Differential production of metalloproteinases after instilling various urban air particle samples to rat lung

Lung injury and inflammation are associated with exposure to various types of particulate air pollutants. The present study was used to determine whether metalloproteinases (MMPs) are secreted after instilling dust samples into the lung, and to relate levels of specific MMPs to different fractions of the ambient air particle sample EHC-93. Rats received an intratracheal injection of 5 mg dust samples in 0.5 ml water and were killed at intervals from 4 hours to 28 days later particle samples were EHC-93 whole dust, and the insoluble, leached, and soluble fractions of the same dust. Samples prepared from EHC-2K dust were also used, as were solutions of zinc and copper chloride. All samples induced inflammation as measured by increased inflammatory cells in bronchoalveolar lavage (BAL) fluid; the highest levels were found 1 to 3 days after instilling the whole dust. This dust also induced production of MMP-2 and MMP-9 as shown in zymograms. The leached dust induced predominantly MMP-9, which was maximal at 4 hours and 1 day. In contrast, the soluble fraction induced almost exclusive 4 MMP-2, also maximal at 4 hours and 1 day; this enzyme was also produced in response to soluble zinc, the most prevalent soluble metal in the EHC samples. The results demonstrate the rapid production and secretion of MMPs in the lung after particle deposition. A differential pattern of MMP production is seen with MMP-9, likely from inflammatory cells, being produced in response to the insoluble particles, and MMP-2, likey from epithelial cells, being produced in response to the water-soluble fraction of the atmospheric dust.     

Comparative pulmonary toxicity of various soluble metals found in urban particulate dusts

The potential toxicity of an atmospheric dust sample EHC-93 has been attributed to the soluble fraction and, more specifically, to the zinc component. The concentration of Zn is the highest among the metals present in the soluble EHC-93 fraction. We now determine whether other metal components of this dust could cause similar lung injury if present at the same concentration as Zn (4.8 mg/g dust). Solutions of Zn, Cu, V, Ni, Fe, and Pb salts in 0.1 mL water were instilled to mouse lung and animals were killed at intervals up to 2 weeks later; each mouse received tritiated thymidine 1 hour before death. Solutions containing Zn and to a lesser degree Cu induced lung injury; in addition, increased numbers of alveolar macrophages and polymorphonuclear leukocytes were found in the lavage fluid, which also contained increased protein levels up to 1 week later. The magnitude of response was similar to that seen after administering EHC-93 dust at 1 mg in 0.1 mL water, whereas the response to other metal solutions containing Ni, Fe, Pb, and V was minimal. Morphologic evidence of lung injury and inflammation was also seen after EHC dust and the Zn or Cu solutions only. Reparative cell proliferation was measured after thymidine uptake and autoradiographs showed increased labeling of lung cells, particularly at 3 and 7 days. Labeling was confined to bronchiolar and type 2 alveolar epithelial cells, indicating previous epithelial cell necrosis in response to Zn or Cu. The results indicate that atmospheric contaminant metals Zn and Cu are most likely to cause lung injury and inflammation as compared to metals such as Ni, Fe, Pb, and V at the same concentrations. It appears that similar toxicity occurs when both redox (Cu) and nonredox (Zn) reactions are involved.     

Supernatants of HIV-infected immune cells affect the barrier function of human HT-29/B6 intestinal epithelial cells

OBJECTIVES: Characterization of the diarrhoea-inducing effect of altered cytokine production in HIV infection. METHODS: Monocyte-derived macrophages (MDM) were infected with macrophagetropic (SF162) and lymphocytotropic (IIIB) HIV-1 strains and cocultured with autologous peripheral blood mononuclear cells (PBMC). After 24 h the supernatants were collected and tested for their immunoreactive levels of cytokines by enzyme-linked immunosorbent assay. The effects of the supernatants and the respective recombinant human cytokines on barrier function of HT-29/B6 cells were determined. RESULTS: Infection of MDM with HIV-1 SF162 or IIIB led to increased production of tumour necrosis factor-alpha (TNFalpha), interleukin-1-beta, interferon-alpha and interferon-gamma after cell-cell contact with PBMC. Supernatants of infected cells decreased transepithelial resistance (R(t)), with higher effects on R(t) in HIV IIIB infection, which was due to higher cytokine concentrations. The effect was not due to cytotoxicity (negative LDH assay) or epithelial monolayer disruption [zonula occludens protein-1 (ZO-1) immunofluorescence staining]. The effect of HIV-1 IIIB coculture supernatants could be mimicked by the respective recombinant human cytokines. TNFalpha is an effector cytokine, because inhibition of TNFalpha by its soluble receptor decreased the effect of the supernatants on transepithelial resistance. Conductance scanning indicated the cytokine-induced barrier defect to be due to both, induction of epithelial apoptoses and tight junction alterations. CONCLUSIONS: Cell-cell interaction of HIV-infected macrophages with PBMC leads to a release of cytokines sufficient to alter intestinal epithelial barrier function. The main effect was mediated by TNFalpha inducing a leak-flux which may contribute to the diarrhoea by HIV per se (HIV-enteropathy).     

