Regulated Upon Activation, Normal T Cell Expressed And Secreted mRNA Research Articles

Reduction of RANTES expression in lesional psoriatic skin after narrow band ultraviolet therapy: a possible marker of therapeutic efficacy

The regulated upon activation, normal T cell expressed and secreted (RANTES) production in psoriatic lesions may amplify the inflammation in these lesions. Narrow band ultraviolet B (NB-UVB), a therapeutic modality for psoriasis, affects the expression of inflammatory cytokines and chemokines. Our aim was to evaluate RANTES mRNA expression in skin lesions of psoriasis before and after NB-UVB phototherapy. This study included 25 psoriatic patients who received 24 sessions of NB-UVB. Skin biopsies were taken before and after phototherapy for real time PCR evaluation of RANTES mRNA. The relative quantitation values (RQ) of RANTES mRNA expression was significantly reduced after treatment. A significant negative correlation was found between pre-treatment RQ RANTES mRNA expression and post-treatment PASI score. We found a significant negative correlation between dRQ RANTES mRNA expression (difference between RQ RANTES mRNA expression before and after phototherapy) and PASI score after phototherapy. We found significant negative correlations between pre-treatment RQ RANTES mRNA expression and both initial response session number and total NB-UVB dose at the end of phototherapy. NB-UVB reduces RANTES mRNA expression in psoriatic lesions. Pre-treatment RQ RANTES mRNA expression could be considered as a marker for clinical improvement and NB-UVB phototherapy efficacy.

Correlations between chemokines CXCL12, CCL2, RANTES and early abortion

To explore the correlations between chemokines stromal cell-derived factor-1 (CXCL12), monocyte chemotactic protein-1 (CCL2), regulated upon activation normal T cell expressed and secreted (RANTES) and early abortion. The villus and decidua tissues of recurrent spontaneous abortion group (n = 26), medical abortion group (n = 30) and normal early pregnancy women (control group, n = 30) were collected at our hospital from 2008 to 2009. And their expressions of CXCL12, CCL2, RANTES mRNA were determined by real-time fluorescence quantitative PCR (polymerase chain reaction). The CXCL12, CCL2 and RANTES mRNA gene expressions were detected in first-trimester placental villus and decidua tissues. And the gene expressions of CXCL12 mRNA in villus and decidua tissues in spontaneous abortion and medical abortion groups were significantly lower than that in the control group (P < 0.01). But the gene expressions of CCL2 and RANTES mRNA in villus and decidua tissues were significantly higher than that in the control group (P < 0.01). CXCL12, CCL2 and RANTES are co-expressed in human villus and decidua tissues. The lower CXCL12 mRNA expression and the higher expressions of CCL2 and RANTES mRNA play important roles in the pathogenesis of early abortion.

Inhibition of TNF-α/IFN-γ induced RANTES expression in HaCaT cell by naringin

Context: Naringin is a bioflavonoid derivative and is predominantly found in Citrus paradisi Macf., Citrus sinensis (Linn.) Osbeck, Citrus unshiu Marc., Citrus reticulata Blanco cv. Nobilis, Citrus tachibana (Makino) Tanaka, Citrus junos Sieb. ex Tanaka (Rutaceae), and related citrus species. It has anti-inflammatory effects that have been well-documented, but the mechanism is poorly characterized.Objective: The effect of naringin on production of RANTES (regulated upon activation normal T-cell expressed and secreted) in human HaCaT cells was investigated here for the first time.Materials and methods: The HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and the proliferation of cell was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The cells were divided into three groups including control group, tumor necrosis factor alpha (TNF-α)/interferon gamma (IFN-γ)-stimulated group, and naringin pretreatment group (first incubated in the presence of naringin and then exposed to TNF-α/IFN-γ). The concentration of RANTES in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA). The expression of RANTES mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The expression of nuclear factor kappa B (NF-κB) P65 protein was detected with immunocytochemical method and western blot method.Results: Naringin hardly inhibits HaCaT cells growth at concentrations rising from 0.25 to 1 mmol/L. However, RANTES expression detected in supernatant stimulated with TNF-α/IFN-γ reduced 15 and 16%, respectively, when cultured with 0.25, 0.5 mmol/L naringin. Furthermore, 1 mmol/L naringin significantly decreased RANTES mRNA level. Finally, naringin decreased the expression of NF-κB P65 protein in nuclei.Discussion and conclusion: Naringin can inhibit the increased production of RANTES, which is partially via NF-κB-dependent signal pathway.

