A self-assembled peptide hydrogel for cytokine sequestration.

Cytokine-directed monocyte infiltration is involved in multiple pathological processes. Immuno-isolating matrices that can sequester cell-released chemokines in a microenvironment may prolong the viability and functionality of implanted materials. We describe a self-assembling peptide-based hydrogel that can capture the cytokine CCL2 released in the extracellular space by immune cells and stromal cells. The shear-responsive matrix can absorb and retain this signaling molecule needed for the chemotaxis of the infiltrating monocytes and their differentiation into phagocytic macrophages. Such cytokine-sequestering biomaterials may be useful as adjunctive materials with the delivery of exogenous implants or cell suspensions for tissue regeneration, without the administration of systemic immunosuppressants. Our work highlights the versatility of nanofibrous peptide hydrogels for modulating the biological response in tissue niches.

A sensitive sandwich-type immunosensor for the detection of MCP-1 based on a rGO-TEPA-Thi-Au nanocomposite and novel RuPdPt trimetallic nanoalloy particles.

A novel electrochemical immunosensor was proposed for the detection of monocyte chemoattractant protein-1 (MCP-1), a biomarker of cardiovascular disease. Due to thionine (Thi) possessing electroactive redox properties, a one-step approach was utilized to synthesize a reduced graphene oxide-tetraethylene-thionine-Au (rGO-TEPA-Thi-Au) nanocomposite at room temperature using the synergistic effect of Thi and rGO-TEPA towards HAuCl4. We obtained the excellent matrix material, which immobilized more primary antibody MCP-1-Ab1 on rGO-TEPA on a modified glassy carbon electrode (GCE). To further enhance the sensitivity of the sensor, a novel signal generation and amplification strategy was developed for detection. RuPdPt trimetallic nanoalloy particles (RuPdPt TNPs), a novel nanomaterial, were synthesized by a one-pot method, displayed a uniform morphology as well as good electrochemical activity and bound with the secondary antibodies against MCP-1 via the Pt-NH2 bond. Most importantly, RuPdPt TNPs have a significant ability to catalyze H2O2 to produce an electron. The electrochemical signal was highly amplified because the electrochemical signal was primarily derived from the synergistic catalysis of H2O2 by RuPdPt TNPs and recorded by chronoamperometry. Under the optimal conditions, this newly designed biosensor exhibited sensitive detection of MCP-1 in the range from 20 fg mL-1 to 1000 pg mL-1, with a detection limit of 8.9 fg mL-1 (based on a S/N = 3). Additionally, the designed immunosensor showed acceptable selectivity, reproducibility and stability. This immunosensor is a promising strategy for analyzing clinical serum samples in the future.

Expression and purification of biologically active recombinant rabbit monocyte chemoattractant protein1 in Escherichia coli.

Monocyte chemoattractant protein 1 (MCP1) with recruiting monocytes is an important factor at the beginning of inflammatory disorders such as atherosclerosis which seems its blocking preclude this process and help improvement of related diseases. To perform clinical research in this field, MCP1 protein is required but firstly, animal studies should be done. As the rabbit is a suitable model for many inflammatory disorders, and Escherichia coli BL21(DE3) (BL21) cell is a high-efficiency host for protein expression, we decided to produce recombinant rabbit MCP1 (rRMCP1) in BL21/pET28a system. After codon usage, a construct containing RMCP1 sequence was synthesized, cloned into the pET28a plasmid, and overexpressed in BL21 cells. Followed that, with changing expression condition such as cell concentration before the induction, time period, temperature, shaking rate and inducer concentration (IPTG), rRMCP1 expression was optimized, and purified by Ni-NTA. The biological activity of the expressed protein was verified using monocyte migration assay. Using this expression system, nearly 28 mg/mL rRMCP1 was produced at 26°C/180 rpm for 24 h in LB broth medium with 1 mM IPTG. Therefore, we were succeeded to express the intermediate level of rRMCP1 with this method. This amount of protein is sufficient for biological researches in the laboratory.

