Gestational Exposure to Air Pollution Alters Cortical Volume, Microglial Morphology, and Microglia-Neuron Interactions in a Sex-Specific Manner.

Microglia are the resident immune cells of the brain, important for normal neural development in addition to host defense in response to inflammatory stimuli. Air pollution is one of the most pervasive and harmful environmental toxicants in the modern world, and several large scale epidemiological studies have recently linked prenatal air pollution exposure with an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD). Diesel exhaust particles (DEP) are a primary toxic component of air pollution, and markedly activate microglia in vitro and in vivo in adult rodents. We have demonstrated that prenatal exposure to DEP in mice, i.e., to the pregnant dams throughout gestation, results in a persistent vulnerability to behavioral deficits in adult offspring, especially in males, which is intriguing given the greater incidence of ASD in males to females (∼4:1). Moreover, there is a striking upregulation of toll-like receptor (TLR) 4 gene expression within the brains of the same mice, and this expression is primarily in microglia. Here we explored the impact of gestational exposure to DEP or vehicle on microglial morphology in the developing brains of male and female mice. DEP exposure increased inflammatory cytokine protein and altered the morphology of microglia, consistent with activation or a delay in maturation, only within the embryonic brains of male mice; and these effects were dependent on TLR4. DEP exposure also increased cortical volume at embryonic day (E)18, which switched to decreased volume by post-natal day (P)30 in males, suggesting an impact on the developing neural stem cell niche. Consistent with this hypothesis, we found increased microglial-neuronal interactions in male offspring that received DEP compared to all other groups. Taken together, these data suggest a mechanism by which prenatal exposure to environmental toxins may affect microglial development and long-term function, and thereby contribute to the risk of neurodevelopmental disorders.

Nonpeptide CXCR2 antagonist prevents neutrophil accumulation in hyperoxia-exposed newborn rats.

Neutrophil influx in lung injury is controlled in part by chemokines acting through the receptor, CXCR2. To avoid adverse effects of steroids typically used to modify inflammation, we evaluated the effects of competitive blockade of CXCR2 in rats on neutrophil function in vitro and on neutrophil influx in vivo in hyperoxia-induced newborn lung injury, a model of bronchopulmonary dysplasia. In vitro, SB-265610 antagonizes rat cytokine-induced neutrophil chemoattractant-1 (CINC-1)-induced calcium mobilization, IC50 = 3.7 nM, and rat neutrophil chemotaxis in a concentration-dependent manner, IC50 = 70 nM. In vivo, newborn rats exposed to 95% O2 for 8 days had increased lung neutrophil content. Injection with 1 to 3 mg/kg SB-265610 on days 3 to 5 reduced hyperoxia-induced neutrophil accumulation in bronchoalveolar lavage and whole lung myeloperoxidase accumulation at the highest doses. To determine whether these effects might be due in part to increased neutrophil apoptosis, peripheral neutrophils were cultured with and without SB-265610. Apoptosis was assessed by morphology, viability, and terminal transferase deoxyuridine triphosphatidyl nucleotide nick-end labeling. Treatment of neutrophils with CINC-1 reduced apoptosis compared with untreated neutrophils. SB-265610 reduced the antiapoptotic effect of CINC-1 to the levels of those untreated with CINC-1. A selective CXCR2 antagonist may be useful in diseases where neutrophil-mediated exacerbation is present.

Anti-neutrophil chemokine preserves alveolar development in hyperoxia-exposed newborn rats.

Inflammation may contribute to lung injury and impaired alveolar development in bronchopulmonary dysplasia. We treated hyperoxia-exposed newborn rats with antibodies to the neutrophil chemokine cytokine-induced neutrophil chemoattractant-1 (CINC-1) during 95% O2 exposure to reduce adverse effects of hyperoxia-induced inflammation on lung development. Rats were exposed at birth to air, 95% O2, or 95% O2 + anti-CINC-1 (injected on days 3 and 4). Bromodeoxyuridine (BrdU) was injected 6 h before death. Anti-CINC-1 treatment improved weight gain but not survival at day 8. Anti-CINC-1 reduced bronchoalveolar lavage neutrophils at day 8 to levels equal to air controls. Total detectable lung CINC-1 was reduced to air control levels. Lung compliance was improved by anti-CINC-1, achieving air control levels in the 10-microg anti-CINC-1 group. Anti-CINC-1 preserved proliferating cell nuclear antigen expression in airway epithelium despite 95% O2 exposure. BrdU incorporation was depressed by hyperoxia but preserved by anti-CINC-1 to levels similar to air control. Alveolar volume and surface density were decreased by hyperoxia but preserved by anti-CINC-1 to levels equal to air control. Blockade of neutrophil influx in newborns may avert early lung injury and avoid alveolar developmental arrest that contributes to bronchopulmonary dysplasia.