The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 +/- 5.85% (from 162 +/- 10 to 73 +/- 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 +/- 2.7 vs 91.2 +/- 0.9%, P < 0.02; 35.96 +/- 11.64 vs 22.19 +/- 9.65%, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 microM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.

Human V 2V 2 T cells contain cytoplasmic RANTES

The adult human Vgamma2Vdelta2 T cell repertoire is a product of chronic selection in the periphery. Endogenous antigens drive the expansion of cells expressing the Vgamma2Vdelta2 TCR. Thus, we would expect the majority of circulating Vgamma2Vdelta2 T cells to be antigen experienced and to have memory phenotype, in contrast to the alpha/beta TCR+ subsets that include a substantial fraction of naive cells. We sought to characterize functional aspects of Vgamma2Vdelta2 T cells that might show whether circulating cells are memory or naive. For these studies, we focus on the expression of the CC chemokine regulated upon activation normal T cell expressed and secreted (RANTES). In naive alphabeta T cells, an initial stimulus triggers the onset of RANTES transcription followed later by protein expression. In memory CD8+ alphabeta T cells, RANTES mRNA is already present in unstimulated cells and protein expression is triggered immediately by TCR signaling; some cells may also contain RANTES protein in cytoplasmic stores. We show here that the vast majority of circulating human T cells contain RANTES protein in cytoplasmic stores and the chemokine is secreted rapidly after TCR signaling. Primary Vgamma2Vdelta2 T cell lines obtained after in vitro stimulation with phosphoantigens behaved similarly to circulating Vgamma2Vdelta2 T cells and contained both RANTES mRNA and protein, but only very low levels of mRNA or protein for macrophage inflammatory protein (MIP)-1alpha or MIP-1beta. The presence of stored RANTES shows that circulating Vgamma2Vdelta2 T cells are mostly memory phenotype and capable of rapid chemokine responses to phosphoantigen stimulation. Considering that one of 40 circulating CD3+ lymphocytes is Vgamma2Vdelta2+, they comprise the largest circulating memory population against a single antigen, and phosphoantigen stimulation will trigger a rapid activation with immediate release of RANTES.

Immunoglobulin G from Patients with Graves’ Disease Induces Interleukin-16 and RANTES Expression in Cultured Human Thyrocytes: A Putative Mechanism for T-Cell Infiltration of the Thyroid in Autoimmune Disease

Mechanisms underlying lymphocyte infiltration of the thyroid gland and orbit in Graves' disease (GD) are poorly understood. The IGF-I receptor (IGF-IR) is a newly recognized self-antigen that, when activated in GD fibroblasts by IGF-I or GD-IgGs, provokes the expression of IL-16 and RANTES (regulated upon activation, normal T cell expressed and secreted)-dependent T lymphocyte chemoattraction and hyaluronan synthesis. IL-16 is a CD4(+)-specific ligand, and RANTES is a C-C chemokine. Here we report that IGF-I and GD-IgG could induce IL-16 and RANTES in cultured human thyrocytes in a time-dependent manner. Importantly, human TSH failed to induce either chemoattractant. This induction could be attenuated by dexamethasone. Rapamycin, a specific inhibitor of the FRAP/mammalian target of rapamycin/p70(s6k) pathway, prevented GD-IgG-provoked IL-16 synthesis. IH7, a monoclonal antibody directed at IGF-IR also blocked the induction of chemoattraction as well as RANTES mRNA synthesis. Our findings suggest that thyrocytes can be activated by GD-IgG and IGF-I to express powerful T-cell chemoattractants. These actions of GD-IgG appear to be mediated through pathways independent of the TSH receptor. Thus, in GD, thyrocytes may participate directly in lymphocyte recruitment through their expression of IL-16 and RANTES.

