Pulmonary perivascular and interstitial inflammation in MRL/MpJ-lpr/lpr mice. III. Modulation by cyclophosphamide and sex hormones in 4- and 6-month-old animals

Estradiol (E) abolished clearing of pulmonary inflammation in 2-month-old male MRL/MpJ-lpr/lpr (MRL/l) mice treated with cyclophosphamide (CY). To determine if this effect persisted in animals with advanced disease, we studied male and female MRL/l mice, aged 4 and 6 months (4M, 6M, 4F, and 6F, respectively). Mice were treated, beginning at 1 month of age, with saline, CY (12 mg/kg/day), CY + castration, CY + castration + testosterone (T) in females, and CY + castration + E in males. CY had no effect on pulmonary inflammation in 4M, possibly because of the development of relatively mild lesions. However, CY was highly effective in 6M. CY + castration + T significantly reduced overall inflammation in 6F and showed a trend in 4F. CY alone had a variable effect on bronchoalveolar lavage fluid (BALF) cells and BALF IgG in both males and females. However, concurrent treatment with T was required for histologic changes of pulmonary inflammation to fully respond to a high dose of CY in female mice. E-treated males had reduced responsiveness to CY.     

Role of T cells in bronchoalveolar space in the development of interstitial pneumonia induced by superantigen in autoimmune-prone mice

To study the mechanisms underlying the development of interstitial pneumonia in autoimmune disease, we analyzed bronchoalveolar lavage fluid (BALF) in an animal model of interstitial pneumonia in which an intratracheal instillation of staphylococcal enterotoxin B (SEB) induced interstitial pneumonia in autoimmune-prone mice. Increases in the numbers of total cells, macrophages, lymphocytes, and neutrophils were observed in BALF from SEB-treated MRL +/+ mice, and peaked at 3 d after SEB administration (Day 3). Flow cytometric analyses revealed increases in SEB-reactive Vbeta8(+) T cells, indicating that SEB-reactive cells play an important role in bronchoalveolar space. The expressions of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, JE/monocyte chemoattractant protein-1, regulated on activation, normal T cells expressed and secreted, and KC/gro messenger RNA (mRNA) in BALF cells from SEB-treated mice peaked at Day 3. Increased expression of TNF-alpha mRNA was observed mainly in macrophages and CD8(+) T cells, and the increase in IFN-gamma mRNA was observed mainly in CD8(+) T cells in BALF at Day 3. The expression of platelet-derived growth factor mRNA was very weak at Day 3 but strongly expressed at Day 14. An immunosuppressant, FK506, but not corticosteroid, suppressed SEB-induced T-cell expansion in BALF as well as increased cytokine and chemokine production in the bronchoalveolar space of SEB-treated mice. Histologically, FK506 but not corticosteroid significantly reduced both the cell infiltration to alveolar septal walls and the synthesis of pulmonary collagen fibers. Further, transfer of T cells of MRL +/+ mice with SEB into SCID mice gave rise to interstitial pneumonia. These results suggest that superantigen-reactive T cells in the bronchoalveolar space may trigger the development of interstitial pneumonia in this model.     

Metformin Reduces Bleomycin-induced Pulmonary Fibrosis in Mice

Metformin has anti-inflammatory and anti-fibrotic effects. We investigated whether metformin has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis in a murine model. A total of 62 mice were divided into 5 groups: control, metformin (100 mg/kg), BLM, and BLM with metformin (50 mg/kg or 100 mg/kg). Metformin was administered to the mice orally once a day from day 1. We sacrificed half of the mice on day 10 and collected the bronchoalveolar lavage fluid (BALF) from their left lungs. The remaining mice were sacrificed and analyzed on day 21. The right lungs were harvested for histological analyses. The messenger RNA (mRNA) levels of epithelial-mesenchymal transition markers were determined via analysis of the harvested lungs on day 21. The mice treated with BLM and metformin (50 mg/kg or 100 mg/kg) showed significantly lower levels of inflammatory cells in the BALF compared with the BLM-only mice on days 10 and 21. The histological examination revealed that the metformin treatment led to a greater reduction in inflammation than the treatment with BLM alone. The mRNA levels of collagen, collagen-1, procollagen, fibronectin, and transforming growth factor-β in the metformin-treated mice were lower than those in the BLM-only mice on day 21, although statistical significance was observed only in the case of procollagen due to the small number of live mice in the BLM-only group. Additionally, treatment with metformin reduced fibrosis to a greater extent than treatment with BLM alone. Metformin suppresses the inflammatory and fibrotic processes of BLM-induced pulmonary fibrosis in a murine model.     

