Macrophage-derived Chemokine mRNA Research Articles

Oxymatrine Sensitizes the HaCaT Cells to the IFN-γ Pathway and Downregulates MDC, ICAM-1, and SOCS1 by Activating p38, JNK, and Akt.

Decreased interferon (IFN)-γ levels and increased levels of macrophage-derived chemokine (MDC) and intercellular adhesion molecule (ICAM)-1 are known to be involved in allergic skin diseases, such as eczema and atopic dermatitis. Activation of the IFN-γ and its downstream interleukin-12 (IL-12) pathway can correct these diseases. Suppressor of cytokine signaling 1 (SOCS1) is a cytokine signaling inhibitor that blocks downstream pathways of IFN-γ by blocking the mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) signaling pathways. Oxymatrine (OMT), a quinolizidine alkaloid extracted from the herbal medicine Radix Sophorae flavescentis, is used to treat allergic skin diseases in China. The non-cytotoxic concentrations of OMT in HaCaT cells were determined through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Tumor necrosis factor (TNF)-α and IFN-γ were used to stimulate HaCaT cells, and OMT was added to this system with tacrolimus (FK506) as a positive control. The mRNAs of cytokines, MDC, ICAM-1, IL-12p35, IL-12p40, and IFN-γ receptor (IFN-γR)α were detected by RT-PCR. Western blot analyses were performed to assess activation of the MAPK (p38, Jun N-terminal kinase, and extracellular signal-regulated kinase) and Akt signaling pathways. OMT increased the mRNA levels of the IL-12 and IFN-γRα, reduced the mRNA levels of ICAM-1, MDC, and SOCS1. But FK506 increased the mRNA levels of IL12 and inhibited the expression of ICAM-1 mRNAs and had no effects on the IFN-γRα, MDC, and SOCS1 mRNA in HaCaT cells stimulated with TNF-α and IFN-γ. Thus, the mechanisms through which OMT and FK506 ameliorate allergic skin diseases differ.

Effect of Histamine H1 Receptor Antagonists on TARC/CCL17 and MDC/CCL22 Production from CD14+ Cells Induced by Antigenic Stimulation in vitro

Background: Thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) are accepted to be important molecules in the development and maintenance of allergic diseases. Although several types of histamine H₁ receptor antagonist (antihistamine) have been developed and used for the treatment of allergic diseases, the influence of antihistamines on TARC and MDC production is not well understood. Objective: The present study was undertaken to examine the influence of antihistamines on TARC and MDC production from CD14+ cells after antigenic stimulation in vitro. Methods: CD14+ cells prepared from patients with pollinosis to Japanese cedar pollen were stimulated with specific allergen extracted from Japanese cedar pollen (Cry j 1) in the presence of azelastine (AZE), ketotifen (KET), fexofenadine (FEX) and oxatomide (OXA) for 6 days. TARC and MDC levels in culture supernatants were examined by ELISA. We also examined the influence of FEX on TARC and MDC mRNA expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and transcription factor activation in CD14+ cells after Cry j 1 stimulation. Results: FEX at 250 ng/ml, which is almost equal to therapeutic blood levels, caused a significant inhibition of TARC and MDC production.However, AZE, OXA and KET required higher concentrations than their therapeutic blood levels to suppress production of these factors. FEX at 250 ng/ml also suppressed NF-ĸB activation, phosphorylation of p38 MAPK and extracellular signal-regulated kinases 1 and 2 and expression of mRNA for TARC and MDC. Conclusions: These results suggest that antihistamines, especially FEX, suppress CC chemokine production from CD14+ cells through interference with antigen-mediated signaling and result in favorable modification of allergic disease states or conditions.

Anti-macrophage-derived chemokine antibody relieves murine lupus nephritis

We observed the distribution of monocyte's changes in damage to renal tissue and the expression of macrophage-derived chemokine (MDC) in MRL/lpr mice. The 8-week-old MRL/lpr mice were randomly assigned to receive either MDC antibodies (n = 6) or placebo (n = 6) at a once-every-other-day dose of 300 μl from week 4. We then quantified the differences in 24-h urine protein and serum creatinine concentrations, performed a histological evaluation of renal tissue and assessed the expression of MDC protein and mRNA between the two groups 4 weeks after treatment was initiated. Antibody-treated mice demonstrated significantly lower urine protein and serum creatinine concentrations and had fewer renal lesions compared with control mice. The expression of MDC protein and mRNA in renal tissues was significantly lower in the antibody-treated mice than in control mice, suggesting that the elevated expression of MDC, which led to monocyte infiltration in the kidney, may play an important role in the development of lupus nephritis. Furthermore, the anti-MDC antibodies may act to alleviate the renal lesions of murine lupus nephritis by inhibiting the infiltration of monocytes in the renal tissue of the lupus mice.