Estradiol and interleukin-1beta exert a synergistic stimulatory effect on the expression of the chemokine regulated upon activation, normal T cell expressed, and secreted in endometriotic cells.

Endometriosis, commonly associated with intraperitoneal inflammation, is estrogen dependent. Possible links between the immunoinflammatory and endocrine changes observed in endometriotic women have been poorly understood. In this study, we report that estradiol (E(2)) and IL-1beta exert a synergistic stimulatory action on RANTES (regulated upon activation, normal T cell expressed, and secreted) expression by endometriotic cells. Treatment of endometriotic cells with IL-1beta had a dose-dependent effect on RANTES protein secretion and mRNA steady state levels, whereas cell treatment with E(2) or progesterone had no detectable effect. Interestingly, treatment of endometriotic cells with E(2) before stimulation with IL-1beta resulted in a further increase in RANTES protein secretion and mRNA steady state levels, compared with IL-1beta alone, whereas treatment with progesterone did not significantly affect cell responsiveness to IL-1beta. Assessment of RANTES mRNA half-life revealed that cell pretreatment with E(2) enhanced RANTES mRNA stability and increased gene transcription as shown by run-on analysis. Immunohistochemical analysis of RANTES in endometriotic tissue showed immunostaining to be predominant in the stroma with no noticeable differences in tissues from the proliferative and secretory phase of the menstrual cycle. This appears to be consistent with the cell culture data and indicates that RANTES expression in endometriotic tissue is not subject to cyclic variation. These findings reveal a new regulatory mechanism by which IL-1beta produced by activated macrophages can in synergy with ovarian and locally produced E(2) lead to enhanced macrophage and T-lymphocyte recruitment, thereby exacerbating the local immunoinflammatory process. Furthermore, the findings provide a further evidence for a close relationship between the endocrine and immunological changes observed in endometriosis.

Expression of a chemokine by ciliary body epithelium in horses with naturally occurring recurrent uveitis and in cultured ciliary body epithelial cells.

To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis were examined for RANTES expression by use of an ELISA and reverse transcription-polymerase chain reaction (RT-PCR). Expression of RANTES mRNA and protein content of primary cultures of equine ciliary pigmented epithelial cells (RT-PCR) and culture supernatant (ELISA) were measured 6 or 24 hours, respectively, after cultures were stimulated with interleukin-1beta and tumor necrosis factor-alpha. Strong reactions to intradermal hypersensitivity testing and significant chemotaxis of equine leukocytes to rh-RANTES wereas observed. Aqueous humor of eyes from horses with uveitis contained increased concentrations of rh-RANTES-like protein (mean +/- SD, 45.9+/-31.7 pg/ml), compared with aqueous humor from clinically normal horses (0 pg/ml). Ciliary body from horses with uveitis expressed RANTES mRNA, whereas ciliary body from clinically normal horses had low mRNA expression. Stimulated ciliary pigmented epithelial cells expressed increased amounts of rh-RANTES-like protein (506.1+/-298.3 pg/ml) and mRNA, compared with unstimulated samples. Ciliary epithelium may play a role in recruitment and activation of leukocytes through expression of RANTES.

Signaling of gp34 (OX40 ligand) induces vascular endothelial cells to produce a CC chemokine RANTES/CCL5

We previously showed that gp34 (OX40 ligand) expressed on vascular endothelial cells is not only involved in adhesion between activated T cells and endothelial cells but also by itself able to transmit intracellular signals leading to expression of c-fos and c-jun mRNA upon OX40 binding. In the present study, we searched for genes that were induced or upregulated by gp34 signaling in human umbilical vein endothelial cells (HUVECs) to define its downstream biological events. HUVECs expressing high levels of gp34 were stimulated with recombinant soluble OX40 or mock control and subjected to analysis using cDNA expression arrays. We found that a CC chemokine RANTES (regulated upon activation, normal T cell expressed and secreted)/CCL5 is one of such inducible genes. Reverse transcriptase-PCR analysis showed that expression of RANTES mRNA was induced after incubation with soluble OX40 and this induction was inhibited by anti-gp34 mAb. We could detect expression of intracellular RANTES protein by flow cytometry in HUVECs stimulated with soluble OX40 as well as fixed OX40 transfectant cells but not those stimulated with mock supernatants or mock transfectant cells. Again, this induction of RANTES protein was inhibited by anti-gp34 mAb. These results clearly indicate that gp34 signaling induces expression of RANTES at both mRNA and protein levels in HUVECs and suggest a possible link between the OX40/gp34 system and RANTES during the process of T cell adhesion to endothelial cells and subsequent extravasation.

