Monocyte Chemotactic Protein-1 Secretion Research Articles

Reactive oxygen species and ERK 1/2 mediate monocyte chemotactic protein-1-stimulated smooth muscle cell migration

Monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for monocytes, is thought to play a major role in atherosclerosis, but whether its atherogenic effects involve the direct modulation of vascular smooth muscle cell (SMC) functions remains unclear. This study examined the effects of MCP-1 on the migration of cultured A7r5 SMCs and the signaling pathways involved. Addition of recombinant MCP-1 stimulated SMC migration in modified Boyden chambers coated with type I collagen in a concentration-dependent manner, with 10(-9) M being maximally effective. Using untreated A7r5 cells, two MCP-1 receptors, CCR2 and CCR4, were detected and MCP-1 secretion was significantly increased by stimulation with platelet-derived growth factor. MCP-1-stimulated A7r5 migration was completely blocked by the NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI), and dose-dependently inhibited by polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), suggesting a role for reactive oxygen species (ROS) in this process. During MCP-1 stimulation, ROS production increased rapidly, then gradually decayed over 60 min, and this effect was markedly decreased by pretreatment with DPI or PEG-SOD. Interestingly, U0126 and PD98059, which inhibit activation of extracellular signal-regulated kinases 1/2 (ERK 1/2), significantly inhibited MCP-1-activated ROS generation. Furthermore, transfection of an active mutant of MEK1 (ERK 1/2 kinase) markedly increased superoxide production in rat aortic smooth muscle cells, as detected by dihydroethydium staining, suggesting that ERK 1/2 activation stimulates ROS generation. ERK 1/2 activation was increased for at least 30 min in cells incubated with MCP-1, and this effect was abolished by U0126 or DPI pretreatment. These results demonstrate that MCP-1 is a chemoattractant for SMCs and that MCP-1-stimulated migration requires both ROS production and ERK 1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1.

Head and neck squamous cell carcinoma spheroid- and monocyte spheroid-stimulated IL-6 and monocyte chemotactic protein-1 secretion are related to TNM stage, inflammatory state and tumor macrophage density

ConclusionMonocyte fragment (F)-spheroid-stimulated and F-spheroid IL-6 and monocyte chemotactic protein (MCP)-1 secretion are related to inflammatory state, macrophage density and the TNM stage of patients with head and neck squamous cell carcinoma (HNSCC).ObjectiveFragment (F)-spheroids from HNSCC patients in vitro secrete and stimulate autologous monocytes to secrete IL-6 and MCP-1. The aim of this investigation was to study this cytokine secretion in relation to other cytokines, spheroid composition and host factors.Material and methodsIn series I (n=14) the densities of epithelial cells, fibroblasts and macrophages were determined in sections from F-spheroids and donor tissue. In series II (n=17) the TNM stage, donor inflammatory state, macrophage density and the secretion of F-spheroid- and monocyte F-spheroid-stimulated IL-6, MCP-1 and tumor necrosis factor (TNF)-α were determined.ResultsEpithelial cells were partly replaced by interstitial tissue during spheroid formation. Malignant (M) F-spheroids secreted more MCP-1 than benign (B) F-spheroids. No F-spheroid secreted measurable amounts of TNF-α. Monocytes secreted more IL-6 when co-cultured with MF- compared to BF-spheroids. Monocyte IL-6 MF- and MCP-1 MB-spheroid-stimulated secretion correlated with macrophage density. In addition, there was an association between MF- and BF-spheroid-stimulated monocyte cytokine secretion, as well as between BF- and MF-spheroid-stimulated MCP-1 secretion. An inverse relation was also noted between the erythrocyte sedimentation rate at monocyte harvest and the monocyte MCP-1 F-spheroid responses.

The -2518 Promotor Polymorphism in the MCP-1 Gene Is Associated with Systemic Sclerosis

