Biomedical subjects

J N Finkelstein

Publications and source records attributed to J N Finkelstein.

At least 37 records · Page 2Linked to original sources

PubMed1998

Pulmonary cytokine and chemokine mRNA levels after inhalation of lipopolysaccharide in C57BL/6 mice.

Inhaled endotoxin (lipopolysaccharide, LPS) can induce acute lung injury and at high doses may lead to respiratory distress syndrome. Using a mouse model of acute lung inflammation induced by inhalation of low doses of LPS we examined the kinetics of chemokine, proinflammatory cytokine, and metallothionein. Eight-week-old C57BL/6 mice were dosed for 10 min with LPS, resulting in an estimated alveolar dose of < 10 ng LPS/mouse, and euthanized 2,6, or 24 h postexposure. Analysis of bronchoalveolar lavage fluid demonstrated increased polymorphonuclear neutrophils (PMNs) of 6.94, 32.7, and 38.8% after 2, 6, and 24 h, respectively. Examination of proinflammatory cytokine, chemokine, and Mt mRNA in the lung revealed increases for messages encoding IL-1 alpha, IL-1 beta, IL-6, IFN-gamma, TNF alpha, Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, Mt, and IP-10, while messages encoding IL-12, IL-10, IFN-beta, Ltn, MCP-1, TGF beta 1 + 2, and RANTES were unchanged from those of sham-exposed mice 2 h postexposure. By 6 h most messages had returned to near control levels. Comparison to 5 mg/kg body weight intraperitoneal injection and 5 micrograms/mouse intratracheal instillation 2 h postexposure demonstrated similar message responses. Our results demonstrate that low levels of LPS exposure by inhalation induce a strong PMN response and a selective cytokine response in the lung, supporting the hypothesis that PMNs may regulate inflammatory processes via cytokine and chemokine response.

Administration, Inhalation↗

PubMed1998

Clara cell secretory protein deficiency increases oxidant stress response in conducting airways.

Little is known about the molecular basis for differential pulmonary oxidant sensitivity observed between genetically disparate members of the same species. We have generated mice that are deficient in Clara cell secretory protein (CCSP -/-) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and metallothionein (MT)] and epithelial cell-specific [cytochrome P-450 2F2 (CYP2F2)] gene expression to further understand the cellular and molecular basis for altered oxidant sensitivity in 129 strain CCSP -/- mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million ozone and preceded reductions in Clara cell CYP2F2 mRNA expression. Despite being qualitatively similar, increases in IL-6 and MT mRNA expression were enhanced in CCSP -/- mice with respect to coexposed 129 strain wild-type mice. Increased MT mRNA expression, indicative of the stress response, localized to the airway epithelium, surrounding mesenchyme, and endothelium of blood vessels. These results demonstrate a protective role for Clara cells and their secretions and indicate potential genetic mechanisms that may influence susceptibility to oxidant stress.

Animals↗

PubMed1998

Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-alpha.

Recent evidence has suggested that epithelial cells may contribute to the inflammatory response in the lung after exposure to crystalline silica through the production of and response to specific growth factors, chemokines, and cytokines. However, the exact cellular and molecular responses of epithelial cells to silica exposure remains unclear. Using a murine alveolar type II cell line [murine lung epithelial (MLE)-15 cell line], we measured the early changes in various cytokine and chemokine mRNA species after exposure of the cells to 4-35 microgram/cm2 of silica (cristobalite), interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and lipopolysaccharide (LPS) alone or in combination. Total mRNA was isolated and assayed with an RNase protection assay after 6 and 24 h of exposure. Cristobalite exposure alone led to an increase in monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-2, and regulated on activation normal T cells expressed and secreted (RANTES) mRNAs. Treatment with IFN-gamma alone increased MCP-1 mRNA levels. Treatment with TNF-alpha or LPS alone led to an increase in MCP-1 and MIP-2 mRNA. The combination of cristobalite plus TNF-alpha led to an additive increase in MCP-1 and MIP-2, whereas cristobalite plus IFN-gamma or LPS had a synergistic effect. We also found with a TNF-alpha-neutralizing antibody that TNF-alpha plays a major role in mediating the type II cell chemokine response to cristobalite exposure. The results indicate that the cristobalite-induced chemokine response in the lung epithelium is mediated in part by TNF-alpha and can be enhanced by macrophage- and lymphocyte-derived inflammatory mediators in an additive and synergistic fashion.

Animals↗

PubMed1998

Exposure to hyperoxia induces p53 expression in mouse lung epithelium.