Fibrates suppress chenodeoxycholic acid-induced RANTES expression through inhibition of NF-κB activation

Fibrates, hypolipidemic agents, are reported to be effective in treatment of primary biliary cirrhosis. However, the mechanism involved in therapeutic benefits of fibrates in primary biliary cirrhosis remains unknown. In contrast, hepatic regulated upon activation, normal T-cell expressed and secreted (RANTES) is increased in patients with primary biliary cirrhosis and bile acids up-regulate RANTES expression in hepatocytes. The role of fibrates in bile acid-induced RANTES expression was investigated in human hepatoma cells; 100 μM of bezafibrate and fenofibrate decreased expression of chenodeoxycholic acid-induced RANTES mRNA and protein. In addition, luciferase enzyme assay using RANTES promoter-luciferase reporter plasmid revealed that 100 μM of bezafibrate and fenofibrate transcriptionally reduced chenodeoxycholic acid-induced RANTES gene expression. Moreover, bezafibrate clearly repressed DNA-binding activity of nuclear factor-κB (NF-κB) induced by chenodeoxycholic acid. Therefore, fibrates might be inhibitory agents of inflammatory cell migration by RANTES to the liver in patients with primary biliary cirrhosis, possibly indicating that fibrates are therapeutic agents in primary biliary cirrhosis.

Nuclear factor-κB regulates RANTES chemokine expression in response to tumor necrosis factor-α in fibroblast-like synoviocytes

We investigated the role of nuclear factor (NF)-κB on tumor necrosis factor (TNF)-α-induced regulated upon activation, normal T-cell expressed and secreted (RANTES) expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA). Using cultured human fibroblast-like synoviocytes from patients with RA, semiquantitative reverse transcriptase-polymerase chain reaction, electrophoretic mobility shift assay, and Western blot were performed for RANTES expression, NF-κB activation, and degradation of IκB, respectively. In addition, the transcriptional effect of TNF-α on RANTES gene expression was analyzed by reporter gene assay. We found that TNF-α clearly induced RANTES protein production and expression of RANTES mRNA in a time-dependent manner. Furthermore, TNF-α persistently induced NF-κB activation caused by IκBα and IκBβ1 degradation. Supershift analysis revealed that TNF-α-induced DNA-binding complexes were composed principally of the p65 and p50 Rel family members. Moreover, transcriptional activation of the RANTES promoter by TNF-α was dependent on specific NF-κB response elements that were regulated by NF-κB. Results herein indicate that NF-κB activation caused by degradation of IκBα and IκBβ1 by TNF-α increased RANTES gene expression in fibroblast-like synoviocytes, suggesting that NF-κB plays an important role in the migration of inflammatory cells by RANTES to the synovium in patients with rheumatoid arthritis.

IL-1beta induction of RANTES (regulated upon activation, normal T cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-kappaB site in the proximal promoter.

