Nystatin, a polyene antifungal antibiotic, is a cholesterol sequestering agent. The antifungal agent alters composition of the plasma membrane of eukaryotic cells, whereas its effects on cells are poorly investigated. In the current study, we investigated the question of whether nystatin was able to induce expression of macrophage inflammatory protein-1 (MIP-1). THP-1 cells rarely express MIP-1alpha and MIP-1beta, however, upon exposure to nystatin, significantly elevated expression of MIP-1alpha and MIP-1beta was observed in a dose-dependent fashion at the messenger and protein levels. Cellular factors activated by nystatin as well as involved in nystatin-induced expression of MIP-1 proteins were identified in order to understand the molecular mechanisms of action of the anti-fungal agent. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK, p38 MAPK, and JNK. Abrogation or significant attenuation of nystatin-induced expression of MIP-1alpha and MIP-1beta was observed by treatment with Akt inhibitor IV, LY294002, and SP6001250. Inhibition of ERK or p38MAPK using U0126 and SB202190 did not lead to attenuation of MIP-1 expression. In addition, inhibitors of protein kinase C, such as GF109203X and Ro-318220, also attenuated expression of MIP-1. These results indicate that nystatin is able to activate multiple cellular kinases and, among them, Akt and JNK play primary roles in nystatin-induced expression of MIP-1 proteins.
Cell Membrane ; Chemokine CCL3 ; Chemokine CCL4 ; Cholesterol ; Eukaryotic Cells ; Macrophage Inflammatory Proteins* ; Macrophages* ; Nystatin* ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases ; Protein Kinase C

UNLABELLEDBACKGROWND: Macrophage inflammatory protein-1α (MIP-l α/CCL3) belongs to the C-C chemokine family (CCL3), which can be secreted by macrophages, other types of hematopoietic cells and bone marrow stromal cells. Higher levels of MIP-1α were found to be associated with several kinds of hematologic malignancies, including multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Moreover, MIP-1α has been reported to be an adverse prognostic factor for CLL. However, the impact of MIP-1α on acute myeloid leukemia (AML) has been poorly investigated.

OBJECTIVETo investigate the influence of MIP-1α on proliferction of AML cells.

METHODSUsing MLL-AF9 induced AML mouse model, the expression of MIP-1α was measured by real time quantitative RT-PCR. AML cell proliferation was examined by cell counting and colony forming assay (CFC). The influence of blocking the MIP-1α action on the growth and pathogenic ability of AML cells was explored by using the small molecule antagonist for interfering interaction of MIP-1α with its receptor CCR1.

RESULTSThe MIP-1α could promote the proliferation and colony formation of AML cells, the blocking MIP-1a could inhibit the growth of AML cells and delay onset of AML.

CONCLUSIONThe MIP-1a promotes the occurence and progression of AML, therefore blocking the MIP-1α signal pathway may be served as a strategy to inhibit the growth of AML cells, and MIP-1α can be a potential target for treatment of AML.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Chemokine CCL3 ; Chemokine CCL4 ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukemia, Myeloid, Acute ; Macrophage Inflammatory Proteins ; Mice ; Multiple Myeloma ; Receptors, CCR1

OBJECTIVETo observe the in vivo antitumor activity of murine liver tumor vaccine expressing MIP-1alpha mediated by recombinant adenoviral vector.

METHODSThe infection efficacy was measured by GFP expression 48 hours after infection of Hepa1-6, and the number of cells was counted daily for 14 days. 5 x 10(6) modified Hepa1-6 cells were inoculated subcutaneously to C57BL/6 mice and the tumor-free animals were rechallenged by 2 x 10(6) wild-type Hepa1-6 cells or syngenic EL4 cells four weeks later. The tumor volume was measured twice a week.

RESULTSAdenoviral vectors could efficiently infect Hepa1-6 cells in vitro, and the in vitro growth rate of AdmMIP-1alpha modified Hepa1-6 cells was not affected; however the in vivo tumorigenicity was significantly decreased, compared with that of control vector modified Hepa1-6. Rechallenge of the tumor-free mice four weeks after administration of AdmMIP-1alpha with the parental Hepa1-6 cells resulted in significant inhibition of tumor growth, but there was no significant difference when rechallenged with EL4.