Factors influencing the initiation or progression of sclerosis in patients with systemic sclerosis (SSc) are poorly understood. Monocyte chemotactic protein-1 (MCP-1) is a potent chemokine, which is upregulated in fibroblasts during development of sclerosis. In this study, we investigated the frequency of the functional -2518G MCP-1 promoter polymorphism in 18 patients with SSc and 139 healthy controls. In the lesional skin of the same SSc patients, expression of MCP-1 protein was examined by immunohistochemistry. To investigate a genotype/phenotype correlation, basal as well as tumor necrosis factor (TNF)-induced MCP-1 expression was analyzed in fibroblasts isolated from the skin of SSc patients with different MCP-1 genotypes by quantitative RT-PCR and ELISA. Genotyping for the -2518 (A/G) MCP-1 promotor polymorphism showed that GG homozygotes were significantly more frequent in patients with SSc than in controls (28%vs 6%). Results of immunohistochemistry revealed that MCP-1 was expressed in keratinocytes, infiltrating inflammatory cells, fibroblasts, and endothelial cells in scleroderma skin, whereas normal control skin showed no MCP-1 expression. MCP-1 expression in fibroblasts from GG-homozygote individuals tended to be stronger as compared to AG or AA genotypes. Furthermore, basal as well as TNF-induced MCP-1 expression of fibroblasts isolated from a GG-homozygote SSc patient was significantly higher than MCP-1 expression of fibroblasts isolated from heterozygote or AA-homozygote donors. The A -2518G polymorphism of the MCP-1 gene appears to affect MCP-1 expression of skin fibroblasts of patients with SSc. In accordance, the G/G genotype may predispose patients to SSc.

Non-Complement- and Complement-Activating Antibodies Synergize to Cause Rejection of Cardiac Allografts

Alloantibodies (AlloAbs) are a clinically significant component of the immune response to organ transplants. In our experimental model, B10.A (H-2a) cardiac transplants survived significantly longer in C57BL/6 (H-2b) immunoglobulin knock-out (IgKO) recipients than in their wild-type (WT) counterparts. Passive transfer of a single 50–200-μg dose of complement-activating IgG2b AlloAbs to IgKO recipients reconstituted acute rejection of cardiac allografts. Although passive transfer of a subthreshold dose of 25 μg of IgG2b or a single 100–200-μg dose of non-complement-activating IgG1 AlloAbs did not restore acute rejection to IgKO recipients, a combination of these AlloAbs did cause acute graft rejection. Histologically, rejection was accompanied by augmented release of von Willebrand factor from endothelial cells. IgG1 AlloAbs did not activate complement on their own and did not augment complement activation by IgG2b AlloAbs. However, IgG1 AlloAbs stimulated cultured mouse endothelial cells to produce monocyte chemotactic protein 1 (MCP-1) and neutrophil chemoattractant growth-related oncogene α (KC). TNF-α augmented IgG1 induced secretion of MCP-1 and KC. These findings indicate that non-complement-activating AlloAbs can augment injury to allografts by complement-activating AlloAbs. Non-complement-activating AlloAbs stimulate endothelial cells to produce chemokines and this effect is augmented in the milieu of proinflammatory cytokines.

Investigation of the effects of anthocyanins on rubratoxin B-treated HL60 cells

The effects of anthocyanins on hepatotoxin rubratoxin B-treated HL60 cells were investigated. Anthocyanins are known to have an anti-oxidant activity and corresponding protective effects on hepatic injury. While cyanidin 3-O-β-D-glucoside (Cy 3-glc) and peonidin 3-O-β-D-glucoside (Pn 3-glc) slightly impaired the inhibitory effect of rubratoxin B on cell proliferation, their effects were not significant. In contrast, malvidin 3-O-β-D-glucoside (Mv 3-glc) drastically strengthened the effect of rubratoxin B. Structural difference between Mv 3- glc and the others is the presence of an R group in the 5' position at the B ring, suggesting that the group in this position affected the proliferation of rubratoxin B-treated cells. The results for cytokine secretion were apparently different from those for cell proliferation. The effects of the three anthocyanins on rubratoxin B-induced secretion of interleukin-8 and tumor necrosis factor-α were not substantial. At 100 μM, all the anthocyanins markedly boosted the secretion of monocyte chemotactic protein (MCP)-1, indicating that the common structure shared by the three anthocyanins affected rubratoxin B-induced MCP-1 secretion. In this study, we thus found that anthocyanins exert their activities in ways that either do or do not depend on the identity of the R group in the 5' position at their B ring.

