Biomedical subjects

A M Manning

Publications and source records attributed to A M Manning.

At least 37 records · Page 2Linked to original sources

PubMed1999

IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

Amino Acid Sequence↗

PubMed1999

Jun N-terminal kinase in rheumatoid arthritis.

Potential mechanisms of joint destruction in rheumatoid arthritis (RA) were examined by studying the regulation of mitogen-activated protein kinases and collagenase gene expression in fibroblast-like synoviocytes (FLS). The three main mitogen-activated protein kinase families [p38, Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases (ERKs)] were constitutively expressed in RA and osteoarthritis (OA) FLS. p38 and ERK1/2 were readily phosphorylated in both RA and OA FLS after interleukin-1 (IL-1) stimulation. JNK was phosphorylated in RA FLS but not OA FLS after IL-1 stimulation. Reverse transcription-polymerase chain reaction studies suggested that JNK2 is the major isoform of the JNK family expressed by FLS. Northern blot analysis of collagenase gene expression demonstrated that RA FLS contained significantly more collagenase mRNA than OA FLS after IL-1 stimulation. The roles of JNK and p38 kinase were evaluated with the p38/JNK inhibitor SB 203580. Low concentrations of SB 203580 (1 microM, a concentration that only inhibits p38) had no significant effect on IL-1-induced collagenase expression in RA FLS whereas 25 microM (which inhibits p38, JNK2, and c-raf) blocked collagenase mRNA accumulation. IL-1-stimulated AP-1 binding was also inhibited by 25 microM SB 203580 in RA FLS. These studies suggest that OA and RA FLS have a different pattern of JNK phosphorylation, which might lead to enhanced collagenase gene expression in RA.

Arthritis, Rheumatoid↗

PubMed1998

Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

Amino Acid Sequence↗

PubMed1998

Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells.

Mononuclear phagocytes play a major role in immune and inflammatory responses. Bacterial lipopolysaccharide (LPS) induces monocytes to express a variety of genes by activating the NF-kappaB/Rel transcription factor family. Recently, we have reported that the tumor necrosis factor and interleukin 1 signaling pathways activate two kinases, IKK1 and IKK2. Phosphorylation of the IkappaB cytoplasmic inhibitors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, by these kinases triggers proteolytic degradation and the release of NF-kappaB/Rel proteins into the nucleus. At present, the role of the IKKs in LPS signaling has not been investigated. Here, we report that LPS induces IKK activity in human monocytes and THP-1 monocytic cells. The kinetics of activation of kinase activity in monocytic cells are relatively slow with maximal activity observed at 60 min, which coincides with the degradation of IkappaBs and the nuclear translocation of NF-kappaB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced kappaB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of kappaB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of kappaB-dependent gene expression in human monocytic cells requires activation of IKK2.

Cell Line↗

PubMed1998

Selective inhibition of TNF-alpha induced cell adhesion molecule gene expression by tanapox virus.

Poxviruses encode virulence factors that have been identified as proteins that are secreted from infected host cells. Some of these secretory proteins impede host immune defences. We have previously demonstrated that tanapox virus (TPV) infected cells secrete an early 38 kDa glycopeptide that binds to human (h) interferon-gamma, hIL-2, and hIL-5. We now show an additional activity in the supernatant from TPV infected cells that down-regulates the expression of tumour necrosis factor-alpha (TNF-alpha) induced cell adhesion molecule gene expression. This activity was not detected in mock infected cells. Enzyme linked immunosorbent assays (ELISA) on primary human endothelial cells, show the induction of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) following TNF-alpha or IL-1 beta treatment, as expected. Supernatant from TPV infected cells significantly decreased the TNF-alpha but not IL-1 beta-induced expression of these molecules. Mobility shift assays and Northern blot analyses further show that the supernatant from TPV infected cells inhibited TNF-alpha-induced activation of the nuclear transcription factor-kappa B (NF-kappa B) and transcriptional activation of the E-selectin, VCAM-1 and ICAM-1 genes. Based on TNF-alpha affinity chromatography, this activity appears to be associated with a 38 kDa glycopeptide.

Animals↗

PubMed1998

AP-1 and NF-kappaB regulation in rheumatoid arthritis and murine collagen-induced arthritis.

