C. Wayne Smith

The inflammatory response in myocardial infarction

One of the major therapeutic goals of modern cardiology is to design strategies aimed at minimizing myocardial necrosis and optimizing cardiac repair following myocardial infarction. However, a sound understanding of the biology is necessary before a specific intervention is pursued on a therapeutic basis. This review summarizes our current understanding of the cellular and molecular mechanisms regulating the inflammatory response following myocardial ischemia and reperfusion. Myocardial necrosis induces complement activation and free radical generation, triggering a cytokine cascade initiated by Tumor Necrosis Factor (TNF)-alpha release. If reperfusion of the infarcted area is initiated, it is attended by an intense inflammatory reaction. Interleukin (IL)-8 synthesis and C5a activation have a crucial role in recruiting neutrophils in the ischemic and reperfused myocardium. Neutrophil infiltration is regulated through a complex sequence of molecular steps involving the selectins and the integrins, which mediate leukocyte rolling and adhesion to the endothelium. Marginated neutrophils exert potent cytotoxic effects through the release of proteolytic enzymes and the adhesion with Intercellular Adhesion Molecule (ICAM)-1 expressing cardiomyocytes. Despite this potential injury, substantial evidence suggests that reperfusion enhances cardiac repair improving patient survival; this effect may be in part related to the inflammatory response. Monocyte Chemoattractant Protein (MCP)-1 is also markedly upregulated in the infarcted myocardium inducing recruitment of mononuclear cells in the injured areas. Monocyte-derived macrophages and mast cells may produce cytokines and growth factors necessary for fibroblast proliferation and neovascularization, leading to effective repair and scar formation. At this stage expression of inhibitory cytokines such as IL-10 may have a role in suppressing the acute inflammatory response and in regulating extracellular matrix metabolism. Fibroblasts in the healing scar undergo phenotypic changes expressing smooth muscle cell markers. Our previous review in this journal focused almost exclusively on reduction of the inflammatory injury. The current update is prompted by the potential therapeutic opportunity that the open vessel offers. By promoting more effective tissue repair, it may be possible to reduce the deleterious remodeling, that is the leading cause of heart failure and death. Elucidating the complex interactions and regulatory mechanisms responsible for cardiac repair may allow us to design effective inflammation-related interventions for the treatment of myocardial infarction.

T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity

Background— Obesity is associated with chronic inflammation, which includes increased macrophage accumulation in adipose tissue (AT) and upregulation of chemokines and cytokines. T cells also play important roles in chronic inflammatory diseases such as atherosclerosis but have not been well studied in obesity. Methods and Results— Flow cytometric analysis showed higher numbers of T cells and macrophages in AT of diet-induced obese insulin-resistant male mice than in lean mice and obese females (P<0.05). RNase protection assay, ELISA, and flow cytometry indicated gender-dependent upregulation of mRNA and protein levels of regulated on activation, normal T cell expressed and secreted (RANTES) and its receptor CCR5 in AT of obese mice. Adipocytes, stromal/vascular cells from mouse AT, and human and murine adipocytes expressed RANTES. RANTES mRNA levels were negatively correlated with adiponectin in mouse AT. Adiponectin-deficient mice fed high-fat diet showed higher RANTES mRNA levels in AT than wild-type mice. Activated T cells coincubated with preadipocytes in vitro significantly suppressed preadipocyte-to-adipocyte differentiation. Obese humans with metabolic syndrome had higher mRNA levels of RANTES and CCR5 in subcutaneous AT than lean humans. RANTES and CCR5 mRNA levels were significantly higher in visceral than subcutaneous AT of morbidly obese humans. RANTES mRNA levels were positively correlated with CD3 and CD11b in human visceral AT. Conclusions— Obesity is associated with increased accumulation of T cells and macrophages in AT, which may play important roles in obesity-related disease by influencing preadipocyte/adipocyte functions. RANTES is an adipokine that is upregulated in AT by obesity in both mice and humans.

Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-α, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion

BACKGROUND Neutrophil-induced cardiomyocyte injury requires the expression of myocyte intercellular adhesion molecule (ICAM)-1 and ICAM-1-CD11b/CD18 adhesion. We have previously demonstrated interleukin (IL)-6 activity in postischemic cardiac lymph; IL-6 is the primary stimulus for myocyte ICAM- 1 induction. Furthermore, we found that induction of IL-6 mRNA occurred very early on reperfusion of the infarcted myocardium. We hypothesized that the release of a preformed upstream cytokine induced IL-6 in leukocytes infiltrating on reperfusion. METHODS AND RESULTS Constitutive expression of TNF-alpha and not IL-1beta was demonstrated in the normal canine myocardium and was localized predominantly in cardiac mast cells. Mast cell degranulation in the ischemic myocardium was documented by demonstration of a rapid release of histamine and TNF-alpha in the cardiac lymph after myocardial ischemia. Histochemical studies with FITC-labeled avidin demonstrated degranulating mast cells only in ischemic samples of canine myocardium. Immunohistochemistry suggested that degranulating mast cells were the primary source of TNF-alpha in the ischemic myocardium. In situ hybridization studies of reperfused myocardium localized IL-6 mRNA in infiltrating mononuclear cells and in mononuclear cells appearing in the postischemic cardiac lymph within the first 15 minutes of reperfusion. Furthermore, isolated canine mononuclear cells incubated with postischemic cardiac lymph demonstrated significant induction of IL-6 mRNA, which was partially blocked with a neutralizing antibody to TNF-alpha. CONCLUSIONS Cardiac mast cells degranulate after myocardial ischemia, releasing preformed mediators, such as histamine and TNF-alpha. We suggest that mast cell-derived TNF-alpha may be a crucial factor in upregulating IL-6 in infiltrating leukocytes and initiating the cytokine cascade responsible for myocyte ICAM-1 induction and subsequent neutrophil-induced injury.

Cardiac Myocytes Produce Interleukin-6 in Culture and in Viable Border Zone of Reperfused Infarctions

BACKGROUND Previous work from our laboratory demonstrated that interleukin (IL)-6 plays a potentially critical role in postreperfusion myocardial injury and is the major cytokine responsible for induction of intracellular adhesion molecule (ICAM)-1 on cardiac myocytes during reperfusion. Myocyte ICAM-1 induction is necessary for neutrophil-associated myocyte injury. We have previously demonstrated the induction of IL-6 in the ischemic myocardium, and the current study addresses the cells of origin of IL-6. METHODS AND RESULTS In the present study, we combined Northern blot analysis and in situ hybridization to demonstrate IL-6 gene expression in cardiac myocytes. Isolated ventricular myocytes were stimulated with tumor necrosis factor-alpha, IL-1beta, lipopolysaccharide, preischemic lymph, and postischemic lymph. Unstimulated myocytes showed no significant IL-6 mRNA expression. Myocytes stimulated with preischemic lymph showed minimal or no IL-6 mRNA expression, whereas myocytes stimulated with tumor necrosis factor-alpha, IL-1beta, lipopolysaccharide, or postischemic lymph showed a strong IL-6 mRNA induction. Northern blot with ICAM-1 probe revealed ICAM-1 expression under every condition that demonstrated IL-6 induction. We then investigated the expression of IL-6 mRNA in our canine model of ischemia and reperfusion. Cardiac myocytes in the viable border zone of a myocardial infarction exhibited reperfusion-dependent expression of IL-6 mRNA within 1 hour after reperfusion. Mononuclear cells infiltrate the border zone and express IL-6 mRNA. CONCLUSIONS Isolated cardiac myocytes produce IL-6 mRNA in response to several cytokines as well as postischemic cardiac lymph. In addition to its production by inflammatory cells, we demonstrate that IL-6 mRNA is induced in myocytes in the viable border zone of a myocardial infarct. The potential roles of IL-6 in cardiac myocytes in an infarct border are discussed.

Leukocyte activation with platelet adhesion after coronary angioplasty: A mechanism for recurrent disease?