Cells that are exposed to free radicals have increased levels of DNA strand breaks with accumulation of the tumor suppressor protein p53, which induces cell cycle arrest and/or apoptosis. Because oxidants injure pulmonary epithelial cells, it was hypothesized that exposure to hyperoxia promotes DNA strand breaks in lung epithelium, resulting in increased expression of p53 and loss of epithelial cell function. Adult male C57Bl/6J mice were exposed to > 95% oxygen for 72 h and DNA integrity was determined in their lungs by terminal transferase immunoreactivity. Both nonimmunoreactive and lightly stained nuclei were observed in cells comprising the airway and parenchyma. Exposure to hyperoxia resulted in a marked increase in the intensity of nuclear staining in distal bronchiolar epithelium and alveolar epithelial and endothelial cells. Airway epithelial cells from control lungs contained detectable levels of p53 protein, which markedly increased in both nuclei and cytoplasm of distal bronchiolar epithelial cells and to a lesser extent in alveolar epithelial cells that were morphologically consistent with type II cells. Western and Northern blot analyses revealed that hyperoxia increased total lung p53 protein expression but not levels of mRNA. Changes in terminal transferase immunoreactivity and p53 expression were not observed in large airway cells, fibroblasts underlying distal airway, or smooth muscle cells. Expression of SP-B mRNA modestly increased and Clara cell secretory protein and cytochrome P-450 2F2 mRNAs decreased, providing additional evidence that hyperoxia injured pulmonary epithelial cells. These findings support the concept that hyperoxia damages DNA of pulmonary epithelial cells, which respond by accumulating p53 and changes in epithelial cell-specific gene expression.

Animals↗

PubMed1998

Cell-specific gene expression reveals changes in epithelial cell populations after bleomycin treatment.

Epithelial repair following acute lung injury involves proliferation and differentiation of existing Clara cells and type II cells. Other mechanisms of epithelial repair may be involved in particularly severe cases. We used epithelial cell-specific markers to examine changes in the mouse lung epithelium 28 days after bleomycin treatment. The spatial distribution of surfactant proteins A, B, C (SPA, SPB, SPC), and Clara cell-specific protein (CC10) mRNA was compared by in situ hybridization in serial lung sections. CC10 mRNA-containing airway cells were replaced in many areas by SPB mRNA-expressing, ciliated cells that did not contain CC10 mRNA. In distal airway regions, we observed a subpopulation of epithelial cells that appeared to express SPA, SPB, SPC, and CC10 mRNA, and speculated that they may represent a multipotential stem cell population. These cells were found in focal clusters, which suggests that they expanded from a common cell. CC10 mRNA-containing cells were seen in alveolar-like structures thought to be the result of Clara cell migration or outpocketing. Our data suggest that there are repair mechanisms involved in epithelial repair after severe injury that have not previously been described.

Animals↗

PubMed1997

Bleomycin induces strain-dependent alterations in the pattern of epithelial cell-specific marker expression in mouse lung.

Clinical use of the antineoplastic agent bleomycin is restricted due to pulmonary toxicity. Murine models of bleomycin-induced pulmonary fibrosis have been developed in an attempt to understand the mechanisms involved in the fibrotic process. Studies have shown that the alveolar epithelium is damaged early after bleomycin treatment. The purpose of this study was to evaluate the pattern of gene expression in airway and alveolar epithelial cells after bleomycin exposure in mice that vary in susceptibility to bleomycin-induced fibrosis. Surfactant protein C (SPC) and Clara cell-specific protein (CC10) mRNA were used as cell-specific markers of alveolar type II cells and airway Clara cells, respectively. Mice were treated with a single intratracheal dose of bleomycin and the pattern of SPC and CC10 transcripts was examined by in situ hybridization. The pattern of SPC mRNA 28 days after treatment was uniform in controls and resistant mice but exhibited a patchy appearance in sensitive mice. Bleomycin treatment also resulted in a strain-dependent loss of CC10 mRNA-expressing cells. In sensitive mice 28 days after treatment, SPC mRNA was ectopically expressed in the distal bronchiolar epithelium in a morphologically distinct cell type. Serial sections revealed that these cells either coexpressed CC10 mRNA or were located adjacent to CC10 mRNA-containing cells. This unique cell population may represent a progenitor cell type important in epithelial repair. The strain-dependent changes in CC10 and SPC gene expression after bleomycin treatment are suggestive of a role for the epithelium in pulmonary fibrosis versus repair.