OBJECTIVE: To determine the expression and regulation of nuclear transcription factors AP-1 and NF-kappaB in rheumatoid arthritis and in collagen-induced arthritis in mice. METHODS: AP-1 and NF-kappaB expression and function were determined in RA, OA and normal synovial tissue by electrophoretic mobility shift assay (EMSA) and immunohistochemistry. The kinetics of transcription factor expression were then examined in collagen-induced arthritis (CIA) in mice. EMSAs were performed with the nuclear extracts obtained from paws of CIA mice from 10 to 45d after immunization to determine AP-1 and NF-kappaB binding activity. The expression of collagenase-3 (MMP13) and stromelysin (MMP3) mRNA was examined by northern blot analysis. RESULTS: Immunohistochemistry showed that NF-kappaB expression was increased in both RA and OA synovial intimal lining. AP-1 components Jun and Fos were also present in the intimal lining and was significantly greater in RA than OA. The DNA binding activities of both AP-1 and NF-kappaB were significantly higher RA patients compared with OA. In CIA, AP-1 and NF-kappaB expression increased by day 20, which was 1-2 weeks before onset of clinical arthritis. However, collagenase and stromelysin gene expression did not increase until day 35. CONCLUSION: The DNA binding activity of AP-1 and NF-kappaB are markedly increased in both CIA and RA. In CIA, activation of AP-1 and NF-kappaB precede both clinical arthritis and metalloproteinase gene expression. NF-kappaB expression correlated better than AP-1 with metalloproteinase expression.

Animals↗

PubMed1997

Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.

Activation of the transcription factor NF-kappa B is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor I kappa B in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-kappa B, I kappa B is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of I kappa B and its subsequent degradation. The ligase recognition signal is functionally conserved between I kappa B alpha and I kappa B beta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-kappa B activation in response to TNF alpha and the consequent expression of E-selectin, an NF-kappa B-dependent cell-adhesion molecule. Inhibition of NF-kappa B function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

Amino Acid Sequence↗

PubMed1997

Interleukin-4 suppression of tumor necrosis factor alpha-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-kappaB.

Interleukin-4 (IL-4), an immunoregulatory cytokine secreted from activated T-helper 2 lymphocytes, eosinophils, and mast cells, stimulates the expression of a number of immune system genes via activation of the transcription factor, STAT6. However, IL-4 can concomitantly suppress the expression of other immune-related gene products, including kappa light chain, FcgammaRI, IL-8, and E-selectin. We demonstrate that IL-4 activates STAT6 in human vascular endothelial cells and that two STAT6 binding sites are present in the promoter of the E-selectin gene. IL-4-induced STAT6 binding does not activate E-selectin transcription but instead suppresses tumor necrosis factor alpha-induced expression of the E-selectin gene. STAT6 was found to compete for binding to a region in the E-selectin gene promoter containing overlapping STAT6 and NF-kappaB binding sites, effectively acting as an antagonist of NF-kappaB binding and transcriptional activation. This novel mechanism for IL-4-mediated inhibition of inflammatory gene expression provides an example of a STAT factor acting as a transcriptional repressor rather than an activator.

DNA↗

PubMed1997

Management of the uncomplicated canine diabetic.

Managing the uncomplicated canine diabetic may still be a challenging endeavor. Client education is the most important treatment to provide. The client must understand the technique of drug handling and administration, the importance of diet in treating the animal, how to handle an emergency situation, and, most importantly, the disease itself. The clinician must realize that although "tight" glycemic control is the optimal goal; the reality is often a compromise of mild hyperglycemia with resolution of clinical signs. When regulation cannot be maintained it is necessary to investigate the causes of insulin resistance.

Animals↗

PubMed1997

Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse.

P-selectin is an important adhesion molecule involved in leukocyte migration. However, to date, no monoclonal antibodies (MAb) generated against rat P-selectin have been identified which block P-selectin mediated leukocyte adhesion. Most studies in the rat have utilized crossreacting antibodies generated against P-selectin in higher species. In a P-selectin deficient mouse we generated an anti-rat/mouse P-selectin MAb, designated RMP-1, by immunization with activated rat platelets. This IgG2a MAb immunoprecipitates a 140 kDa protein under reducing conditions from rat platelet lysate. By ELISA and immunofluorescence flow cytometry, MAb RMP-1 reacts with thrombin-activated but not unactivated rat platelets. In addition, by ELISA MAb RMP-1 binds to activated mouse platelets and recombinant rat and mouse P-selectin. MAb RMP-1 inhibited adhesion of HL-60 myeloid cells to immobilized mouse P-selectin by 97% and to activated rat and mouse platelets by 100% under static conditions, confirming the adhesion function blocking activity of MAb RMP-1. This novel MAb should be useful for studying P-selectin function in vitro and in vivo in both rat and mouse inflammation models.

