Detection of IL-10 in Murine B Cells: In Vitro and In Vivo Techniques.

With the ever-increasing understanding of the roles of B cells in immune response and autoimmune pathogenesis, various techniques have been optimized for the detection of IL-10 production in B cells. In this chapter, we describe several commonly used methods for the effective detection of IL-10 in B cells at both mRNA and protein levels, including quantitative PCR analysis, intracellular staining of IL-10 in live B cells by flow cytometry, ELISA for secreted IL-10 detection, and ELISPOT assay for enumerating IL-10-producing B cells. We have further co-stained IL-10 with other cytokines and examined the staining efficiency. Moreover, we provide a detailed protocol for the detection of IL-10-producing B cells in situ by immunofluorescence microscopy. Since emerging evidence has suggested the promising strategy of cell therapy, we also provide a protocol to determine CD19+CD1dhiCD5+ B-cell distribution upon adoptive transfer using tile-scan imaging. Together, the application of the described methods for the detection of IL-10 will facilitate the characterization of B-cell subsets with regulatory functions and enhance our current understanding of the critical roles of B cells in immune response and autoimmune development.

Characterization and Activity of TIM-1 and IL-10-Reporter Expressing Regulatory B Cells.

In addition to their role in humoral immunity, B cells can exhibit regulatory activity. Such B cells have been termed regulatory B cells (Bregs). Bregs have been shown to inhibit inflammatory immune responses in a variety of autoimmune, alloimmune, and infectious settings. Breg activity is frequently IL-10-dependent, although a number of other mechanisms have been identified. However, our understanding of Bregs has been hampered by their rarity, lack of a specific phenotypic marker, and poor insight into their induction and maintenance. A variety of B-cell subsets enriched for IL-10+ Bregs have been identified in multiple murine disease models that can adoptively transfer Breg activity. However, most of these B-cell subsets actually contain only a minority of all IL-10+ B cells. In contrast, TIM-1 identifies over 70% of IL-10-producing B cells, irrespective of other markers. Thus, TIM-1 can be considered a broad marker for IL-10-expressing Bregs. Moreover, TIM-1 signaling plays a direct role in both the maintenance and induction of Bregs under physiological conditions, in response to both TIM-1 ligation and to apoptotic cells. TIM-1 expression has also been reported on IL-10+ human B cells. Together, these findings suggest that TIM-1 may represent a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs. Here, we provide the methods to analyze and purify the murine TIM-1+ B-cell subset for further in vitro and in vivo experiments. We also provide methods for in vitro analysis and in vivo tracking of Bregs using IL-10-reporter mice.

A fully integrated electrochemical biosensor platform fabrication process for cytokines detection.

Interleukin-1b (IL-1b) and interleukin-10 (IL-10) biomarkers are one of many antigens that are secreted in acute stages of inflammation after left ventricle assisted device (LVAD) implantation for patients suffering from heart failure (HF). In the present study, we have developed a fully integrated electrochemical biosensor platform for cytokine detection at minute concentrations. Using eight gold working microelectrodes (WEs) the design will increase the sensitivity of detection, decrease the time of measurements, and allow a simultaneous detection of varying cytokine biomarkers. The biosensor platform was fabricated onto silicon substrates using silicon technology. Monoclonal antibodies (mAb) of anti-human IL-1b and anti-human IL-10 were electroaddressed onto the gold WEs through functionalization with 4-carboxymethyl aryl diazonium (CMA). Cyclic voltammetry (CV) was applied during the WE functionalization process to characterize the gold WE surface properties. Finally, electrochemical impedance spectroscopy (EIS) characterized the modified gold WE. The biosensor platform was highly sensitive to the corresponding cytokines and no interference with other cytokines was observed. Both cytokines: IL-10 and IL-1b were detected within the range of 1pgmL-1 to 15pgmL-1. The present electrochemical biosensor platform is very promising for multi-detection of biomolecules which can dramatically decrease the time of analysis. This can provide data to clinicians and doctors concerning cytokines secretion at minute concentrations and the prediction of the first signs of inflammation after LVAD implantation.