The role of high mobility group box1 in pulmonary fibrosis

High mobility group box1 protein (HMGB1) was originally discovered as a nuclear binding protein, and is known to play an important role in acute lung injury. However, the role of HMGB1 in pulmonary fibrosis has not been addressed. Therefore, we measured the HMGB1 levels in serum and bronchoalveolar lavage fluids (BALF) from patients with idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia, interstitial pneumonia associated with collagen vascular diseases, and hypersensitivity pneumonitis (HP) by enzyme-linked immunosorbent assay. We also assessed the HMGB1 expression in bleomycin-induced pulmonary fibrosis in mice, and examined the effect of anti-HMGB1 antibody and ethyl pyluvate, which inhibits the HMGB1 secretion from alveolar macrophages. In addition, we examined the effect of HMGB1 on fibroblast proliferation, apoptosis, and collagen synthesis in vitro. Serum HMGB1 levels were not significantly increased in interstitial lung diseases compared with control subjects. BALF HMGB1 levels were significantly increased in IPF and HP compared with control subjects. HMGB1 protein was predominantly detected in inflammatory cells and hyperplasic epithelial cells in IPF. In bleomycin-induced pulmonary fibrosis in mice, HMGB1 protein was predominantly up-regulated in bronchiolar epithelial cells at early phase and in alveolar epithelial and inflammatory cells in fibrotic lesions at later phase. Intraperitoneal injection of anti-HMGB1 antibody or ethyl pyluvate significantly attenuated lung inflammation and fibrosis in this model. HMGB1 significantly induced proliferation, but not apoptosis or collagen synthesis on cultured fibroblasts. HMGB1 may be a promising target against pulmonary fibrosis as well as acute lung injury.     

Characterization of p40 and IL-10 in the BALF of patients with pulmonary sarcoidosis.

This study investigated cytokine protein levels (interleukin-12 p70 [IL-12p70], p40, and IL-10) in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis (n = 59), healthy control subjects (n = 17), and patients with idiopathic pulmonary fibrosis (IPF) (n = 30). The relationship between cytokine levels and clinical course of sarcoidosis was also examined. Overall, p40 was far more abundant than IL-12p70. p40 levels (pg/ml, mean +/- SEM) were significantly higher in the BALF from patients with sarcoidosis (2.97 +/- 3.69) than in IPF patients (0.83 +/- 1.57) and healthy subjects (0.78 +/- 1.00). Size exclusion chromatography indicated that p40 detected in BALF from sarcoidosis patients corresponded to p40 monomers or (p40)(2) homodimers. Further, p40 levels were associated with (paralleled) the clinical course of sarcoidosis, with the highest levels detected in BALF from patients with persistent disease. Higher p40 levels were also found in the BALF from sarcoid patients who required corticosteroid treatment compared with patients with spontaneous regression (3.51 +/- 3.83 vs. 2.01 +/- 3.43, p = 0.03). IL-10 concentrations paralleled p40 changes. No similar association was found for IL-12p70 levels. In conclusion, this report shows that the BALF from patients with sarcoidosis contains elevated levels of p40, (p40)(2), and IL-10 protein but not of IL-12p70. The present data also suggest that BALF p40 concentrations may be indicative of the sarcoidosis clinical course.     

