A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens.

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and "front and back"' section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.

Effects of combined ozone and air pollution particle exposure in mice.

Epidemiologic studies indicate that ozone (O3*) and air pollution particles can exacerbate asthma symptoms. We investigated whether coexposure to inhaled particles and O3 causes a synergistic effect on airway responsiveness and allergic inflammation in a murine (BALB/c) model of ovalbumin (OVA)-induced asthma. Half of the mice were sensitized by intraperitoneal injection of OVA and then exposed to OVA aerosol on 3 successive days to create the asthmatic phenotype; the other half were sensitized to OVA and exposed to phosphate-buffered saline (PBS) to create the nonasthmatic control group. On the same 3 days that the OVA or PBS challenge was administered, mice were further divided into groups that were exposed for 5 hours to concentrated ambient particles (CAPs; mass values ranging from 63 to 1,569 microg/m3 for 1 day's exposure), 0.3 ppm O3, both, or neither (n > or = 61 total mice per exposure group for all 12 experiments). Whole-body plethysmography was used to measure airway responsiveness after challenge with aerosolized methacholine (MCh). Enhanced pause (Penh), an index that closely correlates with pulmonary resistance (Hamelmann et al 1997), was measured daily in each mouse immediately after pollutant exposure and, for 7 of the 12 experiments (n > or = 36/exposure group), beginning 24 hours after the final OVA or PBS challenge. Using several complementary statistical models, we found that exposure to CAPs alone caused a small but significant increase in Penh in both normal and asthmatic mice immediately after exposure (an increase of approximately 1% per 100-microg/m3 increase in CAPs). No increase in Penh was found in animals exposed to O3 alone or to filtered air. Compared with control animals, no combination of exposure atmosphere plus asthma produced a synergistic effect on Penh. By 24 hours after the last OVA or PBS challenge, any enhanced response induced by pollutant exposure had declined to control levels. The pollutant exposures did not significantly increase airway inflammation (assessed by bronchoalveolar lavage [BAL] fluid analysis beginning 24 or 48 hours after the final OVA or PBS challenge). Because CAPs are a heterogeneous mixture of particles, elemental analysis was conducted and associations between specific elemental groupings (present in daily samples) and airway responsiveness were analyzed. This analysis showed that increased Penh in asthmatic mice exposed to CAPs plus O3 was associated with the AlSi fraction of CAPs. No such association was found in control mice or in asthmatic mice not exposed to O3. We conclude that CAPs exposure causes an immediate, short-lived (< 24-hour), small increase in airway responsiveness in mice and that changes in airway physiology are correlated with specific elements found within the particle mixture.

Insoluble components of concentrated air particles mediate alveolar macrophage responses in vitro.

We sought to characterize the bioactive constituents of concentrated ambient air particles (CAPs) through correlation of alveolar macrophage (AM) biological responses (production of TNF, macrophage inflammatory protein (MIP-2), nitrite; cell viability) to components of particle samples. CAPs samples collected on different days showed a range of bioactivity and a strong correlation was found between AM cytokine release and increased AM light scatter, a flow cytometric measure of relative particle load. Evaluation of soluble and insoluble fractions of CAPs suspensions indicate that 1) most biological effects on AMs are mediated by insoluble components and certain particle adsorbed factors such as endotoxin; 2) the variable bioactivity of CAPs collected on different days arises primarily from differences in the relative proportion of insoluble and soluble mass present in particle suspensions; and 3) the activation state of the AM influences which insoluble components are most bioactive. Use of endotoxin neutralizing agents (e.g., polymyxin-B) showed particle-adsorbed endotoxin in CAPs suspensions causes activation of normal (control) AMs while other (nonendotoxin) components are predominantly responsible for the enhanced cytokine release observed by primed AMs incubated with CAPs. The AM biological response did not correlate with any of a panel of elements quantified within insoluble CAPs samples (Al, Cd, Cr, Cu, Fe, Mg, Mn, Ni, S, Ti, V). These data demonstrate an important role for cell activation and phagocytosis of insoluble particulate matter in the response of AMs to CAPs suspensions.