CONCLUSIONSThe liver cancer cells expressing mMIP-1alpha mediated by recombinant adenoviral vector decrease tumorigenicity and elicit specific immunological protection, and could be used as an effective liver tumor vaccine.

Adenoviridae ; genetics ; Animals ; Cancer Vaccines ; immunology ; Chemokine CCL3 ; Chemokine CCL4 ; Genetic Therapy ; Liver Neoplasms, Experimental ; therapy ; Macrophage Inflammatory Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Vaccines, Synthetic ; immunology

This study was aimed to explore the expression of MIP-1alpha, MCP-1 and their receptors CCR-1, CCR-2 in bcr/abl fusion gene positive CML cells, and to study the effects of P210(bcr/abl) fusion protein tyrosine kinase on expression of MIP-1alpha, MCP-1 and their receptors CCR-1, CCR-2 mRNAs in chronic myeloid leukemia cells. The expression levels of MIP-1alpha, MCP-1 and their receptors CCR-1, CCR-2 mRNA were detected by semi-quantitative RT-PCR in bcr/abl negative cells, bcr/abl positive cells, and P210(bcr/abl)-Rb-C-Box positive cells. The results showed that MIP-1alpha and CCR-1 mRNAs were expressed in bcr/abl negative cells, but not in positive cells. Both MCP-1 and CCR-2 mRNA cannot be detected in both bcr/abl positive and negative cells. After inhibiting P210(bcr/abl) tyrosine kinase activity by Rb-C-Box, expressions of MIP-1alpha and CCR-1 mRNAs were restored to normal (similar to P210(bcr/abl) negative cells). It is concluded that P210(bcr/abl) fusion protein inhibits the expression of MIP-1alpha and CCR-1 in chronic myeloid leukemia cells, but does not inhibit MCP-1 and CCR-2 mRNA expressions in these leukemia cells.
Chemokine CCL2 ; biosynthesis ; genetics ; Chemokine CCL3 ; Chemokine CCL4 ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Receptors, CCR1 ; Receptors, CCR2 ; Receptors, Chemokine ; biosynthesis ; genetics ; Tumor Cells, Cultured

OBJECTIVE: To explore the microvessel counts and the expressions of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1 ( MIP-1) alpha mRNA in the pathological scar tissues. METHODS: Immunohistochemical method of avidin-biotin complex was used for microvessel counts on the routinely formalin-fixed and paraffin-embedded sections of specimens of hypertrophic scars, keloids, normal skin, and surgical scar, and in situ hybridization for the expressions of IL-8, MCP-1, MIP-1alpha mRNA. RESULTS: The microvessel counts as well as the positive rates and the scorings of IL-8, MCP-1, and MIP-1alpha mRNA were significantly higher in pathological scars than those in the normal skin and surgical scar (all P < 0.05). The microvessel counts were significantly higher in the positive cases of IL-8, MCP-1 and MIP-1alpha mRNA than those in the negative ones (P < 0.05). The close positive correlations were found among the microvessel counts and the expressive scorings of 3 factors (P < 0.05). The close positive correlations were also found among the expressive scorings of IL-8, MCP-1, and MIP-1alpha mRNA in pathological scars. Microvessel counts were significantly higher in hypertrophic scars with the course less than 1 year than those with the course more than 1 year. CONCLUSION IL-8, MCP-1 and MIP-1alpha play important roles in promoting the neovascularization of pathological scars.
Adolescent ; Adult ; Burns ; complications ; Capillaries ; metabolism ; Chemokine CCL2 ; biosynthesis ; genetics ; Chemokine CCL3 ; Chemokine CCL4 ; Cicatrix ; etiology ; metabolism ; Female ; Humans ; Interleukin-8 ; biosynthesis ; genetics ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Skin ; blood supply

OBJECTIVETo understand whether endotoxin lipopolysaccharide (LPS) is able to induce the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA and protein in human umbilical vein endothelial cells (HUVECs).

