MIP-1alpha mRNA Research Articles

Bone marrow plasma macrophage inflammatory protein protein-1 alpha(MIP-1 alpha) and sclerostin in multiple myeloma: Relationship with bone disease and clinical characteristics

The aim of the study was to investigate the expression of MIP-1 alpha and sclerostin in bone marrow of patients with multiple myeloma (MM), the possible association of the sclerostin and MIP-1 alpha with MBD and the clinical characteristics. 53 patients (29 M, 24 F), median age 64 years was studied. MIP-1 alpha, sclerostin and bone-specific alkaline phosphatase (bALP) levels were quantified using an enzyme-linked immunosorbent assay (ELISA). Sclerostin and MIP-1 alpha mRNA expression was determined by RT-PCR. PTH and 1,25(OH) 2D3 levels were measured with an electrochemiluminescence immunoassay. The sclerostin and MIP-1 alpha concentrations in patients with MM were higher than those in the controls. RT-PCR analysis verified that the bone marrow mononuclear cells (BMMNCs) of most patients showed sclerostin and MIP-1 alpha mRNA expression. The sclerostin and MIP-1 alpha levels in patients with ISS stage III disease were significantly higher than those in patients with ISS stage II disease (p=0.01 and 0.06). The sclerostin and MIP-1 alpha levels in patients with BMD in group C were significantly higher than those in group A+B. There was positive correlation between sclerostin levels and MIP-1 alpha, beta2-microglobulin and aCa levels. A negative association was seen between sclerostin levels and bALP, HB and ALB levels. The MM patients with high sclerostin levels (>0.72 ng/ml) had significantly shorter median survival than those with low sclerostin levels (≤0.72 ng/ml) (χ(2)=7.574, p=0.006). Our findings support the positive relationship between sclerostin levels and MIP-1alpha levels deserve further detailed research.

Nasal polyp fibroblasts produce MIP-3alpha in response to Toll-like Receptor ligands and cytokine stimulation

Dendritic cells (DCs) play important roles in the development and perpetuation of immune responses. DCs are present in upper airway diseases such as chronic rhinosinusitis with nasal polyps. However, the mechanisms of how DCs migrate into the upper airway mucosa during upper airway inflammatory diseases remains unclear. Macrophage inflammatory protein-3alpha (MIP-3alpha) is known to be a migratory factor for immature DCs. There have been very few reports regarding cells producing this chemokine in the airways. To investigate this, we stimulated fibroblasts cultured from the nasal polyps with various toll-like receptor (TLR) ligands, which are derived from microorganisms, and IL-beta1 and TNF-alpha, which are proinflammatory cytokines, and analyzed their ability to produce MIP-3alpha. Fibroblast lines were established from nasal polyps and stimulated with TLR2, 3, 4, 5, 7/8 and 9 ligands, IL-beta1 and TNF-alpha. MIP-3alpha mRNA expression in nasal polyp fibroblasts (NPF) was evaluated by real-time RT-PCR and the protein levels of MIP-3alpha in the supernatants of stimulated NPF was measured by ELISA. Stimulation with TLR2, 3, 4 and 5 ligands, IL-beta1 and TNF-alpha, induced MIP-3alpha gene expression and protein production in the cultured NPF This response was dose- and time-dependent. NPF possibly play an important role in the recruitment of DCs in upper airway diseases such as chronic rhinosinusitis with nasal polyps through the production of MIP-3alpha.

Over-expression of CCL3 MIP-1α in a blastoid mantle cell lymphoma with hypercalcemia

We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL-BV), a rare subtype of B-cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme-linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone-related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT-PCR) analysis, we found predominant expression of mRNA for MIP-1alpha in addition to those for receptor-activator of nuclear-factor kappa B ligand (RANKL), TNF-alpha, and IL-6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP-1alpha significantly stimulated (3)H-thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP-1alpha. In the present case, MIP-1alpha, an osteoclast-activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T-cell leukemia/lymphoma (ATLL). Furthermore, MIP-1alpha is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP-1alpha is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma.