The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments.

Infiltration of leukocytes is one of the hallmarks of the inflammatory response. Among the leukocyte populations, neutrophils are the first to infiltrate, followed by monocytes and lymphocytes, suggesting the presence of mediators that specifically recruit these cell types. Cytokine-like chemoattractants with monocyte chemotactic activity, such as lymphocyte-derived chemotactic factor (LDCF) or tumor-derived chemotactic factor (TDCF), were reported as molecules that could play a critical role in the recruitment of monocytes into sites of immune responses or tumors; however, their identities remained unclear. In the 1980s, researchers began to test the hypothesis that leukocyte chemotactic activity is a part of the wider activities exhibited by cytokines, such as interleukin-1 (IL-1). In 1987, we demonstrated, for the first time, the presence of a cytokine like chemoattractant with cell type-specificity (now known as the chemokine interleukin-8 or CXC chemokine ligand 8) that was different from IL-1. This led us to the purification of the second such molecule with monocyte chemotactic activity. This monocyte chemoattractant was found identical to the previously described LDCF or TDCF, and termed monocyte chemoattractant protein-1 (MCP-1). Isolation of MCP-1 created a revolution in not only inflammation but also cancer research that continues today, and MCP-1 has become a molecular target to treat patients with many diseases. In this review, I will first describe a history associated with the discovery of MCP-1 and then discuss complex mechanisms regulating MCP-1 production in tumor microenvironments.

Adsorption as a Contributor for Inflammatory Mediators Removal by Different Hemofiltration Membranes: A Pilot Study.

Atherosclerosis is an important predictor of mortality in patients with chronic kidney disease (CKD) and is associated with a wide inflammatory response. The aim of this study is to evaluate in vitro how different membranes can remove mediators associated with this pathology in a closed loop dialysis model. We performed experimental hemofiltration in vitro using three different membrane materials. Human plasma was preliminarily incubated with various inflammatory mediators and filtered in a closed loop circulation model for 240 min. Respective concentrations of 17 different mediators were measured over time to study the removal mechanisms of each membrane, including associated removal time course. The experiment was repeated three times for the assay of tumor necrosis factor (TNF)-α to document the model variability. Means were compared using Mann-Whitney test. Most of the investigated mediators were effectively removed with the different dialysis membranes. Adsorption mechanism was mainly at the origin of the decrease in mediators circulating concentrations and was maximized in the region 10 000-20 000 Da. Especially, the HeprAN membrane showed fast removal capacities of mediators with elevated isoelectric point including complement factors and chemokines or having basic groups located in the protein periphery, plasminogen activator inhibitor (PAI-1), and TNF-α-like. The latter was further significantly removed with HeprAN and polymethylmethacrylate (PMMA) compared to polyethersulfone (PES) material (P < 0.01). We concluded that dialysis using ionic adsorptive membrane could have a beneficial impact for CKD patients with atherosclerosis and would deserve further clinical investigations.

N(pro) fusion technology: On-column complementation to improve efficiency in biopharmaceutical production.

N(pro) fusion technology, a highly efficient system for overexpression of proteins and peptides in Escherichia coli, was further developed by splitting the autoprotease N(pro) into two fragments to generate a functional complementation system. The size of the expression tag is thus reduced from 168 to 58 amino acids, so by 66%. Upon complementation of the fragments auto-proteolytic activity is restored. This process has been shown for three model proteins of different size, a short 16 aa-peptide, MCP-1, and lysozyme. Moreover, the complementation was still functional after immobilization of the N-terminal fragment to a solid support which enables recycling of the immobilized fragment. This strategy enhances overall productivity of N(pro) Fusion Technology and thus allows more efficient production of recombinant proteins with reduced costs and in higher yields. Overall, the N(pro) complementation system has, depending on the size of the target molecule, potential to increase the productivity up to 4 fold for batch refolding and even more for on-column refolding strategies by the proven possibility of regeneration of the immobilized fragment.