A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease

Background & Aims : Factors influencing the progression of chronic hepatitis C virus (HCV) infection are poorly understood. Monocyte chemotactic protein-1 (MCP-1) is a potent chemokine, and its hepatic expression is up-regulated during chronic HCV infection mainly in activated hepatic stellate cells (HSC). In this study, we investigated the correlation of the functional −2518 MCP-1 promoter polymorphism with hepatic MCP-1 expression and the disease outcome in patients with HCV. Methods : MCP-1 genotyping was performed in 206 patients and 139 healthy controls. Hepatic MCP-1 messenger RNA (mRNA) expression was quantified by real-time PCR in 58 HCV patients. Cytokine-induced MCP-1 secretion of activated human HSC (n = 13) was determined by enzyme-linked immunosorbent assay (ELISA). Mobility-shift assays were performed using probes corresponding to the MCP-1 promoter sequence (−2511 to −2528) with or without the A to G mutation at −2518. Results : Frequency of MCP-1 genotypes did not differ between HCV patients and controls. However, carriers of the G allele were significantly more frequent in HCV patients with more advanced fibrosis and severe inflammation. In accordance, hepatic MCP-1 mRNA levels were significantly higher in patients with more advanced fibrosis and in patients carrying the G allele. Furthermore, cytokine-induced MCP-1 secretion of HSC isolated from carriers of the G allele was significantly higher, and there was binding activity in nuclear extracts from activated HSC specifically to the G allele, providing a potential mechanism for the differences seen. Conclusions : Inheritance of the −2518 MCP-1 G allele, which appears to affect hepatic MCP-1 expression, may predispose HCV patients to more severe hepatic inflammation and fibrosis.

Differential effects of staurosporine and its analogues on chemokine release by promyelocytic leukemia cell line NB-4

The protein kinase inhibitor staurosporine elicits multiple responses in various systems. We evaluated nine naturally occurring staurosporine derivatives as modulators of chemokine production by monitoring the secretion of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in the cell line NB-4. Several staurosporines increased, dose- and time-dependently, the IL-8 and MCP-1 concentration in the cell culture supernatants and three derivatives strongly inhibited proliferation of the NB-4 cells. By comparing the efficiency of these analogues at the same concentration, the lead compound staurosporine (STS-1) was the best inducer of chemokine secretion, whereas 3-hydroxystaurosporine (STS-3) was the most potent growth inhibitor. Besides the staurosporines, also 12- O-tetradecanoyl phorbol acetate (TPA) and tumor necrosis factor-α (TNFα) strongly increased the IL-8 and MCP-1 secretion of NB-4 cells. Several staurosporine analogues clearly inhibited the TPA-induced but enhanced the TNFα-mediated chemokine increase. These effects, namely the increase of chemokines in untreated or TNFα-treated cells and the inhibition of chemokine release in TPA-treated cells, cannot be explained by the exclusive inhibition of protein kinase C (PKC). It may indicate that staurosporines are additionally involved in activation of the PKC-triggered chemokine production.

Synthesis and secretion of monocyte chemotactic protein-1 stimulated by the high affinity receptor for IgE.

In prior studies aggregation of the high affinity receptors for IgE, Fc epsilon RI, on a rat mast cell line, RBL-2H3, stimulated transcription of the gene for monocyte chemotactic protein-1 (MCP-1) and secretion of the protein. Unexpectedly, those delayed events appeared much less constrained by kinetic proofreading than had been documented for other receptor-initiated responses. The results of the present experiments are consistent with the proposal that the biosynthesis and secretion of MCP-1 result from a soluble messenger formed in the reaction cascades initiated by the receptor, and that Ca(2+) could serve as that messenger. Interestingly, whereas receptor-mediated signals were required for transcription of the gene for MCP-1 and secretion of the chemokine, such signals were not required for the intervening step of translation of its mRNA.

Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha, interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes.

The role of mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, IL-8, and monocyte chemotactic protein-1 (MCP-1) was investigated in human monocytes that were infected with Mycobacterium tuberculosis H37Rv. Analysis of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase showed rapid phosphorylation of both subfamilies in response to M. tuberculosis H37Rv. Using highly specific inhibitors of p38 (SB203580) and of MAPK kinase-1 (U0126 and PD98059), we found that both p38 and ERK were essential for M. tuberculosis H37Rv-induced TNF-alpha production, whereas activation of the p38 pathway, but not that of ERK, was essential for M. tuberculosis H37Rv-induced IL-10 production. Interestingly, the ERK pathway, but not that of p38, was critical for MCP-1 secretion from human monocytes that were infected with M. tuberculosis H37Rv. However, IL-8 secretion was not regulated by ERK1/2 or p38 MAPK. Collectively, these results suggest that induction of the MAPK pathway is required for the expression of TNF-alpha, IL-10, and MCP-1 by human monocytes during M. tuberculosis H37Rv infection.