Interferon regulatory factor-1 is a key transcription factor in murine beta cells under immune attack

IFN-gamma, together with other inflammatory cytokines such as IL-1beta and TNF-alpha, contributes to beta cell death in type 1 diabetes. We analysed the role of the transcription factor interferon regulatory factor (IRF)-1, a downstream target of IFN-gamma/signal transducer and activator of transcription (STAT)-1, in immune-mediated beta cell destruction. Islets from mice lacking Irf-1 (Irf-1 (-/-)) and control C57BL/6 mice were transplanted in overtly diabetic NOD mice. Viability and functionality of islets were evaluated in vitro. Chemokine expression by Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 short interfering RNA (siRNA) was measured by real-time PCR as well as in functional assays in vitro. IRF-1 deletion in islets was associated with higher prevalence of primary non-function (63% vs 25%, p <or= 0.05) and shorter functioning graft survival (6.0 +/- 2.6 vs 10.4 +/- 4.8 days, p <or= 0.05) in contrast to similar skin graft survival. Although Irf-1 (-/-) islets were resistant to cytokine-induced cell death, insulin secretion by them was lower than that of control C57BL/6 islets under medium and cytokine conditions. IL-1 receptor antagonist partly restored the cytokine-induced secretory defect in vitro and completely prevented primary non-function in vivo. Cytokine-exposed Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 siRNA showed increased expression of Mcp-1 (also known as Ccl2), Ip-10 (also known as Cxcl10), Mip-3alpha (also known as Ccl20) and Inos (also known as Nos2) mRNA and elevated production of monocyte chemoattractant protein-1 (MCP-1) and nitrite compared with controls. In vivo, Irf-1 (-/-) islets displayed a higher potential to attract immune cells, reflected by more aggressive immune infiltration in the grafted islets. These data indicate a key regulatory role for IRF-1 in insulin and chemokine secretion by pancreatic islets under inflammatory attack.

Varicose Veins Show Enhanced Chemokine Expression

Leucocyte infiltration in the wall of varicose veins has been reported previously. This study was designed to investigate the expression of pro-inflammatory cytokines and chemokines in control and in patients with varicose veins and to test the effect of treating varicose vein patients with acetylsalicylic acid (ASA) on cytokine expression prior to removal of varices. Sections of vein were removed during operation from both patient groups, and ribonuclease protection assays (RPAs) were performed to assess the expression of chemokines. Group I included non-varicose saphenous veins from healthy patients undergoing amputation for trauma. Varicose veins were obtained from patients with primary varicose undergoing surgical treatment who received no drug (group II) or treatment with 300 mg day(-1) of ASA for 15 days before surgery (group III). Non-varicose veins constitutively expressed low levels of monocyte-chemoattractant protein (MCP-1) and interleukin (IL)-8 mRNA. Varicose veins had a distinct chemokine expression pattern, since significant up-regulation of MCP-1 and IL-8 and a marked expression of IP-10, RANTES, MIP-1alpha and MIP-1beta mRNA were detected. Removal of the endothelium did not alter this pattern. Varicose veins obtained from patients treated with ASA showed a consistent decrease in chemokine expression, although it did not reach statistical significance. Varicose veins showed increased expression of several chemokines compared to control veins. A non-significant reduction of activation was observed following treatment with ASA for 15 days.

Role of IL-10 Deficiency in Pneumonia Induced by Corynebacterium kutscheri in Mice

IL-10 is an important anti-inflammatory cytokine that can inhibit the production of many pro-inflammatory cytokines. Both human and animal studies have shown that pro-inflammatory cytokines play an important role in pneumonia and other inflammatory lung diseases. In the present study, IL-10 knockout (KO) and wild-type mice were infected with Corynebacterium kutscheri to determine whether the severity of pathogenesis and whether protective immunity could be altered in the absence of IL- 10. The survival rate was significantly lower in IL-10 KO mice than wild-type mice. The number of neutrophils in bronchoalveolar lavage fluid and blood were found to be higher in IL-10 KO mice than wild-type mice. IL-10 KO mice showed greater neutrophil infiltration, excessive inflammation, and weight-loss compared with wild-type mice. Furthermore, upregulation of IFN-gamma in bronchoalveolar lavage fluid, and upregulation of MIP-1alpha and IP-10 mRNA in the lungs of IL-10 KO mice compared with wild-type mice after C. kutscheri infection were observed. These results suggest that IL-10 plays an important role in the anti-inflammatory properties against C. kutscheri infection, and that lack of IL-10 leads to a more severe pulmonary inflammatory response. This increased susceptibility to C. kutscheri pneumonia is at least in part caused by IL-10 deficiency and severe recruitment of neutrophils.