Mononuclear Cell-Infiltrate Inhibition by Blocking Macrophage-Derived Chemokine Results in Attenuation of Developing Crescentic Glomerulonephritis

Glomerular monocyte/macrophage (Mo/M phi) infiltrates play a role in many forms of glomerulonephritis (GN), and the intensity of Mo/M phi trafficking correlates with the loss of renal function and histological damage. We analyzed the functional role of macrophage-derived chemokine (MDC), a potent mononuclear cell chemoattractant, during the progression of anti-glomerular basement membrane (GBM) antibody (Ab) GN, a model of crescentic GN in the WKY rat, and whether the effects of MDC were dependent on its receptor CCR4. MDC mRNA and protein expression were markedly induced in nephritic glomeruli throughout the disease. Blocking the function of MDC did not affect the developing of the disease from days 2 to 7, but it dramatically blocked M omicron/M phi infiltration in the glomeruli, prevented crescent formation, and reversed renal function impairment during days 7 to 14 of the anti-GBM GN. In this study, we also found that MDC activity on M omicron/M phi in this GN was at least partly dependent on a new variant of CCR4. These results suggest that MDC is critically involved in the development of anti-GBM GN from acute glomerular injury to irreversible tissue damage. In addition, an antagonist to MDC may represent a prime drug target for therapeutic application to intervene in the progression of anti-GBM GN and in other M omicron/M phi-dominant GN.

Both IL-4 and IL-13 inhibit the TNF-α and IFN-γ enhanced MDC production in a human keratinocyte cell line, HaCaT cells

Background: Macrophage-derived chemokine (MDC) is a Th2 type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Recent reports demonstrated that MDC is expressed not only by macrophages, dendritic cells and lymphocytes, but also by cultured human keratinocytes (KCs). However, the regulation of MDC production in KCs by various cytokines has not been well documented. Objective: In this study, we investigated how Th1/Th2 cytokines regulate MDC production in a human KC cell line, HaCaT cells. Methods: HaCaT cells were cultured with or without various cytokines for 24 h and RT-PCR was performed using these cells to evaluate MDC mRNA levels. ELISA was carried out using supernatant of HaCaT cells to calculate secreted MDC protein levels. Results: MDC mRNA was weakly expressed in HaCaT cells, and upon stimulation with TNF-α or IFN-γ, MDC expression was strongly upregulated. The supernatant MDC levels when stimulated with TNF-α or IFN-γ were significantly higher than those without stimulation, and were synergistically increased when stimulated with a combination of TNF-α and IFN-γ. Both interleukin-4 (IL-4) and IL-13 inhibited TNF-α and IFN-γ enhanced MDC production in HaCaT cells in a dose-dependent manner. Conclusion: Th2-type cytokines IL-4 and IL-13 downregulate the production of MDC, a Th2 type chemokine, by KCs. This may partially contribute to maintaining Th1/Th2 balance in inflammatory skin diseases like atopic dermatitis.

Macrophage-Derived Chemokine Expression in Classical Hodgkin's Lymphoma: Application of Tissue Microarrays

Hodgkin's disease (HD) is a lymphoid malignancy characterized by the presence of Reed-Sternberg (RS) and Hodgkin's cells in a background of mixed inflammatory cells and stromal reaction. Studies have documented that HD is a neoplasm associated with abnormal cytokine and chemokine production. To define the expression of macrophage-derived chemokine (MDC) in HD, 57 cases (18 lymphocyte predominant, 11 mixed cellularity, 28 nodular sclerosis) were stained for MDC by immunohistochemistry and compared with reactive lymph nodes as controls. MDC was expressed by RS cells in classical HD (CHD) and showed a distinct cytoplasmic and Golgi localization. Accumulating evidence suggests that lymphocyte-predominant HD (LPHD) represents an entity distinct from CHD, with different biological properties and clinical course. On the basis of the high level of MDC staining alone, CHD could be distinguished from LPHD (P <.001), which showed only faint staining of scattered histiocytes similar to control tissues. CHD cases with high MDC mRNA levels showed high levels of MDC protein expression by immunohistochemistry (P <.001) and significant eosinophil infiltration, suggesting that MDC may represent another molecule that plays a critical role in eosinophil recruitment. We also analyzed 102 cases of non-Hodgkin's lymphoma and normal spleen, lymph node, and thymic tissue. High levels of MDC expression were specific to CHD cases because only low levels of MDC were observed in a minor subset of LPHD, NHL or normal lymphoid tissues.