Heparin-immobilized microspheres for the capture of cytokines.

The preparation and characterization of heparin-immobilized microspheres which were used to bind acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein 1 (MCP-1/CCL2), and regulation upon activation normal T cell express sequence (RANTES/CCL5) is described. These beads were used as trapping agents in microdialysis sampling experiments in a separate study. Both free heparin and a synthesized heparin-albumin conjugate were immobilized onto microspheres and compared for their effectiveness. The heparin-albumin conjugate microspheres exhibited significant nonspecific adsorption which appeared to be due to the albumin content. The prepared heparin-immobilized microspheres were stable for 3 months at 4 °C. A bead-based flow cytometric assay was developed to study the binding capacity and specificity of the heparin-immobilized microspheres to cytokines. These heparin-immobilized microspheres exhibited broad dynamic ranges for binding to the four cytokines (aFGF, 1.0-1,000 ng/mL; VEGF, 0.5-1,000 ng/mL; CCL2, 1.95-1,000 ng/mL; CCL5, 1.95-500 ng/mL). Fast binding kinetics of the cytokines to the heparin-immobilized beads suggests that these beads may be useful as affinity agents in microfluidic flow systems.

In vitro and in vivo affinity microdialysis sampling of cytokines using heparin-immobilized microspheres.

Heparin-immobilized microspheres were included in microdialysis sampling perfusion fluids under both in vitro and in vivo conditions to improve the recovery of different cytokines, acidic fibroblast growth factor, vascular endothelial growth factor, monocyte chemoattractant protein-1 (or CCL2), and regulation upon activation normal T cell express sequence (or CCL5). Different strategies to dissociate captured CCL2 and CCL5 from the immobilized heparin were attempted, and both cytokines could be quantitatively eluted from the beads using a phosphate buffer (pH 7.4) containing 25% (v/v) acetonitrile which did not interfere with the subsequent detection of cytokine using an ELISA assay. Using these heparin-immobilized microspheres, a two to fivefold increase of microdialysis relative recovery (RR) was achieved for the four cytokines from a quiescent solution. Enhanced microdialysis RR of CCL2 using the heparin-immobilized microspheres from microdialysis probes implanted into the peritoneal cavity of a rat was performed to test the in vivo application. This work suggests that the heparin-immobilized microspheres provide an alternative affinity agent to the previously used antibody-immobilized microspheres for enhanced microdialysis sampling of cytokines.

Affinity-based microdialysis sampling using heparin for in vitro collection of human cytokines.

Microdialysis sampling is a widely used method to sample from complex biological matrices. Cytokines are important signaling proteins that are typically recovered with low relative recovery values during microdialysis sampling. Heparin was included in the microdialysis perfusion fluid as an affinity agent to increase in vitro recovery of different cytokines through polyethersulfone (PES) microdialysis membranes with 100 kDa molecular weight cutoff. No change in fluid volumes collected from the microdialysis probes occurred when heparin was included in the perfusion fluid up to concentrations of 10 microM. The loss of heparin (10 microM) across the dialysis membrane was minimal (2.7+/-0.9%, n=3). Additionally, heparin at these concentrations did not interfere with the cytokine immunoassays. The control and heparin-enhanced relative recoveries for five human cytokines using 0.1 microM heparin in the microdialysis perfusion fluid flowing at 0.5 microL min(-1) were (n=3): interleukin-4 (IL-4), 4.2+/-0.5% and 7.2+/-3.1%; interleukin-6 (IL-6), 1.4+/-0.8% and 3.6+/-1.3%; interleukin-7 (IL-7), 1.3+/-0.8% and 4.8+/-1.8%; monocyte chemoattractant protein-1 (MCP-1), 9.0+/-1.6% and 19.5+/-2.7%; and tumor necrosis factor-alpha (TNF-alpha), 7.4+/-1.3% and 16.9+/-1.6%, respectively. Heparin increased the microdialysis sampling relative recovery of several human cytokines in vitro.