METHODSThe expression of MIP-1alpha mRNA was determined by dot blotting analysis and by in situ hybridization using a digoxigenin-labeled MIP-1alpha cDNA probe after exposure of the cultured HUVECs to LPS at different concentrations. The expression of MIP-1alpha mRNA was determined by RT-PCR as well. In addition, the expression of MIP-1alpha protein was tested by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human monoclonal MIP-1alpha antibody.

RESULTSDot blotting showed that the absorbance values of the dots on the nitrocellulose membrane were 1.490 and 3.310 when exposed to LPS at the concentrations of 1 micro g/ml and 10 micro g/ml which were 1.97- and 4.38-fold over that of the control group (0.775), respectively. In situ hybridization revealed that exposure to LPS at a concentration of 1 micro g/ml led to a significant increase in the MIP-1alpha mRNA expression in HUVECs as compared to the control group (F = 142.83, P < 0.01), whereas the MIP-1alpha mRNA in HUVECs was somewhat decreased when exposed to LPS at a concentration of 10 micro g/ml. RT-PCR revealed that the expression of MIP-1alpha mRNA in HUVECs were 1.65-, 2.86- and 1.26-fold over that of the control group when exposed to LPS at the concentrations of 1 micro g/ml, 5 micro g/ml and 10 micro g/ml respectively. Cell ELISA showed that after exposure of the HUVECs to LPS at the concentrations mentioned above, the expression of MIP-1alpha protein was strongly increased, especially in the 5 micro g/ml LPS group. Analysis of variance showed that there was a significant difference between groups (F = 15.36, P < 0.05).

CONCLUSIONSLPS may induce a high level of MIP-1alpha mRNA and protein expression in HUVECs, and it might, hereby, play an important role in the recruitment of the monocytes/macrophages into the arterial intima.

Cells, Cultured ; Chemokine CCL3 ; Chemokine CCL4 ; Endothelial Cells ; drug effects ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation ; drug effects ; Humans ; In Situ Hybridization ; Lipopolysaccharides ; toxicity ; Macrophage Inflammatory Proteins ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Veins ; drug effects

OBJECTIVETo study the effect of macrophage inflammatory protein-1alpha(MIP-1alpha) and its mRNA on airway inflammation of mouse with induced asthma.

METHODSSeventy male BALB/C mice were randomly divided into the control group and asthma group (including 7 subgroups, 10 mice each). The control group included group A(24) (the lavaging subgroup was sacrificed 24 h after the last challenge) and group A(0) (the non-lavaging subgroup was sacrificed from 18 h to 24 h after the last challenge); asthma group included group B(3) (the lavaging subgroup was sacrificed 3 h after the last challenge), group B(8) (the lavaging subgroup was sacrificed 8 h after the last challenge), group B(24) (the lavaging subgroup was sacrificed 24 h after the last challenge), group B(36) (the lavaging subgroup was sacrificed 36 h after the last challenge) and group B(0) (the non-lavaging subgroup was sacrificed from 18 h to 24 h after the last challenge). In the experiment, the mice model of asthma was established by the ovalbumin (OVA) challenge methods. Eosinophils (EOS) numbers and differentiated cell numbers in bronchoalveolar lavage fluid (BALF) were counted; the concentrations of MIP-1alpha in serum and BALF were measured by sandwich enzyme-linked immunosorbent assay (sandwich ELISA); the protein expressions of MIP-1alpha were detected by immunohistochemical techniques; the mRNA expressions of MIP-1alpha were determined by in situ hybridization technique.

