Macrophage Inflammatory Protein-1alpha mRNA Research Articles

White matter damage and chemokine induction in developing rat brain after intrauterine infection

In order to investigate the neuropathological effects on the developing rat brain after intrauterine infection, identification of glail fibrillary acidic protein (GFAP), 2', 3'-cyclic nucleotide phosphodiesterase (CNPase), and neurofilament (NF) was observed. Escherichia coli (E. coli) was inoculated into uterine horn of pregnant rats when gestation was 70% complete (15 days) and the control group was inoculated with normal saline. Immunohistochemistry was used for evaluation of GFAP, CNPase, and NF expression in pup brains at postnatal day 7 (P7) and reverse transcriptase-PCR (RT-PCR) to analyze macrophage inflammatory protein-1 alpha mRNA (MIP-1 alpha mRNA), macrophage inflammatory protein-1 beta mRNA (MIP-1beta mRNA), the regulated upon activation normal T expressed and secreted chemokine mRNA (RANTES mRNA) and Eotaxin mRNA expression in pup brains at P1, P3 and P7. The numbers of GFAP-positive cells of the E. coli-treated group pups were marked increased in periventricular white matter and hippocampus at P7 compared with the control group but no significant different levels of GFAP expression in corpus callosum were found between two groups. The integrate density (ID) of CNPase-positive staining of the Escherichia coli-treated group pups were marked decreased in periventricular white matter and corpus callosum at P7 compared with the control group. The ID of NF-positive staining of the Escherichia coli-treated group pups were marked decreased in periventricular white matter at P7 compared with the control group and no significant different levels of NF expression in corpus callosum were found between two groups. The expression of MIP-1 alpha mRNA and MIP-1 beta mRNA in brain of the E. coli-treated pup rat were higher than the control at P1, but the expression of MIP-1 alpha mRNA and MIP-1 beta mRNA in brain of the pup rat at P3 and P7 had no significant difference between two groups. The alteration of expression of GFAP, CNPase, and NF in the brain of neonatal rats after intrauterine infection suggested that intrauterine infection could cause neonatal white matter damage. Moreover, the transient increase in expression of chemokine such as MIP-1 alpha, MIP-1 beta in neonatal brain after intrauterine infection indicated that MIP-1 alpha, MIP-1 beta may be a mechanism mediating between the neonatal white matter damage and the intrauterine infection.

Microsurgical intraluminal middle cerebral artery occlusion model in rodents

Focal brain ischemia induced in rodents by occlusion of the middle cerebral artery (MCA) is a widely used paradigm of human brain infarct. The objective of this study is to compare the effectiveness and reproducibility of MCA filament occlusion model in rats and mice. A total of 140 rodents (69 rats and 71 mice) were operated. Ninety-five animals were subjected to MCA occlusion; the surgical procedure consisted of introducing an uncoated surgical nylon monofilament into the cervical common carotid artery (CCA) and advancing it intracranially to permanently block blood flow into the right MCA. Forty-five sham-occluded rodents underwent CCA ligation. Surgical success, autopsy confirmed success and mortality rate were evaluated. Effective MCA occlusion was confirmed by the evidence of motor neurological deficit, by histopathology, immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR). IHC was performed in a randomly selected number of animals to detect the protein product of monocyte chemoattractant protein-1. The brain tissue in mice was examined by RT-PCR for the expression of macrophage inflammatory protein-1 alpha mRNA. Surgical success rate was 89% in the rats, significantly lower than that in the mice (100%, P < 0.05). Autopsy confirmed success rate in the rats, 60%, was also significantly different from that in the mice (92.5%, P < 0.001). The operative mortality rate was 4.3% in the rats and 15% in the mice. The present study demonstrates that the microsurgical filament occlusion of the MCA can be more successfully performed in mice. The lower rate of success in rats seems to be as a result of the architecture of the carotid canal in this animal. No previous reports, using a considerable number of animals, have compared the feasibility of intraluminal model in the rat with that in the mouse.

