Neuropeptide regulation of proinflammatory cytokine responses

Severe postoperative hypoxaemia during sleep may increase the risk of postoperative cardiovascular complications. We hypothesized that the severity of hypoxic episodes after surgery are related to the presence of preoperative sleep-disordered breathing (SDB). We tested this hypothesis in a multicentre study designed to elucidate the major risk factors for development of postoperative nocturnal desaturations. We performed overnight oximetry before operation and for one night between the second and fourth day after operation in 80 patients undergoing major surgery. We calculated oximetry variables such as oxygen desaturation index (ODI), defined as the number of oxygen desaturations exceeding 4% below baseline, percentage time spent at SpO2 < 90% (CT90, %) and lowest SpO2 value. After operation, although the change in ODI was not significant (P = 0.34), deterioration in CT90 and lowest SpO2 values were significant (P = 0.036 and P = 0.007, respectively). Multivariate analysis of possible risk factors for postoperative desaturations revealed that preoperative hypoxaemia and apnoea witnessed by others were highly correlated with postoperative hypoxaemia.     

Persistence of myocardial injury following brief periods of coronary occlusion

Transient (20 min) occlusion of the left anterior descending coronary artery in open-chest anaesthetized dogs caused immediate aneurysmal bulging of the ischaemic segment, which preceded epicardial ST-segment elevation. Reperfusion after 20 min restored epicardial electrograms to normal, wheras mechanical dysfunction persisted for a least 45 min therafter. The study shows that there is temporal disparity between electrical and mechanical events both at the inception of myocardial ischaemia and during recovery from transient myocardial ischaemia.     

Induction of ischemic tolerance following brief focal ischemia in rat brain

The objective of this study was to determine whether brief focal ischemia induces ischemic tolerance in rat brain. Focal ischemia was produced in Wistar rats by occluding the middle cerebral artery (MCA) for 20 min at a distal site. Following recovery for 24 h, the animals were subjected to a 10-min episode of forebrain ischemia using a combination of bilateral carotid artery occlusion and systemic hypotension. Histologic injury, assessed after a survival period of 3-4 days, consisted of selective neuronal necrosis bilaterally in cerebral cortex, striatum, hippocampus, and thalamus superimposed upon a small cortical infarct adjacent to the site of MCA occlusion. However, the intensity of neuronal necrosis in the MCA territory of the neocortex ipsilateral to MCA occlusion was markedly less than that in the contralateral MCA cortex. In contrast, the extent of neuronal necrosis in subcortical structures was similar in both hemispheres. Unexpectedly, animals in which the MCA was manipulated, but not occluded, also exhibited a marked reduction of neuronal necrosis in the ipsilateral MCA neocortex following forebrain ischemia. However, in animals with craniotomy alone, forebrain ischemia caused a similar extent of neuronal necrosis in the MCA neocortex of both hemispheres. Transient occlusion of the MCA induced the focal expression of the 72-kDa heat-shock protein (hsp72) in the MCA territory of the neocortex. Limited expression of hsp72 was also detected following sham occlusion, but not after craniotomy alone. These results demonstrate focal induction of ischemic tolerance in rat neocortex that may be related to expression of heat-shock proteins.     

Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets

BACKGROUND: Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS: We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS: In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI.     

Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis

Hemangiomas are benign vascular tumors of childhood that can lead to disfigurement and/or life-threatening consequences. The pathogenesis of hemangioma formation is likely to involve increased angiogenesis. Basic fibroblast growth factor and vascular endothelial growth factor are cytokines that stimulate angiogenesis in multiple in vivo and in vitro models. Proliferative hemangiomas have been found to have elevated levels of basic fibroblast growth factor and vascular endothelial growth factor protein, but the gene expression of these cytokines in human specimens has not been previously studied. We examined the gene expression and spatial distribution of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA in proliferative versus involuted human hemangioma specimens using nonisotopic in situ hybridization techniques. Thirteen hemangioma specimens were harvested during initial surgical excision. In situ hybridization was performed on frozen sections of both proliferative and involuted hemangioma specimens using genetically engineered antisense probes specific for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA. Controls were an interleukin-6 sense sequence and a transforming growth factor-beta 1 antisense sequence. A large number of cells within the specimens of proliferative hemangiomas revealed localized gene expression of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (626 +/- 129 and 1660 +/- 371 cells/mm2, respectively). The majority of the cells were endothelial in origin. In contrast, involuted hemangioma specimens revealed significantly lower numbers of cells staining positive for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (44 +/- 11 and 431 +/- 76 cells/mm2, respectively; p < 0.05). Transforming growth factor-beta 1 messenger RNA was slightly more expressed by involuted hemangiomas (117 +/- 30 cells/mm2). There were very low levels of transforming growth factor-beta 1 gene expression from proliferative hemangiomas (37 +/- 24 cells/mm2; p < 0.02). These data demonstrate that (1) in situ hybridization allows identification and relative quantitation of cells expressing messenger RNA for specific growth factors in human hemangioma specimens; (2) basic fibroblast growth factor and vascular endothelial growth factor messenger RNA are up-regulated in proliferative hemangiomas; and (3) transforming growth factor-beta 1 messenger RNA remains low in both proliferative and involuted hemangiomas. Because basic fibroblast growth factor and vascular endothelial growth factor messenger RNA have been implicated in the pathobiology of human hemangioma formation, biochemical modulation of these angiogenic cytokines may eventually help inhibit proliferation and promote regression of hemangiomas.     