RESULTS(1) The concentrations of MIP-1alpha in BALF and serum of group B(3) [(30.2 +/- 4.2) pg/ml, (30.8 +/- 4.6) pg/ml], group B(8) [(35.3 +/- 4.9) pg/ml, (34.9 +/- 5.1) pg/ml], group B(24) [(42.9 +/- 5.8) pg/ml, (41.7 +/- 6.3) pg/ml] and group B(36) [(37.8 +/- 4.7) pg/ml, (35.7 +/- 4.9) pg/ml] were significantly higher than those of group A(24) [(20.9 +/- 3.8) pg/ml, (22.4 +/- 4.3) pg/ml] (P < 0.01); the concentrations of MIP-1alpha in BALF and serum went up at 3 h, reached peak at 24 h, and had descended at 36 h. (2) Immunohistochemistry showed that the protein expressions of MIP-1alpha around the bronchus of group B(0) [(26.4 +/- 6.2)%] were significantly elevated as compared to those of group A(0) [(10.3 +/- 2.5)%] (P < 0.01), the epithelial cell was the chief expression cell. (3) In situ hybridization showed that the mRNA expressions of MIP-1alpha around the bronchus of group B(0) [(23.9 +/- 4.2)%] were significantly increased when compared to those of group A(0) [(8.7 +/- 1.8)%] (P < 0.01), the epithelial cell was the chief expression cell. (4) There was a significant correlation between the concentrations of MIP-1alpha and the numbers of EOS in BALF and between the concentrations of MIP-1alpha and the percentage of EOS numbers in the total cell numbers (EOS%) in BALF.

CONCLUSIONSMIP-1alpha protein and MIP-1alpha mRNA were found strongly expressed in mouse asthma model, the epithelial cell was the chief expression cell; the kinetic characteristic of MIP-1alpha showed that its level increased at 3 h, reached peak at 24 h and declined at 36 h; MIP-1alpha and EOS gathering had a significant correlation.

Animals ; Asthma ; blood ; genetics ; Bronchoalveolar Lavage Fluid ; chemistry ; Chemokine CCL3 ; Chemokine CCL4 ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; In Situ Hybridization ; Macrophage Inflammatory Proteins ; blood ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; genetics ; metabolism

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemokine CCL3 ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; physiology ; Diamide ; pharmacology ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Lipid Peroxidation ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Sulfhydryl Reagents ; pharmacology ; Umbilical Veins ; cytology

Animals ; Chemokine CCL20 ; Chemokine CXCL10 ; Chemokines, CC ; biosynthesis ; Chemokines, CXC ; biosynthesis ; Liver ; metabolism ; Liver Transplantation ; Macrophage Inflammatory Proteins ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Transplantation, Homologous

OBJECTIVETo detect the expression of macrophage inflammatory protein-1alpha (MIP-1alpha), a disintegrin-like and metalloproteinase (ADAM) 8 and 12 and CD68 protein in giant cell lesions of jaw and giant cell tumors of long bone, and to study their effects on the histogenesis of giant cells in such lesions.

METHODSMIP-1alpha, ADAM8, ADAM12 and CD68 were detected by immunohistochemistry in 24 paraffin-embedded specimens of central giant cell lesions of jaw and giant cell tumors respectively.

RESULTSMIP-1alpha positive signal was located in blood vessels and bone. ADAM8, ADAM12 and CD68 positive signals were located in the cell membrane and cytoplasm of all multinucleated giant cells and some round mononuclear cells in the lesions. In addition, some spindle mononuclear stromal cells were positive for ADAM12 in both lesions.

CONCLUSIONMultinucleated giant cells probably originate from CD68-postive round mononuclear cells, which are recruited from monocyte-macrophage system by chemokines, such as MIP-1alpha, followed by cell fusion mediated by ADAM8 and ADAM12.

ADAM Proteins ; metabolism ; ADAM12 Protein ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Bone Neoplasms ; metabolism ; pathology ; Cell Membrane ; metabolism ; Chemokine CCL3 ; Chemokine CCL4 ; Cytoplasm ; metabolism ; Giant Cell Tumor of Bone ; metabolism ; pathology ; Giant Cells ; metabolism ; Granuloma, Giant Cell ; metabolism ; pathology ; Humans ; Jaw Diseases ; metabolism ; pathology ; Macrophage Inflammatory Proteins ; metabolism ; Membrane Proteins ; metabolism