Roles of Superoxide Dismutase in Rat Mast Cell Granules

Background: It has been suggested that in the granules of rat mast cells there is some kind of superoxide dismutase (SOD), but details of this SOD in mast cells remain unclear. In the present study, we studied the mode of existence of SOD in mast cells and its releasing mechanism from the granules. In addition, we discussed the physiological role of SOD in allergic events. Methods: Purified rat mast cells were disrupted with a sonic disrupter and granules (sample I) were separated from supernatant (sample II) by centrifugation. The granules were treated with 1 mM Ca²⁺, and the supernatant (sample III) was separated from the pellet (sample IV). Sample III was applied to a heparin column and the eluate was used as sample V. SOD activity was measured in these samples. Results: SOD existed in mast cell granules as a heparin-binding and an inactive form. However, when granules were released and exposed to high Ca²⁺ concentration, SOD was discharged from heparin and shifted to the active form. The expression of macrophage inflammatory protein-1 alpha mRNA was enhanced when hydrogen peroxide (H₂O₂) or sample III with the xanthine-xanthine oxidase system were added to the culture media. Conclusions: These findings suggest that in stimulated rat mast cells, the released SOD may transform the generated superoxide anion into H₂O₂, and the generated H₂O₂ may enhance the expression of chemokine mRNA in the mast cells.

Chlorpromazine modulates cytokine expression in the liver and lung after burn injury and endotoxemia.

Previous data from our laboratory have demonstrated that alterations in cytokine production occur in the lung and liver as the result of a two-hit model of injury, i.e., burn with subsequent endotoxin administration. The purpose of this study was to determine whether the phenothiazine derivative chlorpromazine would alter cytokine production in a sequential model of injury. By using a sublethal burn/endotoxemia model, B2D6F1 mice (n = 40) were assigned to two groups and subjected to a 15% full-thickness burn. Three days after burn injury, one group (BURN/ETX) received 2.5 mg/kg Escherichia coli endotoxin intraperitoneally, and the other group (CPZ) received 4 mg/kg chlorpromazine 1 hour before the administration of 2.5 mg/kg E. coli endotoxin intraperitoneally. At selected time points, the animals were killed and lung and liver were removed and processed for protein and total RNA. Northern blots and enzyme-linked immunosorbent assays were used to assess the production of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and interleukin-10. Chlorpromazine significantly reduced tumor necrosis factor-alpha mRNA and protein expression in the liver. Macrophage inflammatory protein-1alpha mRNA was reduced by chlorpromazine in both liver and lung. Interleukin-10 production was not altered by chlorpromazine. The reduction of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by chlorpromazine in the liver and lungs may have potential as a pharmaceutical agent that may dampen the inflammatory response in a model of sequential injury.

Corticosteroid inhibition of macrophage inflammatory protein-1 alpha in human monocytes and alveolar macrophages.

One of the major inducible cytokines secreted by mononuclear phagocytes is macrophage inflammatory protein 1 (MIP-1), which consists of two homologous polypeptides, MIP-1 alpha and MIP-1 beta. MIP-1 alpha possesses chemotactic and stimulatory activities for lymphocytes, eosinophils, and monocytes and may play a role in various pulmonary inflammatory conditions. We investigated the expression and release of MIP-1 alpha from human peripheral blood monocytes (PBM) and alveolar macrophages (AM) after stimulation with lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha, and interferon-gamma and the inhibitory effects of corticosteroids. LPS and IL-1 beta only enhanced MIP-1 alpha mRNA and protein in a dose- and time-dependent fashion. Dexamethasone (10(-9) to 10(-4) M) inhibited the basal and induced production and expression of MIP-1 alpha. In PBM, dexamethasone (10(-6) M) reduced LPS- and IL-1 beta-stimulated production of MIP-1 alpha protein by 50 and 63%, respectively, maximally at 24 h, whereas the inhibition of mRNA expression occurred maximally at 4 h. Similar trends were observed for AM. MIP-1 alpha mRNA decay was only slightly decreased in the presence of dexamethasone. Inhibition of LPS-induced MIP-1 alpha mRNA by dexamethasone was attenuated by the protein synthesis inhibitor cycloheximide, indicating the involvement of a protein intermediate. Corticosteroids are a potent inhibitor of IL-1 beta- and LPS-induced expression of MIP-1 alpha through mechanisms involving mainly inhibition of transcription and to a minor degree by reducing mRNA stability. Corticosteroids may be effective anti-inflammatory agents by preventing the expression of chemokines such as MIP-1 alpha.