Intraphagosomal pH in alveolar macrophages after phagocytosis in vivo and in vitro of fluorescein-labeled yeast particles

The pH in phagolysosomes of rabbit alveolar macrophages was studied using yeast particles labeled with fluorescein isothiocyanate (FITC). The yeast particles were added to the macrophages in vitro a few hours or 1 day after they had been lavaged from the lung and in vivo 3 h or 1 day before the lungs were lavaged. Intracellular pH was estimated from the ratio between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths of 495 and 450 nm. In both the in vitro and in vivo experiments pH decreased significantly during the first hours after lavage, but after a few hours reached almost the day-2 levels, i.e., 4.9-5.4. The decrease in pH was related to time after lavage and not to time after phagocytosis of the particles. It is suggested that intracellular measurements of pH in alveolar macrophages should be combined with determinations of lung clearance of metal particles.     

Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression

ABSTRACT The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is dependent on hydrolysis of the biologically inactive intermediate big ET-2 by an endothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACHN). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC(50( congruent with11 microM). To determine whether ET-2 synthesis occurs in parallel with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitative RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumour necrosis factor-alpha (TNFalpha), forskolin and a cell-permeable cAMP analogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 synthesis. Combination of forskolin or dibutyryl cAMP with TNFalpha produced a significantly greater increase in ET-2 production than these agents alone, indicating that adenylate cyclase and TNFalpha induce ET-2 synthesis by separate signalling pathways. Studies using receptor selective TNFalpha mutants, (125(I-TNFalpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNFalpha. PreproET-2 mRNA levels were increased by TNFalpha at 1 h and 2 h, but returned to control levels at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not the ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1b/c/d showed TNFalpha to increase mRNA levels at 2 h and 4 h. Forskolin had no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates.     

Donor CD4+ T-cell production of tumor necrosis factor alpha significantly contributes to the early proinflammatory events of graft-versus-host disease

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is an old foe in allogeneic bone marrow transplantation (allo-BMT) promoting acute graft-versus-host disease (aGVHD). We investigated to what extent donor T cells contribute to TNFalpha production. METHODS: Lethally irradiated B6D2F1 mice were transplanted with bone marrow (BM) and T cells from syngeneic B6D2F1 or allogeneic B6 donors and assessed for cytokine production, aGVHD, and survival. RESULTS: Analysis of serum TNFalpha kinetics in recipients of allogeneic B6 wild-type BM and wild-type T cells revealed that TNFalpha levels peaked around day 7 after allo-BMT, whereas TNFalpha was undetectable in syngeneic controls. TNFalpha was produced by both host and donor cells. Further exploration showed that specifically donor CD4(+) but not CD8(+) T cells were the primary donor cell source of TNFalpha at this early time point; numbers of TNFalpha expressing splenic CD4(+) T cells were higher than CD8(+) T cells 7 days after allo-BMT, and maximal serum TNFalpha levels were detected following allo-BMT with only CD4(+) T cells compared to levels found in allogeneic recipients of only wild-type CD8(+) or to only CD4(+) TNFalpha(-/-) T cells. Concurrent with increased TNFalpha levels, early clinical aGVHD and mortality were more severe following allo-BMT with either wild-type CD4(+) and CD8(+) or CD4(+) T cells only. CONCLUSION: Our data demonstrate that in addition to residual host cells donor CD4(+) T cells significantly contribute to the proinflammatory cytokine milieu engendered early after allo-BMT through the production of TNFalpha. These findings support strategies focusing on TNFalpha neutralization as primary treatment for aGVHD.     