Potential Role of the Chemokine Receptors CXCR3, CCR4, and the Integrin αEβ7 in the Pathogenesis of Psoriasis Vulgaris

Various adhesion molecules have been implicated in T lymphocyte binding to dermal vascular endothelium in psoriasis vulgaris, but the chemotactic signals that promote subsequent homing into the adjacent dermis and overlying epidermis are poorly defined. We studied chemokine receptor (CCR1–CCR5, CXCR1–CXCR3), chemokine (interferon-γ inducible protein 10 [IP-10]), monokine induced by interferon-γ (MIG), thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and adhesion molecule (cutaneous lymphocyte antigen [CLA], E-selectin, lymphocyte function-associated antigen-1 [LFA-1], intercellular adhesion molecule-1 [ICAM-1], very late antigen 4 [VLA-4], vascular cell adhesion molecule-1 [VCAM-1], αEβ7, and E-cadherin) expression in psoriasis by immunohistology, flow cytometry, and molecular techniques. CXCR3 and CCR4 were expressed by dermal CD3+ lymphocytes, and their chemokine ligands, IP-10, MIG, TARC, and MDC, were up-regulated in psoriatic lesions. Keratinocytes stimulated with tumor necrosis factor-α and interferon-γ up-regulated expression of IP-10, MIG, and MDC mRNA, whereas dermal endothelial cells, similarly stimulated, up-regulated expression of IP-10, MDC, and TARC mRNA, suggesting that these cell types were sources of the chemokines detected in biopsies. There was enhanced expression of E-selectin, CLA, LFA-1, ICAM-1, VLA-4, VCAM-1, and αEβ7 in psoriatic lesions versus nonlesional skin. Finally, intra-epidermal CLA+ and αEβ7+ T lymphocytes selectively expressed the chemokine receptor CXCR3. Collectively, these data suggest that CXCR3 and CCR4 may be involved in T lymphocyte trafficking to the psoriatic dermis and that CXCR3 is selectively involved in subsequent T cell homing to the overlying epidermis.

MACROPHAGE-DERIVED CHEMOKINE GENE EXPRESSION IN HUMAN AND MACAQUE CELLS: mRNA QUANTIFICATION USING NASBA TECHNOLOGY

Macrophage-derived chemokine (MDC) is a CC-chemokine that inhibits infection by both macrophage- and T cell-tropic strains of HIV-1. This suppressor activity has led to great interest in fully characterizing the role of MDC in the pathogenesis of HIV-1 infection. Methods for the quantitation of constitutive levels of MDC protein in vivo are lacking. In this report, we describe the development and performance of a NASBA-based assay for the quantification of MDC mRNA expression in human and macaque cells. Although the constitutive in vivo levels of MDC mRNA in macaque and human T lymphocytes were low, in vitro activation of these cells greatly increased MDC transcription. Levels in the human and macaque cells were comparable under all conditions tested. Positive correlations between MDC transcription and protein expression were observed. The results indicate that this assay is extremely sensitive and reproducible over a five log dynamic range, and effectively quantifies MDC mRNA in resting and activated T cells. This assay may therefore permit characterization of the role of MDC in HIV-1/SIV pathogenesis, and in vaccine-induced immune responses.

A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock.

CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4(-/-)) mice by gene targeting. CCR4(-/-) mice developed normally. Splenocytes and thymocytes isolated from the CCR4(-/-) mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1alpha. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4(-/-) mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4(+/+) mice. After high dose LPS treatment, serum levels of tumor necrosis factor alpha, interleukin 1beta, and MIP-1alpha were reduced in CCR4(-/-) mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4(-/)- mice by flow cytometry also revealed a significant decrease in the F4/80(+) cell population. This may reflect a defect in the ability of the CCR4(-/-) macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.

Macrophage-Derived Chemokine Is Localized to Thymic Medullary Epithelial Cells and Is a Chemoattractant for CD3+, CD4+, CD8low Thymocytes

Macrophage-derived chemokine (MDC) is a recently identified CC chemokine that is a potent chemoattractant for dendritic cells, natural killer (NK) cells, and the Th2 subset of peripheral blood T cells. In normal tissues, MDC mRNA is expressed principally in the thymus. Immunohistochemical analysis performed on 5 human postnatal thymuses showed high MDC immunoreactivity, which was selectively localized to epithelial cells within the medulla. To examine the effects of MDC on immature T cells, we have identified cDNA clones for mouse and rat MDC. Expression of MDC in murine tissues is also highly restricted, with significant levels of mRNA found only in the thymus. Thymocytes express high-affinity binding sites for MDC (kd = 0.7 nmol/L), and, in vitro, MDC is a chemoattractant for these cells. MDC-responsive murine thymocytes express mRNA for CCR4, a recently identified receptor for MDC. Phenotypic analysis of MDC-responsive cells shows that they are enriched for a subset of double-positive cells that express high levels of CD3 and CD4 and that have reduced levels of CD8. This subset of MDC-responsive cells is consistent with the observed expression of MDC within the medulla, because more mature cells are found there. MDC may therefore play a role in the migration of T-cell subsets during development within the thymus.