Animals↗

PubMed1997

Temporal changes in expression of TGF-beta isoforms in mouse lung exposed to oxygen.

Oxygen-induced pulmonary injury is associated with cell death and a significant inflammatory response. Because transforming growth factor (TGF)-beta is a potent modulator of the immune response, changes in expression of the three TGF-beta (beta 1, beta 2, beta 3) isoforms was determined in lungs of adult mice exposed to > 95% oxygen. TGF-beta 1 immunostaining within cuboidal nonciliated bronchiolar epithelial cells was increased within 3 h of oxygen exposure and continued to increase for 48 h before decreasing to control levels by 72 h. A similar but less marked change that was morphologically consistent with alveolar type II cells was observed in granulated cells. Immunostaining for TGF-beta 2 and TGF-beta 3 revealed a similar change in bronchiolar epithelium with little change observed in the alveolar epithelium. Immunohistochemical changes in TGF-beta expression were not observed in any other pulmonary cells. Northern blot analysis of total lung RNA revealed that expression of the TGF-beta mRNA was not markedly altered over the 72-h exposure period. Exposure to > 95% oxygen resulted in cell type-specific posttranscriptional changes in TGF-beta isoforms in the lung.

Animals↗

PubMed1997

Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis.

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

Acute Disease↗

PubMed1997

Altered pulmonary response to hyperoxia in Clara cell secretory protein deficient mice.

Clara cell secretory protein (CCSP) is an abundant component of the extracellular lining fluid of airways. Even though the in vivo function of CCSP is unknown, in vitro studies support a potential role of CCSP in the control of inflammatory responses. CCSP-deficient mice (CCSP -/-) were generated to investigate the in vivo function of this protein (13). In this study, we used hyperoxia exposure as a model to investigate phenotypic consequences of CCSP deficiency following acute lung injury. The pathologic response of the mouse lung to hyperoxia, and recovery of the lung, include inflammatory cell infiltrate and edema. Continuous exposure to > 95% O2 was associated with significantly reduced survival time among CCSP -/- mice as compared with strain-, age-, and sex-matched wild-type control mice. Differences in survival were associated with early onset of lung edema in CCSP -/- mice as compared with wild-type controls. To further investigate these differences in response, mice were exposed to > 95% O2 for either 48 h or 68 h with one group receiving 68 h of hyperoxia followed by room-air recovery. Lung RNA was characterized for changes in the abundance of cytokine messenger RNA (mRNA) using a ribonuclease (RNase) protection assay. After 68 h of hyperoxia, interleukin-6 (IL-6), IL-1beta, and IL-3 mRNAs were 14-, 3-, and 2.5-fold higher, respectively, in CCSP -/- mice than in similarly exposed wild-type control mice. Increased expression of IL-1beta mRNA in hyperoxia-exposed CCSP -/- mice was localized principally within the lung parenchyma, suggesting that the effects of CCSP deficiency were not confined to the airway epithelium. We conclude that CCSP deficiency results in increased sensitivity to hyperoxia-induced lung injury as measured by increased mortality, early onset of lung edema, and induction of proinflammatory cytokine mRNAs.

Animals↗

PubMed1997

Analysis of cytokine mRNA profiles in the lungs of Pneumocystis carinii-infected mice.

Severe combined immunodeficient (scid) mice lack functional CD4+ lymphocytes, and therefore develop life-threatening Pneumocystis carinii infection. However, when scid mice are immunologically reconstituted with spleen cells, including CD4+ cells, a protective inflammatory response is mounted against the organism. To determine whether these lymphocytes induce elevated cytokine mRNA levels in response to P. carinii infection, steady-state levels of cytokine mRNAs were measured in the lungs of both reconstituted and unaltered scid mice. Despite significant numbers of organisms and the presence of functional alveolar macrophages in the lungs of 8- and 10-wk-old scid mice, there was neither evidence of pulmonary inflammation, nor increased proinflammatory cytokine expression. However, when 8-wk-old scid mice were immunologically reconstituted, signs of intense, focal pulmonary inflammation were observed, and levels of interleukin (IL)-1alpha, IL-1beta, IL-3, IL-6, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF)-alpha, and TNF-beta mRNAs were all significantly elevated. Cytokine expression was increased at day 10 post-reconstitution (PR), maximal at day 12 PR, and returned to baseline by day 22 PR. In situ hybridization demonstrated that at day 12 PR, increased IL-1beta and TNF-alpha expression was localized to sites of intense inflammation and focal P. carinii colonization. Many of the cells expressing high levels of IL-1beta and TNF-alpha in these regions were in direct contact with organisms, or contained degraded organisms within their cytoplasm. Thus, even though functional macrophages are present in scid mice, CD4+ T cells are required for proinflammatory cytokine expression, which is associated with the generation of a protective inflammatory response at sites of P. carinii infection.