Enzyme activities of lung lavage in silicosis

The cytotoxic effect of quartz on lung cells has been well documented by in vitro and animal studies, but the pertinence of these findings to humans has not yet been documented. We measured lactate dehydrogenase (LDH) activities in the lung lavage of 24 long-term workers in the Québec granite industry and 25 control subjects. We found significant increases in LDH activities in the workers’ lung lavage, even in the absence of established silicosis (9 subjects). We looked at a similar observation in the sheep model of early silicosis, measured quartz content of lung lavage, and found significant correlation with LDH levels (R=0.64, p<0.001). All of the quartz particles in human and sheep lung lavage were in the alveolar macrophages. To test further the relationship of macrophage damage (cytotoxicity of quartz) we measured the release of LDH by sheep alveolar macrophage in 24 h cell culture under control conditions, exposure to inert dust, titanium, minusil-5 quartz, or aluminum-treated quartz. The LDH release was at control levels during titanium exposure and showed a significantly dose-related increase during quartz exposure. The latter cytotoxic effect was largely attenuated by aluminum treatment of quartz. These in vitro data agreed with previous reports. This study presents evidence of a cytotoxic effect of quartz inhalation in humans. The effect is related to the intensity of quartz retention in the lung macrophages; it is not a nonspecific dust exposure effect and can be attenuated by surface modification of the quartz.     

Enzyme activities of lung lavage in silicosis

The cytotoxic effect of quartz on lung cells has been well documented by in vitro and animal studies, but the pertinence of these findings to humans has not yet been documented. We measured lactate dehydrogenase (LDH) activities in the lung lavage of 24 long-term workers in the Québec granite industry and 25 control subjects. We found significant increases in LDH activities in the workers' lung lavage, even in the absence of established silicosis (9 subjects). We looked at a similar observation in the sheep model of early silicosis, measured quartz content of lung lavage, and found significant correlation with LDH levels (R = 0.64, p less than 0.001). All of the quartz particles in human and sheep lung lavage were in the alveolar macrophages. To test further the relationship of macrophage damage (cytotoxicity of quartz) we measured the release of LDH by sheep alveolar macrophage in 24 h cell culture under control conditions, exposure to inert dust, titanium, minusil-5 quartz, or aluminum-treated quartz. The LDH release was at control levels during titanium exposure and showed a significantly dose-related increase during quartz exposure. The latter cytotoxic effect was largely attenuated by aluminum treatment of quartz. These in vitro data agreed with previous reports. This study presents evidence of a cytotoxic effect of quartz inhalation in humans. The effect is related to the intensity of quartz retention in the lung macrophages; it is not a nonspecific dust exposure effect and can be attenuated by surface modification of the quartz.     

L-arginine administration ameliorates serum and pulmonary cytokine response after gut ischemia-reperfusion in immature rats

AIM: Small intestinal ischemia-reperfusion (IR) has been demonstrated to result in both local mucosal injury and systemic injuries. The exact role of nitric oxide (NO) in intestinal IR is unclear. We propose that NO and some other cytokines change in the reperfusion period and these changes are associated with lung injury. The aim of this study was to determine the effect of supplementing NO substrate, L-arginine (L-arg), on serum and pulmonary cytokine production during small intestinal IR in immature rats. METHODS: Immature rats underwent 60 min. of superior mesenteric artery occlusion followed by 90 min of reperfusion. L-arg (250 mg/kg) was given intravenously to the experimental group (IR+L-arg) which received L-arg after 45 min of intestinal ischemia. Serum and lung endothelin-1 (ET-1), NO, malondialdehyde (MDA), and tumor necrosis factor a (TNFα) were measured. Sham operation (SHAM) and intestinal IR (IR) groups were performed as control. The lavage fluid of the lung was collected by bronchoalveolar lavage (BAL) and white blood cells and polymorphonuclear cells (PMNs) were immediately counted to identify lung damage. RESULTS: When L-arg was given during small intestinal IR, serum NO concentration increased significantly in IR+L-arg group (162.17±42.93 μmol/L) when compared with IR group (87.57±23.17 μmol/L, t=3.190, P= 0.008 <0.01). Serum MDA reduced significantly in IR+L-arg group (8.93±1.50 nmol/L) when compared with SHAM (23.78±7.81 nmol/L, t= 3.243, P= 0.007<0.01) and IR (25.54±9.32 nmol/L, t= 3.421, P= 0.006<0.01). ET-1 level in lung tissues was significantly lower in IR+L-arg group (13.81±7.84 pg/mL) than that in SHAM (35.52±10.82 pg/mL, t= 2,571, P= 0,03<0.05) and IR (50.83±22.05 pg/mL, t= 3.025, P= 0.009<0.01) groups. MDA contents in lung tissues were significantly lower in IR+L-arg group (10.73±1.99 nmol/L) than in SHAM (16.62±2.28 nmol/L, t= 3.280, P = 0.007<0.01) and IR (21.90±4.82 nmol/L, t= 3.322, P= 0.007<0.01) groups. Serum and lung TNFα concentrations were not significantly different in three groups. NO contents in lung homogenates and white blood cell counts in BAL had no significant difference in three groups; but the percentage of PMNs in BAL was 13.50±8.92, 33.20±16.59, and 22.50±6.09 in SHAM, IR, and IR+L-arg groups, respectively. CONCLUSION: Small intestinal IR induced increases of pulmonary neutrophil infiltration in immature rats. Neutrophil infiltration in lung tissues was reduced by L-arg administration but remained higher than in SHAM group. L-arg administration during intestinal IR enhances serum NO production, reduces serum MDA and lung ET-1 and MDA levels, resulting in the improvement of systemic endothelial function. L-arg supplementation before reperfusion may act as a useful clinical adjunct in the management of intestinal IR, thus preventing the development of adult respiratory distress syndrome, even multiple organ dysfunction syndrome (MODS).     