Delayed cytokine expression in rat brain following experimental contusion

Proinflammatory cytokines mediate brain injury in experimental studies. This study was undertaken to analyze the production of proinflammatory cytokines in experimental contusion. A brain contusion causing delayed edema was mimicked experimentally in rats using a weight-drop model. Intracerebral expression of the cytokines interleukin (IL)-1 beta, tumor necrosis factor-alpha (TNF alpha), IL-6, and interferon-gamma (IFN gamma) was studied by in situ hybridization and immunohistochemistry. The animals were killed at 6 hours or 1, 2, 4, 6, 8, or 16 days postinjury. In the injured area, no messenger (m)RNA expression was seen during the first 2 days after the trauma. On Days 4 to 6 posttrauma, however, strong IL-1 beta, TNF alpha, and IL-6 mRNA expression was detected in mononuclear cells surrounding the contusion. Expression of IFN gamma was not detected. Immunohistochemical double labeling confirmed the in situ hybridization results and demonstrated that mononuclear phagocytes and astrocytes produced IL-1 beta and that mainly astrocytes produced TNF alpha. The findings showed, somewhat unexpectedly, a late peak of intracerebral cytokine production in the injured area and in the contralateral corpus callosum, allowing for both local and global effects on the brain. An unexpected difference in the cellular sources of TNF alpha and IL-1 beta was detected. The cytokine pattern differs from that seen in other central nervous system inflammatory diseases and trauma models, suggesting that the intracerebral immune response is not a uniform event. The dominance of late cytokine production indicates that many cytokine effects are late events in an experimental contusion: Different pathogenic mechanisms may thus be operative at different times after brain injury.     

Inhibition of nuclear factor kappa B attenuates proinflammatory cytokine and inducible nitric-oxide synthase expression in postischemic myocardium

Nitric oxide (NO), a potent biological mediator, plays a key role in physiological as well as pathological processes, including inflammation and cancer. The role of NO in tumor biology remains incompletely understood. While a few reports indicate that the presence of NO in tumor cells or their microenvironment is detrimental to tumor cell survival and consequently their metastatic ability, a large body of clinical and experimental data suggest a promoting role of NO in tumor progression and metastasis. We suggest that tumor cells capable of very high levels of NO production die in vivo, and those producing or exposed to lower levels of NO, or capable of resisting NO-mediated injury undergo a clonal selection because of their survival advantage; they also utilize certain NO-mediated mechanisms for promotion of growth, invasion and metastasis. The possible mechanism(s) are: (a) a stimulatory effect on tumor cell invasiveness, (b) a promotion of tumor angiogenesis and blood flow in the tumor neovasculature, and (c) a suppression of host anti-tumor defense. In this review, we discuss these mechanisms on the basis of data derived from experimental models, in particular, a mouse mammary tumor model in which the expression of eNOS by tumor cells is positively correlated with invasive and metastatic abilities. Tumor-derived NO was shown to promote tumor cell invasiveness and angiogenesis. The invasion-stimulating effects of NO were due to an upregulation of matrix metalloproteases and a downregulation of their natural inhibitors. Treatment of tumor-bearing mice with NO-blocking agents reduced the growth and vascularity of primary tumors and their spontaneous metastases. We propose that selected NO-blocking drugs may be useful in treating certain human cancers either as single agents or as a part of combination therapies.     

Pulmonary and hepatic gene expression following cecal ligation and puncture: monophosphoryl lipid A prophylaxis attenuates sepsis-induced cytokine and chemokine expression and neutrophil infiltration

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1beta, IL-6, gamma interferon (IFN-gamma), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-gamma-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-alpha, MCP-1, MIP-1alpha, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.     