Animals↗

PubMed1997

Generation and characterization of a novel adhesion function blocking monoclonal antibody recognizing both rat and mouse E-selectin.

The prerequisite for the recruitment of circulating leukocytes to sites of inflammation is adhesion to vascular endothelial cells. Selectins play a significant role in the initiation of this multistep process by mediating "rolling" of the leukocytes on the endothelium. Investigation of selectin-dependent cell interactions using function blocking monoclonal antibodies (MAb) provides insights into the mechanisms involved in leukocyte migration into inflammation. Until now most studies in inflammation models in rats have relied on cross-reactive or polyclonal antibodies against rat E-selectin. In an E-selectin knockout mouse, we aimed to generate an adhesion function blocking MAb to rat E-selectin by immunization with rat E-selectin transfected Chinese hamster ovary cells (RESEC). An IgG1 kappa MAb was identified that reacts with RESEC but not with untransfected Chinese hamster ovary cells, as well as with recombinant mouse E-selectin protein as assessed by ELISA. This MAb is designated RME-1. It does not cross-react with rat L-selectin or rat P-selectin or E-selectin expressed on human umbilical vein endothelium. Adhesion of the HL-60 myeloid cells to immobilized mouse E-selectin was completely inhibited by MAb RME-1 under static conditions and adhesion of rat polymorphonuclear leukocytes to recombinant mouse E-selectin was blocked under rotation condition. This novel antibody thus recognizes a function-related epitope on rodent E-selectin.

Animals↗

PubMed1997

Transcription inhibitors in inflammation.

Advances in molecular medicine have revealed a key role for altered gene expression in the aetiology of many inflammatory diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease and sepsis. Until recently, however, modulation of gene transcription has not been the subject of directed pharmaceutical research efforts. Notwithstanding, it is clear that the efficacy of several well-established anti-inflammatory therapeutics is mediated through their ability to modulate gene transcription. Understanding the mechanisms of action of these therapeutics and defining new gene regulatory pathways has stimulated a new wave of anti-inflammatory drug discovery. This update aims to cover our current understanding of transcription inhibitors in inflammation, including the mechanism of action of established therapeutics and the properties of new chemical entities recently described in the literature.

Journal Article↗

PubMed1996

Identification of signal-induced IkappaB-alpha kinases in human endothelial cells.

Activation of the nuclear transcription factor-kappaB is an early event in endothelial activation. NF-kappaB activation is regulated by the inducible phosphorylation and subsequent degradation of the inhibitory subunit IkappaB-alpha. We identified two discrete kinases of approximately 36 and 41 kDa in the cytoplasm of human umbilical vein endothelial cells that specifically bind to and phosphorylate the IkappaB-alpha subunit. IkappaB-alpha kinase activity is transiently elevated following treatment with either tumor necrosis factor alpha, interleukin-1beta, or bacterial lipopolysaccharides and precedes activation of either mitogen-activated kinase or Jun kinase. Furthermore, activation of the IkappaB-alpha kinases precedes both the appearance of hyperphosphorylated IkappaB-alpha and its subsequent degradation, as well as the translocation of NF-kappaB to the nucleus. Deletion mutagenesis of the IkappaB-alpha polypeptide revealed that these kinases bind in or around the ankyrin repeat domains and phosphorylate residues within the C terminus. These kinases, however, were not identical to casein kinase II and displayed a pharmacologic profile distinct from other known kinases. These kinases may represent components of a signal transduction pathway regulating IkappaB-alpha levels in vascular endothelium.

Ankyrins↗

PubMed1996

Endotoxin-induced activation of the nuclear transcription factor kappa B and expression of E-selectin messenger RNA in hepatocytes, Kupffer cells, and endothelial cells in vivo.