A complex network of cytokines mediates immunoregulatory responses in the pathogenesis of endometriosis. RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemoattractant for monocytes and T cells. Endometriotic lesions express RANTES, and its concentration in peritoneal fluid correlates with the severity of endometriosis. We investigated the influence of IL-1beta, a potent macrophage cytokine, on RANTES production in endometriotic stromal cells and determined the region of the RANTES promoter responsible for IL-1beta action. RANTES mRNA was induced 5-fold in endometriotic stromal cells, and the conditioned medium RANTES protein concentrations were 12-fold higher in IL-1beta-treated endometriotic stromal cells vs. untreated controls (P < 0.05). IL-1beta activated the full-length (-940 bp) RANTES promoter as well as a truncated 456-bp 5'-flanking construct by 2-fold. Mutagenesis of a nuclear factor-kappaB response element at -30 bp abolished the IL-1beta effect, whereas mutation of a nearby TNF response element did not affect the IL-1beta induction. An IL-1beta time-course Western assay revealed a rapid diminution of IkappaB (endogenous inhibitor of nuclear factor-kappaB) in endometriotic stromal cells. Overexpression of IkappaB in endometriotic stromal cells inhibited the IL-1beta response of the RANTES gene promoter. Transcription of RANTES mRNA is up-regulated by IL-1beta via a nuclear factor-kappaB response element in the proximal RANTES gene promoter. These results demonstrate a feed-forward regulatory loop in the pathogenesis of endometriosis by which IL-1beta produced from activated macrophages can lead to further macrophage recruitment via RANTES production in endometriotic stromal cells.

Involvement of Oxidative Stress, NF-IL-6, and RANTES Expression in Dengue-2-Virus-Infected Human Liver Cells

The liver has been suspected to be one of the major targets of dengue virus infection. Here, we detected increasing secretion of the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), which functions to recruit the immune cells, in dengue-virus-infected liver cells and patients. Three luciferase reporter genes with various deletions at the 5′-end of the RANTES promoter were constructed to explore the RANTES activation mechanism in human liver cells. The reporter gene was optimally activated by dengue-2 virus when the RANTES promoter contains the region from the transcription starting site (+1) to the nucleotide at the −181 position. NF-IL-6 and an undefined factor forming DNA–protein complexes in the RANTES promoter E and A/B regions in the infected cells were demonstrated by electrophoretic mobility shift assay. Further analysis showed that oxidative stress was an upstream inducer of NF-IL-6 and RANTES signaling in dengue-virus-infected liver cells. This finding was demonstrated by three antioxidants (N-acetyl-l-cysteine, nitro-l-arginine methyl ester, and pyrrolidine dithiocarbamate) used to suppress the activation. In contrast, the DNA binding activity of the undefined factor was not affected by the antioxidant treatment, indicating the existence of an oxidant-independent pathway. We hypothesize that dengue virus infection of the liver cells may trigger both an oxidant-dependent and an oxidant-independent pathway to up-regulate RANTES mRNA expression through activating NF-IL-6 and an undefined factor, respectively. In conclusion, the present study suggests a new direction for the study of liver pathogenesis involving RANTES in host immune responses during dengue virus infection.

Replenishment of RANTES mRNA expression in activated eosinophils from atopic asthmatics.

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T-cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up-regulated by a number of agonists, including complement-dependent factors (C3b/iC3b) and interferon-gamma (IFN-gamma). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil-specific agonists. We analysed RANTES mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum-coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN-gamma (5 ng/ml) transiently and significantly (P<0.05, n = 3) depleted relative amounts of RANTES PCR product (compared with beta2-microglobulin) after 1-4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN-gamma incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil-active cytokines, interleukin-3 (IL-3), IL-4, IL-5 and granulocyte-macrophage colony-stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN-gamma on RANTES mRNA was reversed by cycloheximide, suggesting that IFN-gamma may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN-gamma may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins.

Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression

We investigated the role of NO on expressional regulation of the chemotactic cytokine RANTES (regulated upon activation, normal T-cell expressed and secreted) during tissue regeneration using an excisional wound-healing model in mice. Wound repair was characterized by a large and sustained induction of RANTES expression, and inhibition of inducible nitric oxide synthase (iNOS) during repair only slightly decreased RANTES expression levels. Immunohistochemical analysis revealed keratinocytes of the wound margins and the hyperproliferative epithelium to be the main RANTES-expressing cell type within the wound. Therefore we analysed the regulation of RANTES expression in vitro in cultured human keratinocytes of the cell line HaCaT. Here we demonstrate that NO very efficiently suppressed interleukin-1beta- and tumour-necrosis-factor-alpha-induced RANTES expression in keratinocytes. Furthermore, down-regulation of cytokine-induced RANTES mRNA in keratinocytes was dependent on endogenously produced NO, as inhibition of the co-induced iNOS by L-N(G)-monomethyl-L-arginine increased cytokine-triggered RANTES expression in the cells. Moreover, we observed strongest RANTES-immunopositive labelling in epithelial areas which were characterized by a NO-mediated low cellularity. Thus our data implicate NO as a negative regulator of RANTES expression during wound repair in vivo, as decreased numbers of keratinocytes observed in the absence of wound-derived NO might compensate for the high levels of RANTES expression which are associated with normal repair.

Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization.

Analysis of the host response to viral infection generally has focused on the capacity of viruses to activate or repress transcription of cellular genes, and this approach is also characteristic of work on RNA viruses such as respiratory syncytial virus (RSV). In the present study, it appeared initially that RSV-driven expression of a critical immune regulator, the beta-chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), in primary-culture airway epithelial cells also depended on inducible gene transcription because expression was accompanied by coordinate increases in transcriptional initiation rate and gene promoter activity. However, RSV-driven increases in RANTES gene transcription and promoter activity were small and transient relative to RANTES expression, and they were no different in size and duration than for inactivated RSV that was incapable of fully inducing RANTES expression. These findings suggested that the increase in RANTES gene transcription was not sufficient for inducible expression and that critical regulatory effects occurred at a posttranscriptional level. This type of mechanism for virus-inducible expression of RANTES was established when we found that replicating (but not inactivated) RSV markedly increased RANTES mRNA half-life (from 0.8 to 6.8 h). In addition, RNase protection assays of heterologous promoter/reporter plasmids indicate that basal instability of RANTES mRNA is mediated at least in part by nucleotides 11-389 of the RANTES gene, and this region is also the target for induction by virus. The distinct pathway for production of RANTES (in combination with cytokine-dependent expression of RANTES and related immune-response genes) may more effectively coordinate immune cell interaction with epithelial barrier cells to mediate host defense.

RANTES, IFN-γ, CCR1, and CCR5 mRNA Expression in Peripheral Blood, Lymph Node, and Bronchoalveolar Lavage Mononuclear Cells during Primary Simian Immunodeficiency Virus Infection of Macaques

Primary infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) (as a model of HIV infection in humans) represents a unique opportunity to study early lentivirus/host interactions. We sought to determine whether there is a temporal relationship linking SIV replication and dissemination and the expression of the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) and the SIV/HIV coreceptor CCR5 in different tissues during acute SIV infection of macaques. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate of SIVmac251. RT-PCR was used to monitor the expression of RANTES and CCR5 mRNA in fresh isolated mononuclear cells from blood, lymph node, and bronchoalveolar lavages. These expressions were compared to those of IFN-γ as an indicator of the development of the immune response and to another receptor for RANTES, CCR1, which is not described as a coreceptor for SIV/HIV-1 entry. An enhancement of CCR1/CCR5 mRNA expression was noticed during primary SIVmac251 infection of macaques, mainly in tissue. In the three different compartments investigated, IFN-γ and RANTES overexpression was noticed by the time of systemic viral replication containment. Our results put CCR5 and RANTES mRNA expression back in the context of inflammatory and immune responses to SIV primary infection.

Chemokines induced by infection of mononuclear phagocytes with mycobacteria and present in lung alveoli during active pulmonary tuberculosis.

