benzimidazoles ; pharmacology ; Female ; Histamine H1 Antagonists ; pharmacology ; Interleukin-5 ; biosynthesis ; Leukotriene B4 ; biosynthesis ; Male ; Mice ; Mice, Inbred BALB C ; Skin ; metabolism ; Substance P ; antagonists & inhibitors

BACKGROUNDExpression of murine calcium-activated chloride channel family member 3 (mCLCA3) has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin (OVA). However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified.

METHODSmCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid (BALF) were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC (MUC5AC) were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 (IL-13) were determined quantitatively.

RESULTSmCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-γ, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group (P < 0.05). The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALF. The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue.

CONCLUSIONThese findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.

Allergens ; Animals ; Asthma ; Chloride Channels ; Female ; Inflammation ; chemically induced ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-4 ; genetics ; metabolism ; Interleukin-5 ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Mucin 5AC ; genetics ; metabolism ; Ovalbumin ; pharmacology

OBJECTIVETo investigate the effects of polyinosinic-polycytidylic acid (polyI:C) on the production of thymic stromal lymphopoietin (TSLP) and airway inflammation in mice with exacerbated asthma induced by respiratory syncytial virus (RSV).

METHODSThirty-two female BALB/c mice were randomly divided into 4 groups, namely the PBS control group, OVA group, OVA/RSV group, and OVA/RSV/polyI:C group. In the latter 3 groups, the mice were sensitized by OVA and stimulated with nebulized OVA. RSV was inoculated into the nasal cavity of the sensitized mice and polyI:C (1 mg/kg) was intramuscularly administered. The airway response to metacholine was examined, and the serum levels of IL-4, IL-5, IL-13, and IFN-γ and TSLP in the supernatants of bronchoalveolar lavage fluid (BALF) were detected using ELISA. The total BALF cells, eosinophils, lymphocytes and neutrophils were counted. The lung specimens were collected to observe the inflammation with HE staining, and immunohistochemistry was employed to determine TSLP production in the airway epithelial cells.

RESULTSThe mice in RSV/OVA/polyI:C group showed a significantly lower airway responsiveness to metacholine than those in OVA/RSV group (P<0.01). Compared with OVA/RSV group, RSV/OVA/polyI:C group showed significantly lower serum levels of IL-4, IL-5, IL-13 and TSLP in BALF (P<0.05), with also lower total BALF cells, eosinophils and lymphocytes (P<0.05) and lessened infiltration of the airway inflammatory cells. Immunohistochemistry of TSLP also demonstrated a lower production of TSLP in the airway epithelial cells in RSV/OVA/polyI:C group than in OVA/RSV group.

CONCLUSIONSpolyI:C can inhibit the increase in TSLP production in the airway epithelial cells after RSV infection and relieve airway inflammation in mice with RSV-induced asthma exacerbation.

Animals ; Asthma ; blood ; metabolism ; virology ; Bronchoalveolar Lavage Fluid ; Cytokines ; secretion ; Female ; Inflammation ; pathology ; Interleukin-13 ; blood ; Interleukin-4 ; blood ; Interleukin-5 ; blood ; Mice ; Mice, Inbred BALB C ; Poly I-C ; pharmacology ; Respiratory Syncytial Virus Infections ; blood ; metabolism ; Respiratory Syncytial Viruses

OBJECTIVETo evaluate the effects of Staphylococcus aureus enterotoxin B (SEB) on proinflammatory cytokine/chemokine releases in primary human nasal epithelial cell (HNEC).

METHODSEpithelial cells of nasal polyps (NP) and inferior turbinate (IT) were cultured without serum under stimulus of SEB 1, 10, 100 ng/ml, IL-1beta 20 ng/ml and SEB 10 ng/ml + dexamethasone 13 ng/ml for 12,24 and 48 h, respectively. The expression of IL-5 and Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA derived from epithelial cells was detected by in situ hybridization.

RESULTS(1) The expression of IL-5 and GM-CSF mRNA was time and dose-dependent, and reached to a peak under SEB 10 ng/ml for 24 h (P < 0.05). The mRNA expressed more intensively in epithelial cells from NP than IT (P < 0.05). (2) The expression of IL-5 and GM-CSF mRNA increased less under the stimulus of IL-1beta than SEB 10 ng/ml (P < 0.05). (3) The mRNA level of IL-5 and GM-CSF decreased under the stimulus of SEB + dexamethasone 13 ng/m when compared with the stimulus of SEB 10 ng/ml (P < 0.05).

CONCLUSIONSEB showed proinflammatory effects on HNEC.

Cells, Cultured ; Dexamethasone ; pharmacology ; Enterotoxins ; pharmacology ; Epithelial Cells ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Interleukin-1beta ; metabolism ; Interleukin-5 ; metabolism ; Nasal Mucosa ; cytology ; metabolism ; pathology ; RNA, Messenger ; metabolism