Endotoxin causes neutrophil sequestration in the liver and severe vascular and parenchymal cell injury. Inducible adhesion molecules such as intercellular adhesion molecule-1 and selectins are involved in neutrophil recruitment to an inflammatory site in vivo. The objective of our study was to characterize the translocation of the nuclear factor kappa B (NF-kappa B) from the cytoplasm to the nucleus (NF-kappa B activation) during endotoxemia using the electrophoretic mobility shift assay and to investigate its role in regulation of E-selectin gene transcription in liver cells in vivo. Administration of 0.5 mg/kg Salmonella enteritidis endotoxin to male Fischer rats induced a drastic but transient activation of NF-kappa B at the whole liver level. Major subunits identified were p50 (NF-kappa B1), p65 (RelA), and c-Rel, but not p52 (NF-kappa B2). Isolation of liver cells from control animals induced substantial NF-kappa B activation in Kupffer cells and endothelial cells and minor activation in hepatocytes. One hour after endotoxin treatment in vivo, NF-kappa B was uniformly activated in all isolated cells. At 5 h, NF-kappa B activation was reduced to various degrees in all cell types, with hepatocytes showing only a moderate decrease. Northern blot analysis indicated no E-selectin mRNA in all control cells; however, at 1 h, endotoxin induced E-selectin gene transcription transiently in endothelial cells and in Kupffer cells but not in hepatocytes. These data support the hypothesis that NF-kappa B activation is important for E-selectin gene transcription in hepatic vascular lining cells. However, the fact that hepatic parenchymal cells, despite NF-kappa B activation do not express E- selectin mRNA, suggests that NF-kappa B activation alone is not sufficient for E-selectin gene transcription in vivo.

Animals↗

PubMed1996

ICAM-1 mediates leukocyte-endothelium adhesive interactions in the reversed passive Arthus reaction.

A murine anti-rat intercellular adhesion molecule 1 (ICAM-1) monoclonal antibody (mAb), 1A29, was used to investigate the importance of blood leukocyte-associated beta 2-integrin (CD11/CD18) vascular endothelium-associated ICAM-1 adhesive interactions in the reversed passive Arthus reaction (RPAR) in rats. An Arthus pleurisy reaction (4 h) was employed in these studies because it permits the accurate quantitation of polymorphonuclear neutrophil (PMN) influx into the pleural space and fluid accumulation. 1A29, which localized within Arthus lung lesions, caused a dose-dependent (0.5-2.0 mg/kg, i.v.) inhibition of PMN influx (19-56%) and exudate volume (9-55%) in the Arthus pleurisy reaction. P7 (2 mg/kg, i.v.), a murine anti-human P-selectin mAb used as an isotype-matched control for 1A29, did not localize at the lung lesion site and was inactive. Immunohistochemical analysis of lung tissue from 1A29-treated rats demonstrated increased granulocyte accumulation in the alveolar capillaries compared with more extensive granulocyte emigration into the lung tissue and pleural space in P7-treated rats and Arthus control rats; however, quantitative image analysis revealed increased numbers of lung granulocytes in 1A29-treated rats compared with controls. Neither ICAM-1 mRNA nor expression, assessed by immunocytochemistry, was increased above control levels in rats during the pleural Arthus reaction. Neutropenia was not observed in either 1A29- or P7-treated rats.

Animals↗

PubMed1996

TGF-beta 1, IL-10 and IL-4 differentially modulate the cytokine-induced expression of IL-6 and IL-8 in human endothelial cells.

Multiple cytokines and growth factors are present at sites of inflammation, and each of these can potentially influence the nature of the inflammatory response. Vascular endothelial cells (ECs) must integrate the signals generated by these multiple factors to effectively regulate the immune response and homeostasis. IL-6 and IL-8 and endothelial-derived products which play an important role as regulators of these processes. As a model for how ECs respond to signals from multiple cytokines, we have examined the effects of pretreatment with TGF-beta 1, IL-10 or IL-4 on TNF-alpha, IL-1 beta- or LPS-induced expression of IL-6 and IL-8 in human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with TGB-beta 1 or IL-10 significantly inhibited, but did not completely abolish, the TNF-alpha-, IL-1 beta- or LPS-induced expression of IL-6 and IL-8 protein. Maximal inhibition was achieved with physiologically relevant doses (1-2 ng/ml) of either TGF-beta 1 or IL-10. The inhibitory effects of TGF-beta and IL-10 were additive in nature. In contrast, pretreatment with IL-4 amplified the production of IL-6 in TNF-alpha-, IL-1 beta- or LPS-activated ECs, but inhibited IL-8 expression. Addition of TGF-beta 1 completely reversed the effects of IL-4 on IL-6 expression, whilst augmenting inhibition of IL-8. These studies demonstrate that multiple cytokines can act in concert to differentially regulate the endothelial expression of cytokines important to the inflammatory response. Modulation of endothelial cytokine production may contribute to the progression or resolution of the inflammatory response.