Abstract 3612: Apelin Prevents Aortic Aneurysm Formation by Inhibiting Macrophage Inflammation and Infiltration

To investigate the hypothesis that Apelin treatment would attenuate aortic injury in a murine model of elastase-induced abdominal aortic aneurysm (AAA) disease, by limiting disease-related vascular wall inflammatory. Male C57BL/6 mice were implanted with osmotic mini-pumps filled with Apelin (2 mg/kg/day) or saline (Control), and subsequently treated with intra-aortic porcine pancreatic elastase to create infrarenal AAA’s. Mice were sacrificed at day 3 for aortic PCR analysis or followed ultrasonographically until day 7 and then sacrificed for histological analysis (Elastic Masson, VCAM, ICAM-1, E-Selectin, and Mac-2 semi-quantitative analysis). The cellular expression of inflammatory cytokines and chemokines in response to Apelin was assessed by TaqMan analysis of both macrophages (J774, RAW, and harvested intraperitoneal cells) and rat aortic smooth muscle cells (A7R5) and compared to other stimuli. Apelin treatment resulted in diminished AAA formation, with a 30% reduction in in vivo aortic diameter by ultrasound (1.04 mm vs 1.48 mm, p &lt; 0.05), and a 59% reduction in maximal aortic cross-sectional area by histology (0.59 mm2 vs 1.43 mm2, p &lt; 0.05) relative to the saline-treated group. The macrophage infiltrate also was significantly reduced in the Apelin-treated mice compared to the Control group (68.6 cells/hpf vs 285.4 cells/hpf, p &lt; 0.001). Apelin treatment was associated with reduced mean aortic MCP-1, MIP 1-alpha, IL-6 and TNF-alpha mRNA levels, although these did not reach statistical significance. Apelin stimulation of cultured macrophage models significantly reduced MCP-1 and TNF-alpha mRNA levels compared to basal conditions (−1.12 and −1.49 fold expression change, p &lt; 0.05, respectively). No difference in endothelial cell adhesion molecule expression or smooth muscle cell cytokine production was found in response to Apelin. Apelin significantly reduces AAA formation in this accepted model of human AAA disease. The mechanism appears to be decreased macrophage infiltration, perhaps related to an apelin-mediated decrease in pro-inflammatory cytokine and chemokine activation. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada &amp; Utah).

Differential anti-inflammatory and anti-oxidative effects of dexamethasone and N-acetylcysteine in endotoxin-induced lung inflammation.

Inhalation of bacterial endotoxin induces an acute inflammation in the lower respiratory tract. In this study, the anti-inflammatory effects of the anti-oxidant N-acetylcysteine (NAC) and the glucocorticoid dexamethasone were investigated in mice exposed to aerosolized endotoxin (lipopolysaccharide (LPS)). Powerful reduction of neutrophils in bronchoalveolar lavage fluid (BALF) was obtained by a single i.p. injection of dexamethasone (10 mg/kg), whereas treatment with NAC only resulted in reduction of neutrophils when administered at a high dose (500 mg/kg). Measurement of cytokine and chemokine expression in lung tissue revealed a significant decrease of tumour necrosis factor-alpha, IL-1alpha, IL-1beta IL-6, IL- 12p40, and MIP-1alpha mRNA when mice where treated with dexamethasone but not when treated with NAC. Analysis of oxidative burst demonstrated a remarkable reduction of oxygen radicals in BALF neutrophils after treatment with dexamethasone, whereas the effect of NAC was not significantly different from that in untreated animals. In conclusion, dexamethasone exerted both anti-inflammatory and anti-oxidative effects in acute airway inflammation, probably by blocking early events in the inflammatory cascade. In contrast, treatment with NAC resulted in a weak reduction of the inflammatory response but no inhibition of proinflammatory cytokines or reduction of oxidative burst in neutrophils. These results demonstrate dramatic differences in efficiency and also indicate that the two drugs have different actions. Combined treatment with NAC and dexamethasone revealed an additive action but no synergy was observed.

