Potentiation of bleomycin-induced lung injury by exposure to 70% oxygen. Morphologic assessment

The effects of a single intratracheal instillation of bleomycin followed by exposure to 70% oxygen for 72 h were studied in hamsters. Mortality increased markedly among hamsters exposed to 70% oxygen for 72 h after bleomycin instillation, compared with animals receiving bleomycin and breathing room air. The lethal dose required to kill 50% of the hamsters at 30 days (LD50, 30 day) for bleomycin alone was 0.73 U/100 g body weight, whereas the LD50, 30 day for bleomycin followed by 70% oxygen fell to 0.23 U/100 g body weight. Using morphometry and light microscopy, we found that the amount of diseased lung increased in hamsters given bleomycin with hyperoxia compared with that in those treated with bleomycin alone. After 0.20 U bleomycin and air, 2.8 +/- 1.6% of the lung was abnormal, but with 0.20 U bleomycin followed by 70% oxygen, 42.7 +/- 17.9% of the lung was abnormal. At bleomycin doses that produced no apparent lesions, the addition of 70% oxygen for 72 h produced focal interstitial fibrosis at 30 days. Neither mortality nor significant histologic changes were seen in hamsters treated with saline followed by exposure to 70% oxygen for 72 h. This study demonstrates that hyperoxia potentiates bleomycin damage and suggests that the use of elevated oxygen concentrations in patients being treated with bleomycin should be minimized.     

Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling.

Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. This study assessed the effect of repeated allergen exposure on ASM and epithelial cell deoxyribonucleic acid (DNA) synthesis, cell recruitment and airway wall pathology. Brown-Norway rats were sensitized and then exposed to ovalbumin or saline aerosol every 3 days on six occasions. After the final exposure, rats were administered twice daily for 7 days with the DNA S-phase marker bromodeoxyuridine (BrdU). Using a triple immunohistochemical staining technique, BrdU incorporation into ASM and epithelium was quantified employing computer-assisted image analysis. There were >3-fold mean increases in BrdU incorporation into ASM from 1.3% of cells (95% confidence interval (CI) 1.0-1.6) in saline controls to 4.7% (95% CI 2.6-6.7) after allergen exposure (p<0.001), and in airway epithelium, from 1.3 (95% CI 0.6-2.0) BrdU-positive cells x mm basement membrane(-1) in saline controls to 4.9 (95% CI 3.0-6.7) after allergen exposure (p<0.001). There was increased subepithelial collagen deposition and mucus secretion along with a significant eosinophil and lymphocyte recruitment to the airways. Increased rates of deoxyribonucleic acid synthesis in both airway smooth muscle and epithelial cells along with changes to the airway wall pathology may precede the establishment of smooth muscle thickening and airway remodelling after repeated allergen exposure in rats. This model seems to be appropriate for studying structural changes within the airways as observed in asthma.     

Effects of drugs that modify brain biogenic amine concentrations on thyroid activation induced by exposure to cold.

L-Dihydroxphenylalanine (L-DOPA) significantly inhibited intrathyroidal colloid droplet formation induced by exposure to cold in the rat. Diethyldithiocarbamate (DDC) also inhibited colloid droplet formation in response to cold. The combined administration of L-DOPA and DDC produced an additive inhibition of the thyroidal endocytotic response to exposure to cold. Pretreatment with chlorpromazine (CPZ) ameliorated the inhibitory effect of L-DOPA. DL-alpha-methyl-p-tyrosine (alpha-MT) also signficantly depressed the thyroidal response. Inhibition of colloid droplet formation induced by alpha-MT was not altered by the administration of DL-dihydroxyphenylserine (DL-DOPS). On the other hand, treatment of the alpha-MT-treated rats with L-DOPA to normalize dopamine synthesis resulted in a dramatic recovery from the inhibition. Blockade of serotonin biosynthesis with p-chlorophenylalanine (p-CPA) failed to produce a significant inhibition of colloid droplet formation. However, 5-hydroxytryptophan (5-HTP) markedly inhibited the thyroidal response to cold. Brocresine phosphate (BP) was another inhibitor of the thyroidal endocytotic response to exposure to cold. Oxotremorine also markedly depressed the thyroidal response to cold. Since these drugs did not interfere with pituitary thyroid responsiveness to exogenous thyrotropin-releasing hormone (TRH), it seems that the throidal endocytotic response to exposure to cold as a reflection of TSH secretion was directly influenced by alterations of brain biogenic amine concentrations or turnover rates.     