Lung inflammation in hyperoxia can be prevented by antichemokine treatment in newborn rats.

Hyperoxia may contribute to lung disease in newborns through effects on alveolar neutrophils which predominate in respiratory distress syndrome and other acute lung injuries. Neutrophil chemokines such as interleukin-8 (IL-8) regulate chemoattraction, and are elevated in tracheal aspirates of newborns who develop bronchopulmonary dysplasia (BPD). Blockade of neutrophil chemokines may reduce hyperoxia-induced inflammatory lung injury and BPD. We therefore tested the hypothesis that hyperoxia contributes to elevations of rat neutrophil chemokines, cytokine-induced neutrophil chemoattractant-1 (CINC-1), and macrophage inflammatory protein-2 (MIP-2) in newborn rat lung. Newborn rats were exposed to air or 95% O(2) for 8 d. CINC-1 and MIP-2 were measured in whole lung homogenates by ELISA. Newborn 95% O(2)-exposed animals were given anti-CINC-1 or anti-MIP-2, 1, 5, or 10 microg on Days 3 and 4 of 95% O(2) exposure. Bronchoalveolar lavage (BAL) was performed after perfusion on day 6 to evaluate airway neutrophils, and myeloperoxidase (MPO) was measured in perfused whole lung. Lungs were examined histologically and immunohistochemically for effects of 95% O(2) +/- antichemokine. CINC-1 and MIP-2 increased nearly tenfold by Day 8 95% O(2) treatment versus air control. CINC-1 and MIP-2 immunolabeling was increased in alveolar macrophages and alveolar epithelium in 95% O(2). Anti-CINC-1 and anti-MIP-2 treatment at every dose reduced neutrophil number > 90% in BAL. Anti-CINC-1 10 microg reduced tissue MPO by 50%. Antichemokine treatment on days 3 and 4 prevented alveolar septal thickening and reduced chemokine immunolabeling on Day 6. Hyperoxia-induced neutrophil influx is mediated in part by CINC-1 and MIP-2 in newborn rats and can be partially prevented by treatment with anti-CINC-1 and anti-MIP-2.

Differential nitric oxide production by microvascular and macrovascular endothelial cells.

Phenotypic heterogeneity among endothelial cell populations may account for important organ-specific behaviors. Experimental evidence suggests that endothelium-derived nitric oxide mediates certain of these unique responses. The purpose of these investigations was to compare rat pulmonary microvascular endothelial cells with pulmonary artery and aortic macrovascular endothelial cells in their ability to generate nitric oxide (NO). Cultures of these microvascular and macrovascular endothelial cells were incubated with interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and Salmonella typhimurium lipopolysaccharide (LPS) alone or in combination, and nitrite production was measured. Single-agent exposure with IFN-gamma (up to 1,000 U/ml), TNF-alpha (up to 60,000 U/ml), or LPS (up to 500 ng/ml) had little effect on nitrite generation. Nitrite production by rat aortic macrovascular endothelial cells (RAEC) was significantly greater than that by the rat lung microvascular endothelial cells (RLMVEC) when stimulated with TNF-alpha + IFN-gamma, LPS + IFN-gamma, or TNF-alpha + LPS. The maximal response by all endothelial cell types (approximately 15-fold increase in RAEC and 8-fold increase in RLMVEC) was observed with LPS + IFN-gamma. The nitrite generation from rat pulmonary artery endothelial cells was intermediate between RAEC and RLMVEC responses when stimulated with IFN-gamma + LPS or TNF-alpha. Similar patterns of heterogeneous inducible nitric oxide synthase mRNA induction occurred when Northern analysis of specimens from the cultured endothelial cell types was done. These data suggest that phenotypic heterogeneity between these endothelial cell populations is substantial and, by inference, that site-specific NO. generation may occur.