Rebamipide affects the efficiency of colchicine for the herpes simplex virus-induced inflammation in a Behcet's disease mouse model

Rebamipide inhibits free radicals derived from activated neutrophils and decreases the inhibiting inflammatory cytokine. Behcet's disease (BD) is a chronic, multi-systemic inflammatory disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. This disease has a chronic course with periodic exacerbations and progressive deterioration. To study the effect of rebamipide treatment to BD-like mice, combination treatment with rebamipide and colchicine was compared to colchicine treatment. Colchicine is one of the most frequently prescribed medicine to the patients with BD. For each BD mouse, 200 μl gastric fluid or 2 μg colchicine or 150 μg rebamipide or 2 μg colchicine plus 150 μg rebamipide was treated orally once per day. Treatment was done for 5 consecutive days. Two hour or 20 days after last administration, spleens were isolated for RT-PCR and real time PCR, and serum was collected for ELISA. In the combination treated group, TNF alpha, MIP-1 alpha, p22 phox, p47 phox, and gp91 phox mRNA expressions were lower than rebamipide treated or colchicine treated groups by reverse transcriptase PCR. NADPH oxidase subunits mRNA were markedly downregulated compared to the colchicine treated group by real time PCR. At 20 days after administration, combination treatment decreased 23.5% of the severity score compared to before administration. In contrast, colchicine treatment decreased 14.3% of the severity score compared to before administration. Rebamipide helped the function of colchicine to improve the HSV induced BD-like symptoms by inhibiting the expression of NADPH oxidase in vivo mouse model.

Serotonin decreases HIV‐1 replication in primary cultures of human macrophages through 5‐HT1A receptors

5-HT (serotonin) is known to be involved in neuroinflammation and immunoregulation. The human immunodeficiency virus (HIV) targets cells such as monocytes/macrophages, which colocalize with 5-HT-releasing cell types, mostly platelets. In this study, we investigated the effects of 5-HT on HIV-1-infected macrophages in vitro. Human macrophages cultured in serum-free medium were treated over 7 days with 5-HT at three concentrations (0.01, 1 and 100 microM) with or without agonists and antagonists of 5-HT(1A) and 5-HT(2) receptors. After 7 days of treatment, macrophages were infected with HIV-1/Ba-L and virus replication was monitored over 16 days and expression of proviral HIV DNA was investigated by PCR after 24 h of infection. Cell surface expression of HIV-1/Ba-L receptor (CD4) and coreceptor (CCR5) was investigated by flow cytometry. The CCR5 ligand, macrophage inflammatory protein-1alpha (MIP-1alpha), was quantified by ELISA in cell culture supernatants and MIP-1alpha mRNA expression was assessed by reverse transcriptase-PCR. In vitro, 5-HT downregulated the membranous expression of CCR5 and led to a decrease of HIV-1 infection, probably through its action on 5-HT(1A) receptors. 5-HT (100 microM) was also able to induce overexpression of MIP-1alpha mRNA leading to an increase of MIP-1alpha secretion by human macrophages. The effects of 5-HT on HIV infection could be a consequence of the increase in MIP-1alpha concentrations and/or CCR5 receptor downregulation. These results suggest that 5-HT can inhibit the replication of HIV-1 in primary culture of human macrophages through its action on 5-HT(1A) receptors.

Expression of mRNA for chemokines and chemokine receptors in tissues of the myometrium and uterine leiomyoma

Tissues samples of leiomyoma and myometrium obtained intraoperatively were analyzed. For evaluation of the synthesis of MIP-lalpha, MIP-1beta, RANTES, eotaxin, eotaxin-2, interleukin-8, CCR1, CCR3, CCR5, CXCR1, and CXCR2, mRNA isolated from tissues samples of leiomyoma and myometrium was subjected to reverse transcription-PCR and assayed by a semiquantitative method (relative to beta-actin). The content of eotaxin, MIP-1alpha, MIP-1beta, and CCR5 mRNA in leiomyoma tissue was lower than in the myometrium. The concentration of MIP-1beta, CCR5, and eotaxin mRNA in common leiomyoma was much lower than in the myometrium. Eotaxin mRNA expression in myometrial tissue of patients with single nodes was much higher than in those with multiple nodes. Moreover, expression of eotaxin mRNA in common leiomyoma was higher than in proliferating leiomyoma. The concentration of mRNA for interleukin-8 in leiomyoma tissue, as well as the content of mRNA for MIP-1alpha and CCR3 in myometrial tissue increased in patients with submucosal nodes (as distinct from nodes of another location). A direct correlation was revealed between the size of the uterus and concentration of mRNA for interleukin-8 and MIP-1beta in myometrial tissue. The concentration of mRNA for MIP-1alpha and MIP-1beta in leiomyoma tissue negatively correlated with the size of the uterus (maximum size of the node) and duration of leiomyoma, respectively. Our results indicate that chemokines play an important role in the pathogenesis of uterine leiomyoma.