Effects of bile acids on biliary epithelial cells: Proliferation, cytotoxicity, and cytokine secretion

Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependant manner (50–500 μM). Cell viability was significantly decreased by TLC and TCDC at 500 μM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases.

IL-17 expression in human herpetic stromal keratitis: modulatory effects on chemokine production by corneal fibroblasts.

Herpetic stromal keratitis (HSK) is an immunopathologic disease triggered by infection of the cornea with HSV. Key events in HSK involve the interaction between cornea-infiltrating inflammatory cells and resident cells. This interaction, in which macrophages, producing IL-1 and TNF-alpha, and IFN-gamma-producing Th1 cells play a crucial role, results in the local secretion of immune-modulatory factors and a major influx of neutrophils causing corneal lesions and blindness. The Th1-derived cytokine IL-17 has been shown to play an important role in several inflammatory diseases characterized by a massive infiltration of neutrophils into inflamed tissue. Here we show that IL-17 is expressed in corneas from patients with HSK and that the IL-17R is constitutively expressed by human corneal fibroblasts (HCF). IL-17 exhibited a strong synergistic effect with TNF-alpha on the induction of IL-6 and IL-8 secretion by cultured HCF. Secreted IL-8 in these cultures had a strong chemotactic effect on neutrophils. IL-17 also enhanced TNF-alpha- and IFN-gamma-induced secretion of macrophage-inflammatory proteins 1alpha and 3alpha, while inhibiting the induced secretion of RANTES. Furthermore, considerable levels of IFN-gamma-inducible protein 10 and matrix metalloproteinase 1 were measured in stimulated HCF cultures, while the constitutive secretion of monocyte chemotactic protein 1 remained unaffected. The data presented suggest that IL-17 may play an important role in the induction and/or perpetuation of the immunopathologic processes in human HSK by modulating the secretion of proinflammatory and neutrophil chemotactic factors by corneal resident fibroblasts.

Soluble interleukin-1 receptor type II blocks monocyte chemotactic protein-1 secretion by u937 cells in response to peripheral blood serum of women with endometriosis

Objective: To assess the ability of peripheral blood serum from women with endometriosis to induce monocyte chemotactic protein-1 (MCP-1) secretion by monocytes and the putative role of the interleukin-1 (IL-1) system in endometriosis-associated monocyte activation. Design: Cultures of U937 monocytic cells exposed to serum from normal women (control group) or women with endometriosis. Setting: Gynecology clinic and human reproduction research laboratory. Patient(s): Seventy-nine women with endometriosis and 38 control women with no evidence of endometriosis at laparoscopy. Intervention(s): Peripheral blood obtained a few days before laparoscopy. Main Outcome Measure(s): MCP-1 secretion in the culture medium and serum concentrations of soluble IL-1 receptor type II (sIL-1RII), IL-1β, and IL-1α by ELISA or by enzyme immunometric assay. Result(s): Serum concentrations of sIL-1RII were significantly lower in women with stage I–II endometriosis than in control women, whereas serum concentrations of IL-1β and IL-1α were comparable between the two groups. The serum of women with endometriosis induced higher secretion of MCP-1 by U937 cells than that of control women, particularly in the initial stages of endometriosis (stages I–II), and recombinant IL-1RII (rIL-1RII) significantly blocked that secretion. Conclusion(s): These findings point toward a deficiency in the mechanisms involved in the down-regulation of IL-1 actions at the systemic level and reveal sIL-1RII as a key factor involved in that process.

Pathogenesis of pneumococcal pneumonia in cyclophosphamide-induced leukopenia in mice.

Streptococcus pneumoniae pneumonia frequently occurs in leukopenic hosts, and most patients subsequently develop lung injury and septicemia. However, few correlations have been made so far between microbial growth, inflammation, and histopathology of pneumonia in specific leukopenic states. In the present study, the pathogenesis of pneumococcal pneumonia was investigated in mice rendered leukopenic by the immunosuppressor antineoplastic drug cyclophosphamide. Compared to the immunocompetent state, cyclophosphamide-induced leukopenia did not hamper interleukin-1 (IL-1), IL-6, macrophage inflammatory protein-1 (MIP-1), MIP-2, and monocyte chemotactic protein-1 secretion in infected lungs. Leukopenia did not facilitate bacterial dissemination into the bloodstream despite enhanced bacterial proliferation into lung tissues. Pulmonary capillary permeability and edema as well as lung injury were enhanced in leukopenic mice despite the absence of neutrophilic and monocytic infiltration into their lungs, suggesting an important role for bacterial virulence factors and making obvious the fact that neutrophils are ultimately not required for lung injury in this model. Scanning and transmission electron microscopy revealed extensive disruption of alveolar epithelium and a defect in surfactant production, which were associated with alveolar collapse, hemorrhage, and fibrin deposits in alveoli. These results contrast with those observed in immunocompetent animals and indicate that leukopenic hosts suffering from pneumococcal pneumonia are at a higher risk of developing diffuse alveolar damage.