Animals↗

PubMed1997

Particulate-cell interactions and pulmonary cytokine expression.

The type II cell plays an important role in the response of the alveolar epithelium after lung injury through its synthesis and secretion of pulmonary surfactant, and by acting as the stem cell for the replacement of damaged type I epithelial cells. The nonciliated bronchiolar epithelial (Clara) cell is thought to play a similar role during repair of the bronchiolar epithelium. Recent evidence has suggested that epithelial cells may participate in aspects of the inflammatory response and regulation of fibroblast growth during pulmonary fibrosis through the production of and response to specific growth factors and cytokines. The cellular and molecular responses of epithelial cells and how they lead to the progression of events that defines the pulmonary parenchymal response to a class of particles is unclear. We used particles differing in size, chemical composition, and fibrogenicity in vivo and in vitro to elucidate early changes in proinflammatory and profibrotic cytokine and antioxidant gene expression in lung cells. Early increases in mRNA and protein for the proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha have been observed in epithelial cells following exposure. These are accompanied by changes in specific epithelial genes including surfactant protein C and Clara cell secretory protein. The data indicate that effects on the epithelium are due to direct interactions with particles, not a result of macrophage-derived mediators, and suggest a more significant role in the overall pulmonary response than previously suspected. These results suggest that type II cell growth factor production may be significant in the pathogenesis of pulmonary fibrosis.

Animals↗

PubMed1996

Characterization of the early pulmonary inflammatory response associated with PTFE fume exposure.

Heating of polytetrafluoroethylene (PTFE) has been described to release fumes containing ultrafine particles (approximately 18 nm diam). These fumes can be highly toxic in the respiratory tract inducing extensive pulmonary edema with hemorrhagic inflammation. Fischer-344 rats were exposed to PTFE fumes generated by temperatures ranging from 450 to 460 degrees C for 15 min at an exposure concentration of 5 x 10(5) particles/cm3, equivalent to approximately 50 micrograms/m3. Responses were examined 4 hr post-treatment when these rats demonstrated 60-85% neutrophils (PMNs) in their lung lavage. Increases in abundance for messages encoding the antioxidants manganese superoxide dismutase and metallothionein (MT) increased 15- and 40-fold, respectively. For messages encoding the pro- and anti-inflammatory cytokines: inducible nitric oxide synthase, interleukin 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6), macrophage inflammatory protein-2, and tumor necrosis factor-alpha (TNF alpha) increases of 5-, 5-, 10-, 40-, 40-, and 15-fold were present. Vascular endothelial growth factor, which may play a role in the integrity of the endothelial barrier, was decreased to 20% of controls. In situ sections were hybridized with 33P cRNA probes encoding IL-6, MT, surfactant protein C, and TNF alpha. Increased mRNA abundance for MT and IL-6 was expressed around all airways and interstitial regions with MT and IL-6 demonstrating similar spatial distribution. Large numbers of activated PMNs expressed IL-6, MT, and TNF alpha. Additionally, pulmonary macrophages and epithelial cells were actively involved. These observations support the notion that PTFE fumes containing ultrafine particles initiate a severe inflammatory response at low inhaled particle mass concentrations, which is suggestive of an oxidative injury. Furthermore, PMNs may actively regulate the inflammatory process through cytokine and antioxidant expression.

Animals↗

PubMed1996

Extracellular glutathione peroxidase in human lung epithelial lining fluid and in lung cells.

The epithelial cells of the lower respiratory tract are exposed to high levels of inhaled oxygen and other oxidants. We hypothesized that lung cells would secrete the antioxidant enzyme, extracellular glutathione peroxidase (eGPx), into epithelial lining fluid (ELF). To investigate this hypothesis, we used specific immunoprecipitations of GPx enzymes from ELF, specific immunoprecipitations of 75Se metabolically labeled proteins from lung cells in culture, and in situ hybridization, Northern blot, and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Fifty-seven percent of ELF GPx activity was due to eGPx and 40% was due to cellular GPx (cGPx). Primary bronchial epithelial cells (BEC), primary alveolar macrophages (AM), and two human bronchial epithelial cell lines, BEP2D and A549, synthesized both eGPx and cGPx and secreted eGPx into the medium. Freshly isolated human AM and BEC expressed eGPx mRNA, while freshly isolated rabbit type 2 pneumocytes did not. In lung tissue, eGPx mRNA was found mainly in interstitial cells of tissue surrounding airways. It is concluded that more than half of GPx activity in BAL is due to eGPx, and that BEC, AM, and interstitial cells are potential sources of pulmonary eGPx.