Nitrogen‐containing bisphosphonate, YM529/ONO‐5920, inhibits macrophage inflammatory protein 1α expression and secretion in mouse myeloma cells

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is detected at high concentrations in patients with multiple myeloma, and it is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, we investigated whether or not YM529/ONO-5920, a new bisphosphonate, inhibited MIP-1 alpha mRNA expression in, and MIP-1 alpha secretion from, mouse myeloma cells. When the cells were stimulated by lipopolysaccharide, increased MIP-1 alpha mRNA expression and MIP-1 alpha secretion were observed. YM529/ONO-5920 inhibited MIP-1 alpha mRNA expression and MIP-1 alpha secretion in a concentration-dependent manner. A transient increase in the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) and Akt was observed after lipopolysaccharide stimulation. After YM529/ONO-5920 was given, there was no transient increase in the phosphorylation of ERK1/2 or Akt. These results indicated that YM529/ONO-5920 inhibited the expression and secretion of MIP-1 alpha through blocking the signaling pathway of the Ras/mitogen-activated protein kinase kinase/ERK and Ras/phosphatidylinositol-3 kinase/Akt. Accordingly, YM529/ONO-5920 appears to have promise for use in effective future therapy for osteolysis and myeloma cell growth that depends on MIP-1 alpha.

Inhibition par la sérotonine de l'infection par VIH-1 des macrophages en culture primaire: rôle du sous-type 5-HT1A des récepteurs sérotoninergiques

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is released at peripheral sites from activated platelets. At inflammatory sites, macrophages and lymphocytes could be exposed to 5-HT concentrations up to 100 microM. Moreover, 5-HT could modulate cytokine secretion by monocytes/macrophages and immune functions through the uptake of 5-HT at these inflammatory sites from T cells and dendritic cells. HIV infection is also under the control of inflammatory processes (including T cell proliferation and cytokines secretion). On this basis, we studied explored herein the effects of 5-HT on HIV-1/Ba-L (macrophage-tropic virus) replication in primary cultures of human macrophages. This pharmacological study with isotype-selective receptor agonists and antagonist allowed us to show that the 100 microM 5-HT concentration via 5-HT(1A) subtype receptors could decrease HIV replication. This observation was associated with an increase of MIP-1alpha secretion such as an increase of MIP-1alpha mRNA production and with a decrease of HIV-coreceptor CCR5 cell surface expression. Our results point out for the first time the inhibitory effects of 5-HT on HIV replication in primary culture of human macrophages via activation of 5-HT(1A) subtype receptors.

Minocycline modulates chemokine receptors but not interleukin‐10 mRNA expression in hypoxic‐ischemic neonatal rat brain

Hypoxic-ischemic (HI) brain injury in the perinatal period causes significant morbidity. Minocycline (MN) is a tetracycline derivative that has reduced brain injury in various animal models of neurodegeneration, including perinatal ischemia. To determine whether MN can modulate the expression of chemokine receptors and interleukin-10 (IL10) in a model of neonatal brain injury, we produced an HI insult to the right cerebral hemisphere (ipsilateral) of the 7-day-old rat (PD7) by right common carotid artery ligation and 2.25 hr of hypoxia in 8% oxygen. MN (45 mg/kg, i.p.) or vehicle (PBS) was injected twice: 2 days and immediately before the HI insult. At 0, 1, 3, and 24 hr and 14 days after HI, total RNA from the ipsilateral and contralateral (exposed to hypoxia only) hemispheres was extracted, reverse transcribed, and amplified with gene-specific primers using a semiquantitative RT-PCR for macrophage inflammatory protein-1alpha), interferon-inducible protein (IP-10), C-C chemokine receptor 5 (CCR5; MIP-1alpha receptor), C-X-C chemokine receptor 3 (CXCR3; IP-10 receptor), and IL10. We found that, in the ipsilateral hemisphere, a significant (P < 0.05) increase in MIP-1alpha, IP-10, CCR5, and CXCR3 mRNA levels was observed. MN treatment decreased mRNA levels for CCR5 and CXCR3. In contrast, the levels of antiinflammatory cytokine IL10 were markedly decreased as a result of HI insult. Treatment with MN, however, had no effect on IL10. We conclude that MN decreased proinflammatory chemokine receptor expression but had little or no influence on the expression of antiinflammatory cytokine IL10. These effects confirm the antiinflammatory effect of MN in neonatal HI brain injury.