Cellular interactions in pulmonary oxygen toxicity in vitro: II. Hyperoxia causes adult rat lung fibroblast cultures to produce apparently autocrine growth factors

Mixed lung cell cultures from adult rats were exposed to 21, 50, or 95% O2. In the presence of serum, actively dividing mixed lung cell cultures acutely exposed to 50% O2 had a reduced rate of cell division, while 95% O2 caused growth arrest and cell death. In the absence of serum, 95% O2 again caused cell death, while cell numbers were stable for up to 1 week in 21 or 50% O2. These cells adapted to the nonlethal 50% O2 environment by a 48% increase in superoxide dismutase activity, which was not seen with the lethal 95% O2 environment. Under serum-free conditions, conditioned medium collected from cells exposed to 50% O2, but not 21% O2, contained transferable factors that increased DNA synthesis in other nonhyperoxic mixed lung cell cultures. In a series of studies to determine both the source and target cell types for this growth factor(s), the lung fibroblast was found to release an apparently autocrine growth stimulator, with an apparent molecular weight of approximately 96,000, over the first 3 days of 50% O2 exposure. A separate apparently autocrine growth stimulator, with molecular weight approximately 7000-9000, was released over the second 3 days of a 6-day exposure to 50% O2.     

Adaptation of the growing lung to increased Vo2: III. The effect of exposure to cold environment in rats

This study was undertaken to further test the hypothesis that increased Vo2 operates as a stimulus for enhanced lung growth leading to a pulmonary diffusing capacity adapted to the body's O2 requirements. Vo2 was augmented by raising 4-week-old rats for 3 weeks at 11 degrees C ambient temperature, with controls kept at 24 degrees C; this led to an increase in Vo2 averaged over 24 h by 64%. In contrast to previous experiments with waltzing mice this regime did not affect body growth, as the final body weights were identical in both groups. In the cold-exposed rats the lung volume was larger by 24%, due to an increase by 26% in air volume (at about TLC), 13% in capillary blood volume and 19% in tissue volume. The alveolar and capillary surface areas were increased by 18%, and Dm and Dl by 17% and 21% respectively. It is concluded that the hypothesis of adaptation of pulmonary gas exchange capacity to increased Vo2 cannot be rejected. Whilst in previous experiments some doubts had to be retained as to the specificity of the stimulus, because of its rather marked effect on body weight, this reservation does not hold in this case. The structural modifications which lead to increased Dl in the various experimental models are discussed.     

Effects of fibroblast and epidermal growth factors on ovarian cell proliferation in vitro. II. Proliferative response of luteal cells to FGF but not EGF.

The effect of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on luteal cell proliferation in vitro has been examined. Luteal cells maintained in the presence of low serum (1%) go through a doubling after 7 days. Addition of EGF induced one more doubling of the cells, after which the cells became resting. In contrast, FGF induced the cells to divide logarithmically with a cell cycle of 48 h. The effect of FGF was dependent on the serum and FGF concentrations. It has been obtained with serum concentrations ranging from 0.1% to 10% and with FGF concentrations ranging from 0.1 ng to 10 ng/ml. The half-maximal FGF response was observed at 1.5 x 10(-11)M. In contrast, EGF has no effect besides causing an initial cell doubline within the same range of serum or FGF concentrations. Since granulosa cells have been shown to be highly sensitive to EGF as well as FGF, it can be concluded that during the luteinization process that sensitivity of the cells to EGF is lost, while the sensitivity of FGF is retained. This demonstrates that although luteal cells and granulosa cells are interrelated cell types their sensitivity to growth factors such as EGF is quite different.     

Characterization of GMP-17, a granule membrane protein that moves to the plasma membrane of natural killer cells following target cell recognition.

Cytotoxic lymphocytes are characterized by their inclusion of cytoplasmic granules that fuse with the plasma membrane following target cell recognition. We previously identified a cytotoxic granule membrane protein designated p15-TIA-1 that is immunochemically related to an RNA-recognition motif (RRM)-type RNA-binding protein designated p40-TIA-1. Although it was suggested that p15-TIA-1 might be derived from p40-T1A-1 by proteolysis, N-terminal amino acid sequencing of p15-TIA-1 immunoaffinity purified from a natural killer (NK) cell line by using monoclonal antibody (mAb) 2G9 revealed that p15-T1A-1 is identical to the deduced amino acid sequence of NKG7 and GIG-1, cDNAs isolated from NK cells and granulocyte-colony-stimulating factor-treated mononuclear cells, respectively. Epitope mapping revealed that mAb 2G9 recognizes the C terminus of p15-T1A-1 and p40-T1A-1. The deduced amino acid sequence of p15-T1A-1/NKG7/GIG-1 predicts that the protein possesses four transmembrane domains, and immuno-electron microscopy localizes the endogenous protein to the membranes of cytotoxic granules in NK cells. Given its subcellular localization, we propose to rename-this protein GMP-17, for granule membrane protein of 17 kDa. Immunofluorescence microscopy of freshly isolated NK cells confirms this granular localization. Target cell-induced NK cell degranulation results in translocation of GMP-17 from granules to the plasma membrane, suggesting a possible role for GMP-17 in regulating the effector function of lymphocytes and neutrophils.     