Monocyte chemotactic protein-1 secretion and expression after Toxoplasma gondii infection in vitro depend on the stage of the parasite

Infection of human fibroblasts with tachyzoites of RH and Prugniaud strains, two different strains of Toxoplasma gondii, significantly increased monocyte chemotactic protein (MCP)-1 secretion contrary to what happened with bradyzoites of the cystogenetic strain. Quantification of MCP-1 mRNA by RT-PCR showed that this phenomenon is regulated at the transcriptional level. Thus, the stage of parasite can be deciding in MCP-1 induction since only tachyzoites induced MCP-1 expression and secretion. MCP-1 induced by tachyzoites could be involved in cell recruitment, as shown by the quantification of MCP1 ARNm by real-time PCR (LightCycler, Roche Diagnostics), in the pathogenesis of T. gondii infection.

Activation of PKC-β isoforms mediates HNE-induced MCP-1 release by macrophages

4-Hydroxynonenal (HNE) in the concentration range detectable in many pathophysiologic conditions is able to modulate signal transduction cascades and gene expression. Here, we report the stimulating effect of 1 μM HNE on the release of the monocyte chemotactic protein-1 (MCP-1) by murine macrophages. MCP-1-increased export following 1-h cell treatment with HNE proved to be comparable to that exerted by standard amounts of bacterial lipopolysaccharide (LPS). However, the key molecular event in HNE-induced secretion of MCP-1 appeared to be the increased activity of β-PKC isoforms, which are recognized as playing a role in the regulation of cell protein transport and secretion. On the other hand, in LPS-stimulated cells, the δ isoform was seen to be involved and was probably related to LPS-mediated effects on MCP-1 expression and synthesis. In conclusion, HNE might interact with other pro-inflammatory stimuli, like LPS, in a concerted amplification of MCP-1 production and secretion.

Spontaneous and stimulated secretion of monocyte chemotactic protein-1 and macrophage migration inhibitory factor by peritoneal macrophages in women with and without endometriosis

Objective: To assess spontaneous and stimulated secretion of monocyte chemotactic protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF) by peritoneal macrophages in women with and without endometriosis. Design: Macrophages were isolated from the peritoneal fluid and cultured for different periods of time (6, 20, and 44 hours) without any stimulation to determine spontaneous secretion of MCP-1 and MIF. Macrophages were also exposed to 1 μg/mL lipopolysaccharide for 6 hours to evaluate the stimulated secretion of these cytokines. Setting: Gynecology clinic and human reproduction research laboratory. Patient(s): Twelve fertile women and 11 women with endometriosis. Intervention(s): Peritoneal fluid obtained at laparoscopy. Main Outcome Measure(s): Monocyte chemotactic protein-1 and MIF concentrations in the culture medium using ELISA. Result(s): Peritoneal macrophages of women with endometriosis demonstrated an increased capacity to secrete MCP-1 either spontaneously or after stimulation with lipopolysaccharide. They also showed a marked tendency for an increased secretion of MIF, but no statistically significant difference was found. Conclusion(s): Monocyte chemotactic protein-1 and MIF production by peritoneal macrophages may contribute to paracrine and autocrine activation and to macrophage accumulation in the peritoneal cavity of women with endometriosis. These mechanisms may exacerbate peritoneal inflammation and favor the growth of endometrial implants.