Expression and purification of rhIL-10-RGD from Escherichia coli as a potential wound healing agent.

Various protocols for recombinant Interleukin-10 (IL-10) purification in wound healing have been reported previously. However, the therapeutic effect was not obvious. Thus, it is of great importance to find new and effective approaches for therapy. In this study, we propose that IL-10 and Arginine-Glycine-Aspartic (RGD) peptide would be a valuable therapeutic for wound healing. To explore a high-efficiency and cost-effective approach for the production of IL-10 and RGD peptide with bioactivity, a synthetic gene was cloned into a recombinant pTWIN1 vector. As a consequence, rhIL-10-RGD and the pH-induced self-cleavable Ssp DnaB mini-intein as a fusion protein was highly expressed by IPTG induction in Escherichia coli Rosetta without extra residues in a bioreactor. After Ni affinity chromatographic purification, rhIL-10-RGD was released by the Ssp DnaB intein-mediated self-cleavage that is triggered by pH shift. SDS-PAGE and silver staining showed a major band with an estimated molecular mass of 19.3kDa. Cell proliferation assay confirmed its potent proliferation activity on MC/9 murine mast cells. In conclusion, we report a novel strategy to produce rhIL-10-RGD mediated by the pH-induced self-cleavable Ssp DnaB mini-intein, and show that rhIL-10-RGD could play an effective role in wound healing of BALB/c mice.

Expression, purification, and characterization of scar tissue neovasculature endothelial cell-targeted rhIL10 in Escherichia coli.

Interleukin 10 (IL10) plays a pivotal role in the anti-inflammatory response and immunosuppressive reactions. It has also been identified as a new promising therapy for scar formation. Treatment of scars with IL10 has significant effects, but there are some shortcomings, including poor tissue-binding specificity and low effectiveness. RGD peptide has been demonstrated to bind specifically to αvß3 integrin on neovasculature endothelial cells, and the excess production of neovasculature is crucial to scar formation. To increase efficacy against scar formation and to decrease the side effects on normal tissues, a novel hybrid protein combining human IL10 with RGD was designed. The DNA sequence encoding the recombinant fusion protein IL10-RGD (rhIL10-RGD) was subcloned into a pET22b (+) vector for protein expression in E. coli strain BL21 (DE3). SDS-PAGE analysis displayed an induced expression product band at a molecular weight of 19.3 kDa, which constituted 30 % of the total bacterial protein. We developed a procedure to purify rhIL10-RGD from inclusion bodies and then renatured the protein using dialysis against urea with a step-down concentration procedure. Hypertrophic scar fibroblasts (HSFs) were treated with rhIL10-RGD, and the fibrosis-related protein levels were assessed by Western blotting. The results indicated that rhIL10-RGD can downregulate the expression levels of Col1 and α-SMA in HSFs and suppress tube formation of HUVECs. These results indicate that rhIL10-RGD has anti-fibrosis effects and can potentially be used to treat the neovasculature in scar formation and improve the abnormal deposition of the extracellular matrix (ECM). Thus, rhIL10-RGD may be a more effective candidate for scar-improvement and anti-fibrosis therapy.

Changes in IL-10 and specific antibodies associated to successful Dermatophagoides pteronyssinus immunotherapy in children during the first year of treatment

Background: Allergen-specific immunotherapy (SIT) is a long-term treatment of respiratory allergy. Objective: To look for early predictors of the effectiveness of Dermatophagoides pteronyssinus SIT. Methods: A prospective multi-centre study was carried out in Spain. Children with D. pteronyssinus rhinitis or asthma were invited to participate. The study was divided into times: T0 (recruitment); T1 (inclusion); T2 a-f (immunotherapy times) and T3 (the end of study). Efficacy of SIT was assessed by clinical scores, visual analogue scales (VAS) and lung function tests. We performed D. pteronyssinus skin tests at T1 and T3, and determined specific serum IgE, IgG4 and IL-10 at T1, T2f and T3.Data were analysed using Mann–Whitney and Kruskal–Wallis tests, compared using Wilcoxon and Chi-square tests, and correlated to Spearman test. All tests had a significance level of 0.05. Results: Thirty-eight children completed the study. At T1 all had rhinitis and 34 also had asthma. At T3, 30 patients had improved, six experienced no changes and two worsened. Improvement was associated to FEV1/FVC and VAS improvement; to a reduction in D. pteronyssinus skin prick test; to a progressive increase in serum levels of D. pteronyssinus IgE, and D. pteronyssinus, Der p1 and Der p2 IgG4. IL-10 levels showed an early increase at T2f (the end of initial build-up immunotherapy phase), and then a reduction at T3 (the end of a year of immunotherapy).Improvement associated to an early increase in IL-10 and was correlated with VAS and specific IgG4 evolution(AU)