Alveolar macrophage cytokine production in response to air particles in vitro: role of endotoxin.

The interaction of air particles and alveolar macrophages (AMs) may result in the release of proinflammatory cytokines. Normal mouse AMs were treated with concentrated air particle (CAPs) suspensions in vitro. After 5 h, cytokine release [macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNF-alpha)] and phagocytosis of ambient air particles were measured. CAPs samples collected from urban air (Boston) on different days were used. The CAPs samples and their soluble and solid components caused significant MIP-2 and TNF-alpha production. Variability in the potency of samples collected on different days was observed. Trace endotoxin was measured in CAPs samples (EU/mg: 2.3 +/- 0.7, mean +/- SE, n = 10). A majority of biologic activity (cytokine induction) and endotoxin content was associated with the solid components. Neutralization of endotoxin by polymyxin B abrogated >80% of TNF-alpha induction by CAPs samples, but inhibited MIP-2 production by only approximately 40%. The trace endotoxin present in CAPs caused much more MIP-2 production than predicted by concentration alone (28 +/- 8-fold increase, n = 9), indicating synergistic interaction with other AM-activating components of the particles. Data suggest that low levels of endotoxin may interact with air particles to activate lung macrophages.

Lipopolysaccharide priming amplifies lung macrophage tumor necrosis factor production in response to air particles.

Elevated concentrations of ambient air particles can result in increased mortality and morbidity, especially in people with preexisting pulmonary disease. We postulate that in the inflammatory milieu of diseased lungs, alveolar macrophages (AMs) may be primed for enhanced responses to stimuli such as inhaled air particles. To test this hypothesis in vitro, we first cultured normal AMs with or without lipopolysaccharide (LPS). We then incubated the cells with particle suspensions (urban air particles (UAP, Washington, D.C.), residual oil fly ash (ROFA), concentrated respirable-size (PM2.5) air particles (CAPs), and inert TiO2) and compared rat and human AM production of the critical proinflammatory mediator, tumor necrosis factor (TNF). LPS priming amplified TNF production by both rat and human AMs in response to UAP and CAPs but not inert TiO2. There were also differences observed between rat and human AM responses to particle suspensions. Striking changes seen only in rat were cytotoxic effects of ROFA and diminished particle uptake in response to LPS priming. The potency of CAPs samples (which are collected on separate days) varied when comparing one day's sample with another. When centrifuged, the majority of bioactivity seen in particle suspensions (TNF release) remained within the pelleted fraction while the supernatant showed minimal bioactivity. The data suggest that AMs activated by extant pulmonary inflammation may promote further inflammation by an enhanced cytokine response to inhaled ambient air particles.

Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles.

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR-/-) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 +/- 5, 77 +/- 2, and 85 +/- 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of approximately 70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1-reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 +/- 5 and 47 +/- 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected approximately 70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 approximately 40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.

Intracellular oxidant production and cytokine responses in lung macrophages: evaluation of fluorescent probes.

The fluorescent probes dichlorofluorescin (DCFH), dihydrorhodamine (DHR), and hydroethidine (HE) allow convenient assay of alveolar macrophage (AM) oxidant responses to enviromental particulates and pathogens. We sought to more precisely define the relationship of these measures of oxidant stress to production of pro-inflammatory cytokines. Normal AMs were challenged in vitro with a panel of soluble or particulate stimuli in the presence of DCFH, HE, or DHR. Flow cytometry measured cell-associated fluorescence and relative particle uptake. Tumor necrosis factor alpha and macrophage inflammatory protein 2 expression were quantitated in the same experiments. We observed variable and complex correlations between intracellular oxidant production as reported by these probes and subsequent cytokine response, including examples of striking discordance (e.g., lipopolysaccharide induced large cytokine responses with minimal probe oxidation, whereas fly ash particles caused marked oxidation of DCFH but trivial TNF release; TiO2 caused oxidation of DHR and HE, but not DCFH, and also did not increase cytokine production). Although fluorescent probes offer many advantages in analysis of intracellular oxidant responses, the data indicate that they cannot be used reliably as quantitative predictors of AM cytokine responses to environmental particulates or other stimuli.