Fractalkine and macrophage-derived chemokine: T cell-attracting chemokines expressed in T cell area dendritic cells

Dendritic cells (DC) are a system of antigen-presenting cells specialized in interaction with T cells. Recently it has been reported that DC can produce CC (beta) chemokines that attract T cells. In this study we isolated mouse fractalkine and macrophage-derived chemokine (MDC) belonging to CX3C (delta) and CC chemokine families, respectively, from bone marrow-derived mature DC. While expression of fractalkine, which has so far been only examined in the brain and in vitro endothelial cells so far, was rather ubiquitous, MDC, which has been reported to be synthesized by macrophages and DC, was expressed specifically in the thymus and lymph node. This is the first report that indicates fractalkine expression by DC. Expression of fractalkine and MDC mRNA increased with maturation of DC during in vitro culture of bone marrow cells. Spleen- and epidermis-derived mature DC in culture also expressed these chemokines. Furthermore, their expression was detected selectively by Northern hybridization in CD11c+ B220- DC freshly purified from lymph nodes, and in large stellate cells in the lymph node T cell areas by in situ hybridization. Conditioned media of 293T cells transfected with these chemokine cDNA were chemotactic to Con A-activated splenic T cells as well as the mouse T cell line EL4. In conclusion, while fractalkine and MDC belong to different families of chemokines, both may be involved in recruitment of T cells for interaction with mature DC in the immune response.

Divergent Effects of Interleukin-4 and Interferon-γ on Macrophage-Derived Chemokine Production: An Amplification Circuit of Polarized T Helper 2 Responses

Macrophage-derived chemokine (MDC) is a CC chemokine that recognizes the CCR4 receptor and is selective for T helper 2 (Th2) versus T helper 1 (Th1) cells. The present study was designed to investigate the effect of the prototypic Th2/Th1 cytokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), on the production of MDC by human monocytes. IL-4 and IL-13 caused a time-dependent (plateau at 24 hours) and concentration-dependent (EC50 2 and 10 ng/mL, respectively) increase of MDC mRNA levels in monocytes. Increased expression of MDC mRNA was associated with protein release in the supernatant. MDC expression and production induced by IL-4 and IL-13 were inhibited by IFN-γ. IFN-γ also suppressed the constitutive expression of MDC in mature macrophages and dendritic cells. These results delineate an amplification loop of polarized Th2 responses based on differential regulation of MDC production by IL-4 and IL-13 versus IFN-γ and on the selectivity of this chemokine for polarized Th2 cells.© 1998 by The American Society of Hematology.

Divergent Effects of Interleukin-4 and Interferon-γ on Macrophage-Derived Chemokine Production: An Amplification Circuit of Polarized T Helper 2 Responses

Macrophage-derived chemokine (MDC) is a CC chemokine that recognizes the CCR4 receptor and is selective for T helper 2 (Th2) versus T helper 1 (Th1) cells. The present study was designed to investigate the effect of the prototypic Th2/Th1 cytokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), on the production of MDC by human monocytes. IL-4 and IL-13 caused a time-dependent (plateau at 24 hours) and concentration-dependent (EC50 2 and 10 ng/mL, respectively) increase of MDC mRNA levels in monocytes. Increased expression of MDC mRNA was associated with protein release in the supernatant. MDC expression and production induced by IL-4 and IL-13 were inhibited by IFN-γ. IFN-γ also suppressed the constitutive expression of MDC in mature macrophages and dendritic cells. These results delineate an amplification loop of polarized Th2 responses based on differential regulation of MDC production by IL-4 and IL-13 versus IFN-γ and on the selectivity of this chemokine for polarized Th2 cells.© 1998 by The American Society of Hematology.

Expression of macrophage-derived chemokine (MDC) mRNA in macrophages is enhanced by interleukin-1 β, tumor necrosis factor α, and lipopolysaccharide

A cDNA encoding the C-C chemokine MDC was isolated from a human macrophage cDNA library by differential hybridization using monocyte- and macrophage-specific cDNA probes. During monocyte to macrophage differentiation in vitro, MDC expression is first detected after 1 day of culturing and reaches maximum levels after 6 days when macrophages have fully matured, as judged from the expression of known macrophage marker genes. Exposure of macrophages to lipopolysaccharide (LPS) results in a dose-dependent increase in MDC mRNA levels, with maximum induction occurring after 6-8 h, whereas expression levels of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-2, interleukin-1beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha) respond much faster to LPS. Furthermore, MDC expression in macrophages is enhanced by the inflammatory mediators TNF-alpha and IL-1beta. Similar to other TNF-alpha/IL-1beta-inducible genes, costimulation of macrophages with both cytokines leads to higher MDC expression levels than stimulation with a single cytokine. By contrast, both resting and activated monocytes do not express MDC mRNA.