活菌卡介苗对哮喘小鼠IL-17的调节作用

目的初步探讨活菌卡介苗（BCG）对哮喘小鼠IL-17的调节作用。方法 4周龄雌性Balb/c小鼠30只随机分为A组（对照组）、B组（哮喘组）和C组（BCG干预组）,每组各10只。B、C两组予OVA腹腔注射致敏、OVA雾化诱发哮喘,C组在致敏前14d接种BCG。HE染色观察小鼠肺部病理改变,计数支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）中细胞总数并分类,酶联免疫吸附试验（ELISA）检测血清及BALF中白介素-17（IL-17）含量。结果肺组织病理观察显示A组气管周围基本无炎症细胞浸润,B组支气管周围大量炎症细胞浸润,杯状细胞增生,C组炎症较B组减轻。B、C组BALF中细胞总数和嗜酸性粒细胞、淋巴细胞百分率均显著高于对照组（P〈0.01）,而C组嗜酸性粒细胞比例则明显低于B组（P〈0.01）。B、C组小鼠血清、BALF中IL-17含量高于A组（P〈0.01）,而C组则明显低于B组（P〈0.01）。各组小鼠血清及BALF中IL-17水平与BALF中嗜酸性粒细胞呈正相关（P〈0.01）。结论 BCG接种可抑制IL-17的产生,减轻哮喘小鼠气道炎症反应。     

Extracellular matrix powder protects against bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis refers to a group of lung diseases characterized by inflammation, fibroblast proliferation, and excessive collagen deposition. Although the mechanisms underlying pulmonary fibrosis are poorly understood, current evidence suggests that epithelial injury contributes to the development of fibrosis. Regenerative medicine approaches using extracellular matrix (ECM) scaffolds have been shown to promote site-specific tissue remodeling. This led to the hypothesis that particulate ECM would promote normal tissue repair and attenuate bleomycin-induced pulmonary fibrosis. C57BL/6 mice were treated intratracheally with bleomycin or saline with or without a particulate form of ECM scaffold from porcine urinary bladder matrix (UBM-ECM) or enzymatically digested UBM-ECM. Mice were sacrificed 5 and 14 days after exposure. Compared to control mice, bleomycin-exposed mice had similar increases in inflammation in the bronchoalveolar lavage fluid regardless of UBM-ECM treatment. However, 14 days after exposure, lung histology and collagen levels revealed that mice treated with bleomycin and the particulate or digested UBM-ECM had negligible fibrosis, whereas mice given only bleomycin had marked fibrosis. Administration of the particulate UBM-ECM 24 h after bleomycin exposure also significantly protected against pulmonary injury. In vitro epithelial cell migration and wound healing assays revealed that particulate UBM-ECM promoted epithelial cell chemotaxis and migration. This suggests that promotion of epithelial wound repair may be one mechanism in which UBM-ECM limits pulmonary fibrosis.     

Susceptible cytotoxicity to ultraviolet B light in fibroblasts and keratinocytes cultured from autoimmune-prone MRL/Mp-lpr/lpr mice

The MRL/Mp-lpr/lpr (MRL/l) mouse is an autoimmune model of spontaneous lupus erythematosus (LE), in addition to lupus nephritis. In order to better understand the mechanisms of photosensitivity in LE, in vitro photocytotoxicity was examined by using fibroblasts and keratinocytes cultured from MRL/l mice, control MRL/Mp- +/+ (MRL/n) mice, and normal BALB/c mice. A colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and the acridine orange/ethidium bromide assay were used for determination of cytotoxicity. Fibroblasts cultured from newborn MRL/l mice showed higher susceptibility to single ultraviolet light B (UVB) light irradiation at a dose of 100-500 mJ than those from MRL/n, F1 hybrid of (MRL/l x MRL/n mice), and BALB/c mice. However, the susceptibility to UVB was not observed in young (1-month-old) and adult (4-month-old) MRL/l mice. UVA light irradiation was not cytotoxic. Keratinocytes cultured from MRL mice showed lower cytotoxicity to UVB irradiation than fibroblasts cultured. However, keratinocytes from newborn MRL/l mice showed higher cytotoxicity to 50 mJ UVB irradiation than cells from MRL/n mice. Syngeneic or allogeneic sera augmented UVB-induced cytotoxicity of fibroblasts cultured. UVB irradiation of spleen cells induced no significant difference of cytotoxicity between MRL/l and MRL/n mice. Based on the results of F1 hybrid of (MRL/l x MRL/n) mice, the susceptibility seemed to be associated with autoimmune traits and to be regulated by genetical background.     