二氧化硫对慢性变应性气道炎症、气道上皮下纤维化的促进作用

目的 研究SO2所造成的急性气道炎症、上皮损伤对卵白蛋白反复激发致敏小鼠慢性变应性气道炎症、气道上皮下纤维化(subepithelial fibrosis,SEF)的影响.方法 吸入50 ppm SO2诱发急性气道炎症;卵白蛋白反复激发4、8周,观察首次激发前吸入、未吸入SO2的小鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)细胞学、内皮素1(endothelin-1,ET-1)和转化生长因子β1(transforming growth factor-β1,TGF-β1)水平、肺组织羟脯氨酸(Hyp)含量、气道上皮基底膜下纤维面积及气道组织学改变.结果吸入SO2导致急性中性粒细胞性气道炎症、上皮损伤,卵白蛋白反复激发诱发慢性变应性气道炎症、SEF,二者均导致BALF中ET-1、TGF-β1水平增高.与同期单纯激发组相比,损伤激发组SEF出现更早,气道炎症、SEF更显著,肺组织Hyp与BALF中TGF-β1呈正相关.结论 预先吸入SO2导致的急性气道炎症、上皮损伤能加剧慢性变应性气道炎症,促进并加重SEF,可能与气道炎症、上皮损伤过程中气道微环境ET-1、TGF-β1表达上调有关.     

Inflammatory mediators and cellular infiltration of the lungs in a guinea pig model of the late asthmatic reaction

Alterations in cell numbers, vascular permeability, and concentrations of various inflammatory mediators in the lung were measured in a guinea pig model of the late asthmatic reaction. Animals sensitized by inhalation of ovalbumin were challenged with an aerosol of ovalbumin or saline, and bronchoalveolar lavage fluid (BALF) and peripheral blood were collected after periods ranging from 5 min to 72 h. Increased vascular leakage within the lungs was indicated by elevated BALF/plasma albumin ratios at all time points, and was maximal 6 h after challenge. There were increased numbers of eosinophils in BALF by 6 h after challenge and they remained elevated at least until 72 h. A corresponding increase in the proportion of blood leukocytes represented by eosinophils was observed at 6 and 17 h, which suggests that these cells may be drawn to the lung following their release into the circulation, but by 72 h the proportion in blood had returned to normal. A transitory neutrophilia was evident in BALF and blood 6 h after allergen exposure, but there were no allergen-induced changes in BALF numbers of macrophages, lymphocytes, epithelial cells, or mast cells (as assessed by concentrations of cell-associated histamine).β-Glucuronidase activity was significantly increased in BALF of guinea pigs at 2 h and 17 h following challenge. The degree to which eicosanoids can be recovered in BALF was investigated by instilling a range of tritiated compounds into the lungs of normal guinea pigs at the time of lavage. Ratio high-performance liquid chromatography revealed that there had been little metabolism of the eicosanoids recovered in BALF. However, there was evidence for a rapid removal of these mediators from the lung, a process which will militate against their accurate quantitation in BALF. Histamine, prostaglandin D₂, and thromboxane B₂ were detected in BALF but did not differ between treatment groups, and levels showed no simple relationship with the other inflammatory changes measured.     