Multiple products derived from two CCL4 loci: high incidence of a new polymorphism in HIV+ patients.

Human CCL4/macrophage inflammatory protein (MIP)-1beta and CCL3/MIP-1alpha are two highly related molecules that belong to a cluster of inflammatory CC chemokines located in chromosome 17. CCL4 and CCL3 were formed by duplication of a common ancestral gene, generating the SCYA4 and SCYA3 genes which, in turn, present a variable number of additional non-allelic copies (SCYA4L and SCYA3L1). In this study, we show that both CCL4 loci (SCYA4 and SCYA4L) are expressed and alternatively generate spliced variants lacking the second exon. In addition, we found that the SCYA4L locus is polymorphic and displays a second allelic variant (hereinafter SCYA4L2) with a nucleotide change in the intron 2 acceptor splice site compared with the one described originally (hereinafter SCYA4L1). Therefore, the pattern of SCYA4L2 transcripts is completely different from that of SCYA4L1, since SCYA4L2 uses several new acceptor splice sites and generates nine new mRNAs. Furthermore, we analyzed the contribution of each locus (SCYA4 and SCYA4L1/L2) to total CCL4 expression in human CD8 T cells by RT-amplified fragment length polymorphism and real-time PCR, and we found that L2 homozygous individuals (L2L2) only express half the levels of CCL4 compared with L1L1 individuals. The analysis of transcripts from the SCYA4L locus showed a lower level in L2 homozygous compared with L1 homozygous individuals (12% vs 52% of total CCL4 transcripts). A possible clinical relevance of these CCL4 allelic variants was suggested by the higher frequency of the L2 allele in a group of HIV(+) individuals (n = 175) when compared with controls (n = 220, 28.6% vs 16.6% (p = 0.00016)).

Correlation between decreased type-II interleukin-1 receptor and increased monocyte chemotactic protein-1 expression in the endometrium of women with endometriosis.

PROBLEM: Monocyte chemotactic protein-1 (MCP-1), a potent inducer of macrophage recruitment and activation, is overexpressed in the eutopic endometrium of women with endometriosis. Eutopic endometrial cells of women with endometriosis secrete higher levels of MCP-1 than those of normal women, following stimulation with interleukin-1 (IL-1). The aim of this study was to examine whether there is any correlation between the expression of IL-1 receptor type II (IL-IRII), a specific downregulator of IL-1 activity, and that of MCP-1 in the eutopic endometrium of women with endometriosis. METHOD OF STUDY: Endometrial biopsies of 46 women with endometriosis and 22 healthy women were evaluated simultaneously for IL-1RII and MCP-1 expression by immunohistochemistry. RESULTS: Our study revealed a highly significant correlation between the decreased expression of IL-1RII and the increased expression of MCP-1 in the endometrial tissue of women with endometriosis (Spearman correlation coefficient r = -0.377, P = 0.002), particularly in the initial stages of the disease (stages I and II; r = - 0.368, P = 0.020 and r = -0.480, P = 0.002, respectively). Furthermore, this correlation was observed in the proliferative (r = -0.366, P = 0.047) and the secretory phases (r = -0.321, P = 0.049) of the menstrual cycle. CONCLUSIONS: These results suggest that the reduced capability of endometrial tissue to downregulate IL-1 proinflammatory effects may be involved in the increased expression of MCP-1 in the endometrium of women with endometriosis and the establishment of an inflammatory state. The results also indicate a sustained process of cell activation throughout the menstrual cycle.

Expression of the chemokines, monocyte chemotactic protein (MCP)-1 and MCP-2 in endometrium of normal women and Norplant users, does not support a central role in macrophage infiltration into endometrium.