OBJECTIVES The purpose of this pilot study was to determine whether leukocyte activation occurs, whether leukocyte-platelet complexes develop and whether there is any association between these findings and clinical outcome after coronary angioplasty. BACKGROUND Increased expression of CD11b on monocytes and neutrophils promotes their adhesion to endothelial cells, extracellular matrix and smooth muscle cells. Thrombin-activated platelets adhere to monocytes and neutrophils through P-selectin. These cell complexes may affect the inflammatory process and, thus, the outcome of coronary angioplasty. METHODS During elective single-vessel coronary angioplasty in 11 men, samples were obtained for flow cytometric detection of CD11b, as well as the percent of leukocytes with adherent platelets and the intensity of bound platelet fluorescence (number of platelets/leukocyte). RESULTS After angioplasty, there was an increase in CD11b (monocytes: p = 0.001, neutrophils: p = 0.02) and leukocytes with adherent platelets (p = 0.02). During follow-up, five patients remained in stable condition and six had subsequent clinical events: restenosis and progression of disease requiring coronary artery bypass grafting (n = 3), myocardial infarction involving the dilated artery (n = 1) and unstable angina (n = 2). Values for leukocyte CD11b expression, the percent of leukocytes with adherent platelets and the intensity of bound platelet fluorescence were higher both before and after angioplasty in the six patients experiencing clinical events. CONCLUSIONS Despite standard aspirin and heparin therapy, leukocyte activation with platelet adherence occurs after coronary angioplasty. The magnitude of leukocyte activation and platelet adherence appears to be higher in patients experiencing late clinical events.

Intercellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver.

The potential role of intercellular adhesion molecule‐1 (ICAM‐1) in the pathogenesis of reperfusion injury was investigated in male Fischer rats subjected to 45 min of hepatic ischemia and 24 h of reperfusion. ICAM‐1 mRNA levels increased during ischemia in the ischemic liver lobes; however, during reperfusion mRNA levels increased in both the ischemic and nonischemic lobes. Immunohistochemical evaluation indicated ICAM‐1 expression only on sinusoidal lining cells in controls; ischemia‐reperfusion enhanced ICAM‐1 expression in the sinusoids and induced some expression on hepatocytes. The monoclonal anti–ICAM‐1 antibody 1A29, but not an immunoglobulin G control antibody, administered at 1 h and 8 h of reperfusion (2 mg/kg) significantly attenuated liver injury as indicated by 51% lower plasma alanine aminotransferase activities and 32–36% less hepatic necrosis at 24 h without affecting reactive oxygen formation by Kupffer cells and hepatic neutrophils. Although 1A29 reduced neutrophil extravasation in a glycogen peritonitis by 60%, the antibody had no significant effect on hepatic neutrophil infiltration during reperfusion. These data suggest that ICAM‐1 plays a significant role during the neutrophil‐dependent injury phase after hepatic ischemia and reperfusion and therefore blocking this adhesion molecule may have therapeutic potential against postischemic acute liver failure. J. Leukoc. Biol. 57: 368–374; 1995.

Neutrophil Tethering on E-Selectin Activates β2 Integrin Binding to ICAM-1 Through a Mitogen-Activated Protein Kinase Signal Transduction Pathway

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of β2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via β2 integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears ≥0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that ∼60% of the interacting cells remained firmly adherent, as compared with ∼10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through β2 integrins binding to ICAM-1.

Motility and Adhesiveness in Human Neutrophils: EFFECTS OF CHEMOTACTIC FACTORS

Human peripheral blood neutrophils (PMN) obtained from healthy adults were examined in vitro with techniques adapted to assess the effects of chemotactic factors (CF) on cellular configuration and adhesiveness. The results were compared with those that use certain conventional techniques for assessing chemotaxis and chemokinesis. Exposure of PMN to N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe), zymosan-activated serum, bacterial chemotactic factor, or a low molecular weight chemotactic factor from activated serum (C5a) in the absence of a gradient resulted in a change in cellular shape from a spherical to a polarized configuration in a high percentage of cells. This occurred rapidly in suspension, under conditions designed to exclude a role for cell adhesiveness, and was reversible upon removal of the CF. Restimulation of cells with the CF resulted in reappearance of the polarized configuration to the same extent as on initial stimulation with one exception: f-Met-Phe pretreated cells failed to respond to f-Met-Phe, though they responded fully to the other CF. Each CF caused a significant increase in PMN attachment to protein-coated glass. This enhanced adhesiveness was not reversible upon removal of the CF when the cells were treated under conditions shown to produce chemotactic deactivation. Cells treated under these conditions also exhibited significantly reduced motility on glass and in micropore filters in the absence of a gradient of CF. Bacterial chemotactic factor, even at high concentrations, failed to produce deactivation and did not cause a sustained enhancement of adhesiveness.