Effects of fibroblast and epidermal growth factors on ovarian cell proliferation in vitro. I. Characterization of the response of granulosa cells to FGF and EGF.

Despite numerous studies on the effects of gonadotropins on ovarian cells in tissue culture, the factors controlling the proliferation of granulosa cells in vitro remain unknown. We have examined the effect of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on granulosa cell proliferation in vitro in an attempt to clarify their possible roles in the control of ovarian development. FGF and EGF both stimulate DNA synthesis in resting populations of granulosa cells. The half-maximal response forthis effect with FGF was observed at 4 X 10(-11)M and with EGF at 1.5 X 10(-13)M. Autoradiography demonstrated that the whole cell population initiated DNA synthesis in the presence of either EGF or FGF, thus precluding an additive effect of the two mitogens. When cells were maintained at low density (100 cells/cm2) in the presence of low serum (1%) they divided with a doubling time of 48-72 h, but addition of either EGF or FGF accelerated their proliferation. The doubling time observed in the presence of FGF was 16 h versus 20 h with EGF and the final cell density reached in the presence of EGF or FGF was 20 times that of cells maintained in the presence of 1% calf serum alone. In the presence of 10% serum, granulosa cells had a doubling time of 24 h and the final density reached was similar to that observed in 1% serum with EGF and FGF. Addition of EGF or FGF to 10% serum resulted in a final density 3 to 4-fold higher than that observed with 10% serum alone. The ultrastructure of the granulosa cells grown in the presence of EGF or FGF was similar to that of cells maintained in the absence of added mitogens. The only marked difference was that cells grown in the presence of FGF or EGF had a high lipid granule content while cells grown in their absence had a low lipid granule content. The effect of various concentrations of FGF and EGF on the proliferation of granulosa cells has been analyzed. The minimal effective dose of EGF was 3 X 10(-14)M and saturation was observed at 3 X 10(-11)M, with a half-maximal response at 6 X 10(-13)M. With FGF the minimal dose stimulating proliferation was 1.5 X 10(-12)M and saturation was achieved at 1.5 X 10(-10)M, with a half-maximal response at 3 X 10(-11)M. Our results show that EGF and FGF are the most potent mitogens ever observed and are mitogenic for granulosa cells at 300 to 3000-fold lower concentrations than for other cell types which have been studied, such as fibroblasts or lens epithelial cells.     

Enhanced DNA synthesis of cultured vascular smooth muscle cells from spontaneously hypertensive rats. Difference of response to growth factor, intracellular free calcium concentration and DNA synthesizing cell cycle

It is widely reported that cultured vascular smooth muscle cells (CVSMCs) from spontaneously hypertensive rats (SHR) show enhanced proliferation compared with cells from Wistar-Kyoto rats (WKY). The present studies were designed to find out whether this exaggerated proliferation in SHR is determined genetically and, if so, to evaluate the mechanism on the cell cycle. (1) Incorporation of [3H]thymidine into DNA was enhanced in CVSMCs from 3- and 12-week-old SHR compared with WKY but not in CVSMCs from DOCA-salt hypertensive rats compared with the cells from sham-operated rats. (2) DNA synthesis in SHR cells was enhanced further by addition of insulin (which is considered to be a progression factor) but not by arginine-vasopressin (AVP; considered to be a competence factor) or by angiotensin II (AII). On the other hand, insulin, AVP and AII significantly augmented DNA synthesis in WKY cells. (3) Intracellular free calcium concentration was slightly, but significantly, higher in SHR cells. (4) An increase in the population of DNA-synthesizing S-phase cells and decrease in (G2 + M)-phase cells in SHR were observed by flowcytometry. These data suggest (1) that enhanced DNA synthesis in CVSMCs from SHR is determined genetically, (2) that enhanced DNA synthesis in CVSMCs from SHR is largely dependent on an increased proportion of S-phase cells and (3) that this increase in S-phase cells in CVSMCs from SHR could be due to enhanced competence gene expression in SHR cells. (4) The increased intracellular free calcium concentration is compatible with an activation of the inositol-trisphosphate pathway.