The endometrium contains many leukocytes, including macrophages, the numbers varying with the time of the menstrual cycle and being maximal peri-menstrually. The long-acting progestogenic contraceptive Norplant, has a high rate of discontinuation due to uterine bleeding; this is associated with large numbers of endometrial macrophages. Monocyte chemotactic proteins (MCP) act to recruit and activate monocytes into sites of inflammation. This study compared the cellular localization of endometrial MCP-1 and MCP-2 across the normal menstrual cycle and in users of Norplant. Both MCP-1 and MCP-2 were present in normal endometrium, but with very different patterns of cellular location and considerable variability between individuals. MCP-1 of epithelial origin was present in 77% of tissues, while stromal staining was present in 52% and vascular staining in 34% of samples. MCP-1 was also released from both epithelial and stromal cells in culture. MCP-2 staining was predominantly epithelial and was found in 52% of tissues while stromal staining was present in only 3/56 samples. Vascular staining of MCP-2 was found in 2/56 samples. The epithelial staining was mostly punctate and sometimes within uterine secretions. No correlation of staining for MCP-1 or -2 with the phase of the cycle was found in any cellular compartment. Very little immunoreactive MCP-1 or MCP-2 was detected in endometrium from Norplant users regardless of morphological subtype. These distributions do not support a role for either MCP-1 or MCP-2 in the migration of macrophages into the endometrium and suggest that these cytokines may have other functions in this tissue.

Inhibitory effect of sanguiin H-11 on chemotaxis of neutrophil.

Neutrophil is accumulated by chemoattractants at inflammatory sites. In order to find a new substance to regulate the chemotaxis of neutrophil, we examined the effects of purified tannins and related compounds (33 species). Among those studied, sanguiin H-11, purified from the plant Sanguisorba officinalis, was the most potent inhibitor of rat cytokine induced neutrophil chemoattractant-1 (CINC-1, a counterpart of human GRO (melanoma growth-stimulating activity)) dependent chemotaxis in rat neutrophils. Sanguiin H-11 inhibited platelet-activating factor- and N-formylmethionyl-leucyl-phenylalanine-dependent chemotaxis of neutrophil in addition to CINC-1-dependent reactions. Sanguiin H-11 also inhibited chemokinesis of neutrophil, but did not drastically affect the increase of intracellular free [Ca2+] level or degranulation monitored by the secretion of elastase, following chemoattractant stimulation. These results indicated that sanguiin H-11 is a potent inhibitor of chemoattractant-dependent and independent neutrophil movement.

Monocyte chemotactic and activating factor/monocyte chemoattractant protein-1-mediated histamine release from human nasal mucosa.

OBJECTIVES: To demonstrate the existence and localization of monocyte chemotactic and activating factor or monocyte chemoattractant protein-1 (MCAF/MCP-1) in human nasal mucosa and to verify its activity as a histamine-releasing factor. DESIGN: Detection of MCAF/MCP-1 in culture supernatants of nasal mucosa using Western blot analysis and assay of histamine release from basophils induced by these culture supernatants. Detection of MCAF/MCP-1 expression in nasal mucosa of patients with perennial allergic rhinitis using immunohistochemistry. PATIENTS: Twenty-one patients with house dust mite allergy, 7 nonallergic patients, and 5 patients with chronic inflammatory sinusitis participated in the study. All the allergic patients had positive test results for mite nasal allergy, detected by a clinical history, a nasal provocation test, and determination of specific mite IgE antibodies by a radioallergosorbent test. RESULTS: In Western blot analysis of supernatants of explant culture of human nasal mucosa, the band corresponding to approximately 13 to 15 kd was observed. This band was considered to be MCAF/MCP-1. These supernatants induced histamine release from basophils (approximately 3%-5% in net histamine release), and anti-MCAF/MCP-1 antibody inhibited this histamine-releasing activity. Immunoreactivity of MCAF/MCP-1 was observed in the nasal submucosa but not in the epithelium. Immunoreactive cells of MCAF/MCP-1 were also stained with the antibody, which recognizes monocytes and macrophages. CONCLUSIONS: These results suggest that MCAF/MCP-1, which is produced constantly by monocytes and macrophages and is stored in human nasal mucosa, possibly participates in the protracted histamine release from basophils and in the pathogenesis of perennial allergic rhinitis.