Inflammatory mediators and cellular infiltration of the lungs in a guinea pig model of the late asthmatic reaction

Alterations in cell numbers, vascular permeability, and concentrations of various inflammatory mediators in the lung were measured in a guinea pig model of the late asthmatic reaction. Animals sensitized by inhalation of ovalbumin were challenged with an aerosol of ovalbumin or saline, and bronchoalveolar lavage fluid (BALF) and peripheral blood were collected after periods ranging from 5 min to 72 h. Increased vascular leakage within the lungs was indicated by elevated BALF/plasma albumin ratios at all time points, and was maximal 6 h after challenge. There were increased numbers of eosinophils in BALF by 6 h after challenge and they remained elevated at least until 72 h. A corresponding increase in the proportion of blood leukocytes represented by eosinophils was observed at 6 and 17 h, which suggests that these cells may be drawn to the lung following their release into the circulation, but by 72 h the proportion in blood had returned to normal. A transitory neutrophilia was evident in BALF and blood 6 h after allergen exposure, but there were no allergen-induced changes in BALF numbers of macrophages, lymphocytes, epithelial cells, or mast cells (as assessed by concentrations of cell-associated histamine). beta-Glucuronidase activity was significantly increased in BALF of guinea pigs at 2 h and 17 h following challenge. The degree to which eicosanoids can be recovered in BALF was investigated by instilling a range of tritiated compounds into the lungs of normal guinea pigs at the time of lavage. Ratio high-performance liquid chromatography revealed that there had been little metabolism of the eicosanoids recovered in BALF. However, there was evidence for a rapid removal of these mediators from the lung, a process which will militate against their accurate quantitation in BALF. Histamine, prostaglandin D2, and thromboxane B2 were detected in BALF but did not differ between treatment groups, and levels showed no simple relationship with the other inflammatory changes measured.     

Intra-alveolar release of a competence-type growth factor after lung injury

Growth factors released by platelets, macrophages, and endothelial and smooth muscle cells have been recognized and characterized using in vitro tests of isolated cell populations. However, their production, secretion, and effects on target cells in situ after tissue injury remains largely presumptive. Alveolar macrophages cultured during acute and chronic lung injury release increased amounts of macrophage-derived growth factor (MDGF). In the present study, we sampled the alveolar lining fluid by lavage for the presence of macromolecular competence factor activity. We report that alveolar lavage fluid obtained following acute lung injury induced by bleomycin in the rat contains large amounts of soluble growth factor activity not found in lung lavage fluid from normal animals. We compared the properties of the growth factor found in fresh lavage fluid to MDGF and platelet-derived growth factor (PDGF). The amount of growth factor in lavage fluid paralleled the ability of cultured alveolar macrophages to release MDGF. Like PDGF and MDGF, lavage fluid growth factor served as a competence factor promoting the reentry of quiescent fibroblasts into the cell cycle rather than as a progression factor. Chromatography on DEAE-Sephacel yielded a single peak of growth factor activity eluting at 0.3 M NaCl. On the basis of these and other physical and biologic properties, we conclude that growth factor activity found in high levels in the alveolar space following acute lung injury resembles MDGF. Growth factor present in the alveolar space may provide the major local stimulus to lung structural cell replication after acute lung injury.     

Quantitation of cellular and extracellular constituents of the pulmonary lining in rats by using bronchoalveolar lavage. Effects of silica-induced pulmonary inflammation

The efficacy of bronchoalveolar lavage in the removal of cellular and extracellular components of the lining layer from the lungs of silica-treated and control rats was determined. Exponential functions were fitted to curves generated by plotting the quantity of lining layer constituent removed from the lungs by bronchoalveolar lavage versus the lavage number. From these exponential functions we determined the total amount of constituent available in the pulmonary extracellular lining and hence the efficacy of the lavage procedure in removing materials from the lungs. With control rats the removal of extracellular phospholipids, soluble protein, alkaline phosphatase, and beta-N-acetylglucosaminidase by bronchoalveolar lavage occurred at significantly different rates. Removal of 95% of the total available extracellular phospholipid, beta-N-acetylglucosaminidase, soluble protein, and alkaline phosphatase from the lungs required 4, 4, 8, and 11 lavages, respectively. Removal of 95% of the total available alveolar macrophages required 18 lavages. The influence of pulmonary inflammation on the efficacy of the lavage procedure was investigated by injecting silica dust intratracheally into the lungs of rats (50 mg/200- to 250-g rat) and after 3 days performing the analyses. Silica caused an inflammatory condition in the lungs resulting in the accumulation of materials in the alveoli. Highly significant increases in soluble protein (16-fold), alkaline phosphatase (9-fold), and beta-N-acetylglucosaminidase (11-fold), polymorphonuclear leukocytes, eosinophils, and lymphocytes were observed. Alveolar macrophages and extracellular phospholipid were not significantly elevated at 3 days after dosing. Silica did not alter the efficacy of the lavage procedure in removing from the lungs any of the extracellular constituents of the lung lining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inorganic particles induce secretion of a macrophage homologue of platelet-derived growth factor in a density-and time-dependent manner in vitro.