Induction of iNOS gene expression by monophosphoryl lipid A: a pharmacological approach for myocardial adaptation to ischemia

PURPOSE: Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)-like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women. METHODS: Lymphocyte proliferation assay and 35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. RESULTS: The rate of incorporation of 35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. CONCLUSIONS: The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.     

Comparison of angiotensin converting enzyme and renin inhibition in rats following myocardial infarction

Renal and systemic hemodynamics were studied in rats 1 month after induction of myocardial infarction by ligation of the left coronary artery. The mean arterial pressure, heart rate, and cardiac index were not different from controls, but there were striking elevations in heart weight (p < 0.001), left ventricular end diastolic pressure (p < 0.002), and renal vascular resistance (p < 0.01). Renal blood flow and the percent of cardiac output perfusing the kidneys were reduced by 18% (p < 0.01) and 14% (p < 0.01), respectively. Acute angiotensin inhibition was studied at a dose of the converting enzyme inhibitor, enalapril, or the renin inhibitor, CP71362, that lowered the mean arterial pressure by 15 mm Hg in normal rats. In normal rats, enalapril and CP71362 were without effect on renal blood flow (RBF), renal vascular resistance (RR), and RBF as a percent of cardiac output. However, in rats with myocardial infarction, enalapril and CP71362 increased the RBF and RBF as a percent of cardiac output and lowered the RR to levels similar to normal controls (p < 0.02). Enalapril and CP71362 were equally effective in reducing the left ventricular end-diastolic pressure and total peripheral resistance in rats with myocardial infarction. These data demonstrate significant intrarenal vasoconstriction following myocardial infarction in the absence of detectable changes in mean arterial pressure or cardiac index. Converting enzyme inhibition or renin inhibition had similar beneficial effects on cardiorenal function, suggesting that both classes of compounds act by a similar mechanism to improve renal hemodynamics in congestive heart failure.     

I/D polymorphism of angiotensin converting enzyme gene and myocardial infarction

The DD genotype of angiotensin converting enzyme gene has been reported to be a risk factor for myocardial infarction. However, this association has not been confirmed in some study populations. We hypothesized that the discrepancies between these studies may be due to variations in the definition of ischemic heart diseases. According to the genotype of the ACE gene, we analyzed the profiles of 320 patients who underwent coronary angiography for suspected ischemic heart disease. We found that the II genotype of the ACE gene was associated with a longer period of time between the first anginal pain and the onset of myocardial infarction. Because higher ACE has been reported to be associated with higher plasminogen activator inhibitor-1 activity, our observation suggests that the genotype of the ACE gene is a marker of fibrinolytic activity.     

Serotype-dependent induction of pulmonary neutrophilia and inflammatory cytokine gene expression by reovirus

Reovirus type 3 Dearing (T3D) causes a prominent neutrophil influx, substantially greater than seen with reovirus type 1 Lang (T1L) in a rat model of viral pneumonia. We sought to measure reovirus-mediated increases in chemokine mRNA expression in pulmonary cells. We found that the neutrophilia induced by T1L and T3D infection in vivo correlated directly with increased levels of chemokine mRNA expression in T3D-infected compared with those of T1IL-infected lungs. In vitro, reovirus-infected normal alveolar macrophages (AMs) and the rat AM cell line NR8383 expressed greater levels of macrophage inflammatory protein 2, KC, and tumor necrosis factor alpha mRNA. A synergism between reovirus and lipopolysaccharide was also detected for macrophage inflammatory protein 2 and KC mRNA expression. Tumor necrosis factor protein secretion was also increased to a greater extent by T3D than by T1L in primary rat AMs and the NR8383 cells. We conclude that the virus-mediated inflammatory cytokine induction suggests a role for these cytokines in the neutrophil influx observed in the rat reovirus pneumonia model.     

Induction of acute phase gene expression by brain irradiation

PURPOSE: To investigate the in vivo acute phase molecular response of the brain to ionizing radiation. METHODS AND MATERIALS: C3Hf/Sed/Kam mice were given midbrain or whole-body irradiation. Cerebral expression of interleukins (IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6), interferon (IFN-gamma), tumor necrosis factors (TNF-alpha and TNF-beta), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), alpha 1-antichymotrypsin (EB22/5.3), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay. The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined. RESULTS: Levels of TNF-alpha, IL-1 beta, ICAM-1, EB22/5.3 and to a lesser extent IL-1 alpha and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain. Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy. Most responses were rapid, peaking within 4-8 h, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsided. Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely. Dexamethasone was more inhibitory than pentoxifylline at the doses chosen. CONCLUSIONS: The initial response of the brain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy. This mechanism explains the beneficial effects of the clinical use of steroids in such circumstances.