Noninvasive measures of radiolabeled dextran transport in in situ rabbit lung.

UNLABELLED: Dextrans are nontoxic and can be obtained in a wide variety of molecular weights. The purpose of this study was to label 6-kDa and 40-kDa dextrans with gamma- (99mTc) and positron- (18F) emitting radioisotopes and monitor their transport across the pulmonary microvascular barrier. METHODS: External scan measurements for radiolabeled uncharged dextrans, albumin and red blood cells were obtained in eight blood-perfused in situ rabbit lung preparations. After 3 hr of external scanning, the lungs were removed for postmortem and extravascular distribution volume calculations. Extravascular distribution volumes were obtained in six additional rabbits following 4 hr of dextran perfusion to compare the effect of time. The normalized slope index (NSI), a measure of transvascular transport rate, was calculated for each diffusible tracer. RESULTS: The mean NSI for albumin (0.001676 +/- 0.000537 min-1) was significantly lower than NSI for the 40-kDa dextran (0.002303 +/- 0.0005426 min-1) as well as the 6-kDa dextran (0.004312 +/- 0.001134 min-1). The difference between the 6-kDa and the 40-kDa dextrans was also significant. After 4 hr of equilibration, distribution volumes were not significantly different than those obtained at 3 hr. CONCLUSION: Dextrans can be radiolabeled with gamma and positron emitters and small dextrans traverse the lung microvascular barrier more rapidly than albumin. Our results suggest that the use of small dextrans rather than albumin can reduce scan times in clinical applications and minimize motion artifact associated with the noninvasive gamma detection method.

Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine.

MRL-lpr/lpr mice spontaneously develop various manifestations of autoimmunity including an inflammatory arthropathy and immune complex glomerulonephritis. This study examines the role of nitric oxide, a molecule with proinflammatory actions, in the pathogenesis of MRL-lpr/lpr autoimmune disease. MRL-lpr/lpr mice excreted more urinary nitrite/nitrate (an in vivo marker of nitric oxide production) than did mice of normal strains and MRL-(+/+) and B6-lpr/lpr congenic strains. In addition, MRL-lpr/lpr peritoneal macrophages had an enhanced capacity to produce nitric oxide in vitro as well as increased nitric oxide synthase activity, and certain tissues from MRL-lpr/lpr mice had increased expression of inducible nitric oxide synthase (NOS) mRNA and increased amounts of material immunoreactive for inducible NOS. Oral administration of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, prevented the development of glomerulonephritis and reduced the intensity of inflammatory arthritis in MRL-lpr/lpr mice. By using interspecific backcross mice, the gene for inducible NOS (Nosi) was mapped to mouse chromosome 11. This chromosomal localization was different from those loci that we have previously demonstrated to be linked to enhanced susceptibility to renal disease in an MRL-lpr/lpr cross. However, the chromosomal location of the NOS gene was consistent with an insulin-dependent diabetes locus identified in an analysis of nonobese diabetic (NOD) mice. These results suggest that elevated nitric oxide production could be important in the pathogenesis of autoimmunity, and that treatments to block the production of nitric oxide or block its effects might be valuable therapeutically.

Nitric oxide modulation of human leukemia cell differentiation and gene expression.

Nitric oxide (NO) functions as an intercellular messenger molecule in such varied contexts as neurotransmission, immune regulation, and the control of vascular tone. We report that NO, delivered as purified gas or released from the pharmacologic NO donors sodium nitroprusside or 6-morpholino-sydnonimine, caused monocytic differentiation of cells of the human myeloid leukemia cell line HL-60 and altered gene expression. The treated cells stopped proliferating, became spread and vacuolated, had increased expression of nonspecific esterase and the monocyte marker CD14, and displayed increased capacity to produce hydrogen peroxide. Furthermore, these treated cells had increased steady-state expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), but decreased expression of mRNA for the proto-oncogenes c-myc and c-myb. The increase in TNF-alpha and IL-1 beta mRNA levels was due (at least in part) to a new transcription of these specific mRNAs. NO elaborated in the bone marrow microenvironment may have a role in normal and malignant hematopoietic cell growth and differentiation.