Adolescent↗

PubMed1996

Early and persistent alterations in the expression of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor alpha mRNA levels in fibrosis-resistant and sensitive mice after thoracic irradiation.

Fibrosis, characterized by the accumulation of collagen, is a consequence of a chronic inflammatory response. The purpose of this study was to determine if tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and IL-1 beta mRNA expression are altered acutely after irradiation, during the so-called "latent" phase of pulmonary injury, and to examine if these alterations persist through the development of pneumonitis and fibrosis. Further, we wished to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Fibrosis-sensitive (C57BL/6) and fibrosis-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by slot blotting and hybridized with radiolabeled cDNA probes encoding for TNF-alpha, IL-1 alpha and IL-1 beta. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase. It was found that TNF-alpha mRNA levels were increased in C57BL/6 mice at days 1 and 7 postirradiation after 5 Gy and day 14 postirradiation after both 5 and 12.5 Gy, and IL-1 alpha mRNA levels were increased in C57BL/6 mice at days 56, 112 and 182 postirradiation after both 5 and 12.5 Gy, and IL-1 beta mRNA levels in the C3H/HeJ mice were increased at days 56 and 182 postirradiation after 12.5 Gy. In summary, these studies demonstrated early and persistent alterations in TNF-alpha, IL-1 alpha and IL-1 beta mRNA levels even at the lower dose (5 Gy). The temporal relationship between the elevation of these cytokines and the strain-dependent variation in fibrosis response suggests that IL-1 alpha and TNF-alpha contribute to the radiation-induced component of pulmonary fibrosis, whereas IL-1 beta may have a protective function.

Animals↗

PubMed1995

A perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis.

PURPOSE: Radiation-induced pulmonary reactions have classically been viewed as distinct phases--acute pneumonitis and, later, fibrosis--occurring at different times after irradiation and attributed to different target cell populations. We prefer to view these events as a continuum, with no clear distinction between the temporal sequence of the different pulmonary reactions; the progression is the result of an early activation of an inflammatory reaction, leading to the expression and maintenance of a cytokine cascade. In the current study, we have examined the temporal and spatial expression of cytokine and extracellular matrix messenger ribonucleic acid (mRNA) abundance in fibrosis-sensitive mice after thoracic irradiation. METHODS AND MATERIALS: Radiation fibrosis-prone (C57BL/6) mice received thoracic irradiation of 5 and 12.5 Gy. At Day 1, and 1, 2, 8, 16, and 24 weeks after treatment, animals were killed and lung tissue processed for light microscopy and isolation of RNA. Expression of cytokine and extracellular matrix mRNA abundance was evaluated by slot-blot analysis and cellular localization by in situ hybridization and immunochemistry. RESULTS: One of the cytokines responsible for the inflammatory phase (IL-1 alpha) is elevated at 2 weeks, returns to normal baseline values, then increases at 8 weeks, remaining elevated until 26 weeks when lung fibrosis appears. Transforming growth factor-beta (TGF beta), a proliferative cytokine, is elevated at 2 weeks, persists until 8 weeks, and then returns to baseline values. In parallel with the cytokine cascade, the fibrogenic markers for CI/CIII/IV (collagen genes) correlate by showing a similar early and then later elevation of activity. For instance, the collagen gene expression of CI/CIII is a biphasic response with an initial increase at 1-2 weeks that remits at 8 weeks, remains inactive from 8 to 16 weeks, and then becomes elevated at 6 months when collagen deposition is recognized histopathologically. CONCLUSION: These studies clearly demonstrate the early and persistent elevation of cytokine production following pulmonary irradiation. The temporal relationship between the elevation of specific cytokines and the histological and biochemical evidence of fibrosis serves to illustrate the continuum of response, which, we believe, underlies pulmonary radiation reactions and supports the concept of a perpetual cascade of cytokines produced immediately after irradiation, prompting collagen genes to turn on, and persisting until the expression of late effects becomes apparent pathologically and clinically.

Animals↗

PubMed1995

Relationship of lead-induced proteins to stress response proteins in astroglial cells.