Measurement of small quantities of alpha 2-macroglobulin in bronchoalveolar lavage fluid by enzyme-labelled immunoassay, and its clinical implication in pulmonary disease

We established an enzyme labelled immunoassay for the determination of alpha 2 macroglobulin (alpha 2M). The assay range was from 2 to 140 ng/ml and the within-assay coefficient of variation (CV) were 5.2% at 31.2 ng/ml and 6.4% at 62.5 ng/ml. Between-day CV ranged from 6.9% to 15.4%. Using this method, alpha 2 M was determined in the bronchoalveolar lavage fluid (BALF) from patients with interstitial lung diseases. Those diseases were active and inactive sarcoidosis, hypersensitivity pneumonitis and idiopathic pulmonary fibrosis (IPF, including collagen disease). We divided the IPF patients into two groups, 'acute type' and 'chronic type', judging from the prognosis. alpha 2 M/Albumin ratio in BALF in the active sarcoidosis and acute type IPF groups is significantly higher than that in the inactive sarcoidosis and chronic type IPF. These findings suggest that alpha 2 M in BALF can be a sensitive marker of the interstitial lung disease.     

甘草酸对博莱霉素诱导的实验性肺纤维化的干预作用

目的:探讨甘草酸(GA)对博莱霉素(BLM)诱导的小鼠肺纤维化的干预作用及其可能机制。方法:将160只雄性C57BL/6J小鼠随机分为生理盐水(NS)组、BLM组、BLM+NS组和BLM+GA组。通过口咽气管吸入法吸入博莱霉素(2.5 mg/kg)建立实验性肺纤维化模型,BLM+GA组及BLM+NS组每天给予40 mg/kg甘草酸或等体积的生理盐水灌胃,于术后第3、7、14、21天取材。采用HE染色和Masson染色观察肺组织病理学变化及纤维化程度,采用流式细胞术检测循环单核细胞和肺泡巨噬细胞的亚群比例变化,采用RT-qPCR检测肺组织中转化生长因子β1(TGF-β1)mRNA的表达水平,采用碱水解法检测肺组织中羟脯氨酸(HYP)含量。结果:与NS组相比,BLM组和BLM+NS组肺组织的炎症浸润及胶原含量明显增多,实验性肺纤维化模型制备成功。与BLM+NS组相比:BLM+GA组肺组织的炎症细胞浸润及胶原纤维沉积较少;第3、7天的Ly6C~(hi)单核细胞亚群比例和第7、14天肺泡巨噬细胞M2表型比例显著降低(P0.01);肺组织TGF-β1 mRNA表达量和HYP含量显著降低(P0.01)。结论:GA可以减轻博莱霉素诱导的小鼠肺组织的炎症反应和胶原纤维沉积,可能与GA对单核巨噬细胞的表型偏移和调控以及肺组织TGF-β1的表达下调有关。     

One-way occurrence of graft-versus-host disease in bone marrow chimaeras between congenic MRL mice.

Bone marrow cells (BMCs) of MRL/Mp-lpr/lpr (MRL/l) mice, when infused into irradiated MRL/Mp-+/+ (MRL/n) mice, induced graft-versus-host disease (GVHD) and recipients died of wasting syndromes beginning around a few weeks later. Protracted appearance of GVHD was observed when (MRL/n X MRL/l) F1 mice were used as recipients of MRL/l BMSs. On the other hand, MRL/n BMCs did not elicit GVHD in irradiated MRL/l mice. Treatment of MRL/l BMCs with anti-Thy-l antiserum plus complement did not eliminate GVHD-provoking capacity. Thymectomy of the recipents reduced the incidence of GVHD. No apparent reaction was observed in mutual mixed lymphocyte culture of spleen cells from MRL/l and MRL/n mice. These data suggest that injected T cell precursors of MRL/l mice become mature through the host (MRL/n mice) thymus and appear in the periphery as functioning T cells, resulting in the appearance of GVHD.     