Expression of Macrophage Inflammatory Protein 1-α is Elevated in Alzheimer's Vessels and is Regulated by Oxidative Stress

Inflammatory mediators are highly expressed in the Alzheimer's disease (AD) brain. We have shown that in AD the cerebral microcirculation is a rich source of cytokines and chemokines including interleukins (IL) 1beta, IL-6, IL-8, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1. However, the factors that regulate expression of these inflammatory proteins have not been defined. The objective of this study is to compare expression of macrophage inflammatory protein 1-alpha (MIP-1alpha) in brain microvessels isolated from AD patients to vessels from age-matched controls and further to determine whether expression of MIP-1alpha in brain endothelial cells is altered by oxidative stress. The data show that brain AD-derived microvessels express high levels of MIP-1alpha mRNA and release high levels of MIP-1alpha protein compared to brain microvessels isolated from controls. Treatment of brain endothelial cell cultures with menadione, a superoxide releasing compound, hydrogen peroxide, lipopolysacharride, or oxidatively modified low density lipoproteins (LDL) (Ox-LDL, HNE-LDL) results in a dose- dependent increase in MIP-1alpha mRNA levels and MIP-1alpha release into the media. These results suggest that oxidative and lipid insults to the brain microvasculature are likely to contribute to the inflammatory milieu of the AD brain.

STATINS REDUCE MACROPHAGE INFLAMMATORY PROTEIN‐1α EXPRESSION IN HUMAN ACTIVATED MONOCYTES

1. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exhibit a wide variety of anti-atherogenic effects that may be independent of their property to lower plasma cholesterol. 2. In order to systematically investigate these effects at a cellular level, we investigated gene expression in phorbol myristate acetate (PMA)-activated and non-activated human THP-1 monocytes in response to statins using cDNA arrays. 3. Of 588 genes tested, 26 were differentially expressed in the presence of statins. A marked reduction was found for the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). The decrease in MIP-1alpha mRNA expression after incubation with statins was confirmed by quantitative reverse transcription-polymerase chain reaction in THP-1 monocytes and human freshly isolated monocytes. Macrophage inflammatory protein-1alpha protein in THP-1 monocytes was reduced from 377 to 299 and 305 pg/mL by 0.1 micro mol/L simvastatin and 0.01 micro mol/L cerivastatin, respectively. The reduction in MIP-1alpha expression by statins was due, at least in part, to transcriptional inhibition of MIP-1alpha promoter activity. 4. The CC receptor ligand MIP-1alpha is a chemokine that has been implicated in atherosclerotic lesion formation. The present findings suggest that statin-mediated immunomodulation by inhibiting MIP-1alpha could contribute to the beneficial effects of statin therapy independent of lowering plasma cholesterol.

Expression of Neutrophil Chemoattractants and their Receptors in Subjects with Atopic Dermatitis and Psoriasis