High-level production of human interleukin-10 fusions in tobacco cell suspension cultures.

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER.

Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris.

Interleukin (IL)-10 is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin-induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30h in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.

Design and analysis of rhesus cytomegalovirus IL-10 mutants as a model for novel vaccines against human cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties. METHODS AND FINDINGS: Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10. CONCLUSION: This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV's ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection.

Biological activity of heterologous murine interleukin-10 and preliminary studies on the use of a dextrin nanogel as a delivery system.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer with two intramolecular disulphide bonds essential for its biological activity. A mutated form of murine IL-10 was successfully expressed in E. coli, recovered and purified from inclusion bodies. Its ability to reduce tumor necrosis factor α synthesis and down-regulate class II major histocompatibility complex molecules expression on endotoxin-stimulated bone marrow-derived macrophages was confirmed, and shown to be similar to that of a commercially available IL-10. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems that can effectively deliver the protein. In this work it is shown that a dextrin nanogel effectively incorporate IL-10, stabilize, and enable the slow release of biologically active IL-10 over time. Altogether, these results demonstrate the suitability of dextrin nanogel to be used as a system for the controlled release of IL-10.

Treatment of severe sepsis and septic shock by CHDF using a PMMA membrane hemofilter as a cytokine modulator.

It has been reported that various types of blood purification intended for the removal of humoral mediators, such as cytokines, were performed in patients with severe sepsis/septic shock. While high-volume hemofiltration, hemofiltration using high cut-off membrane filters, and direct hemoperfusion with a polymyxin-B immobilized column are widely used in the treatment of severe sepsis/septic shock, we perform continuous hemodiafiltration using a polymethylmethacrylate membrane hemofilter (PMMA-CHDF), which shows an excellent cytokine-adsorbing capacity, for the treatment of severe sepsis/septic shock. In our previous study, it was found that PMMA-CHDF could efficiently remove various pro-inflammatory cytokines such as TNFalpha, IL-6 and IL-8 from the bloodstream, resulting in early recovery from septic shock. Furthermore, PMMA-CHDF could remove anti-inflammatory cytokines such as IL-10 from bloodstream, suggesting that it might improve immunoparalysis as well. These findings suggest that PMMA-CHDF is useful for the treatment of patients with severe sepsis/septic shock as a cytokine modulator.

Extraction and purification of human interleukin-10 from transgenic rice seeds.

Recombinant protein production system using transgenic rice grain offers many advantages in higher accumulation, preservation, lower production cost, ease of scale up and low risk of contamination by toxic materials. We developed a transgenic rice strain whose seeds accumulate human interleukin (IL)-10, a cytokine that suppresses inflammation-related immune responses. We also developed a method of extracting and purifying IL-10 from rice seeds. A biochemical crosslinking method was used to detect the biologically active noncovalent dimer of IL-10. This method was useful for developing efficient methods of refolding and purification. The purified IL-10 comprised only noncovalent dimers and showed higher activity than the commercial IL-10. The purified IL-10 had very low endotoxin contamination and is expected to have broad clinical application.

Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco.

BACKGROUND: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10. RESULTS: Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells. CONCLUSION: Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.

A simple novel method for the preparation of noncovalent homodimeric, biologically active human interleukin 10 in Escherichia coli-enhancing protein expression by degenerate PCR of 5' DNA in the open reading frame.