The capacity of Mycobacterium tuberculosis (MTB) to induce production of chemokines with known chemotactic activity for monocytes and lymphocytes, the cellular building blocks of granulomas, was investigated. These chemokines included regulated upon activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha). MTB stimulated production of MCP-1 and MIP-1alpha by blood monocytes (MN) and alveolar macrophages (AM). MTB infection of MN and AM stimulated release but not production of RANTES. AM produced or released significantly higher levels than MN of RANTES (by 2.1-fold), MCP-1 (by 6.9-fold), and MIP-1alpha (by 5. 5-fold) (P < 0.05 for each). This study also confirmed that MTB-infected AM produce the chemokine interleukin (IL)-8. MTB infection of AM resulted in increased steady-state expression of messenger RNA (mRNA) for MCP-1 and MIP-1alpha and minimal increased expression of RANTES mRNA. Both an avirulent (H37Ra) and a virulent (H37Rv) strain of MTB and purified protein derivative of H37Rv but not latex beads induced production of chemokines. Supernatants of MTB-infected cells demonstrated chemotactic activity for both monocytes and lymphocytes partially inhibitable by neutralizing antibodies against the chemokines studied. Bronchoalveolar lavage fluid from patients with active pulmonary tuberculosis as compared with healthy control subjects contained increased levels of RANTES (by 8-fold), MCP-1 (by 2.7-fold), and IL-8 (by 8.9-fold) (P < 0.05), but not MIP-1alpha, as compared with healthy control subjects. Thus, multiple chemokines may be involved in recruitment of cells for granuloma formation in tuberculosis.

L-arginine suppresses lipopolysaccharide-induced expression of RANTES in glomeruli.

Endotoxemia leads to the infiltration of inflammatory cells in glomeruli and the tubulointerstitium of the kidney. The ultimate mechanisms for this infiltration, however, are not entirely clear. In this study, the glomerular formation of the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) was examined in an in vivo model of endotoxemia to evaluate the role the local release of chemokines might play in the regulation of this inflammatory cell infiltrate. Since the beneficial effects of nitric oxide (NO) on immune-mediated tissue injury have been reported, we also examined possible interactions between the chemokine RANTES and the L-arginine/NO pathway. To induce endotoxemia, rats were injected intraperitoneally with lipopolysaccharide (LPS). Glomeruli were isolated over a 24-h time period, and RANTES was assessed by Northern blotting, a chemotactic assay, and a specific enzyme-linked immunosorbent assay. The chemokine release was associated with increased glomerular infiltration of monocytes/macrophages. LPS also stimulated the mRNA expression of inducible NO synthase and increased the release of nitrite into the supernatants of isolated glomeruli. Supplementation of L-arginine intake increased the release of glomerular nitrite and reduced glomerular RANTES expression after the injection of LPS. Inhibition of the L-arginine/NO pathway by the unspecific NO synthase inhibitor N(G)-nitro-L-arginine methylester significantly increased glomerular RANTES mRNA expression and the number of infiltrating glomerular macrophages. These data demonstrate that L-arginine suppresses glomerular RANTES formation and suggest that the chemokine-mediated recruitment of glomerular macrophages in LPS-induced endotoxemia can be modulated by the L-arginine/NO pathway.

Immunolocalization and Regulation of the Chemokine RANTES in Human Endometrial and Endometriosis Tissues and Cells1

Retrograde menstruation is postulated as the initiating event in the histogenesis of endometriosis; however, subsequent steps in the pathogenesis of this common disorder remain poorly characterized. The ip accumulation of activated leukocytes and the infiltration of endometriosis lesions by macrophages and T cells are cytological markers of the inflammatory nature of this syndrome. The apparent recruitment of these leukocytes prompted us to search for chemokine expression by endometriosis cells. We reported previously that pelvic fluid RANTES (regulated upon activation, normal T cell expressed and secreted) concentrations correlated with the stage of endometriosis. In the current study, RANTES messenger ribonucleic acid (mRNA) was identified in normal endometrium and endometriosis lesions, and techniques were developed to localize RANTES protein within these tissues. Using isolated endometrial and endometriosis cell cultures, we demonstrated that RANTES mRNA and protein can be induced by the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma in endometrial stromal, but not in epithelial or adenocarcinoma cells. Immunocytochemical studies confirmed the biochemical findings. Metabolic labeling experiments verified that nascent RANTES secreted by cytokine-stimulated endometriosis stromal cells was the mature, 8-kDa protein predicted by the mRNA encoding this chemokine. The results indicate that RANTES is a normal constituent of the eutopic endometrium. We propose that secretion of RANTES by ectopic endometriosis implants provides a mechanism for peritoneal leukocyte recruitment.