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Biomedical subjects

Paul Kubes

Publications and source records attributed to Paul Kubes.

At least 19 recordsLinked to original sources
PubMed2003

Complexity of inducible nitric oxide synthase: cellular source determines benefit versus toxicity.

BACKGROUND: Inducible nitric oxide synthase (iNOS) has been shown to have both beneficial and detrimental effects in sepsis. We focused on a single organ, the heart, and used 2 distinct cell types that express iNOS-the cardiac myocyte and the infiltrating neutrophil-to study the distinct functional effects of iNOS derived from heterogeneous cellular sources. METHODS AND RESULTS: In the first series of experiments, extravascular neutrophils were exposed to isolated single endotoxemic cardiac myocytes. Adhesion of wild-type neutrophils caused a rapid decrease in myocyte shortening and a concomitant increase in neutrophil-derived intracellular oxidative stress within the myocytes that was not observed with neutrophils from iNOS-deficient animals. We previously demonstrated that neutrophil-derived superoxide was essential for myocyte dysfunction; however, superoxide production was not compromised in the iNOS-deficient neutrophils. Because both superoxide and NO were essential for the neutrophil dysfunction, we probed for but could not detect any peroxynitrite assessed by detection of nitrotyrosine. There was a significant increase in length shortening in response to beta-adrenergic stimulation of wild-type myocytes. Surprisingly, myocyte iNOS activity was essential rather than detrimental for the development of beta-adrenergic receptor-mediated increases in shortening in endotoxemic iNOS-deficient myocytes. CONCLUSIONS: These results demonstrate that iNOS, when expressed in isolated cardiac myocytes, can regulate the response to beta-adrenergic stimulation during sepsis. However, as the neutrophils migrate in proximity to myocytes, iNOS now becomes essential for the ability of neutrophils to damage myocytes. These findings demonstrate that cellular source strongly modulates the beneficial and detrimental effect of iNOS.

Animals↗
PubMed2003

Measuring chemotaxis and chemokinesis: the under-agarose cell migration assay.

Chemotaxis is the primary mechanism by which cell movements are directed within multicellular organisms, and it is a major component of embryonic development, wound healing, and immune responses. Chemotaxis involves a complex cascade of events--formation of signaling complexes, receptor polarization, adhesion molecule activation, and cytoskeletal reorganization. Previous assay methods were limited in several ways that reduced users' abilities to obtain quantitative data or to control conditions precisely. We describe a unique chemotactic assay that can incorporate multiple chemotactic gradients in different spatial and temporal combinations. In addition, this assay is easily adapted for live-cell imaging and fluorescent microscopy. With its relative simplicity, flexibility, and precision, this method is a key tool for the study of cellular chemotactic responses and the signaling processes underlying them.

Cell Movement↗
PubMed2003

Human fractalkine mediates leukocyte adhesion but not capture under physiological shear conditions; a mechanism for selective monocyte recruitment.

Fractalkine is a unique chemokine possessing a long mucin-like stalk and a transmembrane region that has been proposed to act as an adhesion molecule. We investigated the ability of fractalkine to recruit leukocytes from whole blood, using an immobilized fractalkine fusion protein in the parallel-plate flow-chamber assay. Significant adhesion of leukocytes to fractalkine peaked at 2 dynes/cm(2) but was minimal at 10 dynes/cm(2). In contrast, VCAM-1 could recruit cells from whole blood at 10 dynes/cm(2). Co-immobilization of fractalkine and VCAM-1 at 10 dynes/cm(2) resulted in a twofold increase in adherent cells compared with VCAM-1 alone, suggesting that fractalkine can mediate adhesion at high shear if combined with a molecule that can mediate leukocyte tethering. Pretreatment of blood with pertussis toxin eliminated this increase in adhesion, implicating intracellular signaling in fractalkine-mediated mechanisms of adhesion to co-immobilized fractalkine/VCAM-1. Analysis of the cell types recruited to fractalkine alone at low shear, or to fractalkine and VCAM-1 at 10 dynes/cm(2), revealed that monocytes were recruited to fractalkine with the highest specificity. In conclusion, fractalkine is unlikely to act alone at shear forces found in most vascular beds where it most likely co-operates with tethering molecules, e.g. VCAM-1, in the recruitment of monocytes.

Alkaline Phosphatase↗
PubMed2003

Anti-adhesive effect of nitric oxide on Plasmodium falciparum cytoadherence under flow.

Nitric oxide (NO) is widely known to inhibit platelet and leukocyte adhesion to endothelium through its regulatory effect on adhesion molecule expression. The objective of the present study was to investigate if NO affects the cytoadherence of Plasmodium falciparum-infected erythrocytes (IRBCs) to human microvascular endothelium (HDMECs) under flow conditions in vitro. The effect of endogenous NO was studied using the NO synthase inhibitor L-N(G)-nitro-arginine-methyl-ester (L-NAME). Treatment of HDMECs with 3 mmol/L of L-NAME for 4 hours significantly enhanced IRBC adhesion and the effect could be reversed by an anti-P-selectin but not an anti-VCAM-1 antibody. The effect of exogenous NO on cytoadherence was studied by using the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PPN). PPN (300 micro mol/L) treatment reduced the number of adherent IRBCs on resting HDMECs by down-regulating basal ICAM-1 expression, and on tumor necrosis factor-alpha-stimulated HDMECs by inhibition of VCAM-1 induction and down-regulation of ICAM-1 expression. The inhibitory effect of PPN on tumor necrosis factor-alpha-induced VCAM-1 expression at 24 hours was evident when the NO donor was added for as short as 2 hours. These findings suggest that NO may be protective against P. falciparum infection by inhibiting cytoadherence, and underscore the therapeutic potential of NO in the treatment of severe falciparum malaria.

Adult↗
PubMed2003

L-selectin: an emerging player in chemokine function.

The emigration of leukocytes across the blood-endothelium barrier and their subsequent transmigration through the interstitium is a complex process that is vital for maintaining the efficiency of the body's innate and adaptive immunity. The chemokines, a family of low-molecular-weight chemoattractant cytokines, are well recognized to be key players in this process. However, recent investigations have highlighted an important role played by the selectin family of adhesion molecules in enhancing chemokine functions. This review summarizes the in vitro and in vivo studies that support this growing notion. It discusses chemotaxis in the context of the phosphoinositide 3-kinase and p38 mitogen-activated protein kinase pathways, and their relation to several chemoattractants (i.e., interleukin-8, leukotriene-B(4), formyl-methionyl-leucyl-phenylalanine, keratinocyte-derived cytokine, and macrophage inflammatory protein-2), the possible role played by L-selectin, and finally how chemotaxis can be altered in different inflammatory settings, such as lipopolysaccharide-mediated endotoxemia or chronic vasculitis.

Animals↗
PubMed2003

The physiology of S-nitrosothiols: carrier molecules for nitric oxide.

Recent work has demonstrated that inhalation of nitric oxide (NO) can impact the peripheral vasculature, suggesting that an NO-stabilizing moiety may exist in vivo. One possibility is the formation of S-nitrosothiols, which extend the half-life of NO manyfold. In this review, we provide evidence that S-nitrosothiols exist in the vasculature, particularly during NO inhalation. The potential biochemical pathways that have been proposed for the formation of these products are also summarized. Finally, we highlight the limited evidence for the role that these potent vasodilating molecules may play as physiologically and therapeutically important regulators of the vascular system.

Animals↗
PubMed2003

The future of GI and liver research: editorial perspectives: II. Modulating leukocyte recruitment to splanchnic organs to reduce inflammation.

A hallmark feature of intestinal inflammation is the recruitment and extravasation of numerous cell types from the blood to the afflicted site. Much of what we know about the mechanisms of leukocyte recruitment to splanchnic organs comes from an extensive series of studies on neutrophils in the mesenteric microvasculature. In this themes article, we highlight the important findings from these experiments but also emphasize some of the limitations. In fact, there is a growing body of evidence that neutrophil recruitment may be quite different in the mesentery than in other splanchnic organs. For example, the molecular mechanisms underlying neutrophil recruitment into the liver are quite different than the mesentery and are dependent on the type of inflammatory disease. We also discuss the effect of modulating leukocyte recruitment to splanchnic organs in chronic inflammation and emphasize that the approaches that have been successful in acute inflammation may be less effective in such conditions as inflammatory bowel disease (IBD). One obvious reason for this observation is the growing body of evidence to suggest that the initiation and maintenance of IBD is, in part, due to dysregulated or inappropriately activated populations of infiltrating T lymphocyte subsets. Therefore, we also discuss some interesting new approaches to limiting lymphocyte recruitment into the inflamed intestine either by targeting T helper (Th)1 vs. Th2 lymphocytes or perhaps by allowing the recruitment of regulatory T cells. Inhibiting specific adhesion molecules or specific chemokine receptors may work in this regard.

Animals↗
PubMed2003

Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.

The rapid and selective accumulation of neutrophils into the lungs is thought to underlie the pulmonary failure that leads to sepsis-related death. In this study we investigated whether neutrophil TLR4 is important in LPS-induced pulmonary neutrophil recruitment by creating chimeric mice (transferring bone marrow between TLR4(+/+) and TLR4(-/-) mice). In TLR4(+/+) mice receiving TLR4(-/-) bone marrow, 6 weeks after transplant TLR4 was absent in all circulating leukocytes as well as in resident macrophages (these mice were termed LeukocyteTLR4(-/-)), and these cells were completely nonresponsive to LPS. In TLR4(-/-) mice receiving TLR4(+/+) bone marrow, endothelial cells but not leukocytes were deficient in TLR4 (EndotheliumTLR4(-/-)). Surprisingly, systemic LPS (0.5 mg/kg) induced a dramatic increase in neutrophil sequestration into the lungs of LeukocyteTLR4(-/-) mice over the first 4 hours. Concomitantly, numbers of circulating leukocytes decreased by 90%. By contrast, EndotheliumTLR4(-/-) mice showed very little increase in neutrophil sequestration in the lungs, suggesting that endothelium rather than leukocyte TLR4 was important. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte-endothelial cell interactions in LPS-treated EndotheliumTLR4(-/-) mice than in LPS-treated LeukocyteTLR4(-/-) mice. This is consistent with less sequestration of leukocytes into the lungs of EndotheliumTLR4(-/-) mice. In conclusion, our data challenge the view that LPS directly activates neutrophils to trap in lungs and suggest a far more important role than previously appreciated for the endothelial cells.

Animals↗
PubMed2003

Molecular mechanisms of leukocyte recruitment: organ-specific mechanisms of action.

Leukocyte recruitment in the microcirculation is a critical process underlying inflammatory responses in tissues. The mechanism of recruitment is summarized by the generally accepted paradigm: selectin-mediated leukocyte rolling, subsequent integrin activation, followed by integrin-mediated firm adhesion. This simple paradigm may not be able to explain the leukocyte recruitment mechanisms in some organs including the liver and brain. Recent studies suggested that these organs have their own leukocyte recruitment paradigms in acute and even chronic inflammation. The combination of unique hemodynamic patterns and specific structural and functional features of the vessels and endothelium in liver may dictate and select the specific patterns of leukocyte recruitment in this organ. In the brain microvasculature, where shears are high and adhesion molecule expression low, platelets may play an important role as a bridge between the leukocytes and endothelium.

Animals↗
PubMed2002

P38 MAPK: critical molecule in thrombin-induced NF-kappa B-dependent leukocyte recruitment.

Thrombin-stimulated endothelium synthesizes numerous adhesion molecules to recruit leukocytes; however, it is unknown which intracellular pathways are responsible for this event. A recent report from our laboratory has shown that thrombin induces E-selectin expression and that blocking nuclear factor-kappa B (NF-kappa B) activity partially blocked both E-selectin expression (60%) and leukocyte recruitment. In this study, we systematically assessed the importance of p38 MAPK in thrombin-induced NF-kappa B activation and E-selectin-dependent leukocyte recruitment. Thrombin caused phosphorylation of p38 MAPK, its substrate ATF-2, and JNK MAPK, but not ERK MAPK. The p38 MAPK inhibitors, SKF86002 and SB-203580 only reduced ATF-2 activity. We treated human umbilical vein endothelial cells with SKF86002, 1 h before thrombin stimulation, and noted inhibition of NF-kappa B mobilization and complete inhibition of leukocyte rolling and adhesion in a laminar flow chamber. Significant inhibition of leukocyte recruitment and E-selectin expression was also observed with SB-203580. SKF86002 did not affect other systems, including tumor necrosis factor-alpha-induced E-selectin-dependent leukocyte recruitment. Moreover, thrombin-induced rapid mobilization of P-selectin from Weibel Palade bodies was not p38 MAPK dependent. These data suggest that thrombin induces p38 MAPK activation, which leads to NF-kappa B mobilization to the nucleus and causes the upregulation of E-selectin and subsequent leukocyte recruitment.

Adenosine Triphosphate↗
PubMed2002

An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients.

Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium to arrive at a site of infection. By necessity, in the setting of multiple chemoattractants, the neutrophils must prioritize, favoring end target chemoattractants (e.g., fMLP and C5a) emanating from the site of infection over intermediary endogenous chemoattractants (e.g., IL-8 and LTB4) encountered en route to sites of infection. In this study, we propose a hierarchical model of two signaling pathways mediating the decision-making process of the neutrophils, which allows end target molecules to dominate over intermediary chemoattractants. In an under agarose assay, neutrophils predominantly migrated toward end target chemoattractants via p38 MAPK, whereas intermediary chemoattractant-induced migration was phosphoinositide 3-kinase (PI3K)/Akt dependent. When faced with competing gradients of end target and intermediary chemoattractants, Akt activation was significantly reduced within neutrophils, and the cells migrated preferentially toward end target chemoattractants even at 1/1,000th that of intermediary chemoattractants. End target molecules did not require chemotactic properties, since the p38 MAPK activator, LPS, also inhibited Akt and prevented migration to intermediary chemoattractants. p38 MAPK inhibitors not only reversed this hierarchy, such that neutrophils migrated preferentially toward intermediary chemoattractants, but also allowed neutrophils to be drawn out of a local end target chemoattractant environment and toward intermediary chemoattractants unexpectedly in an exaggerated (two- to fivefold) fashion. This was entirely related to significantly increased magnitude and duration of Akt activation. Finally, end target chemoattractant responses were predominantly Mac-1 dependent, whereas nondominant chemoattractants used primarily LFA-1. These data provide support for a two pathway signaling model wherein the end target chemoattractants activate p38 MAPK, which inhibits intermediary chemoattractant-induced PI3K/Akt pathway, establishing an intracellular signaling hierarchy.

Calcium↗
PubMed2002

Recombinant PfEMP1 peptide inhibits and reverses cytoadherence of clinical Plasmodium falciparum isolates in vivo.

The parasite ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and host endothelial receptors represent potential targets for antiadhesive therapy for cytoadherence. In the present study, the major host receptor CD36 was targeted in vitro and in vivo with a recombinant peptide, PpMC-179, corresponding to the minimal CD36-binding domain from the cysteine-rich interdomain region 1 (CIDR1) within the MCvar1 PfEMP1. The in vitro inhibitory effect of PpMC-179 on human dermal microvascular endothelial cells (HDMECs) expressing multiple relevant adhesion molecules was investigated using a parallel-plate flow chamber. Pretreatment of endothelial monolayers with PpMC-179 (2 microM) inhibited the adhesion of infected erythrocytes (IRBCs) from all clinical isolates tested by 84.4% on resting and 62.8% on tumor necrosis factor alpha (TNF-alpha)-stimulated monolayers. Adhesion to stimulated cells was further inhibited (90.4%) when PpMC-179 was administered with an inhibitory anti-intercellular adhesion molecule 1 (ICAM-1) monoclonal antibody 84H10 (5 microg/mL). To determine the in vivo effectiveness of PpMC-179, we used a human/severe combined immunodeficiency (SCID) mouse chimeric model that allowed direct visualization of cytoadherence on intact human microvasculature. In unstimulated skin grafts, PpMC-179 inhibited adhesion by 86.3% and by 84.6% in TNF-alpha-stimulated skin grafts. More importantly, PpMC-179 administration resulted in the detachment of already adherent IRBCs by 80.7% and 83.3% on resting and stimulated skin grafts, respectively. The antiadhesive effect of PpMC-179 was rapid and sustained in vivo for at least 30 minutes. Our data indicate that targeting cytoadhesion in vivo is feasible and may offer a rapid antimalarial therapy.

Adhesiveness↗
PubMed2002

Lipopolysaccharide-induced leukocyte-endothelial cell interactions: a role for CD14 versus toll-like receptor 4 within microvessels.

The objective of this study was to systematically assess leukocyte-endothelial cell interactions in vivo in response to LPS in CD14-deficient (CD14(-/-)) and Toll-like receptor 4-deficient (TLR4(d); C3H/HeJ) mice. Local injection of LPS (0.05 micro g/kg) into muscle at a concentration that did not cause systemic effects produced a significant reduction in the speed with which leukocytes roll and a substantial increase in leukocyte adhesion and emigration 4 h postinjection. There was no response to LPS in the muscle microvasculature of CD14(-/-) mice or TLR4(d) animals. Systemic LPS induced leukopenia and significant sequestration of neutrophils in lungs in wild-type mice but not in CD14(-/-) or TLR4(d) mice. P-selectin expression was examined in numerous mouse organs using a dual radiolabeling mAb technique. The results revealed a 20- to 50-fold increase in P-selectin expression in response to LPS in all wild-type tissues examined but no response in any TLR4(d) tissues. Surprisingly, there was consistently a partial, significant increase in P-selectin expression in numerous microvasculatures including skin and pancreas, but no increase in P-selectin was detected in lung, muscle, and other organs in CD14(-/-) mice in response to LPS. Next, the skin and muscle microcirculation were visualized using intravital microscopy after systemic LPS treatment, and the results confirmed a CD14-independent mechanism of leukocyte sequestration in skin but not muscle. In summary, our results suggest that the LPS-induced leukocyte sequestration to some tissues is entirely dependent on both CD14 and TLR4 but there are CD14-independent, TLR4-dependent endothelial cell responses in some microvascular beds.

Animals↗
PubMed2002

Overlapping roles of P-selectin and alpha 4 integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is mediated by inflammatory cells recruited from the circulation to the CNS. We used intravital microscopy to investigate the mechanisms of this recruitment. No leukocyte rolling and very little adhesion was observed in healthy control mice. In contrast, both rolling and adhesion was observed in brain postcapillary venules before onset of physical symptoms of EAE. Rolling and adhesion remained elevated for 2 wk and returned to near normal levels by 5 wk postsymptom onset. Consistent with a role for P-selectin in recruitment to the CNS, P-selectin protein was detected in the brains and spinal cords of EAE mice. Expression was highest before symptom onset and decreased over the next 2 wk. The importance of alpha(4) integrin increased with time as anti-alpha(4) integrin blocked approximately 20, 50, and 60% of leukocyte rolling 2 days before disease onset, 5 days and 2 wk postonset of symptoms, respectively, and 85% of rolling 5 wk postsymptoms. Addition of anti-P-selectin to alpha(4) integrin Ab-treated mice blocked all remaining rolling at each time point. Interestingly, however, alpha(4) integrin-mediated rolling appeared to be entirely dependent on P-selectin as anti-P-selectin alone was able to completely block all leukocyte rolling. In the absence of rolling (with P-selectin Ab), a 70% reduction in adhesion was noted. A very similar reduction was seen when mice were treated with alpha(4) integrin-blocking Ab. In conclusion, we describe increased leukocyte trafficking in the brains of EAE mice with important overlapping roles for both P-selectin and alpha(4) integrin in mediating leukocyte-endothelial cell interactions.

Animals↗
PubMed2002

Inducible nitric oxide synthase (iNOS) in endotoxemia: chimeric mice reveal different cellular sources in various tissues.

The aim of these experiments was to determine the contribution of leukocyte-derived iNOS to total iNOS expression induced by lipopolysaccharide (LPS). By transferring bone marrow between iNOS+/+ and iNOS-/- mice, we created chimeric mice in which iNOS expression was limited to either circulating leukocytes (leukocyte-iNOS mice) or parenchymal cells (parenchyma-iNOS mice). Analysis of congenic markers demonstrated that >95% of thymocytes in chimeric mice were of donor origin. Also, following LPS treatment, iNOS mRNA was detectable in blood from leukocyte-iNOS mice but not parenchyma-iNOS mice. Together these findings indicated that the host marrow had been replaced entirely by donor cells. In the lung, at least 50% of the LPS-induced iNOS mRNA was derived from leukocytes, and immunohistochemical analysis indicated that leukocytes were the main source of iNOS protein. In contrast in the liver, colon, and muscle, iNOS expression was derived predominantly from parenchymal cells. This divergence is potentially explained by the high level of leukocyte recruitment to the lung, relative to the other tissues. Plasma levels of NOS byproducts indicated that parenchymal iNOS was the dominant source of systemic iNOS activity. These findings indicate that in tissues other than the lung, parenchymal cells are the principal source of iNOS during endotoxemia.

Animals↗
PubMed2002

Profound differences in leukocyte-endothelial cell responses to lipopolysaccharide versus lipoteichoic acid.

We have investigated the effects of LPS from Escherichia coli, lipoteichoic acid (LTA), and peptidoglycan (PepG) from Staphylococcus aureus, and live S. aureus on leukocyte-endothelial interactions in vivo using intravital microscopy to visualize muscle microvasculature. Systemic vs local administration of LPS induced very different responses. Local administration of LPS into muscle induced significant leukocyte rolling, adhesion, and emigration in postcapillary venules at the site of injection. LPS given systemically dramatically dropped circulating leukocyte counts and increased neutrophils in the lung. However, the drop in circulating leukocytes was not associated with leukocyte sequestration to the site of injection (peritoneum) nor to peripheral microvessels in muscles. Unlike LPS, various preparations of LTA had no systemic and very minor local effect on leukocyte-endothelial interactions, even at high doses and for prolonged duration. LPS, but not LTA, potently activated human endothelium to recruit leukocytes under flow conditions in vitro. Endothelial adhesion molecule expression was also increased extensively with LPS, but not LTA. Interestingly, systemic administration of live S. aureus induced leukocyte-endothelial cell responses similar to LPS. PepG was able to induce leukocyte-endothelial interactions in muscle and peritoneum, but had no effect systemically (no increase in neutrophils in lungs and no decrease in circulating neutrophil counts). These results demonstrate that: 1) LPS has potent, but divergent local and systemic effects on leukocyte-endothelial interactions; 2) S. aureus can induce a systemic response similar to LPS, but this response is unlikely to be due to LTA, but more likely to be mediated in part by PepG.

Animals↗
PubMed2002

The role of selectins and integrins in adenovirus vector-induced neutrophil recruitment to the liver.

Adenovirus vectors for human gene therapy induce early host inflammatory responses in transduced tissues that limit gene transfer efficiency and can result in significant morbidity. The present study aimed to elucidate the cellular mechanisms underlying the acute inflammation induced by adenovirus vectors in the liver. Leukocyte rolling and adhesion in response to an intravenously administered adenovirus vector was examined by intravital microscopy in mouse liver. Adenovirus vectors significantly increased leukocyte rolling and adhesion in the postsinusoidal venules within minutes of transduction. Unlike other inflammatory states in the liver, no leukocyte retention was seen in the sinusoids in response to adenovirus vector administration. Inhibition of P-selectin, alpha(4)-integrin, and E-selectin was necessary to completely block leukocyte rolling and subsequent adhesion. The administration of an anti-alpha(4)-integrin antibody alone significantly reduced leukocyte adhesion. In contrast, adenovirus vector-induced leukocyte adhesion was unchanged in CD18-knockout mice. Depletion of circulating neutrophils eliminated leukocyte rolling and adhesion in response to adenovirus vector transduction in the liver. In conclusion, adenovirus vectors induce rapid neutrophil-mediated inflammation in the post-sinusoidal venules by selectins and alpha(4)-integrin but surprisingly not by CD18.

Adenoviridae↗