OBJECTIVEPrevious studies have shown that bacillus calmette-guerin (BCG) can deviate TH2 response toward TH1 response, resulting in a suppressive effect on the development of asthma/atopy. This study examined the effect of BCG treatment on regulatory T cells in asthmatic mice to investigate the possible mechanism.

METHODSKunming mice were sensitized and challenged with ovalbumin (OVA) to establish asthmatic models. Asthmatic mice were injected intradermally with BCG five days before and after sensitization. After 24 hrs of last challenge, bronchoaveolar lavage fluid (BALF) and peripheral blood were collected . The total cells and eosinophils were counted in the BALF. The percentage of CD4(+) CD25(+) in peripheral blood was detected with flow cytometry. Single spleen cell suspension was prepared and cultured in 1640 medium for 48 hrs and then the cytokine IL-10 level in the supernatant was determined using ELISA. The mice which were challenged with normal saline were used as the Normal control group.

RESULTSThe number of total cells and eosinophils in BALF in asthmatic mice [(27.27 +/- 5.36) x 10(7)/L and (6.59 +/- 1.32) x 10(7)/L respectively] were more than in the Normal control group [(1.52 +/- 0.36) x 10(7)/L and zero respectively] (P < 0.01). The number of total cells and eosinophils in BALF in asthmatic mice were reduced after BCG treatment [(13.71 +/- 3.17) x 10(7)/L and (1.43 +/- 0.37) x 10(7)/L respectively] (P < 0.01). The percentage of CD4(+) CD25(+) in peripheral blood of asthmatic mice [(11.59 +/- 1.33)%] was noticeably lower than that of the Control group [(13.66 +/- 1.68)%] (P < 0.01), but increased significantly in asthmatic mice after BCG treatment [(14.40 +/- 2.70)%] (P < 0.05). The IL-10 level in spleen cell supernatant in the BCG-treated group (7.79 +/- 1.34 pg/mL) also increased compared with that in the untreated asthmatic mice (5.54 +/- 0.66 pg/mL) (P < 0.01).

CONCLUSIONSBCG can markedly inhibit the airway inflammation in asthmatic mice possibly by promoting the production of regulatory T cells.

Animals ; Asthma ; immunology ; therapy ; BCG Vaccine ; therapeutic use ; Bronchoalveolar Lavage Fluid ; cytology ; Interleukin-10 ; analysis ; physiology ; Male ; Mice ; T-Lymphocytes, Regulatory ; immunology ; Toll-Like Receptor 2 ; physiology

OBJECTIVETo measure levels of interleukin-4 (IL-4) and interferon-gamma (INF-γ) in the bronchoalveolar lavage fluid (BALF) of children with refractory Mycoplasma pneumoniae pneumonia (RMPP), and to investigate changes in local Th1-Th2-type cytokine levels in children with RMPP and their significance.

METHODSA total of 42 children with RMPP were divided into atopic (n=11) and non-atopic groups (n=31) according to whether they had eczema, allergic rhinitis, urticaria, and family history of allergic disease. The study also included a control group of 12 children with bronchial foreign bodies who underwent foreign body removal and were re-examined by fiberoptic bronchoscopy four weeks later. The different cells in BALF from all children were analyzed, and the levels of IL-4 and INF-γ in BALF were measured using enzyme-linked immunosorbent assay.

RESULTSCompared with the control group, the total number of cells in BALF from children with RMPP increased significantly (P<0.05), the increase mainly accounted for by neutrophils (P<0.01), and levels of IL-4 and INF-γ in BALF from children with RMPP increased significantly (P<0.05). Compared with the control group, levels of IL-4 and INF-γ in BALF in the atopic group increased significantly (P<0.05). The level of INF-γ in BALF in the non-atopic group also increased significantly (P<0.05). There were no significant differences in INF-γ/IL-4 ratio among all groups (P>0.05).

CONCLUSIONSSignificant increase in cell numbers, especially neutrophils, as well as IL-4 and INF-γ levels, can be seen in BALF from children with RMPP, but there is no change to the INF-γ/IL-4 ratio. This indicates a significant local inflammatory response in children with RMPP, but there is no evidence of Th2-dominated inflammatory response.