Intradermal injection of monocyte chemoattractant protein-1 induces emigration and differentiation of blood monocytes in rat skin.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant for blood monocytes in vitro. Recent studies in MCP-1-transgenic mice revealed that the local production of MCP-1 caused monocyte infiltration. However, the kinetics of monocyte infiltration after the production of MCP-1 or the amount of MCP-1 necessary for monocyte recruitment are not known. METHODS: We purified recombinant rat MCP-1 expressed in COS-7 cells, and injected it into rat skin. The infiltrating cells were examined by immunohistochemistry and ultrastructural peroxidase cytochemistry. RESULTS: Rat recombinant MCP-1 had a molecular mass of approximately 30 kD and exhibited the peak monocyte chemotactic activity at 10(-9) M. One microgram of MCP-1 caused intra- and extravascular accumulation of mononuclear cells 3 h after injection. The cells were ED1+, indicating they were blood monocytes. The infiltration of mononuclear cells peaked at 12-24 h, and most of them were TRPM-3+ and ED3+, characteristic to exudate macrophages. None of the cells expressed ED2 or Ki-M2R antigens, markers for resident macrophages, until 3 days after injection. There was no uptake of [3H]thymidine by the infiltrating cells. Ultrastructural peroxidase cytochemistry confirmed that the infiltrating cells were monocytes and exudate macrophages. The number of OX8+ lymphocytes also peaked at 12 h, consisting of approximately 9% of the total infiltrating cells. CONCLUSION: These results indicate that MCP-1 attracts blood monocytes as early as 3 h and the infiltrating monocytes differentiate into exudate macrophages in loco. However, this effect was transient and the infiltration of monocytes did not result in tissue damage.

[30 kD MCP-1 protein produced by mouse thymic epithelial cell line MTEC1].

A medullary type mouse thymic epithelial cell line MTEC1 can produce chemokines. Crude culture supernant of MTEC1 cells was concentrated by controlled-pore glass beads. One of these chemotactic proteins was isolated from MTEC1 supernatant and purified to homogeneity by heparin-Sepharose affinity chromatography, cation-exchange FPLC and Reverse Phase-HPLC. The chemotactic factor for both lymphocytes and monocytes was identified as a 30 kD protein by SDS-PAGE analysis under reducing conditions. After cleavage of the NH2-terminally blocked protein with formic acid, the amino acid sequence of the internal fragment was analyzed and found to be identical to the amino acid sequence of mouse MCP-1/JE. The protein was hence identified as a glycosylated MCP-1/JE-like chemokine secreted by thymic epithelial cells. The characterization of chemokines produced by thymic stromal cells will benefit the analysis of the mechanism of thymus homing and the finding of new chemokines.

Identification of monocyte chemoattractant protein-1 in senile plaques and reactive microglia of Alzheimer's disease.

It has been shown that human monocytes express monocyte chemoattractant protein-1 (MCP-1), an inflammatory factor, in response to non-fibrillar beta-amyloid protein. Reactive microglia and inflammatory factors were reported to be present in beta-amyloid deposits (senile plaques) in Alzheimer's disease, suggesting the presence of MCP-1 in senile plaques. To address this issue, we examined MCP-1 immunoreactivity in senile plaques using a mouse monoclonal anti-MCP-1 antibody. Monocyte chemoattractant protein-1 was found immunohistochemically in mature senile plaques and reactive microglia but not in immature senile plaques of brain tissues from five patients with Alzheimer's disease. These findings suggest that MCP-1-related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques.