Immunomodulatory Properties of Vitamin D in the Intestinal and Respiratory Systems.

Vitamin D plays a crucial role in modulating the innate immune response by interacting with its intracellular receptor, VDR. In this review, we address vitamin D/VDR signaling and how it contributes to the regulation of intestinal and respiratory microbiota. We additionally review some components of the innate immune system, such as the barrier function of the pulmonary and intestinal epithelial membranes and secretion of mucus, with their respective modulation by vitamin D. We also explore the mechanisms by which this vitamin D/VDR signaling mounts an antimicrobial response through the transduction of microbial signals and the production of antimicrobial peptides that constitute one of the body's first lines of defense against pathogens. Additionally, we highlight the role of vitamin D in clinical diseases, namely inflammatory bowel disease and acute respiratory distress syndrome, where excessive inflammatory responses and dysbiosis are hallmarks. Increasing evidence suggests that vitamin D supplementation may have potentially beneficial effects on those diseases.

Lung-Centric Inflammation of COVID-19: Potential Modulation by Vitamin D.

SARS-CoV-2 infects the respiratory tract and leads to the disease entity, COVID-19. Accordingly, the lungs bear the greatest pathologic burden with the major cause of death being respiratory failure. However, organs remote from the initial site of infection (e.g., kidney, heart) are not spared, particularly in severe and fatal cases. Emerging evidence indicates that an excessive inflammatory response coupled with a diminished antiviral defense is pivotal in the initiation and development of COVID-19. A common finding in autopsy specimens is the presence of thrombi in the lungs as well as remote organs, indicative of immunothrombosis. Herein, the role of SARS-CoV-2 in lung inflammation and associated sequelae are reviewed with an emphasis on immunothrombosis. In as much as vitamin D is touted as a supplement to conventional therapies of COVID-19, the impact of this vitamin at various junctures of COVID-19 pathogenesis is also addressed.

COVID-19: Lung-Centric Immunothrombosis.

The respiratory tract is the major site of infection by SARS-CoV-2, the virus causing COVID-19. The pulmonary infection can lead to acute respiratory distress syndrome (ARDS) and ultimately, death. An excessive innate immune response plays a major role in the development of ARDS in COVID-19 patients. In this scenario, activation of lung epithelia and resident macrophages by the virus results in local cytokine production and recruitment of neutrophils. Activated neutrophils extrude a web of DNA-based cytoplasmic material containing antimicrobials referred to as neutrophil extracellular traps (NETs). While NETs are a defensive strategy against invading microbes, they can also serve as a nidus for accumulation of activated platelets and coagulation factors, forming thrombi. This immunothrombosis can result in occlusion of blood vessels leading to ischemic damage. Herein we address evidence in favor of a lung-centric immunothrombosis and suggest a lung-centric therapeutic approach to the ARDS of COVID-19.

Vitamin D and intestinal homeostasis: Barrier, microbiota, and immune modulation.

Vitamin D plays a pivotal role in intestinal homeostasis. Vitamin D can impact the function of virtually every cell in the gut by binding to its intracellular receptor (VDR) and subsequently transcribing relevant genes. In the lumen, the mucus layer and the underlying epithelium serve to keep resident microbiota at bay. Vitamin D ensures an appropriate level of antimicrobial peptides in the mucus and maintains epithelial integrity by reinforcing intercellular junctions. Should bacteria penetrate the epithelial layer and enter the interstitium, immune sentinel cells (e.g. macrophages, dendritic cells, and innate lymphoid cells) elicit inflammation and trigger the adaptive immune response by activating Th1/Th17 cells. Vitamin D/VDR signaling in these cells ensures clearance of the bacteria. Subsequently, vitamin D also quiets the adaptive immune system by suppressing the Th1/Th17 cells and favoring Treg cells. The importance of vitamin D/VDR signaling in intestinal homeostasis is evidenced by the development of a chronic inflammatory state (e.g. IBD) when this signaling system is disrupted.

Reperfusion therapy-What's with the obstructed, leaky and broken capillaries?

Microvascular dysfunction is well established as an early and rate-determining factor in the injury response of tissues to ischemia and reperfusion (I/R). Severe endothelial cell dysfunction, which can develop without obvious morphological cell injury, is a major underlying cause of the microvascular abnormalities that accompany I/R. While I/R-induced microvascular dysfunction is manifested in different ways, two responses that have received much attention in both the experimental and clinical setting are impaired capillary perfusion (no-reflow) and endothelial barrier failure with a transition to hemorrhage. These responses are emerging as potentially important determinants of the severity of the tissue injury response, and there is growing clinical evidence that they are predictive of clinical outcome following reperfusion therapy. This review provides a summary of animal studies that have focused on the mechanisms that may underlie the genesis of no-reflow and hemorrhage following reperfusion of ischemic tissues, and addresses the clinical evidence that implicates these vascular events in the responses of the ischemic brain (stroke) and heart (myocardial infarction) to reperfusion therapy. Inasmuch as reactive oxygen species (ROS) and matrix metalloproteinases (MMP) are frequently invoked as triggers of the microvascular dysfunction elicited by I/R, the potential roles and sources of these mediators are also discussed. The available evidence in the literature justifies the increased interest in the development of no-reflow and hemorrhage in heart and brain following reperfusion therapy, and suggests that these vascular events may be predictive of poor clinical outcome and warrant the development of targeted treatment strategies.

Reperfusion injury and reactive oxygen species: The evolution of a concept.

Reperfusion injury, the paradoxical tissue response that is manifested by blood flow-deprived and oxygen-starved organs following the restoration of blood flow and tissue oxygenation, has been a focus of basic and clinical research for over 4-decades. While a variety of molecular mechanisms have been proposed to explain this phenomenon, excess production of reactive oxygen species (ROS) continues to receive much attention as a critical factor in the genesis of reperfusion injury. As a consequence, considerable effort has been devoted to identifying the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Of the potential ROS sources described to date, xanthine oxidase, NADPH oxidase (Nox), mitochondria, and uncoupled nitric oxide synthase have gained a status as the most likely contributors to reperfusion-induced oxidative stress and represent priority targets for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. Although all four enzymatic sources are present in most tissues and are likely to play some role in reperfusion injury, priority and emphasis has been given to specific ROS sources that are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The possibility that multiple ROS sources contribute to reperfusion injury in most tissues is supported by evidence demonstrating that redox-signaling enables ROS produced by one enzymatic source (e.g., Nox) to activate and enhance ROS production by a second source (e.g., mitochondria). This review provides a synopsis of the evidence implicating ROS in reperfusion injury, the clinical implications of this phenomenon, and summarizes current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue.

Role of reactive oxygen and nitrogen species in the vascular responses to inflammation.

Inflammation is a complex and potentially life-threatening condition that involves the participation of a variety of chemical mediators, signaling pathways, and cell types. The microcirculation, which is critical for the initiation and perpetuation of an inflammatory response, exhibits several characteristic functional and structural changes in response to inflammation. These include vasomotor dysfunction (impaired vessel dilation and constriction), the adhesion and transendothelial migration of leukocytes, endothelial barrier dysfunction (increased vascular permeability), blood vessel proliferation (angiogenesis), and enhanced thrombus formation. These diverse responses of the microvasculature largely reflect the endothelial cell dysfunction that accompanies inflammation and the central role of these cells in modulating processes as varied as blood flow regulation, angiogenesis, and thrombogenesis. The importance of endothelial cells in inflammation-induced vascular dysfunction is also predicated on the ability of these cells to produce and respond to reactive oxygen and nitrogen species. Inflammation seems to upset the balance between nitric oxide and superoxide within (and surrounding) endothelial cells, which is necessary for normal vessel function. This review is focused on defining the molecular targets in the vessel wall that interact with reactive oxygen species and nitric oxide to produce the characteristic functional and structural changes that occur in response to inflammation. This analysis of the literature is consistent with the view that reactive oxygen and nitrogen species contribute significantly to the diverse vascular responses in inflammation and supports efforts that are directed at targeting these highly reactive species to maintain normal vascular health in pathological conditions that are associated with acute or chronic inflammation.

Role of intestinal lymphatics in interstitial volume regulation and transmucosal water transport.

Two of the principal functions of intestinal lymphatics are to assist in the maintenance of interstitial volume within relatively normal limits during alterations in capillary filtration (e.g., acute portal hypertension) and the removal of absorbed water and chylomicrons. The contribution of lymphatics to the prevention of interstitial overhydration or dehydration during alterations in transcapillary filtration is similar in the small intestine and colon. While the lymphatics of the small intestine contribute substantially to the removal of absorbed water (particularly at low and moderate absorption rates), the contribution of colonic lymphatics to the removal of the fluid absorbate is negligible. This difference is attributed to the relative caliber and location of lymphatics in the mucosal layer of the small and large intestines. In the small intestine, large lacteals lie in close proximity to transporting epithelium, while colonic lymph vessels are rather sparse and confined to the basal portion of the mucosa. In the small intestine, the lymphatics assume a more important role in removing absorbed water during lipid absorption than during glucose absorption.

The alarmin cytokine, high mobility group box 1, is produced by viable cardiomyocytes and mediates the lipopolysaccharide-induced myocardial dysfunction via a TLR4/phosphatidylinositol 3-kinase gamma pathway.

High mobility group box 1 (HMGB1) is an alarmin actively secreted by immune cells and passively released by necrotic nonimmune cells. HMGB1 has been implicated in both cardiac contractile dysfunction and the lethality associated with sepsis/endotoxemia. The aim of the current study was to assess whether viable cardiomyocytes could produce HMGB1 and whether HMGB1 can affect myocardial contractility. LPS was used as a model of sepsis/endotoxemia in mice and isolated cardiac myocytes. LPS increased myocardial expression of HMGB1 in vivo (immunohistochemistry) and production and secretion of HMGB1 by viable cardiac myocytes in vitro (Western). LPS increased the phosphorylation status of PI3Kgamma in cardiac myocytes, an effect not observed in TLR4(-/-) myocytes. Genetic (PI3Kgamma(-/-)) or pharmacologic (AS605240) blockade of PI3Kgamma ameliorated the LPS-induced 1) cardiomyocyte production and secretion of HMGB1 in vitro and 2) HMGB1 expression in the myocardium in vivo. The LPS-induced depression of myocardial contractility was prevented by the HMGB1 antagonist, A-box. Genetic (PI3Kgamma(-/-)) or pharmacologic (AS605240) blockade of PI3Kgamma ameliorated the LPS-induced decrease in myocardial contractility. No evidence of inflammatory infiltrate was noted in any of the in vivo studies. The findings of the current study indicate that 1) LPS can induce HMGB1 secretion by viable cardiac myocytes through a TLR4/PI3Kgamma signaling pathway, and 2) HMGB1 plays a role in the LPS-induced myocardial contractile dysfunction. The results of the current study also have broader implications (i.e., that viable parenchymal cells, such as cardiac myocytes, participate in the alarmin response).

NADPH oxidase contributes to conversion of cardiac myocytes to a proinflammatory phenotype in sepsis.

It has been reported that polymorphonuclear leukocyte (PMN) infiltration into the myocardial interstitium is involved in sepsis-induced myocardial dysfunction. The aim of this study was to evaluate the role of NADPH oxidase in the sepsis-induced conversion of cardiomyocytes to a proinflammatory phenotype. Using an in vitro approach we evaluated the role of NADPH oxidase in cardiomyocyte CXC chemokine production and its ability to promote PMN transendothelial migration under septic conditions. Treatment of cardiac myocytes with septic plasma (1) activated NADPH oxidase (p47phox phosphorylation) and increased its activity (O(2)(-) production) and (2) converted them to a proinflammatory phenotype; both effects were prevented by blockade of NADPH oxidase. NF-kappaB nuclear translocation was increased in cardiomyocytes conditioned with septic plasma, a response prevented by blockade of NADPH oxidase. The increase in NF-kappaB activation/translocation was associated with phosphorylation of both IKK and the p65 subunit of NF-kappaB. Blockade of NADPH oxidase prevented phosphorylation of IKK, but not p65. Blockade approaches indicated that p38 MAP kinase (previously implicated in NF-kappaB activation) did not play a role in the NADPH oxidase pathway, either upstream or downstream. Collectively, the results of this study and those of previous reports indicate that the conversion of cardiomyocytes to a proinflammatory phenotype in sepsis involves two distinct pathways: NADPH oxidase-mediated phosphorylation of IKK and p38 MAP kinase-mediated phosphorylation of p65.

Alveolar macrophages from septic mice promote polymorphonuclear leukocyte transendothelial migration via an endothelial cell Src kinase/NADPH oxidase pathway.

Alveolar macrophages (AMphi) have been implicated in the polymorphonuclear leukocyte (PMN) recruitment to the lungs during sepsis. Using an in vivo murine model of sepsis (feces in the peritoneum), we show that peritonitis leads to increased activation of AMphi and PMN migration into pulmonary alveoli. To assess cellular mechanisms, an in vitro construct of the pulmonary vascular-interstitial interface (murine AMphi, pulmonary endothelial cells, and PMN) and a chimera approach were used. Using immunologic (Abs) and genetic blockade (CXCR2-deficient AMphi), we show that CXC chemokines in septic plasma are responsible for the activation of AMphi. The activated AMphi can promote PMN transendothelial migration, even against a concentration gradient of septic plasma, by generating platelet-activating factor and H(2)O(2). Platelet-activating factor/H(2)O(2) induce an oxidant stress in the adjacent endothelial cells, an event that appears to be a prerequisite for PMN transendothelial migration, since PMN migration is abrogated across Cu/Zn-superoxide dismutase overexpressing endothelial cells. Using gp91-deficient endothelial cells, we show that NADPH oxidase plays an important role in the AMphi-induced PMN transendothelial migration. Pharmacologic/small interfering RNA blockade of Src kinase inhibits AMphi-induced endothelial NADPH oxidase activation and PMN migration. Collectively, our findings indicate that the PMN transendothelial migration induced by septic AMphi is dependent on the generation of superoxide in endothelial cells via the Src kinase/NADPH oxidase signaling pathway.

Points of control exerted along the macrophage-endothelial cell-polymorphonuclear neutrophil axis by PECAM-1 in the innate immune response of acute colonic inflammation.

PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1(-/-) mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1(-/-) mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (MPhi) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although MPhi activation (NF-kappaB nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1(-/-) MPhi, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1(-/-) PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1(+/+) PMN, PECAM-1(-/-) PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.

Important role of p38 MAP kinase/NF-kappaB signaling pathway in the sepsis-induced conversion of cardiac myocytes to a proinflammatory phenotype.

Septic plasma can convert murine cardiac myocytes to a proinflammatory phenotype. These myocytes 1) have increased nuclear levels of nuclear factor-kappaB (NF-kappaB), 2) release CXC chemokines, and 3) promote polymorphonuclear neutrophil (PMN) transendothelial migration. The purpose of the present study was to evaluate the role of the mitogen-activated protein (MAP) kinases [p38 MAP kinase, extracellular signal-regulated kinase (ERK) 1/2, and c-Jun NH(2)-terminal kinase (JNK)] as upstream intracellular signaling components involved in this phenomenon. Feces-induced peritonitis (FIP) was employed as a model of sepsis. In vitro, cardiac myocytes were treated with plasma (20%) obtained 6 h after either sham (saline) or FIP procedures. Myocyte supernatants were used for 1) detection of the CXC chemokines (enzyme-linked immunosorbent assay) and 2) assessment of their ability to promote PMN transendothelial migration. In vivo, myocardial PMN accumulation was assessed by measuring myeloperoxidase (MPO) activity and function (dF/dt and heart work). Treatment of cardiac myocytes with septic plasma activated p38 MAP kinase and ERK1/2, but not JNK. Blockade approaches (inhibitors or small-interference RNA) indicated that only p38 MAP kinase played a role in the conversion of the myocytes to a proinflammatory phenotype. Time course studies indicated that phosphorylation of p38 MAP kinase preceded the phosphorylation of NF-kappaB p65. Inhibition of p38 MAP kinase (SB-202190) blocked both NF-kappaB p65 phosphorylation and NF-kappaB nuclear translocation. Confirmatory studies in vivo indicated that FIP resulted in an increase in myocardial MPO activity and dysfunction, events reversed by the inhibitor of p38 MAP kinase. Collectively, these data indicate that the cardiomyocyte p38 MAP kinase/NF-kappaB signaling pathway plays an important role in the sepsis-induced conversion of myocytes to a proinflammatory phenotype.

Role of heme oxygenase in the protection afforded skeletal muscle during ischemic tolerance.

OBJECTIVE: Ischemic tolerance (IT) is known to improve resistance to ischemia/reperfusion (I/R)-induced injury; however, the mechanisms remain unknown. The authors hypothesized that induction of heme oxygenase (HO), a heat shock protein, would provide anti-inflammatory benefits during IT, thereby preventing leukocyte-derived I/R injury. METHODS: Male Wistar rats were randomly assigned to sham (n = 4), I/R (n = 9), preconditioning (PC)+I/R (n = 7), chromium mesoporphyrin, to inhibit HO (CrMP; n = 4), or PC+I/R+CrMP (n = 6) groups. PC consisted of 5 cycles of I/R, each lasting 10 min, induced by tightening a tourniquet placed above the greater trochantor of the hindlimb. Twenty-four hours later, the hindlimb underwent 2 h of no-flow ischemia followed by intravital microscopy during 90 min reperfusion to assess capillary perfusion (#/mm), tissue injury (ratio of ethidium bromide to bisbenzimide labeled cells/100 microm2), leukocyte rolling (Lr, #/1000 microm2), and adhesion (La, #/1000 microm2) in postcapillary venules of the extensor digitorum longus (EDL) muscle. RESULTS: In the I/R group, Lr was significantly increased (7.1 +/- 0.4) compared to sham (3.1 +/- 0.4). PC+I/R increased Lr (10.8 +/- 0.72), which was further exacerbated by the removal of HO (14.2 +/- 1.3). La (7.8 +/- 2.0) was significantly increased compared to sham (2.4 +/- 0.9), while PC returned La back to sham levels (1.9 +/- 0.7). Removal of HO activity, via CrMP, had no significant effect on La (3.9 +/- 0.7). However, CrMP removed the protection to microvascular perfusion (I/R = 9.4 +/- 1.1, PC = 16.6 +/- 1.8, sham = 20.5 +/- 2.8, PC+CrMP+I/R = 12.3 +/- 2.3) and prevented protection from ischemia-induced tissue injury. CONCLUSION: The data suggest that HO is an important protective mechanism during IT in skeletal muscle, but such protection was by mechanisms other than altered leukocyte-endothelial cell interaction.

NFkappaB and AP-1 differentially contribute to the induction of Mn-SOD and eNOS during the development of oxidant tolerance.

Exposure of cardiac myocytes to anoxia/reoxygenation (A/R) increases myocyte oxidant stress and converts the myocytes to a proinflammatory phenotype. These oxidant-induced effects are prevented by pretreatment of the myocytes with an oxidant stress (A/R or H2O2) 24 h earlier (oxidant tolerance). Although NF-kappaB and AP-1 (nuclear signaling) and Mn-SOD and eNOS (effector enzymes) have been implicated in the development oxidant tolerance, the precise relationship between the nuclear transcription factors and the effector enzymes in the development of oxidant tolerance has not been defined. Herein, we show that an initial A/R challenge results in nuclear accumulation of both NF-kappaB and AP-1 (EMSA). In addition, blockade of nuclear translocation of NF-kappaB (SN50) or AP-1 (decoy oligonucleotide) prevents the development of oxidant tolerance, i.e., the second A/R challenge produces the same quantitative effects as the initial A/R challenge. In this model, nuclear translocation of both NF-kappaB and AP-1 is required for induction of Mn-SOD, while nuclear translocation of AP-1, but not NF-kappaB, is a prerequisite for induction of eNOS. Collectively, our findings indicate that NF-kappaB and AP-1 work in concert to ensure the induction eNOS and Mn-SOD, which in turn are important for the development of oxidant tolerance.

Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1.

OBJECTIVE: Erythropoietin (EPO) prevents the myocardial dysfunction induced by ischemia/reperfusion (I/R). Since I/R-induced myocardial dysfunction is associated with an acute inflammatory response, we assessed the anti-inflammatory properties of EPO using in vitro and in vivo models of I/R. METHODS: Isolated cardiac myocytes were exposed to anoxia/reoxygenation (A/R; the in vitro counterpart to I/R). Hearts were challenged with I/R in situ. RESULTS: In vitro, A/R increased myocyte oxidant stress and converted the myocytes to a proinflammatory phenotype (these myocytes induced PMN transendothelial migration). Pretreatment of the myocytes with EPO prevented the A/R-induced proinflammatory effects. EPO increased myocyte (1) nuclear translocation of AP-1 (c-fos/c-jun), (2) eNOS, but not iNOS, protein expression, and (3) NO production. An AP-1 "decoy" oligonucleotide prevented the induction of eNOS by EPO and reversed the beneficial effect of EPO. An inhibitor of phosphatidylinostol 3 (PI3)-kinase prevented the nuclear translocation of AP-1 induced by EPO. In vivo, in wild type mice, I/R induced an increase in myocardial MPO activity (indicative of PMN infiltration); an effect prevented by pretreatment of the mice with EPO. This anti-inflammatory effect of EPO was not observed in cardiac specific c-fos(-/-) mice. CONCLUSIONS: Collectively, these findings indicate that EPO can ameliorate the myocardial inflammatory response in both in vitro and in vivo models of I/R. This beneficial effect of EPO is mediated by eNOS-derived NO via a PI3-kinase-dependent activation of AP-1.

Cardiac myocytes activated by septic plasma promote neutrophil transendothelial migration: role of platelet-activating factor and the chemokines LIX and KC.

Cardiac myocytes isolated from rats with peritonitis (cecal ligation and perforation; CLP) promote PMN transendothelial migration. Herein, we assessed (1) the mechanisms involved in cardiac myocyte activation during peritonitis and (2) the means by which these activated myocytes promote PMN transendothelial migration. Plasma obtained from mice subjected to CLP (septic plasma) activated isolated cardiac myocytes as evidenced by (1) increased nuclear levels of nuclear factor-kappaB (NF-kappaB) and (2) their ability to promote PMN migration across endothelial cell monolayers. Pretreatment of septic plasma with an antibody against tumor necrosis factor-alpha (TNF-alpha), but not interleukin-1beta (IL-1beta), blunted the ability of septic plasma to activate the myocytes. However, septic plasma obtained from TNF-alpha-deficient mice could still activate the myocytes; an effect attenuated by an antibody against IL-1beta. If the myocytes were pretreated with a proteasome inhibitor (MG 132) to prevent NF-kappaB activation, the myocyte-induced PMN transendothelial migration was compromised. The activated myocytes released platelet-activating factor (PAF), and myocyte-induced PMN migration was abrogated by a PAF receptor antagonist (WEB 2086). These myocytes also released the CXC chemokines LIX and KC; an event prevented by MG 132. Antibodies against LIX and KC abrogated the myocyte-induced PMN migration. However, LIX and KC, but not PAF, could promote PMN migration when used at concentrations produced by activated myocytes. These observations indicate that TNF-alpha and IL-1beta are, in part, responsible for the ability of septic plasma to activate cardiac myocytes. The activated myocytes promote PMN transendothelial migration, an effect attributable to LIX and KC, and possibly, PAF.

Tumor necrosis factor-alpha-induced cytokine-induced neutrophil chemoattractant-1 (CINC-1) production by rat gastric epithelial cells: role of reactive oxygen species and nuclear factor-kappaB.

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), a counterpart of the human growth-regulated oncogene product (GRO), has been suggested to participate in neutrophil recruitment in an experimental model of gastritis in rat. However, the mechanism(s) involved in regulation of CINC-1 production by the gastric mucosa remains unclear. The aim of this study was to investigate the mechanism(s) of CINC-1 production by rat gastric mucosa in vitro. All experiments were performed using rat normal gastric mucosal cell line (RGM-1). RGM-1s were stimulated with tumor necrosis factor (TNF)-alpha, and CINC-1 mRNA levels (reverse transcription-polymerase chain reaction) and protein secretion (enzyme-linked immunosorbent assay) were assessed. The production of reactive oxygen species (ROS) and nuclear factor (NF)-kappaB activation (translocation to the nuclei) in response to TNF-alpha stimulation was evaluated using fluorescence microscopy in the presence or absence of the inhibitors of mitochondrial electron flow and NF-kappaB activation. Stimulation of RGM-1 cells with TNF-alpha resulted in an increase in intracellular oxidative stress, NF-kappaB translocation to the nuclei, and up-regulation of CINC-1 mRNA and protein, which was prevented by interfering with mitochondria-dependent ROS production and NF-kappaB activation. Taken together, these findings indicate that CINC-1, a counterpart of the human GRO, production by rat gastric epithelial cells in response to TNF-alpha stimulation is an oxidant stress-mediated and NF-kappaB-dependent event.

Delayed preconditioning in cardiac myocytes with respect to development of a proinflammatory phenotype: role of SOD and NOS.

OBJECTIVE: Both superoxide dismutase (SOD) and nitric oxide synthase (NOS) have been implicated in delayed preconditioning (DP) to ischemia/reperfusion (I/R) in the heart. We used isolated cardiac myocytes to test the hypothesis that SOD and NOS may interact in the development of DP. METHODS: Mouse neonatal cardiac myocytes were challenged with anoxia/reoxygenation (A/R; an in vitro counterpart to I/R) and normoxia/normoxia (N/N) served as the control. Two indices of inflammation were measured: oxidant stress (DHR oxidation) and polymorphonuclear leukocyte (PMN) transendothelial migration (cell culture inserts). The role of SOD was assessed using an antisense approach and the role of NOS was assessed using iNOS and eNOS deficient myocytes. RESULTS: Cardiac myocytes exposed to A/R (1) produced more oxidants (intracellular fluorescence emission from 2.0 +/- 0.1 for N/N to 3.0 +/- 0.3 for A/R; P<0.05) and (2) promoted PMN migration (% migration from 8.4 +/- 0.9 for N/N to 14.1 +/- 1.1 for A/R; P<0.05). DP occurred if the myocytes were pretreated with an A/R challenge 24 h earlier. That is, these A/R-induced responses were significantly reduced (fluorescence emission 1.9 +/- 0.1 and % migration 8.4 +/- 0.7; P<0.05 as compared to A/R with no pretreatment). Myocyte Mn-SOD, but not Cu/Zn-SOD, activity increased 24 h after the initial A/R challenge. A Mn-SOD antisense oligonucleotide prevented the development of DP. DP occurred in iNOS, but not eNOS, deficient myocytes. A/R increased mRNA for eNOS, but not iNOS, in wild-type myocytes. A/R increased Mn-SOD protein in both iNOS and eNOS deficient myocytes. However, Mn-SOD activity increased only in iNOS deficient myocytes. CONCLUSIONS: Collectively, these findings suggest that Mn-SOD and eNOS may act in concert in the development of DP in cardiac myocytes.

PMN transendothelial migration decreases nuclear NFkappaB in IL-1beta-activated endothelial cells: role of PECAM-1.

During the systemic inflammatory response, circulating cytokines interact with the vascular endothelium, resulting in activation and nuclear accumulation of the nuclear transcription factor, nuclear factor kappa B (NFkappaB). In turn, NFkappaB transactivates relevant proinflammatory genes, resulting in an amplification of the inflammatory response. Because this scenario is potentially detrimental to the host, mechanisms exist to limit this amplification. Using an in vitro system that mimics the vascular-interstitial interface during inflammation (cell culture inserts), we provide evidence for the existence of a novel negative feedback mechanism on NFkappaB activity. We show that the interleukin 1beta-induced accumulation of nuclear NFkappaB in human umbilical vein endothelial cell monolayers is dramatically reduced when polymorphonuclear leukocytes (PMN) are allowed to migrate across these monolayers. This effect does not appear to be due to PMN-derived elastase or nitric oxide. Fixed PMN (adhere but do not migrate) did not affect nuclear NFkappaB. Furthermore, cross-linking of platelet-endothelial cell adhesion molecule-1 (PECAM-1), but not intercellular adhesion molecule-1, reduces human umbilical vein endothelial cell nuclear NFkappaB induced by interleukin 1beta. Finally, interaction of PMN with PECAM-1-deficient endothelial cells does not reduce nuclear NFkappaB. These observations indicate that engagement of PECAM-1 by emigrating PMN is a pivotal event in this negative feedback on NFkappaB activity.