The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism

Mice deficient in the plasminogen activator inhibitor-1 gene (PAI-1-/- mice) are relatively protected from developing pulmonary fibrosis from bleomycin administration. We hypothesized that one of the protective mechanisms may be the ability of the plasminogen system to enhance hepatocyte growth factor (HGF) effects, which have been reported to be anti-fibrotic in the lung. HGF is known to be sequestered in tissues by binding to extracellular matrix components. Following bleomycin administration, we found that HGF protein levels were higher in bronchoalveolar lavage fluid from PAI-1-/- mice compared to wild-type (PAI-1+/+) mice. This increase could be suppressed by administering tranexamic acid, which inhibits plasmin activity. Conversely, intratracheal instillation of urokinase into bleomycin-injured PAI-1+/+ mice to activate plasminogen caused a significant increase in HGF within bronchoalveolar lavage and caused less collagen accumulation in the lungs. Administration of an anti-HGF neutralizing antibody markedly increased collagen accumulation in the lungs of bleomycin-injured PAI-1-/- mice. These results support the hypothesis that increasing the availability of HGF, possibly by enhancing its release from extracellular matrix by a plasmin-dependent mechanism, is an important means by which activation of the plasminogen system can limit pulmonary fibrosis.     

Anti-Inflammatory Effect of Pirfenidone in the Bleomycin-Hamster Model of Lung Inflammation

We have previously reported the antifibrotic effects of pirfenidone (PD) in the bleomycin (BL)-hamster model of lung fibrosis. Since the development of fibrosis is generally preceded by acute lung inflammation, the present study was conducted to find out if dietary intake of PD (0.5%) has any effects on BL-induced lung inflammation. In this regard, we evaluated the effects of PD on BL-induced increased pulmonary vascular permeability, increased influx of inflammatory cells and increased levels of TGF- in the bronchoalveolar lavage fluid (BALF). Hamsters were intratracheally (IT) instilled with saline (SA) or BL (5.5 units/kg/5 ml). The animals were fed the control diet (CD) or the same diet containing 0.5% PD 2 days prior to IT instillation and throughout the study. The bronchoalveolar lavage was carried out at different times after IT instillation. Lavage fluid was used for total and differential cell counts and BALF-supernatant for measurement of total protein and TGF-. IT instillation of BL caused significant increases in total cells, neutrophils, macrophages and lymphocytes and in the levels of total protein and TGF- in BALF from hamsters in the BL + CD groups as compared to the corresponding SA + CD control groups. In contrast, treatment with pirfenidone in general, suppressed the BL-induced increases in the levels of proteins and TGF- and in the influx of neutrophils, macrophages and lymphocytes in BALF at the early time points in BL + PD groups. Based on the data reported in this study, we conclude that the anti-inflammatory effects of pirfenidone as evident by suppressions of BL-induced increased pulmonary vascular permeability and increased influx of inflammatory cells in the lung contribute additionally to its inherent anti-fibrotic effect.     

Pulmonary inflammation in autoimmune MRL/Mp-lpr/lpr mice. Histopathology and bronchoalveolar lavage evaluation.

Early detection of lupus pneumonitis is difficult because it requires lung biopsy. The authors describe here in detail the age-related histologic changes in pulmonary inflammation, the age-related changes in bronchoalveolar lavage (BAL), and the effect of cyclophosphamide (8 mg/kg) on pulmonary inflammation and bronchoalveolar lavage in MRL/Mp-lpr/lpr mouse, an animal model of systemic lupus erythematosus. To assess the evolution of pulmonary inflammation and response to cyclophosphamide therapy, they compared the age-related progression of pulmonary inflammation with sequential changes in BAL cell populations in this autoimmune mouse model. A striking similarity was noted between age-related changes in pulmonary inflammation and lymphocyte counts in BAL. A trend to reduction in histologic evidence of inflammation was reflected by lymphocytes in BAL in cyclophosphamide-treated (8 mg/kg/day) males but not in females. There was a striking sex-related difference in that the histologic evidence of pulmonary inflammation and bronchoalveolar lavage lymphocyte count in cyclophosphamide-treated males was significantly lower than cyclophosphamide-treated females of the same age.     