The inhalation of inorganic dust can lead to the development of interstitial pulmonary fibrosis, characterized by the accumulation of fibroblasts and connective tissue matrix in the lung interstitium. The fibrosis causes alterations in the architecture of the lung parenchyma, resulting in abnormal gas exchange and hypoxemia. In a rat model of asbestos exposure, inhaled fibers are deposited on alveolar duct bifurcations, followed by an accumulation of alveolar macrophages at the sites of dust deposition. The alveolar macrophage is thought to be a major mediator of the pulmonary inflammatory response to inhaled dust. Platelet-derived growth factor (PDGF) is a cytokine that has potent chemotactic and mitogenic effects on mesenchymal cells, such as fibroblasts and smooth muscle cells. We studied the secretion of an alveolar macrophage-derived homologue of PDGF in response to carbonyl iron spheres or chrysotile asbestos fibers in vitro. We demonstrate here that rat alveolar macrophages attached to a plastic substrate produce 69 +/- 79 picograms (pg) of PDGF per 10 million macrophages. This is similar to amounts recovered from human platelets. In contrast, macrophages exposed to iron spheres secrete 429 +/- 177 pg of PDGF/10(6) macrophages after 24 h in culture. Exposure to asbestos fibers increased the PDGF production to 628 +/- 213 pg/10(6) cells. PDGF secretion was influenced by the particles in a density- and time-dependent manner. We hypothesize that PDGF and other cytokines secreted by macrophages mediate the development of dust-induced lung disease.     

Biomonitoring for assessment of organic dust-induced lung inflammation.

Inhalation exposure to particulate matter containing endotoxin (or lipopolysaccharide (LPS)) occurs in a variety of occupations. Nasal lavage and induced sputum have been used to evaluate lung inflammation resulting from such exposures. Whole blood assay (WBA) measures cytokine production of leukocytes after ex vivo stimulation with LPS. The present study examined the effectiveness of WBA for evaluating inflammatory responses and susceptibility. C3HeB/FEJ mice were tolerised by LPS injection or sham tolerised with saline. Animals then inhaled either swine barn dust extract containing endotoxin or saline. Bronchoalveolar lavage (BAL) fluid was assayed for leukocyte counts and pro-inflammatory cytokines (interleukin-6, tumour necrosis factor-alpha). Whole blood was stimulated with 10 or 100 ng.mL(-1) of LPS, incubated for 5 or 18 h and assayed for cytokines. Barn dust-exposed groups revealed significantly higher total cells, neutrophils and cytokines in BAL compared with saline-exposed groups. Animals tolerised to LPS and exposed to barn dust demonstrated lower cellular and cytokine BAL responses. Similarly, WBA yielded significantly elevated cytokines with barn dust exposure and reduced responses with tolerisation. This study demonstrates the efficacy of whole blood assay as a biomarker of inhalation exposure to inflammatory agents and its use for assessing susceptibility to organic dust-induced lung inflammation.     

Lung inflammation induced by endotoxin is enhanced in rats depleted of alveolar macrophages with aerosolized clodronate

Clodronate liposomes were given to rats via intratracheal inhalation to investigate the importance of alveolar macrophages (AMs) in inhaled endotoxin-induced lung injury. When AM depletion was maximal (87% to 90%), rats were exposed to lipopolysaccharide (LPS) or saline. Neither clodronate nor saline liposomes induced an influx of neutrophils (PMNs) into the lungs. However, depleted LPS-exposed rats had 5- to 8-fold higher numbers of lavage PMNs and greater lavage cell reactive oxygen species release compared to undepleted rats. Although AM depletion by itself did not significantly increase inflammatory cytokine expression in lung tissue, LPS-induced message levels for interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha were approximately 2-fold higher in AM-depleted rats compared to undepleted rats. These results indicate that cells other than AMs can recruit inflammatory cells into the lungs during acute LPS-induced injury and that AMs play an important suppressive role in the innate pulmonary inflammatory response.