Analysis of air pollution particulate-mediated oxidant stress in alveolar macrophages.

Adverse health effects of urban air pollution particulates may be attributable to particle-mediated oxidant stress and inflammation. Intracellular oxidant production in normal hamster alveolar macrophages (AMs) was measured upon exposure to concentrated ambient particulates (CAPs), residual oil fly ash (ROFA), and their water-soluble and particulate fractions. ROFA and CAPs caused increases in dichlorofluorescin (DCFH) oxidation, a fluorescent measure of intracellular reactive oxygen species (ROS) production, comparable to the positive control, phorbol myristate acetate (PMA). The water-soluble component of both CAPs and ROFA (CAPs, S and ROFA, S) significantly increased AM oxidant production over negative control. CAPs samples and components showed substantial day-to-day variability in their oxidant effects. Metal chelation by desferrioxamine (DF, 1 mM) caused significant inhibition of particulate-induced AM oxidant production. ROFA exposure resulted in increased macrophage inflammatory protein-2 (MIP-2) message in AMs and in increased tumor necrosis factor alpha (TNF-alpha) production by the monocyte-macrophage cell line, RAW 264.7. TNF-alpha production was inhibitable by the antioxidant N-acetylcysteine (NAC). The data suggest that metal components adsorbed to urban air pollution particulates can significantly contribute to particulate ability to cause oxidant stress and cytokine production in AMs.

New insights into interactions between the human PTH/PTHrP receptor and agonist/antagonist binding.

We prepared a polyclonal antiserum [Ab-(88-97)] against residues 8-97 of the NH2-terminal tail of the human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor. Ab-(88-97) bound specifically to the receptor, as assessed by fluorescence-activated cell sorter analysis of HEK C21 cells, which stably express approximately 400,000 hPTH/PTHrP receptors per cell. Unlike PTH, Ab-(88-97) binding did not elicit either adenosine 3',5'-cyclic monophosphate or intracellular calcium concentration signaling responses in these cells. Incubation of C21 cells for 90 min at 4 degrees C with hPTH-(1-34) plus antiserum reduced the Ab-(88-97) binding to the cells by up to 40-50% of control values in a PTH concentration-dependent fashion with a half-maximal effective concentration of approximately 5 nM. The decrease in Ab-(88-97) binding caused by hPTH-(1-34) was completely reversed by coincubation with hPTHrP-(7-34). We conclude that residues 88-97 of the hPTH/PTHrPR are involved, either directly or indirectly, in agonist but not antagonist binding to the receptor.

Alveolar macrophage interaction with air pollution particulates.

We applied flow cytometric analysis to characterize the in vitro response of alveolar macrophages (AM) to air pollution particulates. Normal hamster AM were incubated with varying concentrations of residual oil fly ash (ROFA) or concentrated ambient air particulates (CAP). We found a dose-dependent increase in AM-associated right angle light scatter (RAS) after uptake of ROFA (e.g., mean channel number 149.4 +/- 6.5, 102.5 +/- 4.1, 75.8 +/- 3.5, and 61.0 +/- 4.6 at 200, 100, 50, and 25 mg/ml, respectively) or CAP. A role for scavenger-type receptors (SR) in AM uptake of components of ROFA and CAP was identified by marked inhibition of RAS increases in AM pretreated with the specific SR inhibitor polyinosinic acid. We combined measurement of particle uptake (RAS) with flow cytometric analysis of intracellular oxidation of dichlorofluorescin. Both ROFA and CAP caused a dose-related intracellular oxidant stress within AM, comparable to that seen with phorbol myristate acetate (PMA) (e.g., fold increase over control, 6.6 +/- 0.4, 3.6 +/- 0.4, 4.6 +/- 0.5, 200 mg/ml ROFA, 100 mg/ml ROFA, and 10(-7) M PMA, respectively). We conclude that flow cytometry of RAS increases provides a useful relative measurement of AM uptake of complex particulates within ROFA and CAP. Both ROFA and CAP cause substantial intracellular oxidant stress within AM, which may contribute to subsequent cell activation and production of proinflammatory mediators.