Inhibition of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) messenger RNA (mRNA) expression in HL-60 leukemia cells by pentoxifylline and dexamethasone: dissociation of acivicin-induced TNF-alpha and IL-1 beta mRNA expression from acivicin-induced monocytoid differentiation.

We have previously noted that the glutamine antagonist acivicin (alpha S,5S-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) induces monocytoid differentiation of freshly isolated human myeloid leukemia cells and cells of the myeloid leukemia cell line HL-60, and that the differentiation is accompanied by increases in expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). Because we also showed that TNF-alpha and IL-1 beta can act synergistically to cause monocytoid differentiation of HL-60 cells, we hypothesized that acivicin-induced TNF-alpha and IL-1 beta, in an autocrine manner, caused the differentiation. The purpose of the present study was to determine the causal roles of TNF-alpha and IL-1 beta in the acivicin-induced differentiation of HL-60 cells by the use of dexamethasone (DEX) and pentoxifylline (PTX), two drugs that effectively inhibit expression of TNF-alpha and IL-1 beta. Acivicin caused a monocytoid differentiation of the cells as manifest by diminished cell growth, morphologic maturation of the cells, increased ability to generate hydrogen peroxide in response to acute treatment with phorbol myristate acetate, and increased expression of nonspecific esterase and the surface antigens CD14 and CD11b. Acivicin treatment also caused the cells to have diminished steady-state expression of messenger RNA (mRNA) for c-myc and c-myb, and increased expression of mRNA for TNF-alpha and IL-1 beta. DEX and PTX did not alter cell growth, and did not block the acivicin-induced block in growth. PTX caused a slight increase in nonspecific esterase expression, but DEX had no effect on this, and neither drug diminished the acivicin-induced increase in nonspecific esterase. Although neither drug alone lessened the acivicin enhancement of hydrogen peroxide production, DEX and PTX together reduced this. DEX did not modify the acivicin-induced morphologic maturation of the cells, but PTX alone or PTX with DEX potentiated the acivicin-induced increase in mature cells. Basal CD14 and CD11b expression were slightly reduced by DEX and PTX, but neither drug modified the acivicin-induced increases. DEX and PTX reduced the acivicin-induced increases in TNF-alpha and IL-1 beta mRNA expression, but they had little or no effect on the acivicin-induced decreases in expression of mRNA for c-myc and c-myb. Thus, DEX and PTX effectively block the acivicin-induced expression of TNF-alpha and IL-1 beta, but they have little influence on the acivicin-induced differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of acivicin-induced monocytoid differentiation of human myeloid leukemia cells to acivicin-induced modulation of growth factor, cytokine, and protooncogene mRNA expression.

We have previously noted that the glutamine antagonist acivicin (alpha S,5S-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) induces monocytoid differentiation of freshly isolated human myeloid leukemia cells and HL-60 cells. This study was designed to determine the effects of acivicin on the levels of HL-60 cell mRNA transcripts of several cytokines, growth factors, and protooncogenes implicated in the control of hematopoietic cell proliferation and differentiation. Control HL-60 cells did not express mRNA for granulocyte-colony-stimulating factor, granulocyte-macrophage-colony-stimulating factor, interleukin 3, or interleukin 6, and acivicin or phorbol myristate acetate did not induce their expression. Phorbol myristate acetate reduced expression of c-myc, c-myb, and heat shock protein 70 and enhanced those of macrophage-colony-stimulating factor and c-fms. Acivicin caused a decreased expression of c-myc, and an increased expression of mRNA for interleukin 1 beta and tumor necrosis factor alpha (TNF-alpha). The drug also caused an initial increase in c-myb, followed by a subsequent decrease below baseline levels. Supernatants and lysates of acivicin-treated HL-60 cells contained increased levels of interleukin 1 beta. Both TNF-alpha and interleukin 1 beta have been shown previously to influence hematopoietic cell differentiation. In our experiments, exogenous interleukin 1 added to HL-60 cells did not induce differentiation, but the combination of interleukin 1 and TNF synergistically enhanced the process. Pretreatment of the cells with TNF enhanced their responsiveness to subsequent treatment with interleukin 1. Our results demonstrate that the glutamine antagonist acivicin modulates HL-60 cell expression of TNF-alpha, interleukin 1 beta, c-myc, and c-myb and suggest that interleukin 1 beta and TNF-alpha might (in an autocrine manner) cause the differentiation.