Adolescent ; Bronchoalveolar Lavage Fluid ; cytology ; immunology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Interferon-gamma ; analysis ; Interleukin-4 ; analysis ; Male ; Pneumonia, Mycoplasma ; immunology

BACKGROUNDAsthma is a chronic airway disease with inflammation characterized by physiological changes (airway hyper-responsiveness, AHR) and pathological changes (inflammatory cells infiltration and mucus production). Eosinophils play a key role in the allergic inflammation. But the causative relationship between eosinophils and airway inflammation is hard to prove. One of the reasons is lack of activation marker of murine eosinophils. We investigated the expression of CD69 on murine eosinophils in vitro, the relationship between the expression of CD69 on eosinophils from peripheral blood and bronchoalveolar lavage fluid and on airway inflammation in asthmatic mice.

METHODSEosinophils from peripheral blood of IL-5 transgenic mice (NJ.1638) were purified. Mice were divided into five groups: wild type mice sensitized and challenged with saline (WS group), wild type mice sensitized and challenged with ovalbumin (WO group), IL-5(-/-) mice sensitized and challenged with saline and transferred with purified eosinophils (ISE group), IL-5(-/-) mice sensitized and challenged with OVA and transferred with purified eosinophils (IOE group), IL-5(-/-) mice sensitized and challenged with OVA and transferred with purified eosinophils, pretreated with anti CD4 monoclonal antibody (IOE+antiCD4mAb group). IL-5(-/-) mice were sensitized with OVA at day 0 and day 14, then challenged with OVA aerosol. On days 24, 25, 26 and 27 purified eosinophils were transferred intratracheally to IL-5(-/-) mice. On day 28, blood and BALF were collected and CD69 expression on eosinophils measured by flowcytometry.

RESULTSPurified eosinophils did not express CD69. But eosinophils cultured with PMA + MA, IFN-gamma, IL-5 or GM-CSF expressed CD69 strongly. Eosinophils from blood of WO, WS group did not express CD69 at all. The numbers of eosinophils in BALF of WO group, IOE group, ISE group and IOE + antiCD4mAb group were significantly higher than in mice of WS group which did not have eosinophils at all. CD69 expression on eosinophils in BALF of IOE and WO groups was strong. Eosinophils in BALF of ISE and IOE + antiCDmAb groups did not express CD69. The mucus production result was similar to CD69 expression. There were eosinophils infiltration in lung slides of all groups except WS group.

CONCLUSIONActivation in airway of eosinophils could directly lead to airway inflammation.

Animals ; Antigens, CD ; analysis ; Antigens, Differentiation, T-Lymphocyte ; analysis ; Asthma ; immunology ; physiopathology ; Bronchoalveolar Lavage Fluid ; cytology ; Eosinophils ; immunology ; Inflammation ; physiopathology ; Lectins, C-Type ; Lung ; physiopathology ; Mice ; Mice, Transgenic

The role of alveolar macrophages (AMs) in the pathogenesis of asthma is still unknown. The aim of the present study was to investigate the effects of AM in the murine model of asthma. AMs were selectively depleted by liposomes containing clodronate just before allergen challenges, and changes in inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid were measured. AMs were then adoptively transferred to AM-depleted sensitized mice and changes were measured. Phenotypic changes in AMs were evaluated after in vitro allergen stimulation. AM-depletion after sensitization significantly increased the number of eosinophils and lymphocytes and the concentrations of IL-4, IL-5 and GM-CSF in BAL fluid. These changes were significantly ameliorated only by adoptive transfer of unsensitized AMs, not by sensitized AMs. In addition, in vitro allergen stimulation of AMs resulted in their gaining the ability to produce inflammatory cytokines, such as IL-1beta, IL-6 and TNF-alpha, and losing the ability to suppress GM-CSF concentrations in BAL fluid. These findings suggested that AMs worked probably through GM-CSF-dependent mechanisms, although further confirmatory experiments are needed. Our results indicate that the role of AMs in the context of airway inflammation should be re-examined.
Animals ; Asthma/*immunology ; Bronchoalveolar Lavage Fluid/chemistry/cytology/immunology ; Cytokines/biosynthesis/immunology ; Disease Models, Animal ; Female ; Immunization ; Immunomodulation/*immunology ; Inflammation/*immunology ; Leukocytes/immunology ; Macrophages, Alveolar/*immunology ; Mice ; Mice, Inbred C57BL ; Ovalbumin/immunology