Lack of uncoupling of S phase and mitosis after irradiation in p53- human lymphoblast cell lines.

It has been shown that p53- human colorectal cancer cells arrest after DNA damage in a G2-like state and may then undergo DNA synthesis without intervening mitosis (Waldman et al., Nature 381, 713-716, 1996). To further clarify the role of p53 in the regulation of the G2/M-phase checkpoint, we have studied cells of three closely related human lymphoblastoid cell lines (TK6, WTK1 and TK6E6, an HPV16 E6-transfected TK6 line) with differing p53 status. The cells were irradiated with 1.5-12 Gy gamma rays with or without 2 mM caffeine. There was no evidence of uncoupling of DNA synthesis and mitosis after irradiation in the p53- cell lines, WTK1 and TK6E6, suggesting that this uncoupling may not be a universal phenomenon. The apparent formation of tetraploid cells after irradiation of cells of the p53- WTK1 line was due to the occurrence of a G2-phase block in a pre-existing tetraploid population. These results support the conclusion that control of the G2/M-phase checkpoint after irradiation may differ among different cell types.

Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin.

We investigated the utility of the oxidant-reactive probe dichlorofluorescin (DCFH) for measurement of NO synthase (NOS) activity in rat alveolar macrophages (AMs) activated by culture for 18 h with interferon-gamma (IFN-gamma, 25 U/ml). Using both microplate-based fluorometry and flow cytometric analysis, AMs treated with l (but not d)-arginine showed a dose- and time-dependent increase in DCFH oxidation above buffer control (e.g., DCF production (nM): control, d-arginine 100 microM, l-arginine 100 microM respectively; 91+/-9, 76+/-11, 396+/-45, mean +/- SE, n = 6, 120 min). Furthermore, the NOS inhibitor (nitro-l-arginine) showed complete inhibition of the l-arginine-dependent DCF production. Parallel assays showed a strong correlation in DCFH oxidation with nitrite production in the same samples (e.g., DCF production (nM): 143, 222, 409; nitrite (microM): 2.2, 3.6, 7.1; for 5, 10, 50 microM l-arginine, respectively). In contrast, the alternate oxidant-reactive probes hydroethidine (HE) and dihydrorhodamine (DHR) did not report increased oxidant production in activated AMs incubated with l-arginine, despite their ability to easily detect intracellular superoxide anion production in cells treated with menadione (100 microM). We conclude that DCFH is a useful probe for quantitation of NOS-2 activity in activated rat lung macrophages.

Immunologic effects of acute hyperglycemia in nondiabetic rats.

BACKGROUND: This study was designed to determine the consequences of acute hyperglycemia on the immune function of peripheral neutrophils, peritoneal macrophages, and alveolar macrophages in nondiabetic rats. METHODS: The animals were randomly divided into nonsurgical (normal) and surgical groups. The postoperative rats were further divided into normoglycemic (control) and hyperglycemic (glucose) groups. The hyperglycemic condition was maintained by constant infusion of glucose to raise plasma glucose concentration to 300 mg/dL for 3 hours. The immune cells were then harvested to determine their phagocytic and oxidative capacities via flow cytometry. RESULTS: The results showed that hyperglycemia significantly decreased the respiratory burst of alveolar macrophages (p < .05). In contrast, hyperglycemia enhanced phagocytosis in these cells (p < .002). There was a significant activation of the respiratory burst in peripheral neutrophils by surgery (p < .002), but no effect of hyperglycemia. CONCLUSIONS: We conclude that hyperglycemia itself can influence immune function in some phagocytic cells, which may be an important factor in postsurgical infection.

Lung epithelial cell (A549) interaction with unopsonized environmental particulates: quantitation of particle-specific binding and IL-8 production.