DNA inserts encoding human interleukin 10 (hIL-10), optimized for codon usage and secondary RNA structure, were purchased from several commercial sources and subcloned into a pMon vector. Despite the optimization, protein expression was nil. We therefore subjected the 5' segment of the cDNA encoding N-terminal amino acids 2-11 to degenerate PCR in order to create a small library of 130K theoretical cDNA combinations that would not change the respective amino acid sequence and tested their expression. After screening over 320 colonies 10 hIL-10 clones encoding the original amino acid sequence were identified. Three nucleotide substitutions were sufficient to ensure reasonable protein expression. Subsequently, hIL-10 was expressed in Escherichia coli, refolded and purified to homogeneity, yielding over 95% electrophoretically pure noncovalent homodimeric protein, which was biologically active in MC/9 cells. The yield of recombinant hIL-10 from 10L of fermentation culture was 60mg and a protocol for its long-term storage as a carrier-free lyophilized powder at -20 degrees was developed.

Measurement of protein cytokines in tissue extracts by enzyme-linked immunosorbent assays: application to lipopolysaccharide-induced differential milieu of cytokines.

OBJECTIVES: Determination of protein cytokines in local tissues would help to evaluate their local role in health, sickness behavior and immune-mediated diseases. Therefore, developing a simple quantitative method of protein cytokines in tissues/organs is highly important. METHODS: Mouse tissues were collected following intraperitoneal administration of endotoxin-free PBS or lipopolysaccharide. A mild detergent, 0.1% Igepal, was added in a buffer to enhance cytokines extraction. The tissues were then disrupted, homogenized, centrifuged and the supernatants were collected and assayed using solid-phase immunoassays. RESULTS: The presence of 0.1% Igepal extracted significantly more TNF-alpha from liver (322%: p<0.01), brain (358%: p<0.05), lungs (1600%: p<0.01), and more IL-10 from liver (220%: p<0.001), brain (4650%: p<0.001) than PBS alone. On the other hand, using 0.1% Igepal did not increase IFN-gamma extraction from liver, spleen, brain, lungs, skin and kidneys more than PBS alone. Furthermore, i.p. administration of LPS induced a differential milieu of cytokines. LPS increased significantly the production of TNF-alpha, IFN-gamma, and IL-10 from liver (521%, 123%, 72%: p<0.01, 0.04, 0.04), brain (470%, 122%, 280%: p< 0.01, 0.03, 0.01), peritoneal lavage (p<0.001) and blood (p<0.001). However, the pattern of increase was different for the above cytokines in spleen, skin, lungs and kidneys. CONCLUSIONS: The extraction of protein cytokines from tissues was superior with addition of mild detergent. Furthermore, our results showed a differential cytokines response to LPS with respect to tissue and cytokine type. This method should provide an important tool for studying local protein cytokines in behavioral pattern, sickness behavior, and immune-mediated diseases as well as to determine local therapeutic efficacy of immunomodulatory drugs.

Cytokine removal with a novel adsorbent polymer.

BACKGROUND/AIMS: We sought to characterize a novel adsorbent polymer in terms of cytokine removal. METHODS: We challenged 50 rats with lipopolysaccharide to obtain cytokine-rich blood and circulated this through cartridges containing polymer. In separate experiments, cell-free supernatants were passed through cartridges containing polymer. We measured tumor necrosis factor alpha, interleukin 10 and interleukin 6 concentrations under a variety of conditions to evaluate adsorption kinetics. RESULTS: All three cytokines were rapidly removed from the blood with less than 50% of the initial concentrations present after 1 h of circulation through the cartridge. There was no significant difference in the effect across a range of blood flows and Ca2+ concentrations. Adsorption was decreased somewhat by extremely low temperature (4 degrees C). CONCLUSION: The adsorbent polymer removes cytokines with high efficiency, and binding is relatively unaffected by a variety of physical conditions.

Characterization of the recombinant extracellular domains of human interleukin-20 receptors and their complexes with interleukin-19 and interleukin-20.

The soluble extracellular domains of human interleukin-20 (IL-20) receptors I and II (sIL-20R1 and sIL20R2), along with their ligands IL-19 and IL-20, were expressed in Drosophila S2 cells and purified to homogeneity. Formation of the receptor/receptor and ligand/receptor complexes was studied by size exclusion chromatography. Both ligands and soluble receptors were found to be monomeric in solution; homo- or heterodimers are not formed even at elevated concentrations. Under native conditions, both IL-19 and IL-20 form stable ternary 1:1:1 complexes with the sIL-20R1 and sIL20R2 receptors, as well as high-affinity binary complexes with sIL-20R2. Unexpectedly, sIL-20R1 does not bind on its own to either IL-19 or IL-20. Thus, one of the possible consecutive mechanisms of formation of the signaling ternary complex may involve two steps: first, the ligand binds to receptor II, creating a high-affinity binding site for the receptor I, and only then does receptor I complete the complex.

Rat interleukin-10: production and characterisation of biologically active protein in a recombinant bacterial expression system.

Interleukin-10 is an anti-inflammatory Th1 immunosuppressive cytokine, the active form of which is a non-covalent homodimer, and which exhibits species-specificity both with respect to structure and biological activity. The rat homologue of IL-10 shares 73% identity with human IL-10 at the amino-acid sequence level, and has, in addition to the two disulphide bonds present in human IL-10, a fifth, unpaired cysteine (cys-149). Preparation of rat IL-10 by bacterial expression followed by solubilisation and refolding in a glutathione redox system, results in a molecule in which cys-149 is almost entirely oxidised, existing either as disulphide dimer or as a mixed disulphide with glutathione, and which has less than 1% of the activity of the native (cys-149-SH) form of the molecule. Site directed mutagenesis of rat IL-10 to replace cys-149 with tyrosine produces a molecule which readily adopts the active conformation upon solubilisation and refolding, and which is recoverable in good yield from bacterial expression systems. Comparison of the biological activities of rat IL-10tyr149 and commercial rat IL-10 preparations confirms that the activity of native-sequence rat IL-10 is either reduced or absent. It is proposed therefore that the biosynthetic analogue rat IL-10tyr149 is a more useful molecule to investigate the biological actions of IL-10 in the rat.

IL-10 is induced in the reperfused myocardium and may modulate the reaction to injury.

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-alpha release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96-120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.

RT-PCR amplification of various canine cytokines and so-called house-keeping genes in a species-specific macrophage cell line (DH82) and canine peripheral blood leukocytes.

Total ribonucleic acid (RNA) isolated from a continuous canine macrophage cell line (DH82) was used in reverse transcription polymerase chain reactions (RT-PCR) for the detection of transcripts of interleukin (IL)-8, -12, and tumour necrosis factor-alpha (TNF). Three different methods of RNA isolation (standard guanidinium-thiocyanate method with and without application of RNA matrix, and boiling) were used and compared in regard to RT-PCR results. The most suitable method was used to establish RT-PCR amplification of mRNA transcripts of IL-2, -10, and interferon-gamma (IFN) in RNA isolated from canine peripheral blood leukocytes. Integrity of RNA isolates was ensured by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-actin, IL-8, -12, and TNF were amplified from RNA isolated by various methods. Use of guanidinium-thiocyanate with and without RNA matrix gave the most consistent results. Boiling as a mean of RNA isolation was quick and easy, but the RT-PCR results were extremely variable and multiple smaller bands were observed in the agarose gel in some preparations. IL-2, -10 and IFN transcripts were amplified from RNA isolated with guanidinium-thiocyanate from leukocytes stimulated with concanavalin A. DNase-treatment of RNA isolates was necessary to assure the destruction of genomic DNA and to avoid amplification of genomic sequences. This was especially a problem when using primers for GAPDH, beta-actin, IL-12, and TNF. Lack of DNase-treatment may lead to false positive results. This may be especially a problem when amplification of so-called house-keeping genes is used as internal control for RNA integrity. These findings demonstrated that isolation of total RNA with guanidinium-thiocyanate followed by DNase-treatment gave reliable and consistent results for detection of cytokine transcripts by RT-PCR in a canine macrophage cell line and canine peripheral blood leukocytes.