BACKGROUND: This study was purposed to find out the differences in the lymphocyte subsets and differential cell counts of the bronchoalveolar lavage (BAL) fluid in patients with interstitial lung disease (ILD) and to analyze the differences according to their ages, gender and smoking habits. METHODS: BAL fluid samples of 141 ILD patients were examined for lymphocyte subsets and differential cell counts, and the differences among the patients were analyzed according to their diseases. Then, within the three most common disease groups, the differences were further analyzed by the age, gender and smoking habit of the patients. RESULTS: There were no statistically significant differences in total cell counts (per millimeters of BAL fluid) among the patient groups with each ILD. However, significant differences were observed in the percentages of neutrophils, lymphocytes, eosinophils, and macrophages of BAL fluid. Also, in lymphocyte subset analyses, the percentages of total T cells, B cells, CD4+ T cells, CD8+ T cells, CD4/CD8 T cell ratios, and NK cells were significantly different among the patients with each ILD. However, within the same disease group, there were no differences in differential cell counts and lymphocyte subset analyses according to the age, smoking habit, and gender of the patients. CONCLUSIONS: Although the age, smoking habit and gender did not have an effect on the BAL fluid analyses among the patients with the same ILD, there were significant differences among the patients with each ILD; therefore, the differential cell counts and lymphocyte subset analyses of BAL fluid can be useful in differential diagnosis for determining the types of ILD.
Adult ; Aged ; Bronchoalveolar Lavage Fluid/*cytology ; Diagnosis, Differential ; Female ; Humans ; Lung Diseases, Interstitial/diagnosis/*epidemiology/etiology ; Lymphocyte Count ; Lymphocyte Subsets/immunology ; Male ; Middle Aged ; Smoking

OBJECTIVETo study the role of cell adhesion molecule CD44 in the lung on airway inflammatory response in rats with asthma.

METHODSThirty-two Sprague-Dawley rats were randomly divided into normal control and asthma groups. Asthma was induced by repeated inhalation of ovalbulium. CD44 expression in the lung was detected by semi-quantitatively reverse transcription-polymerase chain reaction (RT-PCR) and immuno-histochemical staining 1 week and 2 weeks after ovalbulium challenge. Differential leukocytes (mononuclear phagocytes, neutrophils, eosinophils, and lymphocytes) in bronchoalveolar lavage fluid (BALF) were counted.

RESULTSCD44 expression in the lung increased 1 week after ovalbulium challenge (<0.05) and increased more significantly 2 weeks after ovalbulium challenge (<0.01) compared with that in the control group. The percentages of lymphocytes and eosinophils in BALF increased, while the percentage of BALF mononuclear phagocytes decreased significantly 1 week and 2 weeks after ovalbulium challenge in the asthma group compared with those in the control group (<0.05). An increased percentage of neutrophils was found 1 week after ovalbulium challenge in the asthma group compared with the control group (<0.05). CD44 expression in the lung was positively correlated with the percentages of lymphocytes and eosinophils in BALF in the asthma group 1 week and 2 weeks after ovalbulium challenge, in contrast, lung CD44 expression was negatively correlated with the percentage of mononuclear phagocytes in the asthma group 1 week after ovalbulium challenge.

CONCLUSIONSCD44 was over-expressed in the lung and closely related to inflammatory infiltration in rats with asthma. CD44 may play an important role in the development of airway inflammatory in asthma.

Animals ; Asthma ; immunology ; Bronchoalveolar Lavage Fluid ; cytology ; Eosinophils ; physiology ; Hyaluronan Receptors ; analysis ; genetics ; physiology ; Inflammation ; etiology ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the changes in the levels of interleukin-17 (IL-17) and transforming growth factor beta 1 (TGF-β1) in serum and bronchoalveolar lavage fluid (BALF) and their clinical significance among children with asthma.

METHODSFifty-six children with asthma were divided into moderate or severe asthma (n=37) and mild asthma groups (n=19) and 18 children without asthma were selected as the control group. Cells in BALF were counted under a microscope. The levels of IL-17 and TGF-β1 in serum and BALF were measured using ELISA.

RESULTSwere no significant differences in total cell count and percentage of macrophages between the two asthma groups and the control group (P>0.05). The percentages of neutrophils, eosinophils and epithelial cells in BALF were significantly higher in the two asthma groups than in the control group (P<0.05). The two asthma groups had significantly higher levels of IL-17 and TGF-β1 in serum and BALF than the control group (P<0.05), and the moderate or severe asthma group had significantly higher levels of IL-17 and TGF-β1 in serum and BALF than the mild asthma group (P<0.05). Levels of IL-17 and TGF-β1 in serum were significantly positively correlated with those in BALF (r=0.935 and 0.943, P<0.05 for both). In children with asthma, serum IL-17 level was significantly positively correlated with the percentage of neutrophils, eosinophils and epithelial cells in BALF (r=0.802, 0.799, and 0.674, P<0.05 for all), and a significant positive correlation was also seen between serum levels of IL-17 and TGF-β1 (r=0.878, P<0.05).