Cell Division↗

PubMed1996

U-73122: a potent inhibitor of human polymorphonuclear neutrophil adhesion on biological surfaces and adhesion-related effector functions.

We have reported that U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole- 2,5-dione) an inhibitor of phospholipase C-dependent processes in human polymorphonuclear neutrophils (PMN) and platelets, potently suppresses the responsiveness of suspended PMN and platelets to receptor agonists. We demonstrate here that U-73122 caused a concentration-dependent (10-800 nM) inhibition of N-formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha (TNF alpha), interleukin-8 and phorbol myristate acetate (PMA)-triggered PMN adhesion on fibronectin, fetal bovine serum or keyhole limpet hemocyanincoated microtiter plates. U-73122 also inhibited PMN adherence to and transmigration through TNF-alpha-activated endothelium (IC50 < 50 nM). Further, U-73122 suppressed interleukin-8, N-formylmethionyl-leucyl-phenylalanine and PMA-stimulated up-regulation of the beta 2-integrin, Mac-1 (CD11b/CD18), on the PMN surface (IC50 < 1.3 microM). U-73122 also caused a time-(15-120 min) and concentration-dependent inhibition (IC50 = 25-100 nM) of the N-formyl-methionyl-leucyl-phenylalanine-, TNF alpha- and PMA-elicited adhesion-dependent, oxidative burst, measured as hydrogen peroxide (H2O2) production, in PMN. The CD18-dependent extracellular release of lactoferrin from PMN activated with these stimuli was also suppressed by U-73122. U-73343 (1-[6-[[17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine dione), a close analog of U-73122, did not affect PMN responsiveness.

Animals↗

PubMed1995

Induction of interleukin-6 synthesis in the myocardium. Potential role in postreperfusion inflammatory injury.

BACKGROUND: Neutrophil-induced injury of myocardial cells requires the expression of intercellular adhesion molecule-1 (ICAM-1) on the myocyte surface and is mediated by ICAM-1-CD11b/CD18 adhesion. We have previously shown that interleukin-6 (IL-6) cytokine activity, present in cardiac lymph, induces ICAM-1 on isolated cardiac myocytes. Furthermore, in previous in vivo studies, we have also shown ICAM-1 mRNA induction in the myocardium within the first hour of reperfusion in the previously ischemic viable zone. We hypothesized that induction of IL-6 synthesis in the myocardium was an integral part of the reaction to injury resulting from ischemia and reperfusion and was associated with induction of ICAM-1 on myocardial cells. METHODS AND RESULTS: In this study, cloned canine IL-6 cDNA was used as a molecular probe to study the regulation of IL-6 in an awake canine model of myocardial ischemia and reperfusion. IL-6 mRNA was induced in ischemic and reperfused segments of myocardium preferentially in segments previously exposed to severe ischemia. Peak levels of IL-6 mRNA were reached within 3 hours of reperfusion. At the same time, IL-6 mRNA and ICAM-1 mRNA were found in the same myocardial segments. In contrast to hearts that were ischemic for 1 hour and reperfused for 3 hours, nonreperfused hearts after 4 hours of persistent ischemia demonstrated minimal induction of ICAM-1 or IL-6 despite similar degrees of injury and blood flow reductions during ischemia. After 24 hours of persistent ischemia, levels of IL-6 mRNA were comparable to those observed in hearts that were ischemic for 1 hour and subsequently reperfused for 24 hours. CONCLUSIONS: Our results demonstrate induction of IL-6 mRNA in the myocardium and that this synthesis is accelerated by reperfusion. Evidence is also provided to show that peak IL-6 mRNA precedes that of ICAM-1 mRNA. These findings are compatible with our hypothesis that IL-6 is important in the induction of ICAM-1 in the area of ischemia. In addition, these studies suggest that the necessary factors to promote adhesive interactions between transmigrated neutrophils and cardiac myocytes are present in reperfused myocardium.

Amino Acid Sequence↗