Soluble Cell Adhesion Molecules in Hypertriglyceridemia and Potential Significance on Monocyte Adhesion

Hypertriglyceridemia may contribute to the development of atherosclerosis by increasing expression of cell adhesion molecules (CAMs). Although the cellular expression of CAMs is difficult to assess clinically, soluble forms of CAMs (sCAMs) are present in the circulation and may serve as markers for CAMs. In this study, we examined the association between sCAMs and other risk factors occurring with hypertriglyceridemia, the effect of triglyceride reduction on sCAM levels, and the role of soluble vascular cell adhesion molecule-1 (sVCAM-1) in monocyte adhesion in vitro. Compared with normal control subjects (n=20), patients with hypertriglyceridemia and low HDL (n=39) had significantly increased levels of soluble intercellular adhesion molecule-1 (sICAM-1) (316+/-28.8 versus 225+/-16.6 ng/mL), sVCAM-1 (743+/-52.2 versus 522+/-43.6 ng/mL), and soluble E-selectin (83+/-5.9 versus 49+/-3.6 ng/mL). ANCOVA showed that the higher sCAM levels in patients occurred independently of diabetes mellitus and other risk factors. In 27 patients who received purified n-3 fatty acid (Omacor) 4 g/d for > or =7 months, triglyceride level was reduced by 47+/-4.6%, sICAM-1 level was reduced by 9+/-3.4% (P=.02), and soluble E-selectin level was reduced by 16+/-3.2% (P<.0001), with the greatest reduction in diabetic patients. These results support previous in vitro data showing that disorders in triglyceride and HDL metabolism influence CAM expression and treatment with fish oils may alter vascular cell activation. In a parallel-plate flow chamber, recombinant sVCAM-1 at the concentration seen in patients significantly inhibited adhesion of monocytes to interleukin-1-stimulated cultured endothelial cells under conditions of flow by 27.5+/-7.2%. Thus, elevated sCAMs may negatively regulate monocyte adhesion.

Complement C5a, TGF-β1, and MCP-1, in Sequence, Induce Migration of Monocytes Into Ischemic Canine Myocardium Within the First One to Five Hours After Reperfusion

BACKGROUND Recent studies suggest that reperfusion promotes healing of formerly ischemic heart tissue even when myocardial salvage is no longer possible. Since monocyte-macrophage infiltration is the hallmark of the healing infarct, we have attempted to identify mechanisms that attract monocytes into the heart after reperfusion of ischemic canine myocardium. METHODS AND RESULTS Isolated autologous 99mTc-labeled mononuclear leukocytes injected into the left atrium localized preferentially in previously ischemic myocardium within the first hour after reperfusion. Histological studies revealed CD64+ monocytes in small venules and the perivascular connective tissue within the first hour after reperfusion. Flow cytometric analysis of cells in cardiac lymph showed systematically increasing numbers of neutrophils and monocytes between 1 and 4 hours after reperfusion; monocyte enrichment was eventually greater than neutrophil enrichment. Monocyte chemotactic activity in cardiac lymph collected in the first hour after reperfusion was wholly attributable to C5a. Transforming growth factor (TGF)-beta 1 contributed significantly to this chemotactic activity after 60 to 180 minutes, and after 180 minutes, monocyte chemotactic activity in lymph was largely dependent on monocyte chemoattractant protein (MCP)-1 acting in concert with TGF-beta 1. CONCLUSIONS Beginning in the first 60 minutes after reperfusion, C5a, TGF-beta 1, and MCP-1, acting sequentially, promote infiltration of monocytes into formerly ischemic myocardium. These events may promote the healing of myocardial injury facilitated by reperfusion.