CONCLUSIONSLevels of IL-17 and TGF-β1 in serum and BALF are elevated in children with asthma. IL-17 and TGF-β1 may be involved in the occurrence and development of asthma, and they play important roles in asthma attack and aggravation.

Asthma ; immunology ; pathology ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Interleukin-17 ; analysis ; blood ; physiology ; Male ; Transforming Growth Factor beta1 ; analysis ; blood ; physiology

OBJECTIVETo investigate the effect of respiratory syncytial virus (RSV) infection on the production of thymic stromal lymphopoietin (TSLP) and Th1/Th2 balance in asthmatic mice.

METHODSThirty-two female BALB/c mice were randomly divided into 4 groups, namely the PBS group, ovalbumin (OVA) group, RSV group and OVA/RSV group. The mice were sensitized by OVA and then stimulated with nebulized OVA, and RSV was inoculated into the nasal cavity of the mice. BUXCO noninvasive lung function detection was performed to examine the airway response to metacholine, and enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of IL-4, IL-5, IL-13, and IFN-gamma in the mice. The cells in the bronchoalveolar lavage fluid (BALF) were counted and classified, and the supernatants of the BALF were used for the detection of TSLP. Histopathological changes in the lung tissues of the mice were examined using HE staining, and immunohistochemistry using anti-mouse TSLP antibody was performed to examine TSLP expressions in the airway epithelial cells.

RESULTSRSV infection promoted the production of TSLP in the asthmatic mice, and the concentration of TSLP in OVA/RSV group (2.13-/+0.05 ng/ml) was significantly higher than that in the other groups (P<0.01). RSV infection increased the serum levels of IL-4, IL-5, IL-13, and IFN-gamma in the mice. The total BALF cells, eosinophils, lymphocytes and neutrophils in OVA/RSV group were significantly higher than those in the other groups; noninvasive lung function examination showed higher Penh value in OVA/RSV group (318.66-/+50.87) than in the other groups when the inhaled metacholine increased to 6.25 mg/ml (P<0.01). More obvious and extensive airway inflammatory cell infiltration in OVA/RSV group were observed, and immunohistochemical staining also showed higher expression of TSLP in the airway epithelial cells of OVA/RSV group.

CONCLUSIONSRSV infection promotes the production of TSLP in the airway epithelial cells and increases the level of Th2 cytokines in asthmatic mice. Concurrent RSV infection can exacerbate Th2 inflammatory reaction in asthmatic mice.

Animals ; Bronchoalveolar Lavage Fluid ; Cytokines ; biosynthesis ; secretion ; Female ; Immunohistochemistry ; Inflammation ; immunology ; virology ; Interferon-gamma ; blood ; Interleukins ; blood ; Lung ; immunology ; metabolism ; virology ; Mice ; Mice, Inbred BALB C ; Respiratory Syncytial Virus Infections ; blood ; immunology ; metabolism ; Th2 Cells ; cytology ; immunology ; virology

BACKGROUNDAsthma is clinically related with the degree of eosinophilic inflammation. How asthmatic airway inflammation is affected is still poorly understood. So the effects of bone marrow-derived hematopoietic cells expressing CD(34) (CD(34)(+)) and interleukin-5 (IL-5) receptor messenger RNA (IL-5R mRNA+) on asthmatic airway inflammation were investigated.

METHODSBalb/c mice were sensitized and challenged by ovalbumin (OVA) to establish an asthmatic model while control mice were sensitized and exposed to sterile saline. The mice were killed at different time points after being challenged by OVA and sterile saline. Then, bronchoalveolar lavage fluid (BALF), peripheral blood (PB) and bone marrow (BM) were prepared. Eosinophils in PB (PBEOS) and BALF (BALFEOS), nuclear cells in BALF, PB and BM were counted. By flow cytometry, the percentage of CD(34)(+) cells to nucleated cells in PB, BM and the relative number of CD(34)(+) cells in PB (PBCD(34)(+)) and BM (BMCD(34)(+)) were calculated. Immunocytochemistry and in situ hybridization were used to investigate the hematopoietic cells with co-localized expression of CD(34) and IL-5R mRNA in BM (BMCD34+IL-5R mRNA+). The percentage of BMCD34+IL-5R mRNA+ to BMCD(34)(+) was calculated.