OBJECTIVE: To investigate the influence of aluminum hydroxide adjuvant on a murine model of allergic rhinitis (AR) and to confirm an appropriate method of establishing a mouse model of AR. METHOD: Establishing two types of BALB/c mice models of AR, one was identified as Local group which was characterized through intranasal sensitization and challenge using ovalbumin (OVA), and the other Systemic group which was made by intraperitoneal sensitization with OVA plus aluminum hydroxide and intranasal challenge through OVA. Then the numbers of sneezing and nasal rubbing were counted after the last challenge and the eosinophils in the nasal mucosa of mice models were observed and counted though Luna stain. Furthermore, morphological hyperplasia was examined in intraepithelial goblet cells and submucosal glands with HE stain. In addition, interlukin (IL) -4, IL-5, OVA specific IgE (sIgE) and interferon (IFN)-gamma in nasal lavage fluid (NLF) and serum of mice were examined u sing enzyme linked immunosorbent assay (ELISA). RESULT: The counts of sneezing and nasal rubbing in local group were more than those in systemic group and eosinophilia in the nasal mucosa of former group was greater than that in the latter one. Morphological hyperplasia was stronger in intraepithelial goblet cells and submucosal glands in local group compared with that in systemic group. Furthermore, the contents of IL-4, IL-5 and sIgE increased in the NLF and serum of mice of local group compared to those of systemic one. However, the production of IFN-gamma of mice in local group decreased when compared with that in Systemic group. CONCLUSION OVA plus aluminum hydroxide adjuvant may promote Th1 type immune response as well as Th2 response. OVA intranasal sensitization and challenge locally is an appropriate way in the establishment of AR mice models.
Adjuvants, Immunologic ; pharmacology ; Aluminum Hydroxide ; pharmacology ; Animals ; Disease Models, Animal ; Immunoglobulin E ; immunology ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Interleukin-5 ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Rhinitis, Allergic ; immunology

We performed this study to investigate the feature of rejection in porcine-to-rat corneal orthotopic transplantation and to evaluate the effect of cyclosporine and mycophenolate on the xeno-rejection. Orthotopic corneal transplantation was done at 91 Sprague-Dawley rats, and they were divided into 10 groups based on the combination of immunosuppressants including dexamethasone, cyclosporine, and mycophenolate mofetil. Graft survival was analyzed and grafted eyes were examined with Hematoxylin & Eosin and CD4 or CD8 staining. Enzyme-linked immunosorbent assays were done for interleukin-2 (IL-2), IL-4, IL-5, IL-10, and interferon (IFN)-gamma in cornea, lacrimal gland, and cervical lymph nodes. The longest median survival of the immune suppressant group was 11.00+/-1.96 days, which showed no statistical differences compared with that of control (8.00+/-1.52 days). The neutrophils were prominent in the early phase but soon gave way to the monocytes. The number of CD8+ cells was higher than that of CD4+ cells. IL-2 and IFN-gamma markedly increased at 10 to13 days in cornea, lacrimal glands, and cervical lymph nodes, which showed a decrease with immunosuppressants except in the cornea. In conclusion, cyclosporine and mycophenolate could not prevent the rejection in porcine to rat orthotopic corneal xenograft associated with infiltraton of CD8+ and innate immune cells.
Animals ; *Corneal Transplantation ; Cyclosporine/*pharmacology ; Cytokines/metabolism ; Graft Rejection/immunology/pathology/*prevention & control ; Graft Survival/*drug effects ; Immunosuppressive Agents/*pharmacology ; Interferon-gamma/metabolism ; Interleukin-10/metabolism ; Interleukin-2/metabolism ; Interleukin-4/metabolism ; Interleukin-5/metabolism ; Mycophenolic Acid/*analogs & derivatives/pharmacology ; Neutrophils/immunology ; Rats ; Rats, Sprague-Dawley ; Swine ; Transplantation, Heterologous

BACKGROUNDCorticosteroids remain the most effective therapy available for asthma. They have widespread effects on asthmatic airway inflammation. However, little is known about the effects of corticosteroids on the production of bone marrow inflammatory cells in asthma. This study observed the effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD34+ hematopoietic cells, so as to explore the possible effectiveness of a bone marrow-targeted anti-inflammatory strategy.

METHODSBalb/c mice were sensitized and challenged with ovalbumin (OVA) to establish an asthmatic model. For two consecutive weeks, asthmatic mice were challenged with OVA while being given either prednisone, montelukast, prednisone plus montelukast, or sterile saline solution. The mice were killed 24 hours after the last challenge with OVA, and bronchoalveolar lavage fluid (BALF), peripheral blood, and bone marrow were collected. Eosinophils in peripheral blood and BALF, and nucleated cells in BALF, peripheral blood, and bone marrow were counted. The percentages of CD34+ cells, CD4+ T lymphocytes and CD8+ T lymphocytes among nucleated cells in peripheral blood and bone marrow were counted by flow cytometry. Immunocytochemistry and in situ hybridization were employed to detect expression of CD34 and interleukin (IL)-5Ralpha mRNA (CD34+ IL-5Ralpha mRNA+ cells) among bone marrow hematopoietic cells.

RESULTSCompared with the sterile saline solution group, the number of eosinophils in BALF and peripheral blood, CD34+ cells in peripheral blood and bone marrow, and CD34+ IL-5Ralpha mRNA+ cells in bone marrow of mice from the prednisone and prednisone plus montelukast groups were significantly lower (P < 0.01). The number of eosinophils in BALF from the montelukast group was also significantly lower (P < 0.05).

CONCLUSIONSThe results suggest that, in this asthmatic mouse model, prednisone probably inhibits proliferation, differentiation, and migration of CD34+ cells in bone marrow, blocks eosinophilopoiesis in bone marrow, and interferes with eosinophil migration into peripheral blood and subsequent recruitment in the airway. In addition, montelukast may suppress eosinophil infiltration into the lungs of asthmatic mice. However, a significant inhibitory effect of montelukast on the proliferation and migration of CD34+ cells and a cooperating effect with prednisone on bone marrow of asthmatic mice were not observed.

Acetates ; pharmacology ; Animals ; Antigens, CD34 ; analysis ; Asthma ; drug therapy ; Cell Count ; Hematopoietic Stem Cells ; drug effects ; Immunohistochemistry ; In Situ Hybridization ; Interleukin-5 ; biosynthesis ; Male ; Mice ; Mice, Inbred BALB C ; Prednisone ; pharmacology ; Quinolines ; pharmacology