The A549 cell line was used to model in vitro the interaction of alveolar epithelium with environmental particulates. Confocal and electron microscopy demonstrated A549 binding and internalization of titanium dioxide (TiO2), iron oxide (Fe2O3), concentrated ambient air particulates (CAPs), and the fibrogenic particle alpha-quartz. Flow cytometry allowed quantitation of particle binding by measuring increased right angle light scatter (RAS) (TiO2) [40 micrograms/mL], Fe2O3 [100 micrograms/mL], alpha-quartz [200 micrograms/mL], or CAPs [40 micrograms/mL] fold increase RAS: 8.1 +/- 0.9, 4.3 +/- 0.4, 2 +/- 0.1, 1.6 +/- 0.1, respectively). With this quantitative assay, binding of particle was found to be calcium-dependent for TiO2 and Fe2O3 (% inhibition, 61.0 +/- 1.9, 40.0 +/- 5.6, respectively), while alpha-quartz binding was calcium-independent. A panel of polyanionic ligands known to inhibit scavenger-type receptors was used to identify binding mechanisms for environmental particulates. Both heparin and polyinosinic acid (polyI), but not the control polyanion chondroitin sulfate, caused marked inhibition of particulate binding by A549 cells (e.g., TiO2 [40 micrograms/mL] binding; polyI, heparin, and chondroitin sulfate: 73.8 +/- 3.5, 75.5 +/- 6.0, 7.5 +/- 6.7% inhibition, respectively; mean +/- SE, n > or = 4), indicating that scavenger receptor(s), albeit those distinct from the heparin-insensitive acetylated-LDL receptor, mediate particulate binding. The particulates ability to stimulate interleukin (IL-8) production in A549 cells was also tested. alpha-quartz, but not TiO2 or CAPs, caused a dose-dependent production of IL-8 (range 1-6 ng/mL), demonstrating a particle-specific spectrum of epithelial cell cytokine (IL-8) response. The results suggest that lung epithelial cell interaction with environmental particles is mediated by distinct receptors and can lead to particle dependent cytokine responses.

Relationship between radiation-induced G1 phase arrest and p53 function in human tumor cells.

Three widely studied cell lines were used to examine the nature of the G1 arrest induced in human tumor cells by ionizing radiation and its relation to p53 status. Cell lines MCF-7 and RKO express wild-type p53, whereas HT29 expresses mutant p53. Exponentially growing cells were irradiated with 6 Gy, and the progression of G1 cells into S phase was monitored at regular intervals by flow microfluorimetric and continuous labeling autoradiographic techniques. In some experiments, cells were incubated with Colcemid prior to irradiation in order to block them in mitosis and to prevent the accumulation of cells in the second post-irradiation G1 phase. No evidence of a significant arrest at the first post-irradiation G1-S checkpoint was observed in any of the three cell lines. These results suggest that p53 function alone does not control the progression of irradiated human tumor cells from G1 into S during the first post-irradiation cell cycle. In particular, we found no evidence that radiation induced a prolonged G1 arrest in tumor cells expressing wild-type p53 as has been reported by some investigators.

Flow cytometric assay of lung macrophage uptake of environmental particulates.

We sought to establish a quantitative method using flow cytometry to study uptake of environmental particulates by alveolar macrophages (AMs). We used right angle light scatter (RAS) to measure uptake of titanium dioxide, quartz, and diesel particulates. After incubation with TiO2 in vitro, AMs showed dose-dependent increases in both cell-associated particles visualized by microscopy and RAS measured by flow cytometry (e.g., fold increase RAS at 4, 8, 16, 32, and 80 micrograms/ml, respectively, = 2 +/- 0.1, 4.0 +/- 0.5, 5.5 +/- 0.5, 9.1 +/- 2.5, 14.3 +/- 0.9; mean +/- SEM). Similar results were obtained with quartz and diesel particles. A strong correlation was observed between particle load per cell and AM RAS after uptake of fluorescent latex beads or fluorescent TiO2 (coated with BODIPY-BSA) (R2 = 0.984, 0.997, respectively). Using this technique, we found AM uptake of environmental particulates to be substantially greater than that of a panel of myelomonocytic and epithelial cell lines, consistent with their physiologic role in pulmonary defenses. RAS measurements have also identified both calcium-dependent and calcium-independent components in AM interactions with inert particles. Although this technique does not allow precise quantitation of particle number or mass per cell, flow cytometric analysis of relative increases in RAS is a useful tool to study AM interactions with a variety of environmental particulates.