RATIONALE: Despite colonization with microbes, the skin of atopic dermatitis (AD) subjects is characterized by the lack of neutrophils in contrast to psoriasis.METHODS: Peripheral blood neutrophils and biopsies were obtained from 4 AD, 4 psoriasis subjects and 4 controls. The expression of neutrophil chemoattractans (IL-8, GRO-alpha and MIP-1alpha) was assessed by Real-time PCR. Neutrophils were analyzed by dual-color flow cytometry (CD16) for expression of receptors relevant for cell migration: CCR1, CXCR1, CXCR2, C3a receptor®, C5aR, C3bR, FMLPR, PAFR and CRTH2(DP2).RESULTS: IL-8, GRO-alpha and MIP-1alpha mRNA was highly expressed in the skin of AD (71-fold, 40-fold and 10-fold; respectively) and psoriasis subjects (96-fold, 42-fold and 5-fold; respectively) compared to controls. No difference between groups was observed for any of the receptors except CRTH2 and PAFR. CRTH2 - thought to identify Th2-lymphocytes was detected on neutrophils of controls and psoriasis (netMFI 24±;7 and 46±;11; respectively) in contrast to AD subjects who expressed little to no CRTH2 (netMFI 1±;0.4, p<0.05). PAFR showed a tendency toward higher expression in psoriatics (netMFI 27±;7) compared to controls (netMFI 8±;1) and AD subjects (netMFI 11±;1).CONCLUSIONS: Despite the lack of neutrophils in AD, IL-8, GRO-alpha and MIP-1alpha were markedly increased in AD skin. Their receptors were equally expressed across subject groups. Unexpectedly, CRTH2 was expressed on neutrophils from controls and psoriasis but not AD subjects. The relevance of this finding and that of PAFR will be the focus of future experiments. This preliminary work suggests that the lack of neutrophils in AD is due at least in part to the lack of priming. RATIONALE: Despite colonization with microbes, the skin of atopic dermatitis (AD) subjects is characterized by the lack of neutrophils in contrast to psoriasis. METHODS: Peripheral blood neutrophils and biopsies were obtained from 4 AD, 4 psoriasis subjects and 4 controls. The expression of neutrophil chemoattractans (IL-8, GRO-alpha and MIP-1alpha) was assessed by Real-time PCR. Neutrophils were analyzed by dual-color flow cytometry (CD16) for expression of receptors relevant for cell migration: CCR1, CXCR1, CXCR2, C3a receptor®, C5aR, C3bR, FMLPR, PAFR and CRTH2(DP2). RESULTS: IL-8, GRO-alpha and MIP-1alpha mRNA was highly expressed in the skin of AD (71-fold, 40-fold and 10-fold; respectively) and psoriasis subjects (96-fold, 42-fold and 5-fold; respectively) compared to controls. No difference between groups was observed for any of the receptors except CRTH2 and PAFR. CRTH2 - thought to identify Th2-lymphocytes was detected on neutrophils of controls and psoriasis (netMFI 24±;7 and 46±;11; respectively) in contrast to AD subjects who expressed little to no CRTH2 (netMFI 1±;0.4, p<0.05). PAFR showed a tendency toward higher expression in psoriatics (netMFI 27±;7) compared to controls (netMFI 8±;1) and AD subjects (netMFI 11±;1). CONCLUSIONS: Despite the lack of neutrophils in AD, IL-8, GRO-alpha and MIP-1alpha were markedly increased in AD skin. Their receptors were equally expressed across subject groups. Unexpectedly, CRTH2 was expressed on neutrophils from controls and psoriasis but not AD subjects. The relevance of this finding and that of PAFR will be the focus of future experiments. This preliminary work suggests that the lack of neutrophils in AD is due at least in part to the lack of priming.

Involvement of MAPK activation in chemokine or COX-2 productions by Toxoplasma gondii

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.

Expression of macrophage inflammatory protein-1α in Kupffer cells following liver ischemia or reperfusion injury in rats

To explore the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) in Kupffer cells (KCs) following liver ischemia/reperfusion injury IRI in rats. Forty male SD rats were divided randomly into five groups. A model of partial warm ischemia/reperfusion injury in the rat liver was established. KCs were isolated and incubated one hour, six hours, 12 h, and 24 h after the reperfusion. Tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the supernatants were measured by ELISA. MIP-1alpha in KCs was detected by immunocytochemical and RT-PCR. No or few MIP-1alpha protein and mRNA were expressed in the KCs of the control group. Its expression in the IRI group had a significant increase after the reperfusion (P < 0.05), which was contrary to the control group. The active behavior of the MIP-1alpha gene in KCs following liver ischemia/reperfusion injury is assumed to be one of the major causes for the hepatic ischemia/reperfusion injury.

Specific induction of macrophage inflammatory protein 1‐α in glial cells of Sandhoff disease model mice associated with accumulation of N‐acetylhexosaminyl glycoconjugates

Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), due to a primary defect of the beta-subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylhexosamine residues at their non-reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). We demonstrated that chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 (monocyte chemotactic protein-1), SLC (secondary lymphoid-tissue chemokine), fractalkine and SDF-1 (stromal derived factor-1), were detected. Significant up-regulation of MIP-1alpha mRNA and protein in the above-mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP-1alpha-immunoreactivity (IR) in the above-mentioned brain regions of SD mice was co-localized in Iba1-IR-positive microglial cells and partly in glial fibrillary acidic protein (GFAP)-IR-positive astrocytes, in which marked accumulation of N-acetylglucosaminyl (GlcNAc)-oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP-1alpha-IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP-1alpha might coincide with the accumulation of GlcNAc-oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.