Astroglial cells are resistant to cell death and morphologic damage following lead (Pb) exposure at concentrations which elicit detrimental effects in neurons. A possible explanation may be that astroglial cells respond to Pb by increasing the expression of specific proteins, such as heat-shock proteins (HSPs), which confer resistance to low levels of Pb. However, there has been relatively limited information regarding the ability of Pb to evoke the synthesis of HSPs. In the current study, pulse-labeling of cultured astroglial proteins with [3H]-leucine was used to evaluate the nature of Pb-induced changes in protein expression. The effect of Pb on newly synthesized proteins was compared to the response elicited by heat-shock and oxidative injury. Immunoblot analysis was utilized to examine alterations in levels of various stress proteins including HSP27, HSP70, HSP90, and heme oxygenase-1 (HO-1). Even though Pb induced the synthesis of proteins with estimated molecular weights of 23 kDa, 32 kDa, 70 kDa, and 90 kDa, the accumulation of HSPs other than HO-1 was not observed. Hyperthermia and treatment with Na arsenite both resulted in enhanced expression of HSP70 and HO-1. In addition, exposure to hydrogen peroxide (H2O2), cadmium (Cd), and lipopolysaccharide (LPS) stimulated a rise in HO-1 levels. Although cellular insult failed to elicit an increase in either HSP27 or HSP90, cultured astroglia expressed readily detectable levels of both these proteins. Furthermore, Pb exposure resulted in the development of crosstolerance to subsequent injury by treatment with either Cd or H2O2. The results of this study indicate that Pb triggers a less conventional stress response in astroglial cells, which may provide enhanced resistance to the toxic effects of Pb.

Animals↗

PubMed1995

Induction of newly synthesized proteins in astroglial cells exposed to lead.

Although astroglial cells accumulate lead (Pb), they appear to be less sensitive to its overt deleterious effects than are neurons. One possible mechanism in which as rocytes adapt to Pb may be by upregulating the expression of particular genes. This study evaluated the effect of exogenous Pb on proteins synthesized in primary rat astroglial cultures. Following incubations with 0-50 microM Pb acetate cells were radiolabeled with leucine for various time intervals. Proteins were subsequently analyzed using SDS-PAGE followed by fluorography. Changes observed within the first 24 hr resembled a classical stress response and included increased synthesis of proteins with apparent molecular weights of 90, 70, 32-35, and 23 kDa. The most notable change was the enhanced synthesis and abundance of the 23-kDa protein (p23) which persisted throughout a 14-day treatment period, whereas the synthesis of the other proteins declined. This protein did not appear to be induced in rat lung fibroblasts treated with similar concentrations of Pb. Subcellular fractionation indicated that p23 was localized to the cytosol. Treatment with actinomycin D or cycloheximide prior to the addition of Pb precluded induction of p23. Pulse labeling of cells following a 24-hr Pb exposure revealed that p23 continued to be synthesized for 12 but not 24 hr following removal of Pb. Pulse-chase experiments indicated that the protein was stable for at least 18 hr following the removal of Pb. Two-dimensional gel electrophoresis revealed that the 23-kDa Pb-induced protein consisted of multiple-charged species with pl values ranging from 4.3 to 7.8.

Animals↗

PubMed1995

Interleukin-8 and monocyte chemoattractant protein-1 mRNAs in oxygen-injured rabbit lung.

The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are neutrophil and monocyte attractants, respectively. We hypothesized that IL-8 and MCP-1 mRNA expression in alveolar macrophages (AM) lavaged from rabbit lung would be increased by oxygen exposure, which is known to induce inflammation. Adult rabbits were exposed to > 95% oxygen for up to 64 h and allowed to recover in room air for up to 72 h before killing and pulmonary lavage. Numbers of lavageable polymorphonuclear cells (PMN) and AM rose during the exposure protocol. Quantitative in situ hybridization with 3H-labeled cRNA probes showed both IL-8 and MCP-1 mRNA expression in AM during oxygen exposure, with peak levels of IL-8 mRNA at 56-h oxygen exposure and of MCP-1 mRNA at 64-h oxygen exposure with 24-h room air recovery. IL-8 mRNA was present in PMN between 48-h oxygen exposure and 64-h oxygen exposure with 24-h room air recovery. MCP-1 mRNA was not expressed in PMN. This pattern of chemokine mRNA expression emphasizes the importance of inflammatory cells as effectors in the pulmonary response to oxygen exposure.

Animals↗