Changes in Gene Expression of ARPE-19 Cells in Response to Vitreous Treatment

Purposes: The purpose of this study is to determine the changes in gene expression by a human retinal pigment epithelium (RPE) cell line (ARPE-19) in response to vitreous treatment, which induces RPE proliferation and phenotypic changes in vitro that mimic the repair response observed in vivo during proliferative vitreoretinopathy. Methods: ARPE-19 cells were grown for more than 4 weeks and their gene expression studied in: (1) subconfluent cultures treated with 50% human vitreous for 72 h; (2) subconfluent cultures without vitreous treatment, and (3) a confluent, nondividing monolayer. Total RNA was extracted from RPE cells and differential gene expression between each condition was determined using gene arrays (Clontech, Palo Alto, Calif., USA). Semiquantitative RT-PCR was used to confirm the upregulation of 4 genes related to vitreous treatment. In addition, the secretion of 1 of these upregulated gene products, monocyte chemotactic protein 1 (MCP-1), was confirmed by ELISA. Results: A greater than threefold increase in the expression of mRNA for cell cycle regulators, intracellular transducers, cell adhesion proteins, growth factors and chemokines was observed following vitreous treatment of the RPE cell line and a corresponding decrease in the expression of genes related to apoptosis. RT-PCR confirmed the increased gene expression of MCP-1, intercellular adhesion molecule-1, vascular cell adhesion protein 1 precursor and vascular endothelial growth factor in vitreous-treated cells. Immunoassays further showed an increased MCP-1 secretion by vitreous-treated RPE. Conclusions: These findings suggest that in this in vitro vitreous treatment model, ARPE-19 cells participate in a mock repair response by upregulating genes encoding for proteins associated with inflammation and wound healing.

Analysis of the secretion pattern of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta 2 (TGF-beta2) by human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells, situated between the neurosensory retina and the vascularized choroid, form part of the blood-eye barrier and are important for homeostasis of the outer retina. These cells are able to produce a variety of cytokines which may play a role in the maintenance of the immunosuppressive milieu inside the eye and in intraocular inflammatory responses. In the present study, we investigated whether RPE cells secreted the anti-inflammatory cytokine TGF-beta2 and the proinflammatory cytokine MCP-1 in a polarized manner. Monolayers of human donor RPE cells were cultured on transwell filters. Secretion of TGF-beta2 and MCP-1 at either the apical or basal side of the RPE cell monolayers, that were not treated or stimulated with IL-1beta (200 U/ml), was analysed by ELISA. All three cell lines examined had a different TGF-beta2 secretion pattern. In two of the three donor RPE cell lines tested, TGF-beta2 secretion was polarized, but not in the same direction. TGF-beta2 secretion was not up-regulated by stimulation with IL-1beta. In contrast, IL-1beta strongly induced MCP-1 secretion preferentially into the basal compartment of all RPE monolayers tested. These data indicate that human RPE cells are able to secrete TGF-beta2 and MCP-1 in a polarized fashion. Our results suggest that MCP-1 can be secreted by RPE cells in the direction of choroidal vessels during inflammatory responses in the posterior part of the eye, which may limit damage to the neurosensory retina.

Low-density lipoproteins enhance transforming growth factor-beta 1 (TGF-beta 1) and monocyte chemotactic protein-1 (MCP-1) expression induced by cyclosporin in human mesangial cells.

Cyclosporin (CsA) is widely used in the treatment of renal disease and transplantation, which are often complicated by alterations of lipid metabolism. Both chronic administration of CsA and hyperlipidaemia have been shown to evoke an early macrophage influx and have progressively led to glomerular and interstitial sclerosis. MCP-1 is the major monocyte chemoattractant secreted by stimulated mesangial cells and TGF-beta 1 is a key mediator of fibrogenesis in chronic progressive renal fibrosis. Thus, the combined effect of CsA and low-density lipoprotein (LDL) on the gene and protein expression of MCP-1 and TGF-beta 1 in cultured human mesangial cells (HMC) was explored. Both agents induced an early and persistent increase of MCP-1 and TGF-beta 1 mRNA levels and protein release. The simultaneous addition of CsA and LDL did not display any additive effect on target gene expression, but it caused a synergistic effect on MCP-1 and TGF-beta 1 protein secretion into culture medium. On the other hand, CsA and LDL had different effects on cell proliferation: the latter increased DNA synthesis, whereas CsA inhibited both spontaneous and mitogen-stimulated mesangial cell growth. The study concludes that CsA and LDL display an additive effect on TGF-beta 1 and MCP-1 synthesis and release by HMC, thus possibly co-operating to induce an early macrophage influx and the subsequent mesangial expansion and increased extracellular matrix deposition. However, in contrast they seem to modulate HMC proliferation differently, which is a further critical event intimately involved in the development of glomerulosclerosis.

The IL-6-soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.