Macrophage inflammatory protein 1-alpha mRNA expression in an immortalized microglial cell line and cortical astrocyte cultures.

Macrophage inflammatory protein 1 (MIP-1) is a recently characterized inflammatory and chemokinetic cytokine. Proinflammatory stimuli have been shown to induce expression of MIP-1 by macrophages. We hypothesized that microglia and astrocytes express MIP-1 alpha because of their many immunologic similarities to macrophages. MIP-1 alpha mRNA was examined with quantitative reverse transcription and polymerase chain reaction in an immortalized mouse microglial cell line (BV-2) and in mouse cortical astrocyte cultures. We found that in both the BV-2 microglial cell line and in astrocyte cultures, MIP-1 alpha mRNA was strongly induced by lipopolysaccharide and the phorbol ester PMA. MIP-1 alpha mRNA was reduced by dBcAMP, interferon-gamma, and PGE1. Dexamethasone decreased MIP-1 alpha mRNA levels in astrocyte cultures, but not in BV-2 microglial cells. Interleukin-1 beta, tumor necrosis factor alpha, and MIP-1 alpha had no effect on MIP-1 alpha mRNA expression. These findings demonstrate that MIP-1 alpha mRNA is expressed by cultured glial cells and is regulated by proinflammatory and anti-inflammatory stimuli. MIP-1 alpha may be expressed by microglia and astrocytes in vivo, and may help modulate cerebral inflammation.

Gene expression of macrophage inflammatory protein-1 alpha from human blood monocytes and alveolar macrophages is inhibited by interleukin-4.

Mononuclear phagocytes are essential cellular mediators of both acute and chronic inflammatory responses. In addition to producing substances that mediate tissue injury directly, such as proteolytic enzymes and oxygen radical species, mononuclear phagocytes can secrete proteins involved in the activation and recruitment of inflammatory cells. One of the major inducible polypeptides secreted by mononuclear phagocytes is macrophage inflammatory protein 1 (MIP-1). Native MIP-1 is a protein with leukocyte chemotactic and stimulatory activity. MIP-1 consists of two highly homologous peptides, MIP-1 alpha and MIP-1 beta. We now characterize the expression of MIP-1 alpha from human peripheral blood monocytes (PBM) and identify the T-lymphocyte product interleukin-4 (IL-4) as an important regulator of MIP-1 alpha expression from PBM. In initial experiments, we demonstrated the production of MIP-1 alpha from lipopolysaccharide (LPS)-, interleukin-1 (IL-1)-, and phytohemagglutinin (PHA)-stimulated PBM. IL-4 inhibited the production of MIP-1 alpha from LPS-, IL-1-, and PHA-challenged PBM by 63, 81, and 88%, respectively. The suppressive effects of IL-4 were operative at the level of MIP-1 alpha mRNA, which was reduced in a dose-dependent fashion by IL-4. The suppression of MIP-1 alpha mRNA by IL-4 was observed within a narrow temporal window and was dependent upon the de novo synthesis of a protein intermediate. As determined by mRNA stability studies, IL-4 decreased steady-state levels of MIP-1 alpha mRNA, in part, by accelerating MIP-1 alpha mRNA decay.(ABSTRACT TRUNCATED AT 250 WORDS)