RESULTSTwelve hours after challenge by OVA, BALFEOS and PBEOS in the experimental group were significantly higher than those in the control group (P < 0.01). Twenty-four hours after OVA challenge, BALFEOS, PBEOS and BMCD34+IL-5R mRNA+ were elevated maximally, significantly different from those in the control group (P < 0.01). Forty-eight hours after OVA challenge, BALFEOS and BMCD34+IL-5R mRNA+ were still significantly higher than those of the controls (P < 0.01). The other markers reverted to normal. In 60 mice, BMCD34+IL-5R mRNA+ was closely correlated with the BALEOS, PBEOS, BMCD(34)(+) and BMCD(34)(+) (%) (P < 0.05).

CONCLUSIONSThe amount of CD(34)(+) cells expressing IL-5R mRNA increased in the BM of asthmatic model mice, which favors eosinophilopoiesis and eosinophilic airway inflammation. A signal pathway exists between the lungs and the bone marrow, which is involved in the initiation and maintenance of asthmatic airway inflammation.

Animals ; Antigens, CD34 ; analysis ; Asthma ; immunology ; Bone Marrow Cells ; cytology ; Bronchoalveolar Lavage Fluid ; cytology ; Inflammation ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; Receptors, Interleukin ; genetics ; Receptors, Interleukin-5

OBJECTIVEEosinophilic airway inflammation is one of the basic characteristics of allergic asthma. Toll-like receptor is one of the most important innate immunity pattern recognition receptors. Glucocorticoids (GCS) are still the most effective treatment for asthma. However, few reports of studies on regulatory mechanism of GCS on the innate immunity system are available. The mechanism of effects of GCS on TLR4 is unclear. The present study aimed at understanding the effect of dexamethasone (DXM) on change of TLR4 and mechanism of regulatory effect of TLR4 on eosinophil (EOS) apoptosis.

METHODSTwenty-seven Sprague-Dawley (SD) rats (age 28 to 42 days, body weight 120 to 180 gram) were randomly divided into the control group, asthma group and DXM group with 9 in each. Asthma model rats were sensitized with the mixture of ovalbumin (OVA, 1 mg) and Al (OH)(3), 100 mg on day 1 and day 8, repeatedly exposed to aerosolized OVA after day 15, once a day for three days and continued for 30 minutes at every time. During the sensitization stage, 100 microg/ml DXM were prepared with DXM group for every other day, and the same doses DXM were prepared for every day on the stage of challenge. The histopathological changes of lung tissues were observed with light microscope (LM). EOS and other inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted; the concentrations of OVA-sIgE in serum were measured by using "sandwich" ELISA; The expressions of TLR4 mRNA were determined by in situ hybridization, the apoptosis of EOS was detected by TUNEL.

RESULTS(1) LM showed many inflammatory cells infiltration around the bronchi and blood vessels, bronchus mucus increased, airway epithelium damage and desquamation, and airway mucous plugs in asthma group, whereas DXM group showed significantly milder changes. (2) Inflammationary cells count in BALF of asthma group was significantly higher as compared to control group (P < 0.01); compared with asthma group, the total cell count, EOS absolute count and EOS% were all significantly decreased in DXM group [(2.14 +/- 0.10) x 10(9)/L, (4.78 +/- 1.23) x 10(7)/L, (2.17 +/- 0.25)%]. (3) Levels of OVA-sIgE in serum of asthma group [(83.40 +/- 6.80) microg/ml] were significantly higher than those of the control group [(14.38 +/- 4.25) microg/ml] (P < 0.01), while those of DXM group [(45.02 +/- 7.47) microg/ml] were significantly lower than asthma group (P < 0.0 1). (4) There were no significant differences in TLR4 mRNA detected by in situ hybridization between control group (24.71 +/- 0.85) and asthma group (25.81 +/- 3.56) (P > 0.05); but it significantly increased in DXM group (29.86 +/- 3.92) as compared to asthma group. (5) The percentages of apoptotic EOS in asthma group [(7.39 +/- 1.93)%] were significantly lower than those in control group [(9.06 +/- 1.52)%] (P < 0.01); and significantly higher in DXM group [(13.33 +/- 1.09)%] than in asthma group (P < 0.01). There were significantly positive correlations between TLR4 mRNA and the percentage of apoptotic EOS (r = 0.612, P < 0.01).

CONCLUSIONDXM can decrease OVA-sIgE level, induce EOS apoptosis, which may correlate with the activation of TLR4 signal transduction.

Animals ; Apoptosis ; Asthma ; chemically induced ; immunology ; Bronchoalveolar Lavage Fluid ; cytology ; Dexamethasone ; pharmacology ; Eosinophils ; immunology ; Glucocorticoids ; pharmacology ; Immunoglobulin E ; blood ; Lung ; pathology ; Ovalbumin ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; immunology ; metabolism