Natural killer T (NKT) cells attenuate bleomycin-induced pulmonary fibrosis by producing interferon-gamma

Pulmonary fibrosis is a progressive illness characterized by interstitial fibrosis. Although the precise mechanism for pulmonary fibrosis is not completely understood, an immune response involving interferon (IFN)-gamma appears to play a role. Therefore, we examined the functional roles of natural killer T (NKT) cells, which produce IFN-gamma and interleukin-4 on activation, in bleomycin-induced pulmonary fibrosis. In NKT cell-deficient mice, pulmonary fibrosis was worse in terms of histology, hydroxyproline levels, and mortality than in control mice. The transforming growth factor (TGF)-beta1 levels were higher in the lung after injecting bleomycin, and blockade of TGF-beta1 by neutralizing monoclonal antibody attenuated the pulmonary fibrosis in CD1d-/- mice. In contrast, the production of IFN-gamma was reduced in lungs from CD1d-/- mice. Moreover, the adoptive transfer of NKT cells into CD1d-/- mice increased IFN-gamma and reduced TGF-beta1 production, attenuating pulmonary fibrosis. An in vitro assay demonstrated that IFN-gamma was involved in suppressing TGF-beta1 production in cells collected from bronchoalveolar lavage. The adoptive transfer of NKT cells from IFN-gamma-/- mice did not reverse pulmonary fibrosis or TGF-beta1 production in lungs of CD1d-/- mice whereas NKT cells from B6 control mice attenuated fibrosis and reduced TGF-beta1 production. In conclusion, IFN-gamma-producing NKT cells play a novel anti-fibrotic role in pulmonary fibrosis by regulating TGF-beta1 production.     

STAT6-mediated signaling in Th2-dependent allergic asthma: critical role for the development of eosinophilia, airway hyper-responsiveness and mucus hypersecretion, distinct from its role in Th2 differentiation

When wild-type BALB/c mice were transferred with OVA-specific Th2 cells followed by OVA inhalation, a severe eosinophilia, mucus hypersecretion and airway hyper-responsiveness (AHR) was induced in parallel with a marked elevation of IL-4, IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF). However, neither eosinophilia, AHR nor mucus hypersecretion was induced in Th2 cell-transferred STAT6-/- mice. The failure of eosinophilia was not due to the defect of Th2 cytokine production in BALF of STAT6-/- mice transferred with Th2 cells, but because of the defect of STAT6-dependent eotaxin production. Indeed, intranasal administration of eotaxin reconstituted pulmonary eosinophilia but not AHR and mucus hypersecretion in OVA-inhalated STAT6-/- mice. These results initially provided direct evidence that STAT6-dependent eotaxin production is essential for pulmonary eosinophilia. We also dissociated the role of STAT6 for eosinophilia from that for AHR and mucus hypersecretion. Thus, STAT6 also plays a critical role at late phase of Th2-dependent allergy induction.     

Localization of plasminogen activator activity within normal and injured lungs by in situ zymography

During inflammatory lung injury, the fibrinolytic activity that is normally present within bronchoalveolar lavage (BAL) fluid (BALF) is often suppressed due to increased levels of inhibitors, including plasminogen activator inhibitor (PAI)-1. Despite this suppression, BALF frequently contains fibrin degradation products, indicating persistence of fibrinolytic activity within the lung. To address this discrepancy and determine the sites where plasminogen activation is occurring, we developed an in situ zymographic technique for frozen sections of lung tissue that localizes plasminogen activator activity at the cellular level. After validating the method using enzyme inhibitors and mice with genetic manipulations of their plasminogen system genes, we applied the technique to lungs of normal and bleomycin-exposed mice. In normal mice, plasminogen activator activity was localized to bronchial epithelial cells, cells of the alveolar walls, and alveolar macrophages. After bleomycin exposure, in situ zymography showed that, despite loss of fibrinolytic activity within BALF, abundant enzymatic activity was associated with aggregates of inflammatory cells. PAI-1-deficient mice that are protected from bleomycin-induced fibrosis had preserved plasminogen activator activity in BALF and increased tissue activity, as determined by in situ zymography. We conclude that analysis of BALF does not adequately reflect the fibrinolytic activity that persists within microenvironments of the lung during inflammation.     

Optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice

To determine the time required to repopulate mouse lungs with donor alveolar macrophages following total body irradiation (TBI) and bone marrow transplantation (BMT), C57Bl/6 mice were subjected to TBI with 900 cGy, followed by transplantation of bone marrow cells from mice expressing green fluorescent protein (GFP) in their somatic cells. The mice were euthanized at either 30 (n=5), 60 (n=5) or 90 (n=5) days following BMT. Thirty days following transplantation, 87.8 +/- 3.9% (mean +/- S.E.M.) circulating leukocytes in recipient mice were derived from the donor, as determined by fluorescence activated cell sorting (FACS) analysis for GFP. However, only 46.9 +/- 7.4% of the resident alveolar cells expressed GFP, indicating incomplete repopulation. By day 60 post-transplantation, the percentage of bronchoalveolar lavage fluid (BALF) cells expressing GFP reached 74.5 +/- 2.4%, remaining stable 90 days after transplantation (80.4 +/- 1.9%). We conclude that 60 days after TBI with 900 cGy and bone marrow transplantation, the majority of the lung resident alveolar macrophages is of donor origin. This study provides useful information regarding the time of reconstitution with donor alveolar macrophages in the pulmonary airspaces of recipient mice following marrow transplantation.     

BALB/c鼠和裸鼠呼吸道合胞病毒感染比较研究

目的 比较普通BALB/c鼠和裸鼠呼吸道合胞病毒(RSV)感染免疫及炎症反应特点.方法 BALB/c鼠和裸鼠感染RSV后不同时间空斑形成试验检测肺组织病毒滴度,计数支气管肺泡灌洗液(BALF)白细胞总数和分类,HE染色分析肺组织病理学改变,免疫组化检测肺组织F4/80+细胞和CD49b+细胞.ELISA检测BALF中TNF-α、IFN-r、IL-12和IL-10浓度.结果 BALB/c鼠和裸鼠感染RSV后肺组织病毒滴度在第3天达峰值,感染裸鼠带毒时间更长,在感染后各天病毒滴度明显高于BALB/c鼠(P＜0.05),肺组织病理改变也更重.感染BALB/c鼠和裸鼠BALF白细胞总数明显升高,分类以淋巴细胞为主.感染裸鼠与感染BALB/c鼠比较,肺组织检测到更多的F4/80+巨噬细胞和CD49b+NK细胞(P＜0.05),BALF中TNF-α、IL-12和IL-10水平更高(P＜0.05).结论 RSV感染裸鼠与BALB/c鼠比较,病毒复制水平更高,时间更持久,炎症反应更重.单核巨噬细胞和NK细胞是RSV感染重要的免疫细胞和炎症细胞,炎症反应强度并不一定与T细胞免疫应答平行.     

Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome

Abundant deposition of bronchoalveolar fibrin and fibronectin occurs during the exudative phase of the adult respiratory distress syndrome (ARDS), promoting hyaline-membrane formation and subsequent alveolar fibrosis. To explore the mechanisms that account for the persistence of bronchoalveolar fibrin and fibronectin, we compared the activity of urokinase, which is necessary for plasminogen activation and fibrin degradation, in cell-free bronchoalveolar-lavage fluid from 8 patients with ARDS, 9 patients with acute pulmonary diseases other than ARDS, and 10 normal subjects. The mean level of urokinase activity in the lavage fluid from the patients with ARDS was 0.003 IU per milliliter of fluid (range, 0 to 0.008), which was significantly lower (P = 0.001) than the level in the fluid from either the patients with pulmonary diseases other than ARDS (0.118 IU per milliliter [range, 0.032 to 0.295]) or the normal subjects (0.129 IU per milliliter [range, 0.045 to 0.198]). The lavage fluid from all the patients with ARDS also had antiplasmin activity, which would promote the persistence of fibrin. A true decrease in urokinase activity was confirmed by the failure of the lavage fluid from the patients with ARDS to convert [125I]plasminogen to plasmin. Despite the low urokinase activity, immunochemical assays revealed normal levels of urokinase antigen in the fluid from the patients with ARDS, suggesting the presence of urokinase inhibitors. Inhibitors were demonstrated directly by a fibrin gel-underlay assay that detects complexes of urokinase with inhibitors. Plasminogen-activator inhibitor type 1 was the principal inhibitor identified. We conclude that increased antifibrinolytic activity due to both urokinase inhibitors and antiplasmins in the bronchoalveolar compartment of patients with ARDS contributes to the formation and persistence of hyaline membranes, a